CYP1 Family Enzymes Research Articles

The Engagement of Cytochrome P450 Enzymes in Tryptophan Metabolism

Tryptophan is metabolized along three main metabolic pathways, namely the kynurenine, serotonin and indole pathways. The majority of tryptophan is transformed via the kynurenine pathway, catalyzed by tryptophan-2,3-dioxygenase or indoleamine-2,3-dioxygenase, leading to neuroprotective kynurenic acid or neurotoxic quinolinic acid. Serotonin synthesized by tryptophan hydroxylase, and aromatic L-amino acid decarboxylase enters the metabolic cycle: serotonin → N-acetylserotonin → melatonin → 5-methoxytryptamine→serotonin. Recent studies indicate that serotonin can also be synthesized by cytochrome P450 (CYP), via the CYP2D6-mediated 5-methoxytryptamine O-demethylation, while melatonin is catabolized by CYP1A2, CYP1A1 and CYP1B1 via aromatic 6-hydroxylation and by CYP2C19 and CYP1A2 via O-demethylation. In gut microbes, tryptophan is metabolized to indole and indole derivatives. Some of those metabolites act as activators or inhibitors of the aryl hydrocarbon receptor, thus regulating the expression of CYP1 family enzymes, xenobiotic metabolism and tumorigenesis. The indole formed in this way is further oxidized to indoxyl and indigoid pigments by CYP2A6, CYP2C19 and CYP2E1. The products of gut-microbial tryptophan metabolism can also inhibit the steroid-hormone-synthesizing CYP11A1. In plants, CYP79B2 and CYP79B3 were found to catalyze N-hydroxylation of tryptophan to form indole-3-acetaldoxime while CYP83B1 was reported to form indole-3-acetaldoxime N-oxide in the biosynthetic pathway of indole glucosinolates, considered to be defense compounds and intermediates in the biosynthesis of phytohormones. Thus, cytochrome P450 is engaged in the metabolism of tryptophan and its indole derivatives in humans, animals, plants and microbes, producing biologically active metabolites which exert positive or negative actions on living organisms. Some tryptophan-derived metabolites may influence cytochrome P450 expression, affecting cellular homeostasis and xenobiotic metabolism.

CYP2 family: physiological enzymes subset in GtoPdb v.2021.2

Compared to the other CYP2 family enzymes, this subset have physiological rather than drug metabolising enzyme activities.

Liquid chromatography-tandem mass spectrometry analysis of oxidation of 2′-, 3′-, 4′- and 6-hydroxyflavanones by human cytochrome P450 enzymes

2′-Hydroxyflavanone (2′OHFva), 3′OHFva, 4′OHFva, and 6OHFva, the major oxidative products of flavanone by human cytochrome P450 (P450, CYP) enzymes, were studied in regard to further oxidation by human CYP1A1, 1A2, 1B1.1, 1B1.3, and 2A6. The products formed were analyzed with LC-MS/MS and characterized by their positive ion fragmentations on mass spectrometry. Several di-hydroxylated flavanone (diOHFva) and di-hydroxylated flavone (diOHFvo) products, detected by analyzing parent ions at m/z 257 and 255, respectively, were found following incubation of these four hydroxylated flavanones with P450s. The m/z 257 products were produced at higher levels than the latter with four substrates examined. The structures of the m/z 257 products were characterized by LC-MS/MS product ion spectra, and the results suggest that 3′OHFva and 4′OHFva are further oxidized mainly at B-ring by P450s while 6OHFva oxidation was at A-ring. Different diOHFvo products (m/z 255) were also characterized by LC-MS/MS, and the results suggested that most of these diOHFvo products were formed through oxidation or desaturation of the diOHFva products (m/z 257) by P450s. Only when 4′OHFva (m/z 241) was used as a substrate, formation of 4′OHFvo (m/z 239) was detected, indicating that diOHFvo might also be formed through oxidation of 4′OHFvo by P450s. Finally, our results indicated that CYP1 family enzymes were more active than CYP2A6 in catalyzing the oxidation of these four hydroxylated flavanones, and these findings were supported by molecular docking studies of these chemicals with active sites of P450 enzymes.

