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The activation of carbon tetrachloride to a free radical product (CCl3) by the NADPH-cytochrome P450 enzyme complex is used as an example to discuss aspects of electron flow in liver endoplasmic reticulum. The CCl3 free radical may bind covalently to neighboring molecules, or may react rapidly with molecular oxygen to yield the highly reactive CCI3O2 species. The kineties of reaction of this radical with various substances have been measured using pulse radiolysis techniques. The production of such a reactive radical in the lipid membrane may result in lipid peroxidation. This is a degradative reaction associated with the formation of a number of pharmacologically active materials. Lipid peroxidation is often much reduced in endoplasmic reticulum from cancer cells, and electron transfer reactions in such membranes can be severely disturbed. Some ideas leading to further experimentation in these areas are presented.

The CHO320 cell line, engineered to produce human interferon gamma was investigated with regard to its susceptibility to oxidative stress. Batch cultures of the cells grown in a bench-top bioreactor exhibited no marked response to changes in oxygen concentration between 6% and 14% whereas cell growth and recombinant protein production were inhibited by increasing the oxygen to 20%. High concentrations of hydrogen peroxide (in excess of 200 microM) were required to inhibit growth of the CHO320 cells whereas concentrations of 50 microns and 100 microM had no effect on recombinant protein production. Buthionine sulphoximine (50 microM and 100 microM) completely depleted the cells of glutathione within 24 h; however, no quantitative effect on recombinant protein production was seen. It is concluded that the CHO320 cells are, possibly as a consequence of the long selection process they have undergone, very resistant to oxidative stress.

Oxidation and glycation are non-enzymatic protein modifications involved in the pathogenesis of aging. We evaluated their possible influences in an in vitro system: albumin was oxidized by gamma-irradiation and then exposed to glycation in vitro. Fluorescence modifications were analysed as signals of protein alterations. Both radiolytic oxidation and in vitro glycation provoked a sharp decrease of tryptophan fluorescence (278 nm ex./340 nm em.); their effects tended to be additive, unless a saturation limit was reached. Both individually and in combination, these two non-enzymatic processes induced the appearance of a new fluorescence (335 nm ex./415 nm em.); in this case as well there was an additive effect, with a trend toward saturation. Radiolytic oxidation and in vitro glycation seem to provoke similar damage to the exposed proteins: the observed fluorescence alterations may be due to similar conformational changes, breaks or the development of fluorophores.

A series of flavonoids, able to inhibit non-enzymic and enzymic lipid peroxidation, were investigated as inhibitors of cytochrome P-450 mediated reactions (aminopyrine N-demethylation and aniline hydroxylation) in rat liver microsomes. The most potent compounds were chrysin, luteolin, amentoflavone and eriodictyol (aminopyrine N-demethylation) and apigenin, kaempferol and quercetin (aniline hydroxylation). Some structure-activity relationships were established for inhibition of aminopyrine N-demethylation and it was observed that the resorcinol configuration of the ring A is a determinant feature, whereas O-glycosylation abolished the activity. Most of the compounds showed lower activity on aniline hydroxylation. The presence of these flavonoids during incubation of microsomes in a medium containing NADPH did not modify the cytochrome P-450 concentration and protected against lipid peroxidation induced degradation of this cytochrome.

beta H-crystallin was exposed to radiolytically generated hydroxyl radicals at defined radical concentrations, and its capacity to act as an amine-acceptor substrate and as an amine-donor substrate for transglutaminase were investigated. [14C]Methylamine was used as a probe for labelling amine-acceptor sites; a novel biotinylated hexapeptide was used to label amine-donor sites. The results demonstrate that both primary amine incorporation and hexapeptide incorporation by transglutaminase are considerably increased after oxidative attack on the crystallin. The identity of the labelled subunits was established, and it is shown that, in both cases, this increased incorporation is not due to the production of new substrates, but that the existing incorporation sites become more susceptible. Moreover, using the newly developed probe, we could identify, for the first time, the major crystallin subunits active as amine-donor substrates (both before and after treatment) to be beta B1-, beta A3- and beta A4-crystallin. These data support the proposal that oxidative stress and transglutaminase activity may be jointly involved in the changes found in lens crystallins with age and in the development of cataract.

A modified method was developed to measure nM levels of a range of n -alkanals and hydroxyalkenals in biological samples such as blood plasma and tissue homogenates and also in Folch lipid extracts of these samples. Butylated hydroxytoluene (BHT) and desferrioxamine (Desferal) were added to samples to prevent artifactual peroxidation. Aldehydes were reacted with 1,3-cyclohexanedione (CHD), cleaned up by solid-phase extraction on a Sep-Pak C18 cartridge and the fluorescent decahydroacridine derivatives resolved by reverse-phase high performance liquid chromatography (HPLC) with gradient elution. A wider range of aldehydes was detected in lipid extracts of plasma and liver homogenate compared to whole (unextracted) samples. Human plasma contained nM levels of acetaldehyde, propanal, butanal, pentanal, hexanal, and heptanal. 4-Hydroxy-nonenal (0.93 nmol/g) and alkanals with two to six carbons (up to 7.36 nmol/g) were detected in rat liver. Recovery of aldehydes added to whole plasma or to lipid extracts of plasma was dependent on carbon chain length, varying from 95% for acetaldehyde to 8% for decanal. Recovery from biological samples was significantly less than that of standards taken through the Sep-Pak clean-up procedure, suggesting that aldehydes can bind to plasma protein and lipid components.

