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Intervention to decelerate, arrest, or reverse the process of carcinogenesis by the use of either natural or synthetic agents individually or in combination has emerged as a promising and pragmatic medical approach to reduce cancer risk. In the present study, we examined the cancer chemopreventive potential of a flavonoid-rich fraction isolated from the seeds of Carica papaya, a plant traditionally referred to as papaw. The flavonoid-enriched benzene fraction of the aqueous extract exerted its anticancer properties in vitro through cytoprotection, antioxidative and antiinflammatory mechanisms and genoprotection in response to isocyanate-induced carcinogenicity. Medium-term anticarcinogenicity and 2-stage skin papillomagenesis studies conducted in benzopyrene-induced lung carcinogenesis and 7,12-dimethyl benz(a)anthracene-mediated skin papillomagenesis mouse models further validated our in vitro observations. This is the first demonstration of chemopreventive activities of papaya seed products, however, further studies to understand the subtle targets of intracellular signaling pathways, pharmacological profile and toxicological safety of this bioactive fraction are essential to pave the way for successful clinical translation. Our study supports the inverse association between dietary flavonoid intake and cancer risk.

The world’s worst industrial disaster, at Union Carbide, Bhopal, India, took place on 2–3 December 1984, leading to the leakage of poisonous methyl-isocyanate into the environment, causing thousands of deaths, pregnancy loss and for some, incapacitation for life. More than a quarter of a century later, the Indian Council of Medical Research undertook to redefine the abysmal consequences of the toxic gas exposure on the exposed population. This invigorated the interest of scientific community in the evaluation of the long-term effects, with reference to cytogenetic parameters. The thrust area was identified in terms of genetic disorders, low birth weight, developmental/growth disorders and congenital malformations. Also the impact on epigenetic factors, which may have contributed to variations in the functional expression of genes, was not negated, stimulating intense scientific research on in utero exposure and the progeny of the exposed population. To accomplish this mammoth task, molecular cytogenetic investigations must be undertaken in conjunction with conventional cytogenetics, using techniques such as FISH, Immuno-FISH, SKY and SNP analysis, to build up a cytogenetic database of the surviving population.

Objective To determine whether the OraQuick® HIV-1/2 Assay (OraSure Technologies, Inc., Bethlehem, PA, USA) in sputum is a valid tool for HIV surveillance among TB patients. Methods A cross sectional study was carried out on sputa of patients diagnosed with tuberculosis. Sputa were tested for antibodies to HIV using OraQuick® HIV-1/2 Assay (OraSure Technologies, Inc., Bethlehem, PA, USA). The results were compared with results of serum ELISA. Results Compared to serum ELISA, the OraQuick® HIV-1/2 Assay in sputum specimens reported 90% sensitivity (9/10) and 100% specificity (307/307), with a positive predictive value of 100% (95% CI : 66.37%–100.00%) and a negative predictive value of 99.68% (95% CI : 98.20%–99.99%). Conclusions This testing method may provide a useful strategy for conducting HIV surveillance in possible co-infected TB patients at peripheral centres. Since there is no investment on infrastructure, it may be possible for paramedical health professionals to carry out the test, particularly in areas with low HIV endemicity.

Liver is often exposed to plethora of chemical toxins. Owing to its profound physiological role and central function in metabolism and homeostasis, pertinent succession of cell cycle in liver epithelial cells is of prime importance to maintain cellular proliferation. Although recent evidence has displayed a strong association between exposures to methyl isocyanate (MIC), one of the most toxic isocyanates, and neoplastic transformation, molecular characterization of the longitudinal effects of MIC on cell cycle regulation has never been performed. Here, we sequentially delineated the status of different proteins arbitrating the deregulation of cell cycle in liver epithelial cells treated with MIC. Our data reaffirms the oncogenic capability of MIC with elevated DNA damage response proteins pATM and γ-H2AX, deregulation of DNA damage check point genes CHK1 and CHK2, altered expression of p53 and p21 proteins involved in cell cycle arrest with perturbation in GADD-45 expression in the treated cells. Further, alterations in cyclin A, cyclin E, CDK2 levels along with overexpression of mitotic spindle checkpoints proteins Aurora A/B, centrosomal pericentrin protein, chromosomal aberrations, and loss of Pot1a was observed. Thus, MIC impacts key proteins involved in cell cycle regulation to trigger genomic instability as a possible mechanism of developmental basis of liver carcinogenesis.

