Light-mediated conversion of nitrogen dioxide to nitric oxide by carotenoids.

Plants are more susceptible to the toxic effects of nitrogen dioxide when exposure takes place in the dark. Beta-carotene and other common carotenoids react with nitrogen dioxide in the dark to yield intermediate nitrosating agents consistent with the formation of nitrate esters. Simultaneous exposure of carotenoids to NO2 and light significantly reduced formation of nitrosating intermediates and resulted in the release of nitric oxide (NO) into the gas phase. Light-mediated reduction of NO2 to NO by carotenoids may be an important mechanism for preventing damage in plants exposed to NO2. The formation of nitrosating agents from the reaction of carotenoids with NO2 suggests that their ability to prevent nirosative damage associated with NO2 exposure in both plants and animals may be limited in the absence of light.

lipids (7, N-nitrosamine formation (8), direct mutation of DNA (9)(10)(11), DNA single-strand breaks (12), and/or other oxidative reactions. Nitrogen dioxide accumulation in grain elevators is associated with "silo filler's disease" which is characterized by massive and often lethal lung damage in exposed individuals (13). The free radical reactions of NO2 generally occur independently of light, while more stable species such as nitrite and metal nitrosyl complexes (e.g., nitroprusside) are converted, upon light exposure, to nitrosating species, such as NO' or nitrogen oxide radicals (14)(15)(16), which can subsequently react with nucleophiles to form nitroso compounds (14,1X. Exposure of plants to nitrogen dioxide in the dark causes acute injury and accumulation of nitrite, whereas simultaneous exposure to light reduces toxicity and nitrite formation (18,19). The mechanism of NO2 toxicity in plants has not been elucidated, but likely involves both direct effects from exposure to this highly reactive free radical and indirect effects from the products formed when NO2 reacts with cellular constituents. Although exposure to nitrogen oxides affects photosynthesis (20), it is not known if this is related to the acute toxicity of NO2. Shimazaki et al. (18) have proposed that the accumulation of nitrite in the dark and subsequent lightmediated reaction of nitrite with chlorophyll accounts for the observed toxicity of NO2. Such an explanation, however, does not provide a mechanistic basis to fully explain either the differential accumulation of nitrite in dark versus light-exposed plants or the manner by which simultaneous exposure to light reduces toxicity.
The high reactivity of carotenoids, found in many green plants, toward oxidants and free radicals suggests that carotenoids may play an important role in preventing free radical damage caused by NO2 exposure. However, the reaction products of highly conjugated molecules, such as the carotenoids, with NO2 have not been well characterized; consequently, the conditions under which carotenoids protect against NO2 damage in plants or animals is unknown. Reaction of NO2 with double bonds, such as those found in linoleic acid, or cholesterol, results in the formation of nitrosating agents, which have been identified as nitrite esters (21). Alpha-tocopherol, like cholesterol and linoleic acid, reacts with NO2 to form a nitrosating agent, while y-tocopherol, lacking a methyl group in the ortho position to the phenol, does not (22). Gamma-tocopherol is also more effective at preventing neoplastic transformation of cultured cells (22), a property that may be related to its unique reactivity with nitrogen oxides.
Recently Bittrich et al. (12) reported that 'y-tocopherol was the only tocopherol ana-logue able to significantly reduce singlestrand DNA breaks in Chinese hamster lung fibroblasts exposed to NO2. The protection afforded by y-tocopherol may be related to its ability to reduce NO2 to the more stable NO molecule without requiring light (22). Nitrosation of primary amines on DNA bases in vivo can result directly in genotoxic damage; therefore, formation of a nitrite ester, capable of nitrosating amines, could limit protection from nitrosative damage associated with NO2 exposure. Conversion of NO2 to NO may additionally protect against oxidative damage by way of the relative antioxidant properties of NO (23). To provide insight into the mechanism of dark-phase NO2 toxicity, we used an in vitro model system to study the reactions of carotenoids with NO2 and the effects of light on the products obtained.
As shown in Figure 1, fl-carotene (10-M in hexane) reacted with NO2 to produce NO in the presence of light, whereas control solutions in the dark showed no significant NO evolution. Measured NO levels in the gas phase exiting n-carotene solutions began to decrease significantly after 6-8 min of exposure to NO2, corresponding to the observed decolorization of the carotenoid solution ( Fig. 2) and destruction of the highly conjugated polyene system (color loss occurred independently of light indicating that the initial reaction between NO2 and n-carotene was not light mediated). Higher concentrations of n-carotene (10-4 M) showed sustained NO emission during the 15-min exposure and were not completely decolorized over this time (data not shown). Although 8 min of exposure to NO2 in the dark resulted in complete loss of color and the disappearance of all n-carotene as measured by HPLC, 24 hr later samples regained some color as well as a small amount of P-carotene in samples exposed less than 10 minutes (Fig. 2). Lutein, the major carotenoid found in dark green, leafy vegetables such as spinach, also reduced NO2 to NO in the light (Fig. 3), as did lycopene, an acyclic carotenoid found in tomatoes, and canthaxanthin, a food colorant containing two keto groups (data not shown). Carotenoid solutions exposed to NO2 formed a nitrosating agent as evidenced by A * : ; . *i e ; *. E their ability to subsequently nitrosate morpholine (Fig. 4). Beta-carotene samples exposed to light during NO2 treatment showed significantly less ability to nitrosate morpholine (54% decrease, p = 0.006 for the difference in means) as well as less Griess-Saltzmann reactive material (42% decrease, p = 0.008), indicating less nitrite and/or nitrite ester present. Lutein accumulated less nitrite/nitrite ester relative to P-carotene under both lighting conditions and showed a corresponding reduction in the ability to nitrosate morpholine (Fig. 4).
