Mutagenesis, Vol. 18, No. 2, 119-126,
March 2003
© 2003 UK Environmental Mutagen Society/Oxford University Press
The principal phenolic and alcoholic components of wine protect human lymphocytes against hydrogen peroxide- and ionizing radiation-induced DNA damage in vitro
1 CSIRO Health Sciences and Nutrition, PO Box 10041, Adelaide BC, South Australia 5000, Australia and 2 Clinical and Experimental Pharmacology, The University of Adelaide, Adelaide, Australia
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Abstract |
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We have tested the hypothesis that the alcoholic and phenolic components of wine are protective against the DNA-damaging and cytotoxic effects of hydrogen peroxide and
-radiation in vitro. The components of wine tested were ethanol, glycerol, a mixture of the phenolic compounds catechin and caffeic acid and tartaric acid, all at concentrations that were 2.5 or 10.0% of the concentration in a typical Australian white wine (Riesling). These components were tested individually or combined as a mixture and compared to a white wine stripped of polyphenols, as well as a Hanks balanced salt solution control, which was the diluent for the wine components. The effect of the components was tested in lymphocytes, using the cytokinesis-block micronucleus assay, after 30 min incubation in plasma or whole blood for the hydrogen peroxide or -radiation challenge, respectively. The results obtained showed that ethanol, glycerol, the catechin–caffeic acid mixture, the mixture of all components and the stripped white wine significantly reduced the DNA-damaging effects of hydrogen peroxide and -radiation (P = 0.043–0.001, ANOVA). The strongest protective effect against DNA damage by -irradiation was observed for the catechin–caffeic acid mixture and the mixture of all components (30 and 32% reduction, respectively). These two treatments as well as ethanol produced the strongest protective effects against DNA damage by hydrogen peroxide (24, 25 and 18%, respectively). The protection provided by the mixture did not account for the expected additive protective effects of the individual components. Ethanol was the only component that significantly increased baseline DNA damage rate, however, this effect was negated in the mixture. In conclusion, our results suggest that the main phenolic and alcoholic components of wine can reduce the DNA-damaging effects of two important oxidants, i.e. hydrogen peroxide and ionizing radiation, in this physiologically relevant in vitro system. |
Introduction |
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Evidence for the protective effect of moderate consumption of alcoholic beverages on cardiovascular disease was provided initially by the epidemiological study of St Leger et al. (1979)
. Later, ‘The French Paradox’ was coined to describe the low incidence of cardiovascular disease in specific populations despite elevated levels of risk, including high fat diets, sedentary lifestyles and prevalent cigarette smoking (Renaud and De Lorgeril, 1993), and was hypothesized to be explained by their moderate consumption of wine. Moderate alcohol intake is also associated with a reduced risk of mortality from cancer, however, risk of cancer and other diseases increases progressively with excessive alcohol intake (Boffeta and Garfinkel, 1990). A study from eastern France reported a reduced risk of death of 22% from all cancers in those people drinking up to 21 g ethanol/day (the majority in the form of wine, 82% of total ethanol consumption) when compared with non-drinkers (Renaud et al., 1998). A plausible explanation for these observations is that wine or some of its components may prevent DNA damage events caused by reactive oxygen species that are implicated in the initiation events of cancer and possibly atherosclerosis (DeFlora et al., 1997; Andreassi et al., 2000; Ferguson, 2001; Ghiselli et al., 1998; Casalini et al., 1999). A common link between cardiovascular disease and cancer is the observation that elevated micronucleus frequency (a marker of DNA damage) is associated with increased severity of coronary artery disease (Botto et al., 2002).
We have previously shown that the genotoxic and cytotoxic effects of reactive oxygen species can be efficiently measured using the cytokinesis-block micronucleus (CBMN) assay following challenge by hydrogen peroxide, superoxide, activated neutrophils and/or ionizing radiation (Fenech and Morley, 1986; Fenech et al., 1997; Umegaki and Fenech, 2000). The assay, in its comprehensive mode, also allows measurement of nucleoplasmic bridges between the two nuclei (an index of chromosome rearrangement), necrosis, apoptosis and cytostasis (i.e. inhibition of the extent and frequency of nuclear division) (Fenech, 2000).
