Microsomal epoxide hydrolase polymorphisms, cigarette smoking and prostate cancer risk in the Slovak population.

Polymorphisms in tobacco carcinogen metabolizing enzymes may generate interindividual variations towards the risk of developing prostate cancer. One of these enzymes is microsomal epoxide hydrolase (EPHX1) which metabolizes polycyclic aromatic hydrocarbons, or PAH, carcinogens found in cigarette smoke. The activity of this enzyme is affected by two polymorphisms, a substitution of Tyr113 by His in exon 3 and a substitution of His139 by Arg in exon 4. The aim of this study was to use a population-based case-control study to investigate whether or not such genetic polymorphisms in EPHX1 gene can modify the relationship between smoking status and the risk of developing prostate cancer. We used restriction fragment length polymorphism, or PCR-RFLP to determine EPHX1 genotypes in subjects comprising 194 patients with histologically verified prostate cancer and 305 healthy individuals as control. We found no overall association between prostate cancer risk and functional polymorphisms of EPHX1 gene in exon 3 and exon 4. We further analysed the association between the EPHX1 genotypes and smoking. Smokers carrying the exon 3 Tyr/Tyr and Tyr/His genotypes were at no significant risk compared to non-smokers with the "rapid" Tyr/Tyr genotype. By contrast, a significant interaction of smoking and the exon 4 polymorphism was present.

[Relationship of the CDKAL1 and KCNQ1 gene polymorphisms to the age at diagnosis of type 2 diabetes in the Slovakian population]. / Vzt'ah polymorfizmov génov CDKAL1 a KCNQ1 k veku diagnózy diabetu 2. typu v populácii slovenska.

BACKGROUND/AIMS: The association of CDKAL1 and KCNQ1 genes with type 2 diabetes mellitus (DM2T) was confirmed by several genome-wide association studies in both Caucasian and Asian populations. For both genes, it is supposed that the risk of DM2T development is related to impaired insulin secretion. Based on assumption that the presence of risk allele might predispose to an earlier onset of DM2T, the aim of the present study was to assess the frequency of risk alleles of CDKAL1 rs7756992 and KCNQ1 rs163184 polymorphisms and to analyze their association with the age at DM2T diagnosis in the Slovakian population. METHODS: CDKAL1 rs7756992 A/G and KCNQ1 rs163184 G/T polymorphisms were genotyped using asymmetric PCR with subsequent melting curve analysis in a group of 538 patients with DM2T. Anthropometric and laboratory parameters were determined by using standard methods. Since two genes were analysed, the required level for statistical significance was defined as p < 0.025. RESULTS: Risk homozygotes (CG) for KCNQ1 polymorphism had higher mean age of DM2T diagnosis by 2 years when compared to T-allele carriers (GT + TT) in a recessive model, but the difference did not reach the predefined level of statistical significance. No relationship of CDKAL1 polymorphism to the age at onset of DM2T diagnosis was observed. CONCLUSIONS: In the present study, no relationship of CDKAL1 and KCNQ1 polymorphisms to the earlier onset of type 2 diabetes was observed.

Relationship of five type 2 diabetes candidate gene polymorphisms to the age at diagnosis of diabetes in the Slovakian population.

OBJECTIVES: To examine the relationship between polymorphisms of five candidate genes for type 2 diabetes mellitus (T2DM) and the age at diagnosis of T2DM. METHODS: 538 Slovakian patients with T2DM were included and their age at diagnosis of T2DM retrieved from their medical records. Polymorphisms of genes encoding peroxisome proliferator activated receptor gamma (PPARG), PPARG-coactivator-1 (PGC1), insulin-receptor substrate 1 (IRS1), the subunit Kir 6.2 of the ATP-dependent potassium-channel (KCNJ11) and transcription factor 7-like 2 (TCF7L2) were detected by PCR-RFLP methods. RESULTS: No significant relationship between the risk alleles of the examined gene polymorphisms to the lower mean age at diagnosis of T2DM was observed. The carriers of the TT-genotype of TCF7L2 rs7903146 polymorphism had significantly increased odds ratio for diagnosis of diabetes before the age of 40 years [OR 3.02 (1.34, 6.81), p = 0.008], in comparison with the CC/CT genotype carriers. CONCLUSION: No significant association of PPARG, PGC1, IRS1, KCNJ11 and TCF7L2 gene polymorphisms and the age at diagnosis of T2DM was observed in the present study. Homozygotes for the risk allele of TCF7L2 had more frequently early onset of T2DM, before age of 40 years (Tab. 4, Ref. 15).

