John E. Stein

Bioavailability and biotransformation of aromatic hydrocarbons in benthic organisms exposed to sediment from an urban estuary

Phylogenetically diverse benthic organisms [amphipods (Rhepoxynius abronius and Eohaustqrius washingtonianus); clams (Macoma nasuta); shrimp (Pandalus platyceros); fish (Parophrys vetulus)] were exposed to an urban estuarine sediment [ 16 ppm of two to six benzenoid ring aromatic hydrocarbons (AHs)] to which trace amounts of [3H]benzo[a]pyrene (BaP) were added. The techniques used to assess uptake and metabolism of AHs were gas chromatography/mass spectrometry (GC/MS) for AHs, high-pressure liquid chromatography/fluorescence spectrometry for AH metabolites in fish bile, and radiometric analyses for biotransformation of [3H]BaP. Generally, the extent of metabolism of [3H]BaP (M. nasuta < E. washingtonianus < R. abronius I P. platyceros N P. vetulus) was negatively correlated to tissue concentrations of AHs (three to six ring), except that amphipod species accumulated higher concentrations of AHs than did clams, indicating that other factors (e.g., feeding strategy and rate of excretion) also influenced accumulation of AHs. Radiometric and GC analyses for BaP in both sediment and tissues suggested that not all of the BaP (and presumably other AHs) extracted chemically from sediment was bioavailable.

Effect of Pollution on Fish Diseases: Potential Impacts on Salmonid Populations

Abstract Anthropogenic factors have contributed to the precipitous decline of wild Pacific salmon stocks, although the mechanisms and processes at work are largely unknown. Pollution may be one of these factors. Sediments in estuaries are known to act as repositories for contaminants, and estuaries are important habitats for ocean- and river-migrating salmon. We have shown that juvenile salmon Oncorhynchus spp. and their prey bioaccumulate chlorinated hydrocarbons and aromatic hydrocarbons—important classes of toxic xenobiotics. Furthermore, we have shown that exposure to these pollutants can lead to immunosuppression and increased disease susceptibility in juvenile salmon. Whether pollution influences natural disease outbreaks in host populations, including salmon, is currently unknown. It is postulated that the occurrence of disease depends on the interaction of the host, the environment, and the pathogen. Absence of pathogens would reduce the potential for adverse environments to influence disease outb...

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.

Simultaneous exposure of English sole (Parophrys vetulus) to sediment-associated xenobiotics: Part 1—uptake and disposition of 14C-polychlorinated biphenyls and 3H-benzo[a]pyrene

Abstract English sole ( Parophrys vetulus ) were exposed to environmentally realistic levels of sediment-associated 3 H-benzo[a]pyrene (3 μg BaP/g sediment, dry weight) and 14 C-Aroclor 1254 (1 μg PCBs/g), separately and together, for up to 10 days. BaP and its metabolites in tissues reached steady-state concentrations by the first day of the exposure, whereas PCBs did not approach steady-state concentrations until the tenth day of exposure. Simultaneous exposure of sole to BaP and PCBs, relative to separate exposure to the xenobiotics, significantly increased the concentrations of BaP-derived radioactivity and decreased the concentrations of PCB-derived radioactivity in some tissues and bile. Accumulation of PCB-derived radioactivity, estimated as the burden in tissues, was significantly greater (4- to 13-fold) than that of BaP-derived radioactivity throughout the experiments and regardless of the type of exposure. The rank of the concentrations of PCB-derived radioactivity in tissues and bile was: bile ∼ liver > brain > kidney ∼ gill > skin ∼ blood ∼ muscle and for BaP-derived radioactivity the order was: bile > liver > gill > kidney > skin ∼ blood > muscle > brain. BaP-derived radioactivity in liver and bile was present primary (85–99%) as metabolites, whereas PCB-derived radioactivity was present equally as parent compounds and metabolites in bile and primarily (98 %) as parent compounds in liver. Hydrolysis of bile from PCB/BaP-exposed sole with β-glucuronidase/arylsulfatase released 35 % of the BaP-derived radioactivity and 32 % of the PCB-derived radioactivity as primary metabolites. A much higher proportion of the BaP-derived radioactivity (64 %) than the PCB-derived radioactivity (13 %) in bile was unaffected by the enzyme treatment, indicating differences in the conjugation of the primary metabolites of PCBs and BaP. The results suggest that in contaminated environments the tissue to sediment concentration ratios for PCBs would exceed that for BaP and its metabolites; however, BaP would be continually absorbed and metabolized by sole to potentially carcinogenic and mutagenic compounds. In addition, the tissue levels of these toxic compounds may be increased by simultaneous exposure to PCBs.

