Loss of lipid peroxidation as a histochemical marker for preneoplastic hepatocellular foci of rats.

Since it is known that tumor cell membranes have lost the capacity to undergo lipid peroxidation, it seemed of interest to investigate whether the loss of susceptibility to lipid peroxidation represents a tumoral marker appearing in preneoplastic cells together with the other known tumoral markers. A histochemical technique was developed to detect lipid peroxidation in individual cells of liver sections exposed to effective prooxidants. The technique was based on the detection of protein-bound aldehydes (alkenals) with the use of the Schiff's reagent. The latter reagent can also detect carbonyl function present in acyl residues of peroxidized phospholipids of cellular membranes. Liver preneoplastic foci were obtained in rats by the i.p. administration of diethylnitrosamine and of 2-acetylaminofluorene in the diet. Frozen sections of the liver, incubated in the presence of reduced nicotinamide adenine dinucleotide phosphate:iron revealed the presence of areas in which lipid peroxidation had not been induced (Schiff-negative areas). These areas corresponded strictly, in serial sections, to areas that were strongly positive to gamma-glutamyltranspeptidase.

Cleavage of Bcl-2 in oxidant- and cisplatin-induced apoptosis of human melanoma cells.

Although the anti-apoptotic effect of Bcl-2 is well established, the role of Bcl-2 in tumour response to therapy and drug resistance is still unclear. The post-translational modifications of Bcl-2 are likely involved in the control of the apoptotic pathway. In the present study we have investigated the role of Bcl-2 in cellular response to oxidative stress (hydrogen peroxide) and cisplatin using a clone of human metastatic melanoma, which, in spite of Bcl-2 (over)expression, exhibited a moderate chemosensitivity. With both treatments melanoma cells died through an apoptotic process, associated with detachment of cells from the monolayer. In the floating apoptotic cells generated by either hydrogen peroxide or cisplatin, along with morphological and biochemical features of apoptosis, we detected a significant Bcl-2 cleavage, yielding the Bax-like fragment of 23 kDa. Preincubation of cells with the caspase-3/-7 inhibitor DEVD-CHO completely suppressed Bcl-2 cleavage, thus confirming that such a specific proteolysis requires activation of caspase-3/-7. The oxidant- and cisplatin-induced processing of Bcl-2 documented in the present study may represent a regulatory mechanism to circumvent the survival function of Bcl-2 upon apoptosis triggering and to enhance apoptotic response. Since the Bcl-2 cleavage should be regarded as a pro-apoptotic event, Bcl-2 expression is expected to increase susceptibility to apoptosis. Thus, such a pathway could be exploited to improve the efficacy of cytotoxic therapy of melanomas expressing Bcl-2.

Identification of 4-hydroxynonenal as a cytotoxic product originating from the peroxidation of liver microsomal lipids.

During the NADPH-Fe induced peroxidation of liver microsomal lipids, products are formed which show various cytopathological effects including inhibition of microsomal glucose-6-phosphatase. The major cytotoxic substance has been isolated and identified as 4-hydroxy-2,3-trans-nonenal. The structure was ascertained by means of ultraviolet, infrared and mass spectrometry and high-pressure liquid chromatographic analysis. Moreover, 4-hydroxynonenal, prepared by chemical synthesis, was found to reproduce the biological effects brought about by the biogenic aldehyde. Preliminary investigations suggest that as compared to 4-hydroxynonenal very low amounts of other 4-hydroxyalkenals, namely 4-hydroxyoctenal, 4-hydroxydecenal and 4-hydroxyundecenal are also formed by actively peroxidizing liver microsomes. In the absence of NADPH-Fe liver microsomes produced only minute amounts of 4-hydroxyalkenals. The biochemical and biological effects of synthetic 4-hydroxyalkenals have been studied in great detail in the past. The results of these investigations together with the finding that 4-hydroxyalkenals, in particular 4-hydroxynonenal, are formed during NADPH-Fe stimulated peroxidation of liver microsomal lipids, may help to elucidate the mechanism by which lipid peroxidation causes deleterious effects on cells and cell constituents.

Calcium sequestration activity in rat liver microsomes. Evidence for a cooperation of calcium transport with glucose-6-phosphatase.

