Vitamin E protects against iron-hexachlorobenzene induced porphyria and formation of 8-hydroxydeoxyguanosine in the liver of C57BL/10ScSn mice.

The effect of vitamin E treatment on total porphyrin content, lipid peroxidation (LOOH) and 8-hydroxydeoxyguanosine (8-OHdG) was studied in the livers of C57BL/10ScSn mice following hexachlorobenzene (HCB) and iron treatment. HCB was administered i.p. (totalling 300 mg/kg) twice, with 1 week interval. Three days after the first HCB injection iron-dextran was given i.p. (500 mg Fe per kg). Vitamin E was administered weekly (20 mg/kg) by s.c. injection. Both total hepatic porphyrin and LOOH levels were significantly (P<0.001) increased in the HCB-iron treated group as compared with the control group. Mice treated additionally with vitamin E had significant (P<0.001) lower levels as compared with the HCB-iron group. Similarly, the levels of 8-OHdG were significantly (P<0.001) increased above controls after HCB-iron treatment and this increase was reduced after co-treatment with vitamin E (P<0.02). The data support the hypothesis that the mechanism of hepatic porphyrinogenicity of HCB with iron overload is an oxidative free radical process.

The effect of oxidative stress on the production of the recombinant protein, interferon gamma, produced by Chinese hamster ovary cells in stirred-batch culture.

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.

Free radical reactions involving the angiotensin converting enzyme inhibitor captopril.

Using the pulse radiolysis technique, absolute rate constants have been obtained for the reaction of captopril with several free radicals. The results demonstrate that although captopril reacts rapidly with a number of free radicals, such as the hydroxyl radical (k = 5.1 × 10(9) dm(-3)mol(-1)s(-1)) and the thiocyanate radical anion (k = 1.3 × 10(7) dm(-3)mol(-1)s(-1)), it is not exceptional in this ability. Similarly, the reactions with carbon centred radicals although rapid are an order of magnitude slower than those observed with glutathione. Additional lipid peroxidation studies further demonstrate that captopril is a much less effective antioxidant than glutathione. The data go some way to supporting the view that any attenuation of reperfusion injury by captopril is not through a direct free radical scavenging mechanism but may be afforded by other, non-radical-mediated mechanisms.

Equally efficacious asthma management with budesonide 800 micrograms administered by Turbuhaler or with beclomethasone dipropionate > or = 1500 micrograms given through a pressurized metered-dose inhaler with spacer. The French Budesonide Trial Group.

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.

Depletion of alpha-tocopherol in human atherosclerotic lesions.

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.

Biokinetics in humans of RRR-alpha-tocopherol: the free phenol, acetate ester, and succinate ester forms of vitamin E.

The bioavailability of RRR-alpha-tocopherol from the oral administration of RRR-alpha-tocopherol itself and its acetate and succinate esters was determined in healthy human subjects. Venous blood samples were withdrawn periodically over a 51-h period following oral administration of a gelatin capsule containing an equimolar mixture of RRR-alpha-tocopherol and RRR-alpha-tocopheryl acetate. In a second study, subjects received a capsule containing an equimolar mixture of RRR-alpha-tocopheryl acetate and RRR-alpha-tocopheryl succinate. In Study 1, RRR-alpha-tocopherol was absorbed at similar rates from both the free phenol, and the acetate ester and maximum plasma levels occurred at 12 h in most subjects. The extent of absorption of RRR-alpha-tocopherol varied considerably between subjects in absolute terms, but the relative absorption from the two forms was remarkably consistent, and a ratio of 1.0 was found for parameters of relative bioavailability in plasma. The concentration of RRR-alpha-tocopherol from each form was maximal at approximately 27 h in red blood cells and, as seen with the plasma data, there was a large interindividual variability. In Study 2, there was no significant difference in the extent of absorption of RRR-alpha-tocopherol from the acetate ester and the succinate ester, although there was an apparently higher initial rate of absorption from the acetate ester.

Exposure of beta H-crystallin to hydroxyl radicals enhances the transglutaminase-susceptibility of its existing amine-donor and amine-acceptor sites.

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.

Measurement of n-alkanals and hydroxyalkenals in biological samples.

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-Hydroxynonenal (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.

Distribution of vitamin E between tissues during periods of hyperoxic and nutritional stress in the preterm guinea pig.

1. Using a preterm guinea pig model we examined the impact of oxidative and nutritional stress on the vitamin E status of a number of immature tissues. 2. Liver, brain, lung, red blood cell and plasma alpha-tocopherol concentrations were measured in fed and fasted, preterm guinea pigs exposed to 21 or 95% oxygen for 48 hr. 3. Exposure to hyperoxia did not influence plasma, red blood cell, lung or brain vitamin E status of preterm pups. Liver alpha-tocopherol content was reduced 20% following 48 hr hyperoxic exposure (P < 0.05). 4. Food restriction (48 hr) alone reduced liver (41%) and red blood cell (32%) alpha-tocopherol concentrations in preterm animals while plasma, lung and brain vitamin E levels were unaffected. Combined with hyperoxic exposure, food restriction resulted in a (50%) fall in liver alpha-tocopherol concentration (P < 0.01). 5. The findings of this study suggest that the liver acts as a reservoir for vitamin E in the perinatal period, releasing increased quantities of this lipid soluble antioxidant for use by non-hepatic tissues during periods of hyperoxic and nutrient stress.

An introduction to free radical biochemistry.

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.

Measuring free radical reactions in vivo.

