Inactivation of NADP(+)-dependent isocitrate dehydrogenase by reactive oxygen species

Recently, we demonstrated that the control of cytosolic and mitochondrial redox balance and the cellular defense against oxidative damage is one of the primary functions of NADP(+)-dependent isocitrate dehydrogenase (ICDH) through supply of NADPH for antioxidant systems. When exposed to various reactive oxygen species such as hydrogen peroxide, singlet oxygen generated by photoactivated dye, superoxide anion, and hydroxyl radical produced by metal-catalyzed Fenton reactions, ICDH was susceptible to oxidative modification and damage, which was indicated by the loss of activity, fragmentation of the peptide as well as by the formation of carbonyl groups. Oxidative damage to ICDH was inhibited by antioxidant enzymes, free radical scavengers, and spin-trapping agents. The structural alterations of modified enzymes were indicated by the increase in thermal instability and binding of the hydrophobic probe 8-anilino-1-naphthalene sulfonic acid (ANSA). The reactive oxygen species-mediated damage to ICDH may result in the perturbation of cellular antioxidant defense mechanisms and subsequently lead to a pro-oxidant condition.     

The effect of different antioxidants on glutathione turnover in human cell lines and their interaction with hydrogen peroxide

BACKGROUND: Glutathione plays crucial roles in antioxidant defence and glutathione deficiency contributes to oxidative stress and may therefore play a key role in the pathogenesis of many diseases. The objectives of the present study were to evaluate the effects on glutathione turnover of thiol and non-thiol antioxidants in human cell cultures and if any of the antioxidant had a short-term cellular effect against different levels of hydrogen peroxide. METHODS: We have investigated the effect on the total glutathione amount in HeLa and hepatoma cell cultures of thiol antioxidants in comparison with non-thiol antioxidants, such as a copper chelator, Vitamin C, and a flavonoid. Furthermore, we have investigated the short-term (within 24h) interaction of the different antioxidants with hydrogen peroxide. RESULTS AND CONCLUSION: Lipoic acid and quercetin (Quer) were the two antioxidants that showed the highest stimulation of glutathione synthesis in cell cultures as judged by the total glutathione amount. However, no antioxidant protected against hydrogen peroxide present in concentrations that lowered cell protein. This finding may be attributed to the fact that it is necessary to incubate cell cultures with antioxidants or small doses of oxidants for a period before the cultures are exposed to hydrogen peroxide in order to enhance the antioxidant defence. The presence of Quer and Vitamin C lowered cell protein and total glutathione even in cell cultures containing hydrogen peroxide in concentrations that did not lower cell protein. This finding might be attributed to pro-oxidant properties and formation of excess reactive oxygen species in the presence of Quer and Vitamin C.     

Mechanism of vitamin C inhibition of cell death induced by oxidative stress in glutathione-depleted HL-60 cells

Vitamin C is a well known antioxidant whose precise role in protecting cells from oxidative challenge is uncertain. In vitro results have been confounded by pro-oxidant effects of ascorbic acid and an overlapping role of glutathione. We used HL-60 cells as a model to determine the precise and independent role of vitamin C in cellular protection against cell death induced by oxidative stress. HL-60 cells do not depend on glutathione to transport or reduce dehydroascorbic acid. Depletion of glutathione rendered the HL-60 cells highly sensitive to cell death induced by H2O2, an effect that was not mediated by changes in the activities of glutathione reductase, glutathione peroxidase, catalase, or superoxide dismutase. The increased sensitivity to oxidative stress was largely reversed when glutathione-depleted cells were preloaded with ascorbic acid by exposure to dehydroascorbic acid. Resistance to H2O2 treatment in cells loaded with vitamin C was accompanied by intracellular consumption of ascorbic acid, generation of dehydroascorbic acid, and a decrease in the cellular content of reactive oxygen species. Some of the dehydroascorbic acid generated was exported out of the cells via the glucose transporters. Our data indicate that vitamin C is an important independent antioxidant in protecting cells against death from oxidative stress.     

