Dietary vitamin E and selenium and toxicity of nitrite and nitrate

Toxicology 180 (2002) 195 /207 www.elsevier.com/locate/toxicol

Dietary vitamin E and selenium and toxicity of nitrite and nitrate C.K. Chow a,*, C.B. Hong b a

Graduate Center for Nutritional Sciences, University of Kentucky, Lexington, KY 40506-0054, USA b Department of Veterinary Sciences, University of Kentucky, Lexington, KY 40506-0099, USA

Abstract Nitrites and nitrates are important antimicrobial and flavoring/coloring agents in meat and fish products. However, nitrites and nitrates may cause methemoglobinemia and other illness, and may react with certain amines to form carcinogenic nitrosamines. The nutritional status of vitamin E and selenium has long been associated with nitrite and nitrate toxicity, although the mechanism involved is not yet clear. Information available recently shows that nitrites and nitrates are both oxidation products and ready sources of nitric oxide (NO+ ), that NO+ reacts rapidly with superoxide to form highly reactive peroxynitrite (ONOO
/), and that vitamin E may mediate the generation and availability of superoxide and NO+ . Increased formation of ONOO
/ resulting from nitrite treatment and low intake of vitamin E and selenium may thus be the critical event leading to tissue damage and animal mortality observed previously. The protection against the adverse effects of nitrites/nitrates by vitamin E is attributed to its ability to reduce ONOO
/ formation, while selenium exerts its protective effects via seleno-enzymes/compounds, which reduce ONOO
/ formed. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Vitamin E; Selenium; Superoxide; Nitric oxide; Peroxynitrite; Nitrite; Nitrate

1. Introduction Changes in patterns of agricultural practice, food processing and industrialization have impacted accumulation of nitrates/nitrites in the environment. Intensive farming practice has resulted in an increasing use of nitrogen-based fertilizers, particularly with corn, vegetables, other row crops and forages. Livestock and poultry production as well as urban sewage treatment * Corresponding author. Tel.: /1-859-257-7783; fax: /1859-257-4311 E-mail address: [email protected] (C.K. Chow).

also contribute nitrogenous wastes to the soil and water. Bacteria present in the soil, water, feed and foods are capable of utilizing these nitrogenous compounds to synthesize nitrates (and nitrites) de novo via heterotrophic nitrification (and nitrate reduction) (Tannenbaum et al., 1978). Nitrates and nitrites, used in combination with salt, serve as important antimicrobial agents in meat to inhibit the growth of bacterial spores that cause botulism, a deadly food-borne illness. Nitrites are also used as preservatives and for flavoring and fixing color in a number of red meat, poultry, and fish products. Additionally, nitrites have been employed as a vasodilator, or a

0300-483X/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 0 - 4 8 3 X ( 0 2 ) 0 0 3 9 1 - 8

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circulatory depressant to relieve smooth muscle spasm, and an antidote for treating cyanide poisoning (Nickerson, 1970), and potassium nitrate has been used therapeutically as a diuretic agent. Other precursors or sources of nitrites include N-containing foods, nitrogen containing drugs/chemicals (e.g. nitroglycerin, C3H5N3O9, an anti-anginal agent and coronary vasodilator; sodium nitroprusside, Na2[Fe(CN)6NO], an antihypertensive agent) and nitric oxide (NO+ ). Nitric oxide, which is synthesized in vivo from Larginine, can be oxidized to nitrite or nitrate. Nitric oxide is also a component of cigarette smoke and automobile exhaust. Thus, man and animals are subjected to significant nitrate and nitrite levels in foods, feed and water, as well as those formed in vivo. Nitrites and nitrates formed from nitrogenous sources by microorganisms in saliva and intestine, however, are the major source of human exposure under normal conditions (White, 1975; Tannenbaum et al., 1978). Nitrites and nitrates occupy a unique position in human toxicology. They are both ubiquitous in the environment and can be formed from nitrogenous compounds by microorganisms present in the soil, water, saliva and the gastrointestinal tract. Nitrites and nitrates are important antimicrobial agents against botulism, as well as food preservatives and flavoring/coloring agents in meat and fish products. These compounds, on the other hand, can cause adverse cellular effects, although the extent and significance of health risk resulting from exposure of nitrites and nitrates remain unclear. Also, the mechanism involved in the protection against nitrate and nitrite toxicity by dietary vitamin E and selenium has yet to be delineated. The information available recently provides a plausible explanation on the role of vitamin E and selenium in protecting against the adverse effects of nitrates and nitrites.

2. Adverse effects of nitrites/nitrates Formation of methemoglobin, a substance that interferes with the ability of blood cells to carry oxygen when concentrations reach 30/40% of total hemoglobin concentrations, is the most

recognizable and detectable sign of nitrite and nitrate toxicity in humans. Fatal toxic methemoglobinemia due to high levels of nitrite in drinking water or occupational exposure to nitrite has been reported (Ger et al., 1996). Also, nitrites may react with certain amines in foods to produce carcinogenic N-nitroso compound nitrosamines (Van Maanen et al., 1998; Atanasova-Goranova et al., 1997; Mirvish et al., 2000), and many of which are known to cause cancer. A high dietary intake of nitrite and nitrate has been implicated as a risk factor for human cancer, and formation of nitroso-compounds in the stomach during inflammation is correlated with nitrate in food and water (Ames, 1983; Mirvish, 1995). However, the reported association between the risk of cancer and nitrate/nitrate intake are inconsistent. Weyer et al. (2001), for example, have shown that among a cohort of 21 977 Iowa women using the same water supply for more than 10 years, there are positive associations between nitrate intake and incidence of bladder cancer and ovarian cancer, and inverse associations for uterine cancer and rectal cancer after adjustment for a variety of cancer risk/protective factors, agents that affect nitrosation (smoking, vitamin C, and vitamin E intake), dietary nitrate, and water source. However, the incidence of cancer is not associated with increased nitrate in drinking water, nor is there clear and consistent associations for non-Hodgkin’s lymphoma, leukemia, melanoma, or cancers of the colon, breast, lung, pancreas, or kidney. Also, nitrate intake is not related to urinary excretion of volatile nitrosamine of non-smokers (Levallois et al., 2000). Similarly, concurrent administration of fishmeal and sodium nitrite does not promote renal carcinogenesis in rats after initiation with N -ethyl-N -hydroxyethylnitrosamine (Furukawa et al., 2000). A number of biochemical changes, functional impairments and histopathological lesions have been observed in nitrite-treated rats. Orally administered 25 /100 mg of sodium nitrite/kg diet to Balb/c mice for 21 days results in a dose dependent decrease in lymphocyte percentages, concanavalin A- and lipopolysaccharide-induced lymphocyte proliferation, natural killer cell activity against WEHI-164 target cells, as well as IgM and IgG

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titers against injected sheep erythrocytes, and the immunosuppressive effect of sodium nitrite is reversible after cessation of exposure (Abuharfeil et al., 2001). Based on the incidence of hypertrophy, zona glomerulosa of the adrenal in rats treated with nitrite for 13 weeks, the no-effect level is lower than 100 mg KNO2/liter in the drinking water, which is equivalent to a level lower than 10 mg KNO2/kg body weight per day (Til et al., 1988). While nitrates are not as toxic as nitrites, ingestion of large amount of nitrates may cause severe gastroenteritis. Methemoglobinemia, anemia and nephritis caused by prolonged exposure to small amounts of nitrates are likely resulting from its reduction to nitrite by bacteria. The LD50 orally for KNO3 in rabbit is 1.166 g/kg, and is 108 mg/kg for KNO2 (Dollahite and Rowe, 1974). Due to the potential harmful effects of nitrite and nitroso-compounds, a food manufacturer wanting to use nitrite salts must show that nitrosamines will not form in hazardous amounts in the product under the additive’s intended conditions of use. The currently allowable daily intakes for nitrites and nitrates (8 and 220 mg/ person per day, respectively) are based on studies with rats (Joint FAO/WHO Expert Committee on Food Additives, 1974). As infants do not possess efficient methemoglobin reduction systems as adults, nitrites and nitrates are not permitted in baby, junior or toddler foods.

