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Articles

Embryonic Treatment with Xenobiotics Disrupts Steroid Hormone Profiles in


Hatchling Red-Eared Slider Turtles (Trachemys scripta elegans)
Emily Willingham, Turk Rhen, Jon T. Sakata, and David Crews
Institute of Reproductive Biology, The University of Texas, Austin, Texas, USA

After hatch, some turtles in the assay were


Many compounds in the environment capable of acting as endocrine disruptors have ben assayed sexed immediately, whereas others were kept
for their developmental effcts on morphogenesis; however, few studies have addressed how such for blood sampling for radioimmunoassay
xenobiotics affect physiology. In the current study we examine the effects of three endocrine-dis- (RIA), after which they also were sexed.
rupting compounds, chlordane, tra"nnonadlor, and the polychlorinated biphenyl (PCB) mix- Hatchling FSH challenge and RM4. Blood
ture Arocdor 1242, on the steroid hormone concentrations of red-eared slider turle (Trachemys samples were drawn from 195 turtles (59
scnpta elegans) hatchlings treated in ovo. Basal steroid concentrations and steroid concentrations in temperature control, 51 aroclor-treated, 43
response to follide-stimulating hormone were examined in both male and female turtles treated chlordane-treated, and 42 trans-nonachlor-
with each of the three compounds. Treated male turtles exposed to Arodor 1242 or chlordane treated) at 6 weeks of age. Turtles from each
exibited sigificntly lower testosterone concentrations than controls, whereas chlordane-treated xenobiotic treatment group were randomly
females had significantly lower progesterone, testosterone, and 5c-dihydrotestosterone concentra- assigned to one of three groups for RIA:
tions relative to controls. The effects of these endocrine disruptors extend beyond embryonic nontreated control, vehicle-treated control
development, altering sex-steroid physiology in exposed animals. Key wordx Aroclor 1242, chlor- (0.5 mL nanopure water), and follicle-stimu-
dane, PCBs, steroid hormone, xenobiotic. Environ Health Perect 108:329-332 (2000). [Online lating hormone (FSH)-treated (50 pg ovine
22 February 2000] FSH dissolved in 0.5 mL nanopure water; 1
/rnp://ehpnetl.niehs. nih.gov/docs/2OOO/O8p329-332willngham/abstract.html U/mg; Sigma, St. Louis, MO). Nontreated
turtles were anesthetized on ice and then
pithed, whereas vehicle-treated and FSH-
Endogenous hormones exert effects through- that their sex steroid physiology might have challenged turtles were first injected
out embryonic development, after birth, and been affected as compared to unexposed intraperitoneally with either water vehicle
into adulthood. Compounds in the environ- hatchlings from the same temperature. (0.5 mL H20) or FSH, maintained at room
ment that operate as endocrine disruptors temperature for 3 hr, anesthetized on ice,
may also have the capacity to affect organ- Materials and Methods and then pithed. To obtain an adequate vol-
isms from conception to death (1). Embryonic xenobiotic treatment. We pur- ume of blood from each euthanized turtle
Endocrine-disrupting compounds (EDCs) chased turtle eggs from a commercial supplier (0.15-0.4 mL whole blood), the heart was
found in alligator eggs from Lake Apopka, (Robert Kliebert, Hammond, LA) and can- exposed by removal of a small window from
Florida (2), have the ability to affect sex deter- dled the eggs in the laboratory to establish via- the plastron, the ventricle and atria were cut
mination in the red-eared slider turtle bility. Viable eggs were placed randomly (to with microdissection scissors, and blood was
(Trachemys scripta elegans), a species with tem- separate clutches) in groups of 35 into trays collected directly from the hemorrhaging
perature-dependent sex determination (TSD) containing 1:1 vermiculite:water (wt/vol) and heart with a heparinized 1-cc syringe. Blood
(3). In TSD, the temperature of the incubat- incubated at 28.6C in computer-controlled was then transferred to 0.5-mL microfuge
ing egg determines the sex of the animal; low incubators. We also monitored the tempera- tubes and centrifuged at 3,000 rpm for 10
temperatures (< 28.2C) produce male-biased ture using HOBO temperature loggers min at 40C. Plasma was drawn off and
sex ratios, with temperatures < 27.8C pro- (Onset Computer Corporation, Bourne, MA) frozen at -80C. Upon sacrifice, the gonadal
ducing all males; higher temperatures and by daily readings of in-incubator shelf sex of each turtle was diagnosed as described
(29.4C) produce female-biased sex ratios, thermometers. Temperature variation was previously (6). Briefly, the plastron was
with temperatures > 31C producing all slight, with a minimum temperature of 28.5 removed and the gonads were examined
females (4). Temperature exerts its effects in and a maximum of 28.70C. All turtles exam- under a dissection microscope. Appearance
the middle third of embryonic development, ined in this study were incubated simultane- of gonads (size, shape, vascularization) and
but application of hormones or EDCs during ously. We selected the 28.60C temperature assignment of sex was noted, as was presence
this period can alter the temperature-induced because it is a "threshold temperature" at or absence of oviducts.
