Aflatoxin B1 aldehyde reductase (AFAR) genes cluster at 1p35-1p36.1 in a region frequently altered in human tumour cells

Alterations of the distal portion of the short arm of chromosome 1 (1p) are among the earliest abnormalities of human colorectal tumours. Recently, we have cloned the Aflatoxin B1 aldehyde reductase (AFAR) gene from a smallest region of overlapping deletion that is frequently (48%) hemizygously deleted in sporadic colorectal cancer. AFAR is expressed in a broad range of tissues. Its closely related rat protein is the major factor conferring resistance of rats towards aflatoxin B1-induced liver carcinogenesis. Here, we have identified cDNAs covering two additional human AFAR-related genes localized in close proximity to the previously described AFAR at 1p35-36. We have analysed their structure and tissue-related expression. One of them, AFAR3, carries a Selenocysteine-Insertion Element (SECIS)-like structure that during translation may recode an in-frame TGA-stop codon to a selenocysteine. Two additional AFAR-pseudogenes are localized at Xq25 and 1p12, respectively. AFAR exon sequences share an identity of DNA and amino acids of more than 78%. Also large blocks of intronic sequences can be up to 98.6% identical. Knowledge of the AFAR genes and their structure will be essential in genetic and functional studies, where discrimination of the genes and proteins is a prerequisite for evaluating their individual functions.     

Allelic effects of mouse Pas1 candidate genes in human lung cancer cell lines

Four of the six genes constituting the mouse Pulmonary adenoma susceptibility 1 (Pas1) locus haplotype carry amino acid variants: Lrmp, Casc1, Ghiso, and Lmna-rs1. In vitro colony formation assay of human lung cancer cell lines A549 and NCI-H520 transfected with the allelic variants of the four genes revealed allele-specific modulations of colony numbers by Lmna-rs1 and Casc1, but not by Lrmp or Ghiso. In A549 and NCI-H520 cells, the A/J allele of Lmna-rs1 produced approximately 4- and approximately 2-fold, respectively, more transfectants than did the C57BL/6J allele, whereas the A/J allele of Casc1 produced approximately 6- and approximately 5-fold fewer transfectants, respectively, as compared to the C57BL/6J allele. Inhibition of clonogenicity by allelic forms of Pas1 candidate genes was not mediated by induction of apoptosis. These findings provide evidence that allelic variants of mouse Pas1 candidate genes differentially modulate growth of human cancer cells.     

Susceptibility to aflatoxin B1-related primary hepatocellular carcinoma in mice and humans

The genetic basis of disease susceptibility can be studied by several means, including research on animal models and epidemiological investigations in humans. The two methods are infrequently used simultaneously, but their joint use may overcome the disadvantages of either method alone. We used both approaches in an attempt to understand the genetic basis of aflatoxin B(1) (AFB(1))-related susceptibility to hepatocellular carcinoma (HCC). Ingestion of AFB(1) is a major risk factor for HCC in many areas of the world where HCC is common. Whether humans vary in their ability to detoxify the active intermediate metabolite of AFB(1), AFB(1)-exo-8,9-epoxide, is not certain but may explain why all exposed individuals do not develop HCC. To determine whether human variability in detoxification may exist, in a study of 231 HCC cases and 256 controls, we genotyped eleven loci in two families of AFB(1) detoxification genes; the glutathione S-transferases (GSTs) and the epoxide hydrolases (EPHX). After adjustment for multiple comparisons, only one polymorphism in the epoxide hydrolase family 2 locus remained significantly associated with HCC (odds ratio = 2.06, 95% confidence interval = 1.13-3.12). To determine whether additional susceptibility loci exist, we developed a mouse model system to examine AFB(1)-induced HCC. Susceptibility of 7-day-old mice from two common inbred strains (C57BL/6J, DBA/2J) was assessed. DBA/2J animals were 3-fold more sensitive to AFB(1)-induced HCC and significantly more sensitive to AFB(1) acute toxicity than were C57BL/6J animals. Analysis of the xenobiotic metabolizing genes in the two strains revealed single nucleotide polymorphisms in three genes, Gsta4, Gstt1, and Ephx1. Although the GSTT1 and EPHX1 loci did not appear to be related to HCC in the total population of the human study, a polymorphism in GSTA4 was significantly related to risk in the male subset. The mouse model also demonstrated that absent or compromised p53 was not necessary for the development of carcinogenesis. These results indicate that the comparison of results from human studies and the AFB(1)-susceptible mouse model may provide new insights into hepatocarcinogenesis.     

