Chromosomal aberrations in cultured human lymphocytes treated with the fungicide,Thiram

In vitro effects of different concentrations of Thiram were tested on human lymphocytes to determine, by means of the chromosome aberrations (CAs) assay, whether this fungicide could induce clastogenic damage. Evidences of the effect of Thiram on human lymphocytes were limited to sister chromatid exchange, micronuclei formation, and comet assays. We evaluated 0.01, 0.1, 1.2, and 12.0 μg/mL of Thiram, where 0.01 μg/mL represent the acceptable daily intake dose set by the World Health Organization and the Food and Agriculture Organization for fruit and vegetables, whereas 0.1, 1.2, and 12.0 μg/mL are its multiple values. Results indicated that human lymphocytes treated in vitro with Thiram at concentrations of 1.20 and 12.0 μg/mL significantly increased CAs frequency, compared with the negative control, whereas at lower concentrations (0.01 and 0.1 μg/mL), this effect was not observed. However, Thiram showed a clastogenic effect also at the concentration value of 1.2 μg/mL that represents a lower value with respect to the residue limits found in Italy for grapes, strawberries, potatoes, tobacco, and other fruits and vegetables. Finally, according to some evidence obtained from the study of other fungicides, Thiram produced a significant reduction in the mitotic index with increasing concentration.     

Chromosomal aberrations in cultured human lymphocytes treated with the fungicide, Thiram

In vitro effects of different concentrations of Thiram were tested on human lymphocytes to determine, by means of the chromosome aberrations (CAs) assay, whether this fungicide could induce clastogenic damage. Evidences of the effect of Thiram on human lymphocytes were limited to sister chromatid exchange, micronuclei formation, and comet assays. We evaluated 0.01, 0.1, 1.2, and 12.0 μg/mL of Thiram, where 0.01 μg/mL represent the acceptable daily intake dose set by the World Health Organization and the Food and Agriculture Organization for fruit and vegetables, whereas 0.1, 1.2, and 12.0 μg/mL are its multiple values. Results indicated that human lymphocytes treated in vitro with Thiram at concentrations of 1.20 and 12.0 μg/mL significantly increased CAs frequency, compared with the negative control, whereas at lower concentrations (0.01 and 0.1 μg/mL), this effect was not observed. However, Thiram showed a clastogenic effect also at the concentration value of 1.2 μg/mL that represents a lower value with respect to the residue limits found in Italy for grapes, strawberries, potatoes, tobacco, and other fruits and vegetables. Finally, according to some evidence obtained from the study of other fungicides, Thiram produced a significant reduction in the mitotic index with increasing concentration.     

Chromosomal aberrations and sister-chromatid exchange frequencies in workers occupationally exposed to textile dyes.

The peripheral lymphocytes of 11 male and seven female workers occupationally exposed to textile dyes were studied for cytogenetic change. A significant increase in the frequency of chromosomal aberrations and sister chromatid exchanges were recorded regardless of the duration of the workers' exposure to the dyes.     

Chromosomal aberrations, micronuclei and nuclear buds induced in human lymphocytes by 2,4-dichlorophenoxyacetic acid pesticide formulation

Pesticides of worldwide application are used in agriculture in vast amounts each year, of which herbicides are the most prominent class. Phenoxyacetic herbicides constitute one of the largest groups of herbicides sold in the world. Among them, for many years 2,4-dichlorophenoxyacetic acid (2,4-D) has been the one most used. In this study we used Deherban A, a commercial formulation of 2,4-D to determine its possible genotoxic effect on human lymphocytes in vitro by chromosomal aberration analysis and micronucleus assay including the scoring of nuclear buds. Two different concentrations of pesticide formulation were used so that final concentrations of 2,4-D were 0.4 and 4 microg/ml, both in the presence and in the absence of the liver microsomal fraction as metabolic activator. Both concentrations of pesticide caused an increase in chromatid and chromosome breaks, number of micronuclei and number of nuclear buds. Presence of the S9 mix additionally elevated the number of chromatid breaks and micronuclei in treated lymphocytes.     

