Real-life organochlorine mixture-induced lipid dysregulation and oxidative stress in Leydig TM3 cells: Mechanistic insights into male reprotoxicity

Abstract

Persistent environmental pollutants such as organochlorines (OCs) remain a global concern due to their widespread distribution, bioaccumulative nature, and endocrine-disrupting potential. While associations with male reproductive toxicity are well documented, the underlying mechanisms, particularly those involving lipid metabolism in testicular cells, are not fully understood. This study investigates the mechanistic basis of male reprotoxicity induced by a real-life OC mixture (OC-MIX), modeled after the contaminant profile of ringed seal blubber and comprising 20 environmentally relevant OCs, including pesticides (e.g., dichlorodiphenyltrichloroethane) and industrial compounds (e.g., polychlorinated biphenyls). We applied a mechanistic in vitro test battery that combines receptor-specific reporter gene assays with functional profiling in immature murine Leydig TM3 cells exposed to OC-MIX concentrations ranging from 0.04 to 50 µg/mL. OC-MIX exhibited strong antiandrogenic and dioxin-like activities. Functional assays revealed reduced testosterone and progesterone levels, increased oxidative stress, and impaired mitochondrial function. These effects were driven by broad lipid dysregulation, including enhanced fatty acid degradation and acylcarnitine depletion, which was evident even at the lowest tested concentration (2.5 µg/mL). These lipid alterations were not primarily mediated via androgen receptor antagonism or aryl hydrocarbon receptor agonism. Instead, the lipidomic signature closely resembled that of the lipotoxic drug amiodarone, rather than a non-cytotoxic fatty acid mixture. Our findings underscore the central role of lipid metabolism in testicular function and demonstrate that OC-MIX exerts reproductive toxicity via complex, non-classical endocrine mechanisms. This study highlights the value of integrating lipidomics with mechanistic in vitro models to assess the reproductive toxicity of environmental chemical mixtures.