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I describe lessons learned and the people and principles that influenced six decades of professional endeavours from graduate schools to ascending, often unexpectedly, the science and management ladder in National Oceanic and Atmospheric Administration (NOAA) fisheries, which manages US living marine resources. For this woman chemist from India, the twists of fate and love of adventure presented amazing opportunities as well as challenges. My research on cetacean biosonar as well as on the impact of fossil fuel pollution on seafood safety and the health of marine organisms taught me the value of multidisciplinary approaches and unusual alliances. Transitioning into management, and eventually as the director of Northwest Fisheries Science Center, I learned the value of transparency and empathy while communicating our results to impacted communities, and the resolve to support the science regardless of the consequences. My advice to young professionals is that the journey should be as fulfilling as reaching the goalpost. At the twilight of my own journey, I networked with NOAA Fisheries and India’s marine science community to encourage scientist exchanges and training. My participation in University of Washington’s nature and human health programme confirms my conviction that conserving healthy ecosystems is a powerful and practical approach for people and our planet.

In industrialized and urbanized societies, medical science focuses primarily on trauma and diseases, and most environmental work attempts to remediate natural and anthropogenic degradation. This es...

The most proactive approach to resolving current health and climate crises will require a long view, focused on establishing and fostering partnerships to identify and eliminate root causes of the disconnect between humans and nature. We describe the lessons learned through a unique scientific partnership that addresses a specific crisis, harmful algal blooms (HABs), along the northeast Pacific Ocean coast, that blends current-day technology with observational knowledge of Indigenous communities. This integrative scientific strategy resulted in creative solutions for forecasting and managing HAB risk in the Pacific Northwest as a part of the US Ocean and Human Health (OHH) program. Specific OHH projects focused on: (1) understanding genetic responses of tribal members to toxins in the marine environment, (2) knowledge sharing by elders during youth camps; (3) establishing an early warning program to alert resource managers of HABs are explicit examples of proactive strategies used to address environmental problems. The research and monitoring projects with tribal communities taught the collaborating non-Indigenous scientists the value of reciprocity, highlighting both the benefits from and protection of oceans that promote our well-being. Effective global oceans and human health initiatives require a collective action that gives equal respect to all voices to promote forward thinking solutions for ocean health.

Objectives Chronic stress and burnout are key health issues for office workers that may contribute to a myriad of poor health outcomes. The presence of natural elements may improve psychological well-being in workers but the number of existing studies is relatively low, and more longitudinal research is specifically needed to assess how characteristics of workers’ day-to-day environments may impact mental health outcomes like affect, depression and stress. This report outlines a multi-study investigation of workers at Amazon, a multinational e-commerce company based in Seattle, Washington, USA, and the mental health benefits associated with exposure to nature. Methods In Study 1, participants (n = 153) responded to a cross-sectional survey that assessed the association of self-reported visitation to an indoor company greenspace with psychological well-being including symptoms of depression, anxiety, positive and negative affect, and stress. In Study 2, a subset of participants from Study 1 (n = 33) completed multiple surveys in a 2-week period that assessed the association of the naturalness of their current environments with their state levels of psychological well-being. Results We found contact with more natural outdoor environments was significantly associated with reduced state anxiety, after adjusting for activity type, location, and participants’ trait levels of nature relatedness. Conclusions Findings demonstrate that nature contact in everyday life is significantly associated with decreased levels of state anxiety. More research is needed to investigate the role of nature contact as a potential intervention in the workplace for improved mental health.

At a time of increasing disconnectedness from nature, scientific interest in the potential health benefits of nature contact has grown. Research in recent decades has yielded substantial evidence, but large gaps remain in our understanding.We propose a research agenda on nature contact and health, identifying principal domains of research and key questions that, if answered, would provide the basis for evidence-based public health interventions.We identify research questions in seven domains:Nature contact may offer a range of human health benefits. Although much evidence is already available, much remains unknown. A robust research effort, guided by a focus on key unanswered questions, has the potential to yield high-impact, consequential public health insights. https://doi.org/10.1289/EHP1663.

An important goal of synthetic biology involves the extension and standardization of novel biological elements for applications in medicine and biotechnology. Transcriptional interference, occurring in sets of convergent promoters, offers a promising mechanism for building elements for the design of tunable gene regulation. Here, we investigate the transcriptional interference mechanisms of antisense roadblock and RNA polymerase traffic in a set of convergent promoters as novel modules for synthetic biology. We show examples of elements, including antisense roadblock, relative promoter strengths, interpromoter distance, and sequence content that can be tuned to give rise to repressive as well as cooperative behaviors, therefore resulting in distinct gene expression patterns. Our approach will be useful toward engineering new biological devices and will bring new insights to naturally occurring cis-antisense systems. Therefore, we are reporting a new biological tool that can be used for synthetic biology.

Pilot whales strand periodically along the U.S. coast, and these strandings offer an opportunity for the collection of tissues for biomonitoring of contaminant exposure in cetaceans, as well as for specimen archiving. Concentrations of organochlorine (OC) contaminants (e.g., PCB congeners, pesticides, DDTs) were measured in tissue samples from pilot whales that stranded in 1986 and 1990 along the Massachusetts coast. Adult and fetal samples of blubber, liver, brain, and kidney were collected, as well as ovaries from mature female whales. Many of the OCs found in maternal tissues were detected in corresponding fetal tissues indicating maternal transfer of OCs to the fetus. The concentrations of individual OCs in tissues varied considerably among the animals. Statistically significant differences were found between females and males for the concentrations of certain analytes (e.g., SigmaPCBs, p,p'-DDE) and these differences may be partially due to contaminants being transferred by the female whales during gestation and lactation. The concentrations of OCs in different tissues were similar when based on total lipid weight, except for the brain, which contained the lowest lipid-normalized OC concentrations. The low concentrations in brain may be related to the disparate lipid compositions in this tissue as well as the presence of the blood-brain barrier. The availability of data on these archived and biomonitoring samples provides a baseline for future retrospective studies.

The relationship between hemic neoplasia, a blood cell disorder in bivalve molluscs, and chemical contaminants was evaluated in the common mussel (Mytilus edulis complex). Hemic neoplasia (HN) is endemic to mussel populations in Puget Sound. The prevalence of hemic neoplasia ranged from 0 to 30% in mussels from nine sites in Puget Sound, Washington. Organic chemical contamination in sediment from these sites range from 0.1 to 64.0 ppm of polycyclic aromatic hydrocarbons (PAHs) and 0.07 to 0.50 ppm chlorinated hydrocarbons. No relationship between the body burden of environmental contaminants and the prevalence of HN in mussels was identified. To evaluate the shortterm ability of chemical contaminants to induce HN in mussels, mussels, from a site where mussels were previously determined to be HN free, were fed microencapsulated PAHs (composed of a mixture of phenanthrene, flouranthene, and benzo[a]pyrene) or PCBs (Aroclor 1254) and the prevalence of HN was assessed after 30 days of exposure. Although an apparent increase in HN prevalence (20 to 30%) was observed in all treatments groups except the untreated controls, no significant difference in the prevalence of HN was observed between the control group of mussels fed corn oil (vehicle) and mussels fed either PAHs or PCBs in corn oil. A long-term (180-day) exposure study was conducted to evaluate the influence of PAHs or PCBs in modulating the prevalence of HN in a mussel population already exhibiting a moderate HN prevalence. Mussels, from a site where mussels were previously determined to exhibit a background prevalence of HN, fed microencapsulated PAHs, PCBs, and corn oil (vehicle) over a long time period (180 days), revealed an apparent increased prevalence of HN (30 to 40%) above the low levels (20%) initially present. However, no significant difference in the prevalence of HN was observed between the control group of mussels fed corn oil (vehicle) and mussels fed either PAHs or PCBs in corn oil. Although chemical contaminants have been proposed as a modulating factor in the development and promotion of HN in bivalve molluscs from environmentally stressed and degraded habitats, we find no evidence that chemical contaminants induce or promote the development of HN in the mussel M. edulis complex. r 1999 Academic Press

