Cytochrome P450 1A1 mRNA Levels Research Articles

Transcriptional regulation of human CYP3A4 basal expression by CCAAT enhancer-binding protein alpha and hepatocyte nuclear factor-3 gamma.

Cytochrome P450 3A4 (CYP3A4) is involved in the metabolism of more than 50% of currently used therapeutic drugs, yet the mechanisms that control CYP3A4 basal expression in liver are poorly understood. Several putative binding sites for CCAAT/enhancer-binding protein (C/EBP) and hepatic nuclear factor 3 (HNF-3) were found by computer analysis in CYP3A4 promoter. The use of reporter gene assays, electrophoretic mobility shift assays, and site-directed mutagenesis revealed that one proximal and two distal C/EBP alpha binding sites are essential sites for the trans-activation of CYP3A4 promoter. No trans-activation was found in similar reporter gene experiments with a HNF-3 gamma expression vector. The relevance of these findings was further explored in the more complex DNA/chromatin structure within endogenous CYP3A4 gene. Using appropriate adenoviral expression vectors, we found that both hepatic and nonhepatic cells overexpressing C/EBP alpha had increased CYP3A4 mRNA levels, but no effect was observed when HNF-3 gamma was overexpressed. In contrast, overexpression of HNF-3 gamma simultaneously with C/EBP alpha resulted in a greater activation of the CYP3A4 gene. This cooperative effect was hepatic-specific and also occurred in CYP3A5 and CYP3A7 genes. To investigate the mechanism for HNF-3 gamma action, we studied its binding to CYP3A4 promoter and the effect of the deacetylase inhibitor trichostatin A. HNF-3 gamma was able to bind CYP3A4 promoter at a distal position, near the most distal C/EBP alpha binding site. Trichostatin A increased C/EBP alpha effect but abolished HNF-3 gamma cooperative action. These findings revealed that C/EBP alpha and HNF-3 gamma cooperatively regulate CYP3A4 expression in hepatic cells by a mechanism that probably involves chromatin remodeling.

Effect of genotype on steady-state CYP1A1 gene expression in human peripheral lymphocytes

We have analyzed the steady-state levels of cytochrome P-450 1A1 (CYP1A1) mRNA in peripheral blood lymphocytes of 177 individuals with various CYP1A1 genotypes using a quantitative reverse transcriptase–polymerase chain reaction technique that makes use of a homologous internal standard for accurate quantitation. We found no effects of ethnicity, age, or smoking status on CYP1A1 gene expression in this population. We did see a significant 2-fold increase in the mean level of CYP1A1 mRNA in women compared with men for both Caucasians and African Americans. We observed no effect of the African American-specific polymorphism ( CYP1A1 ∗3 ) on expression of the gene. However, we found a significant 3-fold decrease in expression associated with the homozygous MspI restriction fragment length polymorphism ( CYP1A1 ∗2A/ ∗2A ).

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 (CYP1A). 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 CYP1A 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 (1770±1693.2 DPM) relative to Flax fish (6082.5±1709.9 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.

Resistance of MCF-7 cells to dimethylbenz(a)anthracene-induced apoptosis is due to reduced CYP1A1 expression

