Search

In order to investigate the effects of antioxidant supplementation on chromosome damage, a 3 month antioxidant supplementation trial was conducted on groups of 28 myocardial infarction survivors and 57 rural controls, all male. The supplement consisted of vitamin C (100 mg/day), vitamin E (100 mg/day), β-carotene (6 mg/day) and selenium (50 μmg/day). Dietary antioxidants in plasma were measured, as well as the ferric reducing ability of plasma (a measure of total plasma antioxidant status) and the concentration of malondialdehyde as an indicator of oxidative stress. Lymphocytes collected at the beginning and end of the supplementation period were stimulated to proliferate and metaphases accumulated for scoring of chromosome aberrations: per cent aberrant cells and chromatid and chromosome breaks. Supplementation with antioxidants was associated with a decrease in the percentage of cells with chromosome aberrations in the group of rural controls (0.63% before compared with 0.27% after supplementation; P = 0.03). The largest effect of supplementation was seen in smokers in this group (0.12% aberrant cells in supplemented compared with 0.81% in placebo group; P > 0.001). The results support the hypothesis that antioxidants decrease genetic damage. [ABSTRACT FROM AUTHOR]

Objective: This study investigated the association of the Leu432Val and Asn453Ser CYP1B1 polymorphisms and selected environmental biomarkers with hypertension (HT) in Slovak midlife women., Methods: We studied 575 women. Divided according to their blood pressure status: 255 with HT and 320 without HT. All data was obtained by using standard anthropometric, genetic methods and analyzed by regression models to adjust for HT risk factors such as age, obesity, smoking, and level of education., Results: Our findings revealed that CYP1B1 Leu432Val polymorphism was associated with HT, whereas no association was found between Asn453Ser polymorphism and HT. Women with at least one Val allele had significantly higher odds of HT compared to women with the Leu/Leu genotype in the total sample (Exp(B) = 1.82, CI 1.16-2.84, P = 0.009). After dividing women by menopausal status and the presence of HT environmental risk factor, the association between CYP1B1 polymorphism and HT was observed in pre/perimenopausal women (Exp(B), 2.36; 95% CI 1.13-4.92; P = 0.02), smokers (Exp(B), 3.40; 95% CI 1.48-7.82; P = 0.004), abdominal obesity (Exp(B), 2.41; 95% CI 1.23-4.75; P = 0.01) and in women with only basic education (Exp(B), 4.20, 95% CI 1.12-15.71; P = 0.03). However, general linear models did not reveal a statistically significant interactions between CYP1B1, menopausal status, and HT risk factors and their common association with HT (P > 0.05)., Conclusions: In this pilot study, we have provided novel data that supports the significant association of CYP1B1 Leu432Val gene polymorphism with HT in Slovak midlife women.

Established risk factors for cardiovascular diseases (CVD) may be moderated by genetic variants. In 2403 unrelated individuals from general practice (mean age 40.5 years), we evaluated the influence of 15 variants in 12 candidate genes on quantitative traits (QT) associated with CVD (body mass index, abdominal obesity, glucose, serum lipids, and blood pressure). Prior to multiple testing correction, univariate analysis associated APOE rs429358, rs7412 and ATG16L1 rs2241880 variants with serum lipid levels, while LEPR rs1137100 and ATG16L1 rs2241880 variants were linked to obesity related QTs. After taking into account confounding factors and correcting for multiple comparisons only APOE rs429358 and rs7412 variants remained significantly associated with risk of dyslipidemia. APOE rs429358 variant almost tripled the risk in homozygous subjects (OR = 2.97; 95% CI 1.09-8.10, p < 0.03) and had a lesser but still highly significant association also in heterozygous individuals (OR = 1.67; 95% CI 1.24-2.10; p < 0.001). Associations with hypertension, diabetes mellitus, and metabolic syndrome were not significant after Bonferroni correction. The influence of genetic variation is more evident in dyslipidemia than in other analyzed QTs. These results may contribute to strategic research aimed at including genetic variation in the set of data required to identify subjects at high risk of CVD., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: Metabolic syndrome (MetS) comprises a cluster of risk components which pre-dispose individuals to cardiovascular mortality., Aim: The purpose of this study is to investigate the variability of biochemical and anthropometric characteristics, apolipoprotein E (APOE) and angiotensin converting enzyme (ACE) genes and their contribution to MetS manifestation., Subjects and Methods: A total of 438 adult women were recruited from different localities in Slovakia. All data was established by standard anthropometric, biochemical and genetic methods., Results: The logarithm of the ratio of plasma concentration of triglycerides to HDL-cholesterol [log(TG-to-HDL-C)], waist circumference, systolic blood pressure, apolipoprotein A1, glucose and alanin aminotransferase accounted for most of the differences in MetS manifestation. Logistic regression showed that participants with risk values of the atherogenic index log(TG-to-HDL-C) had a 15.62-fold higher risk of MetS compared to those with lower values for this index (95% CI = 8.3-29.1). Women with hyperglycaemia (or formerly diagnosed diabetes mellitus) had an 8.82-times higher risk of MetS (95%CI = 3.22-24.16). Women with hyper-uricaemia had the same risk of MetS incidence as women with abdominal obesity, Exp (B) = 4.05.Hypercholesterolaemia, ACE and APOE genotypes did not influence MetS., Conclusion: MetS may involve many risk factors that can cause serious disorders in multiple organs. However, women with risk values involving plasma atherogenic index log (TG-to-HDL-C) experienced the highest risk of developing MetS.

