Hepatic Cytochrome P4501A1 mRNA Research Articles

Combined effects of high temperature and pesticide mixture exposure on free-swimming behaviors and hepatic cytochrome P450 1A expression in goldfish, Carassius auratus

ABSTRACT The synergy between multiple compounds and other stressors, including heat, creates volatility and greater unpredictability than standard single-chemical toxicity testing, especially in the case of pesticides and metabolites which might contain several noxious ingredients resulting in adverse ecological effects. To address this, the aim of this study was to examine the dose- and time-dependent effects of low- and high-dose pesticide mixture (metalachlor, linuron, isoproturon, tebucanazole, aclonifen, atrazine, pendimethalin, azinphos-methyl) and heat stress co-exposure (22°C control/32°C treatment for 4-week) on free-swimming behaviors and cumulative actionless time (CAT) of goldfish. Behavioral analysis showed a dose- and time-dependent decrease in distance swam, as well as a subsequent increase in CAT. Vertical and horizontal spatial behavioral use were affected under heat and pesticides co-exposure conditions. In 3- and 4-week(s) exposure groups, horizontal spatial behavioral use demonstrated elevated time spent in the lower third of the aquarium. Similarly, during 3- and 4-week(s) exposure (32°C control and 32°C high doses) vertical spatial behavioral use was found to increase time spent in the outermost edges of the aquarium. In all treatment groups, the final condition factor (KM) showed significant attenuation when compared to the initial KM. However, there was an unclear relationship between heat/pesticide co-exposure and growth most notably in 32°C high-dose groups. In addition, the expression of hepatic cytochrome P450 1A mRNA was significantly higher in pesticide-exposed groups. Taken together, data demonstrated that co-exposure with low- or high-dose pesticide mixture and heat stress significantly impacted natural swimming patterns, which over time might result in the broader population and ecological effects.

Interactive effects of hypoxia and PCB co-exposure on expression of CYP1A and its potential regulators in Atlantic croaker liver.

Although marine and coastal environments which are contaminated with xenobiotic organic compounds often become hypoxic during the summer, the interactive effects of hypoxia and xenobiotic exposure on marine species such as teleost fishes remain poorly understood. The expression and activity of monooxygenase enzyme cytochrome P450-1A (CYP1A) in fishes are upregulated by exposure to polychlorinated biphenyls (PCBs), whereas they are down-regulated during hypoxia exposure. We investigated the interactive effects of hypoxia and PCB co-exposure on hepatic CYP1A expression in Atlantic croaker and on potential regulators of CYP1A. Croaker were exposed to hypoxia (1.7 mg/L dissolved oxygen), 3,3',4,4'-tetrachlorobiphenyl (PCB 77, dose: 2 and 8 µg/g body weight), and Aroclor 1254 (a common PCB mixture, dose: 0.5 and 1 µg/g body weight), alone and in combination for 4 weeks. PCB 77 exposure markedly increased hepatic CYP1A mRNA and protein expression, and ethoxyresorufin-O-deethylase (EROD, an indicator of CYP1A enzyme) activity and increased endothelial nitric oxide synthase (eNOS) protein expression. PCB 77 treatment also increased interleukin-1β (IL-1β, a cytokine) mRNA levels and protein carbonyl (PC, an indicator of reactive oxygen species, ROS) contents. These marked PCB 77- and Aroclor 1254-induced increases in CYP1A mRNA levels and EROD activity were significantly attenuated by co-exposure to hypoxia, whereas the increases in hepatic eNOS protein and IL-1β mRNA expression, and PC contents were augmented by hypoxia co-exposure. The results suggest that biotransformation of organic xenobiotics by CYP1A is reduced in fish during co-exposure to hypoxia and is accompanied by alterations in eNOS, ROS, and IL-1β levels.

Effects of Hypoxia Exposure on Hepatic Cytochrome P450 1A (CYP1A) Expression in Atlantic Croaker: Molecular Mechanisms of CYP1A Down-Regulation