3. Nitrate/nitrite and reactive oxygen/nitrogen species Oxidative damage is a consequence of excessive oxidative stress and/or insufficient antioxidant potential (Chow, 1979, 1991; Halliwell, 1987; Sies, 1986). Oxidative damage induced by reactive oxygen species (ROS) is an important contributing factor in the pathogenesis of cancer, cardiovascular diseases, aging and neurodegenerative diseases (Ames et al., 1993; Freeman and Crapo, 1982; Halliwell, 1987; Halliwell et al., 2000; Sies, 1986; Yu, 1994). Increasing evidence indicates that superoxide plays a central role in the generation and action of ROS. In the presence of superoxide

197

dismutase (SOD), superoxide is readily converted to hydrogen peroxide (H2O2). Hydrogen peroxide in turn can be reduced to water by glutathione peroxidase (GPx) or catalase, or be converted to highly reactive hydroxyl radicals (+ OH) via the iron-catalyzed Haber /Weiss reaction. Hydroxyl radical is the most oxidizing radical that is likely to arise in biological systems (Buettner, 1993). Also, superoxide may interact readily with NO+ to form another reactive free radical peroxynitrite (ONOO
/). Peroxynitrite is a relatively stable free radical produced by activated macrophages and neutrophils. It is 1000 times more oxidizing than H2O2, and its half-life in solution is only 1/2 s (Van Dyke, 1997). Additionally, the protonated form of ONOO
/ may be decomposed to form highly reactive.+ OH and nitrogen dioxide (Beckman et al., 1990; Augusto et al., 1994). Peroxynitrite is a potent cytotoxic oxidant (Pryor and Squadrito, 1995; Squadrito and Pryor, 1998) and is implicated in a variety of free radical-induced tissue injury, including inhibition of mitochondrial electron transport chain, which leads to more ROS generation (Wallace et al., 1998), reperfusion injury (Liu et al., 1998; Nossuli et al., 1998), vascular hyporesponsiveness to constrictor agents seen in experimental sepsis (Chabot et al., 1997), inflammatory cell-mediated tissue injury (Greenacre et al., 1997; Uesugi et al., 1998), chronic renal failure patients with septic shock (Fukuyama et al., 1997), and low density lipoprotein oxidation in atherogenesis (Pannala et al., 1998; Thomas et al., 1998). The ability of ONOO
/ to cross cell membranes contributes to its toxicity by allowing access to intracellular target molecules (Macfadyen et al., 1999). Peroxynitrite has also been shown to inhibit/inactivate the activities of ornithine decarboxylase (Seidel et al., 2001), catalase (Keng et al., 2000) and cytochrome P450BM-3 (Daiber et al., 2000), GPx (Padmaja et al., 1998) and promote the formation of phospholipid hydroperoxides (Shi et al., 1999), nitration of cytochrome c (Cassina et al., 2000) and modification of GSH reductase (Francescutti et al., 1996). Nitric oxide, also known as the endothelium derived relaxation factor, is an important free radical mediator (Ignarro et al., 1987) character-

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ized by a high spontaneous chemical reactivity with many other molecules notably with superoxide to form ONOO
/. The steady-state concentration of NO+ is determined, not only by its rate of formation, but also by its rate of decomposition (Kelm, 1999). The biosynthesis of NO+ from Larginine is catalyzed by a family of NO+ synthase. Under hypoxic conditions this mechanism of NO+ synthesis may be impaired, and NO+ formed by a NO+ synthase-independent mechanism, including the generation of NO+ by a nitrite reductase activity of xanthine oxidase (Zhang et al., 1998; Millar et al., 1998). The ability of NO+ to react with iron complexes renders the cytochrome P450 series of microsomal enzymes natural targets for inhibition by NO+ , and may provide negative feedback control of NO+ synthesis. Nitric oxide released in small quantities decreases tumor cell growth and levels of prostaglandin E2 and F2 alpha (proinflammatory products) and increases protein synthesis and DNA-repair enzymes in isolated hepatocytes. Thus, NO+ possesses both cytoprotective and cytotoxic properties depending on the amount and the isoform of NO+ synthase by which it is produced (Alexander, 1998). Recently both nitrates and nitrites are recognized as key oxidation products of NO+ , which can be generated in tissues by either direct disproportionation or reduction of nitrate/nitrite under the acidic and highly reduced conditions such as ischemia (Zweier et al., 1999). This NO+ formation is not blocked by NO+ synthase inhibitors and is likely responsible for NO+ formation with long periods of ischemia progressing to tissue necrosis. Also, NO+ has a high affinity for hemoglobin which catalyzes the degradation of NO+ to nitrate (Reid, 2000). The vasodilating property of nitrites recognized for sometime (Nickerson, 1970), can now be attributed to its ability to serve as a precursor of NO+ . As NO+ can react readily with superoxide, by releasing NO+ sodium nitrite serves to scavenge superoxide generated (Dalloz et al., 1997). Using electron paramagnetic resonance and spin trapping techniques, Carmichael et al. (1993) have shown that the reactions of H2O2 with nitrite and of superoxide with NO+ have a common ONOO
/ intermediate.

Nitration of tyrosine residues in proteins occurs in a wide range of inflammatory processes involving neutrophil and macrophage activation. Peroxynitrite formed from nitrite and H2O2 (superoxide) may be responsible for nitrotyrosine generated in activated neutrophils and macrophages (Sampson et al., 1998). Formation of nitrotyrosine is associated with DNA strand breaks and mutations, and with interference of protein tyrosine-based signaling and other protein functions. Sodium nitrite, as in ONOO
/, has also been shown to greatly increase H2O2-dependent covalent cross-linking of immune complexes (Uesugi et al., 1998). The findings that sodium nitrite administration reduces myocardial GPx activity (Yang and Wang, 1991) suggest that formation of ONOO
/ is associated with nitrite toxicity (Padmaja et al., 1998).

