A pharmacological and toxicological study of heptachlor

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Title:
A pharmacological and toxicological study of heptachlor
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v, 91 leaves : ill. ; 29 cm.
Language:
English
Creator:
Greene, Frank E., 1933-
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Subjects

Subjects / Keywords:
Heptachlor   ( mesh )
Insecticides   ( mesh )
Insecticides -- pharmacology   ( mesh )
Pesticides -- pharmacology   ( mesh )
Pharmacology thesis Ph.D   ( mesh )
Dissertations, Academic -- Pharmacology -- UF   ( mesh )
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bibliography   ( marcgt )
non-fiction   ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 1962.
Bibliography:
Bibliography: leaves 88-90.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Frank E. Greene.

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University of Florida
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All applicable rights reserved by the source institution and holding location.
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aleph - 029848194
oclc - 25700640
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AA00022900:00001

Table of Contents
    Title Page
        Page i
    Acknowledgement
        Page ii
    Table of Contents
        Page iii
    List of Tables
        Page iv
    List of Figures
        Page v
    Chapter 1. Introduction
        Page 1
        Page 2
    Chapter 2. The chemistry of heptachlor
        Page 3
        Page 4
    Chapter 3. Toxicity studies
        Page 5
        Page 6
        Page 7
        Page 8
        Page 9
        Page 10
        Page 11
        Page 12
        Page 13
        Page 14
        Page 15
        Page 16
        Page 17
        Page 18
        Page 19
        Page 20
        Page 21
        Page 22
        Page 23
        Page 24
        Page 25
        Page 26
        Page 27
        Page 28
        Page 29
        Page 30
        Page 31
        Page 32
        Page 33
        Page 34
    Chapter 4. Investigation of the nature of the difference in susceptibility to the acute toxic effects of heptachlor in male and female rats
        Page 35
        Page 36
        Page 37
        Page 38
        Page 39
        Page 40
        Page 41
        Page 42
        Page 43
        Page 44
        Page 45
        Page 46
        Page 47
        Page 48
        Page 49
        Page 50
        Page 51
        Page 52
        Page 53
        Page 54
        Page 55
        Page 56
        Page 57
        Page 58
    Chapter 5. Localization of the site of action of heptachlor
        Page 59
        Page 60
        Page 61
        Page 62
        Page 63
        Page 64
        Page 65
        Page 66
        Page 67
        Page 68
        Page 69
        Page 70
        Page 71
        Page 72
        Page 73
        Page 74
        Page 75
        Page 76
        Page 77
        Page 78
        Page 79
    Chapter 6. Summary and conclusions
        Page 80
        Page 81
    Appendices
        Page 82
    Appendix I. Preparation of the corn oil solution of heptachlor
        Page 83
    Appendix II. Preparation of the heptachlor emulsion for parenteral and isolated tissue studies
        Page 84
    Appendix III. Calculation of the LD50 by the Litchfield and Wilcoxon method
        Page 85
        Page 86
        Page 87
    Bibliography
        Page 88
        Page 89
        Page 90
    Biographical sketch
        Page 91
        Page 92
        Page 93
Full Text












A PHARMACOLOGICAL AND

TOXICOLOGICAL STUDY OF HEPTACHLOR













By
FRANK E. GREENE












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 February, 1962















ACKNOWLEDGMENTS


The author would like to express his sincere appreciation to the members of his supervisory committee, Dr. Sidney Cassin, Dr. Laurett.a Fox, Dr. Melvin Fried, Dr. Thomas Malewitz, and Dr. Elbert Voss, Chairman. Their encouragement, advice, and above all, patience are gratefully acknowledged.

The author is indebted to the Graduate Council and the American Foundation for Pharmaceutical Education for financial support which made possible the completion of this study.

To his fellow graduate students the author extends special thanks for their helpful suggestions, encouragement, and technical assistance.















TABLE OF CONTENTS

Page

ACKNOWLEDGMENTS . . . . . . .

LIST OF TABLES . . . . . . . . . . iv

LIST OF FIGURES . . . . . . . . . . . v

CHAPTER

I. INTRODUCTION . . . . . . . . .

II. THE CHEMISTRY OF HEPTACHLOR . . . . . . . 3

III. TOXICITY STUDIES . . . . . . . . . 5

IV. INVESTIGATION OF THE NATURE OF THE DIFFERENCE IN
SUSCEPTIBILITY TO THE ACUTE TOXIC EFFECTS OF HEPTACHLOR
IN MALE AND FEMALE RATS . . . . . . . 35

V. LOCALIZATION OF THE SITE OF ACTION OF HEPTACHLOR . 59

VI. SUMMARY AND CONCLUSIONS . . . . . . . . 80

APPENDICES . . . . . . ... . . . . 4 . . 82

BIBLIOGRAPHY . . . . . . . . . . . . 88

BIOGRAPHICAL SKETCH . . . . . . . . . . . 91















LIST OF TABLES

Table Page

1. AVERAGE SURVIVAL TINES OF RATS FED HIGH CONCENTRATIONS
OF HEPTACHLOR . . . . . . . . . . . 10

2. AVERAGE BODY WEIGHTS (IN GRAMS) OF MALE AND FEMALE RATS
FED VARYING CONCENTRATIONS OF HEPTACHLOR IN THEIR DIET

3. STATISTICAL COMPARISON OF INITIAL AND FINAL AVERAGE BODY
WEIGHTS OF CONTROLS AND OF RATS USED FOR CHRONIC FEEDING
STUDIES OF HEPTACHLOR . . . . . . . . . 14

4. STATISTICAL COMPARISON OF SOME AVERAGE ORGAN WEIGHTS OF
RATS FED VARYING CONCENTRATIONS OF HEPTACHLOR IN THEIR
DIET . . . . . . . . . . . . 15

5. SOLUTION OF THE DOSE MORTALITY CURVE OF THE CORN OIL
SOLUTION OF HEPTACHLOR IN MALE RATS . . . . . o 29

6. SOLUTION OF THE DOSE MORTALITY CURVE OF THE CORN OIL
SOLUTION OF HEPTACHLOR IN FEMALE RATS . . . . . 31

7o STRUCTURAL FORMULAS OF SOME INHIBITORS OF DRUG METABOLISM 41

8. THE EFFECT OF CASTRATION AND ADMINISTRATION OF SEX HORMONES
ON THE ACUTE TOXICITY OF HEPTACHLOR IN RATS . . . . 45

9. THE EFFECT OF HORMONE ADMINISTRATION ON THE ACUTE TOXICITY
OF REPTACHLOR IN NORMAL RATS . . . . . o o 48

10. COMBINED DATA FROM EXPERIMENTS CONCERNING THE ENDOCRINE INFLUENCE ON THE ACUTE TOXICITY OF HEPTACHLOR IN RATS o 54 11, THE EFFECT OF SOME EXPERIMENTAL PROCEDURES ON THE BODY WEIGHT OF MALE AND FEMALE RATS . . . . . . . 56












iv















LIST OF FIGURES
Figure Page

1. EFFECTS ON THE GROWTH RATE OF VARIOUS CONCENTRATIONS OF
pHEPTACHLOR IN THE DIETS OF MALE AND FEMALE RATS . . . 13

2. CONTROL LIVER% H AND E, 70X. .. .. .. ... . . . 17

3. LIVER OF FEMALE RAT FED 0.01 PER CENT HEPTACHLOR, H AND E,
90X . . . .. .. .. .. .. .. .. .. .. .19

4. HIGH MAGNIFICATION OF CONTROL LIVER, H AND E. 350X . . 21

5. HIGH MAGNIFICATION OF LIVER FROM A FEMALE RAT FED 0.01 PER
CENT HEPTACHLOR, H AND E, 500X . .. .. .. .. .. 23

6.* LUNG OF CONTROL ANIMAL SHOWING CHRONIC INFLAMMATORY CHANGES,
H AND E, 1OOX .. .. .. .. .. .. .. .. ... . . 25

7. THE DOSE-PER CENT MORTALITY CURVE FOR HEPTACHLOR IN MALE
RATS . . .. .. .. .. .. .. .. .. .. .. ... 30

8. THE DOSE-PER CENT MORTALITY CURVE FOR HEPTACHLOR IN FEMALE
RATS....................... . . .32

9. THE EFFECT OF SKF-525-A ON THE ACUTE TOXICITY OF HEPTACHLOR
IN MALE RATS............... .. .. . . . 51

10, THE EFFECT OF THE CONTROL EMULSION ON THE BLOOD PRESSURE
AND RESPIRATION OF A PENTOBARBITALIZED DOG. ...... . 63

11. THE EFFECT OF HEPTACHLOR ON THE BLOOD PRESSURE AND
RESPIRATION OF A PENTOBARBITALIZED DOG. ........... 64

12. THE EFFECT OF HEPTACHLOR AND ACETYLCHOLINE ON ISOLATED
RABIT ILEUM...... ..................67








Iv















CHAPTER I


INTRODUCT ION


There is little information in the literature concerning the

toxicology and pharmacology of Heptachlor (l,4,5,6,7,8,8-Heptachloro-3a, 4,7 ,7a-tetrahydro-4,7 ,-methanoindene). Frequently the information required by law before an insecticide can be marketed is filed with the United States Department of Agriculture and with the Food and Drug Administration and never published (1). other observations concerning toxicity which may be made during field trials may wind up as progress reports of some governmental agency where they are unavailable to a literature searcher. Pharmacological studies may be undertaken later when hazards of poisoning to warm-blooded animals become apparent,, pointing to the need for more basic knowledge of the mechanism of action of the compound. With this information a rational approach to treatment of accidental poisoning and to the development of safe means of application of the compound by field personnel may be formulated.

In 1957, Heptachlor was selected as one of two pesticides to be

used in the fire ant extermination program undertaken in the Southeastern United States. Shortly after initiation of this program it became apparent that, in addition to its expected activity against insects, the compound was highly toxic to wild and domestic animals.

Lack of knowledge of the pharmacologic effects produced by this compound has forced those treating cases of accidental poisoning to resort to empirical methods, which have not alwd~ys been successful.






2





This investigation was undertaken in order to extend the information concerning the pharmacologic and toxicologic properties of Heptachlor. It was hoped that this additional information would lead to a better understanding of the mechanisms of action of this compound. To this end the activity of Heptachlor on the central and autonomnic nervous system and some factors influencing acute toxicity were studied.














CHAPTER II


THE CHEMISTRY OF HEPTACHLOR


Heptachlor, a member of the cyclodiene family of insecticides, was first discovered as a constituent of technical Chlordan, under a patent assigned to Hyman (2, p 60). Chemically, it is 1,4,5,6,7,8,8Heptachloro-3a,4,7,7a-tetrahydro-4,7,-methanoindene. The structural formula was shown to be:

Cl



CI-C-CI

Cr HH
riC

Other members of this family include Chlordan, Aldrin, and

Dieldrin whose structural formulas are given below. Endrin, a sterioisomer of Dieldrin; Isodrin, a sterioisomer of Aldrin; and Toxaphene, a chlorinated camphene mixture, complete the family of cyclodiene insecticides.