Hypoxia-inducible factor-α (HIF-α) and cytochrome P450 1A (CYP1A) are biomarkers of environmental exposure to hypoxia and organic xenobiotic chemicals that act through the aryl hydrocarbon receptor, respectively. Many aquatic environments heavily contaminated with organic chemicals, such as harbors, are also hypoxic. Recently, we and other scientists reported HIF-α genes are upregulated by hypoxia exposure in aquatic organisms, but the molecular mechanisms of hypoxia regulation of CYP1A expression have not been investigated in teleost fishes. As a first step in understanding the molecular mechanisms of hypoxia modulation of CYP1A expression in fish, we characterized CYP1A cDNA from croaker liver. Hypoxia exposure (dissolved oxygen, DO: 1.7 mg/L for 2 to 4 weeks) caused significant decreases in hepatic CYP1A mRNA and protein levels compared to CYP1A levels in fish held in normoxic conditions. In vivo studies showed that the nitric oxide (NO)-donor, S-nitroso-N-acetyl-DL-penicillamine, significantly decreased CYP1A expression in croaker livers, whereas the competitive inhibitor of NO synthase (NOS), Nω-nitro-L-arginine methyl ester, restored CYP1A mRNA and protein levels in hypoxia-exposed (1.7 mg DO/L for 4 weeks) fish. In vivo hypoxia exposure also markedly increased interleukin-1β (IL-1β, a cytokine), HIF-2α mRNA and endothelial NOS (eNOS) protein levels in croaker livers. Pharmacological treatment with vitamin E, an antioxidant, lowered the IL-1β, HIF-2α mRNA and eNOS protein levels in hypoxia-exposed fish and completely reversed the down-regulation of hepatic CYP1A mRNA and protein levels in response to hypoxia exposure. These results suggest that hypoxia-induced down-regulation of CYP1A is due to alterations of NO and oxidant status, and cellular IL-1β and HIF-α levels. Moreover, the present study provides the first evidence of a role for antioxidants in hepatic eNOS and IL-1β regulation in aquatic vertebrates during hypoxic stress.

Development and Use of Real-Time Reverse Transcription-Polymerase Chain Reaction Assay to Quantify Cytochrome P4501A1 Expression in American Mink

The distribution of natural populations of American mink is restricted to locales that are in proximity to aquatic ecosystems. Because of the lipophilicity and persistence of polychlorinated biphenyls (PCBs) and reliance of mink on aquatic-based diets, mink at contaminated locales often bioacccumulate high levels of PCBs. In addition, in controlled laboratory studies, mink are highly sensitive at reproductive and developmental end points to the toxic effects of environmental PCB mixtures. It is believed that most, if not all, toxic effects of PCBs occur through activation of the aryl hydrocarbon receptor (AHR) pathway. Transcription of cytochrome P4501A1 (CYP1A1) by PCBs is also mediated through activation of AHR. Thus, levels of CYP1A1 mRNA provide a quantitative assay of exposure to and early biologic effect of PCBs on mink and may be predictive of toxicity at higher levels of biologic organization. We developed polymerase chain reaction (PCR) primers to amplify CYP1A1 as well as identified a housekeeping gene from mink cDNA. We used real-time reverse transcription-PCR to quantify and compare levels of hepatic CYP1A mRNA among groups of ranched mink kits and juveniles, which were fed diets or exposed in utero to fish that were low in PCBs (Atlantic herring) or to diets that were contaminated with three different levels of PCBs (carp) from Saginaw Bay, Lake Michigan. We found significant differences in CYP1A1 mRNA expression between mink fed the control diet and those fed a PCB-contaminated carp diet at all three treatment levels and exposure times. CYP1A1 mRNA was significantly induced 5.3- to 6.6-fold and 3.7- to 4.7-fold at 6 and 27 weeks, respectively. In previous studies, dietary exposures to PCB-contaminated carp were shown to cause mild to moderate lesions in the mandible and maxilla of these animals. This study demonstrates that hepatic CYP1A1 mRNA may be a sensitive biomarker of exposure of mink to environmentally relevant levels of PCBs and may be predictive of their effects in natural populations.

High dose of commercial products of kava ( Piper methysticum) markedly enhanced hepatic cytochrome P450 1A1 mRNA expression with liver enlargement in rats

Commercial products containing the kava plant ( Piper methysticum), known to have the anxiolytic activity, are banned in several European countries and Canada because of the suspicion of a potential liver toxicity. In some reports, kava and kavalactones (major constituents of kava) inhibited activities of cytochrome P450 (CYP) isoforms including CYP1A2. On the other hand, a few studies showed that administration of kava to rats moderately increased CYP1A2 proteins in the liver. CYP1A isoforms are likely responsible for the metabolic activation of potent carcinogenic environmental toxins such as aflatoxins, benzo[ a]pyrene, and others. On these bases, we have investigated the effects of administration of commercial kava products on gene expression of hepatic CYP1A isoforms in rats. A high dose (equivalent to approximately 380 mg kavalactones/kg/day; 100 times of the suggested dosage for human use) of two different types of kava products for 8 days significantly increased liver weights. CYP1A2 mRNA expression was moderately increased (2.8–7.3 fold). More importantly, the high dose of kava markedly enhanced CYP1A1 mRNA expression (75–220 fold) as well as ethoxyresorufin O-deethylase activities and CYP1A1 immunoreactivities. Thus, no observed adverse effect levels of kavalactones would be lower than 380 mg/kg/day. When the safety factor of kavalactones is assumed to be 100, a value most often used upon the risk analysis of chemicals and designed to account for interspecies and intraspecies variations, a number of kava product users likely ingest more kavalactones than acceptable daily intakes. Based on overall evidence, we should pay considerable attention to the possibility that kava products induce hepatic CYP1A1 expression in human especially in sensitive individuals.

