P450 2S1 Research Articles

Eicosanoid-Regulated Myeloid ENaC and Isolevuglandin Formation in Human Salt-Sensitive Hypertension.

The mechanisms by which salt increases blood pressure in people with salt sensitivity remain unclear. Our previous studies found that high sodium enters antigen-presenting cells (APCs) via the epithelial sodium channel and leads to the production of isolevuglandins and hypertension. In the current mechanistic clinical study, we hypothesized that epithelial sodium channel-dependent isolevuglandin-adduct formation in APCs is regulated by epoxyeicosatrienoic acids (EETs) and leads to salt-sensitive hypertension in humans. Salt sensitivity was assessed in 19 hypertensive subjects using an inpatient salt loading and depletion protocol. Isolevuglandin-adduct accumulation in APCs was analyzed using flow cytometry. Gene expression in APCs was analyzed using cellular indexing of transcriptomes and epitopes by sequencing analysis of blood mononuclear cells. Plasma and urine EETs were measured using liquid chromatography-mass spectrometry. Baseline isolevuglandin+ APCs correlated with higher salt-sensitivity index. Isolevuglandin+ APCs significantly decreased from salt loading to depletion with an increasing salt-sensitivity index. We observed that human APCs express the epithelial sodium channel δ subunit, SGK1 (salt-sensing kinase serum/glucocorticoid kinase 1), and cytochrome P450 2S1. We found a direct correlation between baseline urinary 14,15 EET and salt-sensitivity index, whereas changes in urinary 14,15 EET negatively correlated with isolevuglandin+ monocytes from salt loading to depletion. Coincubation with 14,15 EET inhibited high-salt-induced increase in isolevuglandin+ APC. Isolevuglandin formation in APCs responds to acute changes in salt intake in salt-sensitive but not salt-resistant people with hypertension, and this may be regulated by renal 14,15 EET. Baseline levels of isolevuglandin+ APCs or urinary 14,15 EET may provide diagnostic tools for salt sensitivity without a protocol of salt loading.

CYP2S1 rs338599 polymorphism confers reduced risk to anti-tuberculosis drug-induced liver injury and may be a novel marker for its risk prediction.

PurposeIn the present study, we would like to explore whether Cytochrome P450 2S1 (CYP2S1) rs338599 polymorphism confers risk to anti‐tuberculosis drug‐induced liver injury (ADLI) and provide evidence of being used as novel marker for ADLI risk prediction.Patients and methodsA total of 162 pulmonary tuberculosis patients admitted to Affiliated Hospital of Hebei University from August 2018 to March 2021 were selected. Patients who developed into ADLI were assigned as ADLI group (n = 50), and those who did not developed into ADLI were assigned as non‐ADLI group (n = 112). The CYP2S1 rs338599 polymorphism was detected by polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) method using binary logistic regression analyses through adjusting for age and sex.ResultsNo difference was detected in age, sex, smoking status, profession, education level, marital status, alcohol consumption, or using liver‐protecting drugs (p > 0.05). Compared with non‐ADLI group, GG genotype and G allele were significantly higher in ADLI group (p < 0.05).ConclusionOur results indicated that CYP2S1 rs338599 polymorphism conferred reduced risk to ADLI. The tuberculosis patients who had GG genotype or G allele were not susceptible to ADLI. CYP2S1 rs338599 polymorphism may be a novel marker for ADLI risk prediction.

Circular RNA circ_0023404 serves as a miR-636 sponge to promote malignant behaviors in cervical cancer cells through upregulation of CYP2S1.