HH
CI CI H CI H
H C l H
CI /H C1 H ,H Cg H
ICI
-C-C Cl-C-CI CH2 C-C-Cl C 0

C I H H
Ci H2 CI H CI H
Chlordan Aldrin Dieldrin




3





4




Heptachlor may be prepared by the action of sulfuryl chloride on the condensation product of cyclopentadiene and hexachloro-cyclopentadiene in carbon tetrachloride in the presence of benzoyl peroxide (2, p 60).

All of the cyclodiene insecticides may be prepared by the

Diels-Alder reaction except Toxaphene, which is made by chlorinating camphene to a chlorine content of 67 to 69 per cent (3, p 239).

Pure Heptachlor is a white crystalline solid, which has a

melting point of 95-960C. The technical material available commercially contains about 72 per cent Heptachlor and 28 per cent related materials and is a soft, waxy solid, light tan in color, with a melting range of 46 to 74.90C. Heptachlor is insoluble in water, but soluble in most organic solvents (2, p 236). A recrystallized product is also available for research purposes, which has a purity of about 90 per cent.















CHAPTER III


TOXICITY STUDIES


Review of the Literature

The available literature on the cyclodiene group of pesticides

which does not pertain to agricultural applications is concerned chiefly with descriptions of toxic reactions occurring as a result of accidental or experimental poisoning in animals. However, a few cases of fatal poisoning in humans by Chiordan (4), Aldrin (5, p 5), and Toxaphene (6), have been reported. The effects of these compounds on humans appear to be identical with those observed in lower animals. No human fatalities resulting from Heptachlor poisoning have been reported, but it must be considered capable of producing severe toxic effects in humans if precautionary measures are not used, as judged by reports of toxicity to animals.

Gross symptoms of acute intoxication following a single exposure are essentially the same for all members of the cyclodiene group of pesticides. Among these symptoms are signs of central nervous system stimulation which may progress to a series of clonic and tonic convulsions. Evidence of increased activity of the parasympathetic nervous system is usually present, Symptoms reported following acute Heptachlor poisoning were increased salivation and lacrimation, generalized tremors, increased respiratory rate and volume, violent clonic and tonic convulsions and opisthotonus. In terminal stages, respiration was irregular with dyspnea and cyanosis as prominent features (7).


5






6




By varying the size and number of doses, three separate types of

response to Dieldrin have been obtained (5, p 222). A few large doses

resulted in one or more convulsions. Unless the animal died, there was

relatively prompt recovery without permanent damage or great weight loss.

Many doses of moderate size have produced a complete loss of appetite, weight loss, and convulsions. Without treatment, death was seemingly inevitable. Many small doses produced one or more convulsions without

any other apparent effect.

In addition to these effects, Aldrin (8) and Dieldrin (9) have

been reported to produce bradycardia, vasodepression, and miosis.

Lethargy and anorexia are common findings following Aldrin (10), Chlordan

(5, p 164), Dieldrin (5, p 226) and Endrin (11) poisoning. Partial to

total blindness, increased response to light tactle stimuli, and

convulsions induced by auditory stimuli have been attributed to Chlordan poisoning (12). These effects have not been noted in cases of poisoning

by other members of this group.

It has been shown that absorption of these compounds through the

skin and mucous membranes in amounts sufficient to produce the toxic

symptoms previously described, is possible if the proper combination of

concentration and exposure time is provided (13).

p Unpublished chronic feeding studies (14) have shown that rats

require about 10 parts per million (ppm) of Heptachlor in their diets

for production of tissue damage, when maintained at that level for 120

weeks. Such animals were reported to have manifested a slightly

depressed rate of growth, but their mortality was no greater than that of the controls. However, females maintained on 30 ppm of Heptachlor






7




prior to mating bad an increased rate of mortality of their offspring. Animals given a diet containing 300 ppm died within eleven days, and those given 100 ppm showed greater mortality than controls.

Pathological changes reported in the above study were: degenerative changes in the cells of the central zone of the liver lobule, kidney lesions involving degeneration of the epithelium of the proximal and distal convoluted tubules, and some nonspecific changes in the neuronal cells of the central nervous system. These effects are essentially the same as those reported for other chlorinated hydrocarbons.

Toxicity data are greatly influenced by the specific experimental conditions under which they were obtained. For this reason the results of a particular experiment may be meaningless unless the exact experimental conditions are also made known. This situation prevails particularly in the literature on pesticides where the original data may lie in a file of some governmental agency, with only the results, stripped of details of experimental conditions, appearing in print. With this in mind, it was considered advisable to determine the LD50 of Heptachlor under known conditions.

Since there was such a pronounced sex difference in acute toxicity of Heptachlor, it seemed likely that in chronic feeding experiments some sex differences might also appear. This aspect seemed worthy of investigation and rats of both sexes were maintained on diets containing various amounts of Heptachlor for six months. This period of time was suggested by Barnes and Denz (15), who felt that any pathology which will develop with chronic exposure to a drug should appear within this period.






8




Experimental

Chronic Toxicity Studies

Materials and Methods

For chronic toxicity studies, immature rats between 50 and 100 ans were selected. Groups of ten rats aof each sex were maintained on ground Purina laboratory chow containing 1.0, 0.1, 0.01, and 0.001 per cent Heptachlor. bOne group given ground lab chow alone served as controls. The period of feeding was six months for the survivors. During this period, the animals were given food and water ad libitum.

These rats were individually weighed and numbered at the onset of the experiment. They were then weighed weekly for the first five weeks, at the end of the seventh week, and again at the termination of the experiment. At this time they were killed by stunning and exsanguination. In addition to the final body weights, organ weights were taken for liver, kidneys, and gonads; and these data were analyzed statistically. These organs, along with the lungs, adrenals, stomach, esophagus, duodenum, colon, ileum, spleen, and heart, were taken for histological examination. Rats which died in the course of the experiments were not examined histologically as they were frequently mutilated by their cage mates or were not found until a number of hours after death.

After fixation in 10 per cent formalin, all tissues, except those used for frozen sections were dehydrated and infiltrated according to



aRats used in all experiments were NLR (Wistar origin) strain, supplied by the National Animal Company, Creve Couer, Missouri. bHeptachlor used in these and all subsequent studies was supplied by the Velsicol Chemical Corporation, Chicago, Illinois, and was labeled "irecrystallized" Heptachlor, 1009-17.






9




the butyl alcohol-paraf in mush method of Johnston et al. (16). The

paraf in embedded tissues were sectioned at 8 microns and stained with

Harris hematoxylin and eosin as modified by Malewitz and Smith (17).

Liver sections were also stained for fat by the oil-red 0 method I (18, p 1.24) and for glycogen by the periodic acid-Schiff (PAS) reaction

(18, p 132).


Results

All animals fed Heptachlor in concentrations of 1. or 0.1 per cent were dead within two weeks. Body weights, taken at the end of the first

week of the experiment showed marked reduction in the growth rate of

these groups, particularly those on the 1 per cent diet. Symptoms of toxicity began to appear for these two groups about the sixth day and

consisted chiefly of lethargy and anorexia with an occasional animal

showing signs of central nervous system stimulation manifested by

hyperexcitability and fighting among cage mates. About the eighth day

this stimulation became more apparent and convulsions of short duration were seen frequently. Many of the animals developed lesions around the

muzzle as a result of fighting with their cage mates. The average

survival time of these groups is found in Table 1.

Average body weights taken for all groups are found in Table 2. Examination of this table shows a slight depression of growth during the first few weeks in the female groups fed the lower concentrations

of Heptachlor. Male rats fed 0.001 per cent Heptachlor also show a

slightly depressed rate of growth during the first two weeks. Growth

curves for the experimental groups surviving the entire period are






10




















TABLE 1

AVERAGE SURVIVAL TIMES OF RATS a
FED HIGH CONCENTRATIONS OF HEPTACHLOR


Average Survival
Diet Sex Time (Days)


1% Heptachlor Male 10.6

1% Heptachlor Female 9.7

0.1% Heptachlor Male 11.9

0.1% Heptachlor Female 12.6


a All groups contained ten animals.



















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found in Figure 1. When comparing groups of the same sex, this graph shows essentially parallel growth for the control and experimental groups with the exception of the females maintained on the 0.01 per cent Heptachlor diet. When t tests (19, p 164) were performed for the initial and final body weights of all groups, this increase in weight was found to be significant. Data obtained from statistical analysis of these weights are found in Table 3. As a test for significance of all comparisons made the confidence limits of 95 per cent probability were arbitrarily selected.

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diet died during the course of the experiment, one death occurring during the fourth, and two during the sixth months of this study. All animals from the other groups survived the entire experimental period.

On autopsy, no gross pathology was found in any of the organs except the lungs, where occasional white nodules were seen. These nodules were also found in the control animals and were considered to result from pulmonary infection rather than exposure to Heptachlor. Results of the statistical analysis of the liver, kidney, and gonad weights are found in Table 4.

These data show that the livers of both male and female rats from the 0.01 and 0.001 per cent groups differ significantly from the controls. This difference in weight was an increase in all instances except for the female group fed the 0.001 per cent diet, where this change was a decrease. The kidneys of the 0.01 per cent male group also show a significant increase in weight when compared with those of the controls. The only significant change observed in gonad weights was an increase found in the male group given 0.01 per cent Heptachlor.







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The most striking changes observed in all tissues studied were found in the livers of female rats fed 0.01 per cent Hleptachlor. aThe cells of the peni-central vein region were greatly enlarged and pale staining. Some cells had undergone karyolysis, while several multinucleate cells were also seen. Margination of the cytoplasmic material and enlarged nuclei were seen in many of the cells in this area. Photomicrographs of control and experimental livers showing these changes are found in Figures 2, 3, 4 and 5. Cells of the peni-central vein region contained neither fat, nor glycogen. Midzonal cells of sections showing the cellular changes previously described showed dense glycogen accumulation. In these same sections, fat droplets were prominent in the peni-portal regions. These later areas were relatively glycogen poor.

These changes were present to a lesser degree in female rats fed

0.001 per cent Heptachlor and were seen only in the 0.01 per cent male rat group. The observation of more extensive pathology in female than in male rat livers offers a possible explanation for the greater number of deaths which occurred in the female group fed 0.01 per cent Heptachlor.

The lungs of practically all animals, experimental and controls, showed evidence of chronic infection. Congestion, lymphocytic infiltration, and areas of necrosis were present in varying degrees in all animals. Hem liderin engorged cells, indicative of chronic congestion were prominent in many lungs (20, p 56). A photomicrograph showing some of these changes is found in Figure 6.


aThe author would like Lo express his appreciation to Drs. J. E. Edwards and M. Waid of the Department of Pathology, J. Hillis Miller Health Center, for their valuable suggestions and interpretations of the pathology found in the livers of these animals.





