Research Note: Quantification of Increase in Hepatic CYP1A1 mRNA by Real-Time RT-PCR after Exposure of the European Eel (Anguilla anguilla) to a Diesel Oil Water Soluble Fraction

Editors' note: Research Notes is a new section now being added to the EBI format. Its purpose is to provide a forum for the publication of short research communications that provide important information but, due to their restricted focus, are not formatted in the manner of full research papers. All papers published under this heading will be peer reviewed. In order to follow the effects of pollutants such as polycyclic aromatic hydrocarbons (PAHs), polychlorobenzenes (PCBs), and dioxins upon the European eel, we have developed an assay that quantifies hepatic cytochrome P4501A1 (CYP1A1) mRNA levels using real-time reverse transcription polymerase chain reaction (real-time RT-PCR). In this study, European eels were exposed to a diesel water soluble fraction (DWSF) in order to mimic environmental exposure to a mixture of organic pollutants. CYP1A1 mRNA levels were measured after 3, 6, 12, 24, 48, 72 and 156 h of exposure. The 7-ethoxyresorufin O-deethylase (EROD) activity was measured in parallel to confir...

Sulindac regulates the aryl hydrocarbon receptor-mediated expression of Phase 1 metabolic enzymes in vivo and in vitro

Sulindac, a widely used non-steroidal anti-inflammatory drug (NSAID), has been shown to inhibit chemically induced carcinogenesis in animal models. In the present study, we have investigated the molecular mechanism by which sulindac affects the activity and expression of the enzymes that mediate the initial detoxification steps of many environmental carcinogens, the cytochromes P450 1A1, 1A2 and 1B1. Sulindac treatment of Sprague-Dawley rats resulted in a dose-dependent increase in hepatic cytochrome P450 (CYP) enzyme activity and in the expression of hepatic CYPs 1A1 and 1B1 mRNA. In the HepG2 human liver cancer cell line, sulindac caused a sustained, dose-dependent increase in CYP enzyme activity. Sulindac treatment resulted in a profound, dose-dependent increase in CYP 1A1 mRNA and a modest increase in 1A2 mRNA. The increase in CYP 1A1 mRNA induced by sulindac was, like enzyme activity, sustained for several days after the initial treatment. Sulindac induced the transcription of the CYP1A1 gene, as measured by the level of heterogeneous nuclear 1A1 RNA and by actinomycin D chase experiment. Since the transcription of CYP1A1 is under the control of the aryl hydrocarbon receptor (AhR), we examined the ability of sulindac to activate the receptor. Sulindac bound to the AhR, as measured by ligand-binding assay, and induced the binding of the AhR with the xenobiotic-responsive element present in the promoter region of the CYP1A1 gene. These results are the first demonstration that NSAIDs modulate carcinogen metabolic enzymes and provide a novel mechanism to explain the established chemopreventive activity of sulindac.

Comparison of hepatic and extra hepatic induction of cytochrome P4501A by graded doses of aryl hydrocarbon receptor agonists in Atlantic tomcod from two populations

Atlantic tomcod Microgadus tomcod from the Hudson River, New York, are exposed to high levels of polycyclic aromatic hydrocarbons (PAHs) and bioaccumulate mixtures of polychlorinated biphenyls (PCBs), polychlorinated dibenzo- p-dioxins and polychlorinatedfurans (PCDD/Fs). Previous studies demonstrated that hepatic cytochrome P4501A (CYP1A) mRNA was not inducible in tomcod from the Hudson River treated with single doses of PCB77 or 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD), but was inducible with PAHs. In this study, we sought to determine if CYP1A mRNA was inducible with higher doses of these and other halogenated aromatic hydrocarbons (HAHs) in Hudson River tomcod and if decreased sensitivity to gene inducibility occurs across all tissues. Tomcod from the Hudson River and the cleaner Miramichi River, New Brunswick, were treated individually with graded doses of TCDD and coplanar PCBs (PCB77, PCB81, PCB126, PCB169) and profiles of hepatic CYP1A mRNA expression were compared between the two populations. CYP1A mRNA inducibility was also compared in multiple tissues of tomcod from the two rivers that were treated with PCB77. Additionally, hepatic CYP1A mRNA was characterized in Miramichi River tomcod treated with pairs of PCB congeners that included aryl hydrocarbon receptor (AHR) agonists and antagonists. Hepatic CYP1A mRNA was significantly inducible by all agonists in tomcod from the Miramichi River and TCDD and two of four PCBs in tomcod from the Hudson River. CYP1A mRNA was also significantly inducible in four of five tissues of tomcod from the Miramichi River but only in liver of Hudson River tomcod. In summary, CYP1A mRNA inducibility was approximately two orders of magnitude less sensitive in tomcod from the Hudson River than in those from the Miramichi River. But when achieved, maximum levels of CYP1A expression were similar in tomcod from the two populations. Co-administration of PCB126 and PCB77 did not produce significantly greater CYP1A mRNA induction than administration of PCB126 alone and co-administration of mono- ortho-substituted PCB105 significantly decreased CYP1A mRNA inducibility by PCB77. These results indicate that CYP1A mRNA expression is significantly inducible by HAHs in tomcod from the Hudson River and suggest that all components of the AHR pathway are present and functional, but that the pathway is less sensitive to activation than in tomcod from the Miramichi River. Our results also indicate that CYP1A mRNA levels in environmentally exposed fish may not reflect additive tissue burdens of PCB congeners.