Molecular epizootiological studies are increasingly being used to investigate environmental effects of genotoxic contaminants. The assessment of damage to DNA and linking the damage to subsequent molecular, cellular, or tissue-level alterations is a central component of such studies. Our research has focused on the refinement of the 32P-postlabeling assay for measuring covalent DNA-xenobiotic adducts arising from exposure to polycyclic aromatic compounds, using DNA adducts as molecular dosimeters of genotoxic contaminant exposure in biomonitoring studies, and investigating the relationship of DNA adduct formation to toxicopathic liver disease, including neoplastic lesions. A combination of field and laboratory studies using the 32P-postlabeling assay has shown that DNA adducts in marine fish are effective molecular dosimeters of genotoxic contaminant exposure. Investigations of the relationship of DNA adduct formation to neoplastic liver disease have shown that elevated levels of DNA adducts in certain fish species from contaminated coastal sites are associated with increased prevalences of toxicopathic hepatic lesions, including neoplasms, and that the ability to assess DNA damage has helped to explain, in part, species differences in lesion prevalence. Moreover, in a study of a site in Puget Sound contaminated with polycyclic aromatic compounds, we have shown, for the first time, that elevated levels of hepatic DNA adducts are a significant risk factor for certain degenerative and preneoplastic lesions occurring early in the histogenesis of hepatic neoplasms in feral English sole (Pleuronectes vetulus). These latter findings coupled with our current studies of mutational events in the K-ras proto-oncogene should provide further mechanistic substantiation that mutagenic events resulting from exposure to complex mixtures of genotoxic polycyclic aromatic compounds are involved in the etiology of hepatic neoplasia in English sole.

Liver neoplasms are rarely detected in young wild fish. Therefore, other lesions occurring early in the histogenesis of hepatic neoplasia need to be considered as biomarkers of chemical contaminant exposure effects in monitoring studies, especially where adult fish are not available. Moreover, exposure effects may be more reliably assessed in younger fish that have not yet migrated extensively. Accordingly, livers of subadult English sole were histologically examined from nine sites in Puget Sound, WA and the same fish were assessed for contaminant exposure by measurement of fluorescent aromatic compounds (FACs) in bile, hepatic levels of polychlorinated biphenyls (PCBs) and cytochrome P4501A (CYP1A) as catalytic activity of aryl hydrocarbon hydroxylase (AHH), and Hydrophobic DNA adducts in liver by 32P-postlabelling. Although neoplasms were rare, higher prevalences of preneoplastic, regenerative, and unique degenerative/ necrotic lesions were detected in sole from contaminated sites. Prevalences of these early histopathologic biomarkers were significantly higher at the more contaminated sites, and concentration of mean biliary FACs at each capture site was a significant risk factor for most lesions, as determined by stepwise logistic regression. By this statistical method, we also demonstrated that several measures of bioaccumulation or biochemical response to contaminants were significant and near-significant risk factors for prevalences of most hepatic lesion categories. For example, mean hepatic AHH activity was a significant risk factor for prevalence of all lesion types, except neoplasms; hepatic PCB and xenobiotic-DNA adduct concentrations were significant risk factors for the most frequently detected lesion category, hepatocellular nuclear pleomorphism/megalocytic hepatosis, and the inclusive category ‘any early toxicopathic lesion’. These findings further support the utility of certain non-neoplastic liver lesions as early indicators of biological damage in subadult as well as adult fish exposed to xenobiotics in the marine environment.

Cytochemical responses in the digestive tissue of Mytilus edulis complex fed a microencapsulated mixture of PAHs (composed of phenanthrene, fluoranthene and benzo[a]pyrene) or PCBs (Aroclor 1254) were evaluated. Lysosomal membrane labilization period and lipofuscin content of digestive tissue were significantly decreased and increased, respectively, after 30 days of exposure to PAHs but not PCBs. NADPH-ferrihemoprotein reductase activity in the digestive tissue was significantly increased after 30 days exposure to both PAHs and PCBs, whereas catalase activity was significantly increased only after exposure to PCBs. Neutral lipid content and gamma-glutamyl transpeptidase activity of digestive tissue of mussels were significantly increased and decreased, respectively, after 30 days exposure to both PAHs and PCBs when compared to untreated mussels, but were not significantly different when compared to mussels treated with com oil representing the vehicle control. Exposure to PAHs induced peroxisome proliferation in the digestive tissue of M. edulis complex. The observed cytochemical responses in the digestive tissue of M. edulis complex exposed to PAHs and PCBs in the laboratory are comparable to the cytochemical responses observed in the digestive tissue of mussels collected in the field from sites contaminated with predominantly PAHs and PCBs.

Experiments with three species of infaunal invertebrates (a polychaete, Armandia brevis and amphipods, Rhepoxynius abronius and Eohaustorius washingtonianus) with different modes of feeding gave a wide range in toxic response to sediment associated tributyltin (TBT), while exhibiting consistent lethal tissue residues. These studies showed that bioaccumulation and toxicity of sediment-associated TBT were strongly controlled by the organic carbon content of the sediment, which we concluded was primarily due to its influence on interstitial water (IW) concentrations of TBT. Major differences in the response to sediment-associated TBT were found between species, which was attributed to differences in their rates of uptake and elimination of this compound. Predictions for bioaccumulation and toxicity for each species based on these toxicokinetic rates were matched closely by observed values. Based on comparisons of water-only and IW exposures (when water and sediment concentrations of TBT were in equilibrium) and predictions made with toxicokinetic rates, the major route of uptake for each of the species tested appeared to be from dissolved TBT. We determined the mean (sd) organic-carbon normalized sediment-water partition coefficient (Koc) to be approximately 25 100 (5500) for TOC values ranging from 0.3 to 1.0%, which was five times higher than the reported Kow. Additionally, we determined the dissolved organic carbon—water partition coefficient (Kdoc) to be 1652, which was three fold lower than the Kow. The results also showed that the Koc could be influenced by infaunal organisms, presumably by reducing IW concentrations below predicted values, which raises questions about the environmental relevance of sediment bioassays using these organisms and the expected IW concentrations present in field sediments.

Puget Sound is a large marine and estuarine ecosystem in Washington State that serves as the habitat for a number of recreationally and commercially important species of groundfish and Pacific salmon Onchorhynchus spp. Over the past several decades, the human population of the Puget Sound drainage basin has increased substantially, resulting in increased habitat degradation and chemical pollution. There is now a body of evidence showing that groundfish and salmon in Puget Sound are experiencing a range of biological effects from chemical contaminant exposure, including impairment of several stages in the reproductive process, increased susceptibility to pathogens resulting from altered immune competence, and development of toxicopathic diseases. Preliminary studies suggest the potential for reduced survival of fish from urban areas of Puget Sound resulting from increased infectious and idiopathic diseases. The question that now must be addressed is whether contaminant-related reductions in reprod...

