The DNA de-methylating agent 5-azacytidine does not restore CYP1A induction in PCB resistant Newark Bay killifish (Fundulus heteroclitus)

Newark Bay (NB) killifish (Fundulus heteroclitus) have been chronically exposed to environmental contaminants that activate the aryl hydrocarbon receptor (AHR) and are tolerant to toxic effects and CYP1A induction provoked by AHR ligands. Resistance to CYP1A induction could be due to an epigenetic mechanism such as DNA methylation. We measured in-ovo CYP1A catalytic activity (ethoxyresorufin-O-deethylase, EROD) in NB and reference site killifish embryos aqueously exposed to various concentrations of the de-methylating agent 5-azacytidine, 5-AC (5, 50 and 500 micro(micro)M) with or without 0.2 micro(micro)g/l of the CYP1A inducer 3,3',4,4',5 pentachlorobiphenyl (IUPAC PCB126). Neither PCB126 alone, nor PCB126 plus 5-AC, induced EROD above levels in vehicle treated Newark Bay fish. In reference site fish, the same PCB126 dose provoked a 7.4-fold EROD induction relative to controls. We conclude that Newark Bay killifish are resistant to CYP1A induction by co-planar PCBs during early embryological development and our data suggests that DNA methylation does not play a critical role in resistance to CYP1A induction in this model.     

The DNA de-methylating agent 5-azacytidine does not restore CYP1A induction in PCB resistant Newark Bay killifish (Fundulus heteroclitus)

Newark Bay (NB) killifish (Fundulus heteroclitus) have been chronically exposed to environmental contaminants that activate the aryl hydrocarbon receptor (AHR) and are tolerant to toxic effects and CYP1A induction provoked by AHR ligands. Resistance to CYP1A induction could be due to an epigenetic mechanism such as DNA methylation. We measured in-ovo CYP1A catalytic activity (ethoxyresorufin-O-deethylase, EROD) in NB and reference site killifish embryos aqueously exposed to various concentrations of the de-methylating agent 5-azacytidine, 5-AC (5, 50 and 500 μ(micro)M) with or without 0.2 μ(micro)g/l of the CYP1A inducer 3,3^′,4,4^′,5 pentachlorobiphenyl (IUPAC PCB126). Neither PCB126 alone, nor PCB126 plus 5-AC, induced EROD above levels in vehicle treated Newark Bay fish. In reference site fish, the same PCB126 dose provoked a 7.4-fold EROD induction relative to controls. We conclude that Newark Bay killifish are resistant to CYP1A induction by co-planar PCBs during early embryological development and our data suggests that DNA methylation does not play a critical role in resistance to CYP1A induction in this model.     

Embryonic exposure to environmentally relevant concentrations of PCB126 affect prey capture ability of Fundulus heteroclitus larvae

Early life stages from a marine fish species, Fundulus heteroclitus, were exposed to sublethal doses of 3,3',4,4',5 pentachlorobiphenyl (PCB126) to evaluate its effects on ecologically relevant responses: growth and behavior. A few hours after fertilisation, eggs were treated topically with PCB126 (2.5-50 pg egg?1). Four days post-hatching (dph), morphological changes (body length and malformations), spontaneous locomotor activity (active swimming speed, rate of travel, % inactivity), prey capture ability (Artemia franciscana nauplii) and whole body EROD activity were evaluated in larvae. Untreated larvae collected at 0.5, 1, 2, 3, 4 dph were also examined. PCB126 did not increase the mortality or malformation rates. Body length and spontaneous locomotor activity were altered only in larvae treated with the highest dose. Treatment with PCB126 caused a dose-responsive reduction in prey capture ability (rate of decline in the number of Artemia) and induction of EROD activity. The lowest observed effective dose for both of these responses was 5.0 pg PCB126 egg?1 or 5.0 TCDD-toxic equivalents pg g?1 egg, using a TCDD-toxic equivalent factor of 0.005 and an egg mass of 5 mg. Prey capture efficiency (number of Artemia captured per feeding strike) was reduced at ≥ 10.0 pg egg?1. In untreated developing larvae, prey capture ability and efficiency increased as post-hatching development progressed and EROD activity remained low. The pattern of behavioral responses observed in PCB126-exposed Fundulus larvae differed from that observed in less-developed larvae indicating that other mechanisms than retarded development were involved. Behavioral dysfunction was a more sensitive response to PCB126 than morphological alterations and it occurred at environmentally relevant concentrations.     

