Genotoxicity of acrylamide in human lymphocytes

Acrylamide is used in the industry and can be a by-product in a high-temperature food processing. It is reported to interact with DNA, but the mechanism of this interaction is not fully understood. In the present study, we investigated the DNA-damaging potential of acrylamide (ACM) in normal human lymphocytes using the alkaline-, neutral- and 12.1 versions of the comet assay and pulsed-field gel electrophoresis. We also investigated effect of acrylamide on caspase-3 activity as well as its influence on the repair process of hydrogen peroxide-induced DNA damage. Acrylamide at 0.5-50 microM induced mainly alkali-labile sites. This damage was repaired during a 60-min repair incubation. Post-treatment of the damaged DNA with repair enzymes: thymine glycol DNA N-glycosylase (Nth) and formamidopyrimidine-DNA glycosylase (Fpg), recognizing oxidized DNA bases, as well as 3-methyladenine-DNA glycosylase II (Alk A), recognizing alkylated bases, caused an increase in the extent of DNA damage, indicating the induction of oxidative and alkylative DNA base modifications by acrylamide. Pre-treatment of the lymphocytes with N-tert-butyl-alpha-phenylnitrone (PBN), a spin trap, as well as vitamins C and E decreased the DNA-damaging effect of acrylamide, which suggest that free radicals/reactive oxygen species may be involved in this effect. Acrylamide impaired the repair of DNA damaged by hydrogen peroxide and increased the activity of caspase-3, which may indicate its potential to induce apoptosis. Our results suggest that acrylamide may exert a wide spectrum of diverse effects on DNA of normal cells, including mostly DNA base modifications and apoptosis. Acrylamide may also impair DNA repair. Free radicals may underline these effects and some dietary antioxidants can be considered as protective agents against genotoxic action of acrylamide. As normal lymphocytes contain cyp2e1 and P450, engaged in the bioactivation of ACM to glicidamide it is uncertain whether acrylamide causes all of measured effect per se or this is the result of the action of its metabolites.     

Toxic effects of the fungicide benomyl on cell membranes

This paper examines the toxicity of the fungicide benomyl towards cell membranes. Approaches to this aim were the study of its acute effects on the stimulatory response of a frog neuroepithelial synapse and on membrane models. The latter consisted of large unilamellar vesicles of dimyristoylphosphatidylcholine (DMPC) and phospholipid multilayers built-up of DMPC and dimyristoylphosphatidylethanolamine (DMPE). Results showed that benomyl at concentrations as low as 10 μM decreased the stimulatory response of the potential difference (PD) and the short-circuit current (SCC) of the frog sympathetic junction. It is concluded that benomyl caused a dose-dependent reduction in the response of a sympathetic junction of the frog to stimulation leading to Cl⁻ channel perturbation. This finding might be explained from those obtained from fluorescence spectroscopy and X-ray diffraction studies on membrane models. In fact, similar (0.01–1.0 mM) concentrations induced structural perturbations in DMPC large unilamellar vesicles and multilayers, respectively. Although it is still premature to define the precise molecular mechanism of benomyl toxicity, the experimental results confirm the important role played by the phospholipid bilayers in the interaction of the pesticide with cell membranes.     