Mechanisms regulating the energy-dependent calcium sequestering activity of liver microsomes were studied. The possibility for a physiologic mechanism capable of entrapping the transported Ca2+ was investigated. It was found that the addition of glucose 6-phosphate to the incubation system for MgATP-dependent microsomal calcium transport results in a marked stimulation of Ca2+ uptake. The uptake at 30 min is about 50% of that obtained with oxalate when the incubation is carried out at pH 6.8, which is the pH optimum for oxalate-stimulated calcium uptake. However, at physiological pH values (7.2-7.4), the glucose 6-phosphate-stimulated calcium uptake is maximal and equals that obtained with oxalate at pH 6.8. The Vmax of the glucose 6-phosphate-stimulated transport is 22.3 nmol of calcium/mg protein per min. The apparent Km for calcium calculated from total calcium concentrations is 31.9 microM. After the incubation of the system for MgATP-dependent microsomal calcium transport in the presence of glucose 6-phosphate, inorganic phosphorus and calcium are found in equal concentrations, on a molar base, in the recovered microsomal fraction. In the system for the glucose 6-phosphate-stimulated calcium uptake, glucose 6-phosphate is actively hydrolyzed by the glucose-6-phosphatase activity of liver microsomes. The latter activity is not influenced by concomitant calcium uptake. Calcium uptake is maximal when the concentration of glucose 6-phosphate in the system is 1-3 mM, which is much lower than that necessary to saturate glucose-6-phosphatase. These results are interpreted in the light of a possible cooperative activity between the energy-dependent calcium pump of liver microsomes and the glucose-6-phosphatase multicomponent system. The physiological implications of such a cooperation are discussed.

Inhibition of calcium sequestration activity of liver microsomes by 4-hydroxyalkenals originating from the peroxidation of liver microsomal lipids.

Aldehydes released during peroxidation of liver microsomal lipids and identified as 4-hydroxyalkenals (4-hydroxynonenal being quantitatively the most significant) strongly inhibited the calcium sequestration activity of liver microsomes. The ID50 for 4-hydroxynonenal was 42 microM. The inhibition appeared to be correlated with the amount of the aldehyde bound to the microsomal protein. In rats intoxicated with BrCCl3 significant amounts of protein-bound aldehydes were formed at only 5 min after poisoning, a time at which the calcium sequestring capacity is markedly inhibited.

Stimulatory effect of glucose 6-phosphate on the non-mitochondrial Ca2+ uptake in permeabilized hepatocytes and Ca2+ release by inositol trisphosphate.

The relationships between Ca2+ transport and glucose-6-phosphatase activity, previously studied in isolated liver microsomes, were investigated in permeabilized hepatocytes in the presence of mitochondrial inhibitors. It was found that the addition of glucose 6-phosphate to the cells markedly stimulates the MgATP-dependent Ca2+ uptake. A progressive increase in the stimulation of Ca2+ uptake was seen with increasing amounts of glucose 6-phosphate up to 5 mM concentrations. Vanadate, when added in adequate concentrations (20-40 microM) to the hepatocytes inhibits both the glucose-6-phosphatase activity and the stimulation of Ca2+ uptake by glucose 6-phosphate, while not affecting the MgATP-dependent Ca2+ uptake. The addition of inositol 1,4,5-trisphosphate to permeabilized hepatocytes in which Ca2+ had been accumulated in the presence of MgATP and glucose 6-phosphate, results in a rapid release of Ca2+.

Cytotoxic aldehydes originating from the peroxidation of liver microsomal lipids. Identification of 4,5-dihydroxydecenal.

During the NADPH-Fe-induced peroxidation of liver microsomal lipids products are formed which are provided with cytopathological activities. In a previous study one of the major products was identified as an aldehyde of the 4-hydroxyalkenal class, namely 4-hydroxynonenal. In the present study another cytotoxic product has been isolated and identified as 4,5-dihydroxy-2,3-decenal. The isolation was performed by means of thin-layer chromatography and high-pressure liquid chromatography and the structure was ascertained mainly by means of mass spectroscopy of the free aldehyde and of its derivatives. In the absence of NADPH-Fe liver microsomes produced no 4,5-dihydroxydecenal. The inhibitory activity of 4,5-dihydroxydecenal on microsomal glucose-6-phosphatase is somewhat lower than that exhibited by 4-hydroxynonenal. This lower inhibitory activity correlates with the lower capacity to bind to the microsomal protein of 4,5-dihydroxydecenal as compared to 4-hydroxynonenal. The reactivities of the two aldehydes with cysteine were comparable. The production of toxic aldehydes may represent a mechanism by which lipid peroxidation causes deleterious effects on cellular functions.

Evidence for aldehydes bound to liver microsomal protein following CCl4 or BrCCl3 poisoning.

