Effects of selenium treatment on endogenous antioxidant capacity in signal crayfish (Pacifastacus leniusculus).

Selenium is an essential element that plays a role in numerous physiological processes and is critical for the maintenance of a strong endogenous antioxidant system. Previous work by our research group reported that the organophosphate pesticide dimethoate decreased glutathione S-transferase activity (GST) in signal crayfish (Pacifastacus leniusculus) collected from the Boise River (Idaho, USA). The goals of this study were to examine whether: 1) sodium selenite modulated the endogenous antioxidants glutathione (GSH), metallothionein (MT), and glutathione S-transferase (GST), thus suggesting a mechanism of antioxidant activity, 2) dimethoate exposure (pro-oxidant stressor) decreased GST activity in a localized population of signal crayfish collected from the Snake River (Idaho, USA), and 3) investigate whether selenium cotreatment ameliorated the adverse effects of dimethoate on GST activity due to the antioxidant properties associated with selenium. Selenium and dimethoate treatments (and co-treatments) did not modulate GSH or MT concentrations at the doses tested in this study. Furthermore, neither selenium nor dimethoate was factors influencing GST activity, and no interaction was found between the treatments. While our results did not support our predictions, they are suggestive and future studies examining the protective role of selenium in pro-oxidant exposure in this species are warranted. Population-specific responses as well as seasonal variations in endogenous antioxidant expression should be considered in future experiments.

Modulation of endogenous antioxidants by zinc and copper in signal crayfish (Pacifastacus leniusculus).

Metal pollution is a long-standing concern and bioindicators are commonly used in ecotoxicological studies to monitor impacted wildlife populations for evidence of sublethal effects. Significant variation in the response of common biomarkers to metals is reported across taxa, thus necessitating careful characterization in model organisms. In this study, we describe the regulation of glutathione S-transferase (GST), glutathione (GSH), and metallothionein (MT) by zinc chloride (0.6, 0.9, 1.2, 2.4, 4.8, 9.6 µg g-1) and copper chloride (0.6, 0.9, 1.2 µg g-1) in signal crayfish (Pacifastacus leniusculus). Zinc chloride did not alter GST activity relative to controls in the hepatopancreas. Crayfish exposed to copper chloride exhibited decreased GST activity at the lowest dose tested (0.6 µg g-1) with no change observed at the higher doses. GSH did not change in response to either metal when sexes were grouped together. MT concentrations increased in response to zinc (2.4, 4.6, and 9.6 µg g-1 doses) and copper (0.6, 0.9, and 1.2 µg g-1 doses) in gill tissue. In tail tissue, MT increased at the 2.4 and 4.8 µg g-1 zinc chloride doses and all the concentrations of copper tested. Sex-specific differences in endogenous antioxidant expression were also analyzed with no clear patterns emerging. We concluded that these endpoints are sensitive to zinc and copper in signal crayfish, although careful interpretation is needed when applying them in field studies given the variation in responses, non-monotonic dose responses, and differences in biotic and abiotic factors that inevitably exist in different aquatic ecosystems.

Response of phase I and II detoxification enzymes, glutathione, metallothionein and acetylcholine esterase to mercury and dimethoate in signal crayfish (Pacifastacus leniusculus).

Metals and pesticides are common pollutants and the modulation of biomarkers can indicate sub-lethal influences on the physiology of organisms inhabiting impacted aquatic systems. We examined the effects of mercury and the organophosphate pesticide dimethoate on EROD, MROD, glutathione S-transferase (GST), acetylcholine esterase (AChE), metallothionein (MT) and glutathione (GSH) in the signal crayfish (Pacifastacus leniusculus). Crayfish were injected with mercury chloride or dimethoate (0.3, 0.6, 0.9 µg kg-1) and dissected after 72 h. EROD activity in the hepatopancreas did not change in response to mercury chloride treatment but exhibited a dose dependent decrease at all concentrations of dimethoate tested. MROD (hepatopancreas) exhibited a significant decrease at the 0.9 µg kg-1 treatment for both chemicals. GST (hepatopancreas) demonstrated a significant dose dependent decrease at all concentrations of both mercury chloride and dimethoate. AChE (tail muscle) decreased at the 0.6 and 0.9 µg kg-1 concentrations of dimethoate and 0.9 µg kg-1 mercury chloride. In gill tissue, MT increased in response to 0.3 and 0.6 µg kg-1 of mercury chloride but no effect was observed at the 0.9 µg kg-1 concentration of mercury chloride or any concentrations of dimethoate tested. MT did not change in response to mercury or dimethoate in tail tissue. Furthermore, neither chemical modulated GSH concentrations. Our results indicate that, apart from GSH, these markers are sensitive to the pollutants tested and that animals exposed in the wild are potentially compromised in their ability to detoxify environmental contaminants and carry out normal cellular processes.

