Ashok K. Giri

Genetic toxicology of a paradoxical human carcinogen, arsenic: a review.

Arsenic is widely distributed in nature in air, water and soil in the form of either metalloids or chemical compounds. It is used commercially, as pesticide, wood preservative, in the manufacture of glass, paper and semiconductors. Epidemiological and clinical studies indicate that arsenic is a paradoxical human carcinogen that does not easily induce cancer in animal models. It is one of the toxic compounds known in the environment. Intermittent incidents of arsenic contamination in ground water have been reported from several parts of the world. Arsenic containing drinking water has been associated with a variety of skin and internal organ cancers. The wide human exposure to this compound through drinking water throughout the world causes great concern for human health. In the present review, we have attempted to evaluate and update the mutagenic and genotoxic effects of arsenic and its compounds based on available literature.

Comparative antimutagenic and anticlastogenic effects of green tea and black tea: a review.

Tea is the most popular beverage next to water, consumed by over two-thirds of the worlds population. It is processed in different ways in different parts of the world to give green, black or oolong tea. Experimental studies have demonstrated the significant antimutagenic and anticlastogenic effects of both green and black tea and its polyphenols in multiple mutational assays. In the present review, we have attempted to evaluate and update the comparative antimutagenic and anticlastogenic effects of green tea, black tea and their polyphenols in different test systems, based on available literature. Existing reports have suggested that the protective effects of black tea is as good as green tea, however, more studies on black tea and its polyphenols are needed before a final conclusion can be made.

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.

Chromosomal aberrations and sister chromatid exchanges in individuals exposed to arsenic through drinking water in West Bengal, India

Arsenic contamination in groundwater has become a worldwide problem. Currently an unprecedented number of people in West Bengal, India and Bangladesh are exposed to the ubiquitous toxicant via drinking water in exposure levels far exceeding the maximum recommended limit laid down by WHO. This arsenic epidemic has devastated nine districts of West Bengal encompassing an area of 38,865 km(2) leading to various clinical manifestations of chronic arsenicosis. We conducted a human bio-monitoring study using chromosomal aberrations (CA) and sister chromatid exchanges (SCE) as end points to explore the cytogenetic effects of chronic arsenic toxicity in the population of North 24 Parganas, one of the arsenic affected districts in West Bengal. Study participants included 59 individuals residing in this district where the mean level (+/-S.E.) of arsenic in drinking water (microg/l) was 211.70+/-15.28. As age matched controls with similar socio-economic status we selected 36 healthy, asymptomatic individuals residing in two unaffected districts--Midnapur and Howrah where the mean arsenic content of water (microg/l) was 6.35+/-0.45. Exposure was assessed by standardized questionnaires and by detecting the levels of arsenic in drinking water, nails, hair and urine samples. In the exposed group the mean arsenic concentrations in nails (microg/g), hair (microg/g) and urine (microg/l) samples were 9.04+/-0.78, 5.63+/-0.38 and 140.52+/-8.82, respectively, which were significantly high (P<0.01) compared to the corresponding control values of 0.44+/-0.03, 0.30+/-0.02 and 5.91+/-0.49, respectively. Elevated mean values (P<0.01) of the percentage of aberrant cells (8.08%) and SCEs per cell (7.26) were also observed in the exposed individuals in comparison to controls (1.96% and 5.95, respectively). The enhanced rates of CAs and SCEs among the residents of North 24 Parganas are indicative of the cytogenetic damage due to long term exposure to arsenic through consumption of contaminated water.

Bitter gourd proteinase inhibitors: potential growth inhibitors of Helicoverpa armigera and Spodoptera litura.

