Rifampicin-induced ER stress and excessive cytoplasmic vacuolization instigate hepatotoxicity via alternate programmed cell death paraptosis in vitro and in vivo.

AIMS: Rifampicin-induced hepatotoxicity is a primary cause of drug-induced liver injury (DILI), posing a significant challenge to its continued clinical application. Moreover, the mechanism underlying rifampicin-induced hepatotoxicity remains unclear. MAIN METHODS: Human hepatocyte line-17 (HHL-17) cells were treated with an increasing dose of rifampicin for 24 h, and male Wistar rats were given rifampicin [150 mg/kg body weight (bw)] orally for 28 days. Viability assay, protein expression, and cell death assays were analyzed in vitro. Moreover, liver serum markers, body/organ weight, H&E staining, protein expression, etc., were assayed in vivo. KEY FINDINGS: Rifampicin induced a dose-dependent hepatotoxicity in HHL-17 cells (IC50; 600 µM), and increased the serum levels of liver injury markers, e.g., alanine transaminase (ALT) and aspartate transaminase (AST) in rats. Rifampicin-induced cell death was non-apoptotic and non-necroptotic both in vitro and in vivo. Further, excessive cellular vacuolization and reduced expression of Alix protein confirmed the induction of paraptosis both in vitro and in vivo. In addition, a significant increase in the endoplasmic reticulum (ER) stress markers (e.g., BiP, CHOP, and total polyubiquitinated proteins) was detected, demonstrating the induction of ER stress and altered protein homeostasis. Interestingly, rifampicin-induced hepatotoxicity was associated with the inhibition of autophagy and enhanced reactive oxygen species (ROS) generation in HHL-17 cells. Furthermore, inhibition of protein synthesis by cycloheximide (CHX) suppressed paraptosis by alleviating rifampicin-induced ER stress and ROS generation. SIGNIFICANCE: Rifampicin-induced hepatotoxicity involves ER stress-driven paraptosis as a novel mechanism of its toxicity that may be targeted to protect liver cells from rifampicin toxicity.

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.

Prenatal arsenic exposure induces immunometabolic alteration and renal injury in rats.

Arsenic (As) exposure is progressively associated with chronic kidney disease (CKD), a leading public health concern present worldwide. The adverse effect of As exposure on the kidneys of people living in As endemic areas have not been extensively studied. Furthermore, the impact of only prenatal exposure to As on the progression of CKD also has not been fully characterized. In the present study, we examined the effect of prenatal exposure to low doses of As 0.04 and 0.4 mg/kg body weight (0.04 and 0.4 ppm, respectively) on the progression of CKD in male offspring using a Wistar rat model. Interestingly, only prenatal As exposure was sufficient to elevate the expression of profibrotic (TGF-ß1) and proinflammatory (IL-1α, MIP-2α, RANTES, and TNF-α) cytokines at 2-day, 12- and 38-week time points in the exposed progeny. Further, alteration in adipogenic factors (ghrelin, leptin, and glucagon) was also observed in 12- and 38-week old male offspring prenatally exposed to As. An altered level of these factors coincides with impaired glucose metabolism and homeostasis accompanied by progressive kidney damage. We observed a significant increase in the deposition of extracellular matrix components and glomerular and tubular damage in the kidneys of 38-week-old male offspring prenatally exposed to As. Furthermore, the overexpression of TGF-ß1 in kidneys corresponds with hypermethylation of the TGF-ß1 gene-body, indicating a possible involvement of prenatal As exposure-driven epigenetic modulations of TGF-ß1 expression. Our study provides evidence that prenatal As exposure to males can adversely affect the immunometabolism of offspring which can promote kidney damage later in life.

Safety studies of Nexrutine, bark extract of Phellodendron amurense through repeated oral exposure to rats for 28 days.

Nexrutine (NX), a marketable herbal extract from a traditional Chinese herbal plant, Phellodendron amurense, is majorly used for the resolution of inflammation, gastroenteritis, and some tissue-specific cancer. Strategies for the identification of the safety of anticancer solutions of plant origin are an important area of study. The present investigation assesses the single and repeated dose (28 days) toxicity of NX following OECD guidelines 425 and 407, respectively. Briefly, to identify acute toxic properties of NX, a dose of 2000 mg/kg b. wt was administered once orally. Simultaneously, repeated dose toxicity was evaluated through daily administration of the three different doses (250, 500, 750 mg/kg b. wt) of NX for 28days. The single administration of NX showed no signs of toxicity and morbidity, suggesting LD50 of NX more than 2000 mg/kg b. wt. Furthermore, repeated dose exposure of NX for 28 days did not show any sign of toxicity. Hematology, serum biochemistry, and histopathological analysis also did not show any significant abnormalities. However, a marginal decrease in triglyceride, cholesterol, and glucose levels along with mild tubular degeneration in the kidney was also noticed in the high dose NX treatment group. Overall, the findings of the study suggest that NX is safe for use up to 500 mg/kg b.wt.

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.