Sulforaphane induces phase II detoxication enzymes in mouse skin and prevents mutagenesis induced by a mustard gas analog
Highlights
► Sulforaphane induces increased levels of glutathione in mouse skin. ► Sulforaphane induces increased levels of GSTA4 in mouse skin. ► Sulforaphane, applied after CEES-treatment, completely abolishes CEES-mutagenesis. ► The therapeutic effect may suggest a long biological half-life for CEES in vivo.
Introduction
Mustard gas (bis-[2-chloroethyl]sulfide, SM) was first used in chemical warfare in 1917 (Papirmeister et al., 1991), and its potential use by terrorists against civilian populations is currently considered a plausible threat by the Department of Homeland Security (http://www.dhs.gov/xlibrary/assets/prep_chemical_fact_sheet.pdf). Dermal exposure results in severe chemical burns affecting all layers of the skin (Papirmeister et al., 1991), and patients require lengthy hospitalization. Despite recent progress in identifying agents that ameliorate some of the effects of mustards (Anumolu et al., 2010, Gordon et al., 2010, O'Neill et al., 2010, Anumolu et al., 2011), there are currently no effective therapies for the acute effects of dermal exposure. Mustard gas and a less toxic analog, 2-(chloroethyl) ethyl sulfide (CEES), induce DNA damage through structurally analogous electrophilic intermediates and are mutagenic (Ogston et al., 1946, Fox and Scott, 1980, Liu et al., 2010, Boulware et al., 2012). Epidemiological evidence suggests that chronic SM exposure leads to increased risk for respiratory malignancies (Wada et al., 1968, Easton et al., 1988), and there have been reports of carcinogenesis in mice following a single exposure (Heston, 1953).
Evidence has been presented (Drasch et al., 1987, Noort et al., 2002, Hattersley et al., 2008) suggesting that sulfur mustard has a relatively long biological half-life in skin, allowing ongoing damage to accumulate over a period of days. We have therefore been interested in identifying potential therapeutic agents that might act to detoxify the active electrophiles in situ and thereby decrease the overall level of macromolecular alkylation. We have recently shown (Boulware et al., 2012) that 2,6-dithiopurine (DTP), a nucleophilic scavenger of many electrophilic DNA-damaging agents (MacLeod et al., 1993, Qing et al., 1996), including CEES (Liu et al., 2010), can block CEES-induced mutagenesis in mouse skin. In this model, DTP was applied therapeutically beginning 1 h after CEES treatment. This suggests that biologically significant DNA damage continues to occur long after the initial treatment, providing a therapeutic window of hours to days. We therefore hypothesized that treatments that increase the organism's endogenous capacity for detoxifying electrophiles, for example through the glutathione (GSH) conjugation pathway (Lu, 2009), might also provide therapeutic benefit.
Sulforaphane (SFN), a natural isothiocyanate found in cruciferous vegetables, blocks DNA damage, mutagenesis and carcinogenesis caused by electrophilic chemicals (Cheung and Kong, 2010, Kwak and Kensler, 2010), by inducing several phase II detoxication enzymes that collectively enhance the conjugation of electrophiles to cellular reduced glutathione (GSH). Effects on both the biosynthesis of GSH, and the expression of GSH S-transferases (GSTs) have been observed (Marrot et al., 2008, Lu, 2009, Higgins and Hayes, 2011). In cultures of normal human epidermal keratinocytes, SFN has shown marginal protective effects against SM toxicity (Gross et al., 2006). Therapeutic and/or protective effects of SFN are currently under study in clinical trials for cancer and other chronic diseases (http://clinicaltrials.gov/ct2/results?term=sulforaphane). We now report the abolition of CEES-induced mutagenesis in an animal model by therapeutic treatment with SFN.
Section snippets
Materials and methods
Husbandry and treatment of C57BL/6 and Big Blue mice with CEES were as described previously (Boulware et al., 2012). Briefly, our standard mutagenesis protocol utilized a single topical treatment of shaved dorsal epidermis of Big Blue mice with 20 μl of 200 mM CEES dissolved in ethanol. This resulted in a total dose of 4 μmol CEES, and a dose density of ~ 2.3 μmol/cm2. Control mice received ethanol only. As described previously, this treatment resulted in a 4–5-fold increase in mutation frequency
Sulforaphane induces phase II detoxification enzymes in mouse skin
SFN is known to increase levels of cellular reduced glutathione (GSH) and of glutathione-S-transferases (GSTs) (Cheung and Kong, 2010). We applied SFN to the skin of C57BL/6 mice and measured levels of reduced GSH in epidermal extracts 24 h later. Increasing single doses of SFN produced increases in epidermal GSH (Fig. 1A, left panel); the increase was statistically significant with doses of 2 or 5 μmol. Significant increases were seen at the 5 μmol dose as early as 12 h post-treatment, and GSH
Discussion
SFN has been widely studied as a cancer chemopreventive agent due to its ability to induce phase II detoxication activities, particularly those involved with conjugation of cellular GSH to electrophilic intermediates of chemical carcinogens (Kwak and Kensler, 2010). Many such phase II genes induced by SFN, including various GSH S-transferases and GSH biosynthetic enzymes, are under the control of the Nrf2 signaling pathway, and the ability of SFN to prevent chemical carcinogenesis is abrogated
Conflict of interest
The authors declare that there are no conflicts of interest.
Acknowledgments
We thank Terry Kavanagh, Ted Mills and Rick Wood for thoughtful comments on the manuscript, Terry Kavanagh for providing antibodies, Jennifer Bubel and Steve Carbajal for technical support, and Rebecca Deen and Joi Holcomb for manuscript preparation. This work was supported by grant U01 NS058191 from NINDS through the CounterACT Program, and by Center Grants from the NCI (P30 CA016672) and the NIEHS (P30 ES007784).
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Current address: Department of Biology, Baylor University, Waco, TX 76798, USA.