Association between heat-shock protein 70 gene polymorphisms and DNA damage in peripheral blood lymphocytes among coke-oven workers

Hsp70 has been shown to act as a chaperone and be associated with cytoprotection against DNA damage caused by environmental stresses. However, it is unknown whether genetic variation in HSP70 plays a role in stress tolerance and cytoprotection against DNA damage. We determined the frequencies of three polymorphisms, HSP70-1 G190C, HSP70-2 G1267A, and HSP70-hom T2437C from 251 steel-plant workers exposed to coke-oven emission and 130 controls. We estimated the association between the HSP70variants/haplotypes and the levels of DNA damage in their peripheral blood lymphocytes detected by single-cell gel electrophoresis assay. Our results showed that overall coke-oven workers had higher levels of the Olive tail moment (Olive TM) (1.27+/-1.12) than that of the controls (0.56+/-0.99, P<0.001). Coke-oven workers with the HSP70-1 C/C genotype had higher levels of Olive TM (2.19+/-0.65), compared with HSP70-1 G/C and G/G carriers (Olive TM=1.34+/-1.09 and 1.14+/-1.08, respectively, P=0.022 and 0.003, respectively). However, the HSP70-2 G1267A and HSP70-hom T2437C polymorphisms were not associated with the levels of Olive TM (P=0.929 and 0.795, respectively). Haplotype analysis showed that carriers of TCG/TCG haplotype pairs had the highest levels of Olive TM among both the exposed subjects (2.04+/-0.59) and the controls (0.81+/-0.59). Our results suggest that the individuals with the homozygous HSP70-1 C/C genotype among the coke-oven workers may be susceptible to DNA damage.     

Associations between XRCC1 and ERCC2 polymorphisms and DNA damage in peripheral blood lymphocyte among coke oven workers.

A wide variety of base damages and single-strand breaks formed by reactive oxygen species during metabolic activation of polycyclic aromatic hydrocarbons (PAHs) have been recognized to be involved in PAH carcinogenesis. In this study, alkaline comet assay was used to detect the DNA damage in peripheral blood lymphocytes among 143 coke-oven workers and 50 non-coke-oven workers, and the effects of genetic polymorphisms of XRCC1 and ERCC2 genes on DNA damage were evaluated. The olive tail moment was significantly higher in coke-oven workers than in non-coke-oven workers (2.6, 95% CI=2.1-3.3 versus 1.0, 95% CI=0.8-1.2, p<0.01), and significant correlation between ln-transformed urinary 1-OHP and ln-transformed olive tail moment was found in total population (n=193, Pearson's r=0.393, p<0.001) and in coke-oven workers (n=143, Pearson's r=0.224, p=0.007). The olive tail moment was significantly higher in coke-oven workers with GA genotype of G27466A polymorphism of XRCC1 than those with GG genotype (4.6, 95% CI=2.5-8.7 versus 2.4, 95% CI=1.9-2.9, p<0.01 with adjustment for covariates). No significant associations between C26304T, G28152A and G36189A polymorphisms of XRCC1 and G23591A and A35931C polymorphisms of ERCC2 and olive tail moment were found in both groups. The study showed that the alkaline comet assay is a suitable biomarker in the detection of DNA damage among coke-oven workers and it suggested that the A allele of G27466A polymorphism of XRCC1 may be associated with decreased DNA repair capacity toward PAH-induced base damage and strand breaks.     

