Nitrotyrosine formation, apoptosis, and oxidative damage: relationships to nitric oxide production in SJL mice bearing the RcsX tumor.

In SJL mice, growth of RcsX lymphoma cells results in activation of macrophages in the spleen and lymph nodes to produce high levels of NO radical (NO.). We used this experimental model system to study the toxicology of NO. in vivo. To characterize spatial relationships between sites of NO. production and tissue damage, immunohistochemical techniques were developed for simultaneous detection of inducible NO. synthase (iNOS), 3-nitrotyrosine, and apoptosis in spleen and lymph nodes of tumor-bearing animals. Elevated expression of iNOS, presumed to reflect increased NO. production, was associated with a significant increase in frequency of apoptotic nuclei. Both apoptotic nuclei and 3-nitrotyrosine staining were found in cells juxtaposed to iNOS-expressing (ie., NO.-producing) macrophages and also within the macrophages themselves. To assess the extent of DNA damage associated with the response, 8-oxoguanine levels were quantified in DNA extracted from spleens of tumor-bearing mice. No increase in levels of this marker of oxidative DNA damage was found in tissues in which apoptosis and 3-nitrotyrosine levels were highly elevated within specific subsets of cells. Collectively, our results indicate that under the pathophysiological conditions existing in the RcsX tumor-bearing SJL mouse, cellular damage caused by NO. and/or other reactive species produced by activated macrophages is highly localized within cells in close proximity to the activated macrophages.