AMMONIUM PERCHLORATE-INDUCED LESIONS

IN ZEBRAFISH KIDNEYS

by

TIM CAPPS, B.S.

A THESIS

IN

ENVIRONMENTAL TOXICOLOGY

Submitted to the Graduate Faculty of Texas Tech University in

Partial Fulfillment of the Requirements for

the Degree of

MASTER OF SCIENCE

Approved

Chairperson of the Committee

Accepted

Dean of the Graduate School

December, 2003

ACKNOWLEDGMENTS

1 would like to especially thank Dr. Reynaldo Patino for being the chairperson of

my committee who made this project possible, and for giving me a positive and enjoyable

educational experience. 1 would also like to thank the other members of my committee:

Dr. Todd Anderson and Professor Victoria Sutton 1 would like to thank Dr. Vickie

Blazer for her histological expertise. I would like to thank The Institute of

Environmental and Human Health at Texas Tech for giving me monetary support, and

being a good organization with great people. I would like to thank the U.S. Geological

Survey through the Texas Cooperative Fish and Wildlife Research Unit at Texas Tech,

and all of the people there, for the use of facilities and for providing a great work

environment. I would also like to thank the U.S. Department of Defense through the

Strategic Environmental Research and Development Program, under a cooperative

agreement with the US. Air Force Institute for Environmental Safety and Occupational

Health, for providing further flinding for this project.

Finally, I would like to thank all of my friends, family, and coworkers for their

support and understanding. Without all of you this would not have been possible.

TABLE OF CONTENTS

ACKNOWLEDGMENTS ii

ABSTRACT v

LIST OF FIGURES vii

CHAPTER

I. BACKGROUND AND OBJECTIVES 1

Introduction 1

Ammonium Perchlorate 2

Biological Effects of Perchlorate 3

Thyroidal Effects of Ammonium Perchlorate 4

Dose-Response Studies of Thyroidal Effects 4

Developmental Effects of Perchlorate 7

Reproductive Effects of Perchlorate 8

Renal Effects of Perchlorate 9

Macrophage Aggregates 11

II. LEGAL ASPECTS OF AMMONIUM PERCHLORATE 13

III. AMMONIUM PERCHLORATE -INDUCED LESIONS

IN ZEBRAFISH KIDNEYS 18

Introduction 18

Materials and Methods 20

Results 22

Discussion 24

IV. CONCLUSIONS 35

LITERATURE CITED 37

ABSTRACT

Ammonium perchlorate (AP) is a highly reactive chemical that is primarily used

as a rocket propellant for military and aerospace industries. Widespread AP

contamination has caused increased concern over its effect on biological systems.

Perchlorate derived from AP, and other perchlorate species, inhibits iodide uptake by the

thyroid follicles, thus impairing the production of thyroid hormones and disrupting the

regulatory feedback mechanisms of the thyroid system. As a result, the secretion of

thyroid-stimulating hormone (TSH) by the pituitary gland increases. Effects of AP on

other physiological systems, such as the renal system, are not well understood. The

mammalian and teleost kidney are both known to contain TSH and thyroid hormone

receptors, as well as iodide transport mechanisms similar to those found in thyroid

follicles. Therefore, the kidney is also a potential target of direct (association with iodide

transporters) or indirect (decreased thyroid hormones and increased TSH) actions of

perchlorate. This study examined the histopathological effects of AP on the zebrafish

kidney. Adult zebrafish were exposed to water-borne perchlorate at concentrations of 18-

ppm for eight weeks and 677-ppm for 4 weeks. At the end of the exposure period, the

fish were processed for histological analyses of the trunk region of the kidney. The

samples were then analyzed for increased presence of macrophage aggregates, focal

granulomas, inflammation, and generating nephrons. Quantitative analysis was

conducted by determining the relative average area covered by a lesion in randomly

selected histological sections. The results indicated that exposure to AP at 18-ppm for

eight weeks caused a significant increase in the incidence of renal macrophage aggregates

when compared with the control fish. The other lesions showed no changes in incidence

at any AP dose. The study also suggests a link between thyroid disruption and increases

in renal macrophage aggregates.

VI

LIST OF FIGURES

1. Hemotoxilyn-Eosin stained macrophage Aggregate 27

2. PAS stained macrophage aggregate 28

3. Iron-stained macrophage aggregate 29

4 Percent kidney area covered by lesions in zebrafish exposed to ammonium perchlorate 30

5. Mean area of kidney histological sections covered by lesions, and /^-values

of statistical comparison 31

6. Hemotoxylin-Eosin stained focal granuloma with basophilic encapsulation 32

7. Hemotoxylin-Eosin stained inflammation with eosinophilic cells and core 33

8. Hemotoxylin-Eosin stained generating tubule cluster 34

Ml

CHAPTER I

BACKGROUND AND OBJECTIVES

Introduction

Ammonium perchlorate (AP) is a chemical primarily used as an ingredient in

soUd fuel rocket engines. In the environment, the perchlorate anion derived from AP

persists for a relatively long period of time, and occurs in drinking water supplies in some

parts of the United States. Perchlorate inhibits thyroid hormone production by limiting

the uptake of iodide, an essential component of thyroid hormones, into the thyroid

follicles. Decreased thyroid hormone production may be associated with some

physiological dysfijnctions.

The purpose of this study is to determine if AP exposure affects the

histopathology of zebrafish kidneys. Effects of perchlorate on the thyroidal system of

zebrafish, and other organisms, are well documented. Due to some similarities between

the thyroid and kidney, namely the presence of iodide transporters, thyroid hormone

receptors, and thyroid stimulating hormone receptors, there exists a potential for a

perchlorate-induced effect on the kidney. Whether or not there is a direct or indirect

eflfect of perchlorate on the kidney will also be analyzed. To my knowledge, this is the

first laboratory-controlled study where the effects of AP at concentrations found in the

environment (Smith et al., 2001) are determined for the kidney of any species.