Genotoxic effects of occupational exposure to lead and cadmium

This study was designed to assess genotoxic damage in somatic cells of workers in a Polish battery plant after high-level occupational exposure to lead (Pb) and cadmium (Cd), by use of the following techniques: the micronucleus (MN) assay, combined with in situ fluorescence hybridization (FISH) with pan-centromeric probes, analysis of sister chromatid exchanges (SCEs), and the comet assay. Blood samples from 44 workers exposed to lead, 22 exposed to cadmium, and 52 unexposed persons were used for SCE and MN analysis with 5'-bromodeoxyuridine (BrdU) or cytokinesis block, respectively. In parallel, the comet assay was performed with blood samples from the same persons for detection of DNA damage, including single-strand breaks (SSB) and alkali-labile sites (ALS). In workers exposed mostly to lead, blood Pb concentrations ranged from 282 to 655 microg/l, while the range in the controls was from 17 to 180 microg/l. Cd concentration in lead-exposed workers fell in the same range as for the controls. In workers exposed mainly to cadmium, blood Cd levels varied from 5.4 to 30.8 microg/l, with respective values for controls within the range of 0.2-5.7 microg/l. Pb concentrations were similar as for the controls. The incidence of MN in peripheral lymphocytes from workers exposed to Pb and Cd was over twice as high as in the controls (P<0.01). Using a combination of conventional scoring of MN and FISH with pan-centromeric probes, we assessed that this increase may have been due to clastogenic as well as aneugenic effects. In Cd- and Pb-exposed workers, the frequency of SCEs as well as the incidence of leukocytes with DNA fragmentation in lymphocytes were slightly, but significantly increased ( P<0.05) as compared with controls. After a 3h incubation of the cells to allow for DNA repair, a clear decrease was found in the level of DNA damage in the controls as well as in the exposed workers. No significant influence of smoking on genotoxic damage could be detected in metal-exposed cohorts. Our findings indicate that lead and cadmium induce clastogenic as well as aneugenic effects in peripheral lymphocytes, indicating a potential health risk for working populations with significant exposures to these heavy metals.     

Follow-up study of the genetic damage in lymphocytes of pharmacists and nurses handling antineoplastic drugs evaluated by cytokinesis-block micronuclei analysis and single cell gel electrophoresis assay

A follow-up study was carried out 4 years after an initial evaluation of the micronucleus frequency in 10 healthy individuals who had been occupationally exposed to antineoplastic drugs in a Brazilian hospital. Upon the first evaluation, these 10 exposed individuals were compared with 10 non-exposed individuals matched for age, sex and smoking habits; the results revealed that the frequency of micronucleated lymphocytes in individuals exposed to antineoplastic drugs was significantly higher (P=0.038) than in controls. The frequency of dicentric bridges was also increased, although not significantly (P=0.0545). After the first analysis, the workers handling antineoplastic drugs were advised to modify their work schedule to limit exposure, and the number of workers in the group was increased from 10 to 12 individuals. In the follow-up study, 12 individuals from the same work area were assessed. In addition to micronucleus frequency, alkaline single cell gel electrophoresis was also used to monitor genetic hazard. This exposed group was compared to 12 non-exposed workers from the same hospital, matched for age, sex and smoking habits. In the follow-up study, no statistical difference was found between exposed workers and controls in terms of micronucleus and dicentric bridge frequency with the Mann--Whitney U-test (P=0.129 and 0.373, respectively). However, the mean value of SCGE analysis was significantly higher in the exposed group than in the controls (P=0.0006). Although the micronucleus analysis seems to be less sensitive to assess DNA damage, it detects chromosome aberrations and not just repairable DNA breakage and alkali-labile sites. Combination of the alkaline single cell gel electrophoresis and cytokinesis blocked micronucleus assay appears to be commendable to monitor populations chronically exposed to genotoxic agents.     

Overexpressed heat shock protein 70 protects cells against DNA damage caused by ultraviolet C in a dose-dependent manner

Heat shock protein 70 (Hsp70) comprises proteins that have been reported to protect cells, tissues, and organisms against damage from a wide variety of stressful stimuli; however, little is known about whether Hsp70 protects against DNA damage. In this study, we investigated the relationship between Hsp70 expression and the levels of ultraviolet C (UVC)-induced DNA damage in A549 cells with normal, inhibited, and overexpressed Hsp70 levels. Hsp70 expression was inhibited by treatment with quercetin or overexpressed by transfection of plasmids harboring the hsp70 gene. The level of DNA damage was assessed by the comet assay. The results showed that the levels of DNA damage (shown as the percentage of comet cells) in A549 cells increased in all cells after exposure to an incident dose of 0, 10, 20, 40, and 80 J/m2 whether Hsp70 was inhibited or overexpressed. This response was dose dependent: a protection against UVC-induced DNA damage in cells with overexpressed Hsp70 was observed at UVC dose 20 J/m2 with a maximum at 40 J/m2 when compared with cells with normal Hsp70 levels and in quercetin-treated cells. This differential protection disappeared at 80 J/m2. These results suggest that overexpressed Hsp70 might play a role in protecting A549 cells from DNA damage caused by UVC irradiation, with a threshold of protection from at UVC irradiation-induced DNA damage by Hsp70. The detailed mechanism how Hsp70 is involved in DNA damage and possible DNA repair warrants further investigation.     

