Polycyclic aromatic hydrocarbons (PAHs) are among the most prevalent environmental pollutants and result from the incomplete combustion of hydrocarbons (coal and gasoline, fossil fuel combustion, byproducts of industrial processing, natural emission, cigarette smoking, etc.). The first phase of xenobiotic biotransformation in the PAH metabolism includes activities of cytochrome P450 from the CYP1 family and microsomal epoxide hydrolase. The products of this biotransformation are reactive oxygen species that are transformed in the second phase through the formation of conjugates with glutathione, glucuronate or sulphates. PAH exposure may lead to PAH-DNA adduct formation or induce an inflammatory atherosclerotic plaque phenotype. Several genetic polymorphisms of genes encoded for enzymes involved in PAH biotransformation have been proven to lead to the development of diseases. Enzyme CYP P450 1A1, which is encoded by the CYP1A1 gene, is vital in the monooxygenation of lipofilic substrates, while GSTM1 and GSTT1 are the most abundant isophorms that conjugate and neutralize oxygen products. Some single nucleotide polymorphisms of the CYP1A1 gene as well as the deletion polymorphisms of GSTT1 and GSTM1 may alter the final specific cellular inflammatory respond. Occupational exposure or conditions from the living environment can contribute to the production of PAH metabolites with adverse effects on human health. The aim of this study was to obtain data on biotransformation and atherosclerosis, as well as data on the gene polymorphisms involved in biotransformation, in order to better study gene expression and further elucidate the interaction between genes and the environment.

The HAP1 (CYP1) gene product of Saccharomyces cerevisiae is known to regulate the transcription of many genes in response to oxygen availability. This response varies according to yeast species, probably reflecting the specific nature of their oxidative metabolism. It is suspected that a difference in the interaction of Hap1p with its target genes may explain some of the species-related variation in oxygen responses. As opposed to the fermentative S. cerevisiae, Kluyveromyces lactis is an aerobic yeast species which shows different oxygen responses. We examined the role of the HAP1-equivalent gene (KlHAP1) in K. lactis. KlHap1p showed a number of sequence features and some gene targets (such as KlCYC1) in common with its S. cerevisiae counterpart, and KlHAP1 was capable of complementing the hap1 mutation. However, the KlHAP1 disruptant showed temperature-sensitive growth on glucose, especially at low glucose concentrations. At normal temperature, 28 degrees C, the mutant grew well, the colony size being even greater than that of the wild type. The most striking observation was that KlHap1p repressed the expression of the major glucose transporter gene RAG1 and reduced the glucose uptake rate. This suggested an involvement of KlHap1p in the regulation of glycolytic flux through the glucose transport system. The DeltaKlhap1 mutant showed an increased ability to produce ethanol during aerobic growth, indicating a possible transformation of its physiological property to Crabtree positivity or partial Crabtree positivity. Dual roles of KlHap1p in activating respiration and repressing fermentation may be seen as a basis of the Crabtree-negative physiology of K. lactis.

One possible mechanism by which diet may reduce cancer risk is through enhancement of metabolic systems that prevent activation of carcinogens or accelerate carcinogen inactivation. We studied the effects of diet and 7,12-dimethylbenz-(a)anthracene (DMBA) on hepatic and mammary gland CYP1A1, CYP1A2 and CYP1B1 enzymes in female Sprague-Dawley rats. Diets (AIN-93G) were fed from conception to adulthood, and DMBA was given by oral gavage at age 48-50 d. The protein sources of diets were casein (CAS), soy protein isolate (SPI) or whey protein hydrolysate (WPH). The DMBA-induced hepatic ethoxyresorufin-O-deethylase and methoxyresorufin-O-demethylase activities and CYP1A1 protein and mRNA expression were lower (P < 0.05) in SPI-fed rats compared with those fed casein. Differences in mammary gland CYP1 expression were also observed with decreased DMBA induction (P < 0.05) of all three CYP1 proteins and mRNAs in rats fed either SPI or WPH compared with those fed CAS. Most notable were the decreased constitutive and DMBA-induced mammary gland expression of CYP1B1 protein of 93 and 96%, respectively, in the SPI-fed rats relative to the CAS-fed controls. The diet-induced changes in CYP1 enzyme expression were consistent with changes in the AhR and ARNT transcription factors that regulate them. Decreased (P < 0.05) mammary constitutive AhR and ARNT proteins were measured in SPI-fed rats. There was also a 100% increase in constitutive AhR protein in the WPH-fed rats that paralleled a 100% increase in constitutive CYP1B1 protein in the mammary gland. These results demonstrate the importance of diet in regulation of phase I metabolism in liver and mammary gland, and suggest a potential mechanism by which soy or whey proteins reduce DMBA-induced mammary tumor incidence.