Cervical cancer is the most common malignant gynecological tumor. Circular RNA (circRNA) circ_0023404 is reported to be upregulated in cervical cancer cells. This aim is to explore the role and mechanism of circ_0023404 in cervical cancer. circ_0023404, microRNA-636 (miR-636), and cytochrome P450 2S1 (CYP2S1) levels were detected by real-time quantitative polymerase chain reaction (RT-qPCR). Cell proliferation, migration, invasion, and apoptosis were detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, 5-ethynyl-2'-deoxyuridine (EDU) assay, colony formation assay, transwell assay, and cytometry assay. Protein levels of cyclin D1, matrix metallopeptidase 9 (MMP9), Bcl-2-associated X protein (Bax), and CYP2S1 were examined by western blot assay. The binding relationship between miR-636 and circ_0023404 or CYP2S1 was predicted by Circinteractome or targetscan, and then verified by a dual-luciferase reporter assay and RNA pull-down assay. circ_0023404 and CYP2S1 expression were increased, and miR-636 was decreased in cervical cancer tissues and cells. Moreover, circ_0023404 knockdown could repress proliferation, migration, invasion, and promote apoptosis of cervical cancer cells in vitro. Mechanically, circ_0023404 could regulate CYP2S1 expression by sponging miR-636. circ_0023404 silencing could attenuate the progression of cervical cancer cells partly by targeting the miR-636/CYP2S1 axis, hinting at a promising therapeutic target for cervical cancer.

Cytochrome b 5 Binds Tightly to Several Human Cytochrome P450 Enzymes.

Numerous studies have been reported in the past 50-plus years regarding the stimulatory role of cytochrome b 5 (b 5) in some, but not all, microsomal cytochrome P450 (P450) reactions with drugs and steroids. A missing element in most of these studies has been a sensitive and accurate measure of binding affinities of b 5 with P450s. In the course of work with P450 17A1, we developed a fluorescent derivative of a human b 5 site-directed mutant, Alexa 488-T70C-b 5, that could be used in binding assays at sub-μM concentrations. Alexa 488-T70C-b 5 bound to human P450s 1A2, 2B6, 2C8, 2C9, 2E1, 2S1, 4A11, 3A4, and 17A1, with estimated K d values ranging from 2.5 to 61 nM. Only weak binding was detected with P450 2D6, and no fluorescence attenuation was observed with P450 2A6. All of the P450s that bound b 5 have some reported activity stimulation except for P450 2S1. The affinity of P450 3A4 for b 5 was decreased somewhat by the presence of a substrate or inhibitor. The fluorescence of a P450 3A4•Alexa 488-T70C-b 5 complex was partially restored by titration with NADPH-P450 reductase (POR) (K d,apparent 89 nM), suggesting the existence of a ternary P450 3A4-b 5-POR complex, as observed previously with P450 17A1. Gel filtration evidence was also obtained for this ternary complex with P450 3A4. Overall, the results indicated that the affinity of b 5 for many P450s is very high, and that ternary P450-b 5-POR complexes are relevant in P450 3A4 reactions as opposed to a shuttle mechanism. SIGNIFICANCE STATEMENT: High-affinity binding of cytochrome b 5 (b 5) (K d < 100 nM) was observed with many drug-metabolizing cytochrome P450 (P450) enzymes. There is some correlation of binding with reported stimulation, with several exceptions. Evidence is provided for a ternary P450 3A4-b 5-NADPH-P450 reductase complex.

Correction to "A Role for the Orphan Human Cytochrome P450 2S1 in Polyunsaturated Fatty Acid ω-1 Hydroxylation Using an Untargeted Metabolomic Approach".

Correction to "A Role for the Orphan Human Cytochrome P450 2S1 in Polyunsaturated Fatty Acid ω-1 Hydroxylation Using an Untargeted Metabolomic Approach".

