Toxicological evaluation of benzophenone-3 and its metabolite benzophenone-1 in TM3 Leydig cells

Recently, a decline in male fertility has been reported, raising concerns about the role of environmental pollutants. Ultraviolet (UV) filters have attracted attention due to their potential effects. Benzophenone-3 (BP-3) is a widely used UV filter. It is frequently detected in environmental and human biomonitoring studies, indicating widespread exposure. After dermal absorption, BP-3 enters systemic circulation and is metabolized to benzophenone-1 (BP-1). Although both compounds have been reported to exhibit endocrine-active properties, including antiandrogenic effects, toxicological data on BP-1 are still limited. Therefore, the potential effects of benzophenone derivatives on Leydig cell function and male reproductive health require further investigation. We aimed to evaluate the potential cytotoxic and genotoxic effects of BP-3 and BP-1 in TM3 mouse Leydig cells. Cytotoxicity was assessed using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) and neutral red uptake assays. DNA damage was evaluated using the alkaline comet assay. Reactive oxygen species (ROS) production was determined using a DCFDA/H₂DCFDA-based assay, and testosterone levels were measured using an enzyme-linked immunosorbent assay (ELISA). Exposure to both compounds resulted in concentration-dependent reductions in cell viability, with a more pronounced decrease for BP-1. BP-1 increased ROS formation at selected concentrations, whereas BP-3 did not induce oxidative response. Assessment of DNA integrity using the comet assay did not reveal measurable DNA damage. Under experimental conditions without hormonal stimulation, no statistically significant difference in testosterone concentrations was observed. The findings indicate BP-1 may exert stronger cytotoxic and oxidative effects than BP-3 in Leydig cells, suggesting that metabolites may contribute to the overall toxicological profile of UV filters in reproductive toxicity evaluations.