Starry flounder (Platichthys stellatus), white croaker (Genyonemus lineatus) and sediments were collected annually from selected sites within San Francisco Bay, and a reference site in Bodega Bay between 1984--1991. Fish livers were examined for toxicopathic lesions and analysed for selected chlorinated hydrocarbons (CHs) such as PCBs, DDTs, chlordanes and dieldrin; sediment and fish stomach contents were analysed for polycyclic aromatic hydrocarbons (PAHs) and CHs; and bile was analysed for PAH metabolites. Sediment concentrations of PAHs, PCBs and DDTs; bile concentrations of PAH metabolites; and liver concentrations of PCBs, dieldrin and chlordanes were generally significantly higher at all San Francisco Bay sites compared to the Bodega Bay reference site. For both species, hydropic vacuolation of biliary epithelial cells was the most prevalent liver lesion detected and was statistically associated with sediment and tissue concentrations of PAHs or their metabolites, PCBs, DDTs, chlordanes and dieldrin. Temporal trends analyses showed that at Hunters Point, sediment PAHs and CHs increased between 1984--1991, while liver concentrations of CHs decreased. Liver concentrations of dieldrin in starry flounder decreased at all three San Francisco Bay sites

Juvenile chinook salmon (Oncorhynchus tshawytscha) were sampled in Puget Sound, Washington, for 2 consecutive years from contaminated urban estuaries, a nonurban estuary, and from the respective hatcheries to assess exposure to anthropogenic chemicals and to determine if biochemical changes were occurring as a consequence of exposure. Exposure to polycyclic aromatic hydrocarbons (PAHs), chlorinated hydrocarbons, and butyltins was determined. The mean concentrations of PAHs and PCBs in stomach contents and PCBs in liver were significantly higher in salmon from the urban estuaries compared to fish from the nonurban estuary in both sampling years. Higher hepatic concentrations of PCBs than DDTs were found in fish from the urban estuaries, but butyltins were rarely detected. Further, mean concentrations of fluorescent aromatic compounds in bile, an estimate of exposure to PAHs, and hepatic cytochrome P4501A and levels of hepatic DNA adducts were also significantly higher in salmon from the urban estuaries compared to either the nonurban estuary or the hatcheries. Results demonstrated increased exposure to chemical contaminants in outmigrant juvenile salmon during their relatively brief residence in urban estuaries of Puget Sound. Moreover, the exposure was sufficient to elicit biochemical responses, which suggest a potential for other biological effects to ensue.

The effects of chemical contaminants on concentrations of hepatic glutathione (GSH), cysteine (L-Cys), and the activity of γ-glutamylcysteine synthetase (γ-GCS) were assessed in English sole (Pleuronectes vetulus) in the laboratory and in fish from the Duwamish Waterway, a contaminated urban site in Puget Sound, Washington. In the laboratory studies, fish from a nonurban (reference) site were exposed to an organic-solvent extract of sediment from the Duwamish Waterway. Hepatic GSH concentrations significantly increased at 3 d after exposure to the extract at 1,000 g sediment extracted/kg fish. At this time point, hepatic GSH showed a dose-dependent increase at dosages ranging from 300 to 1,600 g sediment extracted/kg fish, whereas fish exposed to a reference sediment extract showed no increase. Moreover, the increases in GSH in fish exposed to the contaminated sediment extract were not accompanied by changes in either L-Cys concentrations or γ-GCS activity. Similarly, fish sampled directly from the Duwamish Waterway showed increased GSH concentrations but showed no significant differences in L-Cys levels or γ-GCS activity compared to fish from a reference site. These results substantiate that hepatic GSH in fish is responsive to chemical contaminant exposure and further support its use as a biomarker in environmental monitoring studies. The findings also indicate that L-Cys availability and induction of γ-GCS were not major factors in the increase of hepatic GSH in contaminant-exposed English sole.

Human liver peroxisomes contain two acyl-CoA oxidases, namely, palmitoyl-CoA oxidase and a branched chain acyl-CoA oxidase. The palmitoyl-CoA oxidase (ACOX) oxidizes the CoA esters of straight chain fatty acids and prostaglandins and donates electrons directly to molecular oxygen, thereby producing H2O2. The inducibility of this H2O2-generating ACOX in rat and mouse liver by peroxisome proliferators and the postulated role of the resulting oxidative stress in hepatocarcinogenesis generated interest in characterizing the structure and function of human ACOX. We have constructed a full-length cDNA encoding a 660-amino acid residue human ACOX and produced a catalytically active human ACOX protein at high levels in Spodoptera frugiperda (Sf9) insect cells using the baculovirus vector. Immunoblot analysis demonstrated that the full-length 72-kDa polypeptide (component A) was partially processed into its constituent 51-kDa (component B) and 21-kDa (component C) products, respectively. Recombinant protein (approximately 20 mg/l x 10(9) cells) was purified to homogeneity by a single-step procedure on a nickel-nitrilo-triacetic acid affinity column. Using the purified enzyme, Km and Vmax values for palmitoyl-CoA were found to be 10 microM and 1.4 units/mg of protein, respectively. The maximal activities for saturated fatty acids were observed with C12-18 substrates. The overexpressed human ACOX protein was identified in the cytoplasm of the insect cells by immunocytochemical staining. Individual expression of either the truncated ACOX 51-kDa (component B) or the 21-kDa (component C) revealed lack of enzyme activity, but co-infection of the insect cells with recombinant viruses expressing components B and C resulted in the formation of an enzymatically active heterodimeric B+C complex which could subsequently be inactivated by dissociating with detergent.

Bioaccumulation of polycyclic aromatic hydrocarbons (PAHs) from field-contaminated sediments by 2 infaunal invertebrates, Rhepoxynius abronius (a non-deposit feeding amphipod) and Armandia brevis (a non-selective, deposit-feeding polychaete), was examined. Sediments were selected over a large geographical area of the Hudson-Raritan estuary (New York, USA) to assess the potential for bioaccumulation from a typical urban estuary. Assessment of bioaccumulation in these invertebrates is important because of the need to understand their health and role in ecosystem functioning and because they are vectors of sediment-associated contaminants to demersal fish. Our study compared the response of these 2 species over a wide range of PAH concentrations to learn how feeding mode may affect contaminant accumulation. After 10 d of exposure to sedirnents, we assessed the tissue concentrations of 24 PAHs and found no significant differences for the low molecular weight PAHs (LPAHs) between R. abronius and A. brevis. There was, however, a large differential observed between species for bioaccumulated high molecular weight PAHs (HPAHs). Because we assumed that the amphipod was not feeding and most of its tissue burden was received through ventilation of interstitial water (IW), we concluded that IW was probably the major route of uptake for the LPAHs for both species and that sediment ingestion was a much more important uptake route of HPAHs for the polychaete. Analysis of correlations between amphipod and polychaete tissue burdens found that the species were responding similarly to a gradient of PAH concentrations in sediment; however, when the associations between concentrations in tissue and exposure matrix (e.g. sediment, IW) were examined the interpretations were less clear. Concentrations of PAHs in IW and sediment indicated that the partition coefficient (&,) was generally 2 orders of magnitude higher than expected for LPAHs and highly variable between sites for HPAHs. When K,, was re-calculated using free PAH, i t became much less variable and was uniformly elevated above the predicted values. The BAF,,, (lipid/organic carbon normalized bioaccumulat~on factor) in the polychaete was generally consistent over the series of PAHs, as expected, and when metabolism and pre-steady state conditions were considered, partitioning of PAHs between tissue and sediment was relatively close to the theoretical maximum. Despite much lower than predicted 1W concentrations, the LPAH bioconcentration factors (BCF) for the amphipod were close to expected, but the HPAH BCFs were close to the predicted values only when expressed in terms of the free PAH. From these data, and previous studies, we conclude that there was a significant reduction in bioavailability of HPAHs to R. abronius due to partitioning of HPAHs to dissolved organic carbon.

