Citation
The Effect of ozone inhalation on the frequency of chromosome aberrations observed in irradiated Chinese hamsters

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Title:
The Effect of ozone inhalation on the frequency of chromosome aberrations observed in irradiated Chinese hamsters
Creator:
Zelac, Ronald Edward, 1941-
Publication Date:
Copyright Date:
1970
Language:
English
Physical Description:
xi, 115 leaves. : illus. ; 28 cm.

Subjects

Subjects / Keywords:
Animals ( jstor )
Blood ( jstor )
Chromosome aberrations ( jstor )
Chromosomes ( jstor )
Dosage ( jstor )
Inhalation ( jstor )
Ionizing radiation ( jstor )
Lymphocytes ( jstor )
Ozone ( jstor )
Radiation dosage ( jstor )
Dissertations, Academic -- Environmental Engineering Sciences -- UF
Environmental Engineering Sciences thesis Ph. D
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis -- University of Florida, 1970.
Bibliography:
Bibliography: leaves 98-113.
Additional Physical Form:
Also available on World Wide Web
General Note:
Manuscript copy.
General Note:
Vita.

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University of Florida
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University of Florida
Rights Management:
Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
030302219 ( AlephBibNum )
AEP9635 ( NOTIS )
016441103 ( OCLC )

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Full Text







THE EFFECT OF OZONE INHALATION ON

THE FREQUENCY OF CHROMOSOME ABERRATIONS

OBSERVED IN IRRADIATED CHINESE HAMSTERS














By
RONALD EDWARD ZELAC














A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY










UNIVERSITY OF FLORIDA


1970

































Copyright by

Ronald Edward Zelac

1970









































To Gwen











ACKNOWLEDGEMENTS


A great number of people were involved in this research effort

through its many stages. First, and most importantly, I want to thank

the members of my supervisory committee, Dr. Billy Dunavant, chairman,

Dr. Harvey Cromroy, co-chairman, Dr. Herbert Bevis, Dr. Emmett Bolch,

Jr., and,through most of my program, Dr. Roy McCaldin, for their con-

tinued guidance, encouragement, and numerous efforts of my behalf. I

am particularly grateful to Dr. Cromroy who acted as the main techni-

cal advisor in this research from its beginning to its completion and

who made possible much of the technical assistance and financial support

that the research required. Special thanks are extended to Dr. Bolch

for his extra efforts to further the progress of the research and to

increase the understandableness of the dissertation.

I wish to acknowledge the technical assistance that the follow-

ing persons provided in various aspects of this investigation: Mr.

Steve Bobroff, Miss Barbara Bolt, Miss Eleanor Broome, Mrs. Effie

Galbraith, Mrs. Jenny Hunt, Mr. Erhard Lorenz, Miss Annette Morris, and

Miss Mary Ellen Smedley.

I also want to express my appreciation to the following people

who most generously permitted me to utilize equipment from or in labo-

ratories under their direction: Mr. Wilson Calaway, Dr. Ronald Lowe,

Dr. Walter Mauderli, Dr. Hugh Putnam, Dr. Robert Sholtes, and Dr.

Sherlie West. Special recognition goes to Dr. William Dawson who

graciously permitted me to use equipment and considerable space in







already crowded facilities to house, expose, and sample the animals in

this study.

I am grateful to my parents Mr. and Mrs. Joseph Zelac and my

wife's parents Mr. and Mrs. Robert Motanky for their continued interest,

support, and encouragement.

Financial support for this project came from the following

sources: U. S. Public Health Service Training Grant No. 5 T01 EC00046-

10; National Aeronatics and Space Administration Research Grant No.

NASA-NSA-542, Project A23; and Office of Civil Defense Contract No.

N00228-68-C-2658, Work Unit 3146-A. The x-ray equipment was made avail-

able through U. S. Atomic Energy Commission Division of Biology and

Medicine Research Grant No. AT-(40-1)-3599.












TABLE OF CONTENTS


Page


ACKNOWLEDGEMENTS .............................. ............. ........ iv

LIST OF TABLES .....................................................viii

LIST OF FIGURES .................................................... ix

A BSTRACT .......................................................... x

Chapter

I INTRODUCTION .............................................. 1

The Radiomimetic Nature of Ozone ....................... 2
The Presence of Combincd Ozone and Radiation Environ-
ments and the Problem of Setting Exposure Limits .... 11
Chromosonm Aberrations As an Indicator of Radiation
and of Ozone Exposures .............................. 17
Blod Lywphocytes As the Test Cells ..................... 19
Objectives of the Investigation ........................ 22

II METHODS, ITM TRIALS, AND EQUIPMENT ......................... 21

Chinese Hamsters As the Test Animals .................. 24
The Orbital Bleeding Technique for Blood Uithdrawal .... 26
The Culture of Peripheral Blood Lymphocytes ........... 31
The Production and Quantization of the Ozone Environ-
m en t ..................................... .......... 33
The Production and Quantization of the Radiation Field 44
Scoring Chromosome Aberrations .............. ........... 46
Estimating the Ozone and Radiation Exposures Required .. 49

III PROCEDURES .............................................. 55

Animal Exposures ....................................... 55
Blood Sampling ..................... ................... 57
Tissue Culture Preparation and Maintenance .............. 58
Slide Preparation ......................... ............. 59
Recording of Data ...................................... 62
Analysis of Data ........................ .... ........... 63







Chapter Page

IV RESULTS .................................................. 65

Radiation Exposed Animals .................... ..... ..... 68
Ozone Exposed Animals ................................. 70
Animals Exposed to Combined Ozone and Radiation Environ-
ment ................................................ 70

V DISCUSSION ................................................. 73

Expected vs. Observed Aberration Frequencies with
Radiation ........................................... 73
Chromosome Aberrations and In Vivo Ozone Exposure ...... 75
Interactions Resulting from Simultaneous Ozone and
Radiation Exposure ........................... ..... 77

VI CONCLUSIONS .............................................. 78

Summary of Results ..................................... 78
Significance of Results ................................. 79

APPENDICES ........ .............................................. 81

1 PREPARATION OF CULTURE MEDIA AND SOLUTIONS ............... 82

Heparin Solution for Blood Pipettes ................... 83
Hungerford Culture Media ............................... 83
Mast Ozone Meter Sensing Solution ..................... 84
TC-199 Culture Media with Ileparin ...................... 85
Colchicine Solution for Mitotic Arrest ................. 85
Hypotonic KC1 Solution for Cell Swelling ............... 86

2 ALKALINE KI METHOD FOR OZONE DETERMINATION ............... 88

Introduction ............................... ............ 89
Reagents .......................................... .... 89
Apparatus ............................................ 90
Analytical Procedures ................................... 91
Discussion of Procedure ................................ 94

3 PROCEDURE FOR STAINING SLIDES ........................... 96

LIST OF REFERENCES .................. ...... ........................* 98

BIOGRAPHICAL SKETCH ................................... ............ 114











LIST OF TABLES


Table Page


1 EFFECT OF OZONE INHALrATION ON MORTALITY RESULTING
FROMI RADIATION EXPOSURE ................................ 8

2 SUMMARY OF EXPOSURES ...................................... 66

3 SUMMAIRY OF DATA ............................................ 67

4 SUMMAR' Y 'OF EXPOSURE-ADJ' lS, iI.T BREAK FREQUENCIES ............ 69

5 COMPARISON OF I.. i AND Or fL. ED NUMBERS OF BREAKS
FOR COMBINED OZONE PLUS RADIATION EXPOSURES ............ 72

6 COMPAPIS.'.. OF ESTILIAT D AND OBSERVED EXPOSURE-ADJUSTED
A; '.TION i iS .Q 13 ................................. 74

7 INGREDIENTS h'. SPiCIAL CULTURE MEDIA .................... 84

8 I:; tl r0l1ENTS FOR C-199 CULTURE MEDIA WITH 11EPLRIN ........ 85

9 SLIDE STAINING LIQUIDS AND }: -lUL,! .i TIMES ............... 97













LIST OF FIGURES


Figure Page


1 CHINESE IHAISTER CHROMOSOMES .............................. 25

2 CHINESE AND GOLDEN LHA: ,TEi[,S-COMIPARATIVE SIZE ............. 27

3 DIAGRAM OF BLOOD PIPETTE IN EYE ORBIT .................... 30

4 ANIMAL POSITIONING AND PIPETTE INSERTION FOR THE ORBITAL
BLEEDING TECHNIQUE ...................................... 30

5 OVERALL VIEW OF ANIMAL EXPOSURE SETUP .................... 35

6 THE INTERIOR OF THE EXPOSURE CHAMBER WITHOUT UV SHIELD ... 36

7 THE INTERIOR OF THE EXPOSURE CHAMBER WITH UV SHIELD ...... 37

8 THE EXPOSURE CAGE WITH ANIMALS, R-CHANBER, AND OZONE
SAMPLING PROBE ......................................... 39

9 THE SETUP USED FOR CALIBRATING THE OCCTC;: METER ........... 41

10 ACTUAL OZONE CONCENTRATION VS. MAST METER INDICATED
OZONE CONCENTRATION ..................................... 43

11 DLAGRAM OF THE VARIOUS CHROMOSOME-TYPE CHROMOSOME
ABERRATIONS ............................................. 48





Abstract of Dissertation Presented to the
Graduate Council of the University of Florida in Partial Fulfillment
of the Requirements for the Degree of Doctor of Philosophy

THE EFFECT OF OZONE INHALATION ON THE FREQUENCY
OF CHROMOSOME ABERRATIONS OBSERVED
IN IRRADIATED CHINESE HAMSTERS

By

Ronald Edward Zelac

August, 1970

Chairman: Billy G. Dunavant, Ph.D.
Co-Chairman: Harvey L. Cromroy, Ph.D.
Major Department: Environmental Engineering

Should presently permissible human exposure levels for ionizing

radiation and for respirable ozone be reduced when exposure to both

agents takes place, especially simultaneously? This particular com-

bination of noxious agents warranted consideration for three reasons.

1. In many of its actions on biological materials, ozone mimick-

ed the effects of ionizing radiation; this included the ability

to produce chromosome aberrations in human cells in vitro;

2. The direct effects of inhaled ozone did not appear restricted

to the pulmonary system, but were widely distributed throughout

the body;

3. Situations existed in which people were exposed to these two

agents simultaneously; there were also indications that more

people would be similarly exposed in the future.

Chromosome aberrations produced in circulating blood lymphocytes

were the indicator of biological damage. Effects in these cells would

be a qualitative indicator of similar damage to other cells throughout

the test animal. Exposure-adjusted break frequencies served as the

quantitative measure of effect. Adult female Chinese hamsters,





Cricetulus griseus (2n = 22), were exposed, in groups of four, to x-

radiation (118 keV effective, 70 cm FSD, 230 rad dose), to ozone (0.2

ppm, UV generated), or to both simultaneously. All exposures were of

5 hr duration. There were two groups per treatment and two additional

groups, one serving as a control and the other exposed to 330 rad. A

total of 50 animals were utilized in the investigation, including 18 in

preliminary experiments.

Blood samples (0.2 ml) were obtained by orbital bleeding. Fol-

lowing plasma removal, the cells were cultured in an enriched media for

3 days at 370C in a humidified 5 percent CO2 environment with pokeweed

as the mitogen (lymphocyte transformation and division stimulant).

Division was arrested in metaphase with colchicine. Following hypo-

tonic KCl treatment to swell the cells, they were fixed with a 3:1

methanol-acetic acid solution, transferred to a slide, and rapidly dried

by ignition. The slides were stained with Giemsa and scanned using

bright-field microscopy. Spreads were photographed on 35 mm film and

projected onto a screen for analysis (with subsequent microscope re-

checks of spreads exhibiting breaks). The aberrations scored were dele-

tions, dicentrics, and rings.

The following were the principal results of this investigation:

1. Radiation resulted in an exposure-adjusted break frequency of

5.51 x 10-4 breaks- for cells withdrawn 2 weeks after exposure.
cell-rad
This value appeared reasonable in comparison to available infor-

mation on in vivo exposure of human lymphocytes and Chinese ham-

ster bone marrow cells. Successful lymphocyte cultures could not

be obtained until 2 weeks had elapsed. Break frequency appeared

to vary linearly with dose in the region 230-330 rad.





2. Ozone resulted in an exposure-adjusted break frequency of

1.6 -3 breaks
1.67 x -10-- bres agreeing well with the value expect-
cell-(ppm-min)'
ed from in vitro exposure of human cells. There was no apparent

decrease in break frequency with time for 2 weeks post-treatment.

3. Animals exposed to the two agents simultaneously exhibited

>70 percent of the total number of breaks anticipated assuming

additive actions. Expected contributions from ozone and from

radiation were nearly equal. There was, however, approximately

an 18 percent (15 percent) chance that all the breaks observed

resulted from the radiation (ozone) exposure alone.

Presently permitted human ozone exposures (up to 0.1 ppm, 4

EmAhr) would be expected to result in break frequencies that are orders
week

of magnitude greater than those resulting from permitted human radiation

exposures if the results of this experimental animal study were directly

extrapolated to the human case. Consideration of combined ozone plus

radiation environments is overshadowed by the importance of ozone envi-

ronments alone as long as permitted ozone exposure levels remain at

their present values.















CHAPTER I


INTRODUCTION

Man has become increasingly concerned about his surroundings as

they relate to his health and well-being. The complex environments

which he has created through technology make it necessary for him to

consider how various agents and conditions of these environments affect

him and how they can interact with regard to his health. Ozone and

ionizing radiation are two agents which have been extensively investi-

gated individually and whose combined actions have been under study.

Ozone is a highly reactive three-atom allotrope of oxygen. It

is present in the atmosphere as a natural constituent and is produced

artificially both intentionally and as an unwanted byproduct of various

operations. The biological effects of ozone on man and animals have

received much consideration. Many studies have dealt with the respira-

tory system effects (1-46), as inhalation is the principal path of

entry. It was believed that the action of ozone was restricted to the

respiratory system, for this is where its effects are most noticeable,

particularly at concentrations of 0.3 parts per million (ppm) by volume

and higher (29).

Now there is evidence that the action of ozone is more wide-

spread. Its effects on a wide range of other body functions and

systems have been considered (47-76) as have its effects when present

in combination with other agents including other gases, aerosols, mists,

and bacteria (26, 62, 66, 77-81). A number of general reviews of these









biological effects of ozone on man and animals are available (54, 82-

85). The present occupational exposure limit for ozone has been set at

0.1 ppm.

Ionizing radiation is probably one of the most-studied physical

insults to which man has been exposed. Background sources of irradi-

ation are cosmic rays and emissions from naturally occurring radio-

active materials; technological sources include x-ray generators, arti-

fically produced radioactive materials, nuclear reactors and detona-

tions, and particle accelerators. The effects of ionizing radiation on

biological systems are extremely broad in range. This results from the

penetrating ability of many of the emissions. One of the most colrpre-

hensive publications on this subject is the Report of the United

Nations Scientific Coy iittee on the Effects of Atomic Radiation (86).

This report covers fundamental radiobiology, hereditary and somatic

effects of radiation, sources of irradiation, and comparison of doses

and estimates of risks, and includes nearly 3,000 references. A later

report in 1964 having the same title (87) acts as a supplement to the

comprehensive 1962 work. Therco is, adJitionally, an annotated indexed

biblio i ,.lhy on the biological effects of ionizing radiation that

covers world literature for the time period 1898-1957 and includes al-

most 13,000 entries (88). A supplement to this extensive work covers

1958-1960 and contains nearly 12,000 more entries (89).


Thc Radio imetic Nature of Ozone

The property that incited the coinsiderble].e interest in ozone,

alone and in conjunction \ith other agents, is its large elcctr.,n, -

tirv potential, -2.07 V, (02 + i.20 **: 03 *L 2;i+ + 2c). This value s








only slightly exceeded by fluorine's, -2.1 V (2F- + H20-> F20 + 211+

+ 4e) (90). Thus, ozone is an extremely powerful oxidizing agent. It

has been proposed that the oxidizing action of ozone is via a free

radical mechanism (16, 91).

Ionizing radiation, when interacting with biological systems,

also acts as a powerful oxidizing agent, this through the action of

free radicals formed by the dissociation of water (92). The processes

involved in the initial interactions of ionizing radiation with water

are (93, 94):

Exitation
HOH > io izi g 1 + .OH ................... (1)
radiation

Ionization
HOH -ionizing (H1011)+ + e ................. (2)
radiation

followed by
H+ + e --> IH ......................(3)

or
1120 + e --> H + *OH"- ................... (4)

and possibly
(HOH)+ --> H1 + ..................... (5)

Combination results in the following postulated reactions (93, 94):

11- + .OH --> 1OH .......................(6)

1H. + H. --> H2 .......................(7)

*OH + -OIl --> H202 .....................(8)

or
*01 + -OH -0 1120 + .0 ................... (9)

and
'0 + *0 -> 02 ......................(10)

and if dissolved oxygen is present

*OH + 011 + 02 -->H202 + .0 + *0 ............. (11)

+ 02 > '02H ...................... .. (12)








and possibly
*021H + -0211 -:. H1202 + 02 ................ (13)

Of the above, 6 and 7 remove reducing agents while 8-13 produce oxi-

dizing agents. These oxidizing agents or the OH radical itself (as a

highly reactive electron acceptor, OH" --: *OH + e -3.7 eV) act to oxi-

dize inorganic ions and/or organic compounds that are present in the

aqueous biological system (93).

Thus, through oxidizing processes, ozone and ionizing radiation

might be expected to act similarly on biological materials that are

exposed to them. This raises the question of whether tissues beyond

the pulmonary system are exposed to the action of ozone when ozone is

being inhaled. To put it another way, will ozone inhalation result in

direct extrapulmonary effects as opposed to secondary systemic reac-

tions resulting from pulmonary involvement? If other tissues are

directly affected, then the similarity between exposure to these two

agents, ionizing radiation and ozone, will be even more striking. For

just as an experimental animal in a radiation field would be expected

to have effects of the exposure throughout its body, an animal in an

ozone environment would similarly experience widespread direct effects

of its exposure through inhalation.

There is a variety of observations on the actions of ozone that

demonstrate direct extrapulmonary effects of ozone inhalation. Some of

these effects are similar to those seen after exposure to ionizing ra-

diation. These latter observations have resulted in ozone being widely

considered as a "raeiomineLic" agent. The following is a listiu,' and

discussion of experin ts demonstrating direct extrapulmonary and/or

radiotni tic effects of ozone e-:pofure:




5


1. Cutaneous oxygen consumption in a digit of man has been

shown to be strongly affected by short-term inhalation of moder-

ate ozone concentrations (95). The technique involved the sud-

den occlusion of digital circulation and the estimation of the

ensuing deoxygenation of oxyhemoglobin photoelectrically. Stag-

nant intracapillary deoxygenation normally proceeded logarithmi-

cally to complete dissociation in approximately 10 min. However,

the dissociation immediately following the breathing of 1 ppm

ozone for 10 min only reached the 50 percent level 10 min after

occlusion and appeared to level off. This phenomenon,interpreted

as a reversible intoxication of final heme-enzyme groups in the

redox chain, was also evident following the breathing of pure

oxygen or irradiation of the finger with ultraviolet light. In

the case of ozone, normal deoxygenation was evident if cysteamine

was introduced into the skin immediately after ozone inhalation.

The interpretation of these results was that ozone reacted with

the linings of the airways, and the oxidizing products of this

interaction were distributed by the circulation of the blood.

Cysteamine, as it did with ionizing radiation, provided chemical

protection presumably by inactivating oxidizing agents through

a radical scavenging mechanism. Thus, extrapulmonary oxidizing

action was inferred.

2. The acute toxic action of inhaled ozone has been reduced by

the simultaneous injection or intraperitoneal injection of com-

pounds that furnish -SIH or -SS- bonds (96). Compounds with -S

bonds proved ineffective. As was mentioned previously, these

same chemicals provided protection fiom exposure to ionizing








radiation (86). This further suggests a similarity of action

for ozone and radiation.

3. The effects of ozone inhalation on the visual acuity of man

have been investigated (97). Levels of 0.2-0.5 ppm inhaled for

3 hr or two 3 hr periods with 1 hr rest between resulted in (a)

decrease in visual acuity for dark adaptation and middle vision

ranges, (b) increases in peripheral vision, and (c) change in

the balance of most extraocular muscles. The effect, while not

radiomimetic, demonstrates direct extrapulmonary action of ozone.

4. In animals, including man, red cells start circulating as

flat discs and end up as spherocytes. The process can be great-

ly accelerated by x-irradiation in vitro. If ozonized air is

inhaled before the blood is withdrawn, this process is further

accelerated (98). Distinct effects were seen in man with ozone

exposures as low as 0.25 ppm for 30 min. For example, for blood

drawn 1 hr after this degree of ozone exposure, 7,000 R resulted

in 55 percent of the red cells being spheroid compared to 21

percent for non-ozone-exposed controls. Sphering tendency ac-

celeration by ozone was a reversible reaction, as the amount of

sphering seen decreased with time after ozone administration.

The experiment demonstrated a distinctly radiosensitizing effect

of ozone inhalation and showed ozone action on the blood system.

5. It has been demonstrated that the inhalation of ozone can

result in structural damage of postmitotic nuclei in myocardial

fibers of adult rabbits and mice (98). Exposure to 0.2 ppm,

5 hr/day for 3 weeks,resulted in rupture of nuclear envelopes

and extrusion of contents which are never observed in the nuclei








of normal fibers. This type of damage is also seen in patients

following therapeutic radiation exposure and in irradiated tumor

cells. Here, extrapulmonary tissue damage which mimics that seen

from radiation exposure was shown.

6. Another indication of the deep and radiomimetic effects of

ozone was its effects on the offspring of exposed mice (98).

The exposures were to 0.1 or 0.2 ppm, 7 hr/day, for 3 weeks.

For inbred grey mice, litter sizes were normal, but 3 week neon-

atal mortality was 6.8 percent (0.1 ppm) and 7.5 percent (0.2

ppm) compared to 1.6 percent for controls. For the second lit-

ters from parents exposed to 0.1 ppm, 4.9 percent mortality was

seen against 1.9 percent for controls. For highly inbred C57

black mice, exposure to 0.2 ppm under the same routine resulted

in normal size litters but 34 percent neonatal Lmor:tality com-

pared to 9 percent for controls. Here, then, we have evidence

of a lingering observable effect on gonadal material not unlike

effects of ionizing radiation (86).

To these experiments which demonstrate widespread direct extra-

pulmonary ozone effects along with the oxidizing and radiomimetic na-

ture of ozone action can be added another group of studies involving

the exposure of animals to ozone in conjunction with lethal doses of

ionizing radiation. In these, the effects on radiosensitivity of pre-

exposure to ozone were investigated. These experiments are described

in Table 1.

In each case, ozone inhalation had an effect on mortality. The

radiation doses used were approximately LD50/30's (i.e., doses that

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30 days) or greater. The ozone exposures were all considerably below

lethal levels. (For mice, exposure to 6 ppm for 4 hr results in 50 per-

cent mortality. Rats are even more resistant (84).) Most were below

the approximately 6 ppm-hr at which edema and lympocyte migration into

alveolar spaces begin, i.e., where lung histology changes (68). Some

were even below the 0.7 ppm-hr where lung function temporarily changes

during, exposure (27). (For short term single exposures, the product of

ozone concentration and exposure time produces a constant toxicological

response (84).) Since little if any effect on pulmonary structure or

function could be expected once the ozone exposures ceased, the effect

on mortality of subsequently administered lethal doses of radiation is

an additional demonstration of the direct extrapulmonary action of

inhaled ozone.

It should be noted in Table 1 that two phenomena are exhibited.

The first, radiosensiitzation, acts to increase mortality. This is

seen when the ozone exposure is of short-term duration (1. hr) and im-

nediately prior to the radiation exposure. If the ozone exposure is of

lo r duration ini.cdiately prior to the radiation or of short duration

but with a time lag of 1 day or more between ozone exposure and radia-

tion exposure, the effect is to decrease mortality, radioprotection!

To interpret these apparently opposing observations, it should

be recalled that both ozone and radiation have oxidative processes in

the body as the basis of their action. With ozone as the radiosensi-

tizcr, the oxidation from it (on the premise that it does occur

throi.- v-,i' the body and not just in the respiratory system) adds to

the oxidation result in from the radiation to bring about a level of

d: e greater than that from the radiation alone; hence increased








mortality. To bolster this interpretation, it has been shown that small

doses of ionizing radiation (.65 R) immediately proceeding lethal ozone

exposures increase the mortality observed over that from ozone alone

(104). With ozone as the radioprotective agent, the interpretation is

that the insult from the ozone, i.e., the oxidation from it, was suf-

ficient in magnitude to bring about an antioxidant response by the body

and to stimulate intracellular repair mechanisms before the radiation

exposure began. Through this, some of the oxidizing action of the

radiation was counteracted, resulting in a lower level of damage and

decreased mortality.

This antioxidant response of the body is equally effective if the

source of subsequent oxidant is an additional ozone exposure of lethal

magnitude rather than radiation exposure (34). Likewise, pre-exposure

to ionizing radiation rather than to ozone can initiate the presumably

antioxidant response (106-108). Since this is the case, ozone is also

radiomimetic in these experiments in which it acts as a radioprotective

agent.

Thus, a variety of observations have been made which rather

conclusively indicate that there are direct extrapulmonary effects of

ozone inhalation and that ozone can be generally considered as a

radiomimetic agent.


The Presence of Combined Ozone and Radiation Environments
and the Problem of Setting Exposure Limits

That ozone and radiation appear to act similarly on biological

materials is of practical interest from a health and safely point of

view. Man attempts to provide safe and comfortable environs for himself

by, among other things, limiting his exposures to various agents to








values below damaging or irritating levels, or at least to values for

which the levels or risks of damage are acceptable when weighed against

the overall benefits associated with the exposures. In deciding on ac-

ceptable levels for a particular agent, information from human experi-

ence and from animal and/or human experimentation is utilized. If two

or more agents are present, the general practice is to consider their

actions as additive (unless there is information to the contrary) and

to accordingly reduce the permitted exposure level for each of them.

This has been the case when the agents have some similarity to one an-

other, as mixtures of organic solvents or mixtures of radioactive mate-

rials or radioactive substances and external irradiation in combina-

tion. There has been little if any consideration, however, of cross-

coT'binotions including the one under discussion here, ozone and ioniz-

inz rld5ition .

It appears importanL to consider the action of the ozone-

ionizing radiation combination, particularly, because of the radiomi-

metic nature of ozone previously discussed, because there are presently

situations where people are exposed to both agents simultaneously, and

because there are definite indications that more and more people will

be so exposed in the near future.

There are two broad classifications into which ozone-radiation

environments fall. In the first, the radiation field is the source of

the ozone; for the second, the principal source of the ozone is not the

radiation field but other technological generating means, or the ozone

is present naturally. There are some ozone-radiation environments that

are a co inin tion of these two types.








The irradiation of oxygen, gaseous or liquid, results in ozone

production and in some decomposition of the ozone so formed. Alpha,

fast electron, photon, and neutron irradiation have been investigated

(109-116). The yield of ozone in a closed system increases with dose

(109, 114-116) until an equilibrium value as high as 2,000 ppm (115)

is reached at very high doses of approximately 107 rad (114). The

yield also appears to increase with dose rate (114, 115), although this

point is disputed (113). As would be expected, with a flow-through

system, the concentration is reduced but the total amount of ozone

produced increases (116).

It follows, then, that an ozone plus radiation environment can

be associated with nearly any high-intensity radiation field. Some

examples are research accelerators and isotopic sources, electron beam

irradiators (used in industrial polymerization as for paint curing and

plywood glue hardening), large-scale isotopic food irradiators, high-

radiation areas at nuclear reactors, industrial radiography devices,

and medical therapeutic radiation units.

This problem of ozone production by-radiation has been recog-

nized and investigated at a number of facilities including high-level

gamma installations (117-119) and electron accelerators (117, 120-123).

The principal method of control is usually to provide ventilation ade-

quate for keeping ozone concentrations down to acceptable levels. In

connection with this discussion of radiation as a producer of ozone, it

is interesting to note that a number of experiments with mice, rats,

and monkeys have demonstrated that animals detect radiation by olfac-

tory system response to ozone produced in their nasal passages by the

radiation (124-127).








Other technological production of ozone is by electrical arcs

or corona discharges, silent electric discharges, and shortwave

((2450 A) ultraviolet light (128, 129). It is found industrially at

ozonizers for treatment of sewage, for water purification, and for

control of molds and bacteria at cold storage plants; in conjunction

with high volt ..e electrical apparatus such as at generators, accelera-

tors, x-ray devices, and spectrographs; and with electronically operated

office copy equipment, neon signs, electric motor brushes, quartz ultra-

violet lamps, and inert gas shielded arc welding units (7, 84, 130).

A technolo ically related source of ozone is its environmental produc-

tion in : ,by photo ch 1ical reactions involving air pollutants and

naturally present ultraviolet light (131).

The potential for a combined radiation-ozone situation exists

:whercvr lpople a c: 0posc- to r;'i.ation :h ilc. in th, vicinity of other

technolo ical ozone sources. An example of this type of an environment

is a nuclear suba rine. Here the radiation source is the nuclear

reactor power plant of the vessel; the ozone can be produced both by

the radiation fields associated with the reactor and by the extensive

electrical and electronic equipment aboard. The potential for ozone

production on these ships has been recognized (132), and monitoring

equipment is available or. them (133). Control of the ozone concentra-

tion is complicated by the at ospheric recycling required by the nature

and mission of such vessels.

Another example of an crivironment with a combined ozone-

radiation exposure potential is a spacecraft. Radiation exposure can

result from ctic cosn ic rays, Van Allen belt particles, solar

flares, radioactive d'eris front at os hric nuclear explosions, and








on-board radiation sources (134). Ozone can result from operation of

the space cabin equipment (135) or from irradiation of liquid and gas-

eous oxygen supplied on-board (136). Here, too, the ozone situation is

complicated by the atmospheric recycling required. As with the nuclear

submarine, the ozone production potential has been recognized, as

demonstrated by NASA sponsored experiments dealing with ozone toxicity

(77, 78).

Ozone is also produced naturally in the upper atmosphere by the

action of solar ultraviolet radiation on oxygen present in the strato-

sphere. The vertical concentration profile peaks at about 29.0 km and

decreases rapidly as height decreases; there is also variation with

latitude and season (84, 137). Concentrations up to 15 ppm (by volume)

have been reported; natural ozone levels at ground level at U. S.

Intitides are estijmted at 0.01 ppm or less.

Present-day jet aircraft cruising at 9.2-12.4 km are at the edge

of the so-called ozonosphere, while the supersonic transports (SST's)

cruising at 19.8-24.4 km will be near its center, where the ambient

external ozone level is hazardous to life. As ambient air is used for

cabin pressurization currently and will be used for SST's, ozone levels

in present aircraft have been investigated (138-142) and rigorous ozone

control (decomposition and monitoring) is being planned for the SST

(137, 143-145). The studies of present craft have indicated that the

external air is the principal source of the ozone present (141), al-

though cockpit levels are somewhat higher, suggesting other (electrical)

sources (142). Levels range up to 0.4 ppm. A maximum level of 0.2 ppm

is proposed for the SST (137).








While passengers and crews of present-day jetliners experience

some radiation exposure, primarily from cosmic radiation, people in

SST's will have to contend with a higher radiation level. This is a

result of cosmic radiation being more intense at higher altitudes (6x

higher at 21.4 km that at 9.2 km), protons from solar flares basically

not penetrating the atmosphere to altitudes <18.3 km, and radioactive

debris from high-yield atmospheric nuclear testing being predominantly

concentrated in the stratosphere (above 11.0 km to 30.5 km) (146, 147).

Thus, while jetliners do present a radiation-ozone environment, the

SST will probably involve greater radiation exposure with the ozone

exposures comparable to those presently encountered.

We are therefore confronted with the practical problem of hav-

ing people simultaneously exposed to ionizing radiation and ozone,

along with the prospect of even more people being so exposed in the

future. In view of the apparently radiometric nature of ozone, should

the maximum levels of exposure permitted for each agent when consider-

ed as acting alone still apply in this dual exposure condition? Pre-

sently, the radiation and ozone exposure limits are applied indivi-

dually. A quantitative measure of widespread damage throughout the

body is needed that will serve equally well to evaluate ozone and

radiation exposures. Such a measure would indicate whether the agents

in a combined environment acted in an additive fashion, synergistically,

or antagonistically in their damage production. Then a decision could

be reached as to the adjustments, if any, that should be made in the

individual exposure limits when a combined exposure occurs.








Chromosome Aberrations As an Indicator
of Radiation and of Ozone Exposures

A brief introduction to chromosome organization and structure is

presented here because it forms the basis for the experimental ration-

ale.