Aflatoxin B1-induced DNA damage and its repair

Aflatoxin B(1) (AFB(1))-N(7)-guanine is the predominant adduct formed upon the reaction of AFB(1)-8,9-exo-epoxide with guanine residues in DNA. AFB(1)-N(7)-guanine can convert to the ring-opened formamidopyrimidine, or the adducted strand can undergo depurination. AFB(1)-N(7)-guanine and AFB(1)-formamidopyrimidine are thought to be predominantly repaired by nucleotide excision repair in bacteria, yeast and mammals. Although AFB(1)-formamidopyrimidine is removed less efficiently than AFB(1)-N(7)-guanine in mammals, both lesions are repaired with equal efficiencies in bacteria, reflecting differences in damage recognition between bacterial and mammalian repair systems. Furthermore, DNA repair activity and modulation of repair by AFB(1) seem to be major determinants of susceptibility to AFB(1)-induced carcinogenesis.     

Comparative genomic hybridization and BUB1B mutation analyses in childhood cancers associated with mosaic variegated aneuploidy syndrome

We previously demonstrated that constitutional BUB1B mutations cause mosaic variegated aneuploidy, a condition characterized by constitutional aneuploidies and childhood cancer predisposition. To further investigate the role of BUB1B in cancer predisposition we performed comparative genomic hybridization analysis in an embryonal rhabdomyosarcoma from an MVA case with biallelic BUB1B mutations, revealing aneuploidies typical of sporadic E-RMS, with gain of chromosomes 3, 8, 13 and loss of chromosomes 9, 14, X. To investigate whether somatic BUB1B mutations occur in sporadic childhood cancers we screened 30 Wilms tumours, 10 acute lymphoblastic leukemias, nine rhabdomyosarcomas and 11 rhabdomyosarcoma cell lines for BUB1B mutations. We identified seven exonic and six intronic variants. Six of the exonic variants were synonymous and one resulted in a non-synonymous conservative missense alteration that was also present in a control. These data suggest that the genetic progression in rhabdomyosarcoma from MVA and non-MVA cases may be similar, but that somatic BUB1B mutations are unlikely to be common in sporadic childhood cancers known to be associated with MVA.     

Differential protection against benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide-induced DNA damage in HepG2 cells stably transfected with allelic variants of pi class human glutathione S-transferase.

The pi class glutathione S-transferase (GSTP1-1), which is polymorphic in human populations, is believed to play an important role in detoxification of the ultimate carcinogen of widespread environmental pollutant benzo[a]pyrene [(+)-anti-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide [(+)-anti-BPDE]]. The allelic variants of human GSTP1-1 (hGSTP1-1) differ in their structures by the amino acids in positions 104 (isoleucine or valine) and/or 113 (valine or alanine). Here, we have determined the protective effect of overexpression of allelic variants of hGSTP1-1, through stable transfection in HepG2 cells, against (+)-anti-BPDE-induced DNA modification. Clonal transfectants of HepG2 cells corresponding to the three allelic variants of hGSTP1-1 [(I104,A113), (V104,A113), and (V104,V113), denoted hGSTP1(IA), hGSTP1(VA), and hGSTP1(VV), respectively] with similar levels of hGSTP1 protein were identified and characterized for their GST activity and (+)-anti-BPDE-induced DNA modification. The glutathione S-transferase activity toward (+)-anti-BPDE was significantly higher (approximately 3.0-3.6-fold) in cells transfected with hGSTP1(VA) [HepG2(VA)] and hGSTP1(VV) [HepG2(VV)] compared with hGSTP1(IA) transfectant [HepG2(IA)]. The formation of (+)-anti-BPDE-DNA adducts was significantly reduced in HepG2(VA) and HepG2(VV) cells compared with cells transfected with insert-free vector (HepG2-vect). Maximum protection against (+)-anti-BPDE-induced DNA damage was afforded by the hGSTP1(VV) isoform. The results of this study indicate that the allelic variants of hGSTP1-1 significantly differ in their ability to provide protection against (+)-anti-BPDE-induced DNA damage. Thus, hGSTP1-1 polymorphism may be an important factor in differential susceptibility of individuals to tumorigenesis induced by benzo[a]pyrene.     