A simple and reliable in vivo EROD activity measurement in single Fundulus heteroclitus embryo and larva

Dioxin-like compounds (DLC) induce toxic responses in early life stages of fish through activation of the aryl hydrocarbon receptor (AhR) which is frequently assessed by ethoxyresorufin-O-deethylase (EROD) activity. A novel spectrofluorimetric method was developed to quantitatively assess EROD activity in individual living embryos and prolarvae of a marine model fish species, the mummichog Fundulus heteroclitus. This in vivo method is based on the measurement of the production of resorufin by single live embryos or prolarvae after 5 h incubation with ethoxyresorufin. Freshly fertilized eggs were treated topically from 2.5 to 50 pg egg⁻¹, with 3,3′,4,4′,5-pentachlorobiphenyl (PCB126), a prototypical DLC. EROD activity was assessed in embryos (7 days post-fertilization) and prolarvae (16 days post-fertilization). Resorufin was measured both in the culture medium (25‰ seawater) and in whole fish homogenates, to assess the percentage retained in the body. Approximately 95% and 17% of the resorufin produced in vivo was retained in embryos and prolarvae respectively. EROD activity in homogenates of embryos and in the culture medium of prolarvae increased linearly with dose. EROD activity measured by the in vivo method was highly correlated to that measured by a traditional in vitro technique using S9 fractions for both embryos and prolarvae. Both in vivo and in vitro EROD activity were higher in prolarvae than in embryos pretreated with PCB126. EROD induction measured in prolarvae by the in vivo and in vitro methods were similar whereas higher induction was measured in vivo than in vitro in embryos. The in vivo method was more sensitive and as reliable as the in vitro technique, and required a lower number of fish (4 compared to 3 pools of 5). This in vivo method is useful to link EROD induction in individual embryos or prolarvae to other organism-level responses. Further studies with other categories of xenobiotics should be performed to assess potential toxic effects on resorufin absorption/excretion processes which could affect in vivo measurements.     

菲、芘、苯并(a)芘单一暴露及分别与α-萘黄酮(ANF)联合暴露对海水青鳉(Oryzias melastigma)胚胎发育毒性效应的比较研究

为了解和探讨3~5环PAHs对海水鱼类胚胎发育的毒性及作用方式,比较研究了菲(phenanthrene,Phe)、芘(pyrene,Py)、苯并(a)芘(benzo(a)pyrene,BaP)单一暴露和三者各自与α-萘黄酮(α-naphthoflavone,ANF)联合暴露对海水青鳉(marine medaka, Oryzias melastigma)胚胎发育的毒性效应。胚胎体内EROD活性、发育畸形、孵化率和心律等毒性指标被测定,结果显示:Phe,Py和BaP对海水青鳉胚胎体内EROD活性的诱导能力大小为BaP>Py>Phe,各化合物对EROD诱导与发育畸形之间的关系较为复杂,除Phe所引起的EROD诱导与畸形指数之间呈显著相关(r=0.95,p=0.015)外,Py和BaP均无相关性;在100 μg/dm3 ANF影响下,CYP1A活性诱导被抑制,但胚胎发育的畸形指数被显著提高,ANF分别与Phe,Py和BaP的联合暴露对胚胎发育呈潜在的协同作用。本文研究初步表明,3~5环PAHs化合物对海水青鳉胚胎发育的毒性作用方式可能不同;CYP1A活性抑制在PAHs混合物对海水青鳉胚胎发育的毒性作用过程中未起到缓解毒性的作用,CYP1A抑制剂与PAH型CYP1A诱导剂的混合物对鱼类胚胎发育具有潜在的协同毒性作用,现有的PAHs混合物毒性风险评价方法可能低估了实际环境中PAHs的风险;海水青鳉早期生活阶段的心脏发育对PAHs混合物暴露较为敏感,可推荐其作为生物标志物指示PAHs或溢油污染。     

