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Microcystins (MCs) and nitrites are coexisted in the environment and have reproductive toxicity. The combined toxic effect and mechanism of MCs and nitrite on spermatogenesis remain largely unclear. In the present study, co-exposure to microcystin-leucine arginine (MC-LR) and sodium nitrite (NaNO 2 ) aggravated testicular damage of Balb/c mice and mitochondrial impairment of spermatogonia, Sertoli cells, and sperm. Furthermore, MC-LR and NaNO 2 reduced sperm density with a synergistic effect. In addition, MC-LR and NaNO 2 synergistically induced oxidative stress in the reproductive system by decreasing superoxide dismutase (SOD) activity and glutathione (GSH) levels and increasing levels of mitochondrial reactive oxygen species (mtROS) and reactive oxygen species (ROS). More importantly, mitoquidone mesylate (MitoQ), an inhibitor of mtROS, blocked MC-LR and NaNO 2 -induced spermatogonia and Sertoli cell apoptosis by inhibiting high expression of Bax, Fadd, Caspase-8, and cleaved-Caspase-3. On the other hand, MitoQ suppressed pyroptosis of Sertoli cells by inhibiting the expression of NLRP3, N-GSDMD, and cleaved-Caspase-1. Additionally, MitoQ alleviated co-exposure-induced sperm density reduction and organ index disorders in F1 generation mice. Together, co-exposure of MC-LR and NaNO 2 can enhance spermatogenic disorders by mitochondrial oxidative impairment-mediated germ cell death. This study emphasizes the potential risks of MC-LR and NaNO 2 on reproduction in realistic environments and highlights new insights into the cause and treatment of spermatogenic disorders., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Microcystin-LR (MC-LR) is an intracellular toxin with multi-organ toxicity and the testis is one of its important target organs. Although there is increasing research on MC-LR in male reproductive toxicity, the association between DNA damage and autophagy induced by MC-LR in male germ cells are still unclear. Therefore, it is important to explore the mechanism of MC-LR-induced DNA damage and the role of the activated ATM/p53 signaling pathway in testicular toxicity. The present study showed that MC-LR exposure significantly reduced gonadal index and induced pathological damage of the testes in mice. In addition, MC-LR increased the oxidative stress-related indicator hydroxyl radical, accompanied by increased levels of DNA damage-related indicators gamma-H2AX, 8-hydroxy-2'-deoxyguanosine, the olive tail moment (OTM) and DNA content of comet tail (TailDNA%) in trailing cells. Moreover, MC-LR activated the ATM/p53 pathway by enhancing the phosphorylation levels of ATM, CHK2 and p53 proteins, and then led to cell autophagy, ultimately triggering disrupted testicular cell arrangement, reduced sperm count and spermatogenic cell shedding. Importantly, after pretreatment with the antioxidant NAC, the expression levels of DNA damage-related indicators and the extent of damage in male germ cells were significantly reduced. Furthermore, pretreatment with the ATM inhibitor KU55933 could reduce the occurrence of autophagy and mitigate testicular toxicity of MC-LR through inhibiting the activation of the ATM/p53 pathway. These results indicate that MC-LR-induced oxidative stress can activate the DNA damage-mediated ATM/p53 signalling pathway to induce autophagy in male germ cells. This study provides a novel insight to further clarify the reproductive toxicity caused by MC-LR and to protect male reproductive health., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Microcystin-leucine arginine (MC-LR) is a cyclic heptapeptide, produced by aquatic cyanobacteria such as microcystis, with strong reproductive toxicity which poses greater threat to the reproductive abilities of humans and animals. By exploring the role of trimethylation of histone H3 at lysine 4 (H3K4me3) and the role of oxidative stress in MC-LR-induced apoptosis in testicular Sertoli cells in Sprague-Dawley (SD) rats, this study indicated that MC-LR increased the expression levels of apoptosis-related genes by raising the levels of H3K4me3. 5'-Deoxy-5'-methylthioadenosine (MTA), the inhibitor of H3K4me3, reduced apoptosis, indicating for the first time that epigenetic modification is closely related to the testicular reproductive toxicity induced by MC-LR. MC-LR also induced oxidative stress by stimulating the generation of reactive oxygen species (ROS), and subsequently triggering mitochondria-mediated apoptotic pathway by decreasing mitochondrial membrane potential and increasing the levels of Bax, Bcl-2, Caspase-3, and so on. MC-LR-induced apoptosis of testicular cells could be decreased after pretreatment with oxidative stress inhibitor N-acetyl-cysteine (NAC). Furthermore, the pathological damage to mitochondria and testes were observed in SD rats. These results show that MC-LR can induce apoptosis by raising the levels of H3K4me3, and pretreatment with MTA can ameliorate the MC-LR-induced apoptosis of cocultured cells by lowering the levels of H3K4me3. Furthermore, NAC has a protective effect on MC-LR-induced apoptosis of testicular cells in SD rats by inhibiting the oxidative stress., (© 2019 Wiley Periodicals, Inc.)