The present study evaluated the plausible role of circulating biomarkers in immune pathogenesis of chronic hepatitis considered a priority in clinical hepatology. Total viral load of chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) patients was quantified and correlation studies were performed with circulating levels of Th1/Th2 cytokines; C reactive protein and circulating nucleosomes; glutathione reductase (GR) and superoxide dismutase. To our knowledge, the study is first among its kind that validates strong positive correlation of viral load with IL-4, IL-6, GR in HBV and IL-6, IL-10, GR in HCV infections. Although, multi-centric studies including large cohorts are required for translating our findings to clinical practice, however, role of these biomarkers with potential diagnostic or prognostic significance might be helpful in clinical assessment of high-risk individuals, thereby, designing interventional strategies, towards development of personalized medicare. The results of our study also offer valuable insights of immune signaling mediators engaged in development of hepatocellular carcinoma.

The toxic response of cultured human colon epithelial-FHC cells to methyl isocyanate was investigated with regard to genomic instability. Qualitative and quantitative assessments of the extent of phosphorylation of DNA damage signaling factors such as ATM, gammaH2AX and p53, was increased in treated cells compared to controls. At the same time, many treated cells were arrested at the G2/M phase of the cell cycle, and had an elevated apoptotic index and increased inflammatory cytokine levels. Cytogenetic analyses revealed varied chromosomal anomalies, with abnormal expression of pericentrin protein. Analysis through ISSR PCR demonstrated increased microsatellite instability. The results imply that isocyanates can cause genomic instability in colonocytes.

Gallbladder carcinoma is an uncommon, but highly malignant tumor, with poor prognostic, and diagnostic manifestations in early stages. The Indian Council of Medical Research reported increased incidence of gallbladder carcinoma in the surviving population of the Bhopal gas tragedy that involved exposure of more than 500,000 people to methyl isocyanate gas. The severity of exposure, and increased multi-systemic morbidity in the survivors stimulated us to examine the molecular changes leading to gallbladder carcinoma. Surgically resected samples (N = 40) of gallbladder carcinoma were studied for the p53, Rad50, and cyclin-E expression by immunohistofluorescence bioimaging. Among the 40 samples, 23, 11, and 10 showed p53, Rad50, and cyclin-E expression, respectively, in moderately differentiated adenocarcinomas, demonstrating the prevalence and invasiveness of this disease in the methyl isocyanate-exposed population (P = 0.0009). Nevertheless, co-expression of Rad50, and cyclin-E with p53 was absent in adenomas with dysplasia, demonstrating their independent roles. We conclude that there was altered expression of p53, Rad50, and cyclin-E in the malignant transformation of gallbladder carcinoma in this methyl isocyanate gas-exposed cohort. Hence, these proteins may be useful as markers to identify premalignant lesions that are likely to progress into malignant adenocarcinoma.

Isocyanates, a group of low molecular weight aromatic and aliphatic compounds containing the isocyanate group (-NCO), are important raw materials with diverse industrial applications; however, pathophysiological implications resulting from occupational and accidental exposures of these compounds are hitherto unknown. Although preliminary evidence available in the literature suggests that isocyanates and their derivatives may have deleterious health effects including immunotoxicity, but molecular mechanisms underlying such an effect have never been addressed. The present study was carried out to assess the immunotoxic response of methyl isocyanate (MIC) on cultured human lymphocytes isolated from healthy human volunteers. Studies were conducted to evaluate both dose-dependent and time-course response (n = 3), using N-succinimidyl N-methylcarbamate, a surrogate chemical substitute to MIC. Evaluation of DNA damage by ataxia telangiectasia mutated (ATM) and gamma H2AX protein phosphorylation states; measure of apoptotic index through annexin-V/PI assay, apoptotic DNA ladder assay, and mitochondrial depolarization; induction of oxidative stress by CM-H2DCFDA and formation of 8-hydroxy-2' deoxy guanosine; levels of antioxidant defense system enzyme glutathione reductase; and multiplex cytometric bead array analysis to quantify the secreted levels of inflammatory cytokines, interleukin-8, interleukin-1beta, interleukin-6, interleukin-10, tumor necrosis factor, and interleukin-12p70 parameters were carried out. The results of the study showed a dose- and time-dependent response, providing evidence to hitherto unknown molecular mechanisms of immunotoxic consequences of isocyanate exposure at a genomic level. We anticipate these data along with other studies reported in the literature would help to design better approaches in risk assessment of occupational and accidental exposure to isocyanates.

We present a case of a ring (21) in a mentally challenged patient with mosaicism for trisomy 21 showing karyotype 47, XY,+21/47,XY,+21(r)/46,XY, born to normal parents. The parents and female sibling were phenotypically normal. This is a unique case report from Central India, on occurrence of trisomy 21 and r (21) in the same individual born to normal parents. Also being documented for the first time is the immuno-FISH analysis revealing differential expression of hTERT and a linked over expression of TRF2 in proband, probably corresponding to a high percentage of acrocentric associations.

