Reproductive toxicity mechanisms of PNMC: Evidence from mitochondrial dysfunction and hormone synthesis disruption in the KGN cells.

3-methyl-4-nitrophenol (PNMC), a widespread environmental endocrine-disrupting compound derived from diesel particulate matter and degradation of fenitrothion, poses a potential risk to ovarian function. This study investigated the concentration-dependent effects of PNMC on mitochondrial homeostasis and steroidogenesis using the human ovarian granulosa KGN cell line. Cells were treated with PNMC across a wide range of concentration (1 nM to 1 mM) for 48 h and mitochondrial dynamics and functions as well as steroidogenesis were evaluated. Transcriptional and immunofluorescence analyses showed that 100 μM PNMC treatment significantly upregulated the expression of mitochondrial dynamics-related genes (MFN1/2, OPA1 and DRP1), mitophagy-related genes (PINK1 and PARK2) and antioxidant defense-related genes (SOD1/2 and GPX1). These changes were associated with increased mtDNA copy number, reduced intracellular ROS and increased mitochondrial membrane potential. By contrast, 1 mM PNMC treatment provoked overt mitochondrial toxicity, characterized by diminished mtDNA content, elevated mitochondrial ROS, loss of membrane potential and significant decrease in cell viability. These results indicated that PNMC significantly altered the expression of genes related to mitochondrial dynamics, antioxidant defense, and mitophagy. The expression of CYP19A1 shows significant upregulation at 100 µM PNMC treatment, while the maximal response for other genes (CYP17A1, CYP11A1, HSD3B) is observed at 1 µM PNMC treatment. Correspondingly, the levels of 17β-estradiol and progesterone increased in the culture medium at 100 µM or 1 µM PNMC treatment, respectively. Collectively, these findings reveal that PNMC-associated dysregulation of steroidogenic function is closely linked to concomitant alterations in mitochondrial activity, providing important mechanistic insights into its potential reproductive toxicity.