Chemoprevention of aflatoxin B1 hepatocarcinogenesis by coumarin, a natural benzopyrone that is a potent inducer of aflatoxin B1-aldehyde reductase, the glutathione S-transferase A5 and P1 subunits, and NAD(P)H:quinone oxidoreductase in rat liver

Structurally diverse compounds can confer resistance to aflatoxin B1 (AFB1) hepatocarcinogenesis in the rat. Treatment with either phytochemicals [benzyl isothiocyanate, coumarin (CMRN), or indole-3-carbinol] or synthetic antioxidants and other drugs (butylated hydroxyanisole, diethyl maleate, ethoxyquin, beta-naphthoflavone, oltipraz, phenobarbital, or trans-stilbene oxide) has been found to increase hepatic aldo-keto reductase activity toward AFB1-dialdehyde and glutathione S-transferase (GST) activity toward AFB1-8,9-epoxide in both male and female rats. Under the conditions used, the natural benzopyrone CMRN was a major inducer of the AFB1 aldehyde reductase (AFAR) and the aflatoxin-conjugating class-alpha GST A5 subunit in rat liver, causing elevations of between 25- and 35-fold in hepatic levels of these proteins. Induction was not limited to AFAR and GSTA5: treatment with CMRN caused similar increases in the amount of the class-pi GST P1 subunit and NAD(P)H: quinone oxidoreductase in rat liver. Immunohistochemistry demonstrated that the overexpression of AFAR, GSTA5, GSTP1, and NAD(P)H:quinone oxidoreductase affected by CMRN is restricted to the centrilobular (periacinar) zone of the lobule, sometimes extending almost as far as the portal tract. This pattern of induction was also observed with ethoxyquin, oltipraz, and trans-stilbene oxide. By contrast, induction of these proteins by beta-naphthoflavone and diethyl maleate was predominantly periportal. Northern blotting showed that induction of these phase II drug-metabolizing enzymes by CMRN was accompanied by similar increases in the levels of their mRNAs. To assess the biological significance of enzyme induction by dietary CMRN, two intervention studies were performed in which the ability of the benzopyrone to inhibit either AFB1-initiated preneoplastic nodules (at 13 weeks) or AFB1-initiated liver tumors (at 50 weeks) was investigated. Animals pretreated with CMRN for 2 weeks prior to administration of AFB1, and with continued treatment during exposure to the carcinogen for a further 11 weeks, were protected completely from development of hepatic preneoplastic lesions by 13 weeks. In the longer-term dietary intervention, treatment with CMRN before and during exposure to AFB1 for a total of 24 weeks was found to significantly inhibit the number and size of tumors that subsequently developed by 50 weeks. These data suggest that consumption of a CMRN-containing diet provides substantial protection against the initiation of AFB1 hepatocarcinogenesis in the rat.     

Catalytic efficiencies of allelic variants of human glutathione S-transferase Pi in the glutathione conjugation of alpha, beta-unsaturated aldehydes

The catalytic efficiencies of the allelic variants of human glutathione (GSH) S-transferase Pi (hGSTP1-1), which differ in their primary structures by the amino acids in positions 104 (isoleucine or valine) and/or 113 (alanine or valine), in the GSH conjugation (detoxification) of acrolein and crotonaldehyde have been determined. The k(cat)/K(m) values for hGSTP1-1 isoforms I104,A113 (IA), I104, V113 (IV), V104,A113 (VA) and V104,V113 (VV) toward acrolein were 129+/-3, 116+/-3, 128+/-4 and 92+/-3 mM(-1) s(-1), respectively. The catalytic efficiencies of the hGSTP1-1 variants IA, IV, and VA in the GSH conjugation of acrolein were statistically significantly higher (at P=0.05) compared with the VV isoform. On the other hand, the catalytic efficiencies of the hGSTP1-1 isoforms IA, IV, VA and VV toward crotonaldehyde (16+/-2, 12+/-1, 17+/-2, and 12+/-2 mM(-1)s(-1), respectively) were not statistically significantly different from each other. Our results suggest that hGSTP1-1 polymorphism may be an important factor in differential susceptibility of individuals to the toxic effects of acrolein, which is a widely spread environmental pollutant and generated endogenously during metabolic activation of anticancer drug cyclophosphamide.     

