Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0596263 (carcinogenesis)
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We report that, in a human cell line, human cytochrome P450IIA3 is capable of metabolizing aflatoxin B1, benzo[a]-pyrene, N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) to cytotoxic and mutagenic species. Cytochrome P450IIA3-mediated activation of NDMA and NDEA was compared with human cytochrome P450IIE1-mediated activation in the same cell system. P450IIE1 was more effective at activating NDMA than P450IIA3, while P450IIA3 was more effective at activating NDEA than P450IIE1. Whole cells and microsomal fractions obtained from control cells and from cells expressing the P450IIA3 cDNA were characterized for expression of P450IIA3. Microsomal coumarin 7-hydroxylase activity was some 40 times greater in the transfected cells than in the control cells and was catalyzed by a protein that was immunochemically related to the rat liver cytochrome P450IIA gene family. Immunoblot analysis demonstrated that this protein was readily detectable in transfected cells but barely detectable in control cells. We also report the DNA and deduced amino acid sequence of the P450IIA3 cDNA isolate used in this study. Our isolate encodes a protein 489 amino acids that is five amino acids shorter at the N terminus but otherwise identical to a previously reported human P450IIA3 cDNA sequence.
Carcinogenesis 1990 Aug
PMID:Human cytochrome P450IIA3: cDNA sequence, role of the enzyme in the metabolic activation of promutagens, comparison to nitrosamine activation by human cytochrome P450IIE1. 211 2

Rat liver microsomes metabolized the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) to the genotoxic metabolite 2-hydroxamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (2-hydroxamino-PhIP) and to the detoxified product 2-amino-4'-hydroxy-1-methyl-6-phenylimidazo[4,5-b]pyridine (4'-hydroxy-PhIP). A 25-fold higher rate of metabolism was measured in microsomes from polychlorinated-biphenyl-treated rats (94 nmol/mg proteins/30 min) in comparison with those from untreated rats. Other effective inducers of PhIP metabolism were beta-naphthoflavone and isosafrole (ISF), whereas phenobarbital was ineffective. About twice as much 2-hydroxamino-PhIP as 4'-hydroxy-PhIP was formed in microsomes irrespective of the inducer the rats had been treated with. The metabolism was dependent on NADPH and was abolished by the cytochrome P450 inhibitor alpha-naphthoflavone. In a reconstituted enzyme system purified rat cytochrome P450 IA2 (P450ISF-G) had the highest N-hydroxylation rate (30 nmol/nmol P450/30 min) closely followed by the rat cytochrome P450 IA1 (P450BNF-B). Less activity was seen with rat P450 IIC11 (P450UT-A) and rabbit P450 IA2 (P450 LM4). Rat P450 IIE1 (P450j), P450 IIB1 (P450PB-B) and rabbit P450 IIB4 (P450 LM-2) and P450 IIE1 (P450 LM3a) were essentially inactive. Rat P450 IA1 (P450BNF-B) produced five times more 4'-hydroxy-PhIP (32 +/- 2 nmol/nmol P450/30 min) than did P450 IA2 (P450ISF-G). Hence, the measured ratio of activation to detoxication for rat P450 IA2 (P450ISF-G) enzyme was 7-fold higher than that of the other active P450 enzymes.
Carcinogenesis 1990 Mar
PMID:Differential rates of metabolic activation and detoxication of the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine by different cytochrome P450 enzymes. 231 Nov 93

The Long-Evans rat with a cinnamon-like coat color (LEC rat) is a mutant strain displaying hereditary hepatitis with severe jaundice. The age related difference in microsomal dealkylation of pentoxyresorufin and ethoxyresorufin was examined. The enzyme activity levels of pentoxyresorufin O-depentylase in LEC rats were decreased to 25% of the levels in control [Long-Evans rats with an agouti coat color (LEA rats)]. In contrast, ethoxyresorufin O-deethylase exhibited a much less marked difference between the strains. In parallel with these strain differences in enzyme activities, a decrease in phenobarbital (PB) inducible P450 isozymes, mainly P450b and P450e, was observed by Western blot analysis. The level of P450PB in LEC rats was more markedly depressed than in the LEA strain. On the other hand, microsomes from uninduced LEC rat liver had more 3-methylcholanthrene (MC) inducible P450MC, mainly P450c and P450d, than microsomes from LEA rat liver and these isozymes in the LEC were markedly induced by 3-methylcholanthrene treatment. The great difference in cytochrome P450PB content of the liver microsomes between LEC and LEA rats and the maintained constitutive levels of hepatic cytochrome P450MC in the LEC rats suggest a possible role of these cytochrome isozymes in the onset of spontaneous hepatitis and hepatoma.
Carcinogenesis 1989 Nov
PMID:Selective expression and induction of cytochrome P450PB and P450MC during the development of hereditary hepatitis and hepatoma of LEC rats. 280 35

