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Yasushi Yamazoe - One of the best experts on this subject based on the ideXlab platform.
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constitutive androstane receptor transcriptionally activates human cyp1a1 and CYP1A2 genes through a common regulatory element in the 5 flanking region
Biochemical Pharmacology, 2010Co-Authors: Kouichi Yoshinari, Noriaki Yoda, Takayoshi Toriyabe, Yasushi YamazoeAbstract:Phenobarbital has long been known to increase cellular levels of CYP1A1 and CYP1A2 possibly through a pathway(s) independent of aryl hydrocarbon receptor. We have investigated the role of constitutive androstane receptor (CAR), a xenobiotic-responsive nuclear receptor, in the transactivation of human CYP1A1 and CYP1A2. These genes are located in a head-to-head orientation, sharing a 5'-flanking region. Reporter assays were thus performed with dual-reporter constructs, containing the whole or partially deleted human CYP1A promoter between two different reporter genes. In this system, human CAR (hCAR) enhanced the transcription of both genes through common promoter regions from -461 to -554 and from -18089 to -21975 of CYP1A1. With reporter assays using additional deleted and mutated constructs, electrophoresis mobility shift assays and chromatin immunoprecipitation assays, an ER8 motif (everted repeat separated by eight nucleotides), located at around -520 of CYP1A1, was identified as an hCAR-responsive element and a binding motif of hCAR/human retinoid X receptor alpha heterodimer. hCAR enhanced the transcription of both genes also in the presence of an aryl hydrocarbon receptor ligand. Finally, hCAR activation increased CYP1A1 and CYP1A2 mRNA levels in cultured human hepatocytes. Our results indicate that CAR transactivates human CYP1A1 and CYP1A2 in human hepatocytes through the common cis-element ER8. Interestingly, the ER8 motif is highly conserved in the CYP1A1 proximal promoter sequences of various species, suggesting a fundamental role of CAR in the xenobiotic-induced expression of CYP1A1 and CYP1A2 independent of aryl hydrocarbon receptor.
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omeprazole transactivates human cyp1a1 and CYP1A2 expression through the common regulatory region containing multiple xenobiotic responsive elements
Biochemical Pharmacology, 2008Co-Authors: Kouichi Yoshinari, Rika Ueda, Kazutomi Kusano, Tsutomu Yoshimura, Kiyoshi Nagata, Yasushi YamazoeAbstract:Omeprazole induces human CYP1A1 and CYP1A2 in human hepatoma cells and human liver. Aryl hydrocarbon receptor (AHR) is shown to be involved in this induction. However, its precise molecular mechanism remains unknown because the chemical activates AHR without its direct binding in contrast to typical AHR ligands such as 3-methylcholanthrene (3MC) and beta-naphthoflavone (BNF). Human CYP1A1 and CYP1A2 genes are located in a head-to-head orientation sharing about 23 kb 5'-flanking region. Recently, we succeeded to measure CYP1A1 and CYP1A2 transcriptional activities simultaneously using dual reporter gene constructs containing the 23 kb sequence. In this study, transient transfection assays have been performed using numbers of single and dual reporter constructs to identify omeprazole-responsive region for CYP1A1 and CYP1A2 induction. Reporter assays with deletion constructs have demonstrated that the omeprazole-induced expression of both CYP1A1 and CYP1A2 is mediated via the common regulatory region containing multiple AHR-binding motifs (the nucleotides from -464 to -1829 of human CYP1A1), which is identical with the region for BNF and 3MC induction. Interestingly, omeprazole activated the transcription of CYP1A1 and CYP1A2 to similar extents while BNF and 3MC preferred CYP1A1 expression. We have also found that primaquine is an omeprazole-like CYP1A inducer, while lansoprazole and albendazole are 3MC/BNF-like in terms of the CYP1A1/CYP1A2 preference. The present results suggest that omeprazole as well as BNF and 3MC activates both human CYP1A1 and CYP1A2 expression through the common regulatory region despite that omeprazole may involve a different cellular signal(s) from BNF and 3MC.
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a common regulatory region functions bidirectionally in transcriptional activation of the human cyp1a1 and CYP1A2 genes
Molecular Pharmacology, 2006Co-Authors: Rika Ueda, Frank J Gonzalez, Shioko Kimura, Kazutomi Kusano, Tsutomu Yoshimura, Kiyoshi Nagata, Hiromi Iketaki, Yasushi YamazoeAbstract:The human CYP1A1 and CYP1A2 genes on chromosome 15 are orientated head-to-head and are separated by a 23-kilobase (kb) intergenic spacer region. Thus, the possibility exists for sharing common regulatory elements contained in the spacer region responsible for transcriptional activation and regulation of the CYP1A1 and CYP1A2 genes. In the present study, a reporter gene construct containing -22.4 kb of the 5′-flanking region of the CYP1A2 gene was found to support β-naphthoflavone (BNF) and 3-methylchoranthrene (3-MC)-mediated transcriptional activation. The responsive region was also functional in directing activation of the CYP1A1 promoter, indicating that the region works bidirectionally to govern transcriptional activation of both CYP1A1 and CYP1A2. To simultaneously evaluate transcriptional activation of both genes, a dual reporter vector was developed in which the spacer region was inserted between two different reporter genes, firefly luciferase and secreted alkaline phosphatase. Transient transfection of the dual reporter vector in HepG2 cells revealed increases in both reporter activities after exposure of the cells to BNF and 3-MC. Deletion studies of the spacer region indicated that a region from -464 to -1829 of the CYP1A1 gene works bidirectionally to enhance the transcriptional activation of not only CYP1A1 but also CYP1A2. In addition, a negative bidirectional regulatory region was found to exist from -18,989 to -21,992 of the CYP1A1 gene. These data established that induction of human CYP1A1 and CYP1A2 is simultaneously controlled through bidirectional and common regulatory elements.
