The influence of polymorphisms of xenobiotic-metabolizing and DNA repair genes in DNA damage, telomere length and global DNA methylation evaluated in open-cast coal mining workers.

Coal plants represent one of the main sources of environmental pollution due to the combustion process of this mineral and the consequent release of gases and particles which, in significant quantities, can lead to a potential risk to health and the environment. The susceptibility of individuals to the genotoxic effects of coal mining can be modulated by genetic variations in the xenobiotic detoxification and DNA repair processes. The aim of this study was to evaluate if xenobiotic metabolism polymorphism, base excision repair polymorphisms and non-homologous end joining repair polymorphism, could modify individual susceptibility to genomic instability and epigenetic alterations induced in workers by occupational exposure to coal. In this study, polymerase chain reaction was used to examine the polymorphic sites. The sample population comprising 70 coal mine workers and 71 workers non-exposed to coal. Our results demonstrated the effect of individual genotypes on different biomarkers evaluated. Significant decrease in % of global DNA methylation were observed in CYP1A1 Val/- exposed individuals compared to CYP1A1 Ile/Ile individuals. Coal workers who carried the XRCC4 Ile/Ile genotype showed decrease NBUD frequencies, while the XRCC4 Thr/- genotype was associated with decrease in Buccal micronucleus cells for the group not exposed. No influence of GSTM1 null, GSTT1 null, GSTP1 Ile105Val, hOGG1 Ser326Cys, XRCC1 Arg194Trp polymorphisms was observed. Thus, the current study reinforces the importance of considering the effect of metabolizing and repair variant genotypes on the individual susceptibility to incorporate DNA damage, as these processes act in a coordinated manner to determine the final response to coal exposure.

Shorter telomere length and DNA hypermethylation in peripheral blood cells of coal workers.

Coal is a mixture of several chemicals, mainly inorganic elements and polycyclic aromatic hydrocarbons, many of which have mutagenic and carcinogenic effects. Pneumoconiosis, fibrosis, asbestosis, silicosis, emphysema, loss of lung function and cancer are some examples of coal-related disorders. The aim of this study was to analyze coal miners with respect to telomere length (TL) and percentage (%) of global DNA methylation. The study involved 82 participants divided into two groups: 55 workers exposed to coal and 27 non-exposed individuals. DNA was isolated from peripheral blood samples from all individuals. Telomeres were measured by quantitative real time polymerase chain reaction (qPCR) and global DNA methylation levels were performed by the relative quantitation of 5-methyl-2'-deoxycytidine (5-mdC) by high-performance liquid chromatography (HPLC). TL measurements showed a mean of 9,199 bp (±4,196) for non-exposed and 7,545 bp (±2,703) for exposed groups, and% of global DNA methylation a mean of 2.78% (±0.41) for non-exposed and 3.00% (±0.37) for exposed individuals. Occupationally exposed individuals showed a significant decrease of TL (P < 0.05; Mann-Whitney test) and increase in the percentage of global DNA methylation (P < 0.05; Mann-Whitney test) when compared to the non-exposed group. This study showed that occupational exposure to coal and products of combustion is positively associated with TL and DNA methylation. Previously, we have evaluated the same individuals using comet assay, micronucleus (MN) test, oxidative stress and inorganic elements. No correlations were observed between TL and methylation with previous data in the exposed group. Further studies are needed to determine whether these alterations are associated with induced disease outcomes and if these events could be determinants to identify cancer risk.

Base excision repair (OGG1 and XRCC1) and metabolism (PON1) gene polymorphisms act on modulation of DNA damage and immune parameters in tobacco farmers.