We have developed a series of aryl hydrocarbon (AH)-resistant cell lines derived from MCF-7 human breast epithelial cancer cells by continuous exposure to the AH benzo[a]pyrene. These cell lines display cross-resistance to the mammary carcinogen dimethylbenz[a]anthracene (DMBA). Apoptosis induced by exposure to DMBA is greatly decreased in the resistant cell lines compared to the wild-type, in proportion to the level of resistance. Apoptosis induced by DMBA could be blocked by inhibitors of DMBA metabolism such as alpha-naphthoflavone and diosmetin. We therefore examined the resistant cell lines for their ability to metabolize DMBA and for the formation of DMBA-DNA adducts, and found that both parameters were decreased compared to wild-type cells in proportion to the level of resistance. When exposed to DMBA or 2,3,7,8-tetrachlorodibenzo-p-dioxin, the resistant cell lines have a diminished capacity to carry out ethoxyresorufin-O-deethylation, indicating that the induction of cytochrome P450 1A1 (CYP1A1) enzyme is impaired. We therefore examined the expression of the CYP1A1 gene, and found reduced levels of both CYP1A1 mRNA and CYP1A1-promoter controlled transcription in resistant cells compared to the wild-type. The deleterious effects of AHs are believed to be mediated by the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor which regulates CYP1A1 expression. Resistant cell lines had a reduced expression of the AhR, as measured at the mRNA and protein levels. These data demonstrate that AH resistance in these cells is mediated by changes in the signal transduction pathway which regulates CYP1A1 expression.

CYP3A4 induction by drugs: correlation between a pregnane X receptor reporter gene assay and CYP3A4 expression in human hepatocytes.

Induction of cytochrome P450 3A4 (CYP3A4) is determined typically by employing primary culture of human hepatocytes and measuring CYP3A4 mRNA, protein and microsomal activity. Recently a pregnane X receptor (PXR) reporter gene assay was established to screen CYP3A4 inducers. To evaluate results from the PXR reporter gene assay with those from the aforementioned conventional assays, 14 drugs were evaluated for their ability to induce CYP3A4 and activate PXR. Sandwiched primary cultures of human hepatocytes from six donors were used and CYP3A4 activity was assessed by measuring microsomal testosterone 6beta-hydroxylase activity. Hepatic CYP3A4 mRNA and protein levels were also analyzed using branched DNA technology/Northern blotting and Western blotting, respectively. In general, PXR activation correlated with the induction potential observed in human hepatocyte cultures. Clotrimazole, phenobarbital, rifampin, and sulfinpyrazone highly activated PXR and increased CYP3A4 activity; carbamazepine, dexamethasone, dexamethasone-t-butylacetate, phenytoin, sulfadimidine, and taxol weakly activated PXR and induced CYP3A4 activity, and methotrexate and probenecid showed no marked activation in either system. Ritonavir and troleandomycin showed marked PXR activation but no increase (in the case of troleandomycin) or a significant decrease (in the case of ritonavir) in microsomal CYP3A4 activity. It is concluded that the PXR reporter gene assay is a reliable and complementary method to assess the CYP3A4 induction potential of drugs and other xenobiotics.

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.

Effect of lactational exposure to 1,2,3,4- tetrachlorodibenzo-p-dioxin on cytochrome P-450 1A1 mRNA in the neonatal rat liver: quantitative analysis by the competitive RT-PCR method.

The aim of this study was to assess the effect of lactational exposure to dioxins in neonates on the cytochrome P450 1A1 (CYP1A1) induction in the level of gene expression. Maternal rats were treated with a single dose of 50 or 100 micromol/kg 1,2,3,4-tetrachlorodibenzo-p-dioxin (1,2,3,4-TCDD), a low potent congener of dioxins, on the first day post-partum (day 1). Induction of CYP1A1 mRNA expression was quantitatively analyzed by the competitive reverse transcription-polymerase chain reaction (RT-PCR) method. The CYP1A1 mRNA was detectable at extremely low amounts in the liver of control neonates and mothers. The mRNA ratios of CYP1A1 to beta-actin in neonates were dose-dependently increased by the treatment of 1,2,3,4-TCDD of their mothers. Its peak occurred on day 6 and was sustained at the same level on day 10. Increases of the ratio with 100 micromol/kg 1,2,3,4-TCDD on day 2, 6 and 10 were 26-, 40- and 40-fold of the appropriate controls, respectively. These levels paralleled the activity of ethoxyresorufin-o-deethylase, representing CYP1A mediated monooxygenase. In the mother, the mRNA ratio was increased only to threefold of the control, 10 days after treatment. Current RT-PCR procedure enabled to assess both constitutive and induced levels of CYP1A1 mRNA in the neonatal rat livers. Although the dose of 1,2,3,4-TCDD selected in this study was about 5000 times higher than the daily intake of dioxins in breast-fed infants, CYP1A1 mRNA was highly induced for a longer period of time in neonatal rats receiving 1,2,3,4-TCDD via lactation than the treated maternal rats.

