Heavy metal association with chronic kidney disease of unknown cause in central India-results from a case-control study.

BACKGROUND: Chronic Kidney Disease of unknown cause (CKDu) a disease of exclusion, and remains unexplained in various parts of the world, including India. Previous studies have reported mixed findings about the role of heavy metals or agrochemicals in CKDu. These studies compared CKDu with healthy controls but lacked subjects with CKD as controls. The purpose of this study was to test the hypothesis whether heavy metals, i.e. Arsenic (As), Cadmium (Cd), Lead (Pb), and Chromium (Cr) are associated with CKDu, in central India. METHODS: The study was conducted in a case-control manner at a tertiary care hospital. CKDu cases (n = 60) were compared with CKD (n = 62) and healthy subjects (n = 54). Blood and urine levels of As, Cd, Pb, and Cr were measured by Inductively Coupled Plasma- Optical Emission Spectrometry. Pesticide use, painkillers, smoking, and alcohol addiction were also evaluated. The median blood and urine metal levels were compared among the groups by the Kruskal-Wallis rank sum test. RESULTS: CKDu had significantly higher pesticide and surface water usage as a source of drinking water. Blood As levels (median, IQR) were significantly higher in CKDu 91.97 (1.3-132.7) µg/L compared to CKD 4.5 (0.0-58.8) µg/L and healthy subjects 39.01 (4.8-67.4) µg/L (p < 0.001) On multinominal regression age and sex adjusted blood As was independently associated with CKDu[ OR 1.013 (95%CI 1.003-1.024) P < .05].Blood and urinary Cd, Pb, and Cr were higher in CKD compared to CKDu (p > .05). Urinary Cd, Pb and Cr were undetectable in healthy subjects and were significantly higher in CKDu and CKD compared to healthy subjects (P = < 0.001). There was a significant correlation of Cd, Pb and Cr in blood and urine with each other in CKDu and CKD subjects as compared to healthy subjects. Surface water use also associated with CKDu [OR 3.178 (95%CI 1.029-9.818) p < .05). CONCLUSION: The study showed an independent association of age and sex adjusted blood As with CKDu in this Indian cohort. Subjects with renal dysfunction (CKDu and CKD) were found to have significantly higher metal burden of Pb, Cd, As, and Cr as compared to healthy controls. CKDu subjects had significantly higher pesticide and surface water usage, which may be the source of differential As exposure in these subjects.

Evaluation of interleukin-33 & sST2 levels in type-2 diabetic mellitus patients with or without metabolic syndrome.

Background & objectives: Diabetes mellitus (DM) is characterized by increase in blood glucose levels due to defective insulin secretion or insulin sensitivity. Interleukins (ILs) are known to play an important role in the pathogenesis of DM. The aim of this study was to investigate the serum concentration of IL-33 and its receptor soluble ST2 (sST2) in patients with diabetes and draw a correlation between their serum levels and different standard glycaemic indices of patients affected with type-2 diabetes with or without metabolic syndrome. Methods: Thirty type-2 diabetic individuals and 30 healthy controls were recruited for this study. Serum and plasma were separated by centrifugation of blood for quantitative measurement of IL-33, sST2 and other biochemical parameters. Results: It was observed that serum IL-33 levels were significantly less and sST2 levels were significantly high in type-2 diabetic individuals as compared to healthy controls. A significant correlation between the serum IL-33 concentration and fasting plasma glucose (FPG) and postprandial plasma glucose (PPG) levels were also found. Additionally, data also elucidated that serum levels of high-density lipoprotein, low-density lipoprotein or triglyceride in type-2 diabetics did not influence the serum levels of IL-33 and sST2, thereby excluding these factors as the major drivers of changes in serum IL-33 and sST2 concentration. Interpretation & conclusions: This study demonstrated alteration in serum levels of IL-33 and sST2 in type-2 diabetic individuals. Further mechanistic studies, focusing on the progression of type-2 diabetes could elucidate the involvement of IL-33 in the cellular acquisition of insulin resistance as observed in type-2 diabetics.

Association of Single Nucleotide Polymorphisms in KCNA10 and SLC13A3 Genes with the Susceptibility to Chronic Kidney Disease of Unknown Etiology in Central Indian Patients.