Chromosomes are nuclear organelles. They are in the form of

elongated threads (present in the nuclei of cells) between cell divi-

sions, or in interphase. During division, they contract into short

thick readily observable rods. Chemically, they are characterized as

nucleoproteins having the polynucleic acid DNA (deoxyribonucleic acid)

and two specific proteins as their main components. Functionally,

through subdivisions called genes, they dictate the enzyme and protein

production and hence the metabolic chemical reactions of the cells in

which they occur. This information is transmitted from generation to

generation in a cell line by duplication of each chromosome in the

mother cell prior to division (mitosis) so that at division each daugh-

ter cell gets a complete complement of chromosomes and thereby a com-

plete code for protein production. Through meiotic cell division, each

sexual gamete receives one-half the characteristic number of chromo-

somes. When fertilization occurs, the normal number of chromosomes

for cells of the particular species is restored.

While genes are remarkably stable, alterations do occur. A

change in a gene that is accompanied by a visible change in the struc-

ture of the chromosome of which it is a part is called a chromosomal

mutation; those gene changes that are not accompanied by visible chromo-

somal changes are called point mutations, or gene mutations. As genes

control the metabolic reactions of the cell, gene changes can be

highly detrimental (even lethal) or relatively insignificant.








A chromosomal mutation., or aberration, results when the two ends

created by a single break in a chromosome fail to rejoin with one a-

nother through repair mechanisms. Rejoining can fail to occur or ends

from two different breaks in the same or in separate chromosomes can

join to produce a variety of aberrations. These aberrations often

result in an unequal distribution of chromosomes between daughter cells,

and this usually leads to cell death. If rejoining of the two ends

created by a single break does occur, restitution may be at the mor-

phological level only, and a point mutation due to DNA damage may

appear (84). It is for these reasons that chromosome aberrations are

considered as cellular alterations (damage) with long-term consequences.

There are a variety of agents which have the capacity to bring

about gene changes, i.e., which are mutagenic. Ionizing radiation has

long been recognized as one of these agents (93, 148). Its effective-

ness is dependent on the magnitude of the dose (direct dependence), the

cell type, and the stage of the cell cycle at the time of irradiation,

but all living cells are susceptible to this action (86, 148).

Evidence has accumulated that ozone, too, is a general muta-

genic agent. It has been observed to change the adsorption spectra of

nucleic acids (149); to markedly modify the pyrimidine bases (thymine,

cytosine, and uracil) in E. coli nucleic acids (150); to produce speci-

fic mutants of E. coli exposed in ozonated water (151); to result in

various chromosome aberrations when tissue cultures of embryonic chick

fibroblasts were exposed to gaseous ozone (152); to produce a high

frequency of chromosome aberrations in the root meristems of Vicia

faba (153); and,finally to produce a number of different types of

chromosome aberrations in cultured human cells exposed to gaseous








ozone (154). For this last investigation, the frequency of aberrations

seen in human cells was shown to depend directly on the magnitude of

the ozone exposure. Comparison was made with the frequency of aber-

rations seen with exposure of the cells to x-radiation. For radiation,

the yields also varied directly with the dose.

It appears that ozone, like ionizing radiation, could be expect-

ed to produce chromosome aberrations in all types of cells that are

exposed to it. Because of the radiomimelic nature of ozone, this is

not a surprising observation. Likewise, this action of ozone, produc-

tion of chromosome aberrations, could be expected to occur throughout

the body of an animal inhaling ozone in view of the direct extrapulmo-

nary effects of ozone inhalation that have been demonstrated.

Therefore, it appears that the frequency of chromosome aber-

rations prcduced by C:rOosu'.o of -n anii'l to ionizing radiation and/or

to ozone can serve as the sought-after quantitative measure of wide-

spread d ii-.-e.


Blood Lymphocytes As the Test Cells

There are numerous technical difficulties associated with the

production of mammalian chromosomal preparations. The usual histolo-

gical techniques do not suffice, and squash preparations are not com-

pletely satisfactory, partly because of the small size of mammalian

cells and their relatively large chromosome numbers (155). Since the

early 1950's it has been possible to make suitable preparations from

mammalian cells maintained in tissue cultures by employing a hypotonic

pre-fixation treatment to swell mitotic cells (156). Unfortunately,

this has little value for experiments involving whole animals. Similar-

ly, good preparations have been made from tissue biopsy cells put into








suspension and subjected to the same hypotonic pre-treatment procedure;

a soft, easily dispersed tissue is required. Skin and bone marrow have

been used, but sample collection is difficult, particularly when re-

petitive samples are required.

It was only in 1960 (156) that the cytological technique which

is most widely used today in animal chromosome studies became avail-

able. This technique utilizes the small lymphocytes, the white cells

originating in the lymph system, that are present in the circulating

blood. These cells, 8-10.J in diameter and having a large nucleus, are

the smallest of the white blood cells. They are involved in the carry-

ing and production of antibodies for combating infections and are also

involved in fibrous formation for healing or enclosing clots. They

account for 25-30 percent of the white cells in human blood, and, for

comparison, 80-90 percent of rat blood white cells.

Up until recently, lymphocytes had been thought to have life-

times on the order of 4 hr. These estimates were based on the fact

that the number of lymphocytes entering the blood system through the

lymph ducts is several times greater that the total number present at

any moment (157). But the number of lymphocytes leaving the blood

system and passing through tissue fluids to the lymph system was under-

estimated. In fact, lymphocytes are continuously recirculating be-

tween the blood and the lymph. Lifetimes of 1.00-200 days and longer

have been reported (155, 158).

Lymphocytes were long-considered as mature non-dividing cells.

Since cells undergoing mitosis are required for chromosomes to be

visible, this would imply that lymphocytes should definitely be elimi-

nated from consideration as test cells. But it has been found that a








small fraction of lymphocytes in circulating (peripheral) blood are

engaged in DNA production at a low rate (159). Additionally, and more

importantly, when lymphocytes are exposed to certain drugs in vivo or

in vitro, a transformation takes place, and mature cells grow, taking

on the characteristics of lymphoblasts, which are large immature slight-

ly differentiated precursors of lymphocytes (160, 161). These trans-

formed cells then undergo chromosome duplication and enter into mitosis

(159-162). Therefore, they are suitable for chromosome preparations.

This sequence of events is referred to as "lymphocyte stimulation" and

the drugs as "mitogcns." This action is believed to be related to the

antigen-antibody action of lymphocytes.

The lymphocytes are most often exposed to the mitogenic agent

after a blood sample has been withdrawn from the animal and the cells

put into a suitable culture media. This means that when the animal is

subjected to agents that can produce chromosome breakage, the lympho-

cytes are nearly all at about the same stage of the cell cycle, the

pre-DNA-synthesis resting stage (they are mature cells and would nor-

mally, except for stimulation, not engage in any more division). This

fact greatly simplifies aberration analysis of the chromosome prepara-

tions: First, variations in sensitivity of the cell to the agent with

stage in the cell cycle are absent, so all cells should be equally

sensitive; second, any aberrations that result from the agent will be

of the chromosome type (i.e., will involve both strands of the chromo-

some) since duplication occurs after breakage takes place; this great-

ly reduces the number of aberration types that have to be considered

by eliminating all those of the chromatid type (i.e., that involve

only one strand of the duplicated chromosome).








These are two distinct advantages that chromosome preparations

from lymphocytes have over preparations of cellsfrom other tissues.

Other advantages of using lymphocytes are the relative ease of obtain-

ing the cells (just draw some blood), the option for doing multiple

sampling over a period of time if this is required, and the availabil-

ity of information on chromosome aberration production by ionizing

radiation in the lymphocytes of one animal, man, both in vitro and in

vivo (155, 163-167). Additionally, chromosome aberrations produced in

circulating blood lymphocytes should be indicative, in a qualitative

sense, of similar damage to other cells throughout the entire body,

both for ionizing radiation and for ozone. This follows from demon-

strated body-wide effects of ozone and radiation exposure and the fact

that production of chromosome aberrations by these agents appears to

be-C r action t L is genc a to all cell ty~l -s.


O bicctiv. of _h Tncb Inf.".tri ijn

The basic purpose of this study was to provide experimental

evidence that could be used to help decide whether presently permissi--

ble human exposure levels for ionizing radiation and for respirable

ozone should be reduced when exposure to both agents takes place, par-

ticularly when the exposures are simultaneous. Such evidence should

consist of quantitative information on the production of a specific

type of biological damage in small ma rmals through exposure to each of

the agents alone and to both of them together.

The production of chromosome aberrations in circulating blood

lymphocytes was chosen as the biological indicator of damage. This

choice was i-ade on the basis of known response to radiation (in man)







and probable response to ozone suggested from related information.

Chromosome aberrations in these cells would be indicative of similar

and related damage with long-term consequences in other cells of the

body as well.

As a result of this choice, the specific objectives of this in-

vestigation (all referring to the peripheral blood lymphocytes of a

particular species of small mammal) were the following:

1. determine the frequency of chromosome aberrations produced

by exposure to ionizing radiation; compare this value with those

available for other cells in the same species and for lymphocytes

in the other species;

2. determine whether chromosome aberrations are produced when

the animals undergo exposure to ozone at a concentration approx-

ima--ing thai prr',eni-ly prrmittrtd for human exposure and for a

duration that related information suggests is adequate to pro-

duce sizeable numbers of aberrations; if aberrations are seen,

determine the frequency of production by ozone;

3. determine the frequency of chromosome aberrations produced

when the animals are exposed to ozone and ionizing radiation

simultaneously; compare this value with that which would be

expected based on exposure to each of the agents individually

in order to determine whether synergism, additivity, or antago-

nism occurs with combined exposures;

4. attempt to assess the value of the information obtained by

the experimental study in relation to the overall problem of

deciding on appropriate permissible exposure levels for ozone

and ionizing radiation when exposure to both agents takes place,

especially simultaneously.














CHAPTER II


METHODS, MATERIALS, AND EQUIPMENT


Chinese Hamsters As the Test Animals

The species, of all the small mammals available for laboratory

studies, which seemed most suitable for this investigation was the

Chinese hamster, Cricetulus griseus. This choice was made primarily on

the basis of the low diploid chromosome number of this species, 22 per

somatic cell. See Figure 1 (168). Most other small mammals have many

more; the Syrian or golden hamster, for example, has 44. Small mammals

known to have fewer chromosomes such as the Tasmanian rat kangaroo with

12 or 13 (168) or the mole-vole with 17 (168) are not easily obtainable.

As somatic cells from placental mammals all appear to contain approx-

imately the same amount of genetic material (169), fewer chromosomes

per cell also means larger chromosomes, both of which should facilitate

analysis.

Another reason for choosing the Chinese hamster was the availa-

bility of ample numbers of laboratory-grade animals. Most U. S. com-

mercial suppliers obtained their parent stocks from Children's Cancer

Research Foundation, Boston, which has a well-established colony dating

from 1952. Inbreeding has been accomplished there by brother-sister

matings over many generations (170). The supplierI of the animals for




Chick Line Company, Vineland, New Jersey.








lt) il


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FIGURE 1. CHINESE HAMSTER CHROMOSOMES


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Si
X Y


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3=-X


SI








this investigation has maintained a closed colony since 1964 (171). A

third reason for the choice of this species was the availability of in-

formation on spontaneous and in vivo x-ray induced chromosome aberra-

tions in bone marrow cells (172).

The Chinese hamster is smaller than the more common, golden ham-

ster. See Figure 2 (173). Approximately 10 cm long with a 1-2 cm tail,

it is gray-brown in color with a black dorsal stripe and white under-

side. Maturity is reached at 8-10 weeks, and animals can live at least

3-6 years (171, 173). Its outstanding characteristic is its fearless-

ness. 1'.1 i being picked up, an animal will usually not flee but may

roll over and squeal angrily; handling must be gentle and without nerv-

ousness or the animal will become vicious and bite (173, 174).

Ihe animals in this investigation were all females and had an

avL-.,. _ght of ca ,poxi.i._y 27 g. T'.y \' r obLained at an age of

8-10 w1 cks and were utilized at ages ranging from 13-15 weeks to 19-21

weeks for the preliminary experiments and from 11-13 weeks to 15-17

weeks for the final experiments. As required by their p., 1 a';ous nature

toward one another (173), the animals were housed individually in stand-

ard rat cages and provided with paper towels for nest building. They

were aniantained on standard rat chow in the cage, supplemented with oc-

casional fruit or greens, plus water ad libitum. Lighting was natural.

No evidence of disease or parasites was seen for the 57 animals ulti-

mately maintained over the course of the investigation. Each was ear-

punched for ease in identification.


11 n -1.; 1 I l 11; T. ,-1. iq, rF r ,.1 I I 1-' : 1

Experim ntal design required n utiple sa pling, a minimum of tL.'o

blood samples fro each anivnl. (A pre-1r Latc.Unt culture and at l]




27






















N
H

N
H









C,)





O






1 z

I, C









I
F-1


rr4



k/; *



6i--I '







one post-treatment culture were required.) Because of this, traumatic

techniques were to be avoided. The small size of the animal dictated

that the blood samples would likewise have to be small, on the order of

0.2 ml, which, based on mouse data, could represent approximately 10

percent of the total blood volume (175, 176). Additional requirements,

imposed by the culturing to follow, were that the technique not result

in coagulation, hemolysis, or contamination of the blood.

These requirements, along with hamster body structure and a de-

sire for simplicity, eliminated the more standard techniques for small

animals such as tail bleeding, toe removal, and cardiac or jugular vein

puncture (175). The technique which seemed most satisfactory and which

was employed for the investigation was the orbital bleeding method (175,

176). This technique is based on the fact that a venous plexus lines

the rear of each eye orbit. The capillaries forming this network are

fr-. ile and are easily ruptured. Slight pressure from a thin blood

collecting tube' with polished tp2 results in hemorrhage. Blood accu-

mulates in the orbit, which serves as a reservoir, and the blood tube

fills through capillary action after being withdrawn slightly. See

Figure 3 (176). Blood flow stops when the tube is withdrawn and the

eyeball reestablishes normal ocular pressure on the venous network.

The technique is demonstrated in Figure 4 (176). The loose skin

of the head is drawn tight to make the eye bulge, taking care not to

stop respiration, and the pipette tip is inserted in the inner corner.

It is slid in and back along a bony shelf until the plexus is reached.




2Natelson Blood Collectii- Pipettes, Non-heparinized, Scientific
Products Division of American Hospital Supply Corporation, Evanston,
Illinois.































FIGURE 3.

DIAGRAM OF BLOOD PIPETTE IN EYE ORBIT












FIGURE 4.

ANIMAL POSITIONING AND PIPETTE INSERTION
FOR ORBITAL BLEEDING TECHNIQUE

















































































Uate,.








The tube, 3 mm OD, 1.5 mm ID x 150 mm, fills in a few seconds when a

twisting motion is used. Holding approximately 0.26 ml when filled, it

is held horizontally and then tipped to a vertical position for empty-

ing. The tube interior was moistened with heparin solution (See Appen-

dix 1) immediately before use; the culturing to follow precluded the

use of pre-coated tubes, which included preservative in the heparin.

Bleeding could be done from either eye. Neither vision nor

health appeared to be damaged in any way as a result of this non-

traumatic technique which could be performed repeatedly on an animal,

even on successive days. This agrees with what has been observed for

mice (176).


The Culture of Peripheral Blood Lymphocytes

One of the main difficulties encountered in this investigation

was the development of a technique for the culture of lymphocytes from

the small inoculums of Chinese hamster blood. Principally, this meant

determining what modifications of existing techniques were required in

order to produce successful cultures in this laboratory, for lymphocyte

culturing must still be considered as an art. A brief summary of the

technical developments which led to the procedure utilized in this study

is presented below in order to point out some of the difficulties in-

volved.

The first successful stimulation of lymphocytes from peripheral

blood in culture media was reported in 1960 (156, 177). The technique

used 10 ml inoculums of human blood and required removal of erythlrocytes

before culturing. The mitogen employed was phytohemagglutinin (PHA), an

extract of ordinary beans. Subsequent experimentation with animals in-

dicated that while the method, using somewhat smaller inoculums for








small animals, could be successfully employed for some, considerable

modification was required for success with others, and some defied all

attempts at modification (160). Particular difficulty was experienced

with mice, rats, and hamsters.

Partial success with golden hanisters was reported in 1963 with

a procedure that involved removal of the leucocytes from whole blood,

leaving erythrocytes plus plasma, and the culturing of the leucocytes

in a mndia which employed calf serui~ as a replacement for the hamster

plasma (178). In 1965, using rats, it was discovered that a more com-

plete separation.of the leucocytes from the plasma, done by serial

washings with norn il saline, resulted in greater mitotic index in

cultures (179). But even this did not bring about consistently suc-

cessful nouse and harister cultures.

CoCuriL t ,L.1L, th1ee atitit.;, v., .~~La, also being done to de-

velop culturiin techniques which would epl.oy very small blood samples,

which would not require leucocyte separation from whole blood (with the

loss of mitotically competent cells a frequent occurrence), and which

would be less subject to unpredictable failures. A method was reported

by Hungerford in 1965 that accomplished all these points (180). It

utilized a specially formulated culture media (Sec Appendix 1) and an

improved hypotonic treat nt for swelling the mitotic cells. It was

develop d for hu'an blood and was also used for birds.

Finally, in 1968, complete plasma removal (through high dilution

and centrifu gtion) wrs combined with Hungerford's innovative method to

yield a rmicrote-chnique that was reported by Buckton and Nettesheim to

by consistently successful for rouse blood (181). Through inquiry, it

was earned t1at tl7 se technique had been successfully applied to








Chinese hamsters (182). Consequently, the method of Buckton and

Nettesheim was chosen to serve as a starting point in developing a

method to yield successful cultures in this laboratory. The main mod-

ifications instituted were to substitute orbital bleeding for cardiac

puncture, to halve the amount of whole blood used (0.15-0.25 ml instead

of 0.30-0.50 ml), and to extend the incubation time by 50 percent (3

days instead of 2 days). The complete procedure is presented in Chap-

ter III.

Recently, a preliminary report of an additional method for cul-

turing peripheral blood lymphocytes from Chinese hamsters was issued

(183). It differs most from the method used in this investigation by

its use of extremely small quantities, 1-2 drops, of unseparated whole

blood.


The Production and Quantization of the Ozone Environment

The principal methods of ozone production are electrochemical

dissociation, corona discharge, silent electric discharge, and short-

wave ultraviolet irradiation of oxygen. From these, ultraviolet irra-

diation (UV) was chosen for this investigation. This was to avoid si-

multaneous production of oxides of nitrogen (129), for these can influ-

ence biological response (91).

Both production and decomposition of ozone occur when oxygen is

irradiated with ultraviolet light (129):

Production
02 + h1--:- 2 0 ................. ....(14)

followed by
02 + 0--' 03 ....................... (15)

or
3 0 --- 03 ......................... (16)









Decomposition
2 03 + hv ->- 2 02 + 2 0 ---3 02 ............ (17)
o
For 14, wavelengths <2450 A are required to provide sufficient energy
o
while 17 occurs with a 2537 A wavelength where ozone absorbs strongly.

A mercury vapor discharge produces its strongest (resonance)
o o
radiations at 1849 A and 2537 A. It is a suitable UV source for ozone

production when it is enclosed in quartz rather than glass so that the

shorter waveci-li-lths will be transmitted. Such a bulb3 was utilized.

Ozone production took place within the chamber used for animal

exposures and irradiations. Figure 5 is an overall view of the exper-

imental setup including exposure chamber, x-ray source, ozone detector,

and related equipment. The ozone bulb was placed at the rear and on

the central axis of the 46 cm ID x 92 cm chamber. See Figure 6. The

animals, in a ventilated cage near the center of the chamber, were

shielded fro( the UV light by a 37 cm diameter aluminum sheet. See

Figure 7. Air which flowed continuously from the rear of the chamber

to its front at a rate of 1.5 l./r.in served to transport ozone to the

animal cae.

Ozone concentration was varied by altering the volt.,i.e impressed

across the UV bulb and its series ballast (a 40 W light bulb) by use of

an autotransformir. Response was linear. Unwanted variation was

avoided by operating the autotransformer from the output of a constant

volt. e transformer.

The ozone concentration of interest within the exposure cha ber

was that in the animal cage. This was determined continuously through


Co 1rany, Cleveland, Ohio.


3 o. G4SIJ Ccncral El.





























































OVERALL VIEW OF ANIMAL EXPOSURE SETUP


FIGURE 5.
















A


a r


_rJ" -a


FIGURE 6. THE INTERIOR OF THE EXPOSURE CHAMBER WITHOUT UV SHIELD


























U'r


-s


Sx~


FIGURE 7. THE INTERIOR OF THE EXPOSURE CHAMBER
WITH UV SHIELD


%,or








the use of a Mast Ozone Meter4 connected, through a valve in the chamber,

to a 3.2 mm ID stainless steel sampling tube which terminated in an

unoccupied compartment of the animal cage. See Figure 8.

The Mast Meter utilizes a microcoulomb sensor (184). The sensing

solution containing potassium iodide (KI.) (See Appendix 1) enters the

detector at a fixed rate and flows down an electrode support in a thin

film. Air enters the detector at a fixed rate and comes in intimate

contact with the solution film. When ozone is present and chemical re-

actions occur (See below)), current flows, the anode and cathode having

a constant potential between them. This current is measured on a micro-

amieter in the external circuit. The reactions involved are the follow-

ing (184):

Oxidation of KI in the sensing solution

03 + 2 KT + -20--0 02 + 12 + 2 KOH .........(18)

]Production of 112 layer by polarization at the cathode

2e + 2 II --. 112 ................... (19)

Reaction of 112 and 12 at the cathode

H2 + 12 --- 211I .....................(20)

Equation 20 is followed by a repolarization current of two electrons

flowing in the external circuit. Hence the current is directly propor-

tional to the number of ozone molecules reacting.

Ozone concentrations measured with Mast Meters were reported to

be between 65 percent and 100 percent, depending on their calibration,

of values determined by the neutral KI chemical method of determination,


4Model 724-2, Mast Development Company, Davenport, Iowa.
























a.


4.
*



em.-,
.* b


a
SI


FIGURE 8. THE EXPOSURE CAGE WITH ANIMALS, R-CHAMBER,
AND OZONE SAMPLING PROBE


,,:


*
14,




* *
Am


40

*









considered as the reference method (84, 185). Consequently, it was

necessary to determine the calibration of the particular Mast Meter

being used. For this, another standardized procedure, the alkaline KI

method, was used (185). See Appendix 2. It had some advantages over

the neutral KI method, and data could be converted to standard values

(neutral KI values) by multiplying by 1.54 (84).

For the calibration, ozone was produced by the UV bulb in a 2 1.

beaker. The Mast Meter sampling tube and the inlet for the sampling

train used in determining ozone by the alkaline KI chemical method

both drew air from the beaker at the same position. See Figure 9. The

bypass of the midget impinger containing the sampling reagent was used

to keep the air flow from the beaker, and hence the ozone concentration

in the beaker, from altering drastically when sampling began. A strip

chart recorder was used with the Mast Meter so that average readings

could be determined for the time periods during which samples were

collected. The results of the calibration procedure are shown in

Figure 10.

The autotransformer and ballast bulb for ozone production reg-

ulation, the strip chart recorder for the Mast Meter, and the compressed

air tank and flowmeter for air flow through the chamber were all located

outside the laboratory containing the exposure chamber so that observa-

tion and regulation could be done during an x-ray exposure. An exhaust

pump for the chamber was required to keep it from pressurizing slightly,

which would increase the air flow into the Mast Meter and change its

calibration. This pump was located in the exposure laboratory but did

not require frequent adjustment. Humidity within the chamber was kept

nearly constant by passing the inflowing air through a water bubbler




























































FIGURE 9. THE SETUP USED FOR CALIBRATING THE OZONE METER





































FIGURE 10.

ACTUAL OZONE CONCENTRATION VS. MAST
METER INDICATED OZONE CONCENTRATION
















0
O

a 40-
a.






J


<14
030-
o

Ii
a.
o 25-

z
0


z
0
z
015.
C.


I
/1:


[031 = 1.5 [03] -5.5
[]KI I MAST


/ /
/ /
L /

//
//
//
//
/
/
/ /
/ /
/
/
/
/ I



/
/
/
f/
/
/


10 20 30 40 50 60
MAST METER AVG. OZONE CONCENTRATION, PPM x102









bottle. Temperature re -ul rion was automatic, the sensing thermocouple

being in contact with the animal cage bottom at is center. Air flow

into the Mast Meter was checked periodically both with and without air

flow into the chamber and was found to remain constant.

Many materials react with ozone when first exposed to it or when

exposed to it again after a long period of non-exposure. This reaction

lowers the ozone concentration in the air. The reduction can be very

lar with some substances. Exposure to relatively high concentrations

of ozone (up to 1 ppm) over extended periods (several hours) will elim-

inate the reactivity of many items. This is referred to as "ozone-

conditio. 'l." (186). The exposure chamber, the animal cage, and the

sampling line were "ozone-conditioned" in order to attain the desired

concentrations and to prevent low readings due to reaction in the sam-

pling system.


The Production and Quantization of the Radiation Field

To produce the desired external radiation field, a General

Electric Haximar 250-III therapy x-ray machine5 was employed. The unit

was equipped with an adjustable rectangular collimator to alter field

size plus an internal light to check the field size at the object being

irradiated. There was provision for inserting standard (supplied) fil-

ters into the beam plus a slot for additional filters as required. The

control console was located outside of the exposure room, whose walls

were lin d with 1.6 mrm thich lead sheeting.



5Type 1, Serial No. 457617, General Electric Company, Mil.wauket
Uisconsin.








Radiation was beamed into the exposure chamber through a 10 cm

diameter viewing port. See Figure 5, page 35. The beam axis was per-

pendicular to the central plane of the 15 cm diameter lucite animal

cage, and the target-cage distance was 70 cm. The cage permitted some

animal movement but not toward or away from x-ray source. To monitor

the consistency of the beam during an exposure period, a Victoreen6

Rate Meter, Model 510,was employed in conjunction with a strip chart

recorder. The probe, Model 601 (30-400 keV effective), was placed

outside of the exposure chamber, at the edge of the round viewing port,

and in the corner of the beam (which measured 11 cm x 11 cm there). To

determine the total exposure during a period, a Victoreen R-chamber,

Model 154 (250 R, No. x915),and Model 570 reader were utilized. The

chamber was placed in the unused compartment of the animal cage. See

Figure 8, page 39. A correction factor of 0.98 was applied to all

readings of the chamber. This was a sensitivity factor determined by

comparison of the chamber with one calibrated by the National Bureau of

Standards.

The uniformity of the field over the-cage area was checked in two

ways, with sheet film and with an array of thermoluminescent (TLD) powder

dosimeters. The film, Kodak Type AA Industrial in a leaded cassette,

was placed at the cage position perpendicular to the x-ray beam. The

circular rather than oblong pattern showed the beam definitely was per-

pendicular to the cage; densitometric measurements revealed the 90 per-

cent isodose curve to be very nearly a perfect circle with an area

larger than that of the circular cage. There were two 3.1 mm OD x 2.5 cm


6Victoreen Instrument Company, Cleveland, Ohio.








TLD dosimeters per wedge-shaped animal compartment (with two readings

per dosimeter). They were positioned in the midplane of the cage in

two concentric circles (7 cm and 14 cm diameters) and surrounded with

rice as phantom material. Response was linear with dose in the region

of interest, 9-17 R. The average dose was determined for each animal

compartment; the maximum deviation of animal dose from the mean dose

was 6 percent of the mean.

The x-ray machine was operated with 200 AC volts across the

primary of the high voltage transformer, this corresponding to approxi-

mately 200 KVP. The standard filter employed was 2 mm Cu plus 2 mm Al.

With this combination of voltage and filtration, the half-value layer

(IIVL) was determined to be 2.35 mm Cu, equivalent to an effective beam

energy of approximately 118 keV. This ensured a maximum decrease in dose

of approximately 15 percent over a 4 cm animal depth with the 70 cm

target-to-cage distance used (187). The unit was operated at 5-9 mA with

2 mm Cu additional filtration which served to reduce the intensity of

the beam down to the desired level, approximately 1 R/min at the animal

position. Current was adjusted as necessary during an exposure to keep

the exposure rate, seen on the ratemeter and strip chart, at the desir-

ed value.


Scorii- Chi,-- *o. e Aberrations

The aberrations of interest in this investigation are all of the

chromoso:ie type. They involve both stands of the duplicated chromo-

sormns, since the breaking and rejo:: i associated with exposure of the

animals to ozone and/or radiation occurred before duplication of the

)y1rphocyte chromosomes.








The various chromosome-type aberrations are shown in Figure 11

(93). The following discussion refers to this figure.

The chromosome aberrations of interest in a quantitative invest-

gation are those that require a minimum of subjective judgement on the

part of the observer in order to identify them. Consequently, types

which involve changes in the lengths of the arms (the strand portions

on each side of the constriction, or centromere) should usually not be

included in scoring (counting). These include columns B, C, and F.

Each of these types involves two breaks and an exchange of resulting

arm segments to give chromosomes that in most cases give an outward

appearance of being normal. Columns B and C, involving one chromosome,

are referred to as inversions, and these were never scored (counted).

Column F, involving two separate chromosomes, is referred to as a

tronl T-cati on7. Thl's t W --r"c (-s tim bI-ran) only if the arms of one

of the chroi'osones were decidedly and distinctly much longer than they

should have been. See Figure 1, page 25. Arms shorter than normal

would not necessarily indicate a translocation, but could result from

a simple chromosome break, as shown in Column A.

The types of aberrations which were the basis of scoring are

shown in Columns A, D, E, and F. They are all easily identified.

Column A, called a simple break or a deletion, results from one

chromosome being broken and does not require any type of recombination

in order to appear. It was scored as one break on the basis of finding

acentric fragments (or one fragment as shown in A4), fragments without a

centromere. Deletions were the aberration type seen most frequently.

Columns D, E, and F each involve two breaks and were so scored

when found. Columns D and E, each involving a deletion from a single








SYMMETRICAL
INTRACHANGES


ASYMMETRICAL
INTRACHANGES


rChromosome
break


Inter-arm
inversion


Intra-arm
inversion


Inter-arm
deletion


Intra-arm
deletion


Symmetrical
interchange


Asymmetrical
interchange


0

Al BI C DI El FI GI




A.2 B2 C2 2 D2 2 E2, F J G2




1A BC3 D3 ES F__ G




uC Aor- D4 E4 F4 G4




A4 84 C4
oO


VV
or Do GS


AS BS wC5 D 6 G 6
0 or A or







A6 D_ __
FIGURE 11. DIAGRAM OF THE VARIOUS CHROMOSOME-TYPE CHROMOSOME
ABERRATIONS









chromosome, result in the formation of ring structures, either with or

without a centromere; these aberrations are referred to as rings and

were scored on the basis of finding the rings. Column F, involving an

interchange between two chromosomes, results in the formation of a chro-

mosome with two centromeres; it is called a dicentric and was scored on

the basis of finding dicentric chromosomes.

In summary, a deletion was scored as one break on the basis of

finding eccentric fragments, a gross translocation was scored as two

breaks on the basis of finding a chromosome with exceptionally long arms,

while rings and dicentrics were scored as two breaks each when found.

Figures involving three breaks or more are possible but were not seen.


Estimating the Ozone and Radiation Exposures Required

The principal objective in choosing the ozone and radiation expo-

sure levels was to have exposure to one agent alone or to the two simul-

taneously result in frequencies of chromosome aberration that were sig-

nificantly different statistically.

The production of chromosome aberrations by sparcely ionizing

radiation such as x-ray is a random process. With a given dose, the fre-

quencies of cells in the "one-break," "two-break," etc. classifications

follow the Poisson distribution (155). A given ozone exposure would

likewise be expected to result in a Poisson distribution of chromosome

breaks per cell since ozone, like x-ray, is believed to act primarily

through oxidation events associated with free radical production (154).

The number of breaks per unit of cells (such as 100 cells) should

also follow the Poisson distribution. The number of breaks, n, is a

point estimate of the average number of breaks, u. If u>15, the Poisson









can be approxi',ated by a normal distribution with a mean = u and standard

deviation = u The sample (estimated) mean would be n and the sample

standard deviation would be n2. On a breaks per cell basis, the sample

mean would be n/N, the sample standard deviation n /N, and the 95 per-

cent confidence interval n/N i 2 n2/N, where N = the number of cells in

the unit. The statistical significance of the difference between two

means could be determined through the use of Student's T distribution

or by determining whether the confidence intervals around the means

overlapped, a stricter test.

The ozone and radiation exposure levels were chosen to yield

equal break frequencies, with the total number of breaks for each being

large cri' ,1 for the normal distribution approximation to apply. The

levels were also chosen so that break frequency for combined ozone plus

radiation exposure, expected to be approximately twice that for either

a- .t alone, would be significantly different statistically from the

frequency for either agent alone, as determined by the confidence inter-

val overlap method. The levels were also chosen to be as low as possible

while still meeting these requirements.

