Metallothionein in liver of eels Anguilla anguilla from the Thames Estuary: an indicator of environmental quality?

The purpose of this study was to examine the role of the cysteine-rich protein metallothionein (MT) in the detoxification and metabolism of metals in livers of eels Anguilla anguilla from the Thames Estuary, and to assess the value of MT measurements in environmental monitoring. Hepatic MT levels and associated metal concentrations were determined in A. anguilla collected on three occasions during 1998 at sites along the tidal Thames (from freshwater to the sea) and also from a reference site (Tamar Estuary) in southwest England. MT was present at basal levels of 2 mg g(-1) dry weight (dw), predominantly in association with the essential metals Cu and Zn. MT concentrations were variable within each eel 'population' but were generally highest (up to 11 mg g(-1) in individual eels) at the more contaminated upper- and mid-estuarine sites in the Thames (e.g. Brentford and Kew). Lowest MT levels were those in eels from Blythe Sands at the mouth of the estuary. Season, sex, reproductive status and salinity may have influenced MT levels to some extent, although metals were the most significant factor. Hepatic MT concentrations were highly correlated with associated metal burdens (Zn, Cu, Ag and Cd),--notably reflecting Cu and Ag enrichment in the upper- and mid-estuary. With the exception of Zn, metals in the supernatant fraction of eel livers were primarily associated with MT. Proportions of Cu, Ag and Cd bound to MT increased as a function of accumulated metal burdens, with no indication of saturation. Thus, despite causing induction of MT, excess bioavailable Cu, Ag and Cd appear to be successfully detoxified in eels over the range of environmental contamination encountered along the Thames Estuary. Paradoxically, it may be argued from conventional doctrine that the presence of raised levels of MT, at upstream sites, constitutes a response to contamination and, by definition, signals that the fish are affected by metals; however, obvious signs of deleterious effects were not detected. Superficially, from liver MT data, it would seem that eel populations have become biochemically adapted to metal contamination. The approach tested here has proved viable in helping to interpret trends in bioavailability and the toxicological significance of accumulated metal burdens in Thames eels. A. anguilla is a common inhabitant of estuarine and fresh waters throughout Europe, and related species occur world wide. Therefore, determination of hepatic MT (and associated metals) in eels could be useful for monitoring responses to metal exposure and environmental quality on a much broader basis. Ideally these determinations should be performed in tandem with other sublethal-effects measurements to maximise information.     

Hepatic CYP1A levels and EROD activity in English sole: biomonitoring of marine contaminants in Vancouver Harbour

To assess chemical contaminant stress in the marine environment, ethoxyresorufin-O-deethylase (EROD) activity and cytochrome P450 1A (CYP1A) expression were measured in 88 English Sole (Pleuronectes vetulus) collected during May and June 1999 from four sites in Vancouver Harbour and at an expected reference site outside the harbour. Hepatic microsomes were prepared from the fish and analyzed for total CYP content, EROD activity, and CYP1A protein levels. Hepatic EROD activity and CYP1A protein levels were elevated in fish from two sites in the inner harbour. A comparison with sediment chemistry data showed that fish with increased EROD activity and CYP1A levels came from sites containing relatively high levels of polycyclic aromatic hydrocarbons and polychlorinated biphenyls. Unexpectedly high levels of EROD activity and CYP1A protein were also found in fish from a reference site near Gibsons, in Howe Sound. The elevated EROD activity and CYP1A expression in fish from this site cannot be explained by the chemical analysis data collected.     

Modulation of trout 7-ethoxyresorufin-O-deethylase (EROD) activity by estradiol and octylphenol.

