Heavy metal bioaccumulation triggers oxystress, genotoxicity and immunomodulation in head kidney macrophages of Channa punctatus Bloch.

Studies on heavy metal induced toxicity have been conducted in many water bodies across the globe and such effects have been evaluated in various fish species. The present study was designed to determine the load of some heavy metals in select sites in Southern Assam, India, along with estimating their concentration in tissues of Channa punctatus Bloch. inhabiting those niches. The effect of heavy metals in oxystress generation, genotoxicity and subsequent immune response in fish was also evaluated. In all of these sites, the concentration of Hg, Cd, Pb and Cr were above the permissible ranges while their concentrations were several folds higher in the piscine tissues due to bioaccumulation and possible biomagnification. Kidney showed the highest metal pollution index followed by liver and gills. Generation of ROS was significantly elevated and that in turn triggered oxystress, as is evident from enhanced lipid peroxidation, protein carbonylation and respiratory burst activity. These were in association with the compromised antioxidant enzyme levels with concomitant damage to DNA as evident from Comet parameters. The innate immune potential was significantly impaired as evident from the compromised cell adhesion, phagocytosis, intracellular killing activity in head kidney macrophages (HKM) along with decreased release of nitric oxide (NO) and myeloperoxidase (MPO). Immunosuppression was further validated at protein levels where compromised release of cytokines viz. TNF-α, IL-1ß, IL-6, IL-10 and IL-12 and cell signaling molecules iNOS and NF-κß were noted. Thus the present study indicates genotoxicity along with a compromise in immune status of Channa punctatus Bloch. living in a habitat laden with heavy metals.

GCMS analysis of sadagura (smokeless tobacco), its enhanced genomic instability causing potential due to arsenic co-exposure, and vitamin-C supplementation as a possible remedial measure: a study involving multiple model test systems.

North-eastern states of India, including Assam, have a high prevalence of head and neck cancer cases. In these regions, Sadagura is a unique form of smokeless tobacco (SLT). There are fewer reports regarding the effects of simultaneous sadagura and arsenic co-exposure. Analysis of chemical compounds present in sadagura aqueous extract was done using gas chromatography-mass spectrometry. Estimation of arsenic contamination in groundwater and bioaccumulation in human tissues was performed by using atomic absorption spectroscopy. Buccal micronucleus cytome (BMCyt) assay and analysis of various peripheral blood parameters were performed among study volunteers. Chronic exposure (90 days) experiments were performed in mice test system in vivo to determine any possible protective potential of vitamin C (Vit-C) supplementation against sadagura and arsenic co-exposure. BMCyt assay results revealed a higher incidence of micronucleated cells (p < 0.001), and cell death biomarker among sadagura consumers residing in arsenic affected areas. Comet assay of mice femur bone marrow cells following chronic exposure of the test substances revealed a reduction in DNA damage due to Vit-C supplementation. Histological examination of the hepatic and renal tissues revealed marked improvement due to Vit-C supplementation in mice against sadagura and arsenic chronic co-exposure. Indiscriminate consumption, presence of various harmful compounds in sadagura along with arsenic co-exposure might be a vital link for the higher incidence of oral cancer in the region. Chronic Vit-C supplementation study results in mice show its effective remedial potential against combined sadagura and arsenic co-mediated genotoxicity and ultrastructural changes in major organs.

Aqueous Extract of Moringa oleifera Exhibit Potential Anticancer Activity and can be Used as a Possible Cancer Therapeutic Agent: A Study Involving In Vitro and In Vivo Approach.

OBJECTIVES: New cases of cancers are increasing at an alarming rate globally. It has been hypothesized that modern cancer treatment is associated with lots of side effects and thus evoking the need to develop safer treatment measures. Thus, the present study was undertaken to evaluate the anti-carcinogenic potential of a highly nutricious plant "Moringa oleifera" (MO) in vitro and in vivo. METHODS: GC-MS analysis of aqueous extract of Moringa oleifera (AEMO) was employed to identify the bioactive compound present. Anti-tumor activity of AEMO was assessed in EAC (Ehrlich acites carcinoma) induced solid tumor bearing mice by analyzing tumor weight (TW) and Tumor volume (TV). To assess AEMO induced cytotoxicity, EAC and HEp-2 (Human laryngeal carcinoma) cells were treated with AEMO (0.05, 0.1, 0.25, 0.5 and 1 mg/ml) for both 48 h and 72 h and trypan blue, MTT and LDH released assay was done. Further, cell cycle assay and apoptosis assay was done in EAC cells to understand the mechanism of AEMO induced tumor regression. RESULTS: GC-MS analysis revealed the presence of quinic acid, octadecanoic acid, hexadecanoic acid (palmitic acid), α-tocopherol (Vitamin-E) and É£-sitosterol as major bioactive compounds. AEMO administration reduced the TV and TW of tumor-bearing mice and increases the life span. Side effect analysis showed that AEMO treatment did not induce significant alterations of liver and kidney function and hematological parameters. Further, in vitro cytotoxicity assays revealed that AEMO treatment induced dose and time-dependent toxicity in both the cell lines tested. Flow cytometric analysis confirmed significant induction of apoptotic cells by changing the mitochondrial membrane potential in EAC cell line. CONCLUSION: The results of the present study suggest that AEMO has immense potential to inhibit the tumor progression without affecting the normal physiology and functioning of the body and thus can be used as a cancer therapeutic agent.

