Different response of chicken embryo fibroblasts and hepatocytes to the interference of certain antioxidants on the binding of [G-3H]benzo[a]pyrene to DNA

The effects of treatment with reduced glutathione, cysteine and ascorbic acid on chick embryo fibroblasts and hepatocytes cultured in vitro in the presence of [G-3H]benzo[a]pyrene ([3H]BP) were studied in an attempt to evaluate the capacity of these antioxidants to interfere with the binding of the carcinogen to DNA in these two cell populations. The results revealed that in the absence of treatment with antioxidants, the carcinogen bound to the DNA of the hepatocytes less than it did to that of the fibroblasts, despite the fact that the two cell populations manifest more or less the same uptake of [G-3H]benzo[a]pyrene from the medium. Moreover the hepatocytes, unlike the fibroblasts, seemed to reap little advantage from the known capacity of the antioxidants to interfere with the binding of the carcinogen to DNA.     

Effect of ascorbic acid and curcumin on quercetin-induced nuclear DNA damage, lipid peroxidation and protein degradation.

The effects of ascorbic acid and curcumin on quercetin-induced DNA damage, lipid peroxidation protein degradation were investigated in a model system of isolated rat-liver nuclei under aerobic conditions and in the presence of equimolar concentrations of iron or copper. Neither ascorbic acid nor curcumin inhibited quercetin-induced nuclear DNA damage, lipid peroxidation, or protein degradation. In fact, both antioxidants stimulated the oxidative damage to nuclear macromolecules. Ascorbic acid significantly increased the quercetin-induced nuclear DNA damage in the presence of either iron or copper. The increases in quercetin-induced nuclear lipid peroxidation and protein degradation by ascorbic acid were statistically significant only in the presence of iron or copper, respectively. Similarly, stimulation of quercetin-induced DNA damage and lipid peroxidation by curcumin was statistically significant only in the presence of copper or iron, respectively. Curcumin had no significant effect on nuclear protein degradation. These results demonstrate the pro-oxidant properties of ascorbic acid and curcumin, compounds that also demonstrate antioxidant and anticarcinogenic properties. Ascorbic acid and curcumin may therefore each have a dual role in carcinogenesis.     

The antioxidant conundrum in cancer

The health-related effects of interactions between reactive oxygen species (ROS) and dietary antioxidants and the consequences of dietary antioxidant supplementation on human health are by no means clear. Although ROS, normal byproducts of aerobic metabolism, are essential for various defense mechanisms in most cells, they can also cause oxidative damage to DNA, proteins, and lipids, resulting in enhanced disease risk. Dietary antioxidants (e.g., vitamin E, vitamin C, beta-carotene, and selenium), as well as endogenous antioxidant mechanisms, can help maintain an appropriate balance between the desirable and undesirable cellular effects of ROS. However, any health-related effects of interactions between dietary antioxidants and ROS likely depend on the health status of an individual and may also be influenced by genetic susceptibilities. Clinical studies of antioxidant supplementation and changes in either oxidative status, disease risk, or disease outcome have been carried out in healthy individuals, populations at risk for certain diseases, and patients undergoing disease therapy. The use of antioxidants during cancer therapy is currently a topic of heated debate because of an overall lack of clear research findings. Some data suggest antioxidants can ameliorate toxic side effects of therapy without affecting treatment efficacy, whereas other data suggest antioxidants interfere with radiotherapy or chemotherapy. Overall, examination of the evidence related to potential interactions between ROS and dietary antioxidants and effects on human health indicates that consuming dietary antioxidant supplements has pros and cons for any population and raises numerous questions, issues, and challenges that make this topic a fertile field for future research. Overall, current knowledge makes it premature to generalize and make specific recommendations about antioxidant usage for those at high risk for cancer or undergoing treatment.     