Estrogens appear to have a modulating effect on the expression of cytochrome P4501A (CYP1A) in fish. A number of in vivo studies have demonstrated that hepatic CYP1A expression in females decrease during sexual maturation when plasma levels of 17 beta-estradiol (E2) increase, or in cases when the fish in injected with E2. Since a number of environmental contaminants have weak estrogen-like activities, the question arises if these compounds are able to modulate CYP1A expression as well. In the present study, we used in vitro monolayer cultures of rainbow trout, Oncorhynchus mykiss, liver cells to compare concentration-dependent (10(-9) to 10(-5) M) effects of the natural steroid E2 and the non-steroidal xenoestrogen 4-tert-octylphenol (OP) on CYP1A-catalyzed 7-ethoxyresorufin-O-deethylase (EROD) activity. The concentration dependency of the estrogenic activity of the two test compounds was assessed by determination of hepatocellular vitellogenin (Vg) release into the culture medium. Exposure of hepatocytes to E2 concentrations of 10(-8) M and higher led to a significant inhibition of basal cellular EROD activity. On the contrary, exposure to OP did not result in an inhibition of EROD activity, even at OP concentrations (10(-6) M, 10(-5) M) which were associated with a significant induction of Vg synthesis.     

Semipermeable membrane devices link site-specific contaminants to effects: Part 1 - Induction of CYP1A in rainbow trout from contaminants in Prince William Sound, Alaska

Extracts from semi-permeable membrane devices (SPMDs) deployed on beaches in Prince William Sound (PWS), Alaska, were used to evaluate if complex contaminant mixtures from different sources can be distinguished by the resulting cytochrome P450 1A (CYP1A) activity in exposed test animals. Deployment sites included canneries, salmon hatcheries, and beaches where lingering oil remains from discharges during the 1964 earthquake or the 1989 Exxon Valdez oil spill. Other sites were selected at random to evaluate region-wide contaminant inputs or were located in salmon streams to evaluate contaminants carried and released by migrating salmon carcasses following reproduction. Following standard deployments of approximately 28 d, an aliquot of the accumulated contaminants was intraperitoneally injected without cleanup into juvenile rainbow trout (Oncorhynchus mykiss). After 2 d and 7 d, the activity of CYP1A was measured by the ethoxyresorufin-o-deethylase (EROD) assay. Exposure to extracts from the oiled sites and one hatchery site with numerous creosote pilings elicited strong EROD responses, whereas fish exposed to salmon stream extracts elicited weak but significant responses during late summer compared to late spring. Responses from the other sites were not significant, indicating contaminants from these sources are unlikely to cause CYP1A induction in resident biota. Rather than simply assessing extant contaminants, this method evaluates the potency of the different sites for bringing about aryl hydrocarbon receptor responses in resident biota.     

Increased potential for NAD(P)H-dependent reactive oxygen species production of hepatic subcellular fractions of fish species with in vivo exposure to contaminants.

The present study investigated the proposed involvement of contaminant-stimulated reactive oxygen species (ROS) production in disease processes in fish. NAD(P)H-dependent ROS production of subcellular fractions was determined by the iron/EDTA-mediated oxidation of 2-keto-4-methiolbutyric acid. Hepatic cytosolic NADPH-dependent and microsomal NAD(P)H-dependent ROS production were increased 51-160% (P < 0.05) in rainbow trout (Oncorhynchus mykiss) 15 weeks after a single i.p. injection of polychlorobiphenyl (PCB) (100 mg Clophen A50 kg-1 wet wt.). Hepatic microsomal NADH-dependent ROS production was 114% higher in perch (Perca fluviatilis) from PCB-contaminated Lake J?rnsj?n compared to clean Lake V?nern, Sweden. Hepatic mitochondrial NADH-dependent, cytosolic NADH-dependent and microsomal NADPH-dependent ROS production were variously elevated up to 160% in flounder (Platichthys flesus) at various sites along two pollution transects near to the ports of Rotterdam and Amsterdam, Netherlands. Overall the data indicate increased potential for ROS production in liver of fish exposed to field pollution, and support the hypothesis of oxidative stress as a mechanism of contaminant-mediated disease in fish.     

