Uridine Abrogates Mitochondrial Toxicity Related to Nucleoside Analogue Reverse Transcriptase Inhibitors in Hepg2 Cells

Antiviral Therapy 8:463-470 Uridine abrogates mitochondrial toxicity related to nucleoside analogue reverse transcriptase inhibitors in HepG2 cells Ulrich A Walker1*, Nils Venhoff1, Eva C Koch2, Manfred Olschewski3, Josef Schneider2 and Bernhard Setzer1 1Department of Rheumatology and Clinical Immunology; 2Department of Virology, Institute for Medical Microbiology and Hygiene; and 3Department of Medical Biometry and Statistics, Medizinische Universitätsklinik, Freiburg, Germany *Corresponding author: Tel: +49 761 270 3401; Fax: +49 761 270 3446; E-mail: [email protected] Objective: To assess in vitro if uridine may be suitable to about 65% of NRTI-unexposed control cells. This effect prevent or treat mitochondrial toxicity related to nucleo- was dose-dependent, with a maximum at 200 µM of side analogue reverse transcriptase inhibitors (NRTIs). uridine. Uridine also rapidly and fully restored cell func- Methods: Human HepG2-hepatocytes were exposed to tion when added to cells with established mitochondrial NRTIs with or without uridine for 25 days. Cell growth, dysfunction (zalcitabine for 15 days) despite continued lactate production, intracellular lipids, mitochondrial zalcitabine exposure. Uridine also normalized cell prolif- DNA (mtDNA) and the ratio between the respiratory eration in HepG2 cells exposed to 36 µM of stavudine chain components COX II (mtDNA-encoded) and COX IV and protected HepG2-cells exposed to 7 µM of zidovu- (nuclear-encoded) were measured. dine + 8 µM of lamivudine (pyrimidine analogues), but Results: HepG2 cells exposed to zalcitabine (177 nM) failed to improve cell function or mtDNA in cells exposed without uridine developed a severe depletion of mtDNA to 11.8 or 118 µM of didanosine (a purine analogue). (to 8% of wild-type mtDNA levels), resulting in a decline Conclusions: The pyrimidine precursor uridine may atten- of cell proliferation and COX II levels, with increased uate the mitochondrial toxicity of antiretroviral pyrimidine lactate and lipid accumulation. Uridine fully abrogated NRTIs in vitro, and its supplementation may represent a the adverse effects of zalcitabine on hepatocyte prolifer- promising strategy in the prevention or treatment of ation and normalized lactate synthesis, intracellular mitochondrial toxicities in HIV-infected patients. lipids and COX II levels by adjusting mtDNA levels to Introduction Highly active antiretroviral therapy (HAART), In cases of life-threatening hyperlactataemia and usually comprising nucleoside analogue reverse tran- other forms of severe mitochondrial toxicity, cessation scriptase inhibitors (NRTIs) such as zidovudine of NRTI-treatment is advised [9], but biochemical and (AZT, 3′-azido-3′-deoxythimidine), zalcitabine (ddC, clinical resolution is often of slow offset. Several ′ ′ ′ ′ 2 ,3 -dideoxycytidine), didanosine (ddI, 2 ,3 -dide- dietary supplements, such as coenzyme Q10 oxyinosine), lamivudine (3TC, 2′,3′-dideoxy- (ubiquinone), vitamins or L-carnitine have been advo- 3′-thiacytidine) or stavudine (d4T, 2′,3′-didehydro- cated to improve electron flux, prevent 2′,3′-deoxythymidine), has resulted in a significant radical-mediated respiratory chain damage or to decrease of HIV-associated morbidity and mortality. provide substrates for the respiratory chain [10]. In However, several side effects have been associated vitro models of NRTI-toxicity and isolated HIV cases with the long-term use of such NRTIs and their have lent some support for this recommendation, as ability to inhibit polymerase-γ, which replicates mito- some restoration of mitochondrial damage (such as in chondrial DNA (mtDNA) [1–3]. Decreased mtDNA organelle ultrastructure, markers of oxidative stress or levels result in decreased synthesis of mtDNA- intracellular steatosis) were noted [11,12]. However, encoded respiratory chain subunits and ultimately in no clinical effectiveness has been demonstrated in a defect in oxidative phosphorylation [4]. Many controlled studies of patients with inherited mtDNA organs are thought to be involved [1,2]. The mito- mutations [10]. L-Carnitine, thiamine, riboflavin and chondrial toxicity of NRTIs in the liver has been radical scavengers also failed to demonstrate efficacy in associated with steatosis, steatohepatitis and acute reducing NRTI-related mitochondrial toxicity of liver failure, and may contribute to lactic acidosis in cultured human hepatocytes [13]. Furthermore, many some patients [5–8]. supplements have either a poor pharmacokinetic ©2003 International Medical Press 1359-6535/02/$17.00 463 UA Walker et al. profile (coenzyme Q10) or are less likely to be very concentration of 200 µM. Control HepG2 cells were effective in patients with mtDNA depletion, compared incubated in