Assessment of DNA damage in peripheral blood of heavy smokers with the comet assay and the micronucleus test

The comet assay (single-cell gel electrophoresis, SCG) is being increasingly used in human biomonitoring for the detection of genotoxic exposures. Cigarette smoking is a well-documented source of a variety of potentially mutagenic and carcinogenic compounds. Therefore, smoking should represent a relevant mutagenic exposure and lead to genotoxic effects in exposed cells. However, our previous investigations as well as several other published studies on human biomonitoring failed to show an effect of smoking on DNA migration in the comet assay, while some other studies did indicate such an effect. Although many factors can contribute to the generation of discrepant results in such studies, clear effects should be obtained after high exposure. We therefore performed a comparative study with healthy male heavy smokers (>20 cigarettes per day) and non-smokers (n=12 in each group). We measured the baseline comet assay effects in fresh whole blood samples and isolated lymphocytes. In addition, the amount of 'formamidopyrimidine DNA-glycosylase (FPG)-sensitive sites' was determined by a combination of the standard comet assay with the bacterial FPG protein. Furthermore, the influence of a repair inhibitor (aphidicolin, APC) on baseline DNA damage was comparatively analysed. Duplicate slides from each sample were processed and analysed separately. In all experiments, a reference standard (untreated V79 cells) was included to correct for assay variability. Finally, to compare the comet assay results with another genetic endpoint, all blood samples were investigated in parallel by the micronucleus test (MNT). Baseline and gamma radiation-induced micronucleus frequencies were determined. None of these approaches revealed a significant difference between heavy smokers and non-smokers with regard to a genotoxic effect in peripheral blood cells.     

Vanadate induces DNA strand breaks in cultured human fibroblasts at doses relevant to occupational exposure

To study possible genotoxic effects of occupational exposure to vanadium pentoxide, we determined DNA strand breaks (with alkaline comet assay), 8-hydroxy-2'deoxyguanosine (8-OHdG) and the frequency of sister chromatid exchange (SCE) in whole blood leukocytes or lymphocytes of 49 male workers employed in a vanadium factory in comparison to 12 non-exposed controls. In addition, vanadate has been tested in vitro to induce DNA strand breaks in whole blood cells, isolated lymphocytes and cultured human fibroblasts of healthy donors at concentrations comparable to the observed levels of vanadium in vivo. To investigate the impact of vanadate on the repair of damaged DNA, co-exposure to UV or bleomycin was used in fibroblasts, and DNA migration in the alkaline and neutral comet assay was determined. Although, exposed workers showed a significant vanadium uptake (serum: median 5.38microg/l, range 2.18-46.35microg/l) no increase in cytogenetic effects or oxidative DNA damage in leukocytes could be demonstrated. This was consistent with the observation that in vitro exposure of whole blood leukocytes and lymphocytes to vanadate caused no significant changes in DNA strand breaks below concentrations of 1microM (50microg/l). In contrast, vanadate clearly induced DNA fragmentation in cultured fibroblasts at relevant concentrations. Combined exposure of fibroblasts to vanadate/UV or vanadate/bleomycin resulted in non-repairable DNA double strand breaks (DSBs) as seen in the neutral comet assay. We conclude that exposure of human fibroblasts to vanadate effectively causes DNA strand breaks, and co-exposure of cells to other genotoxic agents may result in persistent DNA damage.     