Coordinate hypermethylation at specific genes in prostate carcinoma precedes LINE-1 hypomethylation

In prostate carcinoma (PCa) increased DNA methylation ('hypermethylation') occurs at specific genes such as GSTP1. Nevertheless, overall methylation can be decreased ('hypomethylation') because methylation of repetitive sequences like LINE-1 retrotransposons is diminished. We analysed DNA from 113 PCa and 36 noncancerous prostate tissues for LINE-1 hypomethylation by a sensitive Southern technique and for hypermethylation at eight loci by methylation-specific PCR. Hypermethylation frequencies for GSTP1, RARB2, RASSF1A, and APC in carcinoma tissues were each >70%, strongly correlating with each other (P<10(-6)). Hypermethylation at each locus was significantly different between tumour and normal tissues (10(-11)82% of PCas. PCa may fall into three classes, that is, with few DNA methylation changes, with frequent hypermethylation, or with additional LINE-1 hypomethylation.     

HapMap-based study of the 17q21 ERBB2 amplicon in susceptibility to breast cancer

ERBB2 is frequently amplified in breast tumours as part of a wide region of amplification on chromosome 17q21. This amplicon contains many candidate genes for breast cancer susceptibility. We used a genetic association study design to determine if common genetic variation (frequency>or=5%) in a 400-kb region surrounding ERBB2 and containing the PPARBP, CRK7, NEUROD2, PPP1R1B, STARD3, TCAP, PNMT, CAB2, ERBB2, C17ORF37, GRB7 and ZNFN1A3 genes, was associated with breast cancer risk. Sixteen tagging single-nucleotide polymorphisms (tSNPs) selected within blocks of linkage disequilibrium from the HapMap database, one HapMap singleton SNP, and six additional SNPs randomly selected from dbSNP were genotyped using Taqman in a large study set of British women (2275 cases, 2280 controls). We observed no association between any of the genotypes or associated haplotypes and disease risk. In order to simulate unidentified SNPs, we performed the leave-one-out cross-validation procedure on the HapMap data; over 90% of the common genetic variation was well represented by tagging polymorphisms. We are therefore likely to have tagged any common variants present in our population. In summary, we found no association between common genetic variation in the 17q21 ERBB2 amplicon and breast cancer risk in British women.     

Role of MC1R variants in uveal melanoma

Variants of the melanocortin-1 receptor (MC1R) gene have been linked to sun-sensitive skin types and hair colour, and may independently play a role in susceptibility to cutaneous melanoma. To assess the role of MC1R variants in uveal melanoma, we have analysed a cohort of 350 patients for the changes within the major region of the gene displaying sequence variation. Eight variants were detected - V60L, D84E, V92M, R151C, I155T, R160W, R163Q and D294H - 63% of these patients being hetero- or homozygous for at least one variant. Standard melanoma risk factor data were available on 119 of the patients. MC1R variants were significantly associated with hair colour (P=0.03) but not skin or eye colour. The frequency of the variants detected in the 350 patients was comparable with those in the general population, and comparison of the cumulative tumour distribution by age at diagnosis in carriers and noncarriers provided no evidence that MC1R variants confer an increased risk of uveal melanoma. We interpret the data as indicating that MC1R variants do not appear to be major determinants of susceptibility to uveal melanoma.     