Since it has been demonstrated in previous studies that peroxidation of liver microsomal lipids leads to the production of aldehydes provided with cytopathological activities--namely 4-hydroxyalkenals--evidence was searched for aldehydes bound to microsomal protein in in vivo conditions (CCl4 and BrCCl3 intoxications) in which peroxidation of lipids of hepatic endoplasmic reticulum had been demonstrated previously. The spectrophotometric analysis of 2,4-dinitrophenylhydrazine-treated non-lipoidal residues of liver microsomes from the intoxicated rats shows absorption spectra similar to those observed for the dinitrophenylhydrazones formed in the reaction of alkenals with -SH groups of proteins or low molecular weight thiols. Similar spectra, although magnified from a quantitative point of view, were obtained either with liver microsomes allowed to react with synthetic 4-hydroxynonenal or with liver microsomes peroxidized in the NADPH-Fe-dependent system. A time-course study of microsomal lipid peroxidation shows that the amount of 2,4-dinitrophenylhydrazine-reacting groups in the non-lipoidal residue of liver microsomes increases with the incubation time and is correlated to the amount of thiobarbituric acid-reacting products formed in the incubation mixture. In both the in vivo conditions (CCl4 and BrCCl3 intoxications) the amount of 2,4-dinitrophenylhydrazine-reacting groups in the non-lipoidal residue of liver microsomes increases from 15 min up to 2 h after poisoning and is higher, in every instance, in the BrCCl3-intoxicated animals compared to the CCl4-poisoned ones. Experiments carried out to ascertain the reliability of the spectrophotometric detection of protein-bound alkenals showed that in the in vitro system in which liver microsomes are allowed to react with 4-hydroxynonenal there is a good agreement between the binding value that can be calculated from the absorption spectrum and the binding value obtained by using labelled 4-hydroxynonenal.

Studies on the mechanism of formation of 4-hydroxynonenal during microsomal lipid peroxidation.

The mechanism of the formation of 4-hydroxynonenal through the NADPH-linked microsomal lipid peroxidation was investigated. The results were as follows: 4-hydroxynonenal arises exclusively from arachidonic acid contained in the polar phospholipids, neither arachidonic acid of the neutral lipids nor linoleic acid of the polar or neutral lipids are substrates for 4-hydroxynonenal generation. This finding results from the estimation of the specific radioactivity of 4-hydroxynonenal produced by microsomes prelabelled in vivo with [U-14C]arachidonic acid. Phospholipid-bound 15-hydroperoxyarachidonic acid would have the structural requirements needed for 4-hydroxynonenal (CH3-(CH2)4-CH(OH)-CH=CH-CHO). Microsomes supplemented with 15-hydroperoxyarachidonic acid and NADPH, ADP/iron converted only minimal amounts (0.6 mol%) of 15-hydroperoxyarachidonic acid into 4-hydroxynonenal; similarly, 15-hydroperoxyarachidonic acid incubated at pH 7.4 in the presence of ascorbate/iron yielded only small amounts of 4-hydroxynonenal with a rate orders of magnitude below that observed with microsomes. Phospholipid-bound 15-hydroperoxyarachidonic acid is therefore not a likely intermediate in the reaction pathway leading to 4-hydroxynonenal. The rate of 4-hydroxynonenal formation is highest during the very initial phase of its formation and the onset does not show a lag phase, suggesting a transient intermediate predominantly formed during the early phase of microsomal lipid peroxidation. After 60 min of incubation, 204 nmol polyunsaturated fatty acids (20 nmol 18:2, 143 nmol 20:4, 41 nmol 22:6) were lost per mg microsomal protein and the incubation mixture contained 206 nmol lipid peroxides, 71.6 nmol malonic dialdehyde and 4.6 nmol 4-hydroxynonenal per mg protein. Under artificial conditions (pH 1.0, ascorbate/iron, 20 h of incubation) not comparable to the microsomal peroxidation system, 15-hydroperoxyarachidonic acid can be decomposed in good yields (15 mol%) into 4-hydroxynonenal. Autoxidation of arachidonic acid in the presence of ascorbate/iron gave after 25 h of incubation 2.8 mol% (pH 7.4) and 1.5 mol% (pH 1.0) 4-hydroxynonenal. The most remarkable difference between the non-enzymic system and the enzymic microsomal system is that the latter forms 4-hydroxynonenal at a much higher rate.

Detection of 4-hydroxynonenal and other lipid peroxidation products in the liver of bromobenzene-poisoned mice.