BACKGROUND

Cytochrome P450 1A1 (CYP1A1) is a member of the CYP1 family, which is a key enzyme in the metabolism of many endogenous substrates and exogenous carcinogens. To date, many studies have examined the association between CYP1A1 MspI and Ile462Val polymorphisms and cancer risk in various populations, but their results have been conflicting rather than consistent.

METHODS

To assess this relationship more precisely, a meta-analysis based on 198 publications was performed. Odds ratios (OR) and corresponding 95% confidence intervals (CIs) were used to assess the association. The statistical heterogeneity across studies was examined with a chi-square-based Q-test.

RESULTS

Overall, a significant elevated risk of cancer was associated with CYP1A1 MspI and Ile462Val polymorphisms for all genetic models studied. Further stratified analysis by cancer types revealed that the MspI polymorphism may increase the risk of lung cancer and cervical cancer whereas the Ile462Val polymorphism may contribute to a higher risk of lung cancer, leukemia, esophageal carcinoma, and prostate cancer. In the subgroup analysis by ethnicity, obvious associations were found in the Asian population for the MspI polymorphism while an increased risk of cancer was observed in Asians and Caucasians for the Ile462Val polymorphism.

CONCLUSIONS

The results of this meta-analysis suggest that CYP1A1 MspI and Ile462Val polymorphisms contribute to increased cancer susceptibility among Asians. Additional comprehensive system analyses are required to validate this association and other related polymorphisms.

Epidemiological studies indicate that Asian women have a lower breast cancer incidence compared with their counterparts in the West, and the difference has been related to soya consumption. Animal studies have suggested that soya may prevent dimethylbenz[a]anthracene (DMBA)-induced carcinogenesis in the breast. In the present study a cell culture model was developed to address the effect of soya isoflavones on the DMBA-induced DNA damage. DMBA is metabolized into a DNA-attacking moiety by two phase I cytochrome P450 (CYP) enzymes CYP1A1 and CYP1B1. DNA mutation caused by this genotoxic agent is a crucial step in cancer initiation. Substances that interfere with the CYP1 enzyme activities can affect the initiation. In the present study, genistein was found to be an effective inhibitor of recombinant human CYP1A1 and CYP1B1 with Ki of 15.35 and 0.68 micromol/l. The other soya isoflavone daidzein, on the other hand, did not demonstrate any significant inhibition of the enzyme activities. At the transcriptional level, DMBA induced the CYP1 enzyme expressions by stimulating the xenobiotic response element (XRE)-dependent transactivation pathway. When genistein (25 micromol/l) was co-administered with DMBA, the XRE-Luc activity the CYP1 mRNA abundances were significantly suppressed. The present study illustrated that the soya isoflavone genistein, but not daidzein, protected against DMBA genotoxicity.

Cigarette smoke is a major risk factor for cardiovascular diseases. It contains thousands of compounds that activate the aryl hydrocarbon receptor (AhR). In addition, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the most potent AhR ligand, has been shown to cause cardiotoxic effects in several in vivo models. Although induction of CYP1 family is the most important effect of AhR activation, the role of CYP1 induction in mediating the cardiotoxic effect of TCDD is usually overlooked. Therefore, we investigated whether AhR activation causes a hypertrophic effect in H9c2 cells and we related this effect to changes in CYP gene expression. In the current study, the cardiac derived H9c2 cells were treated with two AhR ligands, TCDD and beta-naphthoflavone (BNF), for 24 and 48h. The expression of the hypertrophic markers, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), and several CYP genes were measured by real-time PCR. Treatment of H9c2 cells with TCDD or BNF for 24h caused a significant induction of CYP1A1, CYP1B1, and CYP4A1; however, there was no change in the expression of other genes. On the other hand, treatment of the cells with TCDD or BNF for 48h caused a significant induction of the hypertrophic markers, ANP and BNP, and several CYP genes such as CYP1A1, CYP1B1, CYP2E1, CYP2J3, and CYP4F4 parallel to a significant increase in the cell surface area. Neither TCDD nor BNF increased the oxidative stress in H9c2 cells at all concentrations tested. Interestingly, resveratrol, an AhR antagonist, protected the cells from TCDD-induced hypertrophy. In conclusion, AhR ligands caused a hypertrophic effect in H9c2 cells which was associated with induction of several CYP genes which can be prevented by resveratrol.

