Bioaccumulation and speciation of selenium in fish and insects collected from a mountaintop removal coal mining-impacted stream in West Virginia.

A major contaminant of concern for mountaintop removal/valley fill (MTR/VF) coal mining is selenium (Se), an essential micronutrient that can be toxic to fish. Creek chubs (Semotilus atromaculatus), green sunfish (Lepomis cyanellus), and composite insect samples were collected in March-July, 2011-2013 at two sites within the Mud River, West Virginia. One site (MR7) receives MTR/VF coal mining effluent, while the reference site (LFMR) does not. MR7 water had significantly higher concentrations of soluble Se (p < 0.01) and conductivity (p < 0.005) compared to LFMR. MR7 whole insects contained significantly higher concentrations of Se compared to LFMR insects (p < 0.001). MR7 creek chubs had significantly higher Se in fillets, liver, and ovary tissues compared to LFMR samples (p < 0.0001, p < 0.0001, and p < 0.02, respectively). MR7 green sunfish fillets contained significantly higher Se (p < 0.0001). Histological examination showed LFMR creek chub gills contained a typical amount of parasitic infestations; however MR7 gills contained minimal to no visible parasites. X-ray absorption spectroscopic analyses revealed that MR7 whole insects and creek chub tissues primarily contained organic Se and selenite. These two species of Mud River fish were shown to specifically accumulate Se differently in tissues compartments. Tissue-specific concentrations of Se may be useful in determining potential reproductive consequences of Se exposure in wild fish populations.

Immunohistochemical characterization of the hepatic progenitor cell compartment in medaka (Oryzias latipes) following hepatic injury.

Laboratory fish species are used increasingly in biomedical research and are considered robust models for the study of regenerative processes. Studies investigating the response of the fish liver to injury have demonstrated the presence of a ductular reaction and oval-like cells in injured and regenerating liver. To date, however, it is unclear if this cell population is the piscine equivalent of oval cells (OCs) or intermediate hepatobiliary cells (IHBCs) identified in rodents and man, respectively. The present study defines the process of OC differentiation in fish liver using histopathology, immunohistochemistry and transmission electron microscopy. To generate OC proliferation in Japanese medaka (Oryzias latipes), hepatic injury was induced by exposure of adult fish to either microcystin LR or dimethylnitrosamine. A transgenic strain of medaka expressing a red fluorescent protein (RFP) exclusively in hepatocytes was used. The morphological response to injury was characterized by a ductular reaction comprised of cytokeratin (CK) AE1/AE3(+) OCs progressing to IHBCs variably positive for CK and RFP and finally mature RFP(+) hepatocytes and CK(+) cholangiocytes. These observations support a bipotential differentiation pathway of fish OCs towards hepatocytes and cholangiocytes. Ultrastructural morphology confirmed the presence of OCs and differentiation towards hepatocytes. These results demonstrated clear similarities between patterns of reaction to injury in fish and mammalian livers. They also confirm the presence of, and support the putative bipotential lineage capabilities of, the fish OC.

A simple osmium post-fixation paraffin-embedment technique to identify lipid accumulation in fish liver using medaka (Oryziaslatipes) eggs and eleutheroembryos as lipid rich models.

Hepatic lipidosis is a non-specific biomarker of effect from pollution exposure in fish. Fatty liver is often misdiagnosed or overlooked in histological assessments due to the decreasing application of specific fat procedures and stains. For example, ethanol dehydration in standard paraffin processing removes lipids, leaving vacuoles of which the precise nature is unknown. Lipids can be identified using osmium post-fixation in semi-thin resin sections or transmission electron microscopy. However, both are expensive and technically demanding procedures, often not available for routine environmental risk assessment and monitoring programs. The current emphasis to reduce and refine animal toxicity testing, requires refinement of the suite of histopathological techniques currently available to maximize information gained from using fish for toxicity testing and as bio-indicators of environmental quality. This investigation has successfully modified an osmium post-fixation technique to conserve lipids in paraffin-embedded tissues using medaka (Oryzias latipes) eleutheroembryos and eggs (embryos) as lipid rich models.