Ammonium Perchlorate

Ammonium perchlorate has a variety of uses in the public and private sectors of

the United States. Since the 1940s, AP has mainly been used as an oxidizing agent in

military and aerospace solid fuel rocket engines and munitions (Fisher et al., 2000). Non-

military or aerospace uses of AP include airbag inflators, pyrotechnics, industrial

explosives, and batteries. Further, perchlorate salts can be found in products as diverse as

fertilizers, leather tarming solutions, and electrical tubes (Sharp and Walker, 2001). In the

past, perchlorate was used for the treatment of Grave's disease, or hyperthyroidism, in

himians (Fisher et al., 2000).

One major problem associated with AP is the environmental persistence of

perchlorate. In water, the ammonium ion and perchlorate anion of AP dissociate, and

although the perchlorate anion is a strong oxidant, the activation energy required for its

reduction is high (Fisher et al., 2000). As a result, perchlorate can continue in the

envirormient for many years (Urbansky, 2002). Perchlorate is susceptible to various

natural and artificial degradation processes, including induced ion exchange and natural

microbial degradation. However, remediation measures for perchlorate-contaminated

sites have resulted in varying outcomes (Logan, 2001).

A heightened public awareness of AP has stemmed fi-om the increased presence

of perchlorate in ground and surface waters throughout the country. The use of AP in

military munitions and aerospace operations accounts for the majority of environmental

contamination attributed to AP. Nearly all surface and ground waters contaminated by

perchlorate are near or downstream from facilities that manufacture or use AP (Sharp and

Walker, 2001). In the United States, facilities associated with AP occur in at least 39

states, 19 of which have confirmed perchlorate contamination in some ground or surface

water sources (Sharp and Walker, 2001). Perchlorate contamination is a major public

concern in California. As of 2001, perchlorate contamination in California occurred in

197 drinking water sources that serviced over seven million people. Some of the

perchlorate concentrations found in California were above recommended safe levels for

drinking water. Parts of the Colorado River, which flows through and supplies drinking

and irrigation water to people in CaUfomia, Nevada, and Arizona contains levels of

perchlorate as well (Sharp and Walker, 2001). Further, perchlorate contamination is not

always limited to water. Recent studies of a military munitions plant in central Texas

foxmd the presence of perchlorate in ground and surface water, soil, vegetation, aquatic

insects, fish, and mammals (Smith et al, 2001).

Biological Effects of Perchlorate

In recent years, the heightened concern over possible public health effects of

perchlorate contamination led to studies concerning the eflfects of perchlorate on

biological activities such as thyroid fimction, development, and reproduction. However,

studies concerning the effects of perchlorate on other biological flinctions are currently

insufficient. Considering the increase in perchlorate contamination, and consequent

availability to organisms, additional research of all systems conceivably affected by

perchlorate seems justified.

Thvroidal Effects of Ammonium Perchlorate

The longest known effect of perchlorate on biological systems is the alteration of

thyroid gland fiinction (Sellivanova et al., 1968). The primary action of the perchlorate

anion on thyroid follicles (the anatomical units of the thyroid gland that produce thyroid

hormones) is the competitive inhibition of sodium-iodide symporters, which regulate the

amount of iodide taken into the thyroid (Wolf, 1998). Since iodide is an essential

component of thyroid hormones (T3 and T4), the inhibition of iodide uptake results in

decreased thyroid hormone production. Decreased thyroid hormone blood levels disrupt

homeostasis and triggers an increased production and release of thyroid-stimulating

hormones (TSH) by the pituitary gland, which in turn causes the thyroid to produce more

thyroid hormones. Because thyroid follicles are unable to increase thyroid hormone

production, pituitary TSH production continues at relatively high levels eventually

resulting in the hyperactive stimulation of thyroid follicles and excessive circulating TSH

levels (Soldinetal, 2001).

Dose-Response Studies of Thyroidal Effects

Considerable amounts of research have focused on determining the levels of

perchlorate exposure that induce thyroidal effects. An early study of AP toxicity

determined the effects on dogs of AP repeatedly introduced into their stomachs in a

summary dose of 4500 mg/kg over a three-week period (SeUvanova et al, 1969). This

treatment caused an acute and prolonged inhibition of thyroid hormone production as

well as several other severe physiological malfiinctions (Selivanova et al., 1969).

However, more recent studies focus on smaller AP concentrations and their effect on

thyroid health.

Changes in thyroid hormone and TSH levels is one effect of AP. Studies

conducted on rats showed that AP exposure levels in the 3-30 mg/kg/day range (given for

30 days) resuhed in increased TSH levels, and decreased T4 and T3 levels (York et al.,

2001a). A similar thyroid hormone effect occurred in pregnant rabbits dosed with AP

concentrations greater than 30 mg/kg/day (administered on gestation days 6 through 28)

(York et al., 2001b). In frogs, exposures of 14-140 ppb AP result in alterations of thyroid

hormone levels. However, removal from AP reversed the effects on thyroid hormone

levels after 28 days (Goleman et al., 2002a).

