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Food Science and Human Nutrition Department, University of Florida, Gainesville, FL 32611
4To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-diphenyl-ß-picrylhydrazyl and was more than doubled in the
mice that consumed wine compared to control mice that consumed water or
ethanol. Cytochrome P450–2E1 levels and
glutathione-S-transferase activity were modified in such
a way as to be interpreted as protective. An immune response was
elicited by an intraperitoneal injection of lipopolysaccharide. Later
(24 h), natural killer cells and T-lymphocytes derived from the
circulation were quantitated in the leukocyte fraction by flow
cytometry. Ethanol consumption, as ethanol, significantly suppressed
baseline cell numbers relative to the other groups. However, the mice
that consumed the same amount of alcohol as wine had baseline cell
numbers not different from the water-consuming controls. The
lymphocyte response to lipopolysaccharide challenge was inhibited in
the mice that consumed ethanol, but was normal in those that consumed
the same amount of alcohol in the form of wine. We conclude that there
are phytochemicals acting as antioxidants and impacting on the
detoxification pathway in the wine that offset the detrimental effects
of ethanol on immunity.
KEY WORDS: • wine • immunity • alcohol • mice • phytochemical
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, Soleas et al. 1997
).
Ethanol, however, is known to suppress immunity. Chronic alcohol use
predisposes an individual to more frequent and more severe illness.
Although many environmental factors contribute to this, studies
indicate that ethanol is directly immunosuppressive. One underlying
mechanism for immunosuppression may be derived from free radical
damage.
Ethanol is a powerful generator of oxygen free radicals when detoxified
in the liver. A general schematic of ethanol detoxification is shown
below.
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Ethanol
acetaldehyde
Phase II enzymes
+glutathioneacetyl cysteine conjugate
Phase I enzymes are isozymes of the cytochrome P450
family and are one of the main mechanisms by which alcohol is
detoxified and free radicals are generated (Leiber 1997
,
Yang et al. 1991
). The secondary phase II enzymes, such
as glutathione-S-transferase
(GST)4
, are principally detoxifiers by conjugation mechanisms (Tew et al. 1993
). Conjugation increases solubility and excretion. The
balance between the phase I enzymes and the phase II enzymes dictates
detoxification and free radical generation. An increase in the activity
of phase 1 enzymes without a concomitant increase in phase II enzymes
would be potentially more damaging due to an increase in acetaldehyde
formation and subsequent free radical damage. A higher ratio of phase
II activity to phase I activity would be protective.
Studies have shown that alcohol consumption suppresses the immune
system. Reductions in natural killer (NK) cell activity (Blank et al. 1993
) and T-cell dependent reactions have been
documented (Baker and Johnson 1993
). If ethanol
consumption and its subsequent metabolism generate free radicals, and
free radicals damage the immune system, it would follow that
consumption of antioxidants would prevent immunosuppression by ethanol.
In recent studies, red wine has been shown to contain a large amount of
antioxidants in the class of phenolic compounds. Ellagic acid, gallic
acid and catechins are major contributors to the total antioxidant
potential of wine (Formica and Regelson 1995
,
Garrido et al. 1993
,Kanner et al. 1994
).
The objective of this study was to test the hypothesis that ethanol consumption in the form of wine would not suppress an immune response because of the antioxidant activity of the phenolics. We examined red wine consumption and plasma antioxidants, and we compared two wines from two species of grapes having different phenolic composition to explore potential mechanisms of protection.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Feeding studies with mice were begun after the wine had been stored for
about 1 y. Both wines contained ca. 12% alcohol determined by
ebulliometry (Zoecklin et al. 1990
). The cabernet
sauvignon had a titratable acidity (as tartaric acid) of 6.0 g/L and a
pH of 3.60. The muscadine wine had a titratable acidity of 6.9 g/L and
a pH of 3.00. The phenolic composition [as gallic acid and measured by
the Folin-Ciocalteau method (Zoecklin et al. 1990
)]
of the cabernet sauvignon was 912 mg/L and of the muscadine was 1731
mg/L.
Weanling ICR mice were purchased from Harlan Sprague-Dawley (Indianapolis, IN). All procedures were in accordance with the Institutional Animal Care and Use Committee of the University of Florida. The mice were housed in stainless steel cages with 12 h light/dark cycle and fed AIN93G diet (Dyets, Inc., Bethlehem, PA). They were randomly divided into four groups (n = 10) and given water, ethanol or one of the two types of wine in their drinking bottles. No other fluids were provided. Alcohol levels of the wines and the ethanol were adjusted to 6% with water. This adjustment was necessary to equalize the volume of fluid consumed among the four groups. Body weight and food intake were monitored weekly, and fluid intake was monitored daily. Mice were food-deprived overnight before killing. After 8 wk, six mice from each group were injected with 50 µg of lipopolysaccharide (LPS) in saline to produce an immune response. Four mice from each group were injected with saline alone and served as the nonstimulated controls. After injection (24 h), the mice were anesthetized with an i.p. injection of ketamine/xylazine/water (1:1:4), then exsanguinated via the inferior vena cava after opening the body cavity. Mice were then killed by cervical dislocation, organs were removed, flash frozen and stored at -80°C.