Hepatitis C virus (HCV) infects nearly 3% of the population worldwide and is a major cause of acute and chronic infections leading to fibrosis, cirrhosis, and hepatocellular carcinoma. Current laboratory diagnosis of HCV is based on specific antibody detection (anti-hepatitis C virus (anti-HCV)) in serum. As HCV replicates in the liver cells, detection and localization of HCV RNA in liver tissue are vital for diagnosis. Ten biopsy samples diagnosed for cryptogenic liver cirrhosis, negative for the presence of anti-HCV and serum HCV RNA, were studied for analyzing presence of viral nucleic acid in liver tissues. Qualitative screening for HCV was done through ELISA while the nucleic acid analysis was performed through COBAS Amplicor. Detection of HCV RNA in liver tissue biopsies was performed following standard protocol of HCV detection kit (Shenzhen PG Biotech) with modifications using Light Cycler 2.0 (minimum detection limit 10 copies/ml). Quantitative detection in liver biopsies following the modified method showed the presence of HCV RNA in three samples out of the ten studied. The results indicate that using Light Cycler 2.0, following the modified technique described, constitutes a reliable method of quantitative detection and localization of HCV in tissue in “serosilent” HCV infection.

Background/aims We aimed to perform the molecular detection of Mycobacterium tuberculosis in formalin-fixed, paraffin-embedded tissues and biopsies of gastrointestinal specimens using real-time polymerase chain reaction system. Methods The study included three groups: (A) control (n=24), with no previous signs of M. tuberculosis complex (MTBC), (B) patients (n=28) with known TB origin and (C) patients (n=50) with clinical and histopathological signs of TB but who were culture- and acid-fast bacilli (AFB)-negative. The samples were obtained from the Medical and Surgical Gastroenterology Departments of Bhopal Memorial Hospital and Research Centre. We extracted DNA using DNeasy Blood & Tissue kit (QIAGEN, Germany) and performed realtime assay using Roche LightCycler 2.0 with fluorescence resonance energy transfer (FRET) hybridization probes obtained from Roche Molecular Diagnostics (USA) for the specific amplification of the 159 bp region of the mycobacterium genome. Results All the samples (n=24) of Group A were found to be negative, while in Group B, 27 out of the 28 cases studied were found to be positive by LightCycler real-time polymerase chain reaction (LC PCR). In Group C, 18 out of the 50 cases studied were found to be positive, showing a positivity of 36%. The overall positive and negative predictive values of the test for clinical TB (Group C) were 100% and 96.9%, respectively. Conclusions Results of our investigation demonstrated that the real-time detection technology using FRET probes has much higher sensitivity for the detection of MTBC DNA in tissue biopsy samples and formalin-fixed paraffin-embedded surgically resected tissues of the gastrointestinal tract.

The potential toxic effects on the immune system exerted by occupational and accidental environmental exposures and underlying molecular regulatory mechanisms involved in the etiology and progression of infectious diseases are now being characterized. The Bhopal gas tragedy is undoubtedly one of the worst industrial disasters in the history of mankind. After 25 years of accidental exposure to methyl isocyanate (MIC), severe systemic ailments still continue to pre-occupy the lives of the affected population that survived this tragedy. We have performed a molecular surveillance study to characterize hepatitis and tuberculosis infections amongst the first and the second generation of survivors exposed to MIC.Both outdoor and indoor patients referred for molecular diagnosis of hepatitis B virus (HBV), hepatitis C virus (HCV) and Mycobacterium tuberculosis (MTB) were examined. Qualitative analysis for HBsAg, anti-HBc, anti-HCV through ELISA was performed, while BacT/ALERT and Ziehl-Neelson technique were utilized for the assessment of tuberculosis. Detection and quantification of viral and bacterial nucleic acid and characterization of hepatitis genotypes were analyzed using real-time and end-point PCR techniques.The results suggest that HBV infections are most common among the MIC-exposed cohort, followed by extra-pulmonary and pulmonary MTB and HCV infections. Genotype 3 is the most prevalent HCV genotype among the survivors. Failure to detect HBsAg, anti-HBc and anti-HCV through ELISA, and tuberculosis by culture and Ziehl-Neelson stain, indicates higher prevalence of occult hepatitis and latent tuberculosis in the affected population.Our study underscores the importance of hospital-based records used as a data source for monitoring possible environmental health hazards. As the risk of progress of infection is often influenced by conditions and periods of environmental chemical exposure, therefore, insights of interconnected molecular pathways will further illuminate the gene-environment association and might offer valuable information for rational drug design.

