Oxidative injury induced by drinking water disinfection by-products dibromoacetonitrile and dichloroacetonitrile in mouse hippocampal neuronal cells: The protective effect of N-acetyl-L-cysteine

Abstract

Dibromoacetonitrile (DBAN) and dichloroacetonitrile (DCAN) are haloacetonitriles (HANs) produced as by-products of chloramine disinfection of drinking water and can cause neurotoxicity. The molecular pathways leading to HAN-induced neuronal cell death remain unclear. The nuclear factor erythroid 2-related factor 2 (Nrf2) is an important regulator of oxidation reactions. We explored the role of the sequestosome 1 (p62)-Kelch-like ECH-associated protein 1 (Keap1)-Nrf2 pathway in DBAN- and DCAN-induced mouse hippocampal neuronal (HT22) cell injury. DBAN and DCAN reduced cell viability, increased lactate dehydrogenase release rate, and promoted apoptosis. Over the same treatment time, DBAN at lower concentrations caused cell injury, suggesting that DBAN is more cytotoxic than DCAN. DBAN and DCAN triggered oxidative stress by reducing intracellular glutathione and increasing reactive oxygen species concentrations. DBAN and DCAN activated the Nrf2 pathway. Furthermore, Nrf2 inhibitors (all-trans retinoic acid) attenuated DBAN- and DCAN-induced toxicity, whereas Nrf2 activators (tert-Butylhydroquinone) achieved the opposite effect. This indicates that activation of the Nrf2 pathway mediates DBAN- and DCAN-induced cell injury. Notably, the expression of p62, a noncanonical pathway that mediates Nrf2 activation, increased, whereas the expression of Keap1, another regulator of Nrf2, decreased. We noted that high p62 expression activated the Nrf2 pathway, and p62 was regulated through Nrf2, forming a positive feedback loop. N-acetyl-L-cysteine, a mercaptan substance, protected against DBAN- and DCAN-induced toxicity and inhibited the Nrf2 pathway. In summary, Nrf2 pathway inhibition and mercaptan supplementation prevent DBAN- and DCAN-induced HT22 cell injury, accordingly, targeting them is a potential approach to preventing HAN-induced neurotoxicity.

Keywords: By-products of drinking water disinfection; Dibromoacetonitrile; Dichloroacetonitrile; N-acetyl-L-cysteine; p62–Keap1–Nrf2 pathway.