1,3-Butadiene is carcinogenic in B6C3F1 mice and Sprague-Dawley rats, and has been classified as a probable human carcinogen. The genetic basis for butadiene carcinogenicity is likely mediated by its metabolite, 1,2:3,4-diepoxybutane (BDE). Oxidation of butadiene to 1,2-epoxy-3-butene (BMO) and further activation to BDE is catalysed by cytochrome P450 (CYP) isozymes. The production of BMO from butadiene is mediated by CYP2E1 and, at high butadiene concentrations, by CYP2A6. The purpose of the present study was to identify which human CYP isozymes have the ability to oxidize BMO to BDE, and to determine the extent to which this reaction occurs in B6C3F1 mouse, Sprague-Dawley rat, and human liver microsomes. Of the human cDNA-expressed CYP isozymes tested, only CYP2E1 formed detectable concentrations of BDE at 80 microM BMO. CYP2E1 and CYP3A4 were active at 5.0 mM BMO. Interindividual and interspecies variation in the initial rate of oxidation of 80 microM BMO to BDE was determined using 10 samples of human liver microsomes and single pooled samples from rats and mice. Those experiments revealed a 60-fold variation in activity among 10 human liver samples (range: 0.005-0.324 nmol/mg protein/min). Rates of BMO oxidation for mouse and rat liver microsomes were 0.473 and 0.166 nmol/mg protein/min, respectively. Apparent kinetic constants for the oxidation of BMO to BDE by four human microsomal preparations, and pooled samples from mice and rats were estimated from detailed investigations of BMO oxidation at various BMO substrate concentrations. Apparent Km for the human liver samples ranged from 0.304-0.880 mM, and Vmax values ranged from 0.38 to 1.2 nmol/mg protein/min. The apparent values of Km and Vmax for mouse liver microsomes were 0.141 +/- 0.007 mM (mean +/- SE) and 1.303 +/- 0.141 nmol/mg protein/min, respectively. For rat liver microsomes, apparent Km and Vmax were 0.145 +/- 0.036 mM and 0.408 +/- 0.031 nmol/mg protein/min, respectively. Measured rates of BDE formation correlated well with CYP2E1 protein concentrations in the human microsome samples. These results implicate human CYP2E1 as a hepatic isoform responsible for the oxidation of BMO to BDE at low concentrations of BMO. Moreover, our in vitro results reveal that microsomes prepared from human, rat and mouse liver possess the ability to form BDE from BMO. Previous in vitro results suggest that following exposure to butadiene more BMO would probably be present in mice than in rats or humans. Thus, in mice more BMO would be available for activation to BDE.(ABSTRACT TRUNCATED AT 400 WORDS)
Carcinogenesis 1995 Oct
PMID:Oxidation of 1,2-epoxy-3-butene to 1,2:3,4-diepoxybutane by cDNA-expressed human cytochromes P450 2E1 and 3A4 and human, mouse and rat liver microsomes. 758 24