Lipid peroxidation in cellular membranes leads to the formation of toxic aldehydes. One product provided with particular reactivity has been identified as 4-hydroxynonenal and thoroughly studied as one of the possible mediators of the cellular injury induced by pro-oxidants. In the present study we have searched for the presence of 4-hydroxynonenal and other lipid peroxidation products in the liver of bromobenzene-poisoned mice, since under this experimental condition the level of lipid peroxidation is much greater than in the case of CCl4 or BrCCl3 hepatotoxicity. 4-Hydroxynonenal was looked for in liver extracts as either free aldehyde or its 2,4-dinitrophenylhydrazone derivative. In both cases, by means of thin-layer chromatography (TLC) and high-pressure liquid chromatography, a well resolved peak corresponding to the respective standards (free aldehyde or 2,4-dinitrophenylhydrazone derivative) was obtained. Total carbonyls present in the liver of intoxicated animals were detected as 2,4-dinitrophenylhydrazone derivatives. The hydrazones were pre-separated by TLC into three fractions according to different polarity (polar, non-polar, fraction I, and non-polar, fraction II). The amounts of carbonyls present in each fraction were determined by ultraviolet-visible spectroscopy. 'Non-polar carbonyls, fraction II' were further fractionated by TLC. The fraction containing alkanals and alk-2-enals was analyzed by high-pressure liquid chromatography and several aldehydes were identified. In addition, protein bound carbonyls were determined in the liver of bromobenzene-treated mice. The biological implications of the finding of 4-hydroxynonenal and other carbonyls in vivo in an experimental model of hepatotoxicity are discussed.

Detection of carbonyl functions in phospholipids of liver microsomes in CCl4- and BrCCl3-poisoned rats.

Since the peroxidative cleavage of unsaturated fatty acids can result in either the release of carbonyl compounds or the formation of carbonyl functions in the acyl residues, evidence for the presence of carbonyl groups in liver microsomal phospholipids was searched for in in vivo conditions (CCl4 and BrCCl3 intoxications) in which peroxidation of lipids of hepatic endoplasmic reticulum had been previously demonstrated. The spectrophotometric examination of 2,4-dinitrophenylhydrazine-treated phospholipids of liver microsomes from the intoxicated animals showed absorption spectra similar to those observed for the dinitrophenylhydrazones of various carbonyls. Similar spectra, although magnified from a quantitative point of view, were also observed with 2,4-dinitrophenylhydrazine-treated phospholipids of liver microsomes peroxidized in the NADPH-Fe-dependent system. A time-course study of microsomal lipid peroxidation showed that the amount of 2,4-dinitrophenylhydrazine-reacting groups (carbonyl functions) in phospholipids of liver microsomes increases with the incubation time and is correlated to the amount of malonic dialdehyde formed in the incubation mixture. The kinetics of the production of 4-hydroxynonenal was somewhat similar to that of malonic dialdehyde formation. In both the in vivo conditions (CCl4 and BrCCl3 intoxications) the amount of carbonyl functions in microsomal phospholipids, which was higher in the BrCCl3-intoxicated animals as compared to the CCl4-poisoned ones, was close to that found in the vitro condition in which lipid peroxidation is induced by 6 microM Fe2+. The possible pathological significance of formation of carbonyl functions in membrane phospholipids is discussed.

4-Hydroxynonenal and other lipid peroxidation products are formed in mouse liver following intoxication with allyl alcohol.

Some recent reports indicate that lipid peroxidation might play a crucial role in the production of allyl alcohol hepatotoxicity. Previous work from our laboratory has suggested that in the case of bromobenzene, a hepatotoxin sharing the ability of allyl alcohol to induce a marked depletion of liver glutathione, liver injury is likely to be mediated by lipid peroxidation. In particular, we demonstrated that 4-hydroxynonenal and other aldehydes derived from lipid peroxidation can be detected in the liver of bromobenzene-poisoned mice. In the present study, we report also the in vivo formation of 4-hydroxynonenal and other aldehydes after allyl alcohol poisoning. 24-h-fasted mice were intoxicated with allyl alcohol (1.5 mmol/kg body wt., i.p.) and killed 1-3 h later. 4-Hydroxynonenal and other carbonyls were looked for in liver extracts in the form of 2,4-dinitrophenylhydrazone derivatives. After fractionation of liver extracts by means of thin-layer chromatography (TLC), a well-resolved peak corresponding to standard 4-hydroxynonenal was obtained in the high-pressure liquid chromatography analysis. Total carbonyls (as 2,4-dinitrophenylhydrazones) were separated by TLC into three fractions, according to their different polarity. The amounts of carbonyls present in each fraction were determined by ultraviolet-visible spectroscopy. In addition, several products were identified in the fraction of the 'non-polar carbonyls' corresponding to alkanals and alk-2-enals.

