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Here we report a vertically integrated in vitro– in silicostudy that aims to elucidate the molecular initiating events involved in the induction of oxidative stress (OS) by seven diverse chemicals (cumene hydroperoxide, t-butyl hydroperoxide, hydroquinone, t-butyl hydroquinone, bisphenol A, Dinoseb, and perfluorooctanoic acid). To that end, we probe the relationship between chemical properties, cell viability, glutathione (GSH) depletion, and antioxidant gene expression. Concentration-dependent effects on cell viability were assessed by MTT assay in two Hepa-1 derived mouse liver cell lines: a control plasmid vector transfected cell line (Hepa-V), and a cell line with increased glutamate-cysteine ligase (GCL) activity and GSH content (CR17). Changes to intracellular GSH content and mRNA expression levels for the Nrf2-driven antioxidant genes Gclc, Gclm, heme oxygenase-1 (Hmox1), and NADPH quinone oxidoreductase-1 (Nqo1) were monitored after sublethal exposure to the chemicals. In silicomodels of covalent and redox reactivity were used to rationalize differences in activity of quinones and peroxides. Our findings show CR17 cells were generally more resistant to chemical toxicity and showed markedly attenuated induction of OS biomarkers; however, differences in viability effects between the two cell lines were not the same for all chemicals. The results highlight the vital role of GSH in protecting against oxidative stress-inducing chemicals as well as the importance of probing molecular initiating events in order to identify chemicals with lower potential to cause oxidative stress.

Introduction:Hematopoietic stem cell (HSC) gene therapy/editing is a viable treatment option for various hematological diseases and disorders including hemoglobinopathies and HIV/AIDS. Most if not all currently available approaches target CD34-enriched cell fractions, a heterogeneous mix of mostly committed progenitor cells and only very few true HSCs with long-term multilineage engraftment potential. As a consequence, gene therapy/editing approaches are currently limited in their HSC targeting efficiency, very expensive consuming huge quantities of modifying reagents, and can lead to unwanted side-effects in non-target cells.