Aryl Hydrocarbon Receptor-Dependent inductions of omega-3 and omega-6 polyunsaturated fatty acid metabolism act inversely on tumor progression

The Western diet contains a high ratio of omega-6 (ω6) to omega-3 (ω3) polyunsaturated fatty acids (PUFA). The prototypical aryl hydrocarbon receptor (AHR) ligand, 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), induces CYP1 family enzymes, which can metabolize PUFA to epoxides. Mice fed ω3-rich or ω6-rich diets were treated with TCDD and injected subcutaneously with AHR-competent Hepa1-GFP hepatoma cells or AHR-deficient LLC lung cancer cells. TCDD reduced the growth rates of the resulting tumors in ω3-fed mice and inhibited their metastasis to the liver and/or lung, but had the opposite effects in mice fed ω6 PUFA. These responses were likely attributable to the corresponding PUFA epoxides generated in tumor cells and/or host, since many depended upon co-administration of a soluble epoxide hydrolase (EPHX2) inhibitor in males, and/or were associated with increases in epoxide levels in tumors and sites of metastasis. Equivalent effects occurred in females in the absence of EPHX2 inhibition, probably because this sex expressed reduced levels of EPHX2. The responses elicited by TCDD were associated with effects on tumor vascularity, tumor cell proliferation and/or apoptosis. Thus environmental AHR agonists, and potentially also endogenous, nutritional, and microbiome-derived agonists, may reduce or enhance cancer progression depending on the composition of dietary PUFA, particularly in females.

Development of new Coumarin-based profluorescent substrates for human cytochrome P450 enzymes

Cytochrome P450 (CYP) enzymes constitute an essential xenobiotic metabolizing system that regulates the elimination of lipophilic compounds from the body. Convenient and affordable assays for CYP enzymes are important for assessing these metabolic pathways.In this study, 10 novel profluorescent coumarin derivatives with various substitutions at carbons 3, 6 and 7 were developed. Molecular modeling indicated that 3-phenylcoumarin offers an excellent scaffold for the development of selective substrate compounds for various human CYP forms, as they could be metabolized to fluorescent 7-hydroxycoumarin derivatives. Oxidation of profluorescent coumarin derivatives to fluorescent metabolites by 13 important human liver xenobiotic-metabolizing CYP forms was determined by enzyme kinetic assays.Four of the coumarin derivatives were converted to fluorescent metabolites by CYP1 family enzymes, with 6-methoxy-3-(4-trifluoromethylphenyl)coumarin being oxidized selectively by CYP1A2 in human liver microsomes. Another set of four compounds were metabolized by CYP2A6 and CYP1 enzymes. 7-Methoxy-3-(3-methoxyphenyl)coumarin was oxidized efficiently by CYP2C19 and CYP2D6 in a non-selective fashion.The advantages of the novel substrates were (1) an excellent signal-to-background ratio, (2) selectivity for CYP1 forms, and (3) convenient multiwell plate measurement, allowing for precise determination of potential inhibitors of important human hepatic forms CYP1A2, CYP2C19 and CYP2D6.

Comparative liver accumulation of dioxin-like compounds in sheep and cattle: Possible role of AhR-mediated xenobiotic metabolizing enzymes

PCDDs, PCDFs, and PCBs are persistent organic pollutants (POPs) that accumulate in animal products and may pose serious health problems. Those able to bind the aryl hydrocarbon receptor (AhR), eliciting a plethora of toxic responses, are defined dioxin-like (DL) compounds, while the remainders are called non-DL (NDL). An EFSA opinion has highlighted the tendency of ovine liver to specifically accumulate DL-compounds to a greater extent than any other farmed ruminant species. To examine the possible role in such an accumulation of xenobiotic metabolizing enzymes (XME) involved in DL-compound biotransformation, liver samples were collected from ewes and cows reared in an area known for low dioxin contamination. A related paper reported that sheep livers had about 5-fold higher DL-compound concentrations than cattle livers, while the content of the six marker NDL-PCBs did not differ between species. Specimens from the same animals were subjected to gene expression analysis for AhR, AhR nuclear translocator (ARNT) and AhR-dependent oxidative and conjugative pathways; XME protein expression and activities were also investigated. Both AhR and ARNT mRNA levels were about 2-fold lower in ovine samples and the same occurred for CYP1A1 and CYP1A2, being approximately 3- and 9-fold less expressed in sheep compared to cattle, while CYP1B1 could be detectable in cattle only. The results of the immunoblotting and catalytic activity (most notably EROD) measurements of the CYP1A family enzymes were in line with the gene expression data. By contrast, phase II enzyme expression and activities in sheep were higher (UGT1A) or similar (GSTA1, NQO1) to those recorded in cattle. The overall low expression of CYP1 family enzymes in the sheep is in line with the observed liver accumulation of DL-compounds and is expected to affect the kinetics and the dynamics of other POPs such as many polycyclic aromatic hydrocarbons, as well as of toxins (e.g. aflatoxins) or drugs (e.g. benzimidazole anthelmintics) known to be metabolized by those enzymes.

