Deoxynivalenol induced mouse skin tumor initiation: Elucidation of molecular mechanisms in human HaCaT keratinocytes.

Among food contaminants, mycotoxins are toxic to both human and animal health. Our prior studies suggest that Deoxynivalenol (DON), a mycotoxin, behaves as a tumor promoter by inducing edema, hyperplasia, ODC activity and activation of MAPK's in mouse skin. In this study, topical application of DON, 336 and 672 nmol significantly enhanced ROS levels, DNA damage and apoptosis with concomitant downregulation of Ki-67, cyclin D, cyclin E, cyclin A and cyclin-dependent kinases (CDK4 and CDK2) thereby resulting in tumor initiation in mouse skin. Further, the elucidation of molecular mechanisms of tumor initiation by DON (0.42-3.37 nmol/ml) in HaCaT keratinocytes, revealed (i) enhanced ROS generation with cell cycle phase arrest in G0/G1 phase, (ii) increase in levels of 8-OxoG (6-24 hr) and γH2AX protein, (iii) significant enhancement in oxidative stress marker enzymes LPO, GSH, GR with concomitant decrease in antioxidant enzymes catalase, GPx, GST, SOD and mitochondrial membrane potential after DON (1.68 nmol) treatment, (iv) suppression of Nrf2 translocation to nucleus, enhanced phosphorylation with subsequent activation ERK1/2, p38 and JNK MAPK's following DON (1.68 nmol) treatment, (v) overexpression of c-jun, c-fos proteins, upregulation of Bax along with downregulation of Bcl-2 proteins, (vi) increase in cytochrome-c, caspase-9, caspase-3 and poly ADP ribose polymerase levels leads to apoptosis. Pretreatment of superoxide dismutase, mannitol and ethanol to HaCaT cells resulted in significant reduction in ROS levels and apoptosis indicating the role of superoxide and hydroxyl radicals in DON induced apoptosis as an early event and skin tumor initiation as a late event.

Role of oxidative stress in Deoxynivalenol induced toxicity.

Deoxynivalenol (DON) is a Fusarium toxin that causes a variety of toxic effects with symptoms such as diarrhoea and low weight gain. To date, no review has addressed the toxicity of DON in relation to oxidative stress. The focus of this article is primarily intended to summarize the information associated with oxidative stress as a plausible mechanism for DON-induced toxicity. The present review shows that over the past two decades, several investigators have documented the generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in oxidative stress as a result of DON treatment and have correlated them with various types of toxicity. The evidence for induction of an oxidative stress response resulting from DON exposure has been more focused on in vitro models and is relatively lacking in in vivo studies. Hence, more emphasis should be laid on in vivo investigations with doses that are commonly encountered in food products. Since DON is commonly found in food and feed, the cellular effects of this toxin in relation to oxidative stress, as well as effective measures to combat its toxicity, are important aspects to be considered for future studies.

Deoxynivalenol induced mouse skin cell proliferation and inflammation via MAPK pathway.

Several toxicological manifestations of deoxynivalenol (DON), a mycotoxin, are well documented; however, dermal toxicity is not yet explored. The effect of topical application of DON to mice was studied using markers of skin proliferation, inflammation and tumor promotion. Single topical application of DON (84-672nmol/mouse) significantly enhanced dermal hyperplasia and skin edema. DON (336 and 672nmol) caused significant enhancement in [(3)H]-thymidine uptake in DNA along with increased myeloperoxidase and ornithine decarboxylase activities, suggesting tissue inflammation and cell proliferation. Furthermore, DON (168nmol) caused enhanced expression of RAS, and phosphorylation of PI3K/Akt, ERK, JNK and p38 MAPKs. DON exposure also showed activation of transcription factors, c-fos, c-jun and NF-κB along with phosphorylation of IkBα. Enhanced phosphorylation of NF-κB by DON caused over expression of target proteins, COX-2, cyclin D1 and iNOS in skin. Though a single topical application of DMBA followed by twice weekly application of DON (84 and 168nmol) showed no tumorigenesis after 24weeks, however, histopathological studies suggested hyperplasia of the epidermis and hypertrophy of hair follicles. Interestingly, intestine was also found to be affected as enlarged Peyer's patches were observed, suggesting inflammatory effects which were supported by elevation of inflammatory cytokines after 24weeks of topical application of DON. These results suggest that DON induced cell proliferation in mouse skin is through the activation of MAPK signaling pathway involving transcription factors NFκB and AP-1, further leading to transcriptional activation of downstream target proteins c-fos, c-jun, cyclin D1, iNOS and COX-2 which might be responsible for its inflammatory potential.

