Evaluation of selected antioxidants and inhibitors of poly (ADP-ribose) polymerase-1 (PARP-1) as protective agents against mustard toxicity in vitro

Mustards are potent blistering agents. The prototype mustard is sulfur mustard gas, used in World War I and still feared in the present day, due to the absence of an effective antidote. Surrogate agents, such as mechlorethamine (HN2) and 2-chloroethylethyl sulfide (CEES), were used to mimic the toxicity of sulfur mustard gas which is not commercially available. Two related hypotheses, aimed at identifying an effective countermeasure to mustard toxicity, were tested here. The first hypothesis was that antioxidants would protect cultured skin cells from this toxicity. Test antioxidants included butylated hydroxyanisole (BHA), resveratrol (RES), pterostilbene (PTS), vitamin C (VIT C), ebselen (EB-1) and three related organoselenium analogs (EB-2, EB-3, and EB-4). In the absence of test antioxidant, a reproducible and robust cell death was observed in the cells following incubation with HN2 (25 microM, 24 or 48 h) or CEES (1000 microM, 24 h), while cells treated with test antioxidant alone (15, 30 or 60 microM) for similar periods of time were generally not affected. When incubated in the presence of both mustard agents and test antioxidant for 24 h or 48 h, it was found that EB-1, EB-2, EB-3 and EB-4 could spare the cells from death, while the other antioxidants could not. The EB-4 compound was most effective at reducing vesicant toxicity. The organoseleniums were then examined for their effects on reduced (GSH) and oxidized (GSSG) glutathione levels in the skin cells. Among these organoselenium compounds, cells incubated with EB-4 exhibited the highest intracellular levels of GSSG and the lowest GSH/GSSG ratio. In addition, EB-4 completely blocked HN2-induced lipid peroxidation. These studies, taken together, validate the first hypothesis. The second hypothesis proposed that selected inhibitors of PARP-1, including 3-aminobenzamide (3-AB), 3-indazolinone (TT-1), indazole-3-carboxamide (TT-2), and 1-(3-(isatin-1-yl)propyl)-1H-indazole-3-carboxamide (TT-3), would reduce mustard-induced toxicity in the A-431 skin cells. It was found that 3-AB and TT-2 conferred a small but significant protection from HN2 toxicity while TT-1 and TT-3 could not. In general, these compounds demonstrated a weaker ability to protect against HN2 than the organoseleniums. Taken together, the data presented in this work have identified organoselenium antioxidants as promising countermeasures to mustard agents in vitro worthy of future investigation in vivo.