EROD activities of liver in Mugil so-iuy exposed to benzo (a)pyrene, pyrene and their mixture

Abstract-The effects on hepatic EROD (7-ethoxyresorufin O-deethylase) in Mugil so-iuy exposedto benzo(a)pyrene (BaP), pyrene and their mixtures of equal concentration were investigated, at con-centrations of 0.1, 1.0, 5.0, 10.0, 50.0 μg/dm~3, in experimental condition. Time-effects and dose-response of the biochemical indexs were observed. The results showed that the hepatic EROD activitieswere induced by the exposure of BaP, pyrene and their mixtures at high concentration. Dose-responseconnections were that the hepatic EROD activities were elevated with increasing concentration of the pol-lutants. The combined effect of BaP and pyrene at 1:1 concentration ratio on hepatic EROD activity wasantagonism.     

Estrogenic effect of dioxin-like aryl hydrocarbon receptor (AhR) agonist (PCB congener 126) in salmon hepatocytes

Previous studies have shown that aryl hydrocarbon receptor (AhR) agonists possess anti-estrogenic activities and several mechanisms have been proposed to explain the interactions between AhR and estrogen receptor (ER) signalling pathways. In the present study, we show that 3,3'4,4'5-pentachlorobiphenyl (PCB126 - a dioxin-like AhR agonist) produced estrogenic responses in the absence of ER agonist, in fish in vitro system. We exposed salmon primary hepatocytes to PCB126 (1, 10 and 50 pM) and the ER agonist nonylphenol (NP; 5 and 10 microM) singly and also in combination. Vitellogenin (Vtg) and zona radiata proteins (Zr-proteins) levels were analysed by semi-quantitative ELISA. We observed that the protein levels of Vtg and Zr-proteins were significantly induced in a concentration-specific manner in cells treated with PCB126 and NP, singly or in combination. In general, these results show a novel aspect of dioxin-like PCB effect not previously demonstrated in fish system.     

Accumulation and effects of benzo(a)pyrene on cytochrome P450 1A in waterborne exposed and intraperitoneal injected juvenile turbot (Scophthalmus maximus)

Juvenile turbot (Scophthalmus maximus) were exposed to benzo(a)pyrene (BaP) for 14 d using a glass bead generator flow-through system. Exposure was followed by a recovery period of 16 d. The highest BaP concentration in the edible portion of the fish, 16.5 ± 4.3 μg BaP/kg, was observed on the first day. Then concentrations dropped following first-order kinetics. BaP was below detection level at the end of the experiment. A statistically significant increase in bile fluorescence was observed from day 9 until the end of the experiment, suggesting the elimination of BaP metabolites by this route. No significant differences between control and exposed fish in EROD activity and CYP1A concentration, measured by immunodetection method, were observed. Intraperitoneal injection of 2.5 mg BaP/kg in juvenile turbot induced EROD activity. Under waterborne experimental conditions, bile fluorescence was observed to be a more sensitive biomarker of BaP exposure than EROD activity and CYP1A measurement.     

Correlative changes in metabolism and DNA damage in turbot (Scophthalmus maximus) exposed to benzo[a]pyrene

Juvenile turbot (Scophthalmus maximus) were injected intraperitoneally with either corn oil or 5 mg/kg benzo[a]pyrene (BaP) dissolved in corn oil and sampled I and 3 days after injection. After 1 day, no elevation of 7-ethoxyresorufin O-deethylase (EROD) activity was observed, however bile metabolites (BaP-7,8 dihydrodiol representing 70% of the total metabolites) and a single hepatic DNA adduct spot (0.47 adducts/10(8) nucleotides) identified by 32P-postlabelling were formed. No BaP metabolites or DNA adducts were observed in either control or carrier control fish. Fish sampled after 3 days reported 5-fold higher (P < 0.05) levels of EROD activity, a shift in the bile metabolite profile towards BaP phenol formation (1OH and 30H BaP comprising up to 60% of total metabolites detected) and the formation of two adduct spots (0.86 and 0.71 adducts/10(8) nucleotides). These results show that BaP can be metabolised and form hydrophobic DNA adducts in turbot without EROD elevation. Following EROD elevation, a shift in the profile of both BaP metabolites and BaP metabolite-DNA interactions occurs indicative of other oxidative processes.     

