J.P. Kamat

Caffeine as an antioxidant: inhibition of lipid peroxidation induced by reactive oxygen species

Caffeine (1,3,7-trimethyl xanthine), an ingredient of coffee, has been investigated for its potential antioxidant activity against oxidative damage to rat liver microsomes. Such damage was induced by three reactive oxygen species of cardinal importance in causing membrane damage in vivo namely hydroxyl radical (.OH), peroxyl radical (ROO.) and singlet oxygen (1O2). The results obtained showed that caffeine was an effective inhibitor of lipid peroxidation, at millimolar concentrations, against all the three reactive species. The extent of inhibition was high against peroxidation induced by .OH, medium against 1O2 and low against ROO. In general, the antioxidant ability of caffeine was similar to that of the established biological antioxidant glutathione and significantly higher than ascorbic acid. Investigations into the possible mechanisms involved in the observed antioxidant effect reveal that the quenching of these reactive species by caffeine may be one of the possible factor responsible. The rate constant of caffeine with .OH was 7.3 x 10(9) M-1 s-1 and with 1O2 it was 2.9 x 10(7) M-1 s-1. Considering their potential for damage, half-life estimates and generation in biological systems, the ability of caffeine to inhibit oxidative damage induced by these reactive species in membranes suggest one more positive attribute of caffeine, whose daily intake as coffee may be considerable in most populations.

Reactive oxygen species mediated membrane damage induced by fullerene derivatives and its possible biological implications.

Fullerenes have attracted considerable attention in recent years due to their unique chemical structure and potential applications. Hence it is of interest to study their biological effects. Using rat liver microsomes as model systems we have examined the ability of the most commonly used fullerene, C60 and its water-soluble derivative, C60(OH)18 to induce membrane damage on photosensitization. For photoexcitation, UV or tungsten lamps were used. Damage was assessed as lipid peroxidation products like conjugated dienes, lipid hydroperoxides and thiobarbituric acid reactive substances (TBARS). protein oxidation in the form of protein carbonyls, besides loss of membrane bound enzymes. Both fullerene derivatives induced significant oxidative damage. The alterations induced were both time- and concentration-dependent. Role of different reactive oxygen species (ROS) in the damage induced was examined by various scavengers of ROS and by deuteration of the buffer. The changes induced by C60 were predominantly due to 1O2 while that by C60(OH)18 was mainly due to radical species. Biological antioxidants such as glutathione, ascorbic acid and alpha-tocopherol were capable of inhibiting membrane damage induced by both the fullerenes. However, the damage induced by C60(OH)18 was more for both lipids and proteins than that showed by C60. C60 also showed enhancement in the formation of lipid peroxidation in sarcoma 180 ascites microsomes. In conclusion, our studies indicate that fullerene/its derivative can generate ROS on photoexcitation and can induce significant lipid peroxidation/protein oxidation in membranes and these phenomena can be prevented by endogenous/natural antioxidants.

Antioxidant properties of Asparagus racemosus against damage induced by γ-radiation in rat liver mitochondria

The possible antioxidant effects of crude extract and a purified aqueous fraction of Asparagus racemosus against membrane damage induced by the free radicals generated during gamma-radiation were examined in rat liver mitochondria. gamma-Radiation, in the dose range of 75-900 Gy, induced lipid peroxidation as assessed by the formation of thiobarbituric acid reactive substances (TBARS) and lipid hydroperoxides (LOOH). Using an effective dose of 450 Gy, antioxidant effects of A. racemosus extract were studied against oxidative damage in terms of protection against lipid peroxidation, protein oxidation, depletion of protein thiols and the levels of the antioxidant enzyme, superoxide dismutase. An active fraction consisting of polysaccharides (termed as P3) was effective even at a low concentration of 10 microg/ml. Both the crude extract as well as the P3 fraction significantly inhibited lipid peroxidation and protein oxidation. The antioxidant effect of P3 fraction was more pronounced against lipid peroxidation, as assessed by TBARS formation, while that of the crude extract was more effective in inhibiting protein oxidation. Both the crude extract and P3 fraction also partly protects against radiation-induced loss of protein thiols and inactivation of superoxide dismutase. The inhibitory effects of these active principles, at the concentration of 10 microg/ml, are comparable to that of the established antioxidants glutathione and ascorbic acid. Hence our results indicate that extracts from A. racemosus have potent antioxidant properties in vitro in mitochondrial membranes of rat liver.