Members of the cytochrome P450 family 1 (CYP1s) are involved in the detoxification and bioactivation of numerous environmental pollutants and phytochemicals such as polycyclic aromatic hydrocarbons (PAHs), aromatic amines, and flavonoids. The vertebrate CYP1 gene comprises four subfamilies: CYP1A, CYP1B, CYP1C, and CYP1D. Recently, the CYP1D gene was identified in fish, and subsequently in the platypus. These findings indicate the possibility that all vertebrates have a functional CYP1D subfamily. However, there is no information on the mammalian CYP1D gene. In this study we investigated the genomic location of CYP1D genes in mammals and other vertebrates in silico. We also performed phylogenetic analysis and calculated the identities and similarities of CYP1D sequences. The data from synteny and phylogenetic analyses of CYP1D genes demonstrated the evolutionary history of the CYP1 gene family. The results suggested that CYP1D became a nonfunctional pseudogene in human and bovine species; however, several other mammals possess functional CYP1D genes. The promoter regions of CYP1D genes were also examined. Unlike other CYP1 isoforms, few xenobiotic responsive element (XRE)-like sequences were found upstream of the CYP1D genes. Analysis of mammalian CYP1Ds also provided new insight into the relationship between CYP1 genes and the aryl hydrocarbon receptor.

Transcripts of the murine CYP1A1 (cytochrome P1450) mRNA are markedly elevated in mutant hepatoma cell lines that contain missense mutations in the Cyp1a-1 structural gene. This putative derepression extends to other genes in the [Ah] battery. To test whether the Cyp1a-1 gene product is involved in a mechanism of feedback regulation of transcription, we introduced expression plasmids carrying the murine wild-type Cyp1a-1 cDNA into the mutant hepatoma cells. Measurements of steady-state mRNA levels and of transcriptional rates in the transfectants reveal that expression of a functional, exogenous CYP1A1 protein is sufficient to restore the repression of the endogenous gene, as well as restore the inducibility by dioxin, and that this effect takes place primarily at the level of transcription. Similar experiments with expression plasmids that carry the human CYP1A2 cDNA indicate that the CYP1A2 protein (cytochrome P3450) can also function as a transcriptional repressor. In addition, we find that expression of the Nmo-1 [NAD(P)H:menadione oxidoreductase] gene, a third member of the [Ah] gene battery, is also repressed by the exogenous expression of either Cyp1a-1 or CYP1A2 cDNA. These results indicate that the gene product of either member of the mammalian CYP1 family has a previously unrecognized transcriptional regulatory function, which is likely to be exerted by modification of preexisting trans-acting factors. This function may help bring about a fast reprogramming of gene expression, as might be needed during detoxification of toxic foreign chemicals.

Arachidonic acid is biotransformed to metabolites active in signal transduction by cytochrome P450 (CYP) as well as by cyclooxygenase and lipoxygenase enzymes. Inducers of CYP1 enzymes, including 2,3,7,8-tetrachlorodibenzo-p-dioxin and other Ah receptor ligands, markedly increase liver microsomal CYP-dependent arachidonic acid epoxygenation in chicks but depress epoxygenation in rat liver microsomes where they elicit about twofold increases in formation of other CYP products, omega-1 to omega-4-OH arachidonic acid. These studies examined the effect of TCDD on metabolism of [1-14C]-labeled arachidonic acid by mouse liver microsomes. Mouse liver microsomes metabolized arachidonic acid exclusively by a CYP-dependent mechanism as evidenced by lack of metabolism in the absence of NADPH and by formation of specific CYP-dependent metabolites. The major constitutive products were epoxygenase products (EETs and EET-diols) and omega-OH arachidonic acid. Treatment with TCDD increased formation of omega-2- to omega-4-OH arachidonic acid products 23-fold, formation of omega-1-OH arachidonic acid about 5-fold, and formation of epoxygenase products and HETEs each about twofold. In contrast, TCDD treatment decreased formation of omega-OH arachidonic acid by over 70%. EET-diols comprised a greater fraction of total epoxygenase products in mouse liver microsomes than has been found for liver microsomes of other species. The high EET-diol formation was attributable to a non-TCDD-inducible, EET epoxide hydrolase activity in mouse liver microsomes. For comparison, the effect of TCDD on [1-14C]-labeled arachidonic acid was examined in homogenates of spleen, an immune system target of TCDD. While levels of total [1-14C]-arachidonic acid metabolism were comparable in both tissues, virtually all of the metabolism by spleen was CYP-independent, and it was unaffected by TCDD. Western blotting experiments showed that TCDD-induced mouse Cyp1a1 and 1a2 share immunologic epitopes with chick CYP1A4 and 1A5. However, in immunoinhibition studies, an antibody to CYP1A5, the chick arachidonate epoxygenase, was ineffective against TCDD-induced arachidonic acid metabolism in mouse liver microsomes, suggesting that there are differences in the catalytic sites or tertiary structures of CYP1A5 and the CYP-enzyme catalyzing the TCDD-induced arachidonic acid metabolism in mouse liver. This study shows that the effects of TCDD of the profile of CYP-dependent arachidonic acid metabolities and the amounts produced in mouse liver microsomes differ from other species. The findings suggest that species differences in CYP1A catalytic activities including the metabolism of arachidonic acid may contribute to species differences in sensitivity to TCDD toxicity.