Microcystins (MCs) is a class of cyclic heptapeptide compounds with biological activity. There is no effective treatment for liver injury caused by MCs. Hawthorn is a medicinal and edible plant traditional Chinese medicine with hypolipidemic, reducing inflammation and oxidative stress in the liver. This study discussed the protective effect of hawthorn fruit extract (HFE) on liver damage caused by MC‐LR and the underlying molecular mechanism. After MC‐LR exposure, pathological changes were observed and hepatic activity of ALT, AST and ALP were increased obviously, but they were remarkably restored with HFE administration. In addition, MC‐LR could significantly reduce SOD activity and increase MDA content. Importantly, MC‐LR treatment resulted in mitochondrial membrane potential decreased, and Cytochrome C release, eventually leading to cell apoptosis rate increase. HFE pretreatment could significantly alleviate the above abnormal phenomena. To examine the mechanism of protection, the expression of critical molecules in the mitochondrial apoptosis pathway was examined. The levels of Bcl‐2 was inhibited, and the levels of Bax, Caspase‐9, Cleaved Caspase‐9, and Cleaved caspase‐3 were upregulated after MC‐LR treatment. HFE reduced MC‐LR‐induced apoptosis via reversing the expression of key proteins and genes in the mitochondrial apoptotic pathway. Hence, HFE could alleviate MC‐LR induced hepatotoxicity by reducing oxidative stress and apoptosis. [ABSTRACT FROM AUTHOR]

Microcystin-leucine arginine (MC-LR), an important hepatoxin, has the effect of promoting hepatocarcinogenesis. MicroRNA-122 (miR-122), an important tumor suppressor in liver, plays an important role in promoting cell apoptosis. Previous studies found that the expression of miR-122 was reduced after MC-LR exposure in liver. In this study, C57BL/6 mice were exposed to saline, negative control agomir, and MC-LR with or without miR-122 agomir transfection. The results indicated that MC-LR promoted the expressions of tumor suppressor genes and decreased the expressions of anti-apoptotic proteins B cell lymphoma-2 (Bcl-2) and Bcl-2-like 2 (Bcl-w), causing hepatocyte apoptosis. Under MC-LR exposure, miR-122 agomir transfection could further increase the expressions of tumor suppressor genes and the release of cytochrome-c (Cyt-c) and decrease the expressions of Bcl-2 and Bcl-w. In conclusion, miR-122 reduction can mitigate MC-LR-induced apoptosis to a certain extent, which in turn, it is likely to have contributed to MC-LR-induced hepatocarcinogenesis. [ABSTRACT FROM AUTHOR]

Microcystin‐LR (MC‐LR), a potent endotoxin, can induce reproductive toxicity. In order to investigate the role and mechanisms of apoptosis (p53‐dependent and mitochondrial pathways) of germ cells induced by MC‐LR, the co‐cultured primary Sertoli‐germ cells from Sprague‐Dawley rats were used for the experiments. Expression levels of proteins, genes, and mitochondrial membrane potential (MMP) were obtained after exposing co‐cultured Sertoli‐germ cells to MC‐LR with or without the addition of the p53 inhibitor, pifithrin‐α (PFT‐α), and MMP inhibitor, cyclosporin A (CsA). Results indicated that MC‐LR could activate p53‐dependent pathway‐associated proteins in Sertoli‐germ cells, leading to a decrease in MMP (indicating the opening of mitochondrial permeability transition pore [mPTP] and the release of Cytochrome‐c [Cyt‐c]) from the mitochondria into the cytoplasm and eventually the induction of apoptosis. PFT‐α inhibited the expression ofp53, ameliorated the MMP of the co‐cultured Sertoli‐germ cells, and prevented the release of Cyt‐c from the mitochondria into the cytoplasm, which reduces the occurrence of apoptosis. Similarly, the decreased release of Cyt‐c from the mitochondria into the cytoplasm and the declined level of apoptosis in Sertoli‐germ cells induced by MC‐LR were observed after the addition of CsA. These results indicated that the apoptosis of the co‐cultured Sertoli‐germ cells induced by MC‐LR was mediated by the p53‐dependent pathway, with the involvement of the opening of mPTP. [ABSTRACT FROM AUTHOR]