The aim of this study was to investigate several immunologic parameters using of immunonephelometry and adipocytokines by the enzyme immunoassay and their changes in different states of obesity. Obesity is considered to involve a state of chronic low-grade inflammation, with links between adipose cells and the immune system. We found significantly higher complement C3 levels in all obese subjects. Levels of the complement C4 were significantly higher in obese women, but not in men, when compared with the corresponding group of normal weight subjects. The increase in C-reactive protein concentrations was significant in both obese and morbidly obese women, but only in morbidly obese men. No significant differences in tumor necrosis factor-α, interleukin-6, interleukin-10, and soluble intercellular adhesion molecule-1 were found. sE-selectin levels were higher in both overweight and obese women but only in morbidly obese men. We found decreased adiponectin concentrations in obese and morbidly obese women. Concentrations of leptin were significantly higher only in obese men (p < 0.05), whereas in women the increase in leptin levels was significant in overweight, obese, and morbidly obese subjects. In conclusion, our results demonstrate elevated levels of C3, C-reactive protein, sE-selectin, and leptin in obese women and men. In obese women, we also observed increased concentrations of C4 and decreased levels of adiponectin., (Copyright © 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

Slovakia is characterised by an unusually high number of patients affected by genetic Creutzfeldt-Jakob disease (CJD) with E200K mutation at the PRNP gene. Penetrance of the mutation is incomplete (59%). Therefore, for the onset of the clinical manifestation, an influence of other endo- or exogenous factors could not be excluded. Experimental data suggest that copper and manganese levels may play an important role in the pathogenesis of prion diseases. The highest number of Slovak genetic CJD patients originates from Orava - the northern region of central Slovakia. Manganese is a dominant pollutant in Orava. The objective of this study was to clarify a possible exogenous influence of environmental Mn/Cu imbalance on the CJD clustering. Mn and Cu levels were analysed in the brain tissue of genetic CJD cases (from Orava and from control regions of Slovakia), as well as of sporadic CJD patients and controls. Analyses demonstrate i) significantly higher Mn level in focally accumulated, "clustering" genetic CJD cases in comparison to all other groups, ii) Cu status differences between compared groups were without statistical significance; decreased concentrations were found in genetic cases from extrafocal genetic CJD areas, iii) Mn/Cu ratios were increased in all CJD groups in comparison to controls. Metal ratios in clustering gCJD cases were significantly higher in comparison to sporadic cases and also to controls, but not to the extrafocal genetic CJD subgroup. These results indicate that more important than increasing Mn level in pathogenesis of CJD appears to be the role of the Mn/Cu imbalance in the CNS. The imbalance observed in the cluster of genetic CJD cases is probably a result of both: the excessive environmental Mn level and the disturbance of Mn/Cu ratios in the Orava region. Presented findings indicate an environmental Mn/Cu imbalance as a possible exogenous CJD risk co-factor which may, in coincidence with endogenous (genetic) CJD risk, contribute to the focal accumulation (cluster) of genetic CJD in Slovakia.

A large number of functional foods, including those that contain β-d-glucans, have been shown to prevent human DNA against genotoxic effects and associated development of cancer and other chronic diseases. In this paper, carboxymethyl chitin-glucan (CM-CG) isolated from Aspergillus niger was investigated from two standpoints: (1) DNA-protective effects against oxidative DNA damage induced by H(2)O(2) and alkylating DNA damage induced by MMS and MNNG, and (2) a potential effect on rejoining of MMS- and MNNG-induced single strand DNA breaks. The results obtained by the comet assay in human cells cultured in vitro showed that CM-CG reduced significantly the level of oxidative DNA lesions induced by H(2)O(2) but did not change the level of alkylating DNA lesions induced by MMS or MNNG. On the other side, the efficiency of DNA-rejoining of single strand DNA breaks induced by MMS and MNNG was significantly higher in HepG2 cells pre-treated with CM-CG. The antioxidative activity of carboxymethyl chitin-glucan was confirmed by the DPPH assay., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The aim of this paper was to evaluate genotoxic effects of borneol and its ability to change DNA-damaging effects of H2O2 in rat hepatocytes and testicular cells. Both in vitro and ex vivo approaches were used in the case of hepatocytes. Testicular cells were tested only ex vivo, i.e. shortly after isolation from rats supplemented by borneol. Cytotoxicity of borneol increased in in vitro conditions in a concentration-dependent manner and it was associated with DNA-damaging effects at toxic concentrations. While non-toxic concentrations of borneol applied in vitro protected cells against H2O2-induced DNA damage and interfered only partly with rejoining of H2O2-induced DNA strand breaks, cytotoxic concentrations of borneol manifested synergy with H2O2, i.e. enhanced DNA-damaging effects of H2O2. On the other side, borneol given to rats in drinking water decreased the level of DNA damage induced by H2O2 in both hepatocytes and testicular cells. Our results show that though at higher concentrations (2-h treatment with >2 mM borneol >0.3084 mg/ml) borneol acts cytotoxically and genotoxically on primary hepatocytes cultured in vitro, if given to rats during 7 days in a daily concentration of 17.14 or 34.28 mg/kg it reduces genotoxicity of H2O2 in both hepatocytes and testicular cells.

Plant volatiles, which can get into the human organism in food, medicines, or cosmetic preparations, frequently manifest antibacterial, antifungal, antiviral and other effects. We studied anti-oxidative, cytotoxic, genotoxic and possible DNA-protective effects of eugenol and borneol. Anti-oxidative activities of aqueous and ethanolic solutions of these two volatile compounds of plants were determined by a spectrophotometric method by the use of the stable DPPH radical. Borneol did not show any anti-oxidative activity even at the highest concentrations soluble in water or ethanol (<1000mM), while eugenol did manifest anti-oxidative activity, and at much lower concentrations (5-100 microM). The cytotoxicity of eugenol and borneol as well as their DNA-damaging effects and their influence on sensitivity of cells against the DNA-damaging effects of H(2)O(2) were investigated in three different cell lines, i.e. malignant HepG2 hepatoma cells, malignant Caco-2 colon cells, and nonmalignant human VH10 fibroblasts. The trypan-blue exclusion assay showed that in the three cell lines the cytotoxicity of eugenol was significantly higher than that of borneol. Single-cell gel electrophoresis revealed that borneol did not cause any DNA strand-breaks at the concentrations studied, but showed that all concentrations of eugenol (<600 microM) significantly increased the level of DNA breaks in human VH10 fibroblasts and to a lower degree in Caco-2 colon cells. The DNA-damaging effects of eugenol were not observed in metabolically active HepG2 hepatoma cells. Borneol and eugenol differed also with respect to their DNA-protective effects. While borneol protected HepG2 and, to a lesser extent, VH10 cells (but not Caco-2) against H(2)O(2)-induced DNA damage, eugenol either did not change the cellular sensitivity to H(2)O(2) (HepG2 cells) or it even increased the sensitivity (Caco-2 and VH10 cells). These results do not indicate any correlation between the DNA-protective and the anti-oxidative capacities of eugenol and borneol.