Pesticides are one of the most frequently investigated chemical, due to their multiple uses in agricultural and public health areas. This study evaluates lymphocytes CBMN (cytokinesis-block micronucleus cytome assay), inflammatory markers, inorganic elements in blood samples, and the relationship of these parameters with XRCC1Arg194Trp, OGG1Ser326Cys and PON1Gln192Arg polymorphisms in a population of tobacco farmers. The study population comprised 129 agricultural workers exposed to pesticides and 91 nonexposed. Farmers had significantly increased NPB (nuclear plasmatic bridge), MN (micronucleus) and NBUD (nuclear bud) frequencies, as well as IL-6 (interleukin 6) and TNF-α (tumor necrosis factor alpha) serum levels, and decreased cytokines CD4+/CD8+ ratio. In the exposed group, XRCC1 Trp/- was correlated with decreased NDI (nuclear division index), and OGG1 Cys/- was associated with higher levels of NPB and decreased levels of IL-6. The combined effects of PON1 Arg/- and XRCC1 Arg/Arg were associated with increased NPB frequencies. In addition, the combination of PON1 Arg/- with XRCC1 Trp/- or OGG1 Cys/- influenced in increased levels of necrosis in farmers. Furthermore, tobacco farmers showed a positive correlation between TNF-α levels and NPB, CD4+/CD8+ ratio and NBUD; and IL-6 levels with both MN and NDI. The duration of years of work at tobacco fields was correlated positively with NBUD frequency. Sulfur, chlorine and potassium were found at increased levels in the exposed group when compared to the nonexposed one. These findings provide evidence that tobacco farmers' exposure have increased DNA damage and alter the immune system's response, and that XRCC1 and OGG1 polymorphisms could influence both biomarkers results.

Influence of vitamin intake and MTHFR polymorphism on the levels of DNA damage in tobacco farmers.

BACKGROUND: Genetic damage may occur spontaneously under normal metabolic circumstances, inadequate intake of nutrients, and excessive exposure to environmental mutagens. OBJECTIVES: To evaluate the influence of the intake of micronutrients vitamin B12, vitamin B6, and folate and of the polymorphism methylenetetrahydrofolate reductase (MTHFR) C677T on the induction of DNA damage in tobacco farmers. METHODS: The study involved 66 men and 44 women engaged in tobacco cultivation in the region of Venâncio Aires (Rio Grande do Sul state, Brazil). Peripheral blood samples were collected to analyze DNA damage using the Comet assay, the micronucleus (MN) test and MTHFR C677T polymorphism. Dietary intake was evaluated based on the mean values obtained from three 24-h diet recall questionnaires, and nutrient intake data were computerized and estimated in the Food Processor SQL 10.9 program. The statistical tests used to generate the stated results were Kruskal-Wallis test, Exact Fisher's test, and multivariate linear regression analysis. RESULTS: DNA damage was significantly higher in individuals who had an inadequate intake of folate, vitamin B12, and vitamin B6 (P < 0.01) assessed by Comet assay. In relation to MN test results, buccal cells showed MN frequency higher in individuals with inadequate intake of vitamin B6 (P < 0.01). No difference was observed in MN lymphocytes frequency. No significant association was detected between MTHFR C677T polymorphism and DNA damage in tobacco farmers. CONCLUSION: Our results suggest that folate, vitamin B12, and vitamin B6 deficiency may be associated with genotoxic effect in individuals exposed to pesticides.

Polymorphisms in metabolism and repair genes affects DNA damage caused by open-cast coal mining exposure.