Xenobiotic metabolizing enzymes in spawning English sole (Parophrys vetulus) exposed to organic-solvent extracts of marine sediments from contaminated and reference areas

Female English sole (Parophrys vetulus) within 1-2 days of spawning were exposed by i.m. injection to organic-solvent extracts of marine sediments at the following doses: Eagle Harbor (EHSE, contaminated site)--6.8 mg aromatic hydrocarbons (AHs)/kg body wt; Duwamish Waterway (DSE, contaminated site)--0.52 mg AHs and 0.040 mg chlorinated hydrocarbons (CHs)/kg body wt; Hood Canal (HCSE, reference site)--0.00090 mg AHs/kg body wt. Hepatic aryl hydrocarbon hydroxylase (AHH) activity, measured at spawning, was induced 10-, 23-and 2-fold by EHSE, DSE and HCSE, respectively, compared to sham and vehicle controls. Hepatic glutathione-S-transferase and epoxide hydrolase activities were not affected by any treatment. EHSE, but not DSE or HCSE, inhibited spawning (P less than 0.01) in 36% of the exposed fish and hepatic AHH activity in the non-spawning fish was significantly (P less than 0.05) higher than in the fish that did spawn. These results suggest a potential for reproductive toxicity in benthic fish after exposure to sediment-associated contaminants.

Simultaneous exposure of English sole (Parophrys vetulus) to sediment-associated xenobiotics: Part 2—chronic exposure to an urban estuarine sediment with added 3H-benzo[a]pyrene and 14C-polychlorinated biphenyls☆

Abstract 1. 1. The accumulation of aromatic hydrocarbons (AHs) and polychlorinated biphenyls (PCBs) kv English sole (Parophrys vetulus) exposed for up to 108 days to an urban (test) sediment was compared to that for English sole exposed to sediment from a reference area. The concentrations of identified AHs and PCBs in the test sediment were 32 μg/g dry weight and 2·2 μg/g dry weight, respectively. English sole exposed to test and reference sediments for 108 days had biliary concentrations of aromatic compounds fluorescing at BaP wavelengths of 0·6 μg/g, wet weight and hepatic concentrations of PCBs of 1·4 ± 0·6 μg/g wet weight which were ten and eight times, respectively, as great as those in reference sole. These results show that accumulation of AHs and PCBs from sediment by English sole is a significant route of uptake in contaminated environments. 2. 2. 3H-benzo(a)pyrene (BaP) and 14C-PCBs were added to the test sediment to compare the uptake and metabolism of BaP to that of PCBs by sole. Steady-state concentrations of 3H-BaP and 14C-PCBs in tissues and fluids were reached by 4 days and 14 days, respectively. At steady state, the level of covalent binding of 3H-BaP to hepatic proteins in test sole was six times greater than that of 14C-PCBs, and there was significantly greater relative formation of BaP-glutathione (GSH) conjugates than PCB-GSH conjugates. Thus, the amount of BaP metabolized to reactive metabolites was greater than that for PCBs, even though the concentration of 14C-PCBs in liver of test sole was five times as great as that of 3H-BaP. 3. 3. Trace amounts of 3H-BaP were also added to the reference sediment to determine how simultaneous exposure of English sole to those contaminants already present in the test sediment affected the metabolism and disposition of BaP. The 3H-BaP concentration ratios for liver to sediment and bile to sediment, as well as proportions of biliary BaP-GSH conjugates in test sole, were significantly greater than in reference sole. Thus, the formation and accumulation of potentially toxic metabolites of a carcinogenic AH (BaP) in sole liver were increased by simultaneous exposure to contaminants present in an urban sediment.