A study was conducted over the course of a year to determine the induction of hepatic cytochrome P4501A (CYP1A) in three species of benthic fish collected from a contaminated site compared to fish sampled from a less-contaminated site. Juvenile fish were used to minimize effects of reproductive status and migration. CYP1A was determined by two catalytic assays [aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD)] and by an immunoassay (ELISA) utilizing polyclonal antibodies raised against purified CYP1A from cod. AHH activities were measured by a standard method (AHH[sub std]) and by two variations of the standard method. All three primary CYP1A measures (AHH[sub std], EROD, and ELISA) showed consistent between-site differences, indicating that induction of CYP1A can be a reliable biomarker of contaminant exposure in fish if appropriate biological variables are controlled for in field studies. Multiple ANOVA demonstrated that the AHH[sub std] and ELISA data showed less variability due to species or temporal differences, and less unexplained variability, compared to the data from the EROD assay or either variation of the AHH assay. For all measures, variability associated with site differences far outweighed species or temporal variability. Immunoassay, while less sensitive than the AHH[sub std] assay, is nonetheless recommendedmore » to be used in conjunction with catalytic assays because of the potential for samples to lose catalytic activity if not handled properly. The current results suggest that the lower noncontaminant-related variability of AHH[sub std] makes this CYP1A measure potentially more useful for monitoring programs in which analysis of trends is a primary goal.« less

A rapid method has been developed to screen for planar chlorobiphenyl (CB) congeners, as well as certain other CBs and DDTs, in tissue samples from marine biota. The analytes were extracted from tissue matrices with 1:1 hexane/pentane (v/v) and interfering compounds were separated from the CBs on a gravity-flow column packed with acidic, basic and neutral silica gel eluted with 1:1 hexane/methylene chloride (v/v). Subsequently, the planar CB congeners were resolved from the DDTs and other CBs by HPLC on Cosmosil PYE analytical columns cooled to 9 degrees C and were measured by an ultraviolet (UV) photodiode array (PDA) detector. Two important advantages of PDA over conventional UV detection were the ability to identify individual analytes by comparing their UV spectra to those of reference standards and the ability to establish the spectral homogeneity (purity) of the analytes by comparing spectra within a peak to the apex spectrum. The HPLC/PDA method was tested with tissue samples from fish, shellfish and marine mammals; concentrations of certain CBs and DDTs in samples determined by screening compared favorably with those in the same samples analyzed by a comprehensive method (e.g., gas chromatography/high resolution mass spectrometry). However, the HPLC/PDA method was about an order of magnitude less sensitive than determinations by comprehensive methods.

The concentrations of selected chlorinated hydrocarbons (e.g. PCBs, DDTs, DDEs, chlordanes) and essential (e.g. zinc, selenium, copper) and toxic (e.g. mercury, lead, arsenic) elements were measured in tissues and stomach contents from 22 gray whales ( Eschrichtius robustus ) stranded between 1988 and 1991 at sites from the relatively pristine areas of Kodiak Island, AK, to more urbanized areas in Puget Sound, WA, and San Francisco Bay, CA. The majority of animals were stranded at sites on the Washington outer coast and in Puget Sound. The gray whale has the unique feeding strategy among Mysticeti of filtering sediments to feed on benthic (bottom dwelling) invertebrates. Thus, the wide geographical distribution of the stranded whales allowed (1) an initial assessment of whether concentrations of chemical contaminants in these whales exhibited region specific differences and (2) whether toxic chemicals that accumulate in sediments may have contributed to the mortality and stranding of gray whales near the more polluted urban areas. Analyses for chlorinated hydrocarbons in blubber from 22 animals showed no apparent significant differences among stranding sites. The concentrations of ΣPCBs and ΣDDEs in blubber, for example, ranged from 120 to 10000 and 9 to 2100 p.p.b. (ng/g) wet weight, respectively. Additionally, analyses of chlorinated hydrocarbons and selected elements in liver ( n = 10) also showed no apparent significant differences between whales stranded in Puget Sound and whales stranded at more pristine sites (Alaska, Washington outer coast and Strait of Juan de Fuca and Strait of Georgia). For example, the concentrations of ΣPCBs and ΣDDEs in liver ranged from 79 to 1600 and 7 to 280 p.p.b., respectively, and the concentrations of the toxic elements, mercury and lead ranged from 9 to 120 and 20 to 270 p.p.b., respectively. Analyses of stomach contents revealed low concentrations of chlorinated hydrocarbons, but high concentrations (wet weight) of aluminum (1700 000 ± 450 000 p.p.b.), iron (320000 ± 250 000 p.p.b.), manganese (23 000 ± 15 000 p.p.b.), and chromium (3400 ± 1300 p.p.b.), but no significant differences were observed between whales stranded in Puget Sound compared to whales stranded at the more pristine sites. The relative proportions of these elements in stomach contents of stranded whales were similar to the relative proportions in sediments, which is consistent with a geological source of these elements from the ingestion of sediment during feeding. Thus, overall, the concentrations of anthropogenic chemicals in stranded gray whales showed little relation to the levels of chemical contaminants at the stranding sites. Further, the results showed that the concentrations of potentially toxic chemicals in tissues were relatively low when compared with the concentrations in tissues of marine mammals feeding on higher trophic level species, such as fish. The lack of data from apparently healthy gray whales, however, limits the assessment of whether the levels of anthropogenic contaminants found in tissues may have deleterious effects on the health of gray whales.