Increasing evidence suggest that occupational exposure to open-cast coal mining residues like dust particles, heavy metals and Polycyclic Aromatic Hydrocarbons (PAHs) may cause a wide range of DNA damage and genomic instability that could be associated to initial steps in cancer development and other work-related diseases. The aim of our study was to evaluate if key polymorphisms in metabolism genes CYP1A1Msp1, GSTM1Null, GSTT1Null and DNA repair genes XRCC1Arg194Trp and hOGG1Ser326Cys could modify individual susceptibility to adverse coal exposure effects, considering the DNA damage (Comet assay) and micronucleus formation in lymphocytes (CBMN) and buccal mucosa cells (BMNCyt) as endpoints for genotoxicity. The study population is comprised of 200 healthy male subjects, 100 open-cast coal-mining workers from "El Cerrejón" (world's largest open-cast coal mine located in Guajira - Colombia) and 100 non-exposed referents from general population. The data revealed a significant increase of CBMN frequency in peripheral lymphocytes of occupationally exposed workers carrying the wild-type variant of GSTT1 (+) gene. Exposed subjects carrying GSTT1null polymorphism showed a lower micronucleus frequency compared with their positive counterparts (FR: 0.83; P=0.04), while BMNCyt, frequency and Comet assay parameters in lymphocytes: Damage Index (DI) and percentage of DNA in the tail (Tail % DNA) were significantly higher in exposed workers with the GSTM1Null polymorphism. Other exfoliated buccal mucosa abnormalities related to cell death (Karyorrhexis and Karyolysis) were increased in GSTT/M1Null carriers. Nuclear buds were significantly higher in workers carrying the CYP1A1Msp1 (m1/m2, m2/m2) allele. Moreover, BMNCyt frequency and Comet assay parameters were significantly lower in exposed carriers of XRCC1Arg194Trp (Arg/Trp, Trp/Trp) and hOGG1Ser326Cys (Ser/Cys, Cys/Cys), thereby providing new data to the increasing evidence about the protective role of these polymorphisms. This modulation may involve specific and differentiated pathways in different tissues that also may cause a differential sensitivity related to differential induction of some enzymes.

Investigation of potential biomarkers for the early diagnosis of cellular stability after the exposure of agricultural workers to pesticides.

Agricultural workers involved in the harvest of tobacco crops are regularly exposed to large quantities of pesticides. In order to determine how this exposure to pesticides induces genetic alterations in these workers, blood samples were obtained from 77 exposed individuals, as well as from 60 unexposed subjects. DNA damage was analyzed by the Comet assay and by the micronucleus (MN) test. The antioxidant profile was evaluated by activity of superoxide dismutase (SOD), and the polymorphism of gene PON1 was used as a susceptibility biomarker. The content of inorganic elements in the blood samples was determined by PIXE analysis. Our results demonstrated that the damage frequency, damage index, the MN frequency, and the SOD activity were significantly elevated in the exposed relative to the unexposed group. A modulation of the MN results for the PON1 gene was observed in the exposed group. The concentrations of inorganic elements in the exposed group were higher compared to those of the unexposed group. In this study, we observed that genetic damage, and change in oxidative balance were induced by the exposure of workers to complex mixtures of pesticides in the presence of inorganic compounds, whereby an influence of the genotype was evident.

Occupational exposure of workers to pesticides: Toxicogenetics and susceptibility gene polymorphisms.

Farm workers are often exposed to pesticides, which are products belonging to a specific chemical group that affects the health of agricultural workers and is mostly recognized as genotoxic and carcinogenic. The exposure of workers from Piauí, Brazil, to these hazardous chemicals was assessed and cytogenetic alterations were evaluated using the buccal micronucleus assay, hematological and lipid parameters, butyrylcholinesterase (BChE) activity and genetic polymorphisms of enzymes involved in the metabolism of pesticides, such as PON1, as well as of the DNA repair system (OGG1, XRCC1 and XRCC4). Two groups of farm workers exposed to different types of pesticides were evaluated and compared to matched non-exposed control groups. A significant increase was observed in the frequencies of micronuclei, kariorrhexis, karyolysis and binucleated cells in the exposed groups (n = 100) compared to controls (n = 100). No differences were detected regarding the hematological parameters, lipid profile and BChE activity. No significant difference was observed either regarding DNA damage or nuclear fragmentation when specific metabolizing and DNA repair genotypes were investigated in the exposed groups.

Genetic polymorphisms of glutathione S-transferases and cytochrome P450 enzymes as susceptibility factors to systemic lupus erythematosus in southern Brazilian patients.