As an emerging pollutant in the aquatic environment, microcystin-LR (MC-LR) can enter the body through multiple pathways, and then induce apoptosis and gonadal damage, affecting reproductive function. Previous studies focused on male reproductive toxicity induced by MC-LR neglecting its effects on females. The apoptotic signal-regulated kinase 1 (ASK1) is an upstream protein of P38/JNK pathway, closely associated with apoptosis and organ damage. However, the role of ASK1 in MC-LR-induced reproductive toxicity is unclear. Therefore, this study investigated the role of ASK1 in mouse ovarian injury and apoptosis induced by MC-LR. After MC-LR exposure, ASK1 expression in mouse ovarian granulosa cells was increased at the protein and mRNA levels, and decreased following pretreatment by antioxidant N-acetylcysteine, suggesting that MC-LR-induced oxidative stress has a regulatory role in ASK1 expression. Inhibition of ASK1 expression with siASK1 and NQDI-1 could effectively alleviate MC-LR-induced mitochondrial membrane potential damage and apoptosis in ovarian granulosa cells, as well as pathological damage, apoptosis and the decreased gonadal index in ovaries of C57BL/6 mice. Moreover, the P38/JNK pathway and downstream apoptosis-related proteins (P-P38, P-JNK, P-P53, Fas) and genes (MKK4 , MKK3 , Ddit3 , Mef2c) were activated in vivo and vitro , but their activation was restrained after ASK1 inhibition. Data presented herein suggest that the ASK1-mediated P38/JNK pathway is involved in ovarian injury and apoptosis induced by MC-LR in mice. It is confirmed that ASK1 has an important role in MC-LR-induced ovarian injury, which provides new insights for preventing MCs-induced reproductive toxicity in females. [Display omitted] • MC-LR upregulates ASK1 expression via oxidative stress in mouse ovarian cells. • Inhibition of ASK1 alleviates MC-LR-induced ovarian injury and apoptosis in mice. • ASK1-mediated P38/JNK pathway is involved in MC-LR-induced ovarian injury and apoptosis. • ASK1 activates the P38/JNK pathway via MKK3/4 after ovarian cells exposed to MC-LR. [ABSTRACT FROM AUTHOR]

Microcystin-leucine arginine (MC-LR), an intracellular toxin to cause reproduction toxicity, is produced by blooming cyanobacteria and widely distributed in eutrophic waters. It is revealed that MC-LR-induced female reproductive toxicity is more severe than male reproductive toxicity. Previous studies mainly focused on male reproductive toxicity, and the molecular mechanisms of MC-LR-induced apoptosis, follicular atresia and infertility in female remain largely unclear. Here, it was found that MC-LR treatment could induce apoptosis, inflammation, follicular atresia, and decrease of gonadal index in mice ovaries. RNA-Seq data showed that the up-regulation of DNA-damage inducible transcript 3 (Ddit3) under endoplasmic reticulum (ER) stress had predominantly regulatory role in MC-LR-induced apoptotic pathway. Furthermore, MC-LR exposure promoted cleavage of activating transcription factor 6 (ATF6, 50kd), inositol-requiring enzyme 1 (Ire1) expression, phosphorylation of IRE1, mitogen-activated protein kinase 5 (Map3k5) and Ddit3 expression, which was accompanied by the upregulation of death receptor 5 (Dr5) and active-caspase-3, and a decrease in Bcl-2 expression. ER stress inhibitor 4-Phenyl butyric acid (4-PBA) ameliorated these MC-LR-induced changes in protein or mRNA level. More importantly, knockdown of Ddit3 suppressed MC-LR-induced cell apoptosis and follicular atresia by directly regulating Dr5 and Bcl-2. Additionally, it was also found that MC-LR increased Map3k5 phosphorylation by inhibiting protein phosphatase 2A (PP2A) activity, and then promoted Ddit3 expression. In short, our data suggests that Ddit3 promotes MC-LR-induced mice ovarian cells apoptosis and follicular atresia via ER stress activation, which provides a new insight into the relation between infertility in females and the emerging water pollutant MC-LR. Unlabelled Image • MC-LR causes ovarian inflammation and decrease of gonad index. • MC-LR upregulates Ddit3 expression through ER stress and inhibition of PP2A activity. • Ddit3 promotes mouse ovarian cell apoptosis and follicle atresia induced by MC-LR. • Upregulation of Ddit3 accelerates apoptosis through regulating Dr5 and Bcl-2. [ABSTRACT FROM AUTHOR]

