Genotoxic evaluation of the antimicrobial drug, trimethoprim, in cultured human lymphocytes

The antimicrobial drug, trimethoprim, was evaluated for genotoxicity in human peripheral blood lymphocyte cultures set-up from two healthy donors. Sister-chromatid exchanges (SCE) and micronuclei (MN) were scored as genetic endpoints. The treatment was done using different trimethoprim concentrations ranging from 1 to 100 microg/ml. From our results, we can conclude that this drug is able to induce both cytotoxic and moderate genotoxic effects, as revealed by the increases seen in SCE and MN frequencies in cultures from the two donors and, at least, at one of the concentrations tested.     

Genotoxic evaluation of the antimalarial drug,fansidar, in cultured human lymphocytes

Fansidar (pyrimethamine-sulfadoxine) has been used extensively worldwide for the treatment of chloroquine resistant Plasmodium falciparum malaria, toxoplasmosis and Pneumocystis carinii pneumonia in patients with the acquired immunodeficiency syndrome. Because of the wide usage of pyrimethamine-sulfadoxine in developing countries and the lake of information from open literature and reports from manufacturers about the genotoxicity of such antimalarial drug, the present work was suggested. The possible genetic toxicity of fansidar has been evaluated in human peripheral blood lymphocyte cultures. The frequencies of sister-chromatid exchanges (SCE) and micronuclei (MN) were scored as genetic endpoints. Both tests covering a wide range of induced genetic damage as primary DNA damage, clastogenicity and aneugenicity. Cultures were set up by using blood samples from two healthy donors and the treatment was done using different fansidar concentrations ranging from 1:20 to 10:200 g/ml. From our results, it appears that this drug is able to induce moderate genotoxic effects, as revealed by the increases found in SCE and MN frequencies in cultures from the two donors at the two highest concentrations tested (5:100 and 10:200 g/ml). In addition, cyotoxic/cytostatic effects of fansidar were revealed by a decrease in the proliferative rate index (PRI) and in the cytokinesis block proliferation index (CBPI). Our findings suggest that the use of this drug should be restricted to situations where other antimalarial drugs cannot be used. The drug should never be given to pregnant women.     

Genotoxicity studies on the antimicrobial drug sulfamethoxazole in cultured human lymphocytes

The genotoxicity of the antimicrobial drug sulfamethoxazole was evaluated in cultured human peripheral blood lymphocytes. The frequencies of sister-chromatid exchange (SCE) and micronuclei (MN) were scored as genetic endpoints. Both tests cover a wide range of induced genetic damage such as primary DNA damage, clastogenicity and aneugenicity. Cultures were set up with blood samples from two healthy donors and the treatment was done with different sulfamethoxazole concentrations ranging from 10 to 500 microg/ml. From the results obtained it appears that this drug is able to induce weak genotoxic effects, as revealed by the slight increase in the SCE and MN frequencies, at least at one of the two highest concentrations tested. However, the results of the SCE assay should be interpreted with caution because the increase is just significant. In addition, cyotoxic/cytostatic effects of sulfamethoxazole were revealed by a decrease in the proliferative rate index (PRI) and in the cytokinesis block proliferation index (CBPI).     

Cytogenetic damage induced in human lymphocytes by sodium bisulfite.

The frequencies of chromosomal aberrations (CA), sister-chromatid exchanges (SCE), and micronuclei (MN) in human blood lymphocytes exposed to sodium bisulfite (sulfur dioxide) at various concentrations ranging from 5 x 10(-5) M to 2 x 10(-3) M in vitro were studied. It was shown that sodium bisulfite (NaHSO3 and Na2SO3, 1:3 M/M) caused an increase in SCE and MN in human blood lymphocytes in a dose-dependent manner, and also induced mitotic delays and decreased mitotic index. For CA, our results indicated that sodium bisulfite induced an increase of chromatid-type aberrations in lymphocytes from three of four donors in a dose-dependent manner. The chemical at low concentrations induced chromatid-type aberrations, but not chromosome-type aberrations; high concentrations induced both chromatid- and chromosome-type aberrations. No cytogenetic damage in human lymphocytes was induced by sodium sulfate. The results have confirmed that sulfur dioxide is a clastogenic and genotoxic agent.     