Genotoxicity of goniothalamin in CHO cell line

Goniothalamin (GTN) is a styrylpyrrone derivative from Goniothalamus umbrosus and other Annonaceae species. It has been shown to have anti-cancer and apoptosis-inducing properties against various human tumour and animal cell lines. The compound has also been shown to be active in vivo against DMBA-induced rat mammary tumours and was reported as an anti-fertility agent in rats. The aim of our study was to assess the genotoxicity of GTN in CHO cells using the UKEMS guidelines. A metabolic activation fraction (S9) was prepared according to standard methods. The methylthiazoletetrazolium (MTT) screening assay was then carried out to determine the cytotoxicity index (IC50) of GTN. The average IC50 value was 12.45 (+/- 3.63)microM. The mitotic index (MI) assay was then performed to determine the clastogenicity indices (MI(C25), MI(C50) and MI(C100)) of GTN. The chromosome aberration (CA) induction assay using air-dried metaphase spread was then performed to investigate the clastogenic effects of goniothalamin. Benzo[a]pyrene (BaP) and ethylmethanesulphonate (EMS) were used as positive controls in the presence and absence of S9 metabolic activation, respectively. The anti-genotoxicity effect of GTN was also assessed using a combination of GTN and EMS, and GTN and BaP. Dose-responses of CA frequencies were determined for both, the genotoxicity and anti-genotoxicity effects. GTN on its own and when combined with positive controls, was found to induce and enhance CA, respectively. Chromatid and whole chromosome breaks/gaps, as well as interchanges, endoreduplications and ring chromosomes were the main types of aberration induced by GTN. The overall clastogenic effect of GTN was statistically significant. In conclusion, GTN is potentially a genotoxic or clastogenic substance without any anti-genotoxic properties.     

An embryotoxicity study of the fungicide tridemorph and its commercial formulation Calixin

Tridemorph (N-tridecyl-2,6-dimethylmorpholine), the active ingredient of the commercially formulated fungicide Calixin, is a teratogen in rats and mice. The no-effect level for embryotoxic effects was 27.5 mg/kg for mice and 20.6 mg/kg for rats. By contrast, when Calixin, which contains 83% tridemorph, was administered orally at dose levels of 0.156, 0.722, and 3.909 mg/kg, no embryotoxic effects were observed in two strains of rats. Our extensive investigations, carried out under exposure conditions resembling as closely as possible those reported in another study, did not reproduce the previous findings of teratogenicity of Calixin.     

Genotoxicity of vanadium pentoxide evaluate by the single cell gel electrophoresis assay in human lymphocytes

Vanadium compounds are extensively used in modern industry and occupational exposure to high doses of Vanadium is quite common. In this study, the genotoxicity of vanadium pentoxide (V₂O₅) was evaluated directly in whole blood leukocytes and in human lymphocyte cultures using the single-cell gel electrophoresis assay (Comet Assay) to detect DNA damage expressed as DNA strand breaks and alkali labile sites. This chemical produces a clear dose-response in DNA migration in whole blood leukocytes and a significative positive effect only with the highest tested concentration in human lymphocyte cultures. After different recovery times the level of DNA damage returned to the control values. These results indicate that V₂O₅ is capable to induce DNA single-strand breaks and/or alkali-labile damage.     

Pyrimethanil: Between efficient fungicide against Aspergillus rot on cherry tomato and cytotoxic agent on human cell lines

Pyrimethanil, a synthetic fungicide widely used for the treatment of pre‐ and postharvest fungal diseases on different agricultural crops, was explored for its antifungal activity on different fungal strains. The effect of pyrimethanil on fungal ergosterol was tested by using Aspergillus niger as a model organism. Furthermore, it was investigated, if pyrimethanil can effectively reduce the appearance of Aspergillus rot in wounded cherry tomato fruits. The fungicide cytotoxic effect on different human cell lines was evaluated. In addition, its influence on cell proliferation was studied. A. niger was the most resistant fungal strain (MFC 1.88 μg μL^?1) to the effect of pyrimethanil. Addition of ergosterol doubled the MFC on A. niger, indicating that the compound might interfere with ergosterol, main sterol of fungal cell membrane. Disease incidence of A. niger in wounded cherry tomato fruits was not detected with pyrimethanil treatment of 0.75 mg/wound. Some cytotoxic effects of pyrimethanil on human cell lines were recorded already at 50 ng μL^?1, while the expression of Ki67 marker of proliferation was decreased with 150 ng μL^?1. These results altogether indicate that pyrimethanil is effective in reducing various fungal pathogens, but further use of this fungicide should be reevaluated because of its cytotoxicity.     

