Ochratoxin A treated rat derived urinary exosomes enhanced cell growth and extracellular matrix production in normal kidney cells through modulation of TGF-ß1/smad2/3 signaling pathway.

AIMS: Kidney is the main target organ for ochratoxin A (OTA) toxicity; however, the mechanism(s) involved in OTA-induced nephrotoxicity is not fully understood. Recently, exosomes, nano-sized vesicles have been found to play an important role in promotion and progression of disease as well as environmental toxicant-induced patho-physiology of toxicity. Hence, we aimed to investigate the role of exosomes in OTA-mediated nephrotoxicity. MAIN METHODS: Male Wistar rats were divided in to two groups. Rats of one group were treated with OTA (210 µg/kg b. wt) and another with vehicle control through oral gavage (5 days/week) for 270 days. At the end of experiment, exosomes concentrations from rat's urine were measured. To examine the OTA-induced nephrotoxicity, histopathology was performed using H & E, Masson's trichome and PAS staining. For mechanistic study, normal rat kidney (NRK52E) cells were exposed with either vehicles treated rat's urinary exosomes (NEx) or OTA treated rat's urinary exosomes (OEx) and effects on cell proliferation, cell growth, extracellular matrix production and TGF-ß1/smad2/3 pathway was analyzed. KEY FINDINGS: OTA treatment to Wistar rats caused histopathological changes such as tubular degeneration, glomeruli shrinkage and hypercellularity in kidney tissue. Interestingly, OTA treated rat's urine has more exosomes secretion. Moreover, treatment of NRK52E cells with OEx caused increased cell proliferation, cell growth, enhanced the expression of extracellular matrix proteins and activation of TGF-ß1/smad2/3 pathway. SIGNIFICANCE: Our investigations exogenous exposure of OTA derived urinary exosomes caused TGF-ß1/smad2/3 pathway-mediated activation of pro-fibrotic changes in kidney will helpful for deeper understanding the OTA-induced nephrotoxicity.

Transcriptomic and proteomic insights into patulin mycotoxin-induced cancer-like phenotypes in normal intestinal epithelial cells.

Patulin (PAT) is a natural contaminant of fruits (primarily apples) and their products. Significantly, high levels of contamination have been found in fruit juices all over the world. Several in vitro studies have demonstrated PAT's ability to alter intestinal structure and function. However, in real life, the probability of low dose long-term exposure to PAT to humans is significantly higher through contaminated food items. Thus, in the present study, we have exposed normal intestinal cells to non-toxic levels of PAT for 16 weeks and observed that PAT had the ability to cause cancer-like properties in normal intestinal epithelial cells after chronic exposure. Here, our results showed that chronic exposure to low doses of PAT caused enhanced proliferation, migration and invasion ability, and the capability to grow in soft agar (anchorage independence). Moreover, an in vivo study showed the appearance of colonic aberrant crypt foci (ACFs) in PAT-exposed Wistar rats, which are well, establish markers for early colon cancer. Furthermore, as these neoplastic changes are consequences of alterations at the molecular level, here, we combined next-generation RNA sequencing with liquid chromatography mass spectrometry-based proteomic analysis to investigate the possible underlying mechanisms involved in PAT-induced neoplastic changes.

TGF-ß/Smad signaling pathway plays a crucial role in patulin-induced pro-fibrotic changes in rat kidney via modulation of slug and snail expression.

Patulin (PAT) is a mycotoxin that contaminates a variety of food and foodstuffs. Earlier in vitro and in vivo findings have indicated that kidney is one of the target organs for PAT-induced toxicity. However, no study has evaluated the chronic effects of PAT exposure at environmentally relevant doses or elucidated the detailed mechanism(s) involved. Here, using in vitro and in vivo experimental approaches, we delineated the mechanism/s involved in pro-fibrotic changes in the kidney after low-dose chronic exposure to PAT. We found that non-toxic concentrations (50 nM and 100 nM) of PAT to normal rat kidney cells (NRK52E) caused a higher generation of reactive oxygen species (ROS) (mainly hydroxyl (•OH), peroxynitrite (ONOO-), and hypochlorite radical (ClO-). PAT exposure caused the activation of mitogen-activated protein kinases (MAPKs) and its downstream c-Jun/Fos signaling pathways. Moreover, our chromatin immunoprecipitation (ChIP) analysis suggested that c-Jun/Fos binds to the promoter region of Transforming growth factor beta (TGF-ß1) and possibly induces its expression. Results showed that PAT-induced TGF-ß1 further activates the TGF-ß1/smad signaling pathways. Higher activation of slug and snail transcription factors further modulates the regulation of pro-fibrotic molecules. Similarly, in vivo results showed that PAT exposure to rats through gavage at 25 and 100 µg/kg b. wt had higher levels of kidney injury/toxicity markers namely vascular endothelial growth factor (VEGF), kidney Injury Molecule-1 (Kim-1), tissue inhibitor of metalloproteinase-1 (Timp-1), and clusterin (CLU). Additionally, histopathological analysis indicated significant alterations in renal tubules and glomeruli along with collagen deposition in PAT-treated rat kidneys. Overall, our data provide evidence of the involvement of ROS mediated MAPKs and TGF-ß1/smad pathways in PAT-induced pro-fibrotic changes in the kidney via modulation of slug and snail expression.

