Sarbani Giri

The HUman MicroNucleus project on eXfoLiated buccal cells (HUMNXL): The role of life-style, host factors, occupational exposures, health status, and assay protocol

The human buccal micronucleus cytome assay (BMCyt) is one of the most widely used techniques to measure genetic damage in human population studies. Reducing protocol variability, assessing the role of confounders, and estimating a range of reference values are research priorities that will be addressed by the HUMN(XL) collaborative study. The HUMN(XL) project evaluates the impact of host factors, occupation, life-style, disease status, and protocol features on the occurrence of MN in exfoliated buccal cells. In addition, the study will provide a range of reference values for all cytome endpoints. A database of 5424 subjects with buccal MN values obtained from 30 laboratories worldwide was compiled and analyzed to investigate the influence of several conditions affecting MN frequency. Random effects models were mostly used to investigate MN predictors. The estimated spontaneous MN frequency was 0.74‰ (95% CI 0.52-1.05). Only staining among technical features influenced MN frequency, with an abnormal increase for non-DNA-specific stains. No effect of gender was evident, while the trend for age was highly significant (p<0.001). Most occupational exposures and a diagnosis of cancer significantly increased MN and other endpoints frequencies. MN frequency increased in heavy smoking (≥40cig/day, FR=1.37; 95% CI 1.03-.82) and decreased with daily fruit consumption (FR=0.68; 95% CI 0.50-0.91). The results of the HUMN(XL) project identified priorities for validation studies, increased the basic knowledge of the assay, and contributed to the creation of a laboratory network which in perspective may allow the evaluation of disease risk associated with MN frequency.

Genotoxic effects of malathion: an organophosphorus insecticide, using three mammalian bioassays in vivo

The genotoxic effects of malathion was evaluated using chromosome aberration, sister chromatid exchange (SCE) and sperm abnormality assays in mice. All the three acute doses (2.5, 5 and 10mg/kg) of malathion tested in the present study, induced significant dose-dependent increase in the frequency of chromosome aberrations and sperm abnormalities, but did not affect the total sperm count. The highest acute dose induced a >12-fold increase in the frequency of chromosome aberrations, two-fold increase in the frequency of SCEs and four-fold increase in the frequency of sperms with abnormal head morphology following intraperitoneal (i.p.) exposure. Further, a significant increase in the frequency of SCEs was observed, but the increase was not dose-dependent. At higher doses, malathion induced a moderate delay in cell cycle as evident from the increase in average generation time (AGT). The present findings suggest that technical grade malathion is a potent genotoxic agent and may be regarded as a potential germ cell mutagen also.

Mutagenic effects of carbosulfan, a carbamate pesticide

The genotoxic effects of carbosulfan were evaluated using chromosome aberration (CA), bone marrow micronucleus (MN) and sperm abnormality assays in mice. All the three acute doses (1.25, 2.5 and 5mg/kg) of carbosulfan induced significant dose-dependent increase in the frequency of CA (P<0.02), micronucleated polychromatic erythrocytes (PCEs) (P<0.05) and sperm head abnormalities (P<0.05) but did not affect the total sperm count. The highest acute dose of carbosulfan induced >7-fold increase in the frequency of CA, >3.5-fold increase in the frequency of micronucleated PCEs and >4.6-fold increase in the frequency of sperms with abnormal head morphology following intraperitoneal exposure as compared to the untreated controls. The present findings suggest that carbosulfan is a potent genotoxic agent and may be regarded as a potential germ cell mutagen also.

Micronucleus and other nuclear abnormalities among betel quid chewers with or without sadagura, a unique smokeless tobacco preparation, in a population from North-East India.

Genotoxicity is one of the important endpoints for risk assessment of various lifestyle factors. The study is the first report on the genotoxic effect associated with sadagura, a unique smokeless tobacco prepared in southern Assam province of North-East India. Sadagura is consumed with or without betel quid and/or smoking. In the present cytogenetic monitoring study, analysis of micronuceus (MN), nuclear bud, binucleated, karyorrhectic, karyolytic and pyknotic cells tests were performed in the exfoliated buccal cells of 75 habituates and compared to controls matched for gender, age, and habit. Significant increase in the frequency of MN was found in sadagura chewers (0.48%, P < 0.001), smokers (0.46%, P < 0.01), betel quid with sadagura chewers (0.91%, P < 0.001) and smokers chewing betel quid with sadagura (0.53%, P < 0.001) as compared to the unexposed control group (0.07%). Betel quid chewers showed significant increase (1.65%, P < 0.05) in the frequency of binucleated cells as compared to the control group (0.16%). Results of this study demonstrated that sadagura consumed as a single agent or in combination with betel quid, leads to a significant induction of cytogenetic damage in the buccal epithelial cells of habituates. We suggest that analysis of other degenerative nuclear changes in addition to MN can provide valuable information while evaluating potential genotoxic agents.