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.

Specific expression of cytochrome P4501A1 gene in gill, intestine and liver of tilapia exposed to coastal sediments

Toxicological effects of persistent organic pollutants (POPs) in aquatic ecosystems lead to the deterioration of water quality and adversely affect fish and human health. The highly lipophilic nature of these pollutants may enter fish through the diet or by water-borne exposure. In monitoring contamination in aquatic systems, induction of the cytochrome P450 1A1 ( CYP1A1) gene of fish has been evaluated as a sensitive, ‘early warning’ method. The objective of the present study was to characterize the induction of the gene in fish upon exposure to coastal sediments and to determine its specific expression in liver and extrahepatic organs (i.e. gill and intestine) in which the toxicological effects to the corresponding tissues could be addressed. Sediment samples were collected from different sites, including Victoria Harbour (VS), Ma Wan (MW), Tsim Bei Tsui (TBT) and Mai Po (MP). The samples were analyzed for polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs). CYP1A1 mRNA expression was measured in juvenile tilapia exposed experimentally to coastal sediment for 3 and 7 days. A negative control group of fish maintained in seawater was used. Using the primer dropping polymerase chain reaction technique, gill, intestinal and hepatic CYP1A1 mRNAs were quantified. Chemical analysis shows that the samples from VS contaminated with the highest concentration of PCBs (45.24 p.p.b.) and PAHs (1663.7 p.p.b.), followed by MW (16.01 and 347.7 p.p.b.), TBT (14.48 and 235.2 p.p.b.) and MP (14.60 and 242.2 p.p.b.). Fish exposed to sediments were contaminated with various levels of PCBs (VS, 64.14–72.06 p.p.b.; MP, 27.06–31.62 p.p.b.; TBT, 27.29–33.92 p.p.b.; MW, 16.05–17.76 p.p.b.) and PAHs (VS, 124.7–304.9 p.p.b.; MP, 97.57–164.1 p.p.b.; TBT, 25.38–98 p.p.b.; MW, 24.07–68.13 p.p.b.). The control fish displayed moderate expression of CYP1A1 mRNA in liver (1.45 arbitrary units), gill (1.21 arbitrary units) and intestine (0.56 arbitrary units). Following sediment exposure, there was a large increase in CYP1A1 mRNA in intestine and liver but no significant changes in gill tissues. In the first 3 days, there was anywhere from 34–96 and 69–156% induction of the CYP1A1 transcripts in intestine and liver, respectively. Following 7 days of exposure, a continued induction of high level of CYP1A1 mRNA in intestine (73–157%) was observed. The induction of CYP1A1 in liver and intestine provided a defensive mechanism against POPs entering from the external environment.

Accelerated DNA adduct formation in the lung of the Nrf2 knockout mouse exposed to diesel exhaust.

