Assessment of the genotoxicity of imidacloprid and metalaxyl in cultured human lymphocytes and rat bone-marrow

Imidacloprid and metalaxyl are two pesticides that are widely used in agriculture, either separately, or in combination. These agents were studied for their possible genotoxic effects with respect to the following cytogenetic end-points: (1) in vitro micronucleus (MN) formation and sister-chromatid exchange (SCE) induction in human lymphocytes and (2) in vivo micronucleus induction in polychromatic erythrocytes (PCEs) of the rat bone-marrow. The results of the MN analysis indicate that MN frequencies after treatment with both pesticides, separately or as a mixture, do not significantly differ from those in the controls except after treatment with metalaxyl alone at 50 μg/ml (p < 0.05). The results of the SCE analysis show that SCE frequencies after treatment with imidacloprid do not differ significantly from those in the controls. A statistically significant increase (p < 0.05) in SCE frequency resulted from treatments with metalaxyl at 5, 10 and 100 μg/ml and with the combination of imidacloprid and metalaxyl at 100 and 200 μg/ml. Finally, the in vivo micronucleus assay with rat bone-marrow polychromatic erythrocytes showed a statistically significant effect upon separate treatments with imidacloprid and metalaxyl at doses of 300 mg/kg body weight (b.w.) (p < 0.01) or upon combined treatment with 200 mg/Kg b.w. (p < 0.001) and 400 mg/kg b.w. (p < 0.05).     

Methylphenidate is not clastogenic in cultured human lymphocytes and in the mouse bone-marrow micronucleus test

Methylphenidate (MPH) is one of the most frequently prescribed drugs for the treatment of attention deficit hyperactivity disorder (ADHD). A report on cytogenetic effects observed in peripheral lymphocytes from children treated for 3 months with MPH raised questions about the genetic toxicity of this compound. A critical review of this data concluded that the cytogenetic effects in treated children remain unexplained. A literature review showed that MPH was found negative in most genetox studies performed, but no in vitro chromosome aberration data in human lymphocytes have been published. Therefore, we conducted a chromosomal aberration study in cultured human peripheral lymphocytes. The results of this investigation showed that d,l-methylphenidate (MPH, Ritalin) in concentrations up to 10 mM did neither induce structural nor numerical chromosome abnormalities. An oral mouse bone-marrow micronucleus test in B6C3F(1) mice, with doses up to 250 mg/kg bw, was negative too. The data of these studies confirm the absence of clastogenic activity of MPH in non-clinical studies.     

Inhibition of xenobiotic-induced genotoxicity in cultured precision-cut human and rat liver slices

In this study precision-cut liver slices have been used to evaluate the effects of the flavone tangeretin, the flavonoid glycoside naringin and the flavanone naringenin (the aglycone derived from naringin) on xenobiotic-induced genotoxicity. Liver slices were cultured for 24 h in medium containing [3H]thymidine and the test compounds and then processed for autoradiographic determination of unscheduled DNA synthesis (UDS). The cooked food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) markedly induced UDS in cultured human liver slices and both 2-acetylaminofluorene (2-AAF) and aflatoxin B1 (AFB1) induced UDS in cultured rat liver slices. Tangeretin (20 and 50 microM) was found to be a potent inhibitor of 5 and 50 microM PhIP-induced UDS in human liver slices, whereas 20 and 50 microM naringenin was ineffective and naringin only inhibited genotoxicity at a concentration of 1000 microM. In rat liver slices 50 microM tangeretin inhibited 10 and 50 microM 2-AAF-induced UDS, whereas 50 microM naringenin and 100 and 1000 microM naringin were ineffective. None of the three flavonoids examined inhibited 5 microM AFB1-induced UDS in rat liver slices. The inhibition of PhIP- and 2-AAF-induced UDS by tangeretin is probably attributable to the inhibition of the human and rat cytochrome P-450 isoforms which are responsible for the bioactivation of these two genotoxins. Although flavonoids can modulate xenobiotic-induced genotoxicity in human and rat liver slices, any protective effect is dependent on the particular combination of genotoxin and flavonoid examined. These results demonstrate that cultured precision-cut liver slices may be utilised as an in vitro model system to examine the modulation of xenobiotic-induced genotoxicity by flavonoids and other dietary components.     