Joint detection of claudin-1 and junctional adhesion molecule-A as a therapeutic target in oral epithelial dysplasia and oral squamous cell carcinoma.

Abnormal expression of claudin-1 (CLDN-1) and junctional adhesion molecule-A (JAM-A) has been described in certain malignancies but their clinical relevance is poorly understood. The present study aims to elucidate the role of CLDN-1 and JAM-A in oral epithelial dysplasia (OED) and oral squamous cell carcinoma (OSCC). Changes in the expression of these proteins were identified immunohistochemically on tissue sections from patients with OED and OSCC and compared with control. A correlation between the expression level of proteins and clinicopathological features was analyzed by Pearson's correlation χ2 test. The survival curve of the follow-up data was estimated by the Kaplan-Meier method followed by the log-rank test. CLDN-1 and JAM-A were highly expressed in OED and OSCC tissues when compared to control. Also, delocalization of CLDN-1 from the membrane to the cytoplasm to the nucleus was observed as the cell proceeds from normal to malignancy. Increased expression of CLDN-1 and JAM-A in both OED and OSCC were concomitant with histological grades. In addition, increased JAM-A was associated with perineural invasion of cancer cells. A positive correlation between the expression level of proteins was observed in OED (r = 0.733) and OSCC (r = 0.577). Kaplan-Meier analysis in patients with OSCC showed that the survival rate was lower in patients with high CLDN-1 and high JAM-A expression compared to low expressed patients. To conclude, the elevated level and delocalization of CLDN-1 and JAM-A suggest their use as tumor markers. A positive correlation between CLDN-1 and JAM-A suggests joint detection of these proteins as a future diagnostic tool in oral precancerous and cancerous conditions.

Areca nut and smokeless tobacco exposure induces micronucleus, other nuclear abnormalities and cytotoxicity in early chick embryo.

Areca nut (AN) and smokeless tobacco (SLT) are indiscriminately consumed among the populations of Southeast and South Asian countries, even by women during the gestational period. This study aimed to investigate the genotoxic and cytotoxic potentials of AN and Sadagura (SG), a unique homemade SLT preparation, alone and in combination in early chick embryos. Fertile white leghorn chicken eggs were randomly divided into five treatment groups: vehicle control, positive control (Mitomycin C, 20 µg/egg), AN, SG, and AN+SG. AN, SG, and AN+SG were given at dosages of 0.125, 0.25, and 0.5 mg/egg. The hen's egg test for micronucleus induction (HET-MN) was performed in chick embryos to evaluate the genotoxic potential of the test agents. Furthermore, the cytotoxic potential was assessed by studying erythroblast cell populations and the polychromatic erythrocytes (PCEs) to normochromatic erythrocytes (NCEs) ratio. Our results indicated a significant increase (p < .001) in MN frequency and other nuclear abnormalities, suggesting the potential of AN and SG to cause genotoxicity. Also, AN and SG exposure alone and in combination considerably altered the erythroblast cell population (%) and the PCE to NCE ratio in all the treatment periods. Our findings established the genotoxic and cytotoxic potential of both AN and SG alone and in combination during early embryonic development in the chick embryo.

Altered expression of junctional proteins as a potential biomarker in oral precancerous and cancerous patients.

Due to a lower survival rate in patients with advanced clinical stages of oral cancer, discovering a biomarker that could diagnose and predict disease progression is vital. Cell-cell junctional proteins play a crucial role in the maintenance of tissue architecture but are often deregulated in different cancer. The present study investigates the expression of cell-cell junctional proteins viz: e-cadherin (E-cad) and zonula occludens-1 (ZO-1) in oral precancerous (OED) and cancerous (OSCC) patients to monitor if they can serve as practicable molecular markers. The ultrastructural junctional complex was studied by transmission electron microscopy, and the expression of proteins was performed by immunohistochemistry. The relationship between the expression of protein and clinicopathological features of the patients was checked by Pearson's correlation test. Furthermore, the survival curve of the follow-up data was estimated by the Kaplan-Meier method. We observed a disrupted junctional complex and a significantly decreased immunoexpression of E-cad and ZO-1 in OED and OSCC when compared to the adjacent non-cancerous tissues. The expression of ZO-1 was associated with TNM stages, whereas E-cad was associated with histological grades as well as TNM stages. A positive correlation was observed between the expression of ZO-1 and E-cad proteins in OED and OSCC. Further, follow-up studies revealed that high ZO-1 and E-cad expressing patients survived longer than their low expressed counterparts. The present study shows disruption of junctional complex and alteration of junctional proteins expression that could draw the attention of health professionals to explore junctional proteins as a possible therapeutic target in oral cancer.