4. Nitrite/nitrate and antioxidants Direct chemical interaction between antioxidants and nitrites/nitrates has long been recognized. Nitrite has been shown to enhance atocopherol oxidation in the presence of SOD, H2O2 and saturated 1, 2-dilauroyl-sn-glycero-3phosphatidylcholine liposomes, and the major product of a-tocopherol is a-tocopheryl quinone (Singh et al., 1998). When 1,2-diauroyl-sn-glycero3-phosphatidylcholine liposomes containing g-tocopherol are incubated with SOD/H2O2/nitrite, the major product identified is 5-NO2-g-tocopherol. As nitrone spin traps significantly inhibit the formation of a a-tocopheryl quinone and 5-NO2g-tocopherol, it is suggestive that tocopheryl radicals react directly with NO+ and form nitrogen dioxide radical. Exposure of a-tocopherol to NO+ , under aerobic conditions, results in a complex oxidation process whose final outcome is dictated by the nature of the reaction medium (D’Ischia and Novellino, 1996). In a cyclohexane solution, a prevailing route leads to a mixture of relatively unstable polar products positive to Griess reagent. At room temperature these are partially converted to the novel 2,3-dimethyl-4-acetyl-4-hydroxy-5nitroso-2-cyclopentenone derivative. Similarly, reactions of NO+ and a-tocopherol result in the

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formation of a-tocopheryl quinones and nitrite esters (De Groot et al., 1993; D’Ischia and Novellino, 1996; Christen et al., 1997). Based on chemical reactivity, Wolf (1997) suggests that gtocopherol is a more efficient protector against NO+ -initiated peroxidative damage than a-tocopherol. The findings that vitamin E significantly improve the resistance of pancreatic islet cells to toxic doses of NO+ (Burkart et al., 1995) suggest that the vitamin directly interacts with NO+ . In addition to vitamin E, other antioxidants also interact with NO+ . Varma et al. (1997), for example, have shown that GSH oxidation in the presence of sodium nitrite is minimal in the dark, but exposure of GSH to UV (365 nm) in the presence of nitrite substantially accelerates this oxidation, and that ascorbate is effective in preventing thiol oxidation. Similarly, Bednar and Kies (1994) have shown that urinary excretion of nitrate and nitrite is significantly greater at the higher levels of nitrate and nitrite intake than at the lower intake levels, and that increased intake of vitamin C at either level of nitrate and nitrite intake results in apparent decreased urinary excretions of nitrite and nitrate. N -Nitroso compounds can be formed from the precursors nitrate/nitrite as well as secondary and tertiary amines. Wagner et al. (1985) have shown that ingestion of ascorbic acid and a-tocopherol inhibited the incorporation of [15N]nitrate into nitrosoproline by 81 and 59%, respectively, following administration of nitrate and proline in healthy young adults. They also found that ascorbic acid and a-tocopherol blocked this nitrate-induced synthesis of nitrosamino acid, N -nitrosothiazolidine-4-carboxylic acid. The mechanism by which ascorbic acid inhibits the formation of nitrosocompounds appears to be chemical in nature, and results in the formation of NO+ and dehydroascorbic acid. Since NO+ can be oxidized to form [NO]x, which is capable of additional nitrosation, a greater than stochiometric amounts of ascorbate may be needed for effective inhibition in vivo (Tannenbaum, 1989). Experimental data suggest that N -nitroso-compounds can be formed endogenously in the human stomach from the precursors nitrate/nitrite as well as secondary and tertiary amines, and that the formation of these carcino-

199

genic substances may be inhibited by dietary antioxidants (Ames, 1983; Mirvish, 1995). While dietary nitrite may lead to increased generation of reactive nitrogen species and nitroso-compounds in the stomach, epidemiologic studies have not lent supportive evidence for an etiologic role of nitrate intake per se in cancer incidence (Kono and Hirohata, 1996). Also, epidemiological evidence concerning the protective effects of vitamin E and selenium against gastric nitration is sparse and inconsistent.

5. Dietary vitamin E/selenium and nitrite toxicity While the interaction between nitrite/nitrate and vitamin E (and other antioxidants) has long been recognized, relatively little experimental evidence for the role of dietary antioxidants in the toxicity of nitrite/nitrate is available. Rodents maintained on a nutritionally adequate diet are rather resistant against the adverse effects of high concentrations of nitrite/nitrate. Except for increased methemoglobin formation, higher incidence of pulmonary lesions, and lower body weight and plasma levels of vitamin E, no other signs of adverse effects are detected in rats treated with 4000 ppm sodium nitrate or 2000 ppm sodium nitrite in drinking water for 14 months (Chow et al., 1980). As increased formation of methemoglobin is detectable only shortly (within a few hours) after nitrite treatment it is suggestive that the rodent has a highly active methemoglobin reduction system. Also, Hirneth and Classen (1984) have shown that feeding of diets containing 5% NaNO3 daily for 1 h to female Sprague /Dawley rats significantly increases the formation of plasma nitrite and levels of methemoglobin, and that these effects are significantly inhibited by the addition of ascorbic acid or of tocopherol. In an experiment to determine if vitamin E status influences cellular responses of rats to nitrite, unexpectedly over 40% of rats fed a vitamin E deficient and low selenium diet and supplemented with 1000 mg sodium nitrite/kg diet are found dead or moribund by the end of 9 weeks, while no single mortality results from nitrite-treated rats receiving the vitamin E deficient and low selenium

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and important free radicals NO+ , superoxide and ONOO+ , has provided a better insight of the protective effects of vitamin E and selenium against nitrite toxicity observed.

diet supplemented with as low as 10 IU vitamin E or 0.05 mg selenium/kg diet (Chow et al., 1984; Hong and Chow, 1987, 1988). Histopathological examinations revealed that all animals treated with nitrite and maintained on the vitamin E-deficient and low selenium diet developed massive liver necrosis, especially those found dead before the scheduled termination (Table 1). These animals also developed mild to markedly muscular degeneration, tubular nephrosis and eosinophilic enteritis. Only eosinophilic enteritis and mild muscular degeneration are observed in animals from the group fed the basal vitamin E-deficient and low selenium diet alone. No lesions are observed in skeletal muscle, liver, kidney, intestine, brain, lung, heart, pancreas, testis and stomach of rats receiving vitamin E or selenium-supplemented diets with or without nitrite. Additionally, no histopathological lesions are observed in any organs of nitritetreated rats receiving 0.05 ppm selenium or 10 ppm vitamin E supplementation in the diet (Hong and Chow, 1987, 1988). Similarly, Masukawa and Iwata (1979) have shown that selenite decreases nitrite-induced mortality in a dose-dependent manner, and suggested that the protective effect is due to its action in reducing methemoglobin formed by nitrite. These earlier findings indicate a critical role of dietary vitamin E and selenium in preventing the adverse cellular effects of nitrite. The available information, concerning the interrelationship among nitrite, vitamin E, selenium

6. Dietary vitamin E and generation of superoxide and NO+ In addition to being the most important lipidsoluble chain-breaking antioxidant, vitamin E may act as a biological modifier independent of its antioxidant activity (Azzi et al., 1998; Chow, 2001). Mitochondrial electron transport system consumes over 85% of all the oxygen utilized by the cells, and up to 5% of the oxygen consumed by the mitochondrial respiratory chain undergoes one electron reduction to generate superoxide (Chance et al., 1979; Nohl and Hegner, 1978; Shigenaga et al., 1994). The superoxide formed can be converted to H2O2 and other ROS under normal physiological conditions. The findings that increased de novo synthesis of xanthine oxidase in the skeletal muscle of vitamin E-deficient rabbits (Catignani et al., 1974), and higher activity of xanthine oxidase in the liver of vitamin E-deficient rats (Masugi and Nakamura, 1976) suggest that vitamin E may mediate superoxide generation. The view is collaborated by the findings that vitamin E deficientmice have significantly higher rates of mitochondrial generation of H2O2 in skeletal muscle and

