We have previously reported that high accumulation of dioxins and related compounds induced cytochrome P450 (CYP 1s) isozymes in the liver of wild Baikal seals, implying the enhanced hydroxylation of polychlorinated biphenyls (PCBs). The present study attempted to elucidate the residue concentrations and patterns of PCBs and hydroxylated PCBs (OH-PCBs) in the livers of Baikal seals. The hepatic residue concentrations were used to assess the potential effects of PCBs and OH-PCBs in combination with the analyses of serum thyroid hormones, hepatic mRNA levels, and biochemical markers. The hepatic expression levels of CYP1 genes were positively correlated with the concentration of each OH-PCB congener. This suggests chronic induction of these CYP1 isozymes by exposure to PCBs and hydroxylation of PCBs induced by CYP 1s. Hepatic mRNA expression monitoring using a custom microarray showed that chronic exposure to PCBs and their metabolites alters the gene expression levels related to oxidative stress, iron ion homeostasis, and inflammatory responses. In addition, the concentrations of OH-PCBs were negatively correlated with L-thyroxine (T4) levels and the ratios of 3,3',5-triiodo-L-thyronine (T3)/reverse 3,3',5'-triiodo-L-thyroninee (rT3). These observations imply that Baikal seals contaminated with high levels of OH-PCBs may undergo the disruption of mechanisms related to the formation (or metabolism) of T3 and T4 in the liver.

7-Ethoxycoumarin is metabolized by many cytochrome P450 enzymes active in foreign compound metabolism and has been used as a prototypic substrate to monitor P450 (P450) activity in both hepatic and extrahepatic tissues. A spectrofluorometric method is described for determination of P450-catalyzed 7-ethoxycoumarin O-deethylation. Following acidification of the incubation mixture, the enzymatic product, 7-hydroxycoumarin, is recovered by a double-extraction procedure and measured at an excitation wavelength of 370 nm and an emission wavelength of 450 nm. This method is applicable to enzymatic studies to determine the catalytic activity of cDNA-expressed human enzymes in the CYP1, CYP2, and CYP3 families, and 7-ethoxycoumarin O-deethylation activity in microsomes isolated from liver and other tissues.

Koumine is a component of the Chinese medicinal herb Gelsemium elegans and is toxic to vertebrates. We used the ciliate Tetrahymena thermophila as a model to evaluate the toxic effects of this indole alkaloid in eukaryotic microorganisms. Koumine inhibited T. thermophila growth and viability in a dose-dependent manner. Moreover, this drug produced oxidative stress in T. thermophila cells and expressions of antioxidant enzymes were significantly elevated at high koumine levels (p < 0.05). Koumine also caused significant levels of apoptosis (p < 0.05) and induced DNA damage in a dose-dependent manner. Mitophagic vacuoles were present in cells indicating induction of autophagy by this drug. Expression of ATG7, MTT2/4, CYP1 and HSP70 as well as the MAP kinase pathway gene MPK1 and MPK3 were significantly altered after exposed to koumine. This study represents a preliminary toxicological evaluation of koumine in the single celled eukaryote T. thermophila.

Diphenylarsinic acid (DPAA), a chemical warfare-related neurotoxic organic arsenical, is present in the groundwater and soil in some regions of Japan due to illegal dumping after World War II. Inorganic arsenic is carcinogenic in humans and its organic arsenic metabolites are carcinogenic in animal studies, raising serious concerns about the carcinogenicity of DPAA. However, the carcinogenic potential of DPAA has not yet been evaluated. In the present study we found that DPAA significantly enhanced the development of diethylnitrosamine-induced preneoplastic lesions in the liver in a medium-term rat liver carcinogenesis assay. Evaluation of the expression of cytochrome P450 (CYP) enzymes in the liver revealed that DPAA induced the expression of CYP1B1, but not any other CYP1, CYP2, or CYP3 enzymes, suggesting that CYP1B1 might be the enzyme responsible for the metabolic activation of DPAA. We also found increased oxidative DNA damage, possibly due to elevated CYP1B1 expression. Induction of CYP1B1 has generally been linked with the activation of AhR, and we found that DPAA activates the aryl hydrocarbon receptor (AhR). Importantly, the promotion effect of DPAA was observed only at a dose that activated the AhR, suggesting that activation of AhR and consequent induction of AhR target genes and oxidative DNA damage plays a vital role in the promotion effects of DPAA. The present study provides, for the first time, evidence regarding the carcinogenicity of DPAA and indicates the necessity of comprehensive evaluation of its carcinogenic potential using long-term carcinogenicity studies.