Variations in hepatic biomarkers in American alligators (Alligator mississippiensis) from three sites in Florida, USA.

Sub-individual biomarkers are sub-lethal biological responses commonly used in the assessment of wildlife exposure to environmental contaminants. In this study, we examined the activity of glutathione-s-transferase (GST) and lactate dehydrogenase (LDH), and metallothionein (MT) concentrations among captive-raised alligator hatchlings, wild-caught juveniles, and wild-caught adults. Juveniles and adults were collected from three locations in Florida (USA) with varying degrees of contamination (i.e. Lake Apopka (organochlorine polluted site), Merritt Island National Wildlife Refuge (NWR) (metal polluted site), and Lake Woodruff NWR (reference site)). We examined whether changes in the response of these three biomarkers were age and sex dependent or reflected site-specific variations of environmental contaminants. Juvenile alligators from Merritt Island NWR had higher MT concentrations and lower GST activity compared to those from the other two sites. This outcome was consistent with higher metal pollution at this location. Sexually dimorphic patterns of MT and GST (F > M) were observed in juvenile alligators from all sites, although this pattern was not observed in adults. GST activity was lower in captive-raised alligators from Lake Apopka and Merritt Island NWR as compared to animals from Lake Woodruff NWR, suggesting a possible developmental modulator at these sites. No clear patterns were observed in LDH activity. We concluded that GST and MT demonstrate age and sex specific patterns in the alligators inhabiting these study sites and that the observed variation among sites could be due to differences in contaminant exposure.

Effect of low dose exposure to the herbicide atrazine and its metabolite on cytochrome P450 aromatase and steroidogenic factor-1 mRNA levels in the brain of premetamorphic bullfrog tadpoles (Rana catesbeiana).

The transcriptional regulator steroidogenic factor 1 (SF-1) and the enzyme cytochrome P450 aromatase (CYP19) play a central role in modulation of a broad range of tissue-specific developmental processes associated with hormone homeostasis that includes differentiation of the central nervous system. SF-1 and CYP19 expression may be targeted by a variety of endocrine disruptive agents prevalent within the environment. In the present study, we cloned and characterized partial sequences for bullfrog (Rana catesbeiana) SF-1 and CYP19 and examined the effects of a 48h exposure to 1 and 100µg/l of the herbicide atrazine (ATZ) and its major metabolite desethylatrazine (DEA), as well as 5ng/l of the estrogenic chemical, 17α-ethynylestradiol (EE(2)), and 673ng/l of the thyroid hormone, 3,5,3'-triiodothyronine (T(3)), on SF-1 and CYP19 mRNA abundance in the brains of premetamorphic bullfrog tadpoles. Quantitative RT-PCR analysis showed an increase in CYP19 mRNA following a 48h exposure to EE(2) but not T(3) while no significant changes in SF-1 transcript levels occurred. We observed a strong positive correlation between CYP19 and SF-1 transcript abundance in the ATZ-exposed animals which was not evident with DEA- or hormone-exposed tadpoles. Our results are intriguing in light of reported behavioral changes in ATZ-exposed frogs and suggest that further research is warranted to examine the relationship and role of CYP19 and SF-1 in amphibian brain development.

Peering into molecular mechanisms of action with frogSCOPE.