Genotoxicity assessment in Eisenia andrei coelomocytes: a study of the induction of DNA damage and micronuclei in earthworms exposed to B[a]P- and TCDD-spiked soils

Earthworms are useful indicators of soil quality and are widely used as model organisms in terrestrial ecotoxicology. The assessment of genotoxic effects caused by environmental pollutants is of great concern because of their relevance in carcinogenesis. In this work, the earthworm Eisenia andrei was exposed for 10 and 28 days to artificial standard soil contaminated with environmentally relevant concentrations of benzo[a]pyrene (B[a]P) (0.1, 10, 50ppm) and 2,3,7,8-tetrachloro-dibenzo-para-dioxin (TCDD) (1×10(-5), 1×10(-4), 2×10(-3)ppm). Micronucleus (MNi) induction was evaluated in earthworm coelomocytes after DNA staining with the fluorescent dye DAPI. In the same cells, the DNA damage was assessed by means of the alkaline comet assay. Induction of MNi in coelomocytes, identified according to standard criteria, was demonstrated. B[a]P exposure for 10 and 28 days induced a significant increase in MNi frequency. In TCDD-treated earthworms, a significant effect on chromosomal damage was observed at all the concentrations used; surprisingly, greater effects were induced in animals exposed to the lowest concentration (1×10(-5)ppm). The data of the comet assay revealed a significant increase in the level of DNA damage in coelomocytes of earthworms exposed for 10 and 28 days to the different concentrations of B[a]P and TCDD. The results show that the comet and MN assays were able to reveal genotoxic effects in earthworms exposed even to the lowest concentrations of both chemicals tested here. The combined application in E. andrei of the comet assay and the micronucleus test, which reflect different biological mechanisms, may be suggested to identify genotoxic effects induced in these invertebrates by environmental contaminants in terrestrial ecosystems.     

Comparative analysis of cytotoxic, genotoxic and antioxidant effects of 2,2,4,7-tetramethyl-1,2,3,4-tetrahydroquinoline and ethoxyquin on human lymphocytes

2,2,4,7-Tetramethyl-1,2,3,4-tetrahydroquinoline (THQ) is a new synthetic compound with potential antioxidant activity. In this study, cytotoxic, genotoxic and antioxidant activities of THQ were studied on human lymphocytes with the use of the trypan blue exclusion assay, the TUNEL method, the comet assay and the micronucleus test. The activities of THQ were compared with those of a structurally similar compound-ethoxyquin (1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline, EQ), which is used in animal feeds as a preservative. Cytotoxic effects of THQ were observed after 1-h treatment at the concentration of 500 microM and after 24-h treatments at the concentrations of 250-500 microM. Although the micronucleus test did not reveal a genotoxic effect of THQ, in the comet assay the statistically significant increase in DNA damage was observed as compared with the control. On the other hand, the protection of human lymphocytes against DNA damage induced by hydrogen peroxide suggests an antioxidant activity of THQ. The comparative analysis of THQ and EQ activities performed in these studies revealed that THQ was less cytotoxic and less genotoxic than EQ. Slightly lower antioxidant activity of THQ was also shown in the comet assay when it was used at the lower studied doses (1-5 microM), but for the highest one (10 microM) its efficiency was similar to that of EQ. In the micronucleus assay THQ was more effective than EQ in protecting the cultured lymphocytes from clastogenicity of H2O2. We believe that THQ is worthy of further detailed studies on its antioxidant properties to confirm its usefulness as a preservative.     

Genotoxic effects of Roundup on the fish Prochilodus lineatus

Glyphosate-based herbicides, such as Roundup, represent the most extensively used herbicides worldwide, including Brazil. Despite its extensive use, the genotoxic effects of this herbicide are not completely understood and studies with Roundup show conflicting results with regard to the effects of this product on the genetic material. Thus, the aim of this study was to evaluate the genotoxic effects of acute exposures (6, 24 and 96 h) to 10 mg L(-1) of Roundup on the neotropical fish Prochilodus lineatus. Accordingly, fish erythrocytes were used in the comet assay, micronucleus test and for the analysis of the occurrence of nuclear abnormalities and the comet assay was adjusted for branchial cells. The results showed that Roundup produces genotoxic damage in erythrocytes and gill cells of P. lineatus. The comet scores obtained for P. lineatus erythrocytes after 6 and 96 h of exposure to Roundup were significantly higher than respective negative controls. For branchial cells comet scores were significantly higher than negative controls after 6 and 24 h exposures. The frequencies of micronucleus and other erythrocyte nuclear abnormalities (ENAs) were not significantly different between Roundup exposed fish and their respective negative controls, for all exposure periods. In conclusion, the results of this work showed that Roundup produced genotoxic effects on the fish species P. lineatus. The comet assay with gill cells showed to be an important complementary tool for detecting genotoxicity, given that it revealed DNA damage in periods of exposure that erythrocytes did not. ENAs frequency was not a good indicator of genotoxicity, but further studies are needed to better understand the origin of these abnormalities.     

