Arvind Rai

DNA damage and cholinesterase activity in occupational workers exposed to pesticides

The present study was designed to evaluate genotoxicity, acetyl cholinesterase (AChE) activity, hepatic and renal toxicity in occupational workers exposed to mixture of pesticides (n=70) with same number of healthy subjects as controls. The mean comet tail DNA % (TD %) and tail moment (TM) were used to measure DNA damage, while AChE activity and other biochemical parameters such as markers of nephrotoxicity (urea and creatinine) and hepatotoxicity (AST, ALT and ALP) were measured as biomarkers for toxicity due to exposure of pesticides. The occupational workers were continuously exposed to mixture of pirimiphos methyl, chlorpyrifos, temephos and malathion on a regular interval as per usage and activity. The comet assay using lymphocytes of exposed workers showed significantly higher TD percentage value (60.43% vs. 31.86%, p<0.001) and TM value (14.48 μm vs. 6.42 μm, p<0.001) in occupational workers as compared to controls. AChE activity in erythrocytes was found to be decreased (3.45 KAU/L vs. 9.55 KAU/L in controls, p<0.001) and associated with the duration of exposure to pesticides used by the workers. Enzyme levels for hepatic and renal functions were also found significantly different in occupational workers than healthy controls (p<0.001). These results suggest that the exposure to mixture of pirimiphos methyl, chlorpyrifos, temephos and malathion may induce DNA damage, decrease in AChE activity, hepatotoxicity as well as nephrotoxicity. Periodic biomonitoring of these biomarkers along with imparting education and training to occupational workers for safe application of pesticides is recommended for its potential hazards.

Paraoxonase-1 genetic polymorphisms and susceptibility to DNA damage in workers occupationally exposed to organophosphate pesticides

Human paraoxonase 1 (PON1) is a lipoprotein-associated enzyme involved in the detoxification of organophosphate pesticides (OPs) by hydrolyzing the bioactive oxons. Polymorphisms of the PON1 gene are responsible for variation in the expression and catalytic activity of PON1 enzyme. In the present study, we have determined (a) the prevalence of two common PON1 polymorphisms, (b) the activity of PON1 and acetylcholinesterase enzymes, and (c) the influence of PON1 genotypes and phenotypes variation on DNA damage in workers exposed to OPs. We examined 230 subjects including 115 workers exposed to OPs and an equal number of normal healthy controls. The results revealed that PON1 activity toward paraoxon (179.19±39.36 vs. 241.52±42.32nmol/min/ml in controls) and phenylacetate (112.74±17.37 vs. 134.28±25.49μmol/min/ml in controls) was significantly lower in workers than in control subjects (p<0.001). No significant difference was observed in the distribution of genotypes and allelic frequencies of PON1(192)QR (Gln/Arg) and PON1(55)LM (Leu/Met) in workers and control subjects (p>0.05). The PON1 activity toward paraoxonase was found to be significantly higher in the R/R (Arg/Arg) genotypes than Q/R (Gln/Arg) and lowest in Q/Q (Gln/Gln) genotypes in both workers and control subjects (p<0.001). For PON1(55)LM (Leu/Met), PON1 activity toward paraoxonase was observed to be higher in individuals with L/L (Leu/Leu) genotypes and lowest in individuals with M/M (Met/Met) genotypes in both groups (p<0.001). No influence of PON1 genotypes and phenotypes was seen on the activity of acetylcholinesterase and arylesterase. The DNA damage was observed to be significantly higher in workers than in control subjects (p<0.05). Further, the individuals who showed least paraoxonase activity i.e., those with (Q/Q [Gln/Gln] and M/M [Met/Met]) genotypes showed significantly higher DNA damage compared to other isoforms in workers exposed to OPs (p<0.05). The results indicate that the individuals with PON1 Q/Q and M/M genotypes are more susceptible toward genotoxicity. In conclusion, the study suggests wide variation in enzyme activities and DNA damage due to polymorphisms in PON1 gene, which might have an important role in the identification of individual risk factors in workers occupationally exposed to OPs.

The first dominant co-circulation of both dengue and chikungunya viruses during the post-monsoon period of 2010 in Delhi, India.

Independent outbreaks of dengue virus (DENV) infection and sporadic cases of chikungunya virus (CHIKV) have been recorded in the metropolitan city of Delhi on several occasions in the past. However, during a recent 2010 arboviral outbreak in Delhi many cases turned negative for DENV. This prompted us to use duplex reverse transcriptase-polymerase chain reaction (D-RT-PCR) to establish the aetiology of dengue/chikungunya through sequencing of CprM and E1 genes of dengue and chikungunya viruses. Interestingly, for the first time, both DENV and CHIKV co-circulated simultaneously and in equally dominant proportion during the post-monsoon period of 2010. DENV-1 genotype III and the East Central South African genotype of CHIKV were associated with post-monsoon spread of these viruses.