Proteinase inhibitors (PIs) from the seeds of bitter gourd (Momordica charantia L.) were identified as strong inhibitors of Helicoverpa armigera gut proteinases (HGP). Biochemical investigations showed that bitter gourd PIs (BGPIs) inhibited more than 80% HGP activity. Electrophoretic analysis revealed the presence of two major proteins (BGPI-1 and-2) and two minor proteins (BGPI-3 and-4) having inhibitory activity against both trypsin and HGP. The major isoforms BGPI-1 and BGPI-2 have molecular mass of 3.5 and 3.0 kDa, respectively. BGPIs inhibited HGP activity of larvae fed on different host plants, on artificial diet with or without added PIs and proteinases excreted in fecal matter. Degradation of BGPI-1 by HGP showed direct correlation with accumulation of BGPI-2-like peptide, which remained stable and active against high concentrations of HGP up to 3 h. Chemical inhibitors of serine proteinases offered partial protection to BGPI-1 from degradation by HGP, suggesting that trypsin and chymotrypsin like proteinases are involved in degradation of BGPI-1. In larval feeding studies, BGPIs were found to retard growth and development of two lepidopteran pests namely Helicoverpa armigera and Spodoptera litura. This is the first report showing that BGPIs mediated inhibition of insect gut proteinases directly affects fertility and fecundity of both H. armigera and S. litura. The results advocate use of BGPIs to introduce insect resistance in otherwise susceptible plants.

Cytogenetic damage and genetic variants in the individuals susceptible to arsenic‐induced cancer through drinking water

In West Bengal, India, more than 300,000 arsenic‐exposed people are showing symptoms of arsenic toxicity, which include cancers of skin and different internal organs. Since only 15–20% of the exposed population manifest arsenic‐induced skin lesions, it is thought that genetic variation might play an important role in arsenic toxicity and carcinogenicity. A total of 422 unrelated arsenic‐exposed subjects (244 skin‐symptomatic and 178 asymptomatic) were recruited for this study. Cytogenetic damage, as measured by chromosomal aberrations in lymphocytes and micronuclei formation in oral mucosa cells, urothelial cells and binucleated lymphocytes, was studied in unexposed, skin‐symptomatic and asymptomatic individuals with similar socioeconomic status. Identification of null mutations in GSTT1 and GSTM1 genes were carried out by PCR amplification. GSTP1 SNPs, implicated in susceptibility to various cancers, were assessed by PCR‐RFLP method. Symptomatic individuals had higher level of cytogenetic damage compared to asymptomatic individuals and asymptomatic individuals had significantly higher genotoxicity than unexposed individuals. No difference in allelic variants in GSTT1 and GSTP1 was observed between these 2 groups. Incidence of GSTM1 null gene frequencies was significantly higher in the asymptomatic group. Individuals with GSTM1‐positive (at least one allele) had significantly higher risk of arsenic‐induced skin lesions (odds ratio, 1.73; 95% confidence interval, 1.24–2.22). These results show a protective role of GSTM1 null in arsenic toxicity. This study also indicates that asymptomatic individuals are sub clinically affected and are also significantly susceptible to arsenic‐induced genotoxicity.

Enhanced frequency of micronuclei in individuals exposed to arsenic through drinking water in West Bengal, India.

In West Bengal, India arsenic in ground water has been found to be above the maximum permissible limit in seven districts covering an area of 37,493km2. In the present study, evaluation of the micronuclei (MN) formation in oral mucosa cells, urothelial cells and peripheral blood lymphocytes was carried out in the symptomatic individuals exposed to arsenic through drinking water. Forty five individuals with cutaneous signs of arsenicism from four affected districts (368.11 microg/l of As in drinking water) were considered as the exposed group and 21 healthy individuals with no symptoms of arsenic poisoning and residing in two unaffected districts (5.49 microg/l of As) were considered as controls. The exposed and control groups had similar age distribution and socioeconomic status. Standardised questionnaires were utilised and medical examination was conducted to ascertain exposure history, sociodemographic characteristics, diet, health, medication, addiction and chief symptoms in the study participants. Arsenic exposure was confirmed by measuring the arsenic content in the drinking water, nails, hair and urine samples from the volunteers. Arsenic contents in the urine, nail and hair in the exposed group were 24.45 microg/l, 12.58 and 6.97 microg/g, respectively which were significantly high in comparison to corresponding control group values of 4.88 microg/l, 0.51 and 0.34 microg/g, respectively. Exposed individuals showed a statistically significant increase in the frequency of MN in oral mucosa, urothelial cells and lymphocytes (5.15, 5.74 and 6.39/1000 cells, respectively) when compared with the controls (0.77, 0.56 and 0.53/1000 cells, respectively). Thus, the above results indicate that the symptomatic individuals exposed to arsenic through drinking water in this region have significant cytogenetic damage.

