ABSTRACT
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.
Subject(s)
Chemical Warfare Agents/toxicity , Epidermis/drug effects , Glutathione Transferase/metabolism , Keratinocytes/drug effects , Mustard Gas/toxicity , Thiocyanates/pharmacology , Cell Survival/drug effects , Cells, Cultured , Dose-Response Relationship, Drug , Enzyme Induction , Epidermis/enzymology , Glutathione/metabolism , Glutathione Transferase/biosynthesis , Humans , Isothiocyanates , Keratinocytes/enzymology , Sulfoxides , Thiocyanates/toxicity , Time FactorsABSTRACT
Sulfur mustard (SM) is a potent alkylating agent, profoundly cytotoxic, and a powerful vesicant. SM reacts quite extensively with glutathione (GSH) and forms GSH conjugates, which are presumably excreted through the mercapturic acid pathway in mammals. It is unknown whether any enzymes, such as the glutathione-S-transferases (GST), are involved in this detoxification of SM by the formation of conjugates. A prototypic inhibitor (ethacrynic acid, EAA) and a prototypic inducer (Oltipraz, OLT) of GSH-S-transferase, have been used as pretreatment compounds in human epidermal keratinocytes (HEK) to investigate the effect of enzyme levels on cytotoxicity following SM challenge from 50 muM to 300 muM. Pretreatment of HEK for 24 h with EAA doubled survival against 200 muM SM (36% viability in non-pretreated cells vs. 81% in EAA-pretreated cells) and quadrupled survival (17% viability in non-pretreated controls vs. 71% in EAA-pretreated cells), while OLT pretreatment had no effect on cytotoxicity at either SM dose. The role of GST in SM cytotoxicity could not be tested because of the lack of an effect on modulation of GST activities by these 2 drugs. Cellular levels of GSH were increased 250-300% over control values using EAA pretreatment, while OLT pretreatment did not lead to any increase in GSH. Pretreatment of HEK with buthionine sulfoximine (BSO), a known depleter of glutathione levels, reduced glutathione levels and increased cytotoxicity. This large increase in GSH appears to be solely responsible for the enhanced survivability of EAA-pretreated HEK.
ABSTRACT
Sulfur mustard (SM) is a potent vesicating agent that has pronounced cytotoxic effects as well as mutagenic, carcinogenic, and radiomimetic properties. Isolated human peripheral blood lymphocytes (PBLs) and human epidermal keratinocytes (HEKs) have been used as in vitro models for determining SM-induced cytotoxicity. A recently developed colorimetric assay (the CellTiter 96 AQ ueous Non-radioactive Cell Proliferation Assay) was assessed using both of the in vitro models described above. Using 24- or 96-well microplates, reproducible (+/- 10%) SM dose/response curves for both types of human cells were obtained using a spectrophotometric microplate reader set at 490 nm. After a 4-h incubation time, as many as 96 sample wells could be measured within 45 s using this commonly available equipment. Multiple plates of samples can be run immediately. This technique may facilitate cytotoxicity investigations of new candidate compounds for both prophylaxis of and therapy for SM intoxication.