Gene polymorphisms of renin angiotensin system and serotonin transporter gene in patients with vasovagal syncope.

UNLABELLED: Objective of this study was to compare the distribution frequencies of gene polymorphisms of renin-angiotensin and serotonin system in patients with positive and negative head- up tilt test (HUT). METHODS: DNA from 191 patients (mean age 44+ 18 years, 61 men) was collected. HUT was positive in 117 and negative in 74 patients. Following gene polymorphisms were determined by the PCR method: ACE insertion/deletion (I/D ACE), angiotensinogen (AGT) (M 235), angiotensin II receptor (ATR1) (A 1166C) and serotonin transporter (SERT) polymorphism (5HTTLPR). RESULTS: No significant differences in the distribution of gene polymorphisms between syncopal patients with positive and negative HUT were dectected. Distribution of polymorphisms included: I/D ACE: II 19 vs 20%, ID 55 vs 52%, DD 26 vs 28%. Angiotensinogen gene polymorphism MM 27% vs 30%, MT 48% vs 46%, TT 25% vs 24%. ATR1 polymorphism AA 44 vs 32%, AC50 vs 60%, CC 6 vs 8%, 5HTTLPR serotonin transporter gene polymorphism LL 42 vs 43%, SL 41 vs 39%, SS 17 vs 18%. CONCLUSIONS: An association between polymorphisms of ACE, AGT, ATR1 and SERT gene, and predisposition to VVS was not proven by the present study (Tab. 2, Ref. 22). Full Text (Free, PDF) www.bmj.sk.

Relationship among urinary albumin excretion rate, lipoprotein lipase PvuII polymorphism and plasma fibrinogen in type 2 diabetic patients.

Plasma fibrinogen level represents a strong cardiovascular risk factor and is regulated by an interplay of genetic and environmental factors. Hyperfibrinogenemia frequently occurs in cluster with dyslipidemia within the frame of insulin resistance syndrome (IRS) and type 2 diabetes mellitus. Genetic variants with a pleiotropic effect have been proposed to cause IRS features including hyperfibrinogenemia. We studied the influence of polymorphisms in lipoprotein lipase (LPL) gene, beta-fibrinogen gene (FIBB) and environmental factors on plasma fibrinogen levels in type 2 diabetes patients. 131 type 2 diabetes patients (mean age 62+/-10 years, 33% male) were genotyped for polymorphisms in LPL gene (intron 6 PvuII, intron 8 HindIII) and FIBB gene (-148C/T, -455G/A) by PCR-RFLP method. Fibrinogen was measured by thrombin coagulation method, albuminuria by immunoturbidimetric assay. Polymorphism LPL PvuII showed a gene-dose effect on fibrinogen levels, with the highest fibrinogen in P-P- homozygotes (p = 0.05, analysis of variance). P-carriers (P-P- and P+P- combined) had significantly higher fibrinogen levels compared with P+P+ homozygotes (3.74+/-1.40 g/l vs 3.06+/-1.20 g/l, p=0.03). Other studied polymorphisms were not significantly related to fibrinogen levels. Age- and sex-adjusted fibrinogenemia correlated significantly with albuminuria (r = 0.48, p=0.001), serum uric acid (r = 0.42, p=0.006) and serum creatinine (r = 0.32, p=0.04). Multiple stepwise linear regression identified interaction term of LPL PvuII and albuminuria as an independent predictor of fibrinogen level, explaining 18% of fibrinogen variance. Albuminuria thus appears to be the best predictor of fibrinogen plasma levels in type 2 diabetic patients. Relationship between albuminuria and fibrinogenemia may be modified by the genotype LPL PvuII, which also shows a weak association with plasma fibrinogen level in type 2 diabetes patients.