Free radicals are chemical species possessing an unpaired electron that can be considered as fragments of molecules and which are generally very reactive. They are produced continuously in cells either as accidental by-products of metabolism or deliberately during, for example, phagocytosis. The most important reactants in free radical biochemistry in aerobic cells are oxygen and its radical derivatives (superoxide and hydroxyl radical), hydrogen peroxide and transition metals. Cells have developed a comprehensive array of antioxidant defences to prevent free radical formation or limit their damaging effects. These include enzymes to decompose peroxides, proteins to sequester transition metals and a range of compounds to 'scavenge' free radicals. Reactive free radicals formed within cells can oxidise biomolecules and lead to cell death and tissue injury. Establishing the involvement of free radicals in the pathogenesis of a disease is extremely difficult due to the short lifetimes of these species.

1. The effects of free radicals on the capacity of beta L-crystallin to act as a substrate for the enzyme transglutaminase were investigated. 2. beta L-Crystallin was exposed to defined radical species that were generated radiolytically, and transglutaminase activity, using the modified protein as substrate, was subsequently measured by monitoring the incorporation of [14C]putrescine. 3. Exposure of beta L-crystallin to hydroxyl radicals, thymine peroxyl radicals and acetone peroxyl radicals at concentrations of up to 135 microM increased the capacity of the protein to incorporate putrescine. With higher concentrations of these radicals this capacity of beta L-crystallin to act as a transglutaminase substrate declined to control levels or lower. 4. Superoxide radicals were inactive in this regard; hydroperoxyl radicals were active only at high concentrations. 5. It has previously been suggested that changes in the crystallins that occur during aging and with cataract may be due to oxidative reactions and to transglutaminase activity. This study suggests that these phenomena may be considered together rather than separately.

Measurements of the absolute rate constants for the reaction with peroxyl radicals of alpha, beta, gamma and delta-tocopherol and several model compounds are described. The peroxyl radicals were obtained either by the autoxidation of styrene or by the flash photolysis of di-t-butyl ketone in an oxygen-saturated environment. The kinetic data are discussed in stereoelectronic terms. Vitamin E and total lipid-soluble, chain-breaking antioxidant concentrations in some normal and cancerous tissues have been measured. In human blood plasma and erythrocyte ghost membranes vitamin E is the major, and possibly the only, chain-breaking antioxidant. Lipid extracts of Novikoff ascites hepatoma cells contain considerably more vitamin E relative to lipid than do extracts of normal rat liver. These tumour lipids contain relatively fewer highly unsaturated fatty acids and are present at lower lipid/wet tissue ratios than the normal liver lipids. A number of unresolved problems relating to the action of vitamin E in vivo are discussed.

In the present paper we provide a basic enzymatic characterization of biliary epithelial cells (BEC) that have been isolated from normal rat liver. When compared with liver parenchymal cells, BEC display the following major features: (a) a very high specific activity of γ-glutamyltranspeptidase (approx. 200-times higher than the value usually found in hepatocytes); (b) a lack of enzymes that are usually associated with the endoplasmic reticulum in hepatocytes such as cytochrome P -450, aminopyrine demethylase, glucose 6-phosphatase and NADPH cytochrome- c reductase; (c) the presence of enzymes related to the glutathione redox cycle (e.g., GSH-peroxidase, GSSG-reductase and different isozymes of GSH-transferase), but accompanied by a very low content in reduced glutathione. The enzyme pattern of BEC correlates well with histochemical and immunohistochemical studies, as well as with biochemical studies on bile ductular cells isolated from rat liver during cholestasis.