Genotoxicity of imidacloprid in relation to metabolic activation and composition of the commercial product

Imidacloprid is a neonicotinoid insecticide combining excellent efficiency against parasites with low toxicity for mammals. Commercially, it is co-formulated with dimethyl sulfoxide, methylpyrrolidone, propylene carbonate and mineral oil, which can modify its bioavailability and toxicological profile for humans following occupational exposure. A combined in vitro approach employing the comet assay and the micronucleus test was used to assess the genotoxicity of imidacloprid in relation to formulation, metabolic activation and exposure level. Human peripheral blood lymphocytes from unexposed healthy volunteers were treated with imidacloprid (0.2, 2 and 20 μM) and with equimolar concentrations of a commercial product, with and without addition of S9 fraction. Imidacloprid significantly increased the comet score and the frequency of micronuclei only at the highest concentration tested. DNA damage was slightly more severe with the commercial product, and was increased, though not significantly, by metabolic activation. Formation of reactive oxygen species (ROS) does not seem to be involved as a mechanism of genotoxicity, but this result may be explained by the insufficient sensitivity of the 2',7'-dichlorofluorescein diacetate assay at the test concentrations of imidacloprid. These results suggest that at concentrations<20 μM imidacloprid is not genotoxic to human lymphocytes in vitro. Nonetheless, the presence of co-formulants in the commercial product and occupational exposure, along with poor safety procedures, may present an increased risk for DNA fragmentation and chromosomal aberrations.     

Genotoxicity of pemetrexed in human peripheral blood lymphocytes

Pemetrexed (PMX) is an antineoplastic antifolate used in the treatment of non-small cell lung cancer, mesothelioma and several types of neoplasms. Its toxicity in tumor cells has been linked with the potent inhibition of thymidylate synthase, dihydrofolate reductase and glycinamide ribonucleotide formyl transferase, and subsequent depletion of both purine and pyrimidine nucleotides. However, cytogenetic toxicity of PMX in non-diseased cells has not been adequately studied; despite the increasing data on the DNA-damaging potential of antineoplastic agents on normal cells. In the present study, the genotoxic potential of PMX was evaluated in peripheral blood lymphocytes obtained from healthy human subjects using chromosome aberration (CA), sister chromatid exchange (SCE) and micronucleus (MN) assays as the cytogenetic damage markers. Human peripheral blood lymphocytes were exposed to four different concentrations (25, 50, 75 and 100 μg/mL) of PMX for 24- and 48-h treatment periods. PMX significantly increased the formation of CA in 24-h treatment, but not in 48-h treatment. PMX did not increase the mean SCE frequency in 24- and 48-h treatment periods; however, there was a striking increase (although not statistically significant, p > 0.05) in the number of SCEs at 25 μg/mL (24- and 48-h treatment) and 50 μg/mL (24-h treatment) due to an increase of SCE at the single-cell level. Interestingly, PMX did not induce MN formation in either 24- or 48-h treatment periods. PMX strongly decreased the mitotic index (MI), proliferation index (PI) and nuclear division index (NDI) in 24- and 48-h treatment periods. Our results suggest that PMX has a potent cytotoxic effect against human peripheral blood lymphocytes at concentrations which are reached in vivo in the blood plasma.     

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).     

Genotoxicity testing of chloramphenicol in rodent and human cells

The results of this work, carried out to extend the limited information at present available on the genotoxic potential of chloramphenicol (CAP), indicate that in millimolar concentrations this antibacterial agent produced a minimal amount of DNA fragmentation in both V79 cells and metabolically competent rat hepatocytes. Moreover, a level of DNA-repair synthesis indicative of a weak but positive response was detected in primary cultures of liver cells obtained from 2 of 3 human donors, and a borderline degree of repair was present in those prepared from rats. The promutagenic character of CAP-induced DNA lesions was confirmed by a low but significant increase in the frequency of 6-thioguanine-resistant clones of V79 cells, which, however, was absent when the exposure was done in the presence of co-cultured rat hepatocytes. Finally, oral administration to rats of 1/2 LD50 CAP did not increase the incidence of either micronucleated polychromatic erythrocytes or micronucleated hepatocytes. Taken as a whole these findings suggest that CAP should be considered a compound intrinsically capable of producing a very weak genotoxic effect, but only at concentrations about 25 times higher than those occurring in patients treated with maximal therapeutic dosages.     