Development and evaluation of reverse transcription loop-mediated isothermal amplification assay for rapid and real-time detection of the swine-origin influenza A H1N1 virus.

The recent emergence of the swine-origin influenza A H1N1 virus (S-OIV) poses a serious global health threat. Rapid detection and differentiation of S-OIV from seasonal influenza is crucial for patient management and control of the epidemics. A one-step, single-tube accelerated and quantitative S-OIV-specific H1 reverse transcription loop-mediated isothermal amplification (RTLAMP) assay for clinical diagnosis of S-OIV by targeting the H1 gene is reported in this article. A comparative evaluation of the H1-specific RTLAMP assay vis-à-vis the World Health Organization-approved real-time polymerase chain reaction (RTPCR), involving 239 acute-phase throat swab samples, demonstrated exceptionally higher sensitivity by picking up all of the 116 H1N1-positive cases and 36 additional positive cases among the negatives that were sequence-confirmed as S-OIV H1N1. None of the real-time RTPCR-positive samples were missed by the RTLAMP system. The comparative analysis revealed that S-OIV RTLAMP was up to tenfold more sensitive than the World Health Organization real-time RTPCR; it had a detection limit of 0.1 tissue culture infectious dosage of 50/ml. One of the most attractive features of this isothermal gene amplification assay is that it seems to have an advantage in monitoring gene amplification by means of SYBR Green I dye-mediated naked-eye visualization within 30 minutes compared to 2 to 3 hours for a real-time reverse transcription polymerase chain reaction. This suggests that the RTLAMP assay is a valuable tool for rapid, real-time detection and quantification of S-OIV in acute-phase throat swab samples without requiring sophisticated equipment.

Genetic polymorphisms of GSTM1, GSTT1 and GSTP1 and susceptibility to DNA damage in workers occupationally exposed to organophosphate pesticides

GSTM1, T1 and P1 are important enzymes of glutathione S-transferases (GSTs), involved in the metabolism of many endogenous and exogenous compounds. Individual genetic variation in these metabolizing enzymes may influence the metabolism of their substrates. The present study was designed to determine the genotoxic effects using DNA damage and its association with GSTM1, GSTT1, and GSTP1 (Ile105Val) genetic polymorphisms in workers occupationally exposed to organophosphate pesticides (OPs). We examined 230 subjects including 115 workers occupationally exposed to OPs and an equal number of normal healthy controls. The DNA damage was evaluated using the alkaline comet assay and genotyping was done using individual PCR or PCR-RFLP. Significantly higher DNA tail moment (TM) was observed in workers as compared to control subjects (14.41 ± 2.25 vs. 6.36 ± 1.41 tail % DNA, p<0.001). The results revealed significantly higher DNA TM in workers with GSTM1 null genotype than those with GSTM1 positive (15.18 vs. 14.15 tail % DNA, p=0.03). A significantly higher DNA TM was also observed in workers with homozygous Ile-Ile GSTP1 genotype than heterozygous (Ile-Val) and mutant (Val-Val) GSTP1 genotype (p=0.02). In conclusion, the results show that null deletion of GSTM1 and homozygote wild GSTP1 genotype could be related to inter-individual differences in DNA damage arises from the gene-environment interactions in workers occupationally exposed to OPs.

Influence of CYP2C9, GSTM1, GSTT1 and NAT2 genetic polymorphisms on DNA damage in workers occupationally exposed to organophosphate pesticides

Previous studies have revealed that organophosphate pesticides (OPs) are primarily metabolized by xenobiotic metabolizing enzymes (XMEs). Very few studies have explored genetic polymorphisms of XMEs and their association with DNA damage in pesticides-exposed workers. Present study was designed to determine the influence of CYP2C9, GSTM1, GSTT1 and NAT2 genetic polymorphisms on DNA damage in workers occupationally exposed to OPs. We examined 268 subjects including 134 workers occupationally exposed to OPs and an equal number of normal healthy controls. The DNA damage was evaluated using alkaline comet assay and genotyping was done using individual polymerase chain reaction (PCR) or polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Acetylcholinesterase and paraoxonase activity were found to be significantly lowered in workers as compared to control subjects which were analyzed as biomarkers of toxicity due to OPs exposure (p<0.001). Workers showed significantly higher DNA tail moment (TM) compared to control subjects (14.32±2.17 vs. 6.24±1.37 tail % DNA, p<0.001). GSTM1 null genotype was found to influence DNA TM in workers (p<0.05). DNA TM was also found to be increased with concomitant presence of NAT2 slow acetylation and CYP2C9*3/*3 or GSTM1 null genotypes (p<0.05). DNA TM was found increased in NAT2 slow acetylators with mild and heavy smoking habits in control subjects and workers, respectively (p<0.05). The results of this study suggest that GSTM1 null genotypes, and an association of NAT2 slow acetylation genotypes with CYP2C9*3/*3 or GSTM1 null genotypes may modulate DNA damage in workers occupationally exposed to OPs.

