Abstract:

Objective To discuss the effect of pyraclostrobin on ferroptosis in the spermatocytes GC-2 of the mice， and to clarify whether pyraclostrobin can cause male reproductive toxicity. Methods The mouse spermatocytes GC-2 were divided into control group， low dose of pyraclostrobin group （0.8 μmol·L^-1 pyraclostrobin）， medium dose of pyraclostrobin group （1.6 μmol·L^-1 pyraclostrobin） and high dose of pyraclostrobin group （2.4 μmol·L^-1 pyraclostrobin）. After being treated with pyraclostrobin for 24 h，methylthiazolyldiphenyl-tetrazolium bromide （MTT） method was used to detect the proliferation activities of the GC-2 cells after treated with different doses of pyraclostrobin； mitochondrial membrane potential detection kit JC-1 was used to detect the mitochondrial membrane potential in the GC-2 cells in various groups； superoxide dismutase （SOD） activity detection kit was used to detect the activities of SOD in the GC-2 cells in various groups； malondialdehyde （MDA） assay kit was used to determine the levels of MDA in the GC-2 cells in various groups； reduced glutathione （GSH） and oxidized glutathione （GSSG） detection kits were used to detect the levels of GSH and GSSG in the GC-2 cells， and the ratios of GSH/GSSG in various groups were calculated； Western blotting method was used to detect the expression levels of heme oxygenase-1 （HO-1）， ferritin heavy chain 1 （FTH1） and glutathione peroxidase 4 （GPX4） in the GC-2 cells in various groups. Results Compared with control group， the proliferation activities of the GC-2 cells in various pyaclostrobin dose ≥1.0 μg·L^-1 groups were decreased （P<0.01）. The low， medium， and high doses of pyraclostrobin were 0.8， 1.6， and 2.4 μmol·L^-1. The results of JC-1 method showed that compared with control group， the mitochondrial membrane potential of the GC-2 cells in medium， and high doses of pyraclostrobin groups were decreased. Compared with control group， the SOD activity in the GC-2 the cells in low， medium， and high doses of pyraclostrobin groups were decreased （P<0.01）， and the MDA levels in the GC-2 cells were increased （P<0.01）. Compared with control group， the GSH levels in the GC-2 cells in low， medium， and high doses of pyraclostrobin groups were significantly decreased （P<0.01）， the GSSG levels were increased （P<0.01）， and the GSH/GSSG ratios were decreased （P<0.01）. Compared with control group， the expression levels of HO-1 protein in the GC-2 cells in low， medium， and high doses of pyraclostrobin groups were significantly increased （P<0.05 or P<0.01）， while the expression levels of FTH1 and GPX4 proteins were significantly decreased （P<0.05 or P<0.01）. Conclusion Pyraclostrobin can induce the ferroptosis of the spermatocytes GC-2.

Key words: Pyraclostrobin, Mouse spermatocytes, Ferroptosis, Glutathione peroxidase 4, Oxidative stress