Diesel exhaust (DE) has been recognized as a noxious mutagen and/or carcinogen, because its components can form DNA adducts. Mechanisms governing the susceptibility to DE and the efficiency of such DNA adduct formation require clarification. The transcription factor Nrf2 is essential for inducible and/or constitutive expression of a group of detoxification and antioxidant enzymes, and we hypothesized that the nrf2 gene knockout mouse might serve as an excellent model system for analyzing DE toxicity. To address this hypothesis, lungs from nrf2(-/-) and nrf2(+/-) mice were examined for the production of xenobiotic-DNA adducts after exposure to DE (3 mg/m(3) suspended particulate matter) for 4 weeks. Whereas the relative adduct levels (RAL) were significantly increased in the lungs of both nrf2(+/-) and nrf2(-/-) mice upon exposure to DE, the increase of RAL in the lungs from nrf2(-/-) mice exposed to DE were approximately 2.3-fold higher than that of nrf2(+/-) mice exposed to DE. In contrast, cytochrome P4501A1 mRNA levels in the nrf2(-/-) mouse lungs were similar to those in the nrf2(+/-) mouse lungs even after exposure to DE, suggesting that suppressed activity of phase II drug-metabolizing enzymes is important in giving rise to the increased level of DNA adducts in the Nrf2-null mutant mouse subjected to DE. Importantly, severe hyperplasia and accumulation of the oxidative DNA adduct 8-hydroxydeoxyguanosine were observed in the bronchial epidermis of nrf2(-/-) mice following DE exposure. These results demonstrate the increased susceptibility of the nrf2 germ line mutant mouse to DE exposure and indicate the nrf2 gene knockout mouse may represent a valuable model for the assessment of respiratory DE toxicity.

Benzo[g,h,i]perylene Synergistically Transactivates Benzo[a]pyrene-Induced CYP1A1 Gene Expression by Aryl Hydrocarbon Receptor Pathway

Although benzo[g,h,i]perylene (BghiP) has been found to promote the carcinogenesis of benzo[a]pyrene (BaP) in animal models, not much is known about this cocarcinogenic mechanism. In this study, human hepatoma HepG2 cells cotreated with BaP and BghiP were used as a model to investigate the cocarcinogenic mechanism of BghiP in BaP-induced carcinogenesis. DNA adduct formation is thought to initiate carcinogenesis, so the effect of BghiP on BaP–DNA adduct formation was evaluated using a 32P-postlabeling assay. The BaP–DNA adduct levels increased following the addition of BghiP, in a dose-dependent manner. However, no adducts were formed with BghiP alone. Our previous report showed that cytochrome P450 1A1 (CYP1A1) is responsible for the metabolic activation of BaP and the formation of B[a]P adduct in HepG2 cells. Western blot and Northern blot analyses were used to evaluate whether BaP-induced CYP1A1 protein and mRNA levels increased following the addition of BghiP. Our data showed that BghiP enhanced BaP-induced CYP1A1 protein and its mRNA levels. To understand whether BghiP enhances BaP-induced CYP1A1 gene expression through the aryl hydrocarbon receptor (AhR) signaling pathway, a gel retardation assay was performed to elucidate the synergistic mechanism of BghiP in BaP-induced CYP1A1 gene expression. The results showed that BghiP causes an increase in the nuclear accumulation of AhR in cells and/or activation of AhR to a DNA-binding form. There was a concordant increase in the transcription activation of CYP1A1 gene and the induction of AhR signal pathway. Our findings demonstrated that BghiP enhances BaP-induced CYP1A1 transcription by AhR activation and suggested that the induction mechanism of CYP1A1 contributes to the cocarcinogenic potential of BghiP in BaP-induced carcinogenesis.

Effect of 2,3,7,8-Tetrachlorodibenzo-p-dioxin on the Expression of Cytochrome P450 1A1, the Aryl Hydrocarbon Receptor, and the Aryl Hydrocarbon Receptor Nuclear Translocator in Rat Brain and Pituitary