The initial estimates of chromosome break frequencies from x-ray

exposure were made on the basis of available information on the response

of human lrophocytes to x-radiation in vivo and in vitro (155, 165).

Supporting evidence came from chro atid aberration data for the in vivo

irradiation of C',i .1e Mhaster bone Iarrow cells (172), taking into

account that the cells exa ined were in the post-DNA-replication stage

of the cell cycle at the tim of the irradiation and hence had greater

raciosensitivity (dis d:' differences in cell type) (148).









For ozone, deletion frequency was estimated on the basis of the

exposure of human cell cultures to high concentrations of ozone by

Fetner (154), while ring and dicentric frequency was strictly a guess

based on analogy with radiation. Fetner's ozone data were on chromatid

deletions. In arriving at the estimated frequency for this investiga-

tion, the pre-DNA-synthesis stage of the cell cycle (GI) was assumed to

be approximately one-half as sensitive as the post-DNA-synthesis (G2)

stage (148). (Difference in cell. type was disregarded.) Additional

assumptions included linear dependence of break frequency on exposure

time, to be conservative (the data showed exponential dependence), and

validity of concentration x time = constant response (84) from the 8 ppm

used down to 0.1. ppm, the approximate concentration desired.

The following were the initial estimates:

Radiati on

1 -3 deletions
1 1 el0 ons....................(21)
cell-rad


-16 diccntrics I- rn'F (2
6 x 10 --d-- --- ..............(22)
cell-rad-


Ozone
-3 deletions
3 x 10 ................ (23)
cell- (ppnm-mi n)

S-6 dicentrics + rings
7xe10 ---m--n ............... (24)
ccll-(ppm-min)-

The dependence of dicentric or ring frequency on the square of the dose

follows from two breaks b-i.- required for each, the probability for each

break being linearly dependent on dose, and the occurrence of each being

independent of the other. Data on spontaneous aberrations in Chinese

hamster bone marrow (172) and in human blood lyrmphocytes (163, 166) in-

dicated the frequency was so low. (] x 10-3 breaks/coll) that it could








be neglected, but pre-exposure cultures were made anyway as a check.

The data of llungerford (180) for human blood and Buckton and

Nettesheim (1.81) for mouse blood indicated that it would not be unrea-

sonable to expect, per culture, 200 metaphase spreads that would be

suitable for analysis. Assuming 200 cells per culture, it was deter-

mined by trial and error using the coefficients in 21-24 and the ex-

pression for 95 percent confidence interval that 5 hr at 0.1 ppm ozone

and 100 rad (over 5 hr) would be suitable exposures. These levels

would result in confidence intervals of 0.22 _- 0.07 breaks/cell for

ozone or radiation alone and 0.44 4 0.09 breaks/cell for ozone plus

radiation (a.. 'i. additivity) and these intervals do not overlap.

11alving each exposure (to 2.5 hr at 0.1 ppm and 50 rad) would result in

confidence internal that would overlap, so the higher values, 0.5 ppm-

hr I ad 1( raJ, lr tl" o ; of choice.

To determine the suitability of these values, preliminary exper-

iments were conducted utilizing one or two animals at a time exposed

either to ozone or to radiation. The ozone exposure levels wore approx-

imately 0.45 and 0.50 ppm-hr and the radiation doses were 85, 153, and

166 rad (Roentgcn-to-rad conversion followed NBS Handbook 85, Physical

Aspects of Irradiation). Eight animals were involved.

Discounting the 13 and 16 day delays between irradiation and

successful cultures for animals receiving 153 and 166 rad, these delays

in part a result of culturing oven temperature variations, the deletion

yield for irrndiation was only 65 percent of the expected yield. Simi-

larly, for the ozone-exposed animals, the deletion yield was only 62

percent of the expected value. Additionally, it was found that the

aver e nu ber of ri t 'phs spreads per slide that were suitable for








analysis was nearer to 40 than to 200, with values ranging from 10--71.

Since the basic exposure group was to have four animals, there

was a possibility that fewer than 15 deletions would be found for all

the animals pooled. Even if the exposures were raised to correct for

the lower-than-expected yields, only 10 deletions per 100 cells would be

expected, so the average of 160 spreads per animal group would only

give an average of 16 deletions. Fewer than 15 would mean that the

normal distribution approximation to the Poisson distribution could not

be used, and that confidence intervals would not be appropriate. The

breaks that would result from dicentrics and rings were not being

counted on to raise the total breaks observed. This was because the

radiation and the ozone were being administered over a relatively long

time period, 5 hr, which may be longer than the average lifetime of a

chro ouom~ bLcak (155). 'is ,woulJ mn A thai bi;hCs creatcd at the

beginning of the period might not still be available for combination at

the end of the period, so the yields of multiple-break aberrations

would be lowered.

Consequently, the decision was made to raise the ozone and the

radiation levels to correct for the low yields and to increase the ex-

pected number of deletions from 30 up to 15 per 100 cells, for four

animals would certainly be expected to yield 100 analyzable spreads.

It was also decided to increase the ozone concentration rather than

increase the exposure time to >5 hr.

The radiation dose to be used, then, was (100 rads)(1.5/0.65) =

231 rad and the ozone concentration would be (0.1 ppm)(1.5/0.62) = 0.24

ppm but a decision was made to limit the ozone concentration to 0.2 ppm,

for a total exposure of 1,0 ppm-hr. This was done to avoid the








possibility of any observable effect on the well-being of the animals,

i.e., to keep the exposure level at a value known to be- tolerable (84).

For each condition, ozone, radiation, and ozone plus radiation,

there were two exposure groups of four animals each. An additional

group wa.; put thliL'. 1' the entire procedure minus the agents (ozone and/

or radiation) to serve as a control. Also, another group was subjected

to a 335 rad radiation exposure, approximately 50 percent greater than

the dose the others received. This w.as done to demonstrate that break

frequency does in fact increase with radiation dose. A preliminary

report indicated that 335 rad was one-third of the LD50/30 for 100 day

old Chinese hamsters (188), so complications which would interfere with

the procedures to follow were not expected and did not appear.















CHAPTER III


PROCEDURES

This chapter presents a step-by-step description of the numerous

operations that were involved in this investigation. For some of these

operations, details were important, so the entire procedure is present-

ed. Reference is also made to the appendices for various formulations

and supplementary procedures. Operations are presented in their normal

time sequency.


Animal Txposures

The four animals in each exposure group were randomly selected

from among those for which successful pre-treatment cultures were avail-

able. Since the animal quarters were located near the exposure room,

the animals remained in their boarding cages until transferred to indi-

vidual compartments of the exposure cage immediately prior to beginning

an exposure. Each animal occupied approximately 50-70 percent of the

space of its compartment. Exposures began approximately at 9 A.M. and

were of a 5 hr duration.

If the exposure involved ozone, the UV generator remained oper-

ating the previous night so the chamber would have an ozone environment

ready. Similarly, if the exposure involved radiation, the ratemeter

and the R-chamber reader were left operating overnight so they would be

stabilized by irradiation Lime. The response of the ozone meter was

checked with an electric discharge ozone generator outside the chamber

before and after each exposure.








Air flow through the chamber was maintained at 1.5 l./min

throughout ench exposure. This provided a complete air change in the

chamber every 100 min. Since the exposures were conducted during day-

light hours, a light was provided at the second viewing port of the

chamber to illuminate the interior. Temperature was maintained at

15.50C. Relative humidity averaged 70 percent for exposures involving

ozone and 45 percent for exposures to radiation alone. This difference

resulted from ozone exposures being done on consecutive days and radi-

ation exposures having at least one day between them, which gave the

chamber time to air and dry out. The animals of each group were

observed through Lhe viewing port at least twice during their exposure;

they were generally inactive but awake.

The chamber was opened once during each radiation exposure to

read and rclare tbh R-cham ber. This was done so that reading i would

be around mid-scale where they were most accurate. An air density cor-

rection factor of 0.98 was applied to the readings because the exposure

chmb r tct rature : as below 220C. During, each radiation exposure,

the x-ray current was adjusted when necessary to keep the ratemeter

reading at the desired level. An exposure rate of 4.8 R/min (7.0 R/min)

at the rate(cter probe corresponded to the desired 0.82 R/nin (1.15 R/

min) at the cage R-chamber. Values in parentheses were for the high

radiation ; During each ozone exposure, the UV bulb voltage was

adjusted when necessary to keep the cage ozone concentration at the

desired 0.2 ppr Response was relatively rapid, concentration adjust-

ment time vary', inversely ith the size of the voltage change.









Once a steady-state condition was attained, the cage ozone con-

centration was the same as that in the rest of the chamber. This indi-

cated that there was no appreciable ozone degradation by the Lucite

cage after it was ozone-conditioned. There was no detectable ozone

production by the radiation beam, and the x-ray environment appeared

to have no effect on the performance of the ozone meter.

When an exposure was completed, the animal cage was removed from

the exposure chamber immediately; the animals remained in the cage

awaiting the blood sampling procedure, which began promptly.


Blood Sampling

The collection of blood samples by the orbital bleeding technique

was done prior to an animal being accepted for exposure (once, or more

if required by culture failure or low yields of chromosome spreads),

inmvecdiately after exposure, and again at intervals if required or de--

sired. This operation and all those following were the same for pre-

and post-exposure samples.

All materials and equipment contacting the blood were sterile,

from the sampling operation to the cell-killing step of the slide pre-

paration procedure. They were either purchased that way or autoclaved

at 121.50C, 15 psig for 15 min. Sterile technique was employed. Except

for the tissue culture tubes, all glassware was silicone-treated to

reduce cell adhesion. For cl' :-,;- glassware, the detergent employed
7
was non-toxic to tissue cultures.




7 X Glassware Dercrgent, Limbro Che mical Company, New HIaven,
Connect icut.









Leighton-type tissue culture tubes were used. These have an 11

rm x 40 vis flat area on one side and a constriction above this area to

act as a dam. For the procedure being followed, they were reported to

be the only kind in which culturing was successful (181). The tubes

employed were 16 mm OD x 125 mmr with screw-caps. They were filled with

15 ml of heparinized TC-199 culture media (See Appendix 1) and kept on

ice during the sampling procedure.

For each ani 1al, blood was withdrawn with a heparin-wetted

blood pipette and emptied into a culture tube which was then gently

agitated and given a number identifying the animal and the culture from

that animal. Generally, one pipette-full, approximately 0.25 ml, of

blood was put into each culture tube. Occasionally, if <0.20 rml was

transferred in, a second pipette was used in the other eye; the total

ulood puL into cultui ianLid up to 0.35 Al. AfLtr each animal- was

sampled (one culture tube per animal) they were returned to their board-

ing cages and the culture tubes were transported to the culturing labo-

ratory.


Tissue Culture Preparation and 'Naintenance

The purpose of the TC-199 culture media was to greatly dilute

the hamster blood plasma (approximately 1:100). The culture tubes were

spun at 500 g for 10 min in a refrigerated centrifuge (5 C) and the

supernatant liquid (TC-199 plus plasma) was pipetted out. A 4.5 ml

aliquot of culture media was added to cache tube immediately after re-

moval of its supernatant liquid, and the tubes were gently agitated.

The n;dia was prepared following the specifications of Hungerford (180)









(See Appendix 1). This was followed by the addition of 0.05 ml of
8
pokeweed mitogen solution per culture tube.

The tubes were then placed in an incubator at an angle of 50 to

horizontal with their screw-caps slightly open. The incubator was main--

tained at 370C with a 95 percent air plus 5 percent CO2 gas flow through

it that provided a complete change every 100 min. Humidity was main-

tained by a constant-level water pan in the base. The tubes were mo-

mentarily removed twice per day for gentle agitation.

After 67 hr of incubation, 0.4 ml of a 2y g/ml colchicine solu-

tion was added to each tube (See Appendix 1). This drug, a spindle

poison, prevented cells in tle division process from proceeding past

the metaphase stage, where the chromosomes were duplicated and con-

tracted, to the anaphase stage where each chromosome would separate into

two daughter chromoso-es that would migrate to opposite ends of the cell

just prior to its division. In this way cells in metaphase were "stored

up" as cells entered the stage while few, if any, left it (155). After

5 hr exposure to colchicine, 72 hr total incubation, the cultures were

removed from the incubator, and the slide preparation procedures were

begun.


Slide Preparation

After gentle agitation, each culture was transferred by pipette

to a graduated 15 ml centrifuge tube and spun for 4 min at 100 g. The

supernatant liquid was removed by pipetting. A 5 ml aliquot of hepa-

rinized hypotonic potassium chloride (KC1) solution (See Appendix 1),




8Grand Island Biological Company, Grand Is]and, New York.









pre-,warmed to 370C, was added to each culture. Each tube was gently

agitated with a mechanized tube mixer during the KC1 addition to suspend

the cell button. The tubes were covered and placed in the incubator

at 37 C for 15 rin.

The tubes were spun again and the supernatant liquid was removed,

t'"i-; care to leave the thin light-colored overlayer of the button

called the buffy coat. With a gentle mechanical-mixer agitation to

avoid clui:pif a 5 ml portion of fixative was added to each centrifuge

tube. TIh fixative, which caused henolysis and killed the leucocytes,

was made up of three parts absolute ncthanol and one part glacial

acetic acid. The tubes were spun as before (4 min, 100 g), the super-

natant liquid was rc oved, and a second 5 il aliquot of fixative was

added to each tube, again with g ntle mechanical agitation. They were

ccvnr d "n3 pl'.:- in a Pcfrl'cr: ow for 15 nin.

The tubes were spun a.d the supernatant liquid was removed. It

had no color r .ini front heinoglobin. Without agitation, enough

fixative was added to each tube to approximately double the volume of

its cell button, which :-. --I from 0.05-0.10 il.

The 25 nin x 76 in slides to be used had previously been washed,

put into a staining dish to keep them separated, submerged in distilled

water, and placed in the refrigerator. When the centrifuge tubes with

fixative cwre put in the refri lator, the beaker of slides was re-

moved n d plce- on ice, and a flow of CO2 gas that bubbled through the

water wa i un.

Usi a Paste r pip tt- (w ith ru bber bulb) as a rod, the cell

button in a c ntrif e ti be was i.tly dispersed. The 0.1-0.2 ml sus-

pension was thLen dOra into the pipette. A slide .was removed from tie









beaker with tweezers, grasped by the edges, and tilted with one edge

against adsorbent paper to remove excess water. The slide was then

leveled and the suspension from the pipette was transferred to it, con-

fining the delivery to a 24 mm x 50 mm area or less. The slide was then

rapidly passed through an open flame which ignited the methanol. When

it burned off, the slide was again tilted, this time being tapped against

adsorbent paper to remove excess moisture. It was then placed on a

slide warmer at 400C to dry thoroughly. The water film and the burning

were necessary to obtain metaphase figures (spreads) with well-separated

chromosomes that lay flat on the glass.

The slide-making procedure was repeated for each of the cultures

being processed. When the slides were thoroughly dry, the identifying

numbers were etched on them. Each one was then partially but systemati-
9
cally scanned with a phase contrast microscope. This was done to get a

relative measure of how successful the culturing and slide preparation

procedures had been by counting the number of chromosome spreads ob-

served. Few or no spreads seen on a slide indicated that the entire

procedure would have to be repeated for that particular animal. This

did occur in some cases and will be discussed later.

If the quality appeared adequate, the slide was stained with

Giemsa blood stain (See Appendix 3). The 24 mm x 50 mm area with

material was covered with a glass slip fastened with Permount.10 The

stained chromosomes appeared purple.




9AO Phasestar, American Optical Corporation, Buffalo, New York.


1Fisher Scientific Company, Springfield, New Jersey.








Recording of Data

Each slide was scanned usi.; bright-field microscopy. Over-

lapping fields of view were used to reduce the likelihood of missing a

spread near the edge of the field. Scanning was done at 100x total

magnification with spot checks at 950x for questionable figures. The

object was to locate chromosome spreads, record their location (the

microscope st. was -.. .- ted), and photograph them at 950x total

ra i-:;ication for 3ater analysis. So as not to bias the data, each

spread that was located was photographed, without regard to its quality.

Generally, only the 24 mrn x 50 rm portion of the slide covered

with a pla: slip was scanned. Occasionally the preliminary phase

scan indicated that the material had spread beyond the intended area;

these slides were scanned over their entire area.

Th ..C:C.. c:, i a Lcit 11 Ortholux equipped with an

Orthor:at auto zatic 35 rim ca ra. Since the chro: osomes were stained

with Gicmisa, a combination of Wratten filters12 was used to improve the

visual contrast and inage quality (189). With these filters, the

chromosomes appeared black on a green field. Photography was black and

white.

Tie film-diveloper combination chosen was Kodak Panatomic-X and

Kodak D-19 developer. This panchro:mtic film was extremely fine-

grain d for sharpness of detail and high resolving power and combined

moderate contrast with adequate speed (ASA 32) (190). The recom~iended

developers were all mediun-contrast materials. To improve the i -;.ing,



11
E. Leitz, I3c., NeI York, T York.

12\-ratten Nos. 12 (; lid o) aid /!4 (caqua blue) 'Earr n Kodak
CoT'pi ~, R c ster c, Ka~ York.









a high-contrast developer was selected; of those available, D-19 was

chosen since it was usually used for scientific plates and instrumenta-

tion films. Through inquiry, a suitable developing time for this com-

bination was found to be 3.5 min at 200C (191).


Analysis of Data

Films were left in roll form for analysis. A film strip pro-

jector was used to image the films, frame-by-frame, on a screen. ,When

enlarged in this manner, a typical chromosome spread had a diameter of

approximately 25 cm. Detail was not lost because a high degree of en-

largement was possible with the extremely fine grained fine used.

Scoring of aberrations was facilitated by these large-size images.

To avoid the difficulties associated with being reasonably sure

that different people would score the aberrations in a given spread

in the same way, all the scoring was done by one person, the author.

The scoring was conducted as a blind analysis to avoid any biasing of

the data. Each film was identified by number only; there was no indica-

tion as to which exposure group the data had come from. The photo-

graphy log book indicated that the data on a particular film came from

a particular animal, but the exposure group to which the animal be-

longed was not listed there.

The information recorded in the scoring log book for each frame

included the total number of chromosomes, the number of chromosomes in

each to two general morphological classifications, "X"'s and "V"'s (See

Figure 1, page 25), and the number and kinds of chromosome aberrations.

Also noted were chromatid (one-arm) breaks and stain gaps (chromosome

portions that did not take up stain). A group of chromosomes was








counted as a spread if it had 12 or more figures, i.e., greater than

one-half of the 22 chromosomes in a normal spread; sometimes chromo-

somes were lost from a spread during the slide preparation procedures.

A nor ml spread h- 16 "X"-shaped chromosomes and 6 "V"-shaped figures.

IWhen necessary to aid in scoring aberrations, detailed figure counting

for the var ious sizes of "X"-shapes was also done.

Any spread that appeared blurred was later relocated on the

microscope using the photography L book to get its coordinates. This

was to check whetcLer the spread was blurred or the photograph was poor.

If the spread was clear, a new photograph as taken. Similarly, any

spread that exhibited a chromeosomc aberration was relocated on the

microscope. This w.as to determine whether the figure scored as an

aberration was an artifact on tih film or on the slide or was actually

c1 r :L A 1

At the co:' pltion of scoring; the exposure groups were revealed

and the data tabulated on an animal basis and on a group basis, listing

the numbers of spreads analyzed and the numbers of deletions, dicentrics,

and r, i found. (.. t. ri locations were not seen. The production-

frequency coefficients were then formed and comparisons were iade for

the various groups.















CHAPTER IV


RESULTS

Table 2 presents the exposures received by the various treatment

groups. In each case except one (Group 1), the exposure and rate or con-

centration were near the desired values, so variation among the six

principal groups was minimal. The high radiation group (Ii X) dose was

40 percent greater than that received by the other x-ray exposed groups;

also it was delivered at a 40 percent higher rate.

A total of 746 chromosome spreads were examined for breaks in the

final experiment. These spreads displayed a total of 87 chromosome-

type chromosome breaks and 6 aberrations which could be classified as

chromatid-type chromosome breaks or as stain gaps. The results of the

analysis are surnarized in Table 3. In this tabulation, the ozone-

exposed groups were broken down into three categories, "early," "mid,"

and "late." These designations refer to the time interval between treat-

ment and sampling, 0, 6.0, and 15.5 days respectively. The multiple

sampling was done to have ozone data with a delay time comparable to

the delays experienced with the other groups. It was discovered during

the final investigation that the radiation exposure had an inhibitory

effect on the lymphocytes which resulted in unsuccessful cultures (i.e.,

cultures with few if any mitotic chromosome spreads). This effect

appeared to diminish with time after exposure, and satisfactory cul-

tures were obtained in all cases after l days had elapsed.














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The break frequency column was included in Table 3 to permit

rough comparisons. The entries, simply breaks per cell, do not reflect

either differences in exposure among the groups or, for averaging,

differences in numbers of useable spreads per group. Both of these

refines nts w.re incorporated in the exposure-adjusted frequencies

given in Table 4. For each trcatient involving two groups, a weighted

aver. was formed .when the difference between the contributing fre-

quencies vas found to be statistically iN;'i lficant (approximately

normal distribution was assumed for each frequency). Two ozone plus

radiation frequencies were formed, one based on the radiation exposure

only so direct comparison could be made with the frequency of the

radiation treaty' at groups and the other based on the ozone exposure

only so direct co prison could be 1ade with the frequency of the ozone

treat, cn t groo s.


Radiation :posed Animals

Comparison of the exposure-adjusted break frequencies for radi-

ation and for high radiation treationts in Table 4 showed them to be

nearly identical, within 4 percent of one another, and to have compara-

ble sample standard deviations with values 25 percent of the eanI s,

As would be expected, the difference between the frequencies was not

statistically s; "ficant. Th esc data soi ested that the number of

breaks per cell varied linearly with dose in this re ioj, 230-330 rad.

IMiore importantly, since linear dependence had been expected, the high

radiation frequency provided a check on tlhe valid-ty of the radiation

frequency.


























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Ozone Exp osed Anials

The most important fact revealed by the ozone-exposed groups was

that inhaled ozone did result in chromosome breaks in circulating lym--

phocytes. The exposure-adjusted break frequency for 03(late) in Table

4 definitely differed from zero (P<0.001, i.e., <0.1 percent chance of

the frequency being zero). Comparison of the frequency for 03(late)

with that for 03(early) revealed that the difference between them was

not statistically significant. The difference between 03(late) and

03(mid) was significant, the latter frequency being smaller. This per-

haps resulted from 50 percent fewer cells beii, available for examina-

tion for 03(miid). The data suggested, however, that the observed ozone-

induced break frequency did not diminish with time for at least 2 weeks

post-exposure.


Ani- 1 s Exposed to Combined Ozone and Radiation Environ mnt

tl exposure-adjurted break frequency for the ozone plus radia-

tion treatment groups, based on the radiation exposure only, was com-

pared with the frequency for the radiation treatment groups. The dif-

ference between then, while not statistically significant, was a value

that would occur or be exceeded 18 percent of the time (expecting the

03 + X frequency to be higher). In other words, there was an 18 percent

chance that all the breaks obserrved for the ozone plus radiation treat-

went resulted fre the radiation alone even though the 03 + X frequency

was nearly 40 percent greatr than the X frequency. Similarly, there

was a 15 percent chance that all the breaks observed for the ozone plus

radiation treaty t resulted from the ozone exposure alone even t' -_1

the 03 + X f cncy ws 45 percent greater than the 03(laite) frequency.









Another way of examining the ozone plus radiation treatment data

is presented in Table 5. The expected breaks from radiation (computed

from the exposure-adjusted radiation frequency) and the expected breaks

from ozone (computed from the exposure-adjusted ozone frequency) were

compared with the number of breaks observed with combined treatment.

These facts were revealed:

1. The expected contributions from ozone and from radiation were

nearly equal;

2. The number of breaks observed was >70 percent of the total

breaks expected from the two agents combined (assuming additive

effects);

3. The number of breaks observed was 40 percent greater than

the number expected from either agent alone;

(I. E ither o:onc cr radc1;o alone could account for all the

breaks seen (considering I:aximum rather than average expected

values).

The data suggested, but did not show conclusively, that the combined

ozone plus radiation treatment resulted in a higher frequency of chromo-

some breaks than would be expected from radiation or from ozone alone.


























,o

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CHAPTER V


DISCUSSION

The initial estimates of break frequency for radiation and for

ozone presented in Chapter II were based on the frequencies expected

for deletions and for dicentrics and rings. The observed exposure-

adjusted aberration frequencies were compared with these first estimates.
Observed
These data are presented in Table 6. The -sti-ated column facilitated
Es tiuated

comparison, among the four frequencies, of success in estimating.


Expected vs. Observed Aberration Frenuc ncies with Radiation

The estimates for radiation wurie based on frequencies developed

by the irradiation of freshly drawn human whole blood in vitro .with

culturing following ji mediately (155). These frequencies were found to

be reasonably good estimates of in vivo aberration production in humans

(165), generally within a factor of two for deletions and a factor of

six for dicentrics and rings. Frequencies for aberration production in

lymphocytes of other species, either in vitro or in vivo, were not

available for comparison.

Data were available for the production of breaks in other Chinese

hamster cells by radiation. Exposure-adjusted frequencies for the pro-

duction of chromatid-type breaks in bone marrow cells with in vivo

exposure and in cultured "fibroblast-like" (embryo) cells with in vitro

exposure were, nearly equal to one another at 5 x 10-3 beas. For the

bone marrow cells, the frequency decreased exponentially with time after



















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_z. breaks
irradiation to 1 x 10 brcel- at 2 days. The initial frequency was
cell-k

approximately a factor of nine greater than the chromosome-type break

frequency determined in this investigation (172). The dissimilarity

does not seem unreasonable considering the differences in cell type and

stage of division at irradiation (86, 148).

Frequencies involving radiation that were obtained in this in-

vestigation were for cells withdrawn approximately 2 weeks after expo-

sure. The delay before successful cultures could be obtained, perhaps

related to mitotic inhibition by irradiation (86), was not observed in

the human in vivo investigations (155, 165), but the doses were con-

siderably lower, 100 rad or less. In the human studies, the aberration

levels remained relatively constant for 3-4 weeks after irradiation

(155). The observation does not necessarily apply to this investiga-

tion, for the turnover times of the two lymphocyte populations may not

be the same. '[he 2 week frequency for Chinese hamsters would undoubt-

edly be less than or equal to the no-delay frequency (if it could be

determined employing a lower dose).

Considering all these factors, the aberration frequencies for

radiation determined in this investigation appear reasonable in com-

parison to available data. They provide information that is basic to

additional studies of the peripheral lymphocyte technique as a biologi-

cal dosimeter.


Cio', e Aberrations and In Vivo Ozone Exposure

Referring to Table 6, the observed exposure-adjusted aberration

frequencies for ozone were surprisingly close to the initial estimates

considering the numerous assumptions that were involved in comput ;-,








the expected frequencies (See Chapter II). Besides the data that were

used for the initial estimates, from in vitro exposure of cultured

human cells to high concentrations of ozone, there were no frequencies

for comparison. This was the first investigation of chromosome aber-

rations and inhaled ozone to be reported. The results add weight to the

arguments for considering ozone as a radiomimetic agent.

It was instructive to compare the break frequencies that would be

expected from one week of exposure to radiation and to ozone at the

maximum permissible industrial exposure levels. The exposure-adjusted

break frequencies determined in this investigation were presumed to

apply directly to human exposure (thus being conservative for radiation).

In one week the permitted average radiation exposure of 100 mrad would

result in a lymphocyte chromosome break frequency of 5.5 x 10-5 breaks/

cell. One week (40 hr) at the permitted 0.1 ppm for ozone would result

in 0.4 breaks/cell'

Certainly the extension of animal data directly to humans could

have introduced considerable error in the ozone estimate, but the dif-

ference in these two frequencies is too great to be easily explained

away. These data do reflect the fact that the limit for radiation re-

cognized its mutagenic ability while the limit for ozone did not. The

ozone concentration was set at a value chosen to prevent the occurrence

of physical symptoms in most industrially exposed people, namely,nose

and throat irritation (84). The recent "Community Air Quality Guide"

(192) issued for ozone by the American Industrial Hygiene Association

referred to the radiomimetic nature of ozone in stating that "theoreti-

cally, the recommended air limit for ozone . should be zero, or as

close to zero as possible, i.e., less than 0.01 ppm." As a realistic









limit, the Guide recommended an upper concentration limit of 0.05 ppm

and an exposure limit of 0.1 ppm-hr/day on the average during any year

"if human health is not to be significantly impaired during a life-

time of exposure." Projecting again, this level would produce 1,270:

more lymphocyte chromosome breaks than the maximum permitted occupation-

al radiation exposure. The data from this limited investigation suggest

that a lowering of the community and industrial limits for ozone would

be prudent.


Interactions Resulting from Simultaneous Ozone and Radiation Exposure

The data suggested, but did not show conclusively, that the fre-

quency of chromosome aberrations in circulating lymphocytes resulting

from either radiation or ozone exposure would increase if exposure to

the other agent occurred at the same time. The combined effect, however,

would be less than the sum of the effects expected for each agent alone.

Some mode of antagonistic action or protective mechanism is therefore

suggested, but its nature is not apparent (See Chapter I). The impor-

tance of combined effects is overshadowed by the magnitude of the effect

expected for ozone alone at permitted concentrations.















CHAPTER VI


CONCLUSIONS

In this investigation, Chinese ha~,stcrs were exposed to x-

radiation, to ozone, or to both simultaneously. The basic radiation

dose was 230 rad delivered in 5 hr. The average ozone exposure was 5

hr at a concentration of 0.2 ppm. Exposurc-adjusted frequencies of

chromosome aberrations produced in circulating blood lymphocytes served

as the quantititivie insure of widespread d c. The basic purpose

of the study :as to provide inform tion on the combined effect of

c-posurc to these tnts.


Scrt~ r of R islts

The follow.,, 1 re the principal results in this investigation.

1. Radiation exposure resulted in an exposure-adjusted break

frequency 5.51 x 10-4 rl i-a for cells withdr.n 1 2 weeks after
cell -red

exposure. This value appeared reasonable in comparison to avail-

able information on in vivo exposure of human lymphocytes and

Chinese hamster bone nmrro cells. Successful ly)phocyte cul-

tures could not be obtained until 2 weeks had ela sed. The

number of br per cell appeared to vary linearly iith dose

in thi r fo 2 .3-3 rad.

2. actio of in~hled ozone resulted in chromosome abcrra-

tions. c
--^ 1is value NrIs jin iood (, ecent \with the
cell- (i -!in)









expected value calculated on the basis of in vitro exposure of

human cells. There was no apparent decrease in break frequency

with time for 2 weeks post-treatment.

3. Animals exposed to the two agents simultaneously exhibited

>70 percent of the total number of breaks anticipated assuming

the actions of the agents to be additive. The expected contri-

butions fro:. ozone and from radiation were nearly equal. There

was, however, approximately an 18 percent chance that all the

breaks observed resulted from the radiation exposure alone.

Similarly, there was a 15 percent chance that all the breaks

observed resulted from the ozone exposure alone.


Significance of Results

This experiment demonstrated the suitability of lymphocyte chro-

mosome brc-.(i -e as a biological indicator of exposure to an air pollu-

tant, ozone. The technique, introduced by this study, may be useful

for other oxidint pollutants too. Additionally, the investigation pro-

vided data on the in vivo production of lymphocyte chromosome aberrations

in an animal by external ionizing radiation; such information was not

readily available in the literature. The work demonstrated still

another reason for considering ozone as radiomimetic.

Chinese harmstecrs appeared to be excellent laboratory animals for

this type of study when used with the orbital bleeding technique and

the lyi phocyte cultur-e method which was developed during the investi-

gation. This report presented the first detailed description of a

successful method for the short-term culture of circulating blood lym-

phocytes from Chinese hamsters using moderate size blood samples.









The most important single fact to emerge from this investigation

was that inhaled ozone resulted in chromosome aberrations in circula-

ti blood lymphocytes. Because of the body-wide effects of inhaled

ozone and the fact that production of chromosome aberrations by ozone

appeared to be an action that was general to all cell types, the lym-

phocyte result should be indicative, in a qualitative sense, of similar

0d' ... to other cells throughout the entire body. Such aberrations

arc considered as cellular alterations (domagn ) with lo ,-tern conse-

quences.

Presently permitted huiIan ozone exposures would be expected to

result in break frequencies that are orders of magnitude greater than

those result fro1 permitted human radiation exposures if the results

of this animal study were directly extended to the human case. Con-

sideration of combined ozone plus radiation environments is over-

shadow:d by the importance of ozone enviromrnents alone as long as per-

mitted ozone exposure levels remain at their present and seemingly high

values.

Ji't ozone inhalation does result in chromosome aberrations at

the apparent frequencies is an outcome of this limited investigation

which certainly appears worthy of further experimental consideration.