Exposure of critical life stages to harmful chemicals at low, environmentally-relevant concentrations can alter how hormones function, and change metabolic pathways or developmental processes that impact reproduction, behavior, or susceptibility to disease later in life. These alterations can be captured through evaluation of changes to transcriptomes, proteomes, and metabolomes occurring at those critical life stages thereby enabling more effective and earlier identification of mechanism of action, individual susceptibilities and adaptation, and prediction of detrimental sublethal effects. Amphibians are "wet canaries in the coalmine" as indicators for environmental health. There are more than 6000 species living in a variety of ecological niches worldwide yet limited 'omics resources and approaches exist. To provide for a means of addressing this challenge, frogSCOPE (frog Sentinel species Comparative "Omics" for the Environment) combines transcriptomics, proteomics, and metabolomics together to form the foundation for the identification of biological response indicators of harmful effects on a species of wild frog (Rana catesbeiana) at a sensitive tadpole stage. Various exposure and sampling methodologies are possible including standard in vivo exposures, tail fin biopsies, and the C-fin assay. frogSCOPE establishes methodological and analytical approaches applicable to wildlife by using a uniquely-designed frog cDNA array developed to accommodate cross-species hybridization and quantitative real-time polymerase chain reaction (QPCR) assays on poorly genetically-characterized wildlife species. Combination with proteomics (isobaric tags for relative and absolute protein quantitation; iTRAQ) and metabolomics (mass spectrometry) enable the generation of molecular fingerprints to identify mechanisms of action in a more comprehensive fashion to better define suitable indicators of deleterious biological outcomes to wildlife.

Identification of gene expression indicators for thyroid axis disruption in a Xenopus laevis metamorphosis screening assay. Part 1. Effects on the brain.

Thyroid hormones (TH), thyroxine (T(4)) and 3,5,3'-triiodothyronine (T(3)), play crucial roles in regulation of growth, development and metabolism in vertebrates and their actions are targets for endocrine disruptive agents. Perturbations in TH action can contribute to the development of disease states and the US Environmental Protection Agency is developing a high throughput screen using TH-dependent amphibian metamorphosis as an assay platform. Currently this methodology relies on external morphological endpoints and changes in central thyroid axis parameters. However, exposure-related changes in gene expression in TH-sensitive tissue types that occur over shorter time frames have the potential to augment this screen. This study aims to characterize and identify molecular markers in the tadpole brain. Using a combination of cDNA array analysis and real time quantitative polymerase chain reaction (QPCR), we examine the brain of tadpoles following 96 h of continuous exposure to T(3), T(4), methimazole, propylthiouracil, or perchlorate. This tissue was more sensitive to T(4) rather than T(3), even when differences in biological activity were taken into account. This implies that a simple conversion of T(4) to T(3) cannot fully account for T(4) effects on the brain and suggests distinctive mechanisms of action for the two THs. While the brain shows gene expression alterations for methimazole and propylthiouracil, the environmental contaminant, perchlorate, had the greatest effect on the levels of mRNAs encoding proteins important in neural development and function. Our data identify gene expression profiles that can serve as exposure indicators of these chemicals.

The bactericidal agent triclosan modulates thyroid hormone-associated gene expression and disrupts postembryonic anuran development.

We investigated whether exposure to environmentally relevant concentrations of the bactericidal agent, triclosan, induces changes in the thyroid hormone-mediated process of metamorphosis of the North American bullfrog, Rana catesbeiana and alters the expression profile of thyroid hormone receptor (TR) alpha and beta, basic transcription element binding protein (BTEB) and proliferating nuclear cell antigen (PCNA) gene transcripts. Premetamorphic tadpoles were immersed in environmentally relevant concentrations of triclosan and injected with 1 x 10(-11)mol/g body weight 3,5,3'-triiodothyronine (T3) or vehicle control. Morphometric measurements and steady-state mRNA levels obtained by quantitative polymerase chain reaction were determined. mRNA abundance was also examined in Xenopus laevis XTC-2 cells treated with triclosan and/or 10nM T3. Tadpoles pretreated with triclosan concentrations as low as 0.15+/-0.03 microg/L for 4 days showed increased hindlimb development and a decrease in total body weight following T3 administration. Triclosan exposure also resulted in decreased T3-mediated TRbeta mRNA expression in the tadpole tail fin and increased levels of PCNA transcript in the brain within 48 h of T3 treatment whereas TRalpha was unaffected [corrected] Triclosan alone altered thyroid hormone receptor alpha transcript levels in the brain of premetamorphic tadpoles and induced a transient weight loss. In XTC-2 cells, exposure to T3 plus nominal concentrations of triclosan as low as 0.03 microg/L for 24h resulted in altered thyroid hormone receptor mRNA expression. Exposure to low levels of triclosan disrupts thyroid hormone-associated gene expression and can alter the rate of thyroid hormone-mediated postembryonic anuran development.

