In keratinocytes, UVB light stimulates the production of reactive oxygen species (ROS). Lysates of these cells were found to possess a non-dialyzable, trypsin- and heat-sensitive material capable of generating ROS in response to UVB light. Using ion exchange, metal affinity, and size exclusion chromatography, a 240-kDa protein was isolated with ROS generating activity. The protein exhibited strong absorption in the 320-360 nm range with additional soret peaks around 400-410 nm, suggesting the presence of heme. Sequencing using liquid chromatography-ion trap mass spectrometry identified the protein as catalase. Using purified catalases from a variety of species, the ROS generating activity was found to be temperature- and O2-dependent, stimulated by inhibitors of the catalatic activity of catalase, including 3-aminotriazole and azide, and inhibited by cyanide. A marked increase in the production of ROS was observed in UVB-treated cells overexpressing catalase and decreased generation of oxidants was found in UVB-treated keratinocytes with reduced levels of catalase. Our data indicate that catalase plays a direct role in generating oxidants in response to UVB light. The finding that catalase mediates the production of ROS following UVB treatment is both novel and highly divergent from the well known antioxidant functions of the enzyme. We hypothesize that, through the actions of catalase, high energy DNA damaging UVB light is absorbed by the enzyme and converted to reactive chemical intermediates that can be detoxified by cellular antioxidant enzymes. Accumulation of excessive ROS, generated through the action of catalase, may lead to oxidative stress, DNA damage, and the development of skin cancer.

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.

Acetaminophen is a mild analgesic and antipyretic agent known to cause centrilobular hepatic necrosis at toxic doses. Although this may be due to a direct interaction of reactive acetaminophen metabolites with hepatocyte proteins, recent studies have suggested that cytotoxic mediators produced by parenchymal and nonparenchymal cells also contribute to the pathophysiological process. Nitric oxide is a highly reactive oxidant produced in the liver in response to inflammatory mediators. In the present studies we evaluated the role of nitric oxide in the pathophysiology of acetaminophen-induced liver injury. Treatment of male Long Evans Hooded rats with acetaminophen (1 g/kg) resulted in damage to centrilobular regions of the liver and increases in serum transaminase levels, which were evident within 6 hours of treatment of the animals and reached a maximum at 24 hours. This was correlated with expression of inducible nitric oxide synthase (iNOS) protein in these regions. Hepatocytes isolated from both control and acetaminophen-treated rats were found to readily synthesize nitric oxide in response to inflammatory stimuli. Cells isolated from acetaminophen-treated rats produced more nitric oxide than cells from control animals. Production of nitric oxide by cells from both control and acetaminophen-treated rats was blocked by aminoguanidine, a relatively specific inhibitor of iNOS. Arginine uptake and metabolism studies revealed that the inhibitory effects of aminoguanidine were due predominantly to inhibition of iNOS enzyme activity. Pretreatment of rats with aminoguanidine was found to prevent acetaminophen-induced hepatic necrosis and increases in serum transaminase levels. This was associated with reduced nitric oxide production by hepatocytes. Inhibition of toxicity was not due to alterations in acetaminophen metabolism since aminoguanidine had no effect on hepatocyte cytochrome P4502E1 protein expression or N-acetyl-pbenzoquinone-imine formation. Taken together, these data demonstrate that nitric oxide is an important mediator of acetaminophen-induced hepatotoxicity. (HEPATOLOGY 1998; 26:748-754.)

Inhaled nitric oxide is a targeted pulmonary vasodilator that improves clinical outcomes for newborn patients with persistent pulmonary hypertension of the newborn, and may be effective in treating some premature patients with acute respiratory distress syndrome or lung disease of prematurity. Nitric oxide is now recognized as playing an important role in the regulation of diverse physiological processes. However, the pharmacological properties of inhaled nitric oxide are not easy to separate from its toxicological effects. For example, the intended effect of inhaled nitric oxide, vasodilation in the lung, is mediated, in part, by increased cellular cyclic GMP (cGMP). However, increased cGMP can also interfere with normal cellular proliferation. Nitric oxide has also been shown to cause DNA strand breaks and/or base alterations that are potentially mutagenic. Inhaled nitric oxide can rapidly react with oxygen in the lung to form nitrogen dioxide, which is a potent pulmonary irritant. Nitric oxide also reacts with superoxide anion to form peroxynitrite, a cytotoxic oxidant that can interfere with surfactant functioning. The overall effect of inhaled nitric oxide in potentiating or attenuating inflammation and oxidative damage in diseased lung is dependent on the dose administered. Furthermore, despite rapid inactivation by circulating hemoglobin, inhaled nitric oxide exerts effects outside the lung, including blocking platelet aggregation, causing methemoglobinemia, and possibly inducing extrapulmonary vasodilation. The toxicology of inhaled nitric oxide is not completely understood and must be considered in the design of protocols for its safe and effective clinical use.

