192 Inhibition of Ferrous-Induced Lipid Peroxidation by Pyrimido-Pyrimidine Derivatives in Human Liver Membranes J.P. de la Cruz*, T. Carrasco, G. Ortega and F. Sanchez de la Cuesta Department of Pharmacology and Therapeutics, School of Medicine, University of M&laga, 29071 M&laga, Spain The effects of pyrimido-pyrimidine derivatives (dipyrida- mole, RA~L2, and RA-233) on lipid peroxidation, using d- a-tocopherol as standard, were studied in enriched mem- brane fractions from human and rat hepatocytes. Equi- molar concentrations of ferrous sulfate and ascorbic acid were used to induce lipid peroxidation. The amount of peroxidized lipids observed in membrane fractions from human liver was smaller than in those from rat liver. In both species, however, pyrimid~pyrimidine derivatives, ex- cept for RA-233 in rat liver, inhibited lipid peroxidation dose-dependently in the following sequence: RA~2 > di- pyridamole > d-a-tocopherol RA-233. Lipids 27, 192-194 (1992). Oxygen-derived free radicals (ODFR) have been associated with the etiology of certain pathological processes, such as ischemia re-perfusion syndrome (1,2), diabetic vasculopathy (3), cataracts (4), and liver disorders (5). The development of drugs that prevent the formation and/or cell damage by ODFR is a fundamental objective of current research (6). Our group reported that dipyridamole, a pyrirm'do-pyri- midine derivative which acts as a vasodilator and platelet aggregation inhibitor (7), reduces the incidence of post- thrombolysis r~perfusion arrhythmias (PTRA) (8) and of renal excretion of proteins in different types of glomerulone- phritis (GMN) (9); PTRA and GMN are associated with ODFR cell membrane damage. We also reported that di- pyridamole inhibits lipid perexidation induced by hydroxyl anions {HA) in cell membranes of different rat tissue (10), an effect that had been previously observed by Iuliano et {11) using chemical substrates. The aim of the present study was to assess the antiperox- idative effect of dipyridamole in liver cell membranes from humans, as distinct from those of rats, and to compare this effect with that of two other chemical congeners, RA-233 (Mopidamole) and RA~o42,using d-a-tocopherol as standarcL MATERIALS AND METHODS Materials. Dipyridamole (2,6-bis-(diethanolamino)-4,8- dipiperidinopyrimido-[5,4-d]pyrimidine) was obtained from Boehringer Ingelheim S.A. (Barcelona, Spain). RA- 233 (2,6-bis( diethanolamino)-4-piperidinopyrimido[ 5, 4- d]pyrimidine) and RA-642 (2,6-bis(2-hidroxiethyl-2-meto- xiethylamino)- 4,8- b is ( diethylamino)pyrimido[5,4-d] pyri- midine) were donated by Dr. Karl Thomae (Biberach an der Riss, Germany). Malondialdehyde bis-diethylacetal was obtained from Aldrich-Chemie (SteinheindAlbuch, *To whom correspondence should be addressed. Abbreviations: Cyt C, cytochrome C; ED-50, effective concentration, 50%; FeAs, ferroussulfate/ascorbic acid;GMN, glomerulonsphritis; HA, hydroxyl anions; IC50, inhibitoryconcentration,50%; MDA, malondialdehyde; NBT, nitrobluetetrazoliurn; ODFR, oxygen-derived free radicals;PTRA, post-thrombolysis re-perfusionarrhythmias. Germany), and d-a-tocopherol and all other reagents were obtained from Sigma Chemical Co (St. Louis, MO). Preparation of membrane fractions. Enriched mem- brane fractions from human and rat liver were used in this study. Human liver samples (n = 6) were obtained from specimens of partial hepatectomies carried out on patients with traumatic liver injury due to traffic ac- cidents. The normal characteristics of the liver tissue were confirmed by histopathological examination. Informed consent was obtained for all patient samples. Rat liver samples were obtained from six 3- to 4-month-old male Wistar rats. All samples were diluted 1:10 (v/v) in buffer containing 50 mM Tris, 100 mM NaC1, 0.5 mM KC1, 3.1 mM CaC12, 1 mM MgSO4, 0.55 mM KH2PO 4, and 3.20 M sucrose (pH 7.4). The samples were minced and homogenized in a Braun potter at 600 strokes/rain, then centrifuged at 1,000 X g for 10 min at 4~ The pellet was discarded and the supernatant was centrifuged at 20,000 X g for 20 rain at 4 ~ C. The supernatant was removed, and the pellet was diluted (1:10, v/v) in the buffer mentioned above without sucrose Crushed ice surrounded the samples throughout the procedure. Measurement of lipid peroxidation. The products re- sulting from the thiobarbituric acid reaction, most of which were malondialdehyde (MDA) reaction products, were taken as indicators of lipid peroxidation in membrane fractions. A modification of a method described by R. Zim- merman (personal communication) was followed. Briefly, membrane concentrates were diluted (1:4, v/v) in the above ~ mentioned buffer solution but with 20 mM Tris, and were divided into 1.7-mL aliquots. Then 0.1 mL of the buffer (in the assays without inhibitors) or 0.1 mL of different concentrations of pyrimido-pyrimidine derivatives were added. Ferrous sulfate (0.1 mL) and 0.1 mL of ascorbic acid (FeAs) in increasing equimolar concentrations were added in baseline assays (without drugs); 75 ~ of FeAs was used in experiments with drugs. FeAs was used to induce lipid peroxidation via the formation of HA (11,12). Test tubes were incubated at 37~ for 45 min while be- ing continuously shaken. Blanks which contained only tissue were incubated at 4~ Subsequently the reaction was stopped and MDA was analyzed using 1 mL of 0.5% thiobarbituric acid in 20% trichloroacetic acid. The prod- ucts used in the samples were added to the blanks. After agitation, the samples were incubated at 100~ for 15 rain and then centrifuged at 1,000 X g for 15 rain at 4~ The amount of malondialdehyde (MDA) produced was deter- mined by measuring the spectrophotometric absorbance of the supernatant at 532 nm. The absorbances were compared to those of a standard of malondialdehyde bis-diethyl-acetaL The protein content of the samples was determined using the method describ- ed by Lowry et al. (13); the results are expressed in nmol of MDA/mg protein. Statistical analyses were carried out using the Epistat program. The Student's t-test was applied for comparison LIPIDS, VoI. 27, no. 3 (1992)