Contaminant-induced feminization and demasculinization of nonmammalian vertebrate males in aquatic environments.

Many chemicals introduced into the environment by humans adversely affect embryonic development and the functioning of the male reproductive system. It has been hypothesized that these developmental alterations are due to the endocrine-disruptive effects of various environmental contaminants. The endocrine system exhibits an organizational effect on the developing embryo. Thus, a disruption of the normal hormonal signals can permanently modify the organization and future function of the male reproductive system. A wide range of studies examining wildlife either in laboratories or in natural settings have documented alterations in the development of males. These studies have begun to provide the causal relationships between embryonic contaminant exposure and reproductive abnormalities that have been lacking in pure field studies of wild populations. An understanding of the developmental consequences of endocrine disruption in wildlife can lead to new indicators of exposure and a better understanding of the most sensitive life stages as well as the consequences of exposure during these periods.

Developmental alterations as a result of in ovo exposure to the pesticide metabolite p,p'-DDE in Alligator mississippiensis.

The pesticide metabolite p,p'-DDE (1,1-dichloro-2,2-bis (p-chlorophenyl) ethylene), has been implicated in numerous reproductive and developmental abnormalities. Previous work has shown this ubiquitous contaminant to behave in an estrogenic or antiandrogenic manner, depending on the species and endpoints examined. In the current study, we examined the effects of embryonic exposure to p,p'-DDE in the alligator, a species that exhibits temperature-dependent sex determination. We compared sex ratios at an intermediate and all male producing temperature, as well as plasma testosterone (T) and gonad aromatase activity relative to untreated controls and in ovo estradiol-17beta (E(2))-treated neonates that served as positive estrogenic controls. We also compared oviductal epithelial cell height (ECH) and phallus size-estrogen and androgen responsive tissue, respectively. A female biased sex ratio was observed among hatchlings exposed to p,p'-DDE at 100 parts per billion (ppb) wet egg mass at the intermediate incubation temperature. No effect on sex determination was observed for p,p'-DDE at the all male producing temperature. Significant influence on sex determination was observed for E(2) at 100 and 0.1 ppb at the intermediate temperature and 100 ppb at the all male producing temperature. Both p,p'-DDE and E(2) failed to influence plasma T, gonadal aromatase activity, oviductal ECH, and phallus morphology at the concentrations used. Our data show that gonadal differentiation is highly sensitive to chemical perturbation relative to the other endpoints examined, and that the response to the interaction of dose and temperature should be taken into account in similar studies.

Altered neonatal development and endocrine function in Alligator mississippiensis associated with a contaminated environment.

Reduced reproductive success, altered reproductive tract development, and differences in circulating hormones have been documented in American alligators (Alligator mississippiensis) from Lake Apopka, FL, compared to less contaminated sites, such as the Lake Woodruff National Wildlife Refuge, FL. Comparative studies among alligators of varying size and age suggest that in ovo contaminant-induced alterations of endocrine function are further modified during postembryonic development and/or through environmental exposure. In the present study, we examined developmental and endocrine-related indices in neonatal (age, <1 mo) alligators from Lake Apopka in comparison to those of a reference population (Lake Woodruff), thereby limiting contaminant exposure to that derived via maternal contribution. We compared several reproductive and developmental parameters, including hatching success, primary sex determination, and somatic indices. Furthermore, we examined circulating testosterone concentrations and aromatase activity in an effort to establish relative gonadal endocrine function shortly after hatching. Finally, we compared phallus size among males and oviduct epithelial cell height (ECH) among females (androgen- and estrogen-dependent tissues, respectively). Significant differences between populations were noted for body size and spleen somatic index. Neonatal alligators from Lake Apopka exhibited higher plasma testosterone, but no differences were detected in gonadal aromatase activity compared to Lake Woodruff. Phallus tip length and cuff diameter were smaller in males from Lake Apopka, whereas no differences were noted in oviduct ECH. Our data establish basic indices of development and endocrine function in neonatal alligators before environmental exposure to contaminants. These results should begin to help separate developmental abnormalities resulting from in ovo exposure, presumably of maternal origin, from physiological alterations induced through environmental exposure to contaminants.

Seasonal variation in plasma thyroxine concentrations in juvenile alligators (Alligators mississippiensis) from three Florida Lakes.