Effect of gene polymorphisms on lipoprotein levels in patients with dyslipidemia of metabolic syndrome.

Dyslipidemia in the metabolic syndrome (MS) is considered to be one of the most important risk factors for atherosclerosis. It is characterized by hypertriglyceridemia, low concentration of plasma HDL-cholesterol, predominance of small dense LDL particles and an increased concentration of plasma apolipoprotein B (apoB). The pathogenesis of this type of dyslipidemia is partially explained, but its genetic background is still unknown. To evaluate the influence of cholesterol ester transfer protein (CETP) TaqIB polymorphism, lipoprotein lipase (LPL) PvuII and HindIII polymorphisms, hepatic lipase (LIPC) G-250A polymorphism and apolipoprotein C-III (APOC3) SstI gene polymorphism on lipid levels in dyslipidemia of the metabolic syndrome, 150 patients with dyslipidemia of metabolic syndrome were included. 96 % of patients had type 2 diabetes. The patients did not take any lipid lowering treatment. The exclusion criterion was the presence of any disease that could affect lipid levels, such as thyroid disorder, liver disease, proteinuria or renal failure. Gene polymorphisms were determined using the polymerase chain reaction and restriction fragment length polymorphisms. The genotype subgroups of patients divided according to examined polymorphisms did not differ in plasma lipid levels with the exception of apoB. The apoB level was significantly higher in patients with S1S1 genotype of APOC3 SstI polymorphism when compared with S1S2 group (1.10+/-0.26 vs. 0.98+/-0.21 g/l, p=0.02). Similarly, patients with H-H- genotype of LPL HindIII polymorphism had significantly higher mean apoB, compared with H+H- and H+H+ group (1.35+/-0.30 vs. 1.10+/-0.26 g/l, p=0.02). In the multiple stepwise linear regression analysis, apoB level seemed to be influenced by APOC3 SstI genotype, which explained 6 % of its variance. The present study has shown that the S1 allele of APOC3 SstI polymorphism and the H- allele of LPL HindIII polymorphism might have a small effect on apoB levels in the Central European Caucasian population with dyslipidemia of metabolic syndrome.

A pilot study testing the genetic polymorphism of N-acetyltransferase 2 as a risk factor in lung cancer.

NAT2 as phase II enzyme is involved in the detoxification/activation of various drugs, environmental substances and carcinogenic compounds. A genotyping approach has been used to investigate NAT2 genotype with putative relevance in lung cancer in population of 110 Slovak-Caucasians patients and 167 non-malignant individuals from the same region. Slow acetylation was not observed to be a significant risk factor of lung cancer development (OR=1.19; 95% CI: 0.71-1.99). However, one genotype responsible for slow acetylation (NAT2*5B/*6) was observed significantly more frequently in lung cancer patients with squamous cell carcinoma compared with control subjects (OR=2.24; 95% CI: 1.14-4.34). Stratified analysis showed an increasing impact of the specific allelic combination NAT2*5B/*6 in non-smokers (OR=6.5; 95% CI: 1.25-15.08). In the case of squamous lung carcinoma an analysis revealed a tendency to adversely affect cancer risk in the individuals with the mentioned genotype in younger than 60 years (OR=3.14; 95% CI: 0.98-9.72) non-smokers (OR=10.40; 95% CI: 1.35-118.89) and in females (OR=4.25; 1.08-16.25). Additional studies are needed to confirm the results we observed and to assess the impact of other effects (specific allelic combinations, sex differences and histological subtype of lung cancer) on NAT2 susceptibility in lung carcinogenesis.

Combined analysis of polymorphisms in glutathione S-transferase M1 and microsomal epoxide hydrolase in lung cancer patients.