Systemic lupus erythematosus (SLE) is an autoimmune chronic inflammatory disease that presents several clinical manifestations, affecting multiple organs and systems. Immunological, environmental, hormonal and genetic factors may contribute to disease. Genes and proteins involved in metabolism and detoxification of xenobiotics are often used as susceptibility markers to diseases with environmental risk factors. Cytochrome P450 (CYP) enzymes activate the xenobiotic making it more reactive, while the Glutathione S-transferases (GST) enzymes conjugate the reduced glutathione with electrophilic compounds, facilitating the toxic products excretion. CYP and GST polymorphisms can alter the expression and catalytic activity of enzymes. This study aimed to investigate the role of genetic variants of CYP and GST in susceptibility and clinical expression of SLE, through the analysis of GSTM1 null, GSTT1 null, GSTP1*Ile105Val, CYP1A1*2C and CYP2E1*5B polymorphisms. 371 SLE patients from Hospital de Clínicas de Porto Alegre and 522 healthy blood donors from southern Brazil were evaluated. GSTP1 and CYP variants were genotyped using PCR-RFLP and GSTT1 and GSTM1 variants were analyzed by multiplex PCR. Among European-derived individuals, a lower frequency of GSTP1*Val heterozygous genotypes was found in SLE patients when compared to controls (p = 0.005). In African-derived SLE patients, the CYP2E1*5B allelic frequency was higher in relation to controls (p = 0.054). We did not observe any clinical implication of the CYP and GST polymorphisms in patients with SLE. Our data suggest a protective role of the GSTP1*Ile/Val heterozygous genotype against the SLE in European-derived and a possible influence of the CYP2E1*5B allele in SLE susceptibility among African-derived individuals.

Genotoxic assessment in tobacco farmers at different crop times.

Agricultural workers engaged in tobacco cultivation are constantly exposed to large amounts of pesticides as well as to the nicotine present in raw tobacco leaves. Pesticides have been considered potential chemical mutagens: experimental data revealed that various agrochemicals possess mutagenic properties. Studies have affirmed that nicotine absorbed through the skin results in the characteristic green tobacco sickness (GTS), an occupational illness reported by tobacco workers. This study sought to determine genotoxic effects in farmers occupationally exposed to agrochemicals and nicotine. Peripheral blood samples were collected from 30 agricultural workers, at different crop times (off-season, during pesticides application and leaf harvest), and 30 were non-exposed. We obtained data on DNA damage detected by the Comet assay and Micronucleus test as biomarker of occupational exposure and effect. The serum cholinesterase level, which in general present relation with exposition to organophosphates and carbamates, as well as serum cotinine level, which is a metabolite of nicotine, were also evaluated. The results showed a significant increase in Damage index and frequency in tobacco farmers compared to the non-exposed group, for all different crop times; and a significant increase in micronucleated cells in the off-season group. No correlation was found between age and exposure time in relation to biomarker tests. The DNA damage was greater in males than in females, but with a significant difference only in off-season group. No difference, in cholinesterase activity, was seen among the group of farmers and non-exposed group. Elevated level of cotinine was observed in leaf harvest group. This investigation suggests increased DNA damage in all tobacco crop stages, calling attention to the significant increase during the off-season and tobacco leaf harvest.

Genetic and oxidative damage of peripheral blood lymphocytes in workers with occupational exposure to coal.

