Towards the mechanism of spermatotoxicity of p-tert-butyl-alpha-methylhydrocinnamic aldehyde: inhibition of late stage ex-vivo spermatogenesis in rat seminiferous tubule cultures by para-tert-butyl- benzoic acid.

Molecules metabolized to para-tert-butyl-benzoic acid (p-TBBA) affect male reproduction in rats through effects on spermatogenesis. This toxicity is specific to p-TBBA and not observed in meta-substituted analogues. The underlying mode of action was evaluated by comparing effects of p-TBBA and the position isomer m-TBBA (2-50 µM) in an ex vivo 3D primary seminiferous tubule cell culture system from juvenile Sprague Dawley rats (Bio-AlteR). Treated cultures were evaluated for CoA-conjugate formation, cytotoxicity, blood-testis barrier functionality and different germ cell populations to assess effects on spermatogenesis. In addition, an evaluation of the metabolome of treated cultures was performed by using MxP Broad Profiling via a LC-MS/MS and GC-MS platform. Para-TBBA decreased germ cell populations of late stages of spermatogenesis and led to the formation of CoA-conjugates in the ex vivo tissue. In addition, p-TBBA had a pronounced effect on the metabolome by affecting lipid balance and other CoA-dependent pathways contributing to energy production and the redox system. Meta-TBBA did not affect germ cell populations and no m-TBBA related CoA-conjugates were detectable. The metabolic profile of m-TBBA treated cells was comparable to vehicle control treated cultures, indicating that formation of CoA-conjugates, inhibition of spermatogenesis, and effects on the metabolome are mechanistically linked events. Thus, for this specific chemical group an adverse outcome pathway can be postulated, including the formation of benzoic acid metabolites, accumulation of CoA-conjugates to a certain threshold and CoA depletion, which affects the metabolic and lipid profile and leads to tissue specific effects with impaired functionalities such as spermatogenesis.