Oestrogenic compounds and oxidative stress (in human sperm and lymphocytes in the Comet assay)

Reactive oxygen species (ROS) are produced by a wide variety of chemicals and physiological processes in which enzymes catalyse the transfer of electrons from a substrate to molecular oxygen. The immediate products of such reactions, superoxide anion radicals and hydrogen peroxide can be metabolised by enzymes such as superoxide dismutase (SOD) and catalase (CAT), respectively, and depending on its concentration by Vitamin C (Vit C). Under certain circumstances the ROS form highly reactive hydroxyl radicals. We examined human sperm and lymphocytes after treatment with six oestrogenic compounds in the Comet assay, which measures DNA damage, and observed that all caused damage in both cell types. The damage was diminished in nearly all cases by catalase, and in some instances by SOD and Vit C. This response pattern was also seen with hydrogen peroxide. This similarity suggests that the oestrogen-mediated effects could be acting via the production of hydrogen peroxide since catalase always markedly reduced the response. The variable responses with SOD indicate a lesser involvement of superoxide anion radicals due to SOD-mediated conversion of superoxide to hydrogen peroxide generally causing a lower level of DNA damage than other ROS. The variable Vit C responses are explained by a reduction of hydrogen peroxide at low Vit C concentrations and a pro-oxidant activity at higher concentrations. Together these data provide evidence that inappropriate exposure to oestrogenic compounds could lead to free-radical mediated damage. It is believed that the observed activities were not generated by cell free cell culture conditions because increased responses were observed over and above control values when the compounds were added, and also increasing dose-response relationships have been found after treatment with such oestrogenic compounds in previously reported studies.     

Low density lipoprotein cytotoxicity induced by free radical peroxidation of lipid

Low density lipoprotein (LDL) has been reported to be injurious or toxic to cells in vitro. This injurious effect is, in some instances, due to oxidation of the lipid moiety of the lipoprotein. The objectives of this study were to determine if the oxidation rendering the lipoprotein toxic to human skin fibroblasts occurred by free radical mechanisms, and if so, which of the common free radical oxygen species were involved. The selective free radical blockers or scavengers employed included superoxide dismutase for superoxide, catalase for hydrogen peroxide, dimethylfuran for singlet molecular oxygen, and mannitol for hydroxyl radical. The presence during lipoprotein preparation of general free radical scavengers (vitamin E, butylated hydroxytoluene) or the divalent cation chelator ethylenediamine tetraacetic acid prevented the formation of cytotoxic low density lipoprotein, while the simultaneous presence of superoxide dismutase and catalase partially inhibited its formation. The results indicate that superoxide and/or hydrogen peroxide are involved in the formation of the toxic LDL lipid. The toxic action of oxidized LDL could not be prevented by inclusion of antioxidants in the culture medium, indicating that an oxidized lipid was responsible for cell injury rather than free radicals generated in culture by the action of oxidized LDL. Three separate assays for cell injury (enumeration of attached cells, cell loss of lactate dehydrogenase into the culture medium, and trypan blue uptake) indicated a sequence of events in which the fibroblasts are injured, die, and then detach.     

Role of cellular superoxide dismutase against reactive oxygen metabolite injury in cultured bovine aortic endothelial cells.

We examined the protective effect of cellular superoxide dismutase against extracellular hydrogen peroxide in cultured bovine aortic endothelial cells. 51Cr-labeled cells were exposed to hydrogen peroxide generated by glucose oxidase/glucose. Glucose oxidase caused a dose-dependent increase of 51Cr release. Pretreatment with diethyldithiocarbamate enhanced injury induced by glucose oxidase, corresponding with the degree of inhibition of endogenous superoxide dismutase activity. Inhibition of cellular superoxide dismutase by diethyldithiocarbamate was not associated either with alteration of other antioxidant defenses or with potentiation of nonoxidant injury. Enhanced glucose oxidase damage by diethyldithiocarbamate was prevented by chelating cellular iron. Inhibition of cellular xanthine oxidase neither prevented lysis by hydrogen peroxide nor diminished enhanced susceptibility by diethyldithiocarbamate. These results suggest that, in cultured endothelial cells: 1) cellular superoxide is involved in mediating hydrogen peroxide-induced damage; 2) superoxide, which would be generated upon exposure to excess hydrogen peroxide independently of cellular xanthine oxidase, promotes the Haber-Weiss reaction by initiating reduction of stored iron (Fe3+) to Fe2+; 3) cellular iron catalyzes the production of a more toxic species from these two oxygen metabolites; 4) cellular superoxide dismutase plays a critical role in preventing hydrogen peroxide damage by scavenging superoxide and consequently by inhibiting the generation of the toxic species.     

