Bernadita F. Anulacion

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.

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.

Geologically distinct crude oils cause a common cardiotoxicity syndrome in developing zebrafish.

Crude oils from different geological formations vary in composition, yet most crude oils contain a polycyclic aromatic hydrocarbon (PAH) fraction that would be expected to produce cardiotoxic effects in developing fish. To determine whether different crude oils or PAH compositions produce common or distinct effects, we used zebrafish embryos to directly compare two crude oils at different states of weathering. Iranian heavy crude oil (IHCO) spilled in the Yellow Sea following the 2007 Hebei Spirit accident was compared to the intensively studied Alaska North Slope crude oil (ANSCO) using two different exposure methods, water-accommodated fractions containing dispersed oil microdroplets and oiled gravel effluent. Overall, both crude oils produced a largely overlapping suite of defects, marked by the well-known effects of PAH exposure on cardiac function. Specific cardiotoxicity phenotypes were nearly identical between the two oils, including impacts on ventricular contractility and looping of the cardiac chambers. However, with increased weathering, tissue-specific patterns of aryl hydrocarbon receptor (AHR) activation in the heart changed, with myocardial AHR activation evident when alkyl-PAHs dominated the mixture. Our findings suggest that mechanisms of cardiotoxicity may shift from a predominantly AHR-independent mode during early weathering to a multiple pathway or synergistic mode with prolonged weathering and increased proportions of dissolved alkyl-PAHs. Despite continued need for comparisons of crude oils from different sources, the results here indicate that the body of knowledge already acquired from studies of ANSCO is directly relevant to understanding the impacts of other crude oil spills on the early life history stages of fish.

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.

Potent Phototoxicity of Marine Bunker Oil to Translucent Herring Embryos after Prolonged Weathering

Pacific herring embryos (Clupea pallasi) spawned three months following the Cosco Busan bunker oil spill in San Francisco Bay showed high rates of late embryonic mortality in the intertidal zone at oiled sites. Dead embryos developed to the hatching stage (e.g. fully pigmented eyes) before suffering extensive tissue deterioration. In contrast, embryos incubated subtidally at oiled sites showed evidence of sublethal oil exposure (petroleum-induced cardiac toxicity) with very low rates of mortality. These field findings suggested an enhancement of oil toxicity through an interaction between oil and another environmental stressor in the intertidal zone, such as higher levels of sunlight-derived ultraviolet (UV) radiation. We tested this hypothesis by exposing herring embryos to both trace levels of weathered Cosco Busan bunker oil and sunlight, with and without protection from UV radiation. Cosco Busan oil and UV co-exposure were both necessary and sufficient to induce an acutely lethal necrotic syndrome in hatching stage embryos that closely mimicked the condition of dead embryos sampled from oiled sites. Tissue levels of known phototoxic polycyclic aromatic compounds were too low to explain the observed degree of phototoxicity, indicating the presence of other unidentified or unmeasured phototoxic compounds derived from bunker oil. These findings provide a parsimonious explanation for the unexpectedly high losses of intertidal herring spawn following the Cosco Busan spill. The chemical composition and associated toxicity of bunker oils should be more thoroughly evaluated to better understand and anticipate the ecological impacts of vessel-derived spills associated with an expanding global transportation network.

Zebrafish and clean water technology: Assessing soil bioretention as a protective treatment for toxic urban runoff

Urban stormwater contains a complex mixture of contaminants that can be acutely toxic to aquatic biota. Green stormwater infrastructure (GSI) is a set of evolving technologies intended to reduce impacts on natural systems by slowing and filtering runoff. The extent to which GSI methods work as intended is usually assessed in terms of water quantity (hydrology) and quality (chemistry). Biological indicators of GSI effectiveness have received less attention, despite an overarching goal of protecting the health of aquatic species. Here we use the zebrafish (Danio rerio) experimental model to evaluate bioinfiltration as a relatively inexpensive technology for treating runoff from an urban highway with dense motor vehicle traffic. Zebrafish embryos exposed to untreated runoff (48-96h; six storm events) displayed an array of developmental abnormalities, including delayed hatching, reduced growth, pericardial edema, microphthalmia (small eyes), and reduced swim bladder inflation. Three of the six storms were acutely lethal, and sublethal toxicity was evident across all storms, even when stormwater was diluted by as much as 95% in clean water. As anticipated from exposure to cardiotoxic polycyclic aromatic hydrocarbons (PAHs), untreated runoff also caused heart failure, as indicated by circulatory stasis, pericardial edema, and looping defects. Bioretention treatment dramatically improved stormwater quality and reversed nearly all forms of developmental toxicity. The zebrafish model therefore provides a versatile experimental platform for rapidly assessing GSI effectiveness.