Life expectancy in central-Eastern European countries is more than 10 years lower compared with Northern or Western countries which could be the result of complex factors including genetics, nutrition and life style. We conducted a molecular epidemiological study with the aim of investigating links between DNA instability, genetic polymorphisms in nucleotide excision repair genes and ageing. Two groups-151 young people (78 women and 73 men) aged 20-25, and 140 elderly subjects (101 women and 39 men), aged 65-70 have been investigated. Results show elevated levels of micronuclei and chromosome aberrations in elderly compared with young groups (P<0.001); women had more micronuclei than men (P<0.001). Micronucleus frequencies were influenced by age (P<0.001). In the group of elderly people those who were homozygous with C/C or A/A in XPC IVS11 had more aberrant cells compared with C/A heterozygotes (P=0.04). When the dependent variable was break per cell, elderly people A/A homozygous in XPC IVS11 had more breaks per cell compared with C/A heterozygous or C/C homozygous subjects (P=0.03). Significantly the most chromatid breaks were found in elderly people both Lys/Lys homozygous in the XPD Lys751Gln genotype and C/C or A/A homozygous in the XPC IVS11 genotype (P<0.05). A General Linear Model analysis shows a statistically significant effect of interactions between age, sex and genotype XPC IVS11 (P=0.001) and age, sex and genotype XPCin9 (P=0.007) on number of chromatid breaks. When we divided people into two subgroups (without mutant allele and with one or two mutant alleles) we found a significantly higher number of chromosome exchanges in people with one or two variant polymorphism XPCin9 (P=0.04), XPC IVS11 (P=0.004) or XPCex15 (P=0.001). Level of cells with micronuclei was influenced by polymorphisms XPD Lys751Gln (P=0.03). However, we did not find any relationship between XPA polymorphism and studied cytogenetic biomarkers.

The objective of this study was to examine frequency of familial defective apo-B-100 (FDB, R3500Q mutation) in probands with the phenotype of familial hypercholesterolemia (FH) and in the general population of 40-year-old subjects in Slovakia and to characterize their lipid and clinical criteria and to compare the frequency of FDB with other populations. We identified 35 patients with FDB among 362 probands with clinical diagnosis of FH and two cases of FDB in the 40-year-old cohort of 2323 subjects from general Slovak population. Probands with FDB differed from those with FH only in plasma triglyceride concentrations (1.84+/-1.4 mmol/l versus 1.45+/-0.98 mmol/l, respectively, p<0.01). Evaluation of personal history of premature atherosclerosis did not show any differences (11.4% in FDB versus 20% in FH, p<0.16). The FDB patients had similar manifestation of xanthomatosis as the FH patients (17.1% versus 8.25%, p<0.25). The frequency of FDB of 9.7% found in the FH patients is among the highest of those reported to date. The frequency of R3500Q mutation of 0.09% found in Slovak 40-year-old subjects did not differ significantly from published population molecular data. Our comparison of estimated FDB frequencies with those which were found by DNA analysis demonstrated that estimated frequencies were not only wider in range, but also significantly higher than those which were assessed by the analysis. The definitive answer to the prevalence of FDB and its biochemical and clinical characteristics requires screening of unbiased samples of the general population from different ethnic groups based on molecular genetic methods.

The aim of this paper is to evaluate dietary habits and behavioural factors related to atherosclerosis in Slovak Romany, the large minority, characterized by high cardiovascular morbidity. The study involved 150 Romany volunteers (68 males, mean age 42.1 +/- 13.9 y and 82 females, mean age 40.9 +/- 13.7 y). Dietary data were obtained by a validated food-requency questionnaire and a single 24-hour dietary recall. The nutrient intake and health behaviour of the Romany population is not consistent with current guidelines for atherosclerosis prevention. The mean intake of fat is higher than the recommended dietary allowance (RDA), especially in males (155.3 % of RDA). In females the intake of alpha-linolenic acid is low, in males the cholesterol content of the food exceeds the acceptable value. The mean intake of protein is higher than the recommendation (males 153% of RDA, females 122.2%), with a high proportion of animal protein. In both sexes the mean intake of vitamins is below the RDA. In comparison to the general population the diet of the Romany males contains significantly more animal protein (p < 0.05), less plant protein (p < 0.05) and folate (p < 0.01). In the diet of the Romany females a significantly lower intake of plant protein (p < 0.05) and vitamin E (p < 0.05) was observed, as well as a lower intake of linoleic acid and iron in both sexes. The cumulation of ten selected cardiovascular risk factors showed that particularly the Romany males could be considered as having more atherogenic profile.

A vegetarian diet results in higher intake of vitamins and micronutrients, which - although providing antioxidant defence - may lead to deficiency in other micronutrients involved in DNA metabolism and stability (such as vitamins belonging to the B group). The principal difference among various vegetarian diets is the extent to which animal products are avoided. We have performed a pilot study to determine the relationship between the micronucleus frequency in lymphocytes and diet, and we compared the levels of Vitamins C and E, beta-carotene, B(12), folic acid, homocysteine and total antioxidant capacity in healthy vegetarians and non-vegetarians. The vegetarian group, consisting of 24 volunteers (13 women and 11 men), were matched for age and sex with 24 volunteers (12 women and 12 men) with a traditional dietary habit. Among the vegetarians were 13 lacto-ovo-vegetarians with average duration of vegetarian diet 10.8 years (ranging from 5 to 26 years) and 11 lacto-vegetarians with average duration of vegetarian diet 8.2 years (ranging from 3 to 15 years). Homocysteine, Vitamins C and E and beta-carotene levels in plasma were assayed by HPLC, and serum folate and Vitamin B(12) were determined with Elecsys Immunoassay tests. The total antioxidant capacity of plasma was estimated by measuring the ferric-reducing activity in a spectrophotometric assay. Micronuclei were measured in cytokinesis-blocked lymphocytes. Vegetarians had significantly higher levels of Vitamin C and beta-carotene (but not Vitamin E) in plasma compared with non-vegetarians (P<0.001). There were no significant differences in serum levels of folic acid and Vitamin B(12) between the monitored groups. Levels of folic acid in vegetarians correlated with length of vegetarianism (r=0.62, P=0.001, N=24). Vegetarians had elevated levels of homocysteine compared with non-vegetarians (P=0.007), as did vegetarian women compared with non-vegetarian women (P=0.031). We did not find any differences in total antioxidant capacity or in micronucleus frequency between the groups. Micronuclei correlated with age (r=0.62, P<0.001, N=48), women having higher frequencies than men. Multifactorial regression analysis showed significant effects of age, sex and total antioxidant capacity on micronucleus frequency (N=48, P<0.001).