Epidemiological evidence suggests that the presence of human papillomaviruses (HPV), when combined with smoking behaviors, considerably enhances the risk of developing oral, cervical, vulvar, and/or anal carcinomas. It is well established that the cytochrome P450 (CYP), microsomal epoxide hydrolase (mEH), and other biotransformation enzymes are important modulators of the bioactivation and detoxification of many environmental chemicals, including constituents of tobacco smoke such as certain nitrosamines and polycyclic aromatic hydrocarbons (PAH). Since there is little information regarding oral and cervical epithelial-specific expression of these genes, established primary and HPV-immortalized oral and cervical epithelial cell lines were analyzed for morphology, mRNA and protein expression patterns of specific CYPs and mEH. Primary human oral and cervical epithelial cells were immortalized using retroviral infection with HPV-16 E6/E7 genes. Primary human keratinocyte cells were immortalized by transfection of HPV-18 and made tumorigenic with nitrosomethylurea treatment. Expression profiles for mEH, CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2D6, CYP3A, and CYP2E1 were evaluated in these cultures in the presence or absence of a PAH inducer, using reverse transcriptase-coupled polymerase chain reaction analysis. mEH gene expression was evident in all cultures, while CYP2A6 mRNA was not detected in any of the cell lines, regardless of culture conditions. CYP2E1 mRNA expression was greatest in the oral epithelial cultures and detectable in all other epithelial cultures except for the HPV-18 immortalized keratinocyte cell line. Elevated levels of CYP2D6 mRNA existed in both oral epithelial cell lines and the HPV-16 immortalized cervical epithelial cells when compared to the other cell lines examined. CYP1A1 and CYP1A2 mRNAs were detected in all the cells and several cultures were inducible by PAH exposure. To corroborate the RT/PCR data, Western immunoblotting experiments were conducted on selected samples. Using these methods, CYP1A1 and CYP2E1 proteins were detected in primary and HPV-immortalized oral and cervical epithelial cultures. These data indicate that both primary and HPV immortalized cells appear to express certain biotransformation enzymes necessary for the activation of tobacco-specific nitrosamines and PAHs. Although the overall impact of HPV gene infection on expression of these systems remains to be fully elucidated, as in vitro system is characterized which should prove useful in examining interactive mechanisms of HPV with xenobiotic activation in the etiology of squamous cell carcinomas.
Carcinogenesis 1995 Jun
PMID:Expression of cytochrome P450 and microsomal epoxide hydrolase in cervical and oral epithelial cells immortalized by human papillomavirus type 16 E6/E7 genes. 761 6

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone (NNK) is a potential human carcinogen that is known to be metabolized to DNA-reactive intermediates by the cytochromes P450. We have examined the nature of NNK's DNA damaging effects in a mammalian cell system expressing a specific human cytochrome P450 (2A6) and containing a target gene for mutagenesis. Human CYP2A6, which is known to activate NNK to a mutagen, was lipofected via a retroviral vector into the Chinese hamster ovary AS52 cell line, which contains the bacterial gpt gene and can be mutated to 6-thioguanine resistance. AS52 cells expressed negligible CYP2A6-specific coumarin 7-hydroxylase activity (0.7 pmol/mg protein/min), while a CYP2A6 transfected clone (AS52-E8) expressed 30 pmol/mg protein/min. Both cell lines were equally sensitive to the cytotoxic and mutagenic effects of the direct-acting mutagen ethylmethanesulfonate; however, only the AS52-E8 cells exhibited a dose-dependent increase in cytotoxicity and mutant frequency upon treatment with NNK. At the highest NNK dose (1200 micrograms/ml), the mutant frequency in AS52-E8 cells was 14-fold (339 x 10(-6)) greater than the spontaneous frequency of 24 x 10(-6). Ninty-eight mutant clones were isolated following NNK treatment. Based on PCR analysis, 21 clones contained deletions/rearrangements and 77 were putative point mutants. Sequencing potential point mutants showed that 81% contained G:C to A:T transitions. Four of six G:C to A:T hotspots were at the second G of the GGT motif, which is the motif and major mutation found in codon 12 of Ki-ras from NNK-induced lung tumors in strain A mice. Since NNK may be metabolized via different pathways to pyridyloxobutylate or methylate DNA, the data suggest that methylation damage causes the major mutagenic events in AS52-E8 cells when NNK is activated by human CYP2A6.
Carcinogenesis 1994 Dec
PMID:Human CYP2A6 activation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK): mutational specificity in the gpt gene of AS52 cells. 800 Dec 47

