Melatonin alleviates glyphosate-induced testosterone synthesis inhibition via targeting mitochondrial function in roosters.

Glyphosate (GLY) is a widely used herbicide that has been revealed to inhibit testosterone synthesis in humans and animals. Melatonin (MET) is an endogenous hormone that has been demonstrated to promote mammalian testosterone synthesis via protecting mitochondrial function. However, it remains unclear whether MET targets mitochondria to alleviate GLY-inhibited testosterone synthesis in avian. In this study, an avian model using 7-day-old rooster upon chronic exposure to GLY with the treatment of MET was designed to clarify this issue. Data first showed that GLY-induced testicular Leydig cell damage, structural damage of the seminiferous tubule, and sperm quality decrease were mitigated by MET. Transcriptomic analyses of the testicular tissues revealed the potentially critical role of mitophagy and steroid hormone biosynthesis in the process of MET counteracting GLY-induced testicular damage. Also, validation data demonstrated that the inhibition of testosterone synthesis due to GLY-induced mitochondrial dynamic imbalance and concomitant Parkin-dependent mitophagy activation is alleviated by MET. Moreover, GLY-induced oxidative stress in serum and testicular tissue were significantly reversed by MET. In summary, these findings demonstrate that MET effectively ameliorates GLY-inhibited testosterone synthesis by inhibiting mitophagy activation, which provides a promising remedy for the application of MET as a potential therapeutic agent to antagonize reproductive toxicity induced by GLY and similar contaminants.

Glyphosate drives autophagy-dependent ferroptosis to inhibit testosterone synthesis in mouse Leydig cells.

Glyphosate (GLY), a widely used herbicide, can adversely affect the male reproductive health by inhibiting testosterone synthesis. Ferroptosis is a form of iron-dependent oxidative cell death that contributes to inhibition of testosterone secretion. However, it still remains unclear whether ferroptosis is involved in GLY-inhibited testosterone synthesis. Hereby, an in vitro model of 1 mM GLY-exposed testicular Leydig (TM3) cells was established to elucidate this issue. Data firstly showed that GLY causes cytotoxicity and testosterone synthesis inhibition via ferroptosis, while accumulation of lipid peroxides due to intracellular ferrous ion (Fe2+) overload and glutathione depletion is confirmed as a determinant of ferroptosis. Blockage of ferroptosis via chelation of Fe2+ or inhibition of lipid peroxidation can markedly mitigate GLY-induced testosterone synthesis inhibition. Also, autophagy activation is revealed in GLY-treated TM3 cells and nuclear receptor coactivator 4 (NCOA4)-mediated ferritinophagy is involved in ferroptosis through the release of excess Fe2+. GLY-induced cytotoxicity and testosterone synthesis inhibition are significantly alleviated by NCOA4 knockdown, demonstrating the crucial role of NCOA4-mediated ferritinophagy in GLY-inhibited testosterone synthesis. In summary, this study provides solid evidence that NCOA4-mediated ferritinophagy promotes ferroptosis to inhibit testosterone synthesis, highlighting that targeting NCOA4 may be a potential therapeutic approach in GLY-induced male reproductive toxicity.

Persistent activation of Nrf2 in a p62-dependent non-canonical manner aggravates lead-induced kidney injury by promoting apoptosis and inhibiting autophagy.