Lipid peroxidation. Biopathological significance.

Peroxidation of unsaturated lipids, initially studied in the chemistry of oil and fat rancidity, has become a problem of increasing interest in the biological field, because of its proposed role in a variety of pathological conditions. The general mechanism of the process, the formation of toxic aldehydes capable to react with protein and non protein thiols, and the overall effects in cellular membranes are reviewed. The possible implications of lipid peroxidation as one of the main mechanisms of cellular damage in both toxic injury and other pathological conditions are discussed.

Iron released from an erythrocyte lysate by oxidative stress is diffusible and in redox active form.

The incubation of a ghost-free erythrocyte lysate with the oxidizing agent phenylhydrazine resulted in both methemoglobin formation and release of iron in a desferrioxamine (DFO)-chelatable form. The released iron was diffusible, as shown by a dialysis carried out simultaneously with the incubation. When the dialysate was added to erythrocyte ghosts or to microsomes from liver or brain, lipid peroxidation developed in the membranes, indicating that the diffusible iron was in a redox active form. The addition of ATP to the lysate markedly increased both iron diffusion and lipid peroxidation in the membranes subsequently added to the dialysate. The possible implication of these data in some well known pathologies is discussed.

gamma-Glutamyl transpeptidase-dependent lipid peroxidation in isolated hepatocytes and HepG2 hepatoma cells.

Gamma-glutamyltranspeptidase (GGT), a plasma membrane-bound enzyme, provides the only activity capable to effect the hydrolysis of extracellular glutathione (GSH), thus favoring the cellular utilization of its constituent amino acids. Recent studies have shown however that in the presence of chelated iron prooxidant species can be originated during GGT-mediated metabolism of GSH, and that a process of lipid peroxidation can be started eventually in suitable lipid substrates. The present study was undertaken to verify if a GGT-dependent lipid peroxidation process can be induced in the lipids of biological membranes, including living cells, and if this effect can be sustained by the GGT highly expressed at the surface of HepG2 human hepatoma cells. In rat liver microsomes (chosen as model membrane lipid substrate) exposed to GSH and ADP-chelated iron, the addition of GGT caused a marked stimulation of lipid peroxidation, which was further enhanced by the addition of the GGT co-substrate glycyl-glycine. The same was observed in primary cultures of isolated rat hepatocytes, where the lipid peroxidation process did not induce acute toxic effects. GGT-stimulation of lipid peroxidation was dependent both on the concentration of GSH and of ADP-chelated iron. In GGT-rich HepG2 human hepatoma cells, the exposure to GSH, glycyl-glycine, and ADP-chelated iron resulted in a nontoxic lipid peroxidation process, which could be prevented by means of GGT inhibitors such as acivicin and the serine-boric acid complex. In addition, by co-incubation of HepG2 cells with rat liver microsomes, it was observed that the GGT owned by HepG2 cells can act extracellularly, as a stimulant on the GSH- and iron-dependent lipid peroxidation of microsomes. The data reported indicate that the lipid peroxidation of liver microsomes and of living cells can be stimulated by the GGT-mediated metabolism of GSH. Due to the well established interactions of lipid peroxidation products with cell proliferation, the phenomenon may bear particular significance in the carcinogenic process, where a relationship between the expression of GGT and tumor progression has been envisaged.

Iron release, membrane protein oxidation and erythrocyte ageing.

The aerobic incubation of erythrocytes in phosphate buffer for 24-60 h (a model of rapid in vitro ageing) induced progressive iron release and methemoglobin formation. Membrane proteins showed electrophoretic alterations and increase in carbonyl groups (as documented by IR spectroscopy). None of these phenomena were seen when the erythrocytes were incubated under anaerobic conditions. The membranes from aerobically incubated cells bound a much higher amount of autologous IgG than those from anaerobically incubated ones, suggesting that the aerobic incubation gives rise to the senescent antigen. The addition of ferrozine during the aerobic incubation prevented both the IgG binding and the protein alterations seen in the IR spectra, suggesting an intracellular chelation of the released iron by ferrozine.

Protection against oxidative damage of erythrocyte membrane by the flavonoid quercetin and its relation to iron chelating activity.