Role of clozapine in the occurrence of chromosomal abnormalities in human bone-marrow cells in vivo and in cultured lymphocytes in vitro

Summary Bone-marrow chromosomes were examined from 38 mentally and physically retarded and two psychiatric patients who were being treated with a variety of neuropharmacologic drugs. Twenty of these patients used clozapine (Leponex^®). The clastogenic effects of clozapine in vitro were studied in the lymphocyte cultures of three patients-one free of hematologic disease and two who 6 months earlier had had agranulocytosis attributed to the use of clozapine. The mean frequency of cytogenetic abnormalities in the bonemarrow cells of patients who used clozapine was significantly increased (P> 0.05). The two patients who had had agranulocytosis had a greater frequency of cytogenetic abnormalities in their cultured lymphocytes in vivo and in vitro than the patient free of hematologic disease. A clone with a 13/14 chromosome translocation was detected in one of the patients. As all patients received a number of drugs during the in vivo and in vitro studies no definite conclusions could be drawn regarding the role played by clozapine in the occurrence of chromosomal abnormalities.     

Liquid holding effects on methylmercury genotoxicity in human lymphocytes

Methylmercury chloride (MMC) treatment of resting (GO) human lymphocytes resulted in the formation of chromosome and chromatid aberrations. This treatment also induced aneuploidy in second metaphases, suggesting that MMC produces stable damage involved in chromosome segregation errors. The storage of treated cells (liquid holding for 48 h before cell proliferation) did not result in an important recovery from induced cell toxicity or chromosome damage. Therefore, MMC seems to be an X-ray-like agent, able to produce long-lasting damages giving rise to both structural and numerical chromosome abnormalities.     

Nucleoli of cultured human lymphocytes

Summary Nucleolar fusion and nucleolus formation occurred simultaneously, immediately after mitosis, in cultured human lymphocytes. Evidence is presented that in late telophase and post-telophase, the individual nucleolar organising site includes two components, represented in post-telophase by the nucleolus and its attached process; that fusion can be either directly between the nucleoli or via the processes; and that the latter type of fusion persists into metaphase as acrocentric associations, whilst the former is lost.     

Assessment of the genotoxicity of atenolol in human peripheral blood lymphocytes: Correlation between chromosomal fragility and content of micronuclei

The antihypertensive drug atenolol was found to induce chromosome loss, detected as micronuclei in the peripheral lymphocytes of treated patients. The fundamental question which chromosomes the micronuclei were derived from remains to be answered. Analysis of structural chromosomal aberrations (CAs) and expression of fragile sites (FS) were pursued in this study. They revealed a significantly higher incidence of chromosomal aberrations (chromatid and chromosome breaks) in patients compared with controls, where 10 FS emerged as specific. Also, the band 17q12–21, where known fragile sites have not been reported, was only expressed in atenolol-treated patients. Fluorescence in situ hybridization using chromosome-specific probes revealed the preferential involvement of chromosomes 7, 11, 17 and X in the micronuclei (MN) of patients. The results also suggest a correlation between chromosomal fragility and content of MN, and support the findings for a linkage between hypertension and a locus on chromosome 17.     

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.     

Arginine increases genotoxicity induced by methyl methanesulfonate in human lymphocytes

