Adalimumab in Japanese patients with active ulcers of pyoderma gangrenosum: Final analysis of a 52-week phase 3 open-label study.

In this 52-week, phase 3 open-label study, efficacy and safety of adalimumab were evaluated in Japanese patients with active ulcers due to pyoderma gangrenosum (PG) during a 26-week treatment period and another 26-week extension period. Patients received adalimumab 160 mg at week 0, 80 mg at week 2, and 40 mg every week from week 4. At week 26, 12 of 22 patients (54.5%, p < 0.001) achieved the primary efficacy endpoint of PG area reduction 100 (PGAR 100, complete skin re-epithelialization) for the target ulcer. Nine patients with Physician's Global Assessment (PGA) score of 1, 2, or 3, including four patients achieving PGAR 100, continued into the extension period. During the extension period, six of nine patients (66.7%) achieved PGAR 100 for the target PG ulcer at 52 weeks; one patient who achieved PGAR 100 before week 26 experienced a relapse 162 days after achieving this endpoint. Six patients achieved PGA 0 by week 52, and one patient reported new ulcers at day 57 of the extension period. Continued improvements from study baseline to week 52 were observed in pain (mean [95% CI] -4.0 [-6.5 to -1.5] numeric rating scale) and Dermatology Life Quality Index (-7.3 [-15.1 to 0.4]). In addition to the adverse events (AE) reported in 18 patients (including four serious AE) through week 26 (most commonly infections [n = 11]), there was one 1 additional AE (infection) during the extension period. These results suggest that adalimumab is effective and generally well tolerated in Japanese patients with active PG ulcers.

Adalimumab in Japanese patients with active ulcers of pyoderma gangrenosum: Twenty-six-week phase 3 open-label study.

This phase 3 multicenter study, including 26-week treatment and extension periods, evaluated the efficacy and safety of adalimumab in Japanese patients with active ulcers due to pyoderma gangrenosum. Patients received adalimumab 160 mg at week 0, 80 mg at week 2, and then 40 mg every week starting at week 4. Of the 22 enrolled patients, 12 (54.5%, P < 0.001) achieved the primary efficacy end-point of pyoderma gangrenosum area reduction 100 (PGAR 100, defined as complete skin re-epithelialization) for the target ulcer at week 26 assessed by digital planimetry. PGAR 100 response was observed as early as week 6 (13.6%) and continued to increase over time. The mean percent change from baseline in target ulcer area was -31.8% at week 6 and -63.8% at week 26. A Physician's Global Assessment score of 0 (PGA 0, all ulcers completely clear) was achieved by two patients (9.1%) at week 6 and eight (36.4%) at week 26, while PGA 0/1 (completely/almost clear) was achieved by five (22.7%) and 12 patients (54.5%) at week 6 and 26, respectively. Mean changes from baseline in pain numeric rating scale (-1.8 at week 6 and -2.5 at week 26) and the Dermatology Life Quality Index (-3.1 at week 6 and -3.6 at week 26) improved over time. Adverse events were reported by 18 patients, most commonly infections (n = 11), and serious adverse events by four. These results suggest that adalimumab is effective and generally well tolerated in Japanese patients with active ulcers of pyoderma gangrenosum.

Analysis of gene expression for microminipig liver transcriptomes using parallel long-read technology and short-read sequencing.

