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A simple synthetic pathway to obtain glycosilated β-diketo derivatives is proposed. These compounds show a good iron(III) affinity therefore we may suggest the use of their Fe 3+ -complexes as oral iron supplements in the treatment of anaemia. The glycosilated compounds (6-GlcH, 6-GlcOH and 6-GlcOCH 3 ) are characterized by means of spectroscopic (UV, 1 H and 13 C NMR) and potentiometric techniques; they have a good water solubility, are kinetically stable in physiological condition ( t 1/2 > 100 h) and show a low cytotoxicity also in high concentrations (IC 50 > 400 μM). They are able to bind Fe 3+ ion in acid condition (pH ∼ 2) forming complex species thermodynamically more stable than those of other ligands commonly used in the treatment of iron deficiency. The iron complexes show also a good kinetic stability both in acidic and physiological pH and have a good lypophilicity (log P > −0.7) that suggests an efficient gastrointestinal absorption in view of their possible use in oral therapy. In addition they demonstrate a poor affinity for competitive biological metal ion such as Ca 2+ , and in particular 6-GlcOCH 3 is able to inhibit lipid peroxidation.

Allyl alcohol administration in a toxic dose (1.5 mmol/kg) to starved mice causes the development of hemolysis in nearly 50% of the animals. Malonic dialdehyde (MDA) appears in plasma of the animals showing hemolysis. The treatment of mice with desferrioxamine after allyl alcohol intoxication completely prevents lipid peroxidation and hemolysis, suggesting the involvement of iron in the allyl alcohol-induced erythrocyte damage. Erythrocytes obtained from intoxicated mice before the development of hemolysis show, upon incubation, release of iron, lipid peroxidation and lysis. Studies carried out with reconstituted systems of erythrocyte lysates, containing ghosts and different fractions of erythrocyte cytosol and incubated in the presence of acrolein (the major metabolite of allyl alcohol), strongly suggest that iron is released from hemoglobin. This iron appears to promote lipid peroxidation which is accompanied by erythrocyte lysis. Thus, the allyl alcohol-induced hemolysis appears to be a model for iron delocalization from iron stores.

In the iron-loaded liver there may be an increase in the putative intracellular transit pool of iron, components of which could be catalytically active in stimulating lipid peroxidation. To study the levels of low-molecular-weight, catalytically active iron in the liver, cytosolic ultrafiltrates were tested in an assay containing rat liver microsomes and NADPH. Malondialdehyde production was used as an index of lipid peroxidation. This assay system was sensitive enough to detect 0.25 mumol/L ferrous iron; progressive but non-linear increases in malondialdehyde were produced as the iron concentration was increased to 5 mumol/L. Ultrafiltrates from hepatic cytosol of iron-loaded rats had greater prooxidant action than did those from controls. When added to the assay, deferoxamine, an iron chelator, completely suppressed the prooxidant action of hepatic ultrafiltrates, showing that this activity is iron-dependent. Deferoxamine administered intraperitoneally to control animals at a dose of 1 gm/kg completely inhibited the prooxidant effect of hepatic ultrafiltrates prepared from rats killed after 1, 2 and 3 hr. Partial inhibition was observed at 4 hr; by 6 hr the inhibitory effect of deferoxamine was completely lost. Administration of deferoxamine (1 gm/kg intraperitoneally, 1 hr before killing) completely inhibited the prooxidant action of hepatic ultrafiltrates in moderately iron-loaded rats and controls but had no protective effect in heavily iron-loaded rats. These results support the concept that iron overload results in an increase in a hepatic cytosolic pool of low-molecular-weight iron that is catalytically active in stimulating lipid peroxidation. This pool can be chelated transiently in vivo by deferoxamine in moderate, but not heavy, iron overload.