In fact, such investigation appears mandatory!









































APV'NUICLS









































A P1 PI0 1

P11. -R :T : Of C Il] 1. 1,'DI) AI\IND SOLL PCR,-.









Hoparin Solution for Blood Pipettes

To prepare 20 ml of sterile normal (0.85 percent) saline solution

with 103 USP units heparin/ml, do the following in order:

1. Place 0.153 g of desiccator-dried sodium chloride (NaCI) in

a glass screw-cap bottle marked for 20 ml;

2. Add approximately 15 ml of distilled demineralizcd water;

3. Autoclave at 121.50C, 15 psig for 15 min;

4. Add sterile distilled delineralized water to bring the

volume to 18 ml;

5. Add 2 ml of 10 USP units/ml isotonlic aqueous sodium heparin
13
solution and mix.

Steps 4 and 5 must be done with sterile technique. Keep the solution

refrigerated.


li1i .- F i.L'. Cul ture Tedia

To prepare a 115 ml batch of media, add the components listed in

Table 7 (180) to a sterile glass screw-cap bottle, ascptically and in
14
sequence. Keep the final solution refrigerated.




13Organon, Inc., West Orange, New Jersey.

14This sequence was suggested in Price ad_ Reference Nanual,
Grand Island Biological Company, Grand 1 '. .. . (i-'.,. .










TABLE 7

INGREDIENTS FOR SPECIAL CiULTURE 3TMEDIA


Component Quantity, ml

1. Sterile distilled demineralized water 78
2. Earle's balanced salt solution (10x) 10
3. ] : vitamins (100x) 2b
4. 13:: essential amino acids (50:) 4b
5. L-glutamine, 200 rr: (100s) 1
6. Phenol red solution, 0.5% 0.14
7. Sodium bicarbonate solution, 7.5% 3-5c
8. Penicillin-stre to):ycin mix, 5,000
units ach/il 2
9. Fetal bovine scrum (gar a-globulin free) 15
10. Aqueous isotonic sodium hparin
solution, 10_ US, units/mld 0.2
aExcept for 1 and 10, all n trials were obtained from Micro-
biological Associates, Inc. Bothesda, Maryland.

blhi s qu:nti ty I '. d a solution that was twice the normal con-
centration for this copont.

CS fi; Cen I .cJ i -' iL- oii 3..i-i used to produce an orange-
a
dOi n, Inc., West Or. e, New Jersey.


M _J -. M _! r i " _J 7 .li .:,,

To prepare 1 1. of KT sensing solution (184), do the following in

order, u;i..- r( cnt grade chemicals:

1. Add 20 g potassium iodide (KI) to approximately 500 ml of

distilled dcc'ineralized water in a 1 1. beaker;

2. Add 50 g potassium bromide (i ) ; stir to dissolve;

3. Add 2.5 g of sodium phosphate, monobasic (al 2PO4 H20);

4. Add 7.0 g of sodium phosphate, dibasic (Wa2i 14 7 120);

5. Transfer the solution to a i 1. vol etric flask and bring

to 1 1. withisi distilld cmi* 'raliz cd water.

Keep the solution l sto, -ed.








TC-199 Culture Media with Hleparin

To prepare a 100 ml batch of media, add the components listed in

Table 8 to a sterile glass screw-cap bottle, aseptically and in sequence.

Keep the final solution refrigerated.


TABLE 8

INGRELil[;.TS FOR TC-199 CULTURE MEDIA WITH HEPARIN


Component Quantity, ml

1. Medium 199 without NaHCO3 (10x)a 10
2. Sterile distilled demineralized water 90
3. Sodium bicarbonate solution, 7.5%a 0.47b
4. Aqueous isotonic sodium heparin solution,
104 USP units/m1c 0.2
aMlicrobiological Associate, Inc., Bethesda, Maryland.

bThe 10x TC-199 concentrate has phenol red. The NaitCO3 brings
the solution to pll 7.4 (bright orange color).

cOrganon, Inc., West Orange, New Jersey.


Colchicine Solution for Mitotic Arrest

The preparation of the working solution which is 2pg colchicine/

ml of normal (1 percent) buffered saline is done in three steps:

Preparation of Normal Buffered Saline15

To prepare 10 ml of sterile normal (1 percent) buffered saline

solution, do the following in order:

1. Place 0.90 g of desiccator-dried sodium chloride (NaC1) in a

glass screw-cap bottle marked for 100 ml;

2. Add approximaLely 60 rl of distilled demineralized water;



15This is a modification of the procedure suggested by George
Cartwright in Di -. tc T.--.r-t r-V 11-~'tolo-', 4th edition, Grune and
Stratton, New York (1968).








3. Add 0.137 g of anhydrous sodium phosphate, dibasic (Na211P04);

4. Add 0.021 g of sodium phosphate, monobasic (NaH2PO4 1120);

5. Autoclave at 121.5 C, 15 psig for 15 min;

6. Add sterile distilled demineralized water to bring the

volume to 100 ml.

Step 6 must be done with sterile technique. Keep the solution refriger-

ated. The pIl should be 7.4.



To prepare 10 ml of normal saline solution which has a colchicine

concentration of 1 m;g/ml, transfer 10 ml of the normal buffered saline

solution to a sterile screw-cap bottle and add 10 :. of USP colchicine16

(C221125'00) using sterile technique. Keep the solution refrigerated.



To preipre 50 ml of no!ri1! l]ine solution which has a colchicine

concentration of 2 tg/ui, transfer 50 ml of the normal buffered saline

to a sterile screw-cap bottle and add 0.1 ml of the colchicine stock

solution, using sterile technique. Keep the solution refrigerated.


1 ', Y'rl .Slut; ..r 11 S,- 111n

To prepare a 400 ,l batch of 0.075 M potassium chloride (KC1)

solution with 16 USP units heparin/ml, do the following in order:

1. Add 2.24 g KC1 (r. *cnt grade) to approximately 200 ml of

distilled de-inicralized water in a screw-cap bottle marked for

400 ml;

2. Autoclave at 121.50C, 15 psig for 15 min;


16Calbiochc Los I .les, California.





87


3. Add sterile distilled demineralized water to bring the volume

to 400 ml;

4. Add 0.64 ml of 104 USP units/ml istonic aqueous heparin

solutionl7 and mix.

Steps 3 and 4 must be done with sterile technique. Keep the solution

refrigerated.



170rganon, Inc., West Orange, New Jersey.




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PAGE 1

THE EFFECT OF OZONE INHALATION ON THE FREQUENCY OF CHROMOSOME ABERRATIONS OBSERVED IN IRRADIATED CHINESE HAMSTERS By RONALD EDWARD ZELAC A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULRLLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1970

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UNIVERSITY OF FLORIDA 3 1262 08552 6381

PAGE 3

Copyright byRonald Edv/ard Zelac 1970

PAGE 4

To Gwen

PAGE 5

ACKNOisTLE DGEMENT S A great number of people were involved in this research effort through its many stages. First, and most importantly, I want to thank the members of my supervisory committee. Dr. Billy Dunavant, chairman. Dr. Harvey Cromroy, co-chairman, Dr. Herbert Bevis, Dr. Emmctt Bolch, Jr., and, through most of my program, Dr. Roy McCaldin, for their continued guidance, encouragement, and numerous efforts of my behalf. I am particularly grateful to Dr. Cromroy who acted as the main technical advisor in this research from its beginning to its completion and who made possible much of the technical assistance and financial support that the research required. Special thanks are extended to Dr. Bolch for his extra efforts to further the progress of the research and to increase the understandablcness of the dissertation. 1 wish to acknowledge the technical assistance that the follov;ing persons provided in various aspects of this investigation: Mr. Steve Bobroff, Miss Barbara Bolt, Miss Eleanor Broome, Mrs. Effie Galbraith, Mrs. Jenny Hunt, Mr. Erhard Lorenz, Miss Annette Morris, and Miss Mary Ellen Sm.edley. I also V7ant to express my appreciation to the following people who most generously permitted me to utilize equipment from or in laboratories under their direction: Mr. Wilson Calaway, Dr. Ronald Lowe, Dr. Walter >lauderli, Dr. Hugh Putnam, Dr. Robert Sholtes, and Dr. Sherlie West. Special recognition goes to Dr. William Dav7son V7ho graciously permitted mc to use equipment and considerable space in IV

PAGE 6

already crowded facilities to house, expose, and sample the animals in this study. I am grateful to my parents Mr. and Mrs. Joseph Zelac and my wife's parents Mr. and Mrs. Robert Motaiiky for their continued interest, support, and encouragement. Financial support for this project came from the following sources: U. S. Public Health Service Training Grant No. 5 TOl EC0004610; National Aeronatics and Space Administration Research Grant No. NASA-NSA-542, Project A23; and Office of Civil Defense Contract No. N00228-68-C-2658, Work Unit 3146-A. The x-ray equipment was made available through U. S. Atomic Energy Commission Division of Biology and Medicine Research Grant No. AT(40-1) -3599 .

PAGE 7

TABLE OF CONTENTS Page ACKNO'.nTLED CEMENTS ; iv LIST OF TABLES ^i^^ LIST OF FIGURES i^ ABSTPxACT ^ Chapter I INTRODUCTION ^ The Radiomimetic Nature of Ozone 2 The Presence of Combined Ozone and Radiation Environments and the Problem of Setting Exposure Limits .... 11 ChrcmoGo;r« Aberrations As an Indicator of Radiation and of Ozone Exposures •'•' Bleed Lyraphocytes As the Test Cells 19 Objectives of the Investigation 22 II METHODS , MATERIALS , Am EQUIPMENT 24 Chinese Hamsters As the Test Animals 24 The Orbital Bleeding Technique for Blood Uithdrav/al .... 26 The Culture of Peripheral Blood L>Tiiphocytes 31 The Production and Quantization of the Ozone Environmen t • 33 The Production and Quantization of the Radiation Field . 44 Scoring Chromosonie Aberrations • 46 Estimating the Ozone and Radiation Exposures Required .. 49 III PROCEDURES 35 Animal Exposures 55 Blood Sampling 57 Tissue Culture Preparation and Maintenance 58 Slide Preparation 59 Recording of Data 62 Analysis of Data ^3 VI

PAGE 8

Chapter Page IV RESULTS „ 65 Radiation Exposed Animals „ 68 Ozone Exposed Animals « » 70 Animals Exposed to Combined Ozone and E.adiation Environment o 70 V DISCUSSION . . o o o 73 Expected vs. Observed Aberration Frequencies V7ith Radiation 73 Chromosome Aberrations and In Vivo Ozone Exposure 75 Interactions Resulting from Simultaneous Ozone and Radiation Exposure , „ o . . 77 VI CONCLUSIONS 78 Summary of Results . . . , » o 78 Significance of Results , » 79 APPENDICES SI 1 PREPARATION OF CULTURE liEDIA AND SOLUTIONS 82 Heparin Solution for Blood Pipettes 83 Hungerford Culture Media = 83 Mast Ozone Meter Sensing Solution 84 TC-199 Culture Media with Heparin . . o 85 Colchicine Solution for Mitotic Arrest 85 Hypotonic KCl Solution for Cell Swelling 86 2 ALKALINE KI METHOD FOR OZONE DETERl-IINATION 88 Introduction » 89 Reagents « ^ 89 Apparatus 90 Analytical Procedures 91 Discussion of Procedure o 94 3 PROCEDURE FOR STAINING SLIDES , » 96 LIST OF REFERENCES ^ 98 BIOGRAPHICAL SKETCH o « o . . . o » 114 VI 1

PAGE 9

Table LIST OF TABLES Page 1 EFFECT OF OZONE INHALATION ON MORTALITY RESULTING FROM RADIATION EXPOSURE 8 2 SUMM.\RY OF EXPOSURES ^^ 3 SU>E'IARY OF DATA ^^ 4 SWiMARY 'OF EXPOSURE -ADJUSTED BREAK FREQUENCIES 69 5 COMPARISON OF EXPECTED AND OBSERVED NITMBERS OF BREAKS FOR COMBINED OZONE PLUS RADIATION EXPOSURES 72 6 COMPARISON OF ESTIMATED ANT) OBSERVED EXPOSURE -ADJUSTED ABERRATION FREQUENCIES 74 7 INGREDIENTS FOR SPECIAL CULTURE l-EDIA 84 8 INGREDIENTS FOR TC-199 CULTURE MEDU WITH HEPARIN 85 9 SLIDE STAINING LIQUIDS AND REQUIRED TIMES 97 V.l 1 1

PAGE 10

LIST OF FIGURES Figure Page 1 CHINESE HA>ISTER CHROMOSOi^IES 25 2 CHINESE AND GOLDEN HAl^ISTERS -COMPARATIVE SIZE 27 3 DIAGRAJ^I OF BLOOD PIPETTE IN EYE OP.BIT 30 4 ANIMAL POSITIONING AND PIPETTE INSERTION FOR THE ORBITAL BLEEDING TECHNIQUE 30 5 OVERALL VIEW OF ANDIAL EXPOSURE SETUP 35 6 THE INTERIOR OF THE EXPOSURE CHAI-IBER WITHOUT UV SHIELD ... 36 7 THE INTERIOR OF THE EXPOSURE CHA^IBER WITH UV SHIELD 37 8 THE EXPOSURE CAGE WITH ANIMALS, R-CHA>IBER, AND OZONE SAMPLING PROBE 39 9 THE SETUP USED FOR CALIBRATING THE OZONE METER 41 10 ACTUAL OZONE CONCENTPxATION VS. MST METER INDICATED OZONE CONCOCTIUTION 43 11 DMGRfVM OF THE VARIOUS CHROMOSOME -TYPE CHROMOSOME ABERPnATIONS 48 IX

PAGE 11

Abstract of Dissertation Presented to the Graduate Council of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy THE EFFECT OF OZONE INHALATION ON THE FREQUENCY OF CHROMOSOME ABERRATIONS OBSERVED IN IRRADIATED CHINESE tlA[--ISTERS By Ronald Edvyard Zelac August, 1970 Chairman: Billy G. Dunavant, Ph.D. Co-Chairman: Harvey L. Cromroy, Ph.D. Major Department: Environmental Engineering Should presently permissible human exposure levels for ionisiing radiation and for respirable ozone be reduced vjhen exposure to both agents takes place, especially simultaneously? This particular combination of noxious agents v;'arranted consideration for three reasons. 1. In many of its actions on biological materials, ozone mimicked the effects of ionizing radiation; this included the ability to produce cliromosome aberrations in human cells i n vitro ; 2. The direct effects of inhaled ozone did not appear restricted to the pulmonary system, but were widely distributed througliout the body; 3. Situations existed in wliich people v/ere exposed to these t\^70 agents simultaneously; there were also indications that more people V70uld bo similarly exposed in the future. Chromosome aberrations produced in circulating blood IjTnphocytes were the indicator of biological damage. Effects in these cells would be a qualitative indicator of similar damage to other cells throughout the test animal. Exposure -ad justed break frequencies served as the quantitative measure of effect. Adult female Chinese hamsters.

PAGE 12

Cricetulus griseus (2n = 22), were exposed, in groups of four, to xradiation (118 keV effective, 70 cm FSD, 230 rad dose), to ozone (0.2 ppm, UV generated), or to both simultaneously. All exposures were of 5 hr duration. There were two groups per treatment and two additional groups, one serving as a control and the other exposed to 330 rad. A total of 50 animals were utilized in the investigation, including 18 in preliminary experiments. Blood samples (0.2 ml) were obtained by orbital bleeding. Following plasma removal, the cells were cultured in an enriched m.edia for 3 days at 37"C in a humidified 5 percent CO^ environment with pokeweed as the mitogen (lymphocyte transformation and division stimulant). Division was arrested in metaphase witli colchicine. Following hypotonic KCl treatment to swell the cells, they were fixed with a 3:1 methanol-acctic acid solution, transferred to a slide, and rapidly dried by ignition. The slides were stained v^jith Giemsa and scanned using bright-field microscopy. Spreads were photographed on 35 mm film and projected onto a screen for analysis (with subsequent microscope rechecks of spreads exliibiting breaks). The aberrations scored were deletions, dicentrics, and rings. The following were the principal results of tliis investigation: 1. Radiation resulted in an exposure-adjusted break frequency of 5.51 X lO""^' -Jl?iLiil_^ £qjcells withdrav;n 2 v;eeks after exposure, cell-rad This value appeared reasonable in comparison to available information on in vivo exposure of human lymphocytes and Chinese hamster bone marrow cells. Successful lymphocyte cultures could not be obtained until 2 weeks had elapsed. Break frequency appeared to vary linearly with dose in the region 230-330 rad. XI

PAGE 13

2. Ozone resulted in an exposure-adjusted break frequency of --3 breaks tT~7 ' : — \> agreeing well with the value expectcell(ppiri-min) o o r 1.67 X 10' ed from in vitro exposure of human cells. There V7as no apparent decrease in break frequency with time for 2 weeks posttreatment . 3. Animals exposed to the two agents simultaneously exhibited >70 percent of the total number of breaks anticipated assuming additive actions. Expected contributions from ozone and from radiation v;ere nearly equal. There v;as, however, approximately an 18 percent (15 percent) chance that all the breaks observed resulted from the radiation (ozone) exposure alone. Presently permitted human ozone exposures (up to 0.1 ppm, 4 P ' " ." — ) v.'ould be expected to result in break frequencies that are orders week ' ^ of magnitude greater than those resulting from permitted human radiation exposures if the results of this experimental animal study V7ere directly' extrapolated to the human case. Consideration of combined ozone plus radiation environments is overshadov;ed by the importance of ozone environments alone as long as permitted ozone exposure levels remtiin at their present values. XIX

PAGE 14

CHAPTER I INTRODUCTION Man has become increasingly concerned about his surroundings as they relate to his health and well-being. The complex environments which he has created through technology make it necessary for him to consider how various agents and conditions of these environments affect him and how they can interact with regard to his health. Ozone and ionizing radiation are two agents which have been extensively investigated individually and whose combined actions have been under stud}'. Ozone is a highly reactive three -atom allotrope of oxygen. It is present in the atmosphere as a natural constituent and is produced artifically botli intentionally and as an unwanted byproduct of various operations. The biological effects of ozone on ii^an and animals have received much consideration. Many studies have dealt with the respiratory system effects (l--:5), as inhalation is the principal path of entry. It was believed that the action of ozone was restricted to the respiratory system, for this is v/here its effects are most noticeable, particularly at concentrations of 0.3 parts per million (ppm) by volume and higher (29) . Now there is evidence that the action of ozone is more widespread. Its effects on a wide range of other body functions and systems have been considered (47-76) as have its effects v/hen present in com.bination with other agents including other gases, aerosols, mists, and bacteria (26, 62., 66, 77-81). A number of general reviews of these

PAGE 15

biological effects of ozone on man and animals are available (54, 8285). The present occupational exposure limit for ozone has been set at . 1 ppm . Ionizing radiation is probably one of the most-studied physical insults to v;hich man has been exposed. Background sources of irradiation are cosmic rays and emissions from naturally occurring radioactive materials; technological sources include x-ray generators, artifically produced radioactive materials, nuclear reactors and detonations, and particle accelerators. The effects of ionizing radiatici on biological systems are extremely broad in range. lliis results from the penetrating ability of many of the emissions. One of the most comprehensive publications on this subject is the Report of t he United Nations Scientific Cor.imittee o n the Effects of Atomic Radiation (36) . This report covers fundamental ladiobiology , hereditary and somatic effects of radiation, sources of irradiation, and comparison of doses and estimates of risks, and includes nearly .'J, 000 references. A later report in 1964 having the same title (£7) acts as a supplement to the comprehensive 1962 v;ork. There is, additionally, an annotated indexed bibliography on the biological effects of ionizing radiation that covers v.'orld literature for the time period 1898-1937 and includes almost 13,000 entries (88). A supplement to this extensive v.'or]: covers 1958-1960 and contains nearly 12,000 more entries (89). TTic Radiom ime t ic Nature of Ozone The property tliat incited the considerable interest in ozone, alone and in conjunction v.'ith other agents, is its large electronegative potential, -2.07 V, (O2 + lijO • :O3 -IZli"^ -I2c). This value 3 .s

PAGE 16

3 only slightly exceeded by fluorine's, -2.1 V (2F" + H2O —*• F2O + 2H'*" + 4e) (90). Thus, ozone is an extremely powerful oxidizing agent. It has been proposed that the oxidizing action of ozone is via a free radical mechanism (16, 91). Ionizing radiation, v;hen interacting with biological systems, also acts as a powerful oxidizing agent, this through the action of free radicals formed by the dissociation of water (92). The processes involved in the initial interactions of ionizing radiation with water are (93, 94): Exitation . . . HOH i2IllliJ^.%. H+ -OH (1) radiation Ionization HOH i5ili£iil&.> (HOII)+ + e (2) radiation followed by H+ + e — > H(3) or H2O + e — > H+ • Oil" (4) and possibly (HOH)+ ~~> H"'" + -OH (5) Combination results in the following postulated reactions (93, 94): H+ -OH — •>• HOH (6) R. + H-— > H2 o o (7) •OH + "OH — >H2O2 (8) or •OH + -OH — ^ n20 + .0 (9) and • + '0 — ^>O2 (10) and if dissolved oxj-gon is present •OH + 'OH + O2 -'->-H202 + -0 + '0 (U) •H + O2 "-> •'O2H (12)

PAGE 17

and possibly •O2H + -0211 •-:•H2O2 + O2 (13) Of the above, 6 and 7 reraovc reducing agents v/hile 8-13 produce oxidizing agents. These oxidizing agents or the OH radical itself (as a highly reactive electron acceptor, OH" — > -OH + e -3.7 eV) act to oxidize inorganic ions and/or organic compounds that are present in the aqueous biological system (93). Thus, through oxidizing processes, ozone and ionizing radiation might be expected to act similarly on biological materials that are exposed to them. This raises the question of whether tissues beyond the pulmonary systeiri are exposed to the action of ozone V7hon ozone is being inhaled. To put it another v^ay, v.'ill ozone inhalation result in direct extrapulmonary effects as opposed to secondary systemic reactions resulting from pulm.onary involvement? If other tissues are directly affected, then the similarity betv;een exposure to these two agents, ionizing radiation and ozone, will be even more striking. For iust as an experimental animal in a radiation field would be expected to have effects of the exposure throughout its body, an animal in an ozone environment vrould similarly experience v/idespread direct effects of its exposure through inhalation. Tliere is a variety of observations on the actions of ozone that demonstrate direct extrapulmonary effects of ozone inhalation. Some of these effects are sim.ilar to those seen after exposure to ionizing radiation. These latter observations have resulted in ozone being widely considered as a "radiomimetic" agent. The following is a listing and discussion of experiments demonstrating direct extrapulmonary and/or radiomimetic effects of ozone exposure:

PAGE 18

1. Cutaneous oxygen consumption in a digit of man has been shown to be strongly affected by short-term inhalation of moderate ozone concentrations (95) . The technique involved the sudden occlusion of digital circulation and the estimation of the ensuing deoxygenation of oxyhemoglobin photoclectrically , Stagnant intracapillary deoxygenation normally proceeded logarithmically to complete dissociation in approximately 10 min. However, the dissociation immediately following the breathing of 1 ppm ozone for 10 min only reached the 50 percent level 10 min after occlusion and appeared to level off. This phenomenon, interpreted as a reversible intoxication of final heme-enzyme groups in the redox chain, was also evident following the breathing of pure oxygen or irradiation of the finger with ultraviolet light. In the case of ozone, normal deoxygenation was evident if cysteamine was introduced into the skin immediately after ozone inhalation. The interpretation of these results was that ozone reacted with the linings of the airways, and the oxidizing products of this interaction were distributed by the circulation of the blood. Cysteamine, as it did with ionizing radiation, provided chemical protection presumably by inactivating oxidizing agents through a radical scavenging mechanism. Tlius, extrapulmonary oxidizing action was inferred. 2. The acute toxic action of inhaled ozone has been reduced by the simultaneous injection or intraperitoneal injection of codipounds that furnish -SII or -SSbonds (96). Compounds v;ith -S bonds proved ineffective. As was mentioned previously, these same cha:-'icals provided protection from exposure to ionizing

PAGE 19

radiation (86). Tnis further suggests a s5.milarity of action for ozone and radiation. 3. The effects of ozone inhalation on the visual acuity of man have been investigated (97). Levels of 0.2-0.5 ppm inhaled for 3 hr or two 3 hr periods with 1 hr rest between resulted in (a) decrease in visual acuity for dark adaptation and middle vision ranges, (b) increases in peripheral vision, and (c) change in the balance of most extraocular muscles. The effect, while not radiomimetic, demonstrates direct extrapulmonary action of ozone. 4. In animals, including man, red cells start circulating as flat discs and end up as spherocytes. The process can be greatly accelerated by xirradiation in vitro . If ozonized air is inhaled before the blood is withdrawn, this process is further accelerated (98). Distinct effects were seen in man with ozone exposures as low as 0.25 ppm for 30 min. For example, for blood drawn 1 hr after this degree of ozone exposure, 7,000 R resulted in 55 percent of the red cells being spheroid compared to 21 percent for non-ozone-exposed controls. Sphering tendency acceleration by ozone was a reversible reaction, as the amount of sphering seen decreased with time after ozone administration. The experiment demonstrated a distinctly radiosensitizing effect of ozone inhalation and showed ozone action on the blood system. 5. It has been demonstrated that the inhalation of ozone can result in structural damage of postmitotic nuclei in myocardial fibers of adult rabbits and mice (98). Exposure to 0.2 ppm, 5 hr/day for 3 v;eeks, resulted in rupture of nuclear envelopes and extrusion of contents which are never observed in the nuclei

PAGE 20

of normal fibers. This type of damage is also seen in patients following therapeutic radiation exposure and in irradiated tumor cells. Here, extrapulmonary tissue damage which mimics that seen from radiation exposure V7as sho^vn. 6. Another indication of the deep and radiomimctic effects of ozone was its effects on the offspring of exposed mice (98) . Tlie exposures were to 0,1 or 0.2 ppm, 7 hr/day, for 3 weeks. For inbred grey mice, litter sizes V7ere normal, but 3 V7eek neonatal mortality v;as 6.8 percent (0.1 ppm) and 7.5 percent (0,2 ppm) compared to 1.6 percent for controls. For the second litters from parents exposed to 0.1 ppm, 4.9 percent mortality V7as seen against 1.9 percent for controls. For highly inbred C57 black mice, exposure to 0.2 ppm under the same routine resulted in normal size litters but 34 percent neonatal mortality compared to 9 percent for controls. Here, then, we have evidence of a lingering observable effect on gonadal material not unlike effects of ionizing radiation (86). To these experiments vjhich demonstrate widespread direct extrapulmonary ozone effects along with the oxidizing and radiomim.etic nature of ozone action can be added anotlier group of studies involving the exposure of aiiimals to ozone in conjunction with lethal doses of ionizing radiation. In these, the effects on radiosensitivity of preexposure to ozone were investigated. These experiro.ents are described in Table 1. In each case, ozone inhalation had an effect on mortality. The radiation doses used were approximately LlJ50/3o's (i-e-s doses that should result in the deaths of 50 percent of the animals exposed within

PAGE 21

8 o ^\ O n o o n e C.I CJ K CO H 0) r-I g •H r< o • •H C) H O H '-^ u rri O CO Vi t-i VD CS o o > -p G •H O > TJ H Sh CJ r5 W 0J ;>: Pi c' •^ E M O O I o f^

PAGE 22

o u G) to pi n CD H

PAGE 23

10 Low 30 days) or greater. The ozone exposures v/ere all considerably belc lethal levels. (For mice, exposure to 6 ppm for 4 hr results in 50 percent mortality. Rats are even more resistant (84).) Most vrere below the approximately 6 pp:ii-hr at which edema and l>Tr,pocyte migration into alveolar spaces begin, i.e., v;here lung histology changes (68). Some were even below the 0.7 ppm-hr where lung function temporarily changes during exposure (27). (For short term single exposures, the product of ozone concentration and exposure time produces a constant toxicological response (84).) Since little if any effect on pulmonary structure or function could be expected once the ozone exposures ceased, the effect on mortality of subsequently administered lethal doses of radiation is an additional deraonstration of the direct extrapulmonary action of inhaled ozone. It should bo noted in Table 1 that two phenomena are exhibited. The first, radiosensitization, acts to increase mortality. This is seen when the ozone exposure is of short-term duration (1 hr) and immediately prior to the radiation exposure. If the ozone exposure is of longer duration inrr.ediately prior to the radiation or of short duration but with a tiiTie lag of 1 day or more bet^^en ozone exposure and radiation exposure, the effect is to decrease mortality, radioprotection! To interpret these apparently opposing observations, it should be recalled that both ozone and radiation have oxidative processes in the body as the basis of their action. V/ith ozone as the radiosensitizer, the oxidation from it (on the premise that it does occur throughout the body and not just in the respiratory system) adds to the oxidation resulting from the radiation to bring about a level of damage greater than that from the radiation alone; hence increased

PAGE 24

11 mortality. To bolster this interpretation, it has been sho^vm that small doses of ionizing radiation (^S5 R) immediately preceeding lethal ozone exposures increase the mortality observed over that from ozone alone (104). With ozone as the radioprotective agent, the interpretation is that the insult from the ozone, i.e., the oxidation from it, was sufficient in magnitude to bring about an antioxidant response by the body and to stimulate intracellular repair mechanisms before the radiation exposure began. Through this, some of the oxidizing action of the radiation was counteracted, resulting in a lower level of damage and decreased mortality. This antioxidant response of the body is equally effective if the source of subsequent oxidant is an additional ozone exposure of lethal magnitude rather than radiation exposure (34). Likewise, pre-exposure to ionizing radiation rather than to ozone can initiate the presumably antioxidant response (106-108). Since this is the case, ozone is also radiomimetic in these experiments in which it acts as a radioprotective agent. Thus, a variety of observations have been made vjhich rather conclusively indicate that there are direct extrapulmonary effects of ozone inhalation and that ozone can be generally considered as a radiomimetic agent. The Presence of Combined Ozone and Radiation Environments and the Problem of Setting Exposure Limits That ozone and radiation appear to act similarly on biological materials is of practical interest from a health and safely point of view. Man atteiapts to provide safe and comfortable environs for himself by, among other things, limiting his exposures to various agents to

PAGE 25

12 values belov7 damaging or irritating levels, or at least to values for which the levels or risks of damage are acceptable when weighed against the overall benefits associated v.'ith the exposures. In deciding on acceptable levels for a particular agent, information from human experience and from animal and/or himian experimentation is utilized. If two oi" more agents are present, the general practice is to consider their actions as additive (unless there is information to the contrary) and to accordingly reduce the permitted exposure level for each of them. This has been the case v.iien the agents have some similarity to one another, as mixtures of organic solvents or mixtures of radioactive materials or radioactive substances and external irradiation in combination, Tliere has been little if any consideration, hov7ever, of crosscombinations including the one under discussion here, ozone and ionizing radi.ation. It appears important to consider the actic)n of the ozoneionizing radiation combination, particularly', because of the radiomimetic nature of ozone previously discussed, because there are presently situations where people are exposed to both agents simultaneously, and because there are definite indications that more and more people v.'ill be so exposed in the near future. There are two broad classifications into which ozone-radiation environments fall. In the first, the radiation field is the source of the ozone; for the second, the principal source of the ozone is not the radiation field but other technological generating means, or the ozone is present naturally. There are some ozone-radiation environments that are a combination of these two types.

PAGE 26

13 The irradiation of oxygen, gaseous or liquid, results in ozone production and in some decomposition of the ozone so formed. Alpha, fast electron, photon, and neutron irradiation have been investigated (109-116). The yield of ozone in a closed system increases with dose (109, 114-116) until an equilibrium value as high as 2,000 ppm (115) is reached at very high doses of approximately 10' rad (114). The yield also appears to increase with dose rate (114, 115) , although this point is disputed (113). As V7ould be expected, v;ith a flowthrough system, the concentration is reduced but the total amount of ozone produced increases (116) . It follows, then, that an ozone plus radiation environment can be associated with nearly any high-intensity radiation field. Some examples are research accelerators and isotopic sources, electron beam irradiators (used in industrial polymerization as for paint curing and ply^v'ood glue hardening), large-scale isotopic food irradiators, highradiation areas at nuclear reactors, industrial radiography devices, and medical therapeutic radiation units. Tliis problem of ozone production by radiation has been recognized and investigated at a number of facilities including high-level gamma installations (117-119) and electron accelerators (117, 120-123). The principal method of control is usually to provide ventilation adequate for keeping ozone concentrations dov/n to acceptable levels. In connection with this dicussion of radiation as a producer of ozone, it is interesting to note that a number of experiments with mice, rats, and monkeys have demonstrated that animals detect radiation by olfactory system response to ozone produced in their nasal passages by the radiation (124-127).