Estrogen is a major risk factor for endometrial cancer and it has been well-established that smokers have a significantly reduced risk of endometrial cancer. Localized levels of estrogen within the uterus may determine the estrogenic response. The objective of this research was to investigate effects of cigarette smoke related hydrocarbons (benzo(a)pyrene, BP) on uterine CYP1A1/2 and 1B1, enzymes involved in estrogen metabolism. Human endometrium epithelial cells (RL95-2) were incubated with various concentrations (0.05, 0.1, 0.5, 1, and 10mM) of BP for 48h. CYP1 catalytic activity, protein and mRNA levels were determined. Selective chemical and immuno-inhibitors were used to determine the contribution of individual CYP1 isoenzymes. Cells expressing CYP1A1, CYP1A2 and CYP1B1 were used for comparisons. CYP1A1/2 protein and mRNA levels were significantly elevated by BP. Low level of constitutive CYP1 activity was observed in RL95-2 cells, which was significantly induced by BP exposure (12-fold at 1mM). CYP1 activity in BP-induced cells was significantly inhibited by specific anti-CYP1A1 and high concentration of alpha-naphthoflavone (ANF, 100nM), but not by selective CYP1A2 (furafylline) and CYP1B1 (homoeriodictoyl) inhibitors and low concentration of ANF (5nM). These studies suggest that CYP1A2 and CYP1B1 are not induced by BP in the endometrial cells. It also appears that CYP1A1 is one of the major CYP450 enzymes induced by BP.

The results of quantitative structure-activity relationship (QSAR) studies on six series of compounds exhibiting indirect mutagenic activity are reported. These findings demonstrate the importance of frontier orbital energies and, in some cases, frontier orbital electronic populations to overall mutagenicity in diverse polyaromatic hydrocarbons, benzidines and aminobiphenyls, benzonitrofurans, nitrogenous cooked-food mutagens, benzanthracenes, and chrysenes. The correlations between structural parameters and mutagenic potency vary from R=0.81 to R=0.97, and these findings are discussed in the context of possible molecular mechanisms of mutagenicity. In particular, it is generally regarded that cytochrome P450-mediated activation of polyaromatic hydrocarbons and their amino derivatives plays an important role in mutagenic activity. In this respect, it is apparent that enzymes of the cytochrome P4501 (CYP1) family are closely associated with the metabolic activation of polyaromatic mutagens and carcinogens via the generation of reactive intermediates (usually electrophilic in nature) that attack DNA. The findings presented in this study indicate that QSAR analyses on several series of compounds are consistent with the known evidence of procarcinogen activation mechanisms, particularly for polyaromatic hydrocarbons and their heterocyclic/amino derivatives, pointing to the importance of frontier orbital energy values in particular.

Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental contaminants that interact in a complex manner with both the aryl hydrocarbon receptor (AhR) and estrogen receptors (ER). Their potential endocrine-disrupting activities may depend on both inhibitory AhR-ER cross-talk and on AhR-dependent metabolic production of estrogenic PAH metabolites. Here, we analyzed the impact of AhR on estrogen-like effects of PAHs, such as benzo[a]pyrene (BaP), in particular, on control of cell cycle progression/cell proliferation. Using AhR knockout variant of estrogen-sensitive human breast cancer MCF-7 cells (MCF-7 AhRKO cells), we observed that the AhR-dependent control of cytochrome P450 family 1 (CYP1) expression played a major role in formation of estrogenic BaP metabolites, most notably 3-OH-BaP, which contributed to the ER-dependent induction of cell cycle progression/cell proliferation. Both BaP metabolism and the BaP-induced S-phase transition/cell proliferation were inhibited in MCF-7 AhRKO cells, whereas these cells remained sensitive towards both endogenous estrogen 17β-estradiol or hydroxylated BaP metabolites. BaP was found to increase the activity of ER-dependent luciferase reporter gene in wild-type MCF-7 cells; however, unlike its hydroxylated metabolite, BaP failed to stimulate luciferase activity in MCF-7 AhRKO cells. Similarly, estrogen-like effects of other known estrogenic PAHs, such as benz[a]anthracene or 3-methylcholanthrene, were diminished in MCF-7 AhRKO cells. Ectopic expression of human CYP1A1 and CYP1B1 enzymes partly restored both BaP metabolism and its effects on cell proliferation. Taken together, our data suggest that the AhR-dependent metabolism of PAHs contributes significantly to the impact of PAHs on cell proliferation in estrogen-sensitive cells.

Cytochrome P450 (CYP) 1 families including CYP1A1, 1A2 and 1B1 are well known to be deeply involved in the initiation of several cancers, due to the fact that they activate environmental pro-carcinogens to form ultimate carcinogens. Benzo[a]pyrene (BaP) is one of the major classes of prototypical pro-carcinogen. It is activated by the CYP1 family to its ultimate carcinogenic forms, mainly BaP-7,8-diol-9,10-epoxide (BPDE), and it forms adducts with DNA. This has been recognized to be a major initiation pathway for cancer. Our previous study demonstrated that chrysoeriol, which is a dietary methoxyflavonoid, selectively inhibited CYP1B1 enzymatic activity and might protect the CYP1B1 related-diseases such as breast cancer. In the present study, we further examined the effects of chrysoeriol on the other initiation pathway of cancer relating to the CYP1 family with BaP in human breast cancer MCF-7 cells. The effects of chrysoeriol on the formation of BPDE-DNA adducts were analyzed specifically using the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. When MCF-7 cells were incubated with 2 microM BaP for 24h, three types of BPDE-dG adducts, especially (+)-trans-BPDE-dG as the dominant adduct, were detected. Co-treatment of MCF-7 cells with 10 microM chrysoeriol and BaP remarkably reduced (+)-trans-BPDE-dG formation. Chrysoeriol (1-10 microM) dose-dependently inhibited both EROD activity and the gene expressions of CYP1A1, 1B1 and 1A2 stimulated by treatment with BaP. In addition, the same amounts of chrysoeriol significantly inhibited the binding of BaP to the aryl hydrocarbon receptor (AhR), which is the key factor concerning the induction of the CYP1 families. In conclusion, our results clearly indicate that chrysoeriol inhibited the formation of BPDE-DNA adducts via regulation of the AhR pathway stimulated by BaP. As a consequence chrysoeriol may be involved in the chemoprevention of environmental pro-carcinogens such as BaP.

On the basis of studies conducted in animals, it has been established that isothiocyanates suppress cytochrome P450 activity, leading to impairment of the bioactivation of carcinogens, this being a principal mechanism of their chemopreventive activity. However, no studies have been carried out in human tissue to ascertain whether hepatic cytochrome P450 composition is similarly modulated, and this is the objective of the present studies. Precision-cut liver slices from four donors were incubated with a range of concentrations of phenethyl isothiocyanate (PEITC) for 24h, and the expression and activity of cytochrome P450 enzymes were determined; similar studies were performed in rat slices for comparison. PEITC suppressed the O-dealkylation of methoxyresorufin in all human livers and this was accompanied by a parallel drop in CYP1A2 apoprotein levels; the same effect was noted in rat liver slices. The O-dealkylation of ethoxyresorufin was also impaired in the human livers, despite a rise in CYP1A1 apoprotein levels. The CYP3A-mediated benzyloxyquinoline dealkylation was inhibited by PEITC in only two of the four human donors, whereas a rise in CYP3A4 apoprotein levels was noted in all human livers, albeit to different extent. It is concluded that: (a) PEITC can modulate cytochrome P450 composition in human liver, and (b) PEITC, at concentrations that can be achieved by dietary intake, can antagonise the carcinogenicity of chemicals which rely on the CYP1 family for their bioactivation such as heterocyclic amines and polycyclic aromatic hydrocarbons, and this is likely to be a major contributory mechanism to its chemopreventive activity.