Cloning and tissue expression of cytochrome P450 2S1, 4V2, 7A1, 7B1, 8B1, 24A1, 26A1, 26C1, 27A1, 39A1, and 51A1 in marmosets

Common marmoset (Callithrix jacchus) is an attractive animal model primate species for potential use in drug metabolism and pharmacokinetic studies. In this study, marmoset cytochrome P450 (P450) 2S1, 4V2, 7A1, 7B1, 8B1, 24A1, 26A1, 26C1, 27A1, 39A1, and 51A1 cDNAs were isolated from marmoset tissues (brains, lungs, livers, kidneys, and jejunums). Deduced amino acid sequences (89–98% homologous) of the marmoset P450 gene suggested similarity of molecular characteristics of marmoset P450s to human counterparts, compared with those of pig, rabbit, and rodents. Phylogenetic analysis using amino acid sequences indicated 11 marmoset P450 forms clustered with those of human and other primate counterparts, suggesting marmoset P450s have an evolutionary close relationship to human and other primate counterparts. Tissue expression patterns of these P450 mRNAs except for P450 7B1 mRNA were generally similar to those of human P450s in the five tissue types analyzed. These results suggest similarity of molecular characteristics for P450 2S1, 4V2, 7A1, 7B1, 8B1, 24A1, 26A1, 26C1, 27A1, 39A1, and 51A1 between marmosets and humans, in addition to the orthologs of human P450 1, 2, 3, and 4 families previously identified and characterized in marmosets.

A Role for the Orphan Human Cytochrome P450 2S1 in Polyunsaturated Fatty Acid ω-1 Hydroxylation Using an Untargeted Metabolomic Approach.

Cytochrome P450 (P450) 2S1 is one of the orphan P450s, known to be expressed but not having a defined function with an endogenous substrate or in drug oxidations. Although it has been clearly demonstrated to catalyze reductive reactions, its role in NADPH-dependent oxidations has been ambiguous. In our efforts to characterize orphan human P450 enzymes, we used an untargeted liquid chromatography-mass spectromterymetabolomic approach with recombinant human P450 2S1 and extracts of rat stomach and intestine, sites of P450 2S1 localization in humans and animals. The search yielded several candidates, including the product 19-hydroxyarachidonic acid. Subsequent 18O analysis and in vitro studies with commercial arachidonic acid and 19-hydroxyarachidonic acid were used to validate ω-1 hydroxylation of the former molecule as a NADPH- and O2-dependent reaction. Steady-state kinetic assays were done for ω-1 hydroxylation reactions of P450 2S1 with several other long-chain fatty acids, including arachidonic, linoleic, α-linolenic, eicosapentaenoic, and docosapentaenoic acids. Rates of hydroxylation were slow, but no detectable activity was seen with either medium-chain length or saturated fatty acids. P450 2S1 is known to be expressed, at least at the mRNA level, to the extent of some other non-3A subfamily P450s in the human gastrointestinal tract, and the activity may be relevant. We conclude that P450 2S1 is a fatty acid ω-1 hydroxylase, although the physiologic relevance of these oxidations remains to be established. The metabolomic approaches we employed in this study are feasible for orphan P450s and other enzymes, in regard to annotation of function, in mammals and other organisms. SIGNIFICANCE STATEMENT: An untargeted mass spectrometry approach was utilized to identify ω-1 hydroxylation of arachidonic acid as an oxidative reaction catalyzed by human cytochrome P450 2S1. The enzyme also catalyzes the relatively slow ω-1 hydroxylation of several other unsaturated long-chain fatty acids.

Abstract 4545: Quantitative profiling of Cytochrome P450 2S1 in colorectal cancer by PRM assay

Abstract Introduction: Cytochromes P450s (CYPs) constitute a superfamily of xenobiotic metabolising enzymes responsible for metabolism of many pharmaceuticals in the liver. Elevated mRNA levels of specific isoforms, such as CYP2S1, are associated with poor prognosis in colorectal cancer (CRC), and represent novel therapeutic targets for biotransformation of prodrugs to potent cytotoxics at the cancer site. In order to understand the expression of CYP2S1 protein, we have developed a parallel reaction monitoring mass spectrometry (PRM MS) assay to screen CRC samples. Method: Peptides (n=3) uniquely associated with CYP2S1 were synthesized and used as standards to optimise analytical performance (fragmentation conditions, LC retention time, LOQ, LOD, dynamic range) in an Orbitrap Fusion-based PRM MS assay. Levels of CYP2S1 were then determined in protein extracts of CRC, relative to the standards. Results: The PRM MS assay yielded quantitative data over 3 orders of magnitude. CYP2S1 was detected in C106, CaCO2, HCC2998, HT55 and DLD1 cell lines (0.05-1.08pg) and HT55 and DLD1 xenografts (0.29-0.41pg). CYP2S1 was also detected in patient-specific CRC tissues, with elevated levels in Stage III tumours. Conclusions: The PRM MS assay provides a specific, sensitive, high throughput method for identifying CYP2S1 compared to established enzyme and immunoassays. CYP2S1 levels vary considerably across CRC sources highlighting the importance of screening to identify the correct models for new drug development and the right patients for subsequent treatment. Citation Format: Sadr ul Shaheed, Laurence H. Patterson, Klaus Pors, Chris W. Sutton. Quantitative profiling of Cytochrome P450 2S1 in colorectal cancer by PRM assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4545.