Blood was collected into EDTA-containing tubes. Whole blood was layered onto MonoPoly Resolving Medium® (density 1.114 kg/L) (ICN Biomedical, Aurora, OH) and centrifuged at 1000 x g for 30 min. Leukocytes sedimented to one density and were collected, washed in saline and adjusted to 1 x 1010 cells/L.
Oxidant/antioxidant status of the mice was measured by Glavind’s
method using -diphenyl-ß-picrylhydrazyl (DPPH) (Sigma Chemical,
St. Louis, MO) (Fauconneau et al. 1997
, Glavind 1963
). Plasma (100 µL) was mixed with 0.45 mmol/L of DPPH in
95% ethanol at a ratio of 1:5 (v/v) and the resulting loss of color at
517 nm was monitored in a DU64 spectrophotometer (Beckman Instruments,
Carpenteria, CA). Glutathione was measured in the cytosolic fractions
of the liver by the recycling enzyme assay (Baker et al. 1990
, Baker and Hagner 1990
). Liver
malondialdehyde levels were determined by the method recommended by
Jentzsch et al. (1996)
.
Liver microsomes were prepared by homogenization and differential
centrifugation. GST activity was measured with 4 mmol/L of 1-chloro-5,
6-dinitrobenzene and 4 mmol/L of glutathione in phosphate buffer, pH
6.5. Results were obtained kinetically in a microtiter plate reader at
405 nm (Kurata et al. 1992
). Cytochrome P4502E1 was
measured by Western blot analysis using a specific polyclonal rabbit
anti-mouse cytochrome P450–2E1 antibody (Chemicon, Temecula, CA).
Lanes of a 12% polyacrylamide gel were loaded with equal amounts of
microsomal protein, the proteins separated and transferred to
nitrocellulose. The amount of cytochrome P450–2E1 protein was detected
with a chemiluminescence kit and quantified by densitometry.
The percentages of lymphocytes and natural killer (NK) cells were determined by two-color flow cytometry from the isolated leukocyte fraction. Cells (2 x 106) were incubated with fluorescent-labeled antibodies against CD3 and NK1.1 (Pharmingen, San Diego, CA). Cells fluorescing with CD3 were counted as lymphocytes, while cells fluorescing with both NK1.1 and CD3 were counted as NK cells. The immune responses were quantified in the mice by measuring these cell numbers 24 h after an injection of a low dose of LPS.
Significant differences in the rate of body weight gained and the average daily fluid intake among the groups was determined by one-way ANOVA using SigmaStat for Windows, version 1.0. Significant differences in studies involving LPS were assessed by two-way ANOVA, with LPS as one variable and fluid as the other. Post-hoc test for significance used the Student-Newman-Keuls method.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Oxidative stress was not evident in any mice. Neither ethanol, wine nor
LPS stimulation caused any consistent or major changes in the levels of
malondialdehyde or glutathione in the liver (data not shown). The mice
that consumed the wines had a significantly greater plasma total
antioxidant capacity than the mice that consumed water or ethanol
(Fig. 1
). The plasma antioxidant capacity in mice consuming water was
significantly greater after LPS stimulation.
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). When the mice were injected with LPS, the normal response of
lymphocytes is to migrate out of the circulation into tissues and the
lymphatic systems. About 50% of the lymphocytes were gone from the
peripheral blood after water- and wine-consuming mice were injected
with LPS. There was no difference in the percentage of CD3 cells in the
ethanol-consuming mice with or without LPS injection.
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). In mice that consumed water, the NK cell population increased from
1% of the population to 40% of the population after LPS treatment.
The mice consuming cabernet or ethanol had an increase to percentages
not different from that of the water-consuming group. NK cell
numbers in the mice consuming the muscadine increased from 1% to
> 60%, a percentage that was significantly greater than those in
the cabernet-, ethanol- and water-consuming groups.