Summary Background Vitamin E (α-tocopherol) is thought to have a role in prevention of atherosclerosis, through inhibition of oxidation of low-density lipoprotein. Some epidemiological studies have shown an association between high dietary intake or high serum concentrations of α-tocopherol and lower rates of ischaemic heart disease. We tested the hypothesis that treatment with a high dose of α-tocopherol would reduce subsequent risk of myocardial infarction (MI) and cardiovascular death in patients with established ischaemic heart disease. Methods In this double-blind, placebo-controlled study with stratified randomisation, 2002 patients with angiographically proven coronary atherosclerosis were enrolled and followed up for a median of 510 days (range 3-981). 1035 patients were assigned α-tocopherol (capsules containing 800 IU daily for first 546 patients; 400 IU daily for remainder); 967 received identical placebo capsules. The primary endpoints were a combination of cardiovascular death and non-fatal MI as well as non-fatal Ml alone. Findings Plasma α-tocopherol concentrations (measured in subsets of patients) rose in the actively treated group (from baseline mean 34·2 μmol/L to 51·1 μmol/L with 400 IU daily and 64·5 μmol/L with 800 IU daily) but did not change in the placebo group. α-tocopherol treatment significantly reduced the risk of the primary trial endpoint of cardiovascular death and non-fatal Ml (41 vs 64 events; relative risk 0·53 [95% Cl 0·34-0·83; p=0·005). The beneficial effects on this composite endpoint were due to a significant reduction in the risk of non-fatal Ml (14 vs 41; 0·23 [0·11-0·47]; p=0·005); however, there was a non-significant excess of cardiovascular deaths in the α-tocopherol group (27 vs 23; 1·18 [0·62-2·27]; p=0·61). All-cause mortality was 36 of 1035 α-tocopherol-treated patients and 27 of 967 placebo recipients. Interpretation We conclude that in patients with angiographically proven symptomatic coronary atherosclerosis, α-tocopherol treatment substantially reduces the rate of non-fatal MI, with beneficial effects apparent after 1 year of treatment. The effect of α-tocopherol treatment on cardiovascular deaths requires further study.

To avoid the side effects associated with long-term administration of high doses of inhaled glucocorticosteroids, they should be used at the lowest effective dose. This study compared the clinical efficacy of budesonide given via a dry-powder, inspiratory flow-driven device (Turbuhaler), at a daily dose of 800 micrograms, with beclomethasone dipropionate (BDP) 1500 to 2000 micrograms given via pressurized metered-dose inhaler (pMDI) with spacer to adults requiring the latter dose of BDP to control their asthma. The study was performed as a 2-week run-in, 8-week open, randomized, multicenter, parallel-group design. Adult asthmatics with a forced expiratory volume in 1 second 55% or more of predicted normal and receiving BDP 1500 to 2000 micrograms daily entered the study. After a 2-week run-in, one group continued with BDP and the other was switched to budesonide through the Turbuhaler. After 8 weeks, morning peak expiratory flow (PEF) had increased by 5.9 L/min from a mean of 390 L/min in the budesonide group and by 1.9 L/min from a mean of 402 L/min in the BDP group. No clinically or statistically significant differences between groups were evident with regard to the change in this primary variable. Similarly, only small changes in evening PEF and secondary variables of lung function were seen, with no statistically significant difference between groups. The authors concluded that both treatments were equivalent in managing asthma in adult patients with stable asthma.

Estimations of alpha-tocopherol content were made on a series of human necropsy samples of normal arterial wall and of atherosclerotic lesions. The results were compared with stage of lesion, shown by histology, and with the amounts of cholesterol and hydroxycholesterols in the same lesions. The ratio of alpha-tocopherol to cholesterol levels varied widely in normal arterial wall but was consistently low in lesions, especially in lesions rich in macrophage foam cells. The results suggested that significant accumulation of hydroxycholesterols, found almost exclusively in lesions, only occurred when alpha-tocopherol levels were low in relation to the cholesterol content. This suggests that oxidative activity in the lesion may lead to significant oxidation of constituents of low-density lipoprotein only after alpha-tocopherol has been depleted.

It is known that many types of liver tumour cells respond much less readily to pro-oxidant stimuli than does normal liver (1–5).

For many years it has been established that tumour tissue exhibits a strong resistance to lipid peroxidation. Other rapidly dividing cells were also shown to be resistant and it was suggested that low lipid peroxidation activity is a common feature of cell division and that there is actually a biochemical connection between the two processes. The crux of this hypothesis was that possibly lipid peroxidation is a normal cellular process that somehow acts as a coarse regulator of cell division. The mechanisms of resistance of tumours to lipid peroxidation and the relative roles of pro-oxidants such as cytochrome P-450 and antioxidants such as α-tocopherol have been studied. The rapidly dividingnon-transformed regenerating liver has provided interesting observations on the tight temporal connection between cell division and resistance and the role of α-tocopherol in this phenomenon. Foetal liver and intestinal epithelial cells have also been investigated and the overall impression from all these cell types is that there isno common mechanism of resistance.

The antioxidant activity of alpha-tocopherol polyethylene glycol 1000 succinate (TPGS) and of alpha-tocopherol succinate (TS) has been examined in isolated hepatocytes and microsomal fractions from rat liver. Both TPGS and TS require esterase activity to yield free alpha-tocopherol and, hence, antioxidant activity. TPGS and TS consistently exerted a more effective antioxidant protection than an equivalent amount of directly-added free alpha-tocopherol. The low antioxidant efficiency of directly added free alpha-tocopherol in such water-based experimental systems as used here seems to be due to its extreme hydrophobicity. TPGS, on the other hand, is an extremely hydrophilic compound that is being examined as a useful source of alpha-tocopherol in certain clinical situations and is here shown to be a convenient and effective source for experimental studies into lipid peroxidation and antioxidant mechanisms.