Nightmares among Cambodian refugees: the breaching of concentric ontological security.

This article explores the nightmares of Cambodian refugees in a cultural context, and the role of nightmares in the trauma ontology of this population, including their role in generating post-traumatic stress disorder (PTSD). Among Cambodian refugees attending a psychiatric clinic, we found that having a nightmare was strongly associated with having PTSD (chi(2) = 61.7, P < 0.001, odds ratio = 126); that nightmares caused much distress upon awakening, including panic attacks, fear of bodily dysfunction, flashbacks and difficulty returning to sleep; that nightmare content was frequently related to traumatic events; that nightmares resulted in a decrease in the sense of "concentric ontology security" (i.e., in an increased sense of physical and spiritual vulnerability in a culture that conceives of the self in terms of concentric, protective layers), including fears of being attacked by ghosts; and that nightmares frequently led to the performance of specific practices and rituals aiming to extrude and repel attacking forces and to create "protective layers." Cases are presented to illustrate these findings. The Discussion considers some treatment implications of the study.

Embryo toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin to the wood duck (Aix sponsa).

We examined the sensitivity of the wood duck (Aix sponsa) embryo to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) by injecting the toxicant into their eggs. Six groups of wood duck eggs (n = 35 to 211 per trial) were injected with 0 to 4600 pg TCDD/g egg between 2003 and 2005. Injections were made into yolk prior to incubation, and eggs were subsequently incubated and assessed weekly for mortality. Significant TCDD-induced mortality was not observed through day 25 (90% of incubation). Liver, heart, eye, and brain histology were generally unremarkable. Hepatic ethoxyresorufin-O-deethylase activity, a biomarker of dioxin-like compound exposure, was induced by 12-fold in the 4600 pg/g treatment relative to controls. The median lethal dose for chicken (Gallus domesticus) eggs we dosed identically to wood duck eggs was about 100 pg/g, similar to other assessments of chickens. Among dioxin-like compound embryo lethality data for 15 avian genera, the wood duck 4600 pg/g no-observed-effect level ranks near the middle. Because no higher doses were tested, wood ducks may be like other waterfowl (order Anseriformes), which are comparatively tolerant to embryo mortality from polychlorinated dibenzo-p-dioxins and dibenzofurans when exposed by egg injection.

Stage-specific toxicity of cypermethrin to medaka (Oryzias latipes) eggs and embryos using a refined methodology for an in vitro fertilization bioassay.

Using original artificial fertilization methods with medaka (Oryzias latipes), the effects of exposure to cypermethrin on gametes, fertilization, and embryonic development were investigated. The relative sensitivity was studied with 96-hour duration, 24-hour renewal exposures to six nominal concentrations of cypermethrin ranging from 3.1 100.0 microg cypermethrin/L. Tests were initiated at different developmental stages: unfertilized egg (stage 0), late morula (stage 9), eminence of swim bladder (stage 29), and maximum flexion of the atrioventricular region (stage 34). Cypermethrin did not affect the fertilization process. Predominant sublethal effects in embryos included transient visceral edemas intimately associated to the gall bladder with subsequent pericardial edemas. Other sublethal effects were observed in surviving larvae and included spastic movements with or without ability to respond to stimulus (>/=6.3 microg cypermethrin/L), spinal curvatures, and delayed or absence of swim bladder inflation (>/=12.5 microg cypermethrin/L). The exposure of the gametes (stage 0) or animals during earlier embryonic development (stages 9 or 29) was not a critical window for cypermethrin exposure. Although the incidence of edemas in embryos occurred mainly during exposure of these early developmental stages, embryo and larva lethality and the incidence of transient sublethal effects in hatchlings showed that the later exposure window (stage 34) was the most sensitive. The stage 34 group involved advanced organogenetic stages in which the chorion partially degraded before hatching. Our studies reinforced the idea that a combination of morphologic and functional impairment evaluation is a more sensitive response to developmental toxicants than morphologic defects alone.