The genotoxic risk of hospital, pharmacy and medical personnel occupationally exposed to cytostatic drugs--evaluation by the micronucleus assay

The aim of this study was to evaluate the genotoxicity of cytostatic drugs in hospital and pharmacy employees (n=100), occupationally exposed. The micronucleus assay was used to study lymphocytes in 247 peripheral blood samples. Samples were collected at "baseline level" without any cytostatic drugs exposure before recruiting or after at least 3 weeks without cytostatic drugs contact and at three times (cycle 1-3) post-exposure. Samples from 60 office employees served as controls. Furthermore, our results were compared to urinary analyses of cytostatic drugs (oxazaphosporines, anthracyclines, platinum) which were collected in parallel to the cytogenetic investigation. Statistical analyses were performed under consideration of age, gender and X-ray exposure. The frequency of micronuclei was significantly related to the age of the subjects (r(Spearman)=0.16; P<0.05). However, there were no significant differences in micronucleus rates between controls and exposed hospital workers. Similarly, micronucleus rates were not significantly different at the various sampling time points and there was no correlation between duration of employment and micronucleus rates. Furthermore, no correlation between current biomonitoring data of exposure (urine tests) and micronuclei frequency was found. Therefore, significantly increased genotoxic damage of the lymphocytes investigated in this study could not be demonstrated.     

Associations between XRCC1 and ERCC2 polymorphisms and DNA damage in peripheral blood lymphocyte among coke oven workers

A wide variety of base damages and single-strand breaks formed by reactive oxygen species during metabolic activation of polycyclic aromatic hydrocarbons (PAHs) have been recognized to be involved in PAH carcinogenesis. In this study, alkaline comet assay was used to detect the DNA damage in peripheral blood lymphocytes among 143 coke-oven workers and 50 non-coke-oven workers, and the effects of genetic polymorphisms of XRCC1 and ERCC2 genes on DNA damage were evaluated. The olive tail moment was significantly higher in coke-oven workers than in non-coke-oven workers (2.6, 95% CI=2.1–3.3 versus 1.0, 95% CI=0.8–1.2, p<0.01), and significant correlation between ln-transformed urinary 1-OHP and ln-transformed olive tail moment was found in total population (n=193, Pearson's r=0.393, p<0.001) and in coke-oven workers (n=143, Pearson's r=0.224, p=0.007). The olive tail moment was significantly higher in coke-oven workers with GA genotype of G27466A polymorphism of XRCC1 than those with GG genotype (4.6, 95% CI=2.5–8.7 versus 2.4, 95% CI=1.9–2.9, p<0.01 with adjustment for covariates). No significant associations between C26304T, G28152A and G36189A polymorphisms of XRCC1 and G23591A and A35931C polymorphisms of ERCC2 and olive tail moment were found in both groups. The study showed that the alkaline comet assay is a suitable biomarker in the detection of DNA damage among coke-oven workers and it suggested that the A allele of G27466A polymorphism of XRCC1 may be associated with decreased DNA repair capacity toward PAH-induced base damage and strand breaks.     

Protective<Emphasis Type="Italic">in vivo</Emphasis> effect of curcumin on copper genotoxicity evaluated by comet and micronucleus assays

