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Carbonyl compounds released during the NADPH-Fe dependent lipid peroxidation and identified as 4-hydroxyalkenals (almost entirely as 4-hydroxynonenal), when incubated with isolated hepatocytes, produce loss of viability in 95% of the cells, as measured by the trypan blue exclusion test. They also produce an almost complete permeabilization of the plasma-membrane, as measured by the test of the permeability to NADH. concomitantly with the permeabilization of the plasma-membrane, a marked release of enzymes (lactate dehydrogenase and glutamate-pyruvate transaminase) from the hepatocytes occurs. These and other activities of the above mentioned carbonyl compounds suggest the possibility that these products represent some of the effective mediators of the liver injury produced by those toxins, such as CCL4, which promote the peroxidation of membrane lipids.

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.

A line of research from our laboratory has been concerned, for several years, with the pathogenetic mechanisms of the liver damage produced by glutathione (GSH)-depleting agents. The present report deals with the main results obtained in in vivo studies with the use of the prototype aryl halide, bromobenzene.

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.

The mechanisms of bromobenzene toxicity in extrahepatic tissues of mice were studied. Kidney, lung, heart and brain were examined. As observed in this as well as in a previous report for the liver, bromobenzene intoxication caused a progressive decrease in the glutathione content of all the tissues examined. Cellular damage (as assessed by both biochemical determinations and histologic observations) appeared after 6 hours in the case of the kidney and the heart and after 15 hours in the case of the lung. Lipid peroxidation (as assessed by the tissue content of malonic dialdehyde, a parameter correlating with both the diene conjugation absorption and the amount of carbonyl functions in cellular phospholipids) was found to occur at the same times at which cellular damage was observed or even before. As in the case of bromobenzene-induced liver injury, when the individual values for cell damage obtained at 15-20 hours were plotted against the corresponding glutathione contents, a severe cellular damage was generally observed when the glutathione levels reached a threshold value (3.0-0.5 nmol/mg protein). Such a glutathione threshold was also observed for the onset of lipid peroxidation. Glutathione depletion and lipid peroxidation are therefore general phenomena occurring not only in the liver but in all the tissues as a consequence of bromobenzene poisoning. The possibility that lipid peroxidation is the cause of bromobenzene-induced damage to liver and extrahepatic tissues is discussed.

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.

Carbonyl compounds released during the NADPH-Fe dependent peroxidation of liver microsomal lipids and identified as 4-hydroxyalkenals (almost entirely as 4-hydroxynonenal) while inhibiting microsomal enzymes (such as glucose 6-phosphatase and aminopyrine demethylase) which are affected by lipid peroxidation, have no effect on microsomal NADPH-cytochrome c reductase. The latter enzyme activity is unaffected (or even increased) when liver microsomes are allowed to peroxidize in the NADPH-Fe dependent system. NADPH-cytochrome c reductase, contrary to the other enzymes, is similarly unaffected after CCl4 poisoning, that is in a situation in which peroxidation of membrane lipids of liver endoplasmic reticulum has been unequivocally demonstrated. It appears therefore that the effects exherted by lipid peroxidation or by 4-hydroxyalkenals originating from lipid peroxidation parallel the effects of CCl4 intoxication in vivo.

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.

Experimental studies carried out in unselected dogs often face the problem of instabilit of various parameters both in terms of haemodynamics as well as acid-base balance. It is possible, with the injection of a single dose of Fentanyl of 0.35 mg.kg-1 given over a period of ten minutes to obtain, from the 30th minute after the injection, satisfactory cardiovascular stability (confirmed during 120 minutes in 9 dogs and 360 minutes in 2 of them). This haemodynamic state at T + 30 is obtained with a fall in mean blood pressure of -40 per cent, and an increase in peripheral resistance of +38 per cent and stroke volume of +11 per cent. This stability, obtained at the price of a stable normacapnia, correction of any possible metabolic acidosis and maintenance of body temperature, makes it possible to study the cardiovascular effects of certain types of treatment or of induced pathology.

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.

The role of covalent binding of CCl4 radicals to the microsomal protein in the CCl4-induced inhibition of cytochrome P-450 was investigated. The incubation of liver microsomes with NADPH and CCl4 under anaerobic conditions (to avoid lipid peroxidation) results in an inhibition of cytochrome P-450 similar to that observed in the in vivo intoxication with a standard dose (0.25 ml/100 g body wt) of CCl4, when the level of covalent binding is 5-10 fold higher than that observed in the in vivo condition. When in the in vitro experiments the level of covalent binding is of the same magnitude as that observed in vivo, minimal if any inhibition of cytochrome P-450 is seen. Experiments carried out under aerobic conditions seem to indicate that the CCl4-induced inhibition of cytochrome P-450 depends more on lipid peroxidation than on covalent binding.

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.

Porphyria cutanea tarda and haemochromatosis are taken to be spontaneous human models of oxidative cellular damage, with an increased risk of fibrosis and cancer evolution.To define the relative pro-oxidant roles of porphyrin and iron, in their different molecular forms, and their effects on antioxidant biological systems.A group of 17 patients with porphyria cutanea tarda and a group of 14 patients with primary and secondary haemochromatosis, were compared with 21 healthy controls.Plasma retinol, tocopherol, alpha- and beta-carotene, ascorbic acid, glutathione, malonyldialdehyde and red blood cell free iron were determined using high performance liquid chromatography.Only a modest increase in iron stores was demonstrated in the porphyria cutanea tarda group; in the haemochromatosis patients ferritin levels were almost seven times higher. By contrast, there was a sharp and virtually identical increase in red blood cell free iron and malonyldialdehyde in both the patient groups. A significant reduction was observed in retinol, alpha-, beta-carotene and red blood cell glutathione levels being more marked in porphyria cutanea tarda than in haemochromatosis patients.The study confirms the strong pro-oxidant effects of porphyrins in vivo, through an induction of the free toxic iron form, even though the total iron pool is not greatly expanded. The additional free-iron and porphyrin oxidant effects are documented both in red blood cell and plasma in the porphyria cutanea tarda group. It confirmed that aging exerts a negative influence in terms of pro- and antioxidant balance in all cases, but particularly in the haemochromatosis group.