Therapeutic management of avian lead intoxication

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Title:
Therapeutic management of avian lead intoxication
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v, 239 leaves : ill. ; 29 cm.
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English
Creator:
Mautino, Michele, 1959-
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Subjects

Subjects / Keywords:
Lead Poisoning -- drug therapy   ( mesh )
Edetic Acid -- toxicity   ( mesh )
Edetic Acid -- pharmacology   ( mesh )
Edetic Acid -- therapeutic use   ( mesh )
Pentetic Acid -- toxicity   ( mesh )
Pentetic Acid -- pharmacology   ( mesh )
Pentetic Acid -- therapeutic use   ( mesh )
Succimer -- toxicity   ( mesh )
Succimer -- pharmacology   ( mesh )
Succimer -- therapeutic use   ( mesh )
Penicillamine -- toxicity   ( mesh )
Penicillamine -- pharmacology   ( mesh )
Penicillamine -- therapeutic use   ( mesh )
Chelation Therapy   ( mesh )
Copper   ( mesh )
Zinc   ( mesh )
Pigeons   ( mesh )
Genre:
bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1993.
Bibliography:
Includes bibliographical references (leaves 220-238).
Statement of Responsibility:
by Michele Mautino.
General Note:
Typescript.
General Note:
Vita.

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University of Florida
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All applicable rights reserved by the source institution and holding location.
Resource Identifier:
oclc - 50514197
ocm50514197
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AA00011219:00001

Full Text










THERAPEUTIC MANAGEMENT
OF AVIAN LEAD INTOXICATION


















By

MICHELE MAUTINO

















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

UNIVERSITY OF FLORIDA

1993















TABLE OF CONTENTS


ABSTRACT . . ... iv

CHAPTERS

1 INTRODUCTION . . 1


2 THERAPEUTICS OF LEAD INTOXICATION . 8

Chemistry of Chelation . 8
Chelating Agents . .. 10
Polyaminocarboxylic Acids . .. 12
Dithiol Chelators . ... 19
Monothiol Chelators . .. 20
Dimercaptopropanol Derivatives .. 21
Diethyldithiocarbamate . .. 26
Combinations of Chelating Agents .. 26

3 OBJECTIVES . . 29

Use of Chelating Agents in Avian Species .. 30
Assessment of Chelating Agents ... 31
Selection of Chelating Agents .. 31
Chelating Agent Efficacy . .. 33
Chelation of Endogenous Metals .. 37
Chelating Agent Toxicity . .. 40

4 Materials and Methods . .. 45

Experimental Methods . .. 45
Experimental Birds and Induced Lead Intoxication 45
Preparation and Administration of Chelating
Agents . . 45
Sample Analysis . . 46
Lead . . 46
Copper and Zinc . .. 47
Hematological/Serum Chemistry Measurements 48
Experimental Design and Sample
Collection . . 50
Trial I: Chelating Agent Efficacy .. 50
Trial II: Chelation of Endogeous Metals 51








Trial III: Assessment of Subacute Toxicity 52
Statistical Analysis. . .. 54
Trial I and II . .. 50
Trial III . . .. 51


5 RESULTS . . .. ... 57

Trial I: Chelating Agent Efficacy .. 57
Trial II: Chelation of Endogeous Metals .. 79
Trial III: Assessment of Subacute Toxicity 113

6 DISCUSSION . . 177

Chelating Agent Efficacy . .. 177
Chelation of Lead in the Blood .. 177
Chelation of Lead in Tissues ... 179
Bone . .. 179
Soft Tissues . 180
Clinical Efficacy . .. 181
Chelation of Endogenous Metals .. 183
Serum Metal Concentrations ... 183
Tissue Metal Concentrations .. .184
Chelating Agent Toxicity . .. 187
Limitations of Experimentation . 193
Conclusions . ... 195

APPENDIX . . ... 198

REFERENCES . . 220

BIOGRAPHICAL SKETCH . . 239














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

THERAPEUTIC MANAGEMENT OF AVIAN LEAD INTOXICATION

By

Michele Mautino

December 1993


Chairperson: Stephen M. Roberts, PhD
Major Department: Veterinary Medicine

Lead poisoning is the most common intoxication occurring

in avian species and causes significant mortality in

waterfowl, raptors and psittacines. Four chelating agents,

ethylenediamine tetraacetate (EDTA), diethylene triamine

pentaacetic acid (DTPA), dimercaptosuccinic acid (DMSA) and D-

penicillamine (PA), were evaluated to determine efficacy of

lead chelation, effects of chelation on endogenous copper and

zinc and subacute toxicity in birds. The efficacy of DMSA and

PA in removing lead from tissues and lack of subacute

toxicity, in addition to the oral route of administration,

contribute to the potential utility of these drugs for the

treatment of avian lead intoxication.

To assess efficacy of chelation and effects of chelation

on copper and zinc, domestic pigeons (Columba livia domestic,

n=70) were treated with lead acetate daily via oral gavage for








12 days. Experimental groups of lead-intoxicated birds were

subsequently treated with one of four chelating agents at

three dosage levels for each chelating agent; chelation

therapy was administered twice daily for eight days and lead,

copper and zinc concentrations in blood were measured daily.

Sequential necropsies were performed at 48 hour intervals

during chelator treatment to measure lead, copper and zinc

concentrations in liver, kidney and bone tissues. Metal

concentrations in blood and tissues were measured by

electrothermal atomic absorption spectrophotometry. Blood and

tissue concentrations of lead, copper and zinc during

chelation therapy are presented and discussed.

To assess subacute toxicity, pigeons (n=60) were treated

with four chelating agents, each at three dosage levels, for

15 days. Hematological and serum chemistry parameters were

measured at 72 hour intervals during treatment. Significant

differences in several serum chemistry parameters were noted

when comparing chelators and dosage levels; subacute toxicity

was less in birds treated with DMSA or PA than EDTA or DTPA.













CHAPTER 1
INTRODUCTION




The physical and chemical properties of lead and its

derivatives make it suitable for a wide spectrum of

applications; it is the most commonly used nonferrous metal in

the modern industrial age. Metallic lead has been used

extensively by mankind for the last 8,000 years. Lead beads

dating from 6500 BC have been found in Asia Minor and a lead

statue dated at 3800 BC was found in Abydos, Egypt (Gale and

Stos-Gale, 1981). Lead poisoning was reported by ancient

Greek, Roman and Arab physicians; thus, this malady is not

necessarily a byproduct of modern technology. Essential to the

extraction of iron from crude ore, lead smelting was the

harbinger of the iron age (Nriagu, 1983). Present day

environmental lead pollution results primarily from the use of

alkyl lead as a gasoline additive, although many industrial

occupations still entail an exposure to hazardous levels of

lead.

The high incidence of human lead intoxication has

resulted in a large body of scientific literature concerned

with the pathogenisis, diagnosis and treatment of lead

poisoning. The major biochemical effects of lead can be








2

broadly grouped into three classes. First, lead has a high

affinity for sulfhydryl groups and this affinity can result in

the inhibition of biologically essential sulfhydryl-dependent

enzymes. Second, lead mimics calcium in metabolism due to

similar chemical properties. Lead is able to successfully

compete with calcium and exert deleterious effects on

mitochondrial respiration and neurological function. Third,

lead has been shown to affect the synthesis of DNA and RNA

thus altering genetic information. Of all the effects of lead,

this is the least well understood. The response of an organism

to lead exposure is graded and directly related to the

magnitude of exposure; thus, lead intoxication constitutes a

continuous spectrum from subtle psychological changes to

severe physiological derangements and death.

Lead intoxication occurs commonly in wild and

domesticated animals dwelling in our industrialized society.

Cattle and dogs are common victims of lead intoxication but

reports of poisoning in other species fill the literature.

Lead intoxication has been reported in many wild mammalian

species including rodents and raccoons (Kilham et al., 1962;

Sanderson and Thomas, 1961). Confirmed lead intoxication has

occurred in exotic zoo animals including gorillas, baboons,

monkeys, orangutans, mandrills, ferrets, sea lions, seals,

foxes and panthers (Lumeij and Dorrestein, 1986; Zook et al.,

1972). Observations of clinical cases of human lead poisoning

and experimental research on laboratory rodents have been










utilized in an attempt to diagnose and treat lead intoxication

in these domesticated and exotic species. Despite efforts at

eradicating the causal agents of lead intoxication at zoos,

excessive environmental lead exposure is still a significant

cause of mortality in captive species.

Lead intoxication is the most common poisoning occurring

in both caged and wild avian species. Surveys of tissue lead

concentrations reported elevated levels in 52 species of wild

birds and 21 species of marsh birds (Johnson et al., 1982;

Bagley and Locke, 1967; Hall and Fischer, 1985b).

Environmental lead exposure resulting in death also has been

reported in upland game birds (Locke and Bagley, 1967), swans

(Sears et al., 1980; Simpson and Hunt, 1979; Rosen and

Bankowski, 1960; Trainer and Hunt, 1965; Birkhead et al.

1982), geese (Cook and Trainer, 1966; Howard and Penumarthy,

1979), loons (Locke et al., 1982), gulls (Munoz et al., 1976),

albatross (Sileo et al., 1990), egrets and cranes (Honda and

Tatsukawa, 1985; Kennedy et al., 1977). Review of the

literature reveals that waterfowl, raptors and marshbirds, of

all avian species, have the highest mortality rates'due to

lead intoxication (Andrews and Longcore, 1969; Clemens et al.,

1975). Elevated tissue lead concentrations and evidence of

sublethal lead intoxication are often found secondary to other

causes of death in many avian species.








4

Lead toxicosis is a significant waterfowl mortality

factor in many parts of North America, and has received the

attention of wildlife biologists for several decades.

Quantification of waterfowl mortality from elemental lead

ingestion is difficult at best; however, estimates of annual

losses of 2-4 percent of the population are accepted as

accurate (Bellrose, 1959; U.S. Fish and Wildlife Spec. Serv.

Rept., 1976; Baker and Thompson, 1980). This can be translated

into losses of 2-4 million ducks and geese per year in the

United States alone (Roscoe and Neilson, 1979; Wobeser, 1981;

Sanderson and Bellrose, 1986; Feierabend, 1983).

The feeding habits of many species of waterfowl and marsh

birds place these birds at high risk for environmental lead

exposure. The causal factor in this often fatal toxicosis is

the ingestion of lead in the form of spent shot from the

firearms of waterfowl hunters. It has been estimated that

more than 1400 lead pellets are expended for each hunter

bagged bird (Danell et al., 1977; Bellrose, 1964). Several

million hunters in the United States expended more than 6,000

tons of lead shot annually and this shot is dispersed aver the

marshes, lakes and estuaries which constitute waterfowl

habitat (Andrews and Longcore, 1969; U.S. Fish and Wildl.

Serv. Spec. Rept., 1976). In the last decade, several heavily-

hunted areas of the United States have been restricted to

hunters utilizing steel shot to reduce the amount of lead shot

expended in the environment. The steel shot versus lead shot








5

restrictions are a widely debated issue, but even if

restrictions are universally implemented they will not reduce

current environmental lead contamination (Calle et al., 1982).

Despite these efforts to prevent further environmental lead

exposure, the effects of lead shot ingestion are apparent in

the signs of chronic lead intoxication or the annual die-offs

occurring in many species of waterfowl and marsh birds.

Environmental lead exposure has also proven detrimental

to many species of raptors. While the reproductive potential

and high fecundity rate of many avian species reduce the

"impact of lead-related mortality on their populations, the low

numbers and reproduction rates of raptors magnify the effects

of individual mortality. Lead poisoning can occur directly in

raptors by the ingestion of spent lead shot embedded in prey

or secondarily by the ingestion of biologically incorporated

lead in the tissues of prey. Both of these modes of lead

exposure result in significant levels of mortality (Custer et

al., 1984; MacDonald and Randall, 1983; Gilsleider and Oehme,

1982; Redig et al., 1980; Reiser and Temple, 1982). Lead

intoxication has caused fatalities in red-tailed" hawks

(Sikarskie, 1977; Redig et al. 1980), prairie falcons (Benson

et al. 1974), goshawks (Garner, 1991), American kestrels

(Franson et al., 1983; Hoffman et al., 1985a; Hoffman et al.,

1985b; Pattee, 1984), Andean condors (Locke et al., 1969), and

vultures (Lumeij, 1985). Lead toxicosis has also occurred in

endangered species of raptors such as the bald eagle (Mulhern








6

et al., 1970; Jacobsen et al., 1977; Redig et al., 1980;

Kaiser et al., 1980; Hoffman et al., 1981; Pattee et al.,

1981), the golden eagle (Borg, 1975), the California condor

(Redig et al., 1980; Janssen et al., 1986) and the peregrine

falcon (DeMent et al., 1986). Bald eagles and other species

which feed on dead or crippled prey seem especially vulnerable

to lead intoxication. Prey frequenting the same wetland

habitats as waterfowl are likely to contain lead shot or high

levels of tissue-bound lead (Pattee and Hennes, 1983) and be

demonstrating signs of lead toxicity; these animals are easy

prey for raptors. High concentrations of lead have been found

in the blood of many raptor species with and without overt

signs of intoxication; the significance of sublethal levels of

lead in birds of prey is not well understood.

The prevalence of lead intoxication in exotic cage and

aviary birds is well documented in the literature.

Substantiated intoxication has been reported in macaws (Morris

et al., 1985), cockatoos (Panigraphy et al., 1979),

cockatiels, parrots and conures (Beyer et al., 1988; Giddings,

1980; McDonald and Lowenstein, 1983; McDonald, 1986;'Fudge,

1982; Janssen et al., 1979; Petrak, 1982). Lead intoxication

in caged birds results from ingestion of elemental lead

present in materials used to construct aviaries and cages, but

bone meal which is used as a calcium supplement by many

aviculturists may also contain high concentrations of lead.