Co-exposure to metals modulates CYP1A mRNA inducibility in Atlantic tomcod Microgadus tomcod from two populations

Populations from urbanized and industrialized sites are often exposed to mixtures of chemical contaminants including aromatic hydrocarbons (AHs) and heavy metals. The effects of mixtures of these contaminants on these populations are largely unknown. The Hudson River Estuary is highly contaminated with a variety of AHs including, PCBs and PAHs, and metals, and its population of Atlantic tomcod Microgadus tomcod bioaccumulates those which are persistent. The Hudson River's tomcod population exhibits resistance to persistent AHs as exemplified by significantly decreased inducibility of hepatic cytochrome P4501A (CYP1A) mRNA. We used hepatic CYP1A mRNA inducibility in tomcod from the Hudson River and a sensitive population to investigate the effects of acute co-exposure to metals on aryl hydrocarbon receptor (AHR)-mediated gene expression. Adult tomcod from the Hudson River and the cleaner Miramichi River were i.p. injected with one dose of benzo[ a]pyrene (B[ a]P) or coplanar PCB77 and graded doses of four metals, As, Cd, Cr, and Ni, and levels of hepatic CYP1A mRNA and protein were assayed. We observed no effects of metals treatment on basal levels of hepatic CYP1A mRNA expression, but all four metals significantly reduced CYP1A mRNA inducibility in tomcod from one or both populations. The magnitude of the inhibition of CYP1A mRNA inducibility differed among the metals and fish from the two populations. Also, the profile of the metals modulation of induced CYP1A mRNA showed differences that depended on the time after treatment of sacrifice. Our results demonstrate that co-exposure to several metals can impact inducible, but not basal levels of CYP1A expression and perhaps other toxicities mediated by the AHR.

Effect of pyrene on hepatic cytochrome P450 1A (CYP1A) expression in Nile Tilapia (Oreochromis niloticus).

The effect of pyrene on the regulation of the gene expression of cytochrome P4501A ( CYP1A) was studied in tilapia (Oreochromis niloticus), a tropical fish of great ecological and economical importance. To evaluate CYP1A mRNA, tilapia CYP1A cDNA was cloned, sequenced, and compared with those CYP1A reported sequences in the GeneBank DNA database. The top seven matches corresponded to CYP1A from other teleosts. Hepatic CYP1A mRNA levels showed a significant increase at day 1 after pyrene injection (20 mg kg(-1) body weight [BW]), and this CYP1A mRNA levels did not return to basal levels for up to 5 days. The immunoblot analysis of CYP1A protein levels using polyclonal rabbit-anti-trout antibodies in the liver of pyrene-treated (20 mg kg(-1) BW) tilapias showed a 1.9-fold increase at day 3 after injection. Ethoxyresorufin- O-deethylase (EROD) activity increased 18-fold with respect to control fish at day 3 after injection. CYP1A protein and EROD activity remained increased for 5 days after a single pyrene IP administration. Similarly, the highest concentration of 1-hydroxypyrene (1-OH pyrene) in bile was observed in fish sacrificed at day 3 after injection. EROD activity and 1-OH pyrene concentration showed a statistically significant correlation (r = 0.85) according to the Spearman test, suggesting the participation of this cytochrome in the biotransformation of pyrene.

Cytochrome P4501A1 is induced by PCB 77 and benzo[a]pyrene treatment but not by exposure to the Hudson River environment in Atlantic tomcod (Microgadus tomcod) post-yolk sac larvae

In fish, the embryos and larvae are the life-stages most sensitive to damage from environmentally borne dioxin-like compounds and polycyclic aromatic hydrocarbons (PAHs). Methods are not routinely available to measure the body burdens of contaminants in embryos and larvae, thus precluding the investigation of links between exposure and biological effects. Quantification of expression of cytochrome P4501A1 (CYP1A1) provides an index of relative exposure of natural populations to bioavailable aromatic hydrocarbons (AH) and an initial evaluation of their biological effects. We developed a quantitative approach to standardize total RNA loading and then used competitive reverse transcriptase-polymerase chain reaction (RT-PCR) to quantify the CYP1A1 mRNA expression in environmentally exposed Atlantic tomcod (Microgadus tomcod) post yolk-sac larvae (postlarvae) from the Hudson River, New York, and in chemically treated postlarval offspring of controlled laboratory crosses of Hudson River parents. Significant induction of CYP1A1 expression was observed in tomcod postlarvae exposed to waterborne 3,3',4,4'-tetrachlorobiphenyl (PCB 77) (four-fold) and benzo [a] pyrene (eight-fold) compared with vehicle-exposed controls. In contrast, CYP1A1 was not induced in Hudson River-exposed postlarvae compared with vehicle-exposed controls. This study demonstrates the feasibility of using competitive RT-PCR for the measurement of gene expression in environmentally exposed larvae of sentinel species, and is consistent with the hypothesis that postlarvae exposed to the Hudson River environment have not bioaccumulated sufficient levels of AHs to induce CYP1A1 expression. The high levels of hepatic CYP1A1 mRNA expression previously reported in 5-8 month old juvenile tomcod from the Hudson River coincides with their descent to the benthic habitat and the onset of independent feeding on AH-contaminated benthic prey.