Objectives: As asbestos presents a direct genetic hazard for humans, a small-scale molecular epidemiological study was conducted to monitor 61 subjects long-term exposed to asbestos in comparison with 49 town controls and 21 control subjects from administration of the same factory., Results: Asbestos exposed workers had significantly higher numbers of chromosomal aberrations compared with both control groups (P=0.003). Clinical examination showed that 44.3% of exposed workers developed symptoms of asbestosis. We were interested in the relationship between the risk of asbestos-coupled diseases and individual variability in biotransformation enzymes, especially in glutathione S-transferases and microsomal epoxide hydrolase. GSTP1*105Val allele appeared less in the group of workers with asbestosis compared to those without asbestosis (18.5% vs 34.7%, P=0.044), and in subjects with developed asbestosis coupled with bronchitis compared to those without bronchitis (0% vs 25%, P=0.048). Similarly, the genotype corresponding to low activity of microsomal epoxide hydrolase was significantly decreased in workers with fibrotic plaques compared to those without plaques (26.7% vs 56.3%, P=0.045)., Conclusions: Our results suggest that GSTP1*105Val allele and low EPHX1 activity genotype may be protective for people occupationally exposed to asbestos. However, more extensive studies are needed to confirm our results.

Objectives: In humans, epidemiological evidence suggests that increased consumption of fruits and vegetables can substantially enhance the protection against many common types of cancer., Methods & Results: A molecular epidemiological study in 3 Slovak factories producing asbestos, glass fibres and rockwool was conducted. Altogether 388 subjects (239 exposed, 148 controls) were investigated. Food frequency questionnaire was used to ascertain nutrient intake and compared to plasma levels of selected micronutrients, as well as to markers of oxidative stress (MDA, oxidative DNA damage and DNA repair) and antioxidant protection. We found a negative correlation between MDA concentrations and consumption of fruits (p=0.05) and vegetables (p=0.05) in all control subjects. Intake of fruits (p=0.05), vegetables (p=0.01), milk (p=0.01) and cereals (p=0.05) inversely correlated with oxidative DNA damage (net FPG) in all subjects investigated. There was a negative correlation between the intake of fruits (p=0.05) and vegetables (p=0.01) in all exposed subjects., Conclusions: Our results suggest that well balanced food consumption with higher fruits and vegetables intake has a protective effect against oxidative damage.

The influence of apolipoprotein E (ApoE) genotypes on plasma lipid levels and interaction with other environmental factors was determined in two Slovakian population samples; 146 Romany and 351 Slovak individuals. The two samples differ significantly in the distribution of E3/3 genotypes (p<0.014) and E3/2 (p<0.035). Analysis of variance did not reveal any significant effect of the ApoE genotypes on any of the plasma lipid levels in the Romany individuals. In the Slovak sample the variation in plasma low-density lipoprotein cholesterol (LDL-C) levels was significantly associated with the ApoE genotypes (p=0.012). We detected decreased LDL-C concentrations in males with E2 genotype when compared with E3 and E4 carriers (p=0.008). Further, the E2 genotype was found to be associated with high triglycerides levels (p=0.009). The ethnic samples differ significantly in the prevalence of metabolic syndrome and in the case of males of diabetes. Both the Romany and the Slovak males can be considered as having a more atherogenic profile compared with the females.

Participants in a study of occupational exposure to mineral fibres in Slovakia were analysed for the polymorphism 23A-->G in the DNA repair gene XPA. Of the 388 subjects, 239 were exposed to asbestos, stonewool or glass fibre; the rest were unexposed controls. Levels of DNA base alterations (oxidation and alkylation) in lymphocytes were measured using the comet assay with lesion-specific endonucleases. 8-oxoguanine DNA glycosylase (OGG1) DNA repair activity was measured, as incision activity of a lymphocyte extract on DNA containing the OGG1 substrate 8-oxoguanine. Presence of the A allele was associated with higher levels of DNA damage (sites sensitive to formamidopyrimidine DNA glycosylase, endonuclease III or 3-methyladenine DNA glycosylase II) as well as with higher activity of OGG1 repair enzyme. DNA base damage increased with age, showing highly significant correlations when the whole population or subgroups of the population were analysed. OGG1 repair activity also increased with age, but when analysed according to XPA genotype, the increase was observed only in those individuals with an A allele. Although XPA is known as a protein involved in nucleotide excision repair of UV-induced damage and bulky DNA adducts, it may also have a role in the repair of oxidized bases.

The polymorphism at codon 129 (M129V) of the prion protein gene (PRNP) is a recognized genetic marker for susceptibility to Creutzfeldt-Jakob disease (CJD) in the Caucasians. The distribution of this polymorphism in healthy individuals provides an important starting point for the evaluation of CJD risk in the general population. Early studies of reference population cohorts demonstrated that methionine/valine heterozygosity was the most frequent genotype. These studies were performed in relatively small numbers of control subjects and do not correspond with the findings of more recent investigations. In this study, we present an analysis of the codon M129V distribution in 613 corneal donors, representing one of the largest control groups examined to date. Methionine homozygotes represented 48.1%, valine homozygotes 8.7% and methionine/valine heterozygotes 43.2%. While age-related difference was not significant, differentiation according to the gender showed significant difference. The observed highest proportion of methionine homozygotes and statistically significant difference between genders as well as comparison with results obtained in other countries underline the need to re-evaluate the generally used reference data on M129V, including consideration of the gender, age and geographical distribution.

