Broccoli Components as Antioxidants: The Role of Isothiocyanates as Redox Modifiers via Glutathiolation

Abstract

Broccoli is a rich source of antioxidant nutrients. Extracts from broccoli have been shown to have powerful antioxidant capacity in assays that measure reactive oxygen species (ROS) scavenging. However, the relationship between chemical antioxidant assays and cellular measures of antioxidation has yet to be fully evaluated. Broccoli contains another group of dietary bioactive compounds, isothiocyanates, which do not have free radical scavenging capabilities but rather increase cellular antioxidant protection via indirect antioxidant mechanisms. Surprisingly, studies have shown that acutely, rather than acting as antioxidants, isothiocyanates may induce cellular oxidative stress. The role of isothiocyanate-induced oxidative stress in redox regulation by glutathiolation has yet to be evaluated. The objective of this project was to elucidate the role of broccoli components in antioxidant activity, both direct and indirect. Using 22 broccoli genotypes, the variation in phytochemical content was utilized to determine correlations among chemical composition (carotenoids, tocopherols and polyphenolics), chemical antioxidant activity (oxygen radical absorbance capacity; ORAC), and measures of cellular antioxidation (prevention of DNA oxidative damage and oxidation of the biomarker dichlorofluorescein, DCFH, in HepG2 cells) using hydrophilic and lipophilic extracts of broccoli. For lipophilic extracts, ORAC correlated with inhibition of cellular oxidation of DCFH (r = 0.596, p = 0.006). Also, DNA damage inhibition by lipophilic extract was negatively correlated with both chemical and cellular measures of antioxidant activity as measured by ORAC (r = -0.705, p = 0.015) and DCFH (r = -0.671, p = 0.048), respectively. However, no correlations were observed for hydrophilic extracts, except between polyphenol content and ORAC (r = 0.778, p < 0.001). These results indicate that chemical estimates of antioxidant capacity within the plant may not accurately reflect the complex nature of the full antioxidant activity of broccoli extracts within cells. The isothiocyanates sulforaphane and erucin transiently increased ROS and depleted glutathione (GSH) levels in HepG2 cells. Sulforaphane and erucin-treated HepG2 cells also resulted in the glutathiolation of cellular protein. These results indicate that isothiocyanates induce cellular oxidative stress, resulting in glutathiolation which may play a role in redox regulation of cell protein activity.