Dietary choline restriction causes complex I dysfunction and increased H2O2 generation in liver mitochondria

Kenneth Hensley 1 Yashige Kotake 1 Hong Sang 1 Quentin N.Pye 1 Gemma L.Wallis 1 Lisa M.Kolker 1 Tahereh Tabatabaie 1 Charles A.Stewart 1 Yoichi Konishi 0 1 Dai Nakae 0 1 Robert A.Floyd 1 0 Department of Oncological Pathology, Cancer Center, Nara Medical University , 840 Shijo-cho, Kashihara, Nara 634-8521, Japan 1 Free Radical Biology and Aging Research Program, Oklahoma Medical Research Foundation , Oklahoma City, OK 73014, USA 2To whom correspondence should be addressed Email: Removal of choline from the diet results in accumulation of triglycerides in the liver, and chronic dietary deficiency produces a non-genotoxic model of hepatocellular carcinoma. An early event in choline deficiency is the appearance of oxidized lipid, DNA and protein, suggesting that increased oxidative stress may facilitate neoplasia in the choline deficient liver. In this study, we find that mitochondria isolated from rats fed a choline-deficient, L-amino acid defined diet (CDAA) demonstrate impaired respiratory function, particularly in regard to complex I-linked (NADH-dependent) respiration. This impairment in mitochondrial electron transport occurs coincidentally with alterations in phosphatidylcholine metabolism as indicated by an increased ratio of long-chain to short-chain mitochondrial phosphatidylcholine. Moreover, hydrogen peroxide (H2O2) generation is significantly increased in mitochondria isolated from CDAA rats compared with mitochondrial from normal rats, and the NADH-specific yield of H2O2 is increased by at least 2.5-fold. These findings suggest an explanation for the rapid onset of oxidative stress and energy compromise in the choline deficiency model of hepatocellular carcinoma and indicate that dietary choline withdrawal may be a useful paradigm for the study of mitochondrial pathophysiology in carcinogenesis. - Dietary choline deficiency produces a non-genotoxic model of liver cancer in rats (1,2). Choline restriction produces a welldefined pattern of temporal alterations in the liver while leaving other organs largely unaffected (3). Within 24 h of choline restriction, triglycerides begin to accumulate in rat liver with all regions of the liver being affected within 45 days (3). Evidence for membrane peroxidation, specifically the formation of conjugated dienes in nuclear and mitochondrial membranes, has been shown within 15 days (46) and persists throughout choline restriction-induced neoplasia (7). Oxidative damage to DNA, particularly the formation of 8-oxodeoxyguanosine lesions, occurs within the time frame of lipid peroxidation (7). Some hepatocyte death occurs beginning at Abbreviations: CDAA, choline-deficient L-amino acid defined; CSAA, choline-sufficient L-amino acid defined diet; MS, mass spectrometry; NADH, nicotinamide adenine dinucleotide (reduced); PC, phosphatidyl choline. 514 days (8), and by 10 weeks preneoplastic nodules are clearly evident (7,9). True hepatocellular carcinomas are present after one year of prolonged choline deficiency (2). The early appearance of oxidative lesions in choline deficient liver and the observation that antioxidants can inhibit carcinogenesis induced by choline withdrawal (7,9,10), combined with the known ability of oxidants to induce carcinogenesis (11), suggests that choline deficiency either causes increased generation of oxidants in liver or else inhibits the livers ability to detoxify oxidant species. No mechanisms have been proposed to support either alternative. Reasoning that mitochondria are a major source of reactive oxygen species (ROS) in liver (12), and considering that phospholipid composition is altered significantly in choline deficient mitochondria (1315), we hypothesized that mitochondrial respiration might be altered during choline withdrawal so as to produce an increased leakage of ROS which might predispose the liver to neoplasia. Here, we report that mitochondria isolated from rats fed a choline deficient, L-amino acid-defined diet (CDAA) for 37 days are significantly altered with respect to rats fed a choline sufficient, L-amino acid-defined (CSAA) diet and with respect to animals fed a basal diet of normal rat chow. Complex I function (NADH dehydrogenase activity or NADH-linked O2 consumption) was diminished 70% in CDAA rats relative to normal rats, while complex II function was diminished by 30%. CDAA mitochondria produced H2O2 at a 30% faster rate than normal mitochondria, while the NADH-specific yield of H2O2 increased 2.5-fold in the CDAA groups. The time course of complex I alteration correlated with the time course of lipid compositional changes as indicated by mass spectrometric analysis of phosphatidylcholine (PC) and consistent with previously published data. Finally, it was found that lipids extracted from CDAA mitochondria can impair NADH-dehydrogenase activity when reconstituted with protein extracted from normal mitochondria. These data provide a theoretical context for discussing oxidative damage in the CDAA model of hepatocellular carcinoma, and provide a striking example that diet can affect mitochondrial function and oxidant production. Materials and methods Reagents Fluorogenic substrates and standards were purchased from Molecular Probes (Eugene, OR). NADH and succinate were purchased from Sigma (St Louis, MO). All other reagents were of the highest available purity. Animals and diet Male Wistar rats, 100200 g, were purchased from Charles River Laboratories (Wilmington, MA) and maintained in the Oklahoma Medical Research Foundation Laboratory Animal Care Facility until use. In each experiment, animals were divided into two groups of five animals each and fed either a basal diet (normal chow) or a CDAA diet as previously described (16), or a CSAA diet which was identical in every respect to the CDAA diet except that choline was not omitted from the formulation. Separate experiments were conducted with 1, 3 and 7 days of CDAA administration. The basal diet was Purina 5001 (Ralston Purina, St Louis, MO). The CDAA and CSAA diets were purchased from Dyets (Bethlehem, PA). Animals were weighed daily. Isolation of liver mitochondria Mitochondria were isolated similar to previously described methods (12,17,18,2126). Approximately 3 g slices of liver were immersed in ~25 ml ice-cold isolation medium [0.3 M sucrose, 25 mM tris(hydroxymethyl) aminomethane, 2 mM EDTA, pH 7.3] and finely minced by brief disruption (25 s) with a Polytron (Brinkman Instruments, Westbury, NY) motor-driven tissue homogenizer followed by two strokes of a motor-driven glass walled dounce-type homogenizer equipped with a teflon pestel (0.25 mm clearance). Tissue thus homogenized was centrifuged at 1015C and 500 g for 15 min in a fixed-angle rotor. Supernatant was decanted and centrifuged at 9000 g for 15 min. The pellet from the second centrifugation was gently dispersed into 20 ml isolation medium and recentrifuged at 10 000 g for 15 min, and this wash was repeated once more. The final pellet was resuspended (...truncated)