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INDUCTION OF CYP1A2 WITH AROMATIC AMINES
Drug Metabolism and Pharmacokinetics, 1995Co-Authors: Masakuni Degawa, Masafumi Nakayama, Katsuyuki Fukuda, Kouji Senzaki, Yasushi YamazoeAbstract:Male F344 rats were treated with a chemical (aniline, nitrobenzene, 2-methoxy-p-phenylenediamine, 2-methoxy-4-nitroaniline or 2-methoxy-4-nitroazobenzene) produced by the azo-reduction and/or N-oxidation of 2-methoxy-4-aminoazobenzene, a selective inducer of CYP1A2, and effects of their chemicals on CYP1A induction in the liver were examined at the levels of mRNA, protein and enzyme activity. 2-Methoxy-4-nitroaniline and 2-methoxy-4-nitroazobenzene, but not other chemicals used, induced CYP1A enzymes, mainly CYP1A2, as assessed by Western and Northern blots. It is noteworthy that 2-methoxy-4-nitroaniline was more selective than 2-methoxy-4-aminoazobenzene for induction of CYP1A2, and it has the smallest molecular size among known CYP1A2 inducers. Although in induction of CYP1A1 by a polycyclic aryl hydrocarbon (Ah) is thought to be regulated though Ah receptor, cellular component(s) other than Ah receptor would play an important role in the induction of CYP1A2. Presence of a structural unit such as “bay-region” in benzo[a]pyrene molecule or “polyhalogenated aromatic ring” in 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin molecule is reportedly requisite for binding of the chemical to Ah receptor, but such structural unit is absent in 2-methoxy-4-nitroaniline molecule.
Chiehfu Chen - One of the best experts on this subject based on the ideXlab platform.
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oxidative metabolism of the alkaloid rutaecarpine by human cytochrome p450
Drug Metabolism and Disposition, 2006Co-Authors: Yune-fang Ueng, Li Kang Ho, Shuyun Wang, Chiehfu ChenAbstract:Rutaecarpine is the main active alkaloid of the herbal medicine, Evodia rutaecarpa . To identify the major human cytochrome P450 (P450) participating in rutaecarpine oxidative metabolism, human liver microsomes and bacteria-expressed recombinant human P450 were studied. In liver microsomes, rutaecarpine was oxidized to 10-, 11-, 12-, and 3-hydroxyrutaecarpine. Microsomal 10- and 3-hydroxylation activities were strongly inhibited by ketoconazole. The 11- and 12-hydroxylation activities were inhibited by α-naphthoflavone, quinidine, and ketoconazole. These results indicated that multiple hepatic P450s including CYP1A2, CYP2D6, and CYP3A4 participate in rutaecarpine hydroxylations. Among recombinant P450s, CYP1A1 had the highest rutaecarpine hydroxylation activity. Decreased metabolite formation at high substrate concentration indicated that there was substrate inhibition of CYP1A1- and CYP1A2-catalyzed hydroxylations. CYP1A1-catalyzed rutaecarpine hydroxylations had V max values of 1388 to ∼1893 pmol/min/nmol P450, K m values of 4.1 to ∼9.5 μM, and K i values of 45 to ∼103 μM. These results indicated that more than one molecule of rutaecarpine is accessible to the CYP1A active site. The major metabolite 10-hydroxyrutaecarpine decreased CYP1A1, CYP1A2, and CYP1B1 activities with respective IC 50 values of 2.56 ± 0.04, 2.57 ± 0.11, and 0.09 ± 0.01 μM, suggesting that product inhibition might occur during rutaecarpine hydroxylation. The metabolite profile and kinetic properties of rutaecarpine hydroxylation by human P450s provide important information relevant to the clinical application of rutaecarpine and E. rutaecarpa .
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oxidative metabolism of the alkaloid rutaecarpine by human cytochrome p450
Drug Metabolism and Disposition, 2006Co-Authors: Yune-fang Ueng, Li Kang Ho, Shuyun Wang, Chiehfu ChenAbstract:Rutaecarpine is the main active alkaloid of the herbal medicine, Evodia rutaecarpa . To identify the major human cytochrome P450 (P450) participating in rutaecarpine oxidative metabolism, human liver microsomes and bacteria-expressed recombinant human P450 were studied. In liver microsomes, rutaecarpine was oxidized to 10-, 11-, 12-, and 3-hydroxyrutaecarpine. Microsomal 10- and 3-hydroxylation activities were strongly inhibited by ketoconazole. The 11- and 12-hydroxylation activities were inhibited by α-naphthoflavone, quinidine, and ketoconazole. These results indicated that multiple hepatic P450s including CYP1A2, CYP2D6, and CYP3A4 participate in rutaecarpine hydroxylations. Among recombinant P450s, CYP1A1 had the highest rutaecarpine hydroxylation activity. Decreased metabolite formation at high substrate concentration indicated that there was substrate inhibition of CYP1A1- and CYP1A2-catalyzed hydroxylations. CYP1A1-catalyzed rutaecarpine hydroxylations had V max values of 1388 to ∼1893 pmol/min/nmol P450, K m values of 4.1 to ∼9.5 μM, and K i values of 45 to ∼103 μM. These results indicated that more than one molecule of rutaecarpine is accessible to the CYP1A active site. The major metabolite 10-hydroxyrutaecarpine decreased CYP1A1, CYP1A2, and CYP1B1 activities with respective IC 50 values of 2.56 ± 0.04, 2.57 ± 0.11, and 0.09 ± 0.01 μM, suggesting that product inhibition might occur during rutaecarpine hydroxylation. The metabolite profile and kinetic properties of rutaecarpine hydroxylation by human P450s provide important information relevant to the clinical application of rutaecarpine and E. rutaecarpa .