Pet birds often have access to sources of lead such as stained










glass windows or ornaments, batteries, solder, old paint,

metal foil, wire or lead weighted items in the home (Harrison

and Harrison, 1986; Oglesbee, 1991). Many exotic avian species

are no longer imported into the United States and are

endangered in their country of origin; individual birds are of

great value to zoological institutions, aviculturists, or pet

owners.

Review of the literature pertaining to avian lead

intoxication reveals that three groups of birds are at a

distinctly high risk for lead exposure: (1) waterfowl and

marsh birds, (2) raptors, (3) Psittacine and other exotic cage

birds. Most of the literature pertaining to avian lead

intoxication has focused on the ecological impact of lead on

waterfowl. A few isolated case reports on the diagnosis and

treatment of lead toxicity in avian species exist in the

literature; however, no experimental therapeutic trials have

been conducted. Additionally, the kinetics of lead absorption,

storage and excretion in birds have not been fully elucidated.

Currently accepted chelation therapy for acute lead

intoxication in avian species has been extrapolated from dated

human literature. Recent advances in mammalian chelation

therapy have not been explored in avian species. Given the

prevalence of lead intoxication in avian species, the

determination of the kinetics of lead intoxication as well as

the development of effective therapeutic chelation regimes are

significant research objectives.














CHAPTER 2
THERAPEUTICS OF LEAD INTOXICATION


Chemistry of Chelation


If a molecule is to function as a chelating agent, it

must fulfill a minimum of two essential conditions: 1) the

molecule must possess donor atoms capable of donating a pair

of electrons to the metal ion and 2) the donor atoms must be

situated so as to permit formation of a ring with the metal

ion as a closing member. Metal ions have coordination numbers

ranging from 2-10, the most common coordination number being

6. In aqueous solutions the coordination requirements of the

metal ion are satisfied by water molecules. When one of these

water molecules is displaced by a different electron donor, a

metal complex is formed. A metal chelate is a metal complex in

which two or more of the 6 coordinated donor groups are bound

together by chemical bonds. The formation of a chelate tends

to reduce the number of "free" coordination positions through

which metal reactions take place and also reduces the acidity

of the remaining coordinated water molecules. Replacement of

water by other molecules may stabilize an unstable oxidation

state. When this coordination compound possesses specific

structural features (resonance or steric effects) the

stability of the metal chelate is greatly increased.










As a general rule, the metal ion occupies a central

position in the chelate molecule and the chelate may carry a

positive, negative or neutral charge. The central metal atom

is covalently bound to its immediate neighbors as the metal

ion accepts an electron pair from each nonmetal atom; the

former the acceptor and the latter the donor. Another commonly

used convention is calling any negative ion or polar molecule

bound to a metal ion a ligand and the bond between them a

metal-ligand bond. Most ligands are attached to the metal atom

by more than one covalent bond thus forming a heterocyclic

chelate ring.

The chelated metal does not exhibit the reactions common

to the free metal, but the nature and oxidation state of the

central metal ion endow the chelate with its individual

characteristics. Many examples of the individuality of

chelates occur in nature: magnesium in chlorophyll, iron in

hemoprotein, cobalt in vitamin B12, and copper in hemocyanin.

All endow their respective chelate molecules with unique

properties and functions. In biological systems, the important

donor atoms of endogenous ligands are nitrogen, oxygen, sulfur

and phosphorous. Metals can be divided into two classes based

upon their molecular properties: Class A metals such as

calcium, barium and strontium form the most stable complexes

with donor atoms of nitrogen and oxygen while Class B metals

such as mercury, gold or silver form stable complexes with

sulfur and phosphorous. Lead is capable of forming complexes








10

with nitrogen, oxygen or sulfur therefore possessing both

class A and class B characteristics (Chisolm, 1968). Lead is

bonded to nitrogen and oxygen in the EDTA chelate, to sulfur

in the BAL chelate and to sulfur and nitrogen in

D-penicillamine (Martell, 1989).


Chelating Agents



The therapeutic goal in all types of metal intoxication

is to reduce the systemic metal burden below a critical

threshold. To achieve this goal it becomes necessary to study

not only the metabolic behavior of the metal, but also the

possibility of influencing the toxic effects of specific

metals. Acquiring knowledge about the metabolism and toxicity

of a metal is an essential prerequisite for the development of

an optimal treatment regimen as well as for the evaluation of

the therapeutic efficacy of that regimen.

Removal of internal metals by the formulation of soluble

chelates is the most promising approach to the therapeutic

treatment of metal intoxication. While the majority of

research has concentrated on the excretion of metals as a

therapeutic goal, the evaluation of the eventual therapeutic

benefit of chelating agents has been neglected. A failure to

accelerate metal excretion cannot be interpreted as a lack of

therapeutic efficacy; the therapeutic effect may be the










transfer of metal within the organism or the formulation of

insoluble or inert complexes.

The influence of an antidote on the retention or

excretion of a given metal may or may not be paralleled by an

equivalent effect on the toxicity of that metal. A chelating

agent may cause a shift of metal to a critical organ or may

act additively or synergistically to increase the toxicity of

metal exposure. Metals may cause irreversible damage where

subsequent mobilization will be without benefit. The

definition of therapeutic efficacy is crucial; enhanced metal

excretion is meaningless from a therapeutic point of view if

it is not paralleled by a decreased metal concentration in

critical organs.

Treatment of clinical and experimental lead intoxication

has proven that the condition is most responsive when therapy

is initiated early in the course of intoxication.

Establishment of an optimal treatment schedule to provide

maximum therapeutic effects and avoid deleterious side effects

requires that the potential limitations of metal antidotes be

recognized. The distribution, metabolic effects and excretion

of lead must be evaluated as well as the distribution,

metabolism and excretion of each chelating agent. It is

evident that diagnostic tests to guide the dose of metal

antagonists and duration of treatment are essential.

The first chelate molecules discovered were bidentate

chelators possessing two donor atoms; this claw-like mode of










attachment caused early investigators to suggest the name

"chelate" derived from the Greek term for claw. The number and

variety of molecules recognized as having the ability to

chelate metals grew rapidly during the early 1900s. Early

research focused on the chelation of metals by ethylenediamine

and oxalic acid; these chelating agents were utilized by

chemists to study the structure of metal complexes in the

laboratory. In the mid-1900s, chelating agents were first used

therapeutically for metal intoxication; the success in these

initial clinical cases resulted in widespread research

searching for agents possessing increasing levels of efficacy

with decreased toxicity.
Polvaminocarboxylic Acids

Two compounds, ethylenediaminetetraacetate (EDTA) and

diethylenetriamine pentaacetate (DTPA), have been the most

widely investigated and utilized in the treatment of lead

intoxication. Because these compounds form highly stable

chelates with calcium, the free sodium forms of these acids

are highly toxic. The calcium chelates are utilized for

therapeutic purposes, except in isolated exceptional cases

(i.e. hypercalcemia, atherosclerosis and scleroderma). The

chelation properties and pharmacology of the sodium forms will

not be considered in this discussion of therapeutics; thus,

EDTA and DTPA denote Na(CaEDTA) and Na(CaDTPA) respectively.

Numerous research projects have documented that

polyaminocarboxylic acids chelate divalent and trivalent








13

ions binding them to nitrogen and oxygen in a five membered

ring structure. Injection of rats with radio-labeled calcium

chelates of EDTA has demonstrated that EDTA is not

metabolically degraded and is excreted unaltered by the kidney

(Foreman and Trujillo, 1954). By extrapolation of the plasma

concentration to zero time, an initial distribution volume is

obtained which is approximately 20-25 percent of the body

weight. This distribution volume is identical to the volume of

extracellular water and suggests that the chelate ions are

unable to permeate cellular membranes to a significant extent

(Foreman and Trujillo, 1954; Chisolm, 1968; Chisolm, 1970).

Chelates of EDTA are quickly and almost completely

excreted in the urine. In humans, 50 percent of EDTA is

excreted within 1 hour after intravenous administration as

compared with 2.5 hours after intramuscular administration

(Foreman and Trujillo, 1954; Foreman et al., 1956). By 9 hours

after administration, 90 percent of the dosage has been

excreted by the kidneys regardless of the method of

administration. Almost all of the parenterally administered

EDTA (98.8%) could be accounted for in the urine of rats

within 24 hours of dosage (Aronsen and Ahrens, 1971). The

renal plasma clearance of these chelates is identical to that

of inulin; thus, it has been concluded that these chelate

compounds are excreted by glomerular filtration without

tubular secretion. This assumption has been confirmed by

experimental evidence that substances which block tubular








14

transport of organic acids do not affect the excretion of EDTA

(Aronsen and Ahrens, 1971; Forland et al., 1966).

Unlike the near complete absorption and excretion of

these agents when administered parenterally, oral

administration results in absorption of approximately 5

percent of the dosage (Catsch and Harmuth-Hoene, 1975). This

low rate of absorption has been attributed to many factors

including the precipitation of polyaminocarboxylic acids in

low pH gastric juice and the impermeability of the intestinal

wall to the chelate ion (Rubin, 1960; Catsch and

Harmuth-Hoene, 1975). The transcutaneous absorption of EDTA

was also found to be negligible when examined in rats (Furlani

and Vertura, 1970).

The chelation action of the polyaminocarboxylic acids has

been widely debated in the literature. When these chelating

agents were initially developed and utilized in clinical lead

intoxication, it was thought that lead was chelated from the

soft tissues but remained in bone due to bone's high avidity

for lead ions. In the last 20 years this has been disproven as

it has been demonstrated that bone is a significant source of

lead which is mobilized and excreted during EDTA chelation

therapy (Hammond et al., 1967; Hammond, 1971; Hu et al., 1991;

Flood et al., 1988). Almost all of the lead removed in

response to EDTA treatment is from the depletion of osseous

stores; this specific removal has been attributed to the

larger lead burden in bone when compared to soft tissue lead








15

stores. Any loss of lead from soft tissues is due to

redistribution to bone tissue where lead has been previously

vacated by EDTA chelation (Hammond, 1971; Sauk and Somerman,

1991). Thus the degree of mobilization of lead from soft

tissues is dependent upon the lead gradient between the soft

tissues and the EDTA-sensitive bone compartment. EDTA seems

incapable of directly picking up metal moving out of soft

tissues, but it is unknown why bone clears blood of lead

released by soft tissues and yet readily releases it to EDTA

(Hammond et al., 1967; Batuman et al., 1989).

The properties of polyaminocarboxylic acids such as fast

plasma clearance, and rapid and complete renal excretion make

these compounds excellent therapeutic chelation agents.

Unfortunately, therapeutic treatment with polyaminocarboxylic

acids is subject to some limitations. Since absorption through

the gastrointestinal tract is so low, these chelation agents

must be administered parenterally (Foreman and Trujillo,

1954). Intramuscular injections of these agents are painful,

even when administered in conjunction with procaine (Chisolm

and Thomas, 1985). Additionally, these compounds are

nonspecific chelator agents which commonly increase the

urinary output of essential endogenous metal ions (Perry and

Perry, 1959; Antonowicz et al., 1991). Finally, the most well

researched effect of these agents is their nephrotoxicity and

inducement of acute renal failure (Khan et al., 1983). Many

deaths attributed to lead intoxication have actually been the










result of acute nephrosis, increased renal lead burden and

renal failure due to chelation therapy (Chisolm, 1990; Tandon

et al., 1985).

Foreman et al. (1956) provided the first experimental

evidence that administration of EDTA in rodents leads to

nephrotic renal lesions; this has subsequently been confirmed

by Altman et al. (1962), Catsch (1964), Schwartz et al. (1966)

and Weber (1970). Repeated administration of EDTA results in

degeneration of the proximal convoluted tubules, a

simultaneous appearance of hydropic vacuoles, loss of brush

border and tubulorrhexis. Changes involving the distal

convoluted tubule and the glomeruli are much less apparent

(Catsch and Harmuth-Hoene, 1975). Administration of DPTA leads

to nephrotic changes qualitatively similar to those caused by

EDTA; however, renal lesions in DTPA-treated rats are less

pronounced that those seen after equimolar doses of EDTA

(Weber, 1970).

The pathophysiology of these toxic manifestations is not

understood. It is unclear whether the causative agent of soft

tissue toxicity is the chelate compound or the metal ion

itself (Cledes and Allain, 1992). Experimental dosage of

polyaminocarboxylic acids in the absence of metal toxicity

results in nephrotic changes resembling osmotic nephrosis

(Seidel, 1970). It is possible that the administration of

chelates leads to disturbances in electrolyte metabolism

independent of metal intoxication. Batchelor et al. (1964)








17

have demonstrated depression in serum potassium and increased

urinary excretion of potassium in humans treated with EDTA.

Potassium deficiency has been shown to cause hydropic

nephrosis, thus these renal histopathological lesions may be

directly attributed to the action of chelating agents.

As mentioned previously, EDTA and DPTA are nonspecific

metal chelators. The log-stability constant of a metal-chelate

complex determines the efficacy of any chelating agent. In

solution, any metal will displace from its chelate a metal

with a lower log-stability constant. The calcium chelate of

EDTA has a log-stability constant of 10.5, whereas the

log-stability constant of lead-EDTA is 18.04 (Brownie and

Aronson, 1984). The stability of the calcium chelates is much

lower than the stability of the chelates formed with

endogenous essential trace metals. The adverse effects

resulting from the mobilization or binding of those endogenous

metals are not understood.