Many genes involved in the metabolism of carcinogens have been found to be polymorphic in the human population, and specific alleles are associated with increased risk of cancer at various sites. The etiology of most commonly occurring cancers cannot be explained by allelic variability at a single locus. A combined analysis of two polymorphic enzymes, glutathione S-transferase M1 (GSTM1), microsomal epoxide hydrolase (EPHX1)) and their implication as lung cancer risk factors was performed in a case- control study of non small cell lung cancer. Polymerase chain reaction (PCR) or PCR-RFLP-based methods were used to detect variant genotypes of GSTM1 and EPHX1 (113Tyr-113His in exon 3 and 139His-139Arg in exon 4) in 150 controls and group of lung cancer patients (n=121). The slow 113His EPHX1 allele tended to be more frequent among the patients (frequency 0.587) than among the controls (0.320) (Fisher s exact test, p=0.33). The combined EPHX1 homozygote genotype His113/His139 (predicted very slow activity) versus all other genotype combination was associated with an increased risk of lung cancer (OR=2.29; 95% C.I.=0.94- 5.82), particularly in non-smokers (OR=11.23; 95% C.I.=1.48- 88.41). Polymorphism in GSTM1 had no statistically significant impact on lung cancer risk alone (OR=1.09; 95% C.I.: 0.65-1.82). However, obtained the results revealed that combinations GSTM1 null with homozygote His113/His139 genotype (predicted very slow activity EPHX1) significantly increased lung cancer risk (OR=3.65; 95% C.I.: 1.04-16.07). No overall relationship between genotype combinations predicted high EPHX1 activity and lung cancer risk was confirmed in all followed respects. However, the number of investigated individuals in our study was relatively small, therefore these findings should be judged with circumspectness.

[Renin-angiotensin system: molecular biology]. / Renín-angiotenzínový systém: molekulárna biológia.

Renin-angiotensin system (RAS) have been extensively studied in last few decades. RAS regulates blood pressure, water and electrolytes balance. The disorders in function of RAS may play a potential role in development of some complex diseases like: hypertension, myocardial infarction, stroke, nephropathies and renal failure, chronic obstruction pulmonary disease and many more. RAS may take part in formation and progression of these diseases. In this work we focus on molecular biology of RAS and polymorphisms of RAS genes.

Allelic and haplotype frequencies of the p53 polymorphisms in brain tumor patients.

The polymorphisms of the tumor suppressor gene p53 in exon 4 (p53 BstUI) and in intron 6 (p53 MspI) have been suggested to be associated with the genetically determined susceptibility in diverse types of human cancer. In our hospital-based case-control study, we examined the allele and genotype incidence of these polymorphisms as well as their haplotype combinations in 60 brain tumor patients (27 males and 33 females) and 183 controls without malignancies. The genotype characteristics were determined by the PCR-based RFLP method using DNA extracted from peripheral blood. In this study we show that the p53 BstUI and the p53 MspI polymorphisms are not associated with increased risk of brain tumors. Thus, we conclude that the p53 BstUI and the p53 MspI polymorphic sites within the tumor suppressor gene p53 do not represent genetic determinants of susceptibility to brain tumors.

Germ line polymorphisms of the tumor suppressor gene p53 and lung cancer.

Two p53 variable sites (BstUI and MspI SNPs in exon 4 and intron 6, respectively) and their haplotype combinations were studied in 109 patients (84 males and 25 females) with lung cancer and 113 healthy controls from the region of Eastern Slovakia. There were no differences found between lung cancer patients and controls carrying the distribution of p53 BstUI and MspI alleles. However, the genotype distribution showed a significantly higher proportion of MspI heterozygotes in lung cancer patients (P=0.048, OR 1.83, 95% CI 1.00-3.34) than in controls. The analysis based on haplotype frequencies showed the presence of BstUI-MspI 2-1 haplotype in cancer patients (5.4%) in contrast to the absence of this haplotype in healthy controls. The results of this study suggest that the p53 MspI polymorphism may modify the susceptibility to lung cancer as a single factor rather than in combination with BstUI polymorphism.

