Although oxidative stress is implicated in both acute acetaminophen-induced liver failure and chronic liver diseases, cirrhosis and hepatocellular carcinoma (HCC), no common underlying metabolic pathways have been identified. The preliminary results indicate that deficiency of transaldolase (TAL, encoded by the TALDO1 genomic locus), an enzyme of the pentose phosphate pathway (PPP), is an important cause of oxidative stress in the liver that predisposes to acetaminophen-induced liver failure and hepatocarcinogenesis. Recent case reports suggested a link of homozygous mutations in the TAL gene with liver failure and cirrhosis in children. In fibroblasts and lymphoblasts of the first TAL-deficient patient, we found that deletion of S171 caused mis- folding, proteasome-mediated degradation and complete deficiency of enzymatic activity. TAL deficiency resulted in accumulation of sedoheptulose 7-phoshate (S7P) and depletion of glucose 6-phoshate (G6P), indicating a failure to recycle ribose 5-phosphate (R5P) into G6P through the non-oxidative branch of the PPP, thus reducing NADPH production by the oxidative branch. This patient's cells exhibited mitochondrial dysfunction, increased susceptibility to H2O 2 and resistance to Fas apoptosis. To create an animal model of TAL deficiency, we inactivated the TALDO1 genomic locus in the mouse. Cirrhosis and nodular dysplasia were highly prevalent in mice with partial deficiency (TAL+/-;62/229, 27.0%) or complete deficiency of TAL (TAL-/-;77/97, 79.4%), relative to wild-type littermates (TAL+/+;1/102, 1.0%). Cirrhosis invariably followed the formation of microvesicular and macrovesicular lipid droplets indicating nonalcoholic fatty liver disease (NAFLD) and inflammatory changes resembling non-alcoholic steatohepatitis (NASH). HCC was the leading cause of death in both TAL+/- (39/229;17%) and TAL-/- mice (45/97;46%) as compared to TAL+/+ littermates (1/102;1%). Oxidative stress in TAL-deficient livers was characterized by the accumulation of S7P, failure to recycle R5P for the oxidative PPP, depleted NADPH and glutathione levels, and increased production of lipid hydroperoxides (LPO). Reduced 2-catenin phosphorylation and enhanced c-jun expression in TAL-/- livers reflected adaptation to oxidative stress. TAL-/- hepatocytes were resistant to CD95/Fas-mediated apoptosis in vitro and in vivo. Remarkably, lifelong administration of N-acetylcysteine (NAC) blocked acetaminophen susceptibility, restored Fas-dependent apoptosis, phosphorylation of 2-catenin, activation of c-jun and prevented the development of NASH, cirrhosis, and hepatocarcinogenesis in TAL-deficient mice. We also found an increased prevalence of TAL haploinsufficiency in human livers with cirrhosis and HCC. Thus, the preliminary studies identify TAL deficiency as a novel cause of liver cirrhosis and HCC which are preventable by lifelong supplementation of the potent antioxidant NAC. The proposed studies will test the hypothesis that TAL deficiency leads to oxidative stress, through diminished production of NADPH leading to secondary depletion of GSH that underlies the susceptibility of acetaminophen-induced liver failure and chronic liver disease, progressing from NAFLD to NASH, cirrhosis, and HCC.
Under Specific Aim 1, we will determine the molecular mechanisms that cause NADPH depletion in TAL-deficient hepatocytes.
Under Specific Aim 2, we will test the hypothesis that hepatocarcinogenesis of TAL-deficient mice is triggered by NADPH depletion- induced oxidative stress, mediated by the over-expression of aldose reductase (AR) and the activation of 2- catenin and c-jun, and prevented by post-natal treatment with N-acetylcysteine or AR blockade.
Under Specific Aim 3, we will determine the prevalence and molecular basis of TAL deficiency in patients with acetaminophen-induced liver failure, NAFLD, NASH, cirrhosis and HCC. These studies will generate fundamental new information on the pathogenesis and treatment of liver disease.
Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third leading cause of cancer- related death worldwide. Acetaminophen (also known as Tylenol) overdose is the leading cause of acute liver failure in the US. Although oxidative stress has been implicated in HCC and acetaminophen toxicity, the genetic causes that predispose patients to these diseases are unknown. Recently, deficiency of transaldolase (TAL) was discovered in children with liver failure and cirrhosis. TAL is an enzyme that helps the utilization of the sugar glucose in certain cells of the body, such as liver cells, to produce and sustain normal levels of the compounds NADPH and glutathione which protect the integrity of the cells against oxidative stress. We created an animal model of TAL deficiency by a targeted genetic mutation in the mouse. Although mice partially (TAL+/-) or completely deficient of TAL (TAL-/-) develop normally, they are 27-fold and 79-fold more likely to develop cirrhosis and HCC than their siblings having normal TAL gene and activity. TAL-deficient (TAL-/-) mice are also more susceptible to acetaminophen-induced liver failure and death than their siblings carrying the normal TAL gene. Thus, the preliminary studies identify TAL deficiency as a novel cause of liver cirrhosis and HCC as well as acetaminophen-induced liver failure. We hypothesize that NADPH plays critical roles in the survival and multiplication of tumor cells in the liver. The proposed experiments will determine the mechanism of NADPH depletion, whether a correctable defect is responsible for the tumor-causing proliferation of liver cells, and examine the involvement of TAL deficiency in patients with acetaminophen- induced liver failure, fatty liver inflammation, cirrhosis and HCC. These studies should generate fundamental new information on the cause and treatment of potentially fatal diseases of the liver.
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