Relationships between hepatic lesions and chemical contaminant concentrations in sediments, stomach contents, liver tissue, and bile were statistically evaluated in three species of bottomfish, English sole (Pleuronectes vetulus), starry flounder (Platichthys stellatus), and white croaker (Genyonemus lineatus), captured from 27 urban and nonurban sites on the Pacific Coast from Alaska to southern California. Lesions detected were neoplasms, preneoplastic foci of cellular alteration, nonneoplastic proliferative lesions, unique or specific degenerative/necrotic lesions, nonspecific degenerative/necrotic lesions, and hydropic vacuolation of biliary epithelial cells and hepatocytes. In general, lesion prevalences were significantly higher in all three species captured at chemically contaminated urban sites, and certain lesions had significantly higher relative risks of occurrence at urban sites in Puget Sound, San Francisco Bay, the vicinity of Los Angeles, and San Diego Bay. Concentrations of polycyclic aromatic hydrocarbons, polychlorinated biphenyls, DDT and its derivatives, and chlordanes and dieldrin in sediment, stomach contents, liver, and fluorescent aromatic compounds in bile were significant risk factors for the occurrence of neoplastic, preneoplastic, nonneoplastic proliferative, and specific degenerative/necrotic lesions, as well as hydropic vacuolation. Fish age also had a significant influence on occurrence of several hepatic lesions, but gender was rarely a significant risk factor. These relationships provide strong evidence for the involvement of environmental contaminants in the etiology of hepatic lesions in several marine bottomfish species and clearly indicate the utility of these lesions as biomarkers of contaminant-induced effects in wild fish. Images Figure 2. A Figure 2. B Figure 3. Figure 4. A Figure 4. B

The recent development of techniques to measure levels of carcinogens covalently bound to DNA provides the opportunity to use DNA adducts as molecular dosimeters of exposure to environmental carcinogens and mutagens. This is especially important because epizootiologic studies have shown a positive association between environmental carcinogens, such as polycyclic aromatic hydrocarbons, and increased prevalence of neoplasms and related lesions, primarily in liver, of benthic fish species from a wide range of urban and industrialized areas. In studies with wild fish and mammalian species the 32P-postlabeling assay, as developed for aromatic compounds, has been used most extensively because of its high sensitivity and ability to detect structurally uncharacterized adducts. The results to date of field and laboratory studies show that hepatic DNA adducts detected in fish are associated with increased exposure to environmental polycyclic aromatic compounds in the preponderance of species examined, whereas in the limited studies with wild mammals, such a relationship is equivocal at present. The findings with fish suggest that DNA adducts, as measured by 32P-postlabeling, have the potential to be effective molecular dosimeters of exposure to environmental carcinogenic aromatic compounds and thereby may lead to an improved understanding of the etiology of neoplasia in wild teleosts.

The relationship between exposure to xenobiotic compounds and ovarian development was evaluated in prespawning female winter flounder (Pleuronectes americanus) sampled from eleven sites on the northeast coast of the United States during the 1988 and 1989 spawning seasons. Three sites were located in Boston Harbor, MA, four sites were in Raritan Bay, NJ and four sites were in nearby embayments. Sediments from these sites exhibited a wide range in concentrations of xenobiotic compounds (e.g. aromatic hydrocarbon (PAH) concentrations ranged from 20 to 50 000 ng/g dry wt. and polychlorinated biphenyl (PCB) concentrations ranged from 2 to 1400 ng/g dry wt.), with the sites in Boston Harbor and Raritan Bay the most heavily contaminated. The following parameters associated with ovarian development were measured: ovarian developmental stage, ovarian atresia, gonadosomatic index, plasma estradiol, fecundity and egg weight. Contaminant exposure was assessed by measuring concentrations of fluorescent aromatic compounds (FACs) in the bile, hepatic aryl hydrocarbon hydroxylase (AHH) activity, concentrations of PCB in liver, ovary and brain and concentrations of xenobiotic-DNA adducts in liver tissue. Additionally, liver tissue was examined histologically for the presence of suspected toxicopathic lesions. In general, indicators of contaminant exposure were elevated and prevalences of suspected toxicopathic lesions were highest in fish from sites within Boston Harbor and Raritan Bay. Hepatic AHH activity, however, was heavily influenced by the reproductive cycle and showed little correlation with other indicators of contaminant exposure. Evidence of decreased egg weight and increased atresia in fish with high tissue concentrations of PCB or high levels of FACs in bile was observed. However, contaminant exposure had no clear negative impact on gonadal recrudescence, gonadosomatic index, plasma estradiol concentrations, or fecundity in female winter flounder. These results are in contrast to results with another Pleuronectid species, English sole (Pleuronectes vetulus), which shows inhibited gonadal development and lower plasma estradiol concentrations at contaminated sites in Puget Sound, WA. The apparent difference between English sole and winter flounder in susceptibility to contaminant-induced reproductive dysfunction could be related to a number of factors, including the differences in the migratory behavior of the two species during the reproductive season. English sole reside in contaminated estuaries throughout vitellogenesis and move offshore to spawn, while winter flounder often remain offshore for extended periods during early vitellogenesis and move into contaminated estuaries prior to spawning. Because of these contrasting migration patterns, both the duration and timing of exposure to contaminants during gonadal recrudescence may differ substantially in these two species and may contribute, in part, to the observed differences in the prevalence of reproductive dysfunction in fish from contaminated estuaries.

Relationships between hepatic lesions and chemical contaminant concentrations in sediments, stomach contents, and tissues were examined in winter flounder (Pleuronectes americanus) collected from 22 sites in eight major embayments on the Northeast Coast (i.e., Salem Harbor, Boston Harbor, Plymouth Bay, Buzzards Bay, New Bedford Harbor, Narragansett Bay, Long Island Sound, and Raritan Bay). Prevalences of a number ofpathological conditions, including neoplasms, preneoplastic lesions, hydropic vacuolation, and other necrotic and proliferative lesions, were significantly elevated in fish from contaminated urban embayments such as Boston Harbor and Raritan Bay

In order to monitor the quality of coastal waters that provide habitats for living marine resources, samples of sediment and biota must be analyzed to assess the degree and distribution of anthropogenic contamination. Analytical time and costs can be greatly reduced by first employing methods that screen for contaminants before selecting samples for rigorous analyses. In this paper, we review the applications of rapid high-performance liquid chromatographic (HPLC) methods to screen for aromatic compounds in sediment, bile and tissue samples. These methods have been used to assess damage to natural resources after the Exxon Valdez oil spill. In addition, the bile screening method has also been used to evaluate contaminants in fish sampled for a national monitoring program. The rapid screening of sediment or bile provides an estimate of contaminant concentrations that can then be confirmed in selected samples by more complicated and expensive analyses by gas chromatography/3-mass spectrometry (GC-MS). Furthermore, HPLC and GC-MS chromatographic patterns from sediment and bile can provide information about the source of contamination, e.g., crude oil, diesel fuel or pyrogenic contaminants. We also discuss the important role screening methods will play in the future in assessing the quality of aquatic habitats, the safety of seafood, and other important issues related to anthropogenic contamination.

After the grounding of the Exxon Valdez, sediment samples were collected to determine the degree and distribution of the oiling. Sixty sediments from 10 sites in Prince William Sound, AK, were analyzed for Prudhoe Bay crude oil (PBCO) using a rapid HPLC screening method that measured fluorescence at wavelength pairs specific for two- and three-ring petroleum-related aromatic compounds (ACs). Concentrations of individual ACs in the sediments were also determined by GC/MS to compare the results of the two methods. Concentrations of ACs measured by HPLC screening were highly correlated with the sums of ACs determined by GC/MS, thus validating the screening method as an effective tool for estimating concentrations of petroleum-related ACs in sediments. Moreover, differences in HPLC chromatographic patterns among sediments suggested different sources of contamination, e.g., crude oil or diesel fuel. Finally, GC/MS analyses confirmed that PBCO was a primary source of contamination in many sediments. 21 refs., 5 figs., 2 tabs.