P450s in the human brain were originally considered unlikely to contribute significantly to the clearance of drugs and other xenobiotic chemicals, since their overall expression was a small fraction of that found in the liver. However, it is now recognized that P450s play substantial roles in the metabolism of both exogenous and endogenous chemicals in the brain, but in a highly cell type- and region-specific manner, in line with the greater functional heterogeneity of the brain compared to the liver. Studies of brain P450 expression and the characterization of the catalytic activity of specific forms expressed as recombinant enzymes have suggested possible roles for xenobiotic-metabolizing P450s in the brain. It is now possible to confirm these roles through the use of intracerebroventricular administration of selective P450 inhibitors in animal models, coupled with brain sampling techniques to measure drug concentrations in vivo, and modern neuroimaging techniques. The purpose of this review is to discuss the evidence behind the functional importance of P450s from the "xenobiotic-metabolizing" families, CYP1, CYP2 and CYP3 in the brain. Approaches used to define the quantitative and qualitative significance of these P450s in determining tissue-specific levels of xenobiotics in brain will be considered. Finally, the possible roles of these enzymes in brain biochemistry will be examined in light of the demonstrated activity of these enzymes in vitro and the association of particular P450 forms with disease states.

The results of quantitative structure-activity relationships (QSARs) are reported for several series of cytochrome P450 inducers, including those which also act as ligands for the various nuclear receptors involved in regulation of the relevant P450 genes, namely, CYP1, CYP2, CYP3 and CYP4. In several examples presented, the QSARs are consistent with homology modelling studies of the nuclear receptor ligand-binding domains (LBDs) based on available crystal structures of the oestrogen and peroxisome proliferator-activated receptors' LBDs. Good correlations (R=0.91-0.99) are found between various structural parameters and biological activity (either in the form of P450 induction or ligand-binding affinity) for the Ah receptor (AhR), human estrogen receptor alpha (hER alpha), human glucocorticoid receptor (hGR) and the rat peroxisome proliferator-activated receptor alpha (rPPAR alpha).

CYP1(HAP1) is a transcriptional activator involved in the aerobic metabolism of the yeast Saccharomyces cerevisiae. The amino acid sequence of its DNA-binding domain suggests that it belongs to the "zinc cluster" class. This region is indeed characterized by a pattern known to form a bimetal thiolate cluster where two zinc ions are coordinated by six cysteine residues. Structures of two such domains, those from GAL4 and PPR1, have been solved as complexes with DNA. These domains consist of the zinc cluster connected to a dimerization helix by a linker peptide. They recognize, as a dimer, an inverted repeat of a CGG motif that is separated by a specific number of bases. Interestingly, the specificity of that interaction seems not to be due to the interaction between the cluster region and the DNA but rather to a fine tune between the structure of the linker peptide and the number of base-pairs separating the two CGGs. However, the CYP1 target sites fail to display such a consensus sequence. One of the two CGG sites is poorly conserved and some experiments suggest a direct rather than an inverted repeat. Using 1H, 15N and 113Cd NMR spectroscopy, we have undertaken the analysis of the structural properties of the CYP1(56-126) fragment that consists of the zinc-cluster region, the linker peptide and a part of the dimerization helix. We have demonstrated that the six cysteine residues of the peptide chelate two cadmium ions as in GAL4 and PPR1. Fifteen structures of the zinc-cluster region (residues 60 to 100) were calculated, the linker peptide and the dimerization helix being unstructured under the conditions of our study. This region possesses the same overall fold as in GAL4 and PPR1, and most of the side-chains involved in the interaction with DNA are structurally conserved. This suggests that the CYP1 zinc-cluster region recognizes a CGG triplet in the same way as GAL4 and PPR1. In this case, the particular properties of CYP1 seem to be due to the structure of the linker peptide and/or of the dimerization helix.