Toxic and genotoxic effects of Roundup on tadpoles of the Indian skittering frog (Euflictis cyanophlyctis) in the presence and absence of predator stress

Glyphosate, a post emergent herbicide, has become the backbone of no-till agriculture and is considered safe for animals. However, the impact of glyphosate on non-target organisms, especially on amphibians, is the subject of major concern and debate in recent times. We examined the toxic and genotoxic effects of Roundup, a commercial formulation of glyphosate, in the tadpoles of the Indian skittering frog (Euflictis cyanophlyctis). Roundup at different concentrations (0, 1, 2, 3, 4 and 8mg acid equivalent (ae)/L), tested in a 2×6 factorial design in the presence and absence of predator stress, induced concentration-dependent lethality in tadpoles. The 96-h LC50 for Roundup in the absence and presence of predator stress were 3.76mgae/L and 3.39mgae/L, respectively. The 10-day LC50 value for Roundup was significantly lower, 2.12mgae/L and 1.91mgae/L in the absence and presence of predator stress, respectively. Lower concentrations of Roundup (1, 2 and 3mgae/L) induced the formation of micronuclei (MN) in the erythrocytes of tadpoles at 24-h (F3,56=10.286, p<0.001), 48-h (F3,56=48.255, p<0.001), 72-h (F3,56=118.933, p<0.001) and 96-h (F3,56=85.414, p<0.001) in a concentration-dependent manner. Presence of predator stress apparently increased the toxicity and genotoxicity of Roundup; but these effects were not statistically significant. These findings suggest that Roundup at environmentally relevant concentrations has lethal and genotoxic impact on E. cyanophlyctis; which may have long-term fitness consequence to the species.

Role of quercetin on mitomycin C induced genotoxicity: Analysis of micronucleus and chromosome aberrations in vivo

Quercetin, a flavonol group of plant flavonoid, has generated immense interest because of its potential antioxidant, anti-proliferative, chemoprotective, anti-inflammatory and gene expression modulating properties. However, the pro-oxidant chemistry of quercetin is important as it is related to the generation of mutagenic quinone-type metabolites. In the present study, 25mg/kg, 50mg/kg and 100mg/kg of quercetin given through the intra peritoneal (i.p.) route induced 2.31 ± 0.27%, 4.72 ± 0.58% and 6.38 ± 0.68% (control value=0.67 ± 0.30%) respectively, of cells with micronucleus (MN) in polychromatic erythrocytes in bone marrow cells and 10.93 ± 0.98%, 10.00 ± 0.89% and 14.27 ± 3.94% (control 2.61 ± 0.48) of cells with chromosome aberrations (CA) following 24h of the treatments. Higher frequencies of MN and CA were also observed after 48h of the treatments. To verify the effect of route of treatment on the quercetin induced damage, 100mg/kg b.w. was given through oral route which declined frequency of MN (P<0.001) as well as CA (P<0.05) as compared to the i.p. route for the same dose. Quercetin also induced higher frequency of metaphases with sticky chromosomes and C-mitosis. Pre-treatment with quercetin significantly reduced the frequency of mitomycin C (MMC) induced MN as well as CA, but no clear correlation between the dose and effect could be observed. Further studies are required to elucidate the possible interaction of quercetin with DNA as well as with other DNA damaging agents like MMC in vivo. The protective action of quercetin was not enhanced when given orally. Our findings suggest that quercetin may result in genomic instability in the tested dose range and significant reduction in MMC induced genotoxicity in the highest dose tested. These effects of quercetin are to be taken into consideration while evaluating the possible use of quercetin as a therapeutic agent.

Fenvalerate-induced chromosome aberrations and sister chromatid exchanges in the bone marrow cells of mice in vivo

Fenvalerate, a synthetic pyrethroid insecticide, is commonly used in agriculture and other domestic applications due to its high insecticidal activity and low mammalian-, avian- and phyto-toxicities. However, the genotoxic effect of fenvalerate is highly equivocal. In the present study the genotoxic effects of fenvalerate was evaluated using structural chromosome aberration (CA) and sister chromatid exchange (SCE) assays in mice. Out of the three doses (5, 10 and 20 mg/kg) tested, statistically significant increase in CA was found following intra peritoneal (i.p.) treatment of 2 0 mg/kg of fenvalerate for 24 h (P<0.01) and 48 h (P<0.05) only. Neither the acute doses of 5 and 10 mg/kg, nor the sub-acute dose (5x4 mg/kg) of fenvalerate could induce any significant effect. All the three acute doses induced significant increase in the frequency of SCEs (P<0.01) in the bone marrow cells, which showed a significant dose-response correlation (r=0.9541, P<0.05). With certain reservations to possible impurities, from the present findings technical grade fenvalerate may be considered as a weak clastogen and a potent inducer of SCEs in mice.