INTRODUCTION: Lead (Pb) is an environmental toxicant that poses severe health risks to humans and animals, especially renal disorders. Pb-induced nephrotoxicity has been attributed to oxidative stress, in which apoptosis and autophagy are core events. OBJECTIVES: Nuclear factor erythroid 2-related factor 2 (Nrf2) acts as a major contributor to counteract oxidative damage, while hyperactivation or depletion of Nrf2 pathway can cause the redox imbalance to induce tissue injury. This study was performed to clarify the function and mechanism of Nrf2 in Pb-triggered kidney injury. METHODS AND RESULTS: First, data showed that Pb exposure activates Nrf2 pathway in primary rat proximal tubular cells. Next, Pb-induced Nrf2 activation was effectively regulated by pharmacological modulation or siRNA-mediated knockdown in vitro and in vivo assays. Notably, Pb-triggered cytotoxicity, renal injury and concomitant apoptosis were improved by Nrf2 downregulation, confirming that Pb-induced persistent Nrf2 activation contributes to nephrotoxicity. Additionally, Pb-triggered autophagy blockage was relieved by Nrf2 downregulation. Mechanistically, we found that Pb-induced persistent Nrf2 activation is attributed to reduced Nrf2 ubiquitination and nuclear-cytoplasmic loss of Keap1 in a p62-dependent manner. CONCLUSIONS: In conclusion, these findings highlight the dark side of persistent Nrf2 activation and potential crosstalk among Pb-induced persistent Nrf2 activation, apoptosis and autophagy blockage in Pb-triggered nephrotoxicity.

Glyphosate-induced autophagy inhibition results in hepatic steatosis via mediating epigenetic reprogramming of PPARα in roosters.

Glyphosate (Gly) is the most widely used herbicide with well-defined hepatotoxic effects, but the underlying mechanisms of Gly-induced hepatic steatosis remain largely unknown. In this study, a rooster model combined with primary chicken embryo hepatocytes was established to dissect the progresses and mechanisms of Gly-induced hepatic steatosis. Data showed that Gly exposure caused liver injury with disrupted lipid metabolism in roosters, manifested by significant serum lipid profile disorder and hepatic lipid accumulation. Transcriptomic analysis revealed that PPARα and autophagy-related pathways played important roles in Gly-induced hepatic lipid metabolism disorders. Further experimental results suggested that autophagy inhibition was involved in Gly-induced hepatic lipid accumulation, which was confirmed by the effect of classic autophagy inducer rapamycin (Rapa). Moreover, data substantiated that Gly-mediated autophagy inhibition caused nuclear increase of HDAC3, which altered epigenetic modification of PPARα, leading to fatty acid oxidation (FAO) inhibition and subsequently lipid accumulation in the hepatocytes. In summary, this study provides novel evidence that Gly-induced autophagy inhibition evokes the inactivation of PPARα-mediated FAO and concomitant hepatic steatosis in roosters by mediating epigenetic reprogramming of PPARα.

Trehalose prevents glyphosate-induced hepatic steatosis in roosters by activating the Nrf2 pathway and inhibiting NLRP3 inflammasome activation.

Glyphosate (Gly) is a globally spread herbicide that can cause toxic injuries to hepatocytes. Dietary trehalose (Tre) exerts cytoprotective effect in numerous liver diseases through anti-oxidant and anti-inflammatory properties. However, it is yet to be investigated whether Tre affords protection against Gly-induced hepatotoxicity. To evaluate the negative effect of Gly in liver and assess the possible protective role of Tre, sixty Hy-line Brown roosters were allocated into three groups: the first group presented the control with a normal diet, the second group fed normal feed containing 200mg/kg Gly, and the third group fed normal feed containing 200 mg/kg Gly and 5 g/kg Tre. Plasma and liver tissues were collected and analyzed after 120 days. Firstly, Gly-elevated serum levels of hepatic injury markers and liver histopathological damages were evidently alleviated by Tre administration. Also, Tre normalized Gly-altered serum and hepatic lipid profiles and Oil Red O-stained lipid levels, suggesting the improvement of hepatic steatosis. The severely accumulated malondialdehyde levels and impaired antioxidant status in Gly-exposed roosters were markedly improved by administration with Tre. Simultaneously, Gly-inhibited nuclear factor erythroid 2-related factor 2 (Nrf2) level and consequent reduced levels of Nrf2-downstream targets in liver were markedly normalized by Tre treatment. Additionally, Tre treatment evidently mitigated Gly-induced inflammasome response via inhibiting NLRP3 inflammasome activation. Overall, these observations provide novel insights that the protective action of Tre against Gly-induced hepatic steatosis is attributed to activation of Nrf2 pathway and inhibition of NLRP3 inflammasome activation.