The assessment of oil exposure and resulting biological effects in subtidal fish following marine oil spills is a rather challenging task, due to difficulty in sampling and a lack of methodologies that allow direct determination of petroleum exposure. Because fish can rapidly metabolize petroleum hydrocarbons, standard chemical analyses are of little use for assessing exposure of fish to oil. However, methods are now available for measuring the metabolites of petroleum hydrocarbons in fish, as described elsewhere by Krahn and her colleagues. There have also been advances in determining early biochemical effects—such as induction of the cytochrome P450 system—and more severe biological effects—such as histopathological alterations and reproductive dysfunction—in fish species following exposure to petroleum hydrocarbons and related compounds. Accordingly, following the Exxon Valdez oil spill, we have been using these approaches and methodologies to measure oil exposure in subtidal fish species, together with associated biological effects. These studies have been conducted at sites inside Prince William Sound and along the Kenai and Alaska Peninsulas. Our results show a continuing exposure of several fish species, including benthic species, which indicates petroleum contamination of subtidal areas. We have also assessed reproductive function and histopathological alterations in several of these species; and while major effects have not been documented, some suggestion of histopathological alterations of gill exists in one species of benthic fish. Still to be determined are the potential impacts on fishery resources of long-term exposure to petroleum, albeit at moderate to low levels.

A suite of chemical and biochemical parameters was measured in three species of benthic flatfish (English sole, Parophrys vetulus; rock sole, Lepidopsetta bilineata; and starry flounder, Platichthys stellatus) sampled from up to five sites in Puget Sound, WA, USA, to assess the sensitivity of the parameters to differences in levels of contaminant exposure and the relative merit of the use of a suite of indices for assessing exposure and sublethal effects. The indices examined were hepatic levels of polychlorinated biphenyls (PCBs) and biliary fluorescent aromatic-compound concentrations, hepatic activities of aryl hydrocarbon hydroxylase and ethoxyresorufin-O-deethylase, and levels of total hepatic GSH and hydrophobic DNA-xenobiotic adducts; the last-mentioned were determined by using the 32P-postlabeling assay. The results showed that the indices examined could discriminate among sites exhibiting different degrees of chemical contamination; however, species differences in the range of response of some indices were observed. Additionally, the use of multiple indices appeared to enhance the assessment of contaminant exposure and sublethal effects.

Three species of infaunal gammaridean amphipods, Rhepoxynius abronius (Phoxocephalidae), Eohaustorius washingtonianus, and E. estuarius (Haustoriidae) were tested in a water-only system to assess their sensitivity to tributyltin (TBT) without the influence of factors that could affect bioavailability. When mortality (LC50) was the endpoint, the results indicated that R. abronius was ≈20 times more tolerant to tributyltin than either haustoriid species; however, when mortality plus reburial behavior (EC50) was assessed, the difference was only about 10 times. The bioconcentration factor (BCF) was also consistently lower in R. abronius (11.1 to 16.5 times) than in the haustoriids; however, when the LD50 was calculated, the concentration in the tissues associated with 50% mortality for each species was not significantly different. The large disparity in species' response is attributed to reduced uptake and a potentially greater ability to metabolize this compound by R. abronius. An analysis of TBT uptake confirmed that R. abronius was able to accumulate less TBT and hence maintain a low body burden for a given water concentration. The results of a separate uptake study were used to formulate a hypothesis for observed differences in reburial behavior. Because the rate of TBT uptake was lower in R. abronius, we propose that the slower rise in toxicant body burden allowed for a gradual response in this species which included a sublethal effect (non reburial), compared to a rapid rise in the body burden for E. estuarius which caused the response to quickly proceed from no effect to death.

Our studies have shown that early stages of the reproductive process in female English sole (Parophrys vetulus) are disrupted by contaminant exposure. To determine the potential for contaminants to affect later stages of the reproductive cycle, we examined the effects of maternal contaminant exposure on egg and larval viability in this species, in a field study conducted on running ripe fish collected for two consecutive years from spawning grounds. Maternal contaminant exposure was assessed by several methods. Stepwise multiple regression of the data showed that contaminant exposure was only a minor factor in determining egg and larval viability. However, because the range of contaminant exposure in the spawning fish did not encompass the high levels seen in non-spawning fish captured from contaminated areas, it can be hypothesized that female English sole exposed to substantial levels of contaminants might be at least partially excluded from the spawning population.

Field studies on ovarian development in English sole from urban and nonurban sites in Puget Sound, Washington, USA, demonstrated that animals with elevated levels of fluorescent aromatic compounds (FACs) in bile were less likely to enter vitellogenesis and had lower plasma concentrations of estradiol than female sole with low levels of contaminant exposure. Biliary FACs were positively correlated with hepatic P450 activity, which was also elevated in sole showing inhibited ovarian development. These findings suggest that contaminant exposure may disrupt vitellogenesis in female fish. Results of the field study were supported by laboratory experiments showing that pretreatment of gravid female English sole with extracts of contaminated sediment decreased levels of endogenous estradiol. A similar response was observed in rock sole and flathead sole treated with Prudhoe Bay crude oil. However, exposure of English sole to sediment extracts had little effect on the activity of hepatic-steroid-metabolizing enzymes, indicating that some mechanism other than enhanced steroid metabolism may be responsible for reductions in endogenous circulating-steroid levels. Our recent studies suggest that reductions in endogenous estradiol levels may result from depressed ovarian steroidogenesis, as contaminant-associated reductions in in-vitro ovarian estradiol production were observed in English sole, rock sole, and flathead sole. Preliminary studies on winter flounder and white croaker indicate that they may also experience reproductive impairment as a result of exposure to contaminants, but the phase of the reproductive cycle that is most severely impacted varies from species to species.

Approximately 1 year after Kuwaiti crude oil was spilled and burned during the Gulf War, scientists on board the NOAA R/V Mt Mitchell sampled sediment and biota in the Gulf to look for evidence of petroleum contamination. Subtidal sediments and fish bile were analysed for petroleum-related aromatic compounds (ACs) on board the Mt Mitchell using cost-effective and rapid high-performance liquid chromatographic (HPLC) screening methods. Parent ACs and their alkyl homologs were measured in sediments. However, because fish extensively metabolize ACs in their livers to form polar metabolites that are concentrated in bile for excretion, metabolites of ACs were measured in bile. HPLC screening analyses found ACs in many of the sediment and fish bile samples collected. The results of HPLC screening were confirmed in selected samples by gas chromatography/mass spectrometry (GC/MS) analyses that identified petroleum-related ACs (e.g., alkyl phenanthrenes or dibenzothiophenes) in sediments and their metabolites in bile.