Table 1 Effects of dietary vitamin E and selenium on the mortality and incidence of tissue lessons in nitrite-treated rats* Dietary supplement


/Vitamin E
/Selenium

/Vitamin E
/Selenium


/Vitamin E/Selenium

Nitrite treatment

1000 ppm

0

1000 ppm

0

1000 ppm

0

Animal mortality Liver necrosis Myodegeneration Tubular nephrosis

9/22 9/16 (8/8)** 10/12 (5/6) 13/16 (6/8)

0/12 0/8 2/8 0/8

0/12 0/8 0/8 0/8

0/12 0/8 0/8 0/8

0/12 0/8 0/8 0/8

0/12 0/8 0/8 0/8

* Adapted from Chow et al. (1984), Hong and Chow (1987) and Hong and Chow (1988). One-month-old male rats were fed the vitamin E-deficient and low-selenium diet and supplemented with 0 or 1000 ppm sodium nitrite and 0 or 200 IU vitamin E (as RRR-atocopherol acetate) or 0.2 mg selenium (as sodium selenite) per kg diet for 9 weeks. Eight survived animals were killed and examined for each diet and treatment group at the termination of the experiment. ** Number of animals out of 22 in this group examined. Autopsy results on the animals found dead or moribund before scheduled termination are given in parentheses. No histopathological lesions were observed in brain, lung, heart, pancreas, stomach and intestine of any animal groups.

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liver, and that vitamin E supplementation decreases the rates of mitochondrial H2O2 generation in liver and skeletal muscle of female and male rats in a dose-dependent manner (Chow et al., 1999). As the amount of H2O2 detected is the same in the presence or absence of SOD, it is indicative that the measurement of H2O2 is same as measurement of superoxide in situ. Similarly, the rate of superoxide generation by submitochondrial particles is inversely related to the vitamin E content in mouse skeletal muscle, liver, and kidney (Lass and Sohal, 2000). In addition to mitochondria, vitamin E has been shown to mediate superoxide generation by arterial cells, macrophages, neutrophils and monocytes (Ando et al., 1996; Cachia et al., 1998; Devaraj and Jialal, 1999; Kanno et al., 1995). The system responsible for superoxide production in phagocytic cells is the multicomponent enzyme NADPH-oxidase. Recently, Beharka et al. (2002) have shown that vitamin E supplement reduces NO+ production in endotoxin-stimulated macrophages of old mice. These findings support the view that vitamin E is capable of reducing the production and/or availability of not only superoxide, but also of NO+ and ONOO
/. By reducing available superoxide, NO+ , and related ROS, dietary vitamin E not only attenuates oxidative damage resulting from ROS but may also modulate the expression and activation of signal transduction pathways and other redoxsensitive biological modifiers (Azzi et al., 1998; Freedman et al., 1996; Klann et al., 1998; Koya et al., 1998; Kuemerle et al., 1997; Kunisaki et al., 1998; Ozolins and Hales, 1997; Shklar and Schwarz, 1996; Studer et al., 1997) and thereby prevent or delays the onset of degenerative diseases. By reducing available superoxide and NO+ , vitamin E may alleviate nitrite toxicity via reduced formation of reactive ONOO
/. However, it is not yet clear if the action of vitamin E to reduce the generation of superoxide and other ROS is independent of its antioxidant function.

7. Selenium and reduction of peroxynitrite Selenium, an essential trace element in many living organisms, is mainly enriched in nuclei,

201

mitochondria and cytosol (Chen et al., 1999), and its function is interrelated to vitamin E (Chow, 2001). As an integral part of the family of GPx, which catalyzes the reduction of organic hydroperoxides as well as H2O2 (Chow and Tappel, 1974), selenium complements vitamin E in cellular antioxidant defense. However, the protective effect of dietary selenium against mortality in nitrite-treated rats observed previously (Masukawa and Iwata, 1979; Chow et al., 1984; Hong and Chow, 1987, 1988) cannot be explained based on the function of GPx in reducing hydroperoxides alone. As mentioned, ONOO
/ is highly reactive and is capable of reacting with DNA to form strand breaks and mutations, and interfering with protein tyrosine-based signaling and other protein functions. It is, therefore, important that seleniumcontaining enzymes/proteins are capable of catalyzing the reduction of ONOO
/ (Sies et al., 1997). The protection of GPx against dihydrorhodamine 123 oxidation by ONOO
/ is more effective than ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)one), a selenoorganic compound exhibiting a high second-order rate constant for the reaction with ONOO
/, 2 /106/M per s. Carboxymethylation of selenocysteine in GPx by iodoacetate leads to the loss of ‘classical’ GPx activity but maintains protection against ONOO
/-mediated oxidation. Maintenance of protection by GPx against ONOO
/ requires GSH as reductant. When ONOO
/ is infused to maintain a 0.2 mM steadystate concentration, GPx in the presence of GSH, but neither GPx nor GSH alone, effectively inhibits the hydroxylation of benzoate by ONOO
/. Under these steady-state conditions ONOO
/ does not cause the loss of classical GPx activity. Also, GPx, like seleno-methionine, protects against protein 3-nitrotyrosine formation in human fibroblast lysates, and the formation of nitrite rather than nitrate from ONOO
/ is enhanced by GPx or by seleno-methionine. Based on the GSH-dependent protection against ONOO
/-mediated formation of 3-nitrotyrosine in in vitro and in human fibloblast lysates, Sies et al. (1997, 1998) suggested that GPx acts as a peroxynitrite reductase by reducing ONOO
// peroxynitrous acid to nitrite, and protects against

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oxidation and nitration reactions. Briviba et al. (1998) have also shown that the second-order rate constant for the reaction of the reduced form of GPx with ONOO
/ is (8.09/0.8) /106/M per s (per GPx tetramer) at pH 7.4 and 25 8C, and that the rate constant for oxidized GPx is about 10 times lower, (0.79/0.2) /106/M per s. On a selenium basis, the rate constant for reduced GPx is similar to that obtained previously for ebselen. On the other hand, GPx can be inactivated by ONOO
/ under physiologically relevant conditions. Stopped-flow kinetic studies show that the reaction between ONOO
/ and GPx is first-order in each of the reactants, with an apparent secondorder rate constant of 4.59/0.2 /104/M per s per monomer unit of enzyme (Padmaja et al., 1998). The close agreement between the rate constants obtained from enzyme inactivation and from stopped-flow kinetics experiments suggests that the mechanism of the reaction between ONOO
/ and GPx involves the oxidation of the ionized selenol of the seleno-cysteine residue in the enzyme’s active site by ONOO
/. Also, Hondal et al. (1999) have failed to show the protection of seleno-methionine residues in albumin and immunoglobulin G against ONOO
/. Thus, more studies are needed to have a better understanding of the role of seleno-enzymes/proteins in reducing or detoxifying ONOO
/.