Pilot ex vivo experiments in our laboratory demonstrated a reduction in hydrogen peroxide-induced genetic damage levels at 1–3 h post-consumption of 300 ml of red or white wine, with strongest inhibition (>70%) at the 1 h post-consumption time point (Fenech et al., 1997). In vitro experiments in that study showed that lymphocytes incubated in either 12% ethanol solution or phenolic-stripped white wine prior to hydrogen peroxide challenge resulted in a statistically significant 57–87% reduction (P = 0.0354 and P = 0.0053, respectively) in micronucleated (MNed) cell frequency at a concentration of 10% v/v in culture medium. These results of the in vitro and ex vivo studies suggested that both the non-phenolic components as well as the phenolic compounds of wine may exert antioxidant effects. Because the ex vivo experiments did not demonstrate a difference between red and white wine we hypothesized that phenolic and/or non-phenolic components common to red and white wine may be important in prevention of oxidative DNA damage. Candidate molecules abundant in red and white wine include alcohol, glycerol, tartaric acid and the phenolic compounds caffeic acid and catechin (C & C) [Table I lists their concentration in a typical Australian white wine (Riesling) from the Clare Valley, South Australia.] The aim of this study was to determine the relative contribution of these compounds to the protective effect of wine against DNA damage by reactive oxygen species. To answer these questions, two models of genetic damage were used: (i) isolated lymphocyte cultures in autologous plasma challenged with hydrogen peroxide; (ii) lymphocytes in whole blood cultures challenged with ionizing radiation. Effects of the wine components on baseline and induced DNA damage and cell death were determined using the comprehensive CBMN assay.
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Materials and methods |
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Volunteers
The study was advertised to male students by Email after approval by the CSIRO Health Sciences and Nutrition Human Experimentation Ethics Committee and The University of Adelaide Human Research Ethics Committee. Volunteers were not paid to participate. Four healthy male volunteers aged 21–23 years were recruited based upon their responses to a general health, lifestyle and ethanol consumption questionnaire and after giving informed consent. Those who supplemented their diet with antioxidant vitamins and/or multi-vitamins and/or smoked and/or drank in excess of moderate drinking guidelines were excluded from the study. Young adult males were selected for this study because previous studies have shown that this group has the lowest baseline micronucleus frequency in the adult population (Fenech, 1993a
,b).
Wine component preparation
The current study examined the individual components ethanol, glycerol, tartaric acid as well as the mixture C & C at 2.5 and 10% of the concentration found in white wine. The C & C mixture of phenolic compounds was used as they represent the most abundant phenolic compounds in white wine. The wine components were dissolved in Hank’s balanced salt solution (HBSS) and diluted in culture medium as shown in Table I
to achieve final concentrations in vitro equivalent to a 2.5 and 10% dilution of the concentration in white wine. The 2.5% concentration corresponds approximately to the theoretical average concentration of wine components to be found in the blood of a 60 kg person after consuming 300 ml of white wine, assuming that 50% of the components were absorbed and not rapidly excreted from the blood into other tissues and extracellular fluids. For comparison we used the following controls, also at corresponding dilutions: HBSS was the negative control and white wine stripped of phenolic compounds using polyvinyl polypyrollidone (PVPP) (Sigma Chemical Co, St Louis, MO) was a control for the major non-phenolic fraction of white wine. PVPP-stripped white wine was prepared by passing white wine (1997 Deakin Estate Chardonnay, Victoria, Australia) through a column of PVPP (pre-washed with 12% ethanol) under negative pressure (Somers and Ziemalis, 1985).