Laboratory and clinical studies of cancer chemoprevention by antioxidants in berries

Reactive oxygen species (ROS) are a major cause of cellular injury in an increasing number of diseases, including cancer. Most ROS are created in the cell through normal cellular metabolism. They can be produced by environmental insults such as ultraviolet light and toxic chemicals, as well as by the inflammatory process. Interception of ROS or limiting their cellular effects is a major role of antioxidants. Due to their content of phenolic and flavonoid compounds, berries exhibit high antioxidant potential, exceeding that of many other foodstuffs. Through their ability to scavenge ROS and reduce oxidative DNA damage, stimulate antioxidant enzymes, inhibit carcinogen-induced DNA adduct formation and enhance DNA repair, berry compounds have been shown to inhibit mutagenesis and cancer initiation. Berry constituents also influence cellular processes associated with cancer progression including signaling pathways associated with cell proliferation, differentiation, apoptosis and angiogenesis. This review article summarizes laboratory and human studies, demonstrating the protective effects of berries and berry constituents on oxidative and other cellular processes leading to cancer development.     

Protection against radiation-induced oxidative stress in cultured human epithelial cells by treatment with antioxidant agents

PURPOSE: To evaluate the protective effects of antioxidant agents against space radiation-induced oxidative stress in cultured human epithelial cells. METHODS AND MATERIALS: The effects of selected concentrations of N-acetylcysteine, ascorbic acid, sodium ascorbate, co-enzyme Q10, alpha-lipoic acid, l-selenomethionine, and vitamin E succinate on radiation-induced oxidative stress were evaluated in MCF10 human breast epithelial cells exposed to radiation with X-rays, gamma-rays, protons, or high mass, high atomic number, and high energy particles using a dichlorofluorescein assay. RESULTS: The results demonstrated that these antioxidants are effective in protecting against radiation-induced oxidative stress and complete or nearly complete protection was achieved by treating the cells with a combination of these agents before and during the radiation exposure. CONCLUSION: The combination of antioxidants evaluated in this study is likely be a promising countermeasure for protection against space radiation-induced adverse biologic effects.     

Nutritional modulation of DNA repair in a human intervention study

DNA oxidation is a potential cause of cancer in humans. It is well-known that fruits and vegetables protect against cancer, and this may be in part because they contain antioxidants, which decrease the level of oxidation of DNA. However, there are other possible mechanisms, such as an enhancement of cellular repair of this damage. A randomized cross-over study was carried out on healthy human subjects, who were given kiwifruit as a supplement to their normal diet, for 3-week periods at different 'doses', with 2-week washout periods between doses. Endogenous oxidation of bases in lymphocyte DNA, and the resistance of the DNA to oxidation ex vivo, were assessed using single cell gel electrophoresis (the 'comet assay'). The capacity to repair DNA base oxidation was measured with an in vitro test, and levels of expression of repair-related genes OGG1 and APE1 were assessed by semi-quantitative RT-PCR. Concentrations of dietary antioxidants were measured in plasma. The antioxidant status of plasma and of lymphocytes was increased by consumption of kiwifruit. Levels of endogenous oxidation of pyrimidines and purines in DNA were markedly decreased, and DNA repair measured on a substrate containing 8-oxo-7,8-dihydroguanine was substantially increased (without change in levels of OGG1 or APE1 mRNA). The magnitude of these effects was generally not related to the number of kiwifruits consumed per day. Kiwifruit provides a dual protection against oxidative DNA damage, enhancing antioxidant levels and stimulating DNA repair. It is probable that together these effects would decrease the risk of mutagenic changes leading to cancer.     

The influence of high doses of vitamin C and zinc on oxidative DNA damage

BACKGROUND: The influence of interactions between reactive oxygen species (ROS) and dietary antioxidants and their influence on cancer is not clear. It is believed that this effect is mediated by decreased oxidative damage to DNA. The aim of this study was to further investigate the in vitro DNA protective or damaging effects of dietary antioxidants using the single cell gel electrophoresis (Comet) assay. MATERIALS AND METHODS: Stimulated and unstimulated lymphocytes of 10 individuals were cultured with and without different concentrations of vitamins C and zinc and damaged with H2O2. RESULTS: DNA damage measured by Olive tail moment in the Comet assay showed a non-significant trend to reduce DNA strand breaks at low vitamin and trace element concentrations. At higher vitamin C and zinc doses, DNA damage was significantly increased. CONCLUSION: The in vitro data of the present study suggest that high dosage intake of vitamin C and zinc may cause more harm than benefit. There is good evidence that health-related effects of dietary antioxidants strongly depend on individual genetic susceptibilities and health status.     