In addition to alterations in thyroid hormone and TSH levels, studies reveal AP-

induced changes in thyroid follicle histopathology. Most thyroid follicular changes

manifest themselves in the occurrence of thyroid follicle hypertrophy, hyperplasia,

colloid depletion, and angiogenesis. In pregnant rabbits, AP caused increased thyroid

follicular hypertrophy at dose levels greater than 10 mg/kg/day (administered on

gestation days 6 through 28) (York et al, 2001b). Doses of 3-30 mg/kg/day for 14 or 90

days induced increased thyroid weight, follicular cell height (hypertrophy), hyperplasia,

and colloid depletion in rats (Siglin et al, 2000). Further, thyroid adenomas occurred in

rats dosed with 30 mg/kg/day AP for 90 days (Wolf, 2000). In frogs dosed with 14-140

ppb AP, frogs exposed to the higher concentrations of AP exhibited thyroid follicular

hypertrophy (Goleman et al, 2002a).

Several studies suggest a time relevant effect of AP on thyroid histopathology.

One histological study suggests a time-dependent effect of perchlorate on the thyroid

structure of rats. Rats continuously dosed with perchlorate for 1 to 12 months showed a

continuous increase in thyroid weight and increased thyroid hypertrophy, hyperplasia,

colloid depletion, and increased vascularization starting after two months of treatment

(Fernandez Rodriguez, 1991). Further, in a study conducted with zebrafish,

envirormientally relevant concentrations of AP (18 ppm) (Smith et al, 2001),

administered for eight weeks significantly increased thyroid follicular cell hypertrophy

and hyperplasia, angiogenesis, and colloid depletion. However, larger doses (677 ppm)

administered for four weeks resulted in no significant increase in thyroid folUcular

hystopathological parameters (Patiiio et al, 2003).

Human AP toxicity data is less extensive than animal toxicity data. The effects of

perchlorate on human thyroid function have been established through perchlorate's past

use as a treatment for hyperthyroidism (Wolf, 1998). However, information on thyroidal

effects of perchlorate at smaller doses possibly found in the environment is limited to

epidemiological studies, and a small number of laboratory studies.

Studies of thyroidal effects of envirormiental exposures to AP reveal conflicting

effects of perchlorate. An epidemiological study of newborns in Arizona concluded that

those residing in areas with contaminated drinking water from the Colorado River had

higher TSH levels than newborns from areas without perchlorate-contaminated water

(Brechner et al, 2000). Other epidemiological studies yielded contradictory or

inconsistent information. For example, the thyroid function of people from Las Vegas,

Nevada exposed to 4 to 24 |ig/L of perchlorate in their drinking water was not

significantly different from the thyroid function of unexposed people (Li Zet al, 2000; Li

F.X. et al, 2000; Li et al, 2001). Further, children and newborns from a Chilean city with

perchlorate-contamination, at levels up to 120 ng/L, had lower TSH levels than children

and newborns from some uncontaminated areas (Crump et al, 2000).

Human laboratory studies give further data on the thyroid toxicity of AP. A study

with humans dosed wdth AP at 10 mg/day for 14 days found a decrease in radioactive

iodide uptake levels in thyroid follicles without a change in circulating thyroid hormone

or TSH levels (Lawrence et al, 2000). Further, concentrations of perchlorate in the range

of 0.02 to 0.5 mg/kg/day administered in drinking water for 14 days did not affect iodide

uptake in humans, thereby indicating a no-observable-effect level of perchlorate in

drinking water of 250 |xg/L (ppb) (Greer et al, 2000). While these studies administered

low, environmentally relevant doses, the length of exposure is arguably not indicative of

envirormiental exposure through drinking water.

Developmental Effects of Perchlorate

The effects of AP on the thyroid have led to concerns over possible

developmental effects. Thyroid hormones are vital for the proper development of

physiological fiinctions such as neurological and bone development. Thyroid hormone

irregularities during development are a major cause of neurological impairment in

humans (Sharp & Walker, 2001).

Studies reveal varying AP effects on development. In rabbits, the fetuses of

pregnant females continuously dosed via drinking water with 0-102.3 mg/kg/day during

gestation days 6 through 28 showed no increased incidence of skeletal alterations,

neurological abnormaUties, reduced body weight, or increased mortality when compared

with historical incidences of the abnormalities in the test species. These observations led

to a finding of a no-observable-adverse developmental effect level of 100.0 mg/kg/day

(York et al., 2001b). However, in a study with zebrafish exposed to goitrogens

(including sodivmi perchlorate). the inhibition of thyroid hormone production was found

to have substantial developmental effects. The inhibition of transition from larva to

juvenile fish, inhibition of the formation of scales, and stimted fin grow1:h were all found

to occur in the perchlorate treated groups (Brown, 1997). In a study of field mice dosed

with 0, 1 nM, 1 |J,M, or I mM of AP from the time of breeding pair cohabitation to the

time of pup euthanasia, a period of 20 to 70 days, pups from the dosed dams were found

to have lower body and organ weights (Thuett et al, 2002). Further, tadpoles exposed to

AP at concentrations of 0-140 ppb, showed inhibited metamorphosis, and a lower male

sex ratio in groups treated with higher concentrations. However, there were no

incidences of structural abnormalities, and the inhibition of metamorphosis was corrected

after removal of AP (Goleman et al, 2002a).

Reproductive Effects of Perchlorate

The potential of AP to interfere with reproductive fiinctions, through alterations of

thyroid activity, led to experimental determinations of AP's effect on reproductive

performance. In a two-generation study, pregnant rats were exposed to AP at 0-30.0

mg/kg/day for 30 days in drinking water (York et al, 2001a). Reproductive,

developmental and physiological parameters such as body weights, thyroid and major

organ weights, fertility, sperm counts, estrogen levels, litter sizes, and fetus and pup

mortality were recorded. The only adverse effects observed were on thyroid hormone

levels and thyroid weights of both parents and offspring at the higher doses, but there

were no observable reproductive effects on either generation of rats at any of the doses

tested (York et al., 2001a). In a study with zebrafish, groups of eight AP-exposed

females were each paired with four AP-exposed males to determine the effect of AP on

reproductive performance at exposures of 0-ppm, 18- ppm, and 677-ppm for eight weeks

(Patiiio et al, 2003). At 677-ppm, spawned egg volume was decreased immediately, but

the effect was likely due to extrathyroidal effects of AP (either ammonium or

perchlorate). Although the group dosed withl 8-ppm AP for eight weeks exhibited drastic

thyroid follicle histopathological effects, no effect on spawned egg volume or egg

fertilization rate occurred, thereby showing a no observable reproductive performance

effect in zebrafish at environmentally recorded AP concentrations (Smith et al, 2001;

Patiiio et al, 2003).