Inhibited transcription factor EB function induces reactive oxygen species overproduction to promote pyroptosis in cadmium-exposed renal tubular epithelial cells.

Pyroptosis is a pro-inflammatory type of cell death involved in the pathogenesis of multiple kidney diseases, while transcription factor EB (TFEB) is shown to be important for rescuing renal function. Cadmium (Cd) is an omnipresent toxic heavy metal with definite nephrotoxicity, but there is lacking of evidence regarding an interplay between TFEB activity and pyroptosis during Cd exposure. In this study, Cd-exposed NRK-52E cells were used to clarify this issue as an in vitro model of acute kidney injury. First, our results showed that Cd exposure evidently elevated the protein levels involved in pyroptosis, increased lactate dehydrogenase (LDH) release, and disrupted the cell membrane integrity, suggesting the occurrence of pyroptosis in NRK-52E cells. It is also shown that Cd induced a burst of reactive oxygen species (ROS) to mediate pyroptosis. Simultaneously, downregulated TFEB expression with its inhibited nuclear translocation was revealed in Cd-exposed NRK-52E cells. Further investigations have demonstrated that TFEB knockdown promoted Cd-induced ROS production to exacerbate the pyroptosis, while TFEB overexpression inhibited Cd-induced ROS production to alleviate the pyroptosis in NRK-52E cells. In summary, these findings demonstrate that Cd-inhibited TFEB function results in ROS overproduction to promote pyroptosis in NRK-52E cells, which provide new insight into the therapeutic targets for Cd-induced kidney diseases.

Glyphosate-induced mitochondrial reactive oxygen species overproduction activates parkin-dependent mitophagy to inhibit testosterone synthesis in mouse leydig cells.

Glyphosate (GLY), one of the most extensively used herbicides in the world, has been shown to inhibit testosterone synthesis in male animals. Mitochondria are crucial organelles for testosterone synthesis and its dysfunction has been demonstrated to induce the inhibition of testosterone biosynthesis. However, whether low-dose GLY exposure targets mitochondria to inhibit testosterone synthesis and its underlying mechanism remains unclear. Here, an in vitro model of 10 µM GLY-exposed mouse Leydig (TM3) cells was established to elucidate this issue. Data firstly showed that mitochondrial malfunction, mainly manifested by ultrastructure damage, disturbance of mitochondrial dynamics and mitochondrial reactive oxygen species (mtROS) overproduction, was responsible for GLY-decreased protein levels of steroidogenic enzymes, which leads to the inhibition of testosterone synthesis. Enhancement of autophagic flux and activation of mitophagy were shown in GLY-treated TM3 cells, and further studies have revealed that GLY-activated mitophagy is parkin-dependent. Notably, GLY-inhibited testosterone production was significantly improved by parkin knockdown. Finally, data showed that treatment with mitochondria-targeted antioxidant Mito-TEMPO (M-T) markedly reversed GLY-induced mitochondrial network fragmentation, activation of parkin-dependent mitophagy and consultant testosterone reduction. Overall, these findings demonstrate that GLY induces mtROS overproduction to activate parkin-dependent mitophagy, which contributes to the inhibition of testosterone synthesis. This study provides a potential mechanistic explanation for how GLY inhibits testosterone synthesis in mouse Leydig cells.

Enhanced Extracellular Matrix Degradation in Growth Plate Contributes to Manganese Deficiency-Induced Tibial Dyschondroplasia in Broiler Chicks.