Study of the metabolic alterations in patulin-induced neoplastic transformation in normal intestinal cells.

Several surveillance studies have reported significantly high level of patulin (PAT), mycotoxin in fruit juices suggesting the possible exposure to human. In vitro studies have showed that PAT can alter the permeability, ion transport and modulates tight junction of intestine. In real scenario, human can be exposed with low levels of PAT for longer duration through different fruits and their products. Hence, keeping this possibility in view, we conducted a study where normal intestinal cells were exposed with non-toxic levels of PAT for longer duration and found that PAT exposure causes cancer-like properties in normal intestinal cells. It is a well-known fact that cancer cells rewired their metabolism for cell growth and survival and metabolites closely depict the phenotypic properties of cells. Here, metabolomic study was performed in the PAT transformed and passage matched non-transformed cells using 1H HRMAS NMR. We have identified 12 significantly up-regulated metabolites, which, interestingly, were majorly amino acids, suggesting that PAT-induced pre-cancerous cells are involved in acquirement of nutrients for high protein turn-over. Furthermore, pathway analysis of metabolomics data indicated that aminoacyl tRNA biosynthesis, D-glutamate metabolism, glyoxylate and dicarboxylate metabolism and nitrogen metabolism were majorly hampered in PAT-induced pre-cancerous properties in normal intestinal cells.

Occurrence of Alternariol and Alternariolmonomethyl ether in edible oils: Their thermal stability and intake assessment in state of Uttar Pradesh, India.

Alternariol (AOH) and Alternariol monomethyl ether (AME) mycotoxins are found to be present naturally in various food commodities, such as barley, oats, pepper, rye, sorghum, sunflower seeds, tomatoes, and wheat. A few epidemiological studies have correlated the consumption of Alternaria-contaminated cereal grains with higher occurrence of esophageal cancer in Chinese populations. In addition, several studies have reported the toxicological properties of Alternaria mycotoxins. However, surveillance data on AOH and AME occurrence are still limited. Therefore, the goal of this study was to determine the presence of AOH and AME in various commonly consumed, edible oils using HPLC-FLD method. Thirty four percent of samples were found positive for AOH and 35% for AME. Moreover, AOH retained 80% stability, while AME retained 84% stability, after deep frying for 25 min, which is an important factor with respect to Indian cooking style. To the best of our knowledge, this is the first report on the presence of Alternaria mycotoxins in edible oils and their probable dietary intake in Indian population. This surveillance study may help in formulating guidelines for Alternaria mycotoxin levels in India, which are not yet implemented by Food Safety and Standards Authority of India. PRACTICAL APPLICATIONS: At present, no safety guidelines exist for Alternaria mycotoxins in any part of the world. This study will help the regulatory bodies to set permissible levels of Alternaria mycotoxins to safeguard the health of consumers. This study shows that Alternaria mycotoxins are heat stable even after deep frying for 25 min. The data will also help to issue guidelines against exposure of these mycotoxins, keeping in the mind the heat stability factor.

Safety evaluation of Ochratoxin A and Citrinin after 28 days repeated dose oral exposure to Wistar rats.

Mycotoxins, ochratoxin A (OTA), and citrinin (CTN) are toxic metabolites of filamentous fungi. The most common fungal species that produce OTA and CTN belong to genera Aspergillus, Penicillium, Fusarium, and Monascus, and these fungal species are found to be contaminant a wide range of grains, food, and food product. The aim of our study was to evaluate the sub-acute repeated dose oral toxicity of OTA and CTN in experimental rodents by following OECD test guidelines for testing chemicals no. 407 with minor modifications. Twenty-five rats of each sex were divided equally into five groups; vehicle control, OTA 25 µg/kg b. wt., OTA 100 µg/kg b. wt., CTN 25 µg/kg b.wt. and CTN 100 µg/kg b. wt. The results of this study showed no abnormal clinical signs during 28 days of the experimental period. We did not found any significant changes in body weight gain, food consumption pattern, organ weight, hematology except few parameters, and biochemical values in any of the treatment and control groups. However, histopathological observations revealed severe nephrotoxicity and mild follicular depletion in the spleen of 100 µg/kg b. wt. treated groups of both OTA and CTN mycotoxins. The findings of our study are of its first kind that reports the systemic toxicity of OTA and CTN oral exposure to laboratory rodents.