Renal Effects of Perchlorate

Effects of AP on the renal system are relatively unknown. Early studies found that

AP is toxic to the kidney, but these studies often used extremely high doses of AP that

usually ended with injury to several other organs (SeUivanova et al, 1968). Some

epidemiological studies used kidney function as one of the parameters to determine the

risks of AP exposure in the workplace. One such study found that employees exposed to

up to 38 mg/kg/day of AP through inhalation and dermal absorption showed no signs of

kidney injury (Gibbs et al, 1998; Lamm et al, 1999).

Perchlorate has the potential to affect the renal system in several ways. Thyroid

hormones affect and regulate several important renal functions such as kidney growth and

development, plasma flow, filtration rates, and electrolyte metabohsm (Katz et al, 1975;

Capasso et al, 1999; Prasad et al, 1999;). Further, TSH receptors as well as thyroid

hormone receptors occur in mammaUan and teleost kidneys (MacLatchy and Eales, 1992;

Dutton et al, 1997; Sellitti et al, 2000; Vischer and Bogerd, 2003). Although the role of

TSH receptors in kidneys is unknown, it is conceivable that excess amounts of TSH and

decreased levels of thyroid hormones, as during exposure to AP, could affect TSH and

thyroid hormone sensitive renal fiinctions. Further, sodium-iodide symporters occur in

mammalian kidneys (Spitzweg et al, 2001). Human renal iodide symporters are sensitive

to perchlorate, thereby suggesting a similar iodide transport mechanism in the kidney and

thyroid, and similar susceptibility to perchlorate-induced inhibition (Spitzweg et al,

2001). Overall, these fmdings indicate that perchlorate may affect kidney fimction

directly, through interactions with sodium-iodide symporters or indirectly, through

alterations in TSH and thyroid hormone levels.

Considering the current attention on the biological effects of AP and the existence

of similarities between the thyroid and kidney, such as iodide transport, thyroid hormone

receptors, and TSH receptors (Spitzweg et al, 2001: Dutton et al, 1997; Sellitti et al.

10

2000), there is much that remains unknown about the affects of thyroid toxicants, like

AP, on renal function. The possibility that AP causes kidney dysfunction warrants

fiirther research on this topic.

Macrophage Aggregates

A commonly used histopathological indicator of toxic exposure in fish is the

increased number and size of macrophage aggregates (MAs) (Wo Ike, 1992).

Macrophage aggregates most often occur in spleen, kidney, and liver tissues of both

healthy and diseased or distressed fish (Jolly, 1923; Yoffey, 1929). Factors such as fish

age, size, diet, heaUh, and exposure to environmental contaminants can affect the number

and size of MAs (Brown and George, 1985; Blazer et al, 1987). Macrophage aggregates

play key roles in the capture of cell debris and foreign materials for reuse, destruction, or

detoxification (Ellis et al, 1976). Further, macrophage aggregates play important

immunological roles by serving as a primitive form of germinal centers, found in

mammals, which introduce antigens to lymphocytes which produce antibodies to fight

infection (Wolke, 1992). Macrophage aggregates also serve in cellular and tissue

responses to injury. Inflammation is a common response to tissue injury, which releases

chemical signals summoning defensive measures within an organism. Macrophages are

one of the responding defensive agents that act to prevent fiirther damage to tissues by

destroying invading organisms, isolating foreign materials, or removing cellular debris

(Wolke, 1992).

Often, the constituents of MAs can be determined by the pigments they contain.

Melanin containing MAs are common in many fish tissues, including the kidney, and

11

may aid in the neutralization of harmfiil oxidative species, the recycling and removal of

cellular debris, and the sequestering, destruction, and removal of contaminants (Wolke,

1992). The presence of hemosiderin, a protein-bound iron pigment, indicates the

breakdown and storage of iron-containing cellular products (Weeks et al, 1992; Blazer et

al, 1997). PAS-positive MAs indicate the presence of polysaccharide deposits within the

MA.

12

CHAPTER 11

LEGAL ASPECTS OF AMMONIUM PERCHLORATE

Due to the increased environmental occurrence of perchlorate, steps have been

initiated to regulate the amount of perchlorate in water systems. While perchlorate is

currently listed under the Drinking Water Contaminant Candidate List (CCL) (Motzer,

2000), the United States Environmental Protection Agency (USEPA) is compiling data to

determine the threat that perchlorate poses to the public by compiUng extensive

toxicological data as well as data pertaining to perchlorate's distribution and availability

(USEPA, 2003). In 2006, the USEPA will decide the threat perchlorate poses to the

pubUc and if it should be listed under the National Primary Drinking Water Regulation

(NPDWR). If perchlorate becomes listed imder the NPDWR, a rrmximum contaminant

level (MCL) for perchlorate will be determined and used as a standard for regulating

perchlorate contamination in drinking water supplies (Motzer, 2000).

