In vitro genotoxic perspective of Tamiflu

The aim of this study was to investigate the genotoxic and/or cytotoxic effects of Tamiflu, commercial form of the oseltamivir antiviral and most frequently prescribed for the treatment of influenza infections, on cultured human peripheral lymphocytes by using sister chromatid exchange (SCE), chromosomal aberration (CA), and cytokinesis-blocked micronucleus (CBMN) assays. Cells were treated with 0.5, 1, 2 μg/mL oseltamivir, the Tamiflu capsule ingredient, for 24 or 48 h in the absence or presence of an exogenous metabolic activation system (S9 mix). The test chemical did not demonstrate any genotoxic effect dose-dependently but it showed a weak cytotoxicity on cells in this study. On the other hand, some concentrations of Tamiflu (2 μg/mL without S9 mix for 48 h and 1 μg/mL with S9 mix) induced SCE and also decreased significantly the proliferation index (PI) (48 h period) and the nuclear division index (NDI) (24 h period) (P < 0.05) in the absence of S9 mix. Considering the results, Tamiflu did not induce significant increases of CA or micronucleated cells in vitro in cultured peripheral blood lymphocytes under the treatment conditions used but weak SCE induction was observed. On the other hand, the weak cytotoxic effects observed disappeared in the cultures treated in presence of the S9 mix.     

Effect of fungicide Euparen Multi (Tolylfluanid) on the induction of chromosomal aberations in cultivated bovine lymphocytes

The effect of the fungicide Euparen Multi (containing 50% tolylfluanid) was investigated on the induction of chromosomal aberrations (CA) in cultured bovine peripheral lymphocytes. Cultures from two healthy donors were treated with tolylfluanid-based fungicide at concentrations ranging from 1.7 to 17.5 μg/ml for the last 24 and 48 hours of cultivation. Conventional cytogenetic method (CA assay) with Giemsa staining as well as fluorescence in situ hybridization (FISH) with whole bovine chromosomes 1 and 5 painting probes were used in the experiment. In the CA assay, no clastogenic effect of the fungicide was found after Euparen Multi treatment for 24 hours. On the contrary, significant elevation in polyploidy induction was observed with dose-dependence in one of the donors. Using prolonged time of exposure to the fungicide (the last 48 h of the cultivation), a slight clastogenic effect was detected at the doses of 8.75 and 17.5 μg/ml (P < 0.05, P < 0.01, respectively) in donor 1 and at the dose of 8.75 μg/ml (P < 0.05) in donor 2. The highest doses tested caused reduction of the mitotic indices (MI) (P < 0.05, P < 0.01) in both donors as well as both treatment times. The evaluation of stable structural aberrations in lymphocytes by two-colour FISH (48 h exposure) using bovine chromosome painting probes revealed the presence of nonreciprocal translocations at two examined concentrations (3.5 μg/ml and 8.75 μg/ml).     

Genotoxic activity of the commercial herbicide containing bifenox in bovine peripheral lymphocytes

The commercial herbicide with active element bifenox (principal tradename Modown) was tested for the evaluation of genotoxicity in cultured cow peripheral lymphocytes in vitro. Several cytogenetic endpoints as chromosome aberrations (CA), sister chromatid exchanges (SCE), mitotic (MI) and proliferation (PI) indices were investigated in different sampling times. To detect possible metabolic modifications in herbicide genotoxicity, the cultures for SCE determination were also treated with S9 fraction. Cultures of lymphocytes were exposed to the herbicide at concentrations of 25, 50, 250, 500 and 1000 microg/ml. A slight increase of CAs was found after exposure of this agent to doses ranging from 25 to 250 microg/ml for 24 h. In the CA assay no statistical significance was seen. Both higher doses (500 and 1000 microg/ml) caused a decrease of chromosome damage in comparison to the last active dose or control values correlated to induced cytotoxicity. Four concentrations (all except the highest one) of the herbicide were applied into cultures in SCE assays both with and without metabolic activation. Significant elevations of SCE were observed after applications of herbicide tested at doses of 250 and 500 microg/ml in each donor (P<0.001 and P<0.05, respectively) for 24 h. These concentrations also caused a statistically significant decrease in the MI and PI. Treatment for 48 h provided inadequate evidence for the genotoxic activity of the herbicide.     

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.     