Benzo[a]pyrene-Induced Toxicity: Paradoxical Protection in Cyp1a1(−/−) Knockout Mice Having Increased Hepatic BaP–DNA Adduct Levels

Previous studies have shown that cytochrome P450 1A1 (CYP1A1), CYP1B1, and prostaglandin-endoperoxide synthase (PTGS2) are inducible by benzo[a]pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin), and all three metabolize BaP to reactive DNA-binding intermediates and excreted products. Because these three enzymes show differing patterns of basal levels, inducibility, and tissue-specific expression, animal studies are necessary to delineate the role of CYP1A1 in BaP-mediated toxicity. In mice receiving large daily doses of BaP (500 mg/kg i.p.), Cyp1a1(−/−) knockout mice are protected by surviving longer than Cyp1a1(+/−) heterozygotes. We found that a single 500 mg/kg dose of BaP induces hepatic CYP1A1 mRNA, protein, and enzyme activity in Cyp1a1(+/−) but not in Cyp1a1(−/−) mice; TCDD pretreatment increases further the CYP1A1 in Cyp1a1(+/−) but not Cyp1a1(−/−) mice. Although a single 500 mg/kg dose of BaP was toxic to Cyp1a1(+/−) mice (serum liver enzyme elevated about 2-fold above control levels at 48 h), Cyp1a1(−/−) mice displayed no hepatotoxicity. Unexpectedly, we found 4-fold higher BaP–DNA adduct levels in Cyp1a1(−/−) than in Cyp1a1(+/−) mice; TCDD pretreatment lowered the levels of BaP–DNA adducts in both genotypes, suggesting the involvement of other TCDD-inducible detoxification enzymes. BaP was cleared from the blood much faster in Cyp1a1(+/−) than Cyp1a1(−/−) mice. Our results suggest that absence of the CYP1A1 enzyme protects the intact animal from BaP-mediated liver toxicity and death, by decreasing the formation of large amounts of toxic metabolites, whereas much slower metabolic clearance of BaP in Cyp1a1(−/−) mice leads to greater formation of BaP–DNA adducts.

Is hepatic cytochrome P4501A1 expression predictive of hepatic burdens of dioxins, furans, and PCBs in Atlantic tomcod from the Hudson River estuary?

Hepatic cytochrome P4501A1 (CYP1A1) expression in fishes is frequently used to evaluate bioavailable aromatic hydrocarbon contamination of aquatic ecosystems. In controlled laboratory experiments, CYP1A1 expression in naı̈ve fishes is usually dose-responsive to aromatic hydrocarbons and in field studies levels of gene expression in natural populations often correspond with known levels of sediment-borne contaminants. We quantified CYP1A1 mRNA levels in juvenile Atlantic tomcod Microgadus tomcod from 42 sites in the Hudson River estuary to evaluate the correspondence between hepatic CYP1A1 expression and hepatic concentrations of persistent halogenated aromatic hydrocarbons and to determine the utility of CYP1A1 expression as a biomarker in evaluating the microgeographic distribution of bioavailable contaminants within a large aquatic ecosystem. We found significant spatial heterogeneity in CYP1A1 mRNA levels among collection sites with levels of gene expression differing in some cases by 23–34 folds. CYP1A1 mRNA expression was highest in tomcod from the Newark Bay complex and lowest in tomcod from the most upriver collection sites in the main stem of the Hudson River. Although levels of PCDDs, PCDFs, and PCBs expressed as TCDD TEQs and CYP1A1 mRNA were highest in tomcod from the Newark Bay complex, there was no relationship between hepatic halogenated aromatic hydrocarbon levels and hepatic CYP1A1 mRNA in tomcod from sites in the main stem of the Hudson River. These results suggest that levels of CYP1A1 expression in fish from sites highly polluted with mixtures of halogenated aromatic hydrocarbons and other xenobiotics may not always be reflective of levels of bioavailable aromatic hydrocarbon contaminants. Based on these results and earlier controlled laboratory experiments, we hypothesize that elevated levels of CYP1A1 expression in tomcod from the Hudson River may be due primarily to PAHs or other contaminants not measured in this study.