The microminipig is one of the smallest minipigs that has emerged as a possible experimental animal model, because it shares many anatomical and/or physiological similarities with humans, including the coronary artery distribution in the heart, the digestive physiology, the kidney size and its structure, and so on. However, information on gene expression profiles, including those on drug-metabolizing phase I and II enzymes, in the microminipig is limited. Therefore, the aim of the present study was to identify transcripts in microminipig livers and to determine gene expression profiles. De novo assembly and expression analyses of microminipig transcripts were conducted with liver samples from three male and three female microminipigs using parallel long-read and short-read sequencing technologies. After unique sequences had been automatically aligned by assembling software, the mean contig length of 50843 transcripts was 707 bp. The expression profiles of cytochrome P450 (P450) 1A2, 2C, 2E1 and 3A genes in livers in microminipigs were similar to those in humans. Liver carboxylesterase (CES) precursor, liver CES-like, UDP-glucuronosyltransferase (UGT) 2C1-like, amine sulfotransferase (SULT)-like, N-acetyltransferases (NAT8) and glutathione S-transferase (GST) A2 genes, which are relatively unknown genes in pigs and/or humans, were expressed strongly. Furthermore, no significant gender differences were observed in the gene expression profiles of phase I enzymes, whereas UGT2B17, SULT1E1, SULT2A1, amine SULT-like, NAT8 and GSTT4 genes were different between males and females among phase II enzyme genes under the present sample conditions. These results provide a foundation for mechanistic studies and the use of microminipigs as model animals for drug development in the future. Copyright © 2016 John Wiley & Sons, Ltd.

Identification of putative substrates for cynomolgus monkey cytochrome P450 2C8 by substrate depletion assays with 22 human P450 substrates and inhibitors.

Cynomolgus monkeys are widely used in drug developmental stages as non-human primate models. Previous studies used 89 compounds to investigate species differences associated with cytochrome P450 (P450 or CYP) function that reported monkey specific CYP2C76 cleared 19 chemicals, and homologous CYP2C9 and CYP2C19 metabolized 17 and 30 human CYP2C9 and/or CYP2C19 substrates/inhibitors, respectively. In the present study, 22 compounds selected from viewpoints of global drug interaction guidances and guidelines were further evaluated to seek potential substrates for monkey CYP2C8, which is highly homologous to human CYP2C8 (92%). Amodiaquine, montelukast, quercetin and rosiglitazone, known as substrates or competitive inhibitors of human CYP2C8, were metabolically depleted by recombinant monkey CYP2C8 at relatively high rates. Taken together with our reported findings of the slow eliminations of amodiaquine and montelukast by monkey CYP2C9, CYP2C19 and CYP2C76, the present results suggest that these at least four chemicals may be good marker substrates for monkey CYP2C8. Copyright © 2016 John Wiley & Sons, Ltd.

Evaluation of cytochrome P450 inductions by anti-epileptic drug oxcarbazepine, 10-hydroxyoxcarbazepine, and carbamazepine using human hepatocytes and HepaRG cells.

Anti-epileptic drug oxcarbazepine is structurally related to carbamazepine, but has reportedly different metabolic pathway. Auto-induction potentials of oxcarbazepine, its pharmacologically active metabolite 10-hydroxyoxcarbazepine and carbamazepine were evaluated by cytochrome P450 (CYP) 1A2, CYP2B6 and CYP3A4 mRNA levels and primary metabolic rates using human hepatocytes and HepaRG cells. For the CYP1A2 the induction potential determined as the fold change in mRNA levels was 7.2 (range: 2.3-11.5) and 10.0 (6.2-13.7) for oxcarbazepine and carbamazepine, respectively, while 10-hydroxyoxcarbazepine did not induce. The fold change in mRNA levels for CYP2B6 was 11.5 (3.2-19.3), 7.0 (2.5-10.8) and 14.8 (3.1-29.1) for oxcarbazepine, 10-hydroxyoxcarbazepine and carbamazepine, respectively. The fold change for CYP3A4 induction level by oxcarbazepine, 10-hydroxyoxcarbazepine and carbamazepine was 3.5 (1.2-7.4), 2.7 (0.8-5.7) and 8.3 (3.5-14.5), respectively. The data suggest lower induction potential of oxcarbazepine and 10-hydroxyoxcarbazepine relative to carbamazepine. The results in HepaRG cells showed similar trend as the human hepatocytes. After incubation for 72 h in hepatocytes and HepaRG cells, auto-induction was evident for only carbamazepine metabolism. The 10-keto group instead of double bond at C10 position is evidently a determinant factor for limited auto-induction of P450 enzymes by oxcarbazepine.

Similar substrate specificity of cynomolgus monkey cytochrome P450 2C19 to reported human P450 2C counterpart enzymes by evaluation of 89 drug clearances.