PAGE 27

11; Other technological production of ozone is by electrical arcs or corona discharges, silent electric discharges, and shortwave o «2450 A) ultraviolet light (128, 129). It is found industrially at ozonizers for treatment of sev^age, for water purification, and for control of molds and bacteria at cold storage plants; in conjunction v;ith high voltage electrical apparatus such as at generators, accelerators, K-ray devices, and spectrographs; and V7ith electronically operated office copy equipment, neon signs, electric motor brushes, quartz ultraviolet lamps, and inert gas shielded arc welding units (7, 84, 130). A technologicall}' related source of ozone is its environmental production in smog by photochemical reactions involving air pollutants and naturally present ultraviolet light (131). The potential for a combined radiation-ozone situation exists \:hcrcvcr people arc c::poscd to radiation vrhilo in the vicinity of other technological ozone sources. An example of this type of an environment is a nuclear subm.arine. Here the radiation source is the nuclear reactor power plant of the vessel; the ozone can be produced both by the radiation fields associated with the reactor and bj' the extensive electrical and electronic equipment aboard. The potential for ozone production on these ships has been recognized (132) , and monitoring equipment is available on them (133) . Control of the ozone concentration is complicated by the atmospheric recycling required by the nature and mission of such vessels. Another example of an environment witii a combined ozoneradiation exposure potential is a spacecraft. Radiation exposure can result from galactic cosmic rays. Van Allen belt particles, solar flares, radioactive debris from, atmospheric nuclear explosions, and

PAGE 28

15 on-board radialion sources (134). Ozone can result from operation of the space cabin equipment (135) or from irradiation of liquid and gaseous oxygen supplied on-board (136). Here, too, the ozone situation is complicated by the atmospheric recycling required. As with the nuclear submarine, the ozone production potential lias been recognized, as demonstrated by NASA sponsored experiments dealing with ozone toxicity (77, 78). Ozone is also produced naturally in the upper atmosphere by the action of ?olar ultraviolet radiation on oxygen present in the stratosphere. The vertical concentration profile peaks at about 29.0 km and decreases rapidly as height decreases; there is also variation vjith latitude and season (84, 137). Concentrations up to 15 ppm (by volume) have been reported; natural ozone levels at ground level at U. S. latitudes Pre ostimited at 0.01 ppm or ]oss. Present-day jet aircraft cruising at 9.2-12.4 km are at the edge of the so-called ozonosphere, while the supersonic transports (SST's) cruising at 19.8-2^i.4 1cm v/ill be near its center, where the ambient external ozone level is hazardous to life. As aBibicnt air is used for cabin pressurization currently and will be used for SST's, ozone levels in present aircraft have been investigated (138-142) and rigorous ozone control (decomposition and monitoring) is being planned for the SST (137, 143--145). Tlie studies of present craft have indicated that the external air is the principal source of the ozone present (141), although cockpit levels are somewhat higher, suggesting other (electrical) sources (142). Levels range up to 0.4 ppm. A maximum level of 0.2 ppm is proposed for the SST (137) ,

PAGE 29

16 While passengers and crews of present-day jetliners experience some radiation exposure, primarily from cosmic radiation, people in SST's will have to contend with a higher radiation level. This is a result of cosmic radiation being more intense at higher altitudes (6x higher at 21.4 km that at 9.2 km), protons from solar flares basically not penetrating the atmosphere to altitudes <18.3 km, and radioactive debris from high-yield atmospheric nuclear testing being predominantly concentrated in the stratosphere (above 11,0 km to 30.5 km) (146, 147). Thus, while jetliners do present a radiation-ozone environment, the SST will probably involve greater radiation exposure with the ozone exposures comparable to those presently encountered. We are therefore confronted v.'ith the practical problem of having people simultaneously exposed to ionizing radiation and ozone, along with the prospect of even more people being so exposed in the future. In view of the apparently radiometric nature of ozone, should the maximum levels of exposure permitted for each agent when considered as acting alone still apply in this dual exposure condition? Presently, the radiation and ozone exposure limits are applied individually. A quantitative measure of widespread damage throughout the body is needed that will serve equally well to evaluate ozone and radiation exposures. Such a measure v;ould indicate x/hether the agents in a combined environment acted in an additive fashion, synergistically , or antagonistically in their damage production. Then a decision could be reached as to the adjustments, if any, that should be made in the individual exposure limits v^hen a combined exposure occurs.

PAGE 30

IT Chromosome Aberrations As an Indicator of Radiatio n and of Ozone Exposures A brief introduction to chromosome organization and structure is presented here because it forms the basis for the experimental rationale. Chromosomes are nuclear organelles. They are in the form of elongated threads (present in the nuclei of cells) between cell divisions, or in interpliase. During division, they contract into short thick readily observable rods. Chemically, they are characterized as nucleoproteins having the polynuclcic acid DNA (deoxyribonucleic acid) and tv:o specific proteins as their main components. Functionally, through subdivisions called genes, they dictate the enzyme and protein production and hence the metabolic chemical reactions of the cells in which they occur. This information is transmitted from generation to generation in a cell lino by duplication of each chromosome in the mother cell prior to division (mitosis) so that at division each daughter cell gets a complete complement of chromosomes and thereby a complete code for protein production. Through meiotic cell division, each sexual gamete receives one-half the characteristic number of chromosomes. Mien fertilization occurs, the normal nmiber of chromosomes for cells of the particular species is restored. V.^iile genes are remarkably stable, alterations do occur. A change in a gene that is accompanied by a visible change in the structure of the chromosome of which it is a part is called a chromosomal mutation; those gene changes that are not accompanied by visible chromosomal changes are called point mutations, or gene mutations. As genes control the metabolic reactions of the cell, gene changes can be highly detrimental (even lethal) or relatively insignificant.

PAGE 31

18 A chromosomal mutation, or aberration, results when the two ends created by a single break in a chromosome fail to rejoin with one another through repair mechanisms. Rejoining can fail to occur or ends from two different breaks in the same or in separate chromosomes can join to produce a variety of aberrations. These aberrations often result in an unequal distribution of chromosomes between daughter cells, and this usually leads to cell death. If rejoining of the two ends created by a single break does occur, restitution may be at the morphological level only, and a point mutation due to DNA damage may appear (84). It is for these reasons that chromosome aberrations are considered as cellular alterations (damage) with long-term consequences. There are a variety of agents which have the capacity to bring about gene changes, i.e., which are mutagenic. Ionizing radiation has long been recognized as one of these agents (93, 148). Its effectiveness is dependent on the magnitude of the dose (direct dependence), the cell type, and the stage of the cell cycle at the time of irradiation, but all living cells are susceptible to this action (86, 148). Evidence has accumulated that ozone, too, is a general mutagenic agent. It has been observed to change the adsorption spectra of nucleic acids (149); to markedly modify the pyrimidine bases (thymine, cytosine, and uracil) in E. coli nucleic acids (150); to produce specific mutants of E. coli exposed in ozonated water (151) ; to result in various chromosome aberrations when tissue cultures of embryonic chick fibroblasts were exposed to gaseous ozone (152) ; to produce a high frequency of chromosome aberrations in the root meristems of Vicia faba (153); and, finally to produce a number of different types of chromosome aberrations in cultured human cells exposed to gaseous

PAGE 32

19 ozone (154). For this last investigation, the frequency of aberrations seen in human cells was shown to depend directly on the magnitude of the ozone exposure. Comparison was made with the frequency of aberrations seen v/ith exposure of the cells to x-radiation. For radiation, the yields also varied directly with the dose. It appears that ozone, like ionizing radiation, could be expected to produce chromosome aberrations in all types of cells tliat are exposed to it. Because of the radiomimctic nature of ozone, this is not a surprising observation. Likewise, this action of ozone, production of chromosome aberrations, could be expected to occur throughout the body of an animal inhaling ozone in view of the direct extrapulmonary effects of ozone inhalation that have been demonstrated. Therefore, it appears that the frequency of chromosome aberrations produced by cr'posure o£ an animrl to ionizing radiation and/or to ozone can serve as the sought-after quantitative measure of widespread damage. Blood L-^inphocytcs As the Test Cel ls There are numerous technical difficulties associated with the production of mammalian chromosomal preparations. The usual histological techniques do not suffice, and squash preparations are not completely satisfactory, partly because of the small size of mammalian cells and their relatively large chromosome numbers (135). Since the early 1950 's it has been possible to make suitable preparations from mammalian cells maintained in tissue cultures by employing a hypotonic pre-fixation treatment to swell mitotic cells (156). Unfortunately, this has little value for experiments involving whole animals. Similarly, good preparations have been made from tissue biopsy cells put into

PAGE 33

20 suspension and subjected to the same hypotonic pretreatment procedure; a soft, easily dispersed tissue is required. Skin and bone marrow have been used, but sample collection is difficult, particularly when repetitive samples are required. It was only in 1960 (156) that the cytological technique which is most widely used today in animal chromosome studies became available. This technique utilizes the small lymphocytes, the white cells originating in the IjTnph system, that are present in the circulating blood. These cells, 8-lOU. in diameter and having a large nucleus, are the smallest of the white blood cells. They are involved in the carrying and production of antibodies for combating infections and are also involved in fibrous formation for healing or enclosing clots. They account for 25-30 percent of the white cells in human blood, and, for comparison, 80-90 percent of rat blood white cells. Up until recently, l},Tnphocytes had been thought to have lifetimes on the order of 4 hr . These estimates were based on the fact that the number of IjTnphocytes entering the blood system through the lymph ducts is several times greater that the total number present at any moment (157) • But the number of lymphocytes leaving the blood system and passing through tissue fluids to the lymph system was underestimated. In fact, l^TTiphocytes are continuously recirculating between the blood and the lymph. Lifetimes of 100-200 days and longer have been reported (155, 158), Lymphocytes were long-considered as mature non-dividing cells. Since cells undergoing mitosis are required for chromosomes to be visible, this would imply that Ijmiphocytes should definitely be eliminated from consideration as test cells. But it has been found that a

PAGE 34

21 small fraction of Ijmiphocytes in circulating (peripheral) blood are engaged in DNA prodnction at a lov7 rate (159), Additionally, and more importantly, when lymphocytes are exposed to certain drugs in vivo or in vitro , a transformation takes place, and mature cells grow, taking on the characteristics of l^TOphoblasts , v^hich are largo inmnture slightly differentiated precursors of lymphocytes (160, 161). Tlieso transformed cells then undergo chromosome duplication and enter into mitosis (159-162). Therefore, they are suitable for chromosome preparations. This sequence of events is referred to as "lymphocyte stimulation" and the drugs as "mitogens." This action is believed to be related to the antigenantibody action of l^nnphocytes . The Ijiiiphocytes are most often exposed to the mitogenic agent after a blood sample has been wi tlidravm from the animal and the cells put into a suitable culture media. This means that vjhcn the animal is subjected to agents that can produce chromosome breakage, the lyniphocytes are nearly all at about the same stage of the cell cycle, the pre-DNA-synthesis resting stage (they are mature cells and would normally, except for stimulation, not engage in any m.ore division). This fact greatly simplifies aberration analysis of the chromosome preparations: First, variations in sensitivity of the cell to the agent with stage in the cell cycle are absent, so all cells should be equally sensitive; second, any aberrations that result from the agent \;ili be of the chromosome type (i.e., will involve both strands of the chromosome) since duplication occurs after breakage takes place; this greatly reduces the mimber of aberration types that have to be considered by eliminating all those of the chromatidtype (i.e., that involve only one strand of the duplicated chromosome) .

PAGE 35

22 These are two distinct advantages that chromosome preparations from lymphocytes have over preparations of cells from other tissues. Other advantages of using lymphocytes are the relative ease of obtaining the cells (just drav; some blood) , the option for doing mutiple sampling over a period of time if this is required, and the availability of information on chromosome aberration production by ionizing radiation in the l>TTiphocytes of one animal, man, both in vitr o and in vivo (153, 163-167). Additionally, chromosome aberrations produced in circulating blood Ipnphocytes should be indicative, in a qualitative sense, of similar damage to other cells throughout the entire body, both for ionizing radiation and for ozone. This follows from demonstrated body-wide effects of ozone and radiation exposure and the fact that production of chromosome aberrations by these agents appears to be an action that is general to all cell types. Objectives of the Investigation llie basic purpose of ttiis study was to provide experimental evidence that could be used to help decide v;hether presently permissible human exposure levels for ionizing radiation and for respirable ozone should be reduced when exposure to both agents takes place, particularly v.'hen the exposures are simultaneous. Such evidence should consist of quantitative information on the production of a specific type of biological damage in small mammals tlirough exposure to each of the agents alone and to both of them, together. The production of chromosome aberrations in circulating blood lymphocytes V7as chosen as the biological indicator of damage. This choice V7as made on the basis of kno'./n response to radiation (in m.an)

PAGE 36

and probable response to o?one suggested from related information. Chromosome aberrations in these cells would be indicative of similar and related damage with long-term consequences in other cells of the body as well. As a result of this choice, the specific objectives of this investigation (all referring to the peripheral blood IjTiiphocytes of a particular species of small mammal) were the following: 1. determine the frequency of chromosome aberrations produced by exposure to ionizing radiation; compare this value with those available for other cells in the same species and for Ijmiphocytes in the other species; 2. determine v;hether chromosome aberrations are produced v;hen the animals undergo exposure to ozone at a concentration approximating th-'it prp^"^ent]y permitted for human exposure and for a duration that related information suggests is adequate to produce sizeable numbers of aberrations; if aberrations are seen, determine the frequency of production by ozone; 3. determine the frequency of chromosome aberrations produced when the animals are exposed to ozone and ionizing radiation simultaneously; compare this value with that vjhich would be expected based on exposure to each of the agents individually in order to determine whether synergism, additivity, or antagonism occurs with combined exposures; 4. attempt to assess the value of the information obtained by the experimental study in relation to the overall problem of deciding on appropriate permissible exposure levels for ozone and ionizing radiation when exposure to both agents takes place, especially simultaneously .

PAGE 37

CtlAPTER II METHODS, MATERIALS, AND EQUIPl-IENT Chinese Hamsters As the Test Animals The species, of all the small mammals available for laboratory studies, which seemed most suitable for this investigation was the Chinese hamster, Cricetulus griseus . This choice V7as made primarily on the basis of the low diploid chromosome number of this species, 22 per somatic cell. See Figure 1 (168). Most other small mammals have many more; the Syrian or golden hamster, for example, has 44. Small mammals known to have fev;er chromosomes such as the Tasmanian rat kangaroo with 12 or 13 (168) or the mole-vole with 17 (168) are not easily obtainable. As somatic cells from placental mammals all appear to contain approximately the same amount of genetic material (169) , f ev7er chromosomes per cell also means larger chromosomes, both of which should facilitate analysis. Another reason for choosing the Chinese hamster was the availability of ample numbers of laboratory-grade animals. Most U. S. commercial suppliers obtained their parent stocks from Children's Cancer Research Foundation, Boston, v/hich has a well-established colony dating from 1952. Inbreeding has been accomplished there by brothersister matings over many generations (170). The supplier of the animals for Chick Liiie Company, Vineland, New Jersey. 2k

PAGE 38

25 y^^ /JO /)/( oX il X Y AA 6A M IX IS It 6 7 8 9 10 11 )( 1% K :: St 1 2 4 5 3=X ik U kk u It ti 6 7 » 9 10 II FIGURE 1. CHINESE HAMSTER CHROMOSOMES

PAGE 39

26 this investigation has maintained a closed colony since 1964 (171) o A third reason for the choice of this species was the availability of information on spontaneous and in vivo x-ray induced chromosome aberrations in bone marrow cells (172). The Chinese hamster is smaller than the more common, golden hamster. See Figure 2 (173). Approximately 10 cm long with a 1-2 cm tail, it is gray-brov,'n in color vjitli a black dorsal stripe and white underside. Maturity is reached at 8-10 weeks, and animals can live at least 3-6 years (17]-, 173). Its outstanding characteristic is its fearlessness. Mien being picked up, an animal will usually not flee but may roll over and squeal angrily; handling must be gentle and without nervousness or the animal will become vicious and bite (173, 174). The animals in this investigation were all females and had an average weight of approximately 27 g. They \.'ere obtained at an age of 8-10 weeks and were utilized at ages ranging from 13-15 weeks to 19-21 weeks for the preliminary experiments and from 11-13 v.^eeks to 15-17 weeks for the final experiments. As required by their pugnacious nature toward one another (173) , the animals were housed individually in standard rat cages and provided v;ith paper tov.'els for nest building. They were maintained on standard rat chow in the cage, supplemented with occasional fruit or greens, plus v.'ater ad libitum . Lighting was natural. No evidence of disease or parasites was seen for the 57 animals ultimately maintained over the course of the investigation. Each was earpunched for ease in identification. The Orbital Bleeding Technique for Blood VJithdrav;al Experimental design required inutiple sampling, a minimum of two blood samples from each animal. (A pretreatment culture and at least

PAGE 40

27 i w N M CO ta > H H O U W Pi w H w W Q O o w CO w la H w u o H

PAGE 41

2b one posttreatment culture vere required.) Because of this, traumatic techniques were to be avoided. The small size of the animal dictated that the blood samples would likev^ise have to be small, on the order of 0.2 ml, which, based on mouse data, could represent approximately 10 percent of the total blood volume (175, 176). Additional requirements, imposed by the culturing to follov;, were that the technique not result in coagulation, hemolysis, or contamination of the blood. These requirem.ents , along with hamster body structure and a desire for simplicity, eliminated the more standard techniques for small animals such as tail bleeding, toe removal, and cardiac or jugular vein puncture (175). Tlie technique which seemed most satisfactory and v/hich was employed for the investigation was the orbital bleeding metliod (175, 17G). This technique is based on the fact that a venous plexus lines the rear of each eye orbit. The capillaries forming this netv.'ork are fragile and are easily ruptured. Slight pressure from a thin blood collecting tube v.'ith polished tip results in hemorrhage. Blood accumulates in the orbit, v.'hich serves as a reservoir, and the blood tube fills through capillary action after being v^ithdravvn slightly. See Figure 3 (176). Blood flov; stops v-^hen the tube is withdrawn and the eyeball reestablishes normal ocular pressure on the venous network. The technique is demonstrated in Figure 4 (176) . The loose skin of the head is drawn tight to make the eye bulge, taking care not to stop respiration, and the pipette tip is inserted in the inner corner. It is slid in and back along a bony shelf until the plexus is reached. 2 Natelson Blood Collecting Pipettes, Kon-heparinized, Scientific Products Division of American Hospital Supply Corporation, Evanston, Illinois .

PAGE 42

FIGURE 3. DIAGRAl-I OF BLOOD PIPETTE IN EYE ORBIT FIGURE 4. ANIMAL POSITIONING AND PIPETTE INSERTION FOR OPJBITAL BLEEDING TECHNIQUE

PAGE 43

30

PAGE 44

31 The tube, 3 mm OD, 1.5 ram ID x 150 mm, fills in a few seconds when a twisting motion is used. Holding approximately 0.26 ml when filled, it is held hori^ontally and then tipped to a vertical position for emptying. The tube interior was moistened with heparin solution (See Appendix 1) immediately before use; the culturing to follow precluded the use of pre-coated tubes, which included preservative in the heparin. Bleeding could be done from either eye. Neither vision nor health appeared to be damaged in any way as a result of this nontraumatic technique which could be performed repeatedly on an animal, even on successive days. This agrees with v/hat has been observed for mice (176) . The Culture of Peripheral Blood Lymphocytes One of the main difficulties encountered in this investigation was the development of a technique for the culture of lymphocytes from the small inoculums of Chinese hamster blood. Principally, this meant determining what modifications of existing techniques were required in order to produce successful cultures in this laboratory, for lymphocyte culturing must still be considered as an art. A brief summary of the technical developments v.'hich led to the procedure utilized in this study is presented below in order to point out some of the difficulties involved. The first successful stimulation of lyaiphocytes from peripheral blood in culture media was reported in I960 (156, 177). The technique used 10 ml inoculums of human blood and required removal of erythrocytes before culturing. The mitogen eiiiployed was phytohemagglutinin (PilA) , an extract of ordinary beans. Subsequent experimentation vjith aniuials indicated that while the method, using somewhat smaller inoculums for

PAGE 45

32 small animal?, could be successfully employed for some, considerable modification vas required for success vrith others, and some defied all attempts at modification (160) . Particular difficulty v.-'as experienced V7ith mice, rats, and hamsters. Partial success V7ith golden hfir.isters v;as reported in 1963 V7ith a procedure that involved removal of the leucocytes from whole blood, leaving er^'throcytes plus plasma, and the culturing of the leucocytes in a media which employed calf serum as a replacement for the hamster plasma (178). In 1965, using rats, it vjas discovered that a more complete separation . of the leucocytes from the plasma, done by serial washings V7ith normal saline, resulted in greater mitotic index in cultures (179) . But even this did not bring about consistently successful mouse and hamster cultures. ConcuriciiL wiLh these atLi_iupts, v^irk was also being done to develop culturing techniques whicli vjould employ very small blood samples, V7hich v7ould not require leucocj'te separation from V7hole blood (v.'ith the loss of mitotically competent cells a frequent occurrence) , and v/hich v7ould be less subject to unpredictable failures. A method V7as reported by Hungerford in 1965 that accomplished all these poiiits (180) . It utilized a specially formulated culture media (See Appendix 1) and an improved hypotonic treatment for sV7elling the mitotic cells. It V7as developed for human blood and V7as also used for birds. Finall}', in 1968, complete plasma removal (through high dilution and ccntrifugation) V7as combined with Hungerford 's innovative method to yield a microtechnique that V7as reported by Buckton and Nettcshcim to by consistentl}' successful for mouse blood (181). Through inquiry, it was learned that the sam.e technique had been successfully applied to

PAGE 46

33 Chinese hamsters (182). Consequently, the method of Buckton and Nettesheim was chosen to serve as a starting point in developing a method to yield successful cultures in this laboratorj'. The main modifications instituted were to substitute orbital bleeding for cardiac puncture, to lialve the amount of v/nolc blood used (0.15-0.25 ml instead of 0.30-0.50 rill), and to extend the incubation time by 50 percent (3 days instead of 2 days) . The complete procedure is presented in Chapter III. Recently, a preliminary report of an additional method for culturing peripheral blood lymphocytes from Chinese hamsters was issued (183) . It differs most from the method used in this investigation by its use of extremely small quantities, 1-2 drops, of unseparated V7hole blood. Hie Froduction and Quantization of the Ozone Environment Tl-ie principal methods of ozone production are electrochemical dissociation, corona discharge, silent electric discharge, and sliortwave ultraviolet irradiation of oxygen. From these, ultraviolet irradiation (UV) was chosen for this investigation. This \7as to avoid simultaneous production of oxides of iiitrogen (129), for these can influence biological response (91) . Both production and decomposition of ozone occur v/hen oxygen is irradiated v.^ith ultraviolet light (129): Production 02 + Ivj—-2 . (14) follo\ved by O2 + — >O3 (15) or 3 — •;O3 (16)

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3h Decomposition 2 O3 + hi; — > 2 O2 + 2 — ^3 O2 (17) o For 14, wavelengths <2450 A are required to provide sufficient energy o V7hile 17 occurs with a 2537 A wavelength vjhere ozone absorbs strongly. A mercury vapor discharge produces its strongest (resonance) o o radiations at 1849 A and 2537 A. It is a suitable W source for ozone production v.'hen it is enclosed in quartz rather than glass so that the 3 shorter wavelengths v:ill be transmitted. Such a bulb v.'as utilized. Ozone production took place v;ithin the chamber used for animal exposures and irradiations. Figure 5 is an overall view of the experimental setup including exposure char.iber, x-ray source, ozone detector, and related equipment. The ozone bulb v;as placed at the rear and on the central axis of the 46 cm ID x 92 cm chaiiiber. See Figure 6. The animals, in a ventilated cage near the center of the chamber, were shielded fro:n the UY light by a 37 cm diameter aluminum sheet. See Figure 7. Air v.'hich flovred continuously from the rear of the chamber to its front at a rate of 1.5 1 . /min served to transport ozone to the animal cage. Ozone concentration \\ias varied by altering the voltage impressed across the IN bulb and its series ballast (a 40 Vl light bulb) by use of an autotransf ormer . Response vjas linear. Um-Janted variation V7as avoided by operating the autotransformer from the output of a constant voltage transformer. The ozone concentration of interest v.'ithin the exposure chamber was that in the animal cage. This was determined continuously through 3 No. G4S11, General Electric Company, Cleveland, Ohio,

PAGE 48

35 FIGURE 5. OVERALL VIEW OF ANIMAL EXPOSURE SETUP

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36 FIGURE 6. THE INTERIOR OF THE EXPOSURE CHAMBER WITHOUT UV SHIELD

PAGE 50

37 FIGURE 7. THE INTERIOR OF THE EXPOSURE CHAMBER WITH UV SHIELD

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38 the use of a Mast Ozone Meter^ connected, through a valve in the chamber, to a 3.2 mm ID stainless steel sampling tube which terminated in an unoccupied compartment of the animal cage. See Figure 8. The Mast Meter utilizes a microcoulomb sensor (184). The sensing solution containing potassium iodide (Kl.) (See Appendix 1) enters the detector at a fixed rate and flows dov.m an electrode support in a thin film. Air enters the detector at a fixed rate and comes in intimate contact with the solution film. VJhcn ozone is present and chemical reactions occur (See belov:) , current flows, the anode and cathode having a constant potential between them. This current is measured on a microammeter in the external circuit. The reactions involved arc the follov;ing (ISA): Oxidation of KI in the sensing solution Oo + 2 KT HHoO — >• Oo H T., + 2 KOH (18) Troduction of H2 layer by polarization at the cathode 2e + 2 I!+— i-« II2 (19) Reaction of W^ and I2 at the cathode H2 + I2 — :2111 (20) Equation 20 is followed by a repolarization current of two electrons flowing in the external circuit. Hence the current is directly proportional to the number of ozone molecules reacting. Ozone concentrations measured with Mast Meters were reported to be between 65 percent and 100 percent, depending on their calibration, of values determined by the neutral KI chemical method of determination. '^Model 724-2, Mast Developm.ent Com.pany, Davenport, Iov7a.

PAGE 52

39 FIGURE 8. THE EXPOSURE CAGE WITH ANIMALS, R-CHAMBER, AND OZONE SAMPLING PROBE

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1^0 considered as the reference method (84, 185). Consequently, it was necessary to determine the calibration of the particular Mast Meter being used. For this, another standardized procedure, the alkaline KI method, V7as used (185). See Appendix 2. It had some advantages over the neutral KI method, and data could be converted to standard values (neutral KI values) by multiplying by 1.54 (84). For the calibration, ozone was produced by the W bulb in a 2 1. beaker. The Mast Meter sampling tube and the inlet for the sampling train used in determining ozone by the alkaline KI chemical method both drew air from the beaker at the same position. See Figure 9. The bypass of the midget impinger containing the sampling reagent was used to keep the air flow from the beaker, and hence the ozone concentration in the beaker, from altering drastically when sampling began. A strip chart recorder was used v;ith the Mast Meter so that average readings could be determined for the time periods during which samples were collected. The results of the calibration procedure are shovm in Figure 10. The autotransformer and ballast bulb for ozone production regulation, the strip chart recorder for the Mast Meter, and the compressed air tank and floivmeter for air flov; through the chamber were all located outside the laboratory containing the exposure chamber so that observation and regulation could be done during an x-ray exposure. An exhaust pump for the chamber was required to keep it from pressurizing slightly, which would increase the air flow into the Mast Meter and change its calibration. This pump was located in the exposure laboratory but did not require frequent adjustment. Humidity within the chamber was kept nearly constant by passing the inflowing air through a water bubbler

PAGE 54

Ul FIGURE 9. THE SETUP USED FOR CALIBRATING THE OZONE METER

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FIGURE 10. ACTUAL OZONE CONCENTRATION VS. MAST METER INDICATED OZONE CONCENTRATION

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h-i 5045M O K a. 400. g 35< -I < UJ H U UJ cc §25o a: Z 111 o z o u UJ z o N O 201510/|:l / M ='-5[03] -5.5 1 1 1 1 r— 10 20 30 40 50 60 MAST METER AVG. OZONE CONCENTRATION, PPM xlO^

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kk lation was automatic, the sensing thermocouple „.„ ,.„ ... ........ .• .» -' ""'•_, _ „ ., „ „„ ....,-.. ..... .... — • »• "• •"'"• ^ '^ Tn-,-; Q reaction '"""^ . ,,e air The reduction can be very , ,„rs the ozor.e conccntr.->tion in the ax.. ..t^nees E.po3„re to relatively hl.h concentratrons large v.'ith some substances. i^ \ • n plim, ,, 1 pp.) over exteo.ec, Perlo.s (several Hour.) -.U of ozone (up to 1 PPn) ,,,, 1. referred to as "ozone.• l-^^ nf many items. in^-S ^^^ inate the reactivity o£ many chamber the animal cage, and the . . „n nS6^ Tlie exposure chambei , conditioning (1S6) . . ^^^.^^^ /i.-rioned" in order to atCc.iu .Tin', line -were "ozone-conditio.^ea ' , .o prevent low readings due to reaction In the sa.,concentrations and to plt^,cnc pling system. ^^^^^^^^^^.^a^n^^^Oh^-ii-i^^Ll-" .0 produce the de.lred external radiation Held, a Ceneral -,v machines,, -as en>ployed. The unit or^n TTT therapy x-ray macnme Electric Haximar 2^0-111 • ^ ,-^"1 licht to check tlie rici-ii size plus an intein=xl iig^^t , • ^^ f U. for inserting standard (suppUed) fxl ,. . 1 Th-re vas provision foi m.eitmg irradiated. inere ^-^^ ^ _ , , a slot for additional filters as raquxred. ters into the beam plus a slot 1 T7lPrtric Company, Milwaukee T^ 1, serial Ko . 467617, General Electric VJisconsm.

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h5 Radiation was beamed into the exposure chamber through a 10 cm diameter viewing port. See Figure 5, page 35. The beam axis was perpendicular to the central plane of the 15 cm diameter Incite animal cage, and the target-cage distance was 70 cm. The cage permitted some animal movement but not toward or away from x-ray source. To monitor the consistency of the beam during an exposure period, a Victoreen Rate Meter, Model 510, was employed in conjunction with a strip chart recorder. The probe, Model 601 (30-400 keV effective), was placed outside of the exposure chamber, at the edge of the round viewing port, and in the corner of the beam (v.'hich measured 11 cm x 11 cm there) . To determine the total exposure during a period, a Victoreen R-chamber, Model 154 (250 R, No. x915)^ and Model 570 reader were utilized. The chamber was placed in the unused compartment of the animal cage. See Figure 8, page 39. A correction factor of 0.98 v.'as applied to all readings of the chamber. This was a sensitivity factor determined by comparison of the chamber with one calibrated by the National Bureau of Standards. The uniformity of the field over the cage area was checked in two ways, with sheet film and with an array of thermoluminescent (TLD) powder dosimeters. The film, Kodak Type AA Industrial in a leaded cassette, was placed at the cage position perpendicular to the x-ray beam. The circular rather than oblong pattern showed the beam definitely was perpendicular to the cage; densitometric measurements revealed the 90 percent isodose curve to be very nearly a perfect circle with an area larger than that of the circular cage. Tliere were two 3.1 mm OD x 2.5 cm "Victoreen Instrument Company, Cleveland, Ohio.

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h6 TLD dosimeters per wedge-shaped animal compartment (with two readings per dosimeter) « They V7ere positioned in the midplane of the cage in tV7o concentric circles (7 cm and 14 cm diameters) and surrounded V7ith rice as phantom material. Response was linear v.'ith dose in the region of interest, 9-17 R. The average dose was determined for each animal compartment; the maximum deviation of animal dose from the mean dose was 6 percent of the meano The x-ray machine v/as operated with 200 AC volts across the primary of the high voltage transformer, this corresponding to approximately 200 K\T. The standard filter employed was 2 mm Cu plus 2 mm Al. With this combination of voltage and filtration, the half-value layer (}l\a.) was determined to be 2.35 mm Cu, equivalent to an effective beam energy of approximately 118 keV. This ensured a maximum decrease in dose of approximately ]5 percent over a 4 cm animal depth with the 70 cm target-to-cage distance used (187) o The unit was operated at 5-9 mA with 2 mm Cu additional filtration v.-hich served to reduce the intensity of the beam dov.Ti to the desired level, approximately 1 R/min at the animal position. Current was adjusted as necessary during an exposure to keep the exposure rate, seen on the ratemeter and strip chart, at the desired value. Scor ing Chromosom.e Aberrations The aberrations of interest in this investigation are all of the chromosome type. They involve both strands of the duplicated chromosomes, since the breaking and rejoining associated v^ith exposure of the animals to ozone and/or radiation occurred before duplication of the Ijiiiphocyte chromosomes.