The use of fish cell cultures has proven to be an effective tool in the study of environmental and aquatic toxicology. Valuable information can be obtained from comparisons between cell lines from different species and organs. In the present study, specific chemicals were used and biomarkers (e.g. 7-Ethoxyresorufin-O-deethylase (EROD) activity and reactive oxygen species (ROS)) were measured to assess the metabolic capabilities and cytotoxicity of the fish hepatic cell lines Hepa-E1 and RTH-149, and the fish gill cell lines RTgill-W1 and G1B. These cell lines were exposed to β-naphthoflavone (BNF) and benzo[a]pyrene (BaP), the pharmaceutical tamoxifen (TMX), and the organic peroxide tert-butylhydroperoxide (tBHP). Cytotoxicity in gill cell lines was significantly higher than in hepatic cells, with BNF and TMX being the most toxic compounds. CYP1-like associated activity, measured through EROD activity, was only detected in hepatic cells; Hepa-E1 cells showed the highest activity after exposure to both BNF and BaP. Significantly higher levels of CYP3A-like activity were also observed in Hepa-E1 cells exposed to TMX, while gill cell lines presented the lowest levels. Measurements of ROS and antioxidant enzymes indicated that peroxide levels were higher in gill cell lines in general. However, levels of superoxide were significantly higher in RTH-149 cells, where no distinctive increase of superoxide-related antioxidants was observed. The present study demonstrates the importance of selecting adequate cell lines in measuring specific metabolic parameters and provides strong evidence for the fish hepatocarcinoma Hepa-E1 cells to be an excellent alternative in assessing metabolism of xenobiotics, and in expanding the applicability of fish cell lines for in vitro studies.

Flocculosin is an antifungal cellobiose lipid linked to the biocontrol activity of Pseudozyma flocculosa and whose structure is very similar to that of ustilagic acid produced by Ustilago maydis. In this work, homologs of the U. maydis cyp1 gene, involved in the biosynthesis of ustilagic acid, were isolated and sequenced from P. flocculosa and P. fusiformata, the latter species being also known to produce ustilagic acid. Interestingly, no homologs were found in four other closely related Pseudozyma spp. from which no evidence of ustilagic acid production has ever been obtained, thus supporting the specificity of cyp1 with ustilagic acid synthesis. In addition, a homolog of the U. maydis uat1 gene involved in the acetylation of the molecule and located next to the cyp1 gene was partially sequenced from P. flocculosa. All three newly sequenced genes showed strong sequence similarity to their counterparts in U. maydis. Cyp1 expression was monitored in conditions that were either conducive or repressive to flocculosin production. Expression increased markedly (>100x) when P. flocculosa was inoculated in a growth medium conducive to flocculosin production but was rapidly downregulated in a repressive medium (in vitro) or on powdery mildew-infected cucumber leaves (in vivo). This suggests that the molecule was preferentially synthesized early in the process of searching for a growth substrate. This study provides the first identification of genes involved in the production of flocculosin, a molecule potentially associated with the biocontrol properties of P. flocculosa.

