Synthesis of nitric oxide and nitrosamine by immortalized woodchuck hepatocytes.
Woodchuck (Marmota monax) hepatic cells, which were immortalized by the simian virus 40 large T antigen (SV40 Tag) produced nitric oxide (NO; measured as nitrite) in vitro from L-arginine (L-Arg) after lipopolysaccharide (LPS) treatment. NO synthesis was related to L-Arg and LPS concentration and plateaued at 1.0 mM L-Arg and 1.0 microgram/ml LPS. LPS-stimulated cells nitrosated morpholine to form N-nitrosomorpholine (NMOR) in the presence of L-Arg at pH 7.4. NMOR production increased 7-fold in LPS stimulated cells compared to unstimulated hepatocytes. N-nitrosodimethylamine (NDMA) was detected in the cell culture medium in the presence of LPS and L-Arg but without added dimethylamine. NG-monomethyl-L-arginine, a selective inhibitor of nitric oxide synthase, inhibited formation of NO and NMOR, indicating that NO and nitrosating agents were formed via the L-Arg-nitric oxide pathway. These data are the first to report NO and N-nitrosamine production by immortalized hepatocytes and confirm earlier work showing that primary hepatocytes form NO in culture. This suggests that hepatic formation of N-nitroso compounds and/or NO could be an etiologic factor in hepatocellular carcinoma. Immortalized woodchuck hepatic cells may be useful as in vitro models to study the L-Arg-nitric oxide pathway and its possible role in liver carcinogenesis.
Institute of Comparative and Environmental Toxicology, Ithaca, NY.
R H
RH
Liu
J R
JR
Jacob
J H
JH
Hotchkiss
B C
BC
Tennant
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics; D001120:Arginine / Q000378:metabolism; D002472:Cell Transformation, Viral / Q000235:genetics / Q000502:physiology*; D005814:Genes, Viral / Q000235:genetics / Q000502:physiology; D007700:Kinetics; D008070:Lipopolysaccharides / Q000494:pharmacology; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D008392:Marmota / Q000378:metabolism* / Q000502:physiology; D008954:Models, Biological; D009569:Nitric Oxide / Q000378:metabolism*; D009602:Nitrosamines / Q000378:metabolism*; D009857:Oncogenes / Q000235:genetics / Q000502:physiology; D013539:Simian virus 40 / Q000235:genetics / Q000276:immunology; D013268:Stimulation, Chemical
D000952:Antigens, Polyomavirus Transforming; D008070:Lipopolysaccharides; D009602:Nitrosamines; D009569:Nitric Oxide; D001120:Arginine