Mutagenic potential of anti-herpes agents

A number of anti-herpes agents which are either licensed for clinical use (acyclovir) or subject of clinical studies (bromovinyldeoxyuridine, fluoroiodoaracytidine, dihydroxypropoxymethylguanine) or under preclinical investigation (i.e., fluoroiodoarauridine), fluoromethylarauridine, dihydroxybutylguanine, bromovinyldeoxycytidine, bromovinylarauridine and carbocyclic bromovinyldeoxyuridine) were evaluated for their ability to induce sister chromatid exchange (SCE), an indicator of mutagenesis. SCE was scored on metaphase chromosomes of human lymphocytes which had been exposed to 5-bromo-2'-deoxyuridine and varying concentrations of the test compounds. The antiviral assays were based on the inhibition of the cytopathogenicity of herpes simplex virus for human diploid fibroblasts. Most compounds, i.e. acyclovir, bromovinyldeoxyuridine or carbocyclic bromovinyldeoxyuridine, did either not induce SCE or only so at concentrations far above their minimum antiviral concentrations. However, fluoroiodoaracytidine and dihydroxypropoxymethylguanine were found to affect the SCE rate at a concentration (greater than or equal to 4.5 micrograms/ml) that is readily achievable in blood following intravenous injection.     

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.     

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.     

Sensitivity of Bloom syndrome fibroblasts to mitomycin C

Lymphocytes and fibroblasts from people with Bloom syndrome, an autosomal recessive disorder associated with a predisposition to a wide variety of cancers, are known to be hypersensitive to ethylating agents as measured by sister chromatid exchange induction. Recently, hypersensitivity to cell killing by mitomycin C has also been reported in Bloom syndrome fibroblasts from three donors. We report here results which confirm the hypersensitivity of Bloom syndrome fibroblasts as measured by cell killing but show that they have a normal sensitivity to mitomycin C as measured by sister chromatid exchange induction. These results are discussed in terms of their relevance to the diversity of response of Bloom syndrome cells to mutagens, and the nature of the primary defect in Bloom syndrome.     

Benzpyrene-Induced sister chromatid exchanges in lymphocytes of patients with lung cancer

Summary The effect of benzpyrene on sister chromatid exchange was determined in PHA-stimulated lymphocytes of 18 patients with lung cancer and 11 controls without cancer or bronchopulmonary diseases. Patients and controls did not differ either with respect to the spontaneous rate of sister chromatid exchanges or in their response to the carcinogen. We conclude that individual susceptibility to lung cancer cannot be detected by an individual response to benzpyrene, at least in lymphocytes and at the chromosomal level.     

Analysis of toxic and mutagenic activities of antiherpesvirus nucleosides against HeLa cells and herpes simplex virus type 1.

The toxic and mutagenic activities of five antiherpesvirus agents to HeLa cells and herpes simplex virus type 1 (HSV-1) were investigated. 5-Iodo-2'-deoxyuridine (IDU) and 9-beta-D-arabinofuranosyl-adenine (araA) showed very potent inhibitory effects on cell growth and the cloning efficiency of HeLa cells, whereas 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil (BV-araU), E-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and 9-(2-hydroxyethoxymethyl)guanine (ACV) showed less inhibitory effect. 50% inhibitory doses of BV-araU and BVDU for cell growth were 657 and 253 micrograms/ml, respectively. Although the growth inhibitory activity of BVDU was very weak, as above, the mutagenic activity of this drug to the cells, estimated by induction of colchicine-resistant mutants, was observed to be 4 micrograms/ml, which was a markedly smaller dose than the inhibitory dose for cell growth, and the highest frequency of mutation of the cells was shown at 100 micrograms/ml of BVDU. This activity was more potent than that of IDU. No mutagenic activity of BV-araU, araA and ACV to cells was observed within the concentration range of 1-800 micrograms/ml. IDU showed high mutagenic activity to HSV-1 growing in human embryo lung fibroblasts, and IDU-resistant mutants were induced at a high frequency. BVDU also induced a small amount of BVDU-resistant mutant virus, although this drug induced many mutant cells. No mutagenic activity of BV-araU, araA and ACV to HSV-1 was observed.     