Chromosomal aberrations in workers exposed to low levels of benzene: association with genetic polymorphisms

Benzene and its metabolites damage human lymphocytes, resulting in chromosomal aberrations and aneuploidy. Polymorphisms in the genes for benzene-metabolizing enzymes have been implicated in benzene-associated haematotoxicity. In this study, we examined the specificity of benzene-induced aneuploidy and the influence of genetic polymorphisms (GSTM1, GSTT1, GSTP1, NAT2, NQO1 and CYP2E1) on chromosomal aberrations. In total, 82 benzene-exposed workers from a coke oven plant and 76 matched controls were examined. The benzene concentration in the work-place air ranged from 0.014-0.743 p.p.m. (geometric mean 0.557 p.p.m.). Benzene exposure was associated with significant increases in both monosomy and trisomy of chromosomes 8 and 21. Translocations between chromosomes 8 and 21 [t(8:21)] were eight-fold more frequent in the high-level exposure group compared to the control group. Multiple regression analysis indicated that the frequencies of chromosome aberrations were significantly associated with benzene exposure and polymorphisms in the metabolic enzyme genes. A particular subset of genotypes, which included the GSTM1-null and GSTT1-null genotypes, the slow acetylator type of NAT2, a variant of the NQO1 genotype and the CYP2E1 DraI and RsaI genotypes, were either separately, or in combination, associated with increased frequencies of aneuploidy among the benzene-exposed individuals after adjustments for age, alcohol consumption and smoking. These results suggest that polymorphisms in the genes for benzene-metabolizing enzymes influence the susceptibility of individuals to chromosomal aberrations in relation to benzene exposure.     

Chromosomal aberrations and in vitro radiosensitivity: intra-individual versus inter-individual variability

In order to assess the applicability of the micronucleus (MN) and G2 assays as biomarkers of in vitro radiosensitivity and cancer susceptibility, we investigated the inter- and intra-individual variation of these endpoints. For the MN assay unstimulated blood cultures from 57 healthy donors were exposed in vitro to 3.5 Gy Co gamma-rays and for the G2 assay PHA stimulated cultures were irradiated with a dose of 0.4 Gy Co gamma-rays in the G2 phase of the cell cycle. For 14 donors, 2-15 repeat samples were tested over a period of 3 years. The repeat experiments revealed that the intra-individual variability was not significantly different from the inter-individual variability for both G2 and MN assays. As the intra-individual variability determines the reproducibility of the assay, our results highlight the limitations of these endpoints in detecting reproducible differences in radiation sensitivity between individuals within a normal population. Due to the high intra-individual variability and no significant difference with the inter-individual variability found in our study we conclude that care has to be taken when results obtained with chromosomal aberration assays based on one blood sample are used to assess the individual radiosensitivity. Multiple blood sampling may be necessary to draw reliable conclusions. Although more validation studies on the reliability of the G2 and MN assay will be required before they can be used in a confident way as biomarkers of individual radiosensitivity or cancer susceptibility the assays are very valuable to examine population radiosensitivity and the relationship between radiosensitivity, cancer predisposition and genotype.     

Induction of chromosomal aberrations by carbon nanotubes and titanium dioxide nanoparticles in human lymphocytes in vitro

Abstract

We examined if three commercially available nanomaterials – short singlewall carbon nanotubes (SWCNTs), short multiwall carbon nanotubes (MWCNTs) and nanosized titanium dioxide anatase (TiO₂; primary particle size <25 nm) – can induce structural chromosomal aberrations (CAs) in cultures of isolated human lymphocytes. To find a suitable sampling time, the cells were treated with 6.25–300 μg/ml of the nanomaterials for 24, 48 and 72 h. The 48-h treatment was the most effective, inducing a dose-dependent increase in chromosome-type CAs (all materials) and chromatid-type CAs (SWCNTs and TiO₂ anatase). The 72-h treatment yielded a positive result with SWCNTs. None of the treatments significantly affected cell count or the mitotic index. Our results suggest that with nanomaterials a continuous treatment for about two cell cycles is needed for CA induction, possibly reflecting access of nanomaterials to the nucleus during the first mitosis or delayed secondary genotoxic effect associated with the inflammatory process.     

Induction of chromosomal aberrations and sister chromatid exchanges by propane sultone in human lymphocytes in vitro

Genotoxicological investigation of 1,3-propane sultone was undertaken using human lymphocyte cultures as the test system. Concentrations greater than 1 mM induced appreciable chromosomal aberrations, which increased proportionally to the dose or the duration of treatment. It was also observed that treatment administered at 45 degrees C produced almost 3 times more abnormal cells than at 37 degrees C. The chemical also produced a significantly higher frequency of SCEs.     