Lipid peroxidation is an extremely complicated process, especially in biological systems where one is dealing with mixtures of different lipids in matrices also containing proteins, carbohydrates, nucleic acids and catalytically-active enzymes and trace metals. With regard to measuring lipid peroxidation, the complexity of the process may be viewed by an optimist as presenting the investigator with a large number of opportunities: it is a multi-stage process and each stage can be investigated by at least one technique. The view of the pessimist, or perhaps realist, would be that the complexity of lipid peroxidation is a problem, in that no single assay method can fully describe what is happening or has happened in a system undergoing lipid peroxidation. In considering the large range of assay methods concerned with measuring lipid peroxidation, it is readily obvious that some are straightforward to perform, others tedious; some are more sensitive than others, some more prone to interference; some measure end-products, some measure unstable intermediates, and some measure specific identified compounds whilst others measure poorly characterised entities.

(1991). Flavonoids in Biology and Medicine III Current Issues in Flavonoids Research. Free Radical Research Communications: Vol. 14, No. 1, pp. 77-78.

Preparations of rat liver sinusoidal plasma membrane have been tested for their ability to metabolize the hepatotoxin carbon tetrachloride (CCl4) to reactive free radicals in vitro and compared in this respect with standard preparations of rat liver microsomes. The sinusoidal plasma membranes were relatively free of endoplasmic reticulum-associated activities such as the enzymes of the cytochrome P450 system and glucose-6-phosphatase. CCl4 metabolism was measured as (i) covalent binding of [14C]-CCl4 to membrane protein, (ii) electron spin resonance spin-trapping of CCl3· radicals and (iii) CCl4-induced lipid peroxidation. By all of these tests, purified sinusoidal plasma membranes were found unable to metabolize CCl4. The fatty acid composition of the plasma membranes was almost identical to that of the microsomal preparation and both membrane fractions exhibited similar rates of the lipid peroxidation that was stimulated non-enzymically by γ-radiation or incubation with ascorbate and iron. The absence of CCl4-induced lipid peroxidation in the plasma membranes seems to be due, therefore, to an absence of CCl4 activation rather than an inherent resistance to lipid peroxidation. We conclude that damage to the hepatocyte plasma membrane during CCl4 intoxication is not due to a significant local activation of CCl4 to CCl3· within that membrane.

A cell fraction enriched in biliary epithelial cells (BEC) has been isolated from the liver of normal rats. The procedure involved proteolytic digestion by trypsin and mild mechanical disruption of biliary ductular and connective tissue that remained undigested after collagenase-hyaluronidase perfusion. An adherence procedure removed the large majority of contaminating Kupffer cells. The majority (87.4 +/- 3.5%) of the cells were positive to an indirect immunofluorescence staining that used an antiserum against bovine hoof prekeratin that specifically recognizes intermediate filaments of biliary epithelium. Similar results were obtained by histochemical staining for gamma-glutamyltranspeptidase activity. The contamination of the BEC fraction with Kupffer cells and hepatocytes was approximately 7% and 2%, respectively. The viability of the BEC population was always more than 90%. The BEC and hepatocytes were analysed for their lipid composition; the BEC were found to have a cholesterol content approximately 6-times higher than hepatic parenchymal cells, with a cholesterol/phospholipid molar ratio of 0.53 in comparison to a value of 0.11 for hepatocytes. No detectable evidence of cytochrome P-450 or cytochrome P-450-related enzymatic activities was found in the BEC.

The general features of the reduction of nitroblue tetrazolium chloride (NBT) by NADH and phenazine methosulphate (PMS) have been studied under aerobic and anaerobic conditions. Under aerobic condition the reduction appears to be mediated through the intermediate formation of the superoxide anion radical O2-.; this reaction is strongly inhibited by superoxide dismutase and by a number of O2-. scavengers such as propyl gallate, (+)-catechin, manganous ions, reduced glutathione and benzoquinone. Cupric ions inhibited the overall reaction by reoxidising reduced PMS. Under anaerobic conditions, superoxide dismutase had only a small inhibitory action and, with the exception of cupric ions, the other substances mentioned above were ineffective as inhibitors. The data presented show that the use of NBT to detect the presence of O2-. is fraught with difficulties due to an equally rapid reduction of NBT by NADH and PMS under anaerobic conditions.

The degradation of 2-deoxyribose to thiobarbituric acid-reactive material was investigated with two hydroxyl-radical-generating systems: (i) a defined gamma-radiolysis method and (ii) incubation with FeSO4 in phosphate buffer. In each case the thiobarbituric acid-reactive material can be accounted for by malondialdehyde, as measured by an h.p.l.c. method for free malondialdehyde. In the radiolysis system there is a large post-irradiation increase in free malondialdehyde if iron ions are added to the samples. It is proposed that this is due to iron ions catalysing the formation of hydroxyl radicals from radiolytically generated H2O2 as well as stimulating the breakdown of an intermediate deoxyribose degradation product. A mechanism for the formation of malondialdehyde during deoxyribose degradation is proposed.