Cynomolgus monkeys are used widely in preclinical studies as non-human primate species. The amino acid sequence of cynomolgus monkey cytochrome P450 (P450 or CYP) 2C19 is reportedly highly correlated to that of human CYP2C19 (92%) and CYP2C9 (93%). In the present study, 89 commercially available compounds were screened to find potential substrates for cynomolgus monkey CYP2C19. Of 89 drugs, 34 were metabolically depleted by cynomolgus monkey CYP2C19 with relatively high rates. Among them, 30 compounds have been reported as substrates or inhibitors of, either or both, human CYP2C19 and CYP2C9. Several compounds, including loratadine, showed high selectivity to cynomolgus monkey CYP2C19, and all of these have been reported as human CYP2C19 and/or CYP2C9 substrates. In addition, cynomolgus monkey CYP2C19 formed the same loratadine metabolite as human CYP2C19, descarboethoxyloratadine. These results suggest that cynomolgus monkey CYP2C19 is generally similar to human CYP2C19 and CYP2C9 in its substrate recognition functionality.

Comprehensive Evaluation for Substrate Selectivity of Cynomolgus Monkey Cytochrome P450 2C9, a New Efavirenz Oxidase.

Cynomolgus monkeys are widely used as primate models in preclinical studies, because of their evolutionary closeness to humans. In humans, the cytochrome P450 (P450) 2C enzymes are important drug-metabolizing enzymes and highly expressed in livers. The CYP2C enzymes, including CYP2C9, are also expressed abundantly in cynomolgus monkey liver and metabolize some endogenous and exogenous substances like testosterone, S-mephenytoin, and diclofenac. However, comprehensive evaluation regarding substrate specificity of monkey CYP2C9 has not been conducted. In the present study, 89 commercially available drugs were examined to find potential monkey CYP2C9 substrates. Among the compounds screened, 20 drugs were metabolized by monkey CYP2C9 at a relatively high rates. Seventeen of these compounds were substrates or inhibitors of human CYP2C9 or CYP2C19, whereas three drugs were not, indicating that substrate specificity of monkey CYP2C9 resembled those of human CYP2C9 or CYP2C19, with some differences in substrate specificities. Although efavirenz is known as a marker substrate for human CYP2B6, efavirenz was not oxidized by CYP2B6 but by CYP2C9 in monkeys. Liquid chromatography-mass spectrometry analysis revealed that monkey CYP2C9 and human CYP2B6 formed the same mono- and di-oxidized metabolites of efavirenz at 8 and 14 positions. These results suggest that the efavirenz 8-oxidation could be one of the selective markers for cynomolgus monkey CYP2C9 among the major three CYP2C enzymes tested. Therefore, monkey CYP2C9 has the possibility of contributing to limited specific differences in drug oxidative metabolism between cynomolgus monkeys and humans.

Evaluation of 89 compounds for identification of substrates for cynomolgus monkey CYP2C76, a new bupropion/nifedipine oxidase.

Cynomolgus monkeys are widely used in preclinical studies during drug development because of their evolutionary closeness to humans, including their cytochrome P450s (P450s). Most cynomolgus monkey P450s are almost identical (≥90%) to human P450s; however, CYP2C76 has low sequence identity (approximately 80%) to any human CYP2Cs. Although CYP2C76 has no ortholog in humans and is partly responsible for species differences in drug metabolism between cynomolgus monkeys and humans, a broad evaluation of potential substrates for CYP2C76 has not yet been conducted. In this study, a screening of 89 marketed compounds, including human CYP2C and non-CYP2C substrates or inhibitors, was conducted to find potential CYP2C76 substrates. Among the compounds screened, 19 chemicals were identified as substrates for CYP2C76, including substrates for human CYP1A2 (7-ethoxyresorufin), CYP2B6 (bupropion), CYP2D6 (dextromethorphan), and CYP3A4/5 (dextromethorphan and nifedipine), and inhibitors for CYP2B6 (sertraline, clopidogrel, and ticlopidine), CYP2C8 (quercetin), CYP2C19 (ticlopidine and nootkatone), and CYP3A4/5 (troleandomycin). CYP2C76 metabolized a wide variety of the compounds with diverse structures. Among them, bupropion and nifedipine showed high selectivity to CYP2C76. As for nifedipine, CYP2C76 formed methylhydroxylated nifedipine, which was not produced by monkey CYP2C9, CYP2C19, or CYP3A4, as identified by mass spectrometry and estimated by a molecular docking simulation. This unique oxidative metabolite formation of nifedipine could be one of the selective marker reactions of CYP2C76 among the major CYP2Cs and CYP3As tested. These results suggest that monkey CYP2C76 contributes to bupropion hydroxylation and formation of different nifedipine oxidative metabolites as a result of its relatively large substrate cavity.

