Spatial patterns in markers of contaminant exposure, glucose and glycogen metabolism, and immunological response in juvenile winter flounder (Pseudoplueronectes americanus).

Inshore winter flounder (Pseudoplueronectes americanus) populations in NY, USA have reached record low numbers in recent years, and recruitment into the fishery appears to be limited by survival of post-settlement juvenile fish. In order to identify cellular pathways associated with site-specific variation in condition and mortality, we examined differential mRNA expression in juvenile winter flounder collected from six different bays across a gradient in human population density and sewage inputs. Illumina sequencing of pooled samples of flounder from contrasting degraded sites and less impacted sites was used to guide our choice of targets for qPCR analysis. 253 transcripts of >100bp were differentially expressed, with 60% showing strong homology to mostly teleost sequences within the NCBI database. Based on these data, transcripts representing nine genes of interest associated with contaminant exposure, immune response and glucose and glycogen metabolism were examined by qPCR in individual flounder from each site. Statistically significant site-specific differences were observed in expression of all but one gene, although patterns in expression were complex with only one (vitellogenin), demonstrating a west to east gradient consistent with known loadings of municipal sewage effluent. Principal components analysis (PCA) identified relationships among the genes evaluated. Our data indicate that juvenile winter flounder are responding to estrogenic chemicals in more urbanized coastal bays, and suggests potential mechanistic links between immune response, contaminant exposure and energy metabolism.

Measures of immune system status in young-of-the-year winter flounder Pseudopleuronectes americanus.

The immune status of young-of-the-year (YOY) winter flounder Pseudopleuronectes americanus was evaluated in fish collected from six areas around Long Island, NY, U.S.A. representing more urban areas with high population density in the west, to less densely populated more rural areas in to the east. Gene expression markers for innate immunity (pleurocidin) and contaminant exposure (cytochrome P4501A; cyp1a) were measured in liver and fin of fish collected at each site. Expression of pleurocidin was significantly higher in fin than liver, but was highly variable among individuals. Some statistically significant differences in pleurocidin expression among sites were observed, although elevated levels were not associated with degree of urbanization. Expression was related in part to fish size: a positive correlation between expression and total length (LT ) of fish was observed with the largest LT class (>125 mm) exhibiting significantly elevated pleurocidin expression as compared with fish in the smaller LT class. This indicates that immune competency may increase with age. No site-specific differences in cyp1a expression were observed. These data suggest that exposure to aromatic hydrocarbon contaminants is fairly widespread throughout the study area and that any differences in pleurocidin expression in YOY P. americanus are probably due to other factors. Antimicrobial activity was also measured as a functional indicator of immune response. Activity was highly variable, showing no significant site-specific differences, and no significant correlation to pleurocidin expression. The lack of correlation between pleurocidin expression and antimicrobial activity indicates that other antimicrobial peptides may be active against the bacteria tested or that other factors are influencing antimicrobial activity. This is the first quantitative evaluation of pleurocidin expression in YOY P. americanus from an urban area. Further work is needed to characterize factors controlling pleurocidin expression, as well as other indicators of immune response in young fish.

Uptake, metabolism, mutant frequencies and mutational spectra in lambda transgenic medaka embryos exposed to benzo[alpha]pyrene dosed sediments.

The goal of this study was to provide data supporting the use of lambda transgenic medaka (Oryzias latipes) embryos to evaluate mutagens in sediments. Embryos incubated directly on sediments dosed with the reference mutagen, benzo[alpha]pyrene (BaP), were examined for BaP uptake and metabolism. Mutant frequency and mutational spectrum were assessed in the cII transgene recovered from adult medaka livers exposed as embryos. Embryos rapidly accumulated 14C-BaP and metabolized BaP to polar metabolites, indicating sediment-sorbed BaP is available for bioaccumulation and medaka embryos are capable of bioactivating this mutagen. Exposure of embryos to BaP dosed sediments significantly induced cII transgene mutant frequencies with mutations predominantly being in G:C base pairs, consistent with known mechanisms of BaP mutagenesis in transgenic mice and fish.

PCB congener distributions in muscle, liver and gonad of Fundulus heteroclitus from the lower Hudson River Estuary and Newark Bay.