Hydrogen peroxide challenge experiments
The basic protocol is outlined in Figure 1. On two occasions an overnight fasted venous blood sample of 16 ml was collected into lithium–heparin-coated Vacuette® tubes (Greiner Labortechnik, Austria). The whole blood was spun for 10 min at 1400 g and the plasma separated. The pellet was resuspended in an equal volume of HBSS and fresh lymphocytes were isolated using Ficoll gradients (Pharmacia Biotech, Sweden). Samples of 475 µl of plasma were reconstituted with autologous lymphocytes to a concentration of 1x106 cells/ml. Wine components were added in 25 µl aliquots to their respective cultures to achieve the final concentrations shown in Table I. All cultures were incubated at 37°C and 5% CO2 for 30 min. Following this, 15 µl of hydrogen peroxide (Ajax Chemicals, Sydney, Australia) was added to achieve a final concentration of 750 µM. Cultures were then incubated for a further 30 min at 37°C and 5% CO2, after which time the cells were washed in 1 ml of RPMI 1640 (Trace Biosciences, Australia) and resuspended in 500 µl of RPMI 1640 medium supplemented with 10% fetal calf serum (Trace Biosciences, Australia). Similarly prepared matched unchallenged cultures with HBSS only were used as controls. Phytohaemagglutinin (PHA) (Murex Biotech Ltd, Dartford, UK) was added to a final concentration of 22 µg/ml and the cultures placed in a humidified incubator at 37°C and 5% CO2. Forty-four hours after mitogen stimulation, cytochalasin B (final concentration 6 µg/ml; Sigma Chemical Co.) was added to accumulate the dividing lymphocytes at the binucleate phase and 28 h later the cells were harvested to slides using cytocentrifugation (Shandon, Runcorn, UK). The slides were air dried, fixed and stained using Diff-Quik (LabAids, Narrabeen, Australia). All cultures were prepared in duplicate, utilizing single batch reagents to minimize confounding variation.
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-Radiation challenge experimentsThe protocol is outlined in Figure 2
. Briefly, 500 µl of heparinized whole blood was added to a 70 ml flat-bottomed pot (Sarstedt, Australia), prior to the addition of wine components. The components were added in 50 µl aliquots to their respective blood sample. All cultures were incubated at 37°C for 30 min prior to irradiation. Genetic damage was induced by exposure to a 137Cs source (Cis Bio IBL 437 C Blood Product Irradiator, dose rate 5.7 Gy/min). All cultures received a dose of 1.5 Gy, a level known to induce an 
100-fold increase in MNed cell frequency relative to baseline, from preliminary dose–response experiments (Table II). Unirradiated blood samples were used as controls. The CBMN assay was performed according to the protocol of Fenech (1993a) with the following modifications for whole blood culture: 4.5 ml of RPMI 1640 culture medium (Trace Biosciences, Australia) supplemented with 10% FCS (Trace Biosciences) was added to each 500 µl of blood sample. Cultures were kept in a humidified incubator at 37°C and 5% CO2 until PHA (Murex Biotech) was added in a 45 µl aliquot to a final concentration of 200 µg/ml. The cultures were incubated for 44 h prior to addition of cytochalasin B (Sigma Chemical Co.) to a final concentration of 6 µg/ml and were then incubated for a further 28 h. Lymphocytes were harvested by overlaying onto 1.5 ml Ficoll-Paque (Pharmacia Biotech) in 10 ml conical-bottomed centrifuge tubes (Sarstedt) and centrifuged for 25 min at 400 g at 19°C. The isolated lymphocyte (buffy) layer was transferred to a second 10 ml tube and 3 ml of HBSS was added. After centrifugation at 180 g at 19°C for 5 min, the supernatant was removed and the pellet resuspended in 3 ml of HBSS and again the cells were centrifuged for 5 min at 180 g at 19°C. After removal of the supernatant, the cells were resuspended in 300 µl of hypotonic solution (20 mg trypsin in a solution of 5 ml of RPMI 1640, 1.5 ml of dimethyl sulphoxide and 13.5 ml of MilliQ water, prepared within 1 h of addition) and incubated at 37°C for 5 min. Cells were transferred to slides and stained as described above for the hydrogen peroxide experiments.
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Scoring procedure for MNed cells, nucleoplasmic bridges, necrosis and apoptosis
All slides were coded prior to scoring. A single individual (W.G.) scored all slides using a Leitz Dialux 20EB light microscope at 1000x magnification using an oil immersion lens. For the hydrogen peroxide challenge experiments, the frequency of MNed cells in 1000 binucleated cells of mono-, bi- and multi-nucleated cells and of necrotic and apoptotic cells was assessed according to the latest standard procedure (Fenech, 2000
). In the -radiation challenge experiments, MNed cell frequency and nucleoplasmic bridge (NPB) frequency were assessed in 1000 BN cells as markers of chromosome breakage/loss and rearrangements, respectively. Nuclear division index (NDI) was calculated from the ratio of the frequency of mono-, bi- and multi-nucleated cells estimated by scoring 500 cells. It was not possible to reliably score necrotic and apoptotic cells from whole blood cultures in the -irradiation experiment due to the elimination or altered morphology of these cells by the hypotonic treatment.
