Diglycolic acid inhibits succinate dehydrogenase activity, depletes mitochondrial membrane potential, and induces inflammation in an SH-SY5Y neuroblastoma model of neurotoxicity in vitro

Abstract

Diethylene glycol is a toxic industrial solvent resulting in a well-defined toxidrome. Diglycolic acid (DGA) has been identified as the metabolite responsible for the nephrotoxicity and hepatotoxicity. These studies assess the mechanism of DGA-induced neurotoxicity, specifically addressing the known ability of DGA to chelate calcium (Ca2+) in solution and inhibit mitochondrial complex II. SH-SY5Y cells were seeded into 96-well plates to assess intracellular Ca2+ chelation, complex II activity, mitochondrial membrane potential (ΔΨm), ATP production, and release of inflammatory cytokines TNF-α and IL-1β with 2-, 4-, 6-, 24-, and 48-h DGA exposure. Peak Ca2+ chelation occurred at 4 h in cells treated with 6.25–50 mM DGA; however, effects were transient. Complex II activity was significantly decreased at all DGA concentrations tested, with 12.5 mM DGA causing 80% inhibition and 25 and 50 mM DGA causing 97 and 100% inhibition, respectively. Subsequently, 12.5–50 mM DGA concentrations significantly decreased ΔΨm at all time points. 50 mM DGA significantly increased release of TNF-α and IL-1β after 24 and 48 h with significantly decreased ATP production observed at the same time points and concentration. These studies demonstrate that the DGA-induced mechanism of SH-SY5Y cell death involves complex II inhibition leading to mitochondrial depolarization, and subsequent ATP depletion with accompanying inflammatory cytokine release. These results indicate a direct mechanism of DGA-induced neurotoxicity in vitro, similarly observed in other DEG-affected target organs.