Neelam Pathak

Bhopal Gas Tragedy: review of clinical and experimental findings after 25 years

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 infections, 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, inflammatory 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 design 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.

OCCULT HEPATITIS B VIRUS INFECTION WITH LOW VIREMIA INDUCES DNA DAMAGE, APOPTOSIS AND OXIDATIVE STRESS IN PERIPHERAL BLOOD LYMPHOCYTES

Occult HBV infections (OHBI) are often associated with poor therapeutic response and increased risk of developing hepatocellular carcinoma. Despite a decade of research, OHBI still remains an intricate issue and much is yet to be defined about their possible immune implications. As HBV is known to infect peripheral blood lymphocytes, the present study aimed to explore the molecular mechanisms underlying DNA damage response triggered due to OHBI in host cells. The study was divided into three groups i.e. group A (OHBI patients n=30, viral load <or=100 IU/mL); group B (chronic HBV patients, n=30) and group C (controls, n=30). Peripheral blood lymphocytes were isolated and DNA damage response, apoptosis and oxidative stress were the studied parameters. A significant increase in the phosphorylation of DNA damage response proteins (ATM, ATR, H2AX and p53) in OHBI in comparison to controls suggested that OHBI induces DNA damage in peripheral blood lymphocytes and elicit a PI3 kinase mediated cellular response. In addition, increased DNA fragmentation, circulating nucleosome levels and mitochondrial membrane depolarization observed in OHBI group indicated that this damage might lead to cellular demise and immune hypo-responsiveness. Moreover, OHBI was also observed to be strongly associated with oxidative stress as suggested by the augmented levels of DCF fluorescence and depleted GR activity. Collectively, these results provide the basic knowledge about the genotoxic effects of OHBI in peripheral blood lymphocytes. Such studies may possibly open up new avenues for identifying novel therapeutic targets for viral hepatitis.

Role and clinical significance of lymphocyte mitochondrial dysfunction in type 2 diabetes mellitus.

Lymphocyte homeostasis in type 2 diabetes mellitus (T2DM) is associated with increased susceptibility to infections. Mitochondrial oxidative stress is implicated primarily in the immune pathophysiology of diabetes; however, the molecular underpinnings of lymphocyte mitochondrial dysfunction and ensuing downstream cellular effects are hitherto unreported. Both in early diagnosed patients and patients with late complications, we observed an inverse correlation between mitochondrial DNA content in lymphocytes and hemoglobin A1 (HbA1c) levels. This relation established for the first time might serve as a general, yet direct, predictor or indicator for mitochondrial dysfunction in T2DM. Compared with controls, nuclear DNA damage response was higher (P ≤ 0.001) in diabetic subjects with increased accumulation of phospho-ataxia-telangiectasia (ATM), γ-H2AX, along with active recruitment of repair proteins (Mre11, Rad50, and Nbs1). A higher frequency (>2%) of stable chromosomal anomalies with loss of telomere integrity was observed in cases with late complications. A significant decrease (P ≤ 0.001) in enzyme activity of complex II, III, and IV of mitochondrial respiratory chain was evident in both diabetic groups in comparison with healthy controls. Activation in the cascade of nuclear factor kappa-beta (NF-κβ)-mediated feed-forward proinflammatory cytokine response was noted among T2DM subjects. Increased oxidative stress, mitochondrial membrane depolarization, activation of caspase-3, and PARP observed in diabetic groups indicated bax triggered mitochondrial mediated cellular apoptosis. Our results provide the first insights of lymphocyte mitochondrial dysfunction that might be helpful in explaining the clinical significance of immunologic perturbation observed in type 2 diabetic conditions. Our data also indicate that maneuvering through the mitochondrial function might be a viable, indirect method to modulate lymphocyte homeostasis in T2DM.

Evaluation of cytotoxicity and anticarcinogenic potential of Mentha leaf extracts.

We examined the possible molecular mechanisms underlying the cytotoxicity and anticarcinogenic potential of Mentha leaf extracts (petroleum ether, benzene, chloroform, ethyl acetate, methanol, and water extracts) on 6 human cancer (HeLa, MCF-7, Jurkat, T24, HT-29, MIAPaCa-2) and normal (IMR-90, HEK-293) cell lines. Of all the extracts tested, chloroform and ethyl acetate extracts of M piperita showed significant dose- and time-dependent anticarcinogenic activity leading to G1 cell cycle arrest and mitochondrial-mediated apoptosis, perturbation of oxidative balance, upregulation of Bax gene, elevated expression of p53 and p21 in the treated cells, acquisition of senescence phenotype, while inducing pro-inflammatory cytokines response. Our results provide the first evidence of direct anticarcinogenic activity of Mentha leaf extracts. Further, bioassay-directed isolation of the active constituents might provide basis for mechanistic and translational studies for designing novel anticancer drugs to be used alone or as adjuvant for prevention of tumor progression and/or treatment of human malignancies.