Curcumin is a phytochemical with antiinflammatory, antioxidant and anticarcinogenic activities. Apparently, curcumin is not genotoxic in vivo, but in vitro copper and curcumin interactions induce genetic damage. The aim of this study was to test if in vivo copper excess induces DNA damage measured by comet and micronucleus assays in the presence of curcumin. We tested 0.2% curcumin in Balb-C mice at normal (13 ppm) and high (65, 130 and 390 ppm) copper ion concentrations. The comet and micronucleus assays were performed 48 hr after chemical application. Comet tail length in animals treated with 0.2% curcumin was not significantly different from the control. Animals exposed to copper cations (up to 390 ppm) exhibited higher oxidative DNA damage. Curcumin reduced the DNA damage induced by 390 ppm copper. We observed statistically significant increase in damage in individuals exposed to 390 ppm copper versus the control or curcumin groups, which was lowered by the presence of curcumin. Qualitative data on comets evidenced that cells from individuals exposed to 390 ppm copper had longer tails (categories 3 and 4) than in 390 ppm copper + curcumin. A statistically significant increase in frequency of micronucleated erythrocytes (MNE/10000TE) was observed only in 390 ppm copper versus the control and curcumin alone. Also cytotoxicity measured as the frequency of polychromatic erythrocytes (PE/1000TE) was attributable to 390 ppm copper. The lowest cytotoxic effect observed was attributed to curcumin. In vivo exposure to 0.2% curcumin for 48 hr did not cause genomic damage, while 390 ppm copper was genotoxic, but DNA damage induced by 390 ppm copper was diminished by curcumin. Curcumin seems to exert a genoprotective effect against DNA damage induced by high concentrations of copper cations. The comet and micronucleus assays prove to be suitable tools to detect DNA damage by copper in the presence of curcumin.     

Assessment of genotoxic effect of benzo[a]pyrene in endotracheally treated rat using the comet assay

Although benzo[a]pyrene (B[a]P) is a well-known genotoxic agent, little is known about the extent of DNA effects induced by B[a]P in rat tissues after pulmonary exposure. The alkaline single-cell gel electrophoresis (comet assay) was used to measure DNA single-strand breaks in alveolar macrophages, lung cells, peripheral lymphocytes and hepatocytes of OFA Sprague-Dawley rats exposed to a single dose of B[a]P by endotracheal administration.Statistically significant damage was observed in all organs tested after 3, 24 and 48h of pulmonary exposure to 3mg of B[a]P per animal, with a time-dependent relationship. The maximum damage was observed in the four cell types 24h after exposure. The higher level of damage was observed both in lung cells and peripheral lymphocytes; in alveolar macrophages and hepatocytes the level of damage was increased, but at a lower level than in the two other cell types. Furthermore, B[a]P demonstrated a clear dose-related genotoxic activity in the lung cells when tested at doses of 0.75, 1.5 and 3mg.The current study shows that B[a]P caused DNA single-strand breaks in the respiratory tract of endotracheally treated OFA Sprague-Dawley rats. The study also suggests that pulmonary exposure to B[a]P can induce a high level of DNA damage in peripheral lymphocytes. The clear relationship between lung exposure to B[a]P and consequences observed in lymphocytes suggests that the comet assay in peripheral lymphocytes can be used as a sensitive marker in human monitoring studies.     

Lack of adverse effect of smoking habit on DNA strand breakage and base damage, as revealed by the alkaline comet assay

In our preceding papers [M. Wojewódzka, M. Kruszewski, T. Iwanenko, A.R. Collins, I. Szumiel, Application of the comet assay for monitoring DNA damage in workers exposed to chronic low dose irradiation: I. Strand breakage, Mutat. Res., 416 (1998) 21-35; M. Kruszewski, M. Wojewódzka, T. Iwanenko, A.R. Collins, I. Szumiel, Application of the comet assay for monitoring DNA damage in workers exposed to chronic low dose irradiation: II. Base damage, Mutat. Res. , 416 (1998) 37-57.], we evaluated the DNA breakage and base damage with the use of comet assay in a group of 49 workers chronically exposed to low doses of ionizing radiation. There was a statistically significant difference in the damage levels between the hazard and control group. In this paper we describe a confounding lack of effect of the smoking habit on the DNA damage in the tested groups. The genotoxic effect of the smoking habit, as well as its modifying effect on genome damage inflicted by other agents, have been firmly established. However, no statistically significant effect of smoking was found in our study, neither in the control nor in the hazard group. This lack of effect was seen in all DNA damage determinations, both direct (DNA strand breakage and alkali-labile lesions) and enzyme-combined (base damage) and did not depend on the comet parameters, which were taken as damage indicators.     