Polymorphism of the p53 gene within the codon 72 in lung cancer patients.

We tested the codon 72 single nucleotide polymorphism (SNP) of the tumor suppressor gene p53 for association with lung cancer. In our hospital-based case-control study, 168 lung cancer patients (134 males and 34 females) and 148 controls without malignant diseases were recruited. The genotype characteristics were determined by PCR-based RFLP method using DNA extracted from peripheral blood. Only in lung cancer patients but not in the controls we found both significant decrease of A1 allele of the p53 codon 72 (p=0.024, OR 0.56, 95% CI 0.43-0.72) and A1/A1 homozygous genotype (p=0.006, OR 0.27,95% CI 0.15-0.51). The results of this study suggest a protective effect of A1 allele against lung cancer.

p53 single nucleotide polymorphisms and bladder cancer.

Two p53 germline polymorphisms, a BstUI in exon 4 and a MspI in intron 6 were studied using polymerase chain reaction (PCR) based methods in 50 patients with bladder cancer and 145 healthy controls. Increased frequencies of the BstUI and MspI A2 alleles were found to be associated with statistically non-significant (p = 0.2308 and p = 0.5959) but increased odd ratios for bladder cancer (OR 1.44, 95% CI 0.82-2.27 and OR 1.20, 95% CI 0.61-2.33). Statistically significant difference between patients with bladder cancer and controls was found in the distribution of MspI genotypes. There was a significantly lower proportion of the heterozygous A1A2 genotype in all patients but not in controls (p = 0.0354). The results of this study suggest that BstUI and Msp1 germ line polymorphisms of the tumor suppressor gene p53 marginally modify the risk of bladder cancer.

[Genetic polymorphism of enzymes metabolizing xenobiotics as a factor in susceptibility to tumor development]. / Genetický polymorfizmus enzýmov metabolizmu xenobiotík ako faktor susceptibility pri vzniku nádorových ochorení.

Organisms are exposed to a large number of xenobiotics (compounds foreign to the body), such as drugs, pesticides, natural food constituents and so on. To deal with these, usually lipophilic substances, a range of biotransformation enzyme systems are available. Experimental data have shown that metabolic activation and detoxification play an important role in chemical carcinogenesis. There is a considerable interindividual genetic variability in these pathways which might explain the differences in cancer susceptibility and might ultimately enable the identification of people at increased risk and to offer individualized cancer prevention. Recently, molecular genetic bases of many polymorphic enzyme activities involved in xenobiotics metabolism have been elucidated. The purpose of this article is to provide a brief review of recent findings concerning the association of genetically determined metabolic variants with different risks of environmentally induced cancer. (Tab. 6, Fig. 1, Ref. 30.)

Reciprocal adaptive response of human peripheral lymphocytes induced by bleomycine or gamma rays.

The adaptive response and reciprocal adaptive response induced in vitro by exposure to low doses of gamma rays (0.05 Gy) or bleomycin (0.05 microg/ml) in human peripheral blood lymphocytes were assessed by the frequency of chromosome aberrations. Gamma rays (1.5 Gy) or bleomycin (1.5 microg/ml) were used as the challenge doses. In the experiments, blood samples from 5 healthy donors were investigated. It has been found that low doses of bleomycin and gamma rays induced a reciprocal adaptive response to high doses of gamma rays or bleomycin. Moreover, the results confirmed that the adaptive response did not correlate with the radiosensitivity of the peripheral blood lymphocytes.

The role of human glutathione S-transferases M1 and T1 in individual susceptibility to bladder cancer.

