BκF对体外培养人肝细胞诱导细胞色素P4501A的研究

目的: 研究体外培养的人肝细胞经苯并[κ]萤蒽(BκF)诱导后其细胞色素P4501A(CYP1A)亚家族的组成.方法:采用Bis-Tris-HCI缓冲的聚丙烯酰胺凝胶免疫印迹电泳系统和多克隆羊抗人CYP1A1/CYP1A2及兔抗人CYP1A2抗体对经5 μmol/L BκF诱导24 h的6名捐献者的肝细胞中表达的CYP1A1和CYP1A2进行分离鉴定.结果:6份样品中5份仅检出CYP1A1,而另1份则2种CYP均被检出,其CYP1A1/CYP1A2的比值为0.942;溶剂对照诱导的肝细胞CYP1A1和CYP1A2均未检出.结论:新鲜的人肝细胞经BκF诱导后CYP1A亚家族的表达因人而异,与直接从组织制备的人肝微粒体中的CYP1A的水平有明显不同.     

Dibenzo[A,L]pyrene-induced genotoxic and carcinogenic responses are dramatically suppressed in aryl hydrocarbon receptor-deficient mice

Dibenzo[a,l]pyrene (DB[a,l]P), a notorious air pollutant, is the most powerful carcinogenic polycyclic aromatic hydrocarbon (PAH) ever tested. Although the carcinogenicity of PAH may be primarily mediated by the aryl hydrocarbon receptor (AhR), the in vivo role of AhR in skin carcinogenesis remains to be defined. In this context, we investigated the genotoxic and carcinogenic responses of the AhR-deficient mouse skin to DB[a,l]P. A single painting resulted in a striking epidermal hyperplasia in AhR+/+ mice but not in AhR-/- mice. Bromodeoxyuridine-labeling index and accumulation of p53 protein in epidermal cells of AhR+/+ mice were 8- and 33-fold higher than those of AhR-/- mice, respectively. 32P-Postlabeling assay for DB[a,l]P-DNA adducts displayed a 2-fold increase in the AhR+/+ mouse skin. After DB[a,l]P exposure, AhR-/- mice arranged a nearly 60% reduction in the induction of epidermal cytochrome P450 (CYP)1A1, but CYP1B1 was constitutively expressed in both genotypes of mice, irrespective of DB[a,l]P treatment. As compared with AhR+/+ mice, AhR-/- mice had both significantly lower incidence (100% vs. 33%) and multiplicity (2.7 vs. 0.46) of skin tumors by the complete carcinogenesis study. These observations indicate that a reduced tumor yield in AhR-/- mice may be secondary to reduction of inducible CYP1A1 activation and subsequent DNA adduction. It is evident from our continuous work that although AhR is likely to play a central role in epidermal proliferation and possibly neoplastic transformation, the relative importance of AhR for carcinogenesis may be different among PAH examined.     

Polycyclic aromatic hydrocarbon-inducible DNA adducts: evidence by 32P-postlabeling and use of knockout mice for Ah receptor-independent mechanisms of metabolic activation in vivo

There is significant human exposure to polycyclic aromatic hydrocarbons (PAHs), many of which are potent carcinogens in laboratory animals and are suspected human carcinogens. The PAHs are bioactivated by cytochrome P450 (CYP)1A1/1B1 enzymes to reactive intermediates that bind to DNA, a critical step in the initiation of carcinogenesis. The Ah receptor (AHR) plays a critical role in the induction of CYP1 enzymes (i.e., CYP1A1, 1A2 and 1B1) by PAHs such as benzo[a]pyrene (BP) and 3-methylcholanthrene (MC). In our investigation, we tested the hypothesis that AHR-null animals are less susceptible to PAH-induced DNA adduct formation than wild-type animals. Wild-type [AHR (+/+)] mice or mice lacking the gene for the AHR were treated with a single dose (100 micromol/kg) of BP or MC, and hepatic DNA adducts were analyzed by (32)P-postlabeling. BP induced multiple hepatic DNA adducts in wild-type as well as AHR-null animals, suggesting the existence of AHR-independent mechanisms for BP metabolic activation. On the other hand, DNA adduct formation was markedly suppressed in AHR-null animals exposed to MC, although the major MC-DNA adduct was produced in these animals. Hepatic activities and apoprotein contents of 7-ethoxyresorufin O-deethylase (EROD) (CYP1A1) and 7-methoxyresorufin O-demethylase (MROD) (CYP1A2) activities were markedly induced by BP and MC in the wild-type, but not, in AHR-null animals. CYP1B1 expression was also induced, albeit to a lesser extent by the PAH MC, but not BP, in the wild-type animals. In conclusion, these results demonstrate the existence of AHR- and CYP1A1-independent mechanisms of PAH metabolic activation in mouse liver, a phenomenon that may have important implications for PAH-mediated carcinogenesis.     