Prolonged elevated postprandial sugar augments severity in kidney disease: a north Indian hospital-based study.

AIM: Diabetes plays a major role in progression of renal failure. The risk-factor profile changes during the progression of chronic kidney disease (CKD) from mild/moderate to end-stage renal disease. The relationship between glycemic indices, blood pressure, body mass index (BMI) and age at diagnosis in Indians has been less investigated. We assessed association of these risk factors with CKD stages in Indian population. METHODS: This study was carried out on patients (n = 162) who were diagnosed with CKD and normal control group (n = 155). For BMI, National Institutes for Health criteria were used to categorize the patients. RESULT: The mean age of CKD patients were significantly increased with the advancement of stage. BMI, systolic blood pressure (SBP), postprandial sugar level (PP), urea and creatinine were also significantly higher with elevated stages, whereas no differences were observed in diastolic blood pressure (DBP) and fasting blood sugar (FBS). The logistic regression study gave a significant result (p = 0.000) when we compared the group of CKD patients with established/prolonged postprandial blood sugar. It was independently associated with mild CKD [odds ratio (OR) = 5.213, 95% confidence interval (CI) = 2.06-13.21, p = 0.000], moderate CKD (OR = 7.724, 95% CI = 4.05-14.74, p = 0.000) and severe CKD (OR = 7.610, 95% CI = 4.03-14.36, p = 0.000). CONCLUSION: SBP and PP were the best predictors of prevalent nephropathy in this population, while DBP and FBS were found to be less effective. This may have implication for kidney disease risk stratification and protection.

Influence of temperature and pH on the degradation of deoxynivalenol (DON) in aqueous medium: comparative cytotoxicity of DON and degraded product.

Deoxynivalenol (DON), a toxic fungal metabolite, is stable under different processing conditions; however, its stability in aqueous medium at different temperatures and low pH (1-2) (present in the gastrointestinal tract) has not been investigated. In the present study, DON standard was used to study the influence of temperature and pH on DON stability in aqueous medium, the characterisation of the degraded product, and the comparative toxicity profile of the degraded and the parent compound. The results suggest that standard DON was unstable at 125-250°C showing 16-100% degradation whereas DON at pH 1-3 had 30-66% degradation, with a concomitant increase in the formation of a degraded product. Further ESI-MS characterisation of the dominant precursor ion of the HPLC eluate of the DON-degraded product was found to be m/z 279, resembling the known metabolite DOM-1. The degraded product of DON was reconfirmed as DOM-1 by comparison with standard DOM-1 and both gave a similar λmax at 208 nm. Comparative studies of both standard DOM-1 and the degraded product of DON showed no cytotoxicity up to 6400 ng ml(-1) while significant cytotoxicity was observed for DON (400 ng ml(-1)). The results suggest that a highly acidic environment (pH 1-2) could be responsible for the de-epoxydation of DON leading to the formation of DOM-1.

Identification of Th1-responsive leishmanial excretory-secretory antigens (LESAs).