Several genes involved in the metabolism of carcinogens have been found to be polymorphic in the human population, and specific alleles are associated with increased risk of cancer at various sites. This study is focused on the polymorphic enzymes glutathione S-transferase M1 (GSTM1) and T1 (GSTT1) that are involved in the detoxification of many xenobiotics involved in the etiology of bladder cancer. To investigate the role of GSTM1 and GSTT1 in bladder carcinogenesis, the polymerase chain reaction was used to determine GSTM1 and GSTT1 genotypes of cancer patients (n = 76) and controls (n = 248). The proportion of putative risk GSTM1 null genotype in the case group was 52.6%, compared to 49.6% in the control group, but the GSTT1 0/0 frequency in the bladder cancer group was significantly higher (P = 0.04) in comparison with the control group (27.6 vs 16.9%). Individuals lacking the GSTT1 gene are at an approximately 1.9-fold higher risk (OR = 1.87, C.I. 95% = 1.03-3.42) of developing bladder cancer in comparison with individuals with at least one active allele in the GSTT1 locus. A significantly higher incidence of GSTM1 deletion genotype (P = 0.02) was found in smokers with bladder cancer compared to the controls (70.6 vs 49.6%). Smokers lacking the GSTM1 gene are at an approximately 2.4-fold higher risk of bladder cancer (OR = 2.44, C.I. 95% = 1.10-5.30). The effect of smoking associated with the GSTT1 0/0 genotype was not found to affect the risk of bladder cancer.

Cytogenetic monitoring in coke oven workers.

Cytogenetic markers (chromosomal aberrations, sister chromatid exchanges (SCE), cells with high frequency of SCE (HFC), the heterogeneity index SCE (SCE-H) and genetic polymorphism of genotypes GSTM1 and NAT2 were evaluated in the peripheral lymphocytes of 64 coke oven workers and 34 control subjects from the same plant. Personal monitors were used to evaluate exposure to eight carcinogenic (polycyclic aromatic hydrocarbons) PAHs, including B[a]P, during an 8-h working shift. Smoking habits were checked by urinary cotinine measurement. The exposure among coke oven workers ranged widely from 0.6 to 547 microgram/m3 and 2 to 50 137 ng/m3, for carcinogenic PAHs and B[a]P, respectively. The respective values in controls were 0.07 to 1.51 microgram/m3 and from 2 to 63 ng/m3. The results of biomonitoring in exposed vs. control subjects were as follows: frequency of chromosomal aberrations (% AB.C.), 2. 30% AB.C. vs. 1.09% AB.C. (P<0.05); sister chromatid exchanges, 7.47 SCE/cell vs. 5.49 SCE/cell (P<0.05); HFC, 5.94% vs. 2.06% (P<0.05) and SCE-H index, 1.49 vs. 1.01 (P<0.05). All the cytogenetic markers were significantly increased in the exposed vs. control groups. The effect of smoking was observed only in SCE when evaluated as HFC. Using individual exposure data for carcinogenic PAHs, a significant correlation between exposure and %AB.C. (r=0.372, P=0.0002), SCE/cell (r=0.331, P=0.001), HFC (r=0.467, P=0.007) and SCE/H (r=0. 286, P=0.004) was found. No effects of GSTM1 and NAT2 genotypes, individually or in combination, on the cytogenetic markers was observed. It is concluded that occupational exposure of coke oven workers involved in this study resulted in an increased level of chromosomal aberrations and SCE. The frequency of AB.C. and SCE/cell was found to be related to exposure to carcinogenic PAHs.

Genetic polymorphism of glutathione S-transferases M1 and T1 as a risk factor in lung and bladder cancers.

