Cytogenetic biomonitoring of an Italian population exposed to pesticides: chromosome aberration and sister-chromatid exchange analysis in peripheral blood lymphocytes

Chromosome aberrations (CA) and sister-chromatid exchanges (SCE) were measured in lymphocytes of (A) 32 healthy individuals working in the flower industry and exposed to pesticides, (B) 32 individuals exposed as above and hospitalized for bladder cancer, and (C) 31 controls. Compounds to which floriculturists were exposed included 18 nitro-organic herbicides and fungicides, 9 nitro-organic fungicides, 12 organophosphate and organothiophosphate insecticides, 4 hydrocarbon derivative herbicides and 5 inorganic fungicides and insecticides. 150 and 70 metaphases per individual were scored for CA and SCE, respectively. A significant increase in the incidence of CA and SCE was observed in both exposed groups. Cancer patients showed the presence of rare rearrangements (dicentrics, rings and quadriradials) that were not observed in controls and were present at a lower frequency in healthy exposed people. Hyperdiploid and polyploid metaphases were also significantly increased in the 2 exposed groups compared to controls. Stratifying for age or smoking habits, although affecting the significance of individual data, did not change the substance of the results.     

Increased sister-chromatid exchange in bone-marrow cells of mice exposed to whole cigarette smoke

Male Wistar rats were fed diets of varying selenium content in order to obtain selenium-deficient and selenium-supplemented rats. After 5-6 weeks on the respective diet, the rats were used to investigate how selenium influences the effect of dimethylnitrosamine (DMN) on some liver enzymes and related reactions. The selenium-dependent glutathione peroxidase activity in postmicrosomal supernatant from liver was about 1% in selenium-deficient rats as compared to selenium-supplemented rats or rats fed a standard diet. The highest DMN-demethylase activity was observed in postmitochondrial supernatant from selenium-deficient rat liver, and the lowest in selenium-supplemented rats. No dietary effect was observed on hepatic microsomal cytochrome P450 levels. C-Oxygenation of N,N-dimethylaniline (DMA) was not affected by the selenium level. On the other hand, selenium deficiency seemed to reduce N-oxygenation of DMA. The mutagenicity of DMN in Chinese hamster V79 cells after metabolic activation by the isolated perfused rat liver, was approximately doubled when selenium-deficient livers were used as compared to selenium-supplemented livers and livers from rats fed a standard diet. A negative correlation between DMA-N-oxygenation and mutagenicity from DMN was observed, whereas no correlation between DMA-C-oxygenation and mutagenicity from DMN was found.     

Triethylene melamine-induced sister-chromatid exchange in murine lymphocytes exposed in vivo

The induction of sister-chromatid exchange (SCE) by triethylene melamine (TEM), a known animal carcinogen, was investigated in an in vivo exposure/in vitro culture murine lymphocyte assay. Dose-related increases in SCE were observed in B6D2F1 mice following a single i.p. injection of 0.5, 1 or 2 mg/kg TEM. SCE frequencies remained elevated over baseline levels at 24 h post exposure. It is hoped that studies of this nature can determine whether the in vivo/in vitro murine lymphocyte SCE assay is useful for predicting the carcinogenic potential of an agent.     

Chromosome aberrations and sister-chromatid exchange frequencies in pathology staff occupationally exposed to formaldehyde

Past studies have shown that formaldehyde is mutagenic in microbial tests and Drosophila and causes chromosomal aberrations in cultured mammalian cells. Chromosomal analysis of bone marrow cells and spermatocytes from exposed laboratory animals has failed to show any genotoxic effect. Information on individuals occupationally exposed is limited and there is no evidence to date that formaldehyde can induce chromosome damage at occupational levels of exposure. This study examines the chromosome aberration and sister-chromatid exchange frequencies in lymphocytes from a group of 6 pathology workers and 5 unexposed controls. No detectable differences could be found between the groups in either chromosomal aberration induction or sister-chromatid exchange frequencies.     