Biochemical and toxicopathic biomarkers assessed in smallmouth bass recovered from a polychlorinated biphenyl-contaminated river.

Smallmouth bass (Micropterus dolomieu) were collected to quantify the nature and prevalence of biomarker responses, including biochemical indices, toxicopathic lesions and general health indices, among fish collected from polychlorinated biphenyl (PCB)-contaminated and nearby uncontaminated reaches of the Kalamazoo River, Michigan, USA. Blood and tissue samples (gill, liver, spleen, head kidney, trunk kidney, thyroid and gonads) were collected and preserved at necropsy for biochemical and histological analyses. The body condition factor and liver somatic index were significantly lower in fish collected from the downstream, contaminated site. Plasma vitellogenin was not detected in male fish collected from either site. Liver ethoxyresorufin-O-deethylase activity and liver and spleen superoxide dismutase activity were significantly depressed in fish collected from the downstream site. Significant toxicopathic lesions such as glycogen depletion, enhanced macrophage aggregates, hepatic foci of cellular alteration (i.e. preneoplastic lesions) and neoplasia were also detected in the liver of fish collected from the downstream site. This study indicates that many of the biochemical and histopathological biomarker responses were associated with liver and body tissue PCB concentrations. Taken together, the biomarkers of exposure and effect strongly suggest that fish within the downstream site are adversely affected by PCBs and other chemical stressors.

Increasing uptake and bioactivation with development positively modulate diazinon toxicity in early life stage medaka (Oryzias latipes).

Diazinon, an organophosphate pesticide, becomes biotransformed to a more potent oxon metabolite that inhibits acetylcholinesterase (AChE). Early life stages (els) of medaka, Oryzias latipes, were used to determine how development of this teleost affects sensitivity to diazinon. With developmental progression, from day of fertilization to 7-day-old larvae, we found that the 96-h LC50 and AChE IC50 values decreased, indicating greater host sensitivity to diazinon upon continued development. We then examined changes in AChE activity, its inhibition by the active metabolite diazoxon, and uptake and bioactivation of the compound. AChE activity remained low during much of development but increased rapidly just prior to hatch. In addition, in vitro incubation of tissue homogenates from embryos or larvae showed no differences in the sensitivity of AChE to diazoxon. Uptake studies with 14C-diazinon revealed greater body burdens of 14C as medaka developed. In addition, AChE IC50 values determined by in vivo exposure to diazoxon were greater in larvae than in embryos. Because diazinon is bioactivated by the P450 enzyme system, two P450 inhibitors were used in vivo to explore the role of metabolism in sensitivity. When exposure to diazinon occurred in the presence of increasing amounts of piperonyl butoxide (PBO), AChE inhibition decreased in a dose-response fashion and 2.0 x 10(-5) M PBO alleviated any difference in inhibition between larvae and embryos. However, PBO did not alter total 14C uptake when exposed simultaneously with 14C-diazinon, nor did it affect AChE inhibition using diazoxon. Controls ruled out differential effects of PBO on uptake and inhibition. In addition, a second general P450 inhibitor, 1-aminobenzotriazole, also decreased AChE inhibition. Finally, using exogenous acetylcholinesterase as a trap for the oxon metabolite, larval microsomes displayed greater bioactivation of diazinon than did a microsomal preparation from embryos. Taken together, results suggest that uptake and bioactivation are working to enhance diazinon sensitivity in this developmental model of a teleost fish.

Differential biomarker gene and protein expressions in nonylphenol and estradiol-17beta treated juvenile rainbow trout (Oncorhynchus mykiss).