Gradients in sediment polychlorinated biphenyl (PCB) concentrations and PCB congener profiles exist along the Hudson River (NY, USA). We evaluated site and tissue differences in PCB concentration and congener profiles in resident mummichog (Fundulus heteroclitus) collected from PCB-contaminated sites in the lower Hudson River and the New York/New Jersey Harbor Estuary. Fish were collected from three PCB-contaminated sites Piermont Marsh (P), Iona Marsh (I), and Newark Bay (NB), and from two reference sites (Flax Pond, NY; Succotash Salt Marsh, RI). Congener profiles were statistically analyzed using principal component analysis (PCA) and general linear model (GLM) profile analysis. Contaminated fish had PCB tissue concentrations approximately 10-fold higher than those of reference fish. There were no site differences in PCB body burden (all tissues combined) among the contaminated site fish. However, relative PCB concentration did differ between organs: NB fish (gonad=liver>muscle); I and P fish (gonad>liver>muscle). In contrast to PCB content, PCB congener profiles did show site differences; NB mummichog being depleted in the less chlorinated congeners relative to I and P fish, likely reflecting different PCB sources to these populations. Within a site, however, PCB congener patterns were similar between liver, gonad and muscle. In conclusion, PCA and GLM analyses gave complementary results, both analyses indicating differences in site, but not tissue, distributions of PCB congeners. This study also demonstrates that unlike congener profiles, total PCB content does differ dramatically amongst tissues and further, that PCB differences among tissues (gonad vs. liver vs. muscle) can vary with site.

Effects of sewage-impacted sediment on reproduction in the benthic crustacean Leptocheirus plumulosus.

Several organic contaminants in sewage effluent have been shown to elicit an estrogenic response in juvenile fish. Comparatively little emphasis has been placed on assessing these effects in marine invertebrates, particularly benthic organisms inhabiting sediment where lipophilic contaminants tend to persist. The present study examined reproductive effects in the benthic crustacean Leptocheirus plumulosus exposed to sewage-impacted sediment from Jamaica Bay, New York. Data from chronic 28-day tests showed a 50% reduction in the average number of young (juveniles + embryos) produced per surviving female in exposures to sediment from Jamaica Bay (JB). Nonylphenol ethoxylate ('NPEO) concentrations at this site were measured at 44.2 microg/g dw, concentrations two orders of magnitude higher than reference site concentrations in central Long Island Sound (CLIS). Dose-response studies with nonylphenol (NP) amended reference sediment, however, did not significantly affect reproduction suggesting that other contaminants may have contributed to the effects observed.

Determination of steroid estrogens in wastewater by immunoaffinity extraction coupled with HPLC-electrospray-MS.

A new method, based on immunoaffinity extraction coupled with liquid chromatography/electrospray mass spectrometry (LC/ESI-MS) is described for the determination of the steroid estrogens beta-estradiol (E2), estrone (E1), and alpha-ethynylestradiol (E2) in wastewater. The use of highly selective immunosorbents in sample preparation prior to analysis allows the removal of interfering sample matrix compounds present in the wastewater extracts that would otherwise cause severe ionization suppression of the estrogens during the electrospray process. In addition, immunoextraction removes much of the isobaric noise from the selected ion monitoring chromatograms, increasing the signal-to-noise ratios for analytes, and contributing to the low detection limits (0.18 and 0.07 ng/L for E2 and E1, respectively) achieved by the current method. The method was applied to analysis of estrogens in two wastewater effluents. Recoveries of E2 and E1 were excellent (>90%), while the nonimmunogen (but structurally related) analyte EE2 was not retained (recovery <2%) from effluent extracts by the immunosorbent. This illustrates the extreme selectivity of the immunoextraction purification step. Precision of the method was high, with relative standard deviations below 5%. Concentrations of E2 in wastewater varied from 0.77 to 6.4 ng/L, while concentrations of E1 were greater (1.6-18 ng/L).

Relative bioavailability and DNA adduct formation of benzo[a]pyrene and metabolites in the diet of the winter flounder.

1. Radiolabeled metabolites of the carcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP) were shown to be absorbed through the diet of the winter flounder, Pseudopleuronectes americanus. 2. Oral bioavailability of a mixture of naturally produced metabolites was significantly less than that of the parent BaP. 3. Oral bioavailability of a pure metabolite, BaP-7,8-dihydrodiol (7,8-D) was found to be similar to that of BaP. 4. Both metabolites and BaP formed DNA adducts in liver.