The increasing interest in the role of free radicals in the pathogenesis of human disease has led to an increased need for techniques to measure free radicals and their reactions in vivo and, most importantly, in the clinical situation. Free radicals are extremely reactive and thus short lived. Consequently, free radicals are not amenable to direct assay and free radical activity is usually assessed by indirect methods such as measurement of the various end products of reactions with lipids, proteins and DNA. A vast array of analytical techniques has been developed to measure these end products though not all of them are applicable to the clinical situation where the only samples normally available are blood, urine and expired breath. Lipid peroxidation is the most intensively studied process and provides a number of possibilities for assays. Protein and nucleic acid oxidation are attracting increasing attention at the present time. The techniques currently available, however, are limited to semi-quantitative assays of damage to broad classes of biomolecules and there is an urgent need for more specific and informative methods.

Constitutive and inducible profile of glutathione S-transferase subunits in biliary epithelial cells and hepatocytes isolated from rat liver.

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.

Mechanisms and effects of lipid peroxidation.

Lipid peroxidation is an important mechanism in free radical mediated cell injury. It can damage cell membranes directly and the reactive carbonyl products may spread the damage far from the original site of radical production. It has long been considered to be involved in various toxic tissue injuries and in certain disease processes, including cancer. Paradoxically, cancer cells are very resistant to lipid peroxidation. Recently, it has been suggested that lipid peroxidation may exert more subtle effects than was previously thought possible, by influencing gene expression.

Tissue alpha-tocopherol status during late fetal and early neonatal life of the guinea-pig.

The alpha-tocopherol content of a number of different fetal, neonatal and maternal guinea-pig tissues was determined and compared with plasma and erythrocyte alpha-tocopherol values. During late gestation, the fetal liver appears to act as a storage site for alpha-tocopherol, the majority of which is released immediately following birth. In contrast, lung and brain vitamin E levels are relatively constant over the final period of gestation and during early neonatal life. The ontogeny of alpha-tocopherol in brain and lung was similar to that for erythrocytes while plasma alpha-tocopherol content varied considerably and did not accurately reflect tissue alpha-tocopherol status. Surprisingly, fetal and maternal lung alpha-tocopherol concentrations were similar at all time-points considered, whereas fetal liver alpha-tocopherol status was always considerably greater than maternal liver alpha-tocopherol content. These results, if representative of the human fetus, suggest that preterm infants may not have tissue alpha-tocopherol concentrations as low as previously assumed and that during the perinatal period erythrocyte alpha-tocopherol content is a more accurate indicator of tissue alpha-tocopherol concentration than plasma alpha-tocopherol content.

Exposure of beta L-crystallin to oxidizing free radicals enhances its susceptibility to transglutaminase activity.

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.

The reactivity of the SH group of bovine serum albumin with free radicals.

The reactivity of the SH group of bovine serum albumin (BSA) towards free radicals generated by several different systems including gamma-radiolysis and hydrogen peroxide/metal salt mixtures was investigated. On exposure of BSA (1 mg/ml and 5 mg/ml) to HO. radicals generated radiolytically the protein-SH concentration was found to decrease in a dose-dependent manner. At 40 mg/ml albumin no loss of SH was observed. O2-. and HO2. radicals were much less aggressive towards the SH group. The effect of divalent metal salts (copper or iron) plus hydrogen peroxide was studied separately and in combination. H2O2 alone caused a decrease in SH group concentration the rate of which was not decreased by the presence of desferrioxamine and so was apparently not due to reactions catalysed by adventitious metal ions. Copper alone caused a dose-dependent decrease in SH group concentration and the mixture of the two agents caused a greater loss of SH than each separate component. However, this latter effect was again resistant to the effects of desferrioxamine. The SH group of BSA was only moderately sensitive to the presence of ferrous iron alone and in a system containing both ferrous iron and H2O2 rates of SH oxidation were obtained that were identical to those obtained with H2O2 alone. Desferrioxamine again did not alter the rate of SH oxidation in these experiments. We suggest that the highly reactive free radical HO. is not able to reach and to oxidize the SH group of BSA when generated by metal/H2O2 mixtures, in contrast to HO. generated radiolytically. Less reactive radicals and non-radical species such as H2O2 have more potential for doing so.

Menadione and cumene hydroperoxide induced cytotoxicity in biliary epithelial cells isolated from rat liver.

Biliary epithelial cells (BEC) and parenchymal cells isolated from normal rat liver were exposed in vitro to a number of toxic compounds. BEC were found to be highly sensitive to concentrations of menadione (100 microM) and cumene hydroperoxide (10 microM) that are usually not effective as toxic agents in hepatocytes under normoxic conditions. On the other hand, BEC were not affected by concentrations of carbon tetrachloride or 7-ethoxycoumarin that are known to exert toxic effects on hepatocytes. The development of both menadione- and cumene hydroperoxide-induced toxic injury in BEC followed a common and time-correlated pattern, and included a strong depletion of GSH, depletion of protein thiols and an increase in the extent of cell death. The damage induced by cumene hydroperoxide was found to be independent of lipid peroxidative processes and was prevented by a pre-incubation with desferrioxamine. The cytotoxicity of menadione was further exacerbated by dicoumarol but was not prevented by desferrioxamine or promethazine. The mechanisms underlying BEC injury and death induced by the quinone and by the organic hydroperoxide are discussed in relation to the known biochemical characteristics of BEC.

Comparative evaluation of the antioxidant activity of alpha-tocopherol, alpha-tocopherol polyethylene glycol 1000 succinate and alpha-tocopherol succinate in isolated hepatocytes and liver microsomal suspensions.

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.

Biochemical studies on bile duct epithelial cells isolated from rat liver.

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 gamma-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.