Mark S. Myers

Determination of metabolites of xenobiotics in the bile of fish from polluted waterways

An h.p.l.c.-fluorescence technique was used to estimate relative concentrations of metabolites of xenobiotics in bile of 103 English sole (Parophrys vetulus) from both polluted and minimally polluted (reference) sites in Puget Sound, WA. Fish from polluted sites had concentrations of xenobiotics in bile with naphthalene-, phenanthrene- and benzo[a]pyrene-like fluorescence that averaged 9, 14 and 19 times, respectively, those of fish from reference sites. Within a polluted site, fish with liver lesions had significantly higher bile concentrations of xenobiotics with benzo[a]pyrene-like fluorescence than did fish without liver lesions. Individual metabolites of fluorene, phenanthrene, anthracene, biphenyl and dimethylnaphthalene were determined by g.l.c.-mass spectrometry in extracts of hydrolysed bile of three English sole from polluted waterways; concentrations ranged from 90 to 19000 ng/g, wet wt. Other xenobiotics were tentatively identified, but not quantified.

Neoplastic and other diseases in fish in relation to toxic chemicals: an overview☆

Field studies were conducted over a 5-year period in Puget Sound to investigate etiological relationships between prevalences of hepatic neoplasms in bottom-dwelling marine fish, with emphasis on English sole (Parophrys vetulus), and concentrations of toxic chemicals in sediments and bottom fish. Statistically significant correlations were found between prevalences of hepatic neoplasms and (1) sediment concentrations of aromatic hydrocarbons, and (2) concentrations of metabolites of aromatic compounds in the bile. A significant difference was also found between the concentrations of N-oxyl derivatives of nitrogen heterocycles (free radicals) in liver microsomes of English sole with liver lesions compared to sole without liver lesions. These N-oxyl free radicals were apparently metabolically derived from complex suites of nitrogen heterocycles present in Puget Sound environments. Recent evidence suggests that aromatic free radicals (possibly derivatives of aromatic hydrocarbons) may also be bound to hepatic DNA isolated from English sole with liver neoplasms collected in several polluted areas of Puget Sound. Such evidence for xenobiotic-DNA interactions has thus far not been obtained for fish from reference areas and for lesion-free fish from polluted areas. Various laboratory studies designed to evaluate the etiology of the liver neoplasms have also yielded evidence that is consistent with the view that high molecular weight aromatic hydrocarbons play a significant role in the etiology of hepatic tumors in the bottom-dwelling fish. Associations between chemical exposures and diseases in other marine species from southern California and Boston Harbor are also discussed. Similar relationships between liver and skin neoplasms in selected bottom feeding fresh water species and sediment-associated chemicals are also presented.

Overview of studies on liver carcinogenesis in English sole from Puget Sound; evidence for a xenobiotic chemical etiology I: Pathology and epizootiology

Livers of wild English sole (Parophrys vetulus) from polluted waterways and embayments of Puget Sound, Washington, are affected by a spectrum of multiple, co-occurring idiopathic hepatic lesions, including neoplasms, putative preneoplastic foci of cellular alteration, and unique degeneration conditions. Results from a statistical analysis of the patterns of co-occurrence of these lesions in wild English sole indicate that these lesions represent morphologically identifiable steps leading to the development of hepatic neoplasms. This sequence parallels the lesion progression in experimental models of chemically induced liver carcinogenesis in rodents. The hypothesis that these lesions in wild English sole can be caused by exposure to certain xenobiotic hepatotoxic and hepatocarcinogenic compounds in Puget Sound is based on: a) statistical associations between levels of aromatic hydrocarbons (sigma AHs) in sediment and prevalences of these idiopathic liver lesions, b) the contribution of sigma AHs in accounting for the variability in hepatic neoplasm prevalence in a logistic regression model, c) elevated odds ratios for several idiopathic hepatic lesion types in sole from polluted sites in Puget Sound, d) significant correlations between prevalences of idiopathic hepatic lesions and levels of fluorescent metabolites of aromatic compounds (FACs) in bile of English sole, and e) experimental induction of putatively preneoplastic focal lesions in English sole injected with a PAH-enriched fraction of an extract from a contaminated urban sediment from Puget Sound, that were morphologically identical to lesions found in wild English sole from the same site.

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.

Suppression of B-cell mediated immunity in juvenile chinook salmon (Oncorhynchus tshawytscha) after exposure to either a polycyclic aromatic hydrocarbon or to polychlorinated biphenyls