outcome (3,5,6). For example, estradiol (E2) which females first appear in the sex ratio. We pooled plasma from two to three
applied to eggs incubated at a normally male- Treatment occurred at Stage 17, during individuals to make 600 pL samples for RIA
producing temperature causes development of the middle third of development (5). Before
females (5). Mixtures of polychlorinated treatment at Stage 17, all eggs were shifted Address correspondence to D. Crews, Institute of
biphenyls (PCBs) also cause female sex deter- around among the boxes so that no box con- Reproductive Biology, University of Texas at
mination at male-producing temperatures (6), tained eggs that all came from the same Austin, Austin, TX 78712-1064 USA. Telephone:
as do chlordane, the PCB mixture Aroclor clutch. Eggs were treated with one of the (512) 471-1113. Fax: (512) 471-6078. E-mail:
crews@mail.utexas.edu
1242, and trans-nonachlor (3). In the current treatment compounds dissolved in dimethyl- We thank the five anonymous reviewers for their
study we examined the effects of each of three sulfoxide (DMSO), or with DMSO alone as helpful comments and S. Woolley for her technical
compounds-chlordane, the PCB mixture a vehicle control. We selected the concentra- assistance.
Aroclor 1242, and trans-nonachlor- on sex tions based on previously published results This work was supported by NSF IBN-9723617
steroid levels in hatchling red-eared slider tur- with these compounds (3); specific concen- to D.C., an Individual National Research Service
Award (MH 11369) from the National Institute of
tles treated during embryogenesis. Although trations and amounts are shown in Table 1. Mental Health to T.R., and an NSF Predoctoral
the hatchlings from groups with sex ratios Eggs were spotted once with 5 pL dissolved Fellowship to J.T.S.
altered by xenobiotic exposure may appear compound or vehicle and returned to incu- Received 14 July 1999; accepted 29 October
morphologically normal, we hypothesized bators for the duration of development (5). 1999.

Environmental Health Perspectives * Volume 08, Number 4, April 2000 329


Articles * Willingham et al.

Table 1. Concentration and amount of each com- an experimentwise a = 0.05, and a nominal 5a-DHT levels in females (ANOVA, F ratio
pound administered to each turtle egg based on a = 0.008 for individual comparisons. If the = 8.72; df= 3,12; p = 0.002) (Figure 2B).
concentrations found in egg yolks of alligators at overall interaction was not significant, we DHT levels were only significantly lower in
Lake Apopka, Florida (2). then examined the main effects. If the main chlordane-treated females as compared to
Concentration Amount effect of xenobiotic treatment influenced RIA control females (p < 0.001). There was
Xenobiotic (pM) (ng/10 g egg) steroid levels, we determined which xenobi- no significant effect of FSH administration
Aroclor 1242 0.26 0.424 otic groups differed significantly from the or a significant interaction between xenobi-
Chlordane 0.22 0.451 RIA control group, for a total of three otic treatment and FSH challenge on 5a-
trans-Nonachlor 0.25 0.556 comparisons, an experimentwise a = 0.05, DHT levels (p > 0.05 for all).