Antioxidant supplementation decreases lipid peroxidation biomarker F(2)-isoprostanes in plasma of smokers.

Free radicals in cigarette smoke (CS) cause oxidative damage to proteins, DNA, and lipids, contributing to the pathobiology of atherosclerosis, heart disease, and cancer. In vitro studies have shown that antioxidants quench free radicals and ameliorate certain aspects of biomolecular damage caused by CS. It is hypothesized that a combination of antioxidants is more effective than a single antioxidant, due to their interactions. To investigate whether supplemental antioxidants reduce CS-related lipid peroxidation in vivo and whether they are more effective in combination, we conducted an intervention study in smokers. In a randomized double-blind placebo-controlled trial, we investigated whether vitamin C or an antioxidant mixture containing vitamin C, alpha-lipoic acid, and vitamin E decreases plasma F(2)-isoprostane levels, an index of oxidant stress, in smokers. Plasma of 126 smokers (mean age, 46 years; age range, 20-78 years) was analyzed for F(2)-isoprostanes at baseline and after intervention with antioxidants and placebo. In smokers with a body mass index (BMI) above the median, 2 months of daily supplementation with 500 mg of vitamin C decreased plasma F(2)-isoprostane levels by 28.8 pmol/liter when compared with the placebo group (P = 0.001); levels in the mixture group were 7.45 pmol/liter lower after treatment, but this difference was not statistically significant (P = 0.14). There was no treatment effect in smokers with a low BMI. BMI was significantly positively associated with plasma F(2)-isoprostane levels (trend P = 0.001). Antioxidants decrease smoking-related lipid peroxidation markers of oxidative stress in humans with high BMI. Our results do not indicate that an antioxidant combination is more effective than vitamin C alone. The intake of antioxidants may help prevent smoking-related diseases. Smoking cessation should still be considered the most effective way to prevent smoking-related diseases.     

Induction of glutathione-S-transferase activity by antioxidants in hepatocyte culture

Twelve male Sprague-Dawley rats were used for the study. Six rats were injected with benzo(a)pyrene (BP); the other six rats served as the control. Twenty-four hours after injection, hepatocytes were isolated and cultured. The cultured plates were divided into 5 groups and treated with absolute ethanol (control), butylated hydroxytoluene, vitamin E, ascorbic acid or vitamin Elascorbic acid. After 48 hours, the hepatocytes were harvested for enzyme activation determination. With both control and BP-injected rats, each antioxidant treatment significantly increased glutathione-S-transferase activity. The results suggest that antioxidants may have a detoxifying effect against BP-induced carcinogenesis.     

Antioxidant enzymes and cancer

Although oxidation is the most common biological and energy producing reaction, oxidative stress is harmful to cell, because the products of oxidation such as free radicals and peroxides damage the cellular components, causing several diseases. Damage in DNA is responsible for cancer formation and progression. However, several enzymes such as superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glutathione S-transferase etc. act as antioxidants to influence oxidative stress. Polymorphisms in these enzymes are supposed to be associated with DNA damage and subsequently the individual's risk of cancer susceptibility. This review article aims to further elucidate the relationship between antioxidant enzymes and cancers by summarizing the findings of some of the important study concerning expression levels and genetic polymorphisms of antioxidant enzymes in cancer patients.     

Effects of a 6-month vitamin intervention on DNA damage in heavy smokers.

Because their formation is associated with tumor development in specific tissues, DNA adducts have potential usefulness as intermediate end points in chemoprevention studies. To determine the efficacy of a combination of antioxidant vitamins (vitamins C and E and beta-carotene), a randomized clinical trial was conducted among heavy smokers using DNA damage as the end point. Immunological methods were used to measure polycyclic aromatic hydrocarbon-DNA adducts and oxidative DNA damage (8-oxo or hydroxydeoxyguanosine) in mononuclear and oral cells. A total of 121 subjects were randomized to the 6-month intervention and received either vitamins or placebo. Dropout rates were higher in the placebo than in the vitamin group; 65% of subjects in the vitamin group, but only 47% in the placebo group, provided specimens at 6 months. Plasma levels of all three antioxidants rose significantly in the vitamin group but not in the placebo group. All four measures of DNA damage decreased in both groups; the between-group differences were not statistically significant. These data do not provide clear evidence that antioxidant vitamin intake prevents DNA damage. However, the study demonstrates that DNA damage is a useful end point in chemoprevention trials.     

