Tobacco carcinogen induces both lung cancer and non-alcoholic steatohepatitis and hepatocellular carcinomas in ferrets which can be attenuated by lycopene supplementation

Abstract

Early epidemiologic studies have reported that tobacco smoking, which is causally associated with liver cancer, is an independent risk factor for non-alcoholic fatty liver diseases (NAFLD). Lycopene from tomatoes has been shown to be a potential preventive agent against NAFLD and hepatocellular carcinoma (HCC). In the present study, we investigated whether the tobacco carcinogen 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induces lesions in both lungs and livers of ferrets with or without lycopene intervention. Male ferrets (6 groups, n = 8-10) were treated either with NNK (50 mg/kg BW, i.p., once a month for four consecutive months) or saline with or without dietary lycopene supplementation (2.2 and 6.6 mg/kg BW/day, respectively) for 26 weeks. Results demonstrate that NNK exposure results in higher incidences of lung tumors, HCC and steatohepatitis (which is characterized by severe inflammatory cell infiltration with concurrent fat accumulation in liver, hepatocellular ballooning degeneration and increased NF-κB expression), as well as elevations in bilirubin and AST levels in ferrets. Lycopene supplementation at two doses prevented NNK-induced expressions of α7 nicotinic acetylcholine receptor in the lung and NF-κB and CYP2E1 in the liver and attenuated the NNK-induced mortality and pathological lesions in both the lungs and livers of ferrets. The present study provided strong experimental evidence that the tobacco carcinogen NNK can induce both HCC and steatohepatitis in the ferrets and can be a useful model for studying tobacco carcinogen-associated NAFLD and liver cancer. Furthermore, lycopene could provide potential benefits against smoke carcinogen-induced pulmonary and hepatic injury.

Keywords: ferret; liver cancer; lycopene; steatohepatitis; tobacco carcinogen.

Figure 1

Kaplan - Meier survival analysis in ferrets injected with either sham or NNK with or without lycopene supplementation. **P<0.01 (Long rank test). Abbreviations: C+P: control (sham) + placebo group; C+LL: control + low-dose lycopene group; C+HL: control + high-dose lycopene group; N+P: NNK + placebo group; N+LL: NNK + low-dose lycopene group: N+HL: NNK + high-dose lycopene group.

Figure 2

Representative image of histopathologic examination with hematoxylin and eosin staining: normal lung tissue (Panel A), atypical adenomatous hyperplasia (Panel B), squamous metaplasia (Panel C), dysplasia (Panel D), adenocarcinoma (Panel E) and squamous cell carcinoma (Panel F) at 10× magnifications in the ferrets exposed to NNK for 26 weeks.

Figure 3

Representative image of normal liver with very mild steatosis (Panel a); hepatic steatosis with inflammatory cell infiltration (Panel b); steatohepatitis with severe fat accumulation, inflammation and hepatocellular ballooning degeneration (Panel c, The red arrow indicated the hepatocellular ballooning degeneration); hepatic non-cancerous tissue immunostained by cytokeratin 19 (Panel d), HCC (Panels e, f and g), HCC immunostained by cytokeratin 19 (Panel h), hepatic SCC (Panels i, j and k) and hepatic SCC immunostained by cytokeratin 19 (Panel l). The black arrows indicated the border between the non-cancerous tissue and cancerous tissue in Panels e, f, i and j.

Figure 4

The expression of pulmonaryα7 nAChR (a) ,NF-κB (b), Cyclin D1 (c), MMP-2 (d) and hepatic CYP2E1 (e), NF-κB (f) protein levels of ferrets exposed to NNK for 26 weeks with or without lycopene supplementation, measured by western blotting. GAPDH was chosen as internal control for protein equal loading. Values are expressed Mean ± SEM. Different letters for given bars indicate that those values are significantly different from each other (P<0.05, ANOVA (Tukey)).