A cautionary note on using N-acetylcysteine as an antagonist to assess isothiocyanate-induced reactive oxygen species-mediated apoptosis

Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC 20057, USA.
Analytical Biochemistry (Impact Factor: 2.22). 10/2010; 405(2):269-71. DOI: 10.1016/j.ab.2010.06.015
Source: PubMed


N-Acetylcysteine (NAC) has been widely used in cell culture-based studies for the role of reactive oxygen species (ROS) generation in apoptosis induction by isothiocyanates (ITCs). Here we have demonstrated, using [(14)C]phenethyl ITC and [(14)C]sulforaphane, that NAC pretreatment significantly reduces ITC cellular uptake by conjugating with ITCs in the medium, suggesting that reduced uptake of ITCs, rather than the antioxidant activity of NAC itself, is responsible for the diminished downstream apoptotic effects. The study provides a cautionary note on the assay in studying mechanisms of apoptosis by ITCs and other electrophilic and thiol-reactive compounds.

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    • "However, an alternative interpretation of our results is that XB05 can react directly with thiol groups, which are present in NAC or GSH but not AA. Reaction with NAC or GSH could potentially inhibit XB05 activity by sequestering the compound in the medium and prevent it from entering cells, as previously described for another compound [33]. To assess this possibility, we chose to examine the in vitro reactivity of XB05 with relevant thiols using the 5,5′-dithiobis-2-nitrobenzoic acid assay. "
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    • "In addition, NAC and GSH rescued the decrease of the mitochondria membrane potential and restored apoptosis-related proteins, such as capsase-9, caspase-3, PARP, p53 and p21, after physalin F treatment in A498 cells. Because NAC is a precursor of GSH, it has been reported to increase intracellular levels of glutathione, which is important to the prevention of oxidative stress in cells [30]. In addition, NAC and GSH might protect cells from damage caused by conjugation with electrophiles [30], [31]. "
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