On the basis of promising experimental studies, the nitroimidazoles misonidazole (MISO) and benznidazole (BENZO) are under clinical investigation as chemosensitizers in combination with the chloroethylnitrosourea CCNU. We have shown previously that MISO and BENZO can alter the pharmacokinetics of CCNU leading to an improved therapeutic index in mice. Here we demonstrate using optical difference spectroscopy that MISO and BENZO are able to bind to cytochrome P-450 of mouse liver microsomes in vitro. Binding was type II in nature, indicating co-ordination of the free imidazole nitrogen with the heme moiety of cytochrome P-450. This results in an inhibition of CCNU hydroxylation by the hemoprotein. The kinetics of inhibition were of a mixed competitive-non-competitive type. At a CCNU concentration of 0.05 mM the concentrations causing 50% inhibition (I50) were 5.8 and 0.37 mM for MISO and BENZO respectively. At doses producing a similar improvement in therapeutic index in mice (2.5 mmoles/kg MISO and 0.3 mmoles/kg BENZO) the plasma and tissue concentrations achieved would inhibit CCNU hydroxylation by 30%. For BENZO, but not MISO, similar inhibition would also occur at concentrations which can be achieved safely in man.

The effect of various fatty acids on lipid peroxidation of liver microsomes induced by different methods in vitro was studied using oxygen uptake and malonaldehyde (MDA) production. It was observed that fatty acids with a single double bond are effective inhibitors of peroxidation. Stereo and positional isomers of oleic acid were equally effective as oleic acid. There was an absolute requirement for a free carboxyl group, since methyl esters of fatty acids and long-chain saturated and unsaturated hydrocarbons could not inhibit peroxidation. Saturated fatty acids with a chain length of 12-16 carbon atoms showed inhibition, whereas more than 18 carbon atoms reduced the inhibitory capacity. Fatty acids of lower chain length such as capric and caprylic acids did not show inhibition. Fatty acid inhibition was partially reversed by increasing the concentration of iron in the system. Peroxidation induced by methods which were independent of iron was not inhibited by fatty acids. It was observed that intestinal microsomes which were resistant to peroxidation due to the presence of nonesterified fatty acids in their membrane lipids were able to peroxidise by methods which do not require iron. These results suggest that certain fatty acids inhibit peroxidation by chelating available free iron. In addition, they may also be involved in competing with the esterified fatty acids in the membrane lipids which are the substrates for peroxidation.

Lipid peroxidation stimulated by ADP-iron or ascorbate-iron is often considerably less active in many liver tumours than in normal liver (Cockerill et al. 1983; Borrello et al. 1985; Cheeseman et al. 1986a, 1988) and a major contributory factor is an increased concentration of lipid-soluble chainbreaking antioxidant material (Cheeseman et al. 1986a, 1988). The decreased rate of lipid peroxidation in the liver tumours may be due to the high number of dividing cells in the tumour sample; on the other hand it may be a feature of malignancy. In order to distinguish between these possibilities it is of interest to study lipid peroxidation and antioxidant content in regenerating liver after partial hepatectomy, where periodic bursts of cell division are known to occur (Hopkins et al. 1973). We report here that dividing normal cells in regenerating liver have a much reduced rate of lipid peroxidation, and an increased content of lipid-soluble chain-breaking antioxidant, analogous to the situation previously observed in liver tumours (Cheeseman et al. 1986a).

The diene conjugates formed during the autoxidation of microsomal lipid extracts, and in endoplasmic reticulum in vivo after exposing rats to CCl4 have been examined by second derivative absorption spectrophotometry. Within a few minutes after administering CCl4 to a rat there is a pronounced signal in microsomal lipid extracts that is ascribed to the cis-trans diene conjugates of microsomal polyunsaturated fatty acids. Somewhat later a second signal becomes evident that is ascribed to trans-trans isomers. The appearance of the trans-trans isomer is very strongly suppressed by prior administration of vitamin E to the rat. It is concluded that the relative contents of cis-trans and trans-trans dienes in lipid extracts of tissue reflect the tissue contents of hydrogen donors as already established for model experiments with polyunsaturated fatty acids in vitro.

1. Methods using t.l.c. and high-pressure liquid chromatography (h.p.l.c.) have been used to separate the complex variety of substances possessing a carbonyl function that are produced during lipid peroxidation. 2. The major type of lipid peroxidation studied was the ADP-Fe2+-stimulated peroxidation of rat liver microsomal phospholipids. Preliminary separation of the polar and non-polar products was achieved by t.l.c.: further separation and identification of individual components was performed by h.p.l.c. Estimations were performed on microsomal pellets and the supernatant mixture after incubation of microsomes for 30 min at 37 degrees C. 3. The polar fraction was larger than the non-polar fraction when expressed as nmol of carbonyl groups/g of liver. In the non-polar supernatant fraction the major contributors were n-alkanals (31% of the total), alpha-dicarbonyl compounds (22%) and 4-hydroxyalkenals (37%) with the extraction method used. 4. Major individual contributors to the non-polar fraction were found to be propanal, 4-hydroxynonenal, hexanal and oct-2-enal. Other components identified include butanal, pent-2-enal, hex-2-enal, hept-2-enal, 4-hydroxyoctenal and 4-hydroxyundecenal. The polar carbonyl fraction was less complex than the non-polar fraction, although the identities of the individual components have not yet been established. 5. Since these carbonyl compounds do not react significantly in the thiobarbituric acid reaction, which largely demonstrates the presence of malonaldehyde, it is concluded that considerable amounts of biologically reactive carbonyl derivatives are released in lipid peroxidation and yet may not be picked up by the thiobarbituric acid reaction.

