Uma B. Dasgupta

Chronic arsenic toxicity: Studies in West Bengal, India

Chronic arsenic toxicity (arsenicosis) as a result of drinking arsenic‐contaminated groundwater is a major environmental health hazard throughout the world, including India. A lot of research on health effects, including genotoxic effect of chronic arsenic toxicity in humans, have been carried out in West Bengal during the last 2 decades. A review of literature including information available from West Bengal has been made to characterize the problem. Scientific journals, monographs, and proceedings of conferences with regard to human health effects, including genotoxicity, of chronic arsenic toxicity have been reviewed. Pigmentation and keratosis are the specific skin diseases characteristic of chronic arsenic toxicity. However, in West Bengal, it was found to produce various systemic manifestations, such as chronic lung disease, characterized by chronic bronchitis, chronic obstructive and/or restrictive pulmonary disease, and bronchiectasis; liver diseases, such as non cirrhotic portal fibrosis; polyneuropathy; peripheral vascular disease; hypertension; nonpitting edema of feet/hands; conjunctival congestion; weakness; and anemia. High concentrations of arsenic, greater than or equal to 200 μg/L, during pregnancy were found to be associated with a sixfold increased risk for stillbirth. Cancers of skin, lung, and urinary bladder are the important cancers associated with this toxicity. Of the various genotoxic effects of arsenic in humans, chromosomal aberration and increased frequency of micronuclei in different cell types have been found to be significant. Various probable mechanisms have been incriminated to cause DNA damage because of chronic arsenic toxicity. The results of the study in West Bengal suggest that deficiency in DNA repair capacity, perturbation of methylation of promoter region of p53 and p16 genes, and genomic methylation alteration may be involved in arsenic‐induced disease manifestation in humans. P53 polymorphism has been found to be associated with increased occurrence of arsenic‐induced keratosis. Of the various genes involved in the regulation of arsenic metabolism, single‐nucleotide polymorphisms of purine nucleoside phosphorylase, in one study, showed increased occurrence of arsenicosis.

Arsenic exposure induces genomic hypermethylation

Gene‐specific hypermethylation has previously been detected in Arsenic exposed persons. To monitor the level of whole genome methylation in persons exposed to different levels of Arsenic via drinking water, DNA was extracted from peripheral blood mononuclear cells of 64 persons. Uptake of methyl group from 3H labeled S‐Adenosyl Methionine after incubation of DNA with SssI methylase was measured. Results showed statistically significant (P = 0.0004) decrease in uptake of 3H methyl group in the persons exposed to 250–500 μg/L arsenic, indicating genomic hypermethylation.

Association of cytochrome P450, glutathione S-transferase and N-acetyl transferase 2 gene polymorphisms with incidence of acute myeloid leukemia.

The objective of the paper was to study the association of polymorphisms of phases I and II xenobiotic metabolizing enzyme genes cytochrome P450 (CYP-4501A1*2A, *2B, *2C and *4 alleles, CYP-4502D6*4 allele), glutathione-S-transferase (GSTM1 and GSTT1 null genotypes) and N-acetyl transferase 2 (NAT2*6B and *7A alleles) with the incidence of acute myeloid leukemia (AML) in an eastern Indian population. Polymerase chain reaction and restriction fragment length polymorphism of genomic DNA from peripheral blood cells were used to detect CYP-450 and NAT2 gene polymorphisms in 110 AML patients and 144 racially and geographically matched normal controls. Polymerase chain reaction was also applied to detect GST gene polymorphisms in both groups. A statistically significant difference between the AML group and the normal group was observed in the case of glutathione-S-transferase M1 null (odds ratio 3.25, 95% confidence interval 1.9–5.58, P<0.001) and N-acetyl transferase 2*6B (odds ratio 3.04, 95% confidence interval 1.79–5.16, P<0.001) genotypes. Combined deficiency of N-acetyl transferase 2 and glutathione-S-transferase M1 genes produced an odds ratio of 11.91 (95% confidence interval 4.06–34.96, P<0.001). The effect of N-acetyl transferase 2*6B (P<0.001) is significant only at ages ≤40. In the population studied, persons with glutathione-S-transferase M1 null genotype and N-acetyl transferase 2*6B allele are at increased risk of developing AML, and the risk is considerably enhanced in persons with both glutathione-S-transferase M1 and N-acetyl transferase 2 deficiency.

Glutathione S-transferase M1 and T1 null genotype frequency in chronic myeloid leukaemia.

Polymorphisms associated with genes coding for glutathione S-transferase enzymes are known to influence metabolism of different carcinogens and have been associated with incidence of various types of cancer. We have determined the GST M1 and GST T1 ‘null’ genotype frequency in 81 patients with chronic myeloid leukaemia (CML) and 123 racially and geographically matched control individuals by multiplex polymerase chain reaction (PCR). GST M1 null genotype frequencies in CML and controls were 28.4% and 27.7%, respectively. GST T1 null genotype frequencies in CML and controls were 19.8% and 7.3%, respectively. The GST T1 null genotype frequency in CML patients is significantly different from that in controls (odds ratio (OR) 3.12, 95% confidence interval (CI) 1.3–7.45, P=0.008).

