Usha Varanasi

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.

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.

Exposure of two species of deposit-feeding amphipods to sediment-associated [3H]benzo[a]pyrene: Uptake, metabolism and covalent binding to tissue macromolecules

Abstract Two species of deposit-feeding marine gammaridian amphipods, Rhepoxynius abronius and Eohaustorius washingtonianus, were exposed to sediment-associated [3H]benzo[a]pyrene (BaP) at 12±1°C. Concentrations of BaP-derived radioactivity increased with time in both E. washingtonianus and R. abronius, and the levels of radioactivity were similar in both species after 7 days of exposure. A significantly (P

Uptake and release of naphthalene and its metabolites in skin and epidermal mucus of salmonids

Abstract Rainbow trout ( Salmo gairdneri ) exposed to 1,4,5,8-[ 3 H]naphthalene through force feeding, via ip injection, or in flowing water, accumulated significant concentrations of both naphthalene and its metabolic products in skin, regardless of the mode of exposure. Concentrations of naphthalene and its metabolites in skin increased with time initially and subsequently declined. Concentrations of naphthalene decreased more rapidly than the metabolites in all three experiments: as much as 17.6 (force-feeding study), 15.1 (injection study), and 52.0% (water-immersion study) of the total radioactivity in the skin was attributable to the metabolites at the end of each experiment. This tendency of skin to retain metabolites preferentially was also exhibited by the liver. Based on 1 g of dry tissue, skin contained 34 to 84% of naphthalene and 13 to 26% of the metabolites present in the liver 24 hr after the initiation of naphthalene exposure; activity of skin with respect to liver was the highest for the water-immersion study, as may be expected, and lowest for the injection study. Epidermal mucus of the test fish in the injection and force-feeding experiments contained small concentrations of naphthalene and considerably larger concentrations of metabolites for several days after the initial treatment. Because epidermal mucus of fish exists in a state of continuous flux (that is, a small amount of mucus is continuously sloughed off and renewed), these results imply that epidermal mucus is involved in the excretion of hydrocarbons and their metabolites.

Effect of environmental temperature on naphthalene metabolism by juvenile starry flounder (Platichthys stellatus).

Juvenile starry flounder (Platichthys stellatus) maintained at 4° or 12°C were forced-fed3H-1-naphthalene. At 24 hr, after the initiation of exposure, significantly (p < 0.05) higher concentrations (2 to 15 times) of naphthalene were present in tissues of starry flounder at 4°C than those present in fish held at 12°C. The influence of lowering of water temperature on naphthalene retention was even more marked after one week. At this time, muscle and liver of fish at 4°C contained 26 and 34 times, respectively, more naphthalene than did muscle and liver of fish at 12°C. Concentrations of total metabolites, in most tissues were not substantially higher at the lower temperature either 24 or 168 hr after the naphthalene-exposure.Thin-layer Chromatographic separation of the metabolites revealed that at 24 hr, 1,2-dihydro-1,2-dihydroxynaphthalene (dihydrodiol) was the major component in liver (40 to 50% of extracted metabolites) and muscle (∼-80% of extracted metabolites) regardless of the temperature. Bile contained, primarily, conjugates (e.g., glucuronides), which yielded the dihydrodiol as the principal metabolite on enzymatic hydrolysis. From 24 to 168 hr, the concentrations of each metabolite class did not vary directly with the concentrations of total metabolites. Accordingly, at 168 hr, the ratio of total metabolite concentrations in liver of fish at 4°C compared to 12°C was 1.6, whereas the ratios for the dihydrodiol, sulfate/glucoside conjugates and glucuronide conjugates were 4.5, 0.6 and 3.8 respectively.Generally, lowered water temperature increased tissue concentrations of the parent hydrocarbon and its metabolites. However, the magnitude of the increase was dependent upon the compound, the tissue, and the time after the initiation of the exposure. The results emphasize the importance of determining concentrations of individual metabolites together with parent hydrocarbons in tissues of fish when assessing effects of environmental parameters on xenobiotic toxicity.

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.

Using cytochrome P450 to monitor the aquatic environment: Initial results from regional and national surveys

Abstract We are currently analyzing hepatic cytochrome P4501A and associated monooxygenase activities in fish sampled in several regional and national monitoring programs, including the National Benthic Surveillance Project of NOAAs Status and Trends Program, damage assessment studies of the Exxon Valdez oil spill, and intensive surveys of specific embayments, such as Puget Sound, Washington. Thus far, apparent contaminant-related increases in the activities of cytochrome P4501A-dependent monooxygenases have been readily measured in most test species. The results presented in this paper show that, for II species of fish, there is excellent concordance between hepatic activities of aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin-O-deethylase (EROD); moreover, levels of cytochrome P4501A determined by an enzyme-linked immunosorbent assay (ELISA) are also generally concordant with results from catalytic assays. The use of both a catalytic assay and immunoquantitation is recommended, because of the additional quality assurance provided by concurrent use of an immunoquantitation technique, which is desirable in large monitoring programs.