The aim of this study was to examine the effect of antioxidant supplementation on oxidative damage and chromosome stability in middle-aged men, smokers and non-smokers. A total of 124 men aged 48+/-6 years from Bratislava and from the rural population near Bratislava were investigated; 64 men (22 smokers and 42 non-smokers) were supplemented for 12 weeks with antioxidants, while 60 (25 smokers and 35 non-smokers) were given placebo. The daily antioxidant supplementation consisted of vitamin C (100 mg), vitamin E (100 mg), ss-carotene (6 mg), and selenium (50 microg). Samples of blood were taken on two occasions: At the beginning and at the end of the supplementation trial. Concentrations of dietary antioxidants, ferric reducing ability, malondialdehyde as an indicator of lipid peroxidation in plasma, micronuclei and chromosome aberrations in lymphocytes were measured. Antioxidant supplementation significantly increased the levels of vitamin C, ss-carotene, a-tocopherol and selenium in plasma. The overall antioxidant status of plasma measured as ferric reducing ability of plasma (FRAP) increased significantly (p<0.001) after antioxidant supplementation as well. The increase in antioxidant parameters after supplementation were consistently more pronounced in non-smokers than in smokers. There was a significant decrease of malondialdehyde concentration in the non-smokers, while in smokers the decrease of malondialdehyde concentration was not significant. Antioxidant supplementation did not affect the proportion of lymphocytes with micronuclei or the total number of micronuclei; however, there was a significant positive correlation (p<0.001) between the malondialdehyde concentration at the beginning of the supplementation trial and the difference in number of cells with micronuclei before and after the supplementation. The percent of cells with chromosome aberrations decreased significantly after antioxidant supplementation in smokers. These results indicate that a combined antioxidant supplementation (a) is effective even at very moderate doses; (b) significantly diminishes oxidative damage to lipids when it is high initially; and (c) is effective in decreasing chromosomal instability in lymphocytes of middle-aged men.

Risks of carcinogenic and non-carcinogenic effects from asbestos continue owing to the persistence of the fibres in building materials and other products. For this reason, epidemiological and mechanistic research on the toxic effects of asbestos and mineral fibres is still needed. The present molecular epidemiological study was conducted in a former asbestos cement plant in Slovakia. Altogether 82 subjects were investigated, 61 exposed subjects (24 smokers and 37 non-smokers), and 21 factory controls (8 smokers and 13 non-smokers). Workers were exposed to asbestos for between 5 and 40 years. Though the exposure to asbestos during past 40 years was relatively high, at the time of sampling the concentrations of asbestos in the production hall exceeded the Slovak occupational limit (0.001 fibre/cm3) by a factor of only 3-5. The office area levels were below this limit. Biomarkers of exposure, effect and individual susceptibility were measured, including DNA damage (strand breaks [SBs], base oxidation and alkylation, using the comet assay); cytogenetic parameters; and individual DNA repair capacity (incision at 8-oxoguanine measured using a modified comet assay). Oxidised pyrimidines were significantly higher in exposed men compared with non-exposed (P = 0.04). There was also a positive association between SBs (P = 0.04) and age, and alkylation damage to DNA (P = 0.04) and age. Moreover, oxidised pyrimidines (P = 0.01) and alkylated bases (P = 0.001) strongly correlated with years of occupational exposure. Micronucleus frequency did not differ between exposed and control subjects. Repair capacity overall did not show any effect of exposure, though female controls had higher incision rates than did female exposed subjects. However, exposed asbestos workers had significantly higher numbers of chromosomal aberrations (P = 0.01) compared with control group. This finding is consistent with the known association of chromosome aberrations with cancer-risk.

Markers of genetic stability were monitored in lymphocytes from 98 workers employed in rockwool manufacture in a factory in the Slovak Republic, and 43 controls (administrative employees in the same factory). Strand breaks in lymphocyte DNA were higher in exposed compared to control non-smokers, but there was no effect of exposure on specific damage to bases in DNA, nor on chromosome aberrations. The frequency of micronuclei was higher in women in the control group than in rockwool-exposed women. DNA repair (8-oxoguanine DNA glycosylase activity) was unaffected by exposure, but was negatively correlated with micronucleus frequency, implying that unrepaired 8-oxoguanine contributes to micronucleus formation. The conclusion from this study is that, overall, rockwool exposure has no deleterious effect on genetic stability in humans.

Three diterpenoid quinones (royleanone- SAR 3, horminone- SAR 26, and acetyl horminone- SAR 43) isolated from the roots of Salvia officinalis L. were tested for their cytotoxic and DNA-damaging activity in human colon carcinoma cells Caco-2 and human hepatoma cells HepG2 cultured in vitro. Cytotoxicity was measured by the trypan blue exclusion technique and induction of apoptosis was evaluated by flow immunofluorocytometry after 30-300 min. exposure of HepG2 and Caco-2 cells to diterpenoid quinones and following 24 hr post-incubation in the culture medium. Induction of DNA breaks was measured after 60 min. exposure of cells to different concentrations of the compounds studied by the alkaline elution of DNA and by the Comet assay. Though all the quinones tested decreased the viability of the cells studied proportionally to the concentration and to the time of treatment (cytotoxicity= 30-60%), the increased level of apoptotic nuclei comparable to the level of apoptotic nuclei induced by a topoisomerase I inhibitor was proved only in HepG2 cells treated with 1x10(-4) mol/l SAR 26 or SAR 43. Either no or marginal increase of the level of apoptotic nuclei was observed in SAR 3-treated HepG2 cells and in SAR 3-, SAR 26- or SAR 43-treated Caco-2 cells. All compounds tested induced creation of DNA strand breaks in both cell types at concentrations >1x10(-7)-1x10(-6) mol/l. The occurrence of DNA strand breaks at different pH values as well as the kinetics of DNA breaks rejoining were evaluated only in colonic cells Caco-2. The Comet assay processed in parallel at pH 13.0 and pH 12.1 showed that strand breaks detected in SARs-treated colonic Caco-2 cells originated from alkali-labile sites, as induced DNA lesions were converted to DNA strand breaks only under strong alkaline conditions. The kinetics of DNA rejoining revealed that SARs-induced DNA breaks were repaired very slowly.