Statistical analysis
The significance of the dose–response effect for hydrogen peroxide and -radiation and the variation in mean values of the end-points scored for each component treatment relative to the control was evaluated using a General Linear Model Repeated Measures ANOVA. Parametric tests were used for analysis because data for the end-points measured were normally distributed around the mean. Data for cultures treated with hydrogen peroxide and -radiation were analysed separately for observed frequency of genotoxicity/cytotoxicity biomarkers and also for induced frequency of these biomarkers after subtracting baseline values in untreated cultures. Post hoc pairwise t-test comparisons were conducted using a least significant difference adjustment for multiple comparisons. Relationships between variables were assessed using the Spearman rank correlation. Data are expressed as means ± SEM. Statistical calculations were performed using SPSS for Windows 10.0.1 statistical software (© SPSS Inc., 1999) and graphing was performed using Microsoft® Excel 2002.
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Results |
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Dose–response characteristics for gamma rays and hydrogen peroxide
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-radiation dose–response curve was constructed from 0 to 4 Gy (Table II). Significant dose–response effects were observed in the means of MNed BN cells and NPB frequency in BN cells (Pearson correlation coefficient = 0.986, P < 0.001 and P = 0.951, P < 0.001, respectively). From these data we determined that exposure to 1.5 Gy -radiation would provide a readily measurable increment in DNA damage using both the MNed cell and NPB biomarkers.
A significant dose–response effect with hydrogen peroxide dose was observed for frequency of MNed BN cells and necrosis (Table III). From these experiments we determined that 750 µM hydrogen peroxide was the highest appropriate challenge dose because at this concentration we could expect a 6-fold increase in MNed cell frequency without severe cytotoxic or cytostatic effects that could have made it impractical to determine MNed cell frequency in BN cells.
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The doses selected were also based on a power calculation indicating that a 20% change in MNed cell frequency in BN cells would be detectable with statistical significance (P < 0.05) if blood from four subjects was tested in duplicate.
Effect of wine components on baseline genetic damage and cytotoxicity in isolated lymphocyte cultures
Mean baseline MNed cell frequency for the four volunteers was found to be 3.0 ± 0.8 MNed cells/1000 BN cells in the HBSS control. A statistically significant difference in MNed cell frequency in BN cells was found across treatments at 10% (P = 0.05, ANOVA) but not at 2.5% concentration (P = 0.144, ANOVA) (Figure 3a). MNed cell frequency was increased on treatment with ethanol at 10% concentration and although this difference did not reach significance against the HBSS control, it was significant when compared with glycerol, the mixture, phenolic-stripped white wine and tartaric acid (P < 0.02, P < 0.05, P < 0.01 and P < 0.02, respectively). Necrosis was significantly reduced by all wine components except tartaric acid at both concentrations (P < 0.0001, ANOVA) (Figure 4a), however, there were no significant effects on NDI and apoptosis.
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Effect of wine components in whole blood cultures on baseline DNA damage and NDI
The mean background level of MNed cells after whole blood culture was 3.3 ± 0.6 MNed cells/1000 BN cells in HBSS controls (a result that is similar to that obtained for isolated lymphocyte cultures). No NPB were observed in BN cells. There were no significant differences between treatments in MNed cell frequency (Figure 3b
) at either concentration amongst the various wine components (P > 0.05). As in isolated lymphocyte cultures, there was a trend for elevated MNed cell frequency on exposure to ethanol. A significant difference in NDI was observed across treatments at both 10 (P = 0.046, ANOVA) and 2.5% dilution (P = 0.004, ANOVA). At 10%, all treatments except glycerol reduced NDI significantly compared with the HBSS control (P < 0.05), whilst at the lower concentration all treatments except the mixture and tartaric acid reduced NDI values (P < 0.05).