Patterns of heritability of decreased EROD activity and resistance to PCB 126-induced teratogenesis in laboratory-reared offspring of killifish (Fundulus heteroclitus) from a creosote-contaminated site in the Elizabeth River,VA, USA

Killifish (Fundulus heteroclitus) from a highly contaminated site on the Elizabeth River are resistant to the acute toxicity and the cytochrome P4501A (CYP1A)-inducing activity of both the sediments from the site and chemically pure polycyclic aromatic hydrocarbons (PAHs). These effects are highly heritable for one generation, but heritable to a lesser degree by subsequent generations, in clean conditions in the laboratory. We show that offspring of this population of Elizabeth River killifish are also resistant to the teratogenicity and P4501A-inducing activity of PCB congener 126, a prototypical coplanar halogenated aromatic hydrocarbon (HAH). Furthermore, the pattern of greater resistance to acute toxicity and P4501A-inducing activity in the first generation and less in subsequent generations is also observed upon exposure to PCB-126.     

Microsomal estrogen metabolism in channel catfish

Our goal was to study the involvement of cytochrome P450 genes in estrogen metabolism and the extent to which the potentially carcinogenic 4-hydroxyestradiol metabolite is formed by channel catfish (Ictalurus punctatus; CC). Estradiol metabolism and ethoxyresorufin-O-deethylase (EROD) activity were assessed in several tissues from fish collected from three variably contaminated sites in the Mississippi River Delta, from laboratory control fish, and from fish exposed to 20 mg/kg benzo(a)pyrene (BaP) i.p. for 4 days. Liver EROD activity was induced by BaP, but Delta fish EROD activities were not statistically higher than activities in control fish. Gill microsomal EROD activity was also induced by BaP, but activities were 8- to 77-fold lower than those from liver. The predominant estrogen metabolites formed by CC liver, gill and gonad microsomes were 2-hydroxyestradiol and estrone as detected by GC/MS. Liver and gill 2-hydroxyestradiol formation was induced in BaP-dosed fish. The trends in hydroxyestradiol formation in field collected fish were more variable. In all fish liver microsomes there was more 2- compared to 4-hydroxyestradiol formed. However, BaP-treatment increased the 4:2 hydroxyestradiol ratio from 0.04 in control fish to 0.2 in BaP-exposed fish, suggesting that BaP induces the formation of the potentially genotoxic estrogen metabolite. No detectable 4-hydroxyestradiol was produced by gill and gonad microsomes. These results will ultimately help in determining which fish P450 genes are susceptible to environmental contamination and may be involved in estrogen genotoxicity.     

Interactive effects of cadmium and benzo[a]pyrene on metallothionein induction in mummichog (Fundulus heteroclitus).

Previous experiments demonstrated that exposure of mummichog to cadmium (Cd) in combination with benzo[a]pyrene (BaP) caused a higher mortality than would be expected from simple additive effects. Experiments are described here that investigated whether BaP exposure inhibits the induction of metallothionein (MT), a major detoxifying protein for Cd, or if reactive BaP metabolites compete with Cd for binding sites on MT. Fish were injected with or without BaP (18 mg/kg) in combination with a low (1 mg/kg) or high (3.2 mg/kg) dose of Cd, and in one treatment BP was dosed 4 days after Cd. The results showed a rapid induction of MT to 1.5 mg/g wet weight liver, 1 day after injecting the low Cd dose. Simultaneous BaP exposure significantly delayed the induction of MT, for both low and high Cd doses, and BaP temporarily lowered the induced MT concentration when dosed 4 days after induction by Cd. To test if binding of BaP metabolites to MT reduces the detoxification potential for Cd, microsomes of CYP1A-induced fish were incubated with MT and radiolabeled BaP. Active metabolism of BaP was observed by high-performance liquid chromatography analysis, but no association of BaP metabolites with MT was found. Neither could this be demonstrated in vivo, in liver MT isolated from mummichog dosed with 3H-BaP and Cd. These results suggest that increased toxicity of Cd in combination with BaP exposure is likely to be caused by inhibited MT synthesis, rather than by interference of BaP metabolites with Cd binding on MT.     

