Deregulated production of nitric oxide (NO) has been implicated in the development of certain human diseases, including cancer. We sought to assess the damaging potential of NO produced under long-term conditions through the development of a suitable model cell culture system. In this study, we report that when murine macrophage-like RAW264.7 cells were exposed continuously to bacterial lipopolysaccharide (LPS) or mouse recombinant interferon-␥ (IFN-␥) over periods of 21-23 days, they continued to grow, but with doubling times 2 to 4 times, respectively, longer than the doubling time of unstimulated cells. Stimulated cells produced NO at rates of 30 to 70 nmol per million cells per day throughout the stimulation period. Within 24 hr after removal of stimulant, cells resumed exponential growth. Simultaneous exposure to LPS and IFN-␥ resulted in decreased cell number, which persisted for 2 days after removal of the stimulants. Exponential growth was attained only after an additional 4 days. Addition of N-methyl-L-arginine (NMA), an NO synthase inhibitor, to the medium inhibited NO production by 90% of all stimulated cells, partially reduced doubling time of cells stimulated with LPS or IFN-␥, and partially increased viability and growth rates in those exposed to both LPS and IFN-␥. However, when incubated with LPS and IFN-␥ at low densities both in the presence and in the absence of NMA, cells grew at a rate slower than that of unstimulated cells, with no cell death, and they resumed exponential growth 24 hr after removal of stimulants. Results from cell density experiments suggest that macrophages are protected from intracellularly generated NO; much of the NO damaging activity occurred outside of the producer cells. Collectively, results presented in this study suggest that the type of cellular toxicity observed in macrophages is markedly inf luenced by rate of exposure to NO: at low rates of exposure, cells exhibit slower growth; at higher rates, cells begin to die; at even higher rates, cells undergo growth arrest or die. The ability of RAW264.7 cells to produce NO over many cell generations makes the cell line a useful system for the study of other aspects of cellular damage, including genotoxicity, resulting from exposure to NO under long-term conditions.

To better understand the mechanisms through which persistent infections͞inf lammation increase cancer risks, we assessed the potential genotoxic properties of NO produced by macrophages. We recently showed that mouse macrophage RAW264.7 cells were capable of resuming exponential growth after stimulation for NO production by interferon-␥ (IFN-␥) and͞or lipopolysaccharide. Here, we report that increases in mutant fraction (MF) in the endogenous, X-linked, hprt gene of the cells are associated with NO exposure. Cells stimulated with 100 units͞ml IFN-␥ continuously for 14 and 23 days produced a total of 9.8 and 14 mol of NO per 10 7 cells, respectively. MFs in the hprt gene of NO-producing cells were 16.6 and 31.3 ؋ 10 ؊5 , respectively, compared with 2.2 and 2.5 ؋ 10 ؊5 in untreated cells. Addition of an NO synthase inhibitor, N-monomethyl-L-arginine, to the culture medium decreased NO production and MF by 90% and 85%, respectively. Reverse transcription-PCR and DNA sequencing revealed that NO-associated hprt mutations did not differ significantly from those arising spontaneously, with the exception that certain small deletions͞insertions and multiple exon deletions were observed only in the former. MF also increased significantly in cells stimulated for only 4 days with lipopolysaccharide plus IFN-␥ for higher rates of NO production. The types and proportion of hprt mutations induced under these conditions were strikingly similar to those associated with long-term NO exposure. These results indicate that NO exposure results in gene mutations in RAW264.7 cells through mechanisms yet to be identified and may also contribute to spontaneous mutagenesis.A large fraction of cancer cases globally may be associated with chronic microbial infection or parasitic infestation (1). Although precise mechanisms through which persistent infections and the accompanying inflammation increase cancer risks remain unidentified, it is well established that macrophages and neutrophils infiltrate inflamed tissues, where they produce large quantities of reactive oxygen species and NO. Reactive oxygen species produced by inflammatory cells have been shown to induce gene mutations and cell transformation in target cells cocultured with them (reviewed in ref.2). Recent studies of several in vitro experimental systems have demonstrated that NO is capable of inducing DNA damage and mutations (reviewed in refs. 3 and 4). Recent work by our group showed colocalization of genotoxicity with increased NO production in transgenic SJL mice (5), providing evidence for the involvement of NO in mutagenesis in vivo.We also reported recently that growth and viability of stimulated mouse macrophage-like RAW264.7 cells are strongly influenced by NO (6). Under certain stimulation conditions, cells continued to divide and to produce NO over many generations, a capability making them a useful experimental system for characterization of toxicity resulting from long-term exposure to NO. Furthermore, because RAW264.7 cells were initially isolated from a m...

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Characterization of mutations induced by NO in different experimental systems will facilitate elucidation of mechanisms underlying its genotoxicity. The mutagenic specificity of NO in human cells is of particular interest in view of its potential role in inflammation-associated carcinogenesis. We compared mutagenesis in human lymphoblastoid TK6 cells and in Salmonella typhimurium induced by exposure to NO delivered into the medium at rates approximating its production by activated macrophages. Exposure of TK6 cells continuously for 60 min decreased viability by 88%, and survivors exhibited a sixfold increase in mutant fraction in the hprt gene. Independent mutants were isolated and mutations characterized by RT-PCR and DNA sequencing. Among a total of 68 mutants analyzed, RT-PCR products were obtained in 41 (60%), and cDNA sequencing revealed that 26 (63%) of them contained mutations located in the hprt coding region. Base substitutions were present in 18 mutants, 12 occurring at A:T base pairs. Seven mutants contained deletions of 1-27 bp and one a 13-bp insertion; the 15 remaining RT-PCR products contained whole-exon deletions, 14 involving single exons. Six tester strains of S. typhimurium, each containing one of the six possible point mutations in the target codon of a gene in the histidine biosynthetic pathway, were similarly treated with NO and induction of mutation was detected by reversion to histidine auxotrophy. Significant increases were observed in frequencies of each of the six possible base mutations, with the highest occurring in G:C --> A:T transitions. The pattern of NO-induced hprt mutations in TK6 cells was similar to a recently published spectrum in spontaneous mutants, suggesting that reactive species derived from NO may contribute to spontaneous mutagenesis of the endogenous hprt gene in human cells.