Effect of wine components in plasma on hydrogen peroxide-induced DNA damage and cytotoxicity
A significant difference (P = 0.015, ANOVA) in induced (observed – baseline) MNed cell frequency on challenge with hydrogen peroxide was shown for those samples containing wine components at 10% (v/v) concentration (Figure 5a). Samples supplemented with C & C, ethanol or the mixture of all components showed the largest and most significant reductions in MNed cell frequency (P < 0.05). A similar significant effect was observed for components at 2.5% dilution for induced MNed cell frequency (P = 0.043, ANOVA) (Figure 5a). Significant differences in induced necrosis between treatments were found at both concentrations (P < 0.0001, ANOVA) (Figure 4b). Necrosis significantly increased relative to the HBSS control with all components at both concentrations in all cases (P < 0.05 in all cases) except tartaric acid (P > 0.2), which was in fact significantly different from all other treatments (P < 0.05). At the 10% (v/v) concentration, C & C produced an increase in hydrogen peroxide-induced necrosis that was significantly greater than that observed for all other treatments except ethanol. NDI and apoptosis did not change significantly across treatments on hydrogen peroxide challenge at either concentration (P > 0.05, ANOVA).
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Effect of wine components in whole blood on
-radiation-induced DNA damage and NDIThere was a significant difference in
-ray-induced (observed – baseline) MNed cell frequency between treatments at 2.5 (P = 0.037, ANOVA) and 10% (P < 0.001, ANOVA) dilution (Figure 5b). All components significantly reduced MNed cell frequency (P < 0.05) compared with the HBSS control at 10% (v/v) concentration, however, post hoc analysis demonstrated that the most significant reductions in MNed cell frequency were achieved with the mixture of all components, ethanol or C & C (P < 0.001, P = 0.001 and P = 0.001, respectively). C & C and the mixture were not significantly different from each other (P > 0.19). However, C & C was significantly more protective than ethanol (P < 0.01). At 2.5% dilution, all treatments except tartaric acid significantly reduced induced MNed cell frequency (P < 0.05). Likewise, all components significantly reduced NPB formation relative to the HBSS control (P < 0.02 in all cases) at the 10% dilution (Figure 6). The most significant reduction in NPB frequency was in cultures supplemented with the mixture of all components or C & C (P < 0.005 and P <0.001, respectively); these two treatments were significantly different from all other components (P < 0.02), but not from each other (P > 0.125). The NDI was significantly reduced at the 10% concentration on treatment with ethanol, the mixture of components and phenolic-stripped wine (P < 0.05).
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Cross-correlation of measures (Table IV)
For cross-correlations between assay end-points, data from baseline and challenge experiments (observed frequency data only) were combined. In isolated lymphocyte cultures (i.e. control and hydrogen peroxide challenge), a strong positive correlation was observed between MNed cell frequency and necrotic cell frequency (r = 0.665, P < 0.001), and NDI was significantly positively correlated with necrotic cell and MNed cell frequency (r = 0.410 and r = 0.277, respectively, P < 0.005). For whole blood cultures (i.e. control and
-radiation challenge), NPB frequency was significantly and positively correlated with MNed cell frequency (r = 0.895, P < 0.001). Both markers of DNA damage were negatively correlated with NDI (r = –0.284 and r = –0.305, respectively, P < 0.005), which was opposite to the observation for MNed cells in isolated lymphocyte cultures. |
Discussion |
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The epidemiological evidence linking moderate wine consumption with reduced cardiovascular disease risk is compelling and evidence is also accumulating for potential protective effects against cancer (Boffetta and Garfinkel, 1990
; Renaud et al., 1998). Because increased oxidative stress is a recognized risk factor for both cardiovascular disease and cancer some investigators have examined oxidative damage to DNA. A human intervention study suggested that moderate wine consumption reduces the concentration of 8-hydroxydeoxyguanosine (Leighton et al., 1999). A cross-sectional study of pre-menopausal, non-smoking women showed a significant inverse correlation between alcoholic drink consumption and oxidation of lymphocyte DNA (Bianchini et al., 2001). These results are also supported by experiments in rodents which showed that ingestion of wine solids reduces DNA oxidation in the colon (Giovanelli et al., 2000).
