Two T^pes of 4-(Methylnitrosamino)-l-(3-pyridyl)-l-butanone Hemoglobin Adducts, from Metabolites Which Migrate into or Are Formed in Red Blood Cells1

The tobacco-specific nitrosamine, 4-(methylnitrosamino)-l-(3-pyridyl)1-butanone (NNK), is considered to play an important role in the induc tion of lung cancer in tobacco users. In rats treated with [5--'H]NNK, 20 to 40% of the tritium bound to hemoglobin (Hb) is released by base hydrol ysis as 4-hydroxy-l-(3-pyridyl)-l-butanone (HPB). This HPB-releasing adduct has been quantified in tobacco users and is considered a biochemical marker for uptake and activation of tobacco-specific nitrosamines. In this paper we report the formation of this adduct in red blood cells (RBC) cultured for 2 h with hepatocytes and 5 MMNNK (6.35 ±0.21 fmol HPB/mg Hb). The HPB-releasing adduct was not formed in RBC in the absence of hepatocytes (<0.5 fmol/mg Hb). Therefore, the HPB-releasing adduct must form from a pyridyloxobutylating metabolite of NNK which traveled out of the hepatocytes and into RBC where it reacted with Hb. Other distinct Hb adducts were formed when NNK was incubated with RBC alone. 4-Oxo-4-(3-pyridyl)butyric acid was detected by radio flow high-performance liquid chromatography in the media of these incuba tions. The Hb isolated from RBC incubated with | <"I I ,|NNK contained as much as 10 times more covalently bound tritium than the Hb from [5--'H]NNK-treated cells. [C3H3]-l-Methylhistidine and [C3H3]-S-methylcysteine were formed when [C'H.,]NNK was incubated with the 25,000 x g supernatant from RBC. This supernatant contains 50 mg Hb/ml. We propose that Hb mediates a hydroxylation of NNK at the méthylà ̈ne carbon. The a-hydroxynitrosamine formed decomposes to methanediazohydroxide and 4-oxo-l-(3-pyridyl)butanal. The former would methylate nucleophilic sites in Hb, i.e., cysteine and histidine. The latter would bind to Hb or be further oxidized to 4-oxo-4-(3-pyridyl)butyric acid. The ability of the RBC to activate NNK to Hb-binding species stresses the importance of understanding how a particular adduct is formed prior to its use as a biochemical marker or internal dose monitor.