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the alkaloid rutaecarpine is a selective inhibitor of cytochrome p450 1a in mouse and human liver microsomes
Drug Metabolism and Disposition, 2002Co-Authors: Yune-fang Ueng, Taliang Chen, Peter F Guengerich, Chiehfu ChenAbstract:Rutaecarpine, evodiamine, and dehydroevodiamine are quinazolinocarboline alkaloids isolated from a traditional Chinese medicine, Evodia rutaecarpa. The in vitro effects of these alkaloids on cytochrome P450 (P450)-catalyzed oxidations were studied using mouse and human liver microsomes. Among these alkaloids, rutaecarpine showed the most potent and selective inhibitory effect on CYP1A-catalyzed 7-methoxyresorufin O-demethylation (MROD) and 7-ethoxyresorufin O-deethylation (EROD) activities in untreated mouse liver microsomes. The IC50 ratio of EROD to MROD was 6. For MROD activity, rutaecarpine was a noncompetitive inhibitor with aKi value of 39 ± 2 nM. In contrast, rutaecarpine had no effects on benzo[a]pyrene hydroxylation (AHH), aniline hydroxylation, and nifedipine oxidation (NFO) activities. In human liver microsomes, 1 μM rutaecarpine caused 98, 91, and 77% decreases of EROD, MROD, and phenacetinO-deethylation activities, respectively. In contrast, less than 15% inhibition of AHH, tolbutamide hydroxylation, chlorzoxazone hydroxylation, and NFO activities were observed in the presence of 1 μM rutaecarpine. To understand the selectivity of inhibition of CYP1A1 and CYP1A2, inhibitory effects of rutaecarpine were studied using liver microsomes of 3-methylcholanthrene (3-MC)-treated mice and Escherichia coli membrane expressing bicistronic human CYP1A1 and CYP1A2. Similar to the CYP1A2 inhibitor furafylline, rutaecarpine preferentially inhibited MROD more than EROD and had no effect on AHH in 3-MC-treated mouse liver microsomes. For bicistronic human P450s, the IC50 value of rutaecarpine for EROD activity of CYP1A1 was 15 times higher than the value of CYP1A2. These results indicated that rutaecarpine was a potent inhibitor of CYP1A2 in both mouse and human liver microsomes.
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induction of cytochrome p450 dependent monooxygenase in mouse liver and kidney by rutaecarpine an alkaloid of the herbal drug evodia rutaecarpa
Life Sciences, 2001Co-Authors: Yune-fang Ueng, Jongjing Wang, Sang Shin Park, Chiehfu ChenAbstract:Rutaecarpine is one of the main alkaloids of an herbal remedy, Evodia rutaecarpa, which has been used for the treatment of gastrointestinal disorder and headache. Effects of rutaecarpine on hepatic and renal cytochrome P450 (CYP)-dependent monooxygenase were studied in C57BL/6J mice. Treatment of mice with rutaecarpine by gastrogavage at 50 mg/kg/day for three days resulted in 57%, 41%, 6-, and 6-fold increases of hepatic microsomal benzo(a)pyrene hydroxylation, 7-ethoxycoumarin O-deethylation, 7-ethoxyresorufin O-deethylation, and 7-methoxyresorufin O-demethylation activities, respectively. However, the treatment had no effects on hepatic oxidation activities toward benzphetamine, N-nitrosodimethylamine, nifedipine, and erythromycin. In the kidney, rutaecarpine-treatment resulted in 2-fold and 42% increases of microsomal benzo(a)pyrene hydroxylation and 7-ethoxycoumarin O-deethylation activities, respectively. The treatment also increased renal 7-ethoxyresorufin O-deethylation activity to a detectable level. Immunoblot analysis of microsomal proteins showed that rutaecarpine-treatment increased the protein levels of CYP1A1 and CYP1A2 in the liver, whereas hepatic level of CYP3A-immunoreacted protein was not affected by rutaecarpine. These CYPs were not detectable in the immunoblot analyses of control and rutaecarpine-treated mouse kidney microsomes. These results indicated that rutaecarpine was a CYP1A inducer and showed potent inductive effects on both CYP1A1 and CYP1A2 in the liver.
Bhagavatula Moorthy - One of the best experts on this subject based on the ideXlab platform.
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newborn mice lacking the gene for cyp1a1 are more susceptible to oxygen mediated lung injury and are rescued by postnatal β naphthoflavone administration implications for bronchopulmonary dysplasia in premature infants
Toxicological Sciences, 2017Co-Authors: Paramahamsa Maturu, Bhagavatula Moorthy, Lihua Wang, Weiwu Jiang, Yanhong Weiliang, Krithika Lingappan, Roberto Barrios, Yao Liang, Xanthi I CouroucliAbstract:Prolonged hyperoxia contributes to bronchopulmonary dysplasia (BPD) in preterm infants. β-Naphthoflavone (BNF) is a potent inducer of cytochrome P450 (CYP)1A enzymes, which have been implicated in hyperoxic injuries in adult mice. In this investigation, we tested the hypothesis that newborn mice lacking the Cyp1a1 gene would be more susceptible to hyperoxic lung injury than wild-type (WT) mice and that postnatal BNF treatment would rescue this phenotype by mechanisms involving CYP1A and/or NAD(P)H quinone oxidoreductase (NQO1) enzymes. Newborn WT or Cyp1a1-null mice were treated with BNF (10 mg/kg) or the vehicle corn oil (CO) i.p., from postnatal day (PND) 2 to 14 once every other day, while being maintained in room air or hyperoxia (85% O2) for 14 days. Both genotypes showed lung injury, inflammation, and alveolar simplification in hyperoxia, with Cyp1a1-null mice displaying increased susceptibility compared to WT mice. BNF treatment resulted in significant attenuation of lung injury and inflammation, with improved alveolarization in both WT and Cyp1a1-null mice. BNF exposed normoxic or hyperoxic WT mice showed increased expression of hepatic CYP1A1/1A2, pulmonary CYP1A1, and NQO1 expression at both mRNA and protein levels, compared with vehicle controls. However, BNF caused greater induction of hepatic CYP1A2 and pulmonary NQO1 enzymes in the Cyp1a1-null mice, suggesting that BNF protects against hyperoxic lung injury in WT and Cyp1a1-null mice through the induction of CYP1A and NQO1 enzymes. Further studies on the protective role of flavonoids against hyperoxic lung injury in newborns could lead to novel strategies for the prevention and/or treatment of BPD.