Zebrafish embryos are being increasingly used as model organisms for the assessment of single substances and complex environmental samples for regulatory purposes. Thus, it is essential to fully understand the xenobiotic metabolism during the different life-stages of early development. The aim of the present study was to determine arylhydrocarbon receptor (AhR)-mediated activity during selected times of early development using qPCR, enzymatic activity through measurement of 7-ethoxyresorufin-O-deethylase (EROD) activity, and protein expression analysis. In the present study, gene expression of cyp1a, cyp1b1, cyp1c1, cyp1c2, and ahr2 as well as EROD activity were investigated up to 120 h postfertilization (hpf) after exposure to either β-naphthoflavone (BNF) or a polycyclic aromatic hydrocarbons (PAH)-contaminated sediment extract from Vering Kanal in Hamburg (VK). Protein expression was measured at 72 hpf after exposure to 20 μg/L BNF. Altered proteins were identified by matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) peptide mass fingerprinting. Distinct patterns of basal messenger RNA (mRNA) expression were found for each of the cyp1 genes, suggesting specific roles during embryonic development. All transcripts were induced by BNF and VK. ahr2 mRNA expression was significantly upregulated after exposure to VK. All cyp1 genes investigated showed a temporal decline in expression at 72 hpf. The significant decline of Hsp 90β protein at 72 hpf after exposure to BNF may suggest an explanation for the decline of cyp1 genes at this time point as Hsp 90β is of major importance for the functioning of the Ah-receptor. EROD activity measured in embryos was significantly induced after 96 hpf of exposure to BNF or VK. Together, these results demonstrate distinct temporal patterns of cyp1 genes and protein activities in zebrafish embryos as well as show a need to investigate further the xenobiotic biotransformation system during early development of zebrafish.

In animals, exposure to polyaromatic hydrocarbons (PAHs) such as 3-methylcholanthrene (3-MC) is known to induce the expression of two unique cytochrome P450 genes, CYP1A1 and CYP1A2. These genes are thought to have originated by a gene duplication event and diverged no more than 250 million years ago (D. W. Nerbert and F. J. Gonzalez, 1987, Annu. Rev. Biochem. 56, 945-993). Lower vertebrates, such as fish, diverged from land animals before this time and are thought to express only a single CYP1 gene. In this paper, we present evidence to refute this hypothesis and report the isolation and complete genomic nucleotide sequence of two distinct CYP1 genes in rainbow trout. Genomic clones encoding the entire CYP1A1 and CYP1A2 genes were characterized. DNA sequence analysis revealed that both genes contained seven exons and six introns. Exons 1-7 of CYP1A1 and CYP1A2 were highly similar in length and nucleotide sequence. In contrast, the 5'-flanking region and introns 1, 2, 5, and 6 of both genes were significantly less conserved. Two xenobiotic regulatory elements (XREs) were identified in the 5'-flanking region of CYP1A1 but not in that of CYP1A2. The 5'-most start site of transcription was determined to begin at a cytosine residue 27 bases downstream of the putative TATA box of both genes. Northern blot analysis demonstrated that exposure to 3-MC resulted in an increase in CYP1 mRNA levels in the liver. RNase protection assays conducted with riboprobes specific for either CYP1A1 or CYP1A2 confirmed that the transcripts of both genes were expressed in rainbow trout liver in response to 3-MC treatment.