Mutagenicity and genotoxicity of dicapthon insecticide

Mutagenic and genotoxic effects of dicapthon were investigated by using the bacterial reverse mutation assay in Salmonella typhimurium TA97, TA98, TA100 and TA102 strains with or without metabolic activation system (S9 mix), and chromosome aberrations (CAs), sister chromatid exchanges (SCEs), and micronucleus (MN) tests in human peripheral blood lymphocytes in vitro. Dicapthon was dissolved in dimethyl sulfoxide for all test systems. 0.1, 1, 10 and 100 μg/plate doses of dicapthon were found to be weakly mutagenic on S. typhimurium TA 98 without S9 mix. The human peripheral lymphocytes were treated with four experimental concentrations of dicapthon (25, 50, 100, and 200 μg/mL) for 24 and 48 h. Dicapthon increased the frequency of SCE only at the 100 μg/mL concentration for the 24 and 48 h applications. Dicapthon also induced abnormal cell frequency, CA/cell ratio and frequency of MN dose dependently for 24 and 48 h. Dicapthon showed a statistically significant cytotoxic effect by decreasing the mitotic index in all concentrations and a cytostatic effect by decreasing nuclear division index in 100 and 200 μg/mL concentrations for both treatment periods when compared with both untreated and solvent controls. These values decreased also in a dose dependent manner.     

Assessment of the genotoxicity of propineb in mice bone marrow cells using micronucleus assay

Propineb, a dithiocarbamate fungicide, is commonly used for the control of disease in a wide range of crops in agriculture. The genotoxic effects of commercial formulation of propineb in bone marrow cells of mice was investigated in vivo by micronucleus (MN) assay. The three different concentrations of propineb (12.5, 25 and 50 μg/mL; 0.01 mL per gram) were injected intraperitoneally (i.p.) to mice for 24 and 48 h. The results of the MN assay indicated that propineb induced a significant increase in frequency of micronucleated polychromatic erythrocytes (MNPCE) at 25 and 50 μg/mL concentrations for 24 h and at the highest (50 μg/mL) concentration for 48 h when compared with negative control. Also significant reduction for the polychromatic erythrocyte/normochromatic erythrocyte (PCE/NCE) ratio which is indicative for bone marrow cytotoxicity was observed at the same concentrations for 24 and 48 h. These results lead us to the conclusion that propineb may have genotoxic and cytotoxic potential due to induction in the frequency of MN and a reduction in PCE/NCE ratio in the bone marrow cells of mice.     

Synthesis and mutagenicity of 2-aryl-substitute ( o -hydroxy-, m -bromo-, o -methoxy-, o -nitro-phenyl or 4-pyridyl) benzothiazole derivatives on Salmonella typhimurium and human lymphocytes exposed in vitro

Derivatives of 2-aryl-substitute (o-hydroxy-, m-bromo-, o-methoxy-, o-nitro-phenyl or 4-pyridyl) benzothiazole were synthesized and tested for their mutagenicity in in vitro assays: (i) in the Ames test with Salmonella typhimurium TA98 and TA100 strains; and (ii) in the sister chromatid exchange (SCE) in cultured human lymphocytes. The four of compounds (BT-11, B-12, BT-14 and BT-15) caused statistically significant increase in revertant colonies of TA98 and TA100. Treatment of lymphocytes with compounds also caused a significant increase in SCE/cell in association with high levels and long exposure (300 μg/mL and 48 h) of the four compounds. It can be concluded that benzothiazole derivatives showed mutagenic activity and were also able to exert a genotoxic effect reducing both the replication index and mitotic index.     

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.     

Evaluation of the genotoxicity of the imidazole antifungal climbazole: comparison to published results for other azole compounds