FIGURE 2. CONTROL LIVER, H AND E, 70X






18

IL











































I v 4 c'.
ka
































FIGURE 3. LIVER OF FEMALE RAT FED 0.01 PER CENT
HEPTACHLOR, H AND E, 90X








20 c ,tk 0,9, V7
AQA 'I Ilk w



































































Ir


































FIGURE 4. HIGH MAGNIFICATION OF CONTROL LIVER, H AND E, 350X








22
































j
0



.0 f




























',low
.40











Ilk.




































FIGURE 5. HIGH MAGNIFICATION OF LIVER FROM A FEMALE RAT FED 0.01 PER CENT HEPTACHLOR.
H AND E, 50OX








24 Al I..:
1w



Sl
owl .1 At.







A ti,
ft





420
L 4 !10













Apw



iw.












lip





14 1


-Ok








10






&A 4






































FIGURE 6.* LUNG OF CONTROL ANIMAL SHOWING CHRONIC
INFLAMMATORY CHANGES,
H AND E, oox







26


or .Not IP






MIA







-41 bn i














ff



VAW-






27




The results of histological examination of the other tissues and organs were non-remarkable.


Acute Toxicity Studies

Determination of the Oral LD50 of the Corn Oil a Solution of Heptachlor Materials and Methods

Preliminary investigation had shown the dosage range for 0 to 100 per cent fatalities in male rats to be from 40 to 100 mg/Kg, and in females from 60 to 190 mg/Kg. Doses at graded increments were administered orally to groups of eight rats each using a Phipps and Byrd oral needle following a period of fasting of 12 to 18 hours. The rats bad free access to water during the period of fasting and were given food immediately following the administration of Heptachlor. These rats were then observed for a period of eight days, and the LD50 was calculated from the number of deaths occurring during this period using the method of Litchfield and Wilcoxon (21).

The general procedure as outlined by this method is followed

in the calculation of the LD50 for the male rats found in Appendix III. For determination of the female LD503 only the results are shown.


Results

Deaths resulting from a single oral dose of Heptachlor usually fell into two categories: 1) those dying within the first 24 hours, and 2) those dying from about the fifth to the eighth day. These intervals represent the period of early and delayed toxicity respectively. a The method used for the preparation of the corn oil solution is found in Appendix I.






28




In both periods the symptoms prior to death were similar and consisted of a series of short spasms which appeared in increasing frequency, ultimately terminating in a clinic and tonic convulsion. Following these convulsions, the muzzles of all animals were wet, indicating that an increase in secretion had occurred during the seizure. This finding was indicative of parasympathetic nervous system stimulation, and was seen following convulsions only. The LD50's and their confidence limits are given below.

Male rats.- The data obtained from the determination of the LD50 are given in Table 5. The graph for the data is found in Figure 7. The LD50 and its 19/20 confidence limits calculated from these data is 59 (49 to 71) mg/Kg.

Female rats.- The data obtained from this experiment are found in Table 6, and presented graphically in Figure 8. The calculated LD50 and its 19/20 confidence limits is 132 (114 to 154) mg/Kg.


Discussion

The LD50 values determined for male rats are in reasonable

agreement with those previously reported (14). The periods of early and delayed toxicity as described by Lehman (13) were also seen in this study. As in previous studies (14) male rats proved to be much more susceptible to a single oral dose of Heptachlor than females-. The nature of this difference is investigated in the following chapter.

The difference in susceptibility of male and female rats was reversed in the acute and chronic toxicity studies. While males were more susceptible to the acute effects of Heptachlor, females were more






29


















TABLE 5

SOLUTION OF THE DOSE MORTALITY CURVE OF THE CORN OIL
SOLUTION OF HEPTACHLOR IN MALE RATS



Observed'* Expected b Observed Contributions Dose Killed Per Cent Per Cent Minus to 2
mg/Kg Tested Mortality Mortality Expected (chi)


40 0/8 4.4 13 8.6 0.060
50 1/8 12.5 34 21.5 0.190
60 4/8 50.0 52 2.0 0.002
70 5/8 62.5 68 5.5 0.025
80 7/8 87.5 81 6.5 0.028
100 8/8 98.4 93.5 4.9 0.039




a The observed values listed for 0 and 100 per cent effect represent corrected values obtained by the Litchfield and Wilcoxon method.
b The expected values were obtained from Figure 7.






30





3O
99.9 !





99
0



95 90




0

I
w50
U
w






10








20 30 50 70 100 200
DOSE, MG/KG

Fig. 7.--The dose-per cent mortality
curve for Heptachlor in male rats.






31

















TABLE 6

SOLUTION OF THE DOSE MORTALITY CURVE OF THE CORN OIL
SOLUTION OF HEPTACHLOR IN FEMALE RATS



Observed* Expected b Observed Contributions
Dose Killed Per Cent Per Cent Minus to2
mg/K~g Tested Mortality Mortality Expected (Chi)2


60 0/8 0.6 1.8 1.2 0.008
80 1/8 12.5 9.0 3.0 0.011
100 1/8 12.5 22.0 9.5 0.050
120 2/8 25.0 39.0 14.0 0.080
140 4/8 50.0 55.0 5.0 0.010
150 7/8 87.5 62.0 25.5 0.275
160 6/8 75.0 70.0 5.0 0.011
170 8/8 92.6 74.0 .18.60.8




a The observed values listed for 0 and 100 per cent effect represent corrected values obtained by the Litchfield and Wilcoxon method.
bThe expected values were obtained from Figure 8.






32




98


95


90
0

IQ



00


S50

-J

oo
0

Iz 0


w





0.1






0.01



0.0! I K11 ,,,
40 70 100 200 300
DOSE, MG/KG

Fig. 8.--The dose-per cent mortality
curve for Heptachlor in female rats.






33




susceptible to chronic exposure to Heptachlor. The only pathologic differences observed in the surviving animals was a more extensive liver damage in female rats, when compared to males fed the same concentration of Heptachlor. If the death of the three animals in the female group fed 0.01 per cent Heptachlor resulted from this type of damage, it is possible that the lower mortality rate observed in male rats fed the same diet could be due to some protective action of this organ by testosterone. This protective role of testosterone has been proposed by Seyle (22). The significant increase in the weight of the testes of rats fed 0.01 per cent Heptachlor could then represent a functional response to an increased utilization of testosterone. This event could arise following an increased demand of long duration for this hormone if it should be involved in such a role in the liver.

The over-all activity of Heptachlor falls into two, apparently unrelated, categories: 1) central nervous system stimulation seen in acute poisoning with a single large dose, or frequently administered smaller doses, and 2) pathological changes in organs such as the liver and kidney, following continuous exposure to small amounts of this compound. The period of delayed toxicity may contain elements of both types of action.

The anorexic effect of Heptachlor and the property of lipids of the central nervous system to resist mobilization in the face of protracted starvation suggested the following sequence of events in the period of delayed toxicity of Heptachlor.

Following ingestion of Heptachlor in an amount less than the quantity necessary to cause death in the early toxicity period,






34




Heptachlor and the epoxide are stored in body fat. During the period of anorexia which may develop, the animal will mobilize this fat when glycogen deposits are depleted, but lipids of the central nervous system would not be involved in this process. Heptacblor, which is stored in body fat would be mobilized as the fat in which it is stored is broken down. The Heptachlor displaced in this manner would tend to relocate in other lipid tissue. The unmobilized lipid of the central nervous system is a likely site of such events. Through such a process Heptachlor concentration in the nervous system could gradually build up, leading to the symptoms of central nervous system stimulation and ultimately, convulsions in these animals.

A logical treatment of Heptachlor poisoning should be directed toward the two phases of the action of Heptachlor: 1) control of the convulsions during the period of early toxicity, and 2) prevention of the secondary toxic effects. Control of the later effects could possibly be accomplished by supportive therapy directed toward protection of the liver and other organs from further damage. A low fat, high protein diet, plus one or more lipotropic substances (23, p 928), is recommended for treatment of damaged livers, and would, at the same time provide nutritional support for the animal. Such a regimen would be expected to prevent mobilization of body fat and secondary deposition of Heptacblor in the central nervous system, if this event should actually take place. The central nervous system effects could probably be controlled by cautious administration of barbiturates.















CHAPTER IV


INVESTIGATION OF THE NATURE OF THE DIFFERENCE IN
SUSCEPTIBILITY TO THE ACUTE TOXIC EFFECTS OF
HEPTACHLOR IN MALE AND FEMALE RATS Review of the Literature

In the course of work directed toward determination of tissue

residues fromn Heptachlor, Davidow and Radomski (24) found a metabolically altered derivative, an epoxide, along with Heptachlor in the fat of Heptachior-fed dogs. They reported the following as the structure of this compound:







Thseauhos taedtht hi ws yp o bolgialoxdaio podc






stabe iteormediatedo thet prosws at of biological d oxation hich ct



due to its solubility in fat, tended to accumulate in adipose tissue. Since this initial discovery, the epoxide of Heptachlor, along with the parent compound, has been isolated and identified in the body fat of rats and rabbits (25), (26), and in the whole insect in the case of the housefly, Musca domesticus (27). The female rat stores the epoxide to a much greater extent in fat than does the male, the biological


35






36




multiplication ratio being 6.2 for the female and 1.2 for the male when both are maintained on 30 ppm of Heptachlor in their diet (25).

A particularly interesting aspect of the toxicity of Heptachlor in the rat is the difference in response of the male and female to a single oral dose, the reported LD50 for females being 142 mg/Kg, and that for males, 60 mg/Kg (14). A possible explanation for this difference could be a different rate of conversion of Heptachlor to its

epoxdea compound which has an intravenous toxicity distinctly greater than the parent compound. When given a dose of 10 mg/Kg, 100 per cent of epoxide treated mice died, while Heptachlor at the same dosage level caused no deaths (25). If male rats were capable of converting Heptachlor to its epoxide at a faster rate, this might explain the higher mortality produced in males than females given the same dose.

Similar differences in sex responses have been observed with other compounds, particularly the barbiturates, in which it was noted that male rats consistently slept for shorter times than females given the same dose of pentobarbital. Jarcho (28) noted that this sex difference could be demonstrated only with the barbiturates which are known to be detoxified in the liver. Holck et al. (29) investigated the effects of testosterone propionate and estradiol dipropionate administered prior to hexobarbital treatment in normal male and female rats and was able to shorten the sleeping time of testosterone-treated females to that of the males. They also were able to reduce the sleeping


a The biological multiplication ratio is a measure of the ability to concentrate a substance in the body fat and is calculated by dividing the concentration of the substance in the diet into the concentration of the substance found in the fat,






37




time in the testosterone-treated male below that of the nontreated control males. Cameron et al. (30) reported an increase in mean barbiturate-induced sleeping time following castration in male rats. Tureman et al. (31) found that gonadectomy increased sleeping time for both sexes, and that testosterone given to male gonadectomized rats and intact females decreased sleeping time. Changes in sleeping times have provided a convenient method for the determination of the effects of various experimental procedures on metabolic transformations which resulted in the inactivation of certain barbiturates. Conditions which produce changes in sleeping time may also be expected to affect the rate of biotransformation of other substances whose metabolism does not provide so convenient an endpoint.