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related substances are ubiquitous environmental pollutants causing a wide variety of pathological alterations, with the most severe being progressive anorexia and body weight loss. These features suggest a possible involvement of the nervous system and neuroendocrine-related organs including the pituitary gland. However, so far there is little evidence for direct effects of TCDD on these areas. In the present study, male Sprague–Dawley rats were treated with a single oral dose of TCDD (10 μg/kg) and euthanized 1, 3, or 28 days after treatment. The expression of cytochrome P450 1A1 (CYP1A1), the aryl hydrocarbon receptor (AHR), and the aryl hydrocarbon receptor nuclear translocator (ARNT) were analyzed in different brain regions and pituitaries using semiquantitative RT–PCR and Western blotting. Relative levels of CYP1A1 mRNA and protein were dramatically increased in the pituitary. A significant increase in CYP1A1 mRNA was also detected in all the brain regions examined including olfactory bulb, striatum–caudate, hypothalamus, hippocampus, cortex, cerebellum, and substantia nigra. The increase in the expression was time-dependent with the highest level observed 1 day after TCDD treatment. The AHR and ARNT mRNAs were detected in the same areas but in contrast to CYP1A1 the changes in AHR and ARNT mRNA expression were limited to the 28-day time point. The present results provide evidence for the presence of CYP1A1, AHR, and ARNT in the central nervous system and in the pituitary, suggesting that TCDD may exert a direct effect on these regions.

Hemodynamic Dysfunction and Cytochrome P4501A mRNA Expression Induced by 2,3,7,8-Tetrachlorodibenzo-p-dioxin during Embryonic Stages of Lake Trout Development

Lake trout embryos exposed to [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) manifest toxicity after hatching by subcutaneous edema of the yolk sac, pericardial edema, meningeal edema, subcutaneous hemorrhages, and a marked congestion of blood flow in various vascular beds culminating in death. Our objective was to determine if this syndrome was associated temporally with morphologic lesions in the vascular endothelium, increased vascular permeability, and cytochrome P4501A (CYP1A) mRNA induction. Lake trout embryos exposed as fertilized eggs to TCDD were found to exhibit marked reductions in perfusion of the peripheral vasculature during the early sac fry stage of development (stage F19), which consistently preceded other gross lesions and mortality observed later in sac fry development (stage F210). This reduction in blood flow was manifested as severe capillary congestion and hemoconcentration in certain vascular beds. Transmission electron microscopic (TEM) examination of endothelial cells in these vascular beds failed to reveal cellular necrosis at hatching (stage E58) and throughout sac fry development (stages F19–F210). Rather, only subtle ultrastructural changes in endothelial cells were found consisting of increased vacuolation, separation of intercellular junctions, and cytoplasmic blebbing, consistent with the TCDD dose and time course for developmental cardiovascular toxicity, which began to manifest itself in some embryos approximately 1 week prior to hatching (E58). To assess permeability of yolk sac vasculature to certain constituents in blood, sac fry (stage F210) were analyzed for the presence of plasma proteins, granulocytes, and serum creatine kinase activity in yolk sac subcutaneous edema fluid from control and TCDD-exposed treatment groups. TCDD dose- and time-related increases in yolk sac edema volume, plasma protein content of edema fluid, granulocyte concentration, and creatine kinase activity in the fluid were observed in midstage and late stage of sac fry development (stage F210). Thus, yolk sac subcutaneous edema fluid is an ultrafiltrate of blood and results from increased vascular permeability. In contrast to the changes in vascular blood flow and permeability induced by TCDD during stages F19 and F210 of sac fry development, respectively, CYP1A mRNA levels were induced by TCDD as early as the 10-somite embryo (stage E25). TCDD also caused a dose-related increase in CYP1A mRNA levels in sac fry at hatching (stage E58) and throughout sac fry development (stages F19–F210). We conclude that subtle, ultrastructural changes in vascular endothelial cells consistently precede increases in vascular permeability and sac fry mortality; however, induction of CYP1A mRNA occurs prior to any observable morphological lesions, changes in vascular permeability, or sac fry mortality.

