Enzymatic Responses to Alcohol and Tobacco Nicotine- Derived Nitrosamine Ketone Exposures in Long Evans Rat Livers

Research Article

Austin Liver. 2016; 1(1): 1003.

Enzymatic Responses to Alcohol and Tobacco Nicotine- Derived Nitrosamine Ketone Exposures in Long Evans Rat Livers

Yalcin EB¹, Tong M¹ and de la Monte SM1,2*

¹Liver Research Center, Division of Gastroenterology and Department of Medicine, Rhode Island Hospital and the Alpert Medical School of Brown University, USA

²Departments of Neurology, Neurosurgery and Pathology, Rhode Island Hospital and the Alpert Medical School of Brown University, USA

*Corresponding author: de la Monte SM, Departments of Neurology, Neurosurgery and Pathology, Rhode Island Hospital and the Alpert Medical School of Brown University, USA

Received: September 13, 2016; Accepted: November 25, 2016; Published: December 01, 2016


Background: Chronic feeding plus binge administration of ethanol causes very high blood alcohol concentrations. However, its co-administration with tobacco Nicotine-Derived Nitrosamine Ketone (NNK) results in somewhat lower blood alcohol levels, suggesting that NNK and therefore smoking, alters alcohol metabolism in the liver. To explore this hypothesis, we examined effects of ethanol and/or NNK exposures on the expression and activity levels of enzymes that regulate their metabolism in liver.

Methods: This study utilized a 4-way model in which Long Evans rats were fed liquid diets containing 0% or 26% ethanol for 8 weeks, and respectively i.p injected with saline or 2 g/kg of ethanol 3 times/week during Weeks 7 and 8. The control and ethanol-exposed groups were each sub-divided and further i.p treated with 2 mg/kg of NNK or saline (3x/week) in Weeks 3-8. ADH, catalase and ALDH activities were measured using commercial kits. CYP450 mRNA levels (17 isoforms) were measured by qRT-PCR analysis.

Results: Ethanol significantly increased hepatic ADH but not catalase or ALDH activity. NNK had no effect on ADH, ALDH, or catalase, but when combined with ethanol, it increased ADH activity above the levels measured in all other groups. Ethanol increased CYP2C7, while NNK increased CYP2B1 and CYP4A1mRNA levels relative to control. In contrast, dual ethanol + NNK exposures inhibited CYP2B1 and CYP4A1 expression relative to NNK. Conclusion: Dual exposures to ethanol and NNK increase hepatic ethanol metabolism, and ethanol and/or NNK exposures alter the expression of CYP450 isoforms that are utilized in NNK and fatty acid metabolism.

Keywords: Alcoholic liver disease; Ethanol metabolism; NNK; Alcohol dehydrogenase; Aldehyde dehydrogenase; Cytochrome P450


Actβ: Beta-Actin; ADH: Alcohol Dehydrogenase; ALD: Alcoholic Liver Disease; ALDH: Aldehyde Dehydrogenase; ANOVA: Analysis of Variance; CYP: Cytochrome P450; DCA: Dicarboxylic Acid; ER: Endoplasmic Reticulum; HPRT: Hypoxanthine-Guanine Phosphoribosyl transferase; MEOS: Microsomal Ethanol Oxidizing System; NNK: Nicotine Derived Nitrosamine Ketone; qRT-PCR: Quantitative Reverse Transcriptase Polymerase Chain Reaction


Severity of alcohol induced liver injury correlates with dose and duration of alcohol exposure and the levels of its toxic metabolites [1,2]. Acute alcohol-mediated liver injury is characterized by reversible steatosis. High levels of chronic or binge alcohol exposures cause simple steatosis to progress through stages including steatohepatitis, fibrosis, and finally cirrhosis [3]. An important mediator of alcoholinduced liver injury is the accumulation of its highly toxic metabolite, acetaldehyde [1]. However, one relatively recent consideration regarding the pathogenesis of progressive alcohol-mediated liver injury is that a very high percentage of heavy drinkers also smoke tobacco products (mainly cigarettes) [4]. Of note is that chronic exposures to ethanol, tobacco smoke/products, and tobacco specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) all cause steatohepatitis with oxidative damage [5-9]. In our efforts to examine the role of NNK as a co-factor in alcoholic liver disease, we made the unexpected observation that dual exposures to NNK and chronic plus binge ethanol administration results in lower blood alcohol concentrations compared with the same ethanol treatments alone [8]. These observations suggested that hepatic enzyme activities needed to metabolize alcohol could be modulated by NNK exposures and therefore probably smoking.

Ethanol is metabolized in the liver through three distinct oxidative pathways including Alcohol Dehydrogenase (ADH), Cytochrome P450 2E1 (CYP2E1), and catalase (Figure 1). ADH is main enzyme responsible for the metabolism of ethanol. ADH is an abundantly expressed NAD+ dependent cytosolic enzyme [10]. The CYP2E1 microsomal ethanol oxidizing pathway requires NADPH as a cofactor [11]. Catalase, localized in peroxisomes, utilizes hydrogen peroxide to catalyze alcohol metabolism [12]. Acetaldehyde, a highly toxic and carcinogenic metabolite is generated by all three oxidative pathways; acetaldehyde can covalently bind and form adducts with DNA [13], phospholipids [14], hepatic microsomal proteins, hemoglobin [15], and erythrocyte membrane proteins [16,17]. Aldehyde Dehydrogenase (ALDH) detoxifies acetaldehyde to acetate in mitochondria [18]. Acetaldehyde accumulations caused by unbalanced increases in ADH or inhibition of ALDH activity lead to increased oxidative and ER stress and cellular injury [19,20]. ADH metabolism of ethanol also yields reduced NADH which promotes steatosis by stimulating fatty acid synthesis [20].