Roberta Pastorelli

CYP1A1 T3801 C polymorphism and lung cancer: A pooled analysis of 2,451 cases and 3,358 controls

CYP1A1 is involved in the metabolism of benzopyrene, a suspected lung carcinogen; it is therefore conceivable that genetically determined variations in its activity modify individual susceptibility to lung cancer. The role of the CYP1A1 MspI polymorphism in lung cancer has been widely studied but has not been fully clarified. We have included 2,451 cases and 3,358 controls in a pooled analysis of 22 case‐control studies on CYP1A1 and lung cancer risk. We found a clear association between the CYP1A1 homozygous MspI restriction fragment length polymorphism (RFLP) and lung cancer risk in Caucasians (age‐ and gender‐adjusted odds ratio = 2.36; 95% confidence interval 1.16–4.81); other associations were weaker or not statistically significant. The association with the homozygous variant was equally strong for squamous cell carcinomas and adenocarcinomas among Caucasians. We analyzed the risk by duration of smoking: for Caucasian subjects with the MspI RFLP combined variants (homozygotes plus heterozygotes), the increase in the risk of lung cancer was steeper than among the individuals with the homozygous reference allele. Our analysis suggests that Caucasians with homozygous variant CYP1A1 polymorphism have a higher risk of lung cancer. The data were more consistent among Caucasians, with a strong association between the homozygous variant in both squamous cell carcinomas and adenocarcinomas, and a stronger association in men than in women. The analyses were more inconsistent and failed to reach statistical significance in Asians. This observation might be due to design specificities or unknown effect modifiers in the Asian studies.

Effect of dna repair gene polymorphisms on BPDE-DNA adducts in human lymphocytes

To determine whether variations in DNA repair genes are related to host DNA damage, we investigated the association between polymorphism in the XPD gene (codon 199, 312, 751) and the XRCC1 gene (codon 194, 399) and the presence of benzo(a)pyrene diolepoxide adducts to lymphocyte DNA (BPDE‐DNA) in a group of male patients with incident lung cancer, all current smokers. BPDE‐DNA adducts were analyzed by high‐resolution gas chromatography‐negative ion chemical ionization‐mass spectrometry. XPD and XRCC1 genotypes were identified by PCR‐RFLP. XRCC1 and XPD genotypes did not affect the levels and proportion of detectable BPDE‐DNA adducts. The patients were also genotyped for the GSTM1 polymorphism, given its role in the detoxification of BPDE. Individuals with the GSTM1 deletion had significantly higher levels of BPDE‐DNA adducts when they were XPD‐Asp312Asp+Lys751Lys than carriers of at least one variant allele. No such association was found with the XRCC1 genotypes. Because of the small study population (n = 60), further statistical analysis of possible gene‐gene and gene‐environment would not be informative. This is the first study analysing the specific BPDE‐DNA adduct in vivo with regard to polymorphic repair genes (XPD, XRCC1) and xenobiotic metabolizing gene (GSTM1). Our results raise the possibility that the XPD‐Asp312Asp+Lys751Lys genotype may increase BPDE‐DNA damage; this effect might be evident in individuals who are especially likely to have accumulated damage, probably because of lower detoxification capacity and high environmental exposure.

Genetic polymorphisms and modulation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-DNA adducts in human lymphocytes.

2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP) is the most abundant heterocyclic amine derived from food, possibly involved in human carcinogenesis. We evaluated the formation of PhIP‐DNA adducts in lymphocytes from 76 incident colorectal cancer patients likely to be exposed to dietary PhIP. To address the role of the metabolic polymorphisms relevant to PhIP‐DNA adduct formation, the patients were genotyped for common polymorphisms in the N‐acetyltransferase (NAT1 and NAT2), sulfotransferase (SULT1A1) and glutathione S‐transferase (GSTM1 and GSTA1) genes. PhIP released from adducted DNA after hydrolysis was quantitated by liquid chromatography‐tandem mass spectrometry. Overall, adducts were 3.24 ± 3.58/108 nucleotides (mean ± SD); they were not related to sex, smoking habits or age, though levels were not significantly higher in smokers, young subjects and high meat consumers. High vegetable intake significantly reduced PhIP‐DNA adducts (Mann‐Whitney U, p = 0.044). Individuals with the GSTM1 null genotype showed colon cancer onset at earlier age (58.8 ± 1.8 vs. 63.5 ± 1.6 years; Mann‐Whitney U, p = 0.047). None of the genetic polymorphisms studied significantly affected PhIP‐DNA adducts. However, individuals carrying 2 mutated GSTA1 alleles and younger than the median age had higher adduct levels than homozygous wild‐type and heterozygous ones (Kruskal‐Wallis p = 0.0008). In conclusion, these preliminary data indicate that PhIP‐DNA adducts are formed in people likely to be exposed to this carcinogen through the diet, suggesting this biomarker may be useful to detect human exposure and DNA damage. Overall, the genetic polymorphisms considered had limited effect on PhIP‐DNA levels, but young people with lower detoxification capacity may form a subgroup particularly susceptible to dietary carcinogen.