In vitro enrofloxacin binding in human fecal slurries

Most antibiotic inactivation studies have been conducted through in vitro incubations of human use aminoglycosides, beta-lactams, and fluoroquinolones, usually at fecal concentrations expected with therapeutic dose regimens in humans and animals. Less is known about the inactivation of these molecules when ingested at concentrations consistent with residue levels present in animal-derived foods from antibiotic treated animals. In this investigation, we used the fluoroquinolone, enrofloxacin which is specifically marketed for veterinary medicine as test compound. Fecal suspensions at 10%, 25%, and 50% (w/v) were subjected to physicochemical and molecular characterization and used in the drug binding studies. The fecal binding of enrofloxacin added at concentrations of 0.06, 0.1, 1, 5, 15, 50, and 150 mg/L was determined in various fecal slurry suspensions using analytical chemistry and microbiological assay methods. There was consistent correlation between both assay methods. By the analytical chemistry assay, the 10%, 25% and 50% diluted autoclaved fecal samples dosed with enrofloxacin showed binding of 50±4.6%, 54±6.5% and 56±6.8% of the enrofloxacin, respectively. Binding of enrofloxacin to fecal contents occurred rapidly within 10 min and remained constant over the incubation period. Denaturing gradient gel electrophoreses and pyrosequencing analysis showed varied profiles of the bacterial composition of the human intestinal microbiota for fecal samples from different individuals. This study provided information on methodological questions that have concerned regulatory authorities on in vitro testing to determine if concentrations of veterinary antimicrobial agent residues entering the human colon remain microbiologically active.     

Chromosomal aberrations in peripheral lymphocytes of children as biomarkers of environmental exposure and life style

The original purpose of our study was to determine if the detection of chromosomal aberrations in peripheral lymphocytes of children might be used as a biomarker of environmental pollution and life style. We compared the results of cytogenetic analyses performed in children and adolescents in the periods 1984-1993 and 1994-1999, in a total of 3402 subjects. The frequency of aberrant cells (AB.C.) markedly decreased in the period 1994-1999 compared with the period 1984-1993. The decreases in AB.C. were significant in the age groups 7-15 and 16-19 years: 1.63% AB.C. versus 1.14% AB.C. and 2.02% AB.C. versus 1.08% AB.C., respectively (P<0.01). No difference in the frequency of AB.C. was observed in newborns. Based on our experience, we believe that monitoring the spontaneous level of chromosomal aberrations in children over 5 year periods may be used to examine the general changes in environmental pollution in larger geographic areas.     

Sister-chromatid exchanges and chromosome aberrations in lymphocytes from monkeys exposed to ethylene oxide and propylene oxide by inhalation

The ability of long-term exposures to inhaled ethylene oxide (EO) and propylene oxide (PO) to induce sister-chromatid exchanges (SCEs) and chromosome aberrations in peripheral lymphocytes of monkeys was investigated. Five groups of adult male cynomolgus monkeys were exposed at 0 (shared control), 50, or 100 ppm EO, and at 100 or 300 ppm PO (7 hr/day, 5 days/week) for 2 years. EO exposures at 50 and 100 ppm resulted in statistically significant increases in sister-chromatid exchange rates and in the incidence of chromosome aberrations in monkey lymphocytes. Both EO-exposed groups had increased numbers of SCEs/metaphase compared to controls, with the SCEs/metaphase of the EO 100 ppm group also significantly elevated versus the EO 50 ppm group. Variability of SCEs/metaphase within each monkey increased even more than the increase in total SCEs/metaphase group with increasing EO exposure. Chromatid-type aberrations were also significantly increased for both EO 50 and EO 100 ppm groups compared to controls. Statistically significant increases in the number of chromosome-type aberrations (excluding gaps) were found only in the EO 100 ppm group. Combined chromatid- and chromosome-type aberrations were increased in both EO 50 and EO 100 ppm groups. No group differences in the number of gaps were found. In lymphocytes from monkeys exposed at 100 and 300 ppm PO, there were no group differences compared to controls for any variable-chromatid or chromosome-type aberrations, gaps, or SCEs/metaphase. These results indicate that EO is a more potent clastogen than PO and demonstrate, for the first time, statistically significant effects of EO on both SCEs and chromosome aberrations in lymphocytes of nonhuman primates.     