Effect of quercetin on nicotine-induced biochemical changes and DNA damage in rat peripheral blood lymphocytes

We elucidated the protective effect of quercetin, a polyphenolic flavonoid, on lipid peroxidation, endogenous antioxidant status and DNA damage during nicotine-induced toxicity in cultured rat peripheral blood lymphocytes as compared to N-acetylcysteine (NAC), a well-known antioxidant. Lymphocytes were exposed to nicotine (3 mM) with and without quercetin and NAC (1 mM) in RPMI-1640 medium for 1 h. In preliminary experiments to fix the effective dose of quercetin, different doses of quercetin (25, 50, 75, 100 and 200 microM) were administered to lymphocytes with nicotine, and lipid peroxidation markers (thiobarbituric acid reactive substances and hydroperoxides) were analysed. A 75 microM dose of quercetin was found to be effective as evidenced by decreased lipid peroxidation. To evaluate the protective potential of quercetin against genotoxic effects of nicotine we used comet and micronucleus assays, which are valid parameters to assess genetic damage. In addition, biochemical changes including lipid peroxidation and antioxidant status were assessed. There were significant increases in the levels of lipid peroxidation, comet parameters and micronuclei frequencies, followed by decrease in the endogenous antioxidant status, in nicotine-treated lymphocytes, which were brought back to near normal by quercetin or NAC treatment. The protective effect of quercetin against nicotine toxicity was comparable to that of NAC. These findings suggest that quercetin can be as effective as NAC in protecting rat peripheral lymphocytes against nicotine-induced cellular and DNA damage.     

Serum and lymphocyte levels of heat shock protein 70 in aging: a study in the normal Chinese population

Heat shock proteins (Hsps) have been reported to play an important role in both physiological and pathological processes. Hsps also may serve as biomarkers for evaluating disease states and exposure to environmental stresses. Whether Hsp levels in serum and lymphocytes are correlated with age and sex is largely unknown. In this study, we analyzed serum Hsp70 (the most abundant mammalian Hsp) levels by using Western dot blot in 327 healthy male donors aged between 15 and 50 years. We also investigated the association between Hsp70 levels and age in lymphocytes of 80 normal individuals aged between 40 and 77 years because various chronic diseases increase after the age of 40 years. Our data showed that serum Hsp70 levels were positively correlated with age in subjects aged between 15 and 30 years (P < 0.05) but negatively correlated with age in subjects aged between 30 and 50 years (P < 0.05). Serum Hsp70 levels were the highest in individuals aged between 25 and 30 years among all age groups. In the lymphocyte study there also was a significant age-related decrease in Hsp70 levels in lymphocytes of individuals older than 40 years. The Hsp70 levels were negatively correlated with age (r = -3.708, P < 0.0001) but not with sex (r = -10.536, P = 0.452). This suggests that both serum and lymphocyte Hsp70 levels are age-related and that these may be linked to age-related stress. Thus, age is an important factor in using serum and lymphocyte Hsp70 as biomarkers to evaluate the disease states or exposure to environmental stresses (or both).     

Evaluation of DNA damage in a population of bats (Chiroptera) residing in an abandoned monazite mine

Ionising radiation has the ability to induce DNA damage. While the effects of high doses of radiation of short duration have been well documented, the biological effects of long-term exposure to low doses are poorly understood. This study evaluated the clastogenic effects of low dose ionising radiation on a population of bats (Chiroptera) residing in an abandoned monazite mine. Bats were sampled from two chambers in the mine, where external radiation levels measured around 20 microSv/h (low dose) and 100 microSv/h (higher dose), respectively. A control group of bats was sampled from a cave with no detectable radiation above normal background levels. The micronucleus assay was used to evaluate residual radiation damage in binucleated lymphocytes and showed that the micronucleus frequency per 500 binucleated lymphocytes was increased in the lower radiation-exposed group (17.7) and the higher radiation-exposed group (27.1) compared to the control group (5.3). This study also showed that bats exposed to radiation presented with an increased number of micronuclei per one thousand reticulocytes (2.88 and 10.75 in the lower and high radiation-exposed groups respectively) when compared to the control group (1.7). The single-cell gel electrophoresis (comet) assay was used as a means of evaluating clastogenecity of exposure to radiation at the level of individual cells. Bats exposed to radiation demonstrated increased DNA damage as shown by the length of the comet tails and showed an increase in cumulative damage. The results of the micronucleus and the comet assays indicated not only a statistically significant difference between test and control groups (P<0.001), but also a dose-dependent increase in DNA damage (P<0.001). These assays may thus be useful in evaluating the potential clastogenecity of exposure to continuous low doses of ionising radiation.     

