[Frontiers in Bioscience 2, d189-196, May 1, 1997]

	[Frontiers in Bioscience 2, d189-196, May 1, 1997] Reprints PubMed CAVEAT LECTOR
	INTERACTIONS BETWEEN SUPEROXIDE AND NITRIC OXIDE: IMPLICATIONS IN DNA DAMAGE AND MUTAGENESIS David Jourd'heuil, David Kang and Matthew B. Grisham Department of Molecular and Cellular Physiology, Louisiana State University Medical Center, Shreveport, LA 71130, USA Received 4/3/97; Accepted 4/7/97; On-line 5/1/97 4. SUPEROXIDE-NITRIC OXIDE INTER-ACTIONS: OXIDATIVE REACTIONS Nitric oxide reacts with O₂^- to yield ONOO^- with a second order rate constant of 6.7 x 10⁹ M^-¹.s^-¹ (23). Once formed, ONOO^- is protonated resulting in its rapid decomposition to nitrate (NO₃^-) in the absence of any oxidizable substrate. Therefore, O₂^- will rapidly inhibit NO-mediated N-nitrosation reactions in favor of reactions catalyzed by OONO^-. Peroxynitrite exists in equilibrium with its conjugate acid peroxynitrous acid (ONOOH, pKa 6.6): NO+O₂^- --> ONOO^-+H⁺ <==> ONOOH --> ONOOH* --> NO₃^- Peroxynitrous acid is very unstable and at physiological pH decomposes to form derivatives with potent oxidizing properties. This may involve the homolysis of ONOOH to nitrogen dioxide radical (NO₂ ^. ) and hydroxyl radical (OH^.) within a solvent cage, the two free radicals diffusing out of the solvent cage to mediate oxidation reactions (24). Hydroxyl radical is an extremely reactive species, interacting with virtually all biomolecules at diffusion limited rates (~10⁷ -10⁹ M.sec^-¹) (25). Nitrogen dioxide can initiate lipid peroxidaton and N-nitrosate certain amines to yield nitrosamines (26). However, a second more probable mechanism that may account for the oxidative properties of ONOO^- is suggested to involve the formation of an activated isomer of peroxynitrous acid, ONOOH* which would possess NO₂ ^. and OH-like properties (27). From the standpoint of biologically relevant reactions, ONOO^- is a potent oxidizing, hydroxylating, and nitrating agent. In regard to DNA modifications, ONOO^- has been found to oxidize and nitrate isolated DNA resulting in DNA strand breaks (28). Little is known regarding the detailed reactions of ONOO^- with DNA. Because of the multiplicity of DNA modifications produced during oxidative reactions, it has been difficult to establish the specificity of mutations engendered by individual oxidants such as ONOO^-. Among oxidative-mediated DNA base modifications, a major product is 8-hydroxydeoxyguanosine (8-OHdG) (Fig 3) (29). The occurrence of this alteration has been associated with a number of conditions leading to increased oxidative stress including higher basal metabolic rate, gamma-irradiation, and hydrogen peroxide-mediated oxidative stress (30). Nitric oxide and iron have also been implicated in the formation of 8-OHdG in asbestos-treated human lung epithelial cells (31). Peroxynitrite also mediates the oxidation of deoxyguanosine (32). In all of those conditions, the formation of 8-OHdG might lead to mutations by inducing misreading of the base itself and of the adjacent bases (29) which may represent an important source of mutations (33). Peroxynitrite induces G:C toT:A mutations for the supF gene in E. coli and in human AD293 cells (34). Figure 3: Hydroxyl radical (OH^.) and peroxynitrite (ONOO^-)-mediated oxidation of guanine. In addition to oxidative reactions, recent data suggest that the interaction of ONOO^- with DNA results in the nitration of guanine to form 8-nitroguanine (Fig 4) (35). Figure 4: Peroxynitrite (ONOO^-)-mediated nitration of guanine to form 8-nitroguanine. This modification is potentially mutagenic, the epurination of 8-nitroguanine yielding apurinic sites with the resultant possibility of G:C to T:A transversions (36). However, whether ONOO^- mediates such DNA damage in cells and tissues is yet to be determined. The identification of 8-nitroguanine as a marker of ONOO^- -induced nitration may represent an important tool for the detection of such alteration in vivo. It is important to note that the evaluation of ONOO^--mediated mutagenic properties has been assessed in vitro using bolus amounts of chemically generated oxidant. However, it is becoming increasingly evident that the formation of ONOO^- at sites where both O₂^- and NO are produced may depend upon the relative fluxes of NO and O₂^-. Using the hypoxanthine/ xanthine oxidase system to generate both O₂^- and H₂O₂ and the spermine/NO adduct to generate various fluxes of NO, we found that the simultaneous production of equimolarfluxes of O₂^- and NO dramatically increased the oxidation of the oxidant-sensitive probe dihydrorhodamine (DHR) (22). The oxidation of DHR was inhibited by superoxide dismutase but not catalase suggesting that O₂^- and not NO interacted with NO to form ONOO^- (25). In these same experiments, ONOO^--mediated oxidation of DHR was quenched by excess formation of either NO or O₂^- suggesting that excess production of either radical may act as an endogenous modulator of ONOO^- chemistry. Subsequent experiments demonstrated that NO (or O₂^-) interacted with and decomposed ONOO^-. Although this hypothesis suggests that NO and O₂^- may modulate steady state concentrations of ONOO^-, there has yet to be any direct evidence demonstrating such modulation of ONOO^--mediated oxidative and/or nitrating reactions under physiological conditions.