Incubation of glutathione (GSH) depleted mouse erythrocytes with the oxidants phenylhydrazine, acrolein, divicine and isouramil resulted in the release of free iron and in lipid peroxidation and hemolysis. The addition of the flavonoid quercetin, which chelates iron and penetrates erythrocytes, resulted in remarkable protection against lipid peroxidation and hemolysis. The protection seems to be due to intracellular chelation of iron, since a semi-stoichiometric ratio between released iron and the amount of quercetin necessary to prevent lipid peroxidation and hemolysis was found. Incubation of GSH depleted human erythrocytes with divicine and isouramil did not induce lipid peroxidation and hemolysis in spite of a substantial release of iron. However, divicine and isouramil produced alterations of membrane proteins, such as spectrin and band 3, as well as formation of senescent cell antigen. The addition of quercetin prevented these alterations.

Redox modulation of cell surface protein thiols in U937 lymphoma cells: the role of gamma-glutamyl transpeptidase-dependent H2O2 production and S-thiolation.

The expression of gamma-glutamyl transpeptidase (GGT), a plasma membrane ectoenzyme involved in the metabolism of extracellular reduced glutathione (GSH), is a marker of neoplastic progression in several experimental models, and occurs in a number of human malignant neoplasms and their metastases. Because it favors the supply of precursors for the synthesis of GSH, GGT expression has been interpreted as a member in cellular antioxidant defense systems. However, thiol metabolites generated at the cell surface during GGT activity can induce prooxidant reactions, leading to production of free radical oxidant species. The present study was designed to characterize the prooxidant reactions occurring during GGT ectoactivity, and their possible effects on the thiol redox status of proteins of the cell surface. Results indicate that: (i) in U937 cells, expressing significant amounts of membrane-bound GGT, GGT-mediated metabolism of GSH is coupled with the extracellular production of hydrogen peroxide; (ii) GGT activity also results in decreased levels of protein thiols at the cell surface; (iii) GGT-dependent decrease in protein thiols is due to sulfhydryl oxidation and protein S-thiolation reactions; and (iv) GGT irreversible inhibition by acivicin is sufficient to produce an increase of protein thiols at the cell surface. Membrane receptors and transcription factors have been shown to possess critical thiols involved in the transduction of proliferative signals. Furthermore, it was suggested that S-thiolation of cellular proteins may represent a mechanism for protection of vulnerable thiols against irreversible damage by prooxidant agents. Thus, the findings reported here provide additional explanations for the envisaged role played by membrane-bound GGT activity in the proliferative attitude of malignant cells and their resistance to prooxidant drugs and radiation therapy.

Allyl alcohol-induced hemolysis and its relation to iron release and lipid peroxidation.

Allyl alcohol administration to starved mice produced, along with liver necrosis, a high incidence (about 50%) of hemolysis. A marked decrease in erythrocyte glutathione (GSH) was seen in all the intoxicated animals. Such a decrease was significantly higher in the animals showing hemolysis. In these animals a substantial amount of malonic dialdehyde (MDA) was detected in plasma and a marked decrease in arachidonic and docosahexaenoic acids was found in erythrocyte phospholipids. These data suggest that the allyl alcohol-induced hemolysis is mediated by lipid peroxidation. In vitro studies have shown that the addition of acrolein to mouse erythrocytes produces a dramatic GSH depletion, which is followed by the appearance of lipid peroxidation and, after an additional 30 min of incubation, by the development of hemolysis. Prevention of lipid peroxidation by an antioxidant (Trolox C) or an iron chelator (desferrioxamine, DFO), prevented hemolysis even if the erythrocyte GSH level was dramatically decreased. In vitro, allyl alcohol and acrylic acid were ineffective in inducing GSH depletion, lipid peroxidation and hemolysis. Studies of possible induction of lipid peroxidation in erythrocytes showed that a progressive increase in "free" (desferal chelatable) iron occurs in the erythrocytes during the incubation with acrolein. It seems, therefore, that a release of iron from iron-containing complexes occurs in acrolein-treated erythrocytes and that such "free" iron promotes lipid peroxidation.

Loss of membrane protein thiols and lipid peroxidation in allyl alcohol hepatotoxicity.

The data reported suggest that--following initiation of lipid peroxidation--membrane protein thiols can be attacked by lipid-derived radicals and/or reactive, lipid-soluble aldehydes like 4-hydroxynonenal and other hydroxyalkenals originated within the lipid core of cell membranes, resulting in a membrane protein thiol loss which is in turn associated with the development of hepatocellular injury.