Genotoxic activity of the insecticide Nuvacron (Monocrotophos) detected by the micronucleus test in bone marrow erythrocytes of mice and in CHO cells

The organophosphorus insecticide Nuvacron (Monocrotophos) is a very toxic agent widely utilized in Brazilian agriculture. To evaluate the clastogenic potential of this insecticide, in vivo and in vitro micronucleus (MN) assay experiments were carried out on Swiss mice and on Chinese hamster ovary (CHO) cells, respectively. Nuvacron administered at doses of 2.5 and 5.0 mg/kg induced a statistically significant increase in the frequencies of MN detected in polychromatic bone marrow erythrocytes from animals (six/group) treated ip 24 h before. Exponentially growing CHAO cells were treated continuously (16h) with Nuvacron diluted in water to final concentrations of 1, 10, 100, 200, and 400 mug/ml. Three experiments were carried out using the cytokinesis-block method and a total of 6000 binucleated cells were scored to determine MN frequencies. A statistically significant increase in the frequencies of MN was observed for the cells treated with 1 and 10 mug/ ml Nuvacron. A marked decrease in cell proliferation rates was observed for CHO cultures treated with higher concentrations. These data demonstrate that Nuvacron has a genotoxic effect on both in vivo and in vitro mammalian test systems.

Clastogenic activity of integerrimine determined in mouse micronucleus assays

The pyrrolizidine alkaloid integerrimine, obtained from Senecio brasiliensis, was tested by acute dosing at two concentrations (18.75 and 37.5 mg/kg), and at different times, to establish its ability to induce micronuclei in mouse erythrocytes. This alkaloid was able to increase the frequency of micronucleated polychromatic erythrocytes in both, bone marrow and peripheral blood erythrocytes

Efficacy of HUMN criteria for scoring the micronucleus assay in human lymphocytes exposed to a low concentration of p,p'-DDT

The cytokinesis-block micronucleus (CBMN) assay is one of the standard cytogenetic tools employed to assess chromosomal damage subsequent to exposure to genotoxic/cytotoxic agents, and is widely applicable to plant, animal and human cells. In the present study, the CBMN assay was used to assess the baseline damage in binuclear human peripheral blood lymphocytes exposed to 25 µg/L p,p'-DDT for 1, 2, 24, and 48 h by measuring the frequency of micronuclei, nucleoplasmic bridges and nuclear buds. These new scoring criteria facilitated the detection of different types of clastogenic and aneugenic effects induced by this type of pollutant. With these criteria, CBMN can also be used to measure nucleoplasmic bridges which are considered to be consequences of chromosome rearrangements and nuclear buds which are biomarkers of altered gene amplification and gene dosage. The total number of micronuclei observed in binuclear human peripheral blood lymphocytes of the exposed samples (ranging from 32 to 47) was significantly greater (P < 0.05) than that detected in the unexposed (0 time) control sample, where the total number of micronuclei was 7. The number of nucleoplasmic bridges and nuclear buds obtained after 24 and 48 h was also significantly (P < 0.05) greater in the samples treated with p,p'-DDT than in the unexposed control samples. Thus, our results confirmed the usefulness of the new criteria applicable for the CBMN assay employed in measuring the DNA damage and its role of a sensitive cytogenetic biomarker.

Increased frequency of micronuclei in the lymphocytes of patients chronically infected with hepatitis B or hepatitis C virus