Catalase is a highly conserved heme-containing antioxidant enzyme known for its ability to degrade hydrogen peroxide into water and oxygen. In low concentrations of hydrogen peroxide, the enzyme also exhibits peroxidase activity. We report that mammalian catalase also possesses oxidase activity. This activity, which is detected in purified catalases, cell lysates, and intact cells, requires oxygen and utilizes electron donor substrates in the absence of hydrogen peroxide or any added cofactors. Using purified bovine catalase and 10-acetyl-3,7-dihydroxyphenoxazine as the substrate, the oxidase activity was found to be temperature-dependent and displays a pH optimum of 7-9. The K m for the substrate is 2. Mammalian catalase belongs to a family of Fe-protoporphyrin IX containing proteins that include a variety of cytochromes, globins, and peroxidases and is one of the best characterized antioxidant enzymes (1). As a homotetrameric heme-containing enzyme, it is known for its ability to convert hydrogen peroxide into water and oxygen (catalatic activity), and in the presence of low concentrations of hydrogen peroxide to oxidize low molecular weight alcohols (peroxidatic activity). The conversion of hydrogen peroxide to water and oxygen by catalase is a two-step process whereby catalase heme Fe 3ϩ reduces one molecule of hydrogen peroxide to water, generating a covalent Fe 4ϩ ϭO oxyferryl species and a porphyrin cation radical. This reaction intermediate, referred to as compound I, then oxidizes a second hydrogen peroxide molecule forming molecular oxygen and water (1-3) (see Fig. 1). The peroxidatic activity of catalase results from the ability of compound I to oxidize alcohols to aldehydes and water (4 -6) ( Fig. 1). Each catalase monomer binds one molecule of heme; the holoenzyme also binds two molecules of NADPH, although the precise role of this cofactor in enzymatic activity is unclear, because hydrogen peroxide provides both oxidative and reductive potential during catalysis. Recent studies suggest that NADPH may be important in maintaining catalase in an active state (7).In mammalian cells, catalase is found at high concentrations in peroxisomes, along with a variety of oxidases and peroxidases (8). It has been suggested that the enzyme protects cells by removing hydrogen peroxide produced by flavin containing oxidases in the peroxisome, thereby preventing the accumulation of toxic levels of this reactive oxygen intermediate (9). However, hydrogen peroxide is important for an array of activities, including peroxidase-mediated metabolism, in cells, and potentially, without this reactive oxygen intermediate, cellular functioning would be limited. In addition, the K m for the catalatic activity of catalase is Ͼ10 mM, therefore, at low intracellular concentrations of hydrogen peroxide, this reaction is not kinetically favored, and it is assumed that peroxidases such as glutathione peroxidase or the recently discovered l-Cys peroxiredoxins effectively lower intracellular concentrations of hydrogen peroxide (10). In...

No abstract

Sulfur mustard (SM) is a chemical weapon that targets the skin, eyes, and lung. It was first employed during World War I and it remains a significant military and civilian threat. As a bifunctional alkylating agent, SM reacts with a variety of macromolecules in target tissues including nucleic acids, proteins and lipids, as well as small molecular weight metabolites such as glutathione. By alkylating subcellular components, SM disrupts metabolism, a process that can lead to oxidative stress. Evidence for oxidative stress in tissues exposed to SM or its analogs include increased formation of reactive oxygen species, the presence of lipid peroxidation products and oxidized proteins, and increases in antioxidant enzymes such as superoxide dismutase, catalase, and glutathione-S-transferase. Inhibition of antioxidant enzymes including thioredoxin reductase by SM can also disrupt cellular redox homeostasis. Consistent with these findings, SM-induced toxicity has been shown to be reduced by antioxidants in both in vitro and in vivo models. These data indicate that drugs that target oxidative stress pathways may represent important candidates for reducing SM-induced tissue injury.

No abstract