An evaluation of the etiology of reduced CYP1A1 messenger RNA expression in the Atlantic tomcod from the Hudson River, New York, USA, using reverse transcriptase polymerase chain reaction analysis

Adult Atlantic tomcod, Microgadus tomcod, from the Hudson River, New York State, USA, exhibit reduced inducibility of hepatic cytochrome P4501A1 (CYP1A1) mRNA compared with adult tomcod from the cleaner Miramichi River, New Brunswick, Canada, when treated with coplanar polychlorinated biphenyl (PCB) congeners or 2,3,7,8-tetrachlorodibenzo-p-dioxin. In contrast, little difference in CYP1A1 inducibility is observed between tomcod from these two rivers when treated with polycyclic aromatic hydrocarbons (PAHs). We sought to determine if impaired hepatic CYP1A1 inducibility in Hudson River tomcod results from a multigenerational, genetic adaptation or a single generational, physiological acclimation. Embryos and larvae from controlled experimental crosses of Hudson River and Miramichi River parents were exposed for 24 h to water-borne PCB congener 77 (10 ppm), benzo[a]pyrene (BaP; 10 ppm), or dimethysulfoxide, and CYP1A1 expression was assessed in individual larva using competitive reverse transcriptase polymerase chain reaction (RT-PCR) analysis. The CYP1A1 mRNA was significantly induced in larvae from both populations by BaP (47- and 52-fold) and PCB 77 (9- and 22-fold), although levels of expression were higher in offspring of Miramichi matings. Most important, CYP1A1 mRNA was significantly induced by PCB 77 in larvae from Hudson River parents. Concentrations of dioxin, furan, and PCB congeners were measured in livers and eggs of female tomcod from these two locales to quantify the extent of maternal transfer of contaminants. For both rivers, wet-weight contaminant concentrations were significantly higher (4-7 times) in livers than in eggs of the same females, suggesting that a threshold level of contaminants may have to be reached before CYP1A1 transcription is impaired. We conclude that reduced inducibility of hepatic CYP1A1 mRNA in adult tomcod from the Hudson River is most consistent with single-generational acclimation.

Ecotoxicological assessment of persistent organic and heavy metal contamination in Hong Kong coastal sediment.

The aim of the present project is to determine the feasibility of measuring hepatic cytochrome P4501A1 (CYP1A1) and metallothionein (MT) mRNA in fish as an integrative measurement of persistent organic pollutants (POPs) and heavy metal contamination in sediment arising in Hong Kong. Sediment samples were collected from different sites, including Victoria Harbour (VS6), Yim Tin Tsai (YTT) at Tolo Harbour, Mai Po marshes (MPM) at Deep Bay, and Southern Waters (SS6) of coastal waters. The samples were analyzed for total and extractable concentrations of Cd, Cu, Ni, Zn, and Pb, as well as PCBs and PAHs. In addition, biomarker responses were studied in tilapia exposed experimentally to coastal sediment for 7 days. Using RT-PCR technique, hepatic CYP1A1 and MT mRNA were measured. Three control groups were used, including one negative control group maintained in sea water only; the second and third positive control groups were in sea water but were intraperitoneally injected with either beta-naphthoflavone (40 microg/g body weight) or cadmium chloride (10 microg/g body weight), respectively. The chemical data showed that VS6, YTT, and MPM were classified as Class C sediment according to the sediment quality criteria defined by the Hong Kong Environmental Protection Department, indicating the sites were heavily polluted. The exposure of tilapia to the sediment induced hepatic CYP1A1 (VS6 > YTT > MPM > SS6) and MT (VS6 > MPM > YTT > SS6) levels. The induction patterns were comparable to the levels of POPs and metal contamination in the sediment, indicating that the biomarker responses could be used to differentiate low to high levels of contamination among sediment.

A comparison of the dose and time response of CYP1A1 mRNA induction in chemically treated Atlantic tomcod from two populations

Quantification of cytochrome P4501A1 (CYP1A1) mRNA levels in environmentally exposed Atlantic tomcod ( Microgadus tomcod) has revealed significantly induced gene expression in fish from contaminated locales including the Hudson River, New York, and the Miramichi River, New Brunswick. In order to calibrate this response, determine its sensitivity and dose-responsiveness, levels of hepatic CYP1A1 mRNA were quantified in depurated Atlantic tomcod intraperitoneally (i.p.) injected with various concentrations of: β-naphthoflavone (β-NF), the PAH benzo[ a]pyrene (B[ a]P), the non- ortho coplanar PCB congener-3,3′,4,4′- tetrachlorobiphenyl (IUPAC: PCB-77), and the dioxin 2,3,7,8-tetrachlorodibenzo -p-dioxin (2,3,7,8-TCDD). Additionally, the rates of CYP1A1 mRNA induction and disappearance were quantified in depurated Atlantic tomcod i.p. injected with single doses of these chemicals and sacrificed at times ranging up to 72 days. Levels of CYP1A1 mRNA were dose-responsive for all four chemicals with maximum induction ranging from 50- to 460-fold and first significant induction being observed in the low mg per kg fish (wet weight) range for β-NF and B[ a]P, μg/kg range for PCB-77 and ng/kg range for 2,3,7,8-TCDD. However, while tomcod from the Miramichi River responded to both PAHs and halogenated aromatic hydrocarbons (HAHs), Hudson River tomcod responded only to PAHs indicating population level differences in CYP1A1 mRNA inducibility in tomcod. Furthermore, differences in the responsiveness to PAHs and HAHs suggest that more than one molecular mechanism mediates CYP1A1 transcription in Atlantic tomcod. Kinetic profiles of CYP1A1 mRNA induction differed greatly between tomcod treated with HAHs and PAHs. Initial induction occurred within hours of treatment with PAHs and peaked after 1–3 days, compared to initial induction 4–7 days after treatment with HAHs, and maximum induction not occurring for up to 72 days after exposure. Quantification of halogenated aromatic hydrocarbons (HAH) in the livers of tomcod caught in the Hudson and Miramichi Rivers confirmed exposure and accumulation of known CYP1A1 inducing chemicals including 2,3,7,8-TCDD at concentrations as high as 1.5 μg/kg lipid (554 ng/kg w.w.) and PCB-77 at concentrations as high as 108 μg/kg lipid (15 μg/kg w.w.). These results suggest that hepatic CYP1A1 mRNA concentration can be a useful bioindicator of exposure to some aromatic hydrocarbon compounds in the aquatic environment and that profiles of gene induction and disappearance may help identify environmental inducers provided that gene responsiveness is also evaluated under controlled laboratory conditions.