and a nominal a = 0.017 for individual Xenobiotic treatment during embryonic
because a pilot study indicated that hormone comparisons. All statistics were calculated development significantly affected plasma
levels were very low. Consequently, pooled using Version 3.1 of JMP (SAS Institute, progesterone levels in female turtles, which
samples included 23 temperature control, 19 Cary, NC) (9) for Apple Macintosh (Apple was similar to its effect on testosterone and
Aroclor 1242-treated, 18 chlordane-treated, Computer, Inc., Cupertino, CA). 5a-DHT (ANOVA, F ratio = 19.94; df= 3,
and 15 trans-nonachlor-treated turtles, half 12; p < 0.001) (Figure 2C). Progesterone
of which were FSH-treated. We then Results levels were significantly lower only in chlor-
assayed the samples for 5a-dihydrotestos- Sex ratio. Each of the three compounds dane-treated females as compared to RIA
terone (DHT), E2, progesterone, and testos- altered the male:female ratio as compared to control females (p < 0.001). There was no
terone using the methods of Tousignant that of the temperature controls (108 significant effect of FSH administration or a
and Crews (7). Assay sensitivity was 13 pg males:24 females). The male:female sex ratio significant interaction between xenobiotic
5a-DHT/mL plasma, 13 pg E2/mL plasma, in this study for each compound was 54:30 treatment and FSH challenge on proges-
106 pg progesterone/mL plasma, and 10 pg for Aroclor 1242 (p = 0.003; X2 = 8.261); terone levels (p > 0.05 for all).
testosterone/mL plasma. Intra-assay coeffi- 29:14 for chlordane (p = 0.041; X2 = 3.705); Concentrations of E2 were not detectable
cients of variation were 16% for 5a-DHT, and 51:23 for trans-nonachlor (p = 0.027; X2 in any group of females (i.e., concentrations
18% for E2, 12% for progesterone, and 17% = 4.364). were < 13 pg E2/mL plasma).
for testosterone. Interassay coefficients of Males. Circulating levels of testosterone
variation were 18% for 5a-DHT, 17% for in hatchlings were influenced by xenobiotic Discussion
E2, 20% for progesterone, and 13% for treatment during embryonic development Altered hormone levels in response to embry-
testosterone. [ANOVA, p = 0.003; F ratio = 5.31; degrees onic exposure to xenobiotics have been
Statistical analysis. We analyzed sex ratio of freedom (df) = 3, 45] (Figure 1). identified in alligators (10) and in male rats
data using two-by-two contingency tables. Testosterone concentrations were signifi- (11); however, the present study provides the
All experimental groups for the sex ratio cantly lower in Aroclor- and chlordane-treat- first direct link between effects on sex deter-
analysis were compared to simultaneously ed males as compared to RIA control males mination and sexual differentiation during
incubated controls using Fisher's exact one- (p = 0.003 and p = 0.001, respectively). embryogenesis and subsequent effects follow-
tailed tests. We used one-tailed tests because Although males treated with trans-nonachlor ing birth. The three compounds used in this
the results of Willingham and Crews (3) tended to have reduced testosterone levels as study significantly influenced the normal
demonstrated that the compounds used in compared to RIA control males, the differ- process of gonadal differentiation, which sup-
this experiment alter temperature-dependent ence did not reach the statistical criteria of a ports previous results (3). Moreover, two of
gonadal sex determination, increasing the = 0.017 (i.e., p = 0.039). There was no sig- these compounds also exert significant effects
ratio of females. nificant effect of FSH administration or a after hatch by altering circulating hormone
Plasma steroid levels were log-trans- significant interaction between xenobiotic 70.
formed to eliminate unequal variances and treatment and FSH administration for
compared using separate two-way analyses of testosterone levels (p > 0.05 for all). 6B0
variance (ANOVAs) for males and females Neither 5a-DHT nor progesterone levels
for each steroid. Because the nontreated and were influenced by embryonic xenobiotic
vehicle-treated groups for RIA did not differ, treatment or FSH treatment (p > 0.05 for all). -40
E 300 .; :i . ... ... . :.. ... ..
they were combined in the analysis and are Concentrations of E2 were not detectable
......

hereafter described as "RIA control." Thus, in many (72%) of the samples (i.e., < 13 pg 20
independent variables in the two-way E2/mL plasma).