Common gene polymorphisms in the metabolic folate and methylation pathway and the risk of acute lymphoblastic leukemia and non-Hodgkin's lymphoma in adults.

Folate and methionine metabolism is involved in DNA synthesis and methylation processes. Polymorphisms in the genes of folate metabolism enzymes have been associated with some forms of cancer. In a case-control study, we evaluated whether four common polymorphisms in methylenetetrahydrofolate reductase (MTHFR C677T and A1298C), methionine synthase (MS A2756G), and methionine synthase reductase (MTRR A66G) genes may have a role in altering susceptibility to adult acute lymphoblastic leukemia (ALL) and non-Hodgkin's lymphoma (NHL). We analyzed DNA of 120 adult ALL, 200 NHL, and 257 healthy control subjects. Individual carrying the MTHFR 677TT genotype showed a 3.6-fold decreased ALL risk [odds ratio (OR) 0.28, 95% confidence interval (95% CI) 0.12-0.72] than wild-types. Similarly, MS 2756GG individuals showed a 5.0-fold decreased ALL risk (OR 0.20, 95% CI 0.02-1.45) than wild-types. In combined results, subjects with the MTHFR 677CT/TT and MS 2756AG/GG genotypes revealed a 3.6-fold ALL risk reduction (OR 0.28, 95% CI 0.14-0.58) and those with the MTHFR 677TT and MTRR 66AG genotypes revealed a 4.2-fold ALL risk reduction (OR 0.24, 95% CI 0.06-0.81). Finally, those with the MS 2756AG/GG and MTRR 66AG/GG genotypes revealed a 2.2-fold ALL risk reduction (OR 0.45, 95% CI 0.10-0.85). Single analysis for NHL did not show any significant difference for all the polymorphisms investigated, but in the low-grade NHL subgroup, we found a 2.0-fold risk reduction for the MTRR 66GG homozygous genotype (OR 0.50, 95% CI 0.25-0.99), which was higher (OR 0.37, 95% CI 0.14-0.85) when analyzed in combination with MS 2756AA genotype. These data are in accordance with the hypothesis that polymorphisms in the genes for folate and methionine metabolism might play a greater role in the occurrence of ALL than NHL by influencing DNA synthesis and/or DNA methylation.     

Antioxidant properties of Rajgira (Amaranthus paniculatus) leaves and potential synergy in chemoprevention

In recent years there has been a substantial increase in the use of functional foods for disease control. Fruits and vegetables produce phytochemicals such as flavonoids and antioxidants which can lower oxidative stress and reduce the risk of chronic ailments like cancer. The aim of the present study was to investigate the antioxidant capacity and the possible protective effects of Amaranthus paniculatus leaves on the antioxidant defense system in Ehrlich's ascites carcinoma (EAC) -treated mice. Oral administration of the leaf extract at different doses caused a significant decrease in tumor volume, viable cell count and tumor weight and elevated the life span of EAC bearing mice. It also showed an improved antioxidant potential as evidenced by a significant increase in the cellular antioxidant defense system such as catalase, superoxide dismutase and reduced glutathione and also significantly reduced the levels of TBARS. The levels of RBC, hemoglobin and lymphocyte count were altered in EAC bearing mice and were reverted back to near normal levels after the treatment with the leaf extracts. Their adequate content of total phenolics and flavonoids, DPPH scavenging activity which further suggests that the extracts exert a significant protection against oxidative stress conditions.     

The effects of antioxidants on skin tumor initiation and aryl hydrocarbon hydroxylase.