A Review on the Expression and Metabolic Features of Orphan Human Cytochrome P450 2S1 (CYP2S1).

Cytochrome P450 2S1 (CYP2S1) is one of the 'orphan' CYPs, which is expressed primarily among extra-hepatic tissues and it is inducible by dioxin. Although the contribution of extra-hepatic CYPs in drug metabolism is considered less significant, they play more important roles in leading to in situ toxicity in organs with higher expression. A non-systemic search was performed to review articles relevant to CYP2S1 in literature. This review will update the findings related to the expression and regulation of CYP2S1 gene and protein, substrate profiles and metabolism mechanisms, genetic polymorphisms, and their association with diseases. The expression of CYP2S1 was mainly in the epithelium of portal of entry organs such as respiratory and gastrointestinal tract. Aryl Hydrocarbon Receptor (AHR) is believed to be partly involved in the induction of CYP2S1. CYP2S1 was found to activate and deactivate pro-drugs which resulted in toxicity and detoxification of carcinogens. The current knowledge of the endogenous functions of CYP2S1 is largely related to cell proliferation and lipid metabolisms. Several polymorphic alleles of CYP2S1 have been reported and documented to date. Molecular-based investigations should be performed to better understand the regulation mechanism of CYP2S1 in various cells and tissues. It is pivotal to establish optimum expression and incubation systems in vitro to elucidate the substrate specificity of CYP2S1 and characterise the genetic consequences of variant CYP2S1 in vitro.

High-Fat Diets Alter the Modulatory Effects of Xenobiotics on Cytochrome P450 Activities.

Cytochrome P450 monooxygenase (P450) enzymes metabolize critical endogenous chemicals and oxidize nearly all xenobiotics. Dysregulated P450 activities lead to altered capacity for drug metabolism and cellular stress. The effects of mixed exposures on P450 expression and activity are variable and elusive. A high-fat diet (HFD) is a common exposure that results in obesity and associated pathologies including hepatotoxicity. Herein, we report the effects of cigarette smoke on P450 activities of normal weight and HFD induced obese mice. Activity-based protein profiling results indicate that HFD mice had significantly decreased P450 activity, likely instigated by proinflammatory chemicals, and that P450 enzymes involved in detoxification, xenobiotic metabolism, and bile acid synthesis were effected by HFD and smoke interaction. Smoking increased activity of all lung P450 and coexposure to diet effected P450 2s1. We need to expand our understanding of common exposures coupled to altered P450 metabolism to enhance the safety and efficacy of therapeutic drug dosing.

Heterologous expression of human cytochrome P450 2S1 in Escherichia coli and investigation of its role in metabolism of benzo[a]pyrene and ellipticine.