1. We review here the molecular mechanisms underlying the xenobiotic induction of genes encoding cytochrome P450 (CYP) enzymes in the liver and other tissues. We will focus on four major families of CYP genes. 2. Members of the CYP1 gene family are induced by polycyclic aromatic hydrocarbons and this process is mediated by the basic helix-loop-helix proteins: the Ah receptor and its heterodimeric partner Arnt. Considerable progress has been made in elucidating the molecular details of this induction process. 3. CYP4 genes are activated by peroxisomal proliferators, a group of structurally diverse chemicals that also induce peroxisome proliferation. The transcriptional response is dependent on the peroxisome proliferator-activated receptor and its partner RXR, both members of the nuclear receptor superfamily; their role in the induction process has been well characterized at the molecular level. 4. In contrast, the mechanism of gene induction of CYP2 genes by phenobarbital and other structurally diverse inducers is not well understood and a specific phenobarbital-responsive receptor has not been identified. 5. Induction of the CYP3 gene family by the glucocorticoid dexamethasone appears to involve the glucocorticoid receptor, but this receptor is not apparently required for induction by metapyrone and a complete molecular understanding of the induction processes is lacking at present.

Aryl hydrocarbon receptor (AhR) plays important roles in the regulation and induction of xenobiotic-metabolizing enzymes including the cytochromes P450 1 family (CYP1) and UDP-glucuronosyltransferases 1A (UGT1As) by polycyclic aromatic hydrocarbons as well as chlorinated aromatic hydrocarbons. To determine whether pyrene-induced xenobiotic-metabolizing enzymes are regulated by AhR, male AhR (+/+) and (-/-) mice were used. Both genotyped mice were exposed to 0, 205, 300 or 410 mg/(kgday pyrene), once daily, for four consecutive days by gavage. Exposure to pyrene did not influence hepatic CYP1A1-mRNA in mice of both genotypes, whereas it induced hepatic CYP1A2 protein and mRNA expression and associated 7-ethoxyresorufin O-deethylase and pyrene 1-hydroxylation activities in both AhR (+/+) and (-/-) mice. Similar effects were also found with sulfotransferase 1A1 expression and the associated 1-hydroxypyrene sulfation activity. In contrast, pyrene exposure increased expression of the UGT1A1 and 1A6, and glucuronidation activities associated with 1-hydroxypyrene and 1-naphthol in the liver only in AhR (-/-) mice, although pyrene treatment dose-dependently decreased the latter activity. Pyrene exposure did not increase AhR-mRNA expression in AhR (+/+) mice. In contrast, pyrene-induced expression of the hepatic constitutive androstane receptor (CAR) and one of its target genes, CYP2B10, in both AhR (+/+) and (-/-) mice. These results strongly suggest that pyrene-induced CYP1A2 and SULT1A1 are regulated by CAR, not by AhR. However, the mechanisms of UGT1A1 and 1A6 induction by pyrene were not elucidated in this study.

The Saccharomyces cerevisiae HEM13 gene codes for coproporphyrinogen oxidase (CPO), an oxygen-requiring enzyme catalysing the sixth step of heme biosynthesis. Its transcription is increased 40-50-fold in response to oxygen- or heme-deficiency. We have analyzed CPO activity and HEM13 mRNA levels in a set of isogenic strains carrying single or double deletions of the CYP1 (HAP1), ROX1, SSN6, or TUP1 genes. The cells were grown in the presence or absence of oxygen and under heme-deficiency (hem1 delta background). Both Rox1p and Cyp1p partially repressed HEM13 in aerobic heme-sufficient cells, probably in an independent manner. In the absence of heme, Cyp1p activated HEM13 and strongly repressed ROX1, allowing de-repression of HEM13. Cyp1p had no effect on HEM13 expression in anaerobic cells. Deletions of SSN6 or TUP1 dramatically de-repressed HEM13 in aerobic cells. A series of deletions in the HEM13 promoter identified at least four regulatory regions that are required for HEM13 regulation. Two regions, containing motifs similar to the Rox1p consensus sequences, act as repression sites under aerobic growth. The two other sites act as activation sequences required for full induction under oxygen- or heme-deficiency. Taken together, these results suggest that induction of HEM13 occurs in part through relief of repression exerted by Rox1p and Cyp1p, and in part by activation mediated partly by Cyp1p under heme-deficiency and by unknown factors under oxygen-deficiency.