ANOVAs were embryonic xenobiotic treat- Females. Although sample sizes were
ment, FSH challenge, and their interaction. smaller for females, xenobiotic treatment dur- Control Aroclor Chlordane trans-Nonachlor
Because the following hierarchical set of ing embryonic development also significantly Embryonic xenobiotic treatment
comparisons among treatment groups was influenced plasma testosterone levels in
planned a priori, we used the Dunn-Sidak female turtles (ANOVA, F ratio = 4.44; df= Figure 1. Plasma testosterone (T) concentrations
correction for multiple comparisons (8). We 3,1 1; p = 0.028) (Figure 2A). Testosterone in male hatchling red-eared slider turtles
(Trachemys scripta elegans) as a function of
first determined if the interaction between levels were significantly lower in chlordane- embryonic xenobiotic treatment. Testosterone
embryonic xenobiotic treatment and hatch- treated females as compared to RIA control concentrations are shown as least-squares
ling FSH administration was significant. If females (p = 0.005). There was, however, no means ( 1 SE) from the ANOVA, as described in
this interaction was significant, we deter- significant effect of FSH challenge or a signifi- "Materials and Methods." These means repre-
mined whether basal steroid levels differed cant interaction between xenobiotic treatment sent the means for each treatment group while
between RIA controls and xenobiotic-treated and FSH administration on testosterone levels controlling for all other independent variables in
the experiment. The number in the bar is the sam-
turtles and whether FSH-induced steroid lev- (p > 0.05 for all). ple size. Each sample represents plasma pooled
els differed between control and xenobiotic- Xenobiotic treatment during embryonic from two to three individuals.
treated turtles, for a total of six comparisons, development significantly influenced plasma *Significant relative to control (p > 0.05).

330 Volume 108, Number 4, April 2000 * Environmental Health Perspectives


~~~~~~~~~~~~~~~~~~~~~~~~~~ Articles * Xenobiotics disrupt steroid hormone
. profiles in turtles

levels in both male and female turtles. In male proposed that dioxin affects these changes by applied E2, causing a significant increase in
hatchlings exposed during embryogenesis, altering the testosterone synthetic pathway female hatchlings as compared to the effects
Aroclor and chlordane lowered testosterone (11). Thus, changing the pattern of steroid of E2 alone (3). Additionally, female
levels; in female hatchlings exposed during enzyme activity is one possible mechanism hatchlings from chlordane-treated groups
embryogenesis, chlordane caused a reduction by which EDCs can affect reproductive sys- exhibited lower testosterone, DHT, and
in testosterone, DHT, and progesterone. tem function after hatch. progesterone concentrations. These results,
Although much of the research in this Two key steroidogenic enzymes in red- taken together, suggest that chlordane is an
field has focused on mammals, studies using eared slider turtle sex development are anti-androgen which complements the effects
TSD reptiles are informative because of the reductase, which biosynthesizes nonaromati- of E2; furthermore, chlordane may exert its
many similarities between the patterns of zable androgens from testosterone, and aro- effects on the sex ratio by reducing androgen
gonadal development and the possibilities for matase, which metabolizes precursors into concentrations, resulting in an altered
manipulation that TSD reptiles offer (5). estrogens (15). A change in steroidogenic steroidal milieu. The subsequent reduction in
When gonadal sex is altered by administra- enzyme activity causes a shift in the hormon- androgen concentrations could be extreme
tion of a natural estrogen (e.g., 170-estradiol) al milieu of an organism; in the red-eared enough to keep them significantly below
or an aromatase inhibitor during embryogen- slider turtle, these alterations may result in those found in normal females.
esis, steroid profiles do not change detectably the change from one gonadal pathway to the In some cases, turtles that did not undergo
in the young snapping turtle (Chelydra ser- other (5). Applying E2 to eggs during incu- a change to a female pathway still exhibited
pentina), another TSD species (12). Similarly, bation at a male-producing temperature altered levels of circulating steroid hormones
no morphologic or histologic differences have results in females, thus mimicking the end after hatch. Both chlordane and the Aroclor
been observed between temperature-deter- point of the pathway triggered at a female- mixture caused a decrease in testosterone con-
mined female red-eared slider turtles and producing temperature (5). Xenobiotics may centrations in males. Morphologically, the
females resulting from treatment with natural cause a similar shift by altering the sex gonads of these hatchlings appeared normal.