Induction of CYP1A1, CYP1A2, CYP1B1, increased oxidative stress and inflammation in the lung and liver tissues of rats exposed to incense smoke

Incense smoke is increasingly being recognized as a potential environmental contaminant and is linked to malignant and non-malignant respiratory diseases. The detoxification of environmental contaminants including polycyclic aromatic hydrocarbons (PAHs) involves the induction of cytochrome P-450 family enzymes (CYPs) by PAHs. However, the detoxification of PAHs also results in the generation of reactive and unstable intermediary metabolites which are implicated in the oxidative stress, DNA damage, and inflammation. It is unclear whether CYPs are similarly induced by incense smoke, which incidentally contains substantial amounts of PAHs. Here, we examined the impact of long-term incense smoke exposure on the induction of CYPs in male Wister Albino rats. Incense smoke exposure significantly induced the expression of CYP1A1, CYP1A2, and CYP1B1 mRNAs in both lung and liver tissues. The extent of CYP1A1 and CYP1B1 induction was significantly higher in the liver compared to that in the lung, while that of CYP1A2 was greater in the lung than in liver. Incense smoke exposure also increased malondialdehyde and reduced glutathione levels in lung and liver tissues, and the catalase activity in the liver tissues to significant levels. Furthermore incense smoke exposure led to a marked increase in TNF-α and IL-4 levels. The data demonstrate for the first time the capacity of incense smoke to induce CYP1 family enzymes in the target and non-target tissues. Induction of CYPs increased oxidative stress and inflammation appear to be intimately linked to promote the carcinogenesis and health complications in people chronically exposed to incense smoke.

Cytochrome P450 CYP2 genes in the common cormorant: Evolutionary relationships with 130 diapsid CYP2 clan sequences and chemical effects on their expression

Cytochrome P450 CYP2 family enzymes are important in a variety of physiological and toxicological processes. CYP2 genes are highly diverse and orthologous relationships remain clouded among CYP2s in different taxa. Sequence and expression analyses of CYP2 genes in diapsids including birds and reptiles may improve understanding of this CYP family. We sought CYP2 genes in a liver cDNA library of the common cormorant ( Phalacrocorax carbo), and in the genomes of other diapsids, chicken ( Gallus gallus), zebra finch ( Taeniopygia guttata), and anole lizard ( Anolis carolinensis), for phylogenetic and/or syntenic analyses. Screening of the cDNA library yielded four CYP2 cDNA clones that were phylogenetically classified as CYP2C45, CYP2J25, CYP2AC1, and CYP2AF1. There are numerous newly identified diapsid CYP2 genes that include genes related to the human CYP2Cs, CYP2D6, CYP2G2P, CYP2J2, CYP2R1, CYP2U1, CYP2W1, CYP2AB1P, and CYP2AC1P. Syntenic relationships show that avian CYP2Hs are orthologous to CYP2C62P in humans, CYP2C23 in rats, and Cyp2c44 in mice, and suggest that avian CYP2Hs, along with human CYP2C62P and mouse Cyp2c44, could be renamed as CYP2C23, based upon the nomenclature rules. Analysis of sequence and synteny identifies cormorant and finch CYPs that are apparent orthologs of phenobarbital-inducible chicken CYP2C45. Transcripts of all four cormorant CYP2 genes were detected in the liver of birds from Lake Biwa, Japan. The transcript levels bore no significant relationship to levels of chlorinated organic pollutants in the liver, including polychlorinated biphenyls and dichlorodiphenyltrichloroethane and its metabolites. In contrast, concentrations of perfluorooctane sulfonate and perfluorononanoic acid were negatively correlated with levels of CYP2C45 and/or CYP2J25, suggesting down-regulation of expression by these environmental pollutants. This study expands our view of the phylogeny and evolution of CYP2s, and provides evolutionary insight into the chemical regulation of CYP2 gene expression in diapsids including birds.