The cytosolic glutathione S-transferases (GSTs) comprise a pivotal enzyme system protecting the cell from electrophilic compounds. It plays a major role in the detoxication of the primary and dihydrodiol epoxides of polycyclic aromatic hydrocarbons (PAHs), so that modulation of this enzyme system by PAHs will impact on their carcinogenic activity. The potential of six structurally diverse PAHs, namely benzo[a]pyrene (B[a]P), fluoranthene, benzo[b]fluoranthene (B[b]F), dibenzo[a,l]pyrene, dibenzo[a,h]anthracene (D[a,h]A) and 1-methhylphenanthrene, to modulate hepatic GST activity was investigated in human precision-cut slices and compared to rat slices, a species frequently used in long-term carcinogenicity studies; changes were monitored at the activity, using three different substrates, protein and mRNA levels. When activity was monitored using the alpha-class selective 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, B[b]F was the only PAH that caused an increase in activity, which was accompanied by a rise in the Ya immunoreacting band. In rat slices, in addition to B[b]F, B[a]P and D[a,h]A also enhanced activity, being paralleled with increased levels of the Ya immunoreacting band. In the rat, all PAHs elevated mRNA levels. In both human and rat liver slices, only B[b]F enhanced activity when 1-chloro-2,4-dinitrobenzene (CDNB) served as substrate. To investigate tissue differences, similar studies were undertaken in precision-cut rat lung slices, incubated with PAHs under identical conditions, using CDNB, as this was the only substrate for which activity could be detected; none of the PAHs studied stimulated activity. It is concluded that some PAHs have the potential to induce GST activity in human liver tissue and that species and tissue differences exist in the induction of this enzyme system in the rat. However, the extent of induction of GST activity is very modest compared with the effect these compounds have on CYP1 expression, the family responsible for their bioactivation, and it is unlikely to compensate for the enhanced production of reactive intermediates.

Typically, chemopreventive agents either inhibit the cytochrome P450s (CYPs) that are essential for the metabolism of carcinogens or induce phase II detoxifying enzymes. This study examined the chemopreventive effect of eugenol on 7,12-dimethylbenz[a]anthracene (DMBA)-induced DNA damage in MCF-7 cells. Eugenol inhibited the formation of the DMBA-DNA adduct in a dose dependent manner. CYP1A1 and CYP1B1 activity, which catalyze the biotransformation of DMBA, were strongly inhibited by eugenol. Eugenol also suppressed the CYP1A induction by DMBA through decreased aryl hydrocarbon receptor activation and subsequent DNA binding. Furthermore, eugenol increased the expression and activity of NAD(P)H:quinone oxidoreductase (QR), a major detoxifying enzyme for DMBA, through NF-E2 related factor2 binding to antioxidant response element in QR gene. Therefore, eugenol has a potent protective effect against DMBA-induced genotoxicity, presumably through the suppression of the DMBA activation and the induction of its detoxification. These results suggest that eugenol has potential as a chemopreventive.

Cytochrome P450 (CYP) of chicken and other avian species have been studied primarily with microsomes or characterized by cloning and protein expression. However, the overall existing isoforms in avian CYP1-3 families or dominant isoforms in avian xenobiotic metabolism have not yet been elucidated. In this study, we aimed to clarify and classify all of the existing isoforms of CYP1-3 in avian species using available genome assemblies for chicken, zebra finch, and turkey. Furthermore, we performed qRT-PCR assay to identify dominant CYP genes in chicken liver. Our results suggested that avian xenobiotic-metabolizing CYP genes have undergone unique evolution such as CYP2C and CYP3A genes, which have undergone avian-specific gene duplications. qRT-PCR experiments showed that CYP2C45 was the most highly expressed isoform in chicken liver, while CYP2C23b was the most highly induced gene by phenobarbital. Considering together with the result of further enzymatic characterization, CYP2C45 may have a dominant role in chicken xenobiotic metabolism due to the constitutive high expression levels, while CYP2C23a and CYP2C23b can be greatly induced by chicken xenobiotic receptor (CXR) activators. These findings will provide not only novel insights into avian xenobiotic metabolism, but also a basis for the further characterization of each CYP gene.

