Is there a significant role for lipid peroxidation in the causation of malignancy and for antioxidants in cancer prevention?

alpha-Tocopherol (alpha-T) uptake and its relationship to cell proliferation and lipid peroxidation was studied in a baby hamster kidney cell line (BHK-21/C13) and its polyoma virus-transformed malignant counterpart (BHK-21/PyY cells). The principal findings were as follows. (a) The level of lipid peroxidation judged by malondialdehyde (MDA) measurement by HPLC, was higher in the transformed cells than in the nontransformed cells. Oxidative stress by 374 mM Fe3+/10 mM ADP caused a significant increase in the level of MDA of a similar magnitude in both cell types. Addition of 7, 14, and 21 mM alpha-T caused no diminution of the MDA level in the unstressed cells and abolished the increase in MDA seen in the stressed cells. (b) The endogenous level of alpha-T in the transformed cells was lower than in the nontransformed cells and all of the measurable alpha-T in these cells was destroyed by the oxidative stress. Supplementation of the cells with alpha-T caused an increase in the level of alpha-T that was proportional to the level of inclusion of alpha-T in the medium. (c) Growth was stimulated by 7 and 14 mM alpha-T but not by the higher levels of inclusion in the medium. The growth stimulation was much larger in the transformed cells (163% of growth in the unsupplemented medium) than in the nontransformed cells (120%). (d) These results demonstrate that, in this cell system, the growth-stimulating ability of alpha-T is unrelated to the ability of alpha-T to control lipid peroxidation and that the level of peroxidation is increased in the malignant state. The difference between the findings reported here and earlier work showing increased levels of alpha-T and decreased levels of peroxidation in transformed malignant cells is discussed and possible explanations for it are advanced.

Effects of N-methyl hexanoylhydroxamic acid (NMHH) and myoglobin on endothelial damage by hydrogen peroxide.

OBJECTIVE: The aim was to investigate the interaction of the novel antioxidant N-methyl hexanoylhydroxamic acid (NMHH) with myoglobin in protecting endothelial cells against H2O2 mediated damage. METHODS: Cultured bovine aortic endothelial cells were exposed to 50-100 microM H2O2 for 10-60 min with and without NMHH and/or myoglobin, and immediate or delayed damage was assessed by lactate dehydrogenase release, 3H adenine uptake, a tetrazolium reduction assay, and microscopy. RESULTS: Brief exposure to low concentrations of H2O2 caused cell damage, for which the tetrazolium reduction assay was the most sensitive assay, and inhibited subsequent cell division. NMHH in concentrations from 50 to 200 microM protected against damage provided it was present at the time of adding H2O2, and the effect was markedly potentiated by 10 microM oxymyoglobin, which had little protective effect alone. CONCLUSIONS: NMHH is an effective antioxidant which is markedly potentiated by low concentrations of oxymyoglobin. Oxymyoglobin may potentiate NMHH by scavenging H2O2 through the rapid formation of ferrylmyoglobin, which is then reduced by NMHH. This synergism may be particularly relevant to the protection of myoglobin-rich cells such as myocytes.

The identification of flavonoids as glycosides in human plasma.

This study describes evidence for the absorption of flavonoids and their presence in human plasma in the glycosylated form by HPLC analysis with photodiode array detection. Rutin and other quercetin glycosides, phloridzin, as well as an anthocyanin are detected simultaneously. In addition, a compound eluting with the spectral properties of the aurone family is identified. The results reveal that phloretin and quercetin are absorbed from the diet as glycosides. The polyphenols are detected in plasma from non-supplemented humans at individual levels in the range 0.5-1.6 microM.

Current status of antioxidant therapy.

There is evidence that free radical damage contributes to the aetiology of many chronic health problems such as emphysema, cardiovascular and inflammatory diseases, cataracts, and cancer. In this review we are not concerned with tissue damage in vivo induced directly by radicals from exogenous sources, such as air pollutants and tobacco smoke, high-pressure oxygen, irradiation, or through the metabolism of certain solvents, drugs, and pesticides. Rather, we address some of the disease states associated with increased oxidative stress from endogenous sources and the possible therapeutic advantage of the antioxidant treatment. This raises the question of the antioxidant status of individuals and its role in protection against amplification of certain disease processes. We have chosen to concentrate mainly on coronary heart disease, reperfusion injury, and organ storage for transplantation.

The effects of pH on the oxidation of low-density lipoprotein by copper and metmyoglobin are different.