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abstract 4068 role of cytochrome p450 cyp 1a2 in the molecular regulation of cyp1a1 by the carcinogen 3 methylcholanthrene mc in mouse heptoma cells hepa 1
Cancer Research, 2016Co-Authors: Sudha R Kondraganti, Chun Chu, Bhagavatula MoorthyAbstract:Cytochrome P4501A (CYP1A) enzymes play important roles in the activation of PAHs such as 3-methylcholanthrene (MC) to carcinogenic DNA-binding metabolites. We reported earlier that MC causes persistent induction of hepatic and pulmonary CYP1A1 in mice for several weeks after MC withdrawal, and that the phenomenon of sustained hepatic CYP1A1 induction is lost in CYP1A2-null mice. In this study, we tested the hypothesis that MC elicits induction of persistent CYP1A1 induction in hepa-1 cells, and that CYP1A2 contributes mechanistically to this phenomenon. Hepa-1 cells were treated with the MC (2.5 μM), or dimethylsulfoxide (DMSO) as control, and at selected time points, CYP1A1 promoter activity, CYP1A1 enzyme activities, contents, and CYP1A1 mRNA levels were studied. A 24 h treatment induced the CYP1A1 promoter activity, CYP1A1 mRNA, and ethoxyresorufin O-deethylase (EROD) activity by 2-, 1000-, and 15-fold, respectively. The induction was sustained for 4-5 days. MC persistently induced CYP1A1 apoprotein level as well. Electrophoretic mobility shift assay indicated that MC induced a nuclear protein that bound to aryl hydrocarbon response elements in the CYP1A1 promoter region. Transfection of CYP1A2 siRNA resulted in knockdown of CYP1A2 mRNA by 70%, but a statistically significant increase of basal CYP1A1 mRNA by 35-40%. The induction of CYP1A1 promoter activity, CYP1A1 mRNA, CYP1A1 protein, and EROD activity by MC were not affected by CYP1A2 siRNA at the 24 h time point, but was significantly attenuated by CYP1A2 siRNA on day 5. These results suggest that CYP1A2, possibly via a metabolite, contributes to the sustained induction of CYP1A1 by MC in hepa-I cells. Further investigations into the mechanisms of persistent induction of CYP1A1 by MC could lead to novel preventative/therapeutic strategies against PAH-mediated carcinogenesis in humans. Citation Format: Sudha Kondraganti, Chun Chu, Bhagavatula Moorthy. Role of cytochrome P450 (CYP)1A2 in the molecular regulation of CYP1A1 by the carcinogen, 3-methylcholanthrene (MC) in mouse heptoma cells (hepa-1). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4068.
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abstract 4427 role of cytochrome p450 cyp 1a2 in the sustained induction of cyp1a1 by the carcinogen 3 methylcholanthrene mc in mouse heptoma cells hepa 1
Cancer Research, 2012Co-Authors: Bhagavatula Moorthy, Sudha R Kondraganti, Lihua Wang, Weiwu Jiang, Chun ChuAbstract:Humans are constantly exposed to environmental carcinogenic polycyclic aromatic hydrocarbons (PAHs) through cigarette smoke, diesel exhausts, charcoal-broiled meats, etc. Cytochrome P4501A (CYP1A) enzymes play important roles in the activation of PAHs such as 3-methylcholanthrene (MC) to carcinogenic DNA-binding metabolites. We reported earlier that MC causes persistent induction of hepatic and pulmonary CYP1A1 in mice for several weeks after MC ithdrawal, and that the phenomenon of sustained hepatic CYP1A1 induction is lost in CYP1A2-null mice. In this study, we tested the hypothesis that MC elicits induction of persistent CYP1A1 nduction in hepa-1 cells, and that CYP1A2 contributes mechanistically to this henomenon. Hepa-1 cells were treated ith the MC (2.5 µM), or dimethylsulfoxide (DMSO) as control, and at selected ime points, CYP1A1 promoter activity, CYP1A1 enzyme activities, contents, and CYP1A1 mRNA levels were studied. We fund that MC markedly and persistently induced CYP1A1 promoter activity, transcription, apoprotein expression, and the CYP1A1 associated ethoxyresorufin O-deethylase (EROD) activities for up to 5 days. Transfection of CYP1A2 siRNA resulted in knockdown of CYP1A2 mRNA by 70%, but a statistically significant increase of basal CYP1A1 mRNA by 35-40%. The induction of CYP1A1 promoter ctivity, CYP1A1 mRNA, CYP1A1 protein, and EROD activity by MC were not affected by CYP1A2 siRNA at the 24 h time point, but was significantly ttenuated by CYP1A2 siRNA on day 5. These results suggest that CYP1A2, possibly via a metabolite, contributes to the sustained induction of CYP1A1 by MC in hepa-I cells. Further investigations into the mechanisms of persistent induction of CYP1A1 by MC could lead to novel preventative/therapeutic strategies against PAH-mediated carcinogenesis in humans. (Supported in part by 2R01 ES009132.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4427. doi:1538-7445.AM2012-4427