Coal is an important fossil fuel used to generate energy. Coal dust is constituted primarily of hydrocarbons and metals. During coal extraction, large quantities of coal dust particles are emitted, contributing to environmental pollution. Coal miners are constantly exposed to coal dust and its derivatives. The goal of this study was to evaluate the potential genotoxic effects of coal and oxidative stress in individuals from Candiota who were exposed to coal as part of their occupation. The comet assay and micronucleus (MN) test were used to assess these effects. This study involved 128 male participants of whom 71 reported work that included exposure to coal (exposed group) and 57 reported working at different jobs (unexposed group). The exposed group had a significantly increased damage index and damage frequency, as assessed using the comet assay, and increased MN and nucleoplasmic bridge frequencies, as assessed using the MN assay, compared with unexposed individuals. Significant and positive correlations between MN frequencies in the lymphocytes and buccal cells of control and exposed individuals were observed. The exposed individuals presented lower average levels of thiobarbituric acid reactive substances (TBARS) and catalase activity (CAT), while the mean superoxide dismutase activity (SOD) levels were higher in this group. The exposed group also had higher hematocrit levels. No correlation between DNA damage and inorganic elements, as identified using PIXE, was found; however, there was a correlation between the damage index and zinc. The evidence that exposure to coal and its derivatives presents a genetic hazard demonstrates the need for protective measures and educational programs for coal miners.

Evaluation of genetic damage in open-cast coal mine workers using the buccal micronucleus cytome assay.

Coal is the largest fossil fuel source used for the generation of energy. However, coal extraction and its use constitute important pollution factors; thus, risk characterization and estimation are extremely important for the safety of coal workers and the environment. Candiota is located to the southeast of the state of Rio Grande do Sul and has the largest coal reserves in Brazil, and the largest thermal power complex in the state. In the open-cast mines, the coal miners are constantly exposed to coal dust. The human buccal micronucleus cytome (BMCyt) assay has been used widely to investigate biomarkers for DNA damage, cell death, and basal cell frequency in buccal cells. The aim of this study was to assess whether prolonged exposure to coal dust could lead to an increase in genomic instability, cell death, and frequency of basal cells using the BMCyt assay. In the analysis of epithelial cells, the exposed group (n = 41) presented with a significantly higher frequency of basal cells, micronuclei in basal and differentiated cells, and binucleated cells compared to the non-exposed group (n = 29). The exposed group showed a significantly lower frequency of condensed chromatin cells than the non-exposed group. However, we found no correlation between DNA damage and metal concentration in the blood of mine workers. DNA damage observed in the mine workers may be a consequence of oxidative damage resulting from exposure to coal residue mixtures. In addition, our findings confirm that the BMCyt assay can be used to identify occupational risk.

Application of the buccal micronucleus cytome assay and analysis of PON1Gln192Arg and CYP2A6*9(-48T>G) polymorphisms in tobacco farmers.

Tobacco is a major Brazilian cash crop. Tobacco farmers apply large amounts of pesticides to control insect growth. Workers come into contact with green tobacco leaves during the tobacco harvest and absorb nicotine through the skin. In the present study, micronucleus frequency, cell death, and the frequency of basal cells were measured in tobacco farmers using the buccal micronucleus cytome assay (BMCyt), in parallel with measurement of blood butyrylcholinesterase (BChE) and nicotine levels. Polymorphisms in PONIGln192Arg and CYP2A6*9(-48T>G) were evaluated to verify the relationship between genetic susceptibility and the measured biomarkers. Peripheral blood and buccal cell samples were collected from 106 agricultural workers, at two different crop times (during pesticide application and leaf harvest), as well as 53 unexposed controls. BMCyt showed statistically significant increases in micronuclei, nuclear buds, and binucleated cells among exposed subjects in differentiated cells, and in micronuclei in basal cells. In addition, the exposed group showed higher values for condensed chromatin, karyorrhectic, pyknotic, and karyolitic cells, indicative of cell death, and an increase in the frequency of basal cells compared to the unexposed control group. A slight difference in mutagenicity using the BMCyt assay was found between the two different sampling times (pesticide application and leaf harvest), with higher micronucleus frequencies during pesticide application. Elevated cotinine levels were observed during the leaf harvest compared to the unexposed controls, while BChE level was similar among the farmers and controls. PONIGln192Arg and CYP2A6*9(-48T>G) polymorphisms were associated with DNA damage induced by pesticides and cell death.