Effect of exposure to benzo[a]pyrene on SODs, CYP1A1/1A2- and CYP2E1 immunopositive proteins in the blood clam Scapharca inaequivalvis

The effects of water-borne exposure to benzo[a]pyrene (36 h; celite-bound 0.44 mg L(-1) B[a]P) on cytochrome P450 (CYP) and superoxide dismutases (SODs) were examined in digestive gland of the blood clam, Scapharca inaequivalvis. B[a]P accumulation and elimination were rapid, with maximum whole-body concentrations of 1.78 ng g(-1) wet wt after 12 h of treatment, followed by a progressive decline to 0.89 ng g(-1) at 36 h. The presence of B[a]P resulted in an increase in total CYP of digestive gland microsomes from 54+/-14 to 108+/-21 pmol/mg protein (mean+/-SD; p<0.05, 24 h). Increases were also seen in microsomal CYP1A1/1A2-immunopositive protein (50.5 kDa app. mol. wt; p<0.05), but not CYP2E1-immunopositive protein (49 kDa app. mol. wt.), indicating a specific response of the former isoform. Exposure to B[a]P produced a steady increase in Mn-SOD digestive gland activity (p<0.01; p<0.05) but no significant change in Cu/Zn-SOD activity. The respective proteins, measured by western blotting, were not significant induced after B[a]P exposure. Cu/Zn-SOD and Mn-SOD activities were correlated with total CYP levels (r=0.96 and 0.63, respectively), indicating a role for CYP in reactive oxygen species (ROS) production during exposure. Both 'NADPH-independent' and NADPH-dependent metabolism of B[a]P by digestive gland microsomes was seen, producing mainly 1,6-, 3,6- and 6,12-diones, with some phenols and 7,8-dihydrodiol; putative protein adducts were also formed. Redox cycling of the diones may also have contributed to ROS production, leading to the increased SOD activities.     

Natural modulation of hepatic metallothionein and cytochrome P4501A in flounder, Platichthys flesus L.

The use of biomarkers such as cytochrome P4501A (CYP1A) or metallothionein (MT) for pollution monitoring is based on the assumption that an increased activity or concentration is primarily caused by exposure to contaminants. Previous studies have shown that the response of biomarkers may be affected by factors such as season, temperature, gender, nutritional status or size. The objective of the present study was to identify natural factors that affect the hepatic activity of CYP1A and hepatic concentration of MT in flounder (Platichthys flesus L.). Thirty flounder were sampled at each of two sites in the Hvaler archipelago, southern Norway, monthly through one year. A set of variables were recorded for each sampling and individual including water temperature, salinity, external and internal lesions, intestinal content, sex, size and organ weights. Hepatic CYP1A activity (EROD), MT and metal concentrations were determined for each individual flounder. The influence of environmental and endogenous factors on the response of these two biomarkers was assessed in multiple regression models. For both biomarkers, 50–60% of the total variability could be explained from factors directly related to season, gender and maturation. Season contributed significantly in the model for EROD, as did external lesions. Size, condition and diet did not contribute greatly when the above factors were included. The results confirm previous findings that season, gender and maturation must be taken into account in biomarker monitoring, but also indicate that other factors such as external lesions should be considered.     

Biomarkers for endocrine disruptors in three species of Mediterranean large pelagic fish

The hypothesis that Mediterranean top predator species, such as large pelagic fish, are potentially at risk due to endocrine disrupting chemicals (EDCs), is investigated. The potential estrogenic effects of PHAHs in three fish species of commercial interest, the top predators bluefin tuna (Thunnus thynnus thynnus), swordfish (Xiphias gladius), and Mediterranean spearfish (Tetrapturus belone), were investigated using vitellogenin (Vtg), zona radiata proteins (Zrp) and mixed function oxidases (EROD, BPMO) as diagnostic tools. High induction of Vtg and Zrp was detected by western blot and ELISA techniques in adult males of X. gladius and T. thynnus thynnus, suggesting that these species are at high toxicological risk in the Mediterranean sea. Comparison of BPMO and EROD activities in the three species indicated, both in male and female, much higher MFO activity in bluefin tuna. This data suggests high exposure of this species to lipophilic xenobiotic contaminants in the Mediterranean environment.     

Critical evaluation of an intercalibration exercise undertaken in the framework of the MED POL biomonitoring program.

