Expression of keratins in normal, immortalized and malignant oral epithelia in organotypic culture

Keratins have been extensively studied in tissues and cultured keratinocytes but limited information is available on epithelia reconstructed in vitro. The aim of this study was to examine keratin expression in organotypic epithelia with normal (NOK), immortalized (SVpgC2a) and malignant (SqCC/Y1) human buccal cells. Organotypic epithelia were derived from 10 days of culture at the air-liquid interface of collagen gels containing human oral fibroblasts using a standardized serum-free medium. Sections were stained immunohistochemically with selected mono-specific antibodies to a range of keratins. Organotypic epithelia showed sharp differences in keratin expression and distribution. K4/K13, K1/K10, K6/K16 were variably expressed in NOK and SqCC/Y1 but were not detected in SVpgC2a. K5 was expressed in all organotypic epithelia but K14 was absent in SVpgC2a. K7 and K8 showed variable expression while K18 was expressed uniformly in all epithelia. K19 was expressed consistently in NOK and K20 was distributed heterogeneously in SVpgC2a. Overall, organotypic cultures of normal keratinocytes express many of the same keratins as buccal mucosa. Further, the loss of keratins in SVpgC2a and their retention in SqCC/Y1 have several features in common with the respective keratin profile of oral epithelial dysplasia and well-differentiated oral squamous cell carcinoma. Although qualitative and quantitative differences exist compared to keratin expression in vivo, these cell lines in organotypic culture may serve in studies of the multi-step progression of oral cancer.     

Metabolism of mutagens and carcinogens in woodchuck liver and its relationship with hepatitis virus infection

Thirty-six wild-caught woodchucks (Marmota monax) were characterized according to sex, weight, trapping locality, liver pathology, and serum or hepatic markers of woodchuck hepatitis virus. Liver subcellular fractions were assayed for microsomal cytochromes P-450, aryl hydrocarbon hydroxylase, glutathione, cytosolic enzymes involved in its metabolism (glutathione S-transferase, glutathione peroxidase, and glutathione reductase), in the hexose monophosphate shunt (glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase), NADH- and NADPH-dependent diaphorases, and DT diaphorase. Moreover, liver postmitochondrial fractions were assayed for their ability to activate procarcinogens [i.e., a tryptophan pyrolysate product, aflatoxin B1, 2-aminofluorene, and trans-7,8-dihydrobenzo(a)pyrene] to mutagenic metabolites in the Ames reversion test and to decrease the activity of direct-acting mutagens [i.e., 4-nitroquinoline N-oxide, 2-methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine X 2HCl, and sodium dichromate]. A considerable interindividual variability in metabolism was observed among the examined woodchucks. Some of the investigated parameters were more elevated in virus carriers, especially in those suffering from chronic active hepatitis, but only a few of the recorded differences (i.e., oxidized glutathione reductase and NADPH-dependent diaphorase) were statistically significant. The comparison of the monitored activities in woodchucks and in other rodent species (rat and mouse) led to the conclusion that the liver metabolism of mutagens and carcinogens in woodchucks is more oriented in the sense of activation, while detoxification mechanisms are more efficient in rats and mice.     

Suppression of 7,12-dimethylbenz[a]anthracene-induced mammary carcinogenesis by pre-initiation treatment of rats with beta-naphthoflavone coincides with decreased levels of the carcinogen-derived DNA adducts in the mammary gland