The activation of heterocyclic amines to mutagenic products by hepatic microsomal fractions from cynomolgus monkey, marmoset monkey and man was compared with the respective levels of cytochrome P450 enzymes CYP1A1 and CYP1A2. The rate of activation of 2-amino-3,8-dimethylimidazo[4,5-f] quinoxaline (MeIQx), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) to mutagens by hepatic microsomal fraction from cynomolgus monkey was very low. This was associated with a lack of constitutive expression of CYP1A1 and CYP1A2. In contrast, human hepatic microsomal fraction readily activates these heterocyclic amines and this is associated with constitutive expression of CYP1A2. Treatment of cynomolgus monkey with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes a very modest induction of CYP1A2, and a small increase in the activation of MeIQx and IQ. However, there was marked induction of CYP1A1 which was accompanied by > 10-fold increases in PhIP activation and 7-ethoxyresorufin O-deethylase (EROD), 7-methoxyresorufin O-demethylase (MROD) and aryl hydrocarbon hydroxylase activities. Following treatment of cynomolgus monkey with 3-methylcholanthrene, induction of CYP1A1, but not CYP1A2, was evident. In untreated marmoset monkey the activations of MeIQx and PhIP, as well as phenacetin O-deethylase, EROD, MROD and aryl hydrocarbon hydroxylase activities, are similar to those in man, although the activations of IQ and coumarin 7-hydroxylase activity are lower than in man. The presence of constitutive CYP1A2, and the absence of CYP1A1, in the liver of this species correspond to the situation in man. Treatment of marmoset monkey with TCDD results in increased CYP1A2 levels (4-fold), accompanied by proportional increases in the activation of MeIQx and IQ and phenacetin O-deethylase, EROD and MROD activities. The activation of PhIP is increased disproportionately, by 8-fold, most likely due to the activity of CYP1A1 which is also induced by TCDD in this species. Overall, the hepatic metabolism of heterocyclic amines by CYP1A enzymes in the untreated marmoset monkey resembles that in human more closely than that in the cynomolgus monkey. Therefore, marmoset monkey may be a more suitable model than the cynomolgus monkey for carcinogenicity studies involving MeIQx and PhIP, but not IQ.
Carcinogenesis 1994 May
PMID:Contribution of CYP1A1 and CYP1A2 to the activation of heterocyclic amines in monkeys and human. 820 83

Liver tissues were obtained from 20 liver cancer patients from Thailand, an area where the incidence of this tumour is high and where exposure to aflatoxin occurs. The expression of hepatic cytochrome P450s (P450) and glutathione S-transferase (GST) was examined and this expression was compared to the in vitro metabolism of aflatoxin B1 (AFB1). There was a > 10-fold inter-individual variation in expression of the various P450s including CYP3A4 (57-fold), CYP2B6 (56-fold) and CYP2A6 (120-fold). Microsomal metabolism of AFB1 to AFB1 8,9-epoxide (as measured by AFB1 tris-diol formation) and aflatoxin Q1 (AFQ1), the major metabolite produced, was significantly correlated with CYP3A3/4 expression (P < 0.001) and, to a lesser extent, with CYP2B6 expression (P < 0.01). There was a significantly reduced expression of major P450 proteins in microsomes from liver tumours compared to microsomes from the paired normal liver when analysed by Western immunoblot analysis. The production of AFQ1 and AFB1 tris-diol was almost uniformly reduced in tumours, but interestingly, the production of AFP1 was significantly increased. The immunoreactive expression of the major human classes of cytosolic GSTs (alpha, mu and pi) was also analyzed in normal and tumorous liver tissue. The expression of GSTA (alpha) and GSTM (mu) class proteins was markedly decreased and GSTP (pi) increased in the majority of tumour cytosols compared to normal liver. The cytosolic GST activity (1-chloro-2,4-dinitrobenzene conjugation) was significantly lower in liver tumours compared to normal liver (193 +/- 149 versus 875 +/- 299 nmol/min/mg, P < 0.0001), as was glutathione peroxidase (GPx) activity (cumene hydroperoxide) (26 +/- 23 versus 70 +/- 26 nmol/min/mg respectively, P < 0.005). Ten out of 14 individuals (71%) were homozygous null when genotyped for GSTM1. There was no detectable conjugation of AFB1 8,9-epoxide to glutathione by cytosol either from tumorous or normal liver. Thus, capacity of human cytosols to conjugate reactive AFB1 metabolites to GSH resembled AFB1-sensitive species such as rat, trout and duck rather than resistant species such as mouse and hamster. These data indicate a strong capacity of multiple forms of human hepatic P450s to metabolize AFB1 to both the reactive intermediate AFB1 8,9-epoxide and the detoxification product AFQ1. These results suggest that in view of the lack of significant GST-mediated protection against AFB1 in human liver, variations in expression of hepatic P450, due either to genetic polymorphisms or to modulation by environmental factors, may be important determinants in the risk of liver cancer development in AFB1-exposed populations.
Carcinogenesis 1993 Dec
PMID:In vitro metabolism of aflatoxin B1 by normal and tumorous liver tissue from Thailand. 826 34