Articles activation of promutagens in a human bronchial epithelial cell line stably expressing human cytochrome P450 1A2

Normal human bronchial epithelial (NHBE) cells are the putative progenitor cells of all types of lung cancer. NHBE cells immortalized by SV40 T-antigen retain many characteristics of the primary cells and are a useful model for investigating the role of oncogenes, tumor suppressor genes, and certain chemical carcinogens in the molecular pathogenesis of lung cancer. In this study, SV40 T-antigen-positive cells (BEAS-2B) were characterized for their metabolic functions and were shown to continue to express epoxide hydrolase, glutathione S-transferrase π, glutathione peroxidase, and catalase. To increase their metabolic activity towards human procarcinogens, human cytochrome P450 1A2 (CYP1A2) was stably expressed by introducing CYP1A2 cDNA into BEAS-2B cells either by infection with a high-titer recombinant retrovirus (pXT-1A2) or by transfection with a CYP1A2 expression vector (pCMV1A2), which produced the cell lines B-1A2 and B-CMV1A2, and B-CMV1A2, respectively. Cell lines established with either expression system expressed enzymatically active CYP1A2 protein and were 50- to 400-fold more sensitive to the cytotoxic effect of the carcinogen aflatoxin B₁) (AFB₁) than the corresponding control cell lines. The cytotoxic effects of AFB₁ were paralleled by increased metabolism of AFB₁ and enhanced formation of the AFB₁-N⁷ guanine adduct in B-CMV1A2 cells. Cytotoxicity and adduct formation correlated with a significantly higher protein expression of CYP1A2 by the cytomegalovirus promoter-driven plasmid. Since this human epithelial cell line is the precursor cell type of lung cancer, has normal phase II enzymes, and exhibits highly reproducible expression of phase l enzymes, this in vitro model should aid in the evaluation of putative human carcinogens and anticarcinogens. ©1994 Wiley-Liss, Inc.     

CYP1A1 and CYP1B1 gene expression and DNA adduct formation in normal human mammary epithelial cells exposed to benzo[a]pyrene in the absence or presence of chlorophyllin

Benzo[a]pyrene (BP) is a potent pro-carcinogen and ubiquitous environmental pollutant. Here, we examined the induction and modulation of CYP1A1 and CYP1B1 and 10-(deoxyguanosin-N²-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPdG) adduct formation in DNA from 20 primary normal human mammary epithelial cell (NHMEC) strains exposed to BP (4 μM) in the absence or presence of chlorophyllin (5 μM). Real-time polymerase chain reaction (RT-PCR) analysis revealed strong induction of both CYP1A1 and CYP1B1 by BP, with high levels of inter-individual variability. Variable BPdG formation was found in all strains by r7, t8-dihydroxy-t-9, 10 epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE)-DNA chemiluminescence assay (CIA). Chlorophyllin mitigated BP-induced CYP1A1 and CYP1B1 gene expression in all 20 strains when administered with BP. Chlorophyllin, administered prior to BP-exposure, mitigated CYP1A1 expression in 18/20 NHMEC strains (p < 0.005) and CYP1B1 expression in 17/20 NHMEC strains (p < 0.005). Maximum percent reductions of CYP1A1 and CYP1B1 gene expression and BPdG adduct formation were observed when cells were pre-dosed with chlorophyllin followed by administration of the carcinogen with chlorophyllin (p < 0.005 for CYP1A1 and CYP1B1 expression and p < 0.0005 for BPdG adducts). Therefore, chlorophyllin is likely to be a good chemoprotective agent for a large proportion of the human population.     