8. Possible mechanisms of vitamin E/selenium protection against nitrite/nitrate toxicity The information available recently provides a better insight of the occurrence of high mortality rates of nitrite-treated rats maintained in vitamin E-deficient and low selenium diet, as well as the protection by dietary vitamin E and selenium observed previously (Chow et al., 1984; Hong and Chow, 1987, 1988). As a key oxidation product of NO+ , and its ready precursor, nitrite administration leads to increased formation of NO+ , which in turn reacts with superoxide to form reactive ONOO
/. The protective effects of dietary vitamin E can be attributed to its ability to reduce the generation and availability of super-

oxide and NO+ , while seleno-enzymes/proteins reduce ONOO
/ generated. When dietary vitamin E and selenium are adequate increased the formation of NO+ , following nitrite treatment, does not lead to a higher ONOO
/ generation due to the action of vitamin E to reduce superoxide and NO+ , and ONOO
/ formed can be reduced by Seenzymes and seleno-compounds (Table 2). When nitrite-treated rats are fed a vitamin E-deficient but selenium adequate diet, increased ONOO
/ can be reduced by seleno-enzymes/compounds, and only minor adverse effects are expected. Similarly, when nitrite-treated rats are fed a lowselenium but vitamin E adequate diet, relatively smaller amounts of ONOO
/ formed are expected to exert only minor effect. However, when nitritetreated rats are fed a vitamin E-deficient and low selenium diet, the resulting high levels of ONOO
/ , coupled with reduced ONOO
/ reducing capability, may initiate oxidative damage and subsequent cell death and animal mortality. A similar interrelationship among antioxidants and ROS appears to be involved in the pathogenesis of hypertension. Patients with uncontrolled essential hypertension have higher superoxide and H2O2 production by polymorphonuclear leukocytes and the plasma levels of lipid peroxides are higher, while NO+ , vitamin E and SOD are lower (Kumar and Das, 1993). The ability of a-tocopherol to enhance NO+ synthase activity in blood vessels of spontaneously hypertensive rats observed (Newaz et al., 1999) is likely the consequence of its ability to reduce superoxide and NO+ . The findings that both L-arginine and atocopherol are capable of reducing vascular oxidative stress (urinary isoprostane excretion), preserving endothelial function (improve endothelium-dependent vasodilation) and reducing the progression of atherosclerotic plaques in cholesterol-fed hypercholesterolemic rabbits (Boger et al., 1998), that vitamin E reduces the extent of ischemia-reperfusion kidney injury caused by NO+ , and other ROS (Uysal et al., 1998), and that vitamins E and C mitigate GSH depletion and perturbation of the NO+ system (decreased urinary nitrate/nitrite excretion and increased nitrotyrosine accumulation) and severe hypertension in buthionine sulfoximine-treated rats (Vaziri et al.,

C.K. Chow, C.B. Hong / Toxicology 180 (2002) 195 /207

203

Table 2 Possible role of dietary vitamin E and selenium in preventing nitrite toxicity Diet

NO+ formation1

+

O2
formation2

ONOO
/ formation3

ONOO
/ formation4

Resulting ONOO+ 5

Toxicity expected6

/Vitamin E /Selenium
/Vitamin E /Selenium /Vitamin E
/Selenium
/Vitamin E
/Selenium


/

¡/

/


/

/

None


/


/


/
/


/


/

Mild


/

¡/

/

¡/


/

Mild


/


/


/
/

¡/


/
/
/

Severe

1

NO+ formation resulting from nitrite treatment expected. The symbol ‘
/’ denotes increases (‘
/
/’ for bigger increase, and ‘
/
/
/’ for much bigger increase); ‘ ¡/’, decreases; and ‘ /’, little or no changes. 2 Superoxide formation expected. 3 Formation of ONOO
/ expected. 4 ONOO
/ reducing capability expected. 5 Net production of ONOO
/ expected. 6 Toxic effects of nitrite expected.

2000) support the view that both depressed NO+ and increased ONOO
/ are involved in the pathogenesis of hypertension.

9. Conclusion Information available recently provides a better insight of the mechanism by which vitamin E and selenium protect against nitrate/nitrite toxicity. As both nitrites and nitrates are key oxidation products as well as ready sources of NO, which in turn react rapidly with superoxide to form reactive ONOO
/, their adverse cellular effects can be attributed to the action of ONOO
/. Increased formation of ONOO
/ is likely the critical event responsible for the toxic effect resulting from nitrite-treated rats fed a vitamin E-deficient and low selenium diet. The protective effect of dietary vitamin E against nitrite and nitrate toxicity is attributable to its ability to limit the production and availability of superoxide and NO+ , while dietary selenium enhances the level of selenoenzymes/compounds, which reduce or scavenge ONOO
/ formed.

References Abuharfeil, N., Sarsour, E., Hassuneh, M., 2001. The effect of sodium nitrite on some parameters of the immune system. Food Chem. Toxicol. 39, 119 /124. Alexander, B., 1998. The role of nitric oxide in hepatic metabolism. Nutrition 14, 376 /390. Ames, B.N., 1983. Dietary carcinogens and anticarcinogens: oxygen radicals and degenerative diseases. Science 221, 1256 /1264. Ames, B.N., Shigenaga, M.K., Hagen, T.M., 1993. Oxidants, antioxidants, and the degenerative diseases of aging. Proc. Nat. Acad. Sci. USA 90, 7915 /7922. Ando, M., Yoshioka, T., Akiyama, J., Kudo, T., 1996. Regulation of neutrophil superoxide generation by alphatocopherol in human peripheral and umbilical-cord blood. J. Obstet. Gynaecol. Res. 22, 507 /516. Atanasova-Goranova, V.K., Dimova, P.I., Pevicharova, G.T., 1997. Effect of food products on endogenous generation of N-nitrosamines in rats. Br. J. Nutr. 78, 335 /345. Augusto, O., Gatti, R.M., Radi, R., 1994. Spin-trapping studies of peroxynitrite decomposition and 3-morpholinosydnonimide N-ethylcarbamide autooxidation: direct evidence for metal-independent formation of free radical intermediates. Arch. Biochem. Biophys. 310, 118 /125. Azzi, A., Aratri, E., Boscoboinik, D., Clement, S., Ozer, N.K., Ricciarelli, R., Spycher, S., 1998. Molecular basis of alphatocopherol control of smooth muscle cell proliferation. Biofactors 7, 3 /14.