BACKGROUND: Mechanisms underlying prevention by beta-naphthoflavone (beta-NF) of mammary carcinogenesis initiated with 7,12-dimethylbenz[a]anthracene (DMBA) in the rat were elucidated. METHODS AND RESULTS: Treatment of female Sprague-Dawley rats with beta-NF at 40 mg/kg b.wt. for 4 days by oral gavage in corn oil before a single oral dose of DMBA (112 mg/kg b.wt.) suppressed mammary gland carcinogenesis as shown by an increase in the median latent period from 10 to 24 weeks and a 60% decrease in the multiplicity of mammary adenocarcinomas. In contrast, a 20-day treatment with beta-NF starting 3 weeks after DMBA had no significant effects on mammary tumorigenesis. The activities of phase I and phase II enzymes were examined in the liver and mammary gland 24 h after treatment of rats with beta-NF, DMBA, or beta-NF followed by DMBA as in the first bioassay. Treatment with either beta-NF or DMBA increased the hepatic activities of cytochrome P450 (CYP)1A1, 1A2, and 2B1/2, and glutathione S-transferase, and the mammary activity of CYP1A1. The activity of mammary CYP2B1/2 induced by DMBA was decreased by beta-NF. In the liver, the increase of UDP-glucuronosyl transferase (GT) activity in rats treated with beta-NF and DMBA was 2.3-fold greater than in rats treated with DMBA alone. Thus, treatment with beta-NF likely increased the rate of glucuronidation of DMBA dihydrodiols leading to carcinogen detoxification. The levels of the DMBA adducts determined by 32P-postlabeling of the mammary gland DNA were decreased in the beta-NF-pretreated rats. Conclusion: The beta-NF-induced increase in the hepatic UDP-GT activity and decrease in the mammary DNA-DMBA adducts occurred under the same treatment regimen that led to suppression of DMBA-induced mammary carcinogenesis.     

Dehydroepiandrosterone inhibits the expression of carcinogen-activating enzymes in vivo

We investigated the effect of the steroid hormone dehydroepiandrosterone (DHEA) on the hepatic expression and activity of carcinogen-activating enzymes, the cytochromes P450 (CYP) 1A1, 1A2 and 1B1, in Sprague-Dawley rats. In animals fed DHEA at 200 or 400 mg/kg body weight every other day for 2 weeks prior to exposure to the aryl hydrocarbon dimethylbenz[a]anthracene (DMBA, 5 mg/kg), there was a dose-dependent decrease in hepatic CYP activity, as measured by ethoxyresorufin-O (EROD) assay, from 37.1 to 22.9 and 14.7 pmoles/min/10 microg microsomes, respectively. DHEA did not directly inhibit microsomal EROD activity, however, leading us to investigate its effects on enzyme expression. To test this, we examined protein and mRNA levels of the enzymes. Western blot for CYP1A1 and CYP1A2 showed that DHEA inhibited the increase in hepatic CYP1A1 and CYP1A2 enzyme levels that are normally induced by DMBA. DMBA-induced increase in expression of CYP1A1, CYP1A2 and CYP1B1 mRNA was similarly blunted in DHEA-treated animals. DHEA was also able to significantly reduce the basal expression of CYP1A1 and CYP1A2 but not of CYP1B1. These results indicate that DHEA regulates the expression and, hence, the activity of hepatic carcinogen-activating enzymes in vivo, and this may be an important mechanism of its chemopreventive activity.     

Distinct time courses of increase in cytochromes P450 1A2, 2A5 and glutathione S-transferases during the progressive hepatitis associated with Helicobacter hepaticus

Mice naturally infected by Helicobacter hepaticus develop a chronic active hepatitis leading to hepatocellular carcinoma. This mouse model of liver cancer was used to examine the impact of bacterial infection on the hepatic expression and activity of enzymes involved in carcinogen bioactivation (phase I enzymes) and detoxification (phase II enzymes). No major differences in total cytochrome P450 (CYP) content were found between control and infected mice during the course of the study. The most striking modulations of individual isoenzymes were the increases in immunohistochemical staining observed for CYP1A and CYP2A5 in relation to increasing age and liver lesions. The increase in CYP2A5 in mice aged over 12 months was confirmed by the observed increases in coumarin 7-hydroxylation (CYP2A5 substrate) in vitro and CYP2A5 mRNA levels by Northern blot analysis. Immunoblotting confirmed the specific induction of CYP1A2 in infected mice 12 and 18 months of age. Perfusion of liver with nitroblue tetrazolium, an indicator for superoxide formation, demonstrated that in livers of infected mice, hepatocytes often co-expressed CYP2A5 and formazan deposition. Concerning phase II enzymes, an enhancement of glutathione S-transferase (GST) activities, related to the disease process, was observed in infected mice. An age- specific increase of GSTpi and A4.4 (early stage of disease) and GST YaYa (>9 months) expression was also demonstrated by immunohistochemical staining. In contrast, catalase and glutathione- peroxidase activities, as well as reduced glutathione content were decreased in the early stages of disease (3-9 months) in infected mice compared to age-matched control mice. Overall, these results suggest that alterations in CYP and GST expression may contribute to the aetiology of tumour incidence due to H. hepaticus infection via production of reactive oxygen species.      