Major Beta-Globin Gene Mutations in Eastern India and Their Associated Haplotypes

324 alleles of the β-globin gene from unrelated thalassaemia patients native to the eastern region of India (mainly from the state of West Bengal) were analysed for β-globin gene mutations by the amplification refractory mutation system (ARMS). The major mutations that were detected are IVS-1 pos 5 (G–C), codon 26 (G–A) and codon 30 (G–C) with frequencies of 0.45, 0.33 and 0.05, respectively. Haplotype analysis revealed a very strong linkage disequilibrium of IVS-1 pos 5 (G–C) with one particular haplotype. HbE was found to be associated with two major haplotypes. Codon 30 (G–C) was associated with a haplotype that is the same as that found in the African population. Haplotype associated with codon 8/9 (+G) was the same as that found in northwest India. These findings have implications for the use of molecular diagnosis for genetic counselling and prenatal diagnosis of β-thalassaemia in this region.

Induction of apoptosis by Phenothiazine derivatives in V79 cells.

Phenothiazine derivatives chlorpromazine (cpz) and trifluoperazine (tfp) were found to induce apoptosis, abnormal cell cycle and expression of p53 in Chinese hamster lung fibroblast V79 cells. Both the drugs can induce apoptosis when cells are treated with drug at a concentration of 10 microg/ml within 4 h, as detected by propidium iodide staining and DNA fragmentation analysis. Flow cytometric analysis revealed that the apoptotic response is mediated by a loss of G(1) population of cells. In Western blot analysis, p21 is induced and p53 is accompanied by additional bands. Also indirect immunolabeling of single cells revealed that p21 is accumulated from cytoplasm into nucleus after the drug treatment and the intensities of p53 increased. Our findings demonstrate for the first time that phenothiazine derivatives, in addition to their cytotoxic effects, could induce apoptosis, an observation that has important clinical implications.

Analysis of dystrophin gene deletions by multiplex PCR in eastern India.

The most common genetic neuromuscular disease of childhood, Duchenne and Becker muscular dystrophy (DMD/BMD) is caused by deletion, duplication or point mutation of the dystrophin gene located at Xp 21.2. In the present study DNA from seventy unrelated patients clinically diagnosed as having DMD/BMD referred from different parts of West Bengal, a few other states and Bangladesh are analyzed using the multiplex polymerase chain reaction (m-PCR) to screen for exon deletions and its distribution within the dystrophin gene. Out of seventy patients forty six (63%) showed large intragenic deletion in the dystrophin gene. About 79% of these deletions are located in the hot spot region i.e, between exon 42 to 53. This is the first report of frequency and distribution of deletion in dystrophin gene in eastern Indian DMD/BMD population.

Variable severity of β-thalassemia patients of Eastern India: effect of α-thalassemia and XmnI polymorphism

Abstract Sixty-four thalassemia and E-β thalassemia patients were studied for factors that modulate the severity of the disease; i. e., mutation of β-globin gene, presence of α-deletion, and presence of an XnmI site at the −158 position of the Gγ gene. Presence of α-deletion and/or homozygosity for the XmnI site was in general associated with less-severe disease. About 12% of the patients harbored single α-gene deletion, and the gene frequency of the XnmI polymorphism in these patients is 0.48.

Two β-globin cluster-linked polymorphic loci in thalassemia patients of variable levels of fetal hemoglobin

Abstract:  Objective: To correlate different polymorphisms of the β‐globin cluster with fetal hemoglobin (HbF) level in β‐thalassemia and E‐β thalassemia patients. Methods: Fifteen thalassemia patients, seven with high HbF and not requiring transfusion, eight with lower HbF and requiring transfusion were studied for β‐globin mutation, concurrent inheritance of α‐thalassemia, RFLP haplotype, a C→T polymorphism at −158 of Gγ and configuration of an (AT)xTy motif at −540 of β‐globin gene. Results: Senegal 5′β‐haplotype and the polymorphism at −158 of G was (P = 0.063) was linked to the high‐HbF phenotype but the (AT)9T5 configuration of the (AT)xTy motif was not (P = 0.6). Study of 30 chromosomes revealed 7 different configurations of the (AT)xTy motif. Association of these motifs with specific β‐globin mutations of this region has also been determined. Conclusion: The senegal haplotype and the polymorphism at −158 of G was linked to the high‐HbF phenotype.

Cytotoxic and genetic effects of X-irradiation of human cells in the presence of chlorpromazine

Human epidermoid carcinoma cells (Hep-2) were X-irradiated in the presence of 5-10 micrograms/ml of chlorpromazine (CPZ). Survival of the cells decreased with increasing CPZ concentration. Lymphocytes from three normal volunteers exposed to X-irradiation in the presence of CPZ showed an increased frequency of dicentric and ring formation.