Development of ferret as a human lung cancer model by injecting 4-(Nmethyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK)

Abstract

Objective: Development of new animal lung cancer models that are relevant to human lung carcino-genesis is important for lung cancer research. Previously we have shown the induction of lung tumor in ferrets (Mustela putorius furo) exposed to both tobacco smoke and a tobacco carcinogen (4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone, NNK). In the present study, we investigated whether NNK treatment alone induces both preneoplastic and neoplastic lesions in the lungs of ferrets.

Methods: We exposed ferrets to NNK by i.p. injection of NNK (50 mg/kg BW) once a month for four consecutive months and then followed up for 24, 26 and 32 weeks. The incidences of pulmonary pre-neoplastic and neoplastic lesions were assessed by histopathological examination. The expressions of 7 nicotinic acetylcholine receptor ( 7 nAChR, which has been shown to promote lung carcinogenesis)and its related molecular biomarkers in lungs were examined by immunohistochemistry and/or Western blotting analysis.

Results: Ferrets exposed to NNK alone developed both preneoplastic lesions (squamous metaplasia, dysplasia and atypical adenomatous hyperplasia) and tumors (squamous cell carcinoma, adenocarcinoma and adenosquamous carcinoma), which are commonly seen in humans. The incidence of tumor induced by NNK was time-dependent in the ferrets (16.7%, 40.0% and 66.7% for 24, 26 and 32 weeks, respectively). 7 nAChR is highly expressed in the ferret bronchial/bronchiolar epithelial cells, and alveolar macrophages in ferrets exposed to NNK, and in both squamous cell carcinoma and adenocarcinoma of the ferrets. In addition, we observed the tendency for an increase in phospho-ERK and cyclin D1 protein levels (p = 0.081 and 0.080, respectively) in the lungs of ferrets exposed to NNK.

Conclusion: The development of both preneoplastic and neoplastic lesions in ferret lungs by injecting NNK alone provides a simple and highly relevant non-rodent model for studying biomarkers/molecular targets for the prevention, detection and treatment of lung carcinogenesis in humans.

Fig. 1

Representative image of squamous cell carcinoma (Panels A and B), adenocarcinoma (Panels C and D) and preneoplastic lesions (Panels E and F) at 10X and 40X magnifications in the lung of ferrets exposed to NNK for 24 - 32 weeks.

Fig. 2

The expression of α7 nAChR (A), phospho-Akt/total-Akt (B), phospho-ERK/total-ERK (C) and cyclin D1 (D) protein levels in the lung of ferrets that were non-exposed (Cont) and exposed to NNK (NNK) for 26 weeks, measured by western blotting from whole lung homogenate. GAPDH was chosen as internal control for protein equal loading. Values are expressed Mean ± SEM. *p < 0.05.

Fig. 3

Representative image of α7 nAChR immunohistochemistry in the lung of ferrets that were non-exposed (Panel A) and exposed to NNK for 26 weeks [bronchiolar epithelial cells (Panel B), alveolar macrophage (Panel C), adenocarcinoma (Panel D), squamous cell carcinoma (Panel E), and tumor associated macrophage (Panel F)] at 40X magnifications. The arrows indicated the expression of α7 nAChR (strained in reddish-purple) on the cell membranes.