The amplification of low-density lipoprotein (LDL) peroxidation in vitro by copper and myoglobin are well-studied biochemical approaches for investigating the oxidative modification of LDL and its role in the pathogenesis of atherosclerosis. Since the acidity of the environment is increased in inflammatory sites, the aim of this study was to investigate the effects of acidic pH on the oxidisability of LDL mediated by the haem protein myoglobin in comparison with that of copper-mediated LDL oxidation. The results show that acidic pH enhances myoglobin-mediated LDL oxidation as measured by conjugated dienes, lipid hydroperoxides and electrophoretic mobility, whilst a retardation is observed with copper as pro-oxidant; the mechanism probably relates to the effects of pH on the decomposition and formation of lipid hydroperoxides and the relative influences of copper ions and of myoglobin under these conditions.

Structure-antioxidant activity relationships of flavonoids and phenolic acids.

The recent explosion of interest in the bioactivity of the flavonoids of higher plants is due, at least in part, to the potential health benefits of these polyphenolic components of major dietary constituents. This review article discusses the biological properties of the flavonoids and focuses on the relationship between their antioxidant activity, as hydrogen donating free radical scavengers, and their chemical structures. This culminates in a proposed hierarchy of antioxidant activity in the aqueous phase. The cumulative findings concerning structure-antioxidant activity relationships in the lipophilic phase derive from studies on fatty acids, liposomes, and low-density lipoproteins; the factors underlying the influence of the different classes of polyphenols in enhancing their resistance to oxidation are discussed and support the contention that the partition coefficients of the flavonoids as well as their rates of reaction with the relevant radicals define the antioxidant activities in the lipophilic phase.

Urinary excretion of hydroxycinnamates and flavonoids after oral and intravenous administration.

The urinary recoveries of the hydroxycinnamates, ferulic acid (3-methoxy, 4-hydroxy cinnamic acid), and chlorogenic acid (the quinic acid ester of 3,4-dihydroxycinnamic acid), and three structurally related flavonoids were studied in the rat. For the latter, the aglycone quercetin was compared with its 3-glucoside (isoquercitrin) and 3-rhamnoglucoside (rutin). Doses of 50 mg/kg were administered via the oral and intravenous routes and urine collected over the subsequent 24-h period. Reverse phase HPLC with photo-diode array detection was used to analyze the unchanged compound and their metabolites excreted in the urine. Ferulic acid and isoquercitrin were orally absorbed (5.4 and 0.48% of administered dose, respectively) and are therefore bioavailable. In contrast, neither unchanged chlorogenic acid, rutin, quercetin, nor the conjugated metabolites in the form of glucuronide or sulphate were detected in the urine after oral dosing. All the flavonoids studied produced low total urinary recoveries after intravenous administration, 9.2% for quercetin-3-rhamnoglucoside, 6.7% for the 3-glucoside, and 2.4% for the aglycone, indicating that extensive metabolism to low molecular weight compounds or excretion via other routes may be occurring. Overall it can be stated that renal excretion is not a major pathway of elimination for intact flavonoids and hydroxycinnamates in the rat.

Phenolic antioxidants attenuate neuronal cell death following uptake of oxidized low-density lipoprotein.

Oxidative stress is implicated in neuronal loss associated with neurodegeneration such as in Parkinson's disease, Alzheimer's disease and age-related cognitive decline. Recent reports indicate that the consumption of flavonoid-rich fruits partly reverses the age-related neuronal and cognitive decline. In this study, cultured striatal neurons were exposed to oxidized lipids in the form of low-density lipoprotein (oxLDL) as a model for the induction of oxidative injury, and the abilities of phenolic antioxidants, flavonoids and hydroxycinnamic acid derivatives, to attenuate this neuronal damage were examined. OxLDL was demonstrated to enter neuronal cells and to be capable of eliciting neurotoxicity in a dose- and time-dependent manner, inducing DNA fragmentation and cell lysis. Flavonoids exert protective effects, which appear to be related to specific structural characteristics, particularly relevant being those defining their reduction potentials and partition coefficients. In summary, these data suggest a possible role for flavonoids in reducing neurodegeneration associated with chronic disorders in which oxidative stress is implicated.

Antioxidant activities of carotenes and xanthophylls.

The purpose of this study was to assess the relative antioxidant activities of a range of carotenes and xanthophylls through the extent of their abilities to scavenge the ABTS(.+) radical cation. The results show that the relative abilities of the carotenoids to scavenge the ABTS(.+) radical cation are influenced by the presence of functional groups with increasing polarities, such as carbonyl and hydroxyl groups, in the terminal rings, as well as by the number of conjugated double bonds.

Bilirubin and ascorbate antioxidant activity in neonatal plasma.