Genotoxic risk assessment of pathology and anatomy laboratory workers exposed to formaldehyde by use of personal air sampling and analysis of DNA damage in peripheral lymphocytes

A study was conducted to evaluate the genotoxic effect of occupational exposure to formaldehyde on pathology and anatomy laboratory workers. The level of exposure to formaldehyde was determined by use of passive air-monitoring badges clipped near the breathing zone of 59 workers for a total sampling time of 15min or 8h. To estimate DNA damage, a chemiluminescence microplate assay was performed on 57 workers before and after a 1-day exposure. Assessment of chromosomal damage was carried out by use of the cytokinesis-blocked micronucleus assay (CBMN) in peripheral lymphocytes of 59 exposed subjects in comparison with 37 controls matched for gender, age, and smoking habits. The CBMN assay was combined with fluorescent in situ hybridization with a pan-centromeric DNA probe in 18 exposed subjects and 18 control subjects randomized from the initial populations. Mean concentrations of formaldehyde were 2.0 (range <0.1-20.4ppm) and 0.1ppm (range <0.1-0.7ppm) for the sampling times of 15min and 8h, respectively. No increase in DNA damage was detected in lymphocytes after a one-workday exposure. However, the frequency of binucleated micronucleated cells was significantly higher in pathologists/anatomists than in controls (16.9 per thousand+/-9.3 versus 11.1 per thousand+/-6.0, P=0.001). The frequency of centromeric micronuclei was higher in exposed subjects than in controls (17.3 per thousand+/-11.5 versus 10.3 per thousand+/-7.1) but the difference was not significant. The frequency of monocentromeric micronuclei was significantly higher in exposed subjects than in controls (11.0 per thousand+/-6.2 versus 3.1 per thousand+/-2.4, P<0.001), while that of the acentromeric micronuclei was similar in exposed subjects and controls (3.7 per thousand+/-4.2 and 4.1 per thousand+/-2.7, respectively). The enhanced chromosomal damage (particularly chromosome loss) in peripheral lymphocytes of pathologists/anatomists emphasizes the need to develop safety programs.     

Studying the genotoxic effects induced by two kinds of bentonite particles on human B lymphoblast cells in vitro

The aim of the present study was to evaluate the genotoxic effects induced by native and active bentonite particles (BPs) on human B lymphoblast cells using comet assay and cytokinesis-block micronucleus (CBMN) assay in vitro. The cells were exposed to BPs at the concentrations of 30, 60, 120 and 240μg/ml for 24, 48 and 72h, respectively. The quartz contents of native and active BPs were 6.80±0.20 and 6.50±0.10%, respectively. Gypsum and DQ-12 quartz served as negative and positive controls. The results of comet assay showed that DNA damage induced by native and active BPs was significantly higher than that induced by gypsum control (P<0.05 or <0.01), and increased with exposure concentration and duration. When the cells were exposed to BPs at the doses of 120 and 240μg/ml for 72h, DNA damage induced by active BPs and native BPs was significantly higher than that induced by DQ-12 quartz (P<0.01), and DNA damage induced by active BPs enhanced significantly, as compared with native BPs (P<0.01). The results of CBMN assay demonstrated that both native BPs and active BPs could induce significant micronuclei, as compared with gypsum control (P<0.05 or <0.01). However, there was no significant difference of micronucleus frequency (MNF) among native BPs, active BPs and DQ-12 quartz. The water-soluble fractions from two kinds of BPs did not induce significant DNA damage and micronuclei. These findings indicated that the genotoxicity induced by active BPs and native BPs could be detected in comet assay and CBMN assay in vitro, the insoluble particle fractions from BPs may play a main role in the genotoxic effects induced by BPs.