The fragmentation of the membrane protein monoamine oxidase in submitochondrial particles was induced by defined free radicals during radiolysis and by a system dependent on hydrogen peroxide and a transition metal. By injection of alpha-tocopherol in vivo, the levels of this physiological antioxidant in the mitochondrial preparations could be elevated more than ten-fold. In both radical-generating systems the presence of high levels of alpha-tocopherol in the membrane substantially retarded the protein fragmentation, in parallel with lipid peroxidation. It is suggested that membrane-bound proteins are damaged during lipid peroxidation and that alpha-tocopherol protects cells against both types of damage.

alpha-tocopherol is the major lipid-soluble radical-scavenging antioxidant in rat liver. It has long been used as a putative protective agent in CCl4 induced liver injury but with variable results. We have used alpha-tocopherol loaded rat liver microsomes to study the effect of this vitamin on CCl4 metabolism in vitro. As expected, alpha-tocopherol inhibits CCl4-dependent microsomal lipid peroxidation and, at a very high concentration, will inhibit the covalent binding of CCl3 to microsomal protein by up to 50%. No inhibitory effect was observed towards CCl3 production as measured by the electron spin resonance technique of spin-trapping but this apparent discrepancy may represent a limitation of the technique. The high levels required to inhibit covalent binding probably preclude the likelihood of alpha-tocopherol significantly affecting that phenomenon at endogenous concentrations but may be relevant to other experiments employing high doses of alpha-tocopherol as an experimental hepatoprotective agent.

Measurements of the absolute rate constants for the reaction with peroxyl radicals of alpha, beta, gamma and delta-tocopherol and several model compounds are described. The peroxyl radicals were obtained either by the autoxidation of styrene or by the flash photolysis of di-t-butyl ketone in an oxygen-saturated environment. The kinetic data are discussed in stereoelectronic terms. Vitamin E and total lipid-soluble, chain-breaking antioxidant concentrations in some normal and cancerous tissues have been measured. In human blood plasma and erythrocyte ghost membranes vitamin E is the major, and possibly the only, chain-breaking antioxidant. Lipid extracts of Novikoff ascites hepatoma cells contain considerably more vitamin E relative to lipid than do extracts of normal rat liver. These tumour lipids contain relatively fewer highly unsaturated fatty acids and are present at lower lipid/wet tissue ratios than the normal liver lipids. A number of unresolved problems relating to the action of vitamin E in vivo are discussed.

Hydroxyl radicals (OH.), generated by a phosphate-buffered Cu2+/H2O2 system, were detected by lucigenin-amplified chemiluminescence, deoxyribose degradation and benzoate hydroxylation. In each system the buffer, Hepes, was found to stimulate radical generation significantly. There are two main reasons for this effect: Hepes increases Cu2+ solubility in phosphate-buffered systems, and forms a complex with Cu2+ that is effective in generating OH. from H2O2. Pipes, a structurally similar buffer, and histidine, a known Cu2+ chelator, were found to have a similar effect. These data suggest that the crucial factor in such free-radical-generating systems is the availability of Cu2+, and that these actions of Hepes should be considered in the design of studies utilizing such systems.

Oxygen-derived free radicals have been implicated as contributors to inflammatory disorders and it has been suggested that certain anti-inflammatory drugs act by scavenging free radicals. In this paper we have studied the free radical scavenging activity of two such experimental anti-inflammatory drugs MK-447 and ONO-3144. Using the technique of pulse radiolysis we have been able to obtain rate constants for the reactions of these compounds with specific free radicals including OH and O2-. We have also investigated the antioxidant capacity of these compounds using rat liver microsomal lipid peroxidation systems. It is suggested that this approach yielding quantitative data concerning defined free radical species will lead to a better understanding of the role of radical scavenging in anti-inflammatory activity.