It has been demonstrated repeatedly that therapeutic EDTA

treatment leads to increased urinary excretion of iron,

magnesium, copper and zinc (Liberman et al., 1967; Millar et

al., 1954; Brownie and Aronson, 1984; Thomas and Chisolm,

1986). Rats treated with EDTA had the highest concentrations

of plasma lead and urine lead one hour after treatment; plasma

zinc was dramatically decreased and urinary zinc was increased

at this time (Araki et al., 1984; Aono and Araki, 1984).










Beneficial effects of zinc supplementation during EDTA therapy

have been documented (Flora and Tandon, 1990).

If it is assumed that one of the mechanisms of chelating

agent toxicity is the interaction between chelating agents and

essential trace minerals, then those metal chelates with

higher stability constants than calcium chelates would be less

toxic. The decreased toxicity of equimolar dosages of zinc

chelates of EDTA and DTPA have been demonstrated (Catsch,

1964; Brownie and Aronson, 1984; Catsch and Harmuth-Hoene,

1975; Rosenblatt and Aronson, 1978). Experimental evidence

indicates that the acute LD50 of ZnDTPA is 2.5 times larger

than that of CaDTPA, while the chronic cumulative LD50 of

ZnDTPA is about 30 times larger. Renal and gastrointestinal

lesions are less severe or absent in therapeutic trial

utilizing ZnDTPA (Catsch and Harmuth-Hoene, 1975; Weber, 1969;

Weber 1970). Investigations into the chelating efficacy of

ZnEDTA were conducted; unfortunately, it was demonstrated that

ZnEDTA was about 60 percent as effective as CaEDTA in

increasing lead excretion when administered in equimolar

dosages (Brownie and Aronson, 1984). Due to the relative lack

of toxicity associated with ZnEDTA, the dosage was increased

and lead excretion rates which were 76 percent of CaEDTA were

obtained. ZnEDTA usage in treating lead intoxication is still

considered experimental while EDTA is the most common drug

selected for the treatment of clinical lead intoxication

(Glotzer and Bauchner, 1992).








19

Dithiol Chelators

The compound 2,3-Dimercaptopropanol (Dimercaprol, British

anti-lewisite, BAL) was originally synthesized as an

antagonist to vesicant arsenical gasses during World War II.

Many heavy metals exert their toxic effects through the

inhibition of enzymes which depend on sulfhydryl groups for

their activity. A mercaptide bond formed between the metal and

the sulfhydryl group of an essential thiol compound results in

reversible inhibition of enzyme catalysis. The dithiol BAL

competes with protein sulfhydryl groups for binding to heavy

metals and is the chelating agent of choice for mercury,

arsenic, and gold intoxication.

The pharmacological action of BAL has been well studied.

Because of its poor solubility in water, BAL is usually

dissolved in lipid solvents and administered intramuscularly,

subcutaneously, or intraperitoneally. Eighty percent of the

dosage is absorbed from the injection site within one hour and

95 percent is absorbed within six hours. Maximum blood

concentrations are usually attained within one hour of

administration (Peters et al., 1947; Simpson and Young, 1950).

Experimental determination of the volume of distribution

provides evidence that BAL is distributed throughout the

intracellular space (Peters et al., 1947; Simpson and Young,

1950). BAL is rapidly excreted in the urine as a dithiol;

degradation to the monothiol does not occur to any appreciable

extent (Peters et al., 1947).








20

Dithiol chelating agents are markedly more toxic than

polyaminocarboxylic acids due to their ability to inactivate

essential metal containing enzymes such as carbonic anhydrase,

peroxidase, and cytochrome enzymes. The reduction of protein

disulfide bridges by BAL results in the inhibition or

destruction of insulin, hemoglobin and essential liver enzymes

(Barron et al., 1947; Waters and Stock, 1945). Dithiols are

excreted in bile; thus, they can be administered in the face

of complete renal shutdown but not in the presence of any

hepatic insufficiency (Chisolm, 1968, 1970). The acute LD50 of

BAL when administered by intramuscular injection to rats is

0.6 mmol/kg, but dosages much lower than this result in

sublethal intoxication characterized by lacrimation,

conjunctivitis, salivation, vomiting, ataxia, convulsions and

coma. These toxic side effects disappear rapidly in accordance

with the elimination of BAL and no persistent

histopathological lesions are noted (Graham, 1948).
Monothiol Chelators

D-Penicillamine (D-PA) is the primary isomeric form

utilized for therapeutic purposes. The L isomer is

characterized by its ability to substitute for particular

amino acids during protein synthesis and its irreversible

inhibition of essential enzymes (Catsch and Harmuth-Hoene,

1975). The difference in chemical behavior between the two

isomers is consistent with large differences in toxicity. The

acute LD50 of the D,L racemic mixture in rats is approximately








21

2.4 mmol/kg as compared with an LD50 of 17 mmol/kg for the D

isomer (Aposhian, 1958; Aposhian and Aposhian, 1959). The

dose-lethality curve is steep for the L isomer with the LD90

only 1.2 times larger than the LD50. These unique

characteristics of L-Penicillamine have limited its use for

any clinical therapeutic purposes.

D-Penicillamine has demonstrated utility as a chelating

agent for copper, iron, lead, zinc and mercury (Beattie, 1977;

Carton et al., 1985). Long term penicillamine chelation

therapy in rats results in leukopenia and hypochromic anemia

(Catalanotto and Henkin, 1972) while persistent therapy in

humans has been associated with gastrointestinal tract

changes, nephrotoxicity, encephalitis and allergic reaction to

the penicillin component (Rosenberg and Hayslett, 1967).

Despite these adverse reactions, the high rate of absorption

of D-PA through the gastrointestinal tract makes this the drug

of choice for long term treatment of low level lead

intoxication.

Dimercaptopropanol Derivatives

Derivatives of the BAL molecule have been the subject of

research in the Soviet Union, Japan and China for 25 years.

These water soluble chemical analogs of BAL have been

evaluated for therapeutic use in mercury, arsenic and lead

toxicity by several researchers in the United States

(Aposhian, 1983; Gabard, 1978; Liang et al., 1982; Chisolm and

Thomas, 1985; Graziano et al., 1985; Twarog and Cherian,








22

1984). Meso-dimercaptosuccinic acid (DMSA) and 2,3

dimercapto-l-propanesulfonic acid (DMPS) have been found to

form chelation complexes similar to BAL and reported stability

constants indicate these agents bind cadmium, lead, iron,

mercury, zinc and nickel with decreasing stability in this

order (Aposhian, 1983). Of DMSA and DMPS, DMSA appears to be

the more promising potential therapeutic agent for lead

intoxication because of its higher LD50 and wider therapeutic

index (Graziano et al., 1985). The LD50 of DMSA is 12.60

mmol/kg, almost twice that of DMPS (6.53 mmol/kg) (Gabard et

al., 1979).

DMSA was first synthesized in 1954 and was the drug of

choice for the treatment of schistosomiasis (Friedheim et al.,

1954). Although the pharmacokinetics of these compounds are

poorly understood, 95 percent of the dosage is excreted

unchanged in urine within 24 hours of administration. The

volume of distribution of this compound has been the subject

of debate in the literature and no conclusion as to its

ability to permeate cellular membranes can be substantiated

(Tadlock and Aposhian, 1980; Graziano et al., 1985). Oral

administration results in rapid absorption of 60 percent of

the dosage (Liang et al., 1982). When administered

subcutaneously, the maximum blood concentration is obtained in

30 minutes and the half life is 60 minutes (Gabard, 1978;

Liang et al., 1982). The LD50 of DMSA is 30 times that of its

parent compound BAL. Therapeutic blood levels can be








23

maintained when administered at 5mg/kg with few side effects

and no apparent toxicity (Aposhian, 1983). Experimentation

with DMSA treatment of lead poisoning has demonstrated a

linear, dose-dependent decrease in blood lead concentrations

as compared with the logarithmic decrease in blood lead

concentrations characteristic of EDTA or EDTA-BAL therapy

(Chisolm, 1968; Graziano et al., 1985). The logarithmic

decrease in blood lead concentration characteristic of

established chelating agents indicates a diminishing response

to the drug with time. The demonstrated pharmacokinetics of

DMSA have led to the hypothesis that DMSA should be capable of

achieving lower blood lead levels than conventional

therapeutic agents, and it is possible that DMSA is capable of

chelating lead from a compartment larger than that of EDTA

distribution. These speculations have fueled the debate over

whether DMSA is capable of intracellular chelation (Graziano

et al., 1985; Aposhian, 1983; Twarog and Cherian, 1983, 1984).

The use of DMSA in treating occupational lead

intoxication has established that the therapeutic use of this

drug is as effective, if not more so, than treatment with EDTA

(Friedheim et al., 1976; Chisolm and Thomas, 1985; Twarog and

Cherian, 1984; Twarog and Cherian, 1983; Bentur et al., 1987;

Graziano et al., 1988; Cory-Slecta, 1988; Fournier et al.,

1988; Llobet et al., 1990; Thomas and Ashton, 1991; Grandjean

et al., 1991; Graziano et al., 1992; Khalil-Manesh et al.,

1992;). Treatment with DMSA at a dosage of 30 mg/kg/day for










five days was shown to decrease the concentration of blood

lead by 72.5 percent (Graziano et al., 1985). When DMSA is

administered orally or subcutaneously in cases of lead

exposure, removal and excretion of circulating and

tissue-bound lead occurs as well as prevention of pathological

changes in porphyrin metabolism (Friedheim and Graziano,

1978). When DMSA was experimentally compared with EDTA and

D-PA, DMSA was most effective at decreasing the concentrations

of tissue-bound lead.

Despite a lower LD50 than DMSA, DMPS has been proven to

be an additional valuable therapeutic agent for lead

intoxication. Clinical trials utilizing DMPS in humans have

demonstrated a 15 fold increase in urinary lead excretion

after administration (Chisolm and Thomas, 1985). Long term

therapy with DMPS has been proven to significantly decrease

bone lead concentrations (Hoffman and Segewitz, 1975). The

highest concentrations of DMPS during therapeutic

administration have been demonstrated in the kidneys (Twarog

and Cherian, 1983) and experimental trials have demonstrated

that DMPS preferentially removes lead from these organs

(Twarog and Cherian, 1983). This renal specificity is thought

to be related to the hydrophilic properties of this drug;

water-soluble DMSA is filtered by the glomerulus with little

tubular absorption.

Both DMSA and DMPS, like EDTA, have been shown to

increase the excretion of essential endogenous metals during










therapeutic trials (Graziano et al., 1985; Chisolm and Thomas,

1985). Dosage with DMSA resulted in doubling the urinary

excretion of zinc and copper compared with a 100 fold increase

that occurs with EDTA administration (Graziano et al., 1985).

Experimental treatment with DMPS has been associated with a 2

to 30 fold increase in copper excretion and a 1 to 10 fold

increase in zinc excretion when compared with normal values.

In contrast to the dramatic decreases in the plasma

concentrations of these metals seen with EDTA therapy, no

significant changes have been associated with DMPS treatment

(Chisolm and Thomas, 1985) and only slight decreases in serum

concentrations have been noted with the use of DMPS (Gabard et

al., 1979).

The use of DMSA and DMPS for the therapeutic treatment of

lead intoxication has been investigated by several

laboratories and has been approved for clinical use in humans

by the Food and Drug Administration. Features of these

compounds, such as rapid absorption from the gastrointestinal

tract, complete excretion, high specificity for toxic metals,

and affinity for really located lead, have "fueled

investigations of these therapeutic agents for use in human

medicine. It has been well proven that both agents

significantly decrease the body burden of lead and low doses

of these drugs may be the most effective regime for treatment

of moderate lead intoxication with minimal toxic side effects.










Diethyldithiocarbamate

Sodium diethyldithiocarbamate (DDTC) is the chelating

agent of choice for the treatment of nickel and cadmium

intoxication (Gale et al., 1986). This chelating agent, which

has an LD50 of 6.7 mmol/kg when administered intraperitoneally

or orally to rodents, is partially excreted unchanged in the

bile and urine while a portion of the dosage is oxidized to

ethereal and free sulfates (Sunderman et al., 1963).

This compound has been investigated as a potential

therapeutic agent for lead intoxication with equivocal

results. Research has determined that treatment with DDTC

results in decreased lead concentrations in liver and spleen

with a concurrent increased lead concentration in the brain

(Gale et al., 1986). Cantilena and Klaassen (1982) have

demonstrated that elevated concentrations of lead in the brain

are the result of a lipid soluble DDTC:metal complex which is

capable of crossing the blood-brain barrier. The use of DDTC

as a sole therapeutic agent for lead intoxication has been

deemed unacceptable due to transfer of toxic levels of lead to

brain tissue.

Combinations of Chelating Agents

The prevalence of childhood lead intoxication has caused

researchers to explore potential chelating agent combinations

in an effort to reverse the tragic course of this disease. The

usage of multiple chelating agents is justified if they

exhibit sites of action differing from each other or if a more








27

stable mixed ligand compound is formed. Additionally, multiple

ligands should differ with respect to pharmacokinetics and

stability constants. As previously discussed, lead is unique

due to possession of characteristics of both class A and class

B metals. The ability of lead to be bound by nitrogen, sulfur,

oxygen and phosphorous increases the potential utility of

multiple chelating agents, each effective against one

particular class of metal. The current treatment of choice for

lead intoxication utilizes EDTA, a chelating agent for class

A metals, in combination with dithiolcompounds (BAL) which are

chelating agents for class B metals.