Tocotrienols from palm oil as effective inhibitors of protein oxidation and lipid peroxidation in rat liver microsomes

Tocotrienols from palm oil showed significant ability to inhibit oxidative damage induced by ascorbate-Fe2+ and photosensitization, involving different mechanisms, in rat liver microsomes. The tocotrienol-rich fraction from palm oil (TRF), being tried as a more economical and efficient substitute for α-tocopherol, showed time- and concentration-dependent inhibition of protein oxidation as well as lipid peroxidation. It was more effective against protein oxidation. The extent of inhibition by TRF varied with different peroxidation products such as conjugated dienes, lipid hydroperoxides and thiobarbituric acid reactive substances (TBARS). Among the constituents of TRF, γ-tocotrienol was the most effective followed by its α- and δ-isomers. In general, at a low concentration of 5 μM, TRF was able to prevent oxidative damage to significant extent (37% inhibition of protein oxidation and 27–30% of lipid peroxidation at 1 h of incubation). The protective ability of TRF (30.1% at 5 μM with TBARS formation) was significantly higher than that of the dominant form of vitamin E, α-tocopherol (16.5% under same conditions). Hence our studies indicate that this fraction from palm oil can be considered as an effective natural antioxidant supplement capable of protecting cellular membranes against oxidative damage.

Chlorophyllin as an effective antioxidant against membrane damage in vitro and ex vivo

Chlorophyllin (CHL), the sodium-copper salt and the water-soluble analogue of the ubiquitous green pigment chlorophyll, has been attributed to have several beneficial properties. Its antioxidant ability, however, has not been examined in detail. Using rat liver mitochondria as model system and various sources for the generation of reactive oxygen species (ROS) we have examined the membrane-protective properties of CHL both under in vitro and ex vivo conditions. Oxidative damage to proteins was assessed as inactivation of the enzymes, cytochrome c oxidase and succinic dehydrogenase besides formation of protein carbonyls. Damage to membrane lipids was measured by formation of lipid hydroperoxides and thiobarbituric acid reactive substances. The effect of this compound on the antioxidant defense system was studied by estimating the level of glutathione and superoxide dismutase. ROS were generated by gamma-radiation, photosensitization, ascorbate-Fe(2+), NADPH-ADP-Fe(3+) and the peroxyl radical generating agent, azobis-amidopropane hydrochloride. Our results show that CHL is highly effective in protecting mitochondria, even at a low concentration of 10 microM. The antioxidant ability, at equimolar concentration, was more than that observed with ascorbic acid, glutathione, mannitol and tert-butanol. When CHL was fed to mice at a dose of 1% in drinking water, there was a significant reduction in the potential for oxidative damage in cell suspensions from liver, brain and testis. To examine the possible mechanisms responsible for the observed antioxidant ability we have studied the reaction of CHL with the potent ROS in the form of hydroxyl radical and singlet oxygen. The compound shows a fairly high rate constant with singlet oxygen, in the order of 1.3x10(8) M(-1) s(-1). In conclusion, our studies showed that CHL is a highly effective antioxidant, capable of protecting mitochondria against oxidative damage induced by various ROS.

Oxidative damage induced by the fullerene C60 on photosensitization in rat liver microsomes

We have examined the ability of a commonly used fullerene, C60, to induce oxidative damage on photosensitization using rat liver microsomes as model membranes. When C60 was incorporated into rat liver microsomes in the form of its cyclodextrin complex and exposed to UV or visible light, it induced significant oxidative damage in terms of (1) lipid peroxidation as assayed by thiobarbituric acid reactive substances (TBARS), lipid hydroperoxides and conjugated dienes, and (2) damage to proteins as assessed by protein carbonyls and loss of the membrane-bound enzymes. The oxidative damage induced was both time- and concentration-dependent. C60 plus light-induced lipid peroxidation was significantly inhibited by the quenchers of singlet oxygen ((1)O2), beta-carotene and sodium azide, and deuteration of the buffer-enhanced peroxidation. These observations indicate that C60 is an efficient inducer of peroxidation and is predominantly due to (1)O2. Biological antioxidants such as glutathione, ascorbic acid and alpha-tocopherol significantly differ in their ability to inhibit peroxidation induced by C60. Our studies, hence, indicate that C60, on photosensitization, can induce significant lipid peroxidation and other forms of oxidative damage in biological membranes and that this phenomenon can be greatly modulated by endogenous antioxidants and scavengers of reactive oxygen species.

Vanillin as an antioxidant in rat liver mitochondria: inhibition of protein oxidation and lipid peroxidation induced by photosensitization.