Effects of cigarette smoking on the human urinary proteome

In this pilot study we used a proteomic approach to compare the urinary protein patterns of healthy smokers and non-smokers. Proteins were resolved by two-dimensional gel electrophoresis and identified by mass spectrometry. The relative abundance of three inflammatory proteins (S100A8, inter-alpha-trypsin inhibitor heavy chain 4, CD59) and that of two isoforms of pancreatic alpha amylase was significantly higher in smokers. Zinc-alpha-2-glycoprotein was the only protein down-regulated in smokers. Its abundance was significantly correlated with urinary glucocorticoids. Most of the proteins identified may be non-specific biomarkers of tobacco effects, since they are involved in inflammatory responses associated with several diseases. Of greater interest are the changes in abundance of pancreatic alpha amylase and zinc-alpha-2-glycoprotein, which after proper validation, might be candidate biomarkers of diseases resulting from exposure to tobacco smoke. The data also show for the first time that smoking can affect the expression profile of urinary proteins.

Immunomodulatory effects of occupational exposure to mancozeb.

The effects of occupational exposure to ethylene-bis-dithiocarbamate of manganese and zinc on the immune system were evaluated in a group of mancozeb-exposed manufacturers and controls. The immune system tests revealed the following: (a) lymphocyte proliferative responses triggered by different activators and mitogen-induced IL-2 production were higher in exposed subjects than in controls; (b) production of monocyte/macrophage-derived IL-1 and polyclonal IgG and IgM, by beta-lymphocytes, did not differ between exposed subjects and controls; (c) percentages and absolute numbers of total T-cells, T-helper cells, T-suppressor/cytotoxic cells, activated T-cells, total beta-cells, and natural killer cells were similar in exposed subjects and controls; (d) serum immunoglobulin classes and complement fractions were within the range of normality; and (e) rheumatoid factor and non-organ-specific serum autoantibodies were absent in exposed and control subjects. An increase in T-cell functional response was found in the exposed group, suggesting a slight immunomodulator effect of mancozeb in conditions of low-level, prolonged occupational exposure.

Benzo(a)pyrene diolepoxide adducts to albumin in workers exposed to polycyclic aromatic hydrocarbons: association with specific CYP1A1, GSTM1, GSTP1 and EHPX genotypes