The results of an intercalibration exercise among the laboratories participating in the MED POL program for monitoring biological effects of pollutants along the Mediterranean coasts are presented. Three established biomarkers, i.e. lysosomal membrane stability, metallothionein concentration and ethoxyresorufin-O-deethylase (EROD) activity, were intercalibrated. The stability of lysosomal membranes in mussels (Mytilus galloprovincialis Lam.) was assessed with a cytochemical method. The four participating laboratories were able to discriminate between control animals (membrane labilization times ranging from 21 to 35 min) and Cu-exposed animals (40 micrograms/l Cu for 3 days) (labilization times ranging from 4.5 to 7.4 min). The metallothionein concentration was evaluated in digestive gland homogenates of control mussels and of animals exposed to 200 micrograms/l Cd for 7 days. The eight participating laboratories were able to discriminate between controls and treated samples using a spectrophotometric method. The EROD activity was evaluated by 11 laboratories. All laboratories were able to discriminate between liver microsomal preparations obtained from control and from benzo-a-pyrene exposed fish (Dicentrarchus labrax), with values ranging from 0.5 to 15.88 pmol/min/mg protein in controls and from 5.41 to 165.13 pmol/min/mg protein in treated animals. Using S9 fractions, it was possible to correctly identify control and treated fish, with a variation similar to that found using microsomal fractions, albeit with an inevitable difference in specific activity. As a corollary, all laboratories involved produced comparable data and were able to identify pollutant-induced stress syndromes in sentinel organisms. Thus, intercalibration enables the use of biomarkers in large biomonitoring programs.     

The use of polyclonal antibodies raised against rat and trout cytochrome P450 CYP1A1 orthologues to monitor environmental induction in the channel catfish (Ictalurus punctatus)

Induction of hepatic cytochrome P450-dependent microsomal mono-oxygenase by xenobiotics is a well-established phenomenon in teleost fish. As in laboratory mammals, fish possess multiple forms of cytochrome P450 that display overlapping substrate specificity. One such isoform, CYP1A1, which has been cloned and sequenced from rainbow trout, has been shown to be orthologous to rat CYP1A1 and, as in mammals, is inducible up to several hundred-fold by planar aromatic hydrocarbons, PCBs and dioxins. It has been suggested that induction of CYP1A1 orthologues might provide a sensitive biomonitor for environmental pollution by mixtures of such compounds. In the current study, polyclonal antibodies directed against CYP1A1 purified from rat and trout liver were used to monitor induction of the CYP1A1 orthologue in hepatic microsomes from the fresh water species, the channel catfish (Ictalurus punctatus). Catfish from a local fish farm were induced in the laboratory by three daily injections of 50 mg/kg of the PCB mixture Aroclor 1254 and compared with fish taken from a site in central Arkansas—the Bayou Meto, known to be polluted with dioxin. Hepatic microsomal activities towards ethoxyresorufin (EROD) and pentoxyresorufin (PROD) were measured and Western blot analysis carried out with the two antibodies. EROD was elevated in both the Aroclor-treated fish and in the Bayou Meto fish compared with untreated fish farm controls; smaller but significant increases were observed in PROD. Spearman's rank correlations of 0·74 and 0·89 were observed between EROD and immunoquantified cross-reactivity towards the rat CYP1A1 and trout CYP1A1 antibodies.     

Flounder health status in the Seine Bay. A multibiomarker study.

The Seine Bay is used as a pilot area to assess the usefulness of monitoring programmes using a suite of biological measurements. These biomarkers included ethoxyresorfin-O-deethylase (EROD) and acetylcholinesterase (AChE) activities, multixenobiotic resistance (MXR) protein expression level assessment and gonad histopathology. Samples of European flounder collected in three sites close to the Seine Estuary in late September 1998 showed that 8% of the males were intersex, i.e. had gonads with both male and female tissues. Another 10% of individuals, identified as male by morphological observation during sampling, showed only female tissues on histological sections. These dramatic changes were associated with different patterns of EROD activity, MXR expression or AChE activity inhibition that might reflect shorter time effects of xenobiotics and constitute a starting point to integrate biological responses for the assessment of the health status of flounder in the Seine Bay.     

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.     

Biotransformation enzyme activities and PCDD/PCDF levels in pike caught in a Swedish Lake

Recently, analysis of organochlorines in sediment and in pike from Lake Vänern, Sweden, showed a north-south gradient of polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs). In the present study, good correlations were found between muscle 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) equivalents and liver ethoxyresorufin-O-deethylase (EROD) activities (and P4501A levels) in pike caught at three sampling sites along the north-south gradient in the lake. This comparison between tissue levels of PCDD/PCDF and EROD activity (and P4501A) may indicate that TCDD or structural analogs could be responsible for the observed induction of the pike P450 system. It must, however, be taken into account that the analysed contaminants often do not appear alone but are, rather, accompanied by a range of other substances which may be inducers or antagonists.     