Polymorphisms in GSTM1, GSTT1 and CYP1A1 and risk of pancreatic adenocarcinoma

A prospective study of 149 unselected incident cases of pancreatic adenocarcinoma and 146 ethnically-matched controls found no associations between GSTM1 (adjusted odds ratio (AOR) 1.14), GSTT1 (AOR: 1.19) and CYP1A1 (AOR: 1.08) polymorphisms and pancreatic cancer susceptibility. Smoking and drinking status did not affect results. These polymorphisms do not appear to be important gene modifiers in pancreatic cancer.     

Induction of mouse cytochrome P450 2B enzymes by amine metabolites of musk xylene: Contribution of microsomal enzyme induction to the hepatocarcinogenicity of musk xylene

Musk xylene (MX) is a synthetic nitromusk perfume ingredient that, although uniformly negative in genotoxicity testing, causes liver tumors in B6C3F1 mice. MX is also capable of inducing cytochrome P450 enzymes in a manner similar to that of phenobarbital (PB), which suggests that epigenetic mechanisms may be involved in the carcinogenic response. At the same time, MX is metabolized in vivo by nitroreduction, a reaction catalyzed by intestinal flora that yields aromatic amine metabolites. These amine metabolites are also capable of inactivating CYP2B10, the major cytochrome P450 enzyme induced by MX treatment. In the study reported here, the monoamine metabolites of MX, o- and p-NH₂-MX, were evaluated for their potential to induce CYP2B10 and CYP1A2 mRNAs. Northern blot analyses indicated that both amines markedly induced CYP2B10 mRNA, whereas CYP1A2 mRNA, the enzyme implicated in the bioactivation of aromatic amines and frequently induced by aromatic amines, was induced only slightly, a response that was not different from that seen with PB. Induction of CYP2B10 mRNA suggested that the amine metabolites may contribute to the enzyme induction profile seen with MX treatment. To test this hypothesis, mice were treated with broad-spectrum antibiotics (neomycin, tetracycline, and bacitracin) to eliminate the intestinal flora and prevent formation of o- and p-NH₂-MX. In antibiotic-treated mice treated with MX (200 mg/kg) for 4 d, no evidence of microsomal enzyme induction was observed, including no increases in liver weight, total cytochrome P450 content, or CYP2B protein levels. These results indicate that the amine metabolites of MX are responsible for the enzyme induction seen after MX administration. Thus, the biochemical and molecular effects of amine metabolites of MX are markedly different from those of other aromatic amines but very similar to those of PB. Therefore, it appears that MX is a non-genotoxic chemical that may cause mouse liver tumors in a manner analogous to that of PB. Mol. Carcinog. 20:308–316, 1997. © 1997 Wiley-Liss, Inc.     

Specificity of cDNA-expressed human and rodent cytochrome P450s in the oxidative metabolism of the potent carcinogen 7,12-dimethylbenz[a]anthracene

7,12-Dimethylbenz[a]anthracene (DMBA), a potent carcinogen, requires metabolic activation by cytochrome P450s (P450s) to electrophilic metabolites that result in DNA modification, mutagenicity, and carcinogenicity. In this study, we used eight human forms, four rodent forms, and one rabbit form of P450 expressed from recombinant vaccinia or baculovirus vectors to define their specificity for metabolizing DMBA. Of the eight human P450s, 1A1 was the most active (specific activity = 14.7 nmol/min/nmol of P450) in total metabolism of DMBA and showed approximately 6- to 33-fold more activity than other P450s. 2B6, 2C9, and 1A2 were also capable of metabolizing DMBA (2.0–2.5 nmol/min/nmol of P450), whereas 2C8, 2E1, 3A4, and 3A5 exhibited relatively low activities. Among animal P450s, mouse 1A1 exhibited activity similar to that of human 1A1 and had 5.0- to 37-fold more activity than other rodent and rabbit P450s. In regard to enzyme regioselectivity, most human and rodent P450s predominantly formed the 8,9-diol, but human 2B6 and rat 281 preferentially formed the 5,6-diol. In the production of monohydroxymethyl metabolites, all the enzymes yielded more 7-hydroxymethyl-12-methylbenz[a]anthracene (7HOM12MBA) than 12-hydroxymethyl-7-methylbenz[a]anthracene (7M12HOMBA), except for human 1A1, which presented the reverse selectivity. Human liver microsomes from 10 organ donors were shown to metabolize DMBA and in most circumstances generated the metabolic profile DMBA trans-8,9-dihydrodiol > 7HOM12MBA ≥ DMBA trans-5,6-dihydrodiol ≥ 7,12-dihydroxymethylbenz[a]anthracene > 7M12HOMBA > DMBA trans-3,4-dihydrodiol. Thus, the combined activity of hepatic microsomal 2C9, 1A2, and 2B6 may contribute to the metabolic activation and the metabolism of DMBA in normal human liver. © 1996 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Ethanol‐mediated carcinogenesis in the human esophagus implicates CYP2E1 induction and the generation of carcinogenic DNA‐lesions