medium without any NRTI or with to mtDNA mutations [10,13]. uridine alone. In a search for better alternatives, we hypothesized that uridine supplementation may be used as a novel Cellular steatosis approach to prevent or treat NRTI-related mitochon- Intracellular lipid droplets were determined by Oil- drial toxicity, because fibroblasts, depleted of mtDNA Red-O-staining [4]. by long-term exposure to the polymerase-γ inhibitor ethidium bromide, became more dependent upon Lactate production uridine for growth [14]. Uridine also improved the 1.5 ml of culture fluid was collected immediately prior survival and neurite outgrowth of neuronal cells to cell trypsinization and L-lactate was determined exposed to ddC [15], and reversed the toxicity of AZT enzymatically in an automated analyser to bone marrow progenitors [16]. Therefore, we eval- (Roche/Hitachi 917) according to the manufacturers uated if uridine may be protective against instructions. Production of L-lactate was calculated in mitochondrial toxicity in human liver (HepG2) cells mmol per 105 cells [4]. exposed to NRTIs. Quantification of mtDNA Materials and methods mtDNA content was densitometrically quantified by Southern blot using Scion-image (Scion Corporation) Materials as previously described [4,17]. 5 µg of cellular DNA The human hepatoma HepG2 cell line was provided by was digested with PvuII, electrophoresed on agarose the American Type Culture Collection (ATCC HB- and blotted to a nylon membrane. mtDNA was 8065). Cell culture flasks (75 cm3) were purchased probed with a 12.9 kbp, random-prime digoxigenin- from Becton Dickinson and 10% fetal bovine serum labelled fragment, spanning nucleotide positions 3470 from PAA Laboratories, Linz, Austria. AZT, ddI, d4T, and 16379 of human mtDNA. Nuclear DNA (nDNA) uridine and Oil-Red were obtained from Sigma. was detected simultaneously with a second probe GlaxoSmithKline and Roche kindly provided 3TC and directed against 18S ribosomal DNA. The mtDNA and ddC. Pepstatin A was purchased from Boehringer nDNA signals were visualized with an alkaline-phos- Mannheim (Germany). Anti-COX II and anti-COX IV phatase conjugated, anti-digoxigenin monoclonal monoclonal antibodies were from Molecular Probes. antibody (Boehringer Mannheim, Germany) according Abacavir was unfortunately not released for tests on to the manufacturers instructions. mtDNA was HepG2 cells by its manufacturer. normalized for nDNA content by calculating the mtDNA/nDNA ratio. Cell culture ° HepG2 cells were propagated at 37 C and 5% CO2 in Quantification of the mtDNA-encoded COX II Dulbecco’s Modified Eagle Medium (DMEM), respiratory chain subunit containing 4.5 g/l glucose and 110 mg/l pyruvate, The subunit II of cytochrome c-oxidase (COX II) is supplemented with 10% fetal bovine serum, 50 U/ml encoded by mtDNA, whereas the subunit IV of streptomycin, 50 U/l penicillin and 250 µg/l ampho- cytochrome c-oxidase (COX IV) is encoded by tericin B. 2.7×106 HepG2 cells were seeded during nDNA. COX II was quantified by immunoblot logarithmic growth at day 1 and harvested at days 5, densitometry and normalized to the signal of a simul- 10, 15, 20 and 25 when viable cells were counted, taneously used antibody against COX IV as and 2.7×106 cells were replated in new flasks. described in detail elsewhere [4]. The ratios between Medium was renewed at the harvest and on every the COX II and the COX IV signals were calculated third day after plating. and the results expressed in percent of the control NRTIs with or without uridine were added in group mean. concentrations corresponding to the steady state peak plasma levels (Cmax) in humans during HIV therapy, for Statistics example, 7.0 µM of AZT, 177 nM of ddC and 8.3 µM The absolute values at each time point of the cell 5 of 3TC (product data sheets). For d4T, 10-fold Cmax count, of the lactate measurements per 10 cells, of the concentrations (36 µM) were used, because Cmax mtDNA/nDNA ratio and of the COX II/COX IV ratio concentrations did not display discernable mitochon- were compared by repeated measures analysis of vari- drial toxicity. For ddI, two concentrations were tested ance (ANOVA) [4] using the SAS statistical program (11.8 µM corresponding to Cmax, and 118 µM). Unless (vs. 6.12). otherwise indicated, uridine was used at a standard 464 ©2003 International Medical Press Uridine and mtDNA depletion Results Uridine prevents ddC-mediated cytotoxicity When uridine (200 µM) was added at day 1 to the Mitochondrial toxicity of ddC on HepG2 cells medium containing ddC, the cells did not deteriorate HepG2 control cells (without any NRTI) proliferated with respect to cell function (growth and lactate rapidly and had multiplied by an average factor of 4.9 production) or COX II/COX IV ratio. This represented within each 5 day interval,

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