Quinn et al. (32) investigating the biotransformation of

hexabarbital by the microsomal fraction from rat liver homogenate, found close correlation between the rates of biotransformation and the sleeping time. Female rats slept four to five times as long as males and showed a correspondingly lower rate of biotransformation. Further, this difference in the sexes was reduced by administration of testosterone to females for six weeks prior to testing, producing both an increase in biotransformation and a corresponding shortening of sleeping time.

In addition to these drugs, sex differences in the rates of

metabolism in rats have been noted with two non-chlorinated hydrocarbon insecticides, Schradan and Parathion (33). These compounds are relatively weak cholinesterase inhibitors in vitro, but are converted in vivo to powerful cholinesterase inhibitors. Schradan was found to be more toxic for males and Parathion for females. The liver of the






38




male rat has been shown to convert Schradan to its more active oxide better than the livers of females; the reverse holds true for Parathion, which is converted to paraoxone by the liver of the females more rapidly than in that of the male. These reactions were found to occur in the microsomal fraction of liver homogenate and to be DPN and Mg+ dependent. Male and female rats are equally susceptible to the cholinesterase inhibitor TEPP, which does not undergo conversion in vivo.

Holck et al. (34) produced hypothyroidism in rats of both sexes by using 200 mg of propylthiouracil mixed with 1 Kg of food. The animals were maintained on this diet for 20 days prior to testing. This experimental hypothyroidism was expected to decrease liver metabolism and thereby prolong the action of those barbiturates metabolized in this organ. Using pentobarbital, he was able to produce an increase in sleeping time in both sexes, but a significant difference between the male and female response was still present.

The Smith, Kline and French Laboratories have synthesized a

compound which was found to be capable of greatly increasing the effects of certain drugs by affecting the rate at which they are degraded in the body. This compound, beta-diethylaminoethyl-diphenylpropyl acetate, was given the generic name of diphenylpropylacetate and referred to as SKF-525-A. It was sent to the National Institutes of Health where its effects on the biotransformation of a variety of drugs were studied. Axelrod et al. (35) testing the effects of pretreatment with SKF-525-A on hexabarbital sleeping time, found it to be increased four times over that of the saline-treated controls. He also followed blood levels of the barbiturate during this period and found the half-life paralleled






39




this increase. Blood levels of hexobarbital were measured in animals at the time they regained their righting reflex and were found to be the same regardless of whether the animal had been pretreated with SKF-525-A or not. In addition, animals recovering from anesthesia cannot be reinducted by use of SKF-525-A as is the case with chlorpromazine or glucose, which are classified as potentiators (35). On the basis of these findings, SKF-525-A was judged to be a "prolonging" agent rather than a potentiator.

Cook et al. (36) determined that the maximum effect of hexobarbital sleeping time was obtained when SKF-525-A was given 40 minutes prior to administration of the drug. He also found SKF-525-A to be equally effective when given either intraperitoneally or orally. He was able to produce a 35-fold increase in the sleeping time of rats pretreated with SKF-525-A over saline treated controls. In other studies, Cook et al. (37) found that SKF-525-A had no effect on thiopental, ether, or nitrous oxide anesthesia, or on the sleeping times of barbital, or methylparafynol. None of these substances are inactivated by metabolism. However, SKF-525-A did enhance the analgesic properties of morphine sulfate, codeine phosphate, and methorphinan, and prolonged sleeping times of Seconal, Amytal, Butethal, Ortal, pentobarbital and chloral hydrate. Metabolism of members of the later group results in loss of activity.

In vitro studies by Cooper (38), using liver homogenates

demonstrated the inhibition of a variety of biotransformation reactions with SKF-525-A. Some of these were: side chain oxidations, dealkylations, deaminations and cleavage of ether linkages. La Du (39), investigating






40




a group of compounds whose biotransformations could be accomplished with a system comprised of liver microsomes plus reduced TPN and oxygen, found that all could be inhibited by SKF-525-A. The conversion of Parathion and Scaradan to their oxides, cited previously (33), and found to be DPN dependent was also inhibited by SKF-525-A.

Cooper (40) tested the effects of SKF-525-A on enzyme systems involved in generating reduced TPN and in transporting hydrogen from reduced TPN to oxygen via the cytochrome system. He also tested effects on DPN-requiring enzymes, using the alcohol dehydrogenase system. He was unable to demonstrate inhibition in either case and concluded that the action of SKF-525-A in vivo probably lay in its effect on some other, as yet unidentified, system common to all of the biotransformation reactions studied.

Since the discovery of these properties of SKF-525-A, two other compounds have been found which inhibit the same biotransformation reactions. They are 2,4,-dichloro-6-phenoxyethyl diethylamine (Lilly 18947) and iproniazid. Studies on the effects of these compounds on hexobarbital sleeping time by Fouts and Brodie (41), (42) have shown Lilly 18947 to be most effective, followed by SKF-525-A and iproniazid, in that order.

Recently, workers at the Lilly Laboratories have synthesized

another compound designated as Lilly 32391 (2 phenyl-4,6-dichloro-phenoxy) ethylamine HC1, which is said to be about ten times as potent as Lilly compound 18947 (43). The structures of these compounds are shown in Table 7.






41
















TABLE 7

STRUCTURAL FORMULAS OF SOME INHIBITORS OF DRUG METABOLISM




0 C2 H5 0=0-NH-NH-OH-OH3
~/\ 0-0-OH2- OH2-N CN



SKF-525-A Iproniazid





*HBr C2 H5

C1 / -0H2-CH2-N-02H5 01 / O-0H2-0H2-NH2 H01

Lilly Compound 18947 Lilly Compound 32391






42




Metabolic activation of Heptachlor rather than detoxification is strongly suggested by data from the experiments previously discussed concerning the nature of sex difference in response to certain barbiturates. Since male rats are capable of metabolizing barbiturates at a faster rate than females, it is not unreasonable to assume that similar differences in the metabolism of other compounds may exist. If Heptachlor falls into this category, the difference in response by male and female rats could be explained by a more rapid rate of conversion, in the male, to a compound more toxic than the original one. The only known metabolite of Heptachlor, its epoxide, is in fact more toxic than the parent compound. In view of these facts, it was felt that investigations analogous to the studies of sex differences in barbiturate response could yield valuable information concerning some of the factors involved in the mechanism of the toxic action of Heptachlor.


Experimental

The Effect of Gonadectomy and Gonadal Hormones on Acute Toxicity Materials and Methods

To determine the effects on the acute toxicity of Heptachlor

produced by castration and the administration of exogenous hormones to castrated animals, thirty male and thirty female rats weighing 150 to 200 Gms were castrated by the methods of D'Amour and Blood (44, p 44, 45) and allowed one month to recover. At this time, the separate groups of males and females were each divided into three sub-groups of ten animals; and to these, one normal (uncastrated) group of each sex was added for controls.






43




Daily hormone aor sesame oil injections were given subcutaneously according to the schedule given below. These injections were begun two weeks before and continued one week following administration of Heptachlor. The dosage regimen selected was one used by Hoick et al. (29) in their studies on sex differences in pentobarbital sleeping time in rats.

Group Treatment Dosage

Non-castrate Sesame oil 1 ml/Kg/day
Castrate Sesame oil 1 mi/Kg/day
Castrate Estradiol dipropionate 1 mg/Kg/day
Castrate Testosterone propionate 1 mg/Kg/day

On1 the fourteenth day of treatment, the rats were weighed and given an oral dose of 200 mg/Kg of Heptachlor. These rats were older and heavier than those used in the determination of the LD50# and

preliminary trials had shown them to be more resistant to the toxic effects of Heptachlor than younger rats. These trials had indicated that at a dosage level of 200 mg/Kg, one could expect about 80 per cent fatality in males and 40 per cent fatality in females. The animals were observed for fourteen days following administration of Heptachlor and all deaths were recorded. Dead animals were autopsied, and the seminal vesicles of the males, and uteri of the females, were removed and examined for gross effects. These organs were not examined histologically.


a The testosterone used in these studies was manufactured by Charles F. Pfizer and Company, Brooklyn, New York, under the trade name Synandrol (lot no. 88354). Estradiol was supplied by Ciba Pharmaceuticals Incorporated, Summit, New Jersey, as their trade name product, Ovocylin (control no. 242156).






44




Results

The data from this experiment are found in Table 8. The expected sex difference appeared in the non-castrate group with twice as many males as females dying during the first day. Testosterone increased the toxicity of Heptachlor for castrate males and females to the level of normal males and shortened the length of time necessary for symptoms of toxicity to appear. All estradiol-treated animals escaped the early period of toxicity, but some deaths did occur in both male and female rats beginning on the fourth day. At the end of the first week, the sesame-oil-treated, castrate animals showed the same male:female death ratio as the non-castrate, sesame-oil-treated animals; although the actual number of fatalities was less in the former group. During the second week, several fatalities occurred in the castrate sesame-oiltreated and in the castrate, estradiol-treated groups. These deaths were delayed longer than the expected period of delayed toxicity which usually lasts from about the fourth to the eighth day. In this particular experiment, however, this period was very mild for the control animals with only an occasional animal showing signs of central nervous system stimulation. No deaths occurred among the control groups during this period. By the end of the second week, the total number of deaths was essentially the same for all groups except the non-castrate, sesame-oil-treated female group. The latter group had the lowest number of fatalities during the experimental period.

All animals receiving testosterone or estradiol lost weight during the two week period prior to the administration of Heptachlor. Sesame-oil-injected animals showed a slight increase in body weight







45
















0 cc UIN Ln Ln Ln tn
"4
V-4 -. 8 99
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46




during this period. These changes will be tabulated along with the weight changes of other experimental groups at the end of the chapter.

The Effect of Hormone Administration on Acute Toxicity of Heptachlor in Normal Rats

Materials and Methods

This experiment was undertaken in order to determine if the effects observed in castrate animals could be demonstrated in normal animals. An additional point of investigation was to determine if Nilevar (17-ethyl-19-nortestosterone) had any demonstrable effect on the toxicity of Heptachlor. Nilevar is considered to be an anabolic steroid as it has an anabolic:androgenic ratio of 20; whereas this ratio for testosterone is about one (45, p 893). This point of investigation arose when it was observed in the previous experiment that testosterone greatly increased the toxicity of Heptachlor in castrate rats. If this activity was due to an anabolic rather than androgenic effect, Nilevar could be expected to increase toxicity, also, as it shares the anabolic activity of testosterone while having only a fraction of its androgenic activity. The experimental groups and their dosage regimen is listed below:

Group Treatment Dosage

I Sesame oil 1 ml/Kg/day
II Estradiol dipropionate 1 mg/Kg/day
III Testosterone priopionate I mg/Kg/day
IV Nilevara 1 mg/Kg/day

The same series was set up for each sex, and each group contained 8 animals. The injections were given subcutaneously every day according aThe Nilevar (control no. 2041) used in this experiment was manufactured by G. D. Searle and Company, Chicago, Illinois. The other hormone preparations were the same as those used in the previous experiment.