Selected polymorphisms of DNA repair genes and risk of pancreatic cancer

BACKGROUND: Genetic variants of DNA repair genes may contribute to pancreatic carcinogenesis. O(6)-methylguanine-DNA methyltransferase (MGMT) is the major protein that removes alkylating DNA adducts, and apurinic/apyrimidinic endonuclease 1 (APE1) and X-ray repair cross-complementing group 1 (XRCC1) play important roles in the base excision repair pathway. METHODS: We investigated the association between polymorphisms of MGMT (Leu(84)Phe and Ile(143)Val), APE1 (Asp(148)Glu), and XRCC1 (Arg(194)Trp and Arg(399)Gln) and risk of pancreatic cancer in a case-control study. Exposure information from 384 patients with primary pancreatic ductal adenocarcinoma and 357 cancer-free healthy controls were collected and genomic DNAs were genotyped for five markers. Controls were frequency matched to patients by age at enrollment (+/-5 years), gender, and race. We estimated odds ratios (ORs) and 95% confidence intervals (CIs) by using unconditional logistic regression models. RESULTS: There was no significant main effect or interaction with smoking of these genetic variants on the risk of pancreatic cancer. However, the XRCC1(194) polymorphism had a significant interaction with the APE1(148) (p=0.005) or MGMT(84) polymorphism (p=0.02) in modifying the risk of pancreatic cancer. CONCLUSIONS: This study suggests that polymorphisms of genes involved in the repair of alkylating DNA adduct and DNA base damage may play a role in modulating the risk of pancreatic cancer. Larger studies are required to validate these preliminary findings. The mechanism of the combined genotype effects remains to be elucidated.     

Novel adenine adducts, N7-guanine-AFB1 adducts, and p53 mutations in patients with schistosomiasis and aflatoxin exposure

INTRODUCTION: The most frequent mutation in human hepatocellular carcinoma (HCC) in populations exposed to a high dietary intake of aflatoxin B1 (AFB1) is a mutation in codon 249 of the p53 gene. Schistosomiasis is known to cause p53 mutation. We hypothesized that the combination of schistosomiasis and aflatoxin B1 increases the incidence of p53 gene mutation. METHODS: Liver tissue from 21 patients with schistosomiasis and 5 patients without schistosomiasis were analyzed for occurrence of mutations of the p53 gene and levels of N7-guanine-AFB1 adducts. RESULTS: The presence of mutations in codon 249 of p53 gene was higher in patients infected with Schistosoma haematobium (S. haematobium) than in those infected with Schistosoma mansoni (S. mansoni) or a combination of both strains (p < 0.01), compared to control subjects. No mutations were detected in p53 gene in liver DNA from schistosomiasis-free patients. Significant amounts of N7-guanine-AFB1 adducts and novel adenine-adducts (p < 0.01) were detected in patients with schistosomiasis, mostly in patients infected with S. haematobium or a combination of both strains, compared to control subjects. Conclusion: These data suggest that schistosomiasis and exposure to aflatoxin B1 act synergistically to increase the incidence of p53 gene mutation. The increase in p53 mutations may enhance progression of HCC at an early age in patients with schistosomiasis.     

Single-nucleotide polymorphisms in the RB1 gene and association with breast cancer in the British population

A substantial proportion of the familial risk of breast cancer may be attributable to genetic variants each contributing a small effect. pRb controls the cell cycle and polymorphisms within it are candidates for such low penetrance susceptibility alleles, since the gene has been implicated in several human tumours, particularly breast cancer. The purpose of this study was to determine whether common variants in the RB1 gene are associated with breast cancer risk. We assessed 15 tagging single-nucleotide polymorphisms (SNPs) using a case-control study design (n< or = 4474 cases and n < or = 4560 controls). A difference in genotype frequencies was found between cases and controls for rs2854344 in intron 17 (P-trend = 0.007) and rs198580 in intron 19 (P-trend = 0.018). Carrying the minor allele of these SNPs appears to confer a protective effect on breast cancer risk (odd ratio (OR) = 0.86 (0.76-0.96) for rs2854344 and OR = 0.80 (0.66-0.96) for rs198580). However, after adjusting for multiple testing these associations were borderline with an adjusted P-trend = 0.068 for the most significant SNP (rs2854344). The RB1 gene is not known to contain any coding SNPs with allele frequencies > or = 5% but several intronic variants are in perfect linkage disequilibrium with the associated SNPs. Replication studies are needed to confirm the associations with breast cancer.     

