© 2008 The Authors Doi: 10.1111/j.1742-7843.2008.00228.x Journal compilation © 2008 Nordic Pharmacological Society . Basic & Clinical Pharmacology & Toxicology , 103, 36–42 Blackwell Publishing Ltd Deficiency in Cytosolic Malic Enzyme Does Not Increase Acetaminophen-Induced Hepato-Toxicity Su Qian, Sheena Mumick, Peter Nizner, Michael R. Tota, Joseph Menetski, Marc L. Reitman and Douglas J. MacNeil Department of Metabolic Disorders, Merck Research Laboratories, Rahway, NJ, USA (Received June 8, 2007; Accepted November 14, 2007) Abstract: Cytosolic malic enzyme (ME-1) is a nicotinamide adenine dinucleotide phosphate (NADP)-dependent enzyme that generates NADPH. The activity of this enzyme, the reversible oxidative decarboxylation of malate to yield pyruvate, links glycolytic pathway to citric acid cycle. The high level of ME-1 expression in liver, and its involvement in NADPH production, suggests reduced ME-1 activity might compromise hepatic production of reduced glutathione (GSH) by the NADPH-dependent enzyme glutathione reductase, and hence affect xenobiotic detoxification. The role of ME-1 in liver detoxification was evaluated in Mod1 deficient mice (mod1 –/– ) by evaluating their sensitivity to acetaminophen-induced liver injury. The results show that mod1 –/– mice are not more sensitive to acetaminophen hepato-toxicity. Although GSH levels were initially depleted more in the mod1 –/– liver than in wild-type controls, the GSH levels recovered quickly. In conclusion, our data indicate that ME-1 deficiency does not adversely affect GSH-dependent detoxification. Cytosolic malic enzyme (ME-1) is a homotetrameric enzyme catalysing reversible oxidative decarboxylation of malate to yield pyruvate and carbon dioxide. The ME-1 reaction is a step in the pyruvate–citrate shuttle by which acetyl- CoA generated via pyruvate decarboxylation by pyruvate dehydrogenase is exported out of the mitochondria and made available for fatty acid synthesis in the cytosol. In the process, ME-1 generates a significant fraction of the cytosolic reducing agent NADPH, which is important for diverse cellular functions, including de novo synthesis of long-chain fatty acids [1,2]. In mammals, two additional isoforms of malic enzyme reside in mitochondria. One malic enzyme isoform, ME-2, uses nicotinamide adenine dinucleotide as the cofactor, while the other isoform, ME-3, prefers nicotinamide adenine dinucleotide but can also use nicotinamide adenine dinucleotide phosphate (NADP). In lipogenic tissues, like liver and adipose tissue, more than 90% of malic enzyme activity is present in the cytoplasmic fraction [3]. Liver and adipose activity of ME-1 is induced by a high carbohydrate/low-fat diet, and down-regulated by a high- fat diet [2,4–6]. Due to its concerted induction with glucose- 6-phosphate dehydrogenase and 6-phospho-gluconate dehydrogenase, two NADPH producing enzymes important for fatty acid synthesis, ME-1 is also considered a lipogenic enzyme [1–6]. Indeed, higher levels of liver ME-1 activity have been associated with obese mouse and rat models [7,8]. High ME-1 activity has also been observed in certain human carcinoma cell lines [9,10], possibly reflecting altered energy metabolism in cancer cells. Cytosolic malic enzyme may play a significant role in the liver’s detoxification of xenobiotics. Liver detoxification of xenobiotics relies heavily on reduced glutathione (GSH)- mediated peroxide reduction process. To maintain the liver’s detoxification capacity, an adequate supply of NADPH is crucial for GSH regeneration by glutathione reductase. ME-1 is highly expressed in liver, and an increase in NADPH consumption, due to xenobiotic detoxification, increases the specific activity of ME-1 [11]. Thus, the inhibition or loss of ME-1 may compromise the liver’s detoxification capacity, leading to an increase in the sensitivity of liver to toxic agents. To evaluate the effect of a reduction in ME-1 on the liver’s detoxification capacity, we compared mod1 –/– mice to wild- type mice for their sensitivity to liver toxicity induced by acetaminophen (APAP). A strain of spontaneous occurring mod1 –/– mice has been described [12]. The aberrant mod1 mRNA in the mutant mice carries a 522-bp duplication corresponding to nucleotide 1027–1548 in the wild-type mod1 transcript [13]. The aberrant Mod1 protein accumulates to only about 1–2% of the wild-type Mod1 level, presumably due to reduced stability. No ME-1 activity was detected in liver or kidney cytosol preparations from mod1 –/– mice [12]. The mod1 –/– mice do not exhibit overt abnormalities under standard laboratory conditions. Acetaminophen is a commonly used analgestic and antipyretic drug, but this drug can cause life-threatening liver damage when taken in excess. At therapeutic doses, APAP is oxidized by cytochrome P-450 enzymes in the liver to produce N-acetyl-p-benzoquinone imine (NAPQI), which is further detoxified by conjugation with GSH [14]. After an overdose of APAP, GSH is not regenerated fast enough and becomes depleted. The unconjugated NAPQI triggers liver cell necrosis by either covalent binding to cellular proteins and blocking their activities, or increasing oxidative stress [15,16]. Author for correspondence: Su Qian, Department of Metabolic Disorders, Merck Research Laboratories, P.O. Box 2000, RY80M-213, Rahway, NJ 07065, USA (fax 732-594-6708, e-mail su_qian@merck.com).