4 - Effects on Fish of Polycyclic Aromatic HydrocarbonS (PAHS) and Naphthenic Acid Exposures

Polycyclic aromatic hydrocarbons (PAHs) are derived from both natural and anthropogenic sources and are released from a wide range of industries and everyday activities. Unlike many other organic chemical contaminants that are manufactured and regulated, PAHs continue to be released on a global scale because of the worlds dependence on fossil fuels. This chapter briefly reviews the transformation of PAHs in the aquatic environment, highlighting their efficient metabolism in fish and focuses on evidence that links PAH exposure to a wide range of biological dysfunctions in fish. These dysfunctions include neoplasia, reduced reproductive success and other types of endocrine disruption, immunotoxicity, postlarval growth and somatic condition, transgenerational impacts, and finally, recent findings showing that the embryonic development of fish is severely affected by extremely low concentrations of PAH exposure. A brief review of the effects of naphthenic acids on fish is also included because these compounds are increasingly recognized as major factors in the toxicity of process waters from a variety of petroleum sources, most notably the immense oil sands deposits found in Alberta, Canada. It is recommended that future research for understanding and mitigating the effects of PAHs in fish and associated aquatic ecosytems should include the following. • Using models to link molecular-up-to-organismal level effects to population-relevant metrics. • Building on current case studies demonstrating the effects of PAHs on the health of fish in their natural environments in order to derive regulatory approaches. Current approaches that rely on biota to sediment accumulation factors (BSAF) will not work with contaminants that are efficiently metabolized by species of concern. • Focusing considerable resources on better analytical chemistry for both PAHs and naphthenic acids. Currently, our ability to understand and mitigate the effects of these substances is heavily limited by constraints in analysis.

Exposure of juvenile chinook and chum salmon to chemical contaminants in the Hylebos Waterway of Commencement Bay, Tacoma, Washington

The Hylebos Waterway is an industrialized waterway ofCommencement Bay, Tacoma, Washington, that is severelycontaminated with aromatic and chlorinatedhydrocarbons in the sediment. Juvenile chinook (Oncorhynchus keta) and chum salmon (O.tshawytscha) inhabit this waterway for a few days orweeks during their outmigration from freshwaterstreams to saltwater. The purpose of thisinvestigation was to determine to what degree juvenilechum and chinook salmon captured from the HylebosWaterway might bioaccumulate organic contaminants. These levels of exposure will be compared to previousstudies where such exposures have been linked tobiological dysfunction in juvenile salmon. Theresults showed that juvenile chum and chinook salmonfrom the Hylebos Waterway take up a wide range ofchemical contaminants, compared to fish fromhatcheries or reference estuaries. These contaminantsinclude high and low molecular weight polycyclicaromatic hydrocarbons (PAHs), polychlorinatedbiphenyls (PCBs, including the toxic congeners 105 and118), hexachlorobutadiene (HCBD), hexachlorobenzene(HCB), DDTs, heptachlor, and several pesticides. Immunohistochemical examination of the gill and gut injuvenile chum salmon from the Hylebos Waterway showedthe induction of the P450 metabolizing enzyme. Moreover, concentrations of contaminants in juvenilechinook and chum salmon from the Hylebos Waterway arecomparable to levels previously shown to be associatedwith biological injury in juvenile chinook salmon,such as impaired growth, suppression of immunefunction as demonstrated by reduced B cell function,and increased mortality following pathogen exposure.

Persistent Organic Pollutants in Juvenile Chinook Salmon in the Columbia River Basin: Implications for Stock Recovery

Abstract Among the populations of Pacific salmon and steelhead Oncorhynchus mykiss (anadromous Rainbow Trout) that inhabit the Columbia River basin there are currently 13 Evolutionarily Significant Units listed as threatened or endangered under the U.S. Endangered Species Act. While habitat loss, dams, overharvest, and climate change have been implicated in declining abundance of Chinook Salmon O. tshawytscha in the Columbia River, chemical contaminants represent an additional, yet poorly understood, conservation threat. In this study we measured concentrations of persistent organic pollutants in juvenile Chinook Salmon from various Columbia River stocks and life history types to evaluate the potential for adverse effects in these threatened and endangered fish. Polychlorinated biphenyls (PCBs) and dichlorodiphenyltrichloroethane (DDTs), recognized contaminants of concern in the Columbia basin, are the primary focus of this paper; other contaminants found in these fish, such as polybrominated diphenyl eth...

Hepatic CYP1A in winter flounder (Pleuronectes americanus) along the northeast coast : Results from the National Benthic Surveillance project

Abstract As part of the National Benthic Surveillance Project, cytochrome P4501A (CYP1A) and associated enzyme activities were measured in more than 700 samples of liver tissue collected from adult non-spawning winter flounder (Pleuronectes americanus). Between 1988 and 1994, animals were sampled annually from 20 sites, ranging from Penobscot Bay in Maine to Great Bay, New Jersey. Analyses performed were assays of aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufinO-deethylase (EROD) activities, and immunoquantitation of CYP1A by enzyme-linked immunosorbent assay (ELISA). From 1988 through 1990, CYP1A was measured by all three methods, to help determine an appropriate method for assessing temporal trends and to allow for eventual comparisons to methods used in other monitoring efforts. From 1991 through 1994, assays were only done for hepatic AHH activity. Fish from virtually all sites showed induction of hepatic AHH activity, consistent with earlier reports, though fish sampled from sites in the coastal waters of Maine generally showed the lowest mean AHH activities. At sites where fish were sampled during three to six separate years (15 of the 20), individual data were analyzed for monotonic temporal trends. There were trends towards increasing AHH activities over time in fish from the Raritan Bay/Long Island Sound area, and generally increasing trends for sites near Massachusetts (especially Boston Harbor) and Rhode Island. A decreasing trend was noted in fish from Great Bay, New Jersey, and there appeared to be decreasing activities in fish from the nearshore waters of Maine, though these trends were not statistically significant at the α = 0.05 level. The induction of CYP1A is strongly associated with exposure to chemical contaminants, and while the consequences of widespread and increasing induction of CYP1A are not known, these results suggest that the measurement of this enzyme system in benthic fish can be a useful tool for monitoring our coastal ecosystems.