Increasing evidence suggests that iron plays an important role in tissue damage both during chronic iron overload diseases (i.e., hemochromatosis) and when, in the absence of actual tissue iron overload, iron is delocalized from specific carriers or intracellular sites (inflammation, neurodegenerative diseases, postischaemic reperfusion, xenobiotic intoxications, etc.). In the present work, we appropriately modified an iron chelator of the hydroxychromene family in order to obtain a tridentate chelator that would inactivate the iron redox cycle after its complexation, with a view to using this molecule in human therapy and/or in disease prevention. We synthesized such a chelator for the first time and show, by different physicochemical analysis, its tridentate nature and, importantly, its capacity to chelate iron with enough strength to inhibit both iron-dependent H(2)O(2) generation and lipid peroxidation in in vitro biological systems.

Iron is released in a free desferrioxamine-chelatable form when erythrocytes are challenged by an oxidative stress. The release of iron is believed to play an important role in inducing destructive damage (lipid peroxidation and hemolysis) or in producing membrane protein oxidation and generation of senescent cell antigens (SCA). In this report, we further tested the hypothesis that intracellular chelation of iron released under conditions of oxidative stress prevents erythrocyte damage or SCA formation. Fluor-benzoil-pyridoxal hydrazone (FBPH), an iron-chelating molecule of the family of aromatic hydrazones, was prepared by synthesis and used for the above purpose after the capacity of the product to enter cells had been ascertained. GSH-depleted mouse erythrocytes were incubated with the oxidant drug phenylhydrazine in order to produce iron release, lipid peroxidation, and hemolysis. FBPH at a concentration of 200 μM prevented lipid peroxidation and hemolysis in spite of equal values of iron release. FBPH was active even at a lower concentration (100 μM) when the erythrocytes were preincubated with it for 15 min. No preventive effect was seen when FBPH saturated with iron was used. Prolonged aerobic incubation (60 hr) of erythrocytes produced iron release and formation of SCA as determined by autologous immunoglobulin G (IgG) binding. The IgG binding was detected by using an anti-IgG antibody labeled with fluorescein and by examining the cells for fluorescence by confocal microscopy. FBPH prevented SCA formation in a dose-related manner. These results lend further support to the hypothesis that iron release is a key factor in erythrocyte ageing.

Incubation of rat erythrocytes with the hydroxylated metabolites of aniline and dapsone (4-4′-diaminodiphenylsulfone), phenylhydroxylamine and dapsone hydroxylamine, respectively, induced marked release of iron and methemoglobin formation. On the contrary, no release of iron nor methemoglobin formation was seen when the erythrocytes were incubated with the parent compounds (aniline and dapsone). The acute intoxication of rats with aniline or dapsone induced a marked increase in the erythrocyte content of free iron and methemoglobin, indicating that the xenobiotics are effective only after biotransformation to toxic metabolites in vivo. Prolonged administration of aniline or dapsone to rats produced continuos release of iron from erythrocytes. Marked iron overload was seen in the spleen and in the liver Kupffer cells, as detected histochemically. The spleen weight in these subchronically treated animals was significantly increased. The free iron pool was markedly increased in the spleen and to a lower extent in the liver. The possible relationships between iron release in erythrocytes, oxidative damage seen in senescent cells, hemolysis, overwhelmed capacity of spleen and liver to keep iron in storage forms and subsequent increase in low molecular weight, catalitically active iron is discussed.

Reduction of non-protein-bound iron (NPBI) using iron chelators may attenuate hypoxia-ischemia-induced reperfusion injury of the brain. This study investigated whether administration of low-dose deferoxamine and allopurinol, both having NPBI-chelating properties, reduced hypoxia-ischemia-induced NPBI formation in plasma effluent from the brain and in cerebral cortical tissue. Twenty-one newborn lambs underwent severe hypoxia-ischemia. Upon reperfusion and reoxygenation the lambs received either a placebo (n = 7), or deferoxamine 2.5 mg/kg (n = 7) or allopurinol 20 mg/kg (n = 7). The post-hypoxic-ischemic NPBI levels in plasma were significantly lower after deferoxamine but not after allopurinol as compared to placebo-treated lambs. Cortical NPBI levels in both deferoxamine and allopurinol-treated lambs were significantly lower than NPBI levels in placebo-treated lambs. We conclude that deferoxamine effectively lowers NPBI in plasma effluent from the brain, and that both, deferoxamine and allopurinol, lower NPBI in cortical brain tissue.