Juvenile chinook salmon (Oncorhynchus tshawytscha) were injected intraperitoneally with either the polycyclic aromatic hydrocarbon, 7,12-dimethylbenz[a]anthracene (DMBA)1 or with the commercial polychlorinated biphenyl (PCB) mixture, Aroclor 1254, to assess effects on the B-cell mediated immune response. B-cell mediated immunity was assessed by examination of the primary and secondary plaque-forming cell (PFC) responses of anterior kidney and splenic leukocytes to a T-independent antigen, TNP-keyhole limpet hemocyanin (TNP-KLH). Salmon exposed to DMBA at dosages of 20% or 1% of the 96 hr LD50 (12.7 mg and 0.6 mg/kg of salmon, respectively) or to PCBs at a dosage of 20% of the 96 hr LD50 (54.0 mg/kg of salmon) exhibited a suppressed PFC response. The secondary PFC response of anterior kidney and splenic leukocytes to both antigens and the primary splenic PFC response to TNP-LPS were suppressed in salmon exposed to either DMBA or PCBs. However, only the primary PFC response of anterior kidney leukocytes to TNP-LPS was suppressed in salmon exposed to PCBs and no suppression of this response was observed in salmon exposed to DMBA. Neither anterior kidney or splenic leukocytes from salmon exposed to DMBA or PCBs showed an altered primary PFC response to the T-dependent antigen, TNP-KLH. These results suggest that B-cell mediated immunity in salmon is suppressed by known mammalian immunosuppressants and that suppression of the PFC response observed previously in salmon from an urban estuary may be due to contaminant exposure.

Establishing the Causal Relationship between Polycyclic Aromatic Hydrocarbon (PAH) Exposure and Hepatic Neoplasms and Neoplasia-Related Liver Lesions in English Sole (Pleuronectes vetulus)

For almost 25 years our laboratory has studied the impact of PAHs and related industrial contaminants on benthic fish, following an interdisciplinary approach involving chemical exposure assessment linked to synoptic detection of various effects at several levels of biological organization. These data demonstrate a cause-and-effect relationship between neoplastic and neoplasia-related liver lesions in English sole, and exposure to PAHs, and to a lesser degree, chlorinated hydrocarbons such as PCBs. In statistical analyses of data from multiple field studies conducted since 1978, exposure to PAHs measured in various compartments has consistently been identified as a highly significant, major risk factor for neoplasms and related lesions in this species, with PCB exposure shown to be a significant, but less consistent and less strong risk factor for these lesions. A cause-and-effect relationship between PAHs and toxicopathic liver lesions in this species is further supported by the experimental induction of toxicopathic lesions identical to those observed in field-collected fish, in sole exposed in the laboratory to model carcinogenic PAHs such as BaP or to PAH-rich extracts of sediments from Eagle Harbor, a severely PAH-contaminated site in Puget Sound. More recent field studies have identified significant associations between hepatic cytochrome P4501A (CYP1A) induction and xenobiotic-DNA adduct formation, and hepatic lesion prevalences in wild subadult English sole. Field studies in Eagle Harbor subsequent to capping of the most PAH-contaminated region of this harbor with clean dredge spoils have shown a decline in exposure to PAHs as assessed by biliary fluorescent aromatic compounds (FACs) and hepatic xenobiotic-DNA adducts. This decline in PAH exposure has been accompanied by a dramatic decline in risk of occurrence of toxicopathic hepatic lesions in English sole from Eagle Harbor. Further, laboratory studies have induced lesions in English sole by injections of extracts from PAHcontaminated sediments. Overall, these findings relating to exposure to PAHs and chlorinated hydrocarbons and the occurrence of hepatic neoplasms and neoplasiarelated lesions in English sole fulfill the classic criteria for causality in epizootiological or ecological risk assessment studies, including: (1) strength of association, (2) consistency of association, (3) specificity of association, (4) toxicological and biological plausibility, (5) temporal sequence/timing (i.e., exposure precedes disease, effect decreases when the cause is decreased or removed), (6) dose-response or biological gradient, and (7) supportive experimental evidence.

Toxicopathic hepatic lesions as biomarkers of chemical contaminant exposure and effects in marine bottomfish species from the northeast and Pacific coasts, USA

Abstract Relationships between toxicopathic hepatic lesions and chemical contaminants in sediments, stomach contents, liver and bile were evaluated in English sole, starry flounder and white croaker from 27 sites on the Pacific Coast, and winter flounder from 22 sites on the Northeast Coast of the USA, as part of the NOAAs National Benthic Surveillance Program (NBSP). Prevalences of and relative risks for most toxicopathic lesions were significantly higher in fish from contaminated sites in Puget Sound, the Los Angeles area, and San Francisco and San Diego Bays on the Pacific Coast, and in Boston Harbor, Raritan Bay and certain urban sites in Long Island Sound on the Northeast Coast. Exposure to polycyclic aromatic hydrocarbons (PAHs), PCBs, DDTs, chlordanes and dieldrin were significant risk factors for all lesion types in Pacific Coast species. In winter flounder from the Northeast Coast, exposure to PAHs, DDTs or chlordanes were significant risk factors only for hydropic vacuolation, nonneoplastic proliferative and nonspecific necrotic lesions, and less commonly for neoplasms and foci of cellular alteration. Risk of hepatic disease generally increased with fish age, but sex was rarely a risk factor. Temporal trends analyses of hepatic lesion prevalences in starry flounder, white croaker and English sole from NBSP sites on the Pacific Coast failed to detect any significant monotonic increases or decreases in lesion prevalence. Recent studies utilized a two-segment ‘hockey-stick’ regression technique applied to NBSP data to determine threshold levels of sediment PAHs, which are clearly associated with toxicopathic hepatic lesions in English sole. Significant chemical threshold levels for these lesions are in the vicinity of 500–1000 ppb ΣPAHs in sediment, values considerably lower than those reported for other techniques. Application of this dose-response model to these subacute and chronic lesions involved in hepatocarcinogenesis, provides nonlethal sediment quality assessment endpoints for contaminant concentrations that may have long term health implications for chronically exposed native fish populations. Overall, these relationships provide strong evidence for environmental contaminants as etiologic agents for hepatic lesions in several marine bottomfish species, and clearly indicate their utility as biomarkers of contaminant-induced effects in wild fish, whether in national and regional biomonitoring programs or within the injury assessment phase of the legal process of assessing damage to fishery resources.

