Abstract:

Objective To investigate the effect of pyraclostrobin on mitochondrial function and dynamic balance in mouse spermatocyte GC-2 cells， and to elucidate its possible mechanism. Methods Methylthiazolyldiphenyl-tetrazolium bromide（MTT） method was used to detect the cell activity of GC-2 cells in various groups after treatment with different concentrations of pyraclostrobin， so as to determine the appropriate treatment concentration. Mouse spermatocyte cell line GC-2 was divided into control group， low dose of pyraclostrobin （0.8 μmol·L^-1） group， medium dose of pyraclostrobin （1.6 μmol·L^-1） group， and high dose of pyraclostrobin （2.4 μmol·L^-1） group. JC-1 mitochondrial membrane potential assay kit was used to detect the mitochondrial transmembrane potential difference （ΔΨ_m）across the inner membrane of mitochondrial in GC-2 cells in various groups. 2'，7'-dichlorodihydrofluorescein diacetate （DCFH-DA） fluorescent probe method was used to detect the levels of reactive oxygen species （ROS） in GC-2 cells in various groups. Adenosine triphosphate （ATP） assay kit was used to measure the levels of ATP in GC-2 cells in various groups. Mito-tracker fluorescence probe method was used to detect the fusion and fission of mitochondria in GC-2 cells in various groups. Western blotting method was used to detect the expression levels of mitochondrial fusion and fission related proteins， optic atrophy 1 （OPA1） and dynamin-related protein 1 （DRP1）， in GC-2 cells in various groups. Results Compared with control group， the cell activities of GC-2 cells were significantly decreased in groups with pyraclostrobin concentration≥1 μmol·L^-1 （P<0.001）. The half maximal inhibitory concentration （IC₅₀） of pyraclostrobin against GC-2 cells was 4.8 μmol·L^-1. Compared with the control group， the ΔΨ_m in the GC-2 cells were significantly decreased in low dose of pyraclostrobin group， medium dose of pyraclostrobin group， and high dose of pyraclostrobin group （P<0.05 or P<0.01），the levels of ROS in the GC-2 cells were significantly increased（P<0.05 or P<0.01），and the ATP levels in the GC-2 cells were significantly decreased （P<0.001）. The Mito-Tracker staining results showed that the mitochondria in GC-2 cells in control group were distributed in a continuous network pattern with good fusion， while those in low dose of pyraclostrobin group， medium dose of pyraclostrobin group， and high dose of pyraclostrobin group showed fragmented morphology with obvious network structure breakage， decreased fusion， and increased fission. The Western blotting results showed that compared with control group， the expression levels of OPA1 protein in the GC-2 cells were decreased in medium dose of pyraclostrobin group and high dose of pyraclostrobin group （P<0.05 or P<0.01）， and the expression levels of DRP1 protein in the GC-2 cells were significantly increased in low dose of pyraclostrobin group，medium dose of pyraclostrobin group， and high dose of pyraclostrobin group （P<0.001）. Conclusion Pyraclostrobin induced an imbalance between mitochondrial fusion and fission in GC-2 cells， leading to mitochondrial dysfunction. Its toxic effect may be associated with abnormal regulation of mitochondrial dynamics.

Key words: Pyraclostrobin, Mouse spermatocyte, Mitochondrial function, Mitochondrial fusion and fission, Dynamic balance