Lyndal L. Johnson

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.

Effects of the synthetic estrogen, 17α-ethinylestradiol, on aggression and courtship behavior in male zebrafish (Danio rerio)

The synthetic estrogen, 17alpha-ethinylestradiol (EE(2)), is the active component in oral contraceptive pills. It is excreted from the human body in high amounts and released via sewage treatment plant effluent into aquatic environments. In fish, estrogen receptors have strong binding affinities for EE(2), and exposure raises the possibility of adverse neuroendocrine responses in aquatic animals. In the present study we explored the effects of dissolved-phase EE(2) on the dynamics of male-male aggression and courtship behaviors in adult zebrafish. Further, we assessed whether the behavioral effects of EE(2) result in changes in male offspring paternity. We scored the aggressive behaviors of individual unexposed males and categorized these fish as either dominant or subordinate. We then exposed dominant males to EE(2) at doses of 0, 0.5, 5.0, and 50.0ng/L for 48h. Subsequent trials examined the agonistic behaviors of males in two testing scenarios: (1) a dyadic encounter with another male alone, and (2) a competitive spawning interaction with another male and three adult females. Competitive spawning tests were also used to assess the impacts of EE(2) exposure on courtship behavior and paternity using males that were homozygous for green fluorescent protein (GFP) expression under the control of the islet-1 promoter. We found that EE(2) at all exposure concentrations reduced male aggression during male-male dyadic encounters and caused a social dominance reversal in 50% of the fish at the highest exposure dose (50ng/L EE(2)). The frequency of courtship-specific behavior decreased in dominant males exposed to the steroid, though this effect was only significant for the lowest dose group (0.5ng/L EE(2)). In the highest exposure group (50ng/L EE(2)), 50% of dominant males relinquished paternal dominance. Our results show that short-term exposure to EE(2) at environmentally relevant levels can alter aggression, and shift individual social status and reproductive success in male zebrafish.

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.

Inducibility of spawning and reproductive success of female english sole (parophrys vetulus) from urban and nonurban areas of puget sound, Washington

Abstract 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.

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.

Disease susceptibility of salmon exposed to polybrominated diphenyl ethers (PBDEs)

The health effects of the flame retardant polybrominated diphenyl ethers (PBDEs) in fish are not well understood. To determine the potential effects of this ubiquitous contaminant class on fish health, juvenile subyearling Chinook salmon (Oncorhynchus tshawytscha) were fed a diet that reflected the PBDE congeners found in the stomach contents of subyearling Chinook salmon collected from the highly urbanized and industrialized lower Willamette River in the Columbia River Basin of North America. The diet, consisting of five PBDE congeners (BDE-47, BDE-99, BDE-100, BDE-153 and BDE-154), was fed to the salmon at 2% of their body weight in food per day for 40 days. Two concentrations of the diet (1x and 10x PBDE) were fed to the salmon. The 1x PBDE diet reflected the concentration of PBDEs (190 ng PBDEs/g food) found in the stomach contents of juvenile subyearling Chinook salmon; the 10x diet was prepared at 10 times that concentration. The fish were then exposed to the marine bacterial pathogen Listonella anguillarum to assess susceptibility to infectious disease. Juvenile Chinook salmon fed the 1x PBDE diet were more susceptible to L. anguillarum than salmon fed the control diet. This suggests that juvenile salmonids in the lower Willamette River exposed to PBDEs may be at greater risk for disease than nonexposed juvenile salmonids. In contrast, salmon that consumed the 10x PBDE diet were not more susceptible to the pathogen than salmon fed the control diet. The mechanisms for the dichotomous results observed in disease susceptibility between salmon fed the 1x and 10x PBDE diets are currently not known but have also been observed in other species exposed to PBDEs with respect to immune function.

Xenoestrogen exposure and effects in English sole (Parophrys vetulus) from Puget Sound, WA

Vitellogenin, a yolk protein produced in the liver of oviparous animals in response to estrogens, normally occurs only in sexually mature females with developing eggs. However, males can synthesize vitellogenin when exposed to environmental estrogens, making the abnormal production of vitellogenin in male animals a useful biomarker for xenoestrogen exposure. In 1997-2001, as part of the Washington States Puget Sound Assessment and Monitoring Program, we surveyed English sole from a number of sites for evidence of xenoestrogen exposure, using vitellogenin production in males as an indicator. Significant levels of vitellogenin were found in male fish from several urban sites, with especially high numbers of fish affected in Elliott Bay, along the Seattle Waterfront. Intersex fish were rare, comprising only two fish out of more than 2900 examined. Other ovarian and testicular lesions, including oocyte atresia, were also observed, but their prevalence did not appear to be related to xenoestrogen exposure. However, at the Elliott Bay sites where abnormal vitellogenin production was observed in male sole, the timing of spawning in both male and female English sole appeared altered. Sources of xenoestrogens and types of xenoestrogens present in Elliott Bay are poorly documented, but the compounds are likely associated with industrial discharges, surface runoff, and combined sewer outfalls.