GROWTH AND VIABILITY OF MACROPHAGES CONTINUOUSLY STIMULATED TO PRODUCE NITRIC-OXIDE

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 conditio ns 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 mo use recombinant interferon-gamma (IFN-gamma) over periods of 21-23 day s, they continued to grow, but with doubling times 2 to 4 times, respe ctively, longer than the doubling time of unstimulated cells, Stimulat ed cells produced NO at rates of 30 to 70 nmol per million cells per d ay throughout the stimulation period, Within 24 hr after removal of st imulant, cells resumed exponential growth, Simultaneous exposure to LP S and IFN-gamma resulted in decreased cell number, which persisted for 2 days after removal of the stimulants, Exponential growth was attain ed only after an additional 4 days, Addition of N-methyl-L-arginine (N MA), an NO synthase inhibitor, to the medium inhibited NO production b y 90% of all stimulated cells, partially reduced doubling time of cell s stimulated with LPS or IFN-gamma, and partially increased viability and growth rates in those exposed to both LPS and IFN-gamma. However, when incubated with LPS and IFN-gamma at low densities both in the pre sence and in the absence of NMA, cells grew at a rate slower than that of unstimulated cells, with no cell death, and they resumed exponenti al growth 24 hr after removal of stimulants, Results from cell density experiments suggest that macrophages are protected from intracellular ly generated NO; much of the NO damaging activity occurred outside of the producer cells, Collectively, results presented in this study sugg est that the type of cellular toxicity observed in macrophages is mark edly influenced by rate of exposure to NO: at low rates of exposure, c ells exhibit slower growth; at higher rates, cells begin to die; at ev en higher rates, cells undergo growth arrest or die, The ability of RA W264.7 cells to produce NO over many cell generations makes the cell l ine a useful system for the study of other aspects of cellular damage, including genotoxicity, resulting from exposure to NO under long-term conditions.