Effect of predator stress and malathion on tadpoles of Indian skittering frog.

The impact of pesticides on amphibians is of particular concern because their populations appear to be declining on a global scale. We examined the toxic and genotoxic effects of malathion, a commonly used organophosphorus pesticide, in the larvae of Indian skittering frog (Euflictis cyanophlyctis). The different concentrations of malathion (0, 0.5, 1.0, 2.0, 4.0 and 8.0mg/L) tested in a 2×6 factorial design, induced concentration-dependent lethality in tadpoles in the presence and absence of predator cues. The 96 h LC50 for malathion in the presence and absence of predator stress were 3.523 mg/L and 3.588 mg/L, respectively. The 15-day LC50 value for malathion was estimated to be 2.452 mg/L. Lower concentrations of malathion extending into the sublethal range (0.5, 1.0 and 2.0 mg/L) induced micronuclei (MN) in the erythrocytes of tadpoles at 24 h (F₃,₅₆=70.291, p<0.001), 48 h (F₃,₅₆=78.423, p<0.001), 72 h (F₃,₅₆=88.817, p<0.001) and 96 h (F₃,₅₆=64.770, p<0.001) in a concentration-dependent manner. Predator stress significantly enhanced the MN frequency at 48 h following 1.0mg/L malathion treatment (p<0.001). The present report is the first one to analyze genotoxic effect of malathion in the presence of predator stress. These results suggest that predator stress may potentiate the genotoxic effect of lower concentrations of malathion in E. cyanophlyctis tadpoles. These effects may have long-term fitness consequence to the population as a whole.

Sodium arsenite induced changes in survival, growth, metamorphosis and genotoxicity in the Indian cricket frog (Rana limnocharis)

Arsenic contamination of the environment is a matter of great concern. Understanding the effects of arsenic on aquatic life will act as biological early warning system to assess how arsenic could shape the biodiversity in the affected areas. Rapid decline in amphibian population in recent decades is a cause of major concern. Over the years, amphibians have been recognized as excellent bio-indicators of environmental related stress. In the present study, we examined the toxic and genotoxic effects of sodium arsenite in the tadpoles of the Indian cricket frog (Rana limnocharis). Sodium arsenite at different concentrations (0, 50, 100, 200 and 400 μg L(-1)) neither induced lethality nor significantly altered body weight at metamorphosis. However, it accelerated the rate of metamorphosis at higher concentrations, reduced body size (snout-vent length) and induced developmental deformities such as loss of limbs. Besides, at concentration ranges between 100 and 400 μg L(-1), sodium arsenite induced statistically significant genotoxicity at 24, 48, 72 and 96 h of the exposure in a concentration-dependent manner. However, it did not show time effects as the highest frequency was found between 48 and 72 h which remained steady subsequently. The genotoxicity was confirmed by comet assay in the whole blood cells. These findings suggest that arsenic at environmentally relevant concentrations has significant sub-lethal effects on R.limnocharis, which may have long-term fitness consequence to the species and may have similar implications in other aquatic life too.

Effects of low dose radiation and vitamin C treatment on chloroquine-induced genotoxicity in mice.

Chloroquine (CHQ) is a commonly used antimalarial agent. We evaluated the genotoxic potential of CHQ using chromosome aberration (CA), micronucleus (MN), and sperm head abnormality (SA) assays in vivo in Swiss albino mice. The interaction between a low dose of radiation and CHQ, as well as the effect of vitamin C on CHQ‐induced genotoxicity, was also evaluated. It was observed that CHQ induced CA, as well as MN, in the bone marrow cells under certain treatment conditions. Further, CHQ induced significant increase in the frequency of SA both at 24 hr and 21 days of the treatment. In the present study vitamin C pretreatment apparently reduced the frequency of CA, MN, and SA induced by CHQ. In the combination studies with radiation and CHQ, we found that exposure to low doses of radiation (0.5 Gy) either prior to or following CHQ treatment, in the dose ranges tested, has little or no synergistic effect in the mutagenic evaluations in somatic cells. However, radiation exposure along with CHQ treatment resulted in significant increase in the frequency of SA as compared to the groups receiving CHQ alone at 21 days of the treatment. In summary, CHQ has the potential to induce genotoxicity in mammalian cells. Further, germ cells may be relatively more sensitive as compared to the somatic cells. Environ. Mol. Mutagen., 2008.