Genotoxic biomonitoring of tobacco farmers: Biomarkers of exposure, of early biological effects and of susceptibility.

Tobacco farming presents several hazards to those who cultivate and harvest the plant. The genotoxic and mutagenic effects in tobacco farmers were investigated. In order to verify the relationship between genetic susceptibility and biomarkers GSTT1, GSTM1, GSTP1, CYP2A6, PON, OGG1, RAD51, XRCC1, and XRCC4 genes polymorphism were evaluated. Oxidative stress markers and trace elements content were determined. Peripheral blood cells samples were collected from 111 agricultural workers during pesticides application and leaf harvest, and 56 non-exposed subjects. Results show that farmers are exposed to mixture of substances with genotoxic and cytotoxic potential. Only GSTM1 null and CYP2A6*9 showed significant associations with cytokinesis-blocked micronuclei assay results. In pesticide application an increase in trace elements content was observed. The results indicated that exposure to pesticides and nicotine can influence antioxidant enzymes activity. Our study drives the attention once more to the need for occupational training on safe work environment for farm workers.

Susceptibility to DNA damage in workers occupationally exposed to pesticides, to tannery chemicals and to coal dust during mining.

Our mutagenesis group has been studying with important economic drivers of our state, such as agriculture, the foot-wear and leather industry and open-cast coal mining. Working conditions in these sectors have potentially harmful to humans. The aim of these studies is to determine the health risk of workers by biomonitoring subjects exposed to genotoxic agents. The main results of our studies with vineyard farmers we observed a high rate of MN and DNA damage in individuals exposed to pesticides (p < 0.001). In addition, some effects of genetic polymorphisms in the modulation of MN results were observed in this group. Tobacco farmers were also evaluated at different crop times. The results showed a significant increase in the Damage index and frequency in tobacco farmers compared to the non-exposed group, for all crop times. The results for footwear and tannery workers showed a significant increase in the mean ID for the solvent-based adhesive (p < 0.001) group in comparison to the water-based adhesive group and control (p < 0.05). For open-cast coal mine workers, the EBCyt indicated a significant increase in nuclear bud frequency and cytokinetic defects in the exposed group compared to the non-exposed group (p < 0.0001). We were able to associate specific genetic susceptibility with each type of exposure and with the non-use or improper use of personal protection equipment and diet adequacy. These results show how important the continuous education of exposed workers is to minimizing the effect of the occupational exposure and the risk of disease associated with the work.

BER gene polymorphisms (OGG1 Ser326Cys and XRCC1 Arg194Trp) and modulation of DNA damage due to pesticides exposure.

The susceptibility of individuals to the genotoxic effect of pesticides can be modulated by genetic variations in the xenobiotic detoxification and DNA repair processes. This study evaluates if the two BER polymorphisms (XRCC1Arg194Trp and OGG1Ser326Cys) or the combined genotypes of these polymorphisms with PON1Gln192Arg could modify individual susceptibility to pesticide exposure in vineyard workers, as measured by micronucleus formation and DNA damage induction in peripheral leukocytes. The study population comprised 108 agricultural workers exposed to pesticides and 65 nonexposed. Our results demonstrate that individuals with the variant allele (OGG1Cys) showed higher DNA damage, detected by the comet assay, in relation to individuals carrying the wild-type OGG1Ser allele. Considering the combined influence of metabolizing PON1 and the DNA repair OGG1 genes, we observed significantly higher DNA damage in the comet assay in the exposed group when a less efficient OGG1Cys allele was acting independently of the PON1 genotype, reinforcing the importance of the OGG1 repair enzyme in the response to DNA damage by pesticide exposure. The association of the PONGln/Gln genotype with higher MN frequency suggests that the PON1 genotype is a major determinant of genotoxic risk in individuals exposed to pesticides. Analysis of the compared effect of XRCC1 and PON1 genotypes in the exposed group suggested that, among the poorly metabolizing PON1Gln/Gln individuals, the XRCC1Arg/Trp genotype has a protective effect with respect to MN formation. These results indicate that enhanced XRCC1 function may provide some protection from the enhanced genotoxic risk associated with inefficient xenobiotic detoxification in the studied population.