Toxicity of sediments from Bahía de Chetumal, México, as assessed by hepatic EROD induction and histology in nile tilapia Oreochromis niloticus.

The effect of environmental pollutants present in sediments obtained from Bahía de Chetumal, a bay on the border between Mexico and Belize, was studied in nile tilapia (Oreochromis niloticus) intraperitoneally injected with sediment extracts from six different sites of the Bay. Sediment samples used for the study contained a variety of organic chemicals such as organochlorine pesticides, polychlorinated biphenyls (PCBs) and polynuclear aromatic hydrocarbons (PAHs). Total cytochrome P-450 and EROD activity were measured in fish liver. Haematological and histological analyses were also carried out. Hepatic P-450 content in treated fish increased from 43 to 240%, and EROD activity from 85 to 160% compared to controls. Extracts from two sampling sites inhibited EROD activity. There were positive significant correlations between P-450 content and the levels of PCBs 44 and 128. EROD activity correlated to HCB, op'-DDE, pp'-DDE, pp'-DDD, mirex and PCB 18 concentrations. Blood examination showed cell degeneration and binucleated leukocytes with abnormal chromatin. Extract treatment also resulted in foci of hyperplasia on the basement of gill lamellae, hypertrophy and oedema in gills and liver necrosis. Control fish showed no abnormalities. The results demonstrate that sediments from Bahía of Chetumal have the potential to cause histopathological, haematological and biochemical alterations in fish. The administration of sediment extracts to fish may serve as a useful test to screen the toxicity of sediments from different areas.     

In vivo formation of (+)-anti-benzo[a]pyrene diol-epoxide-plasma albumin adducts in fish.

Benzo[a]pyrene (BaP), a procarcinogenic polycyclic aromatic hydrocarbon (PAH), is bioactivated to BaP diol-epoxides (BPDEs) that can form adducts with DNA and blood proteins. We report here for the first time the in vivo formation of adducts between BPDE and plasma albumin (Alb) from two fish species experimentally exposed to BaP. Brook trout (Salvelinus fontinalis) received either a single i.p. dose (10 mg/kg) or two separate i.p. doses (25 mg/kg; 7 days apart) of BaP, and blood was collected 2 (single exposure) or 3 (multiple exposure) days post-treatment. Arctic charr (Salvelinus alpinus) received 10 i.p. doses (3 mg/kg; a single dose every 6 days), and blood was collected 2 days after the second, sixth, and 10th injections. BPDE-Alb adducts were measured by an improved HPLC/fluorescence method developed to detect and quantify BaP-tetrols released after acid hydrolysis of adducted Alb. HPLC/fluorescence chromatograms of Alb from BaP-treated fish revealed only BaP-tetrol I-1, thus indicating the formation of adducts exclusively via the (+)-anti-BPDE metabolite. Levels of (+)-anti-BPDE-Alb adduct ranged from 0.68 to 19.6 ng of tetrol I-1 per gram of Alb. Notably, adduct level was not related to BaP dose and there was no accumulation of adducts with repeated exposure, which may indicate a very short half-life (< 2 days) of plasma Alb in fish. The data suggest that BPDE-Alb adducts in fish could be useful as a non-destructive biomarker of recent exposure to bioactivated BaP.     

32P-postlabelling analysis of DNA adducts and EROD induction as biomarkers of genotoxin exposure in dab (Limanda limanda) from British coastal waters.

Dab (Limanda limanda) were sampled from a number of polluted and unpolluted areas in British coastal waters. The 32P-postlabelling assay was used to analyse the level of aromatic/hydrophobic DNA adducts in pooled samples of liver tissue. The mean levels of DNA adducts detected from areas known to receive anthropogenic pollutants ranged from 4.0 to 26.8 adducts per 10(8) nucleotides, with all sites containing samples displaying DNA adduct profiles consisting of diagonal radioactive zones. In contrast no DNA adducts were detectable in samples from an unpolluted reference site. The ranking of polluted sites based on DNA adduct levels did not correspond with the ranking of sites based on sediment associated polycyclic aromatic hydrocarbon levels, highlighting the problem of linking the presence of contamination with detectable biological responses. No correlation could be found in this study between EROD activity and the level of DNA adducts.     