Effects of copaene,a tricyclic sesquiterpene,on human lymphocytes cells in vitro

In this study, the cytotoxic, genotoxic/antigenotoxic and antioxidant/oxidant activity of copaene (COP), a plant-derived tricyclic sesquiterpene, on human lymphocyte cultures (n = 5) was investigated. COP was added into culture tubes at various concentrations (0, 10, 25, 50, 100, 200 and 400 mg/L). While the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays were used for viability and cytotoxic evaluations, the micronucleus (MN) and sister chromatid exchange (SCE) assays were used for genetic evaluations. Moreover, total antioxidant capacity (TAC) and total oxidative status analysis were used for biochemical evaluations. According to LDH and MTT assays COP significantly reduced cell proliferation at high concentrations (200 and 400 mg/L). In addition, there was no significant increase (P < 0.05) in both SCE and MN frequencies of cultures treated with COP as compared to controls. We have also concluded that concentrations of COP of 50 and 100 mg/L increased TAC level when compared to the controls. In conclusion, in this study it has been reported for the first time that copaene is not genotoxic and it increases the antioxidant capacity in human lymphocyte cultures.     

The genotoxic and cytotoxic effects of ribavirin in rat bone marrow

The genotoxicity of the 2-furylethylene derivative 1-(5-bromofur-2-yl)-2-nitroethene (2-betaNF) has been evaluated in cultured human peripheral blood lymphocytes at concentrations ranging from 0.5 to 15microg/ml. The frequencies of micronuclei (MN) and sister-chromatid exchanges (SCEs) were used and scored as indicators of genetic damage. To asses the role of the metabolism mediated by the enzymes present in the S9 mix, over the possible genotoxic potential of the test agent, the cultures for MN and SCE demonstrations were treated for 3h in presence and in absence of rat liver microsomal fraction. The results indicate that, under the experimental conditions used, the test agent does not induce significant increases in the frequency of micronucleated cells, irrespective of the presence/absence of metabolic fraction. Nevertheless, a slight increase in the SCE frequency was observed in those cultures treated without the S9 mix; although this slight increase disappeared in the experiments carried out with the microsomal fraction. In addition, cytotoxic/cytostatic effects of (2-betaNF) were observed mainly in the cultures treated without the S9 fraction.     

The genotoxic potential of glutaraldehyde in mammalian cells in vitro in comparison with formaldehyde

Glutaraldehyde (GA) induces DNA-protein crosslinks (DPX), but conflicting results have been reported with regard to other genotoxic and mutagenic effects in mammalian cells in vitro. We, therefore, characterized the genotoxic and mutagenic potential of GA in V79 cells. Using the alkaline comet assay we demonstrated the induction of DPX by GA (reduction of gamma ray-induced DNA migration) at a concentration of 10 microM and above. The standard comet assay did not reveal a significant DNA strand-breaking activity of GA. Cross-linking concentrations of GA were also cytotoxic, i.e. inhibited cell growth of treated V79 cultures. Interestingly, a small but statistically significant increase in sister chromatid exchange (SCE) and micronuclei (MN) was already measured at lower concentrations (2 and 5 microM). FISH analysis revealed that the majority of GA-induced MN was due to chromosome breaks. We also compared the genotoxic activity of GA to that of formaldehyde (FA). Similar to GA, FA-induced DPX, SCE and MN, but distinct differences exist with regard to the sensitivity of the endpoints and the relationship between genotoxicity and cytotoxicity. However, the differences in genotoxicity cannot readily explain the different carcinogenic activities of the two compounds.     

Variation in the frequency of sister chromatid exchanges in repeated human lymphocyte cultures

Summary Repeated blood samples from two healthy donors were taken over a period of about one year to determine the temporal variation in human lymphocyte baseline sister chromatid exchange (SCE)-frequencies. The investigations were performed on whole blood cultures and purified lymphocyte cultures using a standardized protocol for blood collection and cultures. Significant differences in the frequencies of SCEs were found between the two cultivation systems and the two blood donors but also between repeated cultures of the same individual. There was no systematic relationship between the proliferation of the cultures and the basal SCE values. The results indicate the necessity of concurrent controls and repeated blood samples whenever SCEs are used as a test for monitoring human exposure to potential mutagens. Temporal variation in human lymphocyte baseline SCE frequencies is a limiting factor for the detection of minor effects of genotoxic agents.     

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.     