2,3,7,8 Tetrachlorodibenzo- p-dioxin induction of cytochrome P4501A in cultured rat and human hepatocytes

We report here a novel observation that 2,3,7,8-tetracholorodibenzo- p-dioxin (TCDD) induced predominantly cytochrome P4501A1 (CYP1A1) in rat hepatocytes and predominantly CYP1A2 in human hepatocytes. As part of our research program to evaluate species-differences in response to CYP inducers, we studied the effects of TCDD on CYP1A activity, protein, and gene expression in primary cultures of rat and human hepatocytes. TCDD was found to induce CYP1A activity, measured as ethoxyresorufin- O-deethylase (EROD) activity, in both rat and human hepatocytes. TCDD induction of EROD activity in human hepatocytes (2–5 fold of concurrent solvent control), was significantly lower than that found in rat hepatocytes (≈20-fold of concurrent solvent control). Two structural analogs of TCDD, 2,3,7,8-tetrachlorodibenzofuran (TCDF) and 6-nitro-1,3,8-trichlorodibenzofuran (6-NCDF), were also evaluated. As observed for TCDD, human hepatocytes consistently showed a lower response than rat hepatocytes. As most TCDD-related effects are believed to be mediated via binding of the TCDD-Ah receptor (AhR) complex to DNA, nuclear AhR levels were measured in rat and human hepatocytes after TCDD treatment. We found that the nuclear AhR levels in TCDD-treated rat hepatocytes were ≈4 times higher than found in TCDD-treated human hepatocytes. However, the estimated binding affinity of [ 3H]TCDD to nuclear AhR from rat hepatocytes was similar. The species difference in response to TCDD was further evaluated by analysis of CYP1A1 and CYP1A2 mRNA levels using Northern analysis, and P4501A1 and 1A2 protein levels using Western immunoblotting. Results showed that, at both gene expression and protein levels, TCDD induced predominantly CYP1A1 in rat hepatocytes and CYP1A2 in human hepatocytes.

The Steroid Hormone Dehydroepiandrosterone InhibitsCYP1A1 Expression in Vitro By a Post-transcriptional Mechanism

The adrenal steroid hormone dehydroepiandrosterone (DHEA) is a potent inhibitor of mammary carcinogenesis induced by polycyclic aromatic hydrocarbons (PAH), though its mechanism is unclear. We examined the effect of DHEA on the expression of the carcinogen-activating enzyme cytochrome P450 1A1 (CYP1A1) in MCF-7 human breast epithelial carcinoma cells. DHEA inhibited the increase in CYP1A1 enzyme activity that occurs when MCF-7 cells are exposed to the PAH dimethylbenzanthracene (DMBA) or 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD). However, DHEA did not directly inhibit enzyme activity as it had no effect when added to the cells after induction by DMBA or TCDD. We observed that the increase of CYP1A1 mRNA in MCF-7 cells caused by DMBA or TCDD was inhibited by DHEA in a concentration-dependent manner. However, DHEA did not inhibit CYP1A1 promoter-driven transcription, indicating that it did not affect the aryl hydrocarbon receptor, which regulates transcription of the CYP1A1 gene. Actinomycin D chase experiments showed that DHEA caused a time- and concentration-dependent decrease in CYP1A1 mRNA levels, indicating that DHEA inhibits CYP1A1 expression by decreasing CYP1A1 mRNA stability. These data demonstrate that DHEA inhibits PAH-induced CYP1A1 mRNA expression and enzyme activity in vitro by a post-transcriptional mechanism. This regulation of the expression of carcinogen-activating enzymes may be responsible for the chemopreventive activity of DHEA and may be one of its physiologic functions in vivo.

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.

CYP1A expression in liver and gill of rainbow trout following waterborne exposure: implications for biomarker determination