Following the subchronic intoxication of rats with phenylhydrazine, resulting in marked anemia, reticulocytosis, methemoglobinemia and increased hemocatheresis, the hepatic content of total iron was increased, as was hepatic ferritin and its saturation by iron. A striking increase (approximately 7-fold) was also observed in free iron which appeared to be redox-active. The increase in liver free iron involved the hepatocellular component of the liver. Since DNA is one of the cellular targets of redox active iron, liver DNA from phenylhydrazine-treated rats was analyzed by electrophoresis and found to be markedly fragmented. Experiments with isolated hepatocytes in culture or in suspension challenged with phenylhydrazine or Fe-nitrilotriacetate strongly suggested that the DNA damage was due to reactive iron rather than to the hepatic metabolism of phenylhydrazine. The levels of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo), a specific marker of oxidative DNA damage, were significantly higher in phenylhydrazine-treated rats as compared to untreated controls. The prolongation of phenylhydrazine treatment over a period of 6 weeks resulted in a persistent damage to DNA and in phenotypic changes such as an increase in hepatocyte γ-glutamyl transpeptidase (γ-GT, EC 2.3.2.2) activity. Possible relationships between iron overload, iron release, DNA damage and tumor initiation are discussed.

Asbestos exposure causes pulmonary fibrosis by mechanisms that remain uncertain. There is increasing evidence that iron from asbestos is responsible for many of its effects. In this paper, we investigated the effect of iron mobilized from crocidolite asbestos on collagen content in rat lung fibroblast cultures under serum-free conditions. Crocidolite (2, 4, 6 μg/cm 2 well) increased collagen content in a dose-dependent manner (+ 42 ± 8, + 92 ± 10, and + 129 ± 13% vs controls). This effect was specific for collagen, since it did not alter total protein content and was inhibited by the iron chelator deferoxamine (DFO). Preincubation of crocidolite with citrate (1 mM) for 48 hr resulted in iron mobilization (51 μM) and increased collagen production (> 3-fold) in treated cells. These effects occurred without the intervention of serum factors. The absence of cell damage, proliferation or lipid peroxidation leads to the supposition that iron from crocidolite per se may act as a profibrogenic agent. Although the in vivo participation of other cells and factors cannot be excluded, we conclude that iron released from crocidolite plays a role in collagen increase occurring during asbestosis.

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.

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.

Iron is released in a free [desferrioxamine (DFO)-chelatable] form in mouse erythrocytes incubated with the oxidizing agents acrolein and phenylhydrazine or in erythrocytes drawn from allyl alcohol-intoxicated mice. The release is accompanied by peroxidation of membrane lipids when the cells are depleted of glutathione. Lipid peroxidation is always followed by the lysis of the cells. The release of iron is also accompanied by methemoglobin formation, but the extent of the release does not correlate with the level of methemoglobin production. The addition of DFO to the incubation mixture or the preincubation of the erythrocytes with DFO in millimolar concentrations completely prevents both lipid peroxidation and hemolysis while not significantly changing the level of iron release. Morphological studies carried out with scanning electron microscopy showed a number of alterations in the shape of the incubated erythrocytes, including echinocyte transformation and the appearance of codocyte, stomatocyte, and cnizocyte like forms. These alterations were more prominent with increasing lipid peroxidation and hemolysis, even if occurring in their absence. On the contrary, the appearance of pits and holes was strictly associated with lipid peroxidation and lysis.

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.