苯并[a]芘(BaP)对褐菖鲉(Sebasticus marmoratus)肝CYP1A1酶活性、基因表达及蛋白表达的影响

为了了解苯并[a]芘(BaP)对鱼类细胞色素P4501A1(CYP1A1)表达的影响,以褐菖鲉(Sebasticus marmoratus)为实验材料,采用体内实验,研究其在经过不同浓度(0.1、1、10、20、50mg/kg鱼体重量)的BaP诱导后,鱼体肝脏研究CYP1A1基因表达的情况,筛选出后续时间-效应实验中BaP注射的最佳浓度,研究BaP诱导6h、12h、1d、3d、7d后(质量浓度为20mg/kg鱼体重量)鱼体肝脏CYP1A1酶活性、基因表达和蛋白表达的情况。结果表明:剂量-效应实验中,20mg/kg鱼体重量为最佳浓度,此浓度下,基因表达在各组中变化最显著。时间-效应实验中,较空白对照组而言,染毒6h、12h和1d后,EROD酶活性显著增加。3d后开始下降,与对照组相比变化不大,7d后酶活性又发生上调。半定量RT-PCR结果表明,各染毒组与对照组相比,CYP1A1基因表达量都发生了上调,呈现先上升后下降的趋势。其中,6h和12h组相对表达量极显著增加,1d后开始下降且与3d和7d组相比变化不明显。Western blot结果表明,蛋白表达量在染毒12h后表现出显著的诱导效应,随着时间的延长略有回落,但与对照组相比仍有显著性差异。研究表明:BaP对褐菖鲉CYP1A1具有较强的诱导作用。一定质量浓度的BaP注射于褐菖鲉不同的时间后,能诱导褐菖鲉活体EROD酶活性、CYP1A1基因m RNA表达及蛋白表达,并随着时间的延长呈现先诱导后抑制的趋势。这说明BaP作为诱导剂对CYP1A1酶活性和蛋白表达的作用机制可能与调控CYP1A1的转录水平有关。     

Evidence for resistance to benzo[a]pyrene and 3,4,3'4'-tetrachlorobiphenyl in a chronically polluted Fundulus heteroclitus population

Halogenated aromatic hydrocarbons (HAHs) and polynuclear aromatic hydrocarbons (PAHs) are major environmental contaminants. Fish species that are chronically exposed to these compounds can develop resistance to their toxic effects. In all fish species studied to date, toxicant resistance has been accompanied by decreased inducibility of the xenobiotic metabolizing enzyme, cytochrome P450 1A (CYPIA). CYP1A induction is mediated through the Aryl Hydrocarbon Receptor (AHR). Although these compounds mediate their effects through this pathway, there have been resistant populations in which one chemical class cannot induce CYPIA expression (HAHs) while the other (PAHs) can. Resistance to PAHs was examined in a HAH-resistant population of Fundulus heteroclitus collected from a site contaminated with both compound classes (Newark Bay, NJ). Fish were injected intraperitoneally with the HAH 3,4,3',4'-tetrachlorobiphenyl (PCB77), benzo[a]pyrene (B[a]P, a PAH) or vehicle and sacrificed after 2 (B[a]P) or 5 days (PCB77, vehicle). We found no significant increase in CYP1A mRNA levels in resistant Newark Bay F. heteroclitus treated with either B[a]P or PCB77, while there was a 3.9 fold (PCB77) and 4.2 fold (B[a]P) increase in CYP1A mRNA in Flax fish relative to controls. AHR labeling studies revealed significantly (P < 0.05) lower levels of hepatic AHR in Newark fish (1,770 +/- 1,693.2 DPM) relative to Flax fish (6,082.5 +/- 1,709.9 DPM). Overall, these data suggest Newark F. heteroclitus are resistant to both PAHs and HAHs at the level of CYP1A mRNA, which might be mediated, in part, though lower expression of AHR. We are currently studying the promoter sequence to determine its role in chemical resistance.     