Caenorhabditis elegans Generates Biologically Relevant Levels of Genotoxic Metabolites from Aflatoxin B1 but Not Benzo[a]pyrene In Vivo

There is relatively little information regarding the critical xenobiotic-metabolizing cytochrome P450 (CYP) enzymes in Caenorhabditis elegans, despite this organism's increasing use as a model in toxicology and pharmacology. We carried out experiments to elucidate the capacity of C. elegans to metabolically activate important promutagens via CYPs. Phylogenetic comparisons confirmed an earlier report indicating a lack of CYP1 family enzymes in C. elegans. Exposure to aflatoxin B(1) (AFB(1)), which is metabolized in mammals by CYP1, CYP2, and CYP3 family enzymes, resulted in significant DNA damage in C. elegans. However, exposure to benzo[a]pyrene (BaP), which is metabolized in mammals by CYP1 family enzymes only, produced no detectable damage. To further test whether BaP exposure caused DNA damage, the toxicities of AFB(1) and BaP were compared in nucleotide excision repair (NER)-deficient (xpa-1) and NER-proficient (N2) strains of C. elegans. Exposure to AFB(1) inhibited growth more in xpa-1 than N2 nematodes, but the growth-inhibitory effects of BaP were indistinguishable in the two strains. Finally, a CYP-nicotinamide adenine dinucleotide phosphate reductase-deficient strain (emb-8) of C. elegans was found to be more resistant to the growth-inhibitory effect of AFB(1) exposure than N2, confirming that the AFB(1)-mediated growth inhibition resulted from CYP-mediated metabolism. Together, these results indicate that C. elegans lacks biologically significant CYP1 family-mediated enzymatic metabolism of xenobiotics. Interestingly, we also found that xpa-1 nematodes were slightly more sensitive to chlorpyrifos than were wild type. Our results highlight the importance of considering differences between xenobiotic metabolism in C. elegans and mammals when using this alternative model in pharmaceutical and toxicological research.

CYP1-mediated antiproliferative activity of dietary flavonoids in MDA-MB-468 breast cancer cells

Among the different mechanisms proposed to explain the cancer-protecting effect of dietary flavonoids, substrate-like interactions with cytochrome P450 CYP1 enzymes have recently been explored. In the present study, the metabolism of the flavonoids chrysin, baicalein, scutellarein, sinensetin and genkwanin by recombinant CYP1A1, CYP1B1 and CYP1A2 enzymes, as well as their antiproliferative activity in MDA-MB-468 human breast adenocarcinoma and MCF-10A normal breast cell lines, were investigated. Baicalein and 6-hydroxyluteolin were the only conversion products of chrysin and scutellarein metabolism by CYP1 family enzymes, respectively, while baicalein itself was not metabolized further. Sinensetin and genkwanin produced a greater number of metabolites and were shown to inhibit strongly in vitro proliferation of MDA-MB-468 cells at submicromolar and micromolar concentrations, respectively, without essentially affecting the viability of MCF-10A cells. Cotreatment of the CYP1 family inhibitor acacetin reversed the antiproliferative activity noticed for the two flavones in MDA-MB-468 cells to 13 and 14 μM respectively. In contrast chrysin, baicalein and scutellarein inhibited proliferation of MDA-MB-468 cells to a lesser extent than sinensetin and genkwanin. The metabolism of genkwanin to apigenin and of chrysin to baicalein was favored by CYP1B1 and CYP1A1, respectively. Taken together the data suggests that CYP1 family enzymes enhance the antiproliferative activity of dietary flavonoids in breast cancer cells, through bioconversion to more active products.

Characterization and expression of extrahepatic CYP2S1

Background: About one-third of the CYP enzymes identified so far, including several novel CYP enzymes such as CYP2S1, CYP2U1 and CYP2W1, belong to the CYP2 family. As with other recently discovered CYP enzymes, detailed information about the catalytic activity and function of CYP2S1 is lacking. Objective: To review and compare the expression of CYP2S1 mRNA and protein in humans, mice and rats, and to critically examine evidence pertaining to CYP2S1 regulation and its catalytic activity. Methods: Information about mouse and human CYP2S1 was summarized from published reports. Data about rat CYP2S1 expression was taken from recent work by the authors. Results/conclusions: CYP2S1 shares molecular characteristics of both CYP1 and CYP2 family enzymes but shows a unique tissue profile of expression. Further studies are needed to identify selective substrates and to measure CYP2S1 protein levels before the role of CYP2S1 in xenobiotic metabolism and its relevance to physiological pathways and disease states can be determined.