In vitro and in vivo effects of melatonin on sister chromatid exchange in human blood lymphocytes exposed to hypoxia

Objective: Many studies have shown that melatonin (MLT) has an anti-genotoxic effect in various tissues and cell lines. The aim of this study was to investigate the anti-genotoxic effect of MLT on normal human peripheral lymphocytes by assessing sister chromatid exchange (SCE) in vitro and in vivo. Materials and methods: Cells were treated with 50 and 200?μM of MLT. The human volunteers (n?=?20) for the in vivo study were administered a single dose of 3?mg MLT daily for 2 weeks. After sufficient time for its clearance, 1.5?mg of MLT daily was then administered to the same volunteers at same the period. Results: Our results demonstrated the anti-genotoxic effect of MLT in human blood lymphocyte in vitro and in vivo. In vitro, hypoxia increased the SCE frequency compared to the control and both doses of MLT significantly decreased the SCE frequency in the hypoxic cells (p?<?0.001). In vivo, oral administration of 3?mg MLT significantly increased the frequency of SCE, yet a small increase of SCE by hypoxia was found. Oral administration of 1.5?mg MLT showed no DNA damage but it had an anti-genotoxic effect. Discussion and conclusion: MLT may prove useful for reducing the genotoxic effects of hypoxia in peripheral lymphocytes and suggest its possible role for ischemic diseases.     

Genotoxic effects in human lymphocytes exposed to arsenic and vitamin A.

Arsenic is a ubiquitous trace element and a well-established human carcinogen. In search for an 'antidote' to this global poison, this work was undertaken to study the probable beneficial effect of vitamin A upon arsenic induced genotoxicity. Peripheral blood lymphocyte culture was carried out to study the effects of arsenic at three different dose levels (0.5, 1 and 2 microg) for 24 h prior to harvesting. In addition, mutagenic in vitro effect of ethyl methanesulphonate was studied as a positive control. Genotoxic variables presented here are sister chromatid exchanges (SCE), cell cycle proliferative index/replicative index (CCPI/RI), average generation time (AGT) and population doubling time (PDT). Inevitably, arsenic treatment showed dose-dependent augmentation in the incidences of SCE and CCPI/RI together with AGT and PDT. However, vitamin A supplemented arsenic cultures demonstrated remarkable resurgence in the described genotoxic parameters. This data shows that vitamin A might be a useful interventional treatment in arsenic poisoning.     

Effects of enrofloxacin on the human intestinal microbiota in vitro

An anaerobic, continuous-flow culture method has been developed to assess the effects of different levels of enrofloxacin (ENR) on the human intestinal microbiota. Chemostats containing human faecal flora were exposed to 1.25, 12.5 and 125 μg/mL ENR. Before and during drug exposure, samples aspirated from culture vessels were analysed using viable cell counting and denaturing gradient gel electrophoresis (DGGE). In addition, the colonisation resistance (CR) of the microbiota to Candida albicans SC5314 was evaluated. When exposed to 1.25 μg/mL ENR, total counts of aerobic bacteria, anaerobic bacteria, Lactobacillus, enterococci, Escherichia coli and Bacteroides fragilis were similar to the control group, except for Bifidobacterium; when exposed to 12.5 μg/mL and 125μg/mL ENR, numbers of categorised microorganisms changed significantly, except for B. fragilis. DGGE indicated that although 1.25 μg/mL ENR had little effect on the total number of microbiota, several bands representing dominant bacteria disappeared. The bands disappeared more quickly when exposed to 12.5 μg/mL and 125 μg/mL ENR. In addition to their influence on microbial diversity, different levels of ENR reduced the CR of the intestinal microbiota to C. albicans SC5314.     

HPLC residues of enrofloxacin and ciprofloxacin in eggs of laying hens

Eggs of 12 laying hens with 5 mg/kg/day oral administration of 5% enrofloxacin (EFX) or ciprofloxacin (CFX) solution during 5 days contained residues from 0.02 to 1.98 μg/g (EFX) or 0.14 to 0.28 μg/g (CFX). At identical dosage regime High Performance Liquid Chromatograhy (HPLC) residues of EFX were 6-fold greater than CFX ones. Maximun concentrations were detected at the second day after the administration withdrawal. The limits of detection were 0.019 μg/g for EFX and 0.156 μg/g for CFX. The recovery was 36–50% for CFX and 49–85% for EFX. The withdrawal treatment periods in hens are six days for EFX and five days for CFX in order to avoid violative levels of egg residues.     