Metabolic proficiency and benzo[a]pyrene DNA adduct formation in APCMin mouse adenomas and uninvolved mucosa.

Tumour formation may involve interactions between genetic factors and environmental carcinogens. Adenoma formation in APCMin/+ mice is associated homozygous adenomatous polyposis coli (APC) gene mutation, but the effects on carcinogen susceptibility are unknown. This study tests the hypothesis that APCMin/+ adenoma formation is accompanied by changes in metabolic proficiency and carcinogen susceptibility. Cytochrome P450 (CYP)1A1/1A2, glutathione S-transferase (GST)alpha, mu and pi classes and DNA adduct formation were assayed in adenomas and uninvolved mucosa from APCMin/+ mice, before and after benzo[a]pyrene (B[a]P) treatment. In untreated adenomas and mucosa, CYP1A1/1A2 and B[a]P-DNA adducts were undetected but GSTalpha, mu and pi class enzymes were constitutively expressed. In adenomas, B[a]P only induced CYP1A1/1A2 to low level while GSTalpha and pi class enzymes were unaffected. A GST mu band which was absent from mucosa, was induced in adenomas. In mucosa, B[a]P induced CYP1A1/1A2 and GSTalpha and pi, to high levels. B[a]P-DNA adduct levels were 56 +/- 15/10(8) nucleotides (median +/- SE) in adenomas versus 89 +/- 19/10(8) nucleotides in mucosa (P < 0.0001). APCMin adenomas show reduced bioactivation capacity and sustain less DNA damage from B[a]P exposure, than APCMin uninvolved mucosa. These properties could influence mutagenesis and subsequent neoplastic transformation of adenomas.     

Ⅰ、Ⅱ相药物代谢酶遗传多态性与晚期非小细胞肺癌化疗疗效的关系

背景与目的目前药物代谢酶遗传多态性与化疗疗效关系的研究结果多不一致,本研究旨在探讨细胞色素P4501A1(cytochrome P450 1A1,CYP1A1)、2E1(cytochrome P450 2E1,CYP2E1)、2D6(cytochrome P450 2D6,CYP2D6)和谷胱甘肽硫转移酶M1(glutathione S-transferase M1,GSTM1)基因多态性与晚期非小细胞肺癌化疗疗效以及与肺癌患者预后的关系。方法采用PCR和PCR-RFLP技术对肺癌患者4种药物代谢酶基因分型,并对他们进行5年跟踪随访。结果携带B型CYP1A1和缺陷性GSTM1肺癌患者比其它基因型患者化疗疗效好(P<0.001)。携带A型CYP1A1肺癌患者接受非铂类化疗药物治疗比B型和C型患者疗效好(P=0.041);携带缺陷性GSTM1肺癌患者接受铂类化疗药物治疗疗效比功能型患者疗效好(P=0.011)。4种酶对晚期非小细胞肺癌患者总生存期(overall survival,OS)没有明显影响(P>0.05)。结论 A型CYP1A1肺癌患者接受非铂类化疗药物治疗比B型和C型患者疗效好;缺陷性GSTM1肺癌患...     