Ligand-activated receptors regulate numerous genes, and mediate effects of a broad set of endogenous and exogenous chemicals in vertebrates. Understanding the roles of these transcription factors in zebrafish (Danio rerio) is important to the use of this non-mammalian model in toxicological, pharmacological, and carcinogenesis research. Response to a potential agonist for the pregnane X receptor (Pxr) [pregnenolone (PN)] was examined in developing zebrafish, to assess involvement of Pxr in regulation of selected genes, including genes in cytochrome P450 subfamilies CYP2 and CYP3. We also examined interaction of Pxr and the aryl hydrocarbon receptor (Ahr) signaling pathways. Pregnenolone caused a dose-dependent increase in mRNA levels of pxr, ahr2, CYP1A, CYP2AA1, CYP2AA12, CYP3A65, and CYP3C1, most of which peaked at 3 µM PN. The well-known Ahr agonist 3,3',4,4',5-pentachlorobiphenyl (PCB126) also upregulated expression of pxr, ahr2, CYP1A, CYP2AA12, CYP3A65, and CYP3C1 in a dose-dependent manner. Inhibition of pxr translation by morpholino antisense oligonucleotides (MO) suppressed PN-induced expression of pxr, ahr2, CYP3A65, and CYP3C1 genes. Levels of CYP2AA1 and CYP2AA12 mRNA were increased in the control-MO group exposed to PN; this was prevented by knocking down Pxr. Similarly, Ahr2-MO treatment blocked PCB126-induced mRNA expression of pxr, CYP1A, CYP2AA12, CYP3A65, and CYP3C1. The present study shows self-regulation of pxr by PN in developing zebrafish. Selected zebrafish CYP1, CYP2 (including several CYP2AAs) and CYP3 genes appear to be under the regulation of both Pxr and Ahr2.

The objective of this study was to identify which anesthetics when used acutely will affect cytochrome P450 (CYP) activity in male Sprague-Dawley rats in vivo. The anesthetics tested were fentanyl citrate, alpha-chloralose, ketamine, urethane (ethyl carbamate), halothane, and ether. CO2 anesthesia was used as the control comparator. Theophylline was used as a probe for CYP1A activity, phenobarbital for CYP2B/2C, flecainide for CYP2D1, and ethosuximide for CYP3A activity. All probes were administered via tail vein injection after anesthetic-induced loss of the righting reflex. Single sample probe clearances were estimated, and used as an index of CYP activity. Fentanyl citrate, alpha-chloralose, halothane, and ether did not have statistically significant effects on any of the CYP activities. Ketamine did not significantly affect CYP1 or CYP2B/2C activity. However, it decreased the clearance of flecainide (i.e. CYP2D1 activity) by 13.4% (p < 0.001) and the clearance of ethosuximide (i.e. CYP3A activity) by 17.6% (p < 0.0001). Urethane increased the clearance of theophylline by 91.5% (p < 0.0001), and decreased the clearance of ethosuximide by 40.5% (p < 0.0001) though it did not affect CYP2B/2C or CYP2D1 activities significantly. From this data, we conclude that a single dose of ketamine mildly inhibits the activity of CYP2D1 and CYP3A, and a single dose of urethane strongly inhibits CYP3A but increases CYP1A activity.

Flavonoids, natural compounds found in plants and in plant-derived foods and beverages, have been extensively studied with regard to their capacity to modulate metabolic enzymes and drug transporters. In vitro, flavonoids predominantly inhibit the major phase I drug-metabolizing enzyme CYP450 3A4 and the enzymes responsible for the bioactivation of procarcinogens (CYP1 enzymes) and upregulate the enzymes involved in carcinogen detoxification (UDP-glucuronosyltransferases, glutathione S-transferases (GSTs)). Flavonoids have been reported to inhibit ATP-binding cassette (ABC) transporters (multidrug resistance (MDR)-associated proteins, breast cancer-resistance protein) that contribute to the development of MDR. P-glycoprotein, an ABC transporter that limits drug bioavailability and also induces MDR, was differently modulated by flavonoids. Flavonoids and their phase II metabolites (sulfates, glucuronides) inhibit organic anion transporters involved in the tubular uptake of nephrotoxic compounds. In vivo studies have partially confirmed in vitro findings, suggesting that the mechanisms underlying the modulatory effects of flavonoids are complex and difficult to predict in vivo. Data summarized in this review strongly support the view that flavonoids are promising candidates for the enhancement of oral drug bioavailability, chemoprevention, and reversal of MDR.