Evaluation of genetic damage in a Brazilian population occupationally exposed to pesticides and its correlation with polymorphisms in metabolizing genes.

Cytogenetic damage in individuals occupationally exposed to pesticides has received the attention of investigators in several countries, but no definitive conclusions can yet be made. The present study aimed at assessing if prolonged exposure to complex mixtures of pesticides leads to an increase in cytogenetic damage. Vineyard workers exposed to pesticides in Caxias do Sul (Brazil) were evaluated using the micronucleus (MN) test in binucleated lymphocytes and the comet assay in peripheral leukocytes. In order to evaluate if genetically determined individual variations in xenobiotic metabolizing capacity could modify individual susceptibility to the possible genotoxic effects of pesticides, the subjects were genotyped for several genes: GSTT1, GSTM1, GSTP1, CYP1A1, CYP2E1 and PON. The study involved a total number of 173 men: 108 were agricultural workers exposed to pesticides and 65 were controls. The present study showed a high rate of MN and DNA damage in pesticide-exposed individuals (P

Evaluation of genetic damage in Brazilian footwear-workers: biomarkers of exposure, effect, and susceptibility.

Employees in the footwear manufacturing industry are routinely exposed to complex mixtures of solvents used in cleaning and as diluents in glues, primers, and degreasers. The objective of this study was to determine the genotoxic effects in a group of footwear-workers occupationally exposed to solvent-based adhesive and solutions containing organic solvents, mainly toluene. Peripheral blood and buccal cells samples were collected from 39 footwear-workers (31 males and 8 females) and 55 controls (44 males and 11 females). As biomarker of exposure, we obtained data on hippuric acid (HA), the main metabolite of toluene in urine, and DNA damage detected by the Comet assay in blood cells. Micronucleus frequencies in binucleated lymphocytes (BNMN) and in epithelial buccal cells (EBCMN) were analyzed as biomarkers of effect, while polymorphisms in genes GSTT1, GSTM1, GSTP1, CYP1A1, and CYP2E1 were used as susceptibility biomarkers. Results of HA and Comet assay showed statistical increased values amongst footwear-workers relative to controls (P < or = 0.001). No differences were observed in BNMN and EBCMN frequencies between the groups, but a correlation test revealed that age was significantly associated with BNMN frequency in both control (r(s)=0.290; P < or = 0.05) and exposed groups (r(s)=0.674; P < or = 0.001). Regarding the results on genetic polymorphisms, GSTM1 null subjects from the control group showed a significant increase in EBCMN frequency relative to GSTM1 non-null subjects (P < or = 0.05). A significant increase in DNA damage detected by Comet assay in leukocytes was obtained for GSTP1 Ile/Val or Val/Val individuals from the exposed group relative to those with GSTP1 Ile/Ile (P < or = 0.05), especially in younger subjects (P < or = 0.01), and a suggestion of interaction with CYP2E1 polymorphism was found. In confirmation of these data, stepwise multiple regression analyses for selecting between the different independent variables showed that about 25% of levels of the DNA damage in footwear-worker can be associated with genetic polymorphisms in GSTP1 and CYP2E1 (P=0.006, F=5.876).

Optimization of an electroporation protocol using the K562 cell line as a model: role of cell cycle phase and cytoplasmic DNAses.