estrogens (5). In juvenile American alligators steroid milieu, either by affecting steroido- These data suggest that chlordane and
(Alligator mississippiensis) exposed to contami- genic enzyme production, activity, or degra- Aroclor 1242 both acted to alter the internal
nants in Lake Apopka, the testes appear nor- dation rates, or by directly mimicking or steroidal milieu, but not to the extent of shift-
mal morphologically; however, the circulating inhibiting sex steroid hormones themselves. ing development to the female sex-determin-
concentration of testosterone in males is Aroclor 1242, the PCB mixture that we ing pathway. Chlordane, once again, could
reduced, as is the activity of aromatase, the used in the current study, lowered concen- cause lowered testosterone concentrations
enzyme that converts testosterone to E2 (10). trations of testosterone in both males and through its activity as an anti-androgen,
These effects have been attributed to contam- females; in males, Aroclor 1242 appeared to whereas Aroclor could have this effect by
ination of the lake by agricultural runoff and cause an increase in E2 levels (data not causing an increase in aromatase activity. One
a pesticide spill. shown). Thus, Aroclor 1242 might act by question that arises, however, is why chlor-
Other species also respond to prenatal increasing aromatase activity, resulting in dane did not cause a reduction in nonaroma-
exposure to EDCs. Aroclor 1254, another lower testosterone concentrations and an tizable androgens in the male hatchlings as it
PCB mixture, causes a decline in steroid increase in E2 concentrations. However, did in the exposed females. Applying increas-
hormone levels in female Atlantic croakers Aroclor 1242 does not increase E2 concen- ing concentrations of testosterone to eggs at
(13). Interestingly, although the testes of alli- trations in females, which are undetectable male-producing temperatures causes concor-
gators experimentally treated in ovo with the in this assay; this may result because the pre- dantly increasing ratios of females to develop;
herbicide atrazine appear normal morpho- ferred estrogen synthetic pathway in female this effect is blocked when testosterone is
logically, the pattern of aromatase activity is red-eared slider turtles is to estrone or estriol, applied in conjunction with aromatase
altered compared to that of normal males rather than to E2. Both estrone and estriol inhibitor (15). Thus, in the presence of excess
(10). Bass exposed to EDCs in two Florida are more powerful estrogens than E2 in this testosterone, aromatase is the preferential
rivers exhibit decreased testosterone levels species when applied exogenously in sex enzyme. But what about lowered concentra-
and are vitellogenic, suggesting altered ratio studies (5). tions of testosterone, as in the case of the
steroidogenic enzyme activity (14). 2,3,7,8- Chlordane caused a significant increase in chlordane-exposed males? A possible explana-
Tetrachlorodibenzo-p-dioxin lowers serum the female sex ratio in this study, a result that tion for the differences in the effects of chlor-
androgen levels in adult male Sprague- supports previous work (3). Chlordane also dane is that for the embryos irreversibly
Dawley rats exposed in utero, and it has been has been shown to interact with exogenously differentiated as males, lower testosterone
0 7i0 700
60 60 !i.
'50 550
.40 ~40
E
E
.30 ~30 3 S,

20 ..~~~~~20 200
100~~~~~~~1
..........

Control Aroclor Chiordane trans-Nonachlor Control Aroclor Chiordane trans-Nonachlor Control Aroclor Chlordane trans-Nonachior
Embryonic xenobiotic treatment Embryonic xenobiotic treatment Embryonic xenobiotic treatment
Figure 2. (A) Plasma testosterone (T), (B) 50c-dihydrotestosterone (DHT), and (C) progesterone (P) concentrations in female hatchling red-eared slider turtles
(Trachemys scripta elegans) as a function of embryonic xenobiotic treatment. Hormone concentrations are shown as least-squares means (+ 1 SE) from the
ANOVA, as described in "Materials and Methods." The number in the bar is the sample size. Each sample represents plasma pooled from two to three individuals.
*Significant relative to control (p > 0.05).

Environmental Health Perspectives * Volume 108, Number 4, April 2000 331


Articles * Willingham et al.