Polymorphisms in CYP1B1, GSTM1, GSTT1 and GSTP1, and susceptibility to breast cancer

Polymorphisms in the cytochrome P450 1B1 (CYP1B1) and glutathione S-transferase (GST) drug metabolic enzymes, which are responsible for metabolic activation/detoxification of estrogen and environmental carcinogens, were analyzed for their association with breast cancer risk in 541 cases and 635 controls from a North Carolina population. Each polymorphism, altering the catalytic function of their respective enzymes, was analyzed in Caucasian and African-American women. As reported in previous studies, individual polymorphisms did not significantly impact breast cancer risk in either Caucasian or African-American women. However, African-American women exhibited a trend towards a protective effect when they had at least one CYP1B1 119S allele (OR=0.53; 95% CI=0.20-1.40) and increased risk for those women harboring at least one CYP1B1 432V allele (OR=5.52; 95% CI=0.50-61.37). Stratified analyses demonstrated significant interactions in younger (age < or =60) Caucasian women with the CYP1B1 119SS genotype (OR=3.09; 95% CI=1.22-7.84) and younger African-American women with the GSTT1 null genotype (OR=4.07; 95% CI=1.12-14.80). A notable trend was also found in Caucasian women with a history of smoking and at least one valine allele at GSTP1 114 (OR=2.12; 95% CI=1.02-4.41). In Caucasian women, the combined GSTP1 105IV/VV and CYP1B1 119AA genotypes resulted in a near 2-fold increase in risk (OR=1.96; 95% CI=1.04-3.72) and the three way combination of GSTP1 105IV/VV, CYP1B1 119AS/SS and GSTT1 null genotypes resulted in an almost 4-fold increase in risk (OR=3.97; 95% CI=1.27-12.40). These results suggest the importance of estrogen/carcinogen metabolic enzymes in the etiology of breast cancer, especially in women before the age of 60, as well as preventative measures such as smoking cessation.     

Chemopreventive effects of 2-(allylthio)pyrazine on hepatic lesion, mutagenesis and tumorigenesis induced by vinyl carbamate or vinyl carbamate epoxide

2-(Allylthio)pyrazine (2-AP), synthesized for its possible use as a hepatoprotective agent, has been found to selectively inhibit rat hepatic cytochrome P450 2E1 (Kim et al., Biochem. Pharmacol., 53, 261- 269, 1997), while it enhances the activities of phase II detoxification enzymes such as glutathione S-transferase and epoxide hydrolase. As part of a program in evaluating the chemopreventive potential of 2-AP, we have determined its effects on hepatotoxicity, mutagenicity and tumorigenicity of vinyl carbamate (VC), a prototypic hepatocarcinogen preferentially activated by P450 2E1 to the ultimate carcinogenic metabolite vinyl carbamate epoxide (VCO), which undergoes detoxification by glutathione conjugation and oxirane hydrolysis. Administration of 2-AP (100 mg/kg body wt) to male Sprague-Dawley rats by gavage, 2 days, 1 day and 4 h prior to VC or VCO, markedly ameliorated the hepatotoxicity of these compounds as determined by decreased serum aspartate aminotransferase and alanine aminotransferase activities. Furthermore, 2-AP pre-treatment significantly suppressed the VC-induced hepatocarcinogenesis in infant male B6C3F1 mice. In a separate experiment, the multiplicities of skin tumors formed in female ICR mice treated with 5.8 micromol of VC or VCO were inhibited 58 and 70%, respectively, by pre-treatment with 2-AP by oral administration. The mutational spectrum of ras-oncogene in papillomas was not altered by 2-AP pre-treatment. 2-AP also inhibited the mutagenicity of VC in the Salmonella-microsome assay. Taken together, these findings suggest that 2-AP is a potential chemopreventive agent.      