Variable sister-chromatid exchange response in human lymphocytes exposed in vitro to gossypol acetic acid

Gossypol has potential for widespread use as a male oral antifertility agent in humans since it appears to be highly efficacious, with reversible spermatostatic effects and minimal side effects. Furthermore, it is both inexpensive and readily available. Therefore, a thorough understanding of gossypol's genotoxic potential is critical. Although genotoxicity studies have produced conflicting reports, increased sister-chromatid exchange (SCE) and DNA-strand breaks have been reported in human cells exposed to gossypol in vitro. In the present study, SCE was examined in purified human lymphocytes and whole blood cultures exposed to gossypol acetic acid at various concentrations in serum-free medium. A small but statistically significant increase in SCE was observed in pooled analysis of 7 donors in whole blood cultures exposed to 0.70 microM gossypol acetic acid (p less than 0.02). Individual analyses revealed only one donor with a significant SCE response (p less than 0.001). In subsequent experiments, exposure at higher doses had no effect on SCE frequencies. A small but significant increase in SCE was observed in ficoll/hypaque purified lymphocytes exposed to 0.07 and 0.70 microM gossypol acetic acid. Interpretation of SCE data with variable response is discussed.     

The persistence of sister-chromatid exchange frequencies in men occupationally exposed to vinyl chloride monomer.

The persistence of sister-chromatid exchange frequencies in a population occupationally exposed to the well known chemical mutagen vinyl chloride monomer was studied. It was shown that increased values of sister-chromatid exchange frequencies were still present in the lymphocytes of workers who had not been exposed for 8-120 days and retired persons for 5-10 years after exposure. The possible ability of vinyl chloride monomer alkylating metabolites to cause long-lasting damage of the DNA molecule is discussed.     

Comet assay for assessment of DNA damage in greenhouse workers exposed to pesticides

Purpose: The main goal of the present study was to determine DNA damage in pesticide-exposed greenhouse workers and pesticides non-exposed controls.

Materials and methods: The DNA damage was measured by alkaline comet assay method (pH?>?13) in 41 greenhouse workers and 45 non-exposed individuals as the control. Pesticide exposure was assessed by duration of working in the greenhouse and pesticide application in the greenhouse time. DNA damage was estimated by arbitrary unit and damage frequency.

Results: Arbitrary unit and damage frequency were consistently significantly higher in greenhouse workers than those of the controls (p?=?0.001). In terms of gender in greenhouse, DNA damage of female workers was significantly higher than those in male workers (p?<?0.05). We found significant correlation between DNA damage and working hours spent. Multiple linear regression analysis showed that working hours in the greenhouse as an indication of pesticide exposure were significantly associated with the DNA damage, which can be attributed to the genotoxic potential of the pesticide mixture.

Conclusions: The comet assay is sensitive to detect the damage exposed to chronic effect of pesticides in greenhouse workers. Significant DNA damage was obtained for the exposed group, which was associated with the pesticide exposure.     

Sister-chromatid exchange analysis in a rural population of Mexico exposed to pesticides.

Cytogenetic damage was evaluated by means of the analysis of sister-chromatid exchange (SCE) in a rural population of Tlaxcala, Mexico, in occupational contact with pesticides. We studied 170 men, 94 exposed and 76 not exposed. It was shown that SCE followed a normal distribution and Student's t test did not present differences between the two groups (P = 0.4). The frequency of SCE was not correlated with the duration of exposure of the rural workers (r = -0.06), the multiple covariance analysis applied to the data of duration of exposure, tobacco intake and alcohol ingestion demonstrated a lack of statistical significance. In the exposed people we observed no symptoms provoked by these compounds.     

Micronuclei in peripheral blood lymphocytes and buccal epithelial cells of Polish farmers exposed to pesticides.

