effects of dietary supplementation of a-tocopherol on ... · antioxidants in plasma; total plasma...

%‘()l. 5. 263-270. April 1996 Cancer Epidemiology, Biomarkers & Prevention 263

Effects of Dietary Supplementation of a-Tocopherol on Plasma

Glutathione and DNA Repair l

Jennifer J. Hu,2 George C. Roush, Marianne Berwick,Neil Dubin, Somdat Mahabir, Monica Chandiramani, andRobert Boorstein

Lombardi Cancer Center and Department of Pharmacology, Georgetown

University Medical Center. Washington. DC 20007 Ii. J. HI: Department of

Epidemiology and Biosiatistics. Memorial Sloan-Kettering Cancer Center, New

York. New York 10021 IM. B., S. Ml; and Departments of Environmental

Medicine Ii. J. H.. G. C. R.. M. B., N. D.. M. C.l and Pathology IR. B.I, New

York University. New York, New York 10010

Abstract

In a randomized double-blind trial of a-tocopherol(vitamin E), we investigated the effects of a-tocopherolsupplementation on lipid- and water-soluble antioxidantsin plasma and DNA repair activities in peripheralmononuclear leukocytes. Baseline levels of antioxidantsand DNA repair activities were assessed twice before a-tocopherol intervention: on day 1 (visit 1) and day 3 (visit2). During the second visit, participants were randomizedto receive one of three dosages of a-tocopherol, 15, 60, or200 mg/day for 4 weeks. The same biochemicalmeasurements as at baseline were repeated twice afterintervention: on day 17 (visit 3) and day 31 (visit 4). Atotal of 31 healthy volunteers were eligible for the study,completed all four visits and were included in the finaldata analysis. At baseline, no appreciable differences ofdietary intake of vitamin E and plasma a-tocopherolwere observed among the three dosage groups. Ingeneral, supplementation of a-tocopherol for 2-4 weeksresulted in a dose-dependent increase of plasma level ofa-tocopherol (compared to baseline); significant increasesof plasma a-tocopherol at visits 3 and 4 were observed inthe two higher dosage groups, 60 and 200 mg, but not inthe lowest dosage group, 15 mg. At visit 4 (but not visit3), plasma glutathione levels were significantly elevated(compared to baseline) in the two higher dosage groups,60 and 200 mg, but not in the lowest dosage group, 15mg. In addition, there was an increase in the lipidprotection ratio by supplementation of a-tocopherol for2-4 weeks in the two higher dosage groups, 60 and 200

mg, but not in the lowest dosage group, 15 mg. Ingeneral, there were no consistent effects of a-tocopherolon DNA repair activities in peripheral mononuclear

Received 6/5/95: revised I 1/28/95; accepted 12/20/95.The costs of publication of this article were defrayed in part by the payment of

page charges. This article must therefore be hereby marked adrertisemen: in

accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

� This study was supported by Preventive Oncology Award CA 01634 (to J. J. H.)and Cancer Center Grant CA 16087 to New York University Medical Center from

the National Cancer Institute, NIH.

2 To whom requests for reprints should be addressed, at Lombardi Cancer Center,

Georgetown University Medical Center, 3970 Reservoir Road. Research Build-

ing. E3l6. Washington. DC 2()007. Fax: (202) 687-5730.

leukocytes after being adjusted for baseline DNA repairactivities. Results from this study demonstrate theinterrelationship between a-tocopherol and otherantioxidants in plasma; total plasma antioxidants can bemodulated by short-term dietary supplementation ofa-tocopherol.