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.     

Apparent lack of CYP1A response to high PCB body burdens in fish from a chronically contaminated PCB site

Chronic exposure to organic contaminants such as polychlorinated biphenyls (PCBs) can lead to the development of resistance to these chemicals, a condition associated with reduced response of CYP1A1, a pollutant-inducible biomarker. We measured CYP1A activity (ethoxyresorufin o-deethylase, EROD) and PCB concentrations in feral fish from the Town Branch/Mud River system (Logan County, KY), a stream historically contaminated with PCBs and partially remediated. As a first step in evaluating the possible development of resistant populations in this system, we measured CYP1A expression and PCB body burdens in resident fish from sites we previously characterized as containing biologically significant levels of CYP1A inducing compounds. Mean PCB concentrations in edible flesh ranged from 75.2 to 16.7 microg/g in fish collected from Town Branch remediated sites and were relatively low (1.23 microg/g) in Town Branch reference site fish. However, hepatic CYP1A activity was similar among individuals of most species collected from reference and contaminated/remediated sites. The absence of elevated CYP1A levels in resident fish species despite the presence of significant PCB body burdens may indicate these fish have developed reduced sensitivity to CYP1A induction, a condition associated with acquired resistance to toxicants.     

苯并(a)芘影响黑鲷肝脏EROD活性变化的动力学研究

研究了黑鲷(Sparusmacro cephalus)在实验生态条件下暴露于不同浓度苯并(a)芘(benzo(a)pyrene,BaP)后,肝脏乙氧基异吩噁唑酮脱乙基酶(EROD)活性在不同暴露时相的动力学变化情况.EROD活性采用动力学方法测定.实验结果表明,暴露于较低质量浓度BaP(0.5,1.0μg/L)黑鲷肝脏的EROD活性在2 d后极显著高于对照组(P<0.01),2.0μg/L组则在2 h后极显著高于对照组,并且随暴露时间的延长,诱导程度表现出不同的下降趋势,而暴露于高质量浓度组(5.0 μg/L)的肝脏EROD活性极显著性诱导出现在12h.总体上较高质量浓度组(2.0,5.0μg/L)EROD活性出现极显著性诱导的时间比低质量浓度组(0.5,1.0μg/L)早,诱导程度也比低质量浓度组的高.在净化实验中各暴露组肝脏EROD活性均下降,与空白对照组比较均没有显著性差异,说明试验浓度没有超出严重损伤肝脏自身恢复系统的范围,在此浓度范围内污染的鱼通过净化可清除BaP污染.     

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.     

Estrogenic and CYP1A response of mummichogs and sunshine bass to sewage effluent

Recent studies demonstrating feminization of effluent-exposed wild-caught male fish in the UK have prompted much research regarding the estrogenic activity of effluent from municipal sewage treatment plants (MSTPs). To investigate the estrogenicity and cytochrome P450 1A (CYP1A) induction potency of MSTP effluent, two species of fish, adult male mummichogs, Fundulus heteroclitus, and juvenile sunshine bass, Morone saxatilis x Morone chrysops, were exposed to un-chlorinated effluent (75% effluent, 25% seawater) from a large MSTP in Yonkers, NY, USA. After a 21-day static-daily (75%) renewal exposure, significant elevations over controls were observed in levels of vitellogenin (VtG) in plasma (1730%) and liver (131%) in effluent-exposed sunshine bass. In contrast, hepatic VtG was not elevated in mummichogs; plasma VtG was not measured in this species. Effluent exposure elevated hepatic CYP1A protein (140-145%) and ethoxyresorufin-O-deethylase (EROD) activity (408-598%) in both species. These findings suggest ontogenetic and/or species differences in response to estrogenic compounds in MSTP effluent. Furthermore, the elevation of CYP1A in response to sewage effluent exposure indicates the presence of additional compounds that may alter xenobiotic and/or steroid biotransformation in fish.     

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.