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Tlie various chromosometype aberrations arc shown in Figure 11 (93). The following discussion refers to this figure. The chromosome, aberrations of interest in a quantitative investgation are those that require a minimum of subjective judgement on the part of the observer in order to identify them. Consequently, types which involve changes in the lengths of the arms (the strand portions on each side of the constriction, or centromere) should usually not be included in scoring (counting). These include columns B, C, and F. Each of these types involves two breaks and an exchange of resulting arm seg-ncnts to give chromosomes that in most cases give an outward appearance of being normal. Colvmns B and C, involving one chromosome, are referred to as inversions, and these were never scored (counted). Coliunn F, involving t^i'o separate chrom.osomes , is referred to as a r tranpl'^catT on. Tliis '.,^:v -^-pd (ns two breaks) only if the arms of one of the clvroDosomes were decidedly and distinctly much longer than they should have been. See Figure 1, page 25. Arms shorter tlian normal would not necessarily indicate a translocation, but could r-esult from a sim.ple chromosome break, as shov/n in Column A. The types of aberrations v.'hich V7ere the basis of scoring are shown in Columns A, D, E, and F. They are all easily identified. Column A, called a simple break or a deletion, results from one chromosome being broken and does not require any type of recombination in order to appear. It v.'as scored as one break on the basis of finding acentric fragments (or one fragment as shown in A^), fragments without a centromere. Deletions were the aberration type seen most frequently. Columns D, E, and F each involve two breaks and were so scored when found. Columns D and E, each involving a deletion from a single

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1^8 SYMMETRICAL INTRACHANGES ASYMMETRICAL INTRACHANGES Chromosome break c A Al A \ A2 Inter-arm inversion Bl -^ Intra-arm inversion / CI o Inter-arm deletion 9 Dl O y\ D2 Intra-arm deletion Symmetrical interchange Fl II Asymmetrical mtcrchange J^ Gl e s m K /o o > B3 C3 D3 ES F3 A or-^ LA, ^^ D4 E4 X F4 i %. A4 B4 G4 C4 60 v.. or A6 o oo B5 A Do 0= K5 or D6 8= 6 ^ FIGURE 11. DIAGRAM OF THE VARIOUS CHROMOSOME -TYPE CHROMOSOME ABERRATIONS

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^9 chromosome, result in the formation of ring structures, either with or without a centromere; these aberrations are referred to as rings and were scored on the basis of finding the rings. Column F, involving an interchange between tv70 chromosomes, results in the formation of a chromosome with two centromeres; it is called a dicentric and was scored on the basis of finding dicentric chromosomes. In summary, a deletion was scored as one break on the basis of finding accentric fragments, a gross translocation was scored as two breaks on the basis of finding a chromosome with exceptionally long arms, while rings and dicentrics Vv'ere scored as two breaks each when found. Figures involving three breaks or more are possible but were not seen. Estimating the Ozone and Radiation Exposures Required The principal objective in choosing the ozone and radiation exposure levels was to have exposure to one agent alone or to the tv70 simultaneously result in frequencies of chromosome aberration that V7ere significantly different statistically. The production of chromosome aberrations by sparcely ionizing radiation such as x-ray is a random process. With a given dose, the frequencies of cells in the "one-break," "two-break," etc. classifications follow the Poisson distribution (155). A given ozone exposure would likewise be expected to result in a Poisson distribution of chromosome breaks per cell since ozone, like x-ray, is believed to act primarily through oxidation events associated witli free radical production (154). The number of breaks per unit of cells (such as 100 cells) should also follow the Poisson distribution. The number of breaks, n, is a point estimate of the average number of breaks, u. If u>l5, the Poisson

PAGE 63

50 can be approxmated by a nonral distribution with a mean == u and standard deviation = n^ . The sample (estimated) mean wonld be n and the sample standard deviation would be n\ On a breaks per cell basis, the sample mean would be n/N, the sample standard deviation n^N, and the 95 per, cent confidence interval n/K t 2 n^/N, where N = the number of cells in the unit. The statistical significance of the difference between two H.eans could be dete^ined through the use of Student's T distribution or by determining whether the confidence intervals around the :neans overlapped, a stricter test. The ozone and radiation exposure levels v.erc chosen to yield •t-v, t'Ur. ^n^^1 nuT-iber of breaks for each being equal break frequencies, with the total nu...Dv,r large enough for the normal distribution approximation to apply. The levels were also chosen so that break frequency for combined ozone plus radiation exposure, expected to be approximately twice that for either agent alone, would be significantly different statistically from the frequency for either agent alone, as determined by the confidence interval overlap method. The levels were also chosen to be as low as possible while still m.eeting these requirements. The initial estimates of chromosome break frequencies from x-ray exposure were made on the basis of available information on the response of hur,.an lymphocytes to x-radiat ion ija vivo and in vitx'o (155, 1G5). Supporting evidence came from chromatid aberration data for the in vivo irradiation of Chinese hamster bone marrow cells (172), taking into account that the cells examined were in the post-DNA-replication stage of the cell cycle at the time of the irradiation and hence had greater radiosensitivity (disregarding differences in cell type) (148).

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For ozone, deletion frequency v;as estimated on the basis of the exposure of hunian cell cultures to higli concentrations of ozone by Fetner (154), while ring and dicentric frequency was strictly a guess based on analogy with radiation. Fetner 's ozone data X7erc on chromatid deletions. In arriving at the estimated frequency for this investigation, the pre-DNA-syntl)csis stage of the cell cycle (GO vjas assum.ed to be approximately one-half as sensitive as the post-DNA-synthesis (G„) stage (148). (Difference in cell type was disregarded.) Additional assumptions included linear dependence of break frequency on exposure time, to be conservative (the data shovred exponential dependence), and validity of concentration x time = constant response (84) from the 8 ppm used down to 0.1 ppm, tlie approximate concentration desired. The following were the initial estimiates: Radiati on 1 ,, 10-3 E!^l£tio^ (21) cell-rad , „-6 dicentrics + rings /oo\ 5x10 r-L — TT '— {l^) cell-rad^ Ozone 3 X 10-^ deletions ^23) cell-(ppm-mi.n) "6 dicent rics -Iring s (j,. 7x10 — — :ri — 7~ ' — \/~ V-^^V cell(ppm-min)"^ The dependence of dicentric or ring frequency on the square of the dose follov7S from tv:o breaks being required for each, the probability for each break being linearly dependent on dose, and the occurrence of each being independent of the other. Data on spontaneous aberrations in Chinese hamster bone marrow (172) and in human blood lymphocytes (163, 166) indicated the frequency vms so low (<,l x 10 breaks/cell) that it could

PAGE 65

52 be neglected, but pre-exposure cultures were made anyi;ay as a check. The data of Ilungcrford (180) for human blood and Buckton and Nettesheim (181) for mouse blood indicated that it would not be unreasonable to expect, per culture, 200 metaphase spreads that would be suitable for analysis. Assuming 200 cells per culture, it was determined by trial and error using the coefficients in 21-24 and the expression for 95 percent confidence interval that 5 hr at 0.1 ppra ozone and 100 rad (over 5 hr) v.'ould be suitable exposures. Tliese levels V7ould result in confidence intervals of 0.22 + 0.07 breaks/cell for ozone or radiation alone and 0.44 + 0.09 breaks/cell for ozone plus radiation (assuming additivity) and these intervals do not overlap. Halving each exposure (to 2.5 lir at 0.1 ppm and 50 rad) would result in confidence intervals tb.at would overlap, so the higher values, 0.5 ppmhr and IGC rad, were the ones of choice. To determine the suitability of these values, preliminary cxpcrim.ents v;ere conducted utilizing one' or two animals at a time exposed either to ozone or to radiation. The ozone exposure levels v.^ere approximately 0.A5 and 0.50 ppra-hr and the radiation doses were 85, 153, and 166 rad (Rocntgen-to-rad conversion followed NBS Handbook 85, Physical Aspects of Irradiation ) . Eight animals were involved. Discounting tlie 13 and 16 day delays between irj^adiation and successful cultures for animals receiving 153 and 165 rad, these delays in part a result of culturing oven temperature variations, the deletion yield for irradiation was only 65 percent of the expected j'ield. Similarly, for the ozone-exposed animals, the deletion yield was only 62 percent of the expected value. Additionally, it v/as found that the average number of metaphase spreads per slide that were suitable for

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53 analysis v.^as nearer to ''lO than to 200, with values ranging from 10-71. Since the basic exposure group v/as to have four animals, there was a possibility that fewer than 15 deletions V70u].cl be found for all the animals pooled. EX^en if the exposures were raised to correct for the lower-than-expectcd yields, only 10 deletions per 100 cells would be expected, so the average of 160 spreads per animal group v/ould only give an. average of 16 deletions. Fewer than 15 vrould mean that the nori'^l distribution approximation to the Poisson distribution could not be used, and that confidence intervals v/ould not be appropriate. The breaks that would' result from dicentrics and rings x%'ere not being counted on to raise the total breaks observed. This was because the radiation and the ozone were being administered over a relatively long time period, 5 hr , whicli may be longer than the average lifetime of a chroiaoboine bi.cak (155). 'jhis v.'oulJ iav:,aik thdl. breaks created at the beginning of the period miglit not still be available for combination at the end of the period, so the yields of multiple-break aberrations would be lowered. Consequently, the decision V7as made to raise the ozone and the radiation levels to correct for the low yields and to increase the expected number of deletions from 10 up to 15 per 100 cells, for four animals v/ould certainly be expected to yield 100 analyzable spreads. It was also decided to increase tlie ozone concentration rather than increase the exposure tim.e to >5 hr. The radiation dose to be used, then, was (300 rads) (1 ,5/0. 65) = 231 rad and the ozone concentration V70uld be (0.1 ppm) (1 .5/0.62) = 0.24 ppm but a decision was made to limit the ozone concentration to 0.2 ppm, for a total exposure of 1,0 ppm-hr. This was done to avoid the

PAGE 67

possibility of any observable effect on the well-being of the animals, i.e., to keep the exposure level at a value knov.ni to be tolerable (84). For each condition, ozone, radiation, and ozone plus radiation, there v.'ere t^.'o exposure groups of four animals each. An additional group v.'as put through the entire procedure minus the agents (ozone and/ ciradiation) to serve as a control. Also, another group was subjected to a 335 rad radiation exposure, approximately 50 percent greater than the dose the others received. This was done to demonstrate that break frequency does in fact increase with radiation dose. A preliminary report indicated that 335 rad was onethird of the LDcq/oq for 100 day old Chinese hamsters (188), so complications v;hich would interfere with the procedures to follow v.^ere not expected and did not appear.

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CHAPTER III PROCEDURES This chapter presents a step-bystep description of the numerous operations that v;ere invo].ved in this investigation. For some of these operations, details were important, so the entire procedure is presented. Reference is also made to the appendices for various formulations and supplementar}' procedures. Operations are presented in their normal time sequency. Animal Exposures The four animals in each exposure group v/ere randomly selected from among those for which successful pretreatment cultures v.'ere available. Since the animal quarters were located near the exposure room, the animals remained in their boarding cages until transferred to individual compartments of the exposure cage icmediately prior to beginning an exposure. Each animal occupied approximately 50-70 percent of the space of its compartment. Exposures began approximately at 9 A.M. and were of a 5 hr duration. If the exposure involved ozone, the W generator remained operating the previous niglit so the chamber would have an ozone environment ready. Similarly, if the exposure involved radiation, the ratemeter and the R-chamber reader were left operating overnight so they V7ould be stabilized by irradiation time. The response of the ozone meter v;as checked with an electric discharge ozone generator outside the chamber before and after each exposure.

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56 Air flow through the chamber was maintained at 1.5 l./min throughout e.ch exposure. This provided a co^nplete air change in the chamber every 100 min. Since the exposures were conducted during daylight hours, a light was provided at the second viewing port of the chamber to illuminate the interior. Temperature was maintained at 15.5°C. Relative humidity averaged 70 percent for exposures involving ozone and 45 percent for exposures to radiation alone. H.is difference resulted from ozone exposures being done on consecutive days and radiation exposures having at least one day between them, which gave the chamber time to air and dry out. The animals of each group were observed through the viewing port at least twice during their exposure; they were generally inactive but awake. The chamber was opened once during each radiation exposure to read and recharge the R-chamber. This was done so that readings would be around mid-scale where they were most accurate. An air density correction factor of 0.98 was applied to the readings because the exposure chamber temperature was below 22°C. During each radiation exposure, the x-ray current was adjusted when necessary to keep the ratemeter reading at the desired level. An exposure rate of 4.8 R/min (7.0 R/min) at the ratemeter probe corresponded to the desired 0.82 R/m.in (1.15 R/ min) at the cage R-chamber. Values in parentheses were for the high radiation group. During each ozone exposure, the UV bulb voltage was adjusted when necessary to keep the cage ozone concentration at the desired 0.2 ppm. Response was relatively rapid, concentration adjustment time varying inversely with the size of the voltage change.

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57 Once a steady-state condition was attained, the cage ozone concentration v/as the same as that in the rest of the chamber. This indicated that there was no appreciable ozone degradation by the Lucite cage after it v?as ozone-conditioned. There V7as no detectable ozone production by the radiation beam, and the x-ray environment appeared to have no effect on the performance of the ozone riieter. l-Jhen ail exposure was completed, the animal cage was removed from the exposure chamber insmcdiately; the animals remained in the cage awaiting the blood sampling procedure, which began promptly. Bloo d Sampling The collection of blood samples by the orbital bleeding technique was done prior to an animal being accepted for exposure (once, or more if required by culture failure or lov.' yields of chrom.osome spreads), immediately after exposure, and again at intervals if required or desired. This operation and all those following were the same for preand post-exposure samples. All materials and equipment contacting the blood v/ere sterile, from the sampling operation to the cell-killing step of the slide preparation procedure. They were either purchased that v;ay or autoclaved at 121. 5°C, 15 psig for 15 min. Sterile technique was employed. Except for the tissue culture tubes, all glassware was silicone-treated to reduce cell adhesion. For cleaning glassv/are, the detergent employed 7 v
PAGE 71

om 58 Leighton-typetissue culture tubes v/ere used. Tliese have an 11 mm X 40 mT^ flat area on one side and a constriction above this area to act as a dam. For the procedure being followed, they were reported to be the only kind in which culturing was successful (181). The tubes employed were 16 mm OD x 125 mii with screw-caps. They were filled witl 15 ml of hcparinized TC-199 culture media (See Appendix 1) and kept on ice during the sampling procedure. For each animal, blood \:as withdrawn with a hepar in-wetted blood pipette and emptied into a culture tube which was then gently agitated and given a number identifying the animal and the culture fr that animal. Generally, one pipette-full, approximately 0.25 ml, of blood v:as put into each culture tube. Occasionally, if <0.20 m.l was transferred in, a second pipette vjas used in the otlier eye; the total Dluou put into culture laug.d up to 0.35 ml. After each anii^l was sam.pled (one culture tube per animal) they were returned to their boarding cages and the culture tubes were transported to the culturing laboratory. Tissue Culture Preparation and Maintenance The purpose of the TC--199 culture media was to greatly dilute the hamster blood plasma (approximately 1:100). The culture tubes were spun at 500 g_ for 10 min in a refri.gerated centrifuge (5 C) and the supernatant liquid (TC-1S9 plus plasm.a) was pipetted out, A 4.5 ml aliquot of culture m,cdia v7as added to each tube immediately after removal of its supernatant liquid, and the tubes were gently agitated. The media V7as prepared following the specifications of Hungerford (180)

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59 (See Appendix 1), This was followed by the addition of 0.05 ml of o pokevjeed mitogen solution per culture tube. The tubes v/ere then placed in an incubator at an angle of 5 to horizontal v/i th their screv.--caps slightly open. The incubator was maintained at 37 C with a 95 percent air plus 5 percent C0„ gas flow through it that provided a complete change every 100 min. Humidity V7as maintained by a constant-level water pan in the base. The tubes were momentarily removed twice per day for gentle agitation. After 67 hr of incubation, 0,4 ml of a 2 ug/m.l colchicine solution vjas added to each tube (See Appendix 1). This drug, a spindle poison, prevented cells in tl-.e division process from proceeding past the m.etaphase stage, v;here the chromosomes were duplicated and contracted, to the anaphase stage v;here each chromosome v7ould separate into tv70 daughter chromosomes that would m.igrate to opposite ends of the cell just prior to its division. In this vray cells in metaphase \:ere "stored up" as cells entered the stage while few, if any, left it (155), After 5 hr exposure to colchicine, 72 hr total incubation, the cultures were removed from the incubator, and the slide preparation procedures were begun. Slide Preparation After gentle agitation, each culture was transferred by pipette to a graduated 15 ml centrifuge tube and spun for 4 min at 100 g_. The supernatant liquid was removed by pipetting. A 5 m.l aliquot of heparinized hypotonic potassium chloride (KCl) solution (See Appendix 1), ^Grand Island Biological Com.pany, Grand Island, New York.

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60 pre-vjarmed to 37 C, X\Tas added to eacli culLuj:e. Each tube V7as gently agitated v/ith a mechanized tube mixer during the KCl addition to suspend the cell button. The tubes were covered and placed in the incubator at 37 C for 15 min. The tubes were spun again and the supernatant liquid was removed, taking care to leave the thin light-colored overlayer of the button called the buffy coat. With a gentle m.echanical -mixer agitation to avoid clumping, a 5 ml portion of fixative v/as added to each centrifuge tube. The fixative, v/hich caused hemolysis and killed the leucocytes, was m^de up of three parts absolute m.ethanol and one part glacial acetic acid. The tubes were spun as before (4 min, 100 g_) , the supernatant liquid V7as removed, and a second 5 ml aliquot of fixative vms added to each tube, again V7ith gentle median ical agitation. They were covered and placed in a rcfrlgeratofor 15 min. The tubes v;ere spun and the supernatant liquid was removed. It had no color remaining from hemoglobin. Without agitation, enough fixative was added to each tube to approximately dovible the volume of its cell button, vjhich ranged from 0.05-0,10 ml. The 25 mm x 76 mjn slides to be used had previously been vrashcd, put into a staining dish to keep them separated, submerged in distilled water, and placed in the refrigerator. Wien the centrifuge tubes with fixative v.'ere put in the refrigerator, tlie beaker of slides v/as removed and pla.ced on ice, and a flow of C0„ gas that bubbled through the v/ater was begun. Using a Pasteur pipette (vrith rubber bulb) as a rod, the cell button in a centrifuge tube was gently dispersed. The 0,1-0.2 ml suspension v/as then drav.-n into the pipette. A slide V7as removed from the

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61 beaker v.'ith tweezers, grasped by the edges, and tilted with one edge against adsorbent paper to remove excess water. The slide was then leveled and the suspension from the pipette was transferred to it, confining the delivery to a 24 mm x 50 mm area or less. The slide was then rapidly passed through an open flame which ignited the methanol. When it burned off, the slide was again tilted, this time being tapped against adsorbent paper to remove excess moisture. It was then placed on a slide warmer at 40 C to dry thoroughly. The V7ater film and the burning were necessary to obtain metaphase figures (spreads) with well-separated chromosomes that lay flat on the glass. The slide-making procedure was repeated for each of the cultures being processed. Wlien the slides were thoroughly dry, the identifying numbers were etched on them. Each one was then partially but systemati9 cally scanned with a phase contrast microscope. This was done to get a relative measure of how successful the culturing and slide preparation procedures had been by counting the number of chromosome spreads observed. Few or no spreads seen on a slide indicated that the entire procedure would have to be repeated for that particular animal. This did occur in some cases and v;ill be discussed later. If the quality appeared adequate, the slide was stained V\rith Giemsa blood stain (See Appendix 3). The 24 mm x 50 mm area with material was covered with a glass slip fastened with Permount. The stained chromosomes appeared purple. AG Phases tar, American Optical Corporation, Buffalo, New York. Fisher Scientific Company, Springfield, New Jersey.

PAGE 75

62 Re cording of Data Each slide was scanned using bright-field microscopy. Overlapping fields of view were used to reduce the likelihood of missing a spread near the edge of the field. Scanning vras done at lOOx total riagnification with spot cliecks at 950x for questionable figures. The object was to locate chromosome spreads, record their location (the microscope stage v/as graduated), and photograph them at 950x total magnification for later analysis. So as not to bias the data, each spread that was located v/as photographed, without regard to its quality. Generally,, only the 24 mm x 50 mm portion of the slide covered with a glass slip was scanned. Occasionally the preliminary phase scan indicated that the material had spread beyond the intended area; these slides were scanned over their entire area. The microscope employed vrac a Leitz Ortholux equipped with an Orthomat autonuitic 35 mm camera , Since tlic chromosomes were stained 12 with Giemsa, a combination of Wratten filters was used to improve the visual contrast and im.age quality (189). V.'ith these filters, the chromosomes appeared black on a green field. Photography was black and white. The film-developer combination chosen vras Kodak Panatomic-X and Kodak D-19 developer. This panchromatic film v.'as extremely finegrained for sharpness of detail and high resolving pov;er and combined moderate contrast with adequate speed (ASA 32) (190). The recommended developers vjere all medium-contrast materials. To improve the im.aging. E. Leit7. , Inc., Nev7 York, New York. 12 Wratten Kos. 12 (yellow) and 44 (aqua blue), Eastm.an Kodak Company, Rochester, Kew York.

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63 a high-contrast developer was selected; of those available, D-19 v;as chosen since it vras usually used for scientific plates and instrumentation films. Tlirough inquiry, a suitable developing time for this combination V7as found to be 3.5 min at 20°C (191). Analysis of Data Films were left in roll form for analysis. A film strip projector was used to image the films, frcmie-by-f rame, on a screen. Wlien enlarged in this manner, a typical chromosome spread had a diaiiieter of approximately 25 cm. Detail v;as not lost because a high degree of enlargement was possible with the extremely fine grained fine used. Scoring of aberrations was facilitated by these large-size images. To avoid the difficulties associated with being reasonably sure that different people would score the aberrations in a given spread in the same v;ay, all the scoring was done by one person, the author. The scoring was conducted as a blind analysis to avoid any biasing of the data. Each film was identified by number only; there Vs-'as i-io indication as to which exposure group the data had come from. The photography log book indicated that the data on a particular film came from a particular animal, but the exposure group to which the animal belonged was not listed there. The information recorded in the scoring log book for each frame included the total number of chromosomes, the number of chrom.osomes in each to two general m.orphological classifications, "X" ' s and "V"'s (See Figure 1, page 25), and the number and kinds of chromosome aberrations. Also noted were chromatid (one-arm) breaks and stain gaps (chromosome portions ' that did not take up stain). A gj-oup of chromosomes V7as

PAGE 77

6k counted as a spread if it had 12 or more figures, i.e., greater than one-half of tlie 22 chromosoiaes in a norraal spread; sometimes chromosomes were lost from a spread during the slide preparation procedures. A normal spread had 16 "X"-shaped chromosomes and 6 "V"-shaped figures. Wlien necessary' to aid in scoring aberrations, detailed figure counting for the various sizes of "X"-shapes was also done. Any spread that appeared blurred v/as later relocated on the microscope, using the photography log book to get its coordinates. This was to ch.eck vjhethcr the spread v;as blurred or the photograph was poor. If the spread was clear, a new photograph v;as taken. Similarly, any spread that exhibited a chromosom.e aberration was relocated on the microscope. This was to determine whether the figure scored as an aberration vjas an artifact on the film or on the slide or V7as actually c Ii r or ; o £ O'.'ia 1 ma. t e r 1 a 1 . » At tl;e completion of scoring, the exposure groups were revealed and the data tabulated on an animal basis and on a group basis, listing the numbers of spreads analyzed and the numbers of deletions, dicentrics, and rings found. Gross translocations were not seen. The productionfrequency coefficients were then formed and cornparisons were made for the various groups.

PAGE 78

CHAPTER IV RESULTS Table 2 prest.nts the exposures received by the various treatment groups. Ill each case except one (Group 1), the exposure and rate or concentration were nea.r the desired values, so variation among the six principal groups was minimal. The high radiation group (111 X) dose was 40 percent greater than that received by the other x-ray exposed groups; also it was delivered at a 40 percent higher rate. A total of 746 chromosome spreads v?ere examined for breaks in tlie final experiment. These spreads displayed a total of 87 chromosometype chromosome breaks and 6 aberrations which could be classified as chromatidtype chrom.osome breaks or as stain gaps. Tlie results of the analysis are summarized in Table 3. In this tabulation, the ozoneexposed groups were broken dov:n into three categories, "early," "mid," and "late." These designations refer to the time interval between treatment and sampling, 0, 6.0, and 15.5 days respectively. Tlie multiple sampling v;as done to have ozone data with a delay time comparable to the delays experienced with the other groups. It was discovered during the final investigation that the radiation exposure had an inhibitory effect on the lymphocytes which resulted in unsuccessful cultures (i.e., cultures with few if any mitotic chromosome spreads). This effect appeared to diminish vjith time after exposure, and satisfactory cultures v.-ere obtained in all cases after ^11 days had elapsed. 65

PAGE 79

66 > < OJ ^1 ;3 to o a. w OJ a o N O X. I p. Pi Q o u o o <^ o o O 1-1 Oi CNl o o ro
PAGE 80

61 1-1 4J C E w o c3 n5 r; t) }-< o o CO (/; O •H 4-) rj Vj O t-t 60 d •r-l O S ttf a CD T) > E C <3 H ^

PAGE 81

68 The break frequency column was included in Table 3 to permit rough comparisons. The entries, simply breaks per cell, do not reflect either differences in exposure among the groups or, for averaging, differences in numbers of useable spreads per group. Both of these refinem.ents were incorporated in the exposure-adjusted frequencies given in Table 4. For eacli treatment involving t\;o groups, a weighted average was formed v/hen the difference between the contributing frequencies v;as found to be statistically insignificant (approximately norm.al distribution V7as assumed for each frequency), IVo ozone plus radiation frequencies were formed, one based on the radiation exposure only so direct comparison could be made witli the frequency of the radiation treatment groups and the other based on tlie ozone exposure only so direct comparison could be m.ade with the frequency of the ozone treatment groups. Radiation I^ose_d_j\nima_]_s Comparison of the exposure-adjusted break frequencies for radiation and for high radiation treatments in Table 4 showed them to be nearly identical, within 4 percent of one another, and to have comparable saraple standard deviations witli values 25 percent of the means. As would be expected, the difference between the frequencies v/as not statistically significant. These data suggested that the nur^ber of breaks per cell varied linearly with dose in this region, 230-330 rad . More importantly, since linear dependence had been expected, the high radiation frequency provided a check on the validity of the radiation frequency.

PAGE 82

69 to -< D CO O Cl X w •H C HO S-i a CJ c (1) Cr CD (U ro

PAGE 83

TO Ozone Exposed Aniinals The Biost Important fact revealed by the ozone -exposed groups was that inhaled ozone did result in chromosome breaks in circulating lymphocytes. The exposure-adjusted break frequency for O^(late) in Table 4 definitely differed from zero (IXO.OOl, i.e., <0.1 percent chance of the frequency being zero). Comparison of the frequency for 03(latc) with that for O3 (early) revealed that the difference between them was not statistically significant. The difference between 03(latc) and O.(mid) was significant, the latter frequency being smaller. This perhaps resulted from 50 percent fewer cells being available for examination for 03(mid). The data suggested, however, that the observed ozoneinduced break frequency did not diminish with tim.c for at least 2 ^./eeks post-exposure , Aninvils Exposed to Comb ine d_Ozone_and_Radiat ion Environment The exposure-adjusted break frequency for the ozone plus radiation treatment groups, based on tlie radiation exposure only, was compared with the frequency for the radiation treatm.ent groups. The difference between them, while not statistically significant, was a value that would occur or be exceeded 18 percent of the time (expecting the + X frequency to be higher). In other words, there was an 18 percent chance that all tlie breaks observed for the ozone plus radiation treatment resulted from the radiation .lone even though the O3 + X frequency was nearly 40 percent greater than the X frequency. Similarly, there was a 15 percent chance that all the breaks observed for the ozone plus radiation treatm.ent resulted from the ozone exposure alone even though the O3 + X frequency was 45 percent greater than the 03(late) frequency.

PAGE 84

71 Another v.'ay of examining the ozone plus radiation treatment data is presented in Table 5. The expected breaks from radiation (computed from the exposure-adjusted radiation frequency) and the expected breaks from o^ione (corr.putcd from the exposure-adjusted ozone frequency) \\'ere compared with the number of breaks observed with combined treatment. These facts were revealed: 1. Tlie expected contributions from ozone and from radiation \\'ere nearly equal; 2. Tlie number of breaks observed was > 70 percent of the total breaks expected from the two agents combined (assum.ing additive effects) ; 3. 'Hie number of brealcs observed V7as 40 percent greater than the nu:Tiber expected from either agent alone; f\ , Either or^or.e or radJ ' '. . alone could account for all the breaks seen (considering maximum rather than average expected values) . The data suggested, but did not sho^\' conclusively, that the combined ozone plus radiation treatment resulted in a higher frequency of chromosome breaks than would be expected from radiation or from ozone alone.

PAGE 85

72

PAGE 86

CHAPTER V DISCUSSION Tlie initial estimates of break frequency for radiation and for ozone presented in Chapter II V7ere based on the frequencies expected for deletions and fo]* dicentrics and rings. The observed exposureadjusted aberration frequencies were compared with these first estimates. „, , n . r,, 1 1 ,rnl ObSBrVed T r • 1 • ^ 1 These data are presented m Table 6. The -— -— —-7 column facilitated ' Estimated comparison, among the four frequencies, of success in estimating. Expected vs. Observed Aberration Frequencies v ;itli Ra diation The estimates for radiation were based on frequencies developed by llie irradiation of freshly drawn humian v?hole blood in vitro v.'ith culturing following iimiediately (155). These frequencies were found to be reasonably good estimates of in vivo aberration production in humans (165), generally v.'ithin a factor of tv.'o for deletions and a factor of six for dicentrics and rings. Frequencies for aberration production in lymphocytes of other species, either in vitro or in vivo , were not available for comparison. Data were available for the production of breaks in other Chinese hamster cells by radiation. Exposure-adjusted frequencies for tlie production of chromatid1. ype breaks in bone m.arro\7 cells with in vivo exposure and in cultured "fibroblastlike" (embryo) cells with in vitro exposure were nearly equal to one another at 5 x 10" _i|~§.. For the bone marrov; cells, the frequency decreased exponentially with time after 7 o

PAGE 87

CM O Ih < w M U ;=> tj Pi o M H Pi W P5 <: p tJ H CO 1:3 < I w Pi CO o w Q CO pq o p < p H 1-1 H CO o :a o CO M O > o a) 4-1 e in a a •H O •H U-l M-l Cj o u o JJ •H > C.i Q rt 00 o > in o a d 7-' Gi > 4J •U -r-l 4-1 14-1 W CD W O u ?>1 o 4J 3 :3 (Li -I u CJ o u -r-: O ^-1 ft Vi X
PAGE 88

irradiation to 1 x 10" " 'ppf V-t; ^^ days. The iiiitial frequency was approximately a factor of nine greater than the chrociosometype break frequency determined in this investigation (172). The dissimilarity does not seem unreasonable considering the differences in cell type and stage of division at irradiation (86, 148). Frequencies involving radiation that were obtained in this investigation were for cells withdrawn approximately 2 weeks after exposure. The delay before successful cultures could be obtained, perhaps related to mitotic inhibition by irradiation (86) , was not observed in the huDian in vi vo investigations (155, 165), but the doses were considerably lower, 100 rad or less. In the human studies, the aberration levels remained relatively constant for 3-4 v/eeks after irradiation (153). Tlie observation does not necessarily apply to this investigation, for the turnover times of the two lymphocyte populations m.ay not be the same. The 2 week frequency for Chinese hamsters v.'ould undoubtedly be less than or equal to the no-delay frequency (if it could be determined employing a lov;er dose) . Considering all these factors, the aberration frequencies for radiation determined in this investigation appear reasonable in comparison to available data. They provide information that is basic to additional studies of the peripheral lymphocyte technique as a biological dosimeter. Chromosome Aber rations and In Vivo Ozone Exposure Referring to Table 6, the observed exposure-adjusted aberration frequencies for ozone V7ere surprisingly close to the initial estimates considering the numerous assumptions that were involved in computing

PAGE 89

76 the expected frequencies (See Chapter II). Besides the data that were used for the initial estimates, from in vitro exposure of cultured human cells to high concentrations of ozone, there were no frequencies for comparison. This vjas the first investigation of chromosome aberrations and inhaled ozone to be reported. The results add weight to the arguments for considering ozone as a radiomimetic agent. It v;as instructive to compare the break frequencies that would be expected from one week of exposure to radiation and to ozone at the maximum permissible industrial exposure levels. The exposure-adjusted break frequencies determined in this investigation were presumed to apply directly to human exposure (thus being conservative for radiation). In one week the permitted average radiation exposure of 100 mrad would result in a ly.nphocy te chromosome break frequency of 5.5 x 10" breaks/ cell. One week (40 hr) at the permitted 0.1 ppm for ozone would result in 0.4 breaks/cell.' CertaiTily the extension of animal data directly to humans could have introduced considerable error in the ozone estimate, but the difference in these two frequencies is too great to be easily explained av7ay. These data do reflect the fact that the limit for radiation recognized its mutagenic ability while the limit for ozone did not. The ozone concentration was set at a value chosen to prevent the occurrence of physical symptoms in most industrially exposed people, namely, nose and throat irritation (84). The recent "Community Air Quality Guide" (192) issued for ozone by the American Industrial Hygiene Association referred to the radiomimetic nature of ozone in stating that "theoretically, the recommended air limit for ozone . , , should be zero, or as close to zero as possible, i,e,, less than 0,01 ppm." As a realistic

PAGE 90

TT limit, the Guide recommended an upper concentration limit of 0.05 ppm and an exposure limit of 0.1 ppm-hr/day on the average during any year "if human health is not to be significantly impaired during a lifetime of exposure." Projecting again, this level would produce l,270x more IjTiiphocyte clironosome breaks than the maximum permitted occupational radiation exposure. The data from this limited investigation suggest that a lov.'ering of the community and industrial limits for ozone would be prudent. Interactions Resulting from Simultaneous Ozone and Radiation Exposure Tiie data suggested, but did not show conclusively, that the frequency of chromosome aberrations in circulating l>Tnphocytes resulting from either radiation or ozone exposure would increase if exposure to the other agent occurred at the same time. The combined effect, however, would be less than the sum of the effects expected for each agent alone. Some mode of antagonistic action or protective mechanism is therefore suggested, but its nature is not apparent (See Chapter I). The importance of combined effects is overshadowed by the magnitude of the effect expected for ozone alone at permitted concentrations.