Genotoxicity testing of fluconazole in vivo and in vitro

The genotoxic effects of the antifungal drug fluconazole (trade name triflucan) were assessed in the chromosome aberration (CA) test in mouse bone-marrow cells in vivo and in the chromosome aberration, sister chromatid exchange (SCE) and micronucleus (MN) tests in human lymphocytes. Fluconazole was used at concentrations of 12.5, 25.0 and 50.0 mg/kg for the in vivo assay and 12.5, 25.0 and 50.0 microg/ml were used for the in vitro assay. In both test systems, a negative and a positive control (MMC) were also included. Six types of structural aberration were observed: chromatid and chromosome breaks, sister chromatid union, chromatid exchange, fragments and dicentric chromosomes. Polyploidy was observed in both the in vivo and in vitro systems. In the in vivo test, fluconazole did not significantly increase the frequency of CA. In the in vitro assays, CA, SCE and MN frequencies were significantly increased in a dose-dependent manner compared with the negative control. The mitotic, replication and cytokinesis-block proliferation indices (CBPI) were not affected by treatments with fluconazole. According to these results, fluconazole is clastogenic and aneugenic in human lymphocytes, but these effects could not be observed in mice. Further studies should be conducted in other test systems to evaluate the full genotoxic potential of fluconazole.     

Genotoxic action of fungicide Conan 5FL (Hexaconazole) on mammalian cells in vivo and in vitro

The genotoxic effects of fungicide Conan 5FL (containing 50 g/L hexaconazole) in mouse bone-marrow cells and human lymphocytes have been evaluated. Three different concentrations of Conan 5FL (17.50, 35.0, and 70.0 μg/mL for human lymphocytes and 17.50, 35.0, and 70.0 mg/kg for mouse bone marrow cells) were studied. Conan 5FL induced significant increases (except 17.5 mg/kg for mouse bone marrow) in the frequency of chromosomal aberrations (CAs) in both test systems. This fungicide caused structural and numerical abnormalities in both mammalian cells. These are sister chromatid union, chromatid and chromosome breaks, fragments, dicentric and ring chromosomes, and polyploidy. Significant increase was found in induction and in minimum-maximum numbers of sister chromatid exchanges (SCEs) at all treatments compared with the negative control. Conan 5FL did not affect the replication index (RI) in human lymphocyte cultures, however, it significantly decreased the mitotic index (MI) in all treatment concentrations in both test systems. Using of Conan 5FL should be reconsidered due to its possible cytotoxic, clastogenic and mutagenic effects. The text was submitted by the authors in English.     

Genotoxic action of fungicide Conan 5FL (hexaconazole) on mammalian cells in vivo and in vitro

The genotoxic effects of fungicide Conan 5FL (containing 50 g/L hexaconazole) in mouse bone-marrow cells and human lymphocytes have been evaluated. Three different concentrations of Conan 5FL (17.50, 35.00 and 70.00 microg/mL for human lymphocytes and 17.50, 35.00 and 70.00 mg/kg for mouse bone marrow cells) were studied. Conan 5FL induced significant increases (except 17.50 mg/kg for mouse bone marrow) in the frequency of chromosomal aberrations (CAs) in both test systems. This fungicide caused structural and numerical abnormalities in both mammalian cells. These are sister chromatid union, chromatid and chromosome breaks, fragments, dicentric and ring chromosomes, and polyploidy. Significant increase was found in induction and in minimum-maximum numbers of sister chromatid exchanges (SCEs) at all treatments compared with the negative control. Conan 5FL did not affect the replication index (RI) in human lymphocyte cultures, however, it significantly decreased the mitotic index (MI) in all treatment concentrations in both test systems. Using of Conan 5FL should be reconsidered due to its possible cytotoxic, clastogenic and mutagenic effects.     

Cytogenetic analysis of human peripheral blood lymphocytes in culture exposed in vitro to styrene and styrene oxide

Styrene and styrene oxide mutagenicity was tested in cultured human lymphocytes treated in vitro with various concentrations of test agents. Styrene alone was found mutagenic at the highest concentration used (5 X 10(-4) mol. l-1, combined with the alkylating agent THIO-TEPA it did not affect the chromosome aberration yield. Exposure to styrene oxide gave a positive result showing a clear-cut dose-effect relationship within the concentration range 5 X 10(-6) to 1 X 10(-3) mol. l-1. In combination with THIO-TEPA its effect on chromosome aberration yields was additive. Styrene oxide proved also to be a very potent inducer of sister chromatid exchanges (SCE) within the concentration range 5 X 10(-6) to 1 X 10(-3) mol. l-1 tested. Combined with THIO-TEPA it exhibited a distinct additive effect in the production of SCEs.     