The main questions that needed to be resolved are essentially the following: (i) which of the major wine components are protective against oxidative stress at concentrations that are physiologically relevant; (ii) what is the relative contribution of the wine components that are protective against oxidative damage to DNA; (iii) are the antioxidant effects of the various wine components additive or synergistic? The results we report in this study provide a first comprehensive assessment of the relative antioxidant properties of tartaric acid, C & C, ethanol and, finally, glycerol. We have used the CBMN assay because we have shown that this method enables the measurement of genotoxic and cytotoxic events to be measured efficiently. In addition, we have also shown that the CBMN assay is amongst the most sensitive methods for measuring DNA damage induced by ionizing radiation, superoxide, hydrogen peroxide and activated neutrophils (Fenech and Morley, 1986; Umegaki and Fenech, 2000). Although the CBMN assay does not provide information on specific DNA base oxidation, it has been shown to be more sensitive than the HPLC assay for 8-hydroxydeoxyguanosine in detecting DNA damage induced by X-rays and UV radiation (Kobus et al., 1993).
The results from our experiments suggest that the wine components may have small but significant effects on baseline DNA damage rate, with ethanol tending to increase micronucleus formation when compared with the other wine components. The other wine components or ethanol in combination with the other wine components tended to reduce DNA damage rate, although the differences were not significant when compared with the HBSS control. The genotoxic effect of ethanol is not surprising given that there is ample evidence in the literature that ethanol can induce chromosome breakage and sister chromatid exchange, particularly in those with polymorphisms that alter the activity of aldehyde dehydrogenase (Morimoto et al., 1994). We have not genotyped the volunteers in our study and therefore we are unable to say whether the genetic background was an important variable, however, future studies should consider this potential confounding effect. The isolated lymphocyte base-line experiments (Figure 4a) suggested a prevention of necrosis by all wine components except tartaric acid, which is consistent with a possible antioxidant action given that necrosis in this in vitro system is efficiently induced by oxidants such as hydrogen peroxide (Fenech et al., 1999). In whole blood cultures wine components reduced the NDI. However, it is difficult to interpret the significance of this observation given that correlation analysis indicates that a reduced NDI is weakly but significantly correlated with increased MNed cell frequency. The effects observed in vitro do not suggest that moderate wine consumption is likely to elevate genotoxicity, cytotoxicity or cell proliferation in vivo.
The results for the hydrogen peroxide challenge suggest that all wine components except tartaric acid significantly reduced the level of induced DNA damage, however, this decrease was at the expense of increased necrosis, with no effect on apoptosis. It is known that hydrogen peroxide challenge can cause cells to switch from apoptosis to necrosis as the main mode of cell death (Hampton and Orrenius, 1997; Fenech et al., 1999). It is therefore possible that the observed reduced MNed cell frequency may have occurred due to improved elimination by apoptosis and necrosis of cells with DNA damage. However, a quenching effect of wine components against reactive oxygen species generated by hydrogen peroxide is also a plausible explanation for the reduced MNed cell frequency. An alternative explanation is the possibility that some of the wine components may have been mildly pro-oxidant under the conditions of the experiment and may have exerted an adaptive response by inducing an up-regulation of antioxidant mechanisms within the cell. Perhaps the most interesting observation was that ethanol, glycerol and C & C were equally effective in reducing DNA damage induced by hydrogen peroxide, however, the mixture of all wine components was not more effective than the individual contribution of ethanol, glycerol or C & C. This suggests that the effect of the components in the mixture is not additive and definitely not synergistic. These results seem to indicate that the protective effects of ethanol, glycerol and C & C may be operating through a similar mechanism and that their protective effects are already at their maximum at the concentrations examined. The latter is also supported by the observation that there is virtually no difference between the extent of protection at the 2.5% dilution relative to the 10% dilution.