As to the clastogenic-, sister-chromatid exchange inducing-and cytotoxic activity of inosine triphosphate in cultures of human peripheral lymphocytes

The influence of commercial inosine triphosphate (ITP) on the chromosome aberration rate, the mitotic rate, sister-chromatid exchange (SCE) frequency, and the proportion of first (X1), second (X2) and third (X3) division metaphases was investigated in 72h cultures of human peripheral lymphocytes. The blood donors had mild inactive arthrosis and a normal health check-up. All cultures of each volunteer were set-up simultaneously. In contrast to a previous report [Arch. Biochem. Biophys. 278 (1990) 238-244], it was demonstrated in two preliminary studies (number of subjects, n=5 each) that ITP at a final concentration of 100 microM does not induce chromosomal aberrations and, furthermore, that not ITP concentrations higher than 100 microM but ITP doses higher than 3.8mM prohibit culture growth.Based on these results, cultures with a final ITP concentration of 3.6mM (max.) and 1.8mM (max./2) were compared with control cultures (number of subjects n=10; three males and seven females, mean age x=57.6 years). Whereas no increase in the chromosomal breakage rate was observed in cultures with an ITP concentration of 1.8mM and only a marginally significant one (P=0.048) for 3.6mM ITP cultures, a highly significant induction of SCEs, not only at an ITP concentration of 3.6mM (P<0.0001) but also at 1.8mM (P<0.0001) was seen. The increase in the SCE frequency was not linear, but steeper from 0 to 1.8mM than from 1.8 to 3.6mM. Nevertheless, the difference between 1.8 and 3.6mM cultures was significant (P=0.027). The distribution of the number of SCEs per metaphase as well as the distribution of SCEs per chromosome correspond to the expected Poisson values. The investigation of the cytotoxic effect of the studied ITP concentrations revealed a highly significant reduction of the mitotic rate from 0 to 1.8mM as well as from 1.8 to 3.6mM in the aberration studies (all P values are equal to smallest possible one for a sample size of 10, namely, 0.002), and in the SCE studies there is a significant decrease in the X3 frequency when ITP is increased (0-1.8mM: P=0.0061 and 1.8-3.6mM: P<0.0001). The proportion of X1 within all X1 and X2 metaphases changes significantly only at the second dose step (0-1.8mM ITP: P=0.22 and 1.8-3.6mM ITP: P<0.0001). The results are discussed.     

Glutathione S-transferase M1 genotype influences sister chromatid exchange induction but not adaptive response in human lymphocytes treated with 1,2-epoxy-3-butene

Induction of sister chromatid exchanges (SCEs) by 1,2-epoxy-3-butene (monoepoxybutene, MEB), an epoxide metabolite of 1,3-butadiene, in human whole-blood lymphocyte cultures has previously been observed to depend on the glutathione S-transferase M1 (GSTM1) and T1 (GSTT1) genotype of the blood donor. Pretreatment of lymphocyte cultures with a low dose of MEB has been shown to reduce the SCE response obtained by later treatment with a higher concentration of MEB. To investigate whether this adaptive response depends on the GSTM1 genotype of the donor, SCE induction by MEB (25 and 250 microM at 48 h for 24 h) was studied from whole-blood lymphocyte cultures of young non-smoking male and female subjects representing GSTM1 positive (n=7) and null (n=7) genotypes, with or without a MEB pretreatment (12.5 microM at 24 h). A higher mean number of induced SCEs per cell at 250 microM MEB was observed in lymphocytes of the GSTM1 null than positive donors, a statistically significant difference being obtained in the presence of the adaptive treatment (9.44 vs. 6.56; results from ethanol-treated controls subtracted). The pretreatment resulted in a statistically significant reduction in the response of the GSTM1 null group at both concentrations of MEB and in the GSTM1 positive group at 250 microM. However, there were no statistically significant differences in the adaptive response of the two genotypes. In conclusion, the present study further supported earlier findings on an increased sensitivity of GSTM1 null donors to SCE induction by MEB, suggesting that GSTM1 is involved in the detoxification of MEB in human lymphocyte cultures. As an adaptive response was observed in both GSTM1 positive and null donors, the phenomenon cannot be explained by GSTM1 induction. It may represent induction of other enzymes operating in MEB detoxification, or activation of DNA repair.     

Genotoxicity of cadmium chloride in human lymphocytes evaluated by the comet assay and cytogenetic tests

Peripheral blood lymphocytes were tested in vitro for genotoxic effects of cadmium chloride. Whole blood samples of four healthy, non-smoking subjects were preincubated with CdCl2 in concentrations of 10(-4), 10(-3), and 5 . 10(-3) mol/L for three hours before the cells were assessed for DNA-damage using the single cell alkaline gel electrophoresis assay (comet assay) or cultivated for chromosomal aberrations (CA), sister chromatid exchanges (SCE), and the micronucleus (MN) test. The comet assay showed notable interindividual differences. The results of the cytogenetic tests showed an increase in the frequency of CA, MN, and SCE with CdCl2 in the treated cultures, yet none was able to show a correlation between concentrations of cadmium chloride and the frequency of damages. The MN slides were stained with Giemsa and with DNA fluorochrome 4', 6'-diamidino-2-phenylindole (DAPI). The frequency of MN in slides stained with DAPI was significantly higher than in those stained with Giemsa, which might be due to an underestimation of small micronuclei in Giemsa-stained slides.     