Epidemiological studies have revealed a protective role of oestrogens against the promotion of colorectal cancer (CRC). Therefore, the oestrogen metabolism status of colonic cells is studied to explain it. Loss of function of adenomatous polyposis coli (Apc) gene product is an early and frequent event in human colorectal carcinogenesis. Normal (Apc(+/+)) and premalignant (Apc(multiple intestinal neoplasia (Min)/+)) mouse colonic epithelial cells were used to compare their respective metabolic capabilities towards oestradiol-17beta (E(2)beta), with or without an inducer of the CYP1 family, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In both cell types, the major metabolite was oestradiol-17beta-3-glucuronide. The formation of catechol (CE) metabolites by cytochromes P450 of the CYP1 family and their derivatives was shown. Among these metabolites, several O-methyl-ether derivatives were detected, as unconjugated metabolites in Apc(+/+) cells and as glucuroconjugates in Apc(Min/+) cells, after TCDD treatment. Apc(Min/+) cells are metabolically more competent than Apc(+/+) cells to produce different hydroxylated metabolites as well as glucuroconjugates. Quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) experiments corroborate these results. Indeed, induction by TCDD has prevailing effects in gene expression of CYP1A1, CYP1A2 and CYP1B1 in Apc(Min/+) cells, compared with Apc(+/+) ones. Apc(Min/+) cells displayed higher rates of oestrogen metabolic biotransformation than Apc(+/+) ones, but exhibited two opposite tendencies. Apc(Min/+) cells were able to detoxify E(2)beta mainly by the formation of glucuronides and displayed at the same time a striking potential to bioactivate E(2)beta by producing only the electrophilic 2-CE derivatives, not the 4-CE ones, even though a significant CYP1B1 mRNA induction was noticed. These specific electrophilic metabolites may form DNA adducts but are not prone to generate new mutations. Interestingly, the ultimate 2-O-methyl-ether metabolite of E(2)beta may be an endogenous protective factor against CRC promotion given its recognised anti-angiogenic and pro-apoptotic properties.

The zebrafish (Danio rerio) embryo is currently explored as an alternative for developmental toxicity testing. As maternal metabolism is lacking in this model, knowledge of the disposition of xenobiotics during zebrafish organogenesis is pivotal in order to correctly interpret the outcome of teratogenicity assays. Therefore, the aim of this study was to assess cytochrome P450 (CYP) activity in zebrafish embryos and larvae until 14 d post-fertilization (dpf) by using a non-specific CYP substrate, i.e., benzyloxy-methyl-resorufin (BOMR) and a CYP1-specific substrate, i.e., 7-ethoxyresorufin (ER). Moreover, the constitutive mRNA expression of CYP1A, CYP1B1, CYP1C1, CYP1C2, CYP2K6, CYP3A65, CYP3C1, phase II enzymes uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1) and sulfotransferase 1st1 (SULT1ST1), and an ATP-binding cassette (ABC) drug transporter, i.e., abcb4, was assessed during zebrafish development until 32 dpf by means of quantitative PCR (qPCR). The present study showed that trancripts and/or the activity of these proteins involved in disposition of xenobiotics are generally low to undetectable before 72 h post-fertilization (hpf), which has to be taken into account in teratogenicity testing. Full capacity appears to be reached by the end of organogenesis (i.e., 120 hpf), although CYP1-except CYP1A-and SULT1ST1 were shown to be already mature in early embryonic development.

CYP1 protein is a yeast transcriptional regulator which contains a zinc cluster in its DNA-binding domain. It was recently shown by selecting random CYP1 binding sites that CYP1 protein recognizes with a higher affinity targets containing the CGGNNNTANCGG consensus sequence. Notably, this ideal sequence is however not found in wild-type CYP1 target sites. In order to investigate how CYP1 protein actually binds to its targets, mutations were introduced in three of them (UAS1-A/CYC1, UAS1-A/CYB2, UAS/CYC7) and the consequences towards the binding of purified CYP1-(1-200)-peptide were analyzed. Our data support the following conclusions: (a) When the sequence element contains two CGGs and no TA, both CGGs are essential for binding. (b) If the sequence element contains only the right CGG and the TA, both are sufficient but indispensable for the binding of CYP1. (c) When two CGCs and the TA are present, the right CGC, and not the left one, is essential for the binding of CYP1. (d) CYP1-(1-200)-peptide is usually a monomer in solution but binds DNA as a dimer. Finally, we found evidence for the presence of two half-sites with different measured affinities in the asymmetric sequences of some CYP1 targets.