Benzo[a]pyrene metabolism and DNA adduct formation mediated by english sole liver enzymes

Tritiated benzo[a]pyrene (BaP) and (+/-)-7,8-dihydroBaP (BaP 7,8-dihydrodiol) were incubated with English sole liver microsomes in the presence of salmon testes DNA. The modified deoxynucleosides were isolated by Sephadex LH-20 column chromatography and analyzed by reverse-phase high-pressure liquid chromatography (HPLC). A single, major adduct (60-68% of the total modified deoxynucleosides) was formed when either BaP or BaP 7,8-dihydrodiol was incubated with sole liver microsomes and DNA. Although other minor BaP-DNA adducts were formed, none represented greater than 3% of the total adducts. The major adduct had a retention time on HPLC identical to that of the N2-[10 beta (7 beta, 8 alpha, 9 alpha-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene)yl]-deoxy-guanosine (7R-anti-BPDE/trans-dG) adduct formed when anti-BPDE, the ultimate carcinogen of BaP in mammals, was incubated with DNA. Analysis of the Bay region tetrols showed that only the 7 alpha, 8 beta, 9 beta, 10 alpha-tetrahydroxy-7,8,9,10-tetrahydroBaP, a hydrolysis product of the anti-BPDE, was formed when BaP was incubated with sole liver microsomes. When BaP 7,8-dihydrodiol was used as the substrate, the 7 alpha, 8 beta, 9 beta, 10 alpha-, 7 alpha, 8 beta, 9 alpha, 10 beta-, and 7 alpha, 8 beta, 9 alpha, 10 alpha-tetrahydroxy-7,8,9,10-tetrahydroBaPs were formed, indicating the formation of both anti- and syn-BPDE. The ratio of tetrols of anti-BPDE/syn-BPDE was 2; however, the ratio of adducts of anti-BPDE/syn-BPDE was 20. Thus, the findings show that hepatic microsomes of English sole, a fish species having a high incidence of liver neoplasia in chemically contaminated estuaries, metabolized BaP and BaP 7,8-dihydrodiol stereoselectively to form predominantly the 7R-anti-BPDE/trans-dG adduct.

Indicators of reproductive development in prespawning female winter flounder (Pleuronectes americanus) from urban and non-urban estuaries in the northeast United States

The relationship between exposure to xenobiotic compounds and ovarian development was evaluated in prespawning female winter flounder (Pleuronectes americanus) sampled from eleven sites on the northeast coast of the United States during the 1988 and 1989 spawning seasons. Three sites were located in Boston Harbor, MA, four sites were in Raritan Bay, NJ and four sites were in nearby embayments. Sediments from these sites exhibited a wide range in concentrations of xenobiotic compounds (e.g. aromatic hydrocarbon (PAH) concentrations ranged from 20 to 50 000 ng/g dry wt. and polychlorinated biphenyl (PCB) concentrations ranged from 2 to 1400 ng/g dry wt.), with the sites in Boston Harbor and Raritan Bay the most heavily contaminated. The following parameters associated with ovarian development were measured: ovarian developmental stage, ovarian atresia, gonadosomatic index, plasma estradiol, fecundity and egg weight. Contaminant exposure was assessed by measuring concentrations of fluorescent aromatic compounds (FACs) in the bile, hepatic aryl hydrocarbon hydroxylase (AHH) activity, concentrations of PCB in liver, ovary and brain and concentrations of xenobiotic-DNA adducts in liver tissue. Additionally, liver tissue was examined histologically for the presence of suspected toxicopathic lesions. In general, indicators of contaminant exposure were elevated and prevalences of suspected toxicopathic lesions were highest in fish from sites within Boston Harbor and Raritan Bay. Hepatic AHH activity, however, was heavily influenced by the reproductive cycle and showed little correlation with other indicators of contaminant exposure. Evidence of decreased egg weight and increased atresia in fish with high tissue concentrations of PCB or high levels of FACs in bile was observed. However, contaminant exposure had no clear negative impact on gonadal recrudescence, gonadosomatic index, plasma estradiol concentrations, or fecundity in female winter flounder. These results are in contrast to results with another Pleuronectid species, English sole (Pleuronectes vetulus), which shows inhibited gonadal development and lower plasma estradiol concentrations at contaminated sites in Puget Sound, WA. The apparent difference between English sole and winter flounder in susceptibility to contaminant-induced reproductive dysfunction could be related to a number of factors, including the differences in the migratory behavior of the two species during the reproductive season. English sole reside in contaminated estuaries throughout vitellogenesis and move offshore to spawn, while winter flounder often remain offshore for extended periods during early vitellogenesis and move into contaminated estuaries prior to spawning. Because of these contrasting migration patterns, both the duration and timing of exposure to contaminants during gonadal recrudescence may differ substantially in these two species and may contribute, in part, to the observed differences in the prevalence of reproductive dysfunction in fish from contaminated estuaries.

Sediment-associated contaminants and liver diseases in bottom-dwelling fish

High concentrations of chemicals have been found in sediments from urban areas of Puget Sound. Hundreds of organic chemicals (including certain aromatic hydrocarbons [AHs] and various chlorinated compounds) were identified. Statistical methods were used to evaluate possible relationships between the chemistry data and fish diseases. Positive correlations were found between the frequencies of liver neoplasms (e. g., hepatocellular carcinoma) and other liver lesions in English sole (Parophrys vetulus) and concentrations of AHs in sediment; such correlations were not found with chlorinated hydrocarbons. Strong evidence was also obtained to show that many organic chemicals in sediment are bioavailable to bottom-dwelling fish. Stomach contents (consisting mainly of benthic invertebrates) from English sole had concentrations of a number of AHs similar to those in the sediment from which the fish were taken. In these same fish, metabolites of many aromatic compounds were found in bile using a procedure combining HPLC with fluorescence detection. Further, the concentrations of certain xenobiotic metabolites in bile were correlated positively with the occurrence of liver neoplasms in English sole.