Formation of singlet oxygen from solutions of vitamin E

Vitamin E offers protection against oxidative stress and is an efficient quencher of singlet oxygen. A recent report suggests that photo-excitation of vitamin E results in the formation of a triplet state (Naqvi et al. Photochem Photobiol Sci 2, 381 (2003)). This leads to the possibility of the triplet state of vitamin E being able to sensitize singlet oxygen and if this is the case it would be counter productive in terms of the biological protective function of vitamin E. We report the production of singlet oxygen, detected by 1270 nm luminescence, from pulsed laser excitation (308 nm) of vitamin E and an analogue, 2,2,5,7,8-pentamethyl-6-hydroxy-chroman (PMHC), with quantum yields between ∼0.1 and 0.2. The luminescence was identified as singlet oxygen from self-quenching by vitamin E with solvent-dependent rate constants similar to published values. Whilst the beneficial antioxidant aspects of vitamin E are well established, these results indicate that vitamin E when directly excited can sensitize singlet oxygen formation and may, therefore, be capable of inducing biochemical and biological damage. The results are discussed in relation to recent reports on the deleterious effects of vitamin E dietary supplementation and pro-oxidant effects of vitamin E.     

Some studies on lipid peroxidation in monomolecular and bimolecular lipid films

Summary Hydrogen peroxide generated from dissolved oxygen through the alloxandialuric acid cycle affected both the permeability and the stability of lipid bilayer membranes. The permeability of the artificial membranes varied directly with the hydrogen peroxide concentration. Membrane stability varied inversely with the hydrogen peroxide concentration. Bilayers formed from solutions containing both phospholipid and the antioxidant vitamin E were less permeable and more stable in the presence of hydrogen peroxide than bilayers generated from solutions containing phospholipid alone. Peroxidation of phospholipid monolayers caused first an expansion of the films presumably through the introduction of peroxide groups. Further oxidation of phospholipid monolayers led to contraction of the films presumably through the formation of water-soluble products. The results of the monolayer studies and a consideration of the possible kinetics for the peroxidation reaction sequence have been used to explain the changes in the permeability and the stability of lipid bilayer membranes. Our data suggest that oxidation of lipid in biological membranes may first increase membrane permeability and then decrease membrane stability.     

Modulation of hOGG1 DNA repair enzyme in human cultured cells in response to pro-oxidant and antioxidant challenge

The putative modulation of the base excision repair enzyme, human 8-oxoguanine glycosylase (hOGG1), important in the removal of the potentially mutagenic lesion 8-oxo-2'-deoxyguanosine (8-oxodG), was investigated in human cell culture models. The expression of specific mRNA and protein was measured following pro-oxidant and antioxidant treatments in one human lymphoblastoid and one keratinocyte line. The measurement of intracellular reactive oxygen species generation was monitored by a fluorogenic assay and potential genotoxic effects confirmed by the dose-dependent increase in formamidopyrimidine-DNA glycosylase (Fpg) sensitive sites by alkaline unwinding following sub-lethal doses of hydrogen peroxide. The generation of a potentially antioxidant environment was assessed by the intracellular increase and extracellular depletion in ascorbic acid, confirmed by capillary electrophoresis. Despite these pro-oxidant and antioxidant treatments no significant change in mRNA of hOGG1 was observed in either cell line. Western analysis revealed that relatively high, yet noncytotoxic, doses of hydrogen peroxide caused a consistent approximate 50% decrease in hOGG1 protein in lymphoblastoid cells. The lack of upregulation of hOGG1 suggests the gene is constitutively expressed, which is further supported by studies examining the sequence of its promoter region. However, hOGG1 protein turnover may be sensitive to intracellular redox changes.     