Role of genetic polymorphisms of CYP1A1, CYP3A5, CYP2C9, CYP2D6, and PON1 in the modulation of DNA damage in workers occupationally exposed to organophosphate pesticides.

Organophosphate pesticides (OPs) are primarily metabolized by several xenobiotic metabolizing enzymes (XMEs). Very few studies have explored genetic polymorphisms of XMEs and their association with DNA damage in pesticide-exposed workers. The present study was designed to determine the role of genetic polymorphisms of CYP1A1, CYP3A5, CYP2C9, CYP2D6, and PON1 in the modulation of DNA damage in workers occupationally exposed to OPs. We examined 284 subjects including 150 workers occupationally exposed to OPs and 134 normal healthy controls. The DNA damage was evaluated using the alkaline comet assay and genotyping was done using PCR-RFLP. The results revealed that the PONase activity toward paraoxonase and AChE activity was found significantly lowered in workers as compared to control subjects (p<0.001). Workers showed significantly higher DNA damage compared to control subjects (14.37±2.15 vs. 6.24±1.37 tail% DNA, p<0.001). Further, the workers with CYP2D6*3PM and PON1 (QQ and MM) genotypes were found to have significantly higher DNA damage when compared to other genotypes (p<0.05). In addition, significant increase in DNA damage was also observed in workers with concomitant presence of certain CYP2D6 and PON1 (Q192R and L55M) genotypes which need further extensive studies. In conclusion, the results indicate that the PON1 and CYP2D6 genotypes can modulate DNA damage elicited by some OPs possibly through gene-environment interactions.

Evaluation of gidB alterations responsible for streptomycin resistance in Mycobacterium tuberculosis

OBJECTIVES To evaluate gidB alterations for possible impact on the cumulative mechanism underlying the acquisition of high-level streptomycin resistance in Mycobacterium tuberculosis. METHODS Fifty-two isolates with high streptomycin resistance and 23 isolates with low streptomycin resistance were sequenced for mutational analysis in the rpsL, rrs and gidB region. As the gidB protein has a complex substrate and no activity assay has yet been formulated, mutants of interest were subjected to in silico modelling and were structurally mapped together with active-site amino acid residues for assessment of the relevance to activity of the mutations found. RESULTS Eight novel sense mutations and four novel mis-sense mutations in gidB were identified. Findings showed that active-site morphology is not only greatly affected by mutants lying in close proximity to the active-site pocket, but also by other mutations altering secondary-structure motifs and having an overall effect on protein structure. CONCLUSIONS We conclude that gidB mutations address many unanswered questions and explain the whole story behind phenotypic streptomycin-resistant strains exhibiting no mutation in rpsL or rrs. They also validate the hypothesis of sequential progression of resistance from low to high due to the existence of gidB alterations in the genetic background.

Epigenetic inactivation of the E-cadherin gene in eyelid sebaceous gland carcinoma.

Background  E‐cadherin and β‐catenin are crucial components of the cell–cell adhesion complex. Their loss has often been associated with tumour metastasis and poor clinical outcome. Both loss of E‐cadherin at the cell membrane and a stabilizing mutation in CTNNB1 (β‐catenin gene) have been associated with ovarian, colorectal, hepatocellular and nonmelanoma skin cancer, such as squamous and basal cell carcinomas. Absence of E‐cadherin may be caused by promoter hypermethylation of the E‐cadherin gene (CDH1).

Targeted detection of 65 kDa heat shock protein gene in endometrial biopsies for reliable diagnosis of genital tuberculosis.

OBJECTIVE To evaluate the clinical utility of PCR compared with other available diagnostic modalities in prompt diagnosis of female genital tuberculosis causing infertility. STUDY DESIGN Prospective case-controlled trial. Premenstrual endometrial biopsy specimens were collected from 150 infertile women of reproductive age group suspected of having genital tuberculosis. All patients underwent diagnostic endoscopy (laparoscopy and hysteroscopy) and the samples obtained were subjected to microscopy, culture by the BACTEC 460 TB System, histopathology and polymerase chain reaction (PCR) for detection of 165 bp region of 65 kDa gene of Mycobacterium tuberculosis. The results were correlated with the laparoscopic findings. RESULTS While the laparoscopy/hysteroscopy findings were indicative of tuberculosis in 12.6% of cases, 14.6% of the specimens showed evidence of 65 kDa gene of M. tuberculosis and only 3.33%, 1.33% and 0.66% were positive by culture, smear and histopathology, respectively. CONCLUSION Since laparoscopy, hysteroscopy other endoscopic procedures are associated with operative risks and may cause flaring of infection, and other conventional laboratory tests including histopathology have poor sensitivity, PCR-based detection of 65 kDa gene of M. tuberculosis in endometrial biopsy specimens could be a promising molecular diagnostic technique compared to conventional methods of diagnosis.