Uridine prevents tamoxifen-induced liver lipid droplet accumulation

BMC Pharmacology and Toxicology Uridine prevents tamoxifen-induced liver lipid droplet accumulation Thuc T Le 0 1 2 Yasuyo Urasaki 0 1 2 Giuseppe Pizzorno 0 1 0 Desert Research Institute , 10530 Discovery Drive, Las Vegas, NV 89135 , USA 1 Nevada Cancer Institute , One Breakthrough Way, Las Vegas, NV 89135 , USA 2 Roseman University of Health Sciences , 11 Sunset Way, Henderson NV 89014 , USA Background: Tamoxifen, an agonist of estrogen receptor, is widely prescribed for the prevention and long-term treatment of breast cancer. A side effect of tamoxifen is fatty liver, which increases the risk for non-alcoholic fatty liver disease. Prevention of tamoxifen-induced fatty liver has the potential to improve the safety of long-term tamoxifen usage. Methods: Uridine, a pyrimidine nucleoside with reported protective effects against drug-induced fatty liver, was co-administered with tamoxifen in C57BL/6J mice. Liver lipid levels were evaluated with lipid visualization using coherent anti-Stokes Raman scatting (CARS) microscopy, biochemical assay measurement of triacylglyceride (TAG), and liquid chromatography coupled with mass spectrometry (LC-MS) measurement of membrane phospholipid. Blood TAG and cholesterol levels were measured. Mitochondrial respiration of primary hepatocytes in the presence of tamoxifen and/or uridine was evaluated by measuring oxygen consumption rate with an extracellular flux analyzer. Liver protein lysine acetylation profiles were evaluated with 1D and 2D Western blots. In addition, the relationship between endogenous uridine levels, fatty liver, and tamoxifen administration was evaluated in transgenic mice UPase1/and UPase1-TG. Results: Uridine co-administration prevented tamoxifen-induced liver lipid droplet accumulation in mice. The most prominent effect of uridine co-administration with tamoxifen was the stimulation of liver membrane phospholipid biosynthesis. Uridine had no protective effect against tamoxifen-induced impairment to mitochondrial respiration of primary hepatocytes or liver TAG and cholesterol export. Uridine had no effect on tamoxifen-induced changes to liver protein acetylation profile. Transgenic mice UPase1/with increased pyrimidine salvage activity were protected against tamoxifen-induced liver lipid droplet accumulation. In contrast, UPase1-TG mice with increased pyrimidine catabolism activity had intrinsic liver lipid droplet accumulation, which was aggravated following tamoxifen administration. Conclusion: Uridine co-administration was effective at preventing tamoxifen-induced liver lipid droplet accumulation. The ability of uridine to prevent tamoxifen-induced fatty liver appeared to depend on the pyrimidine salvage pathway, which promotes biosynthesis of membrane phospholipid. Coherent anti-Stokes Raman scattering microscopy; Drug-induced fatty liver; Lipidomics; Membrane phospholipid; Mitochondrial respiration; Protein lysine acetylation; Pyrimidine; Tamoxifen; Triacylglyceride; Uridine phosphorylase - Background Tamoxifen is an effective drug widely used for the treatment of estrogen receptor-positive breast cancer [1]. Women taking tamoxifen from 5 to 10 years exhibit reduced risks of breast cancer recurrence and mortality [2,3]. While generally well-tolerated, tamoxifen is known to induce fatty liver in 43% of women within the first 2 years of treatment [4-6]. Fatty liver is an established risk factor for non-alcoholic fatty liver disease (NAFLD) [7]. Prolonged tamoxifen treatment increases the risk of NAFLD, particularly in women with pre-existing metabolic condition [8]. The mechanism underlying tamoxifen-induced fatty liver is a topic of active investigation. Evidence from several independent research groups supports tamoxifeninduced impairment of mitochondrial fatty acid oxidation (FAO) as a primary cause of lipid accumulation in the liver [9-11]. Co-administration of tetradecylthioacetic acid, which improves mitochondrial and peroxisomal FAO, prevents tamoxifen-induced fatty liver [12]. Tamoxifen also inhibits hepatic triacylglyceride secretion leading to liver lipid accumulation [10,11]. Therapeutic intervention to prevent tamoxifen-induced fatty liver condition has the potential to improve the safety of long-term tamoxifen usage for breast cancer treatment. Uridine, a pyrimidine nucleoside, has been shown to prevent fatty liver condition induced by several drugs with unrelated therapeutic usages and acting mechanisms [13,14]. Uridine could be salvaged into pyrimidine nucleotides or catabolized into uracil and subsequently -alanine and acetyl-CoA (Figure 1) [15]. Homeostatic regulation of uridine is controlled by uridine phosphorylase, an enzyme that catalyzes the reversible phosphorylitic conversion of uridine to uracil [16]. Genetic knock-out of uridine phosphorylase in UPase1/mice elevates tissues and plasma levels of uridine [17]; whereas, transgenic overexpression of uridine phosphorylase in UPase1-TG mice depletes tissues and plasma levels of uridine [18]. The liver is actively regulating plasma uridine level by continuously degrading plasma uridine and replacing it with de novo uridine synthesis [19]. The interaction between liver uridine homeostasis and lipid metabolism has been reported [18]. Figure 1 Uridine salvage and membrane phospholipid biosynthesis. Dashed arrows indicate multiple enzymatic reactions. However, precise underlying mechanisms have not been determined. Consequently, therapeutic potential of uridine for treatment of fatty liver condition has not been realized. In this study, we examine the effects of uridine coadministration with tamoxifen on liver lipid content in control C57BL/6J and transgenic UPase1/and UPase1TG mice. Specifically, we examine the contribution of pyrimidine salvage and catabolism pathways to the biological activity of uridine. We aim to explore therapeutic potential of uridine for the prevention of drug-induced fatty liver and biological action of uridine on liver lipid metabolism. Methods Ethical statement All animal studies were performed with the ethical approval of the Animal Care and Use Committees at Nevada Cancer Institute, Desert Research Institute, and Touro University Nevada. All experiments conducted on animals were in compliance with the guidelines of the U.S. Office of Laboratory Animal Welfare of the National Institutes of Health and the Public Health Service Policy on Humane Care and Use of Laboratory Animals. Experimental animals Three mice strains were used, C57BL/6J or wildtype mice (Jackson Laboratories, Bar Harbor, ME), UPase1TG mice with ubiquitous genetic knock-in of uridine phosphorylase 1 [18], and UPase1/mice with ubiquitous genetic knock-out of uridine phosphorylase 1 [17]. Transgenic mice described in this study have been deposited into the Mutant Mouse Regional Resource Centers supported by the National Institutes of Health. The MMRRC strains are now known as B6;129-Upp1tm1Gp/ Mmucd (037119-UCD) for UPase1/mice and B6; (...truncated)