Formation of singlet oxygen from solutions of vitamin E

Vitamin E offers protection against oxidative stress and is an efficient quencher of singlet oxygen. A recent report suggests that photo-excitation of vitamin E results in the formation of a triplet state (Naqvi et al. Photochem Photobiol Sci 2, 381 (2003)). This leads to the possibility of the triplet state of vitamin E being able to sensitize singlet oxygen and if this is the case it would be counter productive in terms of the biological protective function of vitamin E. We report the production of singlet oxygen, detected by 1270 nm luminescence, from pulsed laser excitation (308 nm) of vitamin E and an analogue, 2,2,5,7,8-pentamethyl-6-hydroxy-chroman (PMHC), with quantum yields between ～0.1 and 0.2. The luminescence was identified as singlet oxygen from self-quenching by vitamin E with solvent-dependent rate constants similar to published values. Whilst the beneficial antioxidant aspects of vitamin E are well established, these results indicate that vitamin E when directly excited can sensitize singlet oxygen formation and may, therefore, be capable of inducing biochemical and biological damage. The results are discussed in relation to recent reports on the deleterious effects of vitamin E dietary supplementation and pro-oxidant effects of vitamin E.     

Vitamin B6 suppresses apoptosis of NM-1 bovine endothelial cells induced by homocysteine and copper

Hyperhomocysteinemia is an important risk factor for atherosclerosis. We previously reported that formation of early atherosclerosis in the rat aorta was associated with hyperhomocysteinemia and reduction of antioxidant activity caused by low concentration of vitamin B(6)in vivo. In the present study, we examined effects of vitamin B(6) on apoptosis of bovine endothelial cells (NM-1 cells) treated with homocysteine and copper. Homocysteine and copper induced extracellular hydrogen peroxide, intracellular ROS and cellular lipid peroxide levels. Cell viability was reduced to 30% compared to that of control cells. On the other hand, pyridoxal treatment as well as EDTA treatment increased viability of NM-1 cells treated with homocysteine and copper to about 60%, and significantly decreased extracellular hydrogen peroxide, intracellular ROS and cellular lipid peroxide levels. The treatment of catalase recovered cell viability and reduced the level of extracellular hydrogen peroxide and intracellular ROS. Cell death by homocysteine and copper was confirmed to be due to apoptosis by evaluation of DNA fragmentation and by TUNEL assay. However, apoptosis of NM-1 cells induced by homocysteine and copper was due to a caspase-independent pathway as it was not inhibited by the caspase inhibitor, Z-VAD-fmk. Apoptosis of NM-1 cells induced by homocysteine and copper accompanied with mitochondrial permeability but not cytochrome c release. These results suggest that pyridoxal treatment suppresses apoptosis of NM-1 cells induced by homocysteine and copper, most likely through antioxidant effects.     

Different onsets of oxidative damage to DNA and lipids in bone marrow and liver in rats given total body irradiation.

We examined time-dependent changes in antioxidant vitamins and oxidative damage to DNA and lipids in the bone marrow, liver, and plasma of rats given total body irradiation (TBI) with X-rays at 3 Gy. The oxidative damage to DNA and lipids was evaluated by measuring increases of 8-hydroxydeoxyguanosine (8OHdG) in DNA and 4-hydroxy-2-nonenal (HNE), respectively. After the TBI, marked increases in 8OHdG and HNE were detected at 3 to 5 h in the bone marrow, while gradual increases in these parameters were detected after a few days in the liver. These changes in 8OHdG and HNE were well correlated within each tissue. In the bone marrow, levels of both vitamin C and vitamin E were decreased by the TBI; however, the changes in vitamin C were earlier and greater than those in vitamin E. In the liver, the level of vitamin C did not decrease, but that of vitamin E decreased due to the TBI. Changes in HNE, vitamin C, and vitamin E in the plasma were similar to those in the liver. Within each tissue, the time of decrease in antioxidants was almost the same as that of the increase in oxidative damage. An increase in total iron due to the TBI was also detected in these tissues. In particular, the total iron in the bone marrow was markedly increased at a few hours after the TBI, with a slight increase in transferrin and no increase in ferritin. Exposure studies performed on cells or isolated DNA showed that an increase in 8OHdG was detected immediately after irradiation at more than 100 Gy in bone marrow cells and at less than 10 Gy in isolated DNA, suggesting that an increase in 8OHdG is undetectable even in bone marrow immediately after the TBI at 3 Gy. These results indicate that the onset of oxidative damage to DNA and lipids was delayed after TBI at 3 Gy, that it was quite different in the bone marrow and the liver, and that an increase in iron and decrease in antioxidant vitamins were involved in the mechanism of oxidative damage.     