A combined analysis of two polymorphic enzymes, glutathione S-transferase mu (GST M1) and q (GST T1) and their implication as cancer risk factors was performed in a case-control study of lung and bladder cancers. Using a multiplex polymerase chain reaction (PCR) based method, the frequency of the homozygous deleted GSTM1 and GSTT1 genotypes was examined in 117 lung cancer patients, 67 urinary bladder cancer patients, and in a community-based sample of 248 healthy, unrelated individuals. In both cancer groups the frequency of the GSTM1 null genotype was higher in comparison with that of the control group (59% and 59.7% vs. 49.6%), but this increase did not reach statistical significance (p > 0.05). After grouping by the smoking status, among smokers in both cancer groups (62.1% in lung cancer and 71.4% in the bladder cancer group, respectively) there were statistically significantly (p < 0.05) increased frequencies of the GSTM1 deletion genotype as compared to the control group (49.6%). Smokers with absence of the GSTM1 gene were at an approximately 1.7-fold higher risk for lung cancer (odds ratio--OR = 1.67, 95% confidence interval--CI 95% = 1.0-2.7, p = 0.04) and an approximately 2.5-fold higher risk for bladder cancer (OR = 2.54, CI 95% = 1.2-5.5, p = 0.02). As related to GSTT1, our study demonstrated an overall GSTT1 effect on bladder cancer risk. Individuals with absence of the GSTT1 gene were at an approximately 2.5-fold higher risk of developing bladder cancer. In the lung cancer cases, the frequency of the putatively high risk GSTT1 null genotype was not increased as compared with controls. No effect of smoking was found on risk of lung and bladder cancer associated with the GSTT1 0/0 genotype. In combined analysis, the obtained results suggested that individuals who were both GSTM1 null and GSTT1 null may be at increased risk because they lack both enzymes. The findings suggest that the GSTM1 null genotype may be associated with susceptibility to lung and urinary bladder cancer in dependence on the exposure to carcinogens in cigarette smoke and that the GSTT1 null genotype is not a critical factor in mediating the risk of lung cancer, but may be associated with an increased susceptibility to bladder cancer.

The cell cycle positions influence DNA migration as measured with the alkaline comet assay in stimulated human lymphocytes.

Virtually any eukaryotic cell can be processed for analysis of DNA damage using the comet assay. The most commonly examined human cells are lymphocyte populations. However, many parameters can affect the response of lymphocytes to the assay in terms of the ability to detect damage. The response of cultivated lymphocytes in the comet assay indicated cycle-dependent differences. The cell cycle position has been shown to affect the results obtained using both the alkaline and neutral assays. This is primarily a reflection of the complications of including S-phase DNA. In the alkaline assay, replicating structures are interpreted as strand breaks when denatured, increasing the level of detectable damage. We performed the alkaline comet assay to detect differences in the extent of DNA in stimulated human lymphocytes collected at different sample times after mitogen stimulation. Our results clearly indicate that proliferating lymphocytes have a greater migration of DNA (measured with the comet assay as DNA damage) than quiescent lymphocytes. The lymphocytes collected at 36, 42, and 48 h after mitogen stimulation showed a significantly increased extent of DNA migration in comparison to the lymphocytes collected at 0 and 24 h after stimulation. It, probably, can be explained by the higher frequency of the S phase cells in lymphocyte populations collected at 36, 42, and 48 h. Sites of active DNA replication during the S phase behave like single-strand breaks when denatured in alkali, and their presence may result in a significant increase of the tail moments in S phase cells.

The comet assay for the detection of genotoxic damage in the earthworms: a promising tool for assessing the biological hazards of polluted sites.

The comet assay, a relatively new method for DNA strand break detection in individual cells, is becoming a major tool for environmental biomonitoring. One approach for assessing the possible environmental consequences of hazardous waste pollution involves the assessment of genotoxic damage (and other effects) in sentinel organism. The single cell gel electrophoresis (SCGE) technique or comet assay. because of its simplicity, sensitivity, and need for only small numbers of cells, has been suggested as an ideal technique for such studies. An important advantage of the technique is that it is applicable to any eukaryotic organism and cell type. Verschaeve et al. (1993) conducted a pilot study using alkaline comet assay to assess the extent of DNA damage in coelomic leucocytes (coelomocytes) of earthworms (Eisenia foetida) maintained in different soil samples as an indicator of soil pollution. The aim of our study was to evaluate the usefulness of monitoring single strand breaks in coelomocytes for assessing genotoxicity of pollutants in coke oven area. We exposed earthworms to samples of soils obtained from polluted areas of a coke oven. All samples gave a significantly higher comet tail moment that those obtained from worms kept in laboratory conditions (standard black earth = internal controls) and worms kept in soils from control areas (= external controls). Our results show that the comet assay applied to earthworm is very valuable for monitoring and detection of genotoxic compounds in terrestrial ecosystems.