concentrations elicit an increase in reductase shifted to a female developmental pathway gonadal sex affect growth and physiology in the leopard
activity, so that late-stage embryos and (12). Future studies examining the effects of gecko Eublepharis macularius, a lizard with temperature-
dependent sex determination. J Morphol 224:1-12 (1995).
hatchlings have normal concentrations of these compounds on estrogen receptor levels 8. Sokal RR, Rohif FJ. Biometry. 2nd ed. New York:Freeman,
nonaromatizable androgens. For embryos during development will help elucidate this 1981.
irreversibly dedicated to the female pathway, matter. 9. SAS. JMP User's Guide, Version 3.1. Cary, NC:SAS
Institute, 1998.
a mechanism for maintaining androgen con- 10. Crain DA, Guillette LJ Jr, Rooney AA, Pickford DB.
centrations would not be necessary. REFERENCES AND NOTES Alterations in steroidogenesis in alligators (Alligator mis-
Interestingly, in studies of male and sissippiensis) exposed naturally and experimentally to
environmental contaminants. Environ Health Perspect
female hatchlings from different incubation 1. Crisp TM, Clegg ED, Cooper RL, Wood WP, Anderson DG,
Baetcke KP, Hoffman JL, Morrow MS, Rodier DJ, 105:528-533 (1997).
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reduction in the concentration of testosterone an effects assessment analysis. Environ Health Perspect tational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin
(16). Additionally, progesterone concentra- 106(suppl 1):11-56 (1998). (TCDD) on serum androgens and steroidogenic enzyme
2. Heinz GH, Percival HF, Jennings ML. Contaminants in activities in the male rat reproductive tract. J Steroid
tions decrease with increased incubation tem- American alligator eggs from Lake Apopka, Lake Griffin, Biochem Mol Biol 674:347-354 (1998).
perature. Aroclor and chlordane both caused a and Lake Okeechobee, Florida. Environ Monit Assess 12. Rhen TR, Elf PK, Fivizzani AJ, Lang JW. Sex-reversed and
reduction in testosterone, and chlordane 16:277-285 (1991). normal turtles display similar sex steroid profiles. J Exp
3. Willingham EJ, Crews D. Sex reversal effects of environ- Zool 274:221-226 (1996).
treatment resulted in decreased progesterone mentally relevant pesticide concentrations on the red- 13. Thomas P. Teleost model for studying the effects of
and DHT in females. These results suggest eared slider turtle, a species with temperature-dependent chemicals on female reproductive endocrine function. J
that the Aroclor mixture and chlordane affect sex determination. Gen Comp Endocrinol 113:429-435 Exp Zool 4:126-128 (19911.
(1999). 14. Orlando EF, Denslow ND, Folmar LC, Guillette LJ Jr. A
the pathway triggered by incubation tempera- 4. Crews D, Bergeron JM, Bull JJ, Flores D, Tousignant A, comparison of the reproductive physiology of large-
ture, and their effects, at least at the level of Skipper JK, Wibbels T. Temperature-dependent sex mouth bass, Micropterus salmoides, collected from the
circulating steroid hormones, mimic those of determination: proximate mechanisms, ultimate out- Escambia and Blackwater rivers in Florida. Environ
comes, and practical application. Dev Gen 15:297-312 Health Perspect 107:199-204 (1999).
increasing or decreasing temperature. (1994). 15. Crews D, Bergeron JM. Role of reductase and aro-
trans-Nonachlor did not affect circulating 5. Crews D. Temperature-dependent sex determination: the matase in sex determination in the red-eared slider turtle
steroid hormone concentrations in either interplay of steroid hormones and temperature. Zool (Trachemys scripta), a turtle with temperature-depen-
Science 13:1-13 (1996). dent sex determination. J Endocrinol 143:279-289 (1994).
male or female turtles. This result suggests 6. Bergeron JM, Crews D, McLachlan JA. PCBs as environ- 16. Rhen T, Willingham EJ, Sakata JT, Crews D. Incubation
that trans-nonachlor may act as a direct estro- mental estrogens: turtle sex determination as a biomark- temperature influences sex-steroid levels in juvenile red-
gen mimic, possibly without aberrant effects er of environmental contamination. Environ Health eared slider turtles, Trachemys scripta, a species with
temperature-dependent sex determination. Biol Reprod
on the turtle, much like E2 acts in snapping Perspect 102:780-781 (1994).
7. Tousignant A, Crews D. Incubation temperature and 61:1275-1280 (1999).
turtles exposed during embryogenesis and

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332 Volume 108, Number 4, April 2000 * Environmental Health Perspectives

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