Vitamin B6 suppresses apoptosis of NM-1 bovine endothelial cells induced by homocysteine and copper

Hyperhomocysteinemia is an important risk factor for atherosclerosis. We previously reported that formation of early atherosclerosis in the rat aorta was associated with hyperhomocysteinemia and reduction of antioxidant activity caused by low concentration of vitamin B₆in vivo. In the present study, we examined effects of vitamin B₆ on apoptosis of bovine endothelial cells (NM-1 cells) treated with homocysteine and copper. Homocysteine and copper induced extracellular hydrogen peroxide, intracellular ROS and cellular lipid peroxide levels. Cell viability was reduced to 30% compared to that of control cells. On the other hand, pyridoxal treatment as well as EDTA treatment increased viability of NM-1 cells treated with homocysteine and copper to about 60%, and significantly decreased extracellular hydrogen peroxide, intracellular ROS and cellular lipid peroxide levels. The treatment of catalase recovered cell viability and reduced the level of extracellular hydrogen peroxide and intracellular ROS. Cell death by homocysteine and copper was confirmed to be due to apoptosis by evaluation of DNA fragmentation and by TUNEL assay. However, apoptosis of NM-1 cells induced by homocysteine and copper was due to a caspase-independent pathway as it was not inhibited by the caspase inhibitor, Z-VAD-fmk. Apoptosis of NM-1 cells induced by homocysteine and copper accompanied with mitochondrial permeability but not cytochrome c release. These results suggest that pyridoxal treatment suppresses apoptosis of NM-1 cells induced by homocysteine and copper, most likely through antioxidant effects.     

Levels of DNA damage are unaltered in mice overexpressing human catalase in nuclei

Two types of transgenic mice were generated to evaluate the role of hydrogen peroxide in the formation of nuclear DNA damage. One set of lines overexpresses wild-type human catalase cDNA, which is localized to peroxisomes. The other set overexpresses a human catalase construct that is targeted to the nucleus. Expression of the wild-type human catalase transgene was found in liver, kidney, skeletal muscle, heart, spleen, and brain with muscle and heart exhibiting the highest levels. Animals containing the nuclear-targeted construct had a similar pattern of expression with the highest levels in muscle and heart, but with lower levels in liver and spleen. In these animals, immunofluorescence detected catalase present in the nuclei of kidney, muscle, heart, and brain. Both types of transgenic animals had significant increases of catalase activities compared to littermate controls in most tissues examined. Despite enhanced activities of catalase, and its presence in the nucleus, there were no changes in levels of 8OHdG, a marker of oxidative damage to DNA. Nor were there differences in mutant frequencies at a Lac Z reporter transgene. This result suggests that in vivo levels of H(2)O(2) may not generate 8OHdG or other types of DNA damage. Alternatively, antioxidant defenses may be optimized such that additional catalase is unable to further protect nuclear DNA against oxidative damage.     

Caspase-8 dependent trail-induced apoptosis in cancer cell lines is inhibited by vitamin C and catalase

TNF-related apoptosis-inducing ligand (TRAIL/ Apo-2L) is a member of the TNF family of apoptosis-inducing proteins that initiates apoptosis in a variety of neoplastic cells while displaying minimal or absent cytotoxicity to most normal cells. Therefore, TRAIL is currently considered a promising target to develop anti-cancer therapies. TRAIL-receptor ligation recruits and activates pro-caspase-8, which in turn activates proteins that mediate disruption of the mitochondrial membranes. These events lead to the nuclear and cytosolic damage characteristic of apoptosis. Here we report that TRAIL-induced apoptosis is mediated by oxidative stress and that vitamin C (ascorbic acid), a potent nutritional antioxidant, protects cancer cell lines from apoptosis induced by TRAIL. Vitamin C impedes the elevation of reactive oxygen species (ROS) levels induced by TRAIL and impairs caspase-8 activation. We found that the removal of hydrogen peroxide by extracellular catalase during TRAIL-induced apoptosis also impairs caspase-8 activation. These data suggest that hydrogen peroxide is produced during TRAIL-receptor ligation, and that the increase of intracellular ROS regulates the activation of caspase-8 during apoptosis. Additionally we propose a mechanism by which cancer cells might resist apoptosis via TRAIL, by the intake of the nutritional antioxidant vitamin C. This work was supported by grants from the National Institutes of Health (CA 30388), the New York State Department of Health (M020113) and the Lebensfeld Foundation.     