Global rise in the prevalence of endemic chronic kidney disease of unknown etiology (CKDu) possess major health issues. The prevalence of CKDu is also rising in the Indian population. Besides environmental factors, genetic factors play an important role in the predisposition to CKDu. In the present study, we have analyzed the association of single nucleotide polymorphisms (SNPs) in three genes with the susceptibility to CKDu. This was a case-control study with a total of 180 adult subjects (CKD = 60, CKDu = 60, Healthy = 60) from central India. We performed KASP genotyping assay to determine the allele frequency of SNP genotypes. We used the odds ratio (OR) to assess the association of individual SNPs, rs34970857 of KCNA10, rs6066043 of SLC13A3, and rs2910164 of miR-146a with CKDu and CKD susceptibility. In the case of rs34970857 of the KCNA10 gene, we noted a significantly increased OR for CKDu versus healthy control (Dominant model; CKDu versus control, CT + CC versus TT, OR = 3.96, p = 0.004). In the recessive and homozygous model, we observed significantly increased OR for rs6066043 of SLC13A3 gene, CKDu versus healthy control {(Recessive model; CKDu versus control, GG versus AA + GA, OR = 2.41, p = 0.03; homozygous model, GG versus AA, OR = 3.54, p = 0.04)}. CC genotype of rs34970857 of the KCNA10 gene and the GG genotype of the SLC13A3 gene are significantly associated with the susceptibility of CKDu.

Predictive Model for Ambulatory Hypertension Based on Office Blood Pressure in Obese Children.

Background: The epidemic of obesity, along with hypertension (HT) and cardiovascular disease, is a growing contributor to global disease burden. It is postulated that obese children are predisposed to hypertension and subsequent cardiovascular disease in adulthood. Early detection and management of hypertension in these children can significantly modify the course of the disease. However, there is a paucity of studies for the characterization of blood pressure in obese children through ambulatory blood pressure monitoring (ABPM), especially in the developing world. This study aims to characterize ambulatory blood pressure in obese children and to explore feasibility of using office BP that will predict ambulatory hypertension. Methods:In the present study, 55 children with a body mass index (BMI) in the ≥95th percentile for age and sex were enrolled in a tertiary care hospital and underwent 24 h of ABPM and detailed biochemical investigations. Results:Ambulatory hypertension was recorded in 14/55 (25.5%; white coat hypertension in 17/29 (58.6%) and masked hypertension in 2/26 (7.69%). For office SBP percentile the area under curve (AUC) was 0.773 (95% CI: 0.619-0.926, p = 0.005) and for office DBP percentile the AUC was 0.802 (95% CI: 0.638-0.966, p = 0.002). The estimated cut offs (Youden's index) for office blood pressure which predicts ambulatory hypertension in obese children were the 93rd percentile for systolic BP (sensitivity-67% and specificity-78%) and the 88th percentile for diastolic BP (sensitivity-83% and specificity-62%). Conclusion:Ambulatory blood pressure abnormalities are highly prevalent among children with obesity. Office blood pressure did not accurately predict ambulatory hypertension. More than half of the children labeled as "hypertension" on office blood pressure measurement in the study were diagnosed to have white coat hypertension (WCH), thus emphasizing the role of ABPM for evaluation of WCH before the child is subjected to detailed investigations or started on pharmacotherapy.

Role of Sphingosine-1-Phosphate in Mast Cell Functions and Asthma and Its Regulation by Non-Coding RNA.

Sphingolipid metabolites are emerging as important signaling molecules in allergic diseases specifically asthma. One of the sphingolipid metabolite, sphingosine-1-phosphate (S1P), is involved in cell differentiation, proliferation, survival, migration, and angiogenesis. In the allergic diseases, alteration of S1P levels influences the differentiation and responsiveness of mast cells (MCs). S1P is synthesized by two sphingosine kinases (SphKs), sphingosine kinase 1, and sphingosine kinase 2. Engagement of IgE to the FcεRI receptor induces the activation of both the SphKs and generates S1P. Furthermore, SphKs are also essential to FcεRI-mediated MC activation. Activated MCs export S1P into the extracellular space and causes inflammatory response and tissue remodeling. S1P signaling has dual role in allergic responses. Activation of SphKs and secretion of S1P are required for MC activation; however, S1P signaling plays a vital role in the recovery from anaphylaxis. Several non-coding RNAs have been shown to play a crucial role in controlling the MC-associated inflammatory and allergic responses. Thus, S1P signaling pathway and its regulation by non-coding RNA could be explored as an exciting potential therapeutic target for asthma and other MC-associated diseases.