Qualitative de novo analysis of full length cDNA and quantitative analysis of gene expression for common marmoset (Callithrix jacchus) transcriptomes using parallel long-read technology and short-read sequencing.

The common marmoset (Callithrix jacchus) is a non-human primate that could prove useful as human pharmacokinetic and biomedical research models. The cytochromes P450 (P450s) are a superfamily of enzymes that have critical roles in drug metabolism and disposition via monooxygenation of a broad range of xenobiotics; however, information on some marmoset P450s is currently limited. Therefore, identification and quantitative analysis of tissue-specific mRNA transcripts, including those of P450s and flavin-containing monooxygenases (FMO, another monooxygenase family), need to be carried out in detail before the marmoset can be used as an animal model in drug development. De novo assembly and expression analysis of marmoset transcripts were conducted with pooled liver, intestine, kidney, and brain samples from three male and three female marmosets. After unique sequences were automatically aligned by assembling software, the mean contig length was 718 bp (with a standard deviation of 457 bp) among a total of 47,883 transcripts. Approximately 30% of the total transcripts were matched to known marmoset sequences. Gene expression in 18 marmoset P450- and 4 FMO-like genes displayed some tissue-specific patterns. Of these, the three most highly expressed in marmoset liver were P450 2D-, 2E-, and 3A-like genes. In extrahepatic tissues, including brain, gene expressions of these monooxygenases were lower than those in liver, although P450 3A4 (previously P450 3A21) in intestine and P450 4A11- and FMO1-like genes in kidney were relatively highly expressed. By means of massive parallel long-read sequencing and short-read technology applied to marmoset liver, intestine, kidney, and brain, the combined next-generation sequencing analyses reported here were able to identify novel marmoset drug-metabolizing P450 transcripts that have until now been little reported. These results provide a foundation for mechanistic studies and pave the way for the use of marmosets as model animals for drug development in the future.

Hepatic microsomal UDP-glucuronosyltransferase (UGT) activities in the microminipig.

The microminipig, a small minipig, was bred as a novel experimental animal for nonclinical pharmacology/toxicology studies by Fuji Micra Inc. (Shizuoka, Japan). Species differences in drug metabolism between humans and the microminipig need to be elucidated in more detail in order to discuss the results of nonclinical studies. Glucuronidation catalysed by UDP-glucuronosyltransferase (UGT) is an important pathway in the metabolism of a wide variety of compounds. The aim of the present study was to identify the characteristics of hepatic UGT activity in the microminipig and compare them with those in humans and other experimental animals. This study examined in vitro UGT activities using liver microsomes from microminipigs (8 months old and 1 day old), humans, mice, rats, dogs, monkeys and minipigs. The glucuronides of estradiol, imipramine, serotonin, propofol, 3'-azido-3'-deoxythymidine (AZT) and morphine, selective substrates of human UGT1A1, 1A4, 1A6, 1A9 and 2B7 (AZT and morphine), respectively, were measured using LC-MS/MS. Estradiol-3-glucuronidation activity was higher in the microminipig than in humans and the other animals. High levels of estradiol-3-glucuronidation were observed in the microsomes of 1-day-old microminipigs. Imipramine-N-glucuronidation, a distinctive conjugation by human UGT1A4, was catalysed by microminipig liver microsomes, but not by dog liver microsomes. Although AZT-glucuronidation activity was low in the microminipig compared with humans, morphine-3-glucuronidation activity in the microminipig was higher than that in humans. The UGT activities in the microminipig were similar to those in the minipig. The results of the present study have provided useful information for selecting an appropriate animal model for nonclinical studies.