alpha-Pinene inhibits growth and induces oxidative stress in roots

BACKGROUND AND AIMS: Determining the mode of action of allelochemicals is one of the challenging aspects in allelopathic studies. Recently, allelochemicals have been proposed to cause oxidative stress in target tissue and induce an antioxidant mechanism. alpha-Pinene, one of the common monoterpenoids emitted from several aromatic plants including forest trees, is known for its growth-inhibitory activity. However, its mechanism of action remains unexplored. The aim of the present study was to determine the inhibitory effect of alpha-pinene on root growth and generation of reactive oxygen species, as indicators of oxidative stress and changes in activities of antioxidant enzymes. METHODS: Effects of alpha-pinene on early root growth were studied in five test species, Cassia occidentalis, Amaranthus viridis, Triticum aestivum, Pisum sativum and Cicer arietinum. Electrolyte leakage, lipid peroxidation, hydrogen peroxide generation, proline accumulation, and activities of the enzymes superoxide dismutase (SOD), ascorbate peroxidase (APX), guaiacol peroxidase (GPX), catalase (CAT) and glutathione reductase (GR) were studied in roots of C. occidentalis. KEY RESULTS: alpha-Pinene inhibited the radicle growth of all the test species. Exposure of C. occidentalis roots to alpha-pinene enhanced solute leakage, and increased levels of malondialdehyde, proline and hydrogen peroxide, indicating lipid peroxidation and induction of oxidative stress. Activities of the antioxidant enzymes SOD, CAT, GPX, APX and GR were significantly elevated, thereby indicating the enhanced generation of reactive oxygen species (ROS) upon alpha-pinene exposure. Increased levels of scavenging enzymes indicates their induction as a secondary defence mechanism in response to alpha-pinene. CONCLUSIONS: It is concluded that alpha-pinene inhibits early root growth and causes oxidative damage in root tissue through enhanced generation of ROS, as indicated by increased lipid peroxidation, disruption of membrane integrity and elevated antioxidant enzyme levels.     

Cytotoxic, genotoxic, and mutagenic effects of diphenyl diselenide in Chinese hamster lung fibroblasts

Diphenyl diselenide (DPDS) is an electrophilic reagent used in the synthesis of a variety of pharmacologically active organic selenium compounds, and may increase the risk of human exposure to this chemical at the workplace. In a previous study, we demonstrated the pro-oxidant action and the mutagenic properties of this compound on bacteria and yeast. In the present study, we evaluated the putative cytotoxic, pro-oxidant, genotoxic, and mutagenic properties of this molecule in V79 Chinese lung fibroblast cells. When cells were treated with increasing concentrations of DPDS, its cytotoxic activity, as determined using four cell viability endpoints, occurs in doses up to 50 microM. The MTT reduction was stimulated, which may indicate reactive oxygen species (ROS) generation. Accordingly, the treatment of cells for 3h with cytotoxic doses of DPDS increased TBARS levels, and sensitized cells to oxidative challenge, indicating a pro-oxidant effect. The measurement of total, reduced, and oxidized glutathione showed that DPDS can lead to lower intracellular glutathione depletion, with no increase in the oxidation rate in a dose- and time-dependent manner. At the higher doses, DPDS generates DNA strand breaks, as observed using the comet assay. The treatment also induced an increase in the number of binucleated cells in the micronucleus test, showing mutagenic risk by this molecule at high concentrations. Finally, pre-incubation with N-acetylcysteine, which restored GSH to normal levels, annulled DPDS pro-oxidant and genotoxic effects. These findings show that DPDS-induced oxidative stress and toxicity are closely related to intracellular level of reduced glutathione. Moreover, at lower doses, this molecule has antioxidant properties, protecting the cell against oxidative damage induced by hydrogen peroxide.     