Bioactivation of the phytoestrogen diosmetin by CYP1 cytochromes P450

Breast cancer is a major cause of death worldwide. Amongst the various forms of treatment chemoprevention is favoured and natural products such as the dietary flavonoids have been examined for their cancer preventative activity. In this study we investigated the anticancer activity of the flavonoid diosmetin, as a result of cytochrome P450 CYP1 metabolism. Diosmetin was metabolized to luteolin via an aromatic demethylation reaction on the B-ring from CYP1A1, CYP1B1 and the hepatic isozyme CYP1A2. CYP1A1 and CYP1A2 also produced additional unidentified metabolites. CYP1B1 showed the lowest apparent KM and CYP1A1 the highest apparent Kcat. Diosmetin was also metabolized to luteolin in estrogen receptor positive breast cell-line (MCF-7 cells) preinduced for 24 h with the potent CYP1 inducer 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD). Treatment of MCF-7 cells with TCDD caused bioactivation of diosmetin enhancing its cytotoxicity. Taken together these data suggest that the flavonoid diosmetin is metabolised to the more active molecule luteolin by CYP1 family enzymes.

Antiproliferative and cytostatic effects of the natural product eupatorin on MDA-MB-468 human breast cancer cells due to CYP1-mediated metabolism

IntroductionThe natural product eupatorin has been reported to have antiproliferative activity in tumour cell lines, but the exact mechanism is unclear. The cytochromes P450 CYP1B1, CYP1A1, and CYP1A2 have been shown to participate in the activation of various xenobiotics, compounds derived from the diet as well as chemotherapeutic drugs. CYP1B1 and CYP1A1 have also been proposed as targets for cancer chemotherapy for their differential and selective overexpression in tumour cells. In this study, we aimed to identify a possible mechanism of action for the antiproliferative effect of eupatorin, which can be attributed to CYP1 family-mediated metabolism.MethodsThe study focuses on the antiproliferative action of eupatorin on the human breast carcinoma cell line MDA-MB-468 and on a cell line derived from normal mammary tissue, MCF-10A. The cytotoxicity of the flavone, its effect on the cell cycle of the abovementioned cell lines, and its metabolism by CYP1 family enzymes were examined.ResultsEupatorin showed a dose-dependent inhibitory effect of cell growth on MDA-MB-468 cells with a submicromolar median inhibition concentration (IC50) whereas the IC50 of this compound in MCF-10A cells was considerably higher. The antiproliferative effect, as measured by EROD (ethoxyresorufin-O-deethylase) assay and Western immunoblotting, was attributed mainly to CYP1A1 expression in MDA-MB-468 cells but not in MCF-10A cells. Moreover, CYP1 family enzymes were shown to metabolise eupatorin in vitro to the flavone cirsiliol and two other unidentified metabolites. Metabolism of eupatorin was also detected in MDA-MB-468 cell cultures, whereas metabolism by MCF-10A cells was negligible. Eupatorin was further shown to arrest the cell cycle of the CYP1-expressing cell line MDA-MB-468 in G2/M phase, whereas no effect was observed in MCF-10A cells, which do not express CYP1 enzymes. The effect of eupatorin on the MDA-MB-468 cell cycle could be reversed by co-application of the CYP1 inhibitor acacetin.ConclusionThe flavone eupatorin is selectively activated in breast cancer cells, but not in normal breast cells, due to CYP1 family metabolism. This provides a basis for selectivity which is desired against breast tumour cells. In this sense, eupatorin is shown by this study to be a very promising chemopreventative candidate that should be examined further in an in vivo study.

Investigating Human P450s Involved in Drug Metabolism via Homology with High-Resolution P450 Crystal Structures of the CYP2C Subfamily

The important role of high-resolution crystal structures of cytochrome P450 (CYP) enzymes for the generation of P450 models by homology is discussed. The main focus is on human P450 enzymes involved in drug metabolism, where the role of homology modelling has been emphasized in the recent literature. Report of the first human P450 crystal structure has provided an opportunity for comparison between those modelled from other crystallographic templates, and the recent substrate-bound rabbit CYP2C5 structure exemplifies the relevance of high-resolution template structures to generating 3-D models of P450s where the homology is relatively high. In particular, the homology models of human CYP1 and CYP2 family enzymes are presented, where good agreement with experiment findings are apparent.