Occult hepatitis C virus elicits mitochondrial oxidative stress in lymphocytes and triggers PI3-kinase-mediated DNA damage response

Occult hepatitis C viral infection (OHCI) is a newly reported pathological entity associated with increased risk of developing hepatocellular carcinoma and lymphoproliferative disorders. Although hepatocytes are the primary sites of viral replication, hepatitis C virus is potentially lymphotropic, invading and propagating in cells of the immune system. Lymphocytes, the extrahepatic viral reservoirs, are differentially implicated in the occult and the active forms of the disease. This study aimed to elucidate the implications of mitochondrial oxidative stress on the immune pathophysiological mechanisms of OHCI. We herein report that OHCI induces mitochondrial oxidative stress, leading to DNA double-strand breaks and elicitation of a phosphoinositol 3-kinase-mediated cellular response in peripheral blood lymphocytes. Compared to controls, OHCI subjects showed higher accumulation of pATM, pATR, γH2AX, and p-p53, along with active recruitment of repair proteins (Mre11, Rad50, and Nbs1) and altered mitochondrial DNA content. Increased mitochondrial membrane depolarization and circulating nucleosome levels along with chromatid-type aberrations and decreased T-cell proliferative index observed in the OHCI group further indicated that this damage might lead to Bax-triggered mitochondria-mediated cellular apoptosis. Together our results provide the mechanistic underpinnings of mitochondrial dysfunction in OHCI, a previously unknown paradigm, for explaining the immune pathogenesis in a redox-dependent manner.

Molecular mechanisms of isocyanate induced oncogenic transformation in ovarian epithelial cells.

Ovarian surface epithelium is under constant physiological pressure to maintain its integrity. Environmental toxic exposure can contribute to degenerative pathologies including ovarian cancer. Based on our current understanding, we aimed at listing mechanistic insights that contribute to ovarian carcinogenesis after exposure to methyl isocyanate, an ubiquitous environmental pollutant. Ovarian epithelial cells manifested a persistent DNA damage response along with increased accumulation of GADD45, p21, p16(INK4A) and pRb proteins upon treatment. Increase in cell size and β-gal positive staining showing inception of premature senescence with morphological transformation and structural and numerical chromosomal abnormalities were also observed. Immuno-FISH analysis illustrated early loss of TRF2 protein suggestive of telomeric dysfunction due to premature senescence and plausible association with chromosomal and microsatellite instability. Soft-agar assay displayed neoplasticity in treated cells demonstrating onset of malignant transformation. These results indicate that isocyanate exposure alters ovarian epithelial cell proliferation and might lead to ovarian dysfunction and carcinogenesis.

Circulating Biomarkers and their Possible Role in Pathogenesis of Chronic Hepatitis B and C Viral Infections

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.

Mitochondrial Oxidative Stress-Induced Epigenetic Modif ications in Pancreatic Epithelial Cells

Emerging studies have linked prooxidative carbamate compound exposures with various human pathologies including pancreatic cancer. In these studies, our aim was to examine mitochondrial oxidative stress-mediated aberrant chromatin responses in human pancreatic ductal epithelial cells. Posttranslational histone modifications, promoter DNA methylation, and micro-RNA (miRNA) expression patterns were evaluated following induction of mitochondrial oxidative stress by N-succinimidyl N-methylcarbamate exposure. In treated cells, perturbation in mitochondrial machinery led to hypermethylation of p16 and smad4 gene promoters and downregulation of respective gene products. Posttranslational histone modifications that include hypoacetylation of acetylated histone (AcH) 3 and AcH4, hypermethylation of monomethylated histone 3 at lysine 9 and trimethylated histone 4 at lysine 20 ubiquitinated histone (uH) 2A/uH2B, and increased phosphorylation of H2AX and H3 were observed in the treated cells. Altered expression of miRNAs denoted possible location of corresponding genes at oxidatively damaged fragile sites. Collectively, our results provide a direct role of mitochondrial oxidative stress-mediated epigenetic imbalance to perturbed genomic integrity in oxygen radical-induced pancreatic injury. Further, identification and characterization of molecular switches that affect these epigenomic signatures and targets thereof will be imperative to understand the complex role of redox-regulatory network in pancreatic milieu.

Cell cycle deregulation by methyl isocyanate: Implications in liver carcinogenesis

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.

Prevalence of hepatitis C virus genotypes and impact of T helper cytokines in achieving sustained virological response during combination therapy: A study from Central India

Characterisation of host immune response to hepatitis C virus (HCV) genotypes may have an important prognostic and therapeutic implication. Genotype-3 was more prevalent in the examined cohort and demonstrated a significantly higher response to combination therapy than genotype-1. Sustained virological response (SVR) was 94.74% in genotype-3 and 45.45% in genotype-1. The patients who achieved SVR reported higher levels of circulating T helper 1 cytokines in comparison to subjects with no SVR in both the studied groups. Besides providing local prevalence, our study might also assist in understanding the host immune mechanisms involved to achieve SVR during combination therapy in chronic HCV patients.