The time- and dose-dependent transcriptional and translational expression of biomarker genes in nonylphenol (NP) and estradiol-17beta (E(2)) treated juvenile rainbow trout is reported. Fish were exposed to NP (1, 5 and 25 mg/kg) and E(2) (5 mg/kg) and killed at 2, 6, 12, 24, 48 and 72 h after exposure. The estrogen receptor (ER), vitellogenin (Vtg) and eggshell zona radiata protein (Zr-protein) gene expressions were analyzed in total liver RNA using Northern and slot hybridization with specific cDNA probes. Plasma Vtg and Zr-protein levels were evaluated using indirect ELISA. While Zr-protein gene showed an induction only at 24 h post-exposure, the plasma protein levels showed a time-dependent increase in the 25-mg NP treated group. Vtg transcripts showed an apparent time-dependent increase without a concomitant increase in protein levels in the 25-mg NP treated fish. Time-dependent increases in Vtg and Zr-protein gene expressions without the corresponding increases in ER gene transcription was observed in E(2)-treated fish at 2, 6 and 12 h post-exposure. Induction of ER gene transcripts was observed from 24 h and did not change significantly at 48 and 72 h. In the E(2)-treated fish, induction of plasma Vtg levels was observed at 48 and 72 h, while plasma Zr-protein was induced at 24, 48 and 72 h, after exposure. We conclude that the E(2)- and NP-induced Vtg and Zr-protein gene expressions at the early time intervals after exposure are not dependent on increase in the transcriptional activity of the ER gene and that Vtg and Zr-protein gene transcriptions require only basal or minimal ER concentration, in addition to other mechanisms.

Toxicity of methyl-tert-butyl ether to freshwater organisms.

Increased input of the fuel oxygenate methyl-tert-butyl ether (MTBE) into aquatic systems has led to concerns about its effect(s) on aquatic life. As part of a study conducted by University of California scientists for the State of California, the Aquatic Toxicology Laboratory, UC Davis, reviewed existing literature on toxicity of MTBE to freshwater organisms, and new information was generated on chronic, developmental toxicity in fish, and potential toxicity of MTBE to California resident species. Depending on time of exposure and endpoint measured, MTBE is toxic to various aquatic organisms at concentrations of 57-> 1000 mg/l (invertebrates), and 388-2600 mg/l (vertebrates). Developmental effects in medaka (Oryzias latipes) were not observed at concentrations up to 480 mg/l, and all fish hatched and performed feeding and swimming in a normal manner. Bacterial assays proved most sensitive with toxicity to Salmonella typhimurium measured at 7.4 mg/l within 48 h. In microalgae, decreased growth was observed at 2400 and 4800 mg/l within 5 days. MTBE does not appear to bioaccumulate in fish and is rapidly excreted or metabolized. Collectively, the available data suggests that at environmental MTBE exposure levels found in surface waters (< 0.1 mg/l) this compound is likely not acutely toxic to aquatic life. However, more information is needed on chronic and sublethal effects before we can eliminate the possibility of risk to aquatic communities at currently detected concentrations.

Evaluation of fish models of soluble epoxide hydrolase inhibition.

Substituted ureas and carbamates are mechanistic inhibitors of the soluble epoxide hydrolase (sEH). We screened a set of chemicals containing these functionalities in larval fathead minnow (Pimphales promelas) and embryo/larval golden medaka (Oryzias latipes) models to evaluate the utility of these systems for investigating sEH inhibition in vivo. Both fathead minnow and medaka sEHs were functionally similar to the tested mammalian orthologs (murine and human) with respect to substrate hydrolysis and inhibitor susceptibility. Low lethality was observed in either larval or embryonic fish exposed to diuron [N-(3,4-dichlorophenyl), N'-dimethyl urea], desmethyl diuron [N-(3,4-dichlorophenyl), N'-methyl urea], or siduron [N-(1-methylcyclohexyl), N'-phenyl urea]. Dose-dependent inhibition of sEH was a sublethal effect of substituted urea exposure with the potency of siduron < desmethyl diuron = diuron, differing from the observed in vitro sEH inhibition potency of siduron > desmethyl diuron > diuron. Further, siduron exposure synergized the toxicity of trans-stilbene oxide in fathead minnows. Medaka embryos exposed to diuron, desmethyl diuron, or siduron displayed dose-dependent delays in hatch, and elevated concentrations of diuron and desmethyl diuron produced developmental toxicity. The dose-dependent toxicity and in vivo sEH inhibition correlated, suggesting a potential, albeit undefined, relationship between these factors. Additionally, the observed inversion of in vitro to in vivo potency suggests that these fish models may provide tools for investigating the in vivo stability of in vitro inhibitors while screening for untoward effects.