Focused DNA microarray analysis for sex-dependent gene expression of drug metabolizing enzymes, transporters and nuclear receptors in rat livers and kidneys.

Cytochrome P450(CYP)s are known to show a sexual dimorphic expression in rat livers. However, the comprehensive analysis for the sex-dependent gene expressions of drug metabolizing enzymes except for CYPs, transporters and nuclear receptors in rat livers and kidneys has not been investigated yet. The purpose of the present study was to identify the novel drug metabolizing and pharmacokinetics (DMPK)-related gene(s) which show the sex difference in the mRNA expressions in rat livers and kidneys. Total RNAs were prepared from livers and kidneys in both male and female rats (Crl:CD(SD) and Crlj:WI). A DNA microarray analysis using a "GeneSQUARE Multiple Assay DNA Microarray Drug Metabolism Gene Expression for Rat" was performed. DMPK-related genes which showed sex differences in the mRNA expression were identified in rat livers or kidneys. Especially, the female dominant expressions of UDP glucuronosyltransferase (UGT) s were seen in rat livers and kidneys. The sex difference of UGT expressions in rats might be one of the causal factors of the sex difference of the biological response to UGT substrates.

Lung tumorigenesis promoted by anti-apoptotic effects of cotinine, a nicotine metabolite through activation of PI3K/Akt pathway.

We previously found that genetic polymorphism in cytochrome P450 2A6 (CYP2A6) is one of the potential determinants of tobacco-related lung cancer risk. It has been reported that the plasma concentration of cotinine, a major metabolite of nicotine, in carriers of wild-type alleles of CYP2A6 is considerably higher than that in carriers of null or reduced-function alleles of CYP2A6, raising the possibility that cotinine plays an important role in the development of lung cancer. As a novel mechanism of lung tumorigenesis mediated by CYP2A6, we investigated the effects of cotinine on the suppression of apoptosis and promotion of lung tumor growth. In human lung adenocarcinoma A549 cells, cotinine inhibited doxorubicin-induced cell death by suppressing caspase-mediated apoptosis. Enhanced phosphorylation of Akt, a key factor responsible for cell survival and inhibition of apoptosis, was detected after cotinine treatment. These data suggest that cotinine suppresses caspase-mediated apoptosis induced by doxorubicin through activation of the PI3K/Akt pathway. Furthermore, we clarified that cotinine significantly facilitated tumor growth in the Lewis lung cancer model and accelerated development of lung adenomas induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in A/J mice. We herein propose that cotinine induces tumor promotion by inhibiting apoptosis and enhancing cellular proliferation, thus underlining the importance of CYP2A6 in tobacco-related lung tumorigenesis.

Human cytochrome P450 1A1 is a novel target gene of liver X receptor α.

Cytochrome P450 (CYP) 1A1 is involved in the metabolic activation of polycyclic aromatic hydrocarbons (PAHs) and is induced by several compounds, including PAHs. The induction of CYP1A1 mediated by the aryl hydrocarbon receptor (AhR) has been well investigated; however, little has been reported on the mechanisms of CYP1A1 induction mediated by factors other than AhR. In this study, we investigated the involvement of liver X receptor alpha (LXRα) in the induction of CYP1A1. TO-901317, an LXRα ligand, induced CYP1A1 mRNA in a dose-dependent fashion. Luciferase reporter assays using HepG2 cells showed that TO-901317 was capable of activating the promoter of the CYP1A1 gene and that a direct repeat 4 (DR4) motif located in a region from -452 to -467 was required for the induction of CYP1A1 through LXRα. Specific binding of LXRα to this DR4 motif was confirmed by gel shift and chromatin immunoprecipitation assays. Co-treatment of HepG2 cells with TO-901317 and 2,3,7,8-tetrachlorodibenzo-p-dioxin, a typical AhR ligand, caused the synergistic induction of CYP1A1 mRNA. Thus, we propose that the expression of CYP1A1 is regulated by LXRα as well as by AhR, suggesting that exposure to both LXRα and AhR ligands can result in the alteration of individual susceptibility to environmental carcinogens metabolically activated by CYP1A1.

