The Consequences of Lipid Peroxidation in Isolated Hepatocytes

Abstract

Lipid peroxidation was initiated by the addition of either ADP-complexed Fe3 + or cumene hydroperoxide to isolated rat hepatocytes and the resultant biochemical and morphological alterations investigated. As previously observed with microsomes, malonaldehyde formation was associated with the inactivation of glucose-6-phosphatase. Inhibition of microsomal oxidative drug metabolism was correlated with the release and subsequent inactivation of NADPH-cytochrome c reductase, whereas cytochrome P-450 destruction occurred only in the presence of high concentrations of the organic hydroperoxide which were associated with extensive malonaldehyde formation. Under these conditions there were also marked ultrastructural alterations in the hepatocytes which were not apparent after incubation in the presence ofiron (I 187pM Fe3+). The latter treatment was, however, associated with moderate biochemical effects such as glucose-6-phosphatase inactivation and increased membrane permeability. The cellular defence system against lipid peroxidation is discussed and it is concluded that the isolated liver cell system provides a valuable tool for the study of lipid peroxidation and its pathological implications. Because of its widespread toxic effects lipid peroxidation might have pathological implications as has been proposed in connection with iron toxicity [6] and CCI, toxicity [7]. However, the role of lipid peroxidation during the early stages of cell damage leading to cell necrosis has yet to be established.Isolated liver cells produce malonaldehyde, a metabolite of lipid peroxides, upon exposer to ADP-complexed iron or the organic peroxide cumene hydroEn:ymc.s. Collagenase (EC 3.4.24.3); Hyaluronidase or hyaluronate 4-glycanohydrolase (EC 3.2.1.35); NADPH-cytcchrome c reductase (EC 1.6.2.4); Glucose 6-phosphatase (EC 3.1.3.9).peroxide [8]. Malonaldehyde production, which is readily monitored in this isolated-cell system, is probably catalyzed by the drug-hydroxylating enzyme system located in the endoplasmic reticulum and is thus comparable with the enzyme-dependent lipid peroxidation reactions of isolated microsomes [9,10]. The isolated cell system, however, differs from a microsomal system in being fortified with natural inhibitors, which alter the response to agents that stimulate lipid peroxidation [8,11]. The purpose of this work was to characterize some early alterations in isolated hepatocytes in which malonaldehyde production had been induced. MATERIALS AND METHODSThe cell preparation and incubation techniques were the same as described previously [8,11]. Lipid peroxide formation was initiated by addition of ADPcomplexediron(stocksolution50mMADP + 1.87mM FeC1,) or an aqueous solution of cumene hydroperoxide (stock solution 30 mM). The reaction was followed by the withdrawal of aliquots (0.2 ml) of the whole incubation mixture for spectrophotometric measurements of thiobarbituric-acid-reacting substances at 535 nm (malonaldehyde) [12].

Desensitization of Platelet-Derived Growth Factor Receptor-β by Oxidized Lipids in Vascular Cells and Atherosclerotic Lesions

“…This was confirmed by using CHP, which can induce DNA synthesis inhibition 24 and cellular lipid peroxidation. 33 In our experimental system, CHP (used at nontoxic concentration) was able to inhibit PDGFR␤, independently of any peroxidation of cellular lipids (thus independently of 4HNE formation). It is therefore unlikely that in our experiments, 4HNE may result from cellular lipid peroxidation.…”

Evidence for involvement of oxygen free radicals in bile acid toxicity to isolated rat hepatocytes

“…Although this oxygen concentration in the gas mixture may appear high and nonphysiological, it must be pointed out that the oxygen delivered to the hepatocyte in uiuo is mainly ( > 90%) delivered from hemoglobin-bound oxygen (which is absent in our system) and not from that dissolved in plasma. For these reasons, the standard gas mixture used to maintain isolated hepatocyte integrity in in uitro suspensions has been a 95% 0,/5% CO, mixture (58), even in studies of free radical-induced hepatocyte injury (21,(59)(60)(61)(62). However, because of concern that hepatocytes were exposed to hyperoxia under these conditions, experiments were conducted under lower, more physiological 0, concentrations.…”

A Correlation between Glutathione Levels and Cellular Damage in Isolated Hepatocytes

“…97 -100 o/, of the cells in the pellet excluded trypan blue and the yield of viable cells was between 200-300 x lo6 cells/liver. By electron microscopy, hepatocytes were of normal appearance (cj: [6]) and did not exhibit signs of anoxia.…”

“…The activity was measured in the total incubate before and after addition of detergent (sodium deoxycholate, Triton x-100) to estimate the latency of the enzyme. Pyruvate (0.67 mM final concentration) and NADH (0.1 mM final concentration) were included and the rate of NADH oxidation monitored at 340 nm [6]. It was found occasionally that the activity measured after lysis of the cells decreased with time (cc Fig.…”