Extremely low birth weight premature infants have been known for many years to have limited antioxidant protective capacity, especially with reference to those antioxidant components which do not cross the placenta until the third trimester of gestation. In this study the total antioxidant activity and the concentrations of individual antioxidants in plasma from premature neonates (27 +/- 2 weeks gestation) compared to term babies (38-41 weeks gestation) have been examined. The results show elevated levels of ascorbate at birth in the plasma of premature neonates compared with those of term babies, but the total plasma antioxidant status of the premature babies is significantly lower than that of term babies. At 5 days post-partum the ascorbate levels are within the normal adult range and plasma bilirubin levels are considerably enhanced in both groups, while the total plasma antioxidant status of the premature neonates has increased. Analysis of the relationship between the total plasma antioxidant activity and the bilirubin concentration show a direct, highly significant correlation for the term group, r2 = 0.774, consistent with significance of bilirubin as a plasma antioxidant.

Inhibition of peroxynitrite dependent tyrosine nitration by hydroxycinnamates: nitration or electron donation?

Peroxynitrite is a cytotoxic species generated by the reaction between superoxide and nitric oxide. In this study the ability of hydroxycinnamate antioxidants to decrease peroxynitrite-mediated nitration of tyrosine was investigated. The results obtained show that all compounds were able to inhibit nitration of tyrosine. The potency of inhibitory activity was in the order; caffeic acid > or = chlorogenic acid > or = ferulic acid > p-coumaric acid > ocoumaric acid > m-coumaric acid. Trolox, which was included in the study for comparative purposes, had an activity between that of ferulic acid and p-coumaric acid. The data obtained suggest that hydroxycinnamates can act by one of two possible mechanisms: preferential nitration for monophenolates and electron donation by catecholates.

Novel biomarkers of the metabolism of caffeic acid derivatives in vivo.

The purpose of this study was to investigate biomarkers of the bioavailability and metabolism of hydroxycinnamate derivatives through the determination of the pharmacokinetics of their urinary elimination and identification of the metabolites excreted. Coffee was used as a rich source of caffeic acid derivatives and human supplementation was undertaken. The results show a highly significant increase in the excretion of ferulic, isoferulic, dihydroferulic acid (3-(4-hydroxy-3-methoxyphenyl)-propionic acid), and vanillic acid postsupplementation relative to the levels presupplementation. Thus, ferulic, isoferulic, and dihydroferulic acids are specific biomarkers for the bioavailability and metabolism of dietary caffeic acid esters. Isoferulic acid is a unique biomarker as it is not a dietary component, however, dihydroferulic acid may well derive from other flavonoids with a structurally related B-ring. 3-Hydroxyhippuric acid has also been identified as an indicator for bioavailability and metabolism of phenolic compounds, and shows a highly significant excretion increase postsupplementation. The results reveal isoferulic acid (and possibly dihydroferulic acid) as novel markers of caffeoyl quinic acid metabolism.

Total antioxidant activity of low density lipoproteins and the relationship with alpha-tocopherol status.

A rapid method is described for measuring the antioxidant activity of low density lipoproteins. Studies were undertaken on individuals attending a hyperlipidaemia clinic, an unsupplemented group and a group after supplementation with 300 mg dl-alpha-tocopherol acetate for nine weeks. The results show a positive correlation between the antioxidant activity and alpha-tocopherol content of LDL in the supplemented group.

Singlet oxygen quenching and the redox properties of hydroxycinnamic acids.

The singlet oxygen quenching rate constants (kq) for a range of hydroxycinnamic acids in acetonitrile and D2O solutions were measured using time resolved near infrared phosphorescence in order to establish their antioxidant activity. The magnitude of kq observed depends on both the nature of the substituent groups and solvent polarity. The variations in kq depend on the energy of the hydroxycinnamic acid/molecular oxygen charge transfer states, (O2delta- ...HCAdelta+). In D2O the values of kq range from 4x10(7) M(-1) s(-1) to 4x10(6) M(-1) s(-1) for caffeic acid and o-coumaric acid respectively. In acetonitrile, the charge transfer energy levels are raised and this is reflected in lower singlet oxygen quenching rate constants with a kq value of 5x10(6) M(-1) s(-1) for caffeic acid. The phenoxyl radical spectra derived from the hydroxycinnamic acids were determined using pulse radiolysis of aqueous solutions and the reduction potentials were found to range from 534 to 596 mV. A linear correlation is observed between reduction potential, and hence free energy for electron transfer, and log kq. These correlations suggest a charge transfer mechanism for the quenching of singlet oxygen by the hydroxycinnamic acids.

The influence of antioxidant nutrients on platelet function in healthy volunteers.