Quantitative structure–activity relationships (QSARs) for substrates of human cytochromes P450 CYP2 family enzymes

The results of quantitative structure–activity relationship (QSAR) studies on substrates of human CYP2 family enzymes are reported, together with those of a small number of CYP2A6, CYP2C19 and CYP2D6 inhibitors. In general, there are good correlations ( R=0.90–0.99) between binding affinity (based on K m or K D values) and various parameters relating to active site interactions such as hydrogen bonding and π–π stacking. There is also evidence for the role of compound lipophilicity (as determined by either log P or log D 7.4 values) in overall substrate binding affinity, and this could reflect the desolvation energy involved in substrate interaction within the enzyme active site. It is possible to estimate the substrate binding energy for a given P450 from a combination of energy terms relating to hydrogen bonding, π–π stacking, desolvation and loss in rotatable bond energy, which agree closely ( R=0.98) with experimental data based on either K m or K D values. Consequently, it is likely that active site interactions represent the major contributory factors to the overall binding affinities for human CYP2 family substrates and, therefore, their estimation is of potential importance for the development of new chemical entities (NCEs) as this can facilitate an assessment of likely metabolic clearance.

Homology modelling of human CYP2 family enzymes based on the CYP2C5 crystal structure

1. The construction of molecular models for human cytochromes P450 from the CYP2 family are reported, utilizing the recently available crystal structure of CYP2C5, which is also a mammalian (rabbit) form of the enzyme. 2. In particular, selective substrate interactions with CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1 are described in the context of favourable contacts with active site amino acid residues that appear to orientate each substrate for metabolism at the experimentally observed position. 3. The results are consistent with reported findings from site-directed mutagenesis experiments with the CYP2 family, and with published information on substrate metabolism.

Effect of chlorinated hydrocarbons on expression of cytochrome P450 1A1, 1A2 and 1B1 and 2- and 4-hydroxylation of 17β-estradiol in female Sprague–Dawley rats

Chlorinated hydrocarbons (CHCs) are environmental contaminants that bioaccumulate and hence are detected in human tissues. Epidemiological evidence suggests that the increased incidence of a variety of human cancers, such as lymphoma, leukemia and liver and breast cancers, might be attributed to exposure to these agents. The ability of CHCs to disrupt estrogen homeostasis is hypothesized to be responsible for their biological effects. The present study examined the effect of CHCs on the expression of cytochrome P450 (CYP)1A1, CYP1A2 and CYP1B1 mRNAs and the consequent 2- and 4-hydroxylation of 17beta-estradiol (E(2)) in female Sprague-Dawley rats. Animals were administered a single dose of the LD(50) of 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) (25 microg/kg), 2, 4-dichlorophenoxyacetic acid (2,4-D) (375 mg/kg) and dieldrin (DED) (38 mg/kg) by gavage. Seventy-two hours after treatment, increased expression of CYP1A1, CYP1A2 and CYP1B1 was observed in the liver, kidney and mammary tissue. Since CYP1A and CYP1B1 are the major enzymes catalyzing 2- and 4-hydroxylation of E(2), respectively, the effect of these CHCs on the metabolism of E(2) was investigated in rat tissues. Formation of 2- and 4-catechol estrogens was increased in a tissue-specific manner in response to treatment. TCDD was the most potent inducer for CYP1 enzyme mRNA and for the 2- and 4-hydroxylation of E(2). 2,4-D and DED induced similar responses, but less than that of TCDD. These results suggest that induction of CYP1 family enzymes and consequent increases in estrogen metabolism by CHCs in target tissues may be factors contributing to the biological effects associated with exposure to these agents.