Regulation of Cytochrome P4501A Metabolism by Glutathione

Gene expression of cytochrome P4501A (CYP1A) in the rainbow trout Oncorhynchus mykiss is dependent on aromatic hydrocarbon receptor signal transduction, and is markedly sensitive to tissue thiol status. Tissue glutathione (GSH) status was manipulated by exogenous GSH, L-buthionine-[S,R]-sulfoximine (BSO), lipoate or 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Tissue GSH contents were significantly elevated in GSH- and lipoate-supplemented trout. Hepatic, renal and plasma GSH levels were markedly arrested in BSO-treated trout. Oxidized glutathione (oxidized GSH) levels were significantly elevated in the BCNU-supplemented group. Both BCNU treatment and BSO-induced GSH deficiency increased steady-state levels of hepatic CYPIA mRNA. Additional exposure to 0.1 mg/kg 3,3',4,4'-tetrachlorobiphenyl marginally suppressed the tetrachlorobiphenyl-dependent CYP1A induction in BSO-treated livers compared with the respective thiol treatment groups. Tetrachlorobiphenyl exposures altered efficiencies of thiol treatments and increased oxidized GSH content in all but the BSO-treated groups. However, exposure to 5 mg/kg tetrachlorobiphenyl altered effects of thiol treatments on CYP1A mRNA to a small extent, but catalytic activity of CYP1A was many times suppressed in BSO-treated and lipoate-supplemented fish. These results suggest that thiol status interferes with CYPIA metabolism in a two-way mode of action and provide further evidence for a cross-talk between cytochrome P4501A and glutathione.

Strain Differences in Cytochrome P4501A1 Gene Expression Caused by 2,3,7,8-Tetrachlorodibenzo-p-dioxin in the Rat Liver: Role of the Aryl Hydrocarbon Receptor and Its Nuclear Translocator

Rat strain variation in hepatic cytochrome P4501A1 (CYP1A1) gene expression caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was investigated along with possible underlying mechanism. TCDD at a single oral dose of 13.5 ng/kg body weight significantly increased hepatic CYP1A1 mRNA expression in DRH, Long–Evans Cinamon (LEC), Long–Evans (LE), and Holtzman (HO) rats, but not in Sprague–Dawley (SD), Wistar–Imamichi (WI), Lewis (LEW), and Fisher-344 (F344) strains. All showed significant induction of CYP1A1 mRNA at a dose of 40 ng/kg, the relative levels decreasing in the order DRH, LEC, HO, LE, F344, WI, LEW, and SD. A more than 35-fold difference in the induction of CYP1A1 RNA was evident between the DRH and SD strains. Based on CYP1A1 induction, classification into two distinctly separate groups was possible, high responders (DRH, LEC, HO, and LE) and low responders (SD, LEW, WI, and F344). The expression levels closely correlated with the steady-state aryl hydrocarbon receptor (AhR) mRNA expression, this being approximately four-fold higher in the high than in the low responder group. Analysis of the aryl hydrocarbon receptor nuclear translocator (ARNT) showed the presence of a wild type as well as an alternately spliced variant in all strains, with a 45-bp deletion whose sequence corresponded to part of 5′ end of the basic region of the basic helix–loop–helix domain. Expressed levels of both products were almost equal in all the strains except DRH, LEC and HO, where the wild form predominated. The results suggest that differential expression of both AhR and ARNT are responsible for rat strain-specific differences in TCDD induced CYP1A1 expression.

Disruption of endogenous regulator homeostasis underlies the mechanism of rat CYP1A1 mRNA induction by metyrapone.