Introduction

Data from both animal and epidemiological studies suggest that

dietary antioxidants may have protective effects against certaintypes of cancer. Specifically, a-tocopherol (vitamin E), a well-studied lipid-soluble antioxidant, was associated with a reducedrisk of cancer in two longitudinal epidemiologic studies (1, 2)but not in two other studies (3, 4. The potential use of a-to-copherol in human cancer chemoprevention has been evaluatedin lung cancer (5), oral leukoplakia (6), and colorectal polyps

(7, 8). The antimutagenic and antipnoliferative effects of a-to-

copherol may contribute to its chemopreventive property (9).Intracellulanly, a-tocopherol is localized in the lipid-rich mem-

brane and may serve to protect the membrane against LPO3 byreacting with lipid peroxyl and alkoxyl radicals (10). Antioxi-

dants do not function alone; both lipid- and water-solubleantioxidants may interact with each other to achieve overallprotective action against oxidant-induced damage. For exam-ple, upon oxidation, the regeneration of a-tocopherol requires

further coupling in the reducing systems, such as GSH/gluta-thione disulfide and ascorbate (vitamin C; Ref. I I). In a study

of GSH in the red blood cells of humans, rabbits, and rats, oraladministration of vitamin E can increase the levels of GSH(12). Furthermore, in GSH-depleted hepatocytes, a-tocopherolhas protective effects against chemical-induced cellular injury,suggesting an interrelationship between a-tocopherol and GSH

in maintaining the cellular antioxidant defense system (I 3).DNA damage and defective DNA repair are associated

with the development of human cancer (14-16). Although

genetic factors can contribute to genomic instability and defec-tive DNA repair, environmental exposure and dietary factorsmay also play a role in regulating DNA damage and repair by

modulating the antioxidant/oxidant balance ( 17). Antioxidantscan protect cells from DNA damage by directly removingreactive free radicals, thus reducing the amount of DNA dam-age, which may lead to tumorigenesis. Accumulation of DNAdamage and/or cellular reduction/oxidation imbalance maycontribute to decreased DNA repair capacity (18, 19). Further-

more, different studies suggested that a-tocopherol can protect

cells against DNA damage and improve DNA repair activity

3 The abbreviations used are: LPO. lipid peroxidation; PML. peripheral mono-

nuclear leukocyte; GSH, glutathione; ADPRT, ADP-ribosyl transferase; UDS.

unscheduled DNA synthesis; MNNG. N-methyl-N’-nitro-N-nitrosoguanidine;

BMI, body mass index: DTNB, 5,5’-dithiobis-(2-nitrobenzoic acid): TCA. tn-

chloracetic acid; CV. coefficient of variation.

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264 Effects of a-Tocopherol on Glutathione and DNA Repair

count. 4 C. A. Thomas, Jr., personal communication.

(20-22). Markowitz ci a!. (20) showed that pretreatment oflymphocytes with a-tocopherol protected cells against cumen

hydroperoxide-induced activation of ADPRT, a nuclear en-zyme involved in DNA repair. Topinka et a!. (21 ) demonstrated

that vitamin E suppressed LPO induced by ferrous iron andreversed the induction of UDS by LPO in human lymphocytes.Sram et al. (22) showed that simultaneous administration ofvitamin E can prevent the inhibitory effect of fluphenazine onDNA repair activity.

To evaluate the potential role of a-tocopherol in the over-

all antioxidative defense system and DNA repair in humans, weinvestigated the effects of short-term supplementation of a-to-

copherol on lipid- and water-soluble antioxidants in plasma, aswell as DNA repair activities in PMLs, in a randomized double-blind chemoprevention trial.

Materials and Methods

Materials. Lymphocyte Separation Medium was purchasedfrom Organon Teknika Co. (Durham, NC). Dulbecco’s PBS,

RPMI 1640, and fetal bovine serum were obtained from LifeTechnologies-BRL (Gaithersburg, MD). Hydroxyurea, hydro-

gen peroxide (H,O,), DMSO, and MNNG were purchased fromSigma Chemical Co. (St. Louis, MO). [3H]thymidine (specific

activity: 20 Ci/mmol, 1 mCi/mI) was obtained from DuPont-NEN Research Products (Boston, MA). The d/-a-tocopherolacetate powder was provided by Hoffman LaRoche (Nutly, NJ),

and capsules with three dose levels were prepared by PasteurPharmacy (New York, NY).