In this study, we analysed the frequency of micronuclei (MN), nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) and evaluated mutagen-induced sensitivity in the lymphocytes of patients chronically infected with hepatitis B virus (HBV) or hepatitis C virus (HCV). In total, 49 patients with chronic viral hepatitis (28 HBV-infected and 21 HCV-infected patients) and 33 healthy, non-infected blood donor controls were investigated. The frequencies (‰) of MN, NPBs and NBUDs in the controls were 4.41 ± 2.15, 1.15 ± 0.97 and 2.98 ± 1.31, respectively. The frequencies of MN and NPBs were significantly increased (p < 0.0001) in the patient group (7.01 ± 3.23 and 2.76 ± 2.08, respectively) compared with the control group. When considered separately, the HBV-infected patients (7.18 ± 3.57) and HCV-infected patients (3.27 ± 2.40) each had greater numbers of MN than did the controls (p < 0.0001). The HCV-infected patients displayed high numbers of NPBs (2.09 ± 1.33) and NBUDs (4.38 ± 3.28), but only the HBV-infected patients exhibited a significant difference (NPBs = 3.27 ± 2.40, p < 0.0001 and NBUDs = 4.71 ± 2.79, p = 0.03) in comparison with the controls. Similar results were obtained for males, but not for females, when all patients or the HBV-infected group was compared with the controls. The lymphocytes of the infected patients did not exhibit sensitivity to mutagen in comparison with the lymphocytes of the controls (p = 0.06). These results showed that the lymphocytes of patients who were chronically infected with HBV or HCV presented greater chromosomal instability.

Pollen mother cells of Tradescantia clone 4430 and Tradescantia pallida var. purpurea are equally sensitive to the clastogenic effects of X-rays

The Tradescantia micronucleus test is a sensitive bioassay for mutagenesis that may be employed both under field and laboratory conditions. This test has been standardized mostly on the basis of the results obtained with clone 4430. However, this clone is not well adapted to tropical weather, frequently showing problems with growth and flowering. In addition, it is attacked by parasites and insects, a fact that limits its use in field studies aiming at the biomonitoring of air pollution. In the city of São Paulo, Tradescantia pallida (Rose) Hunt. var. purpurea Boom is widely distributed as an ornamental plant in gardens and along roadsides and streets, mostly because of its natural resistance and its easy propagation. In this report, we present dose-response curves indicating that the sensitivity of T. pallida and clone 4430 to X-radiation (1, 10, 25 and 50 cGy) is similar. The results confirm our previous suggestion that T. pallida represents a good alternative for in situ mutagenesis testing in tropical regions, especially biomonitoring studies in which the exposure conditions may not be fully controllable.

Age-related changes in radiation-induced micronuclei among healthy adults

The aim of the present study was to establish the extent of in vitro radioresponse of lymphocytes among 62 healthy adults of both genders and to estimate the distribution of baseline micronuclei and radiosensitivity among individuals of the study population using the cytochalasin block micronucleus test. A younger study group consisted of 10 males (mean age, 22.4 years; range, 21-27) and 12 females (mean age, 24.8 years; range, 20-29), whereas an older study group consisted of 18 males (mean age, 35.1 years; range, 30-44) and 22 females (mean age, 38.5 years; range, 30-48). For evaluation of radiosensitivity blood samples were irradiated in vitro using 60Co g-ray source. The radiation dose employed was 2 Gy, the dose rate 0.45 Gy/min. The study revealed a significant gender effect on baseline micronuclei favoring females (Z = 3.25, P < 0.001), while yields of radiation-induced micronuclei did not differ significantly (Z = 0.56, P < 0.56) between genders. The distribution of baseline micronuclei among the individuals tested followed Poisson distribution in both study groups and in both genders, whereas the distribution of radiosensitivity among individuals of the older study group did not fulfill Poisson expectations (Kolmogorov-Smirnof test, P < 0.01). In contrast to a nonsignificant difference in radiosensitivity between males and females of the same age group (Z = 1.97, P < 0.56), a statistically significant difference in radiosensitivity between younger and older group for both genders was found (Z = 3.03, P < 0.03). Since the individuals tested were healthy, the observed variability in radiation response is considered to be an early effect of ageing.