The influence of GSTM1 and GSTT1 genotypes on the induction of sister chromatid exchanges and chromosome aberrations by 1,2:3,4-diepoxybutane

Cytogenetic tests - chromosome aberrations (CA), sister chromatid exchanges (SCE) and micronuclei (MN) - are most often applied in biomonitoring of the genotoxicity of potentially carcinogenic chemicals in human cells. One of the extensively studied genotoxins is diepoxybutane (DEB) - reactive biometabolite of butadiene (BD). Several studies showed a high SCE induction in human lymphocytes exposed in vitro to various concentrations of DEB. DEB also proved to be a potent inducer of chromosome aberrations and micronuclei. A bimodal distribution of SCE frequency after in vitro DEB treatment was observed. The aim of the present study was to examine the ability of DEB to induce different individual cytogenetic response measured by SCE and CA frequency. The possible influence of genetic polymorphism has also been taken into account, by including donors representing positive or null GSTM1 and GSTT1 genotypes. Our study supported the earlier results showing that DEB is an effective inducer of SCEs and CAs, causing also the decrease in replication index (RI). DEB bioactivity measured by SCE induction - but not by CA test - was significantly higher in GSTT1 negative than in GSTT1 positive donors. GSTM1 polymorphism had no influence on these endpoints. The donors GSTT1-/GSTM1+ were shown to be slightly more sensitive to DEB than GSTT1-/GSTM1- individuals. There was also observed a unimodal distribution of DEB-induced SCEs and CAs in the group, despite the fact that the experiment was performed on the lymphocytes obtained from both GSTT1 positive and negative donors.     

Vicia root-mirconucleus and sister chromatid exchange assays on the genotoxicity of selenium compounds

Selenium (Se) is an important metalloid with industrial, environmental, biological and toxicological significance. Excessive selenium in soil and water may contribute to environmental selenium pollution, and affect plant growth and human health. By using Vicia faba micronucleus (MN) and sister chromatid exchange (SCE) tests, possible genotoxicity of sodium selenite and sodium biselenite was evaluated in this study. The results showed that sodium selenite, at concentrations from 0.01 to 10.0mg/L, induced a 1.9-3.9-fold increase in MN frequency and a 1.5-1.6-fold increase in SCE frequency, with a statistically significantly difference from the control (P<0.05 and 0.01, respectively). Sodium selenite also caused mitotic delay and a 15-80% decrease in mitotic indices (MI), but at the lowest concentration (0.005mg/L), it slightly stimulated mitotic activity. Similarly, the frequencies of MN and SCE also increased significantly in sodium biselenite treated samples, with MI decline only at relatively higher effective concentrations. Results of the present study suggest that selenite is genotoxic to V. faba root cells and may be a genotoxic risk to human health.     

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.     

Paradoxical changes in SCE frequencies persistently elevated in vivo, on exposure to a mutagen in vitro

Lymphocyte cultures from 4 individuals with persistently significantly elevated frequencies of sister-chromatid exchange (SCE) were examined with no treatment, and with 2 concentrations of mitomycin C. In each of the 4 cases, the mean level of SCEs in the untreated lymphocytes exhibited a paradoxical reduction in SCE frequency when exposed to the lower (0.005 microgram/ml) of the two doses of mitomycin C. At the second higher dose of mitomycin C (0.025 microgram/ml) the mean level of SCE/cell exceeded the untreated mean. When the distributions of SCE/cell were examined it appeared that the untreated cultures had two or more populations of cells; one was in the normal SCE frequency range, while the second population was in an elevated SCE frequency range. The paradoxical reduction in SCE frequency was apparently due to elimination of, or mitotic inhibition of cells in the highest range of SCE frequency, while a small elevation in SCEs was initiated in the cells with a normal SCE frequency. Thus, mean levels of SCE/cell can be misleading. This data suggests that new exposure to the same or a different genotoxic agent might possibly result in a misleading lowering of the mean SCE frequency.     

