Vol. 300, Issue 1, 18-25, January 2002

O²-Vinyl 1-(Pyrrolidin-1-yl)diazen-1-ium-1,2-diolate Protection Against D-Galactosamine/Endotoxin-Induced Hepatotoxicity in Mice: Genomic Analysis Using Microarrays

Jie Liu, Joseph E. Saavedra, Tong Lu, Jian-Guo Song, James Clark, Michael P. Waalkes and Larry K. Keefer

Inorganic Carcinogenesis Section, Laboratory of Comparative Carcinogenesis, National Cancer Institute at National Institute of Environmental Health Sciences, (J.L., T.L., M.P.W.) and Comparative Medicine Branch (J.C.), National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina; Academica Sinica Biochemistry Institute, Shanghai, China (J.-G.S.); Science Applications International Corporation-Frederick (J.E.S.) and Chemistry Section, Laboratory of Comparative Carcinogenesis, National Cancer Institute at Frederick (L.K.K.), Frederick, Maryland

O²-Vinyl 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (V-PYRRO/NO), a liver-selective nitric oxide (NO)-donating prodrug, is metabolized by hepatic enzymes to release NO within the liver. This study was undertaken to examine the effects of V-PYRRO/NO on D-galactosamine/lipopolysaccharide (GlaN/LPS)-induced liver injury in mice. Mice were given injections of V-PYRRO/NO (10 mg/kg, s.c. at 2-h intervals) before and after GlaN/LPS (700 mg/30 µg/kg, i.p.). V-PYRRO/NO administration dramatically reduced GlaN/LPS-induced hepatotoxicity, as evidenced by reduced serum alanine aminotransferase activity and improved pathology. To examine the mechanisms of the protection, cDNA microarray was performed to profile the gene expression pattern in livers of mice treated with GlaN/LPS, GlaN/LPS plus V-PYRRO/NO, or controls. V-PYRRO/NO administration greatly ameliorated GlaN/LPS-induced alterations in the expression of genes encoding the stress response, DNA damage/repair response, and drug-metabolizing enzymes in accordance with hepatoprotection. Gel shift assay and Western blot analysis supported microarray results, showing that V-PYRRO/NO suppressed GlaN/LPS-induced activation of nuclear factor-B and GlaN/LPS-induced increases in caspase-1, caspase-8, tumor necrosis factor receptor 1 (TNFR1)-associated death domain, and TNF-related apoptosis-inducing ligand. Immunohistochemical analysis further revealed that GlaN/LPS-induced activation of TNFR1, caspase-3, and hepatocellular apoptosis was ameliorated by V-PYRRO/NO treatment. GlaN/LPS-induced elevation of hepatic caspase-3 activity was diminished by V-PYRRO/NO treatment. In addition, V-PYRRO/NO alone suppressed the basal expression of genes encoding inducible NO synthase and TNF--related components, as revealed by mouse 1.2 array. In summary, this study demonstrates that the liver-selective NO donor, V-PYRRO/NO, is effective in blocking GlaN/LPS-induced hepatotoxicity in mice, and that this protection appears to involve, at least in part, the suppression of the TNF--mediated cell death pathways.