In this biomonitoring study, we investigated whether an occupational exposure to a complex mixture of chemical pesticides produced a significant increase of micronuclei (MN) in both peripheral blood lymphocytes and buccal cells. Forty-nine male workers exposed to pesticides, from an agricultural area of Malopolska Region in Southern Poland, together with 50 men from the same area without indication of exposure to pesticides that served as controls, were used in this investigation.No statistically significant differences in the frequencies of cytogenetic damage were detected between exposed and control individuals, for either type of cells. The multiple linear regression analysis in the case of lymphocytes indicated that the studied cytogenetic endpoints were inversely influenced by alcohol; whilst a negative binomial regression, in the case of buccal cells, indicated that the MN values were directly influenced by the ingestion of red meat. An inverse negative relationship between the cytokinesis-block proliferation index and age, and a significant increase of miscarriages due to the exposure to pesticides were also observed.     

Molecular mechanisms of sister-chromatid exchange

Sister-chromatid exchange (SCE) is the process whereby, during DNA replication, two sister chromatids break and rejoin with one another, physically exchanging regions of the parental strands in the duplicated chromosomes. This process is considered to be conservative and error-free, since no information is generally altered during reciprocal interchange by homologous recombination. Upon the advent of non-radiolabel detection methods for SCE, such events were used as genetic indicators for potential genotoxins/mutagens in laboratory toxicology tests, since, as we now know, most forms of DNA damage induce chromatid exchange upon replication fork collapse. Much of our present understanding of the mechanisms of SCE stems from studies involving nonhuman vertebrate cell lines that are defective in processes of DNA repair and/or recombination. In this article, we present a historical perspective of studies spearheaded by Dr. Anthony V. Carrano and colleagues focusing on SCE as a genetic outcome, and the role of the single-strand break DNA repair protein XRCC1 in suppressing SCE. A more general overview of the cellular processes and key protein "effectors" that regulate the manifestation of SCE is also presented.     

Chromosomal aberrations and sister-chromatid exchanges in workers exposed to styrene

Few studies exist about chromosomal damage in workers occupationally exposed to styrene. In the present study, chromosomal aberrations and SCEs were analyzed from cultures of peripheral lymphocytes of workers employed in 6 different reinforced-plastics industries with styrene air exposure levels ranging from 30 to 400 mg/mc. A control group was selected on the base of sex, age and smoking habit. We examined 50-h cultures (for chromosomal-aberrations) and 72-h cultures (for SCEs) for each individual.All workers exposed to styrene, as compared with controls, showed significantly increased frequencies of chromosomal aberrations, while SCEs were significantly increased at 4 of the 6 plants. High SCE values appeared with styrene air concentrations higher than 200 mg/mc.Apart from the possible presence and role of other interfering chemicals in the various plants, chromosomal aberrations seem to be more sensitive than SCEs for the detection of chromosomal damage caused by exposure to low doses of styrene.     

Chromosomal aberrations and sister-chromatid exchanges in workers exposed to styrene

Few studies exist about chromosomal damage in workers occupationally exposed to styrene. In the present study, chromosomal aberrations and SCEs were analyzed from cultures of peripheral lymphocytes of workers employed in 6 different reinforced-plastics industries with styrene air exposure levels ranging from 30 to 400 mg/mc. A control group was selected on the base of sex, age and smoking habit. We examined 50-h cultures (for chromosomal-aberrations) and 72-h cultures (for SCEs) for each individual. All workers exposed to styrene, as compared with controls, showed significantly increased frequencies of chromosomal aberrations, while SCEs were significantly increased at 4 of the 6 plants. High SCE values appeared with styrene air concentrations higher than 200 mg/mc. Apart from the possible presence and role of other interfering chemicals in the various plants, chromosomal aberrations seem to be more sensitive than SCEs for the detection of chromosomal damage caused by exposure to low doses of styrene.     