Exposure to hydrocarbons 10 years after the Exxon Valdez oil spill: evidence from cytochrome P4501A expression and biliary FACs in nearshore demersal fishes

Three biomarkers of hydrocarbon exposure, CYP1A in liver vascular endothelium, liver ethoxyresorufin O-deethylase (EROD), and biliary fluorescent aromatic compounds (FACs), were examined in the nearshore fishes, masked greenling (Hexagrammos octogrammus) and crescent gunnel (Pholis laeta), collected in Prince William Sound, Alaska, 7-10 years after the Exxon Valdez oil spill (EVOS). All biomarkers were elevated in fish collected from sites originally oiled, in comparison to fish from unoiled sites. In 1998, endothelial CYP1A in masked greenling from sites that were heavily oiled in 1989 was significantly higher than in fish collected outside the spill trajectory. In 1999, fishes collected from sites adjacent to intertidal mussel beds containing lingering Exxon Valdez oil had elevated endothelial CYP1A and EROD, and high concentrations of biliary FACs. Fishes from sites near unoiled mussel beds, but within the original spill trajectory, also showed evidence of hydrocarbon exposure, although there were no correlations between sediment petroleum hydrocarbon and any of the biomarkers. Our data show that 10 years after the spill, nearshore fishes within the original spill zone were still exposed to residual EVOS hydrocarbons.     

Development of hepatic CYP1A and blood vitellogenin in eel (Anguilla anguilla) for use as biomarkers in the Thames Estuary, UK.

The potential of eel (Anguilla anguilla) as a monitoring species for the Thames Estuary, UK, was examined. Hepatic cytochrome P4501A [7-ethoxyresorufin O-deethylase (EROD) activity] and blood vitellogenin (Western analysis) were investigated as biomarkers of exposure to, respectively, organic contaminants and to contaminants showing estrogenic activity. Hepatic microsomal EROD activities in A. anguilla from seven sites in the Thames Estuary in May 1998 varied three-fold (111 +/- 24 to 355 +/- 42 pmol min-1 mg protein-1) (mean +/- S.E.M.) and showed correlation with salinity; however, the latter relationship was not maintained at other times of the year. The range of EROD activities was two- to eight-fold higher than the 37 +/- 8 pmol min-1 mg-1 for A. anguilla from the relatively clean Tamar Estuary. beta-Naphthoflavone treatment (5 mg kg-1 wet wt.; 2 days) of Thames A. anguilla produced a two-fold increase in hepatic microsomal EROD activity. Comparing the Thames EROD data with those for A. anguilla from well-characterised contaminated sites in the Netherlands (Van der Oost, R., Goks?yr, A., Celander, M., Heida, H., & Vermeulen, N. P. E. 1996. Aquatic Toxicology, 36, 189-222), the Thames is suggested to be moderately impacted by polycyclic aromatic hydrocarbons and related contaminants. 17-beta-Estradiol treatment produced the appearance of a plasma protein of 211 Kd app. mol. wt. (recognised by antibodies to vitellogenin of Morone saxatilis), but putative vitellogenin could not be detected in A. anguilla from selected sites in the Thames Estuary.     

Trophic transfer of benzo[a]pyrene metabolites between benthic marine organisms

This study was undertaken to determine the potential for trophic transfer of polycyclic aromatic hydrocarbon (PAH) metabolites from infaunal organisms to bottom-feeding fish. Winter flounder, Pseudopleuronectes americanus, were given single oral doses of ground polychaetes (Nereis virens), either treated with pure [¹⁴C]henzo[a]pyrene (BaP) or containing a mixture of naturally produced radiolabeled BaP and BaP metabolites. Fish were sacrificed 24 h after feeding and total accumulated radioactivity and metabolite class profiles determined in major organs. Metabolites produced by worms were absorbed by flounder, although as a percentage of dose given they were less available than parent BaP. Comparison of metabolite profiles in the worm diet and in target organs in the fish indicated that metabolites accumulated through the diet can be further modified by the prey organism and can lead to the formation of bound residues. These results demonstrate that PAH metabolites in the diet are available for accumulation. Furthermore, metabolites absorbed appear to be susceptible to metabolic alteration by consumer organisms.