Butyrate, a short-chain fatty acid produced by fermentation of dietary fiber, is an important regulator of colonic epithelium homeostasis. In this study, we investigated the impact of this histone deacetylase (HDAC) inhibitor on expression/activity of cytochrome P450 family 1 (CYP1) and on metabolism of carcinogenic polycyclic aromatic hydrocarbon, benzo[a]pyrene (BaP), in colon epithelial cells. Sodium butyrate (NaBt) strongly potentiated the BaP-induced expression of CYP1A1 in human colon carcinoma HCT116 cells. It also co-stimulated the 7-ethoxyresorufin-O-deethylase (EROD) activity induced by the 2,3,7,8-tetrachlorodibenzo-p-dioxin, a prototypical ligand of the aryl hydrocarbon receptor. Up-regulation of CYP1A1 expression/activity corresponded with an enhanced metabolism of BaP and formation of covalent DNA adducts. NaBt significantly potentiated CYP1A1 induction and/or metabolic activation of BaP also in other human colon cell models, colon adenoma AA/C1 cells, colon carcinoma HT-29 cells, or in NCM460D cell line derived from normal colon mucosa. Our results suggest that the effects of NaBt were due to its impact on histone acetylation, because additional HDAC inhibitors (trichostatin A and suberanilohydroxamic acid) likewise increased both the induction of EROD activity and formation of covalent DNA adducts. NaBt-induced acetylation of histone H3 (at Lys14) and histone H4 (at Lys16), two histone modifications modulated during activation of CYP1A1 transcription, and it reduced binding of HDAC1 to the enhancer region of CYP1A1 gene. This in vitro study suggests that butyrate, through modulation of histone acetylation, may potentiate induction of CYP1A1 expression, which might in turn alter the metabolism of BaP within colon epithelial cells.

A compilation of information relating to substrate metabolism via human cytochromes P450 (CYP) from the CYP1 and CYP2 families is reported. The data presented include details of preferred sites of metabolism and Km values (usually for the expressed enzymes) for each reaction for selected substrates of CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1. Although other P450 databases are available, they do not provide such information as is collated here, and which can prove useful for comparing P450 substrate characteristics. This information can be employed in analysing the structural requirements for human P450 enzyme selectivity and for establishing various rules regarding preferred site of metabolism for selective P450 substrates. For example, in most cases it would appear that there is a set number of intervening 'heavy' atoms (atoms other than hydrogen) between sites of metabolism and key hydrogen bond acceptors (or donors) for human P450 substrates, with the number of intervening atoms being dependent upon the type of P450 involved.

Hepatic microsomes derived from Cypla2(-/-) knockout (KO) and parental strains of mice, C57BL/6N and 129Sv, were used to examine the specificity of methoxyresorufin and acetanilide as substrates for CYP1A2 activity. In addition, animals from each group were exposed to CYP1-inducing compounds. As expected, microsomes from untreated 1a2 KO mice did not have immunodetectable CYP1A2 protein; however, methoxyresorufin-O-demethylase (MROD, 25.5+/-6.1 pmol/min/mg protein) and acetanilide-4-hydroxylation (ACOH, 0.64+/-0.04 nmol/min/mg protein) activities were still present. Furthermore, induction of ethoxyresorufin-O-deethylase (EROD) by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in 1a2 KO mice was accompanied by a greater than 70-fold increase in MROD activity. In contrast, ACOH was only induced 2-fold by TCDD. As with 1a2 KO mice, the parental strains exposed to TCDD or 2,3,4,7,8-pentachlorodibenzofuran (4-PeCDF) showed substantial EROD and MROD induction, whereas ACOH activity was induced to a lesser degree. PCB153 (2,2',4,4',5,5'-hexachlorobiphenyl) resulted in low levels of both EROD and MROD induction. Results indicate that both substrates are subject to metabolism by non-CYP1A2 sources, and the apparent contribution of CYP1A1 activity to methoxyresorufin metabolism makes MROD unsuitable for differentiating CYP1A1 and CYP1A2 activities in the mouse.

The mechanisms how environmental compounds influence the human immune system are unknown. The environmentally sensitive transcription factor aryl hydrocarbon receptor (AHR) has immune-modulating functions and responds to small molecules. Cytochrome P4501 enzymes (CYP1) act downstream of the AHR and metabolize small molecules. However, it is currently unknown whether CYP1 activity is relevant for immune modulation. We studied the interdependence of CYP1 and AHR in human primary immune cells using pharmacological methods. CYP1 inhibition increased the expression levels of the stem cell factor receptor (c-Kit) and interleukin (IL)-22 but decreased IL-17. Single cell analyses showed that CYP1 inhibition especially promoted CD4+ helper T (Th) cells that co-express c-Kit and IL-22 simultaneously. The addition of an AHR antagonist reversed all these effects. In addition to T cells, we screened other human immune cells for CYP and found cell-specific fingerprints, suggesting that similar mechanisms are present in multiple immune cells. We describe a feedback loop yet unknown in human immune cells where CYP1 inhibition resulted in an altered AHR-dependent immune response. This mechanism relates CYP1-dependent metabolism of environmental small molecules to human immunity.