Cytochrome P450 (CYP) 2S1 is “orphan” CYP that is overexpressed in several epithelial tissues and many human tumors. The pure enzyme is required for better understanding of its biological functions. Therefore, human CYP2S1 was considered to be prepared by the gene manipulations and heterologous expression in Escherichia coli. Here, the conditions suitable for efficient expression of human CYP2S1 protein from plasmid pCW containing the human CYP2S1 gene were optimized and the enzyme purified to homogeneity. The identity of CYP2S1 as the product of heterologous expression was confirmed by dodecyl sulfate–polyacrylamide gel electrophoresis, Western blotting, and mass spectrometry. To confirm the presence of the enzymatically active CYP2S1, the CO spectrum of purified CYP2S1 was recorded. Since CYP2S1 was shown to catalyze oxidation of compounds having polycyclic aromatic structures, the prepared enzyme has been tested to metabolize the compounds having this structural character; namely, the human carcinogen benzo[a]pyrene (BaP), its 7,8-dihydrodiol derivative, and an anticancer drug ellipticine. Reaction mixtures contained besides the test compounds the CYP2S1 enzyme reconstituted with NADPH:CYP reductase (POR) in liposomes, and/or this CYP in the presence of cumene hydroperoxide or hydrogen peroxide. High performance liquid chromatography was employed for separation of BaP, BaP-7,8-dihydrodiol, and ellipticine metabolites. The results found in this study demonstrate that CYP2S1 in the presence of cumene hydroperoxide or hydrogen peroxide catalyzes oxidation of two of the test xenobiotics, a metabolite of BaP, BaP-7,8-dihydrodiol, and ellipticine. Whereas BaP-7,8,9,10-tetrahydrotetrol was formed as a product of BaP-7,8-dihydrodiol oxidation, ellipticine was oxidized to 12-hydroxyellipticine, 13-hydroxyellipticine, and the ellipticine N2-oxide.Graphical abstractElectronic supplementary materialThe online version of this article (doi:10.1007/s00706-016-1738-2) contains supplementary material, which is available to authorized users.

A transcriptomic approach to elucidate the physiological significance of human cytochrome P450 2S1 in bronchial epithelial cells

BackgroundCytochrome P450 2S1 (CYP2S1) is an orphan P450 with an unknown biological function. Data from our laboratory and others suggest that CYP2S1 may have an important physiological role in modulating the synthesis and metabolism of bioactive lipids including prostaglandins and retinoids. CYP2S1 expression is elevated in multiple epithelial-derived cancers as well as in the chronic hyperproliferative disease psoriasis. Whether CYP2S1 expression in proliferative disease is protective, detrimental, or neutral to disease progression remains to be determined. Two human bronchial epithelial cells (BEAS-2B) were constructed to represent chronic depletion of CYP2S1 using short-hairpin RNA (shRNA) silencing directed toward the 3’UTR (759) and exon 3 (984) of the CYP2S1 gene and compared with a non-targeting shRNA control (SCRAM). Both CYP2S1 mRNA and protein were depleted by approximately 75% in stable cell lines derived from both targeted shRNA constructs (759 and 984). To elucidate the biological significance of CYP2S1, we analyzed transcriptome alterations in response to CYP2S1 depletion in human lung cells.ResultsRNA-sequencing (RNA-seq) analysis was performed to compare the transcriptome of the control (SCRAM) and the CYP2S1-depleted (759) BEAS-2B cell lines. Transcriptomes of the replicates from the two cell lines were found to be distinct populations as determined using Principal Component Analysis and hierarchical clustering. Approximately 1000 genes were differentially expressed in response to CYP2S1 depletion. Consistent with our previous phenotypes, DAVID analysis revealed altered regulation in key pathways implicated in cell proliferation and migration. Transcriptomic profiles were also consistent with the metabolism of proposed endogenous substrates. Pathway analysis also revealed significant expression changes within mTOR signaling, a critical pathway in cell growth. To determine whether these changes manifest as altered cell size, cell diameter and volume were calculated, revealing that CYP2S1 depletion promotes cell growth in BEAS-2B cells.ConclusionsThese data suggest that pathway analysis of sequence-based gene expression is a powerful method to identify pathways and phenotypic alterations in response to changes in orphan enzyme expression. Our results suggest a novel role for CYP2S1-mediated metabolism in modulating BEAS-2B cell size. These findings warrant further studies on CYP2S1 regulated pathways to elucidate potential substrates of CYP2S1.