Cytochrome P450 expression and related metabolism in human buccal mucosa

Constituents in food and fluids, tobacco chemicals and many drugs are candidates for oral absorption and oxidative metabolism. On this basis, the expression of cytochrome P450 isozymes (CYPs) and the conversion of CYP substrates were analysed in reference to buccal mucosa. A RT-PCR based analysis of human buccal tissue from 13 individuals demonstrated consistent expression of mRNA for the CYPs 1A1, 1A2, 2C, 2E1, 3A4/7 and 3A5. CYP 2D6 was expressed in six out of the 13 specimens, whereas all samples were negative for 2A6 and 2B6. Serum-free monolayer cultures of the Siman virus 40 large T-antigen-immortalized SVpgC2a and the carcinoma SqCC/Y1 buccal keratinocyte lines expressed the same CYPs as tissue except 3A4/7 and 3A5 (SVpgC2a), and 2C, 2D6 and 3A4/7 (SqCC/Y1). Dealkylation of ethoxyresorufin and methoxyresorufin in both normal and transformed cells indicated functional 1A1 and 1A2, respectively. SVpgC2a showed similar activity as normal keratinocytes for both substrates, whereas SqCC/Y1 showed about 2-fold lower 7-ethoxyresorufin O-deethylation and 7-methoxyresorufin O-demethylation activities. SVpgC2a showed detectable and many-fold higher activity than the other cell types towards chlorzoxazone, a substrate for 2E1. Absent or minute catalytic activity of 2C9, 2D6 and 3A4 in the various cell types was indicated by lack of detectable diclofenac, dextromethorphan and testosterone metabolism (<0.2-0.5 pmol/min/mg). Metabolic activation of the tobacco-specific N-nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and the mycotoxin aflatoxin B1 (AFB1) to covalently bound adducts was indicated by autoradiographic analysis of both monolayer and organotypic cultures of SVpgC2a. In contrast, SqCC/Y1 showed lower or absent metabolic activity for these substrates. Finally, measurements of various non-reactive AFB1 metabolites indicated rates of formation <0.1 pmol/min/mg in both normal and transformed cells. The results indicate presence of several CYPs of which some may contribute to significant xenobiotic metabolism in human buccal epithelium. Notably, metabolic activation of AFB1 was not previously implicated for oral mucosa. Further, the results show that CYP-dependent metabolism can be preserved or even activated in immortalized keratinocytes. Metabolic activity in SVpgC2a under both monolayer and organotypic culture conditions suggests that this cell line may be useful to pharmaco-toxicological and carcinogenesis studies.     

Estrogen regulates Ah responsiveness in MCF-7 breast cancer cells

Cytochrome P450 (CYP)1A1 and CYP1B1, which are under the regulatory control of the aryl hydrocarbon (Ah) receptor (AhR), catalyze the metabolic activation of numerous procarcinogens and the hydroxylation of 17beta-estradiol (E2) at the C-2 and C-4 positions, respectively. There is evidence of cross-talk between estrogen receptor alpha (ERalpha)- and AhR-mediated signaling in breast and endometrial cells. To further examine these interactions, we investigated the short- and long-term effects of E2 exposure on Ah responsiveness in MCF-7 human breast cancer cells. Short-term exposure to 1 nM E2 elevated the ratio of the 4- to 2-hydroxylation pathways of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced E2 metabolism and the ratio of the induced CYP1B1 to CYP1A1 mRNA levels, as determined by real-time PCR. Cells maintained long-term (9-12 months) in low-E2 medium progressively lost Ah responsiveness, as indicated by diminished rates of TCDD-induced E2 metabolism and ethoxyresorufin O-deethylase activity, and the reduced expression of the CYP1A1 and CYP1B1 mRNAs and proteins levels. These E2-deprived cells showed elevated levels of ERalpha mRNA, depressed levels of AhR mRNA, and unchanged levels of the AhR nuclear translocator mRNA. Transient transfection studies using a CYP1B1-promoter-luciferase reporter construct showed that reduced CYP1B1 promoter activity in E2-deprived cells could be restored by co-transfection with an AhR expression construct, indicating that AhR expression was limiting in these cells. The reduced Ah responsiveness of E2-deprived cells was reversed by culture for four passages in medium supplemented with 1 nM E2; ERalpha and AhR mRNAs returned to near-normal levels and the inducibility of the CYP1A1 and CYP1B1 mRNAs, proteins, and E2 metabolic activities by TCDD was restored. These studies indicate that the continued presence of estrogen is required to maintain high levels of AhR expression and inducibility of the procarcinogen-bioactivating enzymes, CYP1A1 and CYP1B1, in MCF-7 cells.     