Altered Expression of Sphingosine-1-Phosphate Metabolizing Enzymes in Oral Cancer Correlate With Clinicopathological Attributes.

We have determined the gene expression of sphingosine-1-phosphate (S1P) metabolizing enzymes (SphK1, SphK2, SGPL1, SGPP1, SGPP2, PPAP2A, PPAP2B, and PPAP2C) by quantitative real-time polymerase chain reaction in tumor tissues and adjacent normal tissues of 50 oral squamous cell carcinoma (OSCC) patients. Expression of SphK1 and SGPP1 genes was up-regulated significantly in 70% and 75% OSCC tumors respectively. Importantly, expression of SphK2 and PPAP2B was down-regulated in the tumor tissues of 70% OSCC patients. Expression of SphK2 and PPAP2B negatively correlated with tumor-node-metastasis (TNM) staging and tumor volume respectively. Furthermore, LPP1 is an independent predictor of TNM staging and lymph node ratio.

ROS-dependent genotoxicity, cell cycle perturbations and apoptosis in mouse bone marrow cells exposed to formulated mixture of cypermethrin and chlorpyrifos.

Swiss albino mice were exposed to formulated cypermethrin (CMR) and/or or chlorpyrifos (CPF) through oral gavages for 60 days. Test doses of CMR (0.69, 1.38 or 2.76mg/kg/day) or CPF (0.5, 1.0 or 2.0mg/kg/day) or CMR + CPF (0.69 + 0.5, 1.38 + 1.0 or 2.76 + 2.0mg/kg/day) were based on the acute oral median lethal doses of CMR or CPF. Chromosome aberrations (CA), micronucleus (MN) induction, cell cycle perturbations, apoptosis and reactive oxygen species (ROS) generation were analysed in bone marrow cells. To explore the involvement of ROS induction, HaCat cells were exposed in vitro to arbitrary concentrations of CMR and/or CPF. Exposure of CMR (2.76mg/kg/day) induced significant inhibition of mitotic index. Significant (P < 0.01) frequencies of CA and MN were observed with the CMR at 1.38mg/kg/day, whereas CPF or its mixture CMR + CPF showed at highest doses. Chromosome/chromatid breaks and fragments were found to be major aberrations in all the treatment groups. Highest doses of CMR or CMR + CPF revealed significant (P < 0.01 or 0.001) elevation of G0/G1 peak, while CPF-exposed cells revealed significant (P < 0.01) declined in G1 phase. Decline in S phase was observed with highest dose of CMR only. Apoptosis induction measured by gating cell population beside G1 peak showed 3- to 4-fold increase in apoptotic cells in CPF-exposed mice as compared to control or CMR or CMR + CPF-treated mice. Further, all the treatment groups in vivo as well as in vitro revealed significant generation of ROS in comparison with the control group. Present results, together with the earlier reports, which substantiate ROS generation may be major cause of genotoxicity, cell cycle perturbations and apoptosis, nonetheless co-exposure of low doses of CMR and CPF mixture does not potentiate genotoxicity.

Effect of Different Insulin Response Patterns During Oral Glucose Tolerance Test on Glycemia in Individuals with Normal Glucose Tolerance.