Circulating concentrations of thyroxine (T(4)) vary seasonally in many vertebrates. This study examined the seasonal variation in plasma concentrations of T(4) in juvenile American alligators (Alligator mississippiensis) from three populations in central Florida, USA. One site, Lake Woodruff National Wildlife Refuge, is considered a reference site whereas the other two lakes, Lake Apopka and Orange Lake, are significantly impacted by human activity. Juvenile American alligators ranging from 75-150 cm in total length were hand-captured at night from November 2000-April 2002. Plasma thyroxine concentrations were analyzed using a radioimmunoassay (RIA) previously validated for alligator plasma. Juvenile American alligators display seasonal variation in circulating T(4) concentrations. Plasma T(4) concentrations decrease from August/September to November and then begin a slow rise until April, at which point they plateau. Sex of juveniles influenced plasma concentrations of T(4) in some months but did not appear to alter the pattern in seasonal variation. The pattern we observed in plasma T(4) concentrations is not directly related to an environmental factor such as ambient temperature but is similar to that seen in plasma sex steroid concentrations during the reproductive cycle of adult alligators. Although the pattern and plasma concentration of T(4) exhibits significant variation among the three lakes studied, the pattern in seasonal variation appears similar. Comparing the seasonal pattern in plasma T(4) with plasma concentrations of sex steroids (testosterone and estradiol-17beta) or corticosterone could provide important information on the peripubescent life stage of the American alligator.

Variation in sex steroids and phallus size in juvenile American alligators (Alligator mississippiensis) collected from 3 sites within the Kissimmee-Everglades drainage in Florida (USA).

This 3-year study was designed to examine variation in plasma sex steroids, phallus size, and the standard error (S.E.) associated with these endpoints in juvenile alligators collected from 3 sites within the Kissimmee-Everglades drainage (Florida, USA) with varying concentrations of sediment organochlorine contaminants. We hypothesized that decreased plasma sex steroid concentrations and phallus size would be observed in the higher contaminant site when compared to the intermediate and lower contaminant sites. Furthermore, we hypothesized that greater S.E. associated with these endpoints would be observed for the populations from more contaminated sites. We found that differences existed with females from the higher contaminant site exhibiting lower plasma estradiol-17beta (E2) and testosterone (T) concentrations. Males from the higher contaminant site exhibited smaller phallus sizes than males from the intermediate and lower contaminant sites. Smaller phallus size in this case differed from that reported in Lake Apopka male alligators [Gen. Comp. Endocrinol. 116 (1999) 356] in that a significant positive relationship between body size and phallus size existed. No difference among sites was observed in plasma T for males. Lower S.E. was associated with E2 and T concentrations in females from the higher contaminant site and in phallus size in males from the higher contaminant site. This pattern was opposite to what we had hypothesized. We concluded that variation in plasma E2 and T concentrations, phallus size, and the S.E. associated with these endpoints exists among the 3 sites with the patterns matching the patterns of organochlorine contamination, although S.E. patterns were opposite to what was predicted.

Molecular cloning of the estrogen and progesterone receptors of the American alligator.

Steroid hormones perform many essential roles in vertebrates during embryonic development, reproduction, growth, water balance, and responses to stress. The estrogens are essential for normal reproductive activity in female and male vertebrates and appear to have direct actions during sex determination in some vertebrates. To begin to understand the molecular mechanisms of estrogen action in alligators, we have isolated cDNAs encoding the estrogen receptors (ER) from the ovary. Degenerate PCR primers specific to ER were designed and used to amplify alligator ovary RNA. Two different DNA fragments (ERalpha and ERbeta) were obtained and the full-length alligator ERalpha cDNA was obtained using 5' and 3' RACE. The inferred amino acid sequence of alligator ERalpha (aERalpha) was very similar to the chicken ERalpha (91% identity), although phylogenetic analyses suggested profound differences in the rate of sequence evolution for vertebrate ER sequences. We also isolated partial DNA fragments encoding ERbeta and the progesterone receptor (PR) of the alligator, both of which show strong sequence similarities to avian ERbeta and PR. We examined the expression levels of these three steroid receptors (ERalpha, ERbeta, and PR) in the ovary of juvenile alligators and observed detectable levels of all three receptors. Quantitative RT-PCR showed that gonadal ERalpha transcript levels in juvenile alligators decreased after E2 treatment whereas ERbeta and PR transcripts were not changed. These results provide tools that will allow future studies examining the regulation and ontogenic expression of steroid receptors in alligators and expand our knowledge of vertebrate steroid receptor evolution.