Study Subjects. A total of 3 1 healthy subjects (out of a total

of 45) from New York City were eligible for the study; they

completed the study and were included in the final data anal-yses. During the first visit, all the subjects signed informed

consent as approved by the Institutional Review Board ofCancer Prevention Research Institute. Inclusion criteria were:

a) willing to participate and able to give informed consent; h)return for second visit on day 3; and c) over 18 years old.

Exclusion criteria were: a) history ofor active cancer; b) history

of abnormal white cell count (less than 3.0); c) treatment with

chemotherapy, radiation, or hormones within the past month; d)

life-threatening condition of any type; and e) use of a-tocoph-

erol or other micronutnient supplements. Information collectedfrom the baseline questionnaire included diet, alcohol con-sumption, smoking, medications, and vitamin use. Subjectsranged in age from 24 to 78 years and consisted of 23 females

and 8 males.

Study Design. During the first visit, subjects completed abaseline questionnaire that included demographic information,

medical history, family history of cancer, and (for women)reproductive history. Dietary intake was assessed by the NCIFood Frequency Questionnaire (23). Height, weight, and blood

pressure were obtained at the baseline interview. BMI was

computed as weight (kg)Iheight (m)2. All biochemical mea-surements were performed twice at baseline (days I and 3) and

twice after intervention (days I 7 and 3 1 ). After blood was

drawn on day 3 (visit 2), the subjects were randomized to

receive 15, 60 or 200 mg/day of a-tocopherol. The three typesof capsules were assigned three different codes by the statisti-cian (N. D.), who recorded the code in a sealed envelope and

retained the envelope unopened until the data analysis wascompleted. Neither the study participants nor the laboratorytechnicians had knowledge of the dosage level assigned. Corn-

pliance was measured at visits 3 and 4 by calendar and pill

Study Dosage. The average daily intake of a-tocopherol formale and female adults is 9.8 and 7.1 mg, respectively (24).

This is close to the RDA of 10 and 8 mg for male and femaleadults, respectively. To determine the dosage for this trial, wesought the optimal dosage for modulations of overall antioxi-dant defense system and DNA repair. We chose to test threedosages of a-tocopherol in this pilot study, 15 mg (1.5 X

average intake), 60 mg (6 X average intake) and 200 mg (20 X

average intake). The purpose of this study was to test a rangeof dosages that might have an effect on plasma antioxidants and

DNA repair activities.

Initial Sample Handling. At each visit, a total of 40 ml ofvenous blood were obtained from each subject by a trained

phlebotomist. Heparinized whole blood samples were collectedin a nonfasting state between 9:00 and I 1 :00 am., stored in adark container, transported to the laboratory within 2 hours of

phlebotomy, and processed for PML isolation immediately.After the plasma was isolated, the PML fraction was separatedby lymphocyte separation medium (LSM), a formulation basedon Ficoll-sodium diatrizoate as described previously (25).Freshly isolated PMLs were washed once with Dulbecco’s

PBS, resuspended in Dulbecco’s PBS, and used for DNA repairmeasurements (ADPRT and UDS). The plasma for micronu-trient analyses was frozen immediately and stored in liquid

nitrogen until assay.

Profile of Plasma Antioxidants. Frozen plasma samples withequal storage time (I year) were batched and shipped to PantoxLaboratories (San Diego, CA) on dry ice for measurement ofdifferent antioxidants. The lipid-soluble antioxidants were de-

termined by a high-performance liquid chromatography method

as described previously (26, 27) with improvements made byPantox Laboratories. Different aotioxidants were separatedwith a methanol-based mobile phase on a C- 18 column withlarge pore size and uniform particle size. These were monitored

by a programmable wavelength rnonochrometer followed by anelectrochemical detector. In a single run, data were obtained onantioxidants including retinol, a-tocopherol, lycopene, a-caro-

tene, a-carotene, and ubiquinol-lO. Other analyses (i.e., uricacid, total cholesterol, apolipoprotein B, and triglycerides) in-cluded in the Pantox Profile were performed on a Beckman