Chronic alcohol consumption is a major risk factor for esophageal cancer. Various mechanisms may mediate carcinogenesis including the genotoxic effect of acetaldehyde and oxidative stress. Ethanol exerts its carcinogenic effect in the liver among others via the induction of cytochrome P450 2E1 (CYP2E1) and the generation of carcinogenic etheno‐DNA adducts. Here we investigated if such effects can also be observed in the human esophagus. We studied nontumorous esophageal biopsies of 37 patients with upper aerodigestive tract cancer and alcohol consumption of 102.3 ± 131.4 g/day (range: 15–600 g) as well as 16 controls without tumors (12 teetotalers and 4 subjects with a maximum of 25 g ethanol/day). CYP2E1, etheno‐DNA adducts and Ki67 as a marker for cell proliferation were determined immunohistologically. Chronic alcohol ingestion resulted in a significant induction of CYP2E1 (p = 0.015) which correlated with the amount of alcohol consumed (r = 0.6, p < 0.001). Furthermore, a significant correlation between CYP2E1 and the generation of the carcinogenic exocyclic etheno‐DNA adducts 1,N⁶‐ethenodeoxyadenosine (r = 0.93, p < 0.001) and 3,N⁴‐ethenodeoxycytidine (r = 0.92, p < 0.001) was observed. Etheno‐DNA adducts also correlated significantly with cell proliferation (p < 0.01), which was especially enhanced in patients who both drank and smoked (p < 0.001). Nonsmokers and nondrinkers had the lowest rate of cell proliferation, CYP2E1 expression and DNA lesions. Our data demonstrate for the first time an induction of CYP2E1 in the esophageal mucosa by ethanol in a dose dependent manner in man and may explain, at least in part, the generation of carcinogenic DNA lesions in this target organ.     

Ki-ras oncogene mutations in tumors and DNA adducts formed by benz[j]aceanthrylene and benzo[a]pyrene in the lungs of strain A/J mice

Strain A/J mice received intraperitoneal injections of benz[j]aceanthrylene (B[j]A) or benzo[a]pyrene (B[a]P). At 24, 48, and 72 h, lung tissues were removed for analysis of B[a]P- or B[j]A-derived DNA adduct formation during the first 3 d of exposure. One group of mice exposed to these hydrocarbons was kept for 8 mo to determine lung tumor multiplicity, the occurrence of mutations in codons 12 and 61 of the Ki-ras gene in the tumors that arose, the relationship between Ki-ras oncogene mutations in tumors, and the presence and quantity of genomic DNA adducts. The major DNA adduct in the lungs of mice exposed to B[a]P was N²-(10β-[ + B,7α, 9α-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene]yl)-deoxyguanosine (BPDE-l-dGuo) arising from bay-region diolepoxide activation of B[a]P and was consistent with the occurrence of tumors with mutations GGT → TGT (56%), GGT → GTT (25%), and GGT → GAT (19%) in codon 12, all involving mutations of a guanine. B[j]A, a demethylated analogue of 3-methylcholanthrene (3-MCA) with an unsaturated cyclopenta ring, produced 16- to 60-fold more tumors at equivalent doses than did B[a]P; the mutations in tumors were GGT → TGT (4%), GGT → GTT (30%), and GGT → CGT (65%). Analysis of adduction patterns in DNA suggested that B[j]A was activated to form DNA-binding derivatives in A/J mouse lungs primarily at the cyclopenta ring even though B[j]A contains a bay region. As reported in the published literature, the mutation spectrum induced by 3-MCA in Ki-ras codon 12 of mouse cells is similar to that of B[a]P but not to that of its close relative B[j]A. In contrast to B[j]A, 3-MCA is activated mostly via a bay-region diol-epoxide since its cyclopenta ring is saturated and not easily epoxidated. Therefore, we propose that the GGT → CGT mutations produced by B[j]A in Ki-ras codon 12 were mostly the result of cyclopentaring-derived adducts.     