Genotoxicity of microcystin-LR in human lymphoblastoid TK6 cells

Toxic cyanobacteria (blue-green algae) water blooms have become a serious problem in several industrialized areas of the world. Microcystin-LR (MCLR) is a cyclic heptapeptidic toxin produced by the cyanobacteria. In the present study, we used human lymphoblastoid cell line TK6 to investigate the in vitro genotoxicity of MCLR. In a standard 4h treatment, MCLR did not induce a significant cytotoxic response at <80 microg/ml. In a prolonged 24h treatment, in contrast, it induced cytotoxic as well as mutagenic responses concentration-dependently starting at 20 microg/ml. At the maximum concentration (80 microg/ml), the micronucleus frequency and the mutation frequency at the heterozygous thymidine kinase (TK) locus were approximately five-times the control values. Molecular analysis of the TK mutants revealed that MCLR specifically induced loss of heterozygosity at the TK locus, but not point mutations or other small structural changes. These results indicate that MCLR had a clastogenic effect. We discuss the mechanisms of MCLR genotoxicity and the possibility of its being a hepatocarcinogen.     

Clastogenicity of the fungicide afugan in cultured human lymphocytes

The genotoxic effects of the fungicide afugan were analysed by measuring chromosomal aberrations (CAs), sister chromatid exchange (SCE) and micronuclei (MN) in cultured human peripheral lymphocytes. Concentrations of 2.5, 5, 10 and 20 microg/ml of afugan were used during 24 and 48 h. Afugan significantly increased the frequency of CAs at 5, 10 and 20 microg/ml concentrations during a 48 h treatment period. A significant increase was observed for induction of SCE and MN at all treatments compared with the negative control. A significant dose-response correlation was found in all tests. Afugan did not affect the replicative index (RI), however it significantly decreased the mitotic index (MI) at all treatment concentrations except 2.5 microg/ml, and at both treatment times. The present results indicate that afugan is clastogenic and cytotoxic to cultured human lymphocytes.     

Genotoxicity of usnic-acid enantiomers in vitro in human peripheral-blood lymphocytes

Cyto- and genotoxic effects of usnic-acid (+)- and (–)-enantiomers have been studied in human peripheral-blood lymphocytes. It has been shown that usnic-acid enantiomers in concentrations of 0.04–0.30 mM have pronounced cytotoxic action. Usnic-acid enantiomers in concentrations of 0.04–0.30 mM have exhibited a genotoxic effect; moreover, the genotoxicity of usnic-acid (–)-enantiomer in concentrations of 0.15 and 0.30 mM was twice as high as that of (+)-enantiomer. The effect of usnic-acid (+)- and (–)-stereoisomers has been accompanied by the formation of atypical DNA comets. Furthermore, (–)-usnic acid has induced 2.5–3.5 times more atypical comets than its (+)-stereoisomer.     

Genotoxicity of pesticides: a review of human biomonitoring studies

Pesticides constitute a heterogeneous category of chemicals specifically designed for the control of pests, weeds or plant diseases. Pesticides have been considered potential chemical mutagens: experimental data revealed that various agrochemical ingredients possess mutagenic properties inducing mutations, chromosomal alterations or DNA damage. Biological monitoring provides a useful tool to estimate the genetic risk deriving from an integrated exposure to a complex mixture of chemicals. Studies available in scientific literature have essentially focused on cytogenetic end-points to evaluate the potential genotoxicity of pesticides in occupationally exposed populations, including pesticide manufacturing workers, pesticide applicators, floriculturists and farm workers. A positive association between occupational exposure to complex pesticide mixtures and the presence of chromosomal aberrations (CA), sister-chromatid exchanges (SCE) and micronuclei (MN) has been detected in the majority of the studies, although a number of these failed to detect cytogenetic damage. Conflicting results from cytogenetic studies reflect the heterogeneity of the groups studied with regard to chemicals used and exposure conditions. Genetic damage associated with pesticides occurs in human populations subject to high exposure levels due to intensive use, misuse or failure of control measures. The majority of studies on cytogenetic biomarkers in pesticide-exposed workers have indicated some dose-dependent effects, with increasing duration or intensity of exposure.Chromosomal damage induced by pesticides appears to have been transient in acute or discontinuous exposure, but cumulative in continuous exposure to complex agrochemical mixtures.Data available at present on the effect of genetic polymorphism on susceptibility to pesticides does not allow any conclusion.     