It has been proven that inadequate dosages of chelating

agents are capable of increasing some specific toxic effects

of metals; in fact, a considerable molar excess of chelating

agent is essential to result in significant metal excretion

(Chisolm, 1968). By utilizing a combination of chelating

agents, the chelant to metal ratio is doubled without

exceeding the toxic threshold of either agent. Doubling the

chelant to lead ratio is probably the most important

explanation for the increased efficacy of the EDTA-BAL

combination (Chisolm, 1970). Additionally, combination of

these agents is justified because of differing sites of

action, differing pharmacokinetics, and different toxic

effects.

Evaluation of the efficacy of the EDTA-BAL combination as

compared with either agent alone has revealed that the








28

combined therapy resulted in greater decreases in blood and

tissue lead concentrations and increased urinary lead

excretion than either agent alone (Chisolm, 1970). The use of

BAL or EDTA in the treatment of childhood lead intoxication

reduced mortality from 60 percent to 20 percent, but the use

of these agents in combination has decreased lead-related

mortality to below 5 percent (Chisolm, 1968). Unfortunately,

typical lead chelation therapy undertaken with this

combination of drugs involves injections of EDTA or BAL

administered at four hour intervals for five to seven days

for a total of 60 to 80 injections. This course of treatment

must be followed by oral D-PA and often must be repeated

entirely due to rebounds in blood lead (Chisolm and Thomas,

1985; Piomelli et al., 1984; Chisolm, 1968). The toxic side

effects of these compounds combined with this rigorous

treatment schedule have resulted in the investigation and

development of alternative chelating agents discussed in this

review.













CHAPTER 3
OBJECTIVES



The specific objectives of the proposed studies are

directed toward improving the therapeutic management of avian

lead intoxication. Information generated will be used to

enhance the present understanding of the pathophysiology of

lead intoxication and determine optimal therapeutic treatment

regimens for avian lead intoxication.

Specific objectives are

1. Assess the efficacy of four chelating agents (EDTA, DTPA,

DMSA, PA) at multiple dosage levels in lead intoxicated birds

and assess the disposition of lead during dosage and chelation

therapy.

2. Evaluate the effects of these four chelating agents on

essential endogenous metals during chelation therapy.

3. Determine the subacute toxicity of these chelating agents

at multiple dosage levels in birds.

A review of the use of chelating agents in avian species

as well as a description of the rationale and experimental

design to accomplish these objectives follows.













Use of Chelating Agents in Avian Species


Treatment of lead intoxication in avian species has

primarily utilized ethylenediamine tetraacetate (Ca2Na2EDTA;

calcium versenate) administered at empirical dosage rates via

oral or injectable routes (Giddings, 1980; Petrak, 1982;

Woerpel and Rosskopf, 1986; Janssen et al., 1979; Sears et

al., 1980). Variable success has been associated with the

clinical use of this chelating agent in birds; case results

range from excellent therapeutic responses to fatalities

attributable to toxicities associated with chelation. EDTA has

several disadvantages associated with its use in avian

species. This drug must be administered parenterally due to

poor absorption from the gastrointestinal tract and has been

documented to enhance the uptake of lead from the

gastrointestinal tract itself (Piomelli et al., 1984). The

enhanced uptake of lead after oral administration actually

results in increased lead intoxication when administered to

birds with lead shot in the gastrointestinal tract. EDTA has

also been documented to cause the elimination of essential

metals as well as nephrotoxicity (Ishihara et al., 1984; Moel

and Kumar, 1982; Thomas and Chisolm, 1986). A rebound effect

after the cessation of treatment is often noted as the result

of compartmental redistribution of lead and thus several

courses of therapy are often required to reduce the total body








31

burden of lead (Piomelli et al., 1984; David et al., 1985).

These factors suggest that an evaluation of the current use of

EDTA for the treatment of avian lead intoxication is

advisable, and emphasize the need for alternative safe and

efficacious chelating agents.


Assessment of Chelatina Agents



Selection of Chelating Agents

The goal of chelation therapy is to remove a metal ion

from a site at which it is producing a "biochemical lesion";

effective chelation therapy depends on the selection of an

appropriate chelating agent for controlled removal of an

undesirable metal ion.

Four chelating agents were selected as potential

therapeutic agents for use in avian lead intoxication. The

polyaminocarboxylic acid EDTA was investigated as it is the

current therapeutic agent of choice in birds and has been the

standard therapeutic choice for mammalian lead intoxication

for over a century. Although commonly utilized for the

treatment of avian lead intoxication, the efficacy and

toxicity of this agent has not been determined in birds.

Another polyaminocarboxylic acid, DTPA, was chosen for

investigation due to its proven efficacy and relative lack of

deleterious side effects documented in recent research in

mammals.










In addition to the polyaminocarboxylic acids, two drugs

with specific affinity for lead and wider therapeutic indices

for treatment of lead poisoning were investigated. DMSA, a

water soluble analog of BAL, has less toxicity and greater

water solubility than EDTA and can be administered orally for

the treatment of lead poisoning (Aposhian, 1983; Graziano et

al., 1978; Fournier et al., 1988; Graziano et al., 1992;

Thomas and Ashton, 1991). DMSA contains vicinal thioether

groups which effectively enhance the excretion of lead,

mobilizing tissue lead and reversing lead induced biochemical

alterations. The potential utility of DMSA for the treatment

of childhood lead poisoning has prompted the Food and Drug

Administration to

approve this drug for clinical use in humans.

The fourth chelating agent selected for investigation was

D-PA. Oral administration of this drug in mammals has been

proven to reduce blood and tissue concentrations of lead and

copper (Lyle, 1981; Carton et al., 1985; Shannon et al.,

1988). The most extensive use of PA has been for the oral

outpatient treatment of lead poisoning or for oral -therapy

subsequent to parenteral EDTA therapy in children. The oral

route of administration as well as the relative lack of

toxicity of this drug in mammals increase the potential for

the use of this drug in avian lead intoxication

Neither DTPA, DMSA, nor PA have been investigated for use

in avian lead toxicity.










Chelating Agent Efficacy

The concentration of lead in whole blood has

traditionally been the principal biological index of lead

exposure and body burden for epidemiological and experimental

studies. Evidence suggests that the level of lead in the blood

is neither a measure of the absorption of lead, because of

intervening processes of transfer, mobilization and storage

among compartments, nor a direct indicator of concentrations

of lead in other body compartments nor total body burden

(Marcus, 1985). Absorption from the gastrointestinal tract has

been proven to be a monoexponential process with a constant

fraction of the lead absorbed into the blood (Marcus, 1985).

Research in humans has demonstrated that blood lead

concentrations are governed by non-linear mechanisms; the non-

linearity of the dose-response relationship has been

attributed to the formation of lead-binding proteins in

erythrocytes (Barton, 1989; Raghavan et al., 1980).

Once absorbed, lead does not become homogenously

distributed throughout the body but is transported to one of

three physiologically distinct compartments. A rapid exchange

compartment consisting of the blood and a few very well

vascularized organs contain approximately 4% of the lead body

burden and has been documented to have a half life of 36 days

(Rabinowitz et al., 1973). Measurement of blood lead is thus

a sensitive index of recent exposure but correlates poorly

with long term exposure or total lead body burden. The second








34

compartment, of intermediate rate of exchange, consists of

soft tissue organs and contains about 2% of the lead body

burden with a half life of approximately 40 days. The third

compartment, with a slow rate of equilibrium, consists of bone

tissue and contains approximately 94% of the lead body burden

with a half life of over ten thousand days. This reservior of

lead has been documented to be an indicator of cumulative lead

exposure (Rabinowitz et al., 1976).

The precise location of lead pools from which chelatable

lead is derived is unknown and may vary with different

chelating agents and metabolic conditions (Thomas and Chisolm,

1986; David et al., 1985; Osterloh and Becker, 1986;

Rabinowitz et al., .1976; Hammond, 1971). The mobilization of

lead from tissues and enhanced excretion have been the most

common means of evaluating the efficacy of chelating agents in

mammals but have been unresearched in avian species.

The first objective of this investigation was to compare

the efficacy of four chelating agents (EDTA, DTPA, DMSA and

PA) for the treatment of avian lead intoxication. Efficacy of

chelation therapy may be assessed either by measurement of the

amount of metal excreted from the organism during therapy or

by measurement of concentrations of the metal in target organs

during therapy. In human chelation therapy, cumulative

measurement of the amount of lead excreted in urine is often

the only direct assessment of the efficacy of therapy. This

measurement is impractical to assess chelation efficacy in








35

birds due to the urinary excretory physiology of avian

species. Additionally, measurement of excreted metal provides

no information as to the source of the metal which has been

chelated and excreted. In mammals, it has been documented that

different chelating agents have affinities for different

storage pools of lead. Although the compartmental kinetics of

lead in birds have not been elucidated, it is reasonable to

assume that chelating agents which work by different

mechanisms in mammals work by different mechanisms in avian

species as well. As stated previously, efficacy defined as

enhanced excretion of a metal is meaningless from a

therapeutic perspective if it is not paralleled by decreases

in metal concentration in critical organs.

To assess efficacy, the concentrations of lead in the

storage compartments of liver, kidney and bone were measured

in addition to blood lead concentrations. Liver and kidney

were selected as representative of intermediate rate

compartment tissues and bone was selected as representative of

the long rate compartment or cumulative lead burden. One of

the most common clinical signs of avian lead intoxication is

neurological impairment, therefore lead distribution via the

blood to the brain is of interest. The blood has been

hypothesized to act as a "transfer compartment" in addition to

a short term storage compartment, thus blood lead levels may

reflect compartmental redistribution of stored lead. The

concentration of lead in the blood is in direct equilibrium








36

with the concentration of lead in the brain; while the

analytical determination of lead in the blood is common, the

determination of lead in brain tissue is problematic.

Repetitive measurement of blood lead concentrations during

chelation therapy will permit assessment of concentrations of

lead in equilibrium with brain tissue.

Although the precise toxicokinetics of lead during

exposure and chelation in mammals are not known despite

decades of research, the data from this trial would provide

preliminary information in avian species. A knowledge of the

kinetics of lead during exposure and chelation is essential to

the development of an efficacious therapeutic regimen.

The experimental trial assessing efficacy of chelation

was designed to measure the concentrations of lead in the

tissues of intoxicated birds during an eight day chelation

regimen. Three dosage levels of chelating agents were selected

based on metabolic scaling from mammalian dosages (Sedgewick

and Pokras, 1988), empirical clinical experience and

preliminary experimentation. Due to the rapid changes in

tissue concentrations which were expected, blood lead was

measured on a daily basis while concentrations of lead in

liver, kidney and bone were measured at 48 hour intervals in

euthanized birds. The duration of this trial was limited by

repetitive and frequent blood sampling of the birds as well as

the size of the experimental groups which were maintained to

assess three dosage levels and provide birds for euthanasia to








37

obtain tissue measurements. This 8 day regimen approximates

the clinical treatment regimen currently utilized to treat

avian lead intoxication.

Chelation of Endogenous Metals

Essential trace metals are involved in numerous metabolic

activities including neuroconduction, membrane transport,

excretory processes and enzymatic reactions. Endogenous metals

are integral components of many enzyme systems; more than one

third on the enzymes described contain tightly bound metal

ions involved in the catalytic process or involved as

cofactors (Hughs, 1984).

No chelating agent is absolutely specific; there is

always some chelation of endogenous metals other than those

one wishes to remove. Interaction between clinically employed

chelating agents and endogenous metals has been documented in

mammals (Thomas and Chisolm, 1986; Victery et al.,1986;

Victory et al. 1987; Cantilena and Klaasen, 1982; Aono and

Araki, 1984) and has been proven to cause some of the

deleterious effects associated with chelation therapy. Most of

the toxic effects of metal ions are believed to be the- result

of the binding of these metals to metabolically important

groups, often with the displacement of an endogenous trace

metal ion and alteration in trace metal metabolism (Williams

and Halstead, 1983).

Urinary excretion of essential metals has been studied

after treatment with numerous chelating agents (Victery et








38

al., 1981; Araki et al., 1984; Victery et al., 1986), but less

attention has been given to changes in tissue concentration

and metabolism of these metals that may be produced by lead

exposure and chelation. The kidney is a target organ for lead

toxicity and the cells of the proximal renal tubule have an

important role in the homeostasis of essential metals. The

toxic effects of lead on these cells include changes in

cytoplasmic organelles (particularly mitochondria) which are

accompanied by impairment of transport functions (Goyer,

1971). Changes in zinc resorption by the renal tubule of lead-

exposed dogs and rats have been observed (Victery et al.,

1981; Victery et al., 1982) as well as changes in the urinary

excretion of calcium, magnesium, zinc, copper and iron in

lead-exposed rats (Victery et al., 1986).

Lead and zinc have similar affinities or similar

mechanisms of action both at the level of gastrointestinal

tract absorption and at intracellular metabolic sites

involving zinc containing enzymes (Mahaffey, 1980). Increased

urinary excretion of zinc in lead-poisoned mammals may result

from the interaction of lead and zinc at a common transport

pathway in the kidney or may result from the reduction of

renal zinc reabsorption; the mechanism remains unknown.

Each endogenous metal has its own metabolism, metal

binding proteins, interaction with lead and affinity for

chelating ligands. Theoretical chemistry formation constants

provide a rational basis for the selection of therapeutic








39

chelating agents, but the interactions between endogenous

divalent metals and chelating agents are poorly understood in

mammals and have not been investigated in birds. Of all

endogenous metals, it appears that copper and zinc are the

most significantly affected by chelation therapy in mammals.