Microcystin-leucine arginine (MC-LR) is a variant of microcystins (MCs), which poses a serious threat to the reproductive system. Histone acetylation modification can regulate the expressions of apoptosis-related genes. However the mechanisms of histone acetylation involving MC-LR-induced apoptosis were not understood. This study investigated the change of histone acetylation and its role in apoptosis and cell cycle arrest induced by MC-LR. MC-LR enhanced the activity of histone deacetylase (HDAC), decreased the activity of histone acetylase (HAT), up-regulated the expression of HDAC1, and down-regulated the expressions of Ac-H3 and Ac-H4 in vitro and vivo. Meanwhile, MC-LR induced testicular tissue injury and increased the expressions of apoptosis-related genes, such as Bax , Caspase3 and Caspase8 , ultimately causing cells apoptosis in testicular tissues. Furthermore, MC-LR also induced cell cycle arrest in S phase, increased the expression of P21Wif1/Cip1, and inhibited the expressions of cyclinD1, cyclinE1, CDK2 and E2F1. Importantly, HDAC inhibitor Trichostatin A (TSA) could ameliorate MC-LR-induced apoptosis and cell cycle arrest by reverse-regulating the expressions of these proteins. These results indicated that MC-LR could activate the mitochondrial apoptotic pathway and disorder the cell cycle pathway to induce the cell apoptosis by enhancing HDAC activity and reducing histone acetylation of normal testicular cells in SD rats. Hence, histone acetylation has a vital function in MC-LR-induced apoptosis in SD rat testicular cells, which provides a new insight on the reproductive toxicity of male induced by MC-LR. • MC-LR enhanced the activity of HDAC and reduced the acetylation levels of histone H3, H4. • MC-LR induced SD rat testicular cell apoptosis. • MC-LR induced SD rat testicular cell cycle arrest in S phase. • Trichostatin A inhibited MC-LR-induced SD rat testicular cell apoptosis and cell cycle arrest. [ABSTRACT FROM AUTHOR]

Microcystin-leucine arginine (MC-LR), a cyclic heptapeptide produced by cyanobacteria, is a strong reproductive toxin. Studies performed in rat Sertoli cells and Chinese hamster ovary cells have demonstrated typical apoptosis after MC-LR exposure. However, little is known on how to protect against the reproductive toxicity induced by MC-LR. The present study aimed to explore the possible molecular mechanism underlying the anti-apoptosis and protective effects of resveratrol (RES) on the co-culture of Sertoli–germ cells and rat testes. The results demonstrated that MC-LR treatment inhibited the proliferation of Sertoli–germ cells and induced apoptosis. Furthermore, sirtuin 1 (SIRT1) and Bcl-2 were inhibited, while p53 and Ku70 acetylation, Bax expression, and cleaved caspase-3 were upregulated by MC-LR. However, RES pretreatment ameliorated MC-LR-induced apoptosis and SIRT1 inhibition, and downregulated the MC-LR-induced increase in p53 and Ku70 acetylation, Bax expression, and caspase-3 activation. In addition, RES reversed the MC-LR-mediated reduction in Ku70 binding to Bax. The present study indicated that the administration of RES could ameliorate MC-LR-induced Sertoli–germ cell apoptosis and protect against reproductive toxicity in rats by stimulating the SIRT1/p53 pathway, suppressing p53 and Ku70 acetylation and enhancing the binding of Ku70 to Bax. [ABSTRACT FROM AUTHOR]