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disruption of the gene for CYP1A2 which is expressed primarily in liver leads to differential regulation of hepatic and pulmonary mouse cyp1a1 expression and augmented human cyp1a1 transcriptional activation in response to 3 methylcholanthrene in viv
Journal of Pharmacology and Experimental Therapeutics, 2010Co-Authors: Weiwu Jiang, Inayat S Fazili, Sudha R Kondraganti, Lihua Wang, Xanthi I Couroucli, Edward A Felix, Bhagavatula MoorthyAbstract:The cytochrome P4501A (CYP1A) enzymes play important roles in the metabolic activation and detoxification of numerous environmental carcinogens, including polycyclic aromatic hydrocarbons (PAHs). In this study, we tested the hypothesis that hepatic CYP1A2 differentially regulates mouse hepatic and pulmonary CYP1A1 expression and suppresses transcriptional activation of human CYP1A1 (hCYP1A1) promoter in response to 3-methylcholanthrene (MC) in vivo. Administration of wild-type (WT) (C57BL/6J) or CYP1A2-null mice with a single dose of MC (100 μmol/kg i.p.) caused significant increases in hepatic CYP1A1/1A2 activities, apoprotein content, and mRNA levels 1 day after carcinogen withdrawal compared with vehicle-treated controls. The induction persisted in the WT, but not CYP1A2-null, animals, for up to 15 days. In the lung, MC caused persistent CYP1A1 induction for up to 8 days in both genotypes, with CYP1A2-null mice displaying a greater extent of CYP1A1 expression. It is noteworthy that MC caused significant augmentation of human CYP1A1 promoter activation in transgenic mice expressing the hCYP1A1 and the reporter luciferase gene on a CYP1A2-null background, compared with transgenic mice on the WT background. In contrast, the mouse endogenous hepatic, but not pulmonary, persistent CYP1A1 expression was repressed by MC in the hCYP1A1-CYP1A2-null mice. Liquid chromatography–mass spectrometry experiments showed that CYP1A2 catalyzed the formation of 1-hydroxy-3-MC and/or 2-hydroxy-3-MC, a metabolite that may contribute to the regulation of CYP1A1 expression. In conclusion, the results suggest that CYP1A2 plays a pivotal role in the regulation of hepatic and pulmonary CYP1A1 by PAHs, a phenomenon that potentially has important implications for PAH-mediated carcinogenesis.
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persistent induction of cytochrome p450 cyp 1a enzymes by 3 methylcholanthrene in vivo in mice is mediated by sustained transcriptional activation of the corresponding promoters
Biochemical and Biophysical Research Communications, 2009Co-Authors: Weiwu Jiang, Inayat S Fazili, Lihua Wang, Weisheng Zhang, Richard Coffee, Bhagavatula MoorthyAbstract:Abstract There is significant human exposure to polycyclic aromatic hydrocarbons (PAHs), many of which are potent carcinogens. Cytochrome P450 (CYP)1A enzymes play key roles in the metabolic activation of PAHs to carcinogenic metabolites. We previously showed persistent induction of CYP1A enzymes by 3-methylcholanthrene (MC) in vivo in rodents. In this study, we tested the hypothesis that MC elicits persistent induction of CYP1A1 and 1A2 in vivo by mechanisms entailing sustained transcriptional activation of the corresponding promoters. Adult male wild type (WT) (Cd-1) mice, transgenic mice expressing the human CYP1A1 promoter or the mouse CYP1A2 promoter were treated with the vehicle corn oil (CO) or the carcinogenic PAH, 3-methylcholanthrene (MC), once daily for 4 days, and luciferase reporter gene expression was determined at 1, 8, 15, and 22 days after MC withdrawal by bioluminescent imaging. Pulmonary and hepatic endogenous expression of CYP1A1 and 1A2 was also determined at the enzymatic, protein, and mRNA levels. The major findings were that MC elicited marked enhancement in the luciferase expression in the CYP1A1-luc as well CYP1A2-luc transgenic mice that was sustained for up to 22 days, the magnitude of induction being more pronounced in the CYP1A1-luc mice. MC also caused persistent induction of endogenous CYP1A1 and 1A2 expression in the WT, CYP1A1-luc, and 1A2-luc mice for up to 22 days. In conclusion, our results support the hypothesis that MC elicits sustained CYP1A1 and 1A2 expression by sustained transcriptional activation of the corresponding promoters. Thus, these novel transgenic models should be very useful for further understanding of the molecular mechanisms of persistent CYP1A induction, in relation to PAH-mediated carcinogenesis.
Daniel W Nebert - One of the best experts on this subject based on the ideXlab platform.