煤焦沥青烟提取物对BEAS-2B细胞Nrf2及NQO1表达的影响

目的：研究煤焦沥青烟提取物对人肺支气管上皮细胞（BEAS-2B）Nrf2、NQO1 mRNA及Nrf2蛋白表达的影响。方法：煤焦沥青烟提取物以0．00、1．25、2．50、5．00、10．00μg／ml分别染毒BEAS-2B细胞24h后,提取细胞总RNA和总蛋白。采用RT-PCR检测Nrf2、NQO1 mRNA的相对表达量,Western blot测定Nrf2蛋白相对表达量。结果：染毒组BEAS-2B细胞Nrf2 mRNA及蛋白相对表达量均低于对照组,差别均有统计学意义（P均〈0．05）,在1．25—5．00μg／ml浓度范围内,随着染毒浓度的增加,Nrf2 mRNA和蛋白表达呈递增趋势,而当染毒浓度为10．00μg／ml时,Nrf2 mRNA和蛋白表达量均有所减少（P〈0．05）。随着染毒浓度的增加,NQO1基因表达水平升高,在5．00、10．00μg／ml时NQO1基因相对表达量比对照组高（P〈0．05）。结论：在煤焦沥青烟提取物浓度较低时（1．25～5．00μg／ml）,随染毒浓度增加Nrf2表达水平升高,可能上调NQO1基因的表达,增强BEAS-2B细胞氧化应激能力。     

CYP1A1基因多态性与p16基因甲基化对中国人肺癌发病的影响

Objective:To investigate the relationship between the genetic polymorphism of CYP1A1 and the genetic susceptibility to lung cancer as well as to study the effects of the methylation in p16 gene on the risk of lung cancer in a Chinese population.Methods:A case control study was conducted among 47 cases of lung cancer and 94 controls.The genetic polymorphism of CYP1A1 was tested with method of PCR-RFLP,and a methylation-specific PCR(MSP)was performed to detect p16 methylation.Results:It showed that there was no significant difference in frequencies of the genotypes of CYP1A1 between the two groups(P>0.05).Synergistic effects were not found between smoking and CYP1A1.Methylated p16 gene was found in 44.7%(21/47)of lung cancer tissues and in 17.0%(8/47)of normal lung tissues with significant difference(P< 0.05).Conclusion:The genetic polymorphism of CYP1A1 does not increase the risk of lung cancer in a Chinese population. The methylation in p16 gene may be the most common mechanism to inactivate p16 gene in lung cancer,and is not significantly associated with genotype of CYP1A1.     

Induction of CYP1A1 gene expression in H4-II-E rat hepatoma cells by benzo[e]pyrene.

In the rat, expression of the CYP1A1 gene is closely associated with arylhydrocarbon hydroxylase (AHH) enzyme activity. AHH is an inducile enzyme activity known to play an important role in the bioactivation of polycyclic aromatic hydrocarbons (PAHs) to mutagenic and carcinogenic metabolites. PAH-induced expression of the CYP1A1 gene appears to be regulated by several trans-acting factors, including the Ah receptor and the 4S PAH-binding protein. In this study, we used the PAH isomers benzo[a]pyrene (BaP) and benzo[e]pyrene (BeP) to further evaluate the role of the 4S PAH-binding protein in induction of the CYP1A1 gene in H4-II-E rat hepatoma cells. Although BaP is believed to bind to both the Ah receptor and the 4S protein, BeP has been reported to bind exclusively to the 4S protein. The results of the study presented here indicate that BaP and BeP induce the expression of the CYP1A1 gene, as measured by ethoxyresorufin O-deethylase (EROD) activity, in a concentration-dependent manner. However, BaP is about 25 times as potent as BeP in inducing EROD activity in these cells. Slot-blot analysis of total RNA isolated from these cells indicated that BeP, BaP, and 3-methylcholanthrene increased the level of CYP1A1 mRNA expression. Sucrose-gradient analysis of BeP binding activity indicated that BeP bound with high affinity to the 4S PAH-binding protein, but not to the Ah receptor. These results suggest that the 4S protein may play a role in the PAH-induced expression of the CYP1A1 gene in rat H4-II-E cells.