The transcriptional induction of the cytochrome P-450 1A1 (CYP1A1) gene by xenobiotics such as polyaromatic hydrocarbons is dependent on their interaction with the aryl hydrocarbon receptor. Administration of the structurally unrelated compounds metyrapone (a cytochrome P-450 inhibitor) or dexamethasone (a glucocorticoid) to male rats does not induce hepatic CYP1A1 mRNA. However, administration of both metyrapone and dexamethasone to male rats results in the induction of hepatic CYP1A1 mRNA expression. The induction response is mimicked in vitro in cultured rat hepatocytes by the addition of metyrapone and dexamethasone to a serum-free culture medium, suggesting that these compounds act directly on the liver in vivo to effect hepatic CYP1A1 mRNA induction. An examination of the characteristics of CYP1A1 induction by metyrapone and dexamethasone in combination in vitro indicate that at least 6 h of treatment is required for detectable levels of CYP1A1 mRNA to accumulate in hepatocytes. In contrast, beta-naphthoflavone, which is known to bind to the aryl hydrocarbon receptor to effect CYP1A1 gene expression, induces detectable levels of CYP1A1 mRNA within 2 h of treatment. CYP1A1 mRNA is also induced when hepatocytes are treated with metyrapone in combination with the protein synthesis inhibitor cycloheximide but not with dexamethasone in combination with cycloheximide, indicating that CYP1A1 mRNA induction is strictly dependent on the presence of metyrapone and suggesting that the metyrapone-associated induction of CYP1A1 mRNA is dependent on a loss of a constitutively expressed protein that functions to suppress CYP1A1 gene expression. The role of dexamethasone in metyrapone-associated induction of CYP1A1 is probably mediated through the glucocorticoid receptor since the glucocorticoid receptor antagonist RU486 reduces the levels of CYP1A1 mRNA induced by metyrapone and dexamethasone in combination. Increasing the levels of the photosensitizer riboflavin present in the culture medium 10-fold and exposure to light increases the levels of CYP1A1 mRNA induced by metyrapone and dexamethasone in combination in vitro, suggesting that photoactivation of inducing medium constituent(s) might be required for induction. Failure to induce CYP1A1 mRNA by co-administration of metyrapone and dexamethasone in hepatocytes cultured in a balanced salt solution with or without photoactivation indicates that induction is dependent on a photoactivated component of the culture medium and not on metyrapone or dexamethasone alone. The addition of tryptophan in the presence of riboflavin to the balanced salt solution restores CYP1A1 mRNA induction by metyrapone alone and induction is increased when medium is exposed to light, indicating that induction is dependent on tryptophan photoactivation in vitro. Metyrapone failed to compete with 2,3,7,8-tetrachlorodibenzo-p-dioxin for specific binding to the aryl hydrocarbon receptor in rat liver cytosolic fractions. These results suggest that CYP1A1 might be induced in rats by metyrapone through an indirect mechanism associated with an elevation in the level of an endogenously generated inducer such as photoactivated product(s) of tryptophan and not because of metyrapone's interacting with the aryl hydrocarbon receptor. The dependence of CYP1A1 induction on dexamethasone or cycloheximide suggests that derepression by a glucocorticoid receptor-modulated negative-acting factor of CYP1A1 gene expression might be critical to induction by metyrapone.

Induction and post-transcriptional suppression of hepatic cytochrome p450 1a1 by 3,3′,4,4′-tetrachlorobiphenyl

3,3′,4,4′-Tetrachlorobiphenyl (TCB) can induce and inhibit cytochrome P450 1A1 (CYP1A1) in vertebrates. TCB may also suppress CYP1A1 protein levels, but the mechanism is unknown. This study examined transcriptional and translational aspects of hepatic CYP1A1 regulation in the fish scup (Stenotomus chrysops) given single intraperitoneal injections of low (0.1 mg/kg) or high (5 mg/kg) doses of TCB, and sampled over 16 days. The low dose strongly induced hepatic CYP1A1 mRNA (25-fold), protein (12-fold), and activity [ethoxyresorufin O-deethylase (EROD)](15-fold). The high dose also strongly induced CYP1A1 mRNA (29-fold), in a pattern like that at the low dose, but microsomal CYP1A1 protein content was induced only 4-fold and EROD rates were near control levels. Both TCB doses caused similar increases in microsomal cytochrome b 5 content, and in rates of NADPH-cytochrome c (P450) reductase and UDP-glucuronosyltransferase (with p-nitrophenol). The contents of CYP forms other than CYP1A1 (putative CYP2B or CYP3A) were only weakly affected by TCB at either dose. The strong and largely specific post-transcriptional suppression of CYP1A1 content was associated with high concentrations of TCB measured in the liver. Incubation of scup hepatic microsomes with TCB plus NADPH led to a time-dependent inactivation of CYP1A1 that was distinct from catalytic inhibition, and appeared not to involve reactive metabolites of TCB. This in vitro result suggests that TCB may inactivate CYP1A1 in vivo, which could account for the apparent antagonistic effect of TCB on CYP1A1 induction.