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Mitochondrial targeting of mouse NQO1 and CYP1B1 proteins
Biochemical and biophysical research communications, 2013Co-Authors: Hongbin Dong, Mary Beth Genter, Howard G Shertzer, Frank J Gonzalez, Colin R Jefcoate, Vasilis Vasiliou, Daniel W NebertAbstract:Abstract Four dioxin-inducible enzymes––NAD(P)H: quinone oxidoreductase-1 (NQO1) and three cytochromes P450 (CYP1A1, CYP1A2 & CYP1B1)––are implicated in both detoxication and metabolic activation of various endobiotics and xenobiotics. NQO1 is generally regarded as a cytosolic enzyme; whereas CYP1 proteins are located primarily in endoplasmic reticulum (ER), CYP1A1 and CYP1A2 proteins are also targeted to mitochondria. This lab has generated Cyp1a1(mc/mc) and Cyp1a1(mtt/mtt) knock-in mouse lines in which CYP1A1 protein is targeted exclusively to ER (microsomes) and mitochondria, respectively. Comparing dioxin-treated Cyp1(+/+) wild-type, Cyp1a1(mc/mc), Cyp1a1(mtt/mtt), and Cyp1a1(−/−), Cyp1b1(−/−) and Nqo1(−/−) knockout mice, in the present study we show that [a] NQO1 protein locates to cytosol, ER and mitochondria, [b] CYP1B1 protein (similar to CYP1A1 and CYP1A2 proteins) traffics to mitochondria as well as ER, and [c] NQO1 and CYP1B1 targeting to mitochondrial or ER membranes is independent of CYP1A1 presence in that membrane.
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bioactivation versus detoxication of the urothelial carcinogen aristolochic acid i by human cytochrome p450 1a1 and 1a2
Toxicological Sciences, 2012Co-Authors: Marie Stiborova, Daniel W Nebert, Kateřina Levova, Frantisek Barta, Zhanquan Shi, Eva Frei, Heinz H Schmeiser, David H Phillips, Volker M ArltAbstract:Exposure to aristolochic acid (AA) is associated with human nephropathy and urothelial cancer. Individual susceptibility to AA-induced disease likely reflects individual differences in enzymes that metabolize AA. Herein, we evaluated AAI metabolism by human cytochrome P450 (CYP) 1A1 and 1A2 in two CYP1A-humanized mouse lines that carry functional human CYP1A1 and CYP1A2 genes in the absence of the mouse Cyp1a1/1a2 orthologs. Human and mouse hepatic microsomes and human CYPs were also studied. Human CYP1A1 and 1A2 were found to be principally responsible for reductive activation of AAI to form AAI-DNA adducts and for oxidative detoxication to 8-hydroxyaristolochic acid (AAIa), both in the intact mouse and in microsomes. Overall, AAI-DNA adduct levels were higher in CYP1A-humanized mice relative to wild-type mice, indicating that expression of human CYP1A1 and 1A2 in mice leads to higher AAI bioactivation than in mice containing the mouse CYP1A1 and 1A2 orthologs. Furthermore, an exclusive role of human CYP1A1 and 1A2 in AAI oxidation to AAIa was observed in human liver microsomes under the aerobic (i.e., oxidative) conditions. Because CYP1A2 levels in human liver are at least 100-fold greater than those of CYP1A1 and there exists a > 60-fold genetic variation in CYP1A2 levels in human populations, the role of CYP1A2 in AAI metabolism is clinically relevant. The results suggest that, in addition to CYP1A1 and 1A2 expression levels, in vivo oxygen concentration in specific tissues might affect the balance between AAI nitroreduction and demethylation, which in turn would influence tissue-specific toxicity or carcinogenicity.
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for dioxin induced birth defects mouse or human CYP1A2 in maternal liver protects whereas mouse cyp1a1 and cyp1b1 are inconsequential
Journal of Biological Chemistry, 2006Co-Authors: Nadine Dragin, Timothy P Dalton, Marian L Miller, Howard G Shertzer, Daniel W NebertAbstract:Dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin) induces cleft palate and hydronephrosis in mice, when exposed in utero; these effects are mediated by the aryl hydrocarbon receptor. The Cyp1a1, CYP1A2, and Cyp1b1 genes are up-regulated by the aryl hydrocarbon receptor. To elucidate their roles in dioxin-induced teratogenesis, we compared Cyp1a1(-/-), CYP1A2(-/-), and Cyp1b1(-/-) knock-out mice with Cyp1(+/+) wild-type mice. Dioxin was administered (25 microg/kg, gavage) on gestational day 10, and embryos were examined on gestational day 18. The incidence of cleft palate and hydronephrosis was not significantly different in fetuses from Cyp1a1(-/-), Cyp1b1(-/-), and Cyp1(+/+) wild-type mice. To fetuses carried by CYP1A2(-/-) dams, however, this dose of dioxin was lethal; this effect was absolutely dependent on the maternal CYP1A2 genotype and independent of the embryonic CYP1A2 genotype. Dioxin levels were highest in adipose tissue, mammary gland, and circulating blood of CYP1A2(-/-) mothers, compared with that in the Cyp1(+/+) mothers, who showed highest dioxin levels in liver. More dioxin reached the embryos from CYP1A2(-/-) dams, compared with that from Cyp1(+/+) dams. Fetuses from CYP1A2(-/-) dams exhibited a approximately 6-fold increased sensitivity to cleft palate, hydronephrosis, and lethality. Using the humanized hCYP1A1_1A2 transgenic mouse (expressing the human CYP1A1 and CYP1A2 genes in the absence of mouse CYP1A2 gene), the teratogenic effects of dioxin reverted to the wild-type phenotype. These data indicate that maternal mouse hepatic CYP1A2, by sequestering dioxin and thus altering the pharmacokinetics, protects the embryos from toxicity and birth defects; substitution of the human CYP1A2 trans-gene provides the same protection. In contrast, neither CYP1A1 nor CYP1B1 appears to play a role in dioxin-mediated teratogenesis.
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Comparison of mouse hepatic mitochondrial versus microsomal cytochromes P450 following TCDD treatment.
