Molecular profile of 5-fluorouracil pathway genes in colorectal carcinoma.

BACKGROUND: This study addresses involvement of major 5-fluorouracil (5-FU) pathway genes in the prognosis of colorectal carcinoma patients. METHODS: Testing set and two validation sets comprising paired tumor and adjacent mucosa tissue samples from 151 patients were used for transcript profiling of 15 5-FU pathway genes by quantitative real-time PCR and DNA methylation profiling by high resolution melting analysis. Intratumoral molecular profiles were correlated with clinical data of patients. Protein levels of two most relevant candidate markers were assessed by immunoblotting. RESULTS: Downregulation of DPYD and upregulation of PPAT, UMPS, RRM2, and SLC29A1 transcripts were found in tumors compared to adjacent mucosa in testing and validation sets of patients. Low RRM2 transcript level significantly associated with poor response to the first-line palliative 5-FU-based chemotherapy in the testing set and with poor disease-free interval of patients in the validation set irrespective of 5-FU treatment. UPP2 was strongly methylated while its transcript absent in both tumors and adjacent mucosa. DPYS methylation level was significantly higher in tumor tissues compared to adjacent mucosa samples. Low intratumoral level of UPB1 methylation was prognostic for poor disease-free interval of the patients (P = 0.0002). The rest of the studied 5-FU genes were not methylated in tumors or adjacent mucosa. CONCLUSIONS: The observed overexpression of several 5-FU activating genes and DPYD downregulation deduce that chemotherapy naïve colorectal tumors share favorable gene expression profile for 5-FU therapy. Low RRM2 transcript and UPB1 methylation levels present separate poor prognosis factors for colorectal carcinoma patients and should be further investigated.

Polymorphisms in metabolizing enzymes and the risk of head and neck squamous cell carcinoma in the Slavic population of the central Europe.

The question of susceptibility to squamous cell carcinoma of head and neck (SCCHN) in the environmental context was addressed by analysis of functional polymorphisms in enzymes metabolizing smoke constituents and/or alcohol (CYP2A13, CYP1B1, EPHX1, NQO1, GSTM1, GSTP1, GSTT1, ADH1B and ADH1C). Case-control study of 122 age- and sex-matched pairs of subjects was performed using so far unexplored Central European Slavic population with high level of tobacco and alcohol abuse. Age-, gender-, smoking- and alcohol-adjusted logistic regression failed to demonstrate any significant association of the analyzed polymorphisms with the SCCHN risk. When interactions between potential modifiers of effect, i.e. smoking and alcohol were tested, drinkers seemed to be at lower risk than nondrinkers when carrying the heterozygous genotype Ile/Val in codon 432 of CYP1B1 (OR=0.42; 95% CI=0.21-0.83; p=0.013 vs. OR=1.02; 95% CI=0.34-2.94; p=0.977). Similarly, drinkers were at lower risk than nondrinkers when carrying the heterozygous genotype Pro/Ser in codon 187 of NQO1 (OR=0.41; 95% CI=0.19-0.88; p=0.022 vs. OR=0.96; 95% CI=0.29-3.12; p=0.948). More interestingly, drinkers carrying the rare homozygous genotype Val/Val in codon 350 of ADH1C were at significantly higher risk than nondrinkers carrying this genotype (OR=4.01; 95% CI=1.61-10.01; p=0.003 vs. OR=0.93; 95% CI=0.25-3.57; p=0.919). This result confirmed findings of previously published studies. Smoking did not significantly modify the effect of genotypes. Our data thus demonstrate that genetic susceptibility to SCCHN shall be further followed on populations with different genetic background and lifestyle.

The association between glutathione S-transferase gene polymorphisms and pancreatic cancer in a central European Slavonic population.

In the first case-control study on pancreatic cancer conducted on 253 cases and 403 controls in the Czech Republic we observed that the GSTP1-codon 105 Val variant allele and the GSTT1-null genotype were associated with an elevated risk for pancreatic cancer (OR = 1.38; 95%CI = 0.96-1.97 and OR = 1.56; 95%CI = 0.93-2.61, respectively). Combination of GSTT1-null and GSTP1-codon 105 Val variants further increased the risk for pancreatic cancer (OR = 2.50; 95%CI = 1.20-5.20). In conclusion, this study suggests population-specific associations of polymorphisms in key biotransformation genes with elevated risk for pancreatic cancer.

Relationship between the capacity to repair 8-oxoguanine, biomarkers of genotoxicity and individual susceptibility in styrene-exposed workers.