47




to the schedule previously listed. They were given two weeks before and one week after 200 mg/Kg of Heptachlor was given orally. These animals were observed for a period of two weeks after the administration of Heptachlor, and deaths were recorded as they occurred. Dead animals were autopsied, and gonads and accessory sex organs were removed and examined grossly for effects produced by their particular treatment. Results

When compared with controls, the data from this experiment, found in Table 9, do not show a clear-cut effect resulting from the administration of hormones to normal animals. Group 1, the sesame-oil-treated controls, showed the normal pattern of male and female toxicity; but the delayed interval of the acute toxicity period occurred later than usual in the female group.

Estradiol appeared to have increased the early toxicity of

Heptachlor in male rats. Deaths due to delayed toxicity developed more rapidly in females treated with this hormone than in the sesame-oiltreated group.

During the first day mortality was much higher in the testosterone-treated males than in other groups. Four animals from this group died; while only one death occurred in all other groups combined. At the end of the experimental period, all animals from this group were dead. Toxic symptoms appeared sooner in the testosterone-treated females than in the controls. Symptoms of the former group began on the third day and lasted until the tenth day.

The pattern of toxicity of Heptachlor in male rats which were

pretreated with Nilevar differed from that of other experimental groups.







48
















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49




Normally, most of the fatalities which will occur in a group of animals given a standard dose of Heptachlor will fall in a two or three day period. Nilevar-treated animals, however, did not follow this pattern. Deaths occurred in this group from the first through the tenth day following Heptachlor. Mortality of females given this hormone before Heptachlor was slightly lower than in the female control group.

Gross examination of the gonads and accessory sex organs of

those animals which died during the course of the experiment showed a consistent enlargement and fluid distention of the seminal vesicles of the testosterone-treated males and atrophy of these structures in animals receiving estradiol. Uteri of estradiol-treated animals exhibited increased vascularity when compared to controls; while uteri from the testosterone-treated groups were uniformly pale. No effects on either of these tissues were seen in the Nilevar-treated animals.

Changes in body weight will be tabulated along with the weight changes of other experimental groups at the end of this section.


The Effect of SKF-525-A on the Acute Toxicity of Heptachlor in Male Rats

Materials and Methods

This experiment was undertaken in order to test the hypothesis that Heptachlor was metabolized to a more toxic product; and if this metabolism could be slowed or stopped, toxicity would be decreased. SKF-525-Aa was selected for this experiment because of its known ability to inhibit the metabolism of a variety of compounds. aSKF-525-A was supplied by the Smith, Kline and French Laboratories, Philadelphia, Pennsylvania.






50




Twenty-four, 150 to 200 Cm male rats were divided into three groups of eight rats each and were given the following treatment:

Groups Treatment

A Water 10 ml/Kg
B SKF-525-A 100 mg/Kg
C SKF-525-A 100 mg/Kg every twelve hours

This treatment was given orally about forty minutes before the oral administration of 150 mg/Kg of Heptacblor, This period of time was chosen because SKF-525-A has been shown to give maximum prolongation of hexabarbital sleeping time forty minutes after ingestion (36). Since this same work had shown SKF-525-A to be active as long as fifteen hours after administration., one group was given this compound every twelve hours to determine if additional benefits could be gained by multiple dosage. All animals were observed for a period of eight days, and deaths were recorded.


Results

The graph for the data from this experiment is found in Figure 9. Prior treatment of SKF-525-A was shown to produce a marked delay of the onset of toxic symptoms when compared with water-treated controls. Examination of the graph shows that some additional protection was obtained by administration of SKF-525-A every twelve hours. Five animals from the singly treated group died after the first twenty-four hours; while only two deaths occurred in the group given SKF-525-A every twelve hours.







51


X






x






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The Effect of Propylthiouracil (flu) on Acute Toxicity Materials and Methods

This experiment was undertaken in an effort to determine if PTU induced hypothyroidism, and resulting decrease in metabolic activity (34) would effect the acute toxicity of Heptachlor in rats. Twenty-four animals of each sex were placed in three groups of eight rats each, body weights recorded, and two groups of each sex were given 0.15 per cent flU in ground Purina laboratory chow ad libitum for a period of three weeks. As a control, the remaining group of each sex was fed ground laboratory chow, alone. At the end of this period, the animals were weighed, and one group of the flU fed and the control fed group from each sex was given Heptachlor 200 mg/Kg orally. The remaining flU group from each sex was given corn oil, 5 ml/Kg and observed for possible toxic effects which might have been produced by flU. After administration of Heptachlor, the animals were observed for a period of eight days, and deaths were recorded, At death, the animals were autopsied and the thyroid glands removed and examined grossly for the effects of flU.a


Re sults,

Toxic symptoms developed rapidly in both male and female rats

which had been maintained on flU prior to the administration of Heptachlor. All males from these groups were dead within one and one-half hours, Females from the PTU-Heptachlor group developed symptoms of central nervous system stimulation within one hour, and seven were dead in less than twenty-four hours.

aThe propylthiouracil used for this experiment was obtained by crushing 50 mg tablets (control no. Y 563J) manufactured by Parke, Davis, and Company, Detroit, Michigan.






53




Animals fed regular laboratory chow developed symptoms more

slowly, although all males from this group died within twenty-four hours. Only two rats from the female group given regular laboratory chow died within the eight-day observation period. No deaths occurred in the groups fed the PTU diet and given corn oil.

The thyroids of PTU-treated animals which died during this

experiment appeared to be greatly enlarged when compared to non-PTU-fed controls. All male rats, including controls, lost weight during the experimental period. Both groups of females given PTU in their diet showed a weight reduction, while female rats given the control diet gained weight. Changes in body weight will be tabulated along with the weight changes of other experimental groups at the end of this section.


Discussion

Evidence of a possible endocrine basis for the difference in

sex response is presented in Table 10. Castration decreased the number of early deaths in both male and female rats. However, at the end of the experimental period, there was little difference between these groups and the non-castrate males which characteristically demonstrate the highest percentage mortality for a given dose of Heptachlor. Data in this table show only slight differences between any experimental groups at the end of the two week period of observation. Normal females still had fewer deaths than other groups, except for females given Nilevar. The latter group had one less fatality than the control females.

The rate at which the toxic effects of Heptachlor appeared was

accelerated in both castrate and normal animals treated with testosterone.






54







TABLE 10

COMBINED DATA FROM EXPERIMENTS CONCERNING THE
ENDOCRINE INFLUENCE ON THE ACUTE
TOXICITY OF HEPTACHLOR IN RATS


Number of Deaths on Day Indicated Following Oral Administration of Heptachlor, 200 mg/Kg Group Killed Per Cent
Tested Killed
1 2 3 4 5 6 7 8 9 10 11 12 13 14


Female

a
I Normal 0 0 0 0 0 0 22 1 0 0 0 0 0 5/8 62.5
I Normal 0 0 0 2 0 2 1 1 1 0 0 0 0 0 7/8 87.5
IlIl Normalc 0 0 1 1 1 0 1 0 1 1 0 0 0 0 6/8 75.0
IV Normald 0 0 2 0 0 0 0 1 1 0 0 0 0 0 4/8 50.0
V Normala 3 0 0 0 0 0 0 0 0 0 0 0 1 0 4/8 50.0
VI Castrate 0 0 0 0 1 1 0 0 0 2 2 0 0 1 7/8 87.5
VII Castrateb 0 0 0 1 2 0 0 1 0 0 0 1 0 2 7/8 87.5
VIII Castratec 7 0 0 0 0 0 0 0 0 0 0 0 0 0 7/8 87.5

Male
a
INormal 0 4 3 0 0 0 0 0 0 0 0 0 0 0 7/8 87.5
II Normalb 1 5 0 0 0 0 0 0 0 0 0 0 0 0 6/8 75.0
III Normalc 4 3 0 0 0 0 0 0 1 0 0 0 0 0 8/8 100.0
IV Normal d 2 0 0 1 1 1 0 0 0 2 0 0 0 0 7/8 87.5
V Normal a 6 0 0 0 0 0 0 0 0 0 0 0 0 0 6/8 75.0
VI Castrateb 2 0 0 0 1 1 0 2 0 0 0, 0 1 0 7/8 87.5
VII Castrate 0 0 0 0 1 2 1 1 0 1 0 0 0 0 6/8 75.0
VIII Castratec 6 0 0 0 0 1 0 0 0 0 0 0 0 0 7/8 87.5


aPlus Sesame Oil, 1 ml/Kg/day

Plus Estradiol Dipropionate, 1 mg/Kg/day

CPlus Testosterone Propionate, 1 mg/Kg/day

dplus Nilevar, 1 mg/Kg/day






55




This effect was prominent in castrate male and female rats, while in normal rats this response was not as well defined. These observations, together with a slower development of toxicity in the castrate male, suggest that an active role is played by the gonadal hormones of male rats in the acute toxicity of Heptacblor. An additional observation favoring this explanation of the role of male hormones is the significant increase in the testicular weight of male rats fed 0.01 per cent Heptachlor, presented in Chapter III. Since no pathological changes were found in these organs, this increase in weight could have resulted from a physiological hypertrophy caused by an increased demand for gonadal hormones. Such a phenomenon could result from continuous demand for these hormones in the metabolism of Heptachlor. A change in weight has not been previously reported following chronic exposure to Heptachlor.

Estrogenic effects on the toxicity of Heptachlor appear to be

inconsistent. Castration increased the number of deaths in female rats, but estradiol administered to castrate females failed to prevent this increase in mortality.

Changes in body weight observed in the experimental animals from both hormone experiments along with changes which were recorded for propyltbioracil-treated animals are listed in Table 11. It was observed that some of the procedures used in these experiments produced a weight loss while other procedures increased body weight. A weight change brought about by a change of the size of the lipid depot of the body could alter the space available for storage of Heptachlor and its metabolite. This, in turn, could effect the susceptibility of an animal to this compound. However, comparison of the weight changes and the







56













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number of mortalities observed in all groups did not show any relationship between weight changes and toxicity.

The delaying effects of SKF-525-A on the appearance of toxic symptoms provides support for the theory of metabolic activation of Heptachlor. It is difficult to assess the actual benefit obtained by repeated administration of SKF-525-A, due to a higher initial number of deaths in the group which was given this compound every twelve hours. As a result of this there were fewer animals to observe in this group

later in the experiment than in the group which only received a single treatment of SKF-525-A.

