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

3. NITRIC OXIDE AND N-NITROSATION REACTIONS

Exposure of critical genes to mutagenic conditions increases the probability of tumor development. Chronic inflammation is one such environment which promotes malignant transformation. First, tissues neighboring inflammatory foci undergo increased cell division.

Consequently, mutagenic effects associated with chronic inflammation can become multiplicative, as the chance of mis-repair and DNA exposure increase (13). Secondly, certain leukocyte-derived metabolites and inflammatory mediators cause genomic damage, thereby increasing the probability of nicks, deletions, and point mutations (14).

In situations of chronic inflammation, target cells may be exposed to large amounts of NO (as much as 10⁴ molecules/cell/s) (15). Nitric oxide will autooxidize in the presence of molecular oxygen (O₂) to yield a variety of nitrogen oxides:

2NO + O₂ --> 2NO₂

2NO + 2NO₂ --> 2N₂O₃

2N₂O₃ + 2H₂O --> 4NO₂^- + 4H⁺

where NO₂^., N₂O₃, and NO₂^- represent nitrogen dioxide, dinitrogen trioxide, and nitrite, respectively. Of these, NO₂ and N₂O₃ have drawn particular interest due to their ability to N-nitrosate certain nucleophilic substrates such as primary and secondary amines (16).

Such nitrosating species have been shown to promote the nitrosative deamination of primary aromatic amines, including purines and pyrimidines, via the formation of nitrosamine and diazonium ion intermediates (17). Deamination of cytosine, methyl cytosine, adenine, or guanine results in the formation of uracil, thymine, hypoxanthine, and xanthine respectively. Base conversion of cytosine and methyl cytosine can lead ultimately to a base pair substitution mutation, while deamination of adenine and guanine results in transversion mutations. Moreover, the instability of hypoxanthine and xanthine in the DNA structure leads to rapid depurination and consequent single strand breaks. Even crosslinking with other nucleic acids or proteins have been suggested via reaction of a nucleophilic site on an adjacent macromolecule and the diazonium ion of the modified base (18).

Nitrosation of secondary aliphatic and aromatic amines can also produce potentially carcinogenic nitrosamines. Secondary nitrosamines are more stable than their primary amine counterparts:

R₂NH + XNO --> R₂NNO + HX

Such nitrosamines, like many chemical carcinogens, are thought to promote mutagenesis and carcinogenesis via their ability to alkylate specific sites in DNA. For example, these types of nitrosamines undergoe enzymatic alpha-hydroxylation. The alpha-hydroxy nitrosamine decomposes to form the alkyl diaznoium ion and free alkyl carbocation (Fig 1). The alkyl diazonium salt or carbocations then can react with nucleophilic sites in DNA.

Figure 1: Nitrosamine-mediated alkylation of DNA bases. Secondary nitrosamines such as N-nitrosodimethylamine induce point mutations by alkylation of DNA bases such as guanine to form O⁶methylguanine residues.

To date, alkylation of DNA has been noted on the ring-nitrogen positions in the bases (adenine, guanine, cytosine, thymine), the oxygen atoms of hydroxyl or carbonyl groups (guanine, thymine, and cytosine) as well as on the phosphate groups (19).

Thus, the limitation of NO-mediated genomic damage, rests primarily on the localized diffusion of the small molecule, the degree of reactivity and nitrosation, and ultimately the cells' replicative and DNA repair machinery. However, even in the latter case, DNA repair proteins such as O6-methylguanine-DNA-methyltransferase and Fpg, have been shown to be inhibited by NO-derived nitrosating agents such as N₂O₃ in vitro and in vivo (20, 21).

Coincident with the sustained overproduction of NO, inflammatory foci are also sites of enhanced production of reactive oxygen species, such as O₂^- and H₂O₂. Because O₂^- is known to rapidly react with NO, it was of interest to determine whether this reactive oxygen specie may modulate NO-dependent N-nitrosation of primary aromatic amines. Consequently, we have used 2,3-diaminonaphthalene (DAN) as a model to study the effect of O₂^- on the NO-dependent N-nitrosation of this primary aromatic amine. DAN is N-nitrosated by nitrosating agents derived from NO to yield its highly fluorescent triazole derivative 1-naphtho(2,3)triazole (NAT) (Fig 2).

Figure 2: N-nitrosation of 2,3-diaminonaphthalene (DAN) to yield 2,3-naphthotriazole (NAT) by an NO-derived N-nitrosating agent (NO_X).

We have demonstrated that the addition of a O₂^- generator such as hypoxanthine/xanthine oxidase virtually eliminated the NO-dependent N-nitrosation of DAN. Inhibition was maximal when equimolar fluxes of NO and O₂^- were produced (17). We also noted that this inhibition was reversed by the addition of superoxide dismutase, but not catalase suggesting that O₂^- and not H₂O₂ was responsible for the inhibition. We proposed that at equimolar fluxes, O₂^- reacts rapidly with NO to generate products such as peroxynitrite (ONOO^-) or derivatives thereof - which have only a limited ability to N-nitrosate amino compounds (16,17). Although we found that O₂^- inhibits the potentially mutagenic N-nitrosation of primary and secondary amines, the formation of ONOO^- could conceivably promote oxidative (and nitrative) modifications of DNA bases, switching NO-mediated DNA damage from a nitrosative to a more oxidative pattern of mutagenic reactions (22).