Tracy K. Collier

Hepatic activities of xenobiotic metabolizing enzymes and biliary levels of xenobiotics in English sole (Parophrys vetulus) exposed to environmental contaminants.

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 wellstudied 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.

Aryl Hydrocarbon Receptor-Independent Toxicity of Weathered Crude Oil during Fish Development

Polycyclic aromatic hydrocarbons (PAHs), derived largely from fossil fuels and their combustion, are pervasive contaminants in rivers, lakes, and nearshore marine habitats. Studies after the Exxon Valdez oil spill demonstrated that fish embryos exposed to low levels of PAHs in weathered crude oil develop a syndrome of edema and craniofacial and body axis defects. Although mechanisms leading to these defects are poorly understood, it is widely held that PAH toxicity is linked to aryl hydrocarbon receptor (AhR) binding and cytochrome P450 1A (CYP1A) induction. Using zebrafish embryos, we show that the weathered crude oil syndrome is distinct from the well-characterized AhR-dependent effects of dioxin toxicity. Blockade of AhR pathway components with antisense morpholino oligonucleotides demonstrated that the key developmental defects induced by weathered crude oil exposure are mediated by low-molecular-weight tricyclic PAHs through AhR-independent disruption of cardiovascular function and morphogenesis. These findings have multiple implications for the assessment of PAH impacts on coastal habitats.

The Synergistic Toxicity of Pesticide Mixtures: Implications for Risk Assessment and the Conservation of Endangered Pacific Salmon

Background Mixtures of organophosphate and carbamate pesticides are commonly detected in freshwater habitats that support threatened and endangered species of Pacific salmon (Oncorhynchus sp.). These pesticides inhibit the activity of acetylcholinesterase (AChE) and thus have potential to interfere with behaviors that may be essential for salmon survival. Although the effects of individual anticholin-esterase insecticides on aquatic species have been studied for decades, the neurotoxicity of mixtures is still poorly understood. Objectives We assessed whether chemicals in a mixture act in isolation (resulting in additive AChE inhibition) or whether components interact to produce either antagonistic or synergistic toxicity. Methods We measured brain AChE inhibition in juvenile coho salmon (Oncorhynchus kisutch) exposed to sublethal concentrations of the organophosphates diazinon, malathion, and chlorpyrifos, as well as the carbamates carbaryl and carbofuran. Concentrations of individual chemicals were normalized to their respective median effective concentrations (EC50) and collectively fit to a nonlinear regression. We used this curve to determine whether toxicologic responses to binary mixtures were additive, antagonistic, or synergistic. Results We observed addition and synergism, with a greater degree of synergism at higher exposure concentrations. Several combinations of organophosphates were lethal at concentrations that were sublethal in single-chemical trials. Conclusion Single-chemical risk assessments are likely to underestimate the impacts of these insecticides on salmon in river systems where mixtures occur. Moreover, mixtures of pesticides that have been commonly reported in salmon habitats may pose a more important challenge for species recovery than previously anticipated.

Fish embryos are damaged by dissolved PAHs, not oil particles

To distinguish the toxicity of whole oil droplets from compounds dissolved in water, responses of zebrafish embryos exposed to particulate-laden, mechanically dispersed Alaska North Slope crude oil (mechanically dispersed oil (MDO)) were compared to those of embryos protected from direct oil droplet contact by an agarose matrix. Most polycyclic aromatic hydrocarbons (PAHs) in MDO were contained in oil droplets; about 16% were dissolved. The agarose precluded embryo contact with particulate oil but allowed diffusive passage of dissolved PAHs. The incidence of edema, hemorrhaging, and cardiac abnormalities in embryos was dose-dependent in both MDO and agarose and the biological effects in these compartments were identical in character. Although mean total PAH (TPAH) concentrations in MDO were about 5-9 times greater than in agarose, dissolved PAH concentrations were similar in the two compartments. Furthermore, mean differences in paired embryo responses between compartments were relatively small (14-23%, grand mean 17%), typically with a larger response in embryos exposed to MDO. Therefore, the embryos reacted only to dissolved PAHs and the response difference between compartments is explained by diffusion. Averaged over 48 h, the estimated mean TPAH concentration in agarose was about 16% less than the dissolved TPAH concentration in MDO. Thus, PAHs dissolved from oil are toxic and physical contact with oil droplets is not necessary for embryotoxicity.

Sublethal exposure to crude oil during embryonic development alters cardiac morphology and reduces aerobic capacity in adult fish

Exposure to high concentrations of crude oil produces a lethal syndrome of heart failure in fish embryos. Mortality is caused by cardiotoxic polycyclic aromatic hydrocarbons (PAHs), ubiquitous components of petroleum. Here, we show that transient embryonic exposure to very low concentrations of oil causes toxicity that is sublethal, delayed, and not counteracted by the protective effects of cytochrome P450 induction. Nearly a year after embryonic oil exposure, adult zebrafish showed subtle changes in heart shape and a significant reduction in swimming performance, indicative of reduced cardiac output. These delayed physiological impacts on cardiovascular performance at later life stages provide a potential mechanism linking reduced individual survival to population-level ecosystem responses of fish species to chronic, low-level oil pollution.