BACKGROUND: Research is still going on for detecting the earliest glucose homeostasis derangements in individuals, which is crucial for the prevention of glucose intolerance. This cross-sectional study analyzes different insulin response patterns during the oral glucose tolerance test (OGTT) and their implications on glycemia in normoglycemic individuals. SUBJECTS AND METHODS: The sample frame was the "Offspring of Individuals with Diabetes Study" database. All participants underwent OGTT. Blood samples were collected at 0, 30, 60, and 120 min for measurement of insulin, C-peptide, and proinsulin levels. Normal glucose tolerant individuals were selected for analysis. RESULTS: Four hundred fifty subjects (mean age, 25 years) were included and divided into two groups according to timing of plasma insulin peaking during OGTT: Group 1, peaking at 30 min; and Group 2, peaking at 60 or 120 min. Body mass index (BMI) and insulin resistance were comparable between the groups; however, Group 2 showed a significantly higher 60- and 120-min glucose level and lower disposition index. Based on the magnitude of the insulin levels, Group 1 was subdivided into Group N (normal pattern) and Group E (exaggerated pattern) with a 30-min insulin cutoff of 74 µU/mL (Group E, ≥74 µU/mL). Group 2 was subdivided into Group DL (delayed and limited pattern; 60-min insulin <73.0 µU/mL and 120-min insulin <80.0 µU/mL) and Group DE (delayed and exaggerated pattern; 60-min insulin ≥73.0 µU/mL or 120-min insulin ≥80.0 µU/mL). Group DE showed a significantly higher area under the curve (AUC) of glucose compared with the other groups and had a lower disposition index and high-density lipoprotein levels. Group DL had significantly lower insulin resistance and BMI compared with Group E but showed a similar AUC of glucose. CONCLUSIONS: A delayed insulin pattern was associated with higher postprandial glucose levels. Individuals with delayed and exaggerated insulin secretion may have a higher risk for glucose intolerance.

Expression of CCK Receptors in Carcinoma Gallbladder and Cholelithiasis: A Pilot Study.

BACKGROUND: Gastrin and cholecystokinin (CCK) receptors are trophic for various gastrointestinal malignancies. Their role in gallbladder cancer has not been widely studied. OBJECTIVES: To identify expression of CCK-A and CCK-B receptors in the tissue and blood of patients suffering from carcinoma (CA) gallbladder and gallstone disease and to compare expression of CCK A and B receptors in the gall bladder tissue and blood of healthy individuals and patients of CA gallbladder, and gallstone diseases. MATERIALS AND METHODS: Forty nine subjects of both genders were recruited, comprising of 22 patients of CA gall bladder, 19 cases of cholelithiasis and, 8 normal gallbladders obtained from patients operated for trauma of the biliary system or Whipple's procedure. RNA extraction and cDNA formation for CCK-A and CCK-B receptors were carried out. Real Time PCR was performed on cDNA and threshold cycle (Ct) value of each sample was obtained and ΔCt was calculated. Chi-square test for comparing two groups and ANOVA test for comparing multiple groups were applied and if p<0.05 then Dunnett-C test was performed. OBSERVATION AND RESULTS: Both CCK-A and CCK-B receptors were expressed irrespective of its origin in all tissues and blood samples studied; be it normal, Cholelithiasis or CA gallbladder and there was no difference among them (p>0.05). CONCLUSION: This preliminary study showed higher expression of CCK-A receptors in patients of cholelithiasis and decreased expression of CCK-A receptors in patients of CA gallbladder as compared to normal gallbladder although it did not rise to statistical significance.

Promoter region hypermethylation and mRNA expression of MGMT and p16 genes in tissue and blood samples of human premalignant oral lesions and oral squamous cell carcinoma.

Promoter methylation and relative gene expression of O(6)-methyguanine-DNA-methyltransferase (MGMT) and p16 genes were examined in tissue and blood samples of patients with premalignant oral lesions (PMOLs) and oral squamous cell carcinoma (OSCC). Methylation-specific PCR and reverse transcriptase PCR were performed in 146 tissue and blood samples from controls and patients with PMOLs and OSCC. In PMOL group, significant promoter methylation of MGMT and p16 genes was observed in 59% (P = 0.0010) and 57% (P = 0.0016) of tissue samples, respectively, and 39% (P = 0.0135) and 33% (P = 0.0074) of blood samples, respectively. Promoter methylation of both genes was more frequent in patients with OSCC, that is, 76% (P = 0.0001) and 82% (P = 0.0001) in tissue and 57% (P = 0.0002) and 70% (P = 0.0001) in blood, respectively. Significant downregulation of MGMT and p16 mRNA expression was observed in both tissue and blood samples from patients with PMOLs and OSCC. Hypermethylation-induced transcriptional silencing of MGMT and p16 genes in both precancer and cancer suggests important role of these changes in progression of premalignant state to malignancy. Results support use of blood as potential surrogate to tissue samples for screening or diagnosing PMOLs and early OSCC.