Identification, characterization, and ontogeny of a second cytochrome P450 3A gene from the fresh water teleost medaka (Oryzias latipes).

Multiple copies of cytochrome P450 gene family 3 have been identified from numerous mammalian species. Often these genes exhibit differential catalytic activities and gene regulation. To date however, little information is available regarding multiple forms of this gene family in teleost fishes. In this study, a second isozyme of cytochrome P450 3A has been cloned from the teleost fish Oryzias latipes and designated CYP3A40. Screening of a cDNA library to medaka liver resulted in the identification of a full length cDNA clone containing a 2316 base pair (bp) insert with an open reading frame encoding a single peptide of 502 amino acids. Comparisons of the deduced amino acid sequence to other known cytochrome P450 sequences indicate that this gene product is most similar to the CYP3A gene family and shares a 90% identity to CYP3A38 previously identified from medaka liver. Consistent with Northern blot and Western blot analysis, Southern blots of medaka genomic DNA demonstrated the presence of two CYP3A genes. Gene expression studies demonstrated that CYP3A38 and CYP3A40 are differentially regulated according to embryonic development. Northern blot analysis, using a probe to a conserved region of both CYP3A genes, demonstrated the presence of a single CYP3A transcript for early and late embryonic stages and two CYP3A transcripts in larvae and adult liver. Similarly, Western blots show a single faint immunoreactive cytochrome P450 3A protein in microsomes from early and late embryos and two abundant protein bands in microsomes from larval and adult liver. To further examine the transcriptional differences in CYP3A expression, RT-PCR analysis was performed on embryonic stages 11-35, 1- and 14-day-old larvae, and adult liver using primer sets specific for CYP3A38 and CYP3A40. These results demonstrate that CYP3A40 is expressed early in embryonic development and continues throughout adult stages. CYP3A38, however, is tightly regulated during embryonic development and is only expressed post-hatch.

Identification and characterization of a cDNA encoding cytochrome P450 3A from the fresh water teleost medaka (Oryzias latipes).

A new member of the CYP3A gene family has been cloned from the teleost fish medaka (Oryzias latipes) by reverse-transcriptase polymerase chain reaction (RT-PCR). Degenerate primers homologous to highly conserved regions of known CYP3A sequences were used for initial RT-PCRs. Individual PCR products were cloned, sequenced, and identified as those belonging to the cytochrome P450 superfamily based on amino acid sequence similarity and the presence of the highly conserved heme-binding region. PCR products were subsequently used as probes to screen a complementary DNA library. A full-length cDNA clone was identified containing a 1758-base-pair (bp) insert with an open reading frame encoding a single peptide of 500 amino acids. Comparisons of the deduced amino acid sequence to other known cytochrome P450 sequences indicate that this gene product is most similar to the CYP3A gene family and has been designated as CYP3A38 by the cytochrome P450 nomenclature committee. Northern blot analysis identified two abundant CYP3A related transcripts in liver of both male and female adults and demonstrated quantitative differences in abundance according to gender. Similarly, Western blot analysis demonstrated the presence of two abundant cytochrome P450 related proteins in liver of both male and female adults. These results suggests that O. latipes contains multiple forms of CYP3A. Heterologous expression of CYP3A38 cDNA in HEK 293 cells produced a single protein that was reactive with anti-scup P450A (CYP3A) polyclonal antibody. Microsomes of HEK 293 cells expressing recombinant CYP3A38 protein actively catalyzed the hydroxylation of testosterone.

