The shifting perception on antioxidants: the case of vitamin E and β-carotene

Abstract

Antioxidants are vital for aerobic life, and for decades the expectations of antioxidants as health promoting agents were very high. However, relatively recent meta-analyses of clinical studies show that supplementation of antioxidants does not result in the presumed health benefit, but is associated with increased mortality. The dilemma that still needs to be solved is: what are antioxidants in the end, healthy or toxic? We have evaluated this dilemma by examining the presumed health effects of two individual antioxidants with opposite images i.e. the "poisonous" β-carotene and the "wholesome" vitamin E and focused on one aspect, namely their role in inducing BPDE-DNA adducts. It appears that both antioxidants promote DNA adduct formation indirectly by inhibition of the protective enzyme glutathione-S-transferase π (GST π). Despite their opposite image, both antioxidants display a similar type of toxicity. It is concluded that, in the appreciation of antioxidants, first their benefits should be identified and substantiated by elucidating their molecular mechanism. Subsequently, the risks should be identified including the molecular mechanism. The optimal benefit-risk ratio has to be determined for each antioxidant and each individual separately, also considering the dose.

Keywords: DNA damage; Glutathione-S-transferase; Risk–benefit analysis; Vitamin E; β-Carotene.

Graphical abstract

Fig. 1

The effects of β-carotene (10 µM) on BPDE-DNA adduct formation (A), on GST π induced detoxification of BPDE (B) and the inhibitory effect β-carotene on the activity of GST π. The inhibitory effect of β-carotene on GST π induced BPDE detoxification. BPDE-DNA adduct formation was measured by 32P-postlabeling assay. BEAS2B-cells were treated for 1 h with 0.1 µM BPDE in the absence or in the presence of β-carotene, the combination of GST π (50 mU/ml) and GSH (1 mM) or the combination of GST π (50 mU/ml), GSH (1 mM) and β-carotene (10 µM). After treatment, medium was removed and cells were collected and stored at − 20 °C. DNA adduct levels were determined according to the nuclease P1 enrichment technique as described by Reddy and Randerath with minor modifications , . GST π activity measurements were performed as described by Mannervik and Guthenberg with slight modifications . In short, the reaction of 1 mM CDNB with 1 mM GSH in 100 mM potassium phosphate buffer (pH 6.5; 37 °C) was spectrophotometrically monitored by measuring the increase in absorbance at 340 nm. The effect of β-carotene (final concentrations: 5 and 10 µM) on GST enzyme activity (0.05 U/ml in buffer) was determined. The control activity (of 127±3 nmol/min/ml) was set to 100%. Data are shown as means±SEM (n=6). One way analysis of variance (ANOVA) with Bonferroni post hoc correction was used to examine differences in enzyme activities of GST π. In order to determine differences in BPDE-DNA adduct formation, student's t-test was used. Differences were considered to be statistically significant when P<0.05. *P<0.05, **P<0.005, and ***P<0.001.

Fig. 2

The effects vitamin E (30 µM) on BPDE-DNA adduct formation (A), on GST π induced detoxification of BPDE (B) and the inhibitory effect of vitamin E on the activity of GST π. BPDE-DNA adduct formation was measured by 32P-postlabeling assay. HaCaT cells were treated for 1 hour with 0.1 µM BPDE dissolved in DMSO in the absence or in the presence of vitamin E, the combination of GST π (50 mU/ml) and GSH (1 mM) or the combination of GST π (50 mU/ml), GSH (1 mM) and vitamin E. After treatment, medium was removed and cells were collected and stored at − 20 °C. DNA adduct levels were determined according to the nuclease P1 enrichment technique as described by Reddy and Randerath with minor modifications , . The GST π activity was determined by recording the conjugation of 1 mM 1-chloro-2,4-dinitrobenzene (CDNB) by 1 mM GSH. The reaction of 1 mM CDNB with 1 mM GSH in 100 mM potassium phosphate buffer (pH 6.5; 37 °C) was spectrophotometrically monitored by measuring the increase in absorbance at 340 nm. The effect of vitamin E (final concentrations: 1.25, 5 and 10 µM) on GST enzyme activity (0.05 U/ml in buffer) was determined. The control activity (of 127±3 nmol/min/ml) was set to 100%. Data are shown as means±SEM (n=6). One way analysis of variance (ANOVA) with Bonferroni post-hoc correction was used to examine differences in enzyme activities of GST π. In order to determine differences in BPDE-DNA adduct formation, student’s t-test was used. Differences were considered to be statistically significant when P<0.05. *P<0.05, and ***P<0.001.

Fig. 3

Increase (%) in risk for lung cancer in smokers induced by β-carotene (left y-axis) and in skin tumor formation in mice treated with DMBA induced by vitamin E (right y-axis). β-carotene has a relative risk of lung cancer in smokers of 1.36, giving an increase of 36%. Vitamin E increased the number of skin tumors in mice from 1 tumor/25 animals in the control group to 154 tumors/26 animals in DMBA treated mice, giving an increase of 15,400%. The β-carotene data were obtained from the alpha-tocopherol, β-carotene Cancer Prevention Study Group, in which male smokers were daily supplemented with β-carotene (20 mg). Lung cancer incidence was determined during a follow-up of 5–8 years . Vitamin E data were obtained from Mitchel et al. . In this study, DMBA with and without vitamin E were topically applied to the dorsal skin of female SENCAR mice from which the hair was shaved. ninety-eight nd 153 days after DMBA initiation, skin tumor formation was determined.

Box 1

Antioxidant protection: for a long time, it was thought that antioxidants only needed to scavenge radicals to offer protection. Currently, our knowledge on how antioxidants functions has progressed. The mechanism of action of antioxidants consists of multiple steps, as illustrated with the protection of lipoproteins against lipid peroxidation by vitamin E. During the process of lipid peroxidation lipid peroxyl radicals (LOO^●) are formed. In the protection, the first step is the scavenging of LOO^● by the lipophilic antioxidant vitamin E·. This is a chemical reaction in which the reactive radical (LOO^●) is transformed into a non-radical (LOOH) that is relatively unreactive. The second step is that the radical is safeguarded in the antioxidant radical. The vitamin E radical is relatively unreactive due to delocalization of the radical over the antioxidant molecule. In the final step, the radical is transferred safely into the antioxidant network. In sequential reactions, the radical is transferred from one antioxidant to another antioxidant. In the example, the radical located on vitamin E in the lipoprotein is taken over by vitamin C in the plasma ; the vitamin C radical might react with NADH to regenerate vitamin C, a reaction catalyzed by the ascorbate free radical reductase. In the end, the reactivity of the radical is totally absorbed in the 3 steps.

Box 2

The health effect of antioxidants: in the perception of the health effect of an antioxidant, it is often considered that an antioxidant either only provides benefits or that it only poses risks. These opposing, one side views are the main obstacles in the accurate perception of the health effect of antioxidants. A more balanced view is mandatory as any bioactive, antioxidants included, have benefits as well as risks. In fact, the distinction between the two is not as clear-cut as generally assumed; as coined by Adrien Albert, the benefit is a form of selective toxicity. Critical in the risk–benefit analysis is that each individual balance is biased. In people that are expected to profit, the arm of the benefit is relatively long. Contrariwise, in vulnerable people, the arm of the risk is relatively long. The latter is seen for the risk of β-carotene in smokers. Apparently, it has to be determined for each individual separately whether the benefit outweighs the risk. The molecular mechanism is the pulling force that determines the weight of the benefit as well as the risk.