Nitric oxide (NO) is a free radical that is produced in cells from l-arginine. NO is involved in the physiological control of different tissues, but it can act as a toxic mediator in the cells. In this study we investigated the effect of l-arginine on the genotoxicity induced by methyl methanesulfonate (MMS) in human lymphocytes. Blood was treated with N^G-nitro-l-arginine methyl ester (l-NAME) as an inhibitor of nitric oxide synthase for finding out the role of NO in this effect. Human whole blood was treated with l-arginine (50, 100 and 250 μM) and/or l-NAME, then it was treated in vitro with MMS after 24 h of culture. The lymphocytes were stimulated by phytohemagglutinin to find out the micronuclei in cytokinesis-blocked binucleated cells. DNA fragmentation of lymphocytes was detected by using a fluorescence microscope after propidium iodide staining. These data showed that arginine increased the frequency of MMS-induced micronuclei in lymphocytes. However, the genotoxicity was decreased by using l-NAME. Arginine and l-NAME have not shown any DNA damage in cultured human lymphocytes. In conclusion, addition of l-arginine to MMS as an alkylating agent caused an increase of DNA damage in human lymphocytes. This enhancement of genotoxicity was reduced by NAME as NO inhibitor. It is thus cleared that an increase of DNA damage by arginine and MMS is related to NO production.     

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.     

Methionine dependency of cultured human lymphocytes

Human peripheral blood lymphocytes stimulated with phytohemagglutinin and a lymphocyte model consisting of the RPMI 6410 cell, a human virus-transformed B cell, required added methionine (Met) for growth of the cultures. This failure to meet all needs for Met via endogenous synthesis, which is characteristic of oncogenic transformation, occurred even in the presence of adequate homocysteine, methylfolate (5-CH3-H4PteGlu) and cobalamin (Cbl)-dependent methionine synthetase activity. Folinic acid (5-CHO-H4PteGlu), which provides available folate independently of Cbl, improved growth only slightly in the absence of Met. Free Cbl at 222 nM, an amount great enough to alter other intracellular events, failed to increase growth in the absence of Met, but 0.22 nM Cbl bound to transcobalamin II did, however, enhance growth.     

Resveratrol mitigates genotoxicity induced by iodine-131 in primary human lymphocytes

The purpose of this study was to investigate the radioprotective effects of resveratrol as a natural product that protects against genotoxic actions of ¹³¹I in cultured human lymphocytes. Whole-blood samples from human volunteers were treated with resveratrol at doses of 0.5, 1, 5, and 50 μg/mL for 1 h, after which the lymphocytes were incubated with ¹³¹I (100 μCi/1.5 mL) for 2 h. The lymphocyte cultures were then mitogenically stimulated to enable evaluation of the number of micronuclei in cytokinesis-blocked binucleated cells. Incubation of lymphocytes with ¹³¹I induced genotoxicity, which was reflected by an increase in micronuclei frequency. At the doses tested, resveratrol significantly reduced micronuclei frequency. Maximal protective effects occurred at a dose of 1 μg/mL, with total micronuclei values being reduced by 65 % compared to controls. In conclusion, our results indicate protective effects of resveratrol at low doses against genetic damage and adverse effects induced by ¹³¹I administration.     

The in vitro genotoxicity of benzoic acid in human peripheral blood lymphocytes

The chromosomal aberration (CA), sister chromatid exchange (SCE) and micronucleus test (MN) were employed to investigate the in vitro effect of antimicrobial food additive benzoic acid on human chromosomes. Lymphocytes were incubated with various concentrations (50, 100, 200 and 500 μg/mL) of benzoic acid. The results of used assays showed that benzoic acid significantly increased the chromosomal aberration, sister chromatid exchange and micronucleus frequency (200 and 500 μg/mL) without changing the pH of the medium in a dose-dependent manner. Also this additive significantly decreased the mitotic index (MI) at the highest concentration for 24 h and 100, 200 and 500 μg/mL for 48 h. This decrease was dose-dependent as well. However, it did not effect the replication (RI) and nuclear division (NDI) indices.     