Differential expression of cholecystokinin A receptor in gallbladder cancer in the young and elderly suggests two subsets of the same disease?

BACKGROUND: Cholecystokinin type A receptor (CCKAR) is known to be overexpressed in variety of human malignancies but information regarding its expression in gallbladder cancer (GBC) is limited. Attempts were now made to investigate expression pattern of CCKAR mRNA and protein in controls and GBC patients and correlate it with various clinicopathological parameters following surgical resection. MATERIALS AND METHODS: Gallbladder tissue samples from 64 subjects (GBC: 39; control: 25) were studied. Expression of CCKAR mRNA was evaluated by reverse transcriptase-polymerase chain reaction and confirmed using real-time polymerase chain reaction. Protein expression was studied by enzyme-linked immunosorbent assay. RESULTS: Significantly higher expression of CCKAR mRNA (P < 0.0001) and protein (P < 0.0001) was observed in GBC tissues. Overexpression was also observed for stage III and in moderately and poorly differentiated tumors. When the clinicopathological parameters were compared, we found age dependent decrease in CCKAR expression. Relatively higher expression of CCKAR was observed in younger patients (age < 45 years) having more aggressive disease when compared with elderly ones (age ≥ 45 years). CONCLUSIONS: Age related differential expression of CCKAR in GBC may suggest two possible variants of the disease in this endemic belt.

TNF-α, IL-6, and IL-8 cytokines and their association with TNF-α-308 G/A polymorphism and postoperative sepsis.

INTRODUCTION: Early prediction of postoperative sepsis remains an enormous clinical challenge. Association of TNF-α-308 G/A polymorphism with sepsis remains controversial. We, therefore, investigated this polymorphism with serum levels of cytokines TNF-α, IL-6, and IL-8 in relation to development of sepsis following major gastrointestinal surgery. METHODS: Two hundred and thirty-nine patients undergoing major gastrointestinal surgery were enrolled. Polymorphism was studied through the analysis of restriction fragments of Nco1-digested DNA with the polymerase chain reaction. All patients were followed for 1 month following surgery for evidence of sepsis. Levels of serum cytokines TNF-α, IL-6, and IL-8 were measured preoperatively and postoperatively by enzyme-linked immunosorbent assay (ELISA). RESULTS: Forty-seven (19.66 %) patients developed postoperative sepsis. Patients with postoperative sepsis were significantly (p = 0.002) more likely to possess AA homozygous genotype with higher capacity to produce cytokines TNF-α (p < 0.0001), IL-6 (p < 0.0001), and IL-8 (p < 0.0001) as compared to other genotypes. When compared with patients carrying at least one G allele, the AA genotype was associated with a significantly higher probability (odds ratio (OR) = 4.17; p = 0.003; 95 % confidence interval (CI) = 1.5-11.48) of developing sepsis. Compared with the GG genotype, AA was associated with a significantly higher probability (OR = 5.18; p = 0.0008; 95 % CI = 1.82-14.76) of sepsis development. CONCLUSION: TNF-α-308 G/A polymorphism is significantly associated with the development of postoperative sepsis and with increased expression of cytokines TNF-α, IL-6, and IL-8.

Tumor necrosis factor-ß Nco1 polymorphism and susceptibility to sepsis following major elective surgery.