The purpose of this study was to compare the time-course for induction of cytochrome P4501A (CYP1A) mRNA levels between liver and gill tissue from rainbow trout ( Oncorhyncus mykiss) following waterborne exposure to the polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP). Liver and gill CYP1A mRNA levels and CYP1A catalytic activity were rapidly induced following exposure to BaP concentrations of less than 1 μg l −1. The rise of CYP1A mRNA levels ahead of the rise of CYP1A catalytic activity demonstrated the typical pattern of a gene that is under transcriptional control. Nuclear run-on assays with control and BaP-exposed fish demonstrated that transcriptional activation of the CYP1A gene is an important regulator of CYP1A mRNA levels during the first 24 h of exposure. Liver CYP1A mRNA levels were induced between 6 and 72 h of exposure to 1.23±0.08 μg l −1 BaP but decreased to basal levels between 72 and 120 h of exposure. In comparison, gill CYP1A mRNA levels were induced after 6 h of exposure and remained at this high level of induction through 120 h of exposure to 0.78±0.11 μg l −1 BaP. Gill BaP-hydroxylase activity was maximally induced between 24 and 120 h of exposure, and this time period corresponded with the time period when liver CYP1A mRNA levels decreased back to basal levels. Extensive first-pass metabolism of BaP by the gill after 24 h of exposure may have prevented the majority of parent BaP that was cleared by the gill from entering circulation and maintaining induction of liver CYP1A mRNA levels. The comparison between liver and gill CYP1A mRNA levels and CYP1A catalytic activity demonstrated that CYP1A expression should be measured in tissues that are proximate to the environment when assessing fish from contaminated environments.

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.

The flavonoid galangin is an inhibitor of CYP1A1 activity and an agonist/antagonist of the aryl hydrocarbon receptor.

The effect of the dietary flavonoid galangin on the metabolism of 7,12-dimethylbenz[a]anthracene (DMBA), the activity of cytochrome P 450 1A1 (CYP1A1), and the expression of CYP1A1 in MCF-7 human breast carcinoma cells was investigated. Galangin inhibited the catabolic breakdown of DMBA, as measured by thin-layer chromatography, in a dose-dependent manner. Galangin also inhibited the formation of DMBA-DNA adducts, and prevented DMBA-induced inhibition of cell growth. Galangin caused a potent, dose-dependent inhibition of CYP1A1 activity, as measured by ethoxyresorufin-O-deethylase activity, in intact cells and in microsomes isolated from DMBA-treated cells. Analysis of the inhibition kinetics by double-reciprocal plot demonstrated that galangin inhibited CYP1A1 activity in a non-competitive manner. Galangin caused an increase in the level of CYP1A1 mRNA, indicating that it may be an agonist of the aryl hydrocarbon receptor, but it inhibited the induction of CYP1A1 mRNA by DMBA or by 2,3,5,7-tetrachlorodibenzo-p-dioxin (TCDD). Galangin also inhibited the DMBA- or TCDD-induced transcription of a reporter vector containing the CYP1A1 promoter. Thus, galangin is a potent inhibitor of DMBA metabolism and an agonist/antagonist of the AhR, and may prove to be an effective chemopreventive agent. © 1999 Cancer Research Campaign

Annual sex steroid profiles and effects of gender and season on cytochrome P450 mRNA induction in atlantic tomcod (Microgadus tomcod)

AbstractAs a preliminary step in a 4‐year biomonitoring program, sex steroid levels (testosterone and 11‐ketotestosterone in males, and testosterone and estradiol in females), gonad weights, and diameter of vitellogenic oocytes were measured in tomcod collected bimonthly from the Miramichi (industrialized site) and Kouchibouguac (reference site) rivers from September 1993 to September 1994. As well as the reproductive indices, hepatic levels of cytochrome P4501A mRNA (CYP1A mRNA) were also measured. The preparatory period for spawning began in September, with maximal steroid levels in November, and spawning took place from late December to January. The CYP1A mRNA levels in female tomcod appeared inversely related to plasma steroids, with the lowest amounts of CYP1A mRNA coinciding with maximal steroids. The CYP1A mRNA levels in male tomcod did not exhibit this relationship. River–river comparisons of female tomcod showed significantly smaller vitellogenic oocytes in the Miramichi, along with lower plasma testosterone, estradiol, and relative gonad weight. Miramichi CYP1A mRNA levels were higher than Kouchibouguac in the fall but lower in the spring sample. The CYP1A mRNA–sex steroid relationship observed in this study will facilitate meaningful interpretation of data collected during the full 4‐year study.