Argininosuccinate synthetase activity in cultured human lymphocytes

The activity of argininosuccinate synthetase (E.C. 6.3.4.5), a urea cycle enzyme, was measured in cultured human lymphocytes using a new radioactive assay. Control cells had a maximum specific activity of 15.7±8.7 nmoles per hour per milligram of protein and an apparent K _m for citrulline of 2 × 10^–4 m, whereas cells derived from a patient with citrullinemia had no detectable activity. A nutritional variant, selected out of the citrullinemic lymphocyte population by ability to grow in citrulline, had a maximum specific activity of 10.7±3.8 nmoles/hr/mg and an apparent K _m for citrulline of 2 × 10^–2 m. These measurements confirm the observation that citrullinemia is associated with a defect in argininosuccinate synthetase activity and provide further evidence that citrullinemia is expressed in cultured lymphocytes. The emergence of a nutritional variant with a partial defect in argininosuccinate synthetase enzyme suggests that this citrullinemic patient has a heterogeneous population of cells, some totally defective and others only partially defective in argininosuccinate synthetase. The new activity assay is described in detail.This research was supported by a National Institutes of Health Training Grant (5-TO1-GM-0071) and NIH Program Project Grant (2-PO1-GM-15419).     

Evaluation of genotoxicity of combined soil pollution by cadmium and imidacloprid

Cadmium (Cd) is one of the important pollutants of soil and the genotoxicity of Cd-contaminated soil was studied in combination with imidacloprid. The single cell gel electrophoresis or comet assay was used to quantify DNA strand breaks as a measure of DNA damage induced by Cd and imidacloprid contamination in soil. The soil was artificially contaminated by Cd 2 h at 25℃ and were used in the comet assay. DNA damage was measured as the values of percentage of nuclei with tails, tail length, tail DNA, tail moment (TM), and Olive tail moment (OTM). DNA damages of root tips of Vicia faba increased after Cd treatment and there were dose-related increases in DNA damage measured as these parameters. However, the addition of imidacloprid further increased the DNA damage. These data confirmed the genotoxic effect of Cd to plants, and that the combined pollution with imidacloprid can enhance the genotoxicity of Cd.     

Evaluation of genotoxicity of combined soil pollution by cadmium and imidacloprid

Cadmium (Cd) is one of the important pollutants of soil and the genotoxicity of Cd-contaminated soil was studied in combination with imidacloprid. The single cell gel electrophoresis or comet assay was used to quantify DNA strand breaks as a measure of DNA damage induced by Cd and imidacloprid contamination in soil. The soil was artificially contaminated by Cd (0.0, 0.2, 0.5, 1.0, 2.0 mg· kg^?1 dry soil) or Cd (0.0, 0.2, 0.5, 1.0, 2.0 mg · kg^?1 dry soil) and imidacloprid (0.5 mg · kg^?1 dry soil). Roots ofVicia faba were exposed to the contaminated soil for 2 h at 25°C and were used in the comet assay. DNA damage was measured as the values of percentage of nuclei with tails, tail length, tail DNA, tail moment (TM), and Olive tail moment (OTM). DNA damages of root tips ofVicia faba increased after Cd treatment and there were dose-related increases in DNA damage measured as these parameters. However, the addition of imidacloprid further increased the DNA damage. These data confirmed the genotoxic effect of Cd to plants, and that the combined pollution with imidacloprid can enhance the genotoxicity of Cd.     

Evaluation of genotoxicity of combined soil pollution by cadmium and imidacloprid

Cadmium (Cd) is one of the important pollutants of soil and the genotoxicity of Cd-contaminated soil was studied in combination with imidacloprid. The single cell gel electro-phoresis or comet assay was used to quantify DNA strand breaks as a measure of DNA damage induced by Cd and imidacloprid contamination in soil. The soil was artificially contaminated by Cd (0.0, 0.2, 0.5, 1.0, 2.0 mg·kg-1 dry soil) or Cd (0.0, 0.2, 0.5, 1.0, 2.0 mg·kg-1 dry soil) and imidacloprid (0.5 mg-kg~1 dry soil). Roots of Vicia faba were exposed to the contaminated soil for 2 h at 25℃and were used in the comet assay. DNA damage was measured as the values of percentage of nuclei with tails, tail length, tail DNA, tail moment (TM), and Olive tail moment (OTM). DNA damages of root tips of Vicia faba increased after Cd treatment and there were dose-related increases in DNA damage measured as these parameters. However, the addition of imidacloprid further increased the DNA damage. These data confirmed the genotoxic effect of Cd