Women have a higher risk of lung adenocarcinoma than men, suggesting that estrogen pathway may be involved in the pathogenesis of this cancer. This study was designed to determine whether ERα expression, estrogen levels, and endocrine disruptor exposure would influence tumor growth of lung adenocarcinoma cells using a xenograft model in which human lung adenocarcinoma cells with and without transgenic ERα expression were transplanted into female nude mice. Results showed that estrogen promoted tumor growth of ERα(+) lung adenocarcinoma cells but inhibited that of ERα(-) lung adenocarcinoma cells. Endocrine disruptor benzo[a]pyrene stimulated ERα(-) tumor growth dose dependently. Either of ovariectomy and ERα expression abolished the tumor growth-promoting effect of benzo[a]pyrene. The high CYP1B1/CYP1A1 and low COMT/CYP1B1 expression ratios detected in ERα(+) tumors suggested an accumulation of 4-hydroxyestradiol metabolite under high body estrogen, whereas comparable CYP1A1 and CYP1B1 expression plus estrogen-inducible COMT expression might favor the formation of 2-methoxyestradiol in ERα(-) tumors. Inhibition of estrogen on ERα(-) tumor growth might be partly attributable to the anti-proliferative action of 2-methoxyestradiol. Benzo[a]pyrene increased expression of CYP1B1 over CYP1A1 and suppressed estrogen-induced COMT up-regulation in ERα(-) tumor cells, probably switching estrogen metabolism to 4-hydroxyestradiol formation and removing the inhibition of 2-methoxyestradiol on ERα(-) tumors. ERα inhibited AhR from up-regulating CYP1 in response to benzo[a]pyrene exposure, but it increased angiogenic VEGF-A expression with body estrogen levels. Estrogen might increase ERα(+) lung adenocarcinoma growth by up-regulating cancer-related ERα target gene expression.

The expression and inducibility of cytochrome P450 proteins in the liver of chick embryo were investigated using substrate probes and/or immunologically using polyclonal antibodies to the mammalian isoforms. Antibodies to CYP1A1 recognised a single protein which was inducible by structurally diverse chemicals, including aminocompounds, and was parallelled by increases in the O-dealkylations of ethoxy- and methoxyresorufin and in the bioactivation of Glu-P-1. When probed with antibodies to CYP2E1 an immunoreacting protein was revealed which was induced by phenobarbital but not acetone; a second protein became apparent following the treatment with phenobarbital. The increase in apoprotein levels was accompanied by similar increases in p-nitrophenol hydroxylase. Antibodies to CYP2C11 recognised two immunorelated proteins, of which one was inducible by phenobarbital. The same inducer, but not dexamethasone, enhanced the N-demethylation of erythromycin but antibodies to rat CYP3A1 failed to detect any proteins. Finally, lauric acid hydroxylation was not detectable in chick embryo and, moreover, no immunoreacting band was visible following probing of microsomes with anti-CYP4A1. It is concluded that proteins immunorelated to the mammalian CYP1 and CYP2 families are expressed in chick embryo but the regulation of the latter family in the embryo by exogenous chemicals differs markedly from that established for mammals.

This article reviews current research in amphibian and reptilian cytochromes P450, important to the overall understanding of xenobiotic metabolism in the ecosystem and the evolution of P450s. Amphibians and reptilians contain the normal mixed function oxidase system (MFO). In general the MFO content and activities are less than those found in mammals, but only a few of the known activities have been examined in these vertebrate classes. Research to date has focused on two families of cytochromes P450, CYP1 and 2. The isoforms examined catalyze the classic activities but there have been notable absences. The total number of isoforms present and the breadth of substrates metabolized are yet unknown. Induction by foreign compounds (xenobiotics) is lengthier and yields lower levels of induced activity than is typically found in mammals. When these animals are pretreated with 3-methylcholanthrene (3MC) and beta-naphthaflavone (BNF), which are known to induce the same isoform in mammals, multiple isoforms are induced with different activities. Phenobarbital-pretreatment in turtles and alligators induces cytochromes P450 and suggestive data indicates induction in the lizard Agama lizard and the newt Pleurodeles waltl. In amphibians and reptiles a CYP2B protein does appear to be present along with constitutive activities associated with the 2 family of cytochromes P450. The markedly different response to classic inducers combined with lower or absent activities alters the view of how amphibians and reptilians respond to xenobiotic challenges.