Summary The binding of products derived from the peroxidation of liver microsomal lipids to the non-lipid constituents of the microsomes was studied. To this end arachidonic acid labelled with tritium at the positions of the double bonds was given to rats and allowed to incorporate into the membrane lipids of the liver cell. When liver microsomes containing labelled arachidonic acid were incubated aerobically in the NADPH-dependent system, a marked production of malonic dialdehyde (MDA) occurred and, concomitantly, there was a consistent release of radioactivity from the microsoroes into the incubation medium. The addition of EDTA to the incubation medium prevented, to a large extent, both the MDA formation and the release of radioactivity. Chromatographic studies showed that the bulk of the radioactivity released from the incubated microsomes is not MDA. In the incubated microsomes, the radioactivity decreased in total lipids, while it increased by about 15 times in the non-lipoidal residue. A similar increase in radioactivity was seen in microsomal protein, while no increase was observed in microsomal RNA (the radioactivity was negligible in both the incubated and the non-incubated samples). It seems therefore that products originating from lipoperoxidation of arachidonic acid covalentiy bind to the microsomal protein. In order to investigate whether alterations similar to those observed in the in vitro perosidation of liver microsomes could be detected in the In vivo intoxication with carbon tetrachloride, rate given labelled arachidonic acid as above, were poisoned with CCl4. Sixty minutes after poisonings the radioactivity present in the microsomal lipids was generally lower in the intoxicated rats than in the controls, while the labelling of the non-lipoidal residue and of the protein was higher in the CCl4-poisoned rats.

A study was undertaken to investigate whether some of the methods commonly used to detect lipid peroxidation of cellular membranes in vivo correlate with each other. The study was performed with the livers of bromobenzene-intoxicated mice, in which lipid peroxidation develops when the depletion of glutathione (GSH) reaches a threshold value. The methods tested and compared were the following: i) measurement of the malondialdehyde (MDA) content of the liver; ii) detection of diene conjugation absorption in liver phospholipids; iii) measurement of the loss of polyunsaturated fatty acids in liver phospholipids; and iv) determination of carbonyl functions formed in acyl residues of membrane phospholipids as a result of the peroxidative breakdown of phospholipid fatty acids. Correlations among the values obtained with these methods showed high statistical significances, indicating that the procedures measure lipid peroxidation in vivo with comparable reliability. Analogously, the four methods appeared also to correlate when applied to in vitro microsomal lipid peroxidation.

When liver microsomes were incubated with NADPH under anaerobic conditions either in the presence or in the absence of EDTA, no substantial amount of malonic dialdehyde (MDA) was formed and no substantial decrease in glucose-6-phosphatase (G-6-Pase) activity was seen. The addition of CCl4 to these in vitro systems had minor effects on both MDA production and the enzyme activity. When, on the contrary, the incubation was carried out under aerobic conditions in the absence of EDTA, a marked production of MDA occurred and G-6-Pase activity was almost completely destroyed. These results confirm the findings of Glende et al. [(1976) Biochem. Pharmacol.25, 2163–2170]. The presence of EDTA in the aerobic system reduced both the extent of lipoperoxidation and the decline in enzyme activity. The addition of CCl4 to these systems resulted in some increase in both MDA formation and G-6-Pase inactivation. The relationships between various steps of the lipid peroxidation process and the inactivation of G-6-Pase were then studied. It was observed that when the incubation was carried out under anaerobic conditions with carbon tetrachloride, the binding of CCl4 free radicals to microsomal lipids (as evidenced by the g.l.c. analysis of fatty acid methyl esters with an electron-capture detector, ECD) was qualitatively identical to that observed after CCl4 poisoning in vivo. Also, the diene conjugation absorption detected in the samples incubated in vitro was similar to that observed in the in vivo situation. Furthermore, when the fatty acid methyl esters of the lipids of liver microsomes incubated as above were analyzed by thin-layer chromatography, a typical spot (“D” spot), previously reported to occur in the fatty acid methyl esters of liver microsomal lipids of CCl4-poisoned rats, was observed. As in the in vivo situation, the lipids recovered from this spot showed the absorption of conjugated dienes. On the other hand, when the incubation was carried out aerobically in the absence of EDTA either with CCl4 or without it, the g.l.c. analysis of the fatty acid methyl esters of microsomal lipids showed ECD responses which are probably due to the interaction of oxygen with unsaturated fatty acids. Ultraviolet spectra characteristic of peroxidized lipids were also found in this experimental condition. The activity of G-6-Pase, depressed by the aerobic incubation, could not be restored by the addition of different phospholipid fractions (phosphatidylcholine or phosphatidylethanolamine), denoting that during the incubation some irreversible damage to the enzyme activity occurs. The damage does not seem to be related to the alterations of the molecular structure of the membrane lipids (the binding of chlorinated radicals and the presence of conjugated dienes) since these alterations are also present after the anaerobic incubation, which does not cause a decrease in the G-6-Pase activity. Since the only experimental condition which produces an extensive loss of the enzyme activity is aerobic incubation, in which a large MDA formation occurs, it is concluded that some product evolved during the peroxidative breakdown of unsaturated lipids is responsible for the G-6-Pase inactivation.

