UO-126

Abelson murine leukemia viral oncogene homolog 1 (c-Abl, also known as ABL1) is a potent selenium-independent regulator of expression and activity of glutathione peroxidase-1 (GPx1) and extracellular signal-regulated kinase 1/2 (ERK1/2). Since GPx1-ERK1/2 pathway modulates dynamin-related protein 1 (DRP1) serine (S) 616 phosphorylation, we investigated whether c-Abl participates in GPx1-ERK1/2 interaction and DRP1-mediated mitochondrial dynamics in CA1 neurons in response to oxidative stress induced by L-buthionine sulfoximine (BSO, an oxidative stress inducer) and status epilepticus (SE). In the present study, BSO enhanced c-Abl tyrosine (Y) 245 phosphorylation, ERK1/2 activity and GPx1 upregulation in the CA1 region under physiological condition. Imatinib (a c-Abl inhibitor) ameliorated BSO-induced c-Abl Y245, but elicited further ERK1/2 phosphorylation without affecting GPx1 expression. GPx1 knockdown enhanced BSO-induced c-Abl Y245 phosphorylation, but decreased ERK1/2 activity. BSO also facilitated mitochondrial fission in CA1 neurons by augmenting DRP1 expression and its S616 phosphorylation in the CA1 region, which were diminished by GPx1 knockdown and U0126 (an ERK1/2 inhibitor), but reinforced by imatinib. SE increased c-Abl Y245 phosphorylation and mitochondrial length in CA1 neurons, accompanied by reduced GPx1 expression and ERK1/2 phosphorylation. Imatinib and N-acetylcysteine (NAC, an antioxidant) attenuated these post-SE events and CA1 neuronal death. However, GPx1 knockdown deteriorated SE-induced CA1 neuronal degeneration accompanied by augmenting c-Abl Y245 phosphorylation and mitochondrial elongation in CA1 neurons. These findings indicate that the impaired reciprocal regulation between c-Abl and GPx1 may cause CA1 neuronal degeneration in response to oxidative stress by abrogating ERK1/2-DRP1-mediated mitochondrial fission.

The tris (2-chloroethyl) phosphate (TCEP) and tri-n-butyl phosphate (TNBP) are two typical organophosphate esters (OPEs), which have estrogenic activity. However, fewer studies have been conducted on their genotoxicity and mechanisms. In this study, we evaluated the effects of TCEP and TNBP on cellular DNA damage in GT1-7 cells and examined the role of estrogen signaling in regulating DNA damage. The results showed that TCEP and TNBP significantly inhibited the cell viability and elevated intracellular reactive oxygen species (ROS) levels at higher exposure concentrations. TCEP and TNBP at test concentrations significantly increased comet tail length, comet tail DNA percentage (Tail DNA%), and Olive tail moment (OTM) values, indicating that TCEP and TNBP induced cellular DNA damage. In addition, TCEP and TNBP significantly upregulated the mRNA levels of target genes related to DNA damage-repair pathways, and elevated the expression of ataxia-telangiectasia mutated (ATM) and γ-H₂AX proteins associated with DNA double-strand breaks. Furthermore, TCEP and TNBP significantly up-regulated the expression of membrane bound G-protein coupled estrogen receptor 1 (GPER) protein and increased the phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2) at 10 µM. While the pretreatment of GPER1 inhibitor G15 and ERK1/2 inhibitor U0126 significantly inhibited the up-regulation of mRNA expression of the target genes associated with the DNA damage-repair pathways and decreased the DNA damage induced by TCEP and TNBP. These findings indicate that TCEP and TNBP activate GPER1/ERK1/2 signaling pathway which plays an important role in regulating DNA damage. The study will provide a novel insight into the genotoxicity mechanism of OPEs.