BACKGROUND: Post-operative sepsis remains a substantial cause of morbidity and mortality. In injured patients, that a polymorphism of the gene for tumor necrosis factor-ß (TNF-ß) has been related to the development of sepsis. Genetic factors may also have a role in etio-pathogenesis of sepsis following surgery. We investigated the relationship of the polymorphism of the gene for TNF-ß and the serum concentration of TNF-α to the development of sepsis after elective major surgery. METHODS: The study population consisted of 211 patients undergoing major elective surgery. The NcoI polymorphism of TNF-ß was studied in genomic DNA through the analysis of restriction fragments of Nco1-digested DNA with the polymerase chain reaction (PCR). All patients were followed for 1 mo after surgery for any evidence of sepsis. Serum concentrations of TNF-α were measured pre- and post-operatively by enzyme linked immunosorbent assay (ELISA). Genotypes of TNF-ß and the production of TNF-α were related to the occurrence of sepsis. RESULTS: Post-operative sepsis developed in 21.8% (n=46) of the patients. The overall mortality was 4.2% (n=9). The overall allele frequency of the TNF-ß genotype was 0.32 for TNFB1 and 0.68 for TNFB2. Within the TNF-ß genotype, 11.84% (n=25) of the patients were homozygous recessive for TNFB1, 41.23% (n=87) were heterozygous, with TNFB1/TNFB2, and 46.91% (n=99) were homozygous dominant for TNFB2. The incidence of post-operative sepsis was significantly (p=0.01) higher in patients homozygous for the TNFB2 allele. When compared with patients carrying at least one TNFB1 allele (TNFB1 homozygous and heterozygous genotype), the TNFB2 homozygous genotype was associated with an odds ratio (OR) of 2.60 (p=0.005; 95% CI 1.32-5.15) for the development of sepsis. As compared with that for the heterozygous genotype, the OR for the homozygous TNFB2 genotype was 3.00 (p=0.003; 95% CI 1.39-6.44). In patients with post-operative sepsis, serum concentrations of TNF-α were significantly higher (p=0.02) in TNFB2 homozygous individuals than in those of individuals of the other TNF-ß genotypes. CONCLUSION: The development of sepsis was associated with a greater capacity to produce TNF-α after surgery. The Nco1 polymorphism of the TNF-ß gene was associated with the development of post-operative sepsis with an increased serum concentration of TNF-α. In patients without post-operative sepsis, polymorphism of the TNF-ß gene was not related to different levels of TNF-α production. This indicates an association between polymorphism of the TNF-ß gene and post-operative sepsis, suggesting the TNFB2/B2 genotype as a high-risk factor for the development of sepsis after elective surgery.

Role of efflux pumps and intracellular thiols in natural antimony resistant isolates of Leishmania donovani.

BACKGROUND: In view of the recent upsurge in the phenomenon of therapeutic failure, drug resistance in Leishmania, developed under natural field conditions, has become a great concern yet little understood. Accordingly, the study of determinants of antimony resistance is urgently warranted. Efflux transporters have been reported in Leishmania but their role in clinical resistance is still unknown. The present study was designed to elucidate the mechanism of natural antimony resistance in L. donovani field isolates by analyzing the functionality of efflux pump(s) and expression profiles of known genes involved in transport and thiol based redox metabolism. METHODOLOGY/PRINCIPAL FINDINGS: We selected 7 clinical isolates (2 sensitive and 5 resistant) in addition to laboratory sensitive reference and SbIII resistant mutant strains for the present study. Functional characterization using flow cytometry identified efflux pumps that transported substrates of both P-gp and MRPA and were inhibited by the calmodulin antagonist trifluoperazine. For the first time, verapamil sensitive efflux pumps for rhodamine 123 were observed in L. donovani that were differentially active in resistant isolates. RT-PCR confirmed the over-expression of MRPA in isolates with high resistance index only. Resistant isolates also exhibited consistent down regulation of AQP1 and elevated intracellular thiol levels which were accompanied with increased expression of ODC and TR genes. Interestingly, γ-GCS is not implicated in clinical resistance in L. donovani isolates. CONCLUSIONS/SIGNIFICANCE: Here we demonstrate for the first time, the role of P-gp type plasma membrane efflux transporter(s) in antimony resistance in L. donovani field isolates. Further, decreased levels of AQP1 and elevated thiols levels have emerged as biomarkers for clinical resistance.

Micronucleus induction by oxidative metabolites of trichloroethylene in cultured human peripheral blood lymphocytes: a comparative genotoxicity study.