The objective of this study was to evaluate the immunomodulatory role of leishmanial excretory-secretory antigens (LESAs) released by in vitro cultured protozoan parasite Leishmania donovani promastigotes. A total of seventeen excretory-secretory proteins of relative molecular weights 11, 13, 16, 18, 21, 23, 26, 29, 33, 35, 42, 51, 54, 58, 64, 70 and 80 kDa were identified. The proteins were divided into five fractions (F1-F5) along with the whole LESAs, these fractions were evaluated for their potential antigenicity to induce macrophage effector functions, lymphoproliferation and cytokines production capabilities. Two fractions, F1 (11, 13 and 16 kDa) and F3 (26, 29 and 33 kDa), were found to be highly immunogenic as they significantly induced NADPH oxidase and SOD activities as well as NOx, TNF-α, IFN-γ and IL-12 production in stimulated RAW 264.7 macrophages. Further, these antigens also induced significant proliferation of human peripheral blood mononuclear cells along with increased production of IFN-γ and IL-12. The results strongly suggest the potential role of LESAs in the modulation of macrophage effector functions and Th1 immune response that gives a hope to develop potent vaccine for visceral leishmaniasis.

An improved method of bisulfite treatment and purification to study precise DNA methylation from as little as 10 pg DNA.

Methylation of vertebrate DNA is one of the most important epigenetic alterations which have become a center of scientific attraction, especially because of its important role in the regulation of transcription, genomic imprinting, developmental process, and pathogenesis of various diseases. Currently, there are wide ranges of methods available to produce quantitative and qualitative information on genomic DNA methylation. The vast majority of these methods rely on the optimization of the efficient bisulfite treatment. However, all the available methods for bisulfite treatment suffer from major disadvantages, such as large amount of starting material, poor conversion efficiency as well as low recovery and integrity of DNA after bisulfite treatment. Here, we developed a simple, rapid, and convenient column-based bisulfite treatment method by improving the several critical steps, which leads to consistent C-to-U conversion rate 99-100 %, >75 % recovery of DNA after bisulfite treatment. In addition, it is commercially viable and requires very less amount (~10 pg) of DNA.

Identification of TLR inducing Th1-responsive Leishmania donovani amastigote-specific antigens.

Leishmania is known to elicit Th2 response that causes leishmaniasis progression; on the other hand, Th1 cytokines restricts amastigote growth and disease progression. In this study, we report the potential of two leishmanial antigens (65 and 98 kDa, in combination) which enhance strong macrophage effector functions, viz., production of respiratory burst enzymes, nitric oxide, and Th1 cytokines. The identification of antigens were done by resolving the crude soluble antigens on SDS-PAGE and eluted by reverse staining method. Further, RAW264.7 macrophages were challenged with eluted antigens, and the innate immune response was observed by detecting respiratory burst enzymes, nitric oxide (NOx), TNF-α, IFN-γ, IL-12, toll-like receptors (TLRs) gene expression, and TLR-signaling proteins. These antigens increased the production of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, superoxide dismutase, NOx, TNF-α, IFN-γ, IL-12, TLR2, and p38 mitogen-activated protein kinase. These antigens also induced human peripheral blood mononuclear cells proliferation and Th1 cytokine production. This study concludes that these antigens induce innate immune response as well as have prophylactic efficacy.

NADH-oxidase, NADPH-oxidase and myeloperoxidase activity of visceral leishmaniasis patients.

It is believed that the enhanced capability of activated macrophages to resist infection is related to the remarkable increase in the production of oxygen metabolites in response to phagocytosis. Both the production of H2O2 and the oxidation of NAD(P)H are directly dependent upon NAD(P)H-oxidase. It has been established that the respiratory burst is due to activation of NAD(P)H-oxidase localised in the plasmalemma. Myeloperoxidase is believed to be involved in augmenting the cytotoxic activity of H2O2. Low NADH-oxidase, NADPH-oxidase and myeloperoxidase activity were observed in monocytes of patients with active visceral leishmaniasis as compared with healthy controls. These results suggest that low NADH-oxidase, NADPH-oxidase and myeloperoxidase activities may account for persistence of Leishmania parasites in visceral leishmaniasis.