Biochemical and Biophysical Research Communications, 2006Co-Authors: Mary Beth Genter, Corey D. Clay, Hongbin Dong, Timothy P Dalton, Daniel W Nebert, Howard G ShertzerAbstract:Abstract TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) induces cytochromes P450 (CYPs) such as CYP1A1 and CYP1A2 via activation of the aromatic hydrocarbon receptor (AHR). Herein we describe the TCDD-dependent enrichment of CYPs in liver microsomes and mitoplasts from C57BL/6J mice. TCDD-induced accumulation of CYP1A1 and CYP1A2 was observed in microsomes and mitoplasts after treatment with 15 μg TCDD/kg/d for 3 d. While microsomal CYP1 proteins peaked at 1 week and diminished thereafter, mitoplast CYP1 proteins persisted 8 weeks at high levels. TCDD also induced microsomal CYP2A5, but not microsomal proteins immunoreactive to CYP2C11, CYP3A2 or CYP4A1 antibodies. Nevertheless, each of these proteins increased in mitoplasts following TCDD exposure. These results suggest that TCDD increases mitochondrial CYP immunoreactive proteins under the transcriptional control of the AHR, as well as CYPs that are not under AHR control. We speculate that such mitochondrial CYPs may be involved in the generation, or mitigation, of the well-known TCDD-inducible oxidative stress response.
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differential metabolism of 2 amino 1 methyl 6 phenylimidazo 4 5 b pyridine phip in mice humanized for cyp1a1 and CYP1A2
Chemical Research in Toxicology, 2005Co-Authors: Connie Cheung, Timothy P Dalton, Daniel W Nebert, Shioko Kimura, Kristopher W Krausz, Lionel Feigenbaum, Jeffrey R Idle, Frank J GonzalezAbstract:The procarcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is the most abundant heterocyclic amine formed during the cooking of foods. Metabolism of PhIP by CYP1A2 differs substantially between humans and rodents, with more N2-hydroxylation (activation) and less 4'-hydroxylation (detoxication) in humans. Therefore, the human response to PhIP and other heterocyclic amine exposure may not be accurately reflected in the laboratory rodent. By generating mouse models expressing the human genes, species differences in heterocyclic amine metabolism can be addressed. Two transgenic mouse lines were developed, one expressing the human CYP1A1 CYP1A2 transgene in a mouse Cyp1a1-null background (hCYP1A1) and another expressing human CYP1A1 CYP1A2 in a mouse CYP1A2-null background (hCYP1A2). Expression of human CYP1A2 protein was detected in the liver and also at considerably lower levels in extrahepatic tissues such as lung, kidney, colon, and heart. In the hCYP1A1 and hCYP1A2 mice, 3-methylcholanthrene (3-MC) induced both human CYP1A1 and CYP1A2 protein in the liver. Differences in the metabolism of the heterocyclic amine PhIP were observed between wild-type and hCYP1A2 mice. PhIP was preferentially metabolized by N2-hydroxylation in hCYP1A2 mice, whereas in wild-type mice, 4'-hydroxylation was the predominant pathway. Since the N2-hydroxylation pathway for PhIP metabolism has been reported to be predominant in humans, these results illustrate the potential effectiveness of using these transgenic, humanized mice as models for determining human health risks to PhIP and other heterocyclic amines instead of wild-type mice.
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oxidative metabolism of the alkaloid rutaecarpine by human cytochrome p450
Drug Metabolism and Disposition, 2006Co-Authors: Yune-fang Ueng, Li Kang Ho, Shuyun Wang, Chiehfu ChenAbstract:Rutaecarpine is the main active alkaloid of the herbal medicine, Evodia rutaecarpa . To identify the major human cytochrome P450 (P450) participating in rutaecarpine oxidative metabolism, human liver microsomes and bacteria-expressed recombinant human P450 were studied. In liver microsomes, rutaecarpine was oxidized to 10-, 11-, 12-, and 3-hydroxyrutaecarpine. Microsomal 10- and 3-hydroxylation activities were strongly inhibited by ketoconazole. The 11- and 12-hydroxylation activities were inhibited by α-naphthoflavone, quinidine, and ketoconazole. These results indicated that multiple hepatic P450s including CYP1A2, CYP2D6, and CYP3A4 participate in rutaecarpine hydroxylations. Among recombinant P450s, CYP1A1 had the highest rutaecarpine hydroxylation activity. Decreased metabolite formation at high substrate concentration indicated that there was substrate inhibition of CYP1A1- and CYP1A2-catalyzed hydroxylations. CYP1A1-catalyzed rutaecarpine hydroxylations had V max values of 1388 to ∼1893 pmol/min/nmol P450, K m values of 4.1 to ∼9.5 μM, and K i values of 45 to ∼103 μM. These results indicated that more than one molecule of rutaecarpine is accessible to the CYP1A active site. The major metabolite 10-hydroxyrutaecarpine decreased CYP1A1, CYP1A2, and CYP1B1 activities with respective IC 50 values of 2.56 ± 0.04, 2.57 ± 0.11, and 0.09 ± 0.01 μM, suggesting that product inhibition might occur during rutaecarpine hydroxylation. The metabolite profile and kinetic properties of rutaecarpine hydroxylation by human P450s provide important information relevant to the clinical application of rutaecarpine and E. rutaecarpa .