Butylated hydroxytoluene, butylated hydroxyanisole, and vitamins C and E are effective inhibitors of 7,12-dimethylbenz(a)anthracene tumor initiation in a two-stage system of tumorigenesis. These antioxidants did not significantly induce epidermal aryl hydrocarbon [benzo(a)pyrene]hydroxylase, nor did they have any effect when added directly to the in vitro aryl hydrocarbon [benzo(a)pyrene]hydroxylase assay. However, butylated hydroxytolene and butylated hydroxyanisole, when topically to mice, inhibited the in vitro, epidermally mediated, covalent binding of radioactive benzo(a)pyrene and 7,12-dimethylbenz(a)anthracene to DNA. When butylated hydroxytolene and butylated hydroxyanisole were added in vitro, they did not inhibit the epidermally mediated covalent binding of the hydrocarbons to DNA. The inhibition of polycyclic hydrocarbon tumorigenesis by antioxidants may be related to the ability of antioxidants to prevent the in vivo activation of hydrocarbons to carcinogenic epoxides and/or other electrophilic intermediates or may be related to their ability to increase detoxification of the reactive intermediate that requires intact cells to be operational. In any event, the results suggest that the antioxidants have an indirect effect on the epidermal metabolizing system which leads to a decrease in covalent binding to DNA.     

Inhibition of oxidative DNA damage, 8-OHdG, and carbonyl contents in smokers treated with antioxidants (vitamin E, vitamin C, beta-carotene and red ginseng).

The chemopreventive effects of antioxidants (vitamin E, beta-carotene, vitamin C and red ginseng) on oxidative DNA and protein (globin) damages were comparatively investigated in the peripheral blood of smokers (> or = 20 cigarettes/day). Smokers showed a lower baseline level of plasma micronutrients (vitamin C and beta-carotene) (P < 0.01) and higher baseline level of oxidative DNA or protein damage than non-smokers (N = 5; P < 0.05). During daily supplementation of antioxidants (200 IU vitamin of E, 9 mg of beta-carotene, 500 mg of vitamin C, or 1.8 g of red ginseng) for 4 weeks, smokers plasma antioxidant concentrations increased linearly, while their mean levels of 8-hydroxydeoxyguanosine (8-OHdG) and carbonyl contents decreased compared with those in smokers supplemented with a placebo (P < 0.05). Levels of urinary and plasma cotinine remained steady in smokers regardless of supplementation with antioxidants. 8-OHdG and carbonyl content decreased in a time-dependent manner (as the total intake dose increased) after supplementation with vitamin E (8-OHdG, 33.8%; carbonyl content, 43.6%) or red ginseng (8-OHdG, 31.7%; carbonyl content, 21.3%). These preliminary data suggest that supplementation with antioxidants might protect smokers from oxidative damages and could reduce cancer risk or other diseases caused by free radicals associated with smoking.     

The effects of combined antioxidant (beta-carotene, alpha-tocopherol and ascorbic acid) supplementation on antioxidant capacity, DNA single-strand breaks and levels of insulin-like growth factor-1/IGF-binding protein 3 in the ferret model of lung cancer

Insulin-like growth factor 1 (IGF-1) and its major binding protein, IGF binding protein 3 (IGFBP-3) are implicated in lung cancer and other malignancies. We have previously shown that the combination of three major antioxidants [beta-carotene (BC), alpha-tocopherol (AT) and ascorbic acid (AA)] can prevent lung carcinogenesis in a 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-treated and smoke-exposed (SM) ferret model, which is highly analogous to humans. The present study is aimed at determining the effect of the combination of BC, AT and AA on antioxidant capacity, lymphocyte DNA damage, plasma IGF-1 and IGFBP-3 concentrations, as well as on IGF-1/IGFBP-3 mRNA expression in the tissues (lung and liver) of the ferrets. Ferrets were treated with or without combined antioxidant (BC, AT and AA) supplementation (AOX) for 6 months in the following 4 groups: (i) control; (ii) SM+NNK; (iii) AOX; and (iv) SM+NNK+AOX. Combined AOX supplementation significantly attenuated SM+NNK induced lymphocyte DNA damage in the ferret, while increasing resistance to oxidative damage when challenged with H(2)O(2) in vitro. Ferrets treated with SM+NNK had significantly lower IGFBP-3 mRNA expression in lungs, whereas there was significantly higher IGFBP-3 mRNA expression in the liver, as well as higher circulating IGFBP-3 concentrations. Combined AOX supplementation did not affect the plasma or tissue (lung and liver) ratio of IGF-1/IGFBP-3. Combined antioxidant supplementation provides protection against smoke-induced oxidative DNA damage, but does not affect the IGF-1/IGFBP-3 system. Differential expression of IGFBP-3 in different tissues indicates that caution should be taken when using plasma IGFBP-3 as a biomarker of tissue status.     