The improvement of gene therapy protocols is intimately related to the establishment of efficient gene transfer methods. Electroporation has been increasingly employed in in vitro and in vivo protocols, and much attention has been given to increasing its transfection potential. The method is based on the application of an electric field of short duration and high voltage to the cells, forming reversible pores through which molecules can enter the cell. In this work, we describe the optimization of a protocol for the electroporation of K562 cells involving the combination of electric field, resistance and capacitance values. Using RPMI 1640 as pulsing buffer and 30 mug of pEGFP-N1 plasmid, 875 V cm(-1), 500 muF and infinite resistance, we achieved transfection rates of 82.41 +/- 3.03%, with 62.89 +/- 2.93% cell viability, values higher than those reported in the literature. Analyzing cell cycle after electroporation, with three different electric field conditions, we observed that in a heterogeneous population of cells, viability of G(1) cells is less affected by electroporation than that of cells in late S and G(2)/M phases. We also observed that efficiency of electroporation can be improved using the DNAse inhibitor Zn, immediately after the pulse. These results can represent a significant improvement of current methods of electroporation of animal and plant cells.

CYP1A1, GSTM1, GSTT1 and GSTP1 polymorphisms in an Afro-Brazilian group

Gene polymorphisms involved in the metabolism of drugs and chemical carcinogens seem to be responsible for differences in the susceptibility of individuals to cancer, but genetic population studies are needed to characterize these polymorphisms in different ethnic populations. We investigated polymorphisms of the cytochrome P450 (CYP) gene CYP1A1 and the glutathione S-transferase (GSTs) genes GSTM1, GSTT1 and GSTP1 in a sample of Afro-Brazilians from the southern Brazilian city of Porto Alegre to verify if there were ethnic differences compared to the polymorphisms of the same genes in a previously described sample of Brazilians of European descent from the same city. The allele frequencies detected in the Afro-Brazilian population investigated in this study were CYP1A1*2A (30 percent) and GSTP1*Val (42 percent) while the frequency of the GSTM1 null genotype was 34 percent and that of the GSTT1 null genotype was 28 percent. Significant differences in genotype distribution and allelic frequencies were detected between Brazilians of African and of European descent from Porto Alegre in terms of the polymorphisms CYP1A1*2A (p = 0.003), GSTP1-Ile105Val (p = 0.002) and the GSTM1 null genotype (p = 0.01) but there was no detectable significant difference in respect to GSTT1 null genotype frequencies.

CYP1A1, CYP2E1, GSTM1, GSTT1, GSTP1, and TP53 polymorphisms: do they indicate susceptibility to chronic obstructive pulmonary disease and non-small-cell lung cancer?

Gene polymorphisms of phase I (CYP1A1 and CYP2E of cytochrome P,) and phase II (GSTM1, GSTT1 and GSTP1 of glutathione-S-transferase,) enzymes and the TP53 tumor suppressor gene were studied as markers in a sample of 262 Brazilians of European descent, the sample consisting of 97 patients with non-small-cell lung cancer (NSCLC), 75 patients with chronic obstructive pulmonary disease (COPD) and 90 control individuals. For NSCLC, we found no significant relationship between any of the markers studied and susceptibility to this disease. With respect to COPD, although the distribution of the CYP1A1, GSTM1, GSTP1, GSTT1 and TP53 genotypes was similar to that of the controls the frequency of the CYP2E1*1A/*5B heterozygote was about 6 times higher in COPD patients than in controls (OR= 6.3; CI = 1.1-35.5 for p = 0.04). Individuals who presented the GSTT1 null phenotype and GSTP1 Ile/Val genotype had a risk about four times higher (OR= 4.0; CI = 1.2-14.6 for p = 0.02) of having COPD than individuals without these genotypes, the same being true for individuals having the GSTT1 null phenotype and CYP1A1*1A/*2A genotype (OR= 3.7; 1.1-14.6 for p = 0.04).These results suggest that the CYP2E1 and GSTT1 + GSTP or GSTT1 + CYP1A1 polymorphisms may be predictive of susceptibility to COPD, at least in this population of European ancestry.