Evaluating the Endocrine-Disrupting and Oxidative Stress Potential of a 50-Component Human-Relevant Complex Chemical Mixture Using In Vitro Tests

Josefin Engelhardt; Nathalie Struwe; Annika Jansson; Vesna Munic Kos; Maria Larsson; Jana M Weiss

doi:10.1002/jat.70011

Evaluating the Endocrine-Disrupting and Oxidative Stress Potential of a 50-Component Human-Relevant Complex Chemical Mixture Using In Vitro Tests

J Appl Toxicol. 2025 Nov 29. doi: 10.1002/jat.70011. Online ahead of print.

Authors

Josefin Engelhardt¹, Nathalie Struwe², Annika Jansson^{1

3

4}, Vesna Munic Kos⁴, Maria Larsson², Jana M Weiss¹

Affiliations

¹ Department of Environmental Science, Stockholm University, Stockholm, Sweden.
² School of Science and Technology, Örebro University, Örebro, Sweden.
³ Department of Chemical Engineering, Royal Institute of Technology (KTH), Stockholm, Sweden.
⁴ Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden.

PMID: 41317044
DOI: 10.1002/jat.70011

Abstract

Humans are chronically exposed to mixtures of environmental contaminants. Exposure to endocrine-disrupting chemicals (EDCs) contributes to increased health impairment observed globally. This study aimed to evaluate the endocrine-disruptive and oxidative stress potential of a human-relevant, complex chemical mixture in vitro. By testing chemical class subgroup mixtures, the identity of toxicological drivers and mixture additivity could be investigated. A 50-component mixture was compiled based on Swedish human blood concentrations (xHBC), consisting of six subgroup mixtures: polychlorinated biphenyls (PCBs) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (PCB mixture), brominated flame retardants (BFR mixture), per- and polyfluoroalkyl substances (PFAS mixture), pesticide mixture, synthetic phenolic contaminants (phenol mixture), and phthalate mixture. These were tested in four chemically activated luciferase gene expression (CALUX) assays: dioxin responsive (DR-), estrogen receptor α (ERα-), androgen receptor. (AR-), and nuclear factor erythroid 2-related factor 2 (Nrf2)-CALUX, along with an adipocyte cell assay. The total mixture caused significant agonistic activity in DR- and ER-, and antagonistic activity in AR-CALUX at 0.1-15 xHBC, depending on the assay. Mixture additivity was assessed in ERα-, DR-, and anti-AR-CALUX using subgroup mixtures and the concentration addition (CA) model. The total mixture followed the CA model in ERα-, anti-AR- and DR-CALUX. The toxicological drivers of these activities were mainly the PCB and phenol mixture. A significant increase in differentiated adipocytes was observed at 100 xHBC. These results raise concerns regarding potential health effects on the endocrine system. The additive effects at human-relevant concentrations observed in this study motivate considering mixtures in regulatory contexts to protect the well-being of future generations.

Keywords: CALUX; chemical mixtures; concentration additivity; endocrine‐disrupting chemicals; environmental contaminants; human blood concentrations; in vitro; oxidative stress.

Abstract

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