Partial hepatectomy and bile duct ligation in rainbow trout (Oncorhynchus mykiss): histologic, immunohistochemical and enzyme histochemical characterization of hepatic regeneration and biliary hyperplasia.

Hepatic regeneration following partial hepatectomy (PH) and biliary hyperplasia subsequent to bile duct ligation (BDL) were characterized in rainbow trout (Oncorhynchus mykiss) by light microscopy using routine and special (immunohistochemical and enzyme histochemical) stains. Both PH and BDL involved initial hypertrophy and hyperplasia of bile preductular epithelial cells (BPDECs). BPDECs are small oval cells that form junctional complexes with hepatocytes and bile ductular cells and are commonly found in hepatic tubules of teleost liver. Proliferating BPDECs transitioned through intermediate cell types before final differentiation into large basophilic hepatocytes (following PH) or biliary epithelial cells (after BDL). Normal BPDECs and hepatocytes were both negative for cytokeratin intermediate filaments in control fish when screened with the monoclonal antibody AE1/AE3. In contrast, hyperplastic BPDECs and their progeny (intermediate cells, immature hepatocytes, ductal epithelial cells) were all strongly cytokeratin positive. Cytokeratin expression was transient in newly differentiated hepatocytes (expression decreased as hepatocytes acquired characteristics consistent with full differentiation) but was permanent in biliary epithelial cells (expression was very strong in large mature ducts). BPDECs, intermediate cells, and immature ductal cells were also strongly positive for alkaline phosphatase following BDL. Chronology of histologic events and cytokeratin and enzyme expression all support the hypothesis that BPDECs possess the capacity to differentiate into either hepatocytes or biliary epithelial cells. Thus, BPDECs may be the teleost equivalent of a bipolar hepatic stem cell in mammals.

Determining the sensitive developmental stages of intersex induction in medaka (Oryzias latipes) exposed to 17 beta-estradiol or testosterone.

Certain environmentally persistent compounds can adversely affect reproduction by acting as steroid hormone agonists or antagonists. The goal of the present study was to determine the developmental stage most susceptible to exogenous hormone (estradiol and testosterone) exposure using a small teleost model. In the first (pilot study) of two experiments, medaka (Oryzias latipes), at varying developmental stages, were bath-exposed to 5 micrograms/l 17 beta-estradiol for 24 h. At 5 months of age, fecundity, fertility and embryo and larval viability (reproductive success) were investigated in control and exposed groups. Fish at 1, 1.5, 2 and 5.5 months of age were also sampled, processed and examined histologically for gonadal alteration. No significant differences in mortality, gonadal morphology, body weight, sex-ratio or time to maturity were seen between control and exposed fish. At 5 months, however, when exposure groups were compared to controls, significant differences were seen in reproductive success and viability of offspring. A second experiment exposed embryo stage 10, and 1-, 7- and 21-day-old larvae for 6 days to 15 micrograms/l 17 beta-estradiol or 100 micrograms/l testosterone. No significant differences were seen at 5 months in mortality, body weight, or time to sexual maturity. However, sex-ratios were significantly biased toward female in the stage 10, 1- and 7-day post-hatch estradiol exposure groups. No significant changes in sex-ratio were associated with testosterone exposure at any developmental stage. Further, intersex gonads were observed in fish from all groups exposed to 15 micrograms/l estradiol. Only those fish exposed as newly hatched fry or at 1 week post-hatch displayed intersex gonads following 100 micrograms/l testosterone exposure. Data from these experiments show that newly hatched fry are that life stage most sensitive to hormone exposure and the most appropriate to use in determining effects of known endocrine-disrupting compounds.

Tissue and cellular alterations in Asian clam (Potamocorbula amurensis) from San Francisco Bay: toxicological indicators of exposure and effect?