The effects of promethazine (PM) on different aspects of the hepatotoxic action of CCl4 in the rat were investigated with the objective of finding rapid and reliable indicators of hepatoprotective effects. The study was based on definitive histological assessment of liver damage caused by CCl4 in the presence and absence of PM: PM (78 mumol kg-1, i.p.) protected against CCl4-induced hepatic necrosis 24 h after a low dose of CCl4 (1.3 mmol kg-1) but not against a higher dose (13.0 mmol kg-1). The large increases in plasma activities of GOT, GPT and LDH produced by dosing with CCl4 were partially inhibited by the administration of PM. PM and CCl4 caused a synergistic and long-lasting decrease in body temperature (2-3 degrees C for 8-10 h). Modifying the toxicity with PM, together with a low dose of CCl4, helped to minimize secondary effects of CCl4, to clarify the sequence of toxic events, and to assess the sensitivity of some standard tests of hepatotoxicity. Simultaneous measurement of over 20 commonly used biochemical screening tests in individual animals 3 or 6 h after treatment permitted direct correlation of a wide variety of concentrations, activities and effects. For example, liver CHCl3 concentrations (as a measure of CCl4 metabolism) correlate strongly with increases in diene conjugation of microsomal lipids (as a measure of CCl4-induced lipid peroxidation); malonaldehyde production appears to be less sensitive as a measure of lipid peroxidation in vivo than diene conjugation. The changes induced in each parameter and the correlations between them are discussed with reference to the overall nature of the hepatotoxic reaction and its modification by PM.

A study has been made of the factors that contribute to the decreased rates of lipid peroxidation under different pro-oxidant conditions in intact Novikoff tumour cells, and in microsomal suspensions prepared from Novikoff tumour cells, compared with isolated normal rat hepatocytes and microsomal suspensions prepared from normal rat liver. The pro-oxidant conditions were the addition of either NADPH, NADPH + ADP + iron, NADPH + CCl4 or ascorbate+iron to the experimental systems used, or exposure to gamma-radiation. Contributory factors to the lower rates of lipid peroxidation observed include: a significant decrease in the polyunsaturated fatty acid content of Novikoff cells or Novikoff microsomes; the decreases are especially marked for the C20:4 and C22:6 fatty acids; a very marked reduction in NADPH-cytochrome c reductase; and no detectable content of cytochrome P-450. Another, and in our opinion critical, contribution to the diminished rate of lipid peroxidation in the tumour material is the substantial increase in alpha-tocopherol relative both to total lipid and to methylene-interrupted double bonds in fatty acids. Moreover, the alpha-tocopherol is the major contributor to lipid-soluble chain-breaking antioxidant in lipid extracts of normal liver and of Novikoff tumour material.

Promethazine is known to have protective activity in relation to CCl4-induced liver necrosis. This hepatoprotective property has been investigated with regard to the free radical scavenging and antioxidant properties of promethazine using isolated hepatocytes and microsomal suspensions. CCl4 is activated in both systems to free radical metabolites that bind covalently to lipid and protein, and initiate lipid peroxidation. A large number of carbonyl products is produced during CCl4-induced lipid peroxidation; promethazine strongly inhibits the production of all classes of carbonyl compounds in both microsomal suspensions and isolated hepatocytes. In contrast, promethazine is a very weak inhibitor of the covalent binding of metabolites of CCl4. We conclude that promethazine acts by scavenging the trichloromethylperoxyl radical and lipid peroxyl radicals, and is a weak scavenger of the trichloromethyl radical. These data, when considered together with the hepatoprotective effects of promethazine, suggest that lipid peroxidation is of relatively more importance than covalent binding in the pathogenesis of CCl4-induced liver necrosis.

Rats entrained to a strictly regulated lighting and feeding schedule have been subjected to partial hepatectomy or a sham operation. In the partially hepatectomised animals the period of liver regeneration is characterised by regular bursts of thymidine kinase activity. Liver microsomes from rats, at times corresponding to maximum thymidine kinase activity, have much reduced rates of lipid peroxidation compared to control preparations: this is due in part to increased levels of lipid-soluble antioxidant at times of maximal DNA synthesis. This temporal relationship between thymidine kinase and lipid peroxidation is consistent with the view that lipid peroxidation is decreased prior to cell division.

Activities of various components of microsomal suspensions obtained from a considerable number of rat tissues have been studied. The tissue contents reported include NAD(P)H-cytochrome c reductases, NAD(P)H-ferricyanide reductases, cytochrome b5 and P-450, lipid peroxidation and covalent binding of 14CCl4. The reductase activities were found to be widely distributed, and NADH-liinked activities were generally much higher than the corresponding NADPH-dependent activities. NADPH-cytochrome c reductase was most active in liver, adrenal and small intestinal mucosa. Cytochrome b5 was also present in most tissues in easily detectable amounts, but the content of cytochrome P-450 was very low in most tissues with the exception of liver, adrenal and kidney. Values for cytochrome P-450 in lung, spleen, brain, testis, lactating mammary gland are also reported. NADPH-ADP/Fe2+-linked lipid peroxidation showed a rather similar distribution to cytochrome P-450, being most active in liver with very low activities in tissues like lung and spleen. CCl4-stimulated lipid peroxidation was detectable in liver and kidney but not in other tissues to any significant extent; spin trapping of the trichloromethyl free radical and covalent binding from 14CCl4 was mainly associated with liver microsomes, with less than 5% activity relative to liver being observed in other tissues. The very low rate of lipid peroxidation in extrahepatic microsomes is discussed in terms of the fatty acid contents of the suspensions, and of antioxidant status. The activation of CCl4 by the NADPH-cytochrome P-450 chain does not appear to be a simple function of the activities of the NADPH-flavoprotein, cytochrome P-450, or relative content of polyunsaturated fatty acids.