Concentrations of these endogenous metals are affected in

blood and tissues, particularly liver and kidney, during

chelator administration. Most chelation therapies involve

chronic administration of the chelating agent, therefore it is

important to compare the effects that various chelating agents

have on endogenous metals during the course of therapy.

The second purpose of this investigation was to compare

the effects of four chelating agents, at three dosage levels,

on the concentrations of endogenous copper and zinc in the

blood and tissues of lead-intoxicated birds. Alterations in

blood and tissue concentrations of copper and zinc in birds

dosed with lead but untreated with chelating agents are also

reported to separate the effects of lead intoxication versus

chelation on these metals. Zinc was measured due to the

previously discussed body of literature documenting

alterations in zinc metabolism during chelation therapy in

mammals. Despite fewer publications documenting the excretion

of copper during chelation therapy, copper concentrations in

serum and tissues were measured because penicillamine was

selected as one of the chelating agents for evaluation. As

penicillamine has a higher affinity for copper than lead, it








40

is essential to understand the effects of this agent on

endogenous copper before considering it as a potential

therapeutic agent for avian lead intoxication. The

experimental trial extended for a period of 8 days to

approximate the clinical therapeutic regimen; duration of

assessment was again limited by the frequency of blood

sampling and the size of the experimental groups.

Chelating Agent Toxicity

In addition to assessing the effects of chelating agents

on the target metal and on endogenous metals, it is also

necessary to assess the toxic effects of these agents on

birds. The third purpose of this investigation was to assess

the toxicity of four chelating agents each administered at

three dosage levels. Measurement of hematological and serum

chemistry indices at three dosage levels of chelating agents

will permit assessment of dose-dependent toxic effects of

these agents on birds.

Drugs evaluated for clinical application are normally

assessed for acute (lethal) toxicity, subacute toxicity and

chronic toxicity. In this experimentation, assessment of toxic

effects was designed to provide information of maximum

clinical applicability. Preliminary trials established that

acutely toxic dosages of these drugs were well above the

dosages selected for investigation; thus no formal

determination of the lethal dosage or LD50 was conducted.

During normal clinical administration of these chelating








41

agents, the duration of therapy would not result in chronic

toxicity. Thus, although the pathological mechanisms of

chronic intoxication may be of interest to the clinician,

long-term drug administration to determine chronic toxicity

provides no clinically applicable information if the drug is

only administered for shorter periods of therapy.

To obtain information with the maximum clinical

applicability, the subacute toxicity of these agents was

assessed by measurement of clinical pathology parameters

during a 15 day trial at 72 hour intervals. The duration of

this trial is twice the length of the usual clinical

therapeutic treatment regimen allowing detection of

subclinical toxicity. The sampling interval is designed to

assess changes in clinical pathological parameters while

minimizing the changes in these parameters due to the stress

of daily handling and blood sampling. Parameters selected for

measurement of subacute toxicity are measurements commonly

utilized for patient assessment in clinical practice, thus

information gathered during this experimentation will have

applicability to the individual clinical patient.

Determination of the subacute toxicity of these chelating

agents will provide information on target organs and tissues,

cumulative toxicity and dose-response relationships as well as

aiding in the establishment of dosage levels for further

investigation and clinical use.

The use of plasma enzyme activities as a diagnostic index








42

of organ function is a well established procedure in human and

veterinary medicine and research. Increases in plasma enzyme

activities are usually related to leakage of enzymes from

damaged cells. The magnitude of change for a particular enzyme

depends on factors such as the activity of the enzyme in the

cells, the rate of leakage and the rate of clearance of enzyme

from the plasma (Boyd, 1983). Alternatively, organ metabolites

may be measured in plasma; altered organ function is reflected

in the concentration of metabolite in the circulating plasma.

Physiological parameters measured during subacute toxicity

investigations vary; this investigation was complicated by the

use of avian species as experimental subjects. Assessment of

hematological and serum chemistry parameters required the

establishment of "reference ranges" for groups of birds

utilized in this investigation. To avoid confusing

interpretation of data, chelating agents were administered to

birds which had not been treated with lead, thus all apparent

alterations could be attributed to the chelating agents and

not to the effects of lead exposure.

Hematological toxicity was assessed by the measurement of

erythrocyte and granulocyte indices commonly utilized in

mammalian and avian medicine. Erythrocyte indices of packed

cell volume (PCV), red blood cell count (RBC CT), hemoglobin

concentration (HGB) and reticulocyte index (RET IN) were of

interest in treated birds as anemia is often clinically

associated with lead poisoning and chelation therapy. Serum








43

chemistry parameters were selected as measurement of the

effect of chelating agents on target organs, especially liver

and kidney tissue. Multiple serum chemistry parameters were

measured during drug administration in an effort to detect

hepatic and renal toxicity. Assessment of target organ

toxicity through measurement of serum chemistry parameters was

more complex due to the unique physiological attributes of

avian species.

In mammals, the initial diagnostic evaluation for

assessing renal function is the urinalysis. Measurement of the

chemical composition of urine in birds is difficult or

impossible due to the admixing of urine and feces in the

cloaca. Additionally, birds are uricotelic in that they

secrete uric acid as the primary catabolic product of protein,

non-protein nitrogen and purines in contrast to the secretion

of urea in mammals (Lumeij, 1987; Allen, 1989; Campbell and

Coles, 1986). Primary renal disease at the level of the

glomerulus or renal tubule leads to impaired clearance of uric

acid from the blood stream and subsequent hyperuricemia.

Measurement of serum concentrations of uric acid, as well as

concentrations of calcium and phosphorous, are currently

accepted indices of renal function in birds and thus were

selected as indices of renal function in this investigation

(Amand, 1986; Lewandowski et al., 1986; Hawkey and Samour,

1988).

The clinical assessment of hepatic function in birds is








44

also problematic and has been widely debated in avian

literature (Lumeij and Westerhof, 1987). The concentrations of

several hepatic enzymes have been proven to accurately reflect

different stages of hepatic dysfunction in mammals. In birds,

concentrations of aspartate aminotransferase, alanine

aminotransferase, lactate dehydrogenase and alkaline

phosphatase have been measured in attempts to assess hepatic

function. Of these enzymes, aspartate aminotransferase (AST)

has been demonstrated to be the most sensitive and specific

index of hepatic function in birds (Lumeij, 1987).

Unfortunately, AST is not completely specific for hepatic

function; serum elevations have been associated with muscle

damage as well as hepatic insult (Campbell and Coles, 1986;

Lewandowski et al., 1986).

Other parameters selected to assess subacute toxicity of

chelating agents include serum alkaline phosphatase (AP),

lactate dehydrogenase (LDH), creatinine phosphokinase (CPK),

calcium (Ca), phosphorous (Ph) and total protein (TP). AP and

LDH are both cellular enzymes which reflect hepatic function

as well as muscular insult while CPK is a very sensitive and

specific indicator of muscle cell damage. Assessment of AST,

AP, LDH and CPK should permit the differentiation between

hepatic toxic effects and muscular insult in birds. Finally,

the measurement of Ca, Ph and TP will contribute to the

assessment of renal function as measured by serum uric acid.













CHAPTER 4
MATERIALS AND METHODS

Experimental Methods



Experimental Birds and Induced Lead Intoxication

All birds utilized in this experimentation were captive

mature Silver King pigeons (Columba livia domestic) equally

divided as to sex. Blood concentrations of lead were measured

prior to the initiation of each experiment to establish

normative values for these groups of birds. Birds were weighed

and treated with lead acetate dissolved in distilled water at

a dosage rate of 30 mg/kg daily via oral gavage. Treatment was

discontinued after 8-12 days of lead dosage when clinical

signs of intoxication were apparent and when blood lead

concentrations exceeded 5 ug/ml as measured by atomic

absorption spectrophotometry.


Preparation and Administration of Chelating Agents

Calcium disodium ethylenediamine tetraacetic acid

(Ca2Na2EDTA) was dissolved in distilled deionized water at 0.4

M concentration. The calcium trisodium salt of diethylene

triamine pentaacetic acid (Ca2Na3DTPA) was prepared by adding








46

3:1:1 mole ratio amounts of NaOH, DTPA and Ca(OH)2,

respectively, in that order, to distilled deionized water. The

final solution was 0.18 M Ca2Na3DTPA. The chelators

DL-penicillamine and 2,3-dimercaptosuccinic acid were

dissolved in 10% NaHC03 at concentrations of 0.064 and 0.41 M

respectively. All of the above mentioned chemicals were

obtained from Sigma Chemical Co. (St.Louis, MO). Chelator

solutions were prepared fresh daily and administered within 2

hours of preparation.

Each agent was administered at 30 mg/kg, 90 mg/kg and 270

mg/kg with the exception of DTPA which was administered at

dosage rates of 3.3 mg/kg, 10 mg/kg and 30 mg/kg due to

evidence of toxicity noted in a pilot investigation.



Sample Analysis



Lead concentrations in fresh blood and frozen tissues

were determined by electrothermal atomic absorption

spectrophotometry using a model 2380 atomic absorption

spectrophotometer equipped with a HGA 400 graphite furnace and

a deuterium arc background corrector (Perkin-Elmer Corp.,

Norwalk, CT.). Heparinized blood samples were wet digested

(1:4) with nitric acid (Instra-analyzed Fischer Scientific

Co.) at 65 C for 12 hours in stoppered glass tubes and brought

to a constant volume prior to analysis of lead concentrations.








47

Wet weights of liver, kidney and bone (femur) collected

from each bird were determined prior to ashing at 450 C for

twelve hours in a muffle furnace. The ashed tissues were

dissolved in hot nitric acid (Instra-analyzed Fischer

Scientific Co.) and brought up to volume with hydrochloric

acid (Instra-analyzed, Fisher Scientific Co.) prior to

analysis by atomic absorption spectrophotometry. All samples

were calibrated against certified commercially available

standards (Fischer Scientific Co.) and results expressed as ug

of lead per ml of whole blood or ug of lead per g of wet

tissue weight.



Copper and zinc

Copper and zinc concentrations in fresh serum and frozen

tissues were determined by electrothermal atomic absorption

spectrophotometry using a model 2380 atomic absorption

spectrophotometer equipped with a HGA 400 graphite furnace and

a deuterium arc background correcter (Perkin-Elmer Corp.,

Norwalk, CT). Fresh serum was diluted 1:5 utilizing 5%

trichloroacetic acid (TCA) to precipitate serum proteins prior

to analysis. Blanks and standards were prepared in a similar

manner by 1:5 dilution with TCA. Determination of serum copper

and zinc concentrations were made using flameless atomization.

Samples were calibrated against certified commercially

available standards (Fischer Scientific Co.) and results

expressed as ug of metal per dl of serum.








48

Wet weights of liver, kidney and bone (femur) collected

from each bird were determined prior to ashing at 450 C for

twelve hours in a muffle furnace. The ashed tissues were

dissolved in hot nitric acid (Instra-analyzed Fischer

Scientific Co.) and brought up to volume with hydrochloric

acid (Instra-analyzed, Fisher Scientific Co.) prior to

analysis by atomic absorption spectrophotometry. All samples

were calibrated against certified commercially available

standards (Fischer Scientific Co.) and results expressed as ug

of lead per g of wet tissue weight.

The presence of exogenous zinc or copper is problematic

in the analysis of these metals in biological samples; care

must be taken to avoid contamination of samples and glassware

during collection and analysis. All glass and plastic ware

were washed in Acationox detergent solution (American

Scientific Products, McGaw Park, IL), soaked overnight in 1 M

hydrochloric acid, rinsed in de-ionized water and dried. To

minimize metal contamination during sample collection, all

polyethylene-polypropylene syringes were treated using the

same method. Assays were performed in acid-washed borosilicate

tubes which were discarded after a single use.


Hematological and Serum Chemistry Measurements

Hematological parameters were measured utilizing

techniques commonly used in avian medicine and research. The

RBC ct. was determined using a Unopette #5851 (Becton-










Dickinson, Rutherford, NJ) and a Neubaur hemocytometer. HGB

was measured via spectrophotometric analysis of

cyanomethemoglobin. Whole blood was centrifuged for 10

minutes at 1000g after erythrocyte lysis to remove nuclear and

cytoplasmic debree, then absorbance measured at 540 nm and

compared to a standard calibration curve prepared from

commercially available standard hemoglobin solutions (Sigma

Chemical).

Peripheral blood smears were fixed and stained with

Leukostat (Modified Wrights stain; Fischer Scientific Co.,

Orangeburg, NY). The average number of leukocytes per 5

microscopic oil immersion fields was determined and the

following equation used to calculate the white blood cell

count per ul of whole blood:



ESTIMATEDWBC/ulBLOOD= AveNumberWBCper5Fields X3,500,000
1000





If the PCV was outside of normal range, the corrected WBC

count was calculated as follows:


CORRECTEDWBC/ ulBlood=EstWBCX ObservedPCV
NormalPCV








50

Differential white blood cell percentages were determined as

percentage of heterophils, eosinophils, basophils, lymphocytes

and monocytes in 100 counted cells on stained peripheral blood

smears. Peripheral blood smears were also examined to

determine the Reticulocyte Index (Harrison and Harrison 1986)

or degree of polychromasia, and if the PCV was outside of

normal range, the absolute percentage of reticulocytes was

calculated:



Absolute%ofReticulocytes=Observed%X OservePCV
NormalPCV





Clinical chemistry parameters (UA, AST, CA, PH, CPK, LDH, TP)

were measured on a Chemetrics Analyzer II (Chemetrics Corp.