Manganese (Mn) is a crucial trace element for poultry nutrition, and its deficiency compromises tibial cartilage development, leading to perosis and a higher incidence of slipped tendon. Tibial dyschondroplasia (TD) is a metabolic cartilage disease characterized by disruption of endochondral bone formation, which is closely related to extracellular matrix (ECM) degradation, in which Mn deficiency plays an important role. Previous studies have confirmed the role of matrix metalloproteinases (MMPs) in the pathogenesis of TD, but whether dysregulated ECM degradation and MMP expression profiles in growth plate are involved in Mn deficiency-induced avian TD has not been fully elucidated yet. Thus, this study was conducted to clarify these issues. Firstly, we successfully established TD model induced by Mn deficiency in broiler chicks. Mn deficiency decreased the number of chondrocytes, contents of proteoglycan, and type II collagen in tibial growth plate, demonstrating the tibial growth plate damage with enhanced ECM degradation. Also, Mn deficiency inhibited the Nrf2 signaling pathway and enhanced the protein levels of NLRP3, active caspase-1, and active IL-1ß in tibial growth plate, indicating the oxidative stress and inflammatory response in Mn deficiency-induced TD. Additionally, upregulated expression levels of MMPs (MMP1, 9, and 13) were observed in tibial growth plate of Mn deficiency group. In summary, these findings suggest that Mn deficiency-enhanced ECM degradation is involved in avian TD, which may be correlated with oxidative stress, inflammatory response, and upregulation of MMPs.

High fat diet-triggered non-alcoholic fatty liver disease: A review of proposed mechanisms.

Obesity is characterized by the deposition of excessive body fat, and is caused by energy imbalance, especially when consuming fat-rich diets. High fat diet (HFD)-associated obesity is greatly common in patients with non-alcoholic fatty liver disease (NAFLD) that is emerging as one of the most universal causes of liver disease worldwide, especially in Western countries. In spite of its high prevalence, only a small proportion of affected individuals will become inflamed, followed by fibrosis and chronic liver diseases, and most patients only show simple steatosis. In this case, the full comprehension of the mechanisms underlying the progression of NAFLD is of extreme significance; in spite of progress in this field, awareness on the development of NAFLD is still incomplete. Traditionally, liver steatosis is commonly connected with HFD, obesity, and insulin resistance (IR). Recently, various possible mechanisms have been put forward for liver damage, including endoplasmic reticulum stress, perturbation of autophagy, mitochondrial dysfunction, hepatocellular apoptosis, gut microbiota imbalance, dysregulation of microRNAs, and genetic/epigenetic risk factors, as well as an increase in inflammatory responses, among many others. Collectively, these proposed mechanisms allow for a variety of hits acting together on subjects to mediated NAFLD and will offer a more accurate explanation for progression of NAFLD. Therefore, this review summarizes the present information concerning NAFLD after HFD exposure, as well as discusses possible mechanisms through which it may arise.

mTORC1 activation contributes to autophagy inhibition via its recruitment to lysosomes and consequent lysosomal dysfunction in cadmium-exposed rat proximal tubular cells.

Autophagy dysregulation is implicated in cadmium (Cd)-induced nephrotoxicity. The mammalian target of rapamycin complex 1 (mTORC1) is a negative regulator of autophagy, but its role in Cd-induced autophagy inhibition and possible regulatory mechanisms remains poorly understood. In the present study, Cd exposure activated mTORC1 in primary rat proximal tubular (rPT) cells, and two mTORC1 inhibitors (rapamycin and torin 1) were separately utilized to inhibit Cd-induced mTORC1 activation. Data showed that Cd-inhibited autophagic flux was markedly restored by two mTORC1 inhibitors, respectively, as evidenced by immunoblot analysis of autophagy marker proteins and tandem red fluorescent protein-green fluorescent protein-microtubule associated protein light chain 3 (RFP-GFP-LC3) fluorescence microscopy assay. Importantly, Cd exposure triggered the recruitment of mTORC1 onto lysosome membrane assessed by immunofluorescence co-localization analysis, which was obviously inhibited by rapamycin or torin 1. Moreover, Cd-induced lysosomal alkalization, suppressed vacuolar ATPases (V-ATPases) protein levels and impaired lysosomal degradation capacity were markedly reversed by rapamycin or torin 1. In summary, these findings demonstrate that Cd recruits mTORC1 to lysosome membrane to induce its activation, which results in lysosomal dysfunction and resultant autophagy inhibition in rPT cells.