[Frontiers in Bioscience 2, d189-196, May 1, 1997]
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INTERACTIONS BETWEEN SUPEROXIDE AND NITRIC OXIDE: IMPLICATIONS IN DNA DAMAGE AND MUTAGENESIS

David Jourd'heuil, David Kang and Matthew B. Grisham

Department of Molecular and Cellular Physiology, Louisiana State University Medical Center, Shreveport, LA 71130, USA

Received 4/3/97; Accepted 4/7/97; On-line 5/1/97

4. SUPEROXIDE-NITRIC OXIDE INTER-ACTIONS: OXIDATIVE REACTIONS

Nitric oxide reacts with O₂^- to yield ONOO^- with a second order rate constant of 6.7 x 10⁹ M^-¹.s^-¹ (23). Once formed, ONOO^- is protonated resulting in its rapid decomposition to nitrate (NO₃^-) in the absence of any oxidizable substrate. Therefore, O₂^- will rapidly inhibit NO-mediated N-nitrosation reactions in favor of reactions catalyzed by OONO^-.

Peroxynitrite exists in equilibrium with its conjugate acid peroxynitrous acid (ONOOH, pKa 6.6):

NO+O₂^- --> ONOO^-+H⁺ <==> ONOOH --> ONOOH* --> NO₃^-

Peroxynitrous acid is very unstable and at physiological pH decomposes to form derivatives with potent oxidizing properties. This may involve the homolysis of ONOOH to nitrogen dioxide radical (NO₂ ^. ) and hydroxyl radical (OH^.) within a solvent cage, the two free radicals diffusing out of the solvent cage to mediate oxidation reactions (24). Hydroxyl radical is an extremely reactive species, interacting with virtually all biomolecules at diffusion limited rates (~10⁷ -10⁹ M.sec^-¹) (25). Nitrogen dioxide can initiate lipid peroxidaton and N-nitrosate certain amines to yield nitrosamines (26). However, a second more probable mechanism that may account for the oxidative properties of ONOO^- is suggested to involve the formation of an activated isomer of peroxynitrous acid, ONOOH* which would possess NO₂ ^. and OH-like properties (27). From the standpoint of biologically relevant reactions, ONOO^- is a potent oxidizing, hydroxylating, and nitrating agent. In regard to DNA modifications, ONOO^- has been found to oxidize and nitrate isolated DNA resulting in DNA strand breaks (28).

Little is known regarding the detailed reactions of ONOO^- with DNA. Because of the multiplicity of DNA modifications produced during oxidative reactions, it has been difficult to establish the specificity of mutations engendered by individual oxidants such as ONOO^-. Among oxidative-mediated DNA base modifications, a major product is 8-hydroxydeoxyguanosine (8-OHdG) (Fig 3) (29). The occurrence of this alteration has been associated with a number of conditions leading to increased oxidative stress including higher basal metabolic rate, gamma-irradiation, and hydrogen peroxide-mediated oxidative stress (30). Nitric oxide and iron have also been implicated in the formation of 8-OHdG in asbestos-treated human lung epithelial cells (31). Peroxynitrite also mediates the oxidation of deoxyguanosine (32). In all of those conditions, the formation of 8-OHdG might lead to mutations by inducing misreading of the base itself and of the adjacent bases (29) which may represent an important source of mutations (33). Peroxynitrite induces G:C toT:A mutations for the supF gene in E. coli and in human AD293 cells (34).

Figure 3: Hydroxyl radical (OH^.) and peroxynitrite (ONOO^-)-mediated oxidation of guanine.

In addition to oxidative reactions, recent data suggest that the interaction of ONOO^- with DNA results in the nitration of guanine to form 8-nitroguanine (Fig 4) (35).

Figure 4: Peroxynitrite (ONOO^-)-mediated nitration of guanine to form 8-nitroguanine.

This modification is potentially mutagenic, the epurination of 8-nitroguanine yielding apurinic sites with the resultant possibility of G:C to T:A transversions (36). However, whether ONOO^- mediates such DNA damage in cells and tissues is yet to be determined. The identification of 8-nitroguanine as a marker of ONOO^- -induced nitration may represent an important tool for the detection of such alteration in vivo.

It is important to note that the evaluation of ONOO^--mediated mutagenic properties has been assessed in vitro using bolus amounts of chemically generated oxidant. However, it is becoming increasingly evident that the formation of ONOO^- at sites where both O₂^- and NO are produced may depend upon the relative fluxes of NO and O₂^-. Using the hypoxanthine/ xanthine oxidase system to generate both O₂^- and H₂O₂ and the spermine/NO adduct to generate various fluxes of NO, we found that the simultaneous production of equimolarfluxes of O₂^- and NO dramatically increased the oxidation of the oxidant-sensitive probe dihydrorhodamine (DHR) (22). The oxidation of DHR was inhibited by superoxide dismutase but not catalase suggesting that O₂^- and not NO interacted with NO to form ONOO^- (25). In these same experiments, ONOO^--mediated oxidation of DHR was quenched by excess formation of either NO or O₂^- suggesting that excess production of either radical may act as an endogenous modulator of ONOO^- chemistry. Subsequent experiments demonstrated that NO (or O₂^-) interacted with and decomposed ONOO^-. Although this hypothesis suggests that NO and O₂^- may modulate steady state concentrations of ONOO^-, there has yet to be any direct evidence demonstrating such modulation of ONOO^--mediated oxidative and/or nitrating reactions under physiological conditions.