Chemical-Class Submixture Screening Reveals Drivers of Endocrine Disruption in Personalized Human Blood POP Mixtures

Multiple studies demonstrate mixture effects arising from the interactive toxicity of environmental chemicals in human blood, but identifying the main toxic drivers remains challenging. In a recent proof-of-principle <i>in vitro</i> study, we showed that personalized mixtures (PMs), reconstructed from 24 persistent organic pollutant (POPs) concentrations measured in individual blood samples from Swedish adults, induced unique interindividual effects on H295R cell viability and steroidogenesis. Here, we followed up by testing submixtures of four PMs (PM#3, PM#4, PC1-OC-Mix, and PC2-PFAS-Mix), separated by the chemical classes perfluoroalkyl substances (PFASs), organochlorine pesticides (OCPs), polychlorinated biphenyls (PCBs), and polybrominated diphenyl ethers (PBDEs). Submixtures of PFAS and OCPs induced significant effects on testosterone synthesis at low (1×) and medium (10×) concentrations, consistent with effects observed in the corresponding whole PMs, and were therefore likely the primary drivers of the whole-mixture effects on testosterone. Notably, some submixtures altered estradiol and testosterone levels in ways not observed in full PMs, suggesting antagonistic interactions across chemical classes when combined. Potential antagonistic interaction in more complex mixtures, independent of concentration, was also observed within OCP submixtures, as only the less complex OCP mixtures lacking DDE or DDT induced testosterone synthesis. For additional mechanistic insight, we expanded the H295R assay to include oxidative stress analyses, which revealed no effects from the PMs. RT-qPCR analysis showed downregulation of <i>CYP11A1</i> after exposure to PM#3 and PM#4 at high concentrations (100×), suggesting a feedback mechanism contributing to suppressed testosterone synthesis.