外周血中芳香烃受体及芳香烃代谢酶活性与乳腺癌关系的研究

背景与目的：大量研究表明,活化的芳香烃受体(aryl hydrocarbon receptor,AhR)可能参与了由多环芳烃诱导的乳腺癌的发生,AhR作为公认的配体激活的转录因子,可诱导多种酶表达。本研究旨在探讨芳香烃受体和芳香烃代谢酶在人体内蛋白水平与乳腺癌及其病理指标间的关系。方法：以1∶1配对进行病例对照研究,共收集65对血样,采用酶联免疫吸附法检测血液中芳香烃受体及芳香烃代谢酶蛋白水平,同时收集病理资料。计量资料使用秩和检验进行统计分析,相关性分析使用Spearman检验。结果：秩和检验发现对照组与病例组间醛脱氢酶(aldehyde dehydrogenase,ALDH)、细胞色素(cytochrome P450,CYP450)差异无统计学意义(Z=0.196,P=0.845;Z=0.269,P=0.788);AhR、醌氧化还原酶(quinone oxido-ereductase 1,NQO1)、谷胱甘肽-S-转移酶(glutatnione-S-transferases,GST)对照组平均浓度低于病例组,差异有统计学意义(Z=1.956,P=0.041;Z=2.627,P=0.009;Z=3.272,P=0.001)。相关性分析结果显示,病例组中AhR与GST和NQO1相关性较高(r=0.665,P<0.01;r=0.704,P<0.01)。以病理指标(ER、PR、HER-2)-、+、++、+++分组,发现以雌激素受体ER分组的AhR、GST蛋白表达水平差异有统计学意义(P<0.05)。结论：实验结果显示芳香烃受体及芳香烃代谢酶蛋白水平对乳腺癌发生可能有重要影响,检测酶蛋白水平可能对乳腺癌的早期发现有帮助,对治疗方案的选择及预后有一定意义。     

A pharmacogenetic study to investigate the role of dietary carcinogens in the etiology of colorectal cancer

Susceptibility to colorectal cancer, one of the most common forms of cancer in the Western world, has been associated with several environmental and dietary risk factors. Dietary exposure to food derived heterocyclic amine carcinogens and polycyclic aromatic hydrocarbons have been proposed as specific risk factors. Many polymorphic Phase I and Phase II drug metabolizing enzymes are responsible for the metabolism and disposition of these compounds and it is therefore possible that inheritance of specific allelic variants of these enzymes may influence colorectal cancer susceptibility. In a multicenter case-control study, 490 colorectal cancer patients and 593 controls (433 matched case-control pairs) were genotyped for common polymorphisms in the cytochrome P450 (CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2C9, CYP2C19 and CYP2D6), glutathione S-transferase (GSTM1, GSTP1 and GSTT1), sulfotransferase (SULT1A1 and SULT1A2), N-acetyl transferase 2 (NAT2), NAD(P)H:quinone oxidoreductase (NQO1), methylenetetrahydrofolate reductase (MTHFR), and microsomal epoxide hydrolase (EPHX1) genes. Matched case-control analysis identified alleles associated with higher colorectal cancer risk as carriage of CYP1A1*2C (OR = 2.15, 95% CI 1.36-3.39) and homozygosity for GSTM1*2/*2 (OR = 1.53, 95% CI 1.16-2.02). In contrast, inheritance of the CYP2A6*2 (OR = 0.51, 95% CI 0.28-1.06), CYP2C19*2 (OR = 0.72, 95% CI 0.52-0.98) and the EPHX1(His113) alleles were associated with reduced cancer risk. We found no association with colorectal cancer risk with NAT2 genotype or any of the other polymorphic genes associated with the metabolism and disposition of heterocyclic amine carcinogens. This data suggests that heterocyclic amines do not play an important role in the aetiology of colorectal cancer but that exposure to other carcinogens such as polycyclic aromatic hydrocarbons may be important determinants of cancer risk.     