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oxidative metabolism of the alkaloid rutaecarpine by human cytochrome p450
Drug Metabolism and Disposition, 2006Co-Authors: Yune-fang Ueng, Li Kang Ho, Shuyun Wang, Chiehfu ChenAbstract:Rutaecarpine is the main active alkaloid of the herbal medicine, Evodia rutaecarpa . To identify the major human cytochrome P450 (P450) participating in rutaecarpine oxidative metabolism, human liver microsomes and bacteria-expressed recombinant human P450 were studied. In liver microsomes, rutaecarpine was oxidized to 10-, 11-, 12-, and 3-hydroxyrutaecarpine. Microsomal 10- and 3-hydroxylation activities were strongly inhibited by ketoconazole. The 11- and 12-hydroxylation activities were inhibited by α-naphthoflavone, quinidine, and ketoconazole. These results indicated that multiple hepatic P450s including CYP1A2, CYP2D6, and CYP3A4 participate in rutaecarpine hydroxylations. Among recombinant P450s, CYP1A1 had the highest rutaecarpine hydroxylation activity. Decreased metabolite formation at high substrate concentration indicated that there was substrate inhibition of CYP1A1- and CYP1A2-catalyzed hydroxylations. CYP1A1-catalyzed rutaecarpine hydroxylations had V max values of 1388 to ∼1893 pmol/min/nmol P450, K m values of 4.1 to ∼9.5 μM, and K i values of 45 to ∼103 μM. These results indicated that more than one molecule of rutaecarpine is accessible to the CYP1A active site. The major metabolite 10-hydroxyrutaecarpine decreased CYP1A1, CYP1A2, and CYP1B1 activities with respective IC 50 values of 2.56 ± 0.04, 2.57 ± 0.11, and 0.09 ± 0.01 μM, suggesting that product inhibition might occur during rutaecarpine hydroxylation. The metabolite profile and kinetic properties of rutaecarpine hydroxylation by human P450s provide important information relevant to the clinical application of rutaecarpine and E. rutaecarpa .
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the alkaloid rutaecarpine is a selective inhibitor of cytochrome p450 1a in mouse and human liver microsomes
Drug Metabolism and Disposition, 2002Co-Authors: Yune-fang Ueng, Taliang Chen, Peter F Guengerich, Chiehfu ChenAbstract:Rutaecarpine, evodiamine, and dehydroevodiamine are quinazolinocarboline alkaloids isolated from a traditional Chinese medicine, Evodia rutaecarpa. The in vitro effects of these alkaloids on cytochrome P450 (P450)-catalyzed oxidations were studied using mouse and human liver microsomes. Among these alkaloids, rutaecarpine showed the most potent and selective inhibitory effect on CYP1A-catalyzed 7-methoxyresorufin O-demethylation (MROD) and 7-ethoxyresorufin O-deethylation (EROD) activities in untreated mouse liver microsomes. The IC50 ratio of EROD to MROD was 6. For MROD activity, rutaecarpine was a noncompetitive inhibitor with aKi value of 39 ± 2 nM. In contrast, rutaecarpine had no effects on benzo[a]pyrene hydroxylation (AHH), aniline hydroxylation, and nifedipine oxidation (NFO) activities. In human liver microsomes, 1 μM rutaecarpine caused 98, 91, and 77% decreases of EROD, MROD, and phenacetinO-deethylation activities, respectively. In contrast, less than 15% inhibition of AHH, tolbutamide hydroxylation, chlorzoxazone hydroxylation, and NFO activities were observed in the presence of 1 μM rutaecarpine. To understand the selectivity of inhibition of CYP1A1 and CYP1A2, inhibitory effects of rutaecarpine were studied using liver microsomes of 3-methylcholanthrene (3-MC)-treated mice and Escherichia coli membrane expressing bicistronic human CYP1A1 and CYP1A2. Similar to the CYP1A2 inhibitor furafylline, rutaecarpine preferentially inhibited MROD more than EROD and had no effect on AHH in 3-MC-treated mouse liver microsomes. For bicistronic human P450s, the IC50 value of rutaecarpine for EROD activity of CYP1A1 was 15 times higher than the value of CYP1A2. These results indicated that rutaecarpine was a potent inhibitor of CYP1A2 in both mouse and human liver microsomes.
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induction of cytochrome p450 dependent monooxygenase in mouse liver and kidney by rutaecarpine an alkaloid of the herbal drug evodia rutaecarpa
Life Sciences, 2001Co-Authors: Yune-fang Ueng, Jongjing Wang, Sang Shin Park, Chiehfu ChenAbstract:Rutaecarpine is one of the main alkaloids of an herbal remedy, Evodia rutaecarpa, which has been used for the treatment of gastrointestinal disorder and headache. Effects of rutaecarpine on hepatic and renal cytochrome P450 (CYP)-dependent monooxygenase were studied in C57BL/6J mice. Treatment of mice with rutaecarpine by gastrogavage at 50 mg/kg/day for three days resulted in 57%, 41%, 6-, and 6-fold increases of hepatic microsomal benzo(a)pyrene hydroxylation, 7-ethoxycoumarin O-deethylation, 7-ethoxyresorufin O-deethylation, and 7-methoxyresorufin O-demethylation activities, respectively. However, the treatment had no effects on hepatic oxidation activities toward benzphetamine, N-nitrosodimethylamine, nifedipine, and erythromycin. In the kidney, rutaecarpine-treatment resulted in 2-fold and 42% increases of microsomal benzo(a)pyrene hydroxylation and 7-ethoxycoumarin O-deethylation activities, respectively. The treatment also increased renal 7-ethoxyresorufin O-deethylation activity to a detectable level. Immunoblot analysis of microsomal proteins showed that rutaecarpine-treatment increased the protein levels of CYP1A1 and CYP1A2 in the liver, whereas hepatic level of CYP3A-immunoreacted protein was not affected by rutaecarpine. These CYPs were not detectable in the immunoblot analyses of control and rutaecarpine-treated mouse kidney microsomes. These results indicated that rutaecarpine was a CYP1A inducer and showed potent inductive effects on both CYP1A1 and CYP1A2 in the liver.
