[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 5. SUPEROXIDE, FENTON CHEMISTRY AND NITRIC OXIDE In general, O₂^- per se is not thought to be highly toxic to cells and tissues since it is a better reducing agent than an oxidant (37). However, O₂^- will rapidly dismutate to form H₂O₂. Although H₂O₂ is an oxidizing agent, most of the H₂O₂-dependent oxidizing activity is mediated by secondary radicals such as OH^. generated from metal (Mn)-catalyzed reactions. It is known that O₂^- and H₂O₂ interact with chelates of iron or copper to yield the potent oxidant OH^. or OH^.-like species via the superoxide-driven Fenton reaction: O₂^- + Mn⁺² --> O₂ + Mn⁺¹ H₂O₂ + Mn⁺¹ --> OH^. + OH^- + Mn⁺² The interaction of free radicals derived from superoxide-driven Fenton reactions with DNA has received considerable interest (38). Recent reports have focused on the ability of copper to participate in mutagenic reactions in vivo via Fenton-catalyzed reactions. Copper is an important structural metal in chromatin (39) that in fact induces more DNA bases damage in the presence of H₂O₂ than does iron (40). There is now evidence to suggest that NO may modulate Fenton-driven oxidative reactions. We have recently investigated the ability of different fluxes of nitric oxide to modulate iron complex (10) and hemoprotein-catalyzed oxidative reactions (41). We found that generation of O₂^- and H₂O₂ in the presence of 5 µM Fe⁺³-EDTA stimulated oxidation of DHR producing approximately 15 µM rhodamine (10). Catalase and superoxide dismutase were both effective at inhibiting this classic Fenton-driven reaction. Addition of NO increased DHR oxidation such that a 2:1 NO/ O₂^- ratio increased DHR oxidation by 30%. As the ratio NO/ O₂^- further increased to 4.5, DHR oxidation was reduced by 40%. Kanner et al. (42) proposed that NO may inhibit iron-mediated oxidative reactions by forming nitrosyl complexes with ferrous iron: Fe⁺³-EDTA + O₂^- --> Fe⁺²-EDTA + O₂ Fe⁺²-EDTA + NO --> NO-Fe⁺²-EDTA NO-Fe⁺²-EDTA+ H₂O₂ --> Fe⁺³-EDTA + HNO₂ + OH^- The efficiency of such interaction may explain the decrease in DHR oxidation obtained in the presence of high fluxes of NO. We have also assessed the ability of myoglobin to oxidize DHR in the presence or the absence of O₂^-, H₂O₂ and /or NO (33). In the presence of equimolar fluxes of H₂O₂ and O₂^-, the addition of myoglobin dramatically enhanced DHR oxidation via the formation of ferryl (Fe⁺⁴) myoglobin. This oxidative reaction was as expected inhibited by catalase but not Superoxide dismutase. Addition of NO to this system further enhanced DHR oxidation which was inhibited by superosxide dismutase suggesting that O₂^- reacted with NO to form a potent oxidant such as peroxynitrite in addition to the H₂O₂-dependent formation of ferryl myoglobin. Further increases in NO flux dramatically inhibited DHR oxidation which was found to be due to the NO-mediated reduction of ferryl heme to the met form. Taken together, these data suggest that NO may enhance or inhibit iron complex and hemoprotein-catalyzed oxidative reactions depending upon the relative fluxes of O₂^-, H₂O₂ and NO. In accordance with our results, Pacelli et al. have shown that NO can inhibit DNA strand breaks induced by H₂O₂ and certain transition metals (43).

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

5. SUPEROXIDE, FENTON CHEMISTRY AND NITRIC OXIDE

In general, O₂^- per se is not thought to be highly toxic to cells and tissues since it is a better reducing agent than an oxidant (37). However, O₂^- will rapidly dismutate to form H₂O₂. Although H₂O₂ is an oxidizing agent, most of the H₂O₂-dependent oxidizing activity is mediated by secondary radicals such as OH^. generated from metal (Mn)-catalyzed reactions. It is known that O₂^- and H₂O₂ interact with chelates of iron or copper to yield the potent oxidant OH^. or OH^.-like species via the superoxide-driven Fenton reaction:

O₂^- + Mn⁺² --> O₂ + Mn⁺¹

H₂O₂ + Mn⁺¹ --> OH^. + OH^- + Mn⁺²

The interaction of free radicals derived from superoxide-driven Fenton reactions with DNA has received considerable interest (38). Recent reports have focused on the ability of copper to participate in mutagenic reactions in vivo via Fenton-catalyzed reactions. Copper is an important structural metal in chromatin (39) that in fact induces more DNA bases damage in the presence of H₂O₂ than does iron (40).

There is now evidence to suggest that NO may modulate Fenton-driven oxidative reactions. We have recently investigated the ability of different fluxes of nitric oxide to modulate iron complex (10) and hemoprotein-catalyzed oxidative reactions (41). We found that generation of O₂^- and H₂O₂ in the presence of 5 µM Fe⁺³-EDTA stimulated oxidation of DHR producing approximately 15 µM rhodamine (10). Catalase and superoxide dismutase were both effective at inhibiting this classic Fenton-driven reaction. Addition of NO increased DHR oxidation such that a 2:1 NO/ O₂^- ratio increased DHR oxidation by 30%. As the ratio NO/ O₂^- further increased to 4.5, DHR oxidation was reduced by 40%. Kanner et al. (42) proposed that NO may inhibit iron-mediated oxidative reactions by forming nitrosyl complexes with ferrous iron:

Fe⁺³-EDTA + O₂^- --> Fe⁺²-EDTA + O₂

Fe⁺²-EDTA + NO --> NO-Fe⁺²-EDTA

NO-Fe⁺²-EDTA+ H₂O₂ --> Fe⁺³-EDTA + HNO₂ + OH^-

The efficiency of such interaction may explain the decrease in DHR oxidation obtained in the presence of high fluxes of NO. We have also assessed the ability of myoglobin to oxidize DHR in the presence or the absence of O₂^-, H₂O₂ and /or NO (33). In the presence of equimolar fluxes of H₂O₂ and O₂^-, the addition of myoglobin dramatically enhanced DHR oxidation via the formation of ferryl (Fe⁺⁴) myoglobin. This oxidative reaction was as expected inhibited by catalase but not Superoxide dismutase. Addition of NO to this system further enhanced DHR oxidation which was inhibited by superosxide dismutase suggesting that O₂^- reacted with NO to form a potent oxidant such as peroxynitrite in addition to the H₂O₂-dependent formation of ferryl myoglobin. Further increases in NO flux dramatically inhibited DHR oxidation which was found to be due to the NO-mediated reduction of ferryl heme to the met form. Taken together, these data suggest that NO may enhance or inhibit iron complex and hemoprotein-catalyzed oxidative reactions depending upon the relative fluxes of O₂^-, H₂O₂ and NO. In accordance with our results, Pacelli et al. have shown that NO can inhibit DNA strand breaks induced by H₂O₂ and certain transition metals (43).