Pretreatment of human epidermal keratinocytes with D,L-sulforaphane protects against sulfur mustard cytotoxicity.
Sulfur mustard (SM) is a powerful cytotoxic agent as well as a potent vesicant, mutagen, and carcinogen. This compound reacts with glutathione (GSH) and forms GSH-SM conjugates that appear to be excreted through the mercapturic acid pathway in mammals. The question of whether glutathione-S-transferases (GST) are involved in enzymatic formation of these conjugates remains unresolved. In previous studies, ethacrynic acid (EAA), a putative inhibitor of this transferase, and oltipraz, a known inducer,were ineffective in modulating this enzyme in cultured normal human epidermal keratinocytes (NHEK) so this hypothesis could not be tested. Higher levels of intracellular GSH appeared to be solely responsible for resistance of EAA-pretreated cells to SM. A better inducer of GST was needed to test whether this enzyme could be used to modify cytotoxicity following SM exposure. D,L-sulforaphane (DLS), a compound from broccoli extract known to be a potent inducer of this enzyme, was tested for GST induction in cultured NHEK. The enzyme levels increased optimally (40%) in these cells within 4 hours using 0.5 microg DLS/mL over a 48 hour incubation period. When the drug was removed by washing, and pretreated cells were challenged with 0-200 microM SM, there was a 10%-15% increase in survival at 24 hours compared with non-pretreated SM controls. This protective effect due to increased levels of GST was abolished at 300 microM sulfur mustard, where there was no difference in survival between pretreated and non-pretreated controls. Glutathione levels were also assessed and showed no increase at 4 hours in cultured NHEK with DLS pretreatment and appear not to be responsible for this protection against SM.
Gross
Clark L
CL
U.S. Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, Maryland, USA. clark.gross@us.army.mil
Nealley
Eric W
EW
Nipwoda
Mary T
MT
Smith
William J
WJ
D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002619:Chemical Warfare Agents / Q000633:toxicity*; D004305:Dose-Response Relationship, Drug; D004790:Enzyme Induction; D004817:Epidermis / Q000187:drug effects* / Q000201:enzymology; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000378:metabolism*; D006801:Humans; D017879:Isothiocyanates; D015603:Keratinocytes / Q000187:drug effects* / Q000201:enzymology; D009151:Mustard Gas / Q000633:toxicity*; D013454:Sulfoxides; D013861:Thiocyanates / Q000494:pharmacology* / Q000633:toxicity; D013997:Time Factors
D002619:Chemical Warfare Agents; D017879:Isothiocyanates; D013454:Sulfoxides; D013861:Thiocyanates; D005982:Glutathione Transferase; C016766:sulforaphane; D005978:Glutathione; D009151:Mustard Gas