The genotoxic effects of oxidative metabolites of trichloroethylene (TCE), namely chloral hydrate, trichloroacetic acid (TCA), dichloroacetic acid (DCA), and trichloroethanol (TCEOH) were examined in human peripheral blood lymphocytes. In this context, lymphocytes were exposed in vitro to 25, 50, and 100 µg/ml concentrations of these metabolites separately for a period of 48 h and examined for micronucleus (MN) induction through flow cytometer. At 50 µg/ml TCE metabolites, TCA (6.33 ± 0.56 %), DCA (5.06 ± 0.55), and TCEOH (4.70 ± 1.73) induced highly significant (p<0.001) frequency of MN in comparison to control (1.03 ± 0.40) suggestive of their genotoxic potential. However, exposure of 100 µg/ml of all the metabolites consistently declined the frequencies of MN which in some cases was equable to that of observed at 25 µg/ml. Further, cytotoxicity and cell cycle disturbances were also measured to find out the association of these endpoints with the MN induction. DNA content analysis revealed 3-4-fold elevation of S-phase at all the concentrations tested. Particularly, at 100 µg/ml, treatment elevation of S-phase was significantly (p<0.0001) higher as compared to the control. Present findings together with earlier reports indicate that TCE induces genotoxicity through its metabolites. Interaction of these metabolites with DNA, as evident by elevated S-phase, seems to be the major cause of MN induction. However, involvement of spindle disruption cannot be ruled out. This comparative study also suggests that after TCE exposure, the metabolic efficiency of human to generate oxidative metabolites determines the extent of genotoxicity.

Study of intron 22 inversion mutation in north India with review.

The present study assessed the frequency of intron 22 inversion mutation (Inv 22) in north Indian population with a cost analysis of different methods used for Inv 22 detection. We assessed the frequency of intron 22 inversion mutation in a series of 181 cases with hemophilia A and also compared methods used for detection of the mutation including the long-distance PCR, Southern blot analysis, and inverse PCR in terms of cost, infrastructure, and technical input as well as turnaround time. The study group comprised 102 severe cases and 79 moderate cases of hemophilia A from a north Indian population of which 77 cases tested positive for Inv 22. The observed frequency of Inv22 mutation was 42.5%. Inv 22 resulted in a more severe phenotype and lower FVIII bioassay levels as compared to Inv 22 negative cases. Inv 22 positive cases also frequently presented with bleeding episodes at birth and the mean age for commencement of bleeding was lower (19 months) as compared to Inv-negative cases (50 months). The mean frequency of Inv 22 in cases with hemophilia A in a worldwide review is 44.25% of hemophilia A. Inv 22 can be conveniently detected by using the inverse PCR method. This technique is easy to standardize and lowest in cost.

Performance characteristics of bisulfite conversion and SYBR green based quantitative PCR for DNA methylation analysis.

Genomic DNA methylation is one of the most important epigenetic modifications in eukaryotes play vital role in development of severe disease like cancer. Many techniques used for assessment of DNA methylation, bisulfite treatment followed by methylation specific polymerase reaction (MSP) are one of them, which introduce conversion of unmethylated cytosine into uracil. The significant level of bisulfite treated DNA degradation results in the failure of methylation detection. Therefore, this step is to be properly controlled to avoid the degradation of DNA. In the present study, an attempt has been made to access the incubation time of DNA with bisulfate treatment at three time points i.e. 2.5, 4 and 16 hrs to get complete conversion of cytosine to uracil. Currently, the experiments were undertaken using oral cancer tissue, with varying incubation time of bisulfite treatment and 2 representative genes viz MGMT and p16 were selected for the quantitative assessment of methylation by real time PCR. Both genes are frequently methylated at promoter region in carcinogenesis. The short term incubation for 4hrs indicated better real time threshold value for p16 and MGMT gene methylation (Ct 25.55, 27.25) and unmethylation (Ct 18.82, 25.84) in tissue whereas it was 28.16, 37.35 and 21.98, 26.19 in blood sample, respectively as compared to other incubation time which shows less degradation of full length DNA.

Groundwater contaminated with hexavalent chromium [Cr (VI)]: a health survey and clinical examination of community inhabitants (Kanpur, India).