CX5 Autoanalyzer. The lipid protection ratio was calculated asthe sum of the � concentrations of the lipid-soluble antioxi-dants in plasma, which are embedded within the lipoproteinparticles, divided by the m� concentration of cholesterol. Thisratio is considered proportional to the number of antioxidantmolecules protecting the low density lipoprotein and lipopro-tein a particles. Because the lipid soluble antioxidant molecules

are inserted into the lipoprotein particles, the lipid protectionratio may serve as a measure of the “concentration” of theseantioxidants in that solution. It was suggested that lipid pro-

tection ratio may be a better measure of the level of antioxidantsin other compartments than the crude plasma level.4 Levels ofplasma a-tocopherol were adjusted for plasma triglycerides andcholesterol, as suggested by Horwitt (28).

Determination of Plasma GSH. The plasma GSH levels weremeasured by an enzymatic recycling method described by Grif-fith (29) and modified by Hu et a!. (30). The assay was based

on the oxidation of GSH by DTNB and the reduction byNADPH catalyzed by glutathione reductase. The assay wasinitiated by mixing 0.7 ml of 0.3 m�i NADPH, 0. 1 ml of 6 m�iDTNB, and 0. 1 ml of phosphate buffer [all solutions were



Cancer Epidemiology, Biomarkers & Prevention 265

Table I Distribution of study participants

Characteristics A ( 15 mg) B (60 mg) C (200 mg) P value

Age (mean) 41.0 35.5 34.9 0.51’

Sex

Female 8 9 6

Male 2 2 4 0.59”

Race

White 6 8 8

Non-white 4 3 2 0.42”

a-Tocopherol (jiM)’ 0.097 ± 0.020

(n10)

0.103 ± 0.014

(n11)

0.106 ± 0.015

(n=9)”0.41”

Vitamin F/intake (mg)� 7.33 ± 2.89

(a 10)

7.27 t 3.28

(n - 1 1)

1 1.05 ± 6.63

(n = 10)

0.118”

“ P value front ANOVA.

I, p value from Fisher’s exact test.

� Data are presented as mean ± SD of baseline adjusted plasma a-tocopherol levels.

‘I Total of 10 participants were randomized to group C; plasma sample for a-tocopherol measurement was not available from one subject.

� Data are presented as mean ± SD of baseline daily dietary vitamin E intake, estimated by the NC! Food Frequency Questionnaire.

prepared in buffer containing 125 msi sodium phosphate and6.3 rnsi sodium-EDTA (pH 7.5)J, with 0.1 ml plasma or buffer

as reference. The solutions were mixed in a microcentrifugetube at room temperature, and the absorbance was continuously

monitored at 412 nm after the addition of 5 �l glutathionereductase (50 units/ml). Total GSH content was determined byreference to a standard curve. The GSH standard curve wasprepared daily for each batch of the plasma samples to mini-mize the day-to-day variations of temperature, chemicals, andsolutions.

Quantitation of MNNG- and UV-induced UDS. Freshly iso-lated PMLs at the quiescent state were exposed to MNNG or

UVC (254 nm) and allowed to repair in the presence of[3H]thymidine as described previously (17) while hydroxyu-

rea was used to suppress semiconservative DNA replication.For MNNG-induced UDS, PMLs were divided into two

groups: the control group received 10 �l of 0.1% DMSOonly (solvent for MNNG), and the experimental group re-ceived a final concentration of 0. 1 msi of MNNG (dissolvedin 0. 1 % DMSO). The final DMSO concentration was0.002% in a 0.5-mi incubation mixture. For UV-inducedUDS, cells in the experimental group were irradiated withUV (60 i/rn2). The DNA repair reaction was initiated by theaddition of 5 �Ci of [3H]thymidine and hydroxyurea (finalconcentration, 10 mM). After incubation at 37#{176}Cfor 22 h, thereaction was terminated by the addition of 0.5 ml ice-cold

20% TCA/lO% sodium pyrophosphate and washed once with1 ml ice-cold 10% TCA/5% sodium pyrophosphate. RNAwas hydrolyzed with 0.5 ml KOH (0.33 N) at 37#{176}Cfor 60

mm, then 0.5 ml 20% TCA/10% sodium pyrophosphate wasadded, and the tubes were stored at -20#{176}Covernight. After

three more washes with 1 ml ice-cold 10% TCA/5% sodiumpyrophosphate, DNA was extracted with 0.8 ml 5% TCA at90#{176}Cfor 20 mm. The radioactivity (dpm) in 0.5 ml super-natant was measured by using a Wallac 1410 liquid scintil-lation counter (Pharmacia, Gaithersburg, MD).