Effect of chlorinated hydrocarbons on expression of cytochrome P450 1A1, 1A2 and 1B1 and 2- and 4-hydroxylation of 17β-estradiol in female Sprague–Dawley rats

Chlorinated hydrocarbons (CHCs) are environmental contaminants that bioaccumulate and hence are detected in human tissues. Epidemiological evidence suggests that the increased incidence of a variety of human cancers, such as lymphoma, leukemia and liver and breast cancers, might be attributed to exposure to these agents. The ability of CHCs to disrupt estrogen homeostasis is hypothesized to be responsible for their biological effects. The present study examined the effect of CHCs on the expression of cytochrome P450 (CYP)1A1, CYP1A2 and CYP1B1 mRNAs and the consequent 2- and 4-hydroxylation of 17β-estradiol (E 2 ) in female Sprague–Dawley rats. Animals were administered a single dose of the LD 50 of 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD) (25 μg/kg), 2,4-dichlorophenoxyacetic acid (2,4-D) (375 mg/kg) and dieldrin (DED) (38 mg/kg) by gavage. Seventy-two hours after treatment, increased expression of CYP1A1, CYP1A2 and CYP1B1 was observed in the liver, kidney and mammary tissue. Since CYP1A and CYP1B1 are the major enzymes catalyzing 2- and 4-hydroxylation of E 2 , respectively, the effect of these CHCs on the metabolism of E 2 was investigated in rat tissues. Formation of 2- and 4-catechol estrogens was increased in a tissue-specific manner in response to treatment. TCDD was the most potent inducer for CYP1 enzyme mRNA and for the 2- and 4-hydroxylation of E 2 . 2,4-D and DED induced similar responses, but less than that of TCDD. These results suggest that induction of CYP1 family enzymes and consequent increases in estrogen metabolism by CHCs in target tissues may be factors contributing to the biological effects associated with exposure to these agents.

Molecular modelling of CYP1 family enzymes CYP1A1, CYP1A2, CYP1A6 and CYP1B1 based on sequence homology with CYP102

Molecular modelling of a number of CYP1 family enzymes from rat, plaice and human is described based on amino acid sequence homology with the haemoprotein domain of CYP102, a unique bacterial P450 of known structure. The interaction of various substrates and inhibitors within the putative active sites of rat CYP1A1, human CYP1A2, a fish CYP1 enzyme CYP1A6 (from plaice) and human CYP1B1, is shown to be consistent with P450-mediated oxidation in each example or, in the case of inhibitors, mechanism of inhibition. It is reported that relatively small changes between the enzymes’ active site regions assist in the rationalization of CYP1 enzyme preferences for particular substrate types, and a template of superimposed CYP1A2 substrates is shown to fit the putative active site of the human CYP1A2 enzyme.

Immunochemical Analysis of Liver Microsomal Cytochromes P450 of the American Alligator, Alligator mississippiensis

Ten antibodies raised against various mammalian and fish cytochromes P450 (CYP) enzymes were used to probe the effects of xenobiotic pretreatment on liver microsomes of the American alligator, Alligator mississippiensis. Pretreatment with phenobarbital (PB), 3-methylcholanthrene (3MC), and PB plus 3MC elicited significant induction of multiple CYP enzymes in alligator, as detected by antibodies to CYP1A, CYP2B, CYP2C, CYP2E, CYP2K, and CYP3A. In contrast to the rat, 3MC treatment induced alligator liver microsomes that were immunoreactive with antibodies to CYP2 family enzymes. Induction of CYP enzymes was not as apparent with the Aroclor 1254 (ARO), and 2,2′,4,4′ tetrachlorobiphenyl (TCB) pretreatment used; fewer CYP enzymes primarily detected with antibodies against CYP2C or CYP2E were observed. Clofibrate (CLO; 80 mg/kg Days 1–4), markedly induced CYP4A in rat but this induction was not apparent in alligator. A purified PB-induced alligator liver microsomal CYP enzyme cross-reacted with several antibodies raised against CYP2 family enzymes but did not cross-react with antibodies raised against other CYP families. This indicates the PB-inducible CYP in alligator shares some epitope homology with several CYP2-family enzymes from other animals. These experiments demonstrate the usefulness and limitations of using antibodies across phylogenetic classes. While indicating the presence of CYP enzymes that have epitope homology with CYP1A, CYP2, CYP3 and CYP4 enzymes in alligator, it remains to be established whether these CYP forms are alligator orthologues of mammalian enzymes. In all cases, the relative abundance of alligator liver microsomal CYP as determined by immunoblot analysis appeared lower than found in rat. The presence and induction of CYP indicated by immunochemical analysis, corroborated previously reported enzymatic studies of the same microsomal preparations (Ertl et al., 1998a). Thus, increases in CYP protein by the various inducers employed were paralled by the increases in CYP enzyme-specific or selective activities, e.g., induction of CYP1A protein corresponded with induction of EROD.