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CHAPTER VI CONCLUSIONS In this investigation, Chinese hamsters vere exposed to xradiation, to ozone, or to both simultaneously. The basic radiation dose was 230 rad delivered in 5 hr . The average ozone exposure was 5 hr at a concentration of 0.2 ppm. Exposure-adjusted frequencies of chromosome aberrations produced in circulating blood l;>T.iiphocytes served as the quantitative measure of v.'idespread damage. The basic purpose of the study v:as to provide information on the combined effect of exposure to these agents. Stimmary of Results Tlie follov.'ing were the principal results in this investigation. 1. Radiation exposure resulted in an exposure-adjusted break frequency 5.51 x 10""^ breaks — ^^^. ^^^Xs withdrawn 2 weeks after cell-rad exposure. This value appeared reasonable in comparison to available inform.ation on in vivo exposure of human l>Tnphocytes and Chinese hamster bone marrow cells. Successful lymphocyte cultures could not be obtained until 2 weeks had elapsed. The number of breaks per cell appeared to vary linearly V7ith dose in the region 230-330 rad. 2. The action of inhaled ozone resulted in chromosome aberrations. The exposure-adjusted break frequency V7as 1.67 x 10"brcaks ^ This value was in good agreement v/ith the cell(ppm-min) 78

PAGE 92

79 expected value calculated on the basis of in vi tro exposure of hi.iman cells. There v;as no apparent decrease in break frequency with time for 2 weeks post-treatment. 3. Animals exposed to the two agents simultaneously exhibited ^70 percent of the total number of breaks anticipated assuming the actions of the agents to be additive. The expected contributions fro:;t ozone and from radiation V7ere nearly equal. There was, however, approximately an 18 percent chance that all the breaks observed resulted from the radiation exposure alone. Similarly;, there v;as a 15 percent chance that all the breaks observed resulted from the ozone exposure alone. Significance of Results This expei'iment demonstrated the suitability of lymphocyte chromosome breakage as a biological indicator of exposure to an air pollutant, ozone. The technique, introduced by this study, may be useful for other oxidant pollutants too. Additionally, the investigation provided data on the in vivo production of l^miphocyte chromosome aberrations in an animal by external ionizing radiation; such information was not readily available in the literature. Tlie work demonstrated still another reason for considering ozone as radiomimetic . Chinese hamsters appeared to be excellent laboratory animals for this type of study v;hen used with the orbital bleeding technique and the Ij^iiphocyte culture method which V7as developed during the investigation. This report presented the first detailed description of a successful method for the short-term culture of circulating blood lymphocytes from Chinese hamsterS using moderate size blood samples.

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80 Tae most important single fact to emerge from this investigation was that inhaled ozone resulted in chromosome aberrations in circulating blood lymphocytes. Because of the body-wide effects of inhaled ozone and the fact that production of chromosome aberrations by ozone appeared to be an action that was general to all cell types, the lymphocyte result should be indicative, in a qualitative sense, of similar damage to other cells throughout the entire body. Such aberrations are considered as cellular alterations (damage) with long-tern consequences . Presently permitted human ozone exposures would be expected to result in break frequencies that are orders of magnitude greater than those resulting from permitted human radiation exposures if the results of this animal study were directly extended to the hum.an case. Consideration of combined ozone plus radiation environments is overshadowed by tlie importance of ozone environments alone as long as permitted ozone exposure levels remain at their present and seemingly high values . That ozone inhalation does result in chromosome aberrations at the apparent frequencies is an outcome of this limited investigation which certainly appears worthy of further experimental consideration. In fact, such investigation appears mandatory!

PAGE 94

APPENDICES

PAGE 95

APPENDIX 1 PREPi\R,!^TION OF CULTURE MEDIA AKD SOLUTIONS

PAGE 96

83 Heparin Solution for Bloo d P i pettes To prepare 20 ml of sterile normal (0.85 percent) saline solution 3 V7ith 10 USP units hepar in/ml, do the following in order: 1. Place 0.153 g of desiccator -dried sodium chloride (NaCl) in a glass screv7-cap bottle marked for 20 ml; 2. Add approximately 15 ml of distilled dcmineralized V7ater; 3. Autoclave at 121.5 C, 15 psig for 15 min; 4. Add sterile distilled demineralixed vjatcr to bring the volume to 18 ml; 5. Add 2 mil of 10 USP units/ml isotonic aqueous sodium heparin solution and mix. Steps 4 and 5 must be done v;ith sterile technique. Keep the solution refrigerated . Hungerford Culture Media To prepare a 115 ml batch of media, add the components listed in Table 7 (180) to a sterile glass screv7-cap bottle, aseptically and in sequence. Keep the final solution refrigerated. 13 Organon, Inc., West Orange, New Jersey. This sequence V7as suggested in P rice and Reference I^Ianual, Grand Island Biological Company, Grand Island, New York (1969).

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8!^ TABLE 7 INGREDIENTS FOR SPECIAL CULTURE MEDIA Component^ Quantity, ml 1. Sterile distilled deniineralized vater 78 2. Earle's balanced salt solution (lOx) 10 3. MEM vitamins (lOOx) "l^ 4. MEM essential amino acids (50x) 4 5. L-glutamine, 200 irM (lOOx) 1 6. Phenol red solution, 0.57. 0.14 7. Sodium bicarbonate solution, 7.5% 3-5*^ Sc Penicillin-streptomycin mix, 5,000 units each/ml 2 9. Fetal bovine serum (gamma-globulin free) 15 10. Aqueous isotonic sodium heparin so lution, 10^ USP units/ml*^ 0.2 ^Except for 1 and 10, all materials v?ere obtained from Microbiological Associates, Inc., Bcthesda, Maryland. ^This quantity produced a solution that vas tV7ice the normal concentration for this component. '^Sufficient sodiuiii bicarbonate v/as used to produce an orangeamber color at 4°C (pH 7.3). "Organon, Inc., West Orange, New Jersey. Mast Ozone Meter Sensing Solution To prepare 1 1. of IG! sensing solution (154), do the follo\?ing in order, using reagent grade chemicals: 1. Add 20 g potassium iodide (KI) to approximately 500 ml of distilled demineralized water in a 1 1. beaker; 2. Add 50 g potassium bromide (KEr) ; stir to dissolve; 3. Add 2.5 g of sodium phosphate, monobasic (NaH2P04 • H2O) ; 4. Add 7.0 g of sodium phosphate, dibasic (Na2HP0^ ' 7 H2O) ; 5. Transfer the solution to a 1 1 . volLmjetric flask and bring to 1 1. V7ith distilled demineralized v.'ater. Keep the solution well stoppered.

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8:: TC-199 Culture Media XN^ith Heparin To prepare a 100 ml batch of media, add the components listed in Table 8 to a sterile glass screw-cap bottle, aseptically and in sequence, Keep the final solution refrigerated. TABLE 8 INGREDIENTS FOR TC-199 CULTURE ^5£DIA WITH HEPARIN Component Quantity, ml 1. Medium 199 vjithout NaHC03 (lOx)^ 10 2. Sterile distilled demineralized water 90 3. Sodium bicarbonate solution, 7.570.47" 4. Aqueous isotonic sodium heparin solution, 10^ USP units/ml*^ 0^_2 ^Microbiological Associate, Inc., Bethosda, Maryland, blhe lOx TC-199 concentrate has phenol red. The NaHC03 brings the solution to pit 7.4 (bright orange color). CQrganon, Inc., VJest Orange, Nev; Jersey, Colchicine Solution for Mitotic Arrest Tlie preparation of the working solution which is 2 Hg colchicine/ ml of normal (1 percent) buffered saline is done in three steps: Preparation of Normal Buffered Saline To prepare 10 ml of sterile normal (1 percent) buffered saline solution, do the follov/ing in order: 1. Place 0.90 g of desiccator-dried sodium chloride (NaCl) in a glass screw-cap bottle marked for 100 ml; 2. Add approximately 60 ml of distilled demineralized water; ^^This is a modification of the procedure suggested by George Cartwright in Diag nostic Laboratory Hematolo gy, 4th edition, Grune and Stratton, New York" (1958).

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86 3. Add 0.137 g of anhj'drous sodium phosphate, dibasic (Na2HP0^) ; 4. Add 0.021 g of sodium phosphate, monobasic (NaH2P0• 1120); 5. Autoclave at 121.5 C, 15 psig for 15 min; 6. Add sterile distilled dcmineralized vjater to bring the volume to 100 ml, Step 5 must be done with sterile technique. Keep the solution refrigerated. The pH should be 7.4. Preparation of Colchicine Stock Solution To prepare 10 ml of normal saliiio solution V7hich has a colchicine concentration of 1 mg/ml, transfer 10 ml of the normal buffered saline solution to a sterile screw-cap bottle and add 10 mg of USP colchicine (^22^25^^0(3), using sterile technique. Keep the solution refrigerated. Preparation of C olchicine Working Solution To prepare 50 ml of normial saline solution which has a colchicine concentration of 2 ng/ml, transfer 50 ml of tlie normal buffered saline to a sterile screw-cap bottle and add 0.1 ml of the colcliicine stock solution, using sterile teclmique. Keep the solution refrigerated. Hypotonic KCl Solution for Cell Sv^elling To prepare a 400 ml batch of 0.075 M potassium chloride (KCl) solution with 16 USP units heparin/ml, do the following in order: 1. Add 2.24 g KCl (reagent grade) to approxiniatel}' 200 ml of distilled demineralized water in a screw-cap bottle marked for 400 ml; 2. Autoclave at 121. 5°C, 15 psig for 15 min; Calbiochem, Los Angeles, California.

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87 3„ Add sterile, distilled demineralized vjater to bring the volume to 400 ml; 4. Add 0.64 ml of 10 USP units/ml istonic aqueous heparin solution and mix. " Steps 3 and 4 must be done with sterile technique. Keep the solution refrigerated . l^organon, Inc., West Orange, Ncx7 Jersey.

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APPENDIX 2 ALKALINE KI METHOD FOR OZONE DETERI-IINATION

PAGE 102

89 The following method for ozone determination was taken from Selected Methods for the Measurement of Air Pollutants , a 1965 U. S. Public Health Service publication (185) . Introdu ction This method is intended for the manual determination of oxidants (including ozone) in the range of a few parts per hundred million (pphm) to about 20 ppm. Ozone, chlorine, hydrogen peroxide, organic peroxides, and various other oxidants will liberate iodine by this method. The response to nitrogen dioxide is limited to 10 percent b}' the use of sulfamic acid in the procedure to destroy nitrite. It is customary for convenience to express the results as ozone. The advantage of tliis procedure over the neutral iodide procedures is that a delay is permissible between sampling and completion of the analysis. Sampling is conducted in midget impingers containing 1 percent potassium iodide in 1 N sodium hydroxide. A stable product is formed that can be stored with little loss for several days. The analysis is completed in a laboratory by addition of phosphoricsulfamic acid reagent, v^hich liberates the iodine, The yellow iodine color is read in an appropriate instrument. Serious interfering effects occur from reducing gases and dusts. Reagents All reagents are made from analytical-grade chemicals. Tliey are stable for several months in wellstoppered bottles. Double-Distille d Water, Use d for All Reag ents Redistill distilled water in an all-glass still after adding a crystal each of potassium permanganate and barium hydroxide.

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90 Absorbing Reagent Dissolve 40.0 g of sodium hydroxide in almost a liter of vjater, then dissolve 10.0 g of potassium iodide and make the mixture to 1 1 . Store in a glass bottle with a screw-cap (with inert liner) or rubber stopper (previously boiled for 30 min in alkali and \mshed) . Age for at least 1 day before using. Acidifying Reagent Five g of sulfamic acid is dissolved in 100 ml of vzater, then 84 ml of 85 percent phosphoric acid is added, and the mixture is made to 200 ml. Standard Potassium lodate Solution Dissolve 0.758 g of potassium iodate in vjater and dilute to 1 1 . One ml of this stock solution is equivalent to 400y.U of ozone. Prepare a dilute standard solution just before it is required by pipetting exactly 5 ml of stock solution into a 50 ml volumetric flask and making to mark v.-ith water. Ap paratus Absorber All-glass midget impingers with a graduation mark at 10 ml are used. (Other bubblers vjith nozzle or open-end inlet tubes may be used. Fritted bubblers tend to give relatively low results.) Impingers must be kept scrupulously clean and dust free. All traces of grease should be removed by treatment with dichromate-concentrated sulfuric acid solution (cleaning solution) followed by tap and distilled vjater. Air-Metering Device A glass rotameter capable of measuring a flov; of 1-2 l./min v.'itli an accuracy of + 2 percent is recommended.

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91 Air Pump An appropriate suction pujiip capable of drav/ing the required sample flov: for intervals of up to 30 min is suitable. It is desirable to have a trap on the inlet to protect the needle valve and pump against accidental flooding with -absorbing reagent and consequent corrosion. Spectrophotometer A laboratory instrument suitable for measuring the yellow color at 352 mn with stoppered tubes or cuvettes (suitable for ultraviolet use) is recommended. Analytical Procedures Collec tion of Samples Assemble a train composed of a midget impinger, rotameter , and pump. Use ground-glass connections up-stream from the impinger. Buttto-butt glass connections with slightly greased tj'gon tubing may also be used for connections without losses if lengths are kept minimal, Pipet exactly 10 ml of the absorbing solution into the midget im.pinger and sample at a flov; rate of 1-2 l./min. Note the total volume of the air sample. If the sample air temperature and pressure deviate greatly from 25°C and 760 mm hg, m.easure and record these values. Sufficient air should be sampled so that the equivalent of 1-15 JA 1 of ozone is absorbed. If appreciable evaporation has occurred, add distilled water to restore the volume to the 10 ml graduation mark. If the analysis is to be completed later, transfer the solution, v.dthout rinsing, to a clean, dry, glassstoppered, 25 ml, graduated cylinder (or a bottle) for storage. Prolonged storage may cause "freezing" of glass stoppers.

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92 Analysis Measure the volume of exposed absorbing reagent in a 25 ml, glass-stoppered, graduated cylinder. (Do not use rinse water in any transfers.) Add from a rapid (serologicaltype) , graduated pipette exactly one-fifth of this volume of the acidifying reagent and mix thoroughly. Place the stoppered cylinder in a water bath at room tem.perature for 5-10 min to dissipate the heat of neutralization. Transfer a portion of the sample to a cuvette and determine the absorbance at 352 my«.with a cuvette containing distilled water as the reference. D3 not delay the 'reading, since reducing impurities sometimes cause rapid fading of the color. Prepare a reagent blank by adding 2 ml of the acidifying reagent to 10 ml of unexposed absorbing reagent. Cool and determine the blra.k absorbance. Ihis blank absorbance should be determined each day and should be subtracted from the absorbances of the samples. Samples may be aliquoted either before or after acidification if very large concentrations of oxidant are expected. In the former case, dilute the aliquot to 10 ml with unexposed absorbing reagent and proceed in the usual manner. In the latter case, dilute the aliquot to volume with reagent blank mixture. Aliquoting after acidification is not as reliable and should be used only to save a sample when unexpectedly large concentrations of oxidant are encountered. The calculations should include the aliquoting factor. Standardization Add the freshly prepared, dilute, standard iodate solution in graduated am.ounts of 0.10-0.45 ml (measured accurately in a graduated pipet or small buret) to a series of 25 ml, glass-stoppered, graduated

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93 cylinders. Add sufficient alkaline potassium iodide solution to make the total volume of each exactly 10 ml. Acidify and determine the absorbancc of each standard as v;ith the samples. Calculations For convenience, standard conditions are taken as 760 mm of mercury and 25°C; thus, usually only slight correction by means of the well-known perfect gas equation is required to get V, defined as the standard volume of the air sampled in liters. Ordinarily this correction may be omitted. Quantities, customarily expressed in terms of ozone, may be expressed as microliters, defined as V x ppm ozone. It has been determined empirically that 1 mole of ozone liberates 0.65 mole of iodine (I2) by this procedure. Tlie strength of the stock standard potassium iodate solution is computed on the basis of I2 ^^Cr^ ^/-^ ^•'^3' and the follov.'ing: Standard molar volume (25°C, 760 mm) = 24.47 liters; molecular weight KIO^ = 214.02; 1 ml stock solution = 400 UX (0^ = 400 X 10-^ ^. ^65 ^ 214.02 0.758 x lO"^ g KIO^ ^^3^ 24.47 3 3 Plot the absorbances of the standards (corrected for the blank) against milliliters of dilute standard iodate solution. Beer's Law is follov^'ed. Draw the straight line giving the best fit and determine the value in milliliters of the diluted potassium iodate solution intercepted at an absorbance at exactly 1. This value multiplied by 40 gives the standardization factor M, defined as the number of microliters of ozone required by 10 ml of absorbing reagent to give a final absorbance of 1. For 2 cm cells this value is 9.13. llien: M ppm oxidant (expressed as ) = corrected absorbance x —

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9k If the volume ol t'lie air sample, V, is a simple multiple of M, calculations arc simplified. Thus, for the M value of 9.13 previously cited, if exactly 9.13 1. of air is sampled through the impinger, the corrected absorbance is also ppm directly. If other volumes of absorbing reagent are used, V is taken as the volume of air sample per 10 ml of absorbing reagent. Discussion of Procedure Sampling Efficiency Wien tv;o impingers are placed in series, iodine V7ill very rarely be liberated from the solution in tlie second absorber. Thus, the sampling efficiency is very high. Fritted bubblers, V7hich_ also appear to have equally high sam.pling efficicnces, usually give, hov.'ever , less iodine for a giveii amount of ozone and should not, therefore, be used. (This is due to the complex chemistry of ozone in alkaline iodide solution. It appears that hypoiodite is the primary product, but that some is lost through side reactions, with resulting variation in the stoichiometry . Potassium iodate is a convenient chemical to use for standardization, although iodate is probably not produced by adsorption of ozone.) Stability of Ex p osed Absorbing Reagent Studies have indicated that most of the losses in exposed reagent occur in the first day. The reagent may then be stored for as long as a week or more with little further change. Use of analytical-grade reagents and of carefully cleaned glassv.'are reduces losses.

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95 Acidification Step Certain irreversible losses of microquantities of iodine occur during the acidification of alkaline iodide solution. This is probably the explanation for the relationship of 0.65 mole of iodine liberated for each mole of ozone absorbed. Very slov; acidification v?ill yield less iodine. The most reproducible procedure is to add the acid rapidly vith vigorous stirring. Interferences The negative interferences from, reducing gases such as sulfur dioxide and hydrogen sulfide are very serious (probably on a mole-tomole equivalency) . The procedure is very sensitive to reducing dusts that may be present in the air or on the glassv.'are. Losses of iodine also occur even on clean glass surfaces, and thus the manipulations should minimize this exposure.

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APPENDIX 3 PR0CEDUPJ3 FOR STAINING SLIDES

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97 1 o The following procedure vjas employed for staining the Chinese hamster Ij'mphocytc culture slides with Gicmsa blood stain: 1. Prepare the stain solution by mixing 3 parts concentrated Giemsa solution v/ith 5 parts distilled water; mix thoroughly; 2. Place the slide into the liquids listed in Table 9 , in sequence, for the times indicated. TABLE 9 SLIDE STAINING LIQUIDS AND FvEQUIRED TIMES Liquid 1. Stain solution 2. Distilled water 3. Distilled water 4. Acetone 5. Toluene 6 . Xy 1 ene Time Required 20 min quick rinse quick rinse 15 sec v;ith agitation 3 min 3 min 3. Miile the slides are still wetted with xylene, cover them 19 with glass slips fastened with Permount. « niis procedure was used for staining human IjTnphocyte culture slides in the laboratory of Dr. Gerald Bloom, Department of Pediatrics, University of Florida, Gainesville, 19 Fisher Scientific Company, Springfield, New Jersey.

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LIST OF REl^EKENCES 1. Biget, P. L., Pfister, A., Proute, B.^ and Rio, M. , "A New Technic for Studjring the Toxic Action of Ozone in Animals^ " Re -me II(S d. aeronaut, et spat 3 ale , ^(20): 7-11 (1966) (in FrenchJ^ 2. Buell, George C, Tokiva, Y., and M-ueller, Peter K., "Potential Crosslinlcing Agents in Lung Tissue Forn'iation and Isolation After In Vivo S:-rposure to Ozone," Archs envir. Hlth , 10(2): 213-219 3. Clamann, II, G. and Bancroft, R. W,, "Toxicity of Ozone in HighAltitude Fliglit, " Adv. Chem. Ser ., 21: 3523>9 (1959). k. Coffin, D. L., Gardner, D, E., Holzman, R, S., and ^''olcock, F. J., "Influence of Ozone on Pulinonarv Cells," Areas envir. Hlth , 16(5): 633-636 (1968). " 5. Colinet-I^gneau-X, T). and Troquet, J., "Influence of Eserine and Atropine on Pulmonary Edeiiia in the Rat caused by Ozone, " C^ r. Stjanc. Soc. Biol ., 159(2): 506-508 (I965) (in French). 6. Colinet-I^igneaiAj:, D. , Troquet, J., Den:oulin, J. C, and Deheneffe, J., "Effect of Monoamine Oxidase Inhibitors on Acute Rilmonary Ederaa Caused by Ozone Inlialation, " C. r. Sganc. Soc. Biol . , 160(11): 223.1-2213 (1966) (In FrencTrT; 7. Dalhamr., T. and Gerhardsson, G., "Oj^one. Its Toxicity and its Significance as an Occi\pational Hazard," Il ord. hyg. Tidskr ., 39(9-10): IU5-I6U (1958) (In Swedish). 8. Deheneffe, J., Ceir-oulin, J. C, Colinet-La.gneaux, D, , and Troquet, J., "Cardiac Glycosides and Acute Pirlrxnai-y Edema Following Exposure to Ozone in the Rat," C. r. Sdanc. Soc. Biol ., 159(ll): 2279-2231 (1965) (In French). 9. Dixon, J. R. and L^ountain, J. T., "Role of Histamine and Related Substances in Developraent of Tolerance to Edeinagen.ic Gases, " Toxic, appl. Pharinac , 7: 756-766 (I965). 10. Easton, Richard E. and I-Iu.rphi'-, Sheldon D., "Experimental Ozone preexposure and Histamine: Effect on the Aciite Toxicity and Respiratory Function Effects of Histai.iine in Guinea Pigs, " Archs envir. Hlth, 15(2): I6O-I66 (I967). 98

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99 11. Ehrlicher, H. , "Poisoning by Irritant Gases," Zentbl . Arb Med. Arb Schutz , 14(11): 260-265 (1964) (In German). 12. Faircbild, E. J. and Stokinger, H. E., "The Thyroid in Pulmonary Injury of Acute Ozone Poisoning," Fedn Proc. Fedn Am. Socs exp. Biol. , 20(1 pt. 1): 203 (1961). 13. Fair child, E. J., II, "Neurohumoral Factors in Injury from Inhaled Irritants," Archs envir , Hlth , 6i(l): 79-86 (1963). 14. Fairchild, Edward J., II and Graham, Stuart, L. , "Thyroid Influence on the Toxicity of Respiratory Irritant Gases, Ozone and Nitrogen Dioxide," J. Pharmac. exp. Ther. , _139(2): 177-184 (1963). 15. Fairchild, E. J. and Bobb, G. A., "The Effect of Spinal Cord Lesions on the Pathophysiology of a Lung Irritant Ozone," Toxic appl Pharmac. 7(3) : 483 (1965). 16. Fairchild, E. J., II, "Tolerance Mechanisms. Determination of Lung Response to Injurious Agents," Archs envir. Hlth , 14 : 111-126 (1967). 17. Gross, P., Sclieel, L. D., and Stokinger, H. E., "Ozone Toxicity Studies; Destruction of Alveolar Septa: A Precursor of Emphysema," Prog. Res. Emphys. chron. Bronch. , 7_: 376-381 (1965). 18. Hamming, W. J., Mac Beth, V.'illiam G. , and Chass, Robert L. , "The Photochemical Air Pollution Syndrome," Archs envir. Hlth , 14 : 137-149 (1967). 19. llenschler, D., "Protective Effect of Pretreatment with Low Concentration of Gas Against Lethal Lung Edema by Irritating Gas," Arch, exp. Path. Pharmak. , 238: 66--67 (1960) (In German). 20. King, M. E., "Toxicity of Ozone. V. Factors Affecting Acute Toxicity," Ind. Med. Surg. , 32(3): 93-94 (1963). 21. Matzen, Richard N., "Effect of Vitamin C and Hydrocortisone on the Pulmonary Edema Produced by Ozone in Mice," J . appl . Physiol . , 11 : 105-109 (1957). 22. Matzen, R. N., "Development of Tolerance to Ozone in Reference to Pulmonary Edema," Am. J. Physiol. , 190(1): 84-88 (1957). 23. Matzen, Richard N., "Effects of Serotonin on Pulmonary Edema Produced by Ozone in Mice," Guthrie Clin. Bull, , 29 : 102-106 (1959). 24. Mendenhall, R. M. and Stokinger, H. E., "Tolerance and Crosstolerance Development to Atmospheric Pollutants Ketene and Ozone," J. appl. Physiol. , 14(6): 923-926 (1959).

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100 25. Mendenhall^ Richard M. and Stoliinger, Herbert E., "Films from Lung Washings as a Mechanism Model for Lung Injury by Ozone^ " J. app l. Physiol ., 17(1): 28-32 (I962). 26. Miller, Sol and Ehrlich, Richard, S usceptibil ity to Resp iratory Infection of Anims-ls Ex posed to Ozone , U. ~S, A. F. School of Aviation Medicine Report IJo. AF-SP^A-'^QSh (I958), 27. Mittler, Sidney, "Silicone Aerosols and Alcohol Vapor in Ozone Poisoning," Ind. Med. Surg. , 27: i|-3-'^5 (1958). 28. Murphy, Sheldon D. , Ulrich, Charles E. , Frankowitz, Stanley'K. , and Xintaras, Charles, "Altered Pu.nction in Anin-als Inhaling Ijov Concentration of Ozone and Kitrogen Dioxid.e, " Am. ind. Hyg. Ass . J. , 25: 2H6-253 (190^0. 29. Kasr, Ahmed H, M,, "Biochemical Aspects of Ozone Intoxication," J. occup. Med. 9: 589-597 (1967). 30. Purvis, M, R., Miller S., and Ehrlich R., "Effect of Atmospheric Pollutants on Susceptibility to Respiratory Infection. I. Effect of Ozone," J. infect. Pi s. ,^ 109(3) :" 238-2l'r2 (I961), 31. Quilligan, J. J., Boche, Robert D., Fall-;, Hans L,, and Kotin, Paul, "The Toxicity of Ozone for Yo^ong Chicks, " Archs ind. Hlth , 18: 16-22 (1958). 32. Skillen, Ralph G., Txiienes, Clinton H., Cangelosi, Joseph, and Strain, Lorrene, "Lung-5-Hydroxj-tr5''ptamine and Ozone Induced Pulmonary Eder'a in Rats," Proc. Soc. exp. Biol. Med ., 107(1 ): 178-180 (1961). 33So>olic, P., Padovan, I., and Knezevic, M. , "Eyrperimental Study of the Value of Ozone and of Oxygen in Inhalation Treatment of the Respiratory System," Archo ital. Otol. Rinol. Lar ., 7^(3): 311319 (190I) (In Italianji^ 3^r. Stokinger, Herbert E., Wagner, William D,, and Wright, PaulG., "Studies of Ozone Toxicity. 1, Potentiating Effects of Exercise and Tolerance Development," Archs ind. Hlth , 1^: I58-I62 (I956). 35. Stokinger, H. E. , Wagner, W. D. , and Dobrogorski, C. J., "Ozone Toxicity Studies. III. Chronic Injury to Lungs of Animals Folloving Exposure at a Low Ijevel, " Arclis ind. Hlth, l6(6): 51^^-522 (1957). 36. Stokinger, Herbert E. and Scheel, Lester D., "Ozone Toxicity. Imr.iunochemical and Tolerance-Producing Aspects, " Archs envir . Hlth, IK 3): 3??7-33'!(1962).

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101 37. Stokinger, Herbert E., "Factors Modifying Toxicity of Ozone," Adv. Chem. Ser. , n-. 360-369 (1959). 38. Svirbely, J. L. and Saltzman, B. E., "Ozone Toxicity and Substances Associated with Its Production," Archs ind. Hlth , 15(2): 111-118 (1957). 39. Thienes, C. H. , Skillen, R. G., Hoyt , A., and Bogen, E., "Effects of Ozone on Experimental Tuberculosis and on Natural Pulmonary Infection in Mice," Am. ind. Hyg. Ass. J . , 2_6(3): 255-260 (1965). 40. Troquet , J. and T,ccomte, J., "Role of Endogenous Histamine in Pulmonary Edema Due to Ozone," Acta belg. Arte med. Pharm. milit. , n2(4): 563-569 (1959) (In French) . 41. Troquet, J., Delieneffe, J.^ and Demoulin, J. C, "Modification by Certain Antiliistamines of Pulmonary Edema Provoked by Ozone," C. r, Seanc. Soc. Biol. , 158(12): 2<'i94-2495 (1964) (In Frcncli) . 42. Troquet, J. and Cession-Fossion, A., "Pulmonary Edema Induced by Ozone Inhalation and Effect of Prior Administration of Reserpine," C. r. S(^anc. Soc. Bio l., 158(2): 39S-AOO (1964) (In French) . 43. Troquet, J. and Colinet-Lagneaux, D., "Superficial Respiration and Pulmonary Edema Provoked in tlie Rabbit by Inhalation of Ozone," Archs int. Pharmacodyn. Ther. , 157 : 228-230 (1965) (In French). 44. Young, W, A., Shaw, D. B., and Bates, D. V., "Pulmonary Function in Welders Exposed to Ozone," Archs envir . Hlth , 2(3): 337-340 (1963). 45. Young, \-l. A., Shaw, D. B., and Bates, D. V., "Effect of Lo\^ Concentration of Ozone on Pulmonary Function in Man," J. appl. Physiol. , 19(4): 765-768 (1964). 46. Zicot, M., Troquet, J., Boland, J., and Colinet-Lagneaux, D., "Effect of Artificial Ventilation by Ozone of the Upper Air Passages on the Cardiorespiratory Functions of the Rabbit," C. r. Seanc. Soc. Bio l., 161(1): 200-202 (1967) (In French). 47. Beard, Raimon L., "Observation on House Flics in High-Ozone Environments," Ann, ent . Soc. Am. , 58(3): 404-405 (1965). 48. Bobb , GustavuR A. and Fairchild, Edward J., "Neutropliil-toLjmiphocyte Ratio as Indicator of Ozone Exposure," Toxic, appl. Pharm ac, 11(3): 558-564 (1967). 49. Claraann, H. G. and Bancroft, R. W., "Pliysiological Effects of Ozone," Fedn Proc. Fedn Am. Socs exp . Biol. , 16(1, pt . 1): 22 (1957).