Vicia root-mirconucleus and sister chromatid exchange assays on the genotoxicity of selenium compounds

Selenium (Se) is an important metalloid with industrial, environmental, biological and toxicological significance. Excessive selenium in soil and water may contribute to environmental selenium pollution, and affect plant growth and human health. By using Vicia faba micronucleus (MN) and sister chromatid exchange (SCE) tests, possible genotoxicity of sodium selenite and sodium biselenite was evaluated in this study. The results showed that sodium selenite, at concentrations from 0.01 to 10.0 mg/L, induced a 1.9–3.9-fold increase in MN frequency and a 1.5–1.6-fold increase in SCE frequency, with a statistically significantly difference from the control (P < 0.05 and 0.01, respectively). Sodium selenite also caused mitotic delay and a 15–80% decrease in mitotic indices (MI), but at the lowest concentration (0.005 mg/L), it slightly stimulated mitotic activity. Similarly, the frequencies of MN and SCE also increased significantly in sodium biselenite treated samples, with MI decline only at relatively higher effective concentrations. Results of the present study suggest that selenite is genotoxic to V. faba root cells and may be a genotoxic risk to human health.     

Low sensitivity of the comet assay to detect acetaldehyde-induced genotoxicity

Acetaldehyde (AA) is known to induce DNA-protein cross-links (DPX) and other genotoxic and mutagenic effects in cultured mammalian cells. Compared to formaldehyde (FA), AA is a very weak inducer of DPX and increased DPX levels are only measured at high, cytotoxic concentrations by different methods. Besides DPX, AA also induces DNA-DNA cross-links. Because the comet assay is increasingly used for the detection of cross-linking agents, we characterized the effects of AA in the comet assay in relation to cytotoxicity and other genetic endpoints such as the induction of sister chromatid exchange (SCE) and micronuclei (MN). The standard alkaline comet assay did not indicate induction of DNA strand-breaks by AA in a range of concentrations from 0.2 to 20 mM. AA at a concentration of 20 mM was clearly cytotoxic and reduced cell growth and population doubling to less than 50% of the control. Using the comet assay modification with proteinase K, slightly enhanced DNA migration was measured in comparison to treatment with AA only. No significant induction of cross-links by AA (measured as reduction of gamma ray-induced DNA migration) was determined by the comet assay. A small and reproducible but statistically not significant effect was measured for the AA concentration 20 mM. A clear and concentration-related increase in the frequency of sister chromatid exchange (SCE) and micronuclei (MN) was already measured at lower concentrations (0.2 and 0.5 mM, respectively). These results suggest that the comet assay has a low sensitivity for the detection of AA-induced DNA lesions leading to the induction of SCE and MN.     

Resveratrol, a naturally occurring polyphenol, induces sister chromatid exchanges in a Chinese hamster lung (CHL) cell line.

We tested the genotoxicity of 3,5,4'-trihydroxystilbene (resveratrol), a polyphenolic phytoalexin found in grapes, in a bacterial reverse mutation assay, in vitro chromosome aberration (CA) test, in vitro micronucleus (MN) test, and sister chromatid exchange (SCE) test. Resveratrol was negative in the strains we used in the bacterial reverse mutation assay (S. typhimurium TA98 and TA100 and E. coli WP2uvrA) in the absence and presence of a microsomal metabolizing system. It induced structural CAs at 2.5-20 microg/ml and showed weak aneuploidy induction in a Chinese hamster lung (CHL) cell line. It induced MN cells and polynuclear and karyorrhectic cells after 48h treatments in the in vitro MN test. In the SCE test, resveratrol caused a clear cell-cycle delay; at 10 microg/ml, the cell cycle took twice as long as it did in the control. Resveratrol induced SCEs dose-dependently at up to 10 microg/ml, at which it increased SCE six-fold, and the number was almost as large as mitomycin C, a strong SCE inducer. No second mitoses were observed at 20 microg/ml even after 54h. Cell cycle analysis by FACScan indicated that resveratrol caused S phase arrest, and 48h treatment induced apoptosis. Our results suggest that resveratrol may preferentially induce SCE but not CA, that is, it may cause S phase arrest only when SCEs are induced.     

Cytogenetic damages in the cells of allergy patients

The levels of cytogenetic damages in cell cultures of chemical industry workers suffering from different forms of allergy have been investigated. The levels of chromosomal aberrations in cells of allergic patients are shown to reliably increase as against those of healthy donors. Studies on SCE levels have shown no such differences between the patients and donors. The SCE levels have been determined at different periods of BUdR addition and cultures fixation under the effect of environmental factors.