Background: The vegetarian lifestyle is supposedly healthy, and differences between vegetarians and non-vegetarians in biomarkers related to diseases such as cancer might be expected., Aim of the Study: To investigate the possible role of different diets in maintaining genomic stability., Methods: The vegetarian group, consisting of 24 volunteers (13 women and 11 men), were matched for age and sex with 24 volunteers (12 women and 12 men) with a traditional dietary habit. Among vegetarians there were 13 lacto-ovo-vegetarians (8 women, 5 men) with average length of vegetarian diet 10.8 years (ranging from 5 to 26) and 11 lacto-vegetarians (5 women, 6 men) with average length of vegetarian diet 8.2 years (ranging from 3 to15). All volunteers were nonsmokers, non-consumers of alcohol and had similar education and patterns of physical activity. Chromosome aberrations, micronuclei and DNA damage (strand breaks, oxidised bases and H(2)O(2)-sensitivity) were examined in peripheral blood lymphocytes of vegetarians and non-vegetarians. Plasma antioxidant status was assessed with the FRAP assay., Results: We did not find any differences in percentage of cells with chromosome aberrations or in the frequency of micronuclei between vegetarians and non-vegetarians or between lacto-ovo and lacto-vegetarians. There was no statistically significant difference in total antioxidant capacity between the groups. The group with traditional dietary habits had significantly higher levels of oxidative DNA damage (strand breaks and oxidised purines, P = 0.005) compared with vegetarians. A significant positive correlation between age and oxidative DNA damage (net FPG-sensitive sites) was found in non-vegetarians, while there was an opposite trend towards a negative association in vegetarians. On the other hand chromosome aberrations correlated with age in vegetarians (r = 0.48, P = 0.017) but not in non-vegetarians., Conclusions: Our results indicate that a vegetarian diet can lead to a slight decrease in oxidative DNA damage in lymphocytes, but other markers of genetic stability are not affected. The lowest level of DNA damage was found in lymphocytes of lactovegetarians, (especially oxidised pyrimidines, P = 0.0017), suggesting that this diet provides some protection against oxidative stress.

The objective of this study was to examine plasma homocysteine levels and C677T methylenetetrahydrofolate reductase (MTHFR) gene polymorphism in two ethnic groups from Slovakia. The samples consisted of general Slovak-Romany population (68 men and 81 women) from Southwestern Slovakia and the Slovak-Caucasians (174 men and 177 women) who participated in the CINDI project. The homocysteine levels were examined by HPLC, the analysis of MTHFR genotypes was done by PCR. The Slovak-Romany men (12.0+/-5.6 (S.D.) micromol/l) and women (9.2+/-2.6 microol/l) have significantly lower plasma homocysteine levels (p<0.024 and p<0.00001) when compared to Caucasians (13.3+/-5.1 micromol/l in men and 11.3+/-4.3 micromol/l in women). The genetic equilibrium is assumed for the gene frequencies of the MTHFR polymorphism in both samples. The distribution of MTHFR genotypes did not differ between the two populations (TT 13 vs. 10.6 %; CT 46.6 vs. 41.7 %; CC 40.4 vs. 47.7 %, chí(2)2 = 2.315, df=2, ns). The effect of MTHFR genotypes on homocysteine levels was not confirmed in the Slovak-Romanies and TT homozygosity significantly increased plasma homocysteine levels only in Slovak-Caucasians (11.5+/-4.4 micromol/l, ns; vs. 14.8+/-4.8 micromol/l, p 0.002, respectively). To our knowledge, this is the first epidemiological study in the Romany population examining distribution of the MTHFR genotypes and their effect on homocysteine levels. Further studies are needed to establish the variety of cardiovascular risk factors among Romanies in order to evaluate the significance of particular factors.

Living organisms possess a variety of self-protective mechanisms which decrease the free radical attack on DNA and so reduce the risk of cancer. Protection of DNA by endogenous antioxidant systems may be significantly increased by numerous exogenously administered antioxidants. Many of them represent important dietary factors. Biopolymer lignin with its phenolic structure can be included into this group of micronutrients. The aim of the present work was to investigate: 1. the effect of biopolymer lignin, given to Sprague-Dawley (SD) rats in diet, on the level of oxidative DNA lesions induced by oxidative stress in freshly isolated peripheral blood lymphocytes in vitro and 2. the influence of lignin on kinetics of rejoining of DNA strand breaks induced in lymphocytes under these conditions. As model oxidative agents were used H2O2 and visible light in the presence of the photosensitizer Methylene Blue. We found out that dietary intake of lignin caused a significant decrease of H2O2-induced DNA strand breaks and visible light-induced oxidative DNA lesions in freshly isolated rat lymphocytes, but it did not influence the kinetics of rejoining of DNA strand breaks.

In order to investigate the effects of antioxidant supplementation on chromosome damage, a 3 month antioxidant supplementation trial was conducted on groups of 28 myocardial infarction survivors and 57 rural controls, all male. The supplement consisted of vitamin C (100 mg/day), vitamin E (100 mg/day), beta-carotene (6 mg/day) and selenium (50 microg/day). Dietary antioxidants in plasma were measured, as well as the ferric reducing ability of plasma (a measure of total plasma antioxidant status) and the concentration of malondialdehyde as an indicator of oxidative stress. Lymphocytes collected at the beginning and end of the supplementation period were stimulated to proliferate and metaphases accumulated for scoring of chromosome aberrations: per cent aberrant cells and chromatid and chromosome breaks. Supplementation with antioxidants was associated with a decrease in the percentage of cells with chromosome aberrations in the group of rural controls (0.63% before compared with 0.27% after supplementation; P = 0.03). The largest effect of supplementation was seen in smokers in this group (0.12% aberrant cells in supplemented compared with 0.81% in placebo group; P > 0.001). The results support the hypothesis that antioxidants decrease genetic damage.

Dietary antioxidant levels in the blood depend on intake of fruits and vegetables and therefore might be expected to show seasonal variation. A group of healthy male subjects in Bratislava, Slovakia gave blood samples each month for 1 year. Vitamin C, alpha- and gamma-tocopherol and several carotenoids were measured in plasma, and concentrations of essential metals zinc, copper and selenium in serum. Oxidative DNA damage was assessed in lymphocytes using the comet assay. Seasonal variations in antioxidant levels did not follow a common pattern. beta-Cryptoxanthin was highest in the spring. Lycopene peaked in late summer. Lutein/zeaxanthin was higher in summer than in winter. The concentration of zinc in serum was higher in winter than in summer. DNA damage was lower in summer than in winter. Selenium as well as several antioxidants correlated negatively with indices of DNA damage, while zinc levels showed a positive correlation with DNA damage. These results provide some support for a link between consumption of antioxidants and protection against DNA oxidation.