Physical,chemical, and biological factors affecting sister-chromatid exchange induction in human lymphocytes exposed to mitomycin C prior to culture

In experiments to assess the effects of several biological, chemical, and physical variables on sister-chromatid exchange (SCE) induction in cultured lymphocytes exposed to mitomycin C (MMC) before PHA stimulation we observed: (1) high SCE frequencies in female cells, and normal SCE frequencies in Y-bearing metaphases in mixed cultures containing equal numbers of MMC-treated female lymphocytes and untreated male lymphocytes; (2) small, but statistically significant, decreases in SCEs with increasing pH after G₀ exposure in the pH range 6.6–7.6; (3) pronounced reductions in MMC-induced SCEs in lymphocytes exposed at 4°C vs. 37°C. In other studies, SCE induction was evaluated in cultures exposed during G₀ to MMC concentrations ranging from 0.25 to 2.5 μg/ml for varying time intervals ranging from 5 min to 24 h. For all concentrations tested SCE induction varied as a linear function of G₀ exposure time. To compare SCE induction between cultures, we calculated the mean frequencies of SCEs induced per metaphase/unit dose MMC/unit G₀ exposure time (SCE/μg/h). A mean frequency of 20.7 ± 4.8 SCE/μg/h was observed for 41 lymphocyte cultures suggesting that a single term adequately describes the rate of SCE induction following G₀ exposure to a 10-fold range in concentration of MMC for time intervals of 30 min to 24 h.     

DNA topoisomerases and models of sister-chromatid exchange

Pommier et al. (1985) suggested that sister-chromatid exchange (SCE) results from exchange of topoisomerase II subunits. "Homologous displacement", an alternative mechanism, is proposed in which strand switching occurs during removal of parental helical turns by topoisomerases. The steps in the SCE model proposed by Ishii and Bender (1980) for SCE occurring at a blocked replication fork could occur by this mechanism and would require the action of both topoisomerases I and II. Homologous displacement involving topoisomerase II alone provides a mechanism for the strand switching required in the models of Kato (1977) and Cleaver (1981) in which SCE occur between replicated double strands. These mechanisms and models are discussed in relation to current knowledge of the locations and functions of topoisomerases during DNA replication.     

Frequency of sister-chromatid exchange (SCE) in bone-marrow cells of severely malnourished animals during early life

The frequency of sister-chromatid exchange (SCE) was examined in bone-marrow cells of 21-day-old Wistar rats malnourished during lactation and well-nourished controls of the same age. Malnutrition was obtained by increasing the litter size to 15 pups per mother. SCE were scored in 25 consecutive second-division metaphases in the femoral bone marrow cells from each animal. The average SCE in the malnourished animals was significantly higher than in the control group (p less than 0.01). The distribution of SCE per mitosis was also significantly higher in the malnourished animals (p less than 0.001). These results indicate that malnutrition per se during early life can increase SCE in the bone marrow of experimental animals.     

Genotoxic effects of fluoride evaluated by sister-chromatid exchange

The purpose of this investigation was to study the genotoxic potential of fluoride (in the form of sodium fluoride, NaF) using in vitro and in vivo sister-chromatid exchange (SCE) assays with Chinese hamster cells. The NaF concentrations used in cultures of Chinese hamster ovary (CHO) cells ranged from 0 to 6.3 mM, both with and without S9 activation. Fluoride analysis of the culture medium demonstrated that it contained little indigenous fluoride, and the concentration of added fluoride was not affected by the components of the medium or the S9 mix. The CHO cells cultured in 6.3 mM NaF almost vanished, and at the concentration of 5.3 mM NaF in cultures without S9 microsome, only M1 cells were observed. In in vivo studies, Chinese hamsters were intubated with NaF dosages of 0, 0.1, 1.0, 10, 60 and 130 mg/kg, and the bone marrow (CHBM) cells were examined for SCE frequencies. Bone fluoride data showed that the intubated NaF was effectively absorbed. Death occurred in 3 of the 8 animals given 130 mg NaF/kg. The results indicated that NaF, in dosages up to 5.3 mM in CHO cell cultures and 130 mg/kg in in vivo CHBM cells, did not significantly increase the SCE frequencies over those observed in the negative (distilled water) controls. However, examination of the cell cycle revealed an inhibitory effect of NaF on cell proliferation with doses of NaF at or greater than 1.0 mM in cultured CHO cells and at or greater than 60 mg NaF/kg in in vivo CHMB cells. The results of the present study indicated an inhibition of the cell cycle and death of the cells with increasing concentrations of fluoride but not effect of fluoride on SCE frequency in CHO and CHBM cells.     