The dialysate obtained from a system containing actively peroxidizing liver microsomes shows inhibitory effects on glucose 6-phosphatase activity of freshly prepared liver microsomes (test system). The inhibitory factors were recovered in extracts obtained from the dialysate with ethyl ether or ethyl acetate. The extraction procedure completely removed the inhibitory activity from the dialysate. A partial separation of the products present in the dialysate extract was obtained by thin-layer chromatography. When the lipid materials eluted from individual bands or groups of bands were tested for inhibitory activity, it was found that various Chromatographic bands contained various degrees of inhibitory activity, and that the highest inhibitory activity occurs in a well resolved band that is stained yellow by a N , N '-dimethyl- p -phenylene-diamine reagent. Additional studies indicated that this band contains most of the carbonyl functional groups detectable in the unfractionated dialysate extract, while it contains peroxide functional groups in trace amounts only. The peroxide functional groups present in the unfractionated dialysate extract were found to occur in various Chromatographic bands without a well defined relationship with the toxicological activity. It is concluded that lipoperoxidation products highly active in inhibiting microsomal glucose 6-phosphatase activity are fatty aldehydes or other carbonyl compounds, probably provided with a relatively long carbon chain.

The relationships between the peroxidation of musomal lipids and the early liver damage have been investigated in rats pretreated with progressively higher doses of α-tocopherol (vit. E) and intoxicated with various amounts of carbon tetrachloride. Pretreatment of rats with vit. E at 25 mg 100g body wt. has minor effects on both the peroxidation of musomal lipids and the liver triglyceride accumulation in rats poisoned with CC14 at 250 μl 100g body wt. However, a decrease of the peroxidative reaction and of the liver steatosis occurs when the rats are pretreated with progressively higher doses of vit.E. A close correlation exists between the two phenomena, when the intoxication is accomplished with CC14 at 250 μ1 100 g body wt. Also, the musomal concentration of α-tocopherol is strictly correlated to both the decrease of musomal lipoperoxidation and the decrease of liver triglyceride accumulation. The CCl4-induced impairment of musomal glucose-6-phosphatase and the incorporation of 14C from 14CC14 into liver musomal lipids are not affected by vit. E pretreatment. The extent of musomal lipoperoxidation is not correlated to the liver triglyceride accumulation when vit. E-pretreated rats are given CC14 at 25 or 2.5 μ1 100 g body wt. However, a correlation between lipoperoxidation and liver steatosis occurs when non-pretreated rats are challenged with the three different doses of the halogenated hydrocarbon.

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.