A suite of chemical and biochemical variables responsive to contaminant exposure was measured in three species of benthic flatfish (English sole, Parophrys vetulus, rock sole, Lepidopsetta bilineata, and starry flounder, Platichthys stellatus) sampled from up to five sites in Puget Sound, Washington, to assess the sensitivity of the parameters to differences in levels of contaminant exposure The examined indexes were levels of (a) polychlorinated biphenyls (PCBs) in liver, (b) fluorescent aromatic compounds in bile, (c) hepatic aryl hydrocarbon hydroxylase and ethoxy-resorufin O deethylase, (d) total hepatic GSH and (e) hydrophobic DNA xenobiotic adducts in liver as measured by 32P postlabeling The sediment concentrations of PCBs and the sum of selected polycyclic aromatic hydrocarbons at the sampling sites ranged from 21 to 570 and 21 to 5,300 ng/g wet weight sediment, respectively The results showed (a) that all the examined indexes could discriminate among sites exhibiting different degrees of chemical contamination, (b) that species differed in the range of response of most of the measured indexes, and (c) that the use of the indexes in concert appeared to enhance the assessment of contaminant exposure and sublethal effects

Our studies have shown that early stages of the reproductive process in female English sole (Parophrys vetulus) are disrupted by contaminant exposure. To determine the potential for contaminants to affect later stages of the reproductive cycle, we examined the effects of maternal contaminant exposure on egg and larval viability in this species, in a field study conducted on running ripe fish collected for two consecutive years from spawning grounds. Maternal contaminant exposure was assessed by measuring levels of polychlorinated biphenyls (PCBs) in ovarian and hepatic tissue, biliary levels of fluorescent aromatic compounds (FACs), and hepatic aryl hydrocarbon hydroxylase (AHH) activities. Additionally, the presence of significant liver disease or ovarian atresia was documented, and levels of plasma estradiol were measured. Stepwise multiple regression of the data showed that contaminant exposure was only a minor factor in determining both egg and larval viability. Hepatic AHH activity showed a slight negative correlation with fertilization success, explaining only 4% of the variability. Increased levels of PCBs in ovaries of spawning female English sole were not associated with reduced reproductive success. However, because the range of contaminant exposure in the spawning fish did not encompass the high levels seen in non-spawning fish captured from contaminated areas, it can be hypothesized that female English sole exposed to substantial levels of contaminants might be at least partially excluded from the spawning population.

Chemical pollution at sites in or near San Diego Bay was investigated between 1984 and 1988. The mean concentrations of selected polychlorinated biphenyls (PCBs), metals (e.g., copper and lead), and aromatic hydrocarbons in sediments from sites in the southern and central portions of the bay were significantly higher (p≤0.05) than those in sediment samples from nearby nonurban sites

Aromatic hydrocarbons (AHs) are extensively metabolized into oxidized products by fish and mammals. Some metabolites are more toxic than the parent AHs; however, currently there are no methods to screen tissues of fish or other marine species for the presence of AH metabolites. In the present study, a relatively rapid, semiquantitative method that combines enzymatic hydrolysis, methylene chloride extraction and hexane/potassium hydroxide partitioning with HPLC/fluorescence analysis, was developed for the estimation of the concentrations of polar fluorescent aromatic compounds (polar FACs; e.g., AH metabolites) in fish tissues. Liver and muscle from rock sole (Lepidopsetta bilineata) exposed to Prudhoe Bay crude oil in the laboratory were analyzed. Tissues from English sole (Parophrys vetulus) sampled at two sites (one nonurban reference and one AH-contaminated) in Puget Sound, WA were also analyzed. The concentrations of polar FACs in liver and muscle of PBCO-exposed rock sole were linearly proportional to dose. Also, in both species exposed to contaminants, the concentrations of polar FACs in liver were ∼15 times as great as those in muscle. Polar FACs, however, were not detected in muscle of English sole collected at either the contaminated or reference site, suggesting that polar FACs in naturally exposed animals may be present only at low concentrations unless exposed to high levels of contaminants such as AHs. Thus, the present method can detect, estimate and rank the relative concentrations of polar FACs (e.g., AH metabolites) in tissues, such as liver and muscle of fish. The method augments our ability to assess environmental exposure to potentially toxic contaminants such as AHs.

We are currently analyzing hepatic cytochrome P4501A and associated monooxygenase activities in fish sampled in several regional and national monitoring programs, including the National Benthic Surveillance Project of NOAA's Status and Trends Program, damage assessment studies of the Exxon Valdez oil spill, and intensive surveys of specific embayments, such as Puget Sound, Washington. Thus far, apparent contaminant-related increases in the activities of cytochrome P4501A-dependent monooxygenases have been readily measured in most test species. The results presented in this paper show that, for II species of fish, there is excellent concordance between hepatic activities of aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin-O-deethylase (EROD); moreover, levels of cytochrome P4501A determined by an enzyme-linked immunosorbent assay (ELISA) are also generally concordant with results from catalytic assays. The use of both a catalytic assay and immunoquantitation is recommended, because of the additional quality assurance provided by concurrent use of an immunoquantitation technique, which is desirable in large monitoring programs.

Liver neoplasms are rare in young wild fish; therefore, it is essential to consider toxicopathic liver lesions that occur earlier than neoplasms as biomarkers of contaminant exposure in studies where targeted or available fish are not adults. Also some exposure effects may be more reliably assessed in younger fish that have not yet migrated extensively. We addressed these issues by histologically examining subadult English sole, rock sole, and starry flounder captured from eight sites in Puget Sound, and by measuring fluorescent aromatic compounds (FACs) in bile and polychlorinated hiphenyls (PCBs) in liver. Neoplasms were detected rarely; higher prevalences of preneoplastic, regenerative, and degenerative lesions were detected in most species, primarily from contaminated sites, including hydropic vacuolation of hepatocytes and biliary cells in rock sole and starry flounder. These nomeoplastic lesion types are inducible in fish by exposure to various toxicants in the laboratory, and/or (in the case of hydropic vacuolation) are associated with contaminant exposure and liver neoplasia in adult wild fish. Prevalences of these ‘early’ lesions were significantly higher at the more contaminated sites, as determined by several statistical approaches, including logistic regression. Moreover, prevalences of most lesions in all three species correlated with mean bile FAC levels at the sites, and prevalences of some lesions in English sole correlated with liver PCB levels. The present findings further support the utility of certain liver lesions other than neoplasms as early indicators of biological damage in subadult as well as adult fish exposed to xenobiotics in the marine environment.

Obesity is frequently associated with systemic insulin resistance, glucose intolerance, and hyperlipidemia. Impaired insulin action in muscle and paradoxical diet/insulin-dependent overproduction of hepatic lipids are important components of obesity, but their pathogenesis and inter-relationships between muscle and liver are uncertain. We studied two murine obesity models, moderate high-fat-feeding and heterozygous muscle-specific PKC-lambda knockout, in both of which insulin activation of atypical protein kinase C (aPKC) is impaired in muscle, but conserved in liver. In both models, activation of hepatic sterol receptor element binding protein-1c (SREBP-1c) and NFkappaB (nuclear factor-kappa B), major regulators of hepatic lipid synthesis and systemic insulin resistance, was chronically increased in the fed state. In support of a critical mediatory role of aPKC, in both models, inhibition of hepatic aPKC by adenovirally mediated expression of kinase-inactive aPKC markedly diminished diet/insulin-dependent activation of hepatic SREBP-1c and NFkappaB, and concomitantly improved hepatosteatosis, hypertriglyceridemia, hyperinsulinemia, and hyperglycemia. Moreover, in high-fat-fed mice, impaired insulin signaling to IRS-1-dependent phosphatidylinositol 3-kinase, PKB/Akt and aPKC in muscle and hyperinsulinemia were largely reversed. In obesity, conserved hepatic aPKC-dependent activation of SREBP-1c and NFkappaB contributes importantly to the development of hepatic lipogenesis, hyperlipidemia, and systemic insulin resistance. Accordingly, hepatic aPKC is a potential target for treating obesity-associated abnormalities.