Genetic variations in benzene metabolism and susceptibility to multiple myeloma

We have previously shown that deficiency in the biotransformation enzyme glutathione-S-transferase theta (GSTT1) is a risk factor for multiple myeloma (MM). The present case-control study of 102 MM patients and 205 controls revealed a significant trend in increasing risk of MM with inheritance of multiple putative 'high risk' genetic variants in related pathways of benzene detoxification. Individuals who carried polymorphisms for GSTT1 null and/or high activity microsomal epoxide hydrolase (mEH 113YY+139HR or 113YY+139RR or 113YH+139RR) and/or low activity NAD(P)H:quinone oxidoreductase 1 (NQO1 187PS/SS) were 1.65, 2.49 and 13 times more likely to have MM (P(trend)=0.001).     

Efficient activation of aflatoxin B1 by cytochrome P450 2A13, an enzyme predominantly expressed in human respiratory tract

The worldwide human exposure to aflatoxin B1 (AFB1), particularly in developing countries, remains to be a serious public health concern. Although AFB1 is best known as a hepatocarcinogen, epidemiological studies have shown a positive association between human lung cancer occurrence and inhalation exposure to AFB1. Cytochrome P450 (CYP)-catalyzed metabolic activation is required for AFB1 to exert its carcinogenicity. Previous studies have identified CYP1A2 and CYP3A4 as the major enzymes for AFB1 activation in human liver. However, the key CYP enzymes in human lung that can efficiently activate AFB1 in situ are unknown. In the present study, we demonstrate that CYP2A13, an enzyme predominantly expressed in human respiratory tract, has a significant activity in metabolizing AFB1 to its carcinogenic/toxic AFB1-8,9-epoxide and AFM1-8,9-epoxide at both low (15 microM) and high (150 microM) substrate concentrations. Under the same conditions, there was no detectable AFB1 epoxide formation by CYP2A6, which was also reported to be involved in the metabolic activation of AFB1. Consistent with the activity data, there was an approximately 800-fold difference in LC50 values of AFB1 (48-hr treatment) between Chinese hamster ovary (CHO) cells expressing CYP2A13 and CYP2A6 (50 nM versus 39 microM). We further demonstrate that amino acid residues Ala117 and His372 in CYP2A13 protein are important for AFB1 epoxidation and its related cytotoxicity. Our results suggest that CYP2A13-catalyzed metabolic activation in situ may play a critical role in human lung carcinogenesis related to inhalation exposure to AFB1.     

Quantitative analysis of aromatase, sulfatase and 17beta-HSD(1) mRNA expression in soft tissue metastases of breast cancer

Expression of the estrogen-synthesizing genes aromatase, steroid sulfatase (STS) and 17beta-hydroxysteroid dehydrogenase type1 (17beta-HSD(1)) has been shown to be up-regulated in primary breast cancer tissue but their expression status in metastatic tumor tissue has yet to be determined. The mRNA expression levels of the three estrogen-synthesizing genes as well as of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6 and cyclooxygenase (COX)-2, all of which have been reported to up-regulate the estrogen-synthesizing genes, were determined by means of a real-time PCR assay in 100 primary breast cancer tissues and 15 soft tissue metastases. In addition, PCR-gel electrophoresis was used to determine the proportion (%) of promoter (l.4, l.3, Pll and l.7) usage of aromatase. Aromatase and STS mRNA levels were significantly (P=0.04 and P=0.03, respectively) higher in soft tissue metastases than in primary tumors, while 17beta-HSD(1) mRNA levels tended (P=0.09) to be higher. The proportions of the promoter usages were very similar for primary tumors and soft tissue metastases, and the mRNA levels of TNF-alpha, IL-6 and COX-2 were not significantly different. Levels of aromatase, STS and 17beta-HSD(1) mRNA are up-regulated in soft tissue metastases compared to those in primary tumors, suggesting that intra-tumoral estrogen synthesis may play a significant role in the growth stimulation of tumor cells in soft tissue metastases as in primary tumors. TNF-alpha, IL-6 and COX-2, on the other hand, are unlikely to be implicated in this up-regulation.