Mutagenic and genotoxic evaluation of bisphenol F diglycidyl ether (BFDGE) in prokaryotic and eukaryotic systems

The epoxy resin bisphenol F diglycidyl ether (BFDGE), was examined for its mutagenicity in prokaryotic assays (Salmonella typhimurium His(-) and Escherichia coli Trp(-) tests) and its genotoxicity in eukaryotic systems (sister chromatid exchange (SCE) and micronucleus tests in human lymphocytes), in the presence or absence of an exogenous metabolizing system (S9 from rat liver). In the prokaryotic tests, the concentrations of BFDGE ranged between 100 and 5000 micro g per plate, and in the eukaryotic assays from 12.5 to 62.5 micro g/ml. The compound is able to induce mutagenic effects in bacterial strains TA100, TA1535, WP2uvrA and IC3327, as revealed by the increase observed in the number of induced revertants. With respect to the genotoxicity assays, BFDGE induces an increase in the frequency of sister chromatid exchanges and micronuclei in human peripheral blood lymphocytes.     

The genotoxic effect of the new acaricide etoxazole

Etoxazole is a member of the diphenyl oxazoline class of insecticide was newly developed for use on pome fruits, cotton and strawberries as a acaricide. In the present study, genotoxic effects of acaricide etoxazole (ETX) (miticide/ovicide) were investigated using chromosome aberration (CA) test, sister chromatid exchange (SCE) test and micronucleus test in human lymphocytes. ETX induced the CAs at all concentrations (5, 10 and 20 microg/ml) for 24 h and also induced the CA at the highest concentration (20 microg/ml) for 48 h only. The inducing the CAs for 48 hours treatment period was dose-dependent. Besides, it induced the SCE at all concentrations and treatment periods in a dose-dependent manner as well. Although, ETX decreased the mitotic index (MI) at all concentration and treatment periods dose-dependently, while it did not decrease the replication index (RI) when compared to the negative and solvent controls. In addition, ETX induced the micronucleus at all concentrations except 5 microg/ml for 48 h. This inducing was in a dose-dependent manner as well. In conclusion, it can be concluded that ETX has a potential genotoxic effects in cultured human peripheral lymphocytes.     

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.     

Increased sister chromatid exchange in the peripheral blood lymphocytes of young women who smoke cigarettes

Rates of sister chromatid exchange in dividing human peripheral blood lymphocytes were determined and compared between smoking and non smoking young women between the ages of 16 and 25. Chromosomes block-stained with Giemsa were also examined for chromosome aberrations. A striking difference in the frequency of sister chromatid exchange was found between young women who smoked and those who did not. Smokers scored a significantly higher, F(1) = 15.99, p = 0.0015, rate of sister chromatid exchange than non smokers. Smokers scored a higher mean of SCEs per cell (12.771, SD 3.53) than non smokers (9.712, SD 2.53). Smokers also scored a higher range of SCEs (4 to 28) as opposed to non smokers (4 to 17). No statistical difference was found between smokers and non smokers for the frequency of chromosome aberrations. The significantly higher frequency of exchange in young smoking women may indicate that initial damage to the DNA in many of these women has probably already occurred, thus causing an increased risk of developing cancer later in life.     

Participation of DNA repair processes in altering the frequency of sister chromatid exchanges in the peripheral blood lymphocytes in inflammatory diseases

A negative correlation has been established when comparing the data on sister chromatid exchange frequency and DNA repair capabilities of lymphocytes in patients with non-specific inflammatory pulmonary diseases. The role of DNA repair mechanisms in the alteration of sister chromatid exchange frequency in lymphocytes is discussed.     