Other carotenoids examined showed a similar pattern of nitrosation and light-mediated inhibition in spite of their structural differences, although the ability of lycopene exposed to NO2 in the light to subsequently nitrosate morpholine was more variable for undetermined reasons (data not shown). Carotenoids are lipophilic compounds that act as membrane antioxidants in plants to protect against free radical and oxidative damage (1). The double bonds in carotenoids make these compounds highly reactive toward singlet oxygen (25) or free radicals such as NO2 (11). The tocopherols also prevent oxidative damage by reacting with radicals to form less reactive intermediates (26). Interestingly, y-tocopherol is found primarily in seed oils, whereas chloroplasts contain a-tocopherol and carotenoids (27). Whether this differential localization in plant tissues is related to conditions of light and/or NO2 exposure is unknown; however, it is consistent with the observed superiority of y-tocopherol to reduce NO2 to NO in the dark (22).
The light-mediated conversion of NO2 to NO by plant antioxidants offers a plausible mechanism to explain decreased sensitivity to NO2 toxicity in plants exposed to light. In addition to reducing the level of nitrite and/or nitrite esters formed and subsequent nitrosation reactions, the production of nitric oxide may provide additional antioxidant protection through its reaction with NO and other intracellular free radicals to block radical chain propogation reactions. The reaction of NO with NO2 to form N203 might prevent acute free radical damage from NO2, although increasing delayed damage arising from the nitrosation of amines by N203 while the reaction of NO with lipid or oxygen radicals would result in chain termination. Such a mechanism may explain the superior ability of y-tocopherol to prevent single-strand DNA breaks in cells exposed to NO2 (12).
Insight into the chemical mechanism by which y-tocopherol or illuminated carotenoids reduce NO2 to NO is provided in a recent paper by Bosch and Kochi (28) Figure 1. Effect of light on NO formation by NO2exposed carotene. Beta-carotene (1O 5 M in 5 ml hexane) was exposed for the indicated times to 27.5 ppm NO2 in N2 by bubbling at a flow rate of 60 mI/mmn. NO was measured in the exit gas by direct injection of 10O-gI gas samples into a thermal energy analyzer as previously described (22). Samples were kept in total darkness during exposure to NO2 or placed 10 cm from a quartz-halogen 60-watt lamp with an intervening neutral density filter to block infrared light and prevent sample warming. Each point represents the mean of three separate determinations ± SE. Change in A450 for P-ca exposed to NO2. Samples of P-ca hexane), exposed as described were measured immediately a the indicated times for absorb (A450). Samples that were remea showed some recovery of A450, ir loss was in part reversible. Contr carotene in hexane bubbled (60 only in the dark or light are alsi HPLC analysis (22)  Time (mim) Figure 3. NO formation by lutein and NO2 in the presence of light. Lutein (i0 5 M in 5 ml hexane) was exposed for the indicated times to 27.5 ppm NO2 in N2 by bubbling at a flow rate of 60 mI/min and NO measured in the exit gas as described in ml/mmn) with N2 in hexane was added to the 5-ml sample. One plotted,n = 1)2 milliliter of this solution was then placed into an nall quantity of , amber vial and incubated at 370C. A 1-pI sample is none immediwas removed after 24 hr and analyzed by gas chromatography-thermal energy analysis for Nnitrosomorpholine as described previously (22 is well accepted, an analogous function for dietary antioxidants in humans has not been conclusively established, despite numerous epidemiologic studies that show an inverse association between carotenoid consumption and/or serum levels and cancer incidence (3,30). Cigarette smoke, which contains large quantities of nitrogen oxides (31), is strongly associated with lung cancer incidence in humans, and many studies suggest a protective effect for antioxidants against lung cancer (32). Interestingly, a study of Fiji Islanders, who have extremely low lung cancer incidence rates, revealed only weak associations, negatively with carotenoids and positively with a-tocopherol, but did suggest a strong inverse association with plasma y-tocopherol (33).
The observations described here offer a plausible explanation for the differential toxicity of NO2 in plants exposed under various lighting conditions and suggest limitations in the ability of carotenoids to protect against nitrogen oxide-mediated damage in plants in the absence of light. The conditions under which carotenoids and tocopherols protect against oxidative damage in animals, as well as the nature of the oxidative species, remain to be determined. A better understanding of the darkphase reactions of plant antioxidants may ultimately help to elucidate their roles in human health as well.