| As one of the oldest poisons,arsenic(AS)is also a unique gold stone drug in traditional Chinese medicine,which has a very important value in the manufacture of medicine and chemical materials.However,with the increase in the scope of application,the degree of environmental pollution continues to expand,especially in specific occupational environments.Even if arsenic is only a small amount from air and/or drinking water,it also has a huge health hazard by long-term accumulation.At the same time,arsenic pollution also affects the safety of other plants and animals.Many studies have shown that arsenic,an environmental estrogen-like toxic chemical,affects the reproduction of female animals.In recent years,it has been found that arsenic reduced the relative weight of the testis and impairs testicular morphology,sperm mobility and viability decreased,studies on the toxicity mechanism of arsenic are mainly focused on oxidative stress,there are few of studies involved in further mechanism.In view of this,it is of great significance to explore the toxicology of arsenic and to search for effective protective agents.Mouse spermatogonial cells(C-1),treated with 0μg/mL,2μg/mL,3μg/mL,4 μg/mL arsenic trioxide(ATO),were used as the research object,Oxidative stress,mitochondrial structure and function,apoptosis,autophagy and necrosis were evaluated by Transmission electron microscopy,flow cytometry,cytopnea,fluorescence quantitative PCR,western blot methods and techniques.Results showed: The amount of malondialdehyde(MDA)and reactive oxygen species(ROS)treated by ATO and the Lactate dehydrogenase(LDH)activity in cell culture medium both were significantly higher than the control group.Total antioxidant capacity(T-AOC),hydrogen peroxide(CAT),and glutathione(GSH)levels increased first and then decreased as the ATO concentration increased and the difference was remarkable compared with the control group.The addition of L-arginine(L-arg)in the culture medium can significantly inhibited the generation of ROS and MDA induced by ATO,which maintains cell GSH at normal cell level.The above results showed that2μg/mL,3μg/mL,4 μg/mL ATO induced the oxidative stress of mouse spermatogonial cells.Treated with 0μg/mL,2μg/mL,3μg/mL,4 μg/mL arsenic trioxide(ATO)significantly damaged the mitochondrial respiratory function of GC-1.Mitochondrial respiratory control rate(RCR),ATP synthesis and basic oxygen consumption were decreased as ATO concentration increased,and the difference was significant compared with the control group.Furthermore,Mitochondrial membrane potential(MMP)was significantly decreased with the treatment of 2 μg/mL ATO,but no obvious influence while treated with 3μg/mL,4μg/mL ATO.GC-1 cells treated with ATO were evaluated by Apoptosis and necrosis detection kits showed that the proportion of orange-red cells increased gradually as ATO concentration increased,which indicated that the proportion of late apoptosis and necrotic cells increased gradually.Subsequently,the evaluation experimental results of expression levels of genes and proteins related to autophagy,apoptosis and necrosis showed that the relative quantitative expression of pro-apoptotic genes(Bax,Caspase3 and autophagy-related genes such as Beclin1,ATG3,ATG7,P62,LC3 a,LC3b)of GC-1 cells treated with 2 μg/mL ATO was obviously up-regulated,but the expression of Bcl2 gene,which inhibited apoptosis and autophagy,was significantly down-regulated.In addition,the necrotic gene(TNF and RIP3)expression were significantly up-regulated and the inhibited necrotic gene(Caspase 8)expression was decreased.The above results showed that 2 μg/mL ATO induced apoptosis and autophagy of mouse GC-1 cells,while 3 μg/mL ATO resulted in programmed necrosis of mouse GC-1 cells.It can be concluded that low-concentration ATO can induce the occurrence of oxidative stress and mitochondrial damage and mediate autophagy and apoptosis of cell in mouse spermatogonial cells,while high-concentration ATO can lead to programmed necrosis of mouse spermatogonial cells. |
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