We investigated whether the presence of (+)-anti-benzo(a)pyrene diolepoxide adducts to serum albumin (BPDE-SA) among workers exposed to benzo(a)pyrene (BaP) and unexposed reference controls was influenced by genetic polymorphisms of cytochrome P4501A1 (CYP1A1), microsomal epoxide hydrolase (EHPX), glutathione S-transferases M1 (GSTM1) and P1 (GSTP1), all involved in BaP metabolism. Exposed workers had significantly higher levels of adducts (0.124 ± 0.02 fmol BPTmg(-1) SA, mean ± SE) and a higher proportion of detectable adducts (40.3%) than controls (0.051 ± 0.01 fmol BPT mg(-1) SA; 16.1%) (p = 0:014 and p = 0:012). Smoking increased adduct levels only in occupationally exposed workers with the GSTM1 deletion (GSTM1 null) (p = 0:034). Smokers from the exposed group had higher adduct levels when they were CYP1A1 *1/*1 wild-type rather than heterozygous and homozygous for the variant alleles (CYP1A1 *1/*2 plus *2/*2) (p = 0:01). The dependence of BPDE-SA adduct levels and frequency on the CYP1A1 *1/*1 genotype was most pronounced in GSTM1-deficient smokers. Exposed workers with GSTM1 null/GSTP1 variant alleles had fewer detectable adducts than those with the GSTM1 null/GSTP1*A wild-type allele, supporting for the first time the recent in vitro finding that GSTP1 variants may be more effective in the detoxification of BPDE than the wild-type allele. Logistic regression analysis indicated that occupational exposure, wild-type CYP1A1*1/*1 allele and the combination of GSTM1 null genotype+EHPX genotypes associated with predicted low enzyme activity were significant predictors of BPDE-SA adducts. Though our findings should be viewed with caution because of the relatively limited size of the population analysed, the interaction between these polymorphic enzymes and BPDE-SA adducts seems to be specific for high exposure and might have an impact on the quantitative risk estimates for exposure to polycyclic aromatic hydrocarbons.We investigated whether the presence of (+)-anti-benzo(a)pyrene diolepoxide adducts to serum albumin (BPDE-SA) among workers exposed to benzo(a)pyrene (BaP) and unexposed reference controls was influenced by genetic polymorphisms of cytochrome P4501A1 (CYP1A1), microsomal epoxide hydrolase (EHPX), glutathione S-transferases M1 (GSTM1) and P1 (GSTP1), all involved in BaP metabolism. Exposed workers had significantly higher levels of adducts (0.124 ± 0.02 fmol BPTmg−1 SA, mean ± SE) and a higher proportion of detectable adducts (40.3%) than controls (0.051 ± 0.01 fmol BPT mg−1 SA; 16.1%) (p = 0:014 and p = 0:012). Smoking increased adduct levels only in occupationally exposed workers with the GSTM1 deletion (GSTM1 null) (p = 0:034). Smokers from the exposed group had higher adduct levels when they were CYP1A1 *1/*1 wild-type rather than heterozygous and homozygous for the variant alleles (CYP1A1 *1/*2 plus *2/*2) (p = 0:01). The dependence of BPDE-SA adduct levels and frequency on the CYP1A1 *1/*1 genotype was most pronounced in GSTM1-deficient smokers. Exposed workers with GSTM1 null/GSTP1 variant alleles had fewer detectable adducts than those with the GSTM1 null/GSTP1*A wild-type allele, supporting for the first time the recent in vitro finding that GSTP1 variants may be more effective in the detoxification of BPDE than the wild-type allele. Logistic regression analysis indicated that occupational exposure, wild-type CYP1A1*1/*1 allele and the combination of GSTM1 null genotype+EHPX genotypes associated with predicted low enzyme activity were significant predictors of BPDE-SA adducts. Though our findings should be viewed with caution because of the relatively limited size of the population analysed, the interaction between these polymorphic enzymes and BPDE-SA adducts seems to be specific for high exposure and might have an impact on the quantitative risk estimates for exposure to polycyclic aromatic hydrocarbons.

CARCINOGEN-DNA ADDUCTS AS TOOLS IN RISK ASSESSMENT

Genotoxic chemicals are known to react with DNA either directly or after metabolic activation to form adducts, a step thought to be relevant with respect to chemical carcinogenesis. Evaluation of cancer risk due to exposure to chemicals requires information about the internal dose which depends on individual variation in rates of metabolic activation and detoxification. The presence and the amount of specific DNA adducts provide a good indication of chemical exposure and genetic damage resulting from exposure to carcinogens and account for some of the factors affecting individual susceptibility to cancer. Analysis of DNA adducts requires that the sensitivity of the methods be sufficiently high to allow the detection of about 1 adduct/109 normal nucleotides. Most suitable methods are based on 32P-postlabelling, immunoassays or physico-chemical techniques such as HPLC coupled to synchronous fluorescence spectroscopy or gas chromatography-mass spectrometry. These methods have been used to assess human exposure to a variety of chemical carcinogens including polycyclic aromatic hydrocarbons, aromatic amines, heterocyclic aromatic amines or aflatoxins. In some instances, the use of DNA-adducts has given accurate estimates of risk.