Gonadotropin-induced testosterone response in peripubertal male alligators.

Based on the response to three different gonadotropin challenges, we evaluated seasonal production of testosterone in a group of captive-raised four-year-old male alligators that varied in size. To stimulate gonadal steroidogenesis, we injected each alligator with ovine FSH (150 ng/ml plasma). Plasma testosterone concentrations were measured in repeated blood samples taken between 0 and 72 h after FSH injection. To determine if there was seasonal variation in response, we repeated the experiment on the same alligators three times during the breeding season (March, May, and July, 2000). All alligators responded to exogenous FSH by exhibiting increased plasma concentrations of testosterone (p < 0.0001 for all months). However, the degree of the response depended on body size. Thus, larger alligators produced more testosterone and were more affected by changes in season compared to smaller alligators. We have previously observed that juvenile male alligators display seasonal changes in plasma testosterone concentrations that mimic the cycle observed in adult males. Our present data suggest that seasonal changes in plasma testosterone appear to be associated not only with changes in gonadotropin release but in gonadal responsiveness as well. We propose, given these observations, that alligators experience an extended period of puberty, during which the gonads synthesize gradually increasing steroid hormone concentrations. These peripubertal animals are not juveniles but sub-adults capable of responding to the seasonal signals associated with reproductive timing in adults.

Thyroid status in juvenile alligators (Alligator mississippiensis) from contaminated and reference sites on Lake Okeechobee, Florida, USA.

Exposure to environmental contaminants has been shown to alter normal thyroid function in various wildlife species, including the American alligator (Alligator mississippiensis). Abnormalities in circulating levels of the thyroid hormone thyroxine (T4) have been reported in juvenile alligators from several contaminated lakes in Florida. To further elucidate these functional thyroid abnormalities, this study examines the structure of thyroids and circulating T4 concentrations from juvenile alligators collected from three sites of varying contamination on Lake Okeechobee, Florida. The following variables were used to characterize thyroid morphology: epithelial cell height, width and area, percent colloid, and follicle area. These variables were compared among study sites and between genders. No difference was detected in epithelial cell height, epithelial cell area, or follicle area among the sites, whereas significant differences in epithelial cell width (p = 0.02) and percent colloid (p = 0.008) were found. Animals from the most contaminated site (Belle Glade) had significantly greater epithelial cell widths and significantly less colloid present in their follicles compared to animals from the reference site (West). Gender did not have a significant interaction with site for any variable measured. Thyroxine (T4) concentrations were elevated in the intermediately contaminated site (Conservation Area 3A) compared to the other sites (p < 0.0001). It is proposed that the disruptions seen in Lake Okeechobee alligators are due to disruptions at both the thyroid and extra-thyroidal tissues.

Temporal and spatial variation in plasma thyroxine (T4) concentrations in juvenile alligators collected from Lake Okeechobee and the northern Everglades, Florida, USA.

We examined variation in plasma thyroxine (T4) in juvenile American alligators (Alligator mississippiensis) collected from three sites within the Kissimmee River drainage basin (FL, USA). Based on historical sediment data, Moonshine Bay served as the low contaminant exposure site, Water Conservation Area 3A served as an intermediate contaminant exposure site, and Belle Glade served as the high contaminate exposure site. In May 1999, alligators (n = 22) from Water Conservation Area 3A exhibited higher T4 concentrations than animals from both Belle Glade (n = 22; p = 0.0003) and Moonshine Bay (n = 33; p = 0.001). In May 2000, alligators (n = 29) Water Conservation Area 3A again exhibited higher T4 concentrations than those from Belle Glade (n = 49; p = 0.02) but not those from Moonshine Bay (n = 40). No sexual dimorphism was observed among mean T4 concentrations within any of the sites during either year (p > 0.05). Animals within all sites exhibited higher T4 concentrations in May 2000 when compared to May 1999. When variance was examined, animals from Water Conservation Area 3A exhibited higher variance in plasma T4 concentrations than those from either Moonshine Bay or Belle Glade. We concluded that mean plasma T4 concentrations did not match the sediment contaminant mixture data presently available to us, whereas variance seems to be a more reliable indicator of contaminant exposure.