Stage-specific effects of the fungicide carbendazim on Sertoli cell microtubules in rat testis

The aim of the present study is to provide a morphological explanation of carbendazim (CBZ)-induced sloughing of germ cells that occurs in a stage-specific manner. Therefore, very early alterations in the seminiferous tubule epithelium were examined histologically in the rat testis after oral administration of CBZ (400mg/kg). Gaps between the elongated and round spermatids, the first indication of germ cell sloughing (pre-sloughing), were observed in stage late VI-early VII seminiferous tubules at 90-min post-treatment. Tubulin immunoreaction in the Sertoli cells was reduced in intensity in tubules with pre-sloughing. However, electron microscopy demonstrated that there were some intact microtubules in these cells. At 120 min, sloughing was seen in stage late VI-early VII and XIII-XIV. Tubulin immunoreaction in the Sertoli cells was greatly decreased in intensity in tubules where cell sloughing was observed. Electron microscopy showed that there were few microtubules in the body region of these cells. Stages II-V and mid-VII-VIII were exempt from the sloughing effect at 180 min. These changes in microtubules were not observed in Sertoli cells that did not exhibit sloughing characteristics, regardless of the post-treatment intervals. The present results suggest that stage specificity of sloughing is due to the stage-specific susceptibility of Sertoli cell microtubules to CBZ.     

Genotoxicity of acrylamide and glycidamide in human lymphoblastoid TK6 cells

The recent finding that acrylamide (AA), a potent carcinogen, is formed in foods during cooking raises human health concerns. In the present study, we investigated the genotoxicity of AA and its metabolite glycidamide (GA) in human lymphoblastoid TK6 cells examining three endpoints: DNA damage (comet assay), clastogenesis (micronucleus test) and gene mutation (thymidine kinase (TK) assay). In a 4 h treatment without metabolic activation, AA was mildly genotoxic in the micronucleus and TK assays at high concentrations (> 10 mM), whereas GA was significantly and concentration-dependently genotoxic at all endpoints at > or = 0.5 mM. Molecular analysis of the TK mutants revealed that AA predominantly induced loss of heterozygosity (LOH) mutation like spontaneous one while GA-induced primarily point mutations. These results indicate that the genotoxic characteristics of AA and GA were distinctly different: AA was clastogenic and GA was mutagenic. The cytotoxicity and genotoxicity of AA were not enhanced by metabolic activation (rat liver S9), implying that the rat liver S9 did not activate AA. We discuss the in vitro and in vivo genotoxicity of AA and GA.     

Genotoxicity of food preservative sodium sorbate in human lymphocytes in vitro

The genotoxic effects of antimicrobial food additive sodium sorbate (SS) was assessed by using chromosome aberrations (CAs), sister-chromatid exchanges (SCEs), and micronucleus (MN) in cultured human lymphocytes and comet assay in isolated human lymphocytes. Lymphocytes were treated with four concentrations (100, 200, 400 and 800 μg/ml) of SS as well as a negative (sterile distilled water) and a positive control (Mitomycin-C: MMC for cultured lymphocytes and H₂O₂ for isolated lymphocytes). The result of this study indicated that SS increased the frequency of CAs at both 24 and 48 h period compared to control. When gaps were included, this increase was significant at 200, 400 and 800 μg/ml concentrations at 24 h and, at all concentrations at 48 h treatment time. When gaps were excluded, this increase was significant at only 800 μg/ml concentration at both 24 and 48 h treatments. In addition, SS increased SCEs/cell and MN frequency at 400 and 800 μg/ml concentrations at both 24 and 48 h compared to negative control. Furthermore, this additive caused DNA damage at all concentrations in isolated human lymphocytes after 1 h in vitro exposure. The present results show that SS is genotoxic to the human peripheral blood lymphocytes in vitro at the highest concentrations.