Chromosomal aberrations,sister chromatid exchanges,and micronuclei in lymphocytes of oncology department personnel handling anti-neoplastic drugs

Objective: Concern exists regarding the possible hazards to the personnel handling anti-neoplastic drugs. The purpose of the present study was to assess the genotoxicity induced by anti-neoplastic agents in oncology department personnel. Materials and methods: To do this, the frequency of chromosomal aberrations (CAs) induced in peripheral blood lymphocytes was assessed at G₀ phase of the cell cycle using metaphase analysis, cytokinesis block-micronucleus (MN) assay and sister chromatid exchange (SCE) assay. These cytogenetic end points were measured among 71 nurses in oncology department and 10 drugstore personnel handling and preparing anti-neoplastic drugs. The results were compared to those of 74 matched nurses for age and sex not exposed to any anti-neoplastic agents. Results: There was no significant difference between the age of study subjects and control group (p?>?0.05). The results showed that the mean frequency of cytogenetic damages in terms of CAs [chromatid breaks (p?=?0.01), chromosome breaks (p?=?0.005), total CAs (p?=?0.001)], MN formation (p?=?0.001), and SCE (p?=?0.004) in lymphocytes of personnel handling anti-neoplastic drugs were significantly higher than those in control unexposed group. Conclusion: Results of the present study demonstrate the cytogenetic damage in peripheral blood lymphocytes of oncology department personnel. Suitable training and proper knowledge when handling anti-neoplastic drugs are emphasized to avoid potential health hazards caused by cytostatic agents.     

Chromosomal aberrations induced by sodium nitrite in bone marrow of adult rats and liver cells of transplacentally exposed embryos

A commonly used food preservative, sodium nitrite, was administered in the drinking water to pregnant (d 5-18 of gestation) and nonpregnant albino rats. Sodium nitrite induced chromosomal aberrations in bone marrow of both pregnant and nonpregnant adults and liver of transplacently exposed embryos. The magnitude of the effect was greater in embryonic liver cells than in adult bone marrow cells.     

Ecotoxicity of veterinary enrofloxacin and ciprofloxacin antibiotics on anuran amphibian larvae

The ecological risks posed by two β-diketone antibiotics (DKAs, enrofloxacin, ENR and ciprofloxacin, CPX), characterized by their long persistence in aqueous environments and known deleterious effect on model organisms such as zebrafish were analysed using Rhinella arenarum larvae. Sublethal tests were conducted using environmentally relevant concentrations of both ENR and CPX (1–1000 μg L⁻¹) under standard laboratory conditions for 96 h. Biological endpoints and biomarkers evaluated were body size, shape, development and growth rates, and antioxidant enzymes (glutathione-S-transferase, GST; Catalase, CAT). Risk assessment was analysed based on ration quotients (RQ). The size and shape measurements of the larvae exposed to concentrations greater than 10 μg L⁻¹ of CPX were lower compared to controls (Dunnett post hoc p < 0.05) and presented signs of emaciation. Concentrations of 1000 μg L⁻¹of CPX induced GST activity, in contrast with inhibited GST and CAT of larvae exposed to ENR. Risk assessments indicated that concentrations greater than or equal to10 μg L⁻¹ of CPX and ENR are ecotoxic for development, growth, detoxifying, and oxidative stress enzymes. It is suggested that additional risk assessments may provide evidence of bioaccumulation of CPX and ENR in tissues or organs of amphibian larvae by mesocosm sediment test conditions. Finally, intestinal microbiome studies should be considered to establish the mechanisms of action of both antibiotics.     

Cytoprotective activity of amifostine on cultured human lymphocytes exposed to irinotecan

Irinotecan (camptothecin, CAM) is a topoisomerase-I inhibitor with a well established action in the chemotherapy of colorectal and ovarian cancer. Hematological and intestinal toxicity are commonly noted in patients treated with CAM. In this study, we examined the cytoprotective efficacy of amifostine (ethyol, ETH) against chromosomal damage induced by this drug on cultured peripheral human lymphocytes. Cultured lymphocytes were exposed to CAM (50 and 100 ng/ml of final concentrations) without or with ETH (in concentrations varying between 40 and 800 μg/ml of final culture volume). CAM’s genotoxicity was quantified by counting the Sister Chromatid Exchange (SCEs) rate. The mitotic index (MI) and proliferation rate index (PRI) were also assessed. The SCE rate was increased following incubation with CAM, but the combined treatment of CAM with ETH significantly reduced the SCE formation, especially when ETH added at high concentrations. The MIs and PRIs remained also unaltered in cultures with CAM, but MIs were reduced with the combined treatment at high ETH concentrations. Clinical studies are required to assess the predicted benefits from ETH in patients receiving CAM.