The mutagenic and antimutagenic effects of the traditional phytoestrogen-rich herbs, Pueraria mirifica and Pueraria lobata

Pueraria mirifica is a Thai phytoestrogen-rich herb traditionally used for the treatment of menopausal symptoms. Pueraria lobata is also a phytoestrogen-rich herb traditionally used in Japan, Korea and China for the treatment of hypertension and alcoholism. We evaluated the mutagenic and antimutagenic activity of the two plant extracts using the Ames test preincubation method plus or minus the rat liver mixture S9 for metabolic activation using Salmonella typhimurium strains TA98 and TA100 as indicator strains. The cytotoxicity of the two extracts to the two S. typhimurium indicators was evaluated before the mutagenic and antimutagenic tests. Both extracts at a final concentration of 2.5, 5, 10, or 20 mg/plate exhibited only mild cytotoxic effects. The plant extracts at the concentrations of 2.5, 5 and 10 mg/plate in the presence and absence of the S9 mixture were negative in the mutagenic Ames test. In contrast, both extracts were positive in the antimutagenic Ames test towards either one or both of the tested mutagens 2-(2-furyl)-3-(5-nitro-2-furyl)-acrylamide and benzo(a)pyrene. The absence of mutagenic and the presence of anti-mutagenic activities of the two plant extracts were confirmed in rec-assays and further supported by a micronucleus test where both plant extracts at doses up to 300 mg/kg body weight (equivalent to 16 g/kg body weight plant tuberous powder) failed to exhibit significant micronucleus formation in rats. The tests confirmed the non-mutagenic but reasonably antimutagenic activities of the two plant extracts, supporting their current use as safe dietary supplements and cosmetics.

Animal model for age- and sex-related genotoxicity of diethylstilbestrol

Environmental xenoestrogens pose a significant health risk for all living organisms. There is growing evidence concerning the different susceptibility to xenoestrogens of developing and adult organisms, but little is known about their genotoxicity in pre-pubertal mammals. In the present study, we developed an animal model to test the sex- and age-specific genotoxicity of the synthetic estrogen diethylstilbestrol (DES) on the reticulocytes of 3-week-old pre-pubertal and 12-week-old adult BALB/CJ mice using the in vivo micronucleus (MN) assay. DES was administered intraperitoneally at doses of 0.05, 0.5, and 5 µg/kg for 3 days and animals were sampled 48, 72 and 96 h, and 2 weeks after exposure. Five animals were analyzed for each dose, sex, and age group. After the DES dose of 0.05 µg/kg, pre-pubertal mice showed a significant increase in MN frequency (P < 0.001), while adults continued to show reference values (5.3 vs 1.0 MN/1000 reticulocytes). At doses of 0.5 and 5 µg/kg, MN frequency significantly increased in both age groups. In pre-pubertal male animals, MN frequency remained above reference values for 2 weeks after exposure. Our animal model for pre-pubertal genotoxicity assessment using the in vivo MN assay proved to be sensitive enough to distinguish age and sex differences in genome damage caused by DES. This synthetic estrogen was found to be more genotoxic in pre-pubertal mice, males in particular. Our results are relevant for future investigations and the preparation of legislation for drugs and environmentally emitted agents, which should incorporate specific age and gender susceptibility.

Micronucleated lymphocytes in parents of Down syndrome children

Down syndrome (DS) is the most common disease due to an autosomal aneuploidy in live born children and also the major known genetic cause of mental retardation. The risk of a DS pregnancy increases substantially with increasing maternal age. However, several women aged less than 35 years at conception have a child with DS. The micronucleus (MN) assay can identify chromosome breakage or chromosome malsegregation and is an ideal biomarker to investigate genomic instability. The aim of the present study was to determine the frequency of peripheral lymphocytes with MN in the parents of DS individuals. The subjects were 17 couples, 1 father and 9 mothers, and 24 couples who had at least one healthy child formed the control group. For each individual we evaluated the frequency of binucleated micronucleated lymphocytes (BNMN%) as number of binucleated lymphocytes containing one or more MN per 1000 binucleated cells. The mean age of DS parents and controls was 32.6 and 29.8 years, respectively. The frequency of MN in DS parents was significantly higher compared to controls. The higher frequency of MN in DS parents suggests a higher predisposition of DS parents to aneuploidy events in this sample.