Burlingame, CA) utilizing standard photometric techniques

employed by the University of Florida Clinical Pathology

Laboratory for the assay of avian serum chemistries.



Experimental Design and Sample Collection



Trial I: Chelatina Agent Efficacy

Birds (n=70) were weighed and 65 birds were treated with

lead acetate for 12 days as described above. Each

experimental group consisted of 15 birds treated with one of

the four chelating agents, 5 birds at each of three dosages










for each agent. Chelation therapy was initiated 12 hours after

cessation of lead dosage and administered twice per day for 8

days. Two groups of control birds were maintained (n=10).

First, a group of experimental control birds which were not

dosed with lead nor treated with chelating agent were

maintained and handled identically to experimental birds to

monitor for changes in measured parameters relating to

experimental protocol. Secondly, a control group of birds were

dosed with lead but not treated with chelating agent to act as

lead intoxicated but untreated controls; these birds were

handled identically to dosed and treated birds.

Blood concentrations of lead were measured daily during

chelation therapy. Heparinized blood samples (0.3 ml) were

obtained by venipuncture of the cutaneous ulnar vein or the

medial metatarsal vein. Sequential necropsies were performed

at 48 hour intervals during chelation therapy to measure

concentrations of lead in liver, kidney and bone. All

remaining birds were necropsied at the conclusion of 8 days of

chelation therapy.


Trial II: Chelation of Endogenous Metals

Blood concentrations of copper and zinc were measured

prior to initiation of the experiment to establish normative

values for this group of birds. Birds (n=70) were weighed and

treated with lead acetate as described above. Treatment was

discontinued after 12 days of lead dosage when clinical signs










of intoxication were apparent and when blood lead

concentrations exceeded 5 ug/ml as measured by atomic

absorption spectrophotometry.

Experimental groups consisted of 15 birds treated with

one of the four chelating agents, 5 birds at each of three

dosages for each agent. Two control groups were maintained as

above; one group of 5 birds dosed with lead but untreated with

chelating agent and one group of 5 birds undosed with lead and

untreated with chelating agents. Chelation therapy was

initiated 12 hours after cessation of lead dosage and

continued to be administered twice per day for seven days.

Serum concentrations of copper and zinc were measured

daily during chelation therapy. Heparinized blood samples (0.3

ml) were obtained by venipuncture of the cutaneous ulnar vein

or the medial metatarsal vein. Sequential necropsies were

performed at 48 hour intervals during chelation therapy to

measure concentrations of copper and zinc in liver, kidney and

bone. All birds were necropsied at the conclusion of 8 days of

chelation therapy.



Trial III: Assessment of Subacute Toxicity

Birds (n=60) were allowed to acclimate for two weeks

after acquisition and experimental parameters were measured

four times during these two weeks to establish normative

values for this group of birds. Each experimental group

consisted of 12 birds treated with one of the four chelating








53

agents, 4 birds at each of three dosages for each agent.

Chelation therapy was administered twice per day for 15 days

at then previously described dosage levels. Hematological

indices measured prior to and on days 3, 6, 9, 12 and 15 of

chelation therapy included packed cell volume (PCV), red blood

cell count (RBC ct.), white blood cell count (WBC ct.),

differential white blood cell determination, hemoglobin

measurement (HGB) and polychromasia index. Plasma levels of

uric acid (UA), aspartate aminotransferase (AST), lactate

dehydrogenase (LDH), alkaline phosphatase (AP), creatinine

phosphokinase (CK), calcium (CA), phosphorous (PH) and total

protein (TP) were measured prior to and during chelation

therapy. Two control groups of 4 birds each were maintained,

treated with saline and sampled in an identical manner as

experimental birds. All birds were necropsied at the

conclusion of 15 days of chelation therapy.

Heparinized blood samples were collected by venipuncture

of either the medial metatarsal vein or the cutaneous ulnar or

brachial vein. Immediately upon collection, microhematocrit

tubes were filled with whole blood for PCV determination and

blood smears were made utilizing the coverslip method (Schalm

1975). An aliquot of whole blood was retained for

determination of RBC ct. and HGB concentration and the

remainder of the sample centrifuged at 1000 g for 5 minutes to

obtain plasma for the determination of clinical chemistry










parameters. Plasma was frozen at -20 degrees centigrade and

analysis conducted immediately upon thawing.


Statistical Analysis



Experimental Trial I and II

The purpose of statistical analysis was to evaluate the

data to determine if differences in measured parameters were

significant and could be attributed to drug and dosage

effects. The metal concentrations in blood and tissues could

not be subjected to repeated measures analysis due to the

unbalanced experimental design. This problem was recognized

from initial stages of experimental design, but sacrifice of

individuals from experimental groups was the only methodology

to obtain tissue concentrations of metals. Additionally,

tissue metal concentrations represent measurements made on a

single bird sacrificed from the experimental group;

statistical analysis of this small sample size is problematic.

The metal concentrations in tissues were analyzed using

linear regression statistics. A standard linear model was used

as this was found to have the best fit for the majority of the

data sets; for valid comparison purposes, this same model was

applied to all data sets. Comparison of slopes, with respect

to their standard error of estimation, can be used to

determine differences between treatments.










Linear regression statistics were also used to evaluate

the concentrations of metals in the blood and sera. The

repeated measure of metal concentrations in the blood and sera

of birds during treatment added the complication of a temporal

relationship to the unbalanced experimental design. Inter-

individual variation was reduced by adjusting individual

observations by subtraction of initial pre-treatment values

from repeated observations. Linear regression was performed on

the transformed data and the slope of the line used to

estimate drug effect. Slope comparisons were used to assess

treatment differences. All data and regression lines are

graphically represented. Tabular presentation of p values

permit the comparison of the slope for each drug and dosage

combination; differences between treatment groups are

significant if p<0.05.



Experimental Trial III

Measurement of serum chemistry parameters in trial III is

a typical repeated measures experimental design. Each drug-

dosage combination was considered a separate treatment; birds

are nested within treatments and measured repeatedly over

time. When the treatment-time interaction was found to be

significant, multiple comparisons on drug-dosage-time

combinations were performed. The Newman-Keuls test grouping

provided alphabetical designation of statistical significance

at p<0.05. Data are graphically presented as treatment group








56

means, standard errors and statistical grouping denoted by

alphabetical lettering.














CHAPTER 5
RESULTS


Trial I: Chelatina Agent Efficacy



Blood lead concentrations significantly decreased during

chelation therapy with each of the four chelating agents. As

shown in Figure 1, concentrations of lead in the blood during

EDTA therapy were reduced by approximately half of the

original blood lead burden during the 8 days of chelation

therapy. The results of identical measurements made on birds

treated with DTPA are presented in Figure 2. Note that the

decremental pattern of lead in the blood is similar during

chelation therapy with either of the polyaminocarboxyclic

acids.

The concentrations of lead in the blood during therapy

with DMSA and PA are presented in Figures 3 and 4. Compared to

the effects of polyaminocarboxylic acid chelating agents, the

thiol chelating agents caused more rapid decreases i1 blood

lead concentrations. At the conclusion of B days of

treatment, blood lead concentrations were less than twenty

percent of pre-treatment levels.

Table 1 presents statistical comparisons between the

linear regression slopes of blood lead data from each drug and










BLOOD LEAD CONCENTRATIONS
EDTA TREATMENT


7
--e- CONTROL
S--- LOW DOSAGE
S6 -. MEDIUM DOSAGE
......... HIGH DOSAGE
Z

....<. -'-.. .

04


3 -



0 -






0
0 1 2 3 4 5 6 7 8

DAYS TREATMENT




Intercept (+/-SD) Slope (+/-SD)
Control 5.12 (0.31) -0.02 (0:01)
Low Dosage 5.21 (0.35) -0.33 (0.03)
Medium Dosage 5.12 (0.42) -0.40 (0.03)
High Dosage 4.89 (0.27) -0.39 (0.04)

Figure 1.
Blood lead concentrations in pigeons with lead intoxication
during treatment with EDTA administered at low (30 mg/kg),
medium (90 mg/kg) and high (270 mg/kg) dosage levels. Control
birds were dosed with lead but are untreated with chelating
agent. Day 0 represents pre-treatment measurements. Regression
parameters are presented. Sample sizes: n=5 for day 0; n=4 for
days 1,2; n=3 for days 3,4; n=2 for days 5,6; n=1 for days
7,8.











BLOOD LEAD CONCENTRATIONS
DTPA TREATMENT

7
-- CONTROL
--- LOW DOSAGE
6 5 ---- MEDIUM DOSAGE
HIGH DOSAGE
Z
0
S 5'^ ?--e---o --o --9-o a--- --o



I 4 p
















DAYS TREATMENT
H 3 .' .. .










Intercept (+/-SD) Slope (+/-SD)
l 5.12 (. -0.02

1 -




0 1 2 3 4 5 6 7 8

DAYS TREATMENT



Intercept (+/-SD) Slope (+/-SD)
Control 5.12 (0.31) -0.02 (0.01)
Low Dosage 5.16 (0.35) -0.43 (0.01)
Medium Dosage 5.16 (0.33) -0.44 (0.01)
High Dosage 4.83 (0.28) -0.39 (0.03)


Figure 2.
Blood lead concentrations in pigeons with lead intoxication
during treatment with DTPA administered at low (3.3 mg/kg),
medium (10 mg/kg) and high (30 mg/kg) dosage levels. Control
birds were dosed with lead but are untreated with chelating
agent. Day 0 represents pre-treatment measurements. Regression
parameters are presented. Sample sizes: n=5 for day 0; n=4 for
days 1,2; n=3 for days 3,4; n=2 for days 5,6; n=l for days 7,8.











BLOOD LEAD CONCENTRATIONS
DMSA TREATMENT

7
S-- CONTROL
S -- LOW DOSAGE
S- ---6.. MEDIUM DOSAGE
S........ ..- HIGH DOSAGE
Z
0 --
5



4>
Z


0
^ 3
J -


82

........ .. .-



0 1V

0 1 2 3 4 5 6 7 8

DAYS TREATMENT




Intercept (+/-SD) Slope (+/-SD)
Control 5.12 (0.31) -0.02 (0.01)
Low Dosage 4.74 (0.27) -0.48 (0.04)
Medium Dosage 4.49 (0.23) -0.49 (0.03)
High Dosage 4.08 (0.21) -0.47 (0.02)


Figure 3.
Blood lead concentrations in pigeons with lead intoxication
during treatment with DMSA administered at low (30 mg/kg),
medium (90 mg/kg) and high (270 mg/kg) dosage levels. Control
birds were dosed with lead but are untreated with chelating
agent. Day 0 represents pre-treatment measurements. Regression
parameters are presented. Sample sizes: n=5 for day 0; n=4 for
days 1,2; n=3 for days 3,4; n=2 for days 5,6; n=l for days 7,8.










BLOOD LEAD CONCENTRATIONS
PA TREATMENT

7
---0- CONTROL
E --4- LOW DOSAGE
.6 --- -- MEDIUM DOSAGE
S----.......- HIGH DOSAGE



-
z
'* .... "



0










0 1 2 3 4 5 6 7 8

DAYS TREATMENT



Intercept (+/-SD) Slope (+/-SD)
Control 5.12 (0.31) -0.02 (f.01)
Low Dosage 5.25 (0.36) -0.54 (0.04)
Medium Dosage 4.53 (0.22) -0.53 (0.04)
High Dosage 4.14 (0.20) -0.50 (0.03)

Figure 4.
Blood lead concentrations in pigeons with lead intoxication
during treatment with PA administered at low (30 mg/kg),
medium (90 mg/kg) and high (270 mg/kg) dosage levels. Control
birds were dosed with lead but are untreated with chelating
agent. Day 0 represents pre-treatment measurements. Regression
parameters are presented. Sample sizes: n=5 for day 0; n=4 for
days 1,2; n=3 for days 3,4; n=2 for days 5,6; n=l for days 7,8.
days 1,2; n=3 for days 3,4; n--2 for days 5,6; n=1 for days 7,8.


















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dosage level. Comparison of linear regression slopes between

dosage levels of each drug reveals that drug dosage level had

no significant effect on blood lead concentrations during

treatment with any of these chelating agents (all p values for

these comparisons are greater than 0.001 on Table 1).

Comparison of regression slopes between EDTA and DTPA

treatments revealed significant differences only between low

and medium dosages of DTPA when compared with low doses of

EDTA.

Two groups of control birds were utilized in all of these

trials: experimental control birds not dosed with lead nor

treated with chelating agent and control birds which were

dosed with lead but untreated with chelating agent.

Experimental control birds, not dosed with lead nor treated

with chelating agents, had blood and tissue lead

concentrations considered normal for birds not exposed to lead

(less than 0.3 ug/ml). No significant changes occurred in

these undosed and untreated birds during the experimental

trial (data not graphically represented).

The concentration of lead in the liver decreased during

the course of therapy with each of the chelating agents

(Figures 5, 6, 7 and 8). It is apparent that the thiol

chelating agents chelated lead most rapidly from the liver.

The polyaminocarboxylic acids were much less efficacious at

reducing liver lead levels, both at the rate of reduction and

the total reduction of tissue lead burden.










LIVER LEAD CONCENTRATIONS

EDTA TREATMENT


2 4 6 8

DAYS TREATMENT


Control
Low Dosage
Medium Dosage
High Dosage


Intercept (+/-SD)
47.05 (3.00)
43.89 (3.16)
42.09 (3.31)
40.60 (4.11)


Slope (+/-SD)
-3.37 (0.61)
-3.37 (0.64)
-3.64 (0.68)
-4.11 (0.82)


Figure 5.
Liver lead concentrations in pigeons with lead intoxication
during treatment with EDTA administered at low (30 mg/kg),
medium (90 mg/kg) and high (270 mg/kg) dosage levels. Control
birds were dosed with lead but are untreated with chelating
agent. Day 0 represents pre-treatment measurements. Each point
represents a single tissue measurement; regression parameters
are presented.