Dioxins, including polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans, and coplanar polychlorinated biphenyls, are known to be metabolized by enzymes such as cytochrome (CYP) P450, angular dioxygenase, lignin peroxidase, and dehalogenase. It is noted that all of these enzymes have metal ions in their active centers, and the enzyme systems except for peroxidase each have a distinct electron transport chain. Among these enzyme systems, we have focused on cytochrome P450-dependent metabolism of dioxins from the viewpoint of practical use for bioremediation. Mammalian and fungal cytochromes P450 showed remarkable activity toward low-chlorinated PCDDs. In particular, mammalian cytochromes P450 belonging to the CYP1 family showed high activity. Rat CYP1A1 showed high activity toward 2,3,7-trichloro-dibenzo-p-dioxin but no detectable activity for 2,3,7,8-tetrachloro-dibenzo-p-dioxin (2,3,7,8-TCDD). On the basis of these results, we assumed that enlarging the space of the substrate-binding pocket of rat CYP1A1 might generate TCDD-metabolizing enzyme. Large-sized amino acids located at putative substrate-recognition sites and F-G loop were substituted for alanine by site-directed mutagenesis. Finally, we successfully generated 2,3,7,8-TCDD-metabolizing enzyme by site-directed mutagenesis of rat CYP1A1. We hope that recombinant microorganisms harboring genetically engineered cytochrome P450 will be used for bioremediation of soil contaminated with PCDDs, polychlorinated dibenzofurans, and coplanar polychlorinated biphenyls in the future.

1. Molecular orbital calculations, by the Modified Intermediate Neglect of Differential Overlap (MINDO/3) method, of a series of twenty-five 8-acyl-7-hydroxy coumarins show that the inhibition of aryl hydrocarbon hydroxylase (AHH) activities (cytochrome P4501, CYP1 activity, primarily CYP1A1) for 23 of these compounds is related to their structural parameters. The two remaining compounds are the only chlorinated derivatives; these are inactive towards the AHH system and were excluded from the quantitative structure-activity relationship (QSAR) analysis. 2. The results of multiple regression analyses show that AHH activity is dependent on the energy of the highest occupied molecular orbital, E(HOMO), in a single variable expression for the 23 compounds. However, a three-variable expression involving superdelocalizabilities provides a more significant correlation with biological activity. 3. The inactivity of the two chlorinated derivatives can be rationalized in terms of their low degree of molecular planarity, as estimated by the area/depth2 parameter, which presumably precludes them from interaction with CYP1.

Chalcones are phenolic compounds that can be isolated from plants. Previous studies have described some pharmacological applications for these compounds. Making use of our established reporter gene system, we determined the effect of five hydroxychalcones--2-hydroxychalcone, 2'-hydroxychalcone, 4-hydroxychalcone, 4,2',4'-trihydroxychalcone, and 3,4,2',4'-tetrahydroxychalcone--on the cellular xenobiotic responsive element (XRE)-transactivation. The interference of chalcones acting against polycyclic aromatic hydrocarbon (PAH)-DNA binding was also examined. Enzyme inhibition assays of cytochrome P450 (CYP) 1A1 and CYP1B1 were initially performed on recombinant protein expressed in insect microsomes. 2'-Hydroxychalcone and 2-hydroxychalcone were the most effective among the tested hydroxychalcones. The two hydroxychalcones had comparable IC50 values for CYP1A1 and CYP1B1, which were determined to be at the micromolar and submicromolar range, respectively. However, reporter gene assays indicated that 2'-hydroxychalcone suppressed XRE-transactivation, whereas 2-hydroxychalcone induced it when 7,12-dimethylbenz[a]anthracene (DMBA) was co-administered. In the absence of DMBA, 10 microM 2-hydroxychalcone and 2'-hydroxychalcone increased XRE-transactivation by 18- and 2.5-fold, respectively, while other chalcones did not significantly alter the response. Cultures treated with the two hydroxychalcones also displayed separate trends in ethoxyresorufin-O-deethylase (EROD) activity and DMBA-DNA covalent binding. In summary, the present study illustrated that the inhibition of hydroxychalcone on CYP1 enzymes and XRE-transactivation was affected by the position and number of hydroxyl groups in its structure.