Neuroprotective Effects of N,N′bis‐(2‐mercaptoethyl) Isophthalamide (NBMI) Against Lead Induced Toxicity in U‐87 MG Cells

Abstract

Background and ObjectiveLead is one of the most dangerous and ubiquitous environmental toxins with no levels safe for human exposure. Lead causes neurotoxicity primarily by inducing oxidative stress. After crossing the blood brain barrier, lead is taken up by glial cells which serve as protective lead‐binding proteins. While chelation is the mainstay of therapy for lead poisoning, currently FDA approved chelators such as dimercaptosuccinic acid (DMSA) are associated with many adverse effects and safety concerns. N,N′bis‐(2‐mercaptoethyl) isophthalamide (NBMI) is a newer lipophilic water insoluble compound with potential metal chelating and antioxidant properties. The purpose of this study was to determine whether NBMI displayed neuroprotective effects in U‐87 malignant glioblastoma (U‐87 MG) cells exposed to lead acetate (PbAc).Materials and MethodsU‐87 MG cells were treated with either vehicle, NBMI (0–100 μM), or DMSA (0–100 μM) for 24 hours followed by PbAc (0–300 μM) for 48 hours. Cell viability (CellTiter‐Glo® Assay), cell morphology (trypan blue staining), degree of oxidative stress (GSH‐GloTM Glutathione Assay), and impact on apoptosis (Bax and Bcl‐2 protein levels using Western blotting) were determined. Comparisons between multiple groups were performed using one‐way or two‐way analysis of variance (ANOVA) followed by a post‐hoc Tukey’s test. Results with a p value <0.05 were considered statistically significant.ResultsExposure of U‐87 MG cells to PbAc decreased cell viability in both a concentration and time‐dependent manner. Trypan blue staining revealed that PbAc negatively altered the cell morphology. In addition, PbAc exposure led to an increase in total glutathione levels, as well as the Bax/Bcl‐2 ratio, indicating increased oxidative stress and initiation of the cell apoptotic pathway. Pretreatment with NBMI diminished PbAc‐induced cell death, morphology changes, increase in total GSH, and increase in the Bax/Bcl‐2 ratio. Interestingly, the effects of NBMI were more prominent compared to DMSA in attenuating PbAc‐induced cell death.ConclusionLead exposure induces cell death and oxidative stress which are reversed by pretreatment with NBMI implying that NBMI is a new and promising chelator for treating PbAc‐induced neurotoxicity.