Bhopal gas tragedy is considered as one of the world's worst industrial disaster. Approximately, 3,000-6,000 people died and 200,000 injured due to the leak of 40 tons of methyl isocyanate (MIC) gas from a pesticide plant. We aimed to decipher any persistent and subtle immunotoxic effects of MIC in the survivors of the tragedy. The study was divided into 3 groups i.e. group I (n=40); Age and gender matched non-exposed healthy controls recruited from places within the geographical region of Bhopal but from unaffected zones, group II (n=40); Age and gender matched non-exposed healthy controls recruited from places well outside geographical region of Bhopal and group III (n=40); Age and gender matched MIC exposed subjects from affected zones inside geographical region of Bhopal and the status of inflammatory biomarkers (IL-8, IL-1beta, IL-6, TNF, IL-10, IL-12p70 cytokines and C-reactive protein) were analysed. The results displayed a significant increase in the levels of all circulating inflammatory biomarkers in the MIC exposed group in comparison to non-exposed cohorts. A toxin induced genetic and/or epigenetic alteration seems to be the likely underlying cause. However, further studies are essential for both mechanistic understanding and clinical implications of these patterns.

The present study included three groups: (A) age and gender matched control (n=24) with no previous signs of M. tuberculosis complex (MTBC) infection, (B) patients (n=28) diagnosed with gastro-intestinal TB (GITB), (C) patients (n=50) with clinical and histo-pathological signs of GITB, but were culture and AFB negative. Real time assay performed using fluorescence resonance energy transfer hybridization probes showed a positivity index of 36 % in group C, i.e. 18 were found reactive from the total 50 cases studied. In addition, immune characterization of these 18 cases showed depleted CD(4) (+) count and increased levels of IFN-γ and TNF-α cytokines. No positive case was found in group A, while in group B, out of total 28 cases studied 27 were found positive. A combinatorial diagnostic approach for rapid detection and characterization of GITB might provide specific therapeutic strategies for prevention and treatment of the infection in future.

The Bhopal gas tragedy is undoubtedly one of the worst industrial disasters in the history of mankind resulting in mortality of 2500-6000 and debilitating over 200 000 people. Inhabitants in the township were exposed to different degrees and there are more than 500 000 registered victims that survived the tragedy. Clinical studies have shown chronic illnesses such as pulmonary fibrosis, bronchial asthma, chronic obstructive pulmonary disease (COPD), emphysema, recurrent chest infec- tions, keratopathy and corneal opacities in exposed cohorts. Survivors continue to experience higher incidence of reported health problems including febrile illnesses, respiratory, neurologic, psychiatric and ophthalmic symptoms. In-utero exposure to methyl isocyanate in the first trimester of pregnancy caused a persistent immune system hyper-responsiveness, which was in an evident way genetically linked with the organic exposure. Recent experimental studies have provided mechanistic understanding of methyl isocyanate exposure at a molecular level. Immunotoxic implications, toxico-genomic effect, inflam- matory response, elicitation of mitochondrial oxidative stress, chromosomal and microsatellite instability have been studied comprehensively in cultured mammalian cells. Besides providing a framework for understanding potential mechanisms of toxicity of a host of other exposures, these studies may also uncover unique abnormalities thereby stimulating efforts to de- sign newer and effective diagnostic and therapeutic strategies. The authors recommend long-term monitoring of the affected area and use of appropriate methods of investigation that include well-designed cohort studies, case-control studies for rare condition, characterization of personal exposure and accident analysis to determine the possible elements of the gas cloud.

The role of oxidative stress is often attributed in environmental renal diseases. Isocyanates, a ubiquitous chemical group with diverse industrial applications, are known to undergo bio-transformation reactions upon accidental and occupational exposure. This study delineates the role of isocyanate-mediated mitochondrial oxidative stress in eliciting chromosomal instability in cultured human kidney epithelial cells. Cells treated with 0.005 microM concentration of methyl isocyanate displayed morphological transformation and stress-induced senescence. Along the time course, an increase in DCF fluorescence indicative of oxidative stress, depletion of superoxide dismutase (SOD) and glutathione reductase (GR) and consistent accumulation of 8-oxo-dG were noticed. Thus, endogenous oxidative stress resulted in aberrant expression of p53, p21, cyclin E and CDK2 proteins, suggestive of deregulated cell cycle, chromosomal aberrations, centromeric amplification, aneuploidy and genomic instability.