New cytochrome P450 isoforms as cancer biomarkers and targets for chemopreventive and chemotherapeutic agents

Cytochromes P450 (P450) are a multigene family of enzymes possessing a central role in the oxidative metabolism of a wide range of xenobiotics, including anticancer drugs, and endogenous compounds. The activity of different P450 isoforms varies within specific tissues and cell types and is selectively regulated together with their gene expression. Moreover, differential expression of certain P450 isoforms' genes in tumor cells compared to normal tissues can be observed. This creates the potential for the use of these isozymes as tumor markers or selective prodrug activators. This article discusses the characteristics and function of five isoforms of cytochrome P450 (P450 1B1, P450 2W1, P450 2S1, P450 2R1, P450 2U1) that could be potential targets for tumor therapeutic and preventive strategies. These isoforms have been chosen because their level of expression in tumor tissues is definitely higher than in normal tissues.

Reduction of Aromatic and Heterocyclic Aromatic N-Hydroxylamines by Human Cytochrome P450 2S1

Many aromatic amines and heterocyclic aromatic amines (HAAs) are known carcinogens for animals, and there is also strong evidence of some in human cancer. The activation of these compounds, including some arylamine drugs, involves N-hydroxylation, usually by cytochrome P450 enzymes (P450) in Family 1 (1A2, 1A1, and 1B1). We previously demonstrated that the bioactivation product of the anticancer agent 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203), an N-hydroxylamine, can be reduced by P450 2S1 to its amine precursor under anaerobic conditions and, to a lesser extent, under aerobic conditions [Wang, K., and Guengerich, F. P. (2012) Chem. Res. Toxicol. 25, 1740-1751]. In the study presented here, we tested the hypothesis that P450 2S1 is involved in the reductive biotransformation of known carcinogenic aromatic amines and HAAs. The N-hydroxylamines of 4-aminobiphenyl (4-ABP), 2-naphthylamine (2-NA), and 2-aminofluorene (2-AF) were synthesized and found to be reduced by P450 2S1 under both anaerobic and aerobic conditions. The formation of amines due to P450 2S1 reduction also occurred under aerobic conditions but was less apparent because the competitive disproportionation reactions (of the N-hydroxylamines) also yielded amines. Further, some nitroso and nitro derivatives of the arylamines could also be reduced by P450 2S1. None of the amines tested were oxidized by P450 2S1. These results suggest that P450 2S1 may be involved in the reductive detoxication of several of the activated products of carcinogenic aromatic amines and HAAs.

Cytochrome P450 2S1 Depletion Enhances Cell Proliferation and Migration in Bronchial Epithelial Cells, in Part, through Modulation of Prostaglandin E2 Synthesis

Cytochromes P450 (P450s) contribute to the metabolic activation and inactivation of various endogenous substrates. Despite years of research, the physiological role of CYP2S1 remains unknown. CYP2S1 has demonstrated NADPH P450-reductase-independent metabolism of cyclooxygenase (COX)-derived prostaglandins [e.g., prostaglandin G(2) (PGG(2))] at nanomolar concentrations. Arachidonic acid is converted to prostaglandin precursors [PGG(2) and prostaglandin H(2) (PGH(2))] through COX. These precursors are used to synthesize numerous prostanoids, including PGE(2). Prostaglandin E(2) (PGE(2)) promotes cell proliferation and cell migration and inhibits apoptosis. CYP2S1 metabolism of PGG(2) presumably sequesters PGG(2) and PGH(2), making them unavailable for synthesis of prostanoids such as PGE(2). Whether CYP2S1 contributes to prostaglandin metabolism and influences cell physiological remains to be determined. The purpose of this study was to evaluate the physiological role of CYP2S1, if any, in human bronchial epithelial cells [SV40-derived bronchial epithelial cell line (BEAS-2B)]. To do this, we used small interfering RNA to deplete CYP2S1 mRNA and protein by approximately 75% and evaluated the impact of CYP2S1 depletion on cell proliferation and migration. CYP2S1 depletion enhanced both cell proliferation and migration in BEAS-2B cells. Consistent with the proposed role of CYP2S1 in PGE(2) synthesis, the reduction in CYP2S1 expression doubled intracellular PGE(2) levels. Pharmacological administration of PGE(2) enhanced cell proliferation in BEAS-2B cells but failed to promote migration. Our data reveal an important role for CYP2S1 in the regulation of cell proliferation and migration, occurring in part through modulation of prostaglandin synthesis.