The mechanisms of bromobenzene hepatotoxicity in vivo were studied in mice. The relationships among glutathione (GSH) depletion, lipid peroxidation, loss of protein thiols, disturbed calcium homeostasis and liver necrosis were investigated. Liver necrosis (as estimated by the serum glutamate-pyruvate transaminase (SGPT) level) appeared between 9 and 12 hr and increased at 18 hr. Lipid peroxidation which was already detectable at 6 hr in some animals, increased thereafter showing a good correlation with the severity of liver necrosis. Despite a quite fast depletion of hepatic GSH, a significant decrease in protein thiols could be observed at 12-18 hr only. Loss of protein thiols in both whole liver and subcellular fractions (microsomes and mitochondria) was correlated with lipid peroxidation. Also a good inverse correlation was seen between lipid peroxidation and the calcium sequestration activity of liver microsomes and mitochondria. The treatment of mice with desferrioxamine (DFO) after bromobenzene-intoxication completely prevented lipid peroxidation, loss of protein thiols and liver necrosis in the animals sacrificed 15 hr after poisoning. When, however, the animals were examined at 24 hr, although the general correlation between lipid peroxidation and liver necrosis was held, in some animals (about 30% of the survivors) elevation of SGPT was observed in the virtual absence of lipid peroxidation. It seems likely therefore that the liver damage seen during the first phase of bromobenzene-intoxication is strictly related to lipid peroxidation. It is, however, possible that in some animals in which for some reason lipid peroxidation does not develop, another mechanism of liver necrosis unrelated to lipid peroxidation occurs at later times.

The distribution of carbon tetrachloride-induced alterations of membrane lipids in various fractions of liver microsomal lipids was studied. The chromatographic spot (referred to as the “D” spot in the previous paper [1]) which has been shown to contain the compounds responsible for the diene conjugation absorption [1], was found in the fatty acid methyl esters prepared from the fraction containing phosphatidylethanolamine (PE) and also in those obtained from the fraction containing phosphatidylserine (PS) and phosphatidylinositol (PI). The absorption of conjugated dienes was very marked in PE and less intense in PS and PI. The fatty acid methyl esters prepared from the fraction containing phosphatidylcholine (PC) showed no presence of the “D” spot and minimal absorption of conjugated dienes. A decrease in arachidonic acid content was found in the fraction containing PE, while no change in content of this fatty acid was found in the fraction containing PC. Results similar to those observed for PC were also found for neutral lipids (NL). Analysis of the fatty acid methyl esters of the various lipid fractions by gas-liquid chromatography (GLC) with an electron capture detector (ECD) gave a qualitative index of the free radical attack by CCl4 metabolites. Quantitative estimation was attained by study of the irreversible binding of 14C from 14CCl4 to the various lipid fractions. It was found that the fraction containing PS had the highest specific activity, while the fraction containing PC had the lowest specific activity of all the phospholipids. Thin layer chromatography (TLC) of the fraction containing PS revealed that only 11% of the radioactivity was associated with the pure PS moiety, while the remainder was associated with uncharacterized lipids (probably oxidation products). The possible relevance of the alterations induced by carbon tetrachloride in the various phospholipid fractions of liver microsomes to functional changes is discussed.

The effects on cellular structures of products of peroxidation of rat liver microsomal lipids were investigated. A system containing actively peroxidizing liver microsomal fraction was separated from a revealing or target system by a dialysis membrane. The target system, contained in the dialysis tube, consisted of either intact cells (erythrocytes) or subcellular fractions (liver microsomal fraction). When liver microsomal fractions were incubated with NADPH (or an NADPH-generating system), lipid peroxidation, as measured by the amount of malonaldehyde formed, occurred very rapidly. The malon-aldehyde concentration tended to equilibrate across the dialysis membrane. When the target system consisted of erythrocytes, haemolysis occurred abruptly after a lag phase. The lysis was greatly accelerated when erythrocytes from vitamin E-deficient rats were used, but no haemolysis was observed when erythrocytes from vitamin E-treated rats were used. When, in the same system, freshly prepared liver microsomal fractions were exposed to diffusible factors produced by lipid peroxidation, the glucose 6-phosphatase activity markedly decreased. A similar decrease in glucose 6-phosphatase activity, as well as a smaller but significant decrease in cytochrome P-450, was observed when the target microsomal fractions were exposed to diffusible factors derived from the peroxidation of liver microsomal lipids in a separate preincubation step. These and additional experiments indicated that the toxicological activity is relatively stable. Experiments in which the hepatic microsomal fractions destined for lipid peroxidation contained radioactively labelled arachidonic acid, previously incorporated into the membranes, showed that part of the radioactivity released from the microsomal fraction into the incubation medium entered the dialysis tube and was recovered bound to the constituents of the microsomal fractions of the target system. These results indicate that during the course of the peroxidation of liver microsomal lipids toxic products are formed that are able to induce pathological effects at distant loci.