Lungs comprise the primary organ exposed to environmental toxic chemicals, resulting in diverse respiratory ailments and other disorders, including carcinogenesis. Carcinogenesis is a multi-stage phenomenon, which involves a series of genetic alterations that begin with genomic instability provoked by certain factors such as inflammation and DNA damage and end with the development of cancer. Isocyanates such as methyl isocyanate are the chief metabolic intermediates in many industrial settings with diverse applications; exposure to them can lead to severe hypersensitive, mutagenic and genotoxic alterations. We examined the molecular mechanisms underlying isocyanate-mediated inflammatory responses and their probable role in the onset of genomic instability in cultured IMR-90 human lung fibroblasts. The isocyanates induced inflammation, resulting in extensive DNA damage, evidenced by increases in ATM, ATR, gammaH2AX, and p53 expression levels. The apoptotic index also increased. Chromosomal anomalies in treated cells included over-expression of centrosome protein and variable amplification of inter-simple sequence repeats, further demonstrating isocyanate-induced genomic instability. This information could be useful in the design of new approaches for risk assessment of potential industrial disasters.

Implications of environmental toxins on the regulation of neutrophil function are being significantly appraised. Such effects can be varied and markedly different depending on the type and extent of chemical exposure, which results in direct damage to the immune system. Isocyanates with functional group (-NCO), are considered as highly reactive molecules with diverse industrial applications. However, patho-physiological implications resulting from their occupational and accidental exposures have not been well delineated. The present study was carried out to assess the immunotoxic response of isocyanates and their mode of action at a molecular level on cultured human neutrophils isolated from healthy human volunteers. Studies were conducted to evaluate both dose- and time-dependent (n = 3) response using N-succinimidyl N-methylcarbamate, a chemical entity that mimics the effects of methyl isocyanate in vitro. Measure of apoptosis through annexin-V-FITC/PI assay, active caspase-3, apoptotic DNA ladder assay and mitochondrial depolarization; induction of oxidative stress by CM-H(2)DCFDA and formation of 8'-hydroxy-2'-deoxyguanosine; and levels of antioxidant defense system enzyme glutathione reductase, multiplex cytometric bead array analysis to quantify the secreted cytokine levels (interleukin-8, interleukin-1beta, interleukin-6, interleukin-10, interferon-gamma, tumor necrosis factor, and interleukin-12p70) parameters were evaluated. Our results demonstrate that isocyanates induce neutrophil apoptosis via activation of mitochondrial-mediated pathway along with reactive oxygen species production; depletion in antioxidant defense states; and elevated pro-inflammatory cytokine response.

Context: CD14 functions as a multifunctional receptor for bacterial cell wall components including endotoxin and lipopolysaccharide and is likely to influence the cytokine profile and subsequent immunoglobulin E production in response to antigen/allergen contact in allergic phenotypes. Aims: The present study was to investigate genetic polymorphism in CD14 gene - 159C/T, which may be one of the risk factor for increased prevalence of Chronic Lung Diseases in the Central India. Settings and Design: Survivors of Methyl isocyanates toxicity in Bhopal still suffering from various respiratory ailments were examined. Materials and Methods: Polymerase chain reaction-restriction fragment length polymorphism was performed to determine the polymorphism of C-159T. Statistical analysis used: All analysis was done using SPSS software, version 11.5 (SPSS, Chicago, IL, USA). Results: The genotype and allelic frequencies were in Hardy-Weinberg's equilibrium. Prevalence of CC, CT, and TT were 5.5%, 22.2% and 9.25% respectively in asthmatics; 16.6%, 20.3% and 5.5% respectively in chronic obstructive pulmonary disease (COPD) patients and 5.5%, 14.8% and 1.85 respectively among interstitial lung disorder (ILD) patients; whereas the control cohort with no methyl isocyanate exposure displayed (CC, CT, and TT) cytosine,thymine as 2%, 1.6% and 2% respectively. Increased risk of Asthma among those carrying TT genotype and T allele (odds ratio [OR] =2.61 and 2.02 respectively). Conclusion: COPD risk significantly found among those with CC genotype and C allele (OR = 2.81 and 1.50 respectively), whereas ILD risk found significantly among CT genotype and C allele (OR = 1.75 and 1.40 respectively). Therefore, single nucleotide polymorphism (SNP) C-159T polymorphism in CD14 gene might be a risk factor for development of CLD in this population.

pulmonary MPO activity were observed among these three groups. Twelve hours after reperfusion a significant reduction in pulmonary vascular permeability in group HSPTX was observed when compared to groups HS and NS (p