Silibinin as a potential therapeutic for sulfur mustard injuries

• Proven efficacy of silibin prodrug against sulfur mustard toxicity in HaCaT cells.
• Dose–effect relationships evaluated, protection achieved by clinically relevant doses.
• Post-exposure treatment protects against acutely toxic sulfur mustard concentrations.
• Slight amplification of apoptosis and interleukin production only at highest silibinin concentration.
• Biotransformation of prodrug into free silibinin demonstrated by LC–ESI MS/MS.

Sulfur mustard (SM) is a vesicating chemical warfare agent causing skin blistering, ulceration, impaired wound healing, prolonged hospitalization and permanent lesions. Silibinin, the lead compound from Silybum marianum, has also been discussed as a potential antidote to SM poisoning. However, its efficacy has been demonstrated only with regard to nitrogen mustards. Moreover, there are no data on the efficacy of the water-soluble prodrug silibinin-bis-succinat (silibinin-BS).We investigated the effect of SIL-BS treatment against SM toxicity in HaCaT cells with regard to potential reduction of necrosis, apoptosis and inflammation including dose-dependency of any protective effects. We also demonstrated the biotransformation of the prodrug into free silibinin.HaCaT cells were exposed to SM (30, 100, and 300 μM) for 30 min and treated thereafter with SIL-BS (10, 50, and 100 μM) for 24 h. Necrosis and apoptosis were quantified using the ToxiLight BioAssay and the nucleosome ELISA (CDDE). Pro-inflammatory interleukins-6 and -8 were determined by ELISA. HaCaT cells, incubated with silibinin-BS were lysed and investigated by LC–ESI MS/MS.LC–ESI MS/MS results suggest that SIL-BS is absorbed by HaCaT cells and biotransformed into free silibinin. SIL-BS dose-dependently reduced SM cytotoxicity, even after 300 μM exposure. Doses of 50–100 μM silibinin-BS were required for significant protection. Apoptosis and interleukin production remained largely unchanged by 10–50 μM silibinin-BS but increased after 100 μM treatment.Observed reductions of SM cytotoxicity by post-exposure treatment with SIL-BS suggest this as a promising approach for treatment of SM injuries. While 100 μM SIL-BS is most effective to reduce necrosis, 50 μM may be safer to avoid pro-inflammatory effects. Pro-apoptotic effects after high doses of SIL-BS are in agreement with findings in literature and might even be useful to eliminate cells irreversibly damaged by SM. Further investigations will focus on the protective mechanism of silibinin and its prodrug and should establish an optimum concentration for treatment.