Transcriptome analysis reveals the molecular mechanisms of neonicotinoid acetamiprid in Leydig cells.

Abstract

At present, the reproductive toxicology of neonicotinoids has received greater attention, however, its potential mechanisms are still not fully understood. Acetamiprid (ACE) is a new-generation neonicotinoid and has become a ubiquitous contaminant in the environment. This study aimed to investigate the toxic effects of ACE in TM3 Leydig cells based on transcriptome analysis. The viability and apoptosis of TM3 cells exposed to different concentrations of ACE were assessed by CCK8 and flow cytometry, respectively. After ACE exposure, transcriptome analysis was performed to screen differential expression genes (DEGs), followed by qPCR verification. Results showed that ACE exposure resulted in a time- and dose-dependent decrease in the viability of TM3 cells (p < .05). ACE also exerted a dose-dependent pro-apoptotic effect on TM3 cells. Results of RNA-seq showed that 1477 DEGs were obtained, of which 539 DEGs were up-regulated and 938 DEGs were down-regulated. GO and KEGG analyses of DEGs showed that DNA replication and cell cycle might be the key mechanisms for the cytotoxicity of ACE. qPCR results demonstrated that Mdm2, Cdkn1a (p21) and Gadd45 were significantly increased, and Pcna, Ccna2 (CycA), Ccnb1 (CycB), Ccne1 (CycE), and Cdk1 were significantly decreased, indicating that ACE exposure might promote G1/S and G2/M cell cycle arrest. Additionally, FoxO, p53, and HIF-1 signaling pathways and ferroptosis might play important roles in ACE-induced reproductive toxicity. Collectively, this study provides new perspectives into the mechanism of ACE-induced reproductive toxicity and lays a theoretical foundation for the in-depth study of non-target toxicity mechanisms of neonicotinoid insecticides.