Unexpectedly high mortality in Pacific herring embryos exposed to the 2007 Cosco Busan oil spill in San Francisco Bay

In November 2007, the container ship Cosco Busan released 54,000 gallons of bunker fuel oil into San Francisco Bay. The accident oiled shoreline near spawning habitats for the largest population of Pacific herring on the west coast of the continental United States. We assessed the health and viability of herring embryos from oiled and unoiled locations that were either deposited by natural spawning or incubated in subtidal cages. Three months after the spill, caged embryos at oiled sites showed sublethal cardiac toxicity, as expected from exposure to oil-derived polycyclic aromatic compounds (PACs). By contrast, embryos from the adjacent and shallower intertidal zone showed unexpectedly high rates of tissue necrosis and lethality unrelated to cardiotoxicity. No toxicity was observed in embryos from unoiled sites. Patterns of PACs at oiled sites were consistent with oil exposure against a background of urban sources, although tissue concentrations were lower than expected to cause lethality. Embryos sampled 2 y later from oiled sites showed modest sublethal cardiotoxicity but no elevated necrosis or mortality. Bunker oil contains the chemically uncharacterized remains of crude oil refinement, and one or more of these unidentified chemicals likely interacted with natural sunlight in the intertidal zone to kill herring embryos. This reveals an important discrepancy between the resolving power of current forensic analytical chemistry and biological responses of keystone ecological species in oiled habitats. Nevertheless, we successfully delineated the biological impacts of an oil spill in an urbanized coastal estuary with an overlapping backdrop of atmospheric, vessel, and land-based sources of PAC pollution.

Assessing the effects of anthropogenic stressors on Puget Sound flatfish populations

Puget Sound is an estuary in the northwestern United States which serves as the habitat for a number of recreationally and commercially important species of flatfish. Over the past 100 years, there has been substantial urban and industrial development within this region, resulting in heavy inputs of chemical contaminants at selected sites, as well as significant loss or alteration of marine habitat. Studies show that feral flatfish in Puget Sound are experiencing a range of biological effects due to chemical contaminant exposure, including reproductive dysfunction, altered immune competence, and development of toxicopathic diseases, and there is some evidence of reduced survival in fish from urban areas of Puget Sound from increased infectious and toxicopathic disease. Puget Sound sole are also subject to other anthropogenic stressors, such as fishing pressure or alteration of nearshore nursery habitats. The cumulative impact of these stressors on flatfish abundance in Puget Sound, however, is poorly understood. In a series of field and laboratory studies, we determined vital rates and other life history parameters in English sole (Pleuronectes vetulus) subpopulations from urban and non-urban sites in Puget Sound, and are using this information to estimate potential population level impacts of anthropogenic stressors, with age and stage-based Leslie-matrix models. Initial results suggest that declines in the fecundity component of the model, as observed in field studies of fish from contaminated sites, could reduce the size of sub-populations in these areas if the loss of recruits is not offset by density-dependent changes in recruitment, immigration, or other compensating mechanisms. Studies on flatfish species from a variety of sites in Europe and North America suggest that contaminant-related disease and reproductive impairment are widespread in this group of fish, although substantial differences in sensitivity have been observed, even among closely related species. Comparative studies with a variety of Pleuronectid species will enable us to better evaluate the risk posed by anthropogenic stressors to flatfish, and contribute to improved assessment and management of this important fisheries resource.

Toxicopathic liver lesions in English sole and chemical contaminant exposure in Vancouver Harbour, Canada

The prevalence of toxicopathic liver lesions in English sole (Pleuronectes vetulus) was determined along a presumed gradient of chemical contamination in Vancouver Harbour, Canada. Fish were captured from five sites in or near Vancouver Harbour, British Columbia, Canada. No toxicopathic lesions were observed in fish examined at the reference site (Howe Sound outside Vancouver Harbour), or at the outer harbour site. In contrast, 20-23% of the fish from three sites located in the central harbour, Indian Arm and Port Moody Arm had one or more types of toxicopathic lesions. Likewise, aromatic hydrocarbon (AH) metabolites measured in bile exhibited a gradient in levels from lower concentrations at the reference site to significantly higher levels in fish from Indian Arm and Port Moody Arm harbour sites. The occurrence of toxicopathic liver lesions was statistically associated with concentrations of AHs measured in sediment and AH metabolite levels measured in bile.