The clastogenicity of tamoxifen and toremifene was tested in six human lymphoblastoid cell lines each expressing increased monooxygenase activity associated with a specific transfected human cytochrome P450 cDNA (CYP1A1, CYP1A2, CYP2D6, CYP2E1 or CYP3A4). The chemicals were also tested in a cell line (MCL-5) expressing elevated native CYP1A1 and containing transfected CYP1A2, CYP2A6, CYP2E1 and CYP3A4 and epoxide hydrolase, and in a cell line containing only the viral vector (Ho1). Dose-related increases in micronuclei were observed when cells expressing 2E1, 3A4, 2D6 or MCL-5 cells were exposed to tamoxifen. The positive responses in the cell lines were in the order MCL-5 > 2E1 > 3A4 > 2D6. Toremifene also gave positive results with 2E1, 3A4 and MCL-5 cells, although the responses were less marked and the positive effects required higher doses than with tamoxifen. A synthesized epoxide of tamoxifen was also tested in these cell lines and produced similar increases in the incidences of micronucleated cells. The increases in the responses observed with the epoxide were greater than with tamoxifen or toremifene. The P450 isoenzyme activities in these cells were in a range similar to those of human tumour-derived cell lines. Microsomes (1A1, 2A2, 2A6, 2B6, 2E1, 3A4 and 2D6) from these cells all metabolized tamoxifen. The major metabolite detected by HPLC was N-desmethyltamoxifen, and 4-hydroxytamoxifen was also detected in cells with cytochrome P450 2E1 and 2D6. These results are consistent with the following conclusions. (1) Tamoxifen requires metabolic activation to DNA-reactive species by specific CYP monooxygenases in order to exert its genotoxic effects. (2) The positive clastogenic effects elicited in lymphoblastoid cells by tamoxifen epoxide suggest that the genotoxic (and possibly the carcinogenic) effects of tamoxifen may be due to one or more epoxide metabolites that are generated intracellularly, probably in close proximity to the nucleus. (3) Tamoxifen is more genotoxic than toremifene.
Carcinogenesis 1994 Jan
PMID:Genotoxicity of tamoxifen, tamoxifen epoxide and toremifene in human lymphoblastoid cells containing human cytochrome P450s. 829 48

In order to develop more efficient in vitro systems for the study of pro-mutagenic or pro-carcinogenic chemicals, we have produced transgenic C3H/10T1/2 cell lines expressing human cytochrome P450 (CYP) 2A6. A retroviral vector containing the cDNA was packaged in psi-2 cells, and used to infect C3H/10T1/2 cells. From 100 G418-resistant clones initially isolated, three cell lines were chosen for further study based upon their morphologies, growth rates and CYP2A6-dependent coumarin 7-hydroxylase activities. Infected clone 10T1/2-04, like the 10T1/2 cells, had no detectable CYP2A6 enzyme activity, while clones 10T1/2-10 and 10T1/2-29 had microsomal CYP2A6 enzyme activities within the range found in human liver microsomes. CYP2A6 protein levels were in agreement with the observed enzyme activities. Southern blots revealed that cells from clone 10T1/2-04 contained a vector lacking the CYP2A6 cDNA, while cells from clones 10T1/2-10 and 10T1/2-29 contained multiple full-length inserts. Southern analysis also indicated the presence of an endogenous CYP2A6 ortholog in the four cell lines. All cell lines exhibited about equal sensitivity to induction of cytotoxicity and conversion to ouabain resistance by the direct acting mutagen N-methyl-N'-nitro-N-nitrosoguanidine. The four lines were also about equally sensitive to transformation by benzo[a]pyrene, a chemical requiring metabolic activation. However, only clones 10T1/2-10 and 10T1/2-29, which express CYP2A6 activity, were mutated and morphologically transformed by the tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.
Carcinogenesis 1993 Jul
PMID:Retroviral mediated expression of human cytochrome P450 2A6 in C3H/10T1/2 cells confers transformability by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). 833 Mar 60


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