Novel gene markers of immunosuppressive chemicals in mouse lymph node assay.

The murine local lymph node assay (LLNA) is an immunologically based test of the sensitizing potential of immunotoxicants, but also evaluates immunosuppressive potential with good sensitivity and specificity. We conducted the LLNA with calcineurin inhibitors (tacrolimus and cyclosporin A), antimetabolites (methotrexate and azathioprine), steroids (dexamethasone and prednisolone), and an alkylator (cyclophosphamide). We performed a comprehensive analysis of results of gene expression in lymph nodes obtained in the LLNA using a highly sensitive DNA chip, 3D-Gene™, and the quantitative reverse transcription-polymerase chain reaction (qPCR). Zfp459 expression increased in all animals treated with immunosuppressive chemicals. Ltf, Cbll1 and Lias expression changed specifically in response to the calcineurin inhibitors, Fmo2 and 9630033F20Rik in response to the antimetabolites, Krt8, Gjb1, Hmha1 and Sfrs7 in response to the steroids, and Gbp1 and Mup5 in response to the alkylator. Therefore, these genes were considered novel markers for immunosuppression and their expression could be used to evaluate the mechanism of action of immunosuppressive chemicals.

A possible mechanism for hepatotoxicity induced by BIRB-796, an orally active p38 mitogen-activated protein kinase inhibitor.

BIRB-796, a selective inhibitor of p38 mitogen-activated protein kinase, has entered clinical trials for the treatment of autoimmune diseases. Levels of alanine transaminase, a biomarker of hepatic toxicity in clinical pathology, were found to be increased in Crohn's disease patients treated with BIRB-796. The purpose of the present study was to clarify the molecular mechanism(s) of this hepatotoxicity. A toxicogenomic analysis using a highly sensitive DNA chip, 3D-Gene™ Mouse Oligo chip 24k, indicated that BIRB-796 treatment activated the nuclear factor (erythroid-derived 2)-like 2 signaling pathway, which plays a key role in the response to oxidative stress. A reactive intermediate of BIRB-796 was detected by the glutathione-trapping method using mouse and human liver microsomes. The production of this reactive metabolite in the liver may be one of the causes of BIRB-796's hepatotoxicity.

Identification of AhR-regulated genes involved in PAH-induced immunotoxicity using a highly-sensitive DNA chip, 3D-Gene Human Immunity and Metabolic Syndrome 9k.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants with various toxic effects including immune suppression. However, the molecular mechanism of their toxicity has not been fully clarified. The purpose of this study was to identify novel aryl hydrocarbon receptor (AhR)-regulated genes involved in PAH-induced immunotoxicity using a highly-sensitive DNA chip, 3D-Gene(TM) Human Immunity & Metabolic Syndrome 9k. Leucine-rich repeat-containing protein 25, glucosaminyl (N-acetyl) transferase 3 (GCNT3), thyroxine-binding globulin, aldehyde dehydrogenase 8A1, diacylglycerol O-acyltransferase homolog 2 (DGAT2), haptoglobin, neuron navigator 2 isoform 1, hemopexin and bile acid receptor were found to be up- or down-regulated by PAHs via AhR. Among these genes, GCTN3 and DGAT2 were responsible for immune responses. Therefore, disruption of the expression of these genes via AhR may be one of the causes of the immunotoxicity of PAHs.

Two novel CYP2D6*10 haplotypes as possible causes of a poor metabolic phenotype in Japanese.