Climbazole is an imidazole antifungal agent that can provide anti-dandruff benefits when incorporated into a shampoo matrix. A series of genotoxicity studies were performed to support the human safety of this azole antifungal drug. Climbazole was not mutagenic in the Salmonella typhimurium or Escherichia coli Ames assay and did not induce micronuclei in human lymphocytes. In the mouse lymphoma assay (MLA), climbazole was negative (non-mutagenic) with and without metabolic (S9) activation after a 4 h exposure; an increase in small colony mutants was observed without metabolic activation after a 24 h exposure at concentrations of 15 and 17.5 μg/mL. An in vivo mouse micronucleus test was negative up to a maximum tolerated dose (MTD) of 150 mg/kg climbazole administered orally. In the in vivo/in vitro unscheduled DNA synthesis assay, climbazole showed no evidence of DNA damage in the livers of rats at doses up to the MTD of 200 mg/kg orally. A toxicokinetic study was performed in mice with oral administration of [14C]-climbazole (150 mg/kg). Radioactivity (20.42 μg-equiv./g plasma) was detected 15 min after oral administration of [14C]-climbazole, and the peak concentration was 62.96 μg-equiv./g plasma at 8 h after dosing. The measured amounts of radioactivity in plasma, at all sample times from 15 min up to 24 h, exceeded the concentrations that induced increases in mutation frequency after 24 h exposure of mouse lymphoma cells in vitro (15 and 17.5 μg/mL). These observations lend support to the conclusion that climbazole does not present a genotoxic risk in vivo. Furthermore, these data are consistent with the published data for other azole antifungals that work by preventing the synthesis of ergosterol and, as a class, are generally non-genotoxic, except some isolated positive results of questionable significance. Collectively, these data are supportive of the view that climbazole does not present a genotoxic or carcinogenic risk to humans.     

In vitro genotoxicity of rocuronium bromide in human peripheral lymphocytes

Rocuronium bromide (RB), an aminosteroid type neuromuscular blocking agent, acts by reducing or inhibiting the depolarising effect of acetylcholine on the terminal disc of the muscle cell. To our knowledge, there is no adequate information on the genotoxic effects of RB, up to now. In the present study, possible genotoxic effects of RB have been determined by means of sister chromatid exchange (SCE), chromosome aberration (CA) and micronucleus (MN) analyses in human peripheral blood lymphocytes. The human peripheral blood lymphocytes were exposed to three different concentrations of RB (60, 80 and 100 μg/mL) for 24- and 48-h. In this study, RB increased the frequency of CAs, however, did not increase the frequency of SCEs. RB did not decrease the proliferation index (PI) and mitotic index (MI). Accordingly, RB increased the frequency of micronucleus (MN) but did not decrease the nuclear division index (NDI). Findings from this study suggest that rocuronium bromide is clastogenic but not cytotoxic to cultured human peripheral blood lymphocytes.     

Author index

A technique using human lymphocytes together with an Ames-type microsomal (S9) activation system for testig indirect chemical mutagens has been developed and examined. The cytotoxic drug cyclophosphamide (CP), which only displays mutagenic properties after metabolic activation, was used as a test chemical and mutagenicity assessed in terms of sister-chromatid exchange (SCE) induction. Direct exposure of lymphocytes to CP and S9 mix produced increases in the yield of SCE for CP concentrations down to 10^?6 M. Exposure times of 30, 60 and 120 min commencing at the beginning of cell culture or after 48 h gave different ranges of detection and response with later treatment being more effective for SCE induction. Variations in relative proportions of the S9 mix culture medium constituents affected the lymphocytes' tolerance of toxic factors and modifiec the mutagen's effect. CP pre-incubated with S9 mix for 1 h before adding to the lymphocyte cultures also produced increases in SCE levels but the method was complicated and did not reduce toxicity. Direct addition of CP and S9 mix to the lymphocytes without prior pre-incubation showed maximum sensitivity, minimum toxocity and was a simpler, more reliable technique.     

Genotoxic effects of tacrolimus on human lymphocyte cells

We designed in vitro study to determine possible genotoxic effects oftacrolimus (FK-506), which is used as a potent immunosuppressive drug, by using sister chromatid exchange (SCEs), chromosome aberration (CAs), micronuclei tests (MN) and cell growth kinetics such as mitotic index (MI) and replication index (RI) in human lymphocytes. The cells were treated with 5, 25, 50, and 100 ng/mL concentrations of tacrolimus, for 24 h and 48 h treatment periods. Tacrolimus induced CA and MN frequency at all concentrations for 24 and 48 h In additon, it induced the SCE at the highest concantration for 24 h and at 25 and 100 ng/mL for 48 h. Tacrolimus decreased MI at all concentrations (except 5 ng/mL) for all treatment periods. It also inhibited the RI at 50 and 100 ng/mL concentrations for 24 h and at all concentrations for 48 h. Treatments given with tacrolimus result in the enhance of the different endpoints ofgenotoxicity, suggesting its mutagenic action on lymphocytes in vitro.     

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.     