Carbonyl products were separated and identified in suspensions of rat liver microsomal fractions and in isolated hepatocytes, after stimulation of lipid peroxidation by incubation with the pro-oxidants CCl4 and ADP-iron. The carbonyl products were allowed to react with 2,4-dinitrophenylhydrazine, and the derivatives were extracted and separated by t.l.c. into three zones of non-polar materials, and one fraction of polar derivatives that remained at the origin. Separation of the individual non-polar hydrazones in each zone by h.p.l.c. demonstrated that zone I prepared from microsomal fraction or hepatocytes incubated with CCl4 or ADP-iron contained mainly 4-hydroxyhex-2-enal, 4-hydroxynon-2-enal and 4-hydroxynona-2,5-dienal. Zone III consisted mainly of the alkanals propanal, pentanal and hexanal, the 2-alkenals propenal, pent-2-enal, hex-2-enal, hept-2-enal, oct-2-enal and non-2-enal, the ketones butanone, pentan-2-one and pentan-3-one, and deca-2,4-dienal. Incubation of a microsomal fraction with ADP-iron was much more effective in producing malonaldehyde and other carbonyl products than an incubation with CCl4. Despite such quantitative differences, there were no obvious qualitative differences in the h.p.l.c. spectra obtained from zones I and III. However, the stoichiometric evaluation of fatty acid loss and the production of malonaldehyde and other carbonyls suggests that the pathways of lipid peroxidation triggered by CCl4 and ADP-iron are different. The accumulation of carbonyl products of lipid peroxidation in isolated hepatocytes is strongly affected by their metabolism; in particular, 4-hydroxyalkenals were found to be metabolized very rapidly. Nonetheless, both CCl4 and ADP-iron produced stimulation in the production of malonaldehyde and non-polar carbonyl production. After incubation of rat hepatocytes with CCl4 or ADP-iron it was found that approx. 50% of the total amount of non-polar carbonyls produced during incubation escaped into the external medium. This was not leakage from dead cells, as 90-95% of the hepatocytes had retained their integrity at the end of the incubation. Release of carbonyl products from cells stimulated to undergo lipid peroxidation may be a mechanism for spreading an initial intracellular disturbance to affect critical targets outside the parent cell.

The effects of cobaltic protoporphyrin IX (CPP) administration on hepatic microsomal drug metabolism, carbon tetrachloride activation and lipid peroxidation have been investigated using male Wistar rats. CPP (125 mumol/kg, 72 h before sacrifice) profoundly decreased the levels of hepatic microsomal heme, particularly cytochrome P-450. Consequently, the associated mixed-function oxidase systems were equally strongly depressed. An unexpected finding was that CPP administration also greatly decreased the activity of NADPH/cytochrome c reductase, a result not generally found with the administration of the more widely used cytochrome P-450 depleting agents, cobaltous chloride. Activation of carbon tetrachloride, measured as covalent binding of [14C] CCl4, spin-trapping of CCl3 and CCl4-stimulated lipid peroxidation, was much lower in liver microsomes from CPP-treated rats. Other microsomal lipid peroxidation systems, utilising cumene hydroperoxide or NADPH/ADP-Fe2+, were also depressed in parallel with the decrease in microsomal enzyme activities.

The constitutive and inducible cytosolic glutathione S-transferase (EC 2.5.1.18) subunit compositions of parenchymal cells (hepatocytes) and biliary epithelial cells (BEC) from rat liver have been quantitatively analysed using reverse-phase h.p.l.c. Hepatocytes, analysed in the absence of non-parenchymal cells, expressed constitutively the following subunits, in order of their concentration: 3, 4, 2, 1a, 1b, 8, 6 and 10. BEC express constitutively only four of the GST subunits expressed by hepatocytes and these are, in order of their concentration: subunits 2, 7, 4 and 3. Notable differences from hepatocytes are that BEC completely lack the Alpha-class subunits 1a and 1b that are major subunits in hepatocytes, Mu-class subunits make up a very low proportion of the total, and the Pi-class subunit 7 is a major subunit in BEC, whereas it is essentially absent from hepatocytes. For the first time, the effects of the inducing agents phenobarbitone (PB), beta-naphthoflavone (beta-NF) and ethoxyquin (EQ) have been characterized in a comprehensive and quantitative manner in both cell types. PB, beta-NF and EQ increased total GST protein in hepatocytes by approx. 2-fold, 3-fold and 4-fold respectively. Subunits significantly induced in hepatocytes were (in order of fold-induction): by PB, 1b > 8 > 3 > 2 > 4; by beta-NF, 1b > 8 > 2 > 3 > 4; and by EQ, 7 > 1b > 10 > 8 > 3 > 2 > 1a > 4. In BEC, neither PB nor beta-NF had significant effects on the total amount of GST protein, although PB did significantly induce subunit 3 at the expense of other subunits. EQ increased total GST protein nearly 5-fold in BEC, subunits 7 and 3 being induced dramatically above constitutive levels.