Associations of antioxidant nutrients and oxidative DNA damage in healthy African-American and White adults.

High antioxidant intake has been shown to reduce cancer risk and may also mitigate the effects of oxidative DNA damage, which is hypothesized to be causally linked to carcinogenesis. This study examined potential racial differences in (a) dietary intakes and plasma concentrations of vitamin C, vitamin E, and carotenoids and oxidative DNA damage and (b) associations between plasma antioxidants and oxidative DNA damage. Data were from a cross-sectional study of 164 generally healthy nonsmoking African-Americans and Whites in North Carolina, ages 20 to 45 years, equally distributed by race and sex. Participants completed a demographic and health questionnaire, four 24-h dietary recalls, and a dietary supplement inventory; had height and weight measured; and provided a semifasting blood sample. African-Americans had statistically significantly lower plasma concentrations of vitamin E, alpha-carotene, beta-carotene, and lutein + zeaxanthin than Whites, as well as lower self-reported intake of most antioxidants. Levels of oxidative DNA damage, measured using the alkaline comet assay, were lower in African-Americans than Whites. An inverse association between lycopene and oxidative DNA damage (r = -0.20; P = 0.03) was found in the combined study population after adjusting for sex, age, body mass index, passive smoke exposure, physical activity, education, income, and alcohol intake. There was also a positive association of vitamin E with oxidative DNA damage in the total population (r = 0.21; P = 0.02) and in African-American men (r = 0.63; P = 0.01) after adjusting for covariates. This study is among the first to examine these associations in a sample of healthy adults with an adequate representation of African-Americans.     

Antioxidant intervention as a route to cancer prevention

The notion of cancer prevention through antioxidant intervention arises from the fact that fruits and vegetables contain antioxidants and are linked to low cancer rates in those who consume them. Protection against DNA damage by plant food products can be demonstrated in vitro. However, particular care is needed when measuring the damage, since oxidation readily occurs during sample preparation, creating a serious artefact. In the case of DNA oxidation, estimates of background levels in human cells range over 3 orders of magnitude, depending on the method used. Using validated, reliable biomarker assays for DNA oxidation, it is possible to demonstrate a decrease in oxidative damage after supplementation with isolated antioxidants or whole plant foods in humans. In contrast, in several large-scale interventions with disease or death as the endpoint, supplementation with beta-carotene resulted in no effect or an increase in cancer incidence. It is certainly true that we do not yet fully understand the role of phytochemicals as antioxidants, or as modulators of other processes related to carcinogenesis and its prevention.     

Oxidative Stress and Antioxidants in Disease and Cancer: A Review

Reactive oxygen species (ROS), highly reactive molecules, are produced by living organisms as a result ofnormal cellular metabolism and environmental factors, and can damage nucleic acids and proteins, therebyaltering their functions. The human body has several mechanisms to counteract oxidative stress by producingantioxidants. A shift in the balance between oxidants and antioxidants in favor of oxidants is termed as “oxidativestress”. Paradoxically, there is a large body of research demonstrating the general effect of oxidative stress onsignaling pathways, less is known about the initial and direct regulation of signaling molecules by ROS, orwhat we term the “oxidative interface.” This review focuses on the molecular mechanisms through which ROSdirectly interact with critical signaling molecules to initiate signaling in a broad variety of cellular processes,such as proliferation and survival (MAP kinases and PI3 kinase), ROS homeostasis, and antioxidant generegulation (Ref-1 and Nrf-2). This review also deals with classification as well as mechanisms of formation offree radicals, examining their beneficial and deleterious effects on cellular activities and focusing on the potentialrole of antioxidants in preventing and repairing damage caused by oxidative stress. A discussion of the role ofphytochemical antioxidants in oxidative stress, disease and the epigenome is included.