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). Muscadine-consuming mice, on the other hand, had cytochrome
P450–2E1 levels that were 90% lower than the water-consuming
controls. Cytochrome P450–2E1 levels in the ethanol-consuming mice
were not different than those in the controls.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Results of the DPPH-based assay for total plasma antioxidant capacity suggest that wine phenolic phytochemicals increased in the plasma of mice drinking wines. The data suggest that the plasma can accumulate sufficient and significant antioxidant activity in the blood when a moderate amount of wine is consumed on a daily basis. Although the muscadine wine had twice the phenolic content of the cabernet sauvignon, this difference was not apparent in vivo.
Consumption of ethanol lowered the percentage of lymphocytes circulating in the blood. Lymphocytes did not change further after LPS treatment, suggesting impairment in the ability to migrate out of the circulation. Alternatively, consumption of ethanol might have resulted in a situation in which cells were already gone from the periphery. Further migration could not be stimulated. Research will be necessary to distinguish between these explanations. What is the most interesting of these results is that consumption of the same amount of alcohol in the form of wine did not result in the same effects observed in the mice that consumed ethanol only.
A similar observation was made regarding the NK population. Ethanol
consumption lowered baseline percentages of NK cells relative to those
in the wine-consuming mice. LPS caused the percentage of peripheral
NK cells to increase. Why the mice that consumed the muscadine wine had
a significantly greater percentage of NK cells after LPS compared to
the other three groups is not known. Muscadine grapes have a unique
phenolic profile (Auw et al. 1996
) compared to the
cabernet; however, we do not know what component(s) may be responsible
for the higher proportion of NK cells after LPS stimulation. It may be
that this effect of muscadine on NK cells is not biologically
important. Nonetheless, the alteration in NK cell percentages due to
ethanol consumption was not evident in the wine-consuming controls.
Measurements of phase I and phase II enzymes suggest possible mechanisms by which the wines prevented changes in the percentages of the cell subpopulations. The mice consuming cabernet had greater activity of cytochrome P450–2E1 than controls. Although the ratio of GST activity to P450 activity was not different from the water-consuming controls, the higher activity of P450 suggests a greater flux through the pathway, and an overall greater formation rate of the less toxic conjugate. Consumption of muscadine had a much different effect on the phase I and phase II enzymes. Both cytochrome P4502E1 and GST were significantly lower than in the controls, with the reduction in P450 much greater than that in the GST. This resulted in a > 100% greater ratio of GST to P450 compared to water-consuming mice. Therefore, acetaldehyde conjugation was greater than acetaldehyde formation, lessening the formation of potentially damaging free radicals.
Cytochrome P450–2E1 is only one of many of the Phase I isozymes.
Similarly, GST is only one of many phase II enzymes. In this study,
ethanol consumption did not affect either of the two enzymes that were
measured. The induction of P450–2E1 due to alcohol consumption has
been well documented in the literature; however, the alcohol content in
those studies was 20% or more. We used a 6% solution of ethanol. A
10% ethanol concentration did not raise the 2E1 levels to a great
extent (Manson et al. 1997
). In our study, the ratio of
GST/P450 in the liver microsomes of the ethanol-consuming mice was
significantly lower than that of the water-consuming controls. A
lower ratio may imply free radical damage from acetaldehyde, although
we were not able to show damage by crude measures in the liver.
However, it does not rule out that it is possibly the mechanism by
which the immune cell populations were affected by ethanol.
In summary, moderate consumption of ethanol suppressed certain immune system variables. Phase I and phase II enzyme activities were altered such that acetaldehyde levels may have led to free radical damage. These results are interesting because they show that moderate consumption of the food product itself had physiological effects, rather than showing effects due to consumption of purified components, injections or high quantities. In mice consuming ethyl alcohol, the lymphocyte response to LPS was either suppressed or delayed compared to the mice that drank water. When the same amount of ethanol was consumed in the form of wine, the response to LPS was the same as in the water-consuming controls. Wine consumption also led to a significantly greater proportion of circulating NK cells compared to the ethanol-consuming mice. The NK cell response to LPS was significantly greater when muscadine wine was consumed compared to the other groups. In conclusion, it appears that some of the phytochemicals in red wine can overcome the detrimental effects of ethanol on the immune system. The protective effect may be related to alterations in the enzymes responsible for detoxification of alcohol.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 An abstract of this work was presented at the
1999 IFT meeting in Chicago, Illinois. Gordon, L. A., Percival,
S. S. & Sims, C. A. Alcohol, Consumption as Wine Modifies the
Detrimental Effects of Ethanol on Mouse Immunity, 1999.
3 This is Florida Agricultural Experiment Station
Journal Series number 07240.
5 Abbreviations DPPH,
-diphenyl-ß-picrylhydrazyl; GST,
glutathione-S-transferase; LPS lipopolysaccharide; NK,
natural killer.
Manuscript received June 29, 1999. Initial review completed July 29, 1999. Revision accepted November 22, 1999.
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