Bioactivation of Fluorinated 2-Aryl-benzothiazole Antitumor Molecules by Human Cytochrome P450s 1A1 and 2W1 and Deactivation by Cytochrome P450 2S1

Both 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203) and 5-fluoro-2-(3,4-dimethoxyphenyl)-benzothiazole (GW 610) contain the benzothiazole pharmacophore and possess potent and selective in vitro antitumor properties. Prior studies suggested the involvement of cytochrome P450 (P450) 1A1 and 2W1-mediated bioactivation in the antitumor activities and P450 2S1-mediated deactivation of 5F 203 and GW 610. In the present study, the biotransformation pathways of 5F 203 and GW 610 by P450s 1A1, 2W1, and 2S1 were investigated, and the catalytic parameters of P450 1A1- and 2W1-catalyzed oxidation were determined in steady-state kinetic studies. The oxidations of 5F 203 catalyzed by P450s 1A1 and 2W1 yielded different products, and the formation of a hydroxylamine was observed for the first time in the latter process. Liquid chromatography-mass spectrometry (LC-MS) analysis with the synthetic hydroxylamine and also a P450 2W1/5F 203 incubation mixture indicated the formation of dGuo adduct via a putative nitrenium intermediate. P450 2W1-catalyzed oxidation of GW 610 was 5-fold more efficient than the P450 1A1-catalyzed reaction. GW 610 underwent a two-step oxidation process catalyzed by P450 1A1 or 2W1: a regiospecific O-demethylation and a further hydroxylation. Glutathione (GSH) conjugates of 5F 203 and GW 610, presumably through a quninoneimine and a 1,2-quinone intermediate, respectively, were detected. These results demonstrate that human P450s 1A1 and 2W1 mediate 5F 203 and GW 610 bioactivation to reactive intermediates and lead to GSH conjugates and a dGuo adduct, which may account for the antitumor activities of 5F 203 and GW 610 and also be involved in cell toxicity. P450 2S1 can catalyze the reduction of the hydroxylamine to the amine 5F 203 under anaerobic conditions and, to a lesser extent, under aerobic conditions, thus attenuating the anticancer activity.

Species difference in the regulation of cytochrome P450 2S1: lack of induction in rats by the aryl hydrocarbon receptor agonist PCB126

CYP2S1 is an evolutionarily conserved, mainly extra-hepatic member of the CYP2 family and proposed to be regulated by the aryl hydrocarbon receptor (AhR).The present study explores AhR’s regulation of CYP2S1 in male Sprague Dawley rats using PCB126 (3,3′,4,4′,5-pentachlorobiphenyl), the most potent AhR agonist among the PCBs. Additionally, CYP2S1 expression was examined after treatments with the classic CYP-inducers β-naphthoflavone (β-NF, AhR activator), phenobarbital (PB, CAR activator) and dexamethasone (Dex, PXR activator). CYP2S1 and CYP1A1/2, CYP1B1, CYP2B and CYP3A mRNAs were measured in liver, lung, spleen, stomach, kidney, and thymus at different time points.Constitutive CYP2S1 was expressed at comparable levels to other CYPs with the highest expression levels in stomach, kidney and lung. CYP2S1 mRNA was only non-significantly elevated by β-NF in liver tissues. PCB126 did not increase CYP2S1 mRNA in any organ and at any time point examined despite a significant induction of CYP1 genes. PCB126 reduced CYP2S1 mRNA by 40% (not significant) from the 7th post-exposure day in thymus. PB and Dex had no effect on CYP2S1 mRNA levels.These observations show that in this model CYP2S1 is not, or only weakly, regulated by AhR and not induced by CAR or PXR activators.