Part of the data dealing with the persistence and availability of perchlorate the

USEPA is compiling comes from the listing of perchlorate as a List One unregulated

chemical under the Unregulated Contaminant Monitoring Regulation (UCMR). The

UCMR requires the monitoring of chemicals not listed under the NPDWR, but which

may pose a threat to public health, in public water supplies using set analytical methods.

As a List One chemical, the presence of perchlorate is monitored in pubUc drinking water

sources that service over 10, 000 people, as well as in smaller drinking water sources

13

selected to obtain a representative sample of small water systems within a state (Federal

Register, 2000).

In 1999, the USEPA established a reference dose (RfD) for perchlorate based on

doses of perchlorate found to cause thyroidal effects and disruption of neurological

development. The RfD for perchlorate is between .0001 and .0005 mg/kg/day, and a

hypothetical conversion of 4-18 ppb, which assumes 70 kg body weight and 2 liters of

water consumption per day. This RfD is used as an advisory level for perchlorate levels

in water, and as a guideline for RCRA and CERCLA remediation measures (USEPA,

2003).

The pending federal regulation of perchlorate caused several states to take steps to

control perchlorate contamination. California enacted an interim health standard of 18

ppb for perchlorate, which requires the removal of a water source from the public or

public notification of perchlorate levels found above the interim health standard (CDHS,

2003). Further, California enacted legislation calling for a risk assessment of perchlorate

in order to establish a primary drinking water standard that would take effect on January

I, 2004. California's primary drinking water standard will have the same effect of a

national drinking water standard in regards to regulating the cleanup and discharge of

perchlorate, and the removal of contaminated water sources from pubUc use (California

Health & Safety Code, 2003). Other southwestern states have estabUshed interim action

levels based on the USEPA's RfD that require remediation action in areas with

perchlorate contamination exceeding these levels (Logan, 2001). Arizona implemented a

14

health based guidance level of 14-ppb. Nevada implemented an action level at 18-ppb,

while the Texas action level is the highest at 22-ppb (Motzer, 2001).

Though federal drinking water regulation is awaiting review of data on the threat

perchlorate poses, several bills have been introduced in the United States Congress which

would accelerate regulation. The Preventing Perchlorate Pollution Act (The Act),

introduced to the United States House of Representatives on May 15, 2003, would

require the USEPA to list perchlorate as a contaminant under the NPDWR, and establish

an MCL for perchlorate by July 1, 2004. The Act also seeks to increase community

information on perchlorate contamination by requiring parties involved with a perchlorate

discharge into any water source to notify the appropriate state authorities of the

discharge. Further, annual pubUcation in the Federal Register of discharge information

and locations where perchlorate is detected in the groundwater would advance public

awareness of perchlorate contamination. The Act also requires owners or operators of

perchlorate storage facilities to report the storage conditions and amounts of perchlorate

stored at their facihty since 1950. To promote perchlorate remediation. The Act

establishes a fund, augmented by fines resulting from violations of The Act, which used

for perchlorate remediation efforts (H.R.2123, 2003).

Although perchlorate is not a chemical currently regulated by the NPDWR,

perchlorate falls under several other regulations. Among these are the Comprehensive

Environmental Response, Compensation and Liability Act (CERCLA), and the Resource

Conservation and Recovery Act (RCRA). These two acts serve to control the fate of

15

hazardous wastes and chemicals, and makes parties responsible for contamination liable

for remediation measures.

For a chemical to be regulated by RCRA, the chemical must either be listed as a

hazardous waste or have the characteristics of a hazardous waste. Though perchlorate is

not Usted as a hazardous waste, perchlorate has the requisite characteristics, namely its

reactive and explosive nature, to make it a hazardous waste under RCRA (Castaic Lake

Water Agency v. Whittaker Corp., 2003; 40 C.F.R. §§ 261.21-261). By coming under

RCRA regulation, the manufacture, transport, use, and disposal of perchlorate is closely

monitored and regulated (40 C.F.R. §§ 261.21-261).

CERCLA imposes liability on past and present owners of facilities involved in

releases of hazardous wastes. Owners, past and present, responsible for the

contamination of a site become responsible for the clean up and remediation of the

contaminated site (40 C.F.R. §§ 261.21-261). The liability of owners of toxic sites may

extend to areas outside of their facility. In California, an owner of a facihty that used

perchlorate was held liable for the cleanup and remediation of a public water source well

outside of the facility's boundaries after perchlorate seeped into the ground water

(Castaic Lake Water Agency v. Whittaker Corp., 2003).

The threat of civil liability for damages caused by perchlorate contamination may

aid in stemming future perchlorate contamination. California has been the hotbed of

perchlorate related civil Utigation. Most cases filed are class action suits due to the large

number of plaintiffs seeking similar relief Class action suits allow citizens, who

individually would not have the resources to pursue a claim, to pool their monetary

16

resources with others with like afflictions in order to seek legal relief The certification

of plaintiffs as a class is one of many obstacles in class action perchlorate litigation. In

California, plaintiffs from a city sued a corporation that released several harmful

chemicals, including perchlorate, into the water supply. The plaintiffs sought for the

corporation to fiind a health-monitoring program as well as pay punitive damages due to

the health effects caused by the released chemicals. The Superior Court of California

ultimately denied a class action certification of the plaintiffs, who potentially numbered

between 50,000 and 100,000 people, due to a lack of a demonstration of predominant

issues. While all of the plaintiffs experienced exposure to harmfiil chemicals, the level of

exposure was different, either due to differing lengths of exposure or amount of exposure

due to differing quantities of water consumption between the plaintiffs. The plaintiffs'

differing doses made every plaintiffs issues and potential remedies unique to themselves,

thus preventing their certification as a class (Lockhead Martin Corp. v. Superior Court,

2003). This limitation on class action certification severely hinders chemical

contamination Utigation. When a class certification requires plaintiffs to be exposed to

relatively the same cimounts of a chemical in order to obtain class certification, the suit,

along with many of the plaintiffs' chances of bringing a successful claim separately is

denied.