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102 50. Coffin, D. L. and Blomnie'r, E. J., "The Influence of Cold on Mortality from Streptococci Following Ozone Exposure," J . Air Pollut. Control Ass. , _15(11): 523-524 (1965). 51. Coffin, D. L. and Blommer, E. J., "Enhancement of Mortality in Mice From Streptococcal Pneumonia by Irradiated Auto Exhaust," Archs cnvir. Hlth , U: 36-38 (1967). 52. Eglite, M„ E., "Hygienic Assessment of Atmospheric Ozone," Gig. Sanit. , 33.(1)^ 17-22 (1968) (In Russian). 53. Fairchild, Edvjard J., II, Graham, Stuart L. , Hite, Mark, Killens, Richard, and Scheel, Lester D. , "Changes in Thyroid 1-131 Activity in Ozone-Tolerant and Ozone-Susceptible Rats," Toxic, appl. Pharmac . . 5: 607-613 (1964). 54. Freebairn, Hugh T., "The Toxicity of Ozone, a Constituent of Smog,' J. appl. Nutr. , j_2: 2-13 (1959). 55. Gilgcn, A. and Wanner, H. V., "The Toxicological and Hygienic Importance of Ozone," Arch. Hyg. Bakt . , _152(1"2) : 62-78 (1966) (In German) . 56. Goldstein, Bernard D. and Balchum, Oscar J., "Effect of Ozone on Lipid Peroxidation in the Ref Blood Cell," Proc. Soc. exp. Biol. Med. , 126 : 356-358 (1967). 57. Goldstein, Bernard D., Pearson, Brion, Lodi , Charlotte, Buckley, Ramon D., and Balchum, Oscar J., "The Effect of Ozone on Mouse Blood In Vivo ," Archs envir. Hlth , _16(5): 648-650 (1968). 58. Gregory, Robert Arthur, "The Effect of Neonatal Thymectomy on the Development of Ozone Tolerance in Mice," Am. ind. Hyg. Ass. J. , 28: 278-282 (1967). 59. Hathaway, J. A. and Terrill, R. E., "Metabolic Effects of Chronic Ozone Exposure on Rats," Am. ind. Hyg. Ass. Q. , 23(5): 392-395 (1962). 60. Horc, T. and Gibson, D. E., "Ozone Exposure and Intelligence Tests," Archs envir. Hlth , 17: 77-79 (1968). 61. Joubert , J., Fontanges , R. . Guerne, J. M. , Colobert , L. , Eyraud, Ch,, "Investigations on the Deposition of Microorganisms in Ionizing Fields," Staub, 2^: 295-299 (1965) (In German). 62. Lutmer, R. F., Busch, K. A., and De Long, P. L,, "Effect of Nitric Oxide, Nitrogen Dioxide or Ozone on Blood Carboxyhemoglobin Concentration During Low-Level Carbon Monoxide Exposures," Atmosph. Envir, , _1(1): 45-48 (1967).

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103 63. Mittler, Sidney, King, Maurice, and Burkhardt , Betty, "Toxicity of Ozone," Adv. Chera. Ser. , 2j^: 344-351 (1959). 64. Murpliy, Sheldon D., "A Review of Effects on Animals of Exposure to Auto Exhaust and Some of Its Components," J. Air Pollut. Control Ass. , iA(8): 303--308 (1964). 65. Murphy, S. D., Davis, H. V., and Zaratzian, V. L. , "Biochemical Effects in Ra.ts from Irritating Air Contaminants," Toxic, appl . Pharmac . , 6(5): 520-528 (1964). 66. Nevskaya, A. 1. and KocheLkova, T. A., "Toxicology of Ozone and Aerosol of Sulfuric Acid in Their Combined Action," Gig. Truda prof. Zabol. , 5(2): 20-29 (1961) (In Russian). 67. Ordin, L., "Effects of Air Pollutants on Cell Wall Metabolism," Arclis cnvir. Hlth , U^: 189-194 (1965). 68. Pribluda, Samuel, "Subcutaneous Injections of Ozone in Lumbago, Sciatica, and Similar Syndromes," Semana mod., B. Aires , ] 2 3 ( 2 6 ) : 1026-1028 (1963) (In Spanish). 69. Scheel, Lester D. , Dobrogorski, Olga J., Mountain, John T., Svirbcly, Joseph L., and Stokinger, Herbert E., "Physiologic, Biochemical, Immunologic and Pathologic Changes Follov.'ing Ozone Exposure," J. appl. Phy siol ., 14(1) : 67-80 (1959). 70. Skillen, Ralpli C, Thienes, Clinton H., Cangelosi, Joseph, and Strain, Lorrene, "Brain 5-nydroxytryptamiue in Ozone-Exposed Rats," Proc. Soc. cxp. Biol. Med. , KiS(l): 121-122 (1961). 71. Stokinger, H. E., "Toxicologic Interactions of Mixtures of Air Pollutants," Int. J. Air Wat. Pollut ., 2: 313 (1960). 72. Stokinger, Herbert E., "New Concepts and Future Trends in Toxicology," Am. ind. Hyg. Ass. J. , 23: 8-19 (1962). 73. Stokinger, H. E., "Pollutant Gases," Respiration , 7_: 1067-1086 (1965). 74. Troquet, J. and Dresses, A., "Liberation of Adrenal Catechol Amines during Intoxication of the Rat by Ozone," C. r. Seanc. Soc. Biol. , 155 : 1607-1609 (1961) (In French). 75. Troquet, J. and Cession-Fossion, A., "Inhibition of Ozone Poisoning in the Rat by Sodium Thiosulf ate , " C. r. Seanc. Soc. Biol. , 158(12) ; 2492-2494 (1964) (In French). 76. Xintaras, C, Johnson, B. L., Ulriich, C. E., Terril, R. E., and Sobecki, M. F., "Application of the Evoked Response Technique in Air-Pollution Toxicology," Toxic, appl. Pharmac , 8(1): 77-87 (1966).

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10^ 77. MacEwen, James D. and McNerney, James M. , "Effect of a Mixed Cas Atmosphere, at 5 PSIA on the Toxicity of N0„ and 0., in Animals," in Proceedings of the 2nd Annual Conference on Atmospheric Contamination in Confin ed Sp aces, 4 and 5 Ma^^ 1966 , U . S . A , F . Aerospace Medical Research Laboratories Report No. AMRL-TR-66-120 (1966). 78. MacEu'en, J. D. and Gcckler, R. P., Comparative Studies of 90-Day Continuous Exposure to ^, NO^ , and CCl, at Re d uced and Ambient Pressures , U. S. Ac F. Aerospace Medical Research Laboratories Report No. AMRL-TR-67-5S (196S). 79. Stokinger, H. E., "Evaluation of the Hazards of Ozone and Oxides of Nitrogen," Archs ind. Hlth , ]5(3): 181-190 (1957). 80. Svirbcly, J. L. , "Enhanced Toxicity of Ozone-Hydrogen Peroxide Mixtures," Am. ind. Hy g . Ass. J. , 22: 21-26 (1961). 81. V.'agner, V'illlam D., Dobrogorski, Olga J,, and Stokinger, Herbert E., "Antagonistic Action of Oil Mists on Air Pollutants, Effects on Oxidants, Ozone, and Nitrogen Dioxide," Archs envir. Hlth , 2^: 523-534 (1961). 82. Clamann, Hans C, "Physical and Medical Aspects of Ozone," in Proceedings of the 2nd International S>nnposium on the Physics and M edicine of the Atm^osphore and Space, San Antonio, Texas, 1958 , John Wiley and Sons, Inc., Nev7 Yoi-k (1960). 83. Jaffc, Louis So, "The Biological Effects of Photochemical Air Pollutants on Man and Animals," Am. J. publ . Hlth, 57(8) : 1269-1277 (1967). 84. Jaffe, Louis S., "The Biological Effects of Ozone on Man and Animals," Am. ind. Hyg, Ass. J. , 28(3): 267-277 (1967). 85. Stokinger, Herbert Eo, "Ozone Toxicity A Reviev.' of the Literature Through 1953," Archs ind . Hyg., 9: 366-383 (1954). 86 o Report o f the United Nations Scientific Committee on the Eff ects of Atomic Radiat ion, Official Records of the General Assembly, Seventeenth Session, Supplement No. 16 (A/5216), United Nations, Nev7 York (1962) , 8 7 . Repor t of the United Nations Scientific Committee on. the Effects of Atom i c Radiation , Official Records of the General Assembly, Nineteenth Session, Supplement No. 14 (A/5814), United Nations, New York (1964) . 88. Ingram, Mary Lou, compiler, B i.ological Effects of Ionizing Radiat i on An A n notated Bibliography , U. S. Atomic Energy Commission Division of Technical Information, Report No. TID-3098 (1963).

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. 105 89. Pierce, Charlie M. , compiler, The Effects of Radiation and Radioisotopes on the Life Processes An Annotated Bibl1op;raphy , U , S . Atonic Energy Commission Division of Technical Inforr.iation, Report No. Tir)-3093 (I963). 90. Hodgman, Charles D. , editor-in-chief, Handbook of Chemis try and Physics , Ulst edition, Cheiaical Rubber Publishing Co., Cleveland, Ohio (1959). 91. Stokinger, H. E., "Ozone Toxicology: A Review of Research and Industrial Experience: 195^1-196^)-," Archs envir. Hlth , 10(5): 719-731 (1955). 92. Vfeiss, J., "Radiocheniistry in Aqueous Solutions," Nature, Lond ., 153: T'j£-T50 (19^^). 93. LeS; '^' E., Action of Radiation on Living Cells, 2nd edition, Cambridge University Press, London (1956). 9^1-. Bacq, Z. M. and Alexander, Peter, Fi^n damentals of Radiobiology, 2nd edition, Perganion Press, London (I96I). 95. Brihkinan, R. and Lar.iberts, H. B., "Ozone as a Possible Radiommetic Gas," Kature, Lond ., I8I: 3.202-1203 (195S). 96. Fairchild, E. J., Murphy, Sheldon D., and Stokinger, K. E,, "Protection by S-.ilphur Compounds Against Air Pollutants Ozone and Nitrogen Dioxide," Science, IT. Y ., I3O: 86I-B62 (1959). 97. Lagerireif, J. M. , "Prolonged Ozone Inhalation and Its Effects on Visual Parameters," Aerospace i:ed ., 3^(6): liT9-'-B6 (I963). 98. Brirt'vman, B., Lamberts, H. B., and Veninga, T, S., "Radioniinetlc Toxicity of Ozonized Air, " Lancet , 1: I33-I36 (l9o'099. Brinliman, R,, Lsniberts, }I. B., and Veninga, T. S., "A Fev Aspects of Early Radiation Pathology as Guide to Chemcal Protection and Sensitization," P rog, biochem. Pharma-c , 1: 203--203 (19^5)100, Brinliman, R. and Lainberts, H. B. , "Radiopathologjof Extracellular Structures," in Ci.irrent To pics in Radiation Research , Vol. 11 , Foert, Michael and Howard, Alnia, editors, Horth-Holland Publishing Germany, Amsterdam (I966). 101. Brinkrcan, R. and Veninga, T. S,, "Influence of Ozonized Air in Radiosensitivity, " in Progress in Radiology , Vol. 11, Turano, L, , Eatti, A., Biagini, C., editors, Excerpta Medica Foundation, New York (196Y).

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io6 102. Hattori, Keiko, Kato, Norio, Kinoshita, Masahiro, Kinoshita, Syosaku, and Sunada, Tsuj'oshi, "Protective Effect of Ozone in Mice Against Whole-Body X-Irradiation ," Nature, Lon d , 198 : 1220 (1963). 103. Veninga, T. S., "Toxicity of Ozone in Comparison with Ionizing Radiation," Strahlenthe rapic , 134(3): 469-477 (1967). 104. Sanotskii, I. V., Ulanova, I. P., and Shumskaya, N. I., "The Combined Effect of Ozone and Ionizing Radiation," Toksikal. Novykh Prom. Khim. Veshchestv , 6: 116-128 (1964) (In Russian), 105. Sunada, Tsuyoshi , Hattori, Keiko, Kato, Norio, Kinoshita, Masahiro, and Kinoshita, Shosaku, "Development of Tolerance to X-Radiation by Ozone Inhalation in Mice," Jap. J. Hyg . , 18: 268-272 (1963). (In Japanese) . 106. Betz, H., "The Importance of the Resistance Stage of an Organism at the Time a Lethal Dose of X-Radiatjon is Received," C. r. Seanc. Soc. Biol. , 144 : 1439-1442 (1950) (In French). 107. Cronkite, Eugene P., Sipe, Clyde R. , Eltzholtz, Dean C, Chapman, William H. , and Chambers, F. W. , Jr., "Increased Tolerance of Mice to Lethal X-Radiation as a Result of Sublethal Exposures," Proc. Soc. cxp. Biol. Med. , 73: 184-186 (1950). 108. Raper, John R., "Effects of Total Surface Beta Irradiation," Radiology , 4^: 314-324 (1947). 109. Dmitriev, M, T. , "Effect of Ionizing Radiation on the Composition of tlie Atm.osphcre of Industrial Premises," Gig. Sanit . , 30(4) 39-44 (1965) (In Russian). 110. Dmitriev, M. T. , Yurasova, 0. I., Koroleva, No A., "Extent of Ozone Decomposition in Radiation Chambers of Gamma-Facilities," Gig. Sanit. , 33(^): 87-88 (1968) (In Russian). 111. Harteck, P., Dondes , S., and Thompson, B. , "Ozone: Decomposition by Ionizing Radiation," Science, N. Y. , 147 : 393-394 (1965). 112. Johnson, G„ R. Ac and Warman, J. M. , "Formation of Ozone from Oxygen by Action of Ionizing Radiation," Discuss. Faraday Soc , 37.: 87-95 (1964). 113. Lampe, F. W. , Weiner, E. R., and Johnston, W. H., "0Formation in Gaseous Oxygen Radiolysis," Int. J. appl. Radiat . Isotopes , J^(6) : 363-371 (1964). 114. Kircher, John F. and McFiulty, Justin S., The Effects of Radiation on Oxygen Designed for Human ConsLmiption , U. S. A. F. Wright Air Development Center Technical Report No. WADC-TR-618 (1960).

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lOT 115. Sears, John T. and Siitherland, Jarnes V/. , "Radiolytic Formation and Decomposition of Ozone," J. phys. Chem. , Ithaca , 'J2{h): 1166-llTl (1968). 116. Shah, J. and Maxie, E, C, "Gamina Ray Radiosynthesis of Ozone frora ^^^)" Int. J. appl. Radiat. Isotopes , 1?: 155-159 (I966). 117. Less, L. N. a?id Svallov, A. J., "Estimating the Hazard Due to Radiolytic Products from the Air," m-.cleonics , 22(9): 58-6I (196'0' 118. ' Kertesz, z. I. and Parsons, Grace F. , "Ozone Formation in Air Exposed to Cobalt~60 Gamma Radiation," Science, W. Y. , ite: I2891290 (1963). 119. Sobol, II. v., Petushljov, A. A., and Breger, A. Kh., "Principles of Air Circulation in Operational Areas of High-Levol Gamma Installations," Atomn. Energ . , I6: 262-26it(196^) (in Russian). 120. Adley, F, E., Uirson, H. V., Loomis, J. S,, Myers, I, T,, and V/isehart, D. E. , Ozone Hazards Associated vith the Operations of an Electron Van de Gra aif A ccelera tor , U, S. Atonic Energy Commissrori^.eport I:o. K'/-SA-2oS^ (1952). 121. George, Andreas C., Breslin, Ali'red J., Haskins, James W. , Jr., and Ryan, Rooert li. , "Evaluation of the Hazard From Radioactive Gas and Ozone at Linear Electron Accelerators, " in Health Physics Scoi ety Annual Keetirg, Los Angel es, 19*55.U. S. Atorfuc Energy Cosnrjission Report lio. COIi7-6505l5-2S~(1955) . ' 122. Loomis, J. S., Myers, I. T., Larson, H. V., and Wisehart, D. E., "Ozone Hazards Incurred by Electron Van de Graaf f Operations, " Kucl. Instinm. Meth ., 1^:" 2U3-2i^8 (1952). 123. Federoragt, G. W., Van der Korst, A., and Van Duijin, J., "Rapid Ozone Deterrrdnation Kear An Accelerator, " Kature, Lond. , 20S_: 87 (1965). 12-'i-. Gasteiger, Edgar L. and Helling, Sharon A., "X-Ray Detection by the Olfactory System: Ozone as a Mashing Odorant, " S cience, II. Y . , 15^: 1038-10'fl (1956). 125. Gasteiger, E. L. , "Olfactory X-Ray Detection," in Fission Product Metabolis m and Response in Laboratory and Domestic Anir.vals and Planning for E valu ation of Radioa ctive Cont amina tion of the Food ChaJnT^Oe yes'sl^cpo^^t Jan. 1, 19o7-Dec. 3I, 19o7 , Comar, C. L. and Staff, U. 3. AtomJ.c Energy Commission Report llo. ITYO-21'47-13 (1908). 126. Peterson, Dorothy C. and Andrews, Howard L. , "The Role of Ozone in Radiation Avoidance in the Mouse," Radiat. Res ., 19: 331-335 (1953).

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103 127. Taylor, Henry L. , "Factors Influencing the Immediate Response of Rhesus Monkeys to Brief X-Ray Exposures," in Eastern Psychological Meeting , V.'ashin gton, D. C. , 1968 , U. S. Atomic Energy Commission Report No. CONF-680433-1 (1968). 128. Am.erican Industrial Hygiene Association, "Hygienic Guide Series. Ozone (Revised, 1966)," Am. ind. Hy g. Ass. J. , 27: 196-198 (1966). 129. Summer, K. , Methods of Air Deodo r ization , Elsevier Publishing Company, Amsterdam/London/New York (1963). 130. Lunau, F. K. , "Ozone in Arc Welding," Ann, occup. Hyg., 10(3): 175-188 (1967). 131. Katz, Morris, "Some Aspects of the Physical and Chem.ical Nature of Air Pollution," in A ir Pollution , World Health Organization, Geneva (1961).' 132. Siegel, Jac, "Review of Ambient Pressure Anim.al Exposure Data from Selected Nav}' Compounds," in Proceedings of the Conference on Atmospheric Contamination in Confined Spaces, 30 March-1 April 1965 , U. S. A. F. Aerospace Medical Research Laboratories Report No. AI-IRL-TR-65-230 (1965). 133. Eaton, Harold G., Smith, Walter D., Um.stead, Merle, E. , and Johnson, J. Enoch, "Instrumentation Used for the Shipboard Analysis of Submarine Atmospheres," in Proceedings of the 3rd Annual Conference on Atmospheric Contamination in Confined Spaces, 9-11 May 1967, U. S. A. F. Aerospace Medical Research Laboratories Report No. AI^IRL-TR-6 7-200 (1967). 134. Ewing, Dean E. , "Space Radiation Hazard to Manned Space Flight," Res. Tech. Briefs (U. S. A. F.), 4(10): 1-9 (1966). 135. McKee, Herbert C. , Rhoades, John W. , Mieeler , Ralph J., and Burchfield, H. P., Gas Chromatographic Measurement of Trac e Contamination in a Simulated Space Cabin , U. S. National Aeronautics and Space Administration Technical Note No. TND-1825 (1963). 136. Lagerv7erff, John M. , "Space Cabin Atmosphere Trace Contaminants and Their Possible Influence on Visual Parameters," Hum. Factors, 4_: 285-293 (1963). 137. Pierson, V. R. , C rew Compartm.ent /Cabin Atmosphere Control Criteria (Supersonic Transport), Lockheed-California Company Report No. LR 19668 (1966). 138. Bennett, G., "Ozone Contamination of High Altitudes Aircraft Cabins," Aerospace Med. , 21(8): 969-973 (1962).

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109 139. Brabets, Robert I., Hersh, Charles K. , and Klein, Morton J., "Ozone Measureinent Survey in Coirimercial Jet Aircraft," J. Aircr . , 4.: 59-64 (1967). 140. Brabets, Robert I., Ozone Measurement Survey in Commercial Jet Aircraft , U. S. Federal Aviation Agency Report No. FAA ADS5 (1963). 141. Brabets, R. I., Hersh, C. K. , and Klein, M. J., Ozone Measurements in Current Jet Aircraft , American Institute of Aeronautics and Astronautics, Sum.mer Meeting Los Angeles, Calif., June 17-20, 1963, Paper No. 63-235 (1963). 142. Young, \v. A., Shav.', D. B., and Bates, D. V., "Presence of Ozone in Aircraft Flying at 35,000 Feet," Aerospace Med. , 33(3) : 311-318 (1962). 143. Biget, P. L., Fabre, J., and Vauzelle, A., "The Problem of Atmospheric Ozone and Its Significance in Aviation," Revue Med . Aeronaut. , 4(15): 66-76 (1965) (In French). 144. Jaffee, L. S. and Estes, H. D., "Ozone Toxicity Hazards in Cabins of High Altitude Aircraft A Review and Current Program," Aerospace Med. , 34: 633-643 (1963). 145. Langlo, K. , "Atmospheric Ozone and Supersonic Aircraft Operations," \mO Bull., 14: 106-108 (1965). 146. Pierson, William R. , Ioni zing Radiation and the Supersonic Transp ort, Lockheed-California Company Report No. LR 19965 (1966). 147. ICRP Task Group on the Biological Effects of High Energy Radiations, "Radiobiological Aspects of the Supersonic Transport," HJ th Phys . , 12: 209-226 (1966). 148. Evans, H. J., "Chrom.osome Aberrations Induced by Ionizing Radiation," Int. Rev. Cytol. , 13: 221-321 (1962). 149. Christensen, E. and Giese, A. C, "Changes in Absorption Spectra of Nucleic Acids and Their Derivatives Following Exposure to Ozone and Ultraviolet Radiation," Archs Biochem. Biophys . , 51: 208-216 (1954) 150. Prat, R., Nofre, CI., and Cier, A., "Effects of Sodium Hypochlorite , Ozone, and Ionizing Radiations on the Pyrimidine Constituents of Escherichia Coli ," Ann ls Inst. Pasteur , 114: 595-607 (1968) (In French) . 151. Davis, Irving, Microbiologic Studies with Ozone. Mutagenc:sis of Ozone for E s cherichia Coli , U. S. A. F. School of Aerospace Medicine Report No. SAM-TR-61-60 (1961):

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110 152. Sachsenmaier , Wilhelm, Siebs, Wilhelmiiie, and Tjong-An-Tan , "Effects of Ozone Upon Mouse Ascites Tumor Cells and Upon Chick Fibroblasts in Tissue Culture-," Z. Krebsforsch. , 67^(2): 113-126 (1965) (In German) . 153. Fetner, Robert H., Mitotic Inhibition Induced in Grasshopper Neurobla sts by Exposure to Ozone , U. S. A. F. School of Aerospace Medicine Report No. SAM--TDR-63-39 (1963). 154. Fetner, Robert E. , "Ozone Induced Chromosome Breakage in Human Cell Cultures," Nature, Lond . , 194 : 793-794 (1962). 155. Bender, Michael, "Somatic Chromosomal Aberrations-Use in the Evaluation of Human Radiation Exposures," Archs envir. Hlth , 16 : 556-564 (1968). 156. German, Janies, "Studying Human Chromosomes Today," Am. Scientist, 58(2): 182-201 (1970). 157. Ville, Claude, Biology , 4th edition, W. B. Saunders Company, Philadelphia and London (1952). 158. Ferguson, John Hov.'ard, Blood and Body Functions , F. A. Davis Company, Philadelphia (1965). 159. Bender, M. A. and Prescott, D. M. , "DNA Synthesis and Mitosis in Cultures of Human Peripheral Leucocytes," Exp . Cell Res . , 27 : 221-229 (1962). 160. Ling, N. R. , Lymphocy te Stimulation , North Holland Publisliing Company, Amsterdam (1968). 161. Kravis, L. P., Donsky, G. J., Leeks, H. I., Bhagwat, S., "Detection of Tuberculin Sensitivity in Children by Leukocyte Culture," Am. J. Pis. Child. , 115 : 247-252 (1968). 162. Braunsteiner , H. and Zuclcer-Franklin , D., "The Lymphocytes," in The Physiology and Pathology' of L eukocytes, Braunsteiner, Herbert and Zucker-Franklin, Dorthea, editors, Grune and Stratton, New York (1962). 163. Bender, M. A. and Gooch, P. C, "Types and Rates of X-Ray-Induced Chromosome Aberrations in Human Blood Irradiated In Vitro , " Proc. natn. Acad. Sci. U. S. A. , 48^: 522-532 (1962). 164. Nasjleti, C. E. , Walden, J. M. , and Spencer, H. H. , "Polyploidization and Aberration of Human Chromosomes Induced In Vitro and In Vivo v.'ith Ionizing Radiations," J. nucl . Mcd^, 7: 159-176 (1966).

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Ill 165. Bender, M. A. and Gooch, P. C, "Somatic Chromosome Aberrations Induced by Human \sTiole Body Irradiation: the 'Recuplex' Criticality Accident," Radiat. Res. , 29: 568-582 (1966). 166. Schmickel, R. , "Chroraosome Aberrations in Leucocytes Exposed In Vitr o to Diayiostic Levels of X-Rays," Am. J. hum. Ge net. , 19: 1-11 (1967). 167. Bender, M. A., Gooch, P. C., and Kondo, S., "The Gemini XI S-4 Spaceflight-Radiation Interaction Experiment: The Human Blood Experiment," Radiat. Res . . 34: 228-238 (1968). 168. Hsu, T, C. and Benirschke, Kurt, An Atlas of Mammalian Chromosoiaes , Volume 1, Springer-Verlag Inc., Nev; York (1967). 169. Ohono, Susumu, Becak, Willy, and Becak, Maria Luiza, "X-Autosome Ratio and the Behavior Pattern of Individual X-Chromosomes in Placental NammoJs," Chromosoma , 15: 14-30 (1964). 170. Yerganian, G. , "The Chinese Hamster," in The UFAW Handbook on the Care and Management of Laboratory Animals , 3rd edition. The Universities Federation for Animal Welfare Staff, editors, Williams and Wilkins, Baltimore (1967). 171. Institute of Laboratory Animal Resources, National Research Council, Anim.als for Research , 7th edition-revised. National Academy of Sciences, Washington, D. C. (1968). 172. Bender, M. A. and Gooch, P. C, "Spontaneous and X-Ray-Induced Somatic-Chromosome Aberrations in the Chinese Hamster," I nt. J . Radiat. Biol., 4(2): 175-184 (1960). ' ~ 173. Schwentker, Victor, "The Chinese (Striped) Hamster," in The UFAW H andbook on the Care and Management of Laboratory Animals, 2nd edition, Worden, Alistar N. and Lane-Peter, W. , editors. The Universities Federation for Animal Welfare, London (1957). 174. Miitney, Rae , "Hamsters," in Animals for Re search-Principles of Breeding a nd Management , Lane-Peter, VJ. , editor. Academic Press, New York (1963). 175. Moreland, Alvin , "Methods for Collection and Withdrawal of Body Fluids," in Methods of Animal Experi m entation , Volume 1 , Gay, William I., editor. Academic Press, Nevj Y'ork (1965). 176. Riley, Vernon, "Adaptation of Orbital Bleeding Technique to Rapid Serial Blood Studies," Proc . So c. exp. Biol. Med. , 104 : 751-754 (1960). 177. Moorehead, P. S., Novell, P. C, Mellman , W. J., Battips, D. M. , and Hungerford, D. A., "Chromosom.e Preparations of Leukocytes Cultured From Human Peripheral Blood," Exp. Cell Res . , 20: 613616 (1960).

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112 178. Galton, Michael and Holt, Sarah, "Culture of Peripheral Blood Leucocytes of the Golden Ilampster," Proc. Soc. exp. Biol. Med. , 114: 218-219 (1963). 379. Metcalf, W. K. , "Some Experiments on the Phytohaemagglutinln Culture of Leucocytes from Rats and Other Mammals," Exp. C ell Res. , AO: A90-/..98 (1965). 180. Hungerford, David A., "Leococytes Cultured from Small Inocula of Whole Blood and the Preparation of Metaphase Chromosomes by Treatment with hypotonic KCl," Stain Technol. , AO: 333-338 (1965). 181. Buckton, Karin E. and Nettesheim, Paul, "In Vitro and In Vivo Culture of Mouse Peripheral Blood for Chromosome Preparations," Proc. Soc. exp. Biol. Med. , 128: 1106-1110 (1968). 182. Chu, Ernest li. Y., Biology Division, Oak Ridge National Laboratory, Personal Communication (1969). 183. Gillespie, L. J., Leach, V. M. , and VJard , B. C, "A Microtechnique for Chinese Hanpster Blood Cultures," in Radiation B ioef fects , Summary Report, Jan. Dec. 1969 , Hodge, Donald M. , editor, U. S. Public Health Service Bureau of Radiological Health Report No. BRH/DBE 70-1 (1970). 184. Operations and Maintenance Manual. Mas t Models 724-2 Ozone Meter , Mast Development Company, Davenport, Iov:a (1964). 185. Interbranch Chemical Advisory Committee, Selected Methods for th e Measurem.ent of Air Pollutants , U. S. Public Health Service Division of Air Pollution Publication Ko. 999-AP-ll (1965). 186. Altshuller, A. P. and V.'artburg, A. F. , "The Interaction of Ozone V7ith Plastic and Metallic Materials in a Dynamic Flow System," Int. J. Air W a t. Pollut. , 4(1-2): 70-78 (1961). 187. Johns, Harold Elford, The Physics of Radiol ogy, 2nd edition, Charles C. Thomas, Springfield, Illinois (1961). 188. V.axd, Billy C. , Childress, Joe R. , and Jessup, Gordon L. , "Effect of Age on Radiation Mortality in the Chinese Hamster," in Radiation Bioeffects, Summary Report, Jan. Dec. 1969 , Hodge, Donald M. , editor, U. S. Public Health Service Bureau of Radiological Health Report No. BRH/DBE 70-1 (1970). 189. Photography Through the Microscope , Kodak Scientific and Technical Data Book No. P-2, Eastman Kodak Company, Rochester, Nev? York (1966).

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113 190. Fili ns In Rolls for Black and Wliite Photograph y, Kodak Advanced Data Book No. F-13, Eastman Kodak Company, Rochester, New York (1962). 191. Calaway, VJ. T. , Department of Entomology, University of Florida, Personal Coi.-;;unication (1969). 192. American Industrial Hygiene Association, "Community Air Quality Guides. Ozone," Am. ind. Hyg. Ass. J. , 29: 299-303 (1968).

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BIOGRAPHICAL SKETCH Ronald Edv/ard Zelac V7as born January 22, 1941, in Cliicago, Illinois, He attended tlie University of Illinois as a Cook County Scholar and was granted the degree of Bachelor of Science in Engineering Physics v.'ith highest honors in 1962. Ifiiile pursuing his undergraduate v7ork, he V7as recognized by being awaitled Universitj^ Honors and the College of Engineering Bell Award as vjell as by initiation into rhi Eta Sigma, Tau Beta Pi, and Phi Kappa Phi honor societies. He v;as one of ten in the nation to receive a Phi Kappa Phi fellov/ship for graduate study in 1902. Granted tlie Master of Science degree in Physics froni the University of Illinois in 1964, he then continued his graduate v;ork at the University of Kichigan under the U. S. Aton:ic Energy Conmission fellowship program and was awarded the Master of Science degree in Environztiental Health Sciences in 1965. His v7ork experience has been as follows: Argonne National Laboratory, Student A.ide, 1962; Coordinated Science laboratory, U. of Illinois, Research Assistant , 1963-1964; Argonne National Laboratory, Health Physics Trainee, 1965; IIT Research Institute, Chicago, Chief Health Physicist, 1965-1968; and Mercy Hospital Radiation Therapy Center, Chicago, Radiation Phj^sicist, 1967-1968. He has been affiliated v/ith the Health Physics Society, locally and nationally, since 1967, the American Association of Physicists in Medicine, Midwest Chapter, and the American Industrial Hygiene Association, Chicago Section. He has writings in the area of radiological health that have been published in the open literature ?nd distributed as research reports.

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115 His work tov.-ard the degree of Doctor of Philosophj' at the University of Florida, as a U, S. Public Health Service Traiiiee, began in March, 1968. Upon corcpletion of the requirements for this degree, he will assume the position of Radiation Safety Officer and Assistant Professor of Bionucleonics at Temple University, Philadelphia, Pennsylvania. He and his v/ife Ciwen V7ere married in 1961 and have two children, Steven, 7, and Daniel, 5.

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This dissertation was prepared under the direction of the chairman of the candidate's supervisory con.mittee and has been approved by all .nen^bers of that co.,mittee. It was subn.itted to the Dean of the College of Engineering and to the Graduate Council, and was approved as partial fulfillraent of the rcquiren^ents for the degree of Doctor of Philosophy. August, 3 970 BeAn> College o*^ Engineering Dean, Graduate School Supervisory Cor.mittee: y . ^,^=^^^»«ti.>*»j iLLUlA^-

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4r 9 46 B