Bleomycin-Iron Damage To Dna With Formation Of 8-Hydroxydeoxyguanosine and Base Propenals. Indications That Xanthine Oxidase Generates Superoxide From Dna Degradation Products

Bleomycin, in the presence of ferric salts, oxygen and a suitable reductant, degrades DNA with the release of base propenals, detected as thiobarbituric acid (TBA) reactivity, and the formation of 8-hydroxydeo-xyguanosine (80HdG) detected by HPLC. When xanthine oxidase is added to the incubated mixture of DNA degradation products, TBA-reactivity is destroyed but 80HdG formation is increased. EPR Spin trapping experiments show that hydroxyl radicals (OH) are formed in the reaction mixture and can be inhibited by the inclusion of either superoxide dismutase or catalase. These findings suggest that the base propenals and possibly malondialdehyde, formed from them, are aldehydic substrates for xanthine oxidase and, the product of this reaction is superoxide (O₂^-) and hydrogen peroxide (H₂O₂). Thus, TBA reactivity is destroyed in the formation of O₂^- and H₂O₂ which stimulate further oxidative damage to DNA resulting in increased 8OHdG formation.     

Cigarette smoke-induced DNA damage in cultured human lung cells: role of hydroxyl radicals and endonuclease activation.

Cigarette smoke can cause DNA single strand breaks in cultured human lung cells (T. Nakayama et al., Nature, 314 (1985) 462-464) but the mechanisms behind this DNA damage have not been clearly elucidated. In the present study we have investigated the possibility that one of the major constituents in cigarette smoke, hydroquinone, may be important for mediating smoke-induced DNA damage in the human epithelial lung cell line, A 549, and the mechanisms behind this damage. Cells were exposed to cigarette smoke, hydrogen peroxide, or hydroquinone, in the absence and presence of different inhibitors, and the resulting DNA damage was assessed either as DNA single strand break formation or formation of the oxidative DNA adduct, 8-hydroxydeoxyguanosine. It was found that (i) exposure to cigarette smoke, hydrogen peroxide or hydroquinone causes a rapid decrease in the intracellular thiol level and a considerable DNA single strand break formation, (ii) the formation of DNA single strand breaks in cells exposed to cigarette smoke is inhibited by catalase, dimethylthiourea, and o-phenantroline, suggesting that hydroxyl radicals generated from iron-catalyzed hydrogen peroxide dissociation are involved in the DNA damage, (iii) hydroquinone causes considerable DNA strand break formation that is blocked by aurintricarboxylic acid, an inhibitor of endonuclease activation, and by BAPTA, an intracellular calcium chelator, (iv) addition of hydroquinone to a smoke condensate greatly enhances its ability to cause DNA single strand breaks, and (v) smoke, but not hydroquinone, causes formation of 8-hydroxydeoxyguanosine, a DNA damage product induced by the action of hydroxyl radicals on the DNA base, deoxyguanosine. These findings suggest that the ability of cigarette smoke to cause DNA single strand breaks in cultured lung cells is due to mechanisms involving hydroxyl radical attack on DNA and endonuclease activation. They also suggest that hydroquinone is an important contributor to the DNA damaging effect of cigarette smoke on human lung cells.     

Trolox enhances curcumin's cytotoxicity through induction of oxidative stress

Curcumin, a natural polyphenol in the spice turmeric, has been found to exhibit anticancer activity. Although curcumin is generally considered an antioxidant, it is also able to elicit apoptosis through the generation of ROS, thereby functioning as a pro-oxidant in cancer cells. The present study investigated the effects of antioxidant pretreatment on curcumin-induced cytotoxicity in the human cancer cell lines A2780, MCF-7, and MDA-MB-231. Cytotoxicity was enhanced by trolox, vitamin C or vitamin E; trolox, a water soluble vitamin E derivative, was the most potent. The combination of curcumin (10 μM) and trolox (10-50 μM) induced apoptosis of cancer cells as evidenced by PARP cleavage and caspase-3 activation. Furthermore, expression of the pro-apoptotic protein Bad was up-regulated and expression of the anti-apoptotic proteins Bcl-2 and Bcl-xl was down-regulated in cells that had been treated with trolox plus curcumin. ROS generation was detected in curcumin-treated cells and was significantly enhanced when cells were treated with trolox plus curcumin. Exogenous catalase or SOD1 did not alter cytotoxicity, while over-expression of either catalase or SOD1 did, pointing to the importance of intracellular hydrogen peroxide generation in cell killing. In conclusion, we demonstrated for the first time that antioxidants such as trolox can potentiate cancer cell killing by curcumin, a finding which may help in the development of novel drug combination therapies.