Accumulation and metabolism of carbon-14 labeled benzene, naphthalene, and anthracene by young coho salmon (Oncorhynchus kisutch).

Carbon-14 labeled benzene, naphthalene, and anthracene were administered to young coho salmon (Oncorhynchus kisutch) in the food and by intraperitoneal injection. Regardless of the mode of application the accumulated carbon-14 (% administered dose) in key organs (e.g., liver and brain) increased in the order anthracene > naphthalene > benzene over various time periods. The metabolic fate of the hydrocarbons after intraperitoneal injection was studied. It was shown that the highest percentages of metabolites occurred in the gall bladder; however, significant amounts were also found in the liver, brain, flesh, and “carcass.” Solvent partition and thin-layer chromatographic techniques were developed to determine the structure of individual metabolites. In brain, liver, and gall bladder, 1-naphthol and 1-naphthyl glucuronic acid were major products of naphthalene metabolism; however, glycoside/sulfate fractions and mercapturic acid were indicated. The heart and flesh were rich in 1-naphthol and the former organ contained significant amounts of 1,2-dihydro-1,2-dihydroxynaphthalene. The findings indicated that the aromatic hydrocarbons in key organs increased in relation to the number of benzenoid rings. Further, it appears that aromatic metabolites are broadly distributed throughout fish exposed to polynuclear aromatic hydrocarbons.

Toxicopathic hepatic lesions in subadult English sole (pleuronectes vetuls) from Puget Sound, Washington, USA: Relationships with other biomarkers of contaminant exposure

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.

Health of Common Bottlenose Dolphins (Tursiops truncatus) in Barataria Bay, Louisiana, Following the Deepwater Horizon Oil Spill

The oil spill resulting from the explosion of the Deepwater Horizon drilling platform initiated immediate concern for marine wildlife, including common bottlenose dolphins in sensitive coastal habitats. To evaluate potential sublethal effects on dolphins, health assessments were conducted in Barataria Bay, Louisiana, an area that received heavy and prolonged oiling, and in a reference site, Sarasota Bay, Florida, where oil was not observed. Dolphins were temporarily captured, received a veterinary examination, and were then released. Dolphins sampled in Barataria Bay showed evidence of hypoadrenocorticism, consistent with adrenal toxicity as previously reported for laboratory mammals exposed to oil. Barataria Bay dolphins were 5 times more likely to have moderate-severe lung disease, generally characterized by significant alveolar interstitial syndrome, lung masses, and pulmonary consolidation. Of 29 dolphins evaluated from Barataria Bay, 48% were given a guarded or worse prognosis, and 17% were considered poor or grave, indicating that they were not expected to survive. Disease conditions in Barataria Bay dolphins were significantly greater in prevalence and severity than those in Sarasota Bay dolphins, as well as those previously reported in other wild dolphin populations. Many disease conditions observed in Barataria Bay dolphins are uncommon but consistent with petroleum hydrocarbon exposure and toxicity.

Hepatic xenobiotic metabolizing enzymes in two species of benthic fish showing different prevalences of contaminant-associated liver neoplasms

English sole (Parophrys vetulus) and starry flounder (Platichthys stellatus) are closely related benthic fish which show substantial differences in prevalences of contaminant-associated hepatic neoplasms and putatively preneoplastic foci of cellular alteration when captured from estuaries containing a variety of organic chemical contaminants, including polycyclic aromatic hydrocarbons (PAH) and chlorinated hydrocarbons. Because PAH are strongly implicated as causative agents in the etiology of these lesions, several of the hepatic enzymes involved in activation and detoxication of PAH were studied in these two species. Hepatic aryl hydrocarbon hydroxylase (AHH), epoxide hydrolase (EH), and glutathione S-transferase (GST) activities were measured in animals sampled from both contaminated and reference areas. English sole, the species showing higher prevalences of contaminant-associated hepatic lesions, had higher (1- to 2-fold) hepatic activities of AHH and lower activities of EH (0.8-fold) and GST (1.8-fold) than those of starry flounder, regardless of site of capture. These results are largely consistent with increased activation and decreased detoxication of PAH by English sole in comparison to starry flounder. Both laboratory and field data suggested that the observed species differences in enzyme activities were constitutive and not related to differential exposure to contaminants. There were also substantial differences between these species with respect to expression of GST isoenzymes, in that starry flounder expressed two highly anionic GST isoenzymes which did not correspond to any GST isoenzymes expressed in English sole liver; a previous study in an elasmobranch fish showed that an anionic GST was most active toward PAH oxides. These differences in enzyme activities and isoenzyme profiles suggest a toxicological basis which may help to explain, at least in part, the differences in prevalences of contaminant-associated liver neoplasms between these two species.

Molecular epizootiology of genotoxic events in marine fish : Linking contaminant exposure, DNA damage, and tissue-level alterations

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.