LIVER LEAD CONCENTRATIONS
DTPA TREATMENT


--- CONTROL
--- LOW DOSAGE
.----- MEDIUM DOSAGE
. ........ HIGH DOSAGE


0 2 4 6 8

DAYS TREATMENT


Control
Low Dosage
Medium Dosage
High Dosage


Intercept (+/-SD)
47.05 (3.00)
43.50 (3.47)
42.16 (3.44)
40.56 (3.94)


Slope (+/-SD)
-3.37 (0.61)
-3.86 (0.71)
-3.85 (0.70)
-3.86 (0.80)


Figure 6.
Liver lead concentrations in pigeons with lead intoxication
during treatment with DTPA administered at low (3.3 mg/kg),
medium (10 mg/kg) and high (30 mg/kg) dosage levels. Control
birds were dosed with lead but untreated with chelating agent.
Day 0 represents pre-treatment measurements. Each point
represents a single tissue measurement; regression parameters
are presented.










LIVER LEAD CONCENTRATIONS
DMSA TREATMENT


CONTROL
LOW DOSAGE
MEDIUM DOSAGE
HIGH DOSAGE


2 4 6 8

DAYS TREATMENT


Control
Low Dosage
Medium Dosage
High Dosage


Intercep
47.05
42.07
39.23
36.92


t (+/-SD)
(3.00)
(3.20)
(5.06)
(6.77)


Slope (+/-SD)
-3.37 (0.61)
-4.66 (0.65)
-4.98 (1.03)
-5.31 (1.38)


Figure 7.
Liver lead concentrations in pigeons with lead intoxication
during treatment with DMSA administered at low (30 mg/kg),
medium (90 mg/kg) and high (270 mg/kg) dosage levels. Control
birds were dosed with lead but are untreated with chelating
agent. Day 0 represents pre-treatment measurements. Each point
represents a single tissue measurement; regression parameters
are presented.










LIVER LEAD CONCENTRATIONS
PA TREATMENT


---- CONTROL
---- LOW DOSAGE
-------- MEDIUM DOSAGE
-....... HIGH DOSAGE


0 2 4 6 8


Control
Low Dosage
Medium Dosage
High Dosage


DAYS TREATMENT



Intercept (+/-SD)
47.05 (3.00)
38.55 (5.60)
38.41 (5.70)
36.51 (6.70)


Slope (+/-SD)
-3.37 (0:61)
-4.37 (1.14)
-4.84 (1.16)
-5.08 (1.37)


Figure 8.
Liver lead concentrations in pigeons with lead intoxication
during treatment with PA administered at low (30 mg/kg),
medium (90 mg/kg) and high (270 mg/kg) dosage levels. Control
birds were dosed with lead but untreated with chelating agent.
Day 0 represents pre-treatment measurements. Each point
represents a single tissue measurement; regression parameters
are presented.








68

The statistical comparisons of linear regression slopes

of liver lead concentrations are presented in Table 2. During

the course of therapy, the concentrations of lead in the liver

tissue of birds treated with chelation therapy did not

significantly differ from control birds dosed with lead but

untreated with chelating agent. Additionally, there was no

statistical significance between liver lead concentrations in

birds treated with different drugs or different dosages.

The concentrations of lead in the kidneys of birds

treated with these 4 agents were also reduced (Figures 9, 10,

11, 12). Control birds dosed with lead but untreated with

chelating agent had gradual declines in renal lead. Thiol

chelating agents caused a rapid and dramatic reduction in

renal lead, while the polyaminocarboxylic acid therapy

resulted in less rapid reduction of lead in the kidney.

Statistical comparison of linear regression slopes of kidney

lead concentrations are presented in Table 3. Evaluation of p

values presented reveals no significant differences between

drug and dosage combinations and control birds with the

exception of comparison between treatment with EDTA at high

dosages and control birds.

The concentrations of lead in bone were decreased by all

4 chelating agents (Figures 13, 14, 15, 16). Control birds

dosed with lead but untreated with chelating agent

demonstrated a slight increase in bone lead concentrations

during the 8 day trial. Statistical comparison of linear
























a



m a
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KIDNEY LEAD CONCENTRATIONS
EDTA TREATMENT


CONTROL
LOW DOSAGE
MEDIUM DOSAGE
HIGH DOSAGE


4 6 8

DAYS TREATMENT


Control
Low Dosage
Medium Dosage
High Dosage


Intercept (+/-SD)
47.78 (2.39)
46.57 (3.77)
44.34 (1.49)
43.11 (2.61)


Slope (+/-SD)
-3.23 (0:48)
-3.90 (0.77)
-4.48 (0.30)
-5.43 (0.53)


Figure 9.
Kidney lead concentrations in pigeons with lead intoxication
during treatment with EDTA administered at low (30 mg/kg),
medium (90 mg/kg) and high (270 mg/kg) dosage levels. Control
birds were dosed with lead but untreated with chelating agent.
Day 0 represents pre-treatment measurements. Each point
represents a single tissue measurement; regression parameters
are presented.











KIDNEY LEAD CONCENTRATIONS
DTPA TREATMENT


---- CONTROL
-- -- LOW DOSAGE
----4- MEDIUM DOSAGE
..........- HIGH DOSAGE


0 2


4 6 8


Control
Low Dosage
Medium Dosage
High Dosage


DAYS TREATMENT



Intercept (+/-SD)
47.78 (2.39)
48.20 (2.10)
44.22 (3.01)
44.79 (4.15)


Slope (+/-SD)
-3.23 (0-48)
-4.15 (0.43)
-4.46 (0.61)
-5.74 (0.85)


Figure 10.
Kidney lead concentrations in pigeons with lead intoxication
during treatment with DTPA administered at low (3.3 mg/kg),
medium (10 mg/kg) and high (30 mg/kg) dosage levels. Control
birds were dosed with lead but untreated with chelating agent.
Day 0 represents pre-treatment measurements. Each point
represents a single tissue measurement; regression parameters
are presented.










KIDLEY LEAD CONCENTRATIONS
DMSA TREATMENT


---e- CONTROL
---- LOW DOSAGE
..--v-- MEDIUM DOSAGE
. -.......... HIGH DOSAGE


0 2 4 6 8

DAYS TREATMENT


Control
Low Dosage
Medium Dosage
High Dosage


Intercept (+/-SD)
47.78 (2.39)
38.59 (5.50)
37.21 (5.94)
35.71 (7.60)


Slope (+/-SD)
-3.23 (0.48)
-4.70 (1.12)
-5.05 (1.21)
-5.31 (1.55)


Figure 11.
Kidney lead concentrations in pigeons with lead intoxication
during treatment with DMSA administered at low (30 mg/kg),
medium (90 mg/kg) and high (270 mg/kg) dosage levels. Control
birds were dosed with lead but untreated with chelating agent.
Day 0 represents pre-treatment measurements. Each point
represents a single tissue measurement; regression parameters
are presented.










KIDNEY LEAD CONCENTRATIONS
PA TREATMENT


CONTROL
LOW DOSAGE
MEDIUM DOSAGE
HIGH DOSAGE


2 4 6 8


Control
Low Dosage
Medium Dosage
High Dosage


DAYS TREATMENT



Intercept (+/-SD)
47.78 (2.39)
39.75 (4.66)
38.41 (4.88)
36.85 (6.478)


Slope (+/-SD)
-3.23 (0148)
-4.62 (0.95)
-4.62 (1.00)
-5.12 (1.32)


Figure 12.
Kidney lead concentrations in pigeons with lead intoxication
during treatment with PA administered at low (30 mg/kg),
medium (90 mg/kg) and high (270 mg/kg) dosage levels. Control
birds were dosed with lead but untreated with chelating agent.
Day 0 represents pre-treatment measurements. Each point
represents a single tissue measurement; regression parameters
are presented.























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BONE LEAD CONCENTRATIONS
EDTA TREATMENT


CONTROL
LOW DOSAGE
MEDIUM DOSAGE
HIGH DOSAGE


2 4 6 8

DAYS TREATMENT


Control
Low Dosage
Medium Dosage
High Dosage


Intercept (+/-SD)
131.82 (2.66)
121.53 (9.98)
114.11 (16.49)
102.70 (23.64)


Slope (+/-SD)
3.98 (0.54)
-11.17 (2.04)
-13.19 (3.37)
-14.54 (4.82)


Figure 13.
Bone lead concentrations in pigeons with lead intoxication
during treatment with EDTA administered at low (30 mg/kg),
medium (90 mg/kg) and high (270 mg/kg) dosage levels. Control
birds were dosed with lead but untreated with chelating agent.
Day 0 represents pre-treatment measurements. Each point
represents a single tissue measurement; regression parameters
are presented.


200

S180

160
z
0
i- 140
a-
z 120
Li
L)






z 60
0 100


0


Mo
< 80
_i

0
m










BONE LEAD CONCENTRATIONS
DTPA TREATMENT


200
--- CONTROL
--- LOW DOSAGE
I 180 ----. MEDIUM DOSAGE
S ...- HIGH DOSAGE
160 -




z 120






60
z 0

40

20 .


0 2 4 6 8

DAYS TREATMENT



Intercept (+/-SD) Slope (+/-SD)
Control 131.82 (2.66) 3.98 (D.54)
Low Dosage 120.16 (14.68) -14.65 (2.99)
Medium Dosage 112.06 (24.54) -15.54 (3.69)
High Dosage 101.63 (24.54) -14.68 (5.01)


Figure 14.
Bone lead concentrations in pigeons with lead intoxication
during treatment with DTPA administered at low (3.3 mg/kg),
medium (10 mg/kg) and high (30 mg/kg) dosage levels. Control
birds were dosed with lead but untreated with chelating agent.
Day 0 represents pre-treatment measurements. Each point
represents a single tissue measurement; regression parameters
are presented.










BONE LEAD CONCENTRATIONS
DMSA TREATMENT


200
---e- CONTROL
LOW DOSAGE
180 .----- MEDIUM DOSAGE
S ..... HIGH DOSAGE
160




z 120

o 100

80
< 80
-J

z 60 -
0
40 -

20 v %
0
0 I --",'-" -
0 2 4 6 8

DAYS TREATMENT



Intercept (+/-SD) Slope (+/-SD)
Control 131.82 (2.66) 3.97 (0.54)
Low Dosage 147.61 (9.20) -13.27 (1.88)
Medium Dosage 145.73 (12.66) -18.71 (2.58)
High Dosage 124.71 (16.20) -15.47 (3.31)


Figure 15.
Bone lead concentrations in pigeons with lead intoxication
during treatment with DMSA administered at low (30 mg/kg),
medium (90 mg/kg) and high (270 mg/kg) dosage levels. Control
birds were dosed with lead but untreated with chelating agent.
Day 0 represents pre-treatment measurements. Each point
represents a single tissue measurement; regression parameters
are presented.










BONE LEAD CON: L r i NATIONS
PA TREATMENT


--- CONTROL
-- LOW DOSAGE
-------- MEDIUM DOSAGE
..---... HIGH DOSAGE


'a
'V. "-


'0


I "-...
i I 1___________""_T-.


Control
Low Dosage
Medium Dosage
High Dosage


DAYS TREATMENT



Intercept (+/-SD)
131.82 (2.66)
141.58 (5.42)
142.87 (6.71)
134.51 (3.15)


Figure 16.
Bone lead concentrations in pigeons with lead intoxication
during treatment with PA administered at low (30 mg/kg),
medium (90 mg/kg) and high (270 mg/kg) dosage levels. Control
birds were dosed with lead but untreated with chelating agent.
Day 0 represents pre-treatment measurements. Each point
represents a single tissue measurement; regression parameters
are presented.


200

180 -

160-


120

100


80

60


Slope
3.97
-11.47
-16.66
-13.90


(+/-SD)
(D.54)
(1.11)
(1.37)
(0.64)










regression slopes of bone lead concentrations (Table 4)

reveals significant differences between control and treated

birds. No significant differences were noted between drug and

dosage combinations.




Trial II: Chelation of Endogenous Metals



The concentrations of copper and zinc were measured in

the serum of birds during chelation therapy with each of the

4 chelating agents (Figures 17 through 24). No significant

changes in serum copper or zinc were noted in either the

undosed and untreated experimental control group (data not

graphically presented) nor the lead dosed and untreated

control group ("control" on graphs); all control birds

maintained pretreatment concentrations of serum copper and

zinc in both control groups.

No significant changes in serum copper concentrations

occurred during therapy with EDTA or DTPA at three dosage

levels when compared with pretreatment measurements and

untreated control birds (Figures 17 and 18). No significant

changes were noted in serum copper concentrations during

therapy with DMSA at three dosages (Figure 19), while

treatment with PA resulted in reductions of serum copper

concentrations which were significantly different from control

birds at medium and high dosage levels (Figure 20, Table 5).




