The opposing effects of SKF-525-A and testosterone on the

metabolism of certain drugs, as demonstrated by hexobarbital sleeping time studies, were also seen during these experiments. The effect of these compounds were reversed in their influence on Heptachlor toxicity, as testosterone increases toxicity while SKF-525-A delays or decreases toxicity. The opposite holds true for activity of hexobarbital, which is inactivated by metabolic pathways. In the later case, testosterone shortens the activity, while SKF-525-A prolongs the effects of this drug. Since inhibition of drug metabolism has not been reported for testosterone, nor stimulation of this process by SKF-525-A, it may be proposed that testosterone, by increasing the rate of biotransformation to the epoxide, increases the toxicity of Heptachlor, while SKF-525-A, through its inhibitory action on this reaction, delays the appearance of toxic symptoms. One point which must not be overlooked, however, is the probability that only the rate of metabolism is effected by these compounds, and that some metabolic conversion of Heptachlor will occur






58




regardless of the endocrine condition of the animal. In addition, there is no evidence that Heptachlor per se is inactive, and it is quite possible that the toxic effects observed in a particular animal at a given time are produced by the sum of the concentrations of both Heptachlor and its epoxide present at that time.

The action of propylthiouracil in speeding the onset of toxic reactions and increasing the susceptibility of rats to Heptachlor was unexpected, since PTU induced hypothyroidism normally slows metabolism, thereby prolonging pentobarbital sleeping time in rats (34), an effect shared by SKF-525-A. Since evidence which had been obtained in previous studies indicated that a decrease in metabolism resulted in a decreased toxicity, a possible explanation for this increase in toxicity could come from an action of PTU on some other organ or enzyme system which increases the animals susceptibility to Heptachlor or its epoxide. It is also possible that a pathway of metabolism of Heptachlor which has not yet been described may be effected. There is no experimental evidence, however, to support either possibility.














CHAPTER V


LOCALIZATION OF THE SITE OF ACTION OF HEPTACHLOR Review of the Literature

There are no reports in the literature concerning pharmacological investigations of Heptachlor. Studies with Aldrin (8) and Dieldrin (9) have indicated that these compounds produce an apparent potentiation of acetylcholine, and an increase in the sensitivity of spinal centers to this compound. However, neither a direct peripheral effect nor inhibition of cholinesterase could be demonstrated for these two cyclodiene insecticides. From these studies, it was concluded that the parasympathetic activity as well as the convulsant properties of Aldrin and Dieldrin are of central nervous system origin.

Since the principal pharmacologic effects produced by Heptachlor, and other members of the cyclodiene family, seem to consist chiefly of stimulation of the central and autonomic nervous systems, pharmacologic investigation of this compound was confined to those areas. Localization of the principal site of activity in the central nervous system and the nature of the autonomic stimulation were the objects of this phase of the investigation.












59






60




Experimental

Peripheral Effects

The Effect of Heptachlor on Blood Pressure and Respiration

Materials and Methods

The effect of Reptachior on blood pressure and respiration was tested on rabbits, cats, and dogs in the following manner:

Blood pressure measurements were taken from a polyethylene

cannula introduced into the right femoral artery. The system used for these measurements consisted of a Sanborn electrosnanometer model 121-B100, a Sanborn DC amplifier model 64-300B and a Sanborn Twin-Viso recorder model 60-1300B. The femoral vein of the opposite leg was cannulated to provide a site for intravenous injection of Heptachlor or other compounds. All animals were anesthetized with pentobarbital sodium, 35 mg/Kg intraperitoneally and were given additional pentobarbital intravenously as needed.

The amplitude and frequency of respiration, recorded only for dogs, was accomplished by using a displacement transducer, attached by means of a silk suture to the skin of the chest, a few centimeters above the xyphoid process. A Sanborn strain gauge amplifier, model 64-500B and a Twin-Viso recorder comprised the remainder of this system.

Heptachlor emulsion,a 40 mg/mI was given in 1 ml doses initially, and in some experiments in increasing amounts up to 10 ml until signs of central nervous system stimulation appeared. When tremors or convulsions developed, pentobarbital sodium was given intravenously until they were controlled.

a See Appendix II for the method used to prepare the emulsion.






61




The dogs used in these experiments were healthy 8 to 12 Kg mongrels. Healthy alley cats weighing 2 to 4 Kg, and white albino rabbits which weighed 2 to 3 Kg were also used in these studies. Results

Heptachlor, given in amount sufficient to produce tremors or convulsions, produced no perceptible change in the blood pressure in the four rabbits tested. The total amount of Heptachlor necessary to produce these symptoms was dependent on the depth of anesthesia of the animal being tested and varied from four injections of 40 mg, spaced at two-minute intervals, to nine such injections. In trials on three unanestbetized rabbits, convulsions, terminating in death, were produced by a single injection of 40 mg of Heptachlor. Convulsions in anesthetized animals were easily controlled by cautiously injecting pentobarbital sodium intravenously until the convulsions or tremors ceased. This was usually accomplished by I to 2 ml of a 25 mg/ml solution of pentobarbital.

The initial blood pressure studies on cats showed that a profound hypotensive effect was produced by the Heptachlor emulsion; however, the control emulsion a produced a similar drop in blood pressure. This fall in pressure was found to be due to a constituent of the lecithin used to prepare the emulsion. This constituent may be removed by a series of extraction with organic solvents and the success of this extraction procedure may be demonstrated by the failure of the purified product to a The method of preparation of the control emulsion is found in Appendix Ii.






62




produce a fall in the blood pressure of a barbitalized cat (46). Due to the expense and complicated nature of this procedure, purification of the lecithin used in our emulsions was not attempted and blood pressure studies in the cat were discontinued.

Dogs normally did not show a fall in blood pressure after injections with emulsions containing lecithin. All dogs used in these experiments were given 10 ml of the control emulsion intravenously at the beginning of the experiment to determine if they would react to the bypotensive constituent of lecithin. Of eight animals tested only one showed the drop in blood pressure characteristic of the effect produced in cats. The recording of this blood pressure effect is found in Figure 10. This animal was not used for blood pressure and respiration studies.

Heptachlor induced no change in the blood pressure of dogs, when given in an amount producing symptoms of central nervous system stimulation. In some instances, however, a fall in pressure did develop when convulsions were allowed to continue for a few minutes. This fall was probably secondary to the convulsions rather than a direct effect of Heptachlor since a pressure drop did not precede any convulsive episode, but was frequently seen if the convulsions were allowed to continue for a period of time. The transitory drop seen in Figure 11 could be duplicated by injection of a similar volume of the control emulsion.

The intravenous administration of 400 mg of Heptachlor produced sustained increase in respiratory rate in all animals tested. After short delay, most animals began rapid, shallow breathing, which gradually increased in depth while maintaining the increased frequency.








63





















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A second injection produced an additional increase in respiration which followed a similar pattern. In deeply anesthetized animals the first injection did not produce a rapid shallow breathing, but groups of two to three respirations which occurred at about the same frequency as the single respiration before Heptachlor was injected. Subsequent injections produced the type of rapid shallow breathing previously described. These effects are shown in Figure 11. Respiratory stimulation persisted and in most cases the animal developed tremors, fasciculations, and convulsions. The latter effects may easily be controlled with an intravenous injection of pentobarbital 25 mg/ml, administered cautiously. However, this procedure must be repeated frequently as the stimulating effects of Heptachlor may appear again after five to ten minutes. Attempts to lower the respiratory rate with pentobarbital in the two cases attempted ended in death of the animals, before the original rate of respiration had been reached. Attempts to restore respiration in these animals by the immediate injection of 400 mg of Heptachlor were unsuccessful.

These experiments indicate that Heptachlor shares the respiratory stimulating properties of other members of the cyclodiene group, but lacks the vasodepressive and bradycardia producing effects produced by Aldrin and Dieldrin.


The Action of Heptachlor on Isolated Rabbit Ileum

Materials and Methods

To determine if a parasym~pathetic response to Heptachlor could be demonstrated in smooth muscle, sections of ileum, 2 to 3 cm long, taken from a site proximal to the cecum were removed from rabbits freshly






66




killed by a blow on the head. This area of the intestine is recommended by Ludeuna (47, p 142), who states that motility and durability decrease from the cecum to the duodenal end of the small intestine. The intestine strips were placed in warm Ringer's solution at 35 to 38 0 C, and constantly aerated. Activity was recorded by means of a displacement transducer connected to a Sanborn DC amplifier and Twin-Viso recorder, as previously described for the recording of respiration. When amplitude and frequency of contraction became stabilized 1 ml of the control emulsion was introduced into the bath and allowed to remain in contact with the intestinal strip for five minutes. Immediately following this period, 1 ml of the Heptachlor emulsion containing 40 mg/ml was added to the bath and allowed to remain in contact with the intestinal strip for five to thirty minutes. At this time, all of the solution was drained from the bath and replaced with fresh, aerated solution. The preparation was then tested for its ability to respond to acetylcholine by adding enough of the latter compound to the bath, to achieve a final concentration of 1 ppin.


Results

Amplitude, frequency and tone level of these preparations were not altered by contact with Heptachlor for periods of time up to thirty minutes. All strips were found to be reactive to acetylcholine following exposure to Heptachlor. A typical recording obtained in this procedure is found in Figure 12.








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68




Isolated Lung Perfusion

Materials and Methods

In an effort to determine what part, if any, of the respiratory difficulty exhibited by animals acutely poisoned with Heptachlor was due to bronchiolar constriction, isolated rabbit lungs were perfused, using the method of Sollmann and Von Qettingen (48), described as follows: A Mariotte bottle filled with Locke-Ringer's solution was connected by means of a rubber tube to a Woulff bottle, also filled with Locke-Ringer's solution and maintained at 40 0C with a water bath. The outflow of the Woulff bottle was attached by means of a short rubber tube to a"T tube. One free end of the "T" tube was connected by means of a short piece of rubber tube to a cannula tied into the trachea, and the other end to a short section of rubber tubing which was occluded by means of a pinch clamp.

After expelling the air from the lungs, the level of the Mariotte bottle was adjusted so that air entered the bottle at a rate of about twenty bubbles per minute. When this rate appeared to be stabilized, the preparation was considered ready for use. To determine the effect of Heptachlor on the perfusion rate of this preparation, 1 ml of Heptachlor emulsion containing 40 mg of Heptachlor was injected into the rubber tubing, just above the trachea. Immediately following this, 1 ml of perfusion fluid was removed from the tube connecting the Mariotte bottle to the Woulff bottle in order to maintain a constant perfusion pressure. The rate of bubbles entering the Mariotte bottle per minute were counted every two minutes, for a total of ten times. At this time

1 ml of pilocarpine nitrate, 1:1000 solution, was introduced into the






69




trachea as previously described, and after five minutes the inflow of bubbles again recorded for one minute to determine if the preparation would respond to an agent known to produce bronchial constriction. Prior to injection of Heptachlor emulsion, an injection of the control emulsion was made to determine if the perfusion rate could be changed by the emulsion alone.