204

C.K. Chow, C.B. Hong / Toxicology 180 (2002) 195 /207

Beckman, J.S., Beckman, T.W., Chen, J., Marshall, P.A., Freeman, B.A., 1990. Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc. Natl. Acad. Sci. USA 87, 1620 /1624. Bednar, C., Kies, C., 1994. Nitrate and vitamin C from fruits and vegetables: impact of intake variations on nitrate and nitrite excretions of humans. Plant Foods Human Nutr. 45, 71 /80. Beharka, A.A., Wu, D., Serafini, M., Meydani, S.N., 2002. Mechanism of vitamin E inhibition of cyclooxygenase activity in macrophages from old mice: role of peroxynitrite. Free Rad. Biol. Med. 32, 503 /511. Boger, R.H., Bode-Boger, S.M., Phivthong-ngam, L., Brandes, R.P., Schwedhelm, E., Mugge, A., Bohme, M., Tsikas, D., Frolich, J.C., 1998. Dietary L-arginine and alpha-tocopherol reduce vascular oxidative stress and preserve endothelial function in hypercholesterolemic rabbits via different mechanisms. Atherosclerosis 141, 31 /43. Briviba, K., Kissner, R., Koppenol, W.H., Sies, H., 1998. Kinetic study of the reaction of glutathione peroxidase with peroxynitrite. Chem. Res. Toxicol. 11, 1398 /1401. Buettner, G., 1993. The packing order of free radicals and antioxidants: lipid peroxidation, alpha-tocopherol, and ascorbate. Arch. Biochem. Biophys. 300, 535 /543. Burkart, V., Gross-Eick, A., Bellmann, K., Radons, J., Kolb, H., 1995. Suppression of nitric oxide toxicity in islet cells by alpha-tocopherol. FEBS Lett. 364, 259 /263. Cachia, O., Benna, J.E., Pedruzzi, E., Descomp, B., GougertPocidalo, M.A., Leger, C.L., 1998. Tocopherol inhibits the respiratory burst in human monocytes. Attenuation of p47phox membrane translocation and phosphorylation. J. Biol. Chem. 273, 32801 /32805. Carmichael, A.J., Steel-Goodwin, L., Gray, B., Arroyo, C.M., 1993. Reactions of reactive oxygen species studied by EPR and spin trapping. Free Rad. Res. Commun. 19, S1 /S16. Cassina, A.M., Hodara, R., Souza, J.M., Thomson, L., Castro, L., Ischiropoulos, H., Freeman, B.A., Radi, R., 2000. Cytochrome C nitration by peroxynitrite. J. Biol. Chem. 275, 21409 /21415. Catignani, G.L., Chytil, F., Darby, W.J., 1974. Vitamin E deficiency: immunochemical evidence for increased accumulation of liver xanthine oxidase. Proc. Nat. Acad. Sci. USA 71, 1966 /1968. Chabot, F., Mitchell, J.A., Quinlan, G.J., Evans, T.W., 1997. Characterization of the vasodilator properties of peroxynitrite on rat pulmonary artery: role of poly(adenosine 5?diphosphoribose) synthase. Br. J. Pharmacol. 121, 485 /490. Chance, B., Sies, H., Boveris, A., 1979. Hydroperoxide metabolism in mammalian organs. Physiol. Rev. 59, 527 / 605. Chen, C., Zhang, P., Hou, X., Chai, X., 1999. Subcellular distribution of selenium and Se-containing proteins in human liver. Biochim. Biophys. Acta 1427, 205 /215. Chow, C.K., 1979. Nutritional influence on cellular antioxidant defense systems. Am. J. Clin. Nutr. 32, 1066 /1081.

Chow, C.K., 1991. Vitamin E and oxidative stress. Free Rad. Biol. Med. 11, 215 /232. Chow, C.K., 2001. Vitamin E regulation of mitochondrial generation of hydrogen peroxide. Biol. Signals Receptors 10, 112 /124. Chow, C.K., Tappel, A.L., 1974. Response of glutathione peroxidase as a function of dietary selenium in rats. J. Nutr. 104, 444 /451. Chow, C.K., Chen, C.J., Gairola, C., 1980. Effect of nitrate and nitrite in drinking water of rats. Toxicol. Lett. 6, 199 /206. Chow, C.K., Hong, C.B., Reese, M., Gairola, C., 1984. Effect of dietary vitamin E on nitrite-treated rats. Toxicol. Lett. 23, 109 /117. Chow, C.K., Ibrahim, W., Wei, Z., Chan, A.C., 1999. Vitamin E regulates mitochondrial hydrogen peroxide generation. Free Rad. Biol. Med. 27, 580 /587. Christen, S., Woodall, A.A., Shigenaga, M.K., SouthwellKeely, P.T., Duncan, M.W., Ames, B.N., 1997. Gammatocopherol traps mutagenic electrophiles such as NO(X) and complements alpha-tocopherol: physiological implications. Proc. Natl. Acad. Sci. USA 94, 3217 /3222. Daiber, A., Herold, S., Schoneich, C., Namgaladze, D., Peterson, J.A., Ullrich, V., 2000. Nitration and inactivation of cytochrome P450BM-3 by peroxynitrite. Stopped-flow measurements prove ferryl intermediates. Eur. J. Biochem. 267, 6729 /6739. Dalloz, F., Maupoli, V., Lecour, S., Briot, F., Rochette, L., 1997. In vitro studies of interaction on NO+ donor drugs with superoxide and hydroxyl radicals. Mol. Cell. Biochem. 177, 193 /200. De Groot, H., Hegi, U., Sies, H., 1993. Loss of alphatocopherol upon exposure to nitric oxide or the sydnonimine SIN-1. FEBS Lett. 315, 139 /142. Devaraj, S., Jialal, I., 1999. Alpha-tocopherol decreases interleukin-1 beta release from activated human monocytes by inhibition of 5-lipoxygenase. Arterioscler. Thromb. Vasc. Biol. 19, 1125 /1133. D’Ischia, M., Novellino, L., 1996. Nitric oxide-induced oxidation of alpha-tocopherol. Bioorg. Med. Chem. 4, 1747 / 1753. Dollahite, J.W., Rowe, L.D., 1974. Nitrate and nitrite intoxication in rabbits and cattle. Southwest Vet. 27, 246 /248. Francescutti, D., Baldwin, J., Lee, L., Mutus, B., 1996. Peroxynitrite modification of glutathione reductase: modeling studies and kinetic evidence suggest the modification of tyrosines at the glutathione disulfide binding site. Protein Eng. 9, 189 /194. Freeman, B.A., Crapo, J.D., 1982. Biology of disease: free radicals and tissue injury. Lab. Invest. 47, 412 /426. Freedman, J.E., Farhat, J.H., Loscalzo, J., Keaney, J.F., Jr, 1996. Alpha-tocopherol inhibits aggregation of human platelets by a protein kinase C-dependent mechanism. Circulation 94, 2434 /2440. Fukuyama, N., Takebayashi, Y., Hida, M., Ishida, H., Ichimori, K., Nakazawa, H., 1997. Clinical evidence of peroxynitrite formation in chronic renal failure patients with septic shock. Free Rad. Biol. Med. 22, 771 /774.