BACKGROUND: We assessed the health effects of hexavalent chromium groundwater contamination (from tanneries and chrome sulfate manufacturing) in Kanpur, India. METHODS: The health status of residents living in areas with high Cr (VI) groundwater contamination (N = 186) were compared to residents with similar social and demographic features living in communities having no elevated Cr (VI) levels (N = 230). Subjects were recruited at health camps in both the areas. Health status was evaluated with health questionnaires, spirometry and blood hematology measures. Cr (VI) was measured in groundwater samples by diphenylcarbazide reagent method. RESULTS: Residents from communities with known Cr (VI) contamination had more self-reports of digestive and dermatological disorders and hematological abnormalities. GI distress was reported in 39.2% vs. 17.2% males (AOR = 3.1) and 39.3% vs. 21% females (AOR = 2.44); skin abnormalities in 24.5% vs. 9.2% males (AOR = 3.48) and 25% vs. 4.9% females (AOR = 6.57). Residents from affected communities had greater RBCs (among 30.7% males and 46.1% females), lower MCVs (among 62.8% males) and less platelets (among 68% males and 72% females) than matched controls. There were no differences in leucocytes count and spirometry parameters. CONCLUSIONS: Living in communities with Cr (VI) groundwater is associated with gastrointestinal and dermatological complaints and abnormal hematological function. Limitations of this study include small sample size and the lack of long term follow-up.

Genetic predisposition for dermal problems in hexavalent chromium exposed population.

We studied the effect of genetic susceptibility on hexavalent chromium induced dermal adversities. The health status of population was examined from the areas of Kanpur (India) having the elevated hexavalent chromium levels in groundwater. Blood samples were collected for DNA isolation to conduct polymorphic determination of genes, namely: NQO1 (C609T), hOGG1 (C1245G), GSTT1, and GSTM1 (deletion). Symptomatic exposed subjects (n = 38) were compared with asymptomatic exposed subjects (n = 108) along with asymptomatic controls (n = 148) from a non contaminated reference community. Exposed symptomatic group consisted of 36.8% subjects who were GSTM1 null genotyped as compared to asymptomatic where only 19.4% subjects were null. The exposed subjects with GSTM1 null genotype were more susceptible to dermal adversities in comparison with wild genotyped subjects (OR = 2.42; 95% CI = 1.071-5.451). Age, smoking, gender or duration of residence were not found to have any confounding effect towards this association. Association with other genes was not statistically significant, nonetheless, possible contribution by these genes cannot be ruled out. In conclusion, variation in the polymorphic status of GSTM1 gene may influence dermal outcomes among residents from Cr(VI) contaminated areas. Further studies are therefore, needed to examine these observations among different population groups.

Detoxification and antioxidant effects of curcumin in rats experimentally exposed to mercury.

Curcumin, a safe nutritional component and a highly promising natural antioxidant with a wide spectrum of biological functions, has been examined in several metal toxicity studies, but its role in protection against mercury toxicity has not been investigated. Therefore, the detoxification and antioxidant effects of curcumin were examined to determine its prophylactic/therapeutic role in rats experimentally exposed to mercury (in the from of mercuric chloride-HgCl(2), 12 micromol kg(-1) b.w. single intraperitoneal injection). Curcumin treatment (80 mg kg(-1) b.w. daily for 3 days, orally) was found to have a protective effect on mercury-induced oxidative stress parameters, namely, lipid peroxidation and glutathione levels and superoxide dismutase, glutathione peroxidase and catalase activities in the liver, kidney and brain. Curcumin treatment was also effective for reversing mercury-induced serum biochemical changes, which are the markers of liver and kidney injury. Mercury concentration in the tissues was also decreased by the pre/post-treatment with curcumin. However, histopathological alterations in the liver and kidney were not reversed by curcumin treatment. Mercury exposure resulted in the induction of metallothionein (MT) mRNA expressions in the liver and kidney. Metallothionein mRNA expression levels were found to decrease after the pre-treatment with curcumin, whereas post-treatment with curcumin further increased MT mRNA expression levels. Our findings suggest that curcumin pretreatment has a protective effect and that curcumin can be used as a therapeutic agent in mercury intoxication. The study indicates that curcumin, an effective antioxidant, may have a protective effect through its routine dietary intake against mercury exposure.