17

CHAPTER III

AMMONIUM PERCHLORATE-INDUCED LESIONS IN ZEBRAFISH KIDNEYS.

Introduction

Ammonium perchlorate (AP) is a strong oxidant used primarily as a rocket

propellant in military and aerospace industries and operations (Fisher et al, 2000). After

the dissociation of AP's ammonium and perchlorate ions in water, the resulting

perchlorate anion persists in the environment for a relatively long time (Fisher et al,

2000; Urbansky, 2002). In the United States, of the 39 states with facilities where AP is

manufactured or used, indicating possible perchlorate contamination, 19 states have

confirmed perchlorate contamination in ground or surface water (Sharp and Walker,

2001).

Perchlorate affects the fimction of the thyroid gland by inhibiting sodium-iodide

symporters that regulate the amount of iodide taken into thyroid follicles (Wolf, 1998).

Reduced thyroid foUicle uptake of iodide retards the production and release of th5Toid

hormones thereby reducing the amount of circulating thyroid hormones. In response, the

pituitary gland acts to trigger the release of thyroid hormones by releasing thyroid-

stimulating hormones (TSH). However, the impaired production of thyroid hormones

causes the continual hyperactive stimulation of thyroid follicles by TSH, and an excess of

circulating TSH (Katz, 1975; Wolf, 1998; Soldin et al, 2001). Laboratory studies have

confirmed these effects of perchlorate on thyroid function by finding that

environmentally relevant perchlorate exposures decrease circulating thyroid hormones

18

levels and increase circulating TSH levels in mammals and amphibians (Brechner et al,

2000; York et al, 2001b; Goleman et al, 2002a). Further, studies reveal that perchlorate

exposure leads to increased thyroid follicular cell hypertrophy, hyperplasia, angiogenesis,

and colloid depletion in mammals and zebrafish (Fernandez Rodriguez et al, 1991; Siglin

et al, 2000; York et al, 2001b; Patiiio et al, 2003).

Altered thyroid functions caused by perchlorate may consequently affect thyroid-

dependent reproductive and developmental processes such as bone and neurological

development in humans (Sharp and Walker, 2001). Environmentally relevant exposures

to perchlorate cause some developmental abnormaUties in frogs (Goleman et al,

2002a,b), mice (Thuett et al, 2002), and zebrafish (Brown, 1997). However, no clear

effects of environmentally relevant exposures to perchlorate on reproduction function

have been observed in zebrafish (Patino et al, 2003) or rodents (York et al, 2001 a,b).

Whereas the effects of perchlorate on the thyroid, development, and reproduction

are relatively well studied, effects on the renal system are somewhat unknowTi. Thyroid

hormones influence renal fiinctions such as kidney growth, development, plasma flow,

filtration rates, and electrolyte metaboUsm (SelUvanova et al, 1968; Capasso et al, 1999;

Prasad et al, 1999). Further, the discovery of mammaUan and teleost renal thyroid

hormone and TSH receptors (MacLatchy and Eales, 1992; Dutton et al, 1997; SeUitti et

al, 2000; Vischer and Bogerd, 2003), as weU as mammaUan perchlorate-sensitive

sodium-iodide transporters (Spitzweg et al, 2001), opens the possibiUty of perchlorate-

induced effects of renal function by indirectly affecting thyroid hormones and TSH

sensitive renal processes, or by directly inhibiting renal iodide transport.

19

Materials and Methods

Fish and Exposure Regimens

Samples for this study were generated during a previous study of AP effects on

zebrafish reproduction and thyroid function (Patifio et al, 2003). Adult zebrafish were

exposed to AP using a static-renewal procedure at concentrations of 6-ppb (control), 18-

ppm, and 677-ppm. Four to five female tank repUcates of 8 fish, and two male tank

repUcates of twelve fish were used per concentration for a total of 6-7 repUcates per

treatment. Because of the differential effects of AP at different concentrations on

reproductive performance (the focus of the original study; Patifio et al, 2003), the 677-

ppm groups offish were sampled at four weeks of exposure whereas the control and 18-

ppm groups were sampled at eight weeks. For the present study, at least three fish per

treatment repUcate (aquarium) were prepared for histological analysis of the kidney.

Histological Preparations

After euthanasia by immersion in 1 g/L MS-222, the fish were fixed whole in

Bouin's solution for 48 h at 4°C, rinsed in water for several hours, and placed in 70%

ethanol The trunk region of the fish was embedded in paraffin and serial sections of the

kidney (5-6 |im) were obtained with a microtome. SUdes were stained with Harris's

hematoxyUn stain and counterstained with eosin (Sigma Diagnostics, St. Louis, MO).

Select sUdes were also stained with Mason's Trichrome Stain (Sigma Diagnostics, St.

20

Louis, MO), periodic acid Shiff (PAS) reagent (Sigma Diagnostics, St. Louis, MO), and

Wright's Iron Stain (Sigma Diagnostics, St. Louis, MO).

Histological Observations

A preliminary microscopical analysis at lOOx total magnification for general

abnormaUties was performed on all of the fish samples. If these observations resulted in

the finding of lesions or chemges of potential interest, further examination was conducted.

Following these preliminary observations, all subsequent histological observations were

recorded using the same tissue section for each fish sample. The sections were chosen

according to their histological integrity and quaUty when viewing the first row of sections

on the sUde from left to right. To measure the incidence of each histological character of

interest, a 1 x l-mm grid containing 121 crosshairs was positioned over the left lobe of

the kidney (oriented so that the spine was at the top of the view) covering as much kidney

tissue area as possible at lOOx total magnification. Crosshairs of the grid that fell on each

type of lesion were counted. Crosshairs that feU outside the kidney or on large blood

vessels were regarded as misses and subtracted from the total crosshair count.