Lipid peroxidation, detoxification capacity, and genome damage in mice after transplacental exposure to pharmaceutical drugs

Data on genome damage, lipid peroxidation, and levels of glutathione peroxidase (GPX) in newborns after transplacental exposure to xenobiotics are rare and insufficient for risk assessment. The aim of the current study was to analyze, in an animal model, transplacental genotoxicity, lipid peroxidation, and detoxification disturbances caused by the following drugs commonly prescribed to pregnant women: paracetamol, fluconazole, 5-nitrofurantoin, and sodium valproate. Genome damage in dams and their newborn pups transplacentally exposed to these drugs was investigated using the in vivo micronucleus (MN) assay. The drugs were administered to dams intraperitoneally in three consecutive daily doses between days 12 and 14 of pregnancy. The results were correlated, with detoxification capacity of the newborn pups measured by the levels of GPX in blood and lipid peroxidation in liver measured by malondialdehyde (HPLC-MDA) levels. Sodium valproate and 5-nitrofurantoin significantly increased MN frequency in pregnant dams. A significant increase in the MN frequency of newborn pups was detected for all drugs tested. This paper also provides reference levels of MDA in newborn pups, according to which all drugs tested significantly lowered MDA levels of newborn pups, while blood GPX activity dropped significantly only after exposure to paracetamol. The GPX reduction reflected systemic oxidative stress, which is known to occur with paracetamol treatment. The reduction of MDA in the liver is suggested to be an unspecific metabolic reaction to the drugs that express cytotoxic, in particular hepatotoxic, effects associated with oxidative stress and lipid peroxidation.

Compounds used to produce cloned animals are genotoxic and mutagenic in mammalian assays in vitro and in vivo

The compounds 6-dimethylaminopurine and cycloheximide promote the successful production of cloned mammals and have been used in the development of embryos produced by somatic cell nuclear transfer. This study investigated the effects of 6-dimethylaminopurine and cycloheximide in vitro, using the thiazolyl blue tetrazolium bromide colorimetric assay to assess cytotoxicity, the trypan blue exclusion assay to assess cell viability, the comet assay to assess genotoxicity, and the micronucleus test with cytokinesis block to test mutagenicity. In addition, the comet assay and the micronucleus test were also performed on peripheral blood cells of 54 male Swiss mice, 35 g each, to assess the effects of the compounds in vivo. The results indicated that both 6-dimethylaminopurine and cycloheximide, at the concentrations and doses tested, were cytotoxic in vitro and genotoxic and mutagenic in vitro and in vivo, altered the nuclear division index in vitro, but did not diminish cell viability in vitro. Considering that alterations in DNA play important roles in mutagenesis, carcinogenesis, and morphofunctional teratogenesis and reduce embryonic viability, this study indicated that 6-dimethylaminopurine and cycloheximide utilized in the process of mammalian cloning may be responsible for the low embryo viability commonly seen in nuclear transfer after implantation in utero.

Cell cycle synchronization and BrdU incorporation as a tool to study the possible selective elimination of ErbB1 gene in the micronuclei in A549 cells

Lung cancer often exhibits molecular changes, such as the overexpression of the ErbB1 gene that encodes epidermal growth factor receptor (EGFR). ErbB1 amplification and mutation are associated with tumor aggressiveness and low response to therapy. The aim of the present study was to design a schedule to synchronize the cell cycle of A549 cell line (a non-small cell lung cancer) and to analyze the possible association between the micronuclei (MNs) and the extrusion of ErbB1 gene extra-copies. After double blocking, by the process of fetal bovine serum deprivation and vincristine treatment, MNs formation was monitored with 5-bromo-2-deoxyuridine (BrdU) incorporation, which is an S-phase marker. Statistical analyses allowed us to infer that MNs may arise both in mitosis as well as in interphase. The MNs were able to replicate their DNA and this process seemed to be non-synchronous with the main cell nuclei. The presence of ErbB1 gene in the MNs was evaluated by fluorescent in situ hybridization (FISH). ErbB1 sequences were detected in the MNs, but a relation between the MNs formation and extrusion of amplified ErbB1could not be established. The present study sought to elucidate the meaning of MNs formation and its association with the elimination of oncogenes or other amplified sequences from the tumor cells.