Using rat liver mitochondria, as model systems, we have examined the ability of the natural compound and the food-flavoring agent, vanillin to protect membranes against oxidative damage induced by photosensitization at concentrations normally used in food preparations. Vanillin, at a concentration of 2.5 mmol/L, has afforded significant protection against protein oxidation and lipid peroxidation in hepatic mitochondria induced by photosensitization with methylene blue plus light. The effect observed was both time- and concentration-dependent. The inhibitory effect is similar to ascorbic acid and the singlet oxygen quencher, diazabicyclo[2.2.2]octane (DABCO) but less effective than sodium azide and glutathione. Examination of possible mechanisms responsible for the observed protection, showed that vanillin has a significant ability to quench singlet oxygen (1O2), a reactive species responsible for damage induced during photosensitization by Type II mechanism. Hence, this flavoring compound, due to its antioxidant ability, may have potential to prevent oxidative damage to membranes in mammalian tissues and thereby the ensuing diseased states.

Nicotinamide (vitamin B3) as an effective antioxidant against oxidative damage in rat brain mitochondria.

Nicotinamide (vitamin B3) an endogenous metabolite, showed significant inhibition of oxidative damage induced by reactive oxygen species (ROS) generated by ascorbate-Fe2+ and photosensitization systems in rat brain mitochondria. It protected against both protein oxidation and lipid peroxidation, at millimolar concentrations. Inhibition was more pronounced against oxidation of proteins than peroxidation of lipids. Chemically related endogenous compounds, tryptophan and isonicotinic acid, showed comparable inhibitory properties. The protective effect observed, at biologically relevant concentrations, with nicotinamide was more than that of the endogenous antioxidants ascorbic acid and alpha-tocopherol. Hence our studies suggest that nicotinamide (vitamin B3) can be considered as a potent antioxidant capable of protecting the cellular membranes in brain, which is highly susceptible to prooxidants, against oxidative damage induced by ROS.

Methylene blue plus light-induced lipid peroxidation in rat liver microsomes: inhibition by nicotinamide (vitamin B3) and other antioxidants

Methylene blue plus visible light, in the presence of oxygen, induced lipid peroxidation in rat liver microsomes, as assessed by the formation of thiobarbituric acid reactive substances (TBARS), lipid hydroperoxides and the loss of membrane-bound enzymes. Peroxidation was enhanced by deuteration of the buffer and inhibited by scavengers of singlet oxygen (1O2) and superoxide (O2.-). The damage induced seemed to be mainly due to Type II involving 1O2 and to a lesser extent Type I reactions with O2.- and hydroxyl radical (.OH) as intermediates. Nicotinamide or vitamin B3, an endogenous metabolite occurring at high concentrations in tissues, had a relatively high rate constant of 1.8 x 108 M-1 s-1 with 102 and had a significant inhibitory effect on lipid peroxidation induced by photosensitization. This effect was both time- and concentration-dependent, high inhibition being associated with millimolar concentrations. Chemically related endogenous compounds like tryptophan and isonicotinic acid also had significant inhibitory properties. Similar protective effects were observed with natural antioxidants such as beta-carotene, canthaxanthin, lipoic acid, glutathione, alpha-tocopherol and to a lesser extent ascorbic acid. Nicotinamide was a more effective antioxidant than ascorbic acid. It also showed a similar inhibitory effect against NADPH-ADP-FE3(+)-induced lipid peroxidation. Our results suggest that nicotinamide had significant ability to protect against photosensitization-induced cytotoxicity and cell damage and that it may do so by its ability to react with 102 and other reactive oxygen species.

Chlorophyllin as a protector of mitochondrial membranes against γ-radiation and photosensitization

Ionizing radiation and photosensitization are highly damaging events and they generate oxygen-derived free radicals as well as excited species. However, the types as well as extent of reactive oxygen species (ROS) differ. They have been linked to various pathological conditions. Hence natural compounds capable of preventing oxidative damage induced by these agents may have potential applications. Chlorophyllin (CHL), the water-soluble analogue of chlorophyll, has been examined for its ability to inhibit membrane damage induced by y-radiation and photosensitization involving methylene blue plus visible light. Using rat liver mitochondria as model systems the mechanisms of damage induced by these two agents as well as its possible prevention by CHL have been examined. The parameters used were lipid peroxidation as assessed by formation of thiobarbituric acid reactive substances (TBARS) and 4-hydroxynonenal (4-HNE), protein oxidation besides glutathione (GSH) and superoxide dismutase (SOD). Peroxidation increases with radiation dose, in the range of 75-600 Gy. A similar observation also was observed with photosensitization, as a function of time. CHL, at a concentration of 10 microM offered a high degree of protection against radiation and photosensitization as indicated by decreased peroxidation, protein oxidation as well as the restoration of GSH and SOD. When compared with the established antioxidants, ascorbic acid and GSH, CHL offered a much higher degree of protection. Pulse radiolysis studies show that this compound has a relatively high rate constant with hydroxyl radical (*OH), a crucial species generated during y-radiation. Hence the studies show that CHL is a potent antioxidant in mitochondrial membranes.