Results

The rate of perfusion was not altered by either the control or

Heptachlor emulsion in the four preparations tested. Pilocarpine nitrate tested on the same lungs produced an average drop in perfusion rate of above 50 per cent. Data obtained from these experiments indicate that Heptachlor has no direct effect on the bronchial muscles and that respiratory difficulties observed in acutely poisoned animals are probably not of a peripheral nature.


Effect on the Frog Rectus Muscle

Materials and Methods

The purpose of this experiment was threefold: 1) to determine if Heptachlor had a direct effect on skeletal muscle; 2) to determine if Heptachlor would influence the response of skeletal muscle to acetylcholine; and 3) to determine if Heptachlor could inhibit cholinesterase in vitro.

The erectus abdominus muscle of the frog was removed and set up for recording of contractions according to the method of Burn (49, p 1). Contractions were recorded using a displacement transducer and Sanborn Twin-Viso recorder as previously described for smooth muscle. For parts






70




I and 2, contraction was obtained by adding 1 ppm acetylcholine to the bath in which the muscle was immersed. This drug was allowed to act on the muscle for ninety seconds and the height of contraction recorded on stationary drum. At this time the solution was drained from the bath and fresh frog Ringer's solution was added. When the muscle had returned to its original length, the drum was advanced 0.5 cm and stopped again for the next recording. The preparation was allowed to rest five minutes, at which time 40 mg of Heptachlor, contained in 1 ml of the emulsion was added. This emulsion was allowed to remain in contact with the muscle for a period of five minutes and at this time acetylcholine, 1 ppm, was added and allowed to act on the muscle for ninety seconds as previously described.

To determine if cholinesterase could be inhibited by Heptachlor (Part 3), 0.2 ml of horse serum, containing this enzyme was incubated for five minutes with 1 ml of 1:100,000 acetylcholine solution as a control. The same procedure was followed after a ten-minute period of incubation of the horse serum with I ml of Heptachlor, 40 mg/ml. Control contractions produced by 1 ml of 1:100,000 acetylcholine solution added to the bath were recorded, initially and following the addition of the last test solution to the bath, to indicate the responsive state of the muscle. The solutions containing the acetylcholine and the serum mixtures, were allowed to react with the muscle for ninety seconds and the bath was drained and fresh frog Ringer's solution added, immediately following each test. The muscle was allowed five minutes to recover before the next test was performed. The order in which the tests were performed was: 1) acetylcholine, 2) acetylcholine plus horse serum,






71




3) horse serum containing Heptachlor plus acetylcholine, and 4) acetylcholine. A new muscle was used for each series.


Results

In the four rectus abdominus preparations tested, Heptachlor showed neither direct action on the muscle, nor potentiation of the response to a standard concentration of acetylcholine.

Cholinesterase of horse serum was not affected by incubation with Heptachior, and no response was obtained when the horse-serum-Heptachloracetylcholine mixture was added to the muscle bath. Inactivation of acetylcholine was complete in the mixture containing horse serum and Heptachlor, and in the mixture containing horse serum alone.


Central Effects

Localization of the Site of Action in the Central Nervous System of the Froga

Materials and Methods

For beginning studies, the frog is an ideal experimental system, since discrete areas of its central nervous system may be removed quickly and easily with the preparation surviving for several hours after the operation.

Decerebration is performed by severing the head of the frog with a pair of scissors at the level of the posterior margin of the eyes. In order to remove the optic lobes, the part corresponding to the tectum of the midbrain in higher animals, decerebration was first performed as aFrogs used in these studies were Rana pipiens, obtained from J. R. Schettle, Stillwater, Minnesota.






72




above, and the optic lobes, exposed by this procedure, were removed with a blunt probe. A third type of preparation produced by removing the entire brain was obtained by severing the head with scissors just caudal to the posterior border of the tympanic membrane, resulting in a spinal

animal (50, p 256).

This experiment was performed in two parts: 1) determination

of the level of transaction of the brain necessary to prevent development of convulsion and 2) determination of the level of transaction of the brain necessary to abolish convulsions, once they had developed. For Part 1, four groups of three frogs each were placed in battery jars each containing a small amount of frog Ringer's solution. Groups of three decerebratel optic lobectomized, and spinal frogs were prepared as previously described, the remaining group of three frogs served as unoperated controls. After about fifteen minutes, each frog was given 25 mg of Heptachlor in corn oil by injection into the ventral lymph sac. These frogs were then observed for twelve hours for appearance of convulsions.

The procedure for Part 2 was essentially the same as Part 1 except that the operations were not performed until convulsions began to develop. Frogs were divided into four groups of three frogs each and given 25 mg of Heptachlor in corn oil by injection into the ventral lymph sac. The frogs were then placed in battery jars which were labeled as to the type of operation which would be performed in the event that convulsions did develop. The frogs were observed for a period of twelve hours and the appropriate operation performed when indicated.






73





Results

The development of convulsions in normal frogs required from six to ten hours, and consisted of a series of clonic convulsions terminating in a tonic convulsion of a few seconds duration. This pattern was repeated following periods during which the frog appeared normal. The same activity was present in decerebrate and optic lobectomized frogs, but not in spinal preparations.

The abolition of convulsions in frogs was successful only in the spinal preparation, but, in general, this procedure was not as successful as the previous experiment. Since the appearance of convulsions develop over a long period of time, some operations were not performed until the symptoms were well advanced, and some of these animals did not recover from their surgical treatment. Observations on the successful preparations, however, showed that the convulsions could be stopped by sectioning below the brain stem, but not by removal of the cerebrum or optic lobes. These experiments indicate that the principal site of action of Heptachlor in the frog is in the midbrain.


Localization of the Site of Action in the Central Nervous System of Rats

Materials and Methods

In an effort to determine the principal site of convulsive

activity of Heptachlor in warm-blooded animals, a series of decerebratespinal rats was prepared, using a modification of the separate methods of decerebration and production of spinal animals of D'Amour and Blood (44 p 53, 54) as described below.

One day prior to use in the site of action studies, the rats

were anesthetized with chloral hydrate, 400 mg/Kg, and the spinal cord






74





exposed between the sixth and ninth thoracic vertebra. Tension was

applied to the tail, and the cord transected with a scapel. The cord

was checked for completeness of section and the incision closed with silk suture and Michael clips. Spinal section at this level does not

interfere with respiration.

The same animal was then prepared for decerebration by making a mid-line incision over the region of the cerebellum. A hole was drilled just caudal to the transverse sinus, until the dura could be seen. This hole was then filled with bone wax and the incision closed with Michael

clips.

A third operative procedure performed at the same time consisted of disecting the trachea free from overlying muscle and surrounding I fascia, so that a tracheal cannula could later be inserted in a short

period of time.

These procedures resulted in an animal which could be decerebrated, and in which a tracheal cannula could be installed in less than two

minutes. In addition, spinal reflex activity had returned to the areas below the section on the day following the operation and the animal was

suitable for the demonstration of the effects of drugs on spinal activity.

On the day following the preparatory operations the animal was lightly anesthetized with ether and the clips were removed from the
head, exposing the hole caudal to the transverse sinus. A blunt probe

was inserted directly downward and moved from side to side several times

in order to insure complete decerebration. Sectioning at this level is

said to separate all connection rostral to the pons (44, p 54). The

animals were turned on their backs, and the trachea rapidly exposed and

cannulated, using polyethylene tubing of appropriate size.






75




The animals were then observed for the appearance of decerebrate rigidity which appeared, in successful experiments, in five to fifteen minutes. This was comprised chiefly of extensor rigidity of the forelimbs, as the hindlimbs were previously severed from their cerebral connections by spinal sectioning.

When the animal's respiration appeared to be stabilized, a test dose of 0.5 ml of the control emulsion, was administered via the sublingual vein, and the animal was observed for effects produced by the emulsion alone. After two minutes, a dose of 0.5 ml of the Heptachlor emulsion containing 20 mg of Heptachlor was given in a similar manner.

The response to Heptachlor given under these conditions could fall into three broad categories:

1) No effect, indicating origin of the convulsive activity lay in the cerebrum.

2) Convulsive activity in the areas still innervated by the brain stem and upper spinal cord, and not in the areas served by the spinal cord distal to the section, indicating that principal activity

lay in the brain stem.

3) Convulsive activity both above and below the cord section indicating direct action of the spinal cord itself, not dependent on higher centers.

This method has the advantage that the site of action may be found using a single animal instead of two, as would be required for separate decerebrate-spinal animals. One serious drawback, however, is a high number of unsuccessful experiments, since only six out of twenty attempted preparations developed signs of decerebrate rigidity






76




and were considered acceptable f or use in these experiments. In the other animals, respiration ceased shortly after decerebration and the animals died if they were not maintained on a respirator pump. These animals were considered unsatisfactory for use.


Results

Immediately following the injection of Heptachlor, a marked

increase in rate and depth of respiration appeared. After three to five minutes, the convulsant effects of this compound could be seen through the decerebrate rigidity already present in the animal. These effects consisted of tremors, fasciculations and clonic spasms of the muscles of the upper extremities. Occasionally these spasms would terminate in a prolonged tonic extension. During intervening periods, the animal returned to a state of decerebrate rigidity.

There was no increase in tone or spontaneous activity in the muscles of the hindlimb of the animal indicating an absence of spinal or direct skeletal muscle effect of Heptachlor;

All animals were dead within one hour after injection of Heptachlor. Respiration ceased before cardiac arrest occurred.

From these experiments it can be concluded that in the rat, as in the frog, the principal site of activity of Heptachlor lies in the brain stem. It is probable, however, that Heptachlor, as is the case of other centrally acting compounds, effects other areas of the central nervous system, and its classification as a brain stem stimulant is more one of convenience than precise anatomical localization.






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Discussion

The two pharmacologic properties described for Heptachlor -central nervous system stimulation and parasynipathomimetic activity -apparently have similar origins. The absence of a response in vivo by tissues which respond readily to autonomic drugs leads to the conclusion that direct stimulation of autonomically innervated structures plays no part in the symptoms of acute Heptachlor poisoning.

No evidence favoring the inhibition of cholinesterase, direct

action on skeletal muscle, or potentiation of acetylcholine by Heptachlor was obtained on the isolated frog rectus muscle. These findings, coupled with the observation that increased secretory activity in rats was most prominent following periods of convulsions indicates that the central nervous system is the origin of both convulsive and autonomic effects of Heptachlor. This conclusion was also reached in studies of Dieldrin

(9) and Aldrin (8). However, these compounds were reported to have produced bradycardia, vasodepression and potentiation of acetylcholine. None of these effects were seen with Heptachlor.