C.K. Chow, C.B. Hong / Toxicology 180 (2002) 195 /207 Furukawa, F., Nishikawa, A., Miyauchi, M., Nakamura, H., Son, H.Y., Yamagishi, M., Hirose, M., 2000. Concurrent administration of fish meal and sodium nitrite does not promote renal carcinogenesis in rats after initiation with Nethyl-N-hydroxyethylnitrosamine. Cancer Lett. 154, 45 /51. Ger, J., Kao, H., Shih, T.S., Deng, J.F., 1996. Fatal toxic methemoglobinemia due to occupational exposure to methyl nitrite. Clin. Med. J. 57, S78. Greenacre, S., Ridger, V., Wilsoncroft, P., Brain, S.D., 1997. Peroxynitrite: a mediator of increased microvascular permeability? Clin. Exp. Pharmacol. Physiol. 24, 880 /882. Halliwell, B., 1987. Oxidants and human diseases: some new concepts. FASEB J. 1, 358 /364. Halliwell, B., Zhao, K., Whiteman, M., 2000. The gastrointestinal tract: a major site of antioxidant action? Free Rad. Res. 33, 819 /830. Hirneth, H., Classen, H.G., 1984. Inhibition of nitrate-induced increase of plasma nitrite and ethemoglobinemia in rats by simultaneous feeding of ascorbic acid or tocopherol. Arzneimittelforschung 34, 988 /991. Hondal, R.J., Motley, A.K., Hill, K.E., Burk, R.F., 1999. Failure of selenomethionine residues in albumin and immunoglobulin G to protect against peroxynitrite. Arch. Biochem. Biophys. 371, 29 /34. Hong, C.B., Chow, C.K., 1987. Prevention of eosinophilic enteritis and eosinophilia in rats with selenium and vitamin E. Fed. Proc. 46, 1154A. Hong, C.B., Chow, C.K., 1988. Induction of eosinophilic enteritis and eosinophilia in rats by vitamin E and selenium deficiency. Exp. Mol. Pathol. 48, 182 /192. Ignarro, L.J., Buga, G.M., Wood, K.S., Byrns, R.E., Chaudhuri, G., 1987. Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide. Proc. Natl. Acad. Sci. USA 84, 9265 /9269. Joint FAO/WHO Expert Committee on Food Additives, 1974. Seventeenth Report. Toxicological evaluation of some food additives including anticaking agents, antimicrobials, antioxidants, emulsifiers and thickening agents. WHO Food Add. Ser. 5: 97 /109. Kanno, T., Utsumi, T., Kobuchi, H., Takehara, Y., Akiyama, J., Yoshioka, T., Horton, A.A., Utsumi, K., 1995. Inhibition of stimulus-specific neutrophil superoxide generation by alpha-tocopherol. Free Rad. Res. 22, 431 /440. Kelm, M., 1999. Nitric oxide metabolism and breakdown. Biochim. Biophys. Acta 1411, 273 /289. Keng, T., Privalle, C.T., Gilkeson, G.S., Weinberg, J.B., 2000. Peroxynitrite formation and decreased catalase activity in autoimmune MRL-lpr/lpr mice. Mol. Med. 6, 779 /792. Klann, E.M., Roberson, E.D., Knapp, L.T., Sweatt, J.D., 1998. A role for superoxide in protein kinase C activation and induction of long-term potentiation. J. Biol. Chem. 273, 4516 /4522. Kono, S., Hirohata, T., 1996. Nutrition and stomach cancer. Cancer Causes Control. 7, 41 /55. Koya, D., Haneda, M., Kikkawa, R., King, G.L., 1998. DAlpha-tocopherol treatment prevents glomerular dysfunc-

205

tions in diabetic rats through inhibition of protein kinase Cdiacylglycerol pathway. Biofactors 7, 69 /76. Kuemerle, N.B., Brandt, R.B., Chan, W., Krieg, R.J., Jr, Chan, J.C., 1997. Inhibition of transforming growth factor beta-1 induction by dietary vitamin E in unilateral obstruction in rats. Biochem. Mol. Med. 61, 82 /96. Kumar, K.V., Das, U.N., 1993. Are free radicals involved in the pathobiology of human essential hypertension? Free Rad. Res. Commun. 19, 59 /66. Kunisaki, M., Bursell, S.E., Umeda, F., Nawata, H., King, G.L., 1998. Prevention of diabetes-induced abnormal retinal blood flow by treatment with d-alpha-tocopherol. Biofactors 7, 55 /67. Lass, A., Sohal, R.S., 2000. Effect of coenzyme Q(10) and alpha-tocopherol content of mitochondria on the production of superoxide anion radicals. FASEB J. 14, 87 /94. Levallois, P., Ayotte, P., Van-Maanen, J.M., Desrosiers, T., Gingras, S., Dallinga, J.W., Vermeer, I.T., Zee, J., Poirier, G., 2000. Excretion of volatile nitrosamines in a rural population in relation to food and drinking water consumption. Food Chem. Toxicol. 38, 1013 /1019. Liu, P., Yin, K., Nagele, R., Wong, P.Y., 1998. Inhibition of nitric oxide synthase attenuates peroxynitrite generation, but augments neutrophil accumulation in hepatic ischemiareperfusion in rats. J. Pharmacol. Exp. Ther. 284, 1139 / 1146. Macfadyen, A.J., Reiter, C., Zhuang, Y., Beckman, J.S., 1999. A novel superoxide dismutase-based trap for peroxynitrite used to detect entry of peroxynitrite into erythrocyte ghosts. Chem. Res. Toxicol. 12, 223 /229. Masugi, F., Nakamura, T., 1976. Effect of vitamin E deficiency on the levels of superoxide dismutase, glutathione peroxidase, catalase and lipid peroxide in rat liver. Int. J. Vit. Nutr. Res. 46, 187 /191. Masukawa, T., Iwata, H., 1979. Protective effect of selenite on nitrite toxicity. Experientia 35, 1360 /1361. Millar, T.M., Stevens, C.R., Benjamin, N., Eisenthal, R., Harrison, R., Blake, D.R., 1998. Xanthine oxidoreductase catalyses the reduction of nitrates and nitrite to nitric oxide under hypoxia conditions. FEBS Lett. 427, 225 /228. Mirvish, S.S., 1995. Role of N-nitroso compounds (NOC) and N-nitrosation in etiology of gastric, esophageal, nasopharyngeal and bladder cancer and contribution to cancer of known exposures to NOC. Cancer Lett. 93, 17 /48. Mirvish, S.S., Reimers, K.J., Kutler, B., Chen, S.C., Haorah, J., Morris, C.R., Grandjean, A.C., Lyden, E.R., 2000. Nitrate and nitrite concentrations in human saliva for men and women at different ages and times of the day and their consistency over time. Eur. J. Cancer Prev. 9, 335 /342. Newaz, M.A., Nawal, N.N., Rohaizan, C.H., Muslim, N., Gapor, A., 1999. Alpha-tocopherol increased nitric oxide synthase activity in blood vessels of spontaneously hypertensive rats. Am. J. Hypertens. 12, 839 /844. Nickerson, M., 1970. In: Goodman, L.S., Gilm1an, A. (Eds.), The Pharmacological Basis of Therapeutics, 4th edition. London, MacMillan, pp. 745 /763.