Sister chromatid exchange frequency,cellular replication and relative cloning efficiency in human teratocarcinoma-derived cells

To determine the relationships between the induction of specific biological responses and exposure to DNA-damaging agents, human teratocarcinoma-derived cells were exposed to either ethyl methanesulfonate or to methyl methanesulfonate, and sister chromatid exchange, cellular proliferation and relative cloning ability measured. SCE increased while cellular proliferation and relative cloning ability each decreased in a concentration-dependent manner. Methyl methanesulfonate was consistently more efficient in inducing biological responses than was ethyl methanesulfonate. When the individual responses were compared, the decrease in cellular proliferation paralleled the reduction in cloning efficiency. A strong correlation was also observed between the reduction in relative cloning ability and sister chromatid exchange frequency. Because these relationships are similar to those previously described in other mammalian cell lines, the observations in our study suggest that the P3 cell line is an appropriate choice for modeling effects of toxicant exposure in human cells.Abbreviations AGT average generation time - BUdR 5-bromodeoxyuridine - CHO Chinese hamster ovary - EMS ethyl methanesulfonate - ENU N-ethyl-N-nitrosourea - MMS methyl methanesulfonate - MNU N-methyl-Nnitrosourea - SCE sister chromatid exchange     

Induction of sister chromatid exchanges by chemical mutagens and its possible relevance to DNA repair

Several chemical mutagens were found to induce sister chromatid exchanges in Chinese hamster chromosomes. Among them, effects of 4NQO and MMC were very similar to those of UV light in that the exchange frequency increased with increasing dose of chemicals and that it was markedly lowered in the presence of 1 mM caffeine during a post-treatment period. The frequency of proflavin-induced sister chromatid exchanges was also found to be dose dependent, but it was insensitive to the caffeine post-treatment. On the other hand, no appreciable increase was detected in the incidence of sister chromatid exchanges in MNNG-treated cells over a 100-fold range of variation in chemical dose. Caffeine by itself raised the exchange frequency only slightly over a control level. It was found that 4NQO and MMC exerted remarkable delayed effects on the exchange induction, whereas proflavin did not. This seems to suggest that the lesions caused by the former mutagens would be long-lived and repeatedly provoke sister chromatid exchanges. These data imply that there are several possible ways in which the initial DNA lesions ultimately lead to the formation of sister chromatid exchanges, and that at least UV-, 4NQO- and MMC-induced sister chromatid exchanges would have evolved through a caffeine sensitive repair process, probably related to a post-replication repair of DNA damage.     

Human sister chromatid exchange caused by methylazoxymethanol acetate

The incidence of sister chromatid exchange was determined in human leucocyte cultures treated with methylazoxymethanol acetate. In all individuals examined, treated cultures manifested a significantly higher frequency of sister chromatid exchanges than controls. Two concentrations of MAM AC were tested in blood cultures from nine individuals. The concentrations varied from individual to individual since they were determined by means of individual dose-response curves, which involved [3H]-thymidine incorporation in PHA-stimulated short-term lymphocyte cultures versus MAM AC contraction. The lower concentration was less than the TD50 dose. Compared to control cultures, the lower concentration caused a higher incidence of sister chromatid exchange in eight of nine individuals. The cumulative mean value for all control cultures was 5.32 exchanges per cell while that for cultures treated with the higher concentration was 10.73.     

The genotoxic and anti-genotoxic effects of <Emphasis Type="Italic">Stachys petrokosmos</Emphasis> leaf extract in human lymphocytes using microsomal fractions

The genotoxic and anti-genotoxic effects of Stachys petrokosmos leaf extracts (Sp) were investigated in human lymphocytes. The cells were treated with 1.5, 3.0 and 6.0 μL/mL concentrations of Sp leaf extracts for 24 and 48 h treatment periods in the absence and presence of metabolic activator (S9mix). In the absence of S9mix, Sp alone did not induce chromosome aberrations and formation of micronucleus while inducing the mean sister chromatid exchange at the highest concentration. In addition, Sp decreased the mutagenic effect of mitomycin-c. Sp alone showed a cytotoxic effect determined by a decrease in the proliferation index, mitotic index and nuclear division index. On the other hand a mixture of Sp and mitomycin-c resulted in a higher cytotoxic effect especially for 48 h treatment period. In the presence of S9mix, Sp was not genotoxic and cytotoxic however, it showed an anti-genotoxic effect by decreasing the effects of cyclophosphamide.