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SERUM COPPER CONCENTRATIONS
EDTA TREATMENT


--e- CONTROL
--- LOW DOSAGE
---. MEDIUM DOSAGE
.-.....----- HIGH DOSAGE


0 1 2 3 4 5 6 7 8

DAYS TREATMENT


Control
Low Dosage
Medium Dosage
High Dosage


Intercept (+/-SD)
30.84 (1.23)
30.81 (1.20)
30.48 (0.97)
30.14 (0.99)


Slope (+/-SD)
0.11 (0.17)
0.15 (0.24)
-0.22 (0.15)
-0.29 (0.19)


Figure 17.
Serum copper concentrations in pigeons with lead intoxication
during treatment with EDTA administered at low (30 mg/kg),
medium (90 mg/kg) and high (270 mg/kg) dosage levels. Control
birds were dosed with lead but are untreated with chelating
agent. Day 0 represents pre-treatment measurements. Regression
parameters are presented. Sample sizes: n=5 for day 0; n=4 for
days 1,2; n=3 for days 3,4; n=2 for days 5,6; n=l for days 7,8.


50

45


35

30 --- -- -










SERUM COPPER CONCENTRATIONS
DTPA TREATMENT


--e-- CONTROL
---*- LOW DOSAGE
------ MEDIUM DOSAGE
-...- HIGH DOSAGE


U ~ 5 ..~...-. T-
z -,


2 3 4 5 6 7 8

DAYS TREATMENT


Control
Low Dosage
Medium Dosage
High Dosage


Intercept (+/-SD)
30.84 (1.23)
31.54 (1.25)
31.54 (1.26)
31.82 (1.29)


Slope (+/-SD)
0.11 (0.17)
0.19 (D.15)
0.18 (0.13)
0.33 (0.15)


Figure 18.
Serum copper concentrations in pigeons with lead intoxication
during treatment with DTPA administered at low (3.3 mg/kg),
medium (10 mg/kg) and high (30 mg/kg) dosage levels. Control
birds were dosed with lead but are untreated with chelating
agent. Day 0 represents pre-treatment measurements. Regression
parameters are presented. Sample sizes: n=5 for day 0; n=4 for
days 1,2; n=3 for days 3,4; n=2 for days 5,6; n=l for days
7,8.


20 -










SERUM COPPER CONCENTRATIONS
DMSA TREATMENT


--e- CONTROL
-- -LOW DOSAGE
-------- MEDIUM DOSAGE
--. ..HIGH DOSAGE


0 1 2 3 4 5 6 7 8

DAYS TREATMENT


Control
Low Dosage
Medium Dosage
High Dosage


Intercept (+/-SD)
30.84 (1.23)
30.09 (1.19)
30.16 (1.21)
29.86 (0.99)


Slope (+/-SD)
0.11 (0.17)
-0.32 (0.13)
-0.38 (0.13)
-0.26 (0.10)


Figure 19.
Serum copper concentrations in pigeons with lead intoxication
during treatment with DMSA administered at low (30 mg/kg),
medium (90 mg/kg) and high (270 mg/kg) dosage levels. Control
birds were dosed with lead but are untreated with chelating
agent. Day 0 represents pre-treatment measurements. Regression
parameters are presented. Sample sizes: n=5 for day 0; n=4 for
days 1,2; n=3 for days 3,4; n=2 for days 5,6; n=l for days
7,8.
































20 [


SERUM COPPER CONCENTRATIONS
PA TREATMENT



---- CONTROL
---- LOW DOSAGE
MEDIUM DOSAGE
HIGH DOSAGE




0 ,__ 0 -


- *--#--.:
.. .......... --------- --
.... .--........-
v -....

V ***=-***** V V-


V


0 1 2 3 4 5

DAYS TREATMENT


6 7 8


Control
Low Dosage
Medium Dosage
High Dosage


Intercept (+/-SD)
30.84 (1.23)
27.88 (0.89)
27.65 (0.92)
23.21 (0.84)


Figure 20.
Serum copper concentrations in pigeons with lead intoxication
during treatment with PA administered at low (30 mg/kg),
medium (90 mg/kg) and high (270 mg/kg) dosage levels. Control
birds were dosed with lead but are untreated with chelating
agent. Day 0 represents pre-treatment measurements. Regression
parameters are presented. Sample sizes: n=5 for day 0; n-4 for
days 1,2; n=3 for days 3,4; n=2 for days 5,6; n=l for days
7,8.


Slope
0.11
-0.67
-1.30
-1.41


(+/-SD)
(0.17)
(0.13)
(0.14)
(0.13)


S" "".......... ,-.


I I I I I I I









85

SERUM ZINC CONCENTRATIONS
EDTA TREATMENT


140 ----- CONTROL
---*- LOW DOSAGE
.....-- MEDIUM DOSAGE
S...... HIGH DOSAGE
120


z 100


a "*-...
S 80 ... "


0
o --I-
o 60

N
40


20



0 1 2 3 4 5 6 7 8

DAYS TREATMENT


Intercept (+/-SD) Slope (+/-SD)
Control 97.93 (2.33) 0.94 (0.81)
Low Dosage 96.09 (3.95) -4.18 (0.59)
Medium Dosage 93.84 (5.45) -5.23 (0.37)
High Dosage 87.35 (4.99) -8.29 (0.70)


Figure 21.
Serum zinc concentrations in pigeons with lead intoxication
during treatment with EDTA administered at low (30 mg/kg),
medium (90 mg/kg) and high (270 mg/kg) dosage levels. Control
birds were dosed with lead but are untreated with chelating
agent. Day 0 represents pre-treatment measurements. Regression
parameters are presented. Sample sizes: n=5 for day 0; n=4 for
days 1,2; n=3 for days 3,4; n=2 for days 5,6; n=l for days
7,8.










SERUM ZINC CONCENTRATIONS
DTPA TREATMENT


0 1 2 3 4 5 6 7 8

DAYS TREATMENT


Control
Low Dosage
Medium Dosage
High Dosage


Intercept (+/-SD)
97.93 (2.33)
95.86 (3.65)
92.08 (4.00)
85.85 (4.67)


Slope (+/-SD)
0.94 (0.81)
-4.72 (0.32)
-5.50 (0.54)
-8.75 (0.72)


Figure 22.
Serum zinc concentrations in pigeons with lead intoxication
during treatment with DTPA administered at low (3.3 mg/kg),
medium (10 mg/kg) and high (30 mg/kg) dosage levels. Control
birds were dosed with lead but are untreated with chelating
agent. Day 0 represents pre-treatment measurements. Regression
parameters are presented. Sample sizes: n=5 for day 0; n=4 for
days 1,2; n=3 for days 3,4; n=2 for days 5,6; n=l for days
7,8.


140















140 r-


ZU A


SERUM ZINC CONCENTRATIONS
DMSA TREATMENT


----- CONTROL
S-- -LOW DOSAGE
------ MEDIUM DOSAGE
-..... HIGH DOSAGE


--V





- I i I I_


100 --


60


40


20


0 1 2 3 4 5 6 7 8

DAYS TREATMENT


Control
Low Dosage
Medium Dosage
High Dosage


Intercept (+/-SD)
97.93 (2.33)
88.53 (2.62)
86.73 (2.57)
85.45 (2.49)


Slope (+/-SD)
0.94 (0.81)
-2.59 (D.64)
-2.88 (0.26)
-2.59 (0.80)


Figure 23.
Serum zinc concentrations in pigeons with lead intoxication
during treatment with DMSA administered at low (30 mg/kg),
medium (90 mg/kg) and high (270 mg/kg) dosage levels. Control
birds were dosed with lead but are untreated with chelating
agent. Day 0 represents pre-treatment measurements. Regression
parameters are presented. Sample sizes: n=5 for day 0; n=4 for
days 1,2; n=3 for days 3,4; n=2 for days 5,6; n=l for days
7,8.










SERUM ZINC CONCENTRATIONS
PA TREATMENT

140 r 0 CONTROL
LOW DOSAGE
v MEDIUM DOSAGE
7v HIGH DOSAGE
120


z100






8 60 -....
0
z
N
40



20 -

2O


0 1 2 3 4 5 6 7 8

DAYS TREATMENT


Intercept (+/-SD) Slope (+/-SD)
Control 97.93 (2.33) 0.94 (0.81)
Low Dosage 88.66 (2.67) -2.77 (0.25)
Medium Dosage 88.56 (2.65) -3.45 (0.52)
High Dosage 84.46 (2.52) -3.24 (0.37)


Figure 24.
Serum zinc concentrations in pigeons with lead intoxication
during treatment with PA administered at low (30 mg/kg),
medium (90 mg/kg) and high (270 mg/kg) dosage levels. Control
birds were dosed with lead but are untreated with chelating
agent. Day 0 represents pre-treatment measurements. Regression
parameters are presented. Sample sizes: n=5 for day 0; n=4 for
days 1,2; n=3 for days 3,4; n=2 for days 5,6; n=l for days
7,8.



















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90

The concentrations of zinc in the serum of birds during

chelation therapy are presented in Figures 21 through 24.

Statistical comparison of linear regression slopes between

drug and dosage combinations are presented in Table 6.

Treatment with any drug or dosage combination resulted in

serum zinc concentrations which were significantly different

from untreated control birds. Some significant effects of

dosage were noted in treatment with EDTA, DTPA and PA.

The concentrations of copper in kidney tissue was

significantly affected by chelation therapy (Figures 25

through 28, Table 7). No significant changes occurred in

copper concentrations in the kidneys of experimental control

birds (data not graphically represented) or lead dosed but

untreated birds ("Control" on graphs), but all treated birds

differed significantly from controls. In Figures 25 through

28, note that PA caused the greatest reduction in renal copper

concentration at all three dosage levels; significant

differences were noted between dosage levels (Table 7).

In Figures 29 and 30, significant increases in renal zinc

concentrations were noted with treatment by EDTA or DTPA when

compared with untreated control birds (Table 8). No

significant differences were noted between dosage levels of

polyaminocarboxylic acids. In contrast, kidney zinc

concentrations were altered significantly less by thiol

treatment (Figures 31 and 32, Table 8); kidney zinc






















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KIDNEY COPPER CONCENTRATIONS

EDTA TREATMENT

---- CONTROL
--- LOW DOSAGE
--- MEDIUM DOSAGE
...... HIGH DOSAGE





- --
--- ----------

.._ ..... .... ---....


Control
Low Dosage
Medium Dosage
High Dosage


DAYS TREATMENT



Intercept (+/-SD)
7.34 (0.14)
7.48 (0.07)
7.35 (0.16)
7.44 (0.18)


Figure 25.
Kidney copper concentrations in pigeons with lead intoxication
during treatment with EDTA administered at low (30 mg/kg),
medium (90 mg/kg) and high (270 mg/kg) dosage levels. Control
birds were dosed with lead but untreated with chelating agent.
Day 0 represents pre-treatment measurements. Each point
represents a single tissue measurement; regression parameters
are presented.


Slope
0.02
-0.14
-0.24
-0.32


(+/-SD)
(07.03)
(0.01)
(0.03)
(0.04)


I I I I











KIDNEY COPPER CONCENTRATIONS

DTPA TREATMENT


CONTROL
LOW DOSAGE
MEDIUM DOSAGE
HIGH DOSAGE


--- --------- --
----------.. ....
-- ------


0 2


6 8


DAYS TREATMENT


Control
Low Dosage
Medium Dosage
High Dosage


Intercept (+/-SD)
7.34 (0.14)
7.52 (0.08)
7.40 (0.09)
7.56 (0.21)


Figure 26.
Kidney copper concentrations in pigeons with lead intoxication
during treatment with DTPA administered at low (3.3 mg/kg),
medium (10 mg/kg) and high (30 mg/kg) dosage levels. Control
birds were dosed with lead but untreated with chelating agent.
Day 0 represents pre-treatment measurements. Each point
represents a single tissue measurement; regression parameters
are presented.


Slope
0.02
-0.17
-0.25
-0.37


(+/-SD)
(0-.03)
(0.02)
(0.02)
(0.04)


--4-
.-.-.--B.--











KIDNEY COPPER CONCENTRATIONS

DMSA TREATMENT

CONTROL
------ LOW DOSAGE
------V- MEDIUM DOSAGE
HIGH DOSAGE





0 o
........._______ __



----- -- -------
.. ------


Control
Low Dosage
Medium Dosage
High Dosage


DAYS TREATMENT




Intercept (+/-SD)
7.34 (0.14)
7.40 (0.08)
7.32 (0.10)
7.29 (0.16)


Figure 27.
Kidney copper concentrations in pigeons with lead intoxication
during treatment with DMSA administered at low (30 mg/kg),
medium (90 mg/kg) and high (270 mg/kg) dosage levels. Control
birds were dosed with lead but untreated with chelating agent.
Day 0 represents pre-treatment measurements. Each point
represents a single tissue measurement; regression parameters
are presented.


Slope
0.02
-0.23
-0.25
-0.37


(+/-SD)
(0.03)
(0.02)
(0.02)
(0.03)


I I I I









95

KIDNEY COPPER CONCENTRATIONS

PA TREATMENT

-- e,- CONTROL
-- -* LOW DOSAGE
...--- MEDIUM DOSAGE
............... HIGH DOSAGE







_......._.o.-
L"<-;.:^--^.^ _i

"**. --.9..* "


"-,..


DAYS TREATMENT


Control
Low Dosage
Medium Dosage
High Dosage


Intercept (+/-SD)
7.34 (0.14)
7.47 (0.07)
7.43 (0.14)
7.44 (0.14)


Figure 28.
Kidney copper concentrations in pigeons with lead intoxication
during treatment with PA administered at low (30 mg/kg),
medium (90 mg/kg) and high (270 mg/kg) dosage levels. Control
birds were dosed with lead but untreated with chelating agent.
Day 0 represents pre-treatment measurements. Each point
represents a single tissue measurement; regression parameters
are presented.


Slope
0.02
-0.24
-0.43
-0.62


(+/-SD)
(0:03)
(0.02)
(0.03)
(0.03)


I I I I