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4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a tobacco-specific nitrosamine formed from nicotine during tobacco curing and smoking. Its in vivo metabolism to NNAL causes the formation of two enantiomers, (R)- and (S)-NNAL. (S)-NNAL is equipotent to NNK with respect to lung tumorigenicity in the A/J mouse and is significantly more tumorigenic than either (R)-NNAL or racemic NNAL. (S)-NNAL is also selectively retained in the lung tissue of the F344 rat. The present work is a validation of the recirculating isolated perfused lung system for use in studying the lung tissue retention of NNK and its metabolites. The lungs of male Fisher 344 rats (n=6, weight 315 ± 35 g) were perfused in a recirculating system with 50 ml of a modified oxygenated Krebs-Ringers buffer (pH maintained at 7.4) at a flow rate of 8 ml/min. The system was maintained at 37° C. A 50 μCi bolus dose of 3H-NNK was injected into the reservoir and serial perfusate samples were taken over 120 min. At 120 min, the lung was perfused with blank perfusate, then was excised and flash frozen in liquid nitrogen. Perfusate and tissue samples were analyzed by LSC and HPLC with radioflow detection. The major perfusate metabolites were NNK-N-oxide (48 ± 7% of radioactivity present), keto alcohol (14 ± 4%), and keto acid (12 ± 4%) with 4.5% NNK remaining. In the tissue the major metabolites were NNAL-N-oxide (24 ± 3%), and keto acid (21 ± 4%) with 6.5 ± 2% NNK remaining. Although NNK was almost completely metabolized by the perfused lung, its extraction ratio was 0.21 ± 0.5 (n=5). Its clearance was 1.63 ± 0.4 ml/min, with a terminal elimination half-life of 24.8 ± 8.2 min. Oxidative metabolism was clearly predominant in the lung, leading to both detoxified products (N-oxides) as well as the α-hydroxylation products of both NNK (keto acid, keto alcohol) and NNAL (diol, hydroxy acid). A significant portion of the NNK dose passed through the NNAL pathway, as over 40% of the radioactivity in the lung at the end of the perfusion was NNAL or its metabolites. The pattern of metabolites between the perfusate and tissue shows some interesting differences. NNK-N-oxide was the predominant metabolite in the perfusate, while NNAL-N-oxide and keto acid predominated in the lung tissue. More than 50% of the metabolites in the perfusate were the N-oxides, and 28% were products of the α-hydroxylation pathways. In the lung tissue, 33.5% of the metabolites were the N-oxides and about 40% were the α-hydroxylation products. Since the α-hydroxylated metabolites are the end products of the bioactivation pathways responsible for the formation of DNA-adducts, this suggests that the lung tends to retain these activated metabolites. These data indicate the feasibility of the use of the isolated perfused lung system to evaluate formation and retention of NNK and NNAL metabolites. This work was supported by PHS grants NCI CA-81301 and NCI/NIDA P50DA13333 (S.H.), and the 3M Science and Technology Fellowship (L.M.).

[Proc Amer Assoc Cancer Res, Volume 47, 2006]