There is mounting evidence that antioxidants may help to prevent coronary heart disease and modulate some thrombotic events such a platelet adhesion. However, the effects of antioxidant supplementation on platelet function in vivo are controversial. A double-blind, randomised, placebo-controlled study was performed on 40 healthy volunteers (20-50 years) supplemented daily with vitamin E (300 mg), vitamin C (250 mg) or beta-carotene (15 mg) for 8 weeks. Platelet function was assessed by platelet aggregation induced by ADP, arachidonic acid or collagen, platelet responsiveness to the inhibitor PGE1, beta-thromboglobulin release and ATP secretion. Supplementation with vitamin E resulted in a significant increase in platelet alpha-tocopherol level (+68%) reflecting closely the increase in plasma alpha-tocopherol level (+69%). Platelet function was significantly decreased by vitamin E as revealed by the decreased platelet aggregation in response to ADP and arachidonic acid, the increased sensitivity to inhibition by PGE1, the decreased plasma beta-thromboglobulin concentration and the decreased ATP secretion. Supplementation with vitamin C did not affect platelet function significantly although a trend towards a decreased platelet aggregability and an increased sensitivity to the inhibitor PGE1 were observed. No significant changes in platelet function occurred after supplementation with beta-carotene. In conclusion, supplementation of healthy volunteers with vitamin E decreased platelet function whereas supplementation with vitamin C or beta-carotene had no significant effects.

Tea flavonoids and cardiovascular health.

Tea is rich in antioxidant polyphenols (catechins, flavonols, theaflavins and thearubigins). Epidemiological evidence relating regular consumption of tea or related polyphenols to CHD is equivocal. Catechins are absorbed from tea, but low plasma concentrations are attained. The bioavailability of theaflavins and thearubigins is unknown. Tea does not reduce blood pressure or plasma lipids in well-controlled human trials. Tea polyphenols inhibit LDL lipid peroxidation in vitro, but the effect ex vivo is small. The plasma antioxidant potential increases after drinking green but not black tea. Tea consumption tended to reduce the development of aortic atherosclerosis in rabbits. Tea polyphenols exert marked effects on cells, and inhibit neutrophil migration and inflammatory responses, sometimes at low concentrations. These diverging results suggest potential beneficial effects, but emphasize the need for good human trials of tea using early markers of CHD before firm conclusions can be drawn.

Measurement of total antioxidant activity as a marker of antioxidant status in vivo: procedures and limitations.

The ability to apply a simple routine assay to report on the total antioxidant activity of plasma/serum and other body fluids and their responses to dietary intervention and nutritional supplementation is an attractive useful proposition. This article will attempt to address where we are, in the author's view, in this context, setting out the pros and cons of the available methodologies en route.

Factors influencing the antioxidant activity determined by the ABTS.+ radical cation assay.

This study introduces a simple direct antioxidant assay, based on the reduction of the ABTS.+ radical cation, and compares it with the myoglobin/ABTS.+ assay. The methods give closely similar results, establishing that the antioxidants studied to date in the latter assay act by scavenging the ABTS.+ radical cation and not by inhibiting its formation through reduction of ferryl myoglobin or reaction with H2O2.

The effect of the phenolic antioxidant ferulic acid on the oxidation of low density lipoprotein depends on the pro-oxidant used.

The action of ferulic acid during the oxidation of LDL has been investigated using both copper ions and the haem protein metmyoglobin as pro-oxidants. The results demonstrate the ability of ferulic acid to act as a pro-oxidant when LDL oxidation is induced by copper at concentrations of the phenolic acid which are protective when the LDL oxidation is mediated by metmyoglobin. The suggested mechanism involves the reduction of Cu2+ to Cu+ by ferulic acid resulting in the production of the ferulic phenoxyl radical.

The polyphenolic content of fruit and vegetables and their antioxidant activities. What does a serving constitute?

Analysis of the major flavone, flavonol, anthocyanidin and hydroxycinnamic acid constituents (and their glycosides) of onion, tomato, egg plant and apple has been undertaken and the antioxidant activities of the phenolic extracts determined. The major phenolic antioxidant components of egg plant are chlorogenic acid in the flesh and a delphinidin conjugate in the skin. In the case of apple, the major phenolic antioxidants detected are chlorogenic acid, procyanidins/catechin compounds, rutin and phloridzin. Quercetin glycosides are well-known to be the major phenolic components of onion. Assessment of the antioxidant activities of a serving of 100g fresh weight fruit, vegetable and comparison with previously reported findings for 150 ml beverage (500 ml portion in the case of beer), expressed in micromol Trolox equivalents show that the antioxidant activities of 1 glass (150 ml) red wine equivalent to 12 glasses white wine equivalent to 2 cups of tea equivalent to 4 apples equivalent to 5 portions of onion equivalent to 5.5 portions egg plant equivalent to 3.5 glasses of blackcurrant juice equivalent to 3.5 (500 ml) glasses of beer equivalent to 7 glasses of orange juice equivalent to 20 glasses of apple juice (long life).