Hepatocytes freshly isolated from male Wistar rats fed a common diet or a vitamin A- or vitamin E-supplemented diet (each for 21, 28, or 41 days) were assayed for sensitivity to DNA breakage and cytogenetic changes induced by carcinogens. Different indirectly acting carcinogens were assayed. N-nitrosomorpholine (NMOR) was the only agent that induced DNA breaks, chromosomal aberrations, and micronuclei in all experiments. Benzo[a]pyrene (B[a]p) and dimethyldibenzo [c,g]carbazole (diMeDBC) induced only DNA breaks in all experiments. Occasionally, B[a]P induced chromosomal aberrations and micronuclei, and diMeDBC induced micronuclei, but not chromosomal aberrations. These results demonstrated that the tested carcinogens assayed at concentrations highly effective in a hypoxanthine phosphoribosyltransferase/V79 system significantly increased DNA damage, while cytogenetic changes were less frequent. In hepatocytes from rats fed vitamin A, a reduction in the severity of all three end points was observed after NMOR treatment. After B[a]P treatment, we found a reduction in DNA breaks and chromosomal aberrations; after treatment with diMeDBC, we observed a reduction in DNA breaks. Treatment with vitamin E was less effective: it reduced DNA strand breaks induced by B[a]P and partially reduced those induced by diMeDBC and NMOR and the level of micronuclei induced by NMOR and B[a]P. Both vitamins reduced the level of DNA strand breaks induced by the oxidative effect of a visible light-excited photosensitizer.

The impact of environmental pollution at the place of residence of pregnant women and of their smoking habits on the cellular energy metabolism of placental tissue was investigated. Samples of full-term placentas were randomly collected from two environmentally different regions of Slovakia (Bratislava, Stará Lubovna) and the activity of lactate dehydrogenase (LDH) was measured. Our results showed enhanced LDH activity in the placenta that was dependent on both the type of environmental pollutants at the place of residence and the smoking habits during pregnancy. The enhanced LDH activity may reflect hypoxic conditions due to the accumulation of heavy metals and toxic compounds of tobacco smoke in the placental tissue. A high content of heavy metal particles, found in placental samples from Stará Lubovna in our previous studies, might contribute to the increased LDH activity in placentas from this region. We hypothesize that fine metal particles deposited in the placental tissue might be phagocytozed by the syncytiotrophoblast, thus contributing to the decreased oxygen level in placental tissue.

Previous morphological studies failed to show appreciable injury of biliary epithelial cells (BEC) after cold ischemia of rat liver, although recent evidence indicated that BEC integrity and function were impaired in this model. We tested the hypothesis that analysis of bile for enzymes, such as lactate dehydrogenase (LDH), alanine transaminase (ALT), and aspartate transaminase (AST), can be used for assessing cold ischemic injury of BEC. Furthermore, we examined whether biliary gamma-glutamyltransferase (GGT) reflects warm ischemic injury of BEC and whether normothermic reperfusion aggravates the negative effect of cold ischemia on BEC integrity and function. Rat livers were reperfused after different periods of cold or warm ischemia using a blood-free perfusion model. Compared with controls, perfusate LDH, ALT, and AST levels and parameters of hepatocyte function, including hepatocyte tight junction permeability, were not significantly altered by 18-h cold ischemia. On the other hand, 9-h cold ischemia markedly increased biliary LDH, ALT, and AST levels. However, only LDH release into the bile was strongly dependent on the time of cold storage. Biliary GGT, LDH, and glucose levels decreased during the reperfusion period following 18-h cold ischemia. The results suggest that biliary LDH can be used for assessing injury of BEC in cold-preserved livers and that normothermic reperfusion does not aggravate preservation-induced injury of BEC after cold ischemic storage., (Copyright 2000 Academic Press.)

Background: Rat liver transplantation models or isolated liver perfusion models are currently used for assessing efficacy of liver preservation methods. We tested the hypothesis that hepatocellular enzymes released into the washout solution after preservation may predict hepatic function during reperfusion and could thus be alternatively used for evaluating efficiency of liver preservation solutions. Furthermore, we applied this approach for assessing the role of Kupffer cells (KC) in preservation-induced liver damage., Methods: After preservation in University of Wisconsin (UW) or Euro-Collins (EC) solution, rat livers were washed with Ringer-lactate solution. Correlations between enzymes released into the washout solution and hepatocyte functional parameters determined during reperfusion on using a blood-free perfusion model were investigated., Results: In UW-preserved livers, acid phosphatase (ACP) activity correlated negatively with bile flow (R = -0.904), taurocholate intrinsic clearance (R = -0.841), and bromosulfophthalein excretion (R = -0.831). Both alanine transaminase and aspartate transaminase activities correlated with the functional parameters investigated. In EC-stored livers, correlation was also found between ACP activity and bile flow (R = -0.666). Livers stored in UW solution exhibited approximately 3 times lower washout activities of enzymes studied than livers stored in EC solution. Mitochondria isolated from UW-stored livers exhibited significantly better function than those isolated from EC-stored livers. Blockade of KC did not influence enzyme release into the washout solution., Conclusions: Determination of ACP, alanine transaminase, and aspartate transaminase activities in the washout solution can be used as a rapid, simple, and cost-effective way for screening liver preservation solutions. The results also suggest that KC were not involved in preservation-induced liver damage.

The aim of the present study was to investigate the ability of the chloroacetanilide herbicide acetochlor to interact with the endocrine system. The modulation of the binding of [3H]estradiol-17beta to protamine sulphate-precipitated uterine nuclear and cytoplasmic estrogen receptors was analysed for this purpose. Two different stages of reproductive life cycle of female rats, virgin and uniparous, were used. Our results demonstrate that acetochlor interacts in a specific manner with high-affinity limited-capacity uterine estrogen receptors. A significant difference (p < 0.001) in estrogen receptor density was observed between two control groups: uniparous rats (Bmax = 43.634 +/- 9.516) and virgin rats (Bmax = 154.375 +/- 21.462), suggesting an intrinsic difference in the nuclear estrogen receptor levels between female rats in different reproductive life cycle stages. Consequently, a different response to acetochlor treatment was noted. Exposure to acetochlor significantly (p < 0.001) increased estrogen receptor binding activity in the nuclear fraction of uniparous female rats (Bmax = 123.324 +/- 5.666) in comparison to control (Bmax = 43.634 +/- 9.516). In exposed virgin female rats, no significant difference was detected when compared to the corresponding control group. These results should prompt us to more thoroughly evaluate potential hazards of acetochlor to human and wildlife endocrine systems.