Analysis of T-cell proliferation and cytokine secretion in the individuals exposed to arsenic

Over six million people in nine districts of West Bengal, India are exposed to very high levels of arsenic primarily through their drinking water. More than 300,000 people showed arsenic-induced skin lesions in these districts. This is regarded as the greatest arsenic calamity in the world. Chronic arsenicosis causes varied dermatological signs ranging from pigmentation changes, hyperkeratosis to non-melanocytic cancer of skin, and also malignancies in different internal organs. Higher incidences of opportunistic infections are found in the arsenic-exposed individuals, indicating that their immune systems may be impaired somehow. We have thus investigated the effect of arsenic on T-cell proliferation and cytokine secretion in 20 individuals with arsenic-induced skin lesions and compared the results with 18 arsenic-unexposed individuals. A marked dose-dependent suppression of Concanavalin A (Con A) induced T-cell proliferation was observed in the arsenic-exposed individuals compared with the unexposed (P < 0.001) individuals. This correlated with a significant decrease in the levels of secreted cytokines by the T cells (TNF-α, IFN-γ, IL2, IL10, IL5, and IL4) in the exposed individuals (P < 0.001). Thus it can be inferred that arsenic exposure can cause immunosuppression in humans.

High arsenic in rice is associated with elevated genotoxic effects in humans

Arsenic in drinking water may cause major deleterious health impacts including death. Although arsenic in rice has recently been demonstrated to be a potential exposure route for humans, there has been to date no direct evidence for the impact of such exposure on human health. Here we show for the first time, through a cohort study in West Bengal, India, involving over 400 human subjects not otherwise significantly exposed to arsenic through drinking water, elevated genotoxic effects, as measured by micronuclei (MN) in urothelial cells, associated with the staple consumption of cooked rice with >200 μg/kg arsenic. Further work is required to determine the applicability to populations with different dietary and genetic characteristics, but with over 3 billion people in the world consuming rice as a staple food and several percent of this rice containing such elevated arsenic concentrations, this study raises considerable concerns over the threat to human health.

Mechanism of erythrocyte death in human population exposed to arsenic through drinking water.

Arsenic contamination in drinking water is one of the biggest natural calamities, which has become an imperative threat to human health throughout the world. Abbreviation of erythrocyte lifespan leading to the development of anemia is a common sequel in arsenic exposed population. This study was undertaken to explore the mechanism of cell death in human erythrocytes during chronic arsenic exposure. Results revealed transformation of smooth discoid red cells into evaginated echinocytic form in the exposed individuals. Further distortion converted reversible echinocytes to irreversible spheroechinocytes. Arsenic toxicity increased membrane microviscosity along with an elevation of cholesterol/phospholipid ratio, which hampered the flexibility of red cell membrane and made them less deformable. Significant increase in the binding of merocyanine 540 with erythrocyte membrane due to arsenic exposure indicated disruption of lipid packing in the outer leaflet of the cell membrane resulting from altered transbilayer phospholipid asymmetry. Arsenic induced eryptosis was characterized by cell shrinkage and exposure of phosphatidylserine at the cell surface. Furthermore, metabolic starvation with depletion of cellular ATP triggered apoptotic removal of erythrocytes from circulation. Significant decrease in reduced glutathione content indicating defective antioxidant capacity was coupled with enhancement of malondialdehyde and protein carbonyl levels, which pointed to oxidative damage to erythrocyte membrane. Arsenic toxicity intervened into red cell membrane integrity eventually leading to membrane destabilization and hemoglobin release. The study depicted the involvement of both erythrophagocytosis and hemolysis in the destruction of human erythrocytes during chronic arsenic exposure.