Consumption pattern and genotoxic potential of various smokeless tobacco products in Assam, India: A public health concern.

Smokeless tobacco (SLT) consumption is presumed to be one of the major causes of high incidence of oral cancer in India. The present study aimed to document various types of SLT products consumed and their potential impact on the genome instability on the population from Assam state in Northeast India. A cross-sectional study (n = 5000) showed that 60.56 % of the study population consumed at least one of the three forms (sadagura, zarda and khaini) of SLT of which 52.0 % were only sadagura users. Genotoxicity assessment using buccal cytome assay in 240 age and sex matched volunteers revealed that except for zarda, other forms of SLT induced significantly higher incidence micronuclei in the buccal epithelial cells compared to the control individuals. Similar effects were also observed in other cytome parameters related to cell proliferation, cytokinesis defects and cell death. Significantly higher incidence of micronucleus was observed among sadagura and khaini users in lymphocyte cytokinesis-blocked micronucleus assay. The addition of lime in sadagura increased the pH and anion levels which possibly result in higher absorption and may lead to the development of cellular anomalies.

Arsenic and smokeless tobacco exposure induces DNA damage and oxidative stress in reproductive organs of female Swiss albino mice.

Arsenic contamination in the groundwater of Southern Assam, India is well-documented. A specific type of smokeless tobacco (sadagura, SG) is highly prevalent among the local population. Thus, the present study is aimed to evaluate the toxicological implications of arsenic and smokeless tobacco co-exposure on the reproductive health of female mice. The estrous cycle of experimental animals was monitored for 30 days. Histopathological studies and comet assay of ovarian and uterine tissues were performed after 30 days of exposure to SG and arsenic (sodium arsenite, SA). Oxidative stress was estimated biochemically by taking tissue glutathione, lipid peroxidation (LPO), and superoxide dismutase activity as endpoints. Our findings indicated a prolonged diestrus phase in the SG + L + SA group (p < 0.001). Histopathological study revealed abnormal tissue architecture in treated groups. Comet assay study showed that SG + SA exposure significantly induced DNA damage in test animals. The elevated LPO level in the SG + SA group indicated oxidative stress generation in the reproductive tissues. The present study suggests that female reproductive organs are vulnerable to SA and SG and oxidative stress generation may be the possible mechanism behind DNA damage, impaired follicular growth, atresia, and altered estrous cycle in the mice test system.

Effect of nutritional status on arsenic and smokeless tobacco induced genotoxicity, sperm abnormality and oxidative stress in mice in vivo.

BACKGROUND: Recently, high concentrations of arsenic have been documented in ground waters of Southern Assam, India. Indiscriminate smokeless tobacco consumption is a common practice in this region. Correlation between nutritional status and arsenic and smokeless tobacco-induced health effects has not been taken up in humans or other test systems. METHODS: Mice were divided into groups based on protein (casein) content in the diet: High protein (40%), optimum protein (20%), and low protein (5%). Simultaneous chronic exposure (90 days) to arsenic and smokeless tobacco (sadagura) orally was given to evaluate the extent of the cytological and genotoxicological damage. Micronucleus assay and Comet assay of the femur bone marrow cells were conducted. Germ cell toxicity was evaluated by recording the sperm head abnormalities and total sperm count. Cell cycle analysis was performed in femur bone marrow cells using flow cytometer. Hepatic, renal, and intestinal tissues were analyzed for various oxidative stress evaluations. Histological examination of liver and kidney was performed. RESULTS: Notably, high protein diet groups had lower arsenic and sadagura induced genotoxicity, germ cell abnormalities and oxidative stress as compared to optimum protein and low protein diet counterparts. CONCLUSION: Our study indicates that sufficient levels of dietary protein appear to reduce the long-term arsenic and smokeless tobacco-induced toxicity in mice test system, as compared to lower or deficient amount of protein in the diet. This observation has implications and invites further studies especially epidemiological studies in the human population exposed to arsenic in South East Asian countries. Environ. Mol. Mutagen. 59:386-400, 2018. © 2018 Wiley Periodicals, Inc.