Benzo[k] fluoranthene induces male germ cell damage

Abstract

Objective To establish a damage model of mouse spermatocyte GC-2spd cells induced by benzo[k] fluoranthene (BkF), a representative compound of high-molecular weight polycyclic aromatic hydrocarbons, so as to investigate the effect and mechanism of BkF on GC-2 cells. Methods Related genes of male reproductive diseases associated with BkF were screened in Comparative Toxicogenomics Database (CTD), and functional and pathway enrichment analysis is conducted subsequently to explore the possible pathway mechanism. Moreover, GC-2 cells were treated with BkF at a final concentration of 0, 10, 20, 40 or 80 μmol/L for 72 h, and then the cell cycle, oxidative stress and DNA damage effect were detected. Western blotting was used to determine the protein levels of the key genes as well as the core molecules of related pathways. Results The key genes CYP1A1 and CYP17A1 associated with BkF-related reproductive system diseases were screened out from the CTD database. Gene Ontology (GO) enrichment analysis found that they were involved in male reproductive system and cell cycle arrest, obviously enriched in biological processes such as oxidative stress. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that these genes were significantly enriched in cytochrome P450 pathway (P < 0.01). After GC-2 cells were exposed to BkF for 72 h, the proliferation was significantly decreased in a dose-dependent manner, as compared with the control group. When the exposure dose was higher than 40 μmol/L, the cell cycle arrest was obvious (P < 0.05); Both the intracellular reactive oxygen species (ROS) level (P < 0.05) and the protein level of DNA damage marker γ-H2AX (P < 0.01) were remarkably increased in a dose-dependent manner. In addition, the protein levels of CYP1A1 and CYP17A1 were greatly higher in the exposure group than the control group (P < 0.05), while treatment of AhR inhibitor CH223191 resulted in notably down-regulated the expression of the 2 proteins (P < 0.05). Conclusion BkF induces the activation of AhR receptors, then causes elevated expression of downstream genes CYP1A1 and CYP17A1 as well as increased intracellular ROS, and finally leads to oxidative stress and genetic damage in GC-2 cells.

Keywords

• benzo[k] fluoranthene
• gc-2spd cells
• oxidative damage
• ahr-cyp1a1 signaling pathway
• bioinformatics analysis