Genotoxic effects related to exposure to styrene have been a matter of investigation for many years by employing markers of exposure, effect and susceptibility. The role of individual DNA-repair capacity in response to exposure to styrene may explain the controversial results so far obtained, but it is still scarcely explored. In the present study, we measured capacity to repair oxidative DNA damage in cell extracts obtained from 24 lamination workers occupationally exposed to styrene and 15 unexposed controls. The capacity to repair oxidative DNA damage was determined by use of a modified comet assay, as follows: HeLa cells, pre-treated with photosensitizer and irradiated with a halogen lamp in order to induce 7,8-dihydroxy-8-oxoguanine, were incubated with cell extracts from mononuclear leukocytes of each subject. The level of strand breaks reflects the removal of 7,8-dihydroxy-8-oxoguanine from substrate DNA by the enzymatic extract. In styrene-exposed subjects a moderate, non-significant increase in oxidative DNA repair was observed. Stratification for sex and smoking habit showed that unexposed males (P=0.010) and unexposed smokers (P=0.037) exhibited higher DNA-repair rates. The repair capacity did not correlate with parameters of styrene exposure and biomarkers of genotoxic effects (DNA strand breaks, N1-styrene-adenine DNA adducts, chromosomal aberrations and mutant frequencies at the HPRT locus). Significantly higher levels of DNA-repair capacity were observed in carriers of GSTM1-plus, compared to those with a deletion in GSTM1. The DNA-repair capacity was significantly lower in individuals with variant Gln/Gln genotype in XRCC1 Arg399Gln than in those with heterozygous Arg/Gln and wild-type Arg/Arg genotypes. Significantly lower repair capacity was also found in individuals with the wild-type Lys/Lys genotype in XPC Lys939Gln as compared with those homozygous for the Gln/Gln variant genotype.

Protein expression of ATP-binding cassette transporters ABCC10 and ABCC11 associates with survival of colorectal cancer patients.

PURPOSE: This study investigated the prognostic importance of protein expression of ATP-binding cassette (ABC) transporters ABCC10 and ABCC11 in colorectal cancer. METHODS: Protein content of ABCC10 and ABCC11 was assessed in tumor tissue blocks of 140 colorectal cancer patients and associated with survival of patients with regard to 5-fluorouracil-based therapy. RESULTS: Low ABCC10 protein content in tumors increased hazard ratio of patient's death more than three times in comparison with high ABCC10-expressing tumors (P = 0.004). In contrast, the low ABCC11 content increased the hazard ratio of cancer recurrence in patients almost four times (P = 0.016). Analysis of patients treated with regimens based on 5-fluorouracil revealed that patients with low ABCC11 content in their tumors had shorter disease-free interval than those with higher content (P = 0.024). CONCLUSIONS: The present study shows for the first time that the protein expression of ABCC10 significantly associates with overall survival and the expression of ABCC11 with disease-free interval of colorectal cancer patients and provides strong impulse for further validation of their prognostic value in colorectal cancer.

Genetic polymorphisms of biotransformation enzymes: allele frequencies in the population of the Czech Republic.

Polymerase chain reaction-restriction fragment length polymorphism based genotyping assays were used to determine the frequency of polymorphisms in CYP1A1 (3'-flanking region), CYP2E1 (5'-flanking region and intron 6), EPHX (exon 3 and exon 4), GSTM1 (deletion), GSTP1 (exon 5) and GSTT1 (deletion) in a group of 416 Czech individuals. A comprehensive overview of the methodology is also presented. We have found the following frequencies of mutated alleles: CYP1A1-m2, 0.097; CYP2E1-C, 0.077; CYP2E1-c2, 0.023; EPHX(exon 3)-His, 0.381; EPHX(exon 4)-Arg, 0.198; GSTM1-null, 0.51; GSTP1-Val, 0.3; GSTT1-null, 0.164. These values are similar to those presented in the majority of studies on European Caucasians, although a few cases of significant differences in the distribution of genotypes were found. These differences were most probably caused by methodological variations or statistical bias in the analyses of low numbers of samples in the control groups of some authors. Based on the results of EPHX genotyping, the activity of its protein product was deduced and the Czech population was divided into three subgroups with low, medium and high EPHX activity. We found that 43% of the Czech population would fall into the low, 44% into the medium and 13% into the high EPHX activity group. The data obtained may prove to be very useful for epidemiological studies on the influence of genetic polymorphisms of biotransformation enzymes on carcinogenesis or other environment-related diseases.

Comparison of in vitro activities of biotransformation enzymes in pig, cattle, goat and sheep.

In vitro activities of cytochromes P450 (7-alkyl/aryloxyresorufin dealkyl(aryl)ases, testosterone hydroxylase/oxidase, 6-chlorzoxazone hydroxylase, 7-methoxy-4-trifluoromethyl-coumarin demethylase, and lauric acid hydroxylases), reductases of carbonyl group (toward metyrapone, daunorubicin, glyceraldehyde, and 4-pyridine-carboxaldehyde) and conjugation enzymes (p-nitrophenol-UDP-glucuronosyl transferase, 1-chloro-2,4-dinitrobenzene glutathione-S-tranferase) in young adults, males, non-castrated (N=6) farm animals were studied and compared. Presence of proteins cross-reacting with anti-human CYP3A4, CYP2C9, and CYP2E1 IgG was detected in all farm species. Bovine microsomes differed from other microsomes of farm species in very high 7-ethoxyresorufin-O-deethylase activity (CYP1A1/2). Significantly higher 7-methoxy-4-trifluoromethyl-coumarin demethylase (2-3 times) and 12-lauric acid hydroxylases (4-10 times) activities (probably corresponding to CYP2C and CYP4A, respectively) were found in ovine microsomes. The highest 6beta-testosterone hydroxylase activity, which is usually considered to be a CYP3A activity marker, was found in pig. Reductases of all farm animals display considerable ability to reduce carbonyl group of xenobiotics. Significant differences in level and activity of many biotransformation enzymes tested suggest that extrapolation of pharmacokinetic data obtained in one species to another (even related) could be misleading.