Effect of ELF-EMF on antioxidant status and micronuclei in K562 cells and normal lymphocytes

The effect of ELF-EMF on DNA through changes in antioxidative enzyme activities has not been sufficiently explored yet. The aim of this study was to determine ELF-EMF effect on antioxidative enzymes in cancer cell line and genotoxic potential on normal human lymphocytes. K562 cells were exposed to 50 Hz ELF-EMF (40 μT, 100 μT; 3 h, 24 h) and spectrophotometric determination of lipid peroxidation and antioxidative enzyme activities was conducted. Genotoxicity of ELF-EMF (50 Hz, 100 μT) was investigated by cytokinesis-block micronucleus assay in a normal human lymphocytes (exposure 24 h and 48 h). Results demonstrated that ELF-EMF did not alter the process of lipid peroxidation and superoxide dismutase activity. Catalase activity was increased only after application of 100 μT EMF for 24 h. Glutathione-S-transferase and -reductase activities were increased. Treatment with 100 μT ELF-EMF (24 h, 48 h) significantly reduced micronuclei incidence, while cell proliferation was significantly increased. Results indicate that 50 Hz ELF-EMF (40 μT, 100 μT) are week stressors which alone cannot generate enough ROS to induce process of lipid peroxidation in cancer cell line but strong enough to induce response of antioxidative system. Furthermore, 100 μT ELF-EMF in human lymphocytes did not exhibit genotoxic potential during 24 h and 48 h treatment, but stimulated cell proliferation.     

In vivo and in vitro promutagen activation by Vicia faba of thiocarbamate herbicides molinate and butylate to products inducing sister chromatid exchanges in human lymphocyte cultures

Molinate and butylate treatments for 4 h of Vicia faba root tip meristems, showed that both thiocarbamate herbicides increased significantly SCE frequency. Direct treatments of molinate and butylate on human lymphocytes applied 24 h after the beginning of culture did not induce SCE. When S10 extracts of the Vicia roots, treated for 4 h with molinate and butylate (in vivo activation) were added to lymphocytes (24 h after of the beginning of culture), SCE were induced in a concentration-response manner. The in vitro assays, in which molinate and butylate was added at 48 h lymphocyte cultures for 4 h, showed a negative response, however, in the treatment where the S10 metabolic mix was added the SCE frequencies were significantly different to the control, and the concentration-response relationship was not observed with molinate, but it was obtained with butylate. The results showed that both herbicides needed the V. faba metabolism to produce SCE in human lymphocyte culture.     

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.     

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 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.     

Mutagenic and genotoxic effects of Anilofos with micronucleus,chromosome aberrations,sister chromatid exchanges and Ames test

We aimed to evaluate the mutagenic effect of Anilofos, organophosphate pesticide, by using Ames/Salmonella/microsome test. Its cytotoxic and genotoxic effects were also determined by chromosome aberration (CA), sister chromatid exchange (SCE) and micronucleus (MN) test in human peripheral blood lymphocytes. In the Ames test, five different concentrations of Anilofos were examined on TA97, TA98, TA100 and TA102 strains in the absence and presence of S9 fraction. According to the results all concentrations of this pesticide have not shown any mutagenic activity on TA97, TA100 and TA102 strains in the absence and presence of S9 fraction. But, 10, 100 and 1000 µg/plate concentrations of Anilofos were determined to be mutagenic on TA98 strain without S9 fraction. Lymphocytes were treated with various concentrations (25, 50, 100 and 200 µg/ml) of Anilofos for 24 and 48 h. The results of the assays showed that Anilofos did not induce SCE frequency, replication index and MN formation at all concentrations for both treatment periods. Anilofos significantly increased CA frequency at 100 and 200 µg/ml concentrations at 24 h treatment periods and at 50, 100 and 200 µg/ml concentrations in 48 h treatment periods. Additionally, it was determined that this pesticide decreased mitotic index and nuclear division index significantly. It was concluded that Anilofos has genotoxic and cytotoxic effects in human peripheral lymphocytes.     

Induction of sister-chromatid exchanges in cultured human lymphocytes by Aldicarb, a carbamate pesticide

The aim of this work was to investigate the effects of Aldicarb on human lymphocytes in vitro in the presence of an exogenous metabolic activation system. This was done by means of an analysis of SCE and mitotic delay. CP was used to compare the chromosomal effects of Aldicarb with a known genotoxic agent. Our experiments showed that Aldicarb as well as CP induced a significant increase of SCE values in the absence of S9 mix. In vitro metabolic activation of both chemicals increased the SCE values. The addition of a metabolic system slightly decreased the successive mitotic progression of cells in culture.