The US Geological Survey has reported the presence of a metal contamination gradient in clam tissues, decreased condition indices, and irregular reproductive patterns have been reported in the Asian clam, Potamocorbula amurensis, from San Francisco Bay. If metals are driving the observed patterns in the field, then biomarkers of exposure, and possibly deleterious effect, should show a corresponding gradient. In this study, biomarkers from sub-cellular to tissue levels of biological organization were assessed in P. amurensis collected from the Bay or exposed to cadmium in the laboratory. Cellular and tissue alterations were assessed using histopathology and enzyme histochemistry (EH). Alterations in the ovary, testis, kidney, and gill tissues were most common at the most contaminated station when data were averaged over a 12-month sampling period. EH analysis indicated decreased active transport, energy status, and glucose oxidation in kidney and digestive gland at the most contaminated site which may indicate a decreased potential for growth. Ovarian lesions observed in feral Asian clams were experimentally induced in healthy clams by cadmium exposure in laboratory exposures. Our results suggest a contaminant etiology for tissue alterations.

Sublethal effects of chromium-VI in the Asian clam (Potamocorbula amurensis).

Previously, we have shown that Asian clams (Potamocorbula amurensis) with highest metallic body burdens have highest prevalence of disease and lowest reproduction. The present study was designed to assess and validate potential sublethal toxicity of hexavalent chromium (Cr-VI) in clams under controlled laboratory exposure. For 7 days, three replicates of clam (n = 10 per replicate) were exposed to aqueous solution containing 0.00, 0.92, 8.40, or 25.6 mg l-1 of Cr-VI at 15 degrees C and 15 g l-1 salinity. Mortality reached 100% in the 25.6 mg l-1 group within 7 days. There was no significant difference in mortality among the control, 0.92, and 8.40 mg l-1 groups. Western blot analyses revealed significantly elevated stress protein hsp70 levels in the 8.40 mg l-1 treatment group. Histopathologic analyses revealed mild digestive gland (DG) atrophy in the control group. Clams exposed to 0.92 mg l-1 Cr-VI showed moderate DG atrophy, moderate granulomatous inflammation and necrosis in DG, ovary and testis. Lesions observed in the 8.40 mg l-1 treatment group included severe DG atrophy, severe granulomatous inflammation and necrosis in byssal gland, DG, gill, kidney, ovary and testis. In gills and testes of treated groups, apoptotic cells outnumbered mitotic cells. In addition, gills from clams in the 8.40 mg l-1 group showed enhanced hsp70 staining. Our studies support a cause-effect relationship between contaminants and reduced health in Asian clams and indicate the DGs, gills, and reproductive organs are principal targets of Cr-VI toxicity at sublethal concentrations. Results from this study suggest that Cr-VI may have played a role in the increased incidence of diseased clams seen in previous studies and these adverse effects may be working to decrease clam populations at sites with highest metallic contamination in the San Francisco Bay Estuary.

Spatial profiles of hsp70 proteins in Asian clam (Potamocorbula amurensis) in northern San Francisco Bay may be linked to natural rather than anthropogenic stressors.

Multi-year investigations in northern San Francisco Bay by United States Geological Survey have linked reduced condition indices in populations of Asian clam (Potamocorbula amurensis) with elevated cadmium tissue concentrations. Our study seeks to determine whether levels of hsp70 proteins in P. amurensis can be correlated with these findings, and/or are related to histopathologic alterations and concentrations of metallothionein-like proteins. Here we present our results on stress proteins in clams collected monthly from four field stations between July 1996 and January 1998. In addition, animals were exposed in the laboratory to a range of salinities. Stress proteins were analyzed by Western blotting using monoclonal antibodies. Hsp70 protein levels in field-collected clams were significantly higher at the seaward (high salinity/low cadmium) stations (12.5, 8.1) than at the landward (low salinity/high cadmium) stations (6.1, 4.1). Laboratory studies showed that clams exposed to 0.1 ppt salinity had markedly lower hsp70 levels than clams exposed to higher salinities. In view of our previous laboratory studies showing that cadmium induces hsp70 in P. amurensis, our present results indicate that reduced hsp70 protein levels in field-collected clams may be linked to salinity effects rather than cadmium tissue concentrations.