Estimations of the area of kidney sections covered by a particular lesion were determined

by dividing the number of crosshairs faUing on the lesion by the total crosshair count.

These measurements were performed three separate times on each fish sample using the

same tissue section.

21

Statistical Analvsis

For each lesion, the average of the three measurements was designated as the fish

value. The fish values within each aquarium were then averaged to determine the tank

value. The tank values, grouped by treatment, were then subjected to statistical analysis

(Patiiio et al, 2003).

Analyses were conducted using the STATISTICA for Windows 1998 software

package (StatSoft®; Tulsa, OK, USA). Because of the difference in treatment durations

(Patifio et al, 2003), the 18-ppm and 677-ppm groups could not be compared to each

other. Thus, the following plarmed comparisons were made: control versus 18 ppm, and

control versus 677 ppm. The assumption made for the latter comparison is that the kidney

condition in the control group (sampled at 8 weeks) is unlikely to have changed

significantly from the time the 677-ppm group was sampled at 4 weeks. The data was

analyzed using Student's ^tests to determine treatment effects, and a 2-way ANOVA test

to determine effects caused by treatment and sex. Homogeneity of variances was assessed

using the Cochran C statistic and the Bartlett Chi-square test, and appropriate data

transformations were used if needed to correct non-homogeneities. Statistical differences

were considered significant at overaU a of 0.05.

Results

There were 6, 7, and 6 aquarium repUcates for the control, 18-ppm, and 677-ppm

treatments, respectively (Patiiio et al, 2003). Observations from the present study showed

that fish from one male control aquarium and one male 18-ppm aquarium had signs of

22

kidney mycobacterial infection, which exacerbated the incidence of some of the lesions

of interest. These two aquaria were omitted from the statistical analysis, leading to a

final 5, 6, and 6 replicate count for the control, 18-ppm, and 677-ppm treatments,

respectively. The omission of these two male tanks resulted in one male tank for the

control and 18-ppm groups for which to conduct a sex-based analysis. Due to the low

sample size of males in this study, a statistical analysis based on sex was not feasible.

Based on the observations made during the preliminary scan of histological

sections, the specific kidney lesions recorded in subsequent detailed observations were

macrophage aggregates (MAs), focal granulomas, inflammation, and generating tubules.

In sections stained with hematoxylin-eosin, macrophage aggregates were observed as

clear or light-brown clusters of macrophages of varying sizes (Fig. 1). CeUs in some of

the aggregates were positive for PAS (Fig. 2) and Wright's Iron Stain (Fig. 3) indicating

the presence of polysaccharides and iron, respectively. Macrophage aggregates were most

readUy observed in the 18-ppm group, and the results of the Student's /-test indicated

significantly higher levels in the 18-ppm group when compared with the control group {P

= 0.015639, square-root data transformation; Fig. 4, Table 1). However, no significant

differences were observed between the 677-ppm and control groups (P > 0.05; square-

root data transformation; Fig. 4, Table 1). Focal granulomas were characterized by dark-

staining, basophilic, encapsulated, cyst-Uke masses (Fig. 5). The occurrence of focal

granulomas was not statisticaUy significant between the control and 18-ppm groups

(Student's /-test, P > 0.05, square-root data transformation; Fig. 4, Table 1), or the control

and 677-ppm groups (Student's /-test, P > 0.05; square-root data transformation; Fig. 4,

23

Table 1). Inflammation was sometimes associated with focal granulomas and was

characterized by aggregations of eosinophiUc cells around a darkly staining eosinophiUc

core (Fig. 6). The incidence of inflammation was not significantly different between the

control and 18-ppm groups (Student's /-test; P > 0.05, square-root data transformation;

Fig. 4, Table 1), or the control and 677-ppm groups (Student's /-test; P > 0.05; square-

root data transformation; Fig. 4, Table 1). Newly forming kidney tubules were observed

as darkly stained (basophilic) small tubules or cell clusters (Fig. 7). The incidence of

generating kidney tubules was not significantly different between the control and 18-ppm

groups (Student's /-test; P > 0.05; Fig. 4, Table 1), or the control group and the 677-ppm

group (Student's /-test; P > 0.05; Fig. 4, Table 1).

Discussion

The present study showed that kidneys of zebrafish exposed to 18-ppm AP for

eight weeks have an increased incidence of macrophage aggregates (MAs). Increases in

the number and size of MAs are considered indicators of exposure to environmental

contaminants (Wolke, 1992), and may serve as an immunotoxicologic biomarker (Weeks

et al, 1992; Blazer et al, 1997). MAs are a common response to tissue injury (WoUce,

1992); therefore, the increase in kidney MAs in zebrafish exposed to AP suggests that AP

affects renal condition.

The renal MAs of zebrafish in the present study contained some melanin,

hemosiderin, and PAS-positive components. Melanin containing MAs are common in

many tissues of fishes, including the kidney, and aid in several responses to ceUular

24

injury (Weeks et al, 1992; Blazer et al, 1997). The presence of hemosiderin, a protein-

bound iron pigment, indicates the breakdown and storage of iron-containing cellular

products. Therefore, an increase in the amount of hemosiderin-containing MAs could

indicate alterations in the hemopoietic function of kidneys (Wolke, 1992). PAS-positive

MAs indicate the presence of polysaccharide deposits.