Interaction of genetic polymorphisms in cytochrome P450 2E1 and glutathione S-transferase M1 to breast cancer in Taiwanese woman without smoking and drinking habits

P450 (CYP) and glutathione S-transferase (GST) are involved in the activation and detoxification of many potential carcinogens. Although, the interaction between environmental exposure and genetic polymorphisms of cytochrome P450 2E1 (CYP2E1) and glutathione S-transferase M1 (GSTM1) in breast cancer has been assessed, the gene–gene interactions between CYP2E1 and GSTM1 related to breast cancer have not been focused on and reported. We conducted a hospital-based case-control study to investigate whether the genetic interaction effects of CYP2E1 and GSTM1 modify the risk of developing breast cancer independent of the effect of cigarette smoking and alcohol consumption. Individuals with the C2/C2 genotype of CYP2E1 had a lower risk (OR = 0.24, 95% CI = 0.08–0.74) when compared with those with the C1/C1 genotype. However, there was no significant difference (OR = 1.05, 95% CI = 0.73–1.50) in the GSTM1 genotype frequency between the cases with breast cancer and that of the controls. When individuals with the genotype of C1/C1 or C1/C2 of CYP2E1 and the wild-type of GSTM1 were compared with those of C2/C2 of CYP2E1 and the null-type of GSTM1 however, we found a significantly increased risk (OR = 3.50, 95% CI = 1.01–16.55) in the breast cancer patients. Our findings indicated a gene–gene interaction between CYP2E1 and GSTM1 was accessible to developing breast cancer in Taiwanese women without the habits of cigarette smoking and alcohol consumption even though independent effects of CYP2E1 and GSTM1 were weak or non-significant and suggest that environmental carcinogen besides cigarette and alcohol consumption could induce breast cancer.Szu-Hsien Wu and Shih-Meng Tsai contributed equally to this work.     

Distinct effects of ultraviolet B light on antioxidant expression in undifferentiated and differentiated mouse keratinocytes

Ultraviolet (UV) B causes oxidative stress, which has been implicated in carcinogenesis. We determined if the sensitivity of keratinocytes to UVB-induced oxidative stress is dependent on their differentiation state. In primary cultures of undifferentiated and differentiated mouse keratinocytes, UVB (25 mJ/cm(2)) stimulated production of reactive oxygen intermediates. This was associated with increased messenger RNA (mRNA) expression of the antioxidant enzymes glutathione peroxidase, heme oxygenase-1 (HO-1) and the glutathione S-transferase (GST), GSTA1-2. The effects of UVB on GSTA1-2 were greater in undifferentiated when compared with differentiated cells. UVB also induced GSTM1, but only in undifferentiated cells. In contrast, UVB reduced expression of manganese superoxide dismutase, metallothionein-2, GSTA3 and microsomal glutathione S-transferase (mGST)3 in both cell types, whereas it had no major effects on catalase, copper-zinc superoxide dismutase, GSTP1, mGST1 or mGST2. Of note, levels of GSTA4 mRNA were 4- to 5-fold greater in differentiated relative to undifferentiated cells. Moreover, whereas GSTA4 was induced by UVB in undifferentiated cells, it was inhibited in differentiated cells. UVB activated p38 and c-jun N-terminal kinase mitogen-activated protein (MAP) kinases in both undifferentiated and differentiated keratinocytes. Whereas inhibition of these kinases blocked UVB-induced HO-1 in both cell types, GSTA1-2 and GST-4 were only suppressed in undifferentiated cells. In differentiated keratinocytes, p38 inhibition also suppressed GSTA1-2. In contrast, MAP kinase inhibition had no major effects on UVB-induced suppression of GSTA4 in differentiated cells. These data indicate that UVB-induced alterations in antioxidant expression are differentiation dependent. Moreover, MAP kinases are critical regulators of this response. Alterations in antioxidants are likely to be important mechanisms for protecting the skin from UVB-induced oxidative stress.