206

C.K. Chow, C.B. Hong / Toxicology 180 (2002) 195 /207

Nohl, H., Hegner, D., 1978. Do mitochondria produce oxygen radicals in in vivo? Eur. J. Biochem. 82, 563 /567. Nossuli, T.O., Hayward, R., Jensen, D., Sealia, R., Lefer, A.M., 1998. Mechanisms of cardioprotection by peroxynitrite in myocardial ischemia and reperfusion injury. Am. J. Physiol. 275, H509 /H519. Ozolins, T.R., Hales, B.F., 1997. Oxidative stress regulates the expression and activity of transcription factor activator protein-1 in rat conceptus. J. Pharmacol. Exp. Ther. 280, 1085 /1093. Padmaja, S., Squadrito, G.L., Pryor, W.A., 1998. Inactivation of glutathione peroxidase by peroxynitrite. Arch. Biochem. Biophys. 349, 1 /6. Pannala, A.S., Rice-Evans, C., Sampson, J., Singh, S., 1998. Interaction of peroxynitrite with carotenoids and tocopherols within low density lipoprotein. FEBS Lett. 423, 297 / 301. Reid, G., 2000. Association of sudden infant death syndrome with grossly deranged iron metabolism and nitric oxide overload. Med. Hypotheses 54, 137 /139. Pryor, W.A., Squadrito, G.L., 1995. The chemistry of peroxynitrite: a product from the reaction of nitric oxide with superoxide. Am. J. Physiol. 268, L699 /L722. Sampson, J.B., Ye, Y., Rosen, H., Beckman, J.S., 1998. Myeloperoxidase and horseradish catalyze tyrosine nitration in proteins from nitrite and hydrogen peroxide. Arch. Biochem. Biophys. 356, 207 /213. Seidel, E.R., Ragan, V., Liu, L., 2001. Peroxynitrite inhibits the activity of ornithine decarboxylase. Life Sci. 68, 1477 /1483. Shi, H., Noguchi, N., Xu, Y., Niki, E., 1999. Formation of phospholipid hydroperoxides and its inhibition by alphatocopherol in rat brain synaptosomes induced by peroxynitrite. Biochem. Biophys. Res. Commun. 257, 651 /656. Shigenaga, M.K., Hagen, T.M., Ames, B.N., 1994. Oxidative damage and mitochondrial decay in aging. Proc. Natl. Acad. Sci. USA 91, 10771 /10778. Shklar, G., Schwarz, J.L., 1996. Vitamin E inhibits experimental carcinogenesis and tumor angiogenesis. Eur. J. Cancer B. Oral. Oncol. 32, 114 /119. Sies, H., 1986. Biochemistry of oxidative stress. Angew. Chem. Int. Ed. Engl. 25, 1058 /1071. Sies, H., Sharov, V.S., Klotz, L.O., Briviba, K., 1997. Glutathione peroxidase protects against eroxynitrite-mediated oxidations. A new function for selenoproteins as peroxynitrite reductase. J. Biol. Chem. 272, 27812 /27817. Sies, H., Klotz, L.O., Sharov, V.S., Assmann, A., Briviba, K., 1998. Protection against peroxynitrite by selenoproteins. Z. Naturforsch. 53, 228 /232. Singh, R.J., Goss, S.P., Joseph, J., Kalyanaraman, B., 1998. Nitration of gamma-tocopherol and oxidation of alphatocopherol by copper-zinc superoxide dismutase/H2O2/ NO2
: role of nitrogen dioxide free radical. Proc. Natl. Acad. Sci. USA 95, 12912 /12917. Squadrito, G.L., Pryor, W.A., 1998. Oxidative chemistry on nitric oxide: the roles of superoxide, peroxynitrite, and carbon dioxide. Free Rad. Biol. Med. 25, 392 /403.

Studer, R.K., Craven, P.A., De Rubertis, F.R., 1997. Antioxidant inhibition of protein kinase C-signaled increased in transforming growth factor-beta in sesangial cells. Metabolism 46, 918 /925. Tannenbaum, S.R., 1989. Preventive action of vitamin C on nitrosamine formation. Int. J. Vit. Nutr. Res. 30, S109 / S113. Tannenbaum, S.R., Frett, D., Young, V.R., Land, P.D., Bruce, W.R., 1978. Nitrite and nitrate are formed by endogenous synthesis in the human intestine. Science 200, 1487 /1489. Thomas, S.R., Davies, M.J., Stocker, R., 1998. Oxidation and antioxidation of human low-density lipoprotein and plasma exposed to 3-morpholinosyldnonimine and reagent peroxynitrite. Chem. Res. Toxicol. 11, 484 /494. Til, H.P., Falke, H.E., Kuper, C.F., Willems, M.I., 1988. Evaluation of the oral toxicity of potassium nitrite in a 13-week drinking-water study in rats. Food Chem. Toxicol. 26, 851 /859. Uesugi, M., Hayshi, T., Jasin, H.E., 1998. Covalent crosslinking of immune complexes by oxygen radicals and nitrite. J. Immunol. 161, 1422 /1427. Uysal, F., Girgin, F.K., Tuzun, S., Aldemir, S., Sozmen, E.Y., 1998. Effect of vitamin E on antioxidant enzymes and nitric oxide in ischemia-reperfused kidney injury. Biochem. Mol. Biol. Int. 44, 1255 /1263. Van Dyke, K., 1997. The possible role of peroxynitrite in Alzheimer’s disease: a simple hypothesis that could be tested more thoroughly. Med. Hypotheses 48, 375 /389. Van Maanen, J.M., Pachen, D.M., Dallinga, J.W., Kleinjans, J.C., 1998. Formation of nitrosamines during consumption of nitrite- and amine-rich foods, and the influence of the use of mouthwashes. Cancer Detect. Prev. 22, 204 /212. Varma, S.D., Ali, A.H., Devamanoharan, P.S., Morris, S.M., 1997. Nitrite-induced photo-oxidation of thiol and its implications in smog toxicity to the eye: prevention by ascorbate. J. Ocul. Pharmacol. Ther. 13, 179 /187. Vaziri, N.D., Wang, X.Q., Oveisi, F., Rad, B., 2000. Induction of oxidative stress by glutathione depletion causes severe hypertension in normal rats. Hypertension 36, 142 /146. Wagner, D.A., Shuker, D.E., Bilmazes, C., Obiedzinski, M., Baker, I., Young, V.R., Tannenbaum, S.R., 1985. Effect of vitamins C and E on endogenous synthesis of N-nitrosamino acids in humans: precursor-product studies with [15N]nitrate. Cancer Res. 45, 6519 /6522. Wallace, D.C., Brown, M.D., Melov, S., Graham, B., Lott, M., 1998. Mitochondrial biology, degenerative disease and aging. Biofactors 7, 187 /190. Weyer, P.J., Cerhan, J.R., Kross, B.C., Hallberg, G.R., Kantamneni, J., Breuer, G., Jones, M.P., Zheng, W., Lynch, C.F., 2001. Municipal drinking water nitrate level and cancer risk in older women: the Iowa Women’s Health Study. Epidemiology 12, 327 /338. White, J.W., 1975. Relative significance of dietary source of nitrate and nitrite. J. Am. Food Chem. 23, 886 /891. Wolf, G., 1997. Gamma-tocopherol: an efficient protector of lipids against nitric oxide-initiated peroxidative damage. Nutr. Rev. 55, 376 /378.

C.K. Chow, C.B. Hong / Toxicology 180 (2002) 195 /207 Yang, Q., Wang, F., 1991. Effect of sodium nitrite on myocardial glutathione peroxidase and protective action of vitamin E and selenium. Biomed. Environ. Sci. 4, 373 /375. Yu, B.P., 1994. Cellular defenses against damage from reactive oxygen species. Physiol. Rev. 74, 139 /162. Zhang, Z., Naughton, D., Winyard, P.G., Benjamin, N., Blake, D.R., Symous, M.C., 1998. Generation of nitric oxide by a

207

nitric reductase activity of xanthine oxidase: a potential pathway for nitric oxide formation in the absence of nitric oxide synthase activity. Biochem. Biophys. Res. Commun. 249, 767 /772. Zweier, H., Samouilov, A., Kuppusamy, P., 1999. Non-enzymatic nitric oxide synthesis in biological systems. Biochim. Biophys. Acta 1411, 250 /252.