Sulforaphane induces phase II detoxication enzymes in mouse skin and prevents mutagenesis induced by a mustard gas analog

Abstract

Mustard gas, used in chemical warfare since 1917, is a mutagenic and carcinogenic agent that produces severe dermal lesions for which there are no effective therapeutics; it is currently seen as a potential terrorist threat to civilian populations. Sulforaphane, found in cruciferous vegetables, is known to induce enzymes that detoxify compounds such as the sulfur mustards that react through electrophilic intermediates. Here, we observe that a single topical treatment with sulforaphane induces mouse epidermal levels of the regulatory subunit of glutamate-cysteine ligase, the rate-limiting enzyme in glutathione biosynthesis, and also increases epidermal levels of reduced glutathione. Furthermore, a glutathione S-transferase, GSTA4, is also induced in mouse skin by sulforaphane. In an in vivo model in which mice are given a single mutagenic application of the sulfur mustard analog 2-(chloroethyl) ethyl sulfide (CEES), we now show that therapeutic treatment with sulforaphane abolishes the CEES-induced increase in mutation frequency in the skin, measured four days after exposure. Sulforaphane, a natural product currently in clinical trials, shows promise as an effective therapeutic against mustard gas.

Fig. 1. Induction of Phase II detoxication genes in mouse skin by SFN

A. Epidermal extracts were assayed for reduced GSH content 24 h after the mice received various doses of SFN (left panel), applied topically in acetone, or at various times after receiving 5 µmol SFN (right panel). Each graph represents the mean and standard deviation of data from three mice per experimental group in three independent experiments. In all panels, asterisks indicate statistically significant differences from the acetone control, p<0.05. B. Epidermal extracts were assayed for GCLC and GCLM by immunoblotting 0, 12, 24 or 48 h after the mice received 5 µmol SFN; a representative immunoblot is shown. Both proteins were quantitated in the same gels. Data were normalized in each experiment to the level of tubulin seen in a duplicate gel, and data from three independent experiments are plotted in the right-hand panel. Open bars, GCLC; solid bars, GCLM. C. Epidermal extracts were assayed for GST-alpha and GSTA4 by immunoblotting 24 h after the mice received various doses of SFN (left panel); representative gels are shown. Data from three independent experiments were normalized to tubulin controls in the same gel and are presented in the right-hand panel. Open bars, GST-alpha; solid bars, GSTA4.

Fig. 2

Abolition of CEES-induced mutagenesis by sulforaphane. Big Blue mice (3 mice per group) were treated once with either ethanol (Et) or 200 mM CEES in ethanol (6). Beginning one h post-CEES, 4 daily treatments with acetone (Ac) or 5 µmol SFN dissolved in acetone were given. Genomic DNA was isolated from treated skin 4 days post-CEES, and mutation frequency determined; mean and standard deviation are plotted for each group. *, significantly different than Et/Ac control, p<0.0002.