ADPRT Enzyme Activity Assay. Two forms of ADPRT ac-tivity were experimentally determined. The physiological orconstitutive ADPRT activity is associated with cellular growth,expression, and differentiation. The activated ADPRT activityinvolves induction via the introduction of DNA strand breaks

with deoxyribonuclease, radiation, or carcinogens/mutagens.

After a standardized exposure to 100 p.M H2O2, both the con-stitutive and the activated levels of ADPRT were measured.

The procedure was adapted frotn the permeabilized cell

technique of Bergen (3 1 ) with modifications as described

(17, 32). Duplicate samples of 0.5 X 106 PMLs were cul-

tuned in PBS with different concentrations of autologous

plasma (0, 1, and 20%) for 30 mm at 37#{176}Cin the presence

or absence of 100 ,,LM of H2O2. The cells were harvested by

centrifugation at 500 x g for 10 mm and then permeabilized

at 4#{176}Cby suspension for 15 mm in 1.5 ml of ice-coldpermeabilization buffer [10 mrsi Tris-HC1 (pH 7.8)-l msi

EDTA-30 mM 2-mercaptoethanol-4 mrvi MgCl2]. The cy-toskeletons were recovered by centrifugation and then re-

suspended in 91 p1 reaction mixture: 47 pA permeabilization

buffer, 28 �l 100 mM Tnis-HC1 (pH 7.8), 120 mrsi MgCl,,

10.8 p1 ofnicotinamide adenine dinucleotide (NAD) (2 mM),

and 5 �l of [3Hladenine-labeled NAD� (final concentration,

220 jLM). Incubation was conducted at 30#{176}Cfor 15 mm. The

reaction was terminated by addition of 0.5 ml 3 M NaCI at

60#{176}Cfor 10 mm. The ADP-ribose-protein complexes were

precipitated with ice-cold 7% TCA and then collected ontonitrocellulose filters (Millipore HAWP 02500). The data

were recorded as dpm TCA precipitable (ppt) [3Hladenine-labeled NAD�/0.5 x 106 cells in the presence (activated) or

absence (constitutive) of 100 �M H2O2.

Statistical Analysis. All the assays for antioxidants and DNArepair were carried out four times: day I (visit I), day 3 (visit

2), 2 weeks after supplementation (visit 3), and 4 weeks after

supplementation (visit 4). Assay values of visits 1 and 2 were

averaged to provide a baseline value. Fixed-effect ANOVA was

used to assess differences in plasma a-tocopherol and GSH atbaseline among the three dosage groups. Paired t tests and

paired Mann-Whitney-Wilcoxon tests were used to assess aug-

mentation in plasma a-tocopherol or GSH at either visit 3 or 4

compared to baseline, for each of the dosage groups separately.In addition, repeated-measures ANOVA was used simulta-

neously to compare assay values at visits 3 and 4 to baseline for

each of the dosage groups separately. Repeated-measures

ANOVA was also used to compare changes in assay levels overtime among the three dosage groups. In the ANOVA models,

covariate adjustment for baseline levels of plasma-adjusteda-tocopherol was always included. All statistical analyses were

carried out by using the Statistical Analysis System (SAS

Institute, Cary, NC) on a personal computer. Results were

considered significant at the two-sided P < 0.05 level.




Table 2 Effects of ca-tocopherol supplements on plasma-adjusted a-tocopherol levels�’

a-tocopherol dose . . . . . .

