Abstract A01: Lung and pancreatic carcinogenicity and DNA binding of the enantiomers of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in F344 rats
Authors: Balbo Silvia , Johnson Charles S. , Zhao Lijiao , O'Sullivan M Gerard , Stepanov Irina , Wang Mingyao , Kassie Fekadu , Carmella Steven , Upadhyaya Pramod , Le Chap T. , Hecht Stephen S. 
Published: May 2019

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent pulmonary carcinogen in laboratory animals and is believed to play an important role as a cause of tobacco related cancers. NNK is metabolically converted to NNAL in virtually all biological systems. All tobacco users as well as people exposed to secondhand tobacco smoke have NNAL in their urine. NNAL has a chiral center at its 1-position, thus consequently exists in enantiomeric forms. Previous studies have shown differing amounts of (R)- or (S)-NNAL in human tissues and urine after exposure to NNK, depending on the conditions and the system studied. However, little is known about the carcinogenic activity of (R)- or (S)-NNAL. Only one previous study has been reported demonstrating higher lung tumorigenicity of (S)-NNAL than (R)-NNAL in an A/J mouse model. Understanding mechanisms of carcinogenesis by these compounds can provide critical insights relevant for cancer prevention in people exposed to tobacco products. Therefore, in the study reported here, we have evaluated the carcinogenicity and DNA binding of the NNAL enantiomers in F-344 rats chronically treated with these compounds in the drinking water.

Groups of 24 male F-344 rats, 7 weeks of age, were treated chronically with NNK, (R)-NNAL or (S)-NNAL (5 ppm in drinking water). Controls received tap water or racemic NNAL (15 rats, 10 ppm in drinking water). The chronic treatment lasted 90 weeks. The number of animals per group which came to necropsy at the end of the study were: NNK, 23; (R)-NNAL, 23; (S)-NNAL, 22; control, 22; racemic NNAL, 15. Nearly all animals in the (R)-NNAL, (S)-NNAL, racemic NNAL, and NNK-treated groups had lung tumors. In the (R)-NNAL-treated group, there were 22 animals with lung adenoma and 3 with lung carcinoma while in the (S)-NNAL group there were 20 rats with lung adenoma and 5 with lung carcinoma. In the racemic NNAL group there were 15 rats with lung adenoma and 13 with lung carcinoma while in the NNK group there were 23 rats with lung adenoma and 14 with lung carcinoma. There were no lung tumors in the control animals. These results demonstrate that both enantiomers of NNAL are pulmonary carcinogens in the F-344 rat, but less carcinogenic to the lung than NNK (P&lt;0.001). Tumors of the pancreas, predominantly exocrine adenocarcinomas, were also observed in this study: (R)-NNAL, 1 rat with 2 tumors; (S)-NNAL 2 rats with 3 tumors; racemic NNAL, 4 rats with 14 tumors; NNK 3 rats with 3 tumors.

DNA binding studies were performed using tissues harvested at various time-points throughout the experiment. Rats (9 per group) from the NNK, (R)-NNAL, (S)-NNAL, and control groups were euthanized at 10, 30, 50 and 70 weeks. DNA was isolated from the lung and pancreas tissues. The DNA samples were analyzed by LC-ESI-MS/MS with selected reaction monitoring (SRM) analysis for various DNA adducts. O6-Methyl-Gua, POB-DNA adducts, including 7-POB-Gua, O2-POB-dThd and O6-POB-dG and PHB-DNA adducts, including 7-PHB-Gua, O2-PHB-dThd and O6-PHB-dG were measured in lung DNA samples. POB-DNA adducts were measured in pancreas DNA. The preliminary results from these analyses indicate that O6-Methyl-Gua and POB-DNA adducts levels (both in lung and pancreas DNA) were similar in NNK and (S)-NNAL treated rats and significantly higher than in (R)-NNAL treated rats, while PHB-DNA adducts levels were significantly higher in (R)-NNAL treated animals compared to NNK and (S)-NNAL treated groups. The results of this study demonstrate that both (R)-NNAL and (S)-NNAL, at 5 ppm in the drinking water, are effective pulmonary carcinogens in the F-344 rat and are metabolically activated to DNA binding metabolites in the lung. Furthermore, the results presented here confirm the previously observed pancreatic carcinogenicity of racemic NNAL in the F-344 rat. Collectively, these results provide important new insights relevant to mechanisms of carcinogenesis by NNK and NNAL, and to lung and pancreatic cancer in people exposed to tobacco products.

Citation Format: Silvia Balbo, Charles S. Johnson, Lijiao Zhao, M Gerard O'Sullivan, Irina Stepanov, Mingyao Wang, Fekadu Kassie, Steven Carmella, Pramod Upadhyaya, Chap T. Le, Stephen S. Hecht. Lung and pancreatic carcinogenicity and DNA binding of the enantiomers of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in F344 rats. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr A01.