In this present study, juvenile chinook salmon were collected from a contaminated urban estuary, the Duwamish Waterway, as well as from a non-urban estuary, the Nisqually River estuary, to determine if exposure to toxic chemicals affects immunocompetency. Juvenile chinook salmon were also sampled from the two hatcheries that release salmon into these estuaries. The ability of anterior kidney (AK) and splenic (SP) leucocytes from primed and unprimed juvenile salmon from the Duwamish Waterway, Nisqually River estuary, and their respective hatcheries to produce a primary and secondary in vitro plaque-forming cell (PFC) response to the hapten, trinitrophenyl (TNP) was examined. Trinitrophenyl-keyhole limpet haemocyanin (TNP-KLH) was added in vitro to AK leucocytes or as TNP-lipopolysaccharide (TNP-LPS) to both AK and SP leucocytes. The primary AK and SP plaque-forming cell response to TNP in salmon from the estuaries or hatcheries was not significantly different. Primed AK leucocytes from salmon collected from the hatcheries and the non-urban estuary were able to produce a heightened secondary response to TNP-KLH; in contrast, primed AK leucocytes from salmon collected from the urban estuary were unable to produce a secondary PFC response to TNP-KLH. Anterior kidney leucocytes from salmon collected from all four areas were able to produce a heightened secondary PFC response to TNP-LPS. However, the AKs plaque-forming cell response generated in primed chinook salmon collected from the urban estuary to TNP-LPS was significantly lower than that produced in salmon from the hatchery. This suppressed PFC response in primed AK cells to TNP-LPS was not observed in primed AK leucocytes from salmon collected from the non-urban estuary and its hatchery. These results suggest that the cells involved in generating immunological memory to TNP-KLH and TNP-LPS were affected in salmon exposed to contaminants in the urban estuary, with cells which generate memory to TNP-KLH being more affected. The PFC responses of primed SP leucocytes from salmon collected from the Duwamish Waterway and Nisqually River estuaries, stimulated in vitro with TNP-LPS were not significantly different. The consequence of suppressed immunological memory in disease resistance of juvenile salmon is currently unknown.

Epizootiological analyses of field data on prevalances of hepatic neoplasms and other hepatic lesions in English sole (Parophrys vetulus) from Puget Sound suggest a link between these lesions and sediment-associated xenobiotics, particularly aromatic hydrocarbons (AHs). To further investigate this relationship, English sole from a minimally contaminated site were parenterally exposed at monthly intervals for 1 yr to an organic solvent extract of a contaminated urban sediment containing high levels of AHs (30 or 75 mg/kg body weight) or to the model carcinogen benzo[a]pyrene (BaP, 12 mg/kg body weight). Control groups included English sole exposed to an extract of a relatively uncontaminated reference sediment, sole exposed to the carrier only, and untreated sole. Eighteen months after the initial injections, examination of livers revealed a significantly different (increased, [Formula: see text]) incidence of a spectrum of hepatic lesions, including presumptive preneoplastic foci of cellular alteration, in sole exposed to the urban sediment extract or BaP. Sole in control groups did not develop any of these hepatic lesions. These results represent the first direct demonstration of the hepatotoxicity of sediment-associated chemical contaminants in English sole and strengthen the case for a cause-and-effect relationship between exposure to AHs and epizootic neoplastic hepatic lesions in this species.

English sole (Parophrys vetulus) are susceptible to the development of hepatic disease, including neoplasia, as a result of environmental exposure to polycyclic aromatic hydrocarbons (PAHs). The metabolism of PAHs, believed to be an essential factor in the development of neoplasia, has received considerable study in English sole, except that xenobiotic metabolizing enzymes (XMEs) have not been well-studied in this species. In the present work, the activities of hepatic aryl hydrocarbon hydroxylase (AHH), glutathione-S-transferase (GST), and epoxide hydrolase (EH) were measured in English sole exposed to several organic xenobiotics. These studies included an examination of the effects of captivity, the short-term responses of hepatic XME activities to several xenobiotic compounds, and detailed studies of the time- and dose-responses of hepatic XME activities to both a representative carcinogenic PAH (benzo[a]pyrene) and to a complex mixture of contaminants extracted from a sediment collected from a polluted area of Puget Sound, WA. Additionally, during the captivity and time- and dose-response studies, the levels of fluorescent aromatic compounds (FACs) were measured in the bile of the fish, both to provide an estimation of contaminant exposure and to evaluate the time- and dose-responses of this measure. The results of the captivity studies showed that the levels of FACs in bile were most affected by captivity, primarily as a result of changes in feeding status. The results of the exposure studies showed that xenobiotic metabolism, as reflected in hepatic activities of XMEs and levels of FACs in the bile, is altered by exposure to environmental contaminants. Whereas hepatic AHH activity could be rapidly and substantially increased by such exposure, activities of GST and EH were not affected, even up to 42 days after exposure. Moreover, because fish were exposed to a wide range of doses of chemicals or mixtures of chemicals which are known to be present in contaminated estuaries, and the responses of the hepatic AHH system and the levels of FACs in bile were measured at several time periods after exposure, the results provided substantial validation for the use of these two measures as bioindicators of exposure to environmental contamination in benthic fish.

1. Effects of a contaminated sediment extract on the disposition and metabolism of 3H-17β-estradiol in juvenile English sole and on plasma levels of endogenous estradiol in gravid females were examined. 2. Plasma estradiol levels were reduced in both extract-treated juvenile and gravid sole compared to controls, and in juvenile sole this decrease preceeded an increase in bile levels of 3H-17β-estradiol. 3. Extract treatment had no significant effect on types or proportions of 17β-estradiol metabolites or levels of related hepatic enzymes. 4. Thus, lower plasma estradiol levels in extract-treated sole were apparently due to alterations in physiological factors (e.g. increased bile flow) and not to enhanced metabolism of 17β-estradiol.

Vitellogenic female English sole were sampled from four areas in Puget Sound that varied in the nature and degree of chemical contamination. The fish were then injected with an analogue of Luteinizing Hormone Releasing Hormone (LHRH) in the laboratory to induce spawning. Ability to spawn, time to spawn, larval viability, and initial concentrations of plasma estradiol and vitellogenin [measured as alkaline-labile protein associated phosphate (ALP)] were assessed. In general, low initial plasma estradiol and ALP concentrations and subsequent reproductive impairment were most common in English sole from sites where levels of sediment contaminants [e.g. polycyclic aromatic hydrocarbons and polychlorinated biphenyls (PCB)] and measures of contaminant exposure in fish (hepatic PCB concentrations and aryl hydrocarbon hydroxylase activity) were highest, and where pollution-associated liver lesions in fish (i.e. neoplasms, foci of cellular alterations, specific degeneration/necrosis, and storage disorders) were most prevalent. Additionally, the time to spawn was found to be inversely correlated with initial plasma estradiol concentrations. Spawning success was found to be positively correlated with initial plasma estradiol and ALP concentrations as determined by logistic regression analysis; nearly 62% of the variability in the spawning response could be accounted for by these factors. Fertilization success was also found to be positively correlated with initial ALP concentrations, whereas fish captured from contaminated sites produced lower proportions of normal larvae. Overall, these findings suggest that contaminant exposure may result in poor reproductive success of female English sole. This may be related to a hormone imbalance or to slower ovarian development (non-synchronous timing) of female English sole from contaminated sites.