During the course of sequencing for the CYP2D6 gene, we found a novel single nucleotide polymorphism of g.3318G>A (E383K) associated with CYP2D6*10, termed as CYP2D6*72. We also found a g.1611T>A (F120I) in the CYP2D6*49, which was previously identified as a CYP2D6*10-associated allele in an independent Japanese population. To clarify the effects of these novel CYP2D6*10 haplotypes on the functions of CYP2D6, kinetic analysis for dextromethorphan O-demethylation was performed using the Escherichia coli expression system and human liver microsomes. The V(max)/K(m) values for dextromethorphan O-demethylation catalyzed by recombinant CYP2D6 forms encoded by CYP2D6*10, CYP2D6*49, and CYP2D6*72 were 3.0, 0.5, and 1.3%, respectively, compared with that catalyzed by CYP2D6.1. Liver microsomes from a human subject genotyped as CYP2D6*10/*49 also showed a reduced dextromethorphan O-demethylase activity. CYP2D6.49 formed a 7-hydroxydextromethorphan, with a roughly similar V(max)/K(m) value to that of O-demethylation. These results suggest that these two CYP2D6*10 haplotypes are possible causes of interindividual variation in the activities and the substrate specificity of CYP2D6.

Activation of p53 as a causal step for atherosclerosis induced by polycyclic aromatic hydrocarbons.

This study was performed to prove our hypothesis that the metabolite(s) of polycyclic aromatic hydrocarbons (PAHs) caused the activation or phosphorylation of p53 via DNA damage to suppress the liver X receptor (LXR)-mediated signal transductions as a probably more direct mechanism. We found that LXR-mediated trans-activation was inhibited by 3-methylchoranthrene (MC) and doxorubicin (Dox) in HepG2 cells carrying wild-type p53, but not in Hep3B cells possessing mutant p53. The exogenous expression of wild-type p53 suppressed the LXR-mediated trans-activation in Hep3B cells. The expression of mRNA for ATP binding cassette A1 was suppressed by MC and Dox in HepG2 cells. The protein expression of retinoid X receptor (RXR), a partner of LXR to form a heterodimer, was suppressed by MC and Dox in HepG2 cells.

CYP1A1-mediated mechanism for atherosclerosis induced by polycyclic aromatic hydrocarbons.

Polycyclic aromatic hydrocarbons (PAHs) have been known to induce atherosclerosis. It has been reported that the metabolic activation of PAHs by cytochrome P450 (CYP) is an important step for PAH-induced atherosclerosis. We recently reported that PAHs down-regulated the liver X receptor (LXR) alpha-regulated genes via aryl hydrocarbon receptor (AHR) as one of the causes responsible for atherosclerosis induced by PAHs. Thus, the aim of this study was to clarify the role of CYP1A1 in the suppression of LXR-mediated signal transductions by 3-methlychoranthrene (MC), one of the PAHs. We found that LXR-mediated transactivation was inhibited by the PAH, but not by halogenated aromatic hydrocarbon, which is scarcely metabolized by CYP1A1. The repression of LXR-mediated signal transductions by MC was restored by co-treatment of HepG2 cells with a CYP1A1 inhibitor, alpha-naphthoflavone, and by the transfection of short interference RNA for CYP1A1. Based on these lines of evidence, we propose that the metabolic activation of PAHs by CYP1A1, but not the activation of AHR by PAHs, is a direct mechanism for atherosclerosis via the suppression of LXR-mediated signal transductions.

Genetic polymorphism of CYP2A6 as one of the potential determinants of tobacco-related cancer risk.

Analyzing the CYP2A6 gene of subjects who showed a poor metabolic phenotype toward SM-12502, we discovered a novel mutant allele (CYP2A6*4C) lacking the whole CYP2A6 gene. Using genetically engineered Salmonella typhimurium expressing a human CYP, we found that CYP2A6 was involved in the metabolic activation of a variety of nitrosamines such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) contained in tobacco smoke. Taking these results into consideration, we hypothesized that the subjects carrying the CYP2A6*4C allele had lower risk of tobacco-related lung cancer. In accordance with our hypothesis, our epidemiological studies indicated that smokers homozygous for the CYP2A6*4C allele showed much lower odds ratios toward cancer risk. Other mutant alleles reducing the CYP2A6 activity, besides CYP2A6*4C, also reduced the risk of lung cancer in smokers, particularly of squamous-cell carcinoma and small-cell carcinoma, both smoking-related cancers. 8-Methoxypsoralen, an inhibitor of CYP2A6, efficiently prevented the occurrence of adenoma caused by NNK in A/J mice.