High-concentration MEHP triggers mtDNA depletion in undifferentiated HepaRG and C2C12 cultures and disrupts mitochondrial homeostasis in both HepaRG culture states

Phthalates are often called "everywhere chemicals" because they are widely used in consumer products and are detectable in the environment and humans. One of the most studied phthalates, di-2-ethylhexyl phthalate (DEHP), is metabolized to mono-(2-ethylhexyl) phthalate (MEHP), which is known to disrupt metabolic processes through peroxisome proliferator-activated receptor (PPAR) signaling. However, accumulating evidence suggests that lipophilic phthalates also affect mitochondria, key regulators of oxidative metabolism, autophagy, and apoptosis. Based on previous observations that undifferentiated cells are more sensitive to a mitotoxic agent, we hypothesized that MEHP differentially affects mitochondrial function and mtDNA maintenance across hepatic cell states. To test this, we used the human HepaRG hepatoma-derived cell line, which can be cultured in undifferentiated and differentiated states, and assessed viability and mitochondrial function following prolonged 6- and 12-day high-concentration MEHP treatments. Prolonged treatments reduced viability and altered bioenergetics in both states. Short treatments (1-3 days) reduced viability only in differentiated cultures and were associated with mtDNA depletion in undifferentiated cultures. In both states, MEHP increased the expression of the low-molecular-weight mitochondrial genome maintenance exonuclease (MGME1) isoform, altered the levels of autophagy-related factors, and induced apoptosis. In another mitochondrial-competent myoblast model (C2C12 cells), a high concentration of MEHP was associated with mtDNA depletion, whereas lower concentrations were associated with modest reductions in cell density without detectable mtDNA loss. These results demonstrate state-dependent mitochondrial responses to MEHP and indicate that a reduced endpoint cell density is a sensitive outcome occurring independently of, and at lower concentrations than, mtDNA depletion in undifferentiated cells.