Covalent binding of benzo[a]pyrene to DNA in fish liver

Abstract Benzo[a]pyrene became bound to the hepatic DNA in juvenile English sole ( Parophrys vetulus ) force fed tritiated benzo[a]pyrene. No statistically significant change was observed in the level of the binding from 16 h to 2 wk after the single exposure. Specific activities of binding were similar for both DNA and protein. Moreover, a binding index was calculated to represent the number of benzo[a]pyrene molecules bound per 106 nucleotides after administration of a theoretical dose of 1 mmole of hydrocarbon per kg body weight. The value for English sole liver DNA was of the same order of magnitude as the values reported for mouse skin and mammary gland in which benzo[a]pyrene is carcinogenic.

Contaminant effects on reproductive success in selected benthic fish

Abstract 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.

A comprehensive assessment of the impacts of contaminants on fish from an urban waterway

Abstract The Hylebos Waterway, in central Puget Sound in the state of Washington, is severely contaminated by a variety of organic and inorganic contaminants. Studies in the 1970s had shown that flatfish residing in the Hylebos Waterway had increased prevalences of several types of toxicopathic liver disease, including neoplasms. In 1994, studies were initiated to: (i) determine if contaminant source control had resulted in improvements in habitat quality, as determined by prevalences of liver disease in flatfish; (ii) encompass the assessment of reproductive function in flatfish; and (iii) determine whether and to what degree there might be contaminant exposure of juvenile salmonids which migrate through this waterway. The findings show that there have been no appreciable changes in disease prevalences or apparent contaminant exposure of flatfish from this site since the 1970s. Moreover, female flatfish from the Hylebos Waterway are showing evidence of precocious sexual maturation in young animals, and inhibited gonadal development in older fish. The results also showed that two species of juvenile salmon sampled from this site are being exposed to a wide range of chemical contaminants, and the levels of exposure are comparable to levels which have previously been shown to cause impaired growth, immunosuppression, and increased mortality following pathogen exposure. These studies, which were done to provide a scientific and legal basis for cleaning up and restoring this degraded habitat, provide an example of how studies of pollutant responses in marine organisms (PRIMO) are being applied to societal and resource manager needs.

Biochemical responses to sediment-associated contaminants in brown bullhead (Ameriurus nebulosus) from the Niagara River ecosystem

Brown bullhead (Ameriurus nebulosus) were collected from three sites in the Niagara River ecosystem in June and September of 1991, and sediment samples from these sites were obtained in July 1991. The sites were located in the Buffalo River, the Niagara River adjacent to the Love Canal dump site, and in Black Creek, a Canadian tributary of the Niagara River which served as a reference site. Sediment samples from these sites contained measurable concentrations of various polycyclic aromatic hydrocarbons (PAHs) and chlorinated hydrocarbons (CHs). However, the Buffalo River and Love Canal samples were significantly more contaminated than those from Black Creek. Moreover, Buffalo River samples contained greater PAH concentrations than samples from the Love Canal, while the reverse was observed for CHs. Bile and liver of bullhead were used for the following analyses: fluorescent aromatic compounds in bile, a measure of exposure to PAHs, microsomal cytochrome P450 (CYP) and P450IA (CYP1A) contents and ethoxyresorufin-O-deethylase (EROD) activities, total glutathione (TH-GSH) concentrations, concentrations of 8- oxodeoxyguanosine (8-oxo-dG), and concentrations of hydrophobic DNA adducts (as measured by 32P-postlabelling). Additionally, a laboratory experiment was performed to examine CYP1A-associated responses in bullhead exposed to the model inducer, β- naphthoflavone (BNF). Results from the laboratory induction study were generally consistent with those observed in the field study, but the field study results suggested induction of CYP1A in bullhead from the reference site (Black Creek). For both field collections, fish from the Buffalo River displayed the greatest concentrations of fluorescent compounds in bile and hepatic DNA adducts, whilst fish from the Love Canal site displayed the greatest microsomal CYP1A concentrations and EROD activities. TH- GSH concentrations were significantly greater in Buffalo River fish versus Black Creek only for the June sampling. No statistically significant differences in 8-oxo-dG concentrations in bullhead hepatic DNA were observed among the sites at either sampling date. The different patterns in biochemical responses observed were consistent with sediment chemistries, and these results suggest that exposure of feral teleosts to different suites of bioavailable contaminants can be associated with expression of a characteristic array of biochemical responses