The results for effects on -radiation-induced DNA damage show that all the components of wine examined, including tartaric acid, significantly reduced MNed cell frequency and NPB frequency when present at the 10% dilution, while all but tartaric acid significantly protected against radiation-induced DNA damage at the 2.5% dilution, although to a lesser extent than that observed at the 10% dilution. Unlike the hydrogen peroxide challenge experiments, it is evident that the protective effect is not saturated at the 2.5% dilution. However, like the hydrogen peroxide challenge, the protective effect of the mixture was not greater than that of C & C and clearly did not exhibit an additive effect. An additive protective effect should have resulted in a reduction in MNed cell frequency of 64%, however, the observed reduction for the mixture was only 32%, which is similar to the effect of C & C (30% reduction) alone. Unlike hydrogen peroxide challenge, C & C was more effective than ethanol in reducing DNA damage induced by ionizing radiation. Therefore, it is apparent that wine and its components also protect against the genotoxic effect of ionizing radiation, however, it is still uncertain whether this protective effect is saturated at the 10% dilution. The lack of an additive effect suggests that the various wine components examined may be exerting their protective effect via a similar mechanism such as quenching of singlet oxygen, as has been reported recently for caffeic acid (Foley et al., 1999), or hydroxyl radical scavenging, as has been reported for alcohols (Worm et al., 1993). The observation that phenolic-stripped white wine was less protective than the mix also suggests that wine phenolics play an important role in the observed protective effects of the mixture. Our results support previous observations that ethanol and glycerol are protective against the cytotoxic effects of ionizing radiation (Worm et al., 1993) and that plant phenolic compounds prevent chromosome damage induction by ionizing radiation (Shimoi et al., 1994; Parshad et al., 1998; Virinda and Devi, 2001).
Taken together, the results of our experiments support the hypothesis that the major components of white wine (at physiologically relevant concentrations that are in proportion to their concentration in white wine) are protective against oxidative stress generated by hydrogen peroxide and ionizing radiation. The novelty of our results is the observation that both wine phenolics and ethanol/glycerol are protective to significant extents against the DNA-damaging effects of hydrogen peroxide and ionizing radiation and that these effects do not appear to be additive. The latter may have important implications with regard to the ongoing debate about whether the phenolics in wine provide a health advantage over alcoholic drinks that have much lower concentrations of these compounds. Also notable for this discussion is the observation that ethanol was the only wine component increasing baseline MNed cell frequency and that this effect was neutralized in the mixture of all components. The results we have obtained suggest that at physiologically relevant concentrations: (i) ethanol on its own is genotoxic compared with phenolics; (ii) that under conditions of hydrogen peroxide stress ethanol and phenolics are equally protective; (iii) under conditions of ionizing radiation stress phenolics appear to be more protective than ethanol and glycerol. Our results suggest the hypotheses that dealcoholized wines may be equally protective as complete wine but wines stripped of phenolics are likely to be less protective than complete wine and dealcoholized wines. These hypotheses need to be tested in properly controlled intervention studies. If these hypotheses turn out to be correct, they may explain why it has been difficult epidemiologically to distinguish between the health benefits of different alcoholic drinks drunk in moderation and why some studies suggest that wine may afford more protection than other alcoholic drinks with lower phenolic and higher ethanol contents (Klatsky and Armstrong, 1993; Gronbaeck et al., 1995).
Our future studies are now aimed at performing controlled intervention studies in vivo to determine whether alcohol, phenolic-stripped wine and whole wine differ significantly in their capacity to alter the antioxidant properties of blood ex vivo with respect to protection against hydrogen peroxide- and ionizing radiation-induced DNA damage.
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Acknowledgments |
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We are thankful to Creina Stockley and Dr Phil Burcham for their helpful discussions and suggestions during the course of these studies. We are grateful to the Department of Clinical and Experimental Pharmacology of the University of Adelaide for supporting the PhD candidature of Will Greenrod. This study would not have been realised without the help of the volunteers, the staff at the CSIRO Health Sciences and Nutrition Clinic and the occasional technical assistance of Julie Turner, Felicia Bulman and Phil Thomas. This study was supported by a PhD scholarship granted to W.G. by the Grape and Wine Research and Development Corp. (Australia).
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Notes |
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3 To whom correspondence should be addressed. Tel: +61 8 8303 8880; Fax:+61 8 8303 8899; Email: michael.fenech{at}csiro.au
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References |
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Received on July 4, 2002; accepted on September 30, 2002.
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