Respiratory stimulation was the most consistent effect produced by Heptachlor in normal-anesthetized and decerebrate animals. This effect became apparent imediately following the intravenous injection of large doses of this compound. The cumulative nature of this compound is demonstrated by the appearance of symptoms after a series of injections which, individually, would not provoke a response. Respiratory stimulation could be increased further by additional injections of Heptachlor. Tremors and convulsions which usually followed the initial respiratory stimulation were easily controlled by intravenous






78




pentobarbital. However, respiratory rate was more difficult to control. The deaths of two animals which resulted during attempts to restore the respiratory rate to normal suggest that efforts to antidote Heptachlor by intravenous barbiturates must be made with extreme caution. This sudden cessation of respiration in animals which had been hyperventilating as a result of the stimulating effects of Heptachlor, may be due to a sudden removal of this stimulation by the barbiturates, leaving a state of fatigue brought on by the prolonged over-activity. Additional contributory factors would be a lack of the normal carbon dioxide stimulating effect, since hyperventilation would be expected to result in a low partial pressure of circulating carbon dioxide. The blood of such an animal would also be well oxygenated, and an additional means of respiratory stimulation would not be available. Respiration arrested in the manner described was not restored by additional Heptachlor injections.

The principal locus of activity of Heptachlor, in the frog and rat, is the brain stem. In the frog, removal of the cerebrum failed to stop or prevent the convulsions following injections of Heptachlor, while removal of the entire brain successfully blocked the appearance of convulsions. In these respects, and in the general appearance of the convulsion, Heptachlor is similar to the brain stem stimulants such as Metrazole. However, no central nervous stimulant has its effects confined to a discrete area of the central nervous system, but different areas of the brain seem to vary in susceptibility to a given dose. Thus Metrazole) a typical brain stem stimulant, in high doses will also stimulate the spinal cord. In view of the many interconnecting pathways of the central nervous system it is impossible to assign discrete areas






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of the central nervous system as the site of action of Heptachlor, or any other central nervous system stimulant. The respiratory center of the medulla seems to be especially sensitive to Heptachlor as the effects of stimulation are first to appear in this area and are not controlled with measures used successfully for the control of convulsions and tremors.

Evidence favoring medullary action rather than action of the

carotid sinus as the cause of respiratory effects of Heptachlor, arises from the absence of inspiratory gasps characterized by compounds such as lobeline or cyanide which stimulate this area directly (23, p 298).















CHAPTER VI


SUMMARY AND CONCLUSIONS


The LD50 values for male and female rats determined in this study were 59 and 132 mg/Kg respectively. No rats from either sex survived the first two weeks of a six-month chronic feeding period, when fed a diet containing 1 or 0.1 per cent Heptachlor. During the remainder of this period, female rats proved to be more susceptible to the lethal effects of the higher concentration of Heptachlor than male rats.

Significant weight changes were found in the liver of male and female rats fed 0.01 per cent and 0.001 per cent Heptachlor. A significant change in kidney weight was also noted with the higher concentration in both sexes. An increase in testicle weight was found in male rats fed 0.01 per cent Heptachlor.

Pathological findings of tissues from the organs of Heptachlor fed animals consisted chiefly of degenerative changes of the liver. These changes were much more extensive in female than in male rats.

An endocrine basis for the difference in sex response was found using castrate and hormone treated animals. Castration delays the appearance of toxic symptoms of Heptachlor in male rats, while testosterone treated rats developed symptoms more rapidly than other groups. Estradiol was without apparent effect on the toxicity of Heptachlor in either sex.



80






81




Evidence suggesting metabolic activation of Heptachior was

obtained from experiments showing that the appearance of toxic symptoms of Heptachlor could be accelerated by testosterone pretreatment and delayed by SKF-525-A, compounds known to speed up and retard respectively,

the rate of biotransformation of a number of compounds.

Propylthiouracil, given in the diet of male and female rats

increases the speed of onset and mortality of Heptachlor in the female and to a lesser extent, the male.

Heptachlor was shown to have no direct effect on isolated smooth or skeletal muscle. No potentiation of acetylcholine or inhibition of cholinesterase could be demonstrated, and blood pressure was not effected.

Heptachlor given intravenously produced a sustained increase in the respiratory rate in both decerebrate and normal anesthetized animals. This stimulation appeared before other symptoms of central nervous system stimulation could be seen.

Muscle tremors and convulsions of Heptachlor poisoned animals are easily controlled with pentobarbital sodium, but frequent administration are required, due to the persistance of Heptachlor stimulation. Respiration stopped with pentobarbital in Heptachlor poisoned animals could not be reinitiated by this compound.

The activity of Heptachlor in frogs and rats was found to be

confined to the central nervous system, with the principal site of action being the brain stem. This finding coupled with failure of Heptachlor to produce any peripheral response indicates that both the convulsive and autonomic effects of Heptachlor are mediated through the central effects of this compound.







































APPENDICES















APPENDIX I


PREPARATION OF THE CORN OIL SOLUTION OF HEPTACHLOR


Usual organic solvents in which Heptachlor is soluble are either toxic or have undesirable pharmacologic activity. Since Heptachlor is insoluble in water, it was necessary to use a vegetable oil as the solvent for oral administration in the acute toxicity studies. Corn oil was selected because of its ready availability. The solutions were prepared by dissolving a carefully weighed amount of Heptachlor in a small amount of hot corn oil and then diluting to volume. In all cases the quantity of drug to be given per kilogram of body weight was present in 5 ml of solution.




























83















APPENDIX II


PREPARATION OF THE HEPTACHLOR EMULSION FOR p PARENTERAL AND ISOLATED TISSUE STUDIES


For intravenous administration and isolated tissue studies, the

oil solution of Heptachlor was unsuitable because of its immisciblity

with physiologic media. Organic solvents, for reasons previously stated, were also unsuitable. To solve this problem, an emulsion

similar to one used for intravenous administration of lipids to humans

(46) was prepared. The formula of this emulsion is listed below:

Corn oil containing 20% w/v Heptachlor 20 ml
Lecithin 1 Gin
Pluronic F68 0.2 Gm
Normal saline 80 ml

To prepare the emulsion, the lecithin was dissolved in the corn oil

solution of Heptachlor, and this solution was slowly stirred into the

saline in which the Pluronic F68 had previously been dissolved. The

resulting crude emulsion was then passed five times through a MantonGaulin two-stage laboratory homogenizer at a pressure of 2500 pounds

per square inch. This procedure is stated to result in a very stable

emulsion, miscible with physiologic media, having a particle size of

one micron or less (46) and containing 40 mg/ml of Heptachlor. A

control emulsion containing 20 per cent pure corn oil was prepared in

a similar manner.







84















APPENDIX III


CALCULATION OF THE LD5() BY THE LITCHFIELD AND WILCOXON METHOD


Calculation of the LD50 by this method is accomplished by first

plotting all responses exclusive of the 0 and 100 per cent effects on

log probability paper (Codex 3128). A line is then fitted to these points by inspection. The corrected 0 and 100 per cent effects are

obtained using information obtained from the graph and a nomograph

contained in the original article. A (Chi)2 test is then performed to determine if the line fits the data. This method also provides a means for the determination of the 95 per cent confidence limits of the LD50

and the slope of the line. The original paper presenting this method

of calculation contains several nomographs and tables which greatly

simplify the calculations (21).


I'ale Rats

Total contribution of (chi)2 from Table 5 0.335

Total number of animals 48

Number of doses K 6

I Animals/dose 48/6 8

(Chi) 0.335 x 8 2.680

Degrees of Freedom N K-2 = 4

(Chi)2 for N 4 is 9.49. Since 2.680 is less than 9.9 the data are

not significantly heterogenous and the line is a good fit.




85






86




From the graph of LD50 the following values were found:

LD84 84 mg/Kg LD 59 mg/Kg LD16 = 42 mg/Kg

Calculation of the Slope Function S

S- LD84/LD50 + LD50/LD16 1.415
2

Calculation of the confidence limits of the LD50 for 19/20 probability limits

LD50 x FLD50 upper limit D50 I FLD50 lower limit

FLD50- S2.77/ VNN' total number of animals tested at those doses whose expected effects were between 16 and 84 per cent 24

FLD50 1.4152.77/ V2' 1.2

LDs0 x FLD50 59.0 x 1.2 70.8 mg/Kg LD50 / F5 59.0 / 1.2 49.1 mg/Kg

50

Sumary

LD50 59(70.8 to 49.1) mg/Kg

S = 1.415(1.14 to 1.175)


Female Rats

The data obtained from the determination of the LD50 are given in Table 6. Calculations were carried out in the manner previously described, and only the results are given. The graph for these data is found in Figure 8.






87





Total number of animals 483 Number of doses K w 6 Animals/dose 8 Total contribution to (Chi)2 from Table 6 0.625 I (Chi)2 for 6 degrees of freedom 12.6. Since 5.0 is less than 12.6,

the line is a good fit.

The following values were obtained from the graph: LD 84 195 mg/K& LD 50w132 mg/Kg LD 16 95 mg/Kg The calculated 19/20 probability limits for the slope function were: upper limit 1.35 lower limit 1.17 The calculated 19/20 probability limits for the LD 5 were: upper limit 154 lower limit 114 The value of the slope function was found to be 1.475

Summary of data:

LD~ 50 132(114 to 154) mg/Kg S 1.475(l.85 to 1.17)














BIBLIOGRAPHY


I. Smyth, Henry F., Jr., Agric. Food Chem., 4:644 (1956).

2. Frear, Donald E. H., "Chemistry of the Pesticides," 3rd ed., Do Van
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The Blakiston Division, McGraw-Hill Book Co., New York, N. Y.,
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BIOGRAPHICAL SKETCH


The author was born on May 1, 1933 at Holly Springs, Georgia. He graduated from Canton High School, Canton, Georgia, in 1950 and entered Mercer University the fall of that year.

He received a B.S. degree in pharmacy from Southern College of Pharmacy, Atlanta, Georgia, in 1954. Following graduation he served in the army until August, 1956.

1 The author began his graduate studies at the University of

Florida during the Fall Semester of 1956, and has been in attendance at that institution since that time. He expects to receive the Pb.D. degree in February, 1962.

The author is a member of Kappa Psi pharmaceutical fraternity,

Rho Chi honorary pharmaceutical fraternity, Gamma Sigma Epsilon honorary chemical fraternity, Phi Sigma honorary biological fraternity and is an associate member of the American Association for the Advancement of Science. He has been the recipient of a Graduate Council Fellowship and is a fellow of the American Foundation for Pharmaceutical Education.














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This dissertation was prepared under the direction of the Chairman of the candidate's supervisory committee and has been approved by all members of that committee. It was submitted to the Dean of the College of Pharmacy and to the Graduate Council and was approved as partial fulfillment of the requirements for the degree of Doctor of Philosophy.



February 3, 1962



jp,

Dean, College of Pharmacy




Dean, Graduate School

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Chairman























































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