Background/aims: In clinical transplantation, exposure of donors to gut-derived endotoxin occurs frequently and may adversely affect liver transplantation therapy. The aim of this study was to investigate: 1) whether brief exposure of rats to endotoxin before liver procurement aggravates the early phase of reperfusion injury of hepatic explants; and if so 2) whether Kupffer cell activation is a contributing factor to liver injury; and 3) whether heparin and pentoxifylline could minimize this effect., Methods: Male Wistar rats were injected with 0.2-4.0 mg/kg of Escherichia coli lipopolysaccharide 2 h prior to liver harvest. After preservation in University of Wisconsin cold-storage solution, the livers were reperfused using a blood-free perfusion model. To inactivate Kupffer cells, some rats were pretreated with gadolinium chloride or liposome-encapsulated dichloromethylene-diphosphonate before lipopolysaccharide administration. The other rats received lipopolysaccharide with heparin or pentoxifylline., Results: In a dose-independent fashion, lipopolysaccharide impaired portal flow during graft reperfusion. In a dose-dependent way, lipopolysaccharide increased lactate dehydrogenase release into the perfusate and decreased bile flow and bromosulfophthalein excretion. Gadolinium chloride, liposomal dichloromethylene-diphosphonate, heparin, and pentoxifylline reduced lactate dehydrogenase release by 34%, 43%, 59%, and 64%, respectively, and improved functional parameters of the liver. A 52-fold increased neutrophil infiltration in the liver sinusoids after lipopolysaccharide exposure was not affected significantly by the drugs studied; however, heparin reduced markedly neutrophil activation., Conclusions: The results of this investigation provide direct evidence that aggravation of preservation-reperfusion injury of rat liver by endotoxin is mediated by Kupffer cell-dependent mechanism(s) and it can be minimized by heparin and pentoxifylline.

Evaluation of the 50% inhibitory concentration (IC50) of acetylsalicylic acid, ferrous sulfate, amitriptyline, methanol, isopropanol and ethylene glycol was done on human cancer cells cultured in in vitro conditions. Three different in vitro assays were used in this study: the plating efficiency test, the microprotein test and the neutral red uptake test. Obtained results were evaluated by statistical methods. All used methods seem to be useful for screening a cytotoxic potential of the tested chemicals. The knowledge of cytotoxic effects of frequently used chemicals on mammalian cells is important not only for necessary in vitro genotoxicity and carcinogenicity studies but also for assessing the toxicity of chemicals to find out possible hazards to the human health. Results presented in this paper underline the usefulness of the wider methodological approach for the comparison of the different endpoints as well as a necessity for selection of a battery of in vitro cytotoxicity tests allowing to estimate the possible harmful effects of xenobiotics.

Mutagenicity of N-cyclohexylthiophthalimide (Duslin P) was tested first by the Ames test in the bacteria, Salmonella typhimurium. The negative results of the Ames test suggested that this compound does not induce mutations in the genome of S. typhimurium under the conditions used. To estimate the cytotoxicity of Duslin P to human cells, we measured cellular DNA and protein as well as cell proliferation, i.e., the mitotic index of treated and control cells. The genotoxic effects were assayed by two biochemical methods developed for detection of single-strand breaks of DNA in mammalian cells, i.e., by the alkaline single cell gel electrophoresis (comet assay) and by the DNA unwinding method, respectively. The DNA unwinding method showed that this compound did not induce DNA damage at concentrations < 7 micrograms/ml. Alkaline single cell gel electrophoresis revealed approximately double the level of DNA damage (in comparison to untreated control DNA) at a concentration of 2 micrograms/ml, which reduced proliferation to approximately 30%, and triple the level of DNA damage at higher concentrations (6 and 7 micrograms/ml), which inhibited completely both DNA synthesis and proteosynthesis. Cells with moderately damaged DNA were more common than cells with heavily damaged DNA. Parallel experiments with the strong mutagen and carcinogen MNNG showed that MNNG induced in cells a high level of DNA damage at concentrations which did not reduce the mitotic index or proteosynthesis, while DNA synthesis inhibited only partially. After treatment with MNNG, cells with heavily damaged DNA were more common than cells with moderately damaged DNA. Duslin P-treated VH10 cells were also tested cytogenetically, confirming that Duslin P induced neither chromosomal aberrations nor aneuploidy. We conclude that Duslin P has no mutagenic effect on bacteria, does not induce chromosomal aberrations and CREST positive or CREST negative micronuclei in human cells and induces only a small increase of DNA damage in human cells which is consistent with DNA fragmentation due to cell death.

Apoptosis is genetically programmed cell death, an irreversible process of cell senescence with characteristic features (cell shrinkage, chromatin condensation, DNA fragmentation, apoptotic bodies) different from necrosis. Several effective in vitro methods for qualitative and quantitative detection of apoptotic events have been developed. Chromatin degradation, reductions in cell volume and other apoptosis-associated changes in cell morphology and physiology can be quickly analysed by multiparametric flow cytometry (FC) using small numbers of intact cells. One further method used for morphological determination of apoptotic changes is the fluorescent microscopic technique (FM) based on labeling of cells with fluorochromes acridine orange and ethidium bromide. In our experiments FC was used for determination of DNA changes in HeLa cells based on staining of DNA by fluorochrome propidium iodide (PI). Among the tested chemicals (paracetamol and sodium fluoride) apoptotic process could only be detected in paracetamol-treated cells. Apoptosis was induced mainly in cells treated with paracetamol (concentration range 4-5 mg/ml) for 8 h and following incubation for 18 h in fresh medium without paracetamol. The results obtained by the FM method correlated with the results obtained by FC.