Induction of sister-chromatid exchange (SCE) and cell-cycle inhibition in mouse peripheral blood B lymphocytes exposed to mutagenic carcinogens in vivo

To determine the sensitivity of the mouse peripheral blood lymphocyte (PBL) culture system, male B6C3f1 mice were injected i.p. with either 2-acetylaminofluorene (AAF) (20, 40, 80, 160 mg/kg), benzo[a]pyrene (BP) 25, 75, 150, 300 mg/kg), dichlorvos (DCV) (5, 15, 25, 35 mg/kg), ethyl methanesulfonate (EMS) (10, 30, 90, 180, 270 mg/kg), or N-nitrosomorpholine (NM) (37.5, 75, 150, 300 mg/kg) dissolved in either RPMI 1640 (DCV, EMS, NM) or sunflower oil (AAF, BP). 24 h later blood was removed by cardiac puncture, and the lymphocytes were cultured in the presence of lipopolysaccharide for analysis of SCE in B lymphocytes. All 4 mutagenic carcinogens (AAF, BP, EMS, NM) induced significant dose-related increases in SCE frequency. DCV, a potent neurotoxicant, caused no change in the baseline SCE frequency. At the highest concentration of each chemical examined, AAF caused a 1.6-fold increase, EMS a 1.8-fold increase, NM a 3.0-fold increase, and BP a 3.1-fold increase in SCE frequency compared to concurrent controls. A comparison of these results for PBLs with those reported in the literature for bone marrow cells indicates that PBLs offer a good quantitative and qualitative estimate of the SCE-inducing potential for these 5 compounds in bone marrow cells.     

Chromosomal analysis in vinyl chloride exposed workers: Comparison of the standard technique with the sister-chromatid exchange technique

A group of 21 workers occupationally exposed to vinyl chloride and 6 controls were examined for the presence of chromosomal aberrations or sisterchromatid exchanges in their peripheral lymphocytes. These people comprised a second sampling from a group of exposed workers and controls first examined 18 months earlier. The vinyl chloride exposed workers showed levels of chromosomal aberrations elevated above those of the controls, but there was only a slight increase in sister-chromatid exchanges (per cell or per chromosome) and this increase was not statistically significant. Sister-chromatid exchanges (SCEs) were also examined from in vitro cultures of lymphocytes exposed in G₀/early G₁ and late G₁/early S phase to vinyl chloride, both with and without metabolic activation. There was no increase in SCEs in vitro without metabolic activation but there was a marked increase with metabolic activation and this increase was shown to be independent of cell-cylce phase. It thus was apparent that the small increases of SCEs in workers were not due to the inability of vinyl chloride to induce SCEs in human lymphocytes but were probably because of low exposures and SCE levels could have returned to normal relatively quickly after exposure. The present study suggested that the analysis of longer-living conventional chromosomal aberrations appeared to be a more sensitive monitor of exposure to vinyl chloride in exposed workers than the estimation of SCEs; however, it should be noted that in a 3rd sampling taken 24 months later the exposed workers had chromosomal aberration levels similar to the controls.     

Variation in sister-chromatid exchange among 106 members of the general U.K. population

SCE scores of lymphocytes from 106 people revealed that the majority of background variation in SCE was between cells within individuals. Highly significant differences existed between individuals. Lesser, but still highly significant differences also existed between replicate cultures. Inter-individual variation was contributed to by each person's sex and their smoking habits. SCE frequency was not influenced by any of the other factors considered, age, drinking habits and diagnostic X-ray exposure of persons or lymphocyte number and proliferation rate in cultures.