Based on the findings of a similar study conducted on mosquitofish, the increase

in MAs found in zebrafish kidneys in the present study is likely due to perchlorate

exposure and not ammonium exposure. In the mosquitofish study, adult mosquitofish

were exposed to 0, 1, 10, and 100-ppm of sodium perchlorate for eight weeks. The 100-

ppm group, when compared with the control, had a significant increase in MAs, like is

seen in the present study (Mukhi et al, unpubUshed data). Due to the similar effects from

exposure to different perchlorate species, it can be assumed that in the present study,

ammonium did not play a substantial role in the increased incidence of MAs.

The present study showed that perchlorate exposures at 18-ppm for eight weeks,

but not at 677-ppm for four weeks, induced an increase in kidney MAs in zebrafish. This

pattern of AP effects in the kidney closely paraUels the pattern of histopathological

effects in the thyroid reported in a previous study with the same fish (Patino et al, 2003).

The effects of AP on foUicular ceU hypertrophy, hyperplasia, and coUoid depletion in fiish

exposed to 18-ppm for eight weeks were much greater than in fish exposed to 677-ppm

for four weeks (Patiiio et al, 2003).

The similarity in the patterns of AP effects in the thyroid (Patino et al, 2003) and

the kidney (present study) suggests that the effects of AP in both tissues are linked in

25

zebrafish. The presence of thyroid hormone receptors and TSH receptor mRNA in teleost

kidneys (MacLatchy and Eales, 1992; Vischer and Bogerd, 2003) suggests the possibiUty

of perchlorate-dependent effects on the kidneys via the reduction of thyroid hormone

levels or the subsequent increase in circulating TSH. A time-dependent inhibitory effect

of perchlorate on thyroid condition, as seen in mammals (Fernandez Rodriguez et al,

1991), possibly explains the greater thyroid histopathological effects (Patiiio et al, 2003)

and higher incidence of renal MAs (present study) in zebrafish exposed to 18 ppm

perchlorate for eight weeks when compared to zebrafish exposed to 677 ppm for four

weeks. An ahemative scenario is that perchlorate directly affected kidney condition thus

leading to the increased incidence of renal MAs. However, this scenario does not clearly

account for the similar time-concentration patterns of response of the thyroid (Patifio et

al, 2003) and kidney (present study) to AP exposure.

26

Figure 1: Hemotoxilyn-Eosin stained macrophage aggregate. Arrow pomts to melamn containing macrophage aggregate. Bar = 50 |um.

27

r r

k1 Figure 2: PAS-stained macrophage aggregates. Arrow points to red staining polysaccharide deposits. Bar = 100 ̂ im.

28

Figure 3: Iron-stained macrophage aggregate. Arrows point to blue staining hemosiderin deposits (Arrow). Bar = 50 |am.

29

•D «

> 0 U (0

C

i . « Q.

3n

c 2-

1-

^ • 6 ppb ^ ^ 1 8 ppm 1=1677 ppm

i Focal

I ̂ New

Granuloma Nephrons

Inflammation

Lesions

i i i Macrophage

Aggregates

Figure 4: Percent kidney area covered by lesions in zebrafish exposed to ammonium perchlorate. Asterisk indicates significant difference (P < 0.05).

30

Lesion:

Macrophage Aggregates

Focal Granuloma

Inflammation

New Nephrons

Control

0.48

0.0

0.0

0.18

18-ppm

2.385 /'= 0.0156

0.367 P= 0.095

0.185 P= 0.1878

0.98 P= 0.0996

677-ppm

0.39 P= 0.3087

0.0

0.0

0.22 P= 0.7759

Figure 5: Mean area of kidney histological sections covered by lesions, and/?-values of statistical comparison. The 18-ppm group and 677-ppm group were compared to the control group separately.

31

Figure 6: Hemotoxylin-Eosin stained focal granuloma with basophiUc encapsulation. Bar = 40 pm.

32

Figure 6: HemotoxyUn-Eosin stained inflammation with eosinophiUc ceUs and core (arrow). Bar = 50 |um.

33

Figure 7: HemotoxyUn-Eosin stained generating tubule cluster (Circle). Note the highly basophiUc nature of the tubules. Bar = 100 pm.

34

CHAPTER IV

CONCLUSIONS

The presence of MAs in the kidney of zebrafish exposed to AP at 18 ppm may be

an indicator of a toxic renal response to environmentally relevant (Smith et al, 2001),

concentrations of perchlorate. The increase in renal MAs also suggests a link to a

perchlorate inhibited thyroid system. These observations indicate that perchlorate has

effects on organisms beyond its classical effects on the thyroid and general development,

and extends to other physiological systems that may be affected by perchlorate directly,

or through an altered thyroid system. Due to the many systems affected by the thyroid

system, including the renal system, research into the effects of perchlorate on systems

affected by the thyroid system are necessary for establishing safe standards of perchlorate

consumption.

This study also raises several other issues for fiirther study. The increased

incidence of MAs suggests the need for additional and more detailed studies of the effect

of perchlorate on kidney function. Increased MAs are a structural kidney abnormality.

However, their increased presence may not indicate a harmfiil effect of AP on the kidney.

Since an AP induced structural effect on zebrafish kidneys occurs, it is important to

determine if renal function is adversely and irreversibly affected in zebrafish, as well as

mammals. Further, the present study revealed a renal effect at an exposure of 18-ppm for

eight weeks. Whether or not there is a similar effect at smaller doses for longer periods

of time, or whether the same effect can be found in mammals at similar times and doses.

35

or any time and dose, needs to be determined in order to establish a safe level of AP

exposure for humans.

In conclusion, while there is much known about the effects of perchlorate on

certain physiological processes, such as the thyroid and development, the effects of

perchlorate on other systems needs fiirther study.

36

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41

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