Alterations induced by carbon tetrachloride poisoning in fatty acids of liver microsomal lipids were studied. Thin layer chromatography of fatty acid methyl esters prepared from liver microsomal lipids, revealed, in the CCl 4 -treated rats, the presence of a component (the “D” spot) with an R f value lower than that of the methyl esters. The lipids recovered from this component showed a marked diene conjugation absorption when examined spectrophotometrically over the UV range, while the lipids recovered from the spot of the methyl esters showed no absorption of conjugated dienes. Studies carried out with labelled carbon tetrachloride indicated that compounds present in the “D” spot contained 28% of 14 C applied to the chromatoplate. The spot of the methyl esters (the “M” spot) contained 42% of 14 C applied to the chromatoplate. However, specific activity of the “D” spot was about 1000 times greater than specific activity of the “M” spot. The lipids recovered from either the “D” spot or the spot of the methyl esters were analyzed separately by gas-liquid chromatography (GLC) with an electron capture detector (ECD). It was found that the lipids recovered from the “D” spot showed no response, while those recovered from the spot of the methyl esters exhibited the response of the ECD, which was similar to that observed with the unfractionated fatty acid methyl esters. The lack of the response of the ECD for compounds in the “D” spot appears to be due to the fact that they cannot be eluted from the column. On the basis of the analytical results, it can be postulated that the “D” spot contains compounds formed by a chain termination addition reaction of free radicals derived from CCl 4 (probably trichloromethyl free radicals) to fatty acid free radicals containing conjugated dienes. On the other hand, the spot of the methyl esters appears to contain also, together with unmodified fatty acids, the fatty acids in which a simple addition of CCl 4 free radicals to double bonds has occurred.

Carbonyl compounds released during the NADPH-Fe dependent peroxidation of liver microsomal lipids and identified as 4-hydroxyalkenals (almost entirely as 4-hydroxynonenal) inhibit protein synthesis in a rabbit reticulocyte lysate. The ID50 was 0.48 mM. The inhibitory effect was reproduced by synthetic 4-hydroxynonenal. The inhibition was already evident at 1–2 min of incubation. The addition of −SH groups to the incubation medium afforded a marked protection against the inhibition of protein synthesis. The inhibitory effect seems to be due to an interaction of the carbonyl compound with −SH groups essential for the cellular protein synthetic machinery.

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.

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-Fedependent 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.

Increasing evidence suggests that iron plays an important role in tissue damage both during chronic iron overload diseases (i.e., hemochromatosis) and when, in the absence of actual tissue iron overload, iron is delocalised from specific carriers or intracellular sites (inflammation, neurodegenerative diseases, post-ischaemic reperfusion, etc.). In order to be used for therapeutical purposes in vivo, a reliable iron chelator should be capable of preventing the undesired effects that follow the electrochemical activation of iron (see below). Bearing in mind the molecular structure of some flavonols that are able to chelate iron, we synthesised a new oral iron-chelator, 2-methyl-3-hydroxy-4 H -benzopyran-4-one (MCOH). We demonstrate that MCOH chelates iron in a 2:1 ratio showing a stability constant of ∼10 10 . MCOH is able to cross cell membranes (erythrocytes, ascite tumour cells) in both directions. Following intraperitoneal administration to rats, it is quickly taken up by the liver and excreted in the urine within 24 h. A similar behaviour has been documented after oral administration. We propose that MCOH may represent the prototype of a new class of iron chelating agents to be developed for iron-removal therapy in vivo with the goal of preventing tissue damage caused by the iron redox cycle.