Sulfur mustard (SM), a chemical weapon first employed during World War I, targets the skin, eyes, and lung. It remains a significant military and civilian threat. The characteristic response of human skin to SM involves erythema of delayed onset, followed by edema with inflammatory cell infiltration, the appearance of large blisters in the affected area, and a prolonged healing period. Several in vivo and in vitro models have been established to understand the pathology and investigate the mechanism of action of this vesicating agent in the skin. SM is a bifunctional alkylating agent which reacts with many targets including lipids, proteins, and DNA, forming both intra- and intermolecular cross-links. Despite the relatively nonselective chemical reactivity of this agent, basal keratinocytes are more sensitive, and blistering involves detachment of these cells from their basement membrane adherence zones. The sequence and manner in which these cells die and detach is still unresolved. Much has been discovered over the past two decades with respect to the mechanisms of SM-induced cytotoxicity and the intracellular and extracellular targets of this vesicant. In this review, the effects of SM exposure on the skin are described, as well as potential mechanisms mediating its actions. Successful therapy for SM poisoning will depend on following new mechanistic leads to develop drugs that target one or more of its sites of action.

The formation of reactive oxygen species by the cytochrome P450 monoxygenase system is thought to be due to autooxidation of NADPH-cytochrome P450 reductase and the non-productive decay of oxygen-bound cytochrome P450 intermediates. To characterize this process in recombinant microsomal enzymes, we used a highly sensitive hydrogen peroxide assay based on Amplex-Red oxidation. This assay is 20 times more sensitive (LLD = 5.0 pmoles/assay, and LLQ = 30 pmoles/assay) than the standard ferrous thiocyanate assay for detection of hydrogen peroxide. We found low, but detectable spontaneous generation of hydrogen peroxide by recombinant human NADPH-cytochrome P450 reductase complexes (0.034 nmoles hydrogen peroxide/min/100 Units of NADPH-cytochrome P450 reductase). Significantly higher rates of hydrogen peroxide production were observed when recombinant cytochrome P450 enzymes were coexpressed with NADPH-cytochrome P450 reductase (0.31 nmoles of hydrogen peroxide/min/100 Units of NADPH-cytochrome P450 reductase). This was independent of the addition of any exogenous cytochrome P450 substrates. These data demonstrate that cytochrome P450’s are a major source of hydrogen peroxide in the recombinant cytochrome P450 monooxygenase system. Moreover, substrate binding is not required for the cytochrome P450’s to generate reactive oxygen species.

Pulmonary fibrosis is one of the most severe consequences of exposure to paraquat, an herbicide that causes rapid alveolar inflammation and epithelial cell damage. Paraquat is known to induce toxicity in cells by stimulating oxygen utilization via redox cycling and the generation of reactive oxygen intermediates. However, the enzymatic activity mediating this reaction in lung cells is not completely understood. Using self-referencing microsensors, we measured the effects of paraquat on oxygen flux into murine lung epithelial cells. Paraquat (10 -100 M) was found to cause a 2-4-fold increase in cellular oxygen flux. The mitochondrial poisons cyanide, rotenone, and antimycin A prevented mitochondrial-but not paraquat-mediated oxygen flux into cells. In contrast, diphenyleneiodonium (10 M), an NADPH oxidase inhibitor, blocked the effects of paraquat without altering mitochondrial respiration. NADPH oxidases, enzymes that are highly expressed in lung epithelial cells, utilize molecular oxygen to generate superoxide anion. We discovered that lung epithelial cells possess a distinct cytoplasmic diphenyleneiodonium-sensitive NAD(P)H:paraquat oxidoreductase. This enzyme utilizes oxygen, requires NADH or NADPH, and readily generates the reduced paraquat radical. Purification and sequence analysis identified this enzyme activity as thioredoxin reductase. Purified paraquat reductase from the cells contained thioredoxin reductase activity, and purified rat liver thioredoxin reductase or recombinant enzyme possessed paraquat reductase activity. Reactive oxygen intermediates and subsequent oxidative stress generated from this enzyme are likely to contribute to paraquat-induced lung toxicity.Exposure of humans and animals to toxic doses of paraquat (1,1Ј-dimethyl-4,4Ј-bipyridylium) is known to damage the lung leading to pulmonary edema and hypertension, acute respiratory distress syndrome, and progressive lung fibrosis (1). In target cells paraquat undergoes redox cycling which may contribute to its toxic actions. Several mammalian NADPH oxidases have been identified as potential inducers of paraquat redox cycling including cytochrome P450 reductase and nitric-oxide synthase (2, 3). These enzymes generate a reduced paraquat radical that can act as an electron donor (4) (see Reaction 1). Reacting rapidly with molecular oxygen, the paraquat radical recycles back to paraquat and in the process forms highly toxic oxidants including superoxide anion, hydrogen peroxide, hydroxyl radicals, and in the presence of nitric oxide, peroxynitrite (5-7). Cellular damage generated by these oxidants, including lipid peroxidation, may be important in paraquatinduced lung damage (8, 9).Redox cycling reactions are known to consume significant quantities of oxygen (10 -12). In cells, this has the potential to reduce levels of oxygen available for metabolic processes resulting in oxidative stress and toxicity (13). Previous studies have demonstrated that paraquat can stimulate oxygen uptake by microsomes from rat and rabbit liver and rabbit lung as...

The one-electron reduction of redox-active chemotherapeutic agents generates highly toxic radical anions and reactive oxygen intermediates (ROI). A major enzyme catalyzing this process is cytochrome P450 reductase. Because many tumor cells highly express this enzyme, redox cycling of chemotherapeutic agents in these cells may confer selective antitumor activity. Nitrofurantoin is a commonly used redox-active antibiotic that possesses antitumor activity. In the present studies we determined whether nitrofurantoin redox cycling is correlated with cytochrome P450 reductase activity and cytotoxicity in a variety of cell lines. Recombinant cytochrome P450 reductase was found to support redox cycling of nitrofurantoin and to generate superoxide anion, hydrogen peroxide, and, in the presence of redox-active iron, hydroxyl radicals. This activity was NADPH dependent and inhibitable by diphenyleneiodonium, indicating a requirement for the flavin cofactors in the reductase. Nitrofurantoin-induced redox cycling was next analyzed in different cell lines varying in cytochrome P450 reductase activity including Chinese hamster ovary cells (CHO-OR) constructed to overexpress the enzyme. Nitrofurantoin-induced hydrogen peroxide production was 16-fold greater in lysates from CHO-OR cells than from control CHO cells. A strong correlation between cytochrome P450 reductase activity and nitrofurantoin-induced redox cycling among the cell lines was found. Unexpectedly, no correlation between nitrofurantoininduced ROI production and cytotoxicity was observed. These data indicate that nitrofurantoininduced redox cycling and subsequent generation of ROI are not sufficient to mediate cytotoxicity and that cytochrome P450 reductase is not a determinant of sensitivity to redox-active chemotherapeutic agents.

Background: Enzymes mediating chemical redox cycling in the lung are poorly defined. Results: Sepiapterin reductase was identified as a key mediator of redox cycling and was analyzed using inhibitors and sitedirected mutagenesis. Conclusion: Sepiapterin reductase generates reactive oxygen species during redox cycling in a mechanism distinct from sepiapterin reduction. Significance: This is the first report demonstrating that sepiapterin reductase mediates chemical redox cycling.