Cytochrome P450 2S1 is Reduced by NADPH-Cytochrome P450 Reductase

Cytochrome P450 (P450) 2S1 is one of the orphan P450s without a clear physiological function. Controversy has arisen as to whether it can interact with NADPH-P450 reductase and accept electrons. The reduction of 1,4-bis{[2-(dimethylamino-N-oxide)ethyl]amino}-5,8-dihydroxyanthracene-9,10-dione (AQ4N) by P450 2S1 was confirmed, and the NADPH consumption rates were measured aerobically and anaerobically in the absence and presence of the drug. The reduction kinetics of P450 2S1 were rapid, as measured by stopped-flow kinetics. These results confirm that P450 2S1 can be reduced by NADPH-P450 reductase and suggest normal mixed-function oxidase roles of P450 2S1 to be revealed.

Functional Characterization of Human Cytochrome P450 2S1 Using a Synthetic Gene-Expressed Protein inEscherichia coli

Human cytochrome P450 2S1 was recently identified and shown to be inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin and hypoxia. It is highly expressed in epithelial cells of tissues that are exposed to the environment and in many tumors of epithelial origin. The biological function of CYP2S1 has not yet been determined, although its possible role in carcinogen metabolism has been suggested. In this report, we investigated its ability to metabolize carcinogens. To obtain a large quantity of active enzyme for substrate screening, we overexpressed CYP2S1 in Escherichia coli (200 nM culture), using a synthetic gene approach. High-level expression allowed us to achieve purification of CYP2S1 with high specific content and purity (16 nmol/mg). Despite high-level expression, we found that CYP2S1 was not readily reduced by cytochrome P450 reductase, and thus no activity was found using NADPH. However, the oxidative activity of CYP2S1 was supported by cumene hydroperoxide or H(2)O(2), such that CYP2S1 oxidized many important environmental carcinogens, including benzo[a]pyrene, 9,10-dihydro-benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene, benzo[a]pyrene-7,8-dihydrodiol, aflatoxin B1, naphthalene, and styrene, with high turnover. Most substrates tested were converted to detoxification products, except in the case of benzo[a]pyrene-7,8-dihydrodiol, which was converted into the very potent carcinogenic metabolite 7,8-dihydrodiol-trans-9,10-epoxide at a relatively efficient rate (K(m) = 12.4 +/- 2 microM, turnover = 2.3 min(-1)). This metabolite formation was also supported both in vitro and in vivo by fatty acid hydroperoxides described in the accompanying report (p. 1044). Together, these data indicate that CYP2S1 contributes to the metabolism of environmental carcinogens via an NADPH independent activity.

CYP2S1 Gene Polymorphisms in a Korean Population

Cytochrome P450 2S1 (CYP2S1) is a drug-metabolizing enzyme that shows interindividual variations in response to treatments for psoriasis and is regarded as a putative prognostic marker for colorectal cancer treatment. To gain insight into the genetic basis for the interindividual variations, CYP2S1 gene polymorphisms were analyzed in Korean subjects. Using direct sequencing of the CYP2S1 gene, 12 genetic variations, which included the two novel nonsynonymous mutations CYP2S1 S61N (0.3%) and CYP2S1 L230R (0.8%), were identified in 50 Korean subjects. Homology modeling predicted the location of the L230R change to be near two conserved alpha-helices in the hood of the substrate-binding site. Linkage disequilibrium (LD) analysis with seven common CYP2S1 variations showed two discrete LD blocks in CYP2S1 gene and consequently a limited number of haplotypes. Although the importance of novel CYP2S1 variants and haplotypes remains to be discovered, CYP2S1 polymorphisms would provide useful information on interindividual variations with respect to CYP2S1 function, which facilitates drug response predictions and disease prognosis.