. 11 Baseline, (SM Visit 3, �zM % of baseline Visit 4, �.sM(mg/day)

.of baseline

15 10 0.097 ± 0.020 (0.091 )“ 0.09l ± 0.023 (0.090) 101’ 0.1 10 ± 0.027 (0.107)

fit) II 0.103±0.014(0.103) 0.132±0.027(0.136)” 131 0.129±0.031(0.124)”

200 9 0.106 ± 0.015 (0.1 14) 0.161 ± 0.031 (0.15IY 146 0.146 ± 0.035 (0.141)”

118

127

138

“Repeated measures ANOVA, P = 0.022, for testing dose effects over time.

I, Data are presented as mean ± SD (median).‘ (‘�f of baseline, mean of the paired difference within each individual.

‘I p < o.os. visit 3 or visit 4 compared to baseline, by paired Wilcoxon test.

� P < 0.01, visit 3 or visit 4 compared to baseline, by paired Wilcoxon test.

Table 3 The effects of a-tocopherol supplements on plasma glutathione levels”

a-tocopherol . . . . . .Ft Baseline. p.� Visit 3, j.�M % of baseline Visit 4, �iM

(mg/day).

% of baseline

15 10 31.7 ± 13.4 (32.4)” 29.2 ± 16.3 (33.6) 89’ 29.5 ± 14.9 (34.1)

60 1 1 33.7 ± 7.1 (34.6) 35.5 ± I 1.5 (36.3) 1 10 40.4 ± 12.0 (39.9)”

200 10 33.1 ± 13.5 (38.2) 37.3 ± 6.6 (39.5) 141 42.2 ± 6.6 (39.9)”

106

126

167

“ Repeated measures ANOVA, P = 0.024, for testing dose effects over time.

S Data are presented as mean ± SD (median).

‘� C/� of baseline is the mean of the paired difference within each individual.

‘I p < 0.05, visit 3 or visit 4 compared to baseline, by paired Wilcoxon test.

Results

Quality control procedures were applied to all laboratory anal-yses. Pantox Laboratories measured a “high” and “low” control

every day before or after running samples. These controls wererecorded, and if they moved out of range, corrective action was

undertaken. The CV for repeated determinations on subsequentdays range from 2% (ascorbate) to 10% (u-carotene). For DNArepair measurements, repeated determinations could not be

performed because freshly isolated PMLs were used; however,a human lymphoblastoid cell line, CCRF, was used as externalquality control sample. The week-to-week variation of ADPRT

assay was determined over a 35-week period; the CV rangedfrom 3 1% (constitutive) to 37% (activated). The CV for dupli-cate samples ranged from 7% (UV-UDS) to 22% (ADPRT).

A total of 31 (23 females and 8 males) healthy volunteers

completed the study and were included in the final data anal-ysis. Characteristics of study subjects by dosage group, includ-ing age, sex, race, baseline plasma a-tocopherol levels, andbaseline dietary intake of vitamin E are summarized in Table 1.There were no significant differences in sex, race, age, andbaseline dietary intake of vitamin E by dosage group. The mean

adjusted plasma a-tocopherol level was 0. 102 ,.tM for all par-ticipants in the study, and there was no evidence of appreciable

differences among the three dosage groups at baseline(ANOVA, P = 0.41). The average age for females was slightlyolder than for males (38 versus 33 years, respectively); how-

ever, the difference was not significant (t test, P = 0.34). Therewas no difference in BMI between males and females (t test,P = 0.91). Most of the plasma micronutrients at baseline weresimilar by sex; however, mean uric acid levels were signifi-

cantly higher (P = 0.0072) in males (336.8 j.�M) than females(250.7 .LM). Most measures of baseline DNA repair capacitywere similar in males and females (induced ADPRT with 0, 1,

and 20% plasma and UV-induced UDS); however, meanMNNG-induced UDS was significantly higher in males (3341

dpm/l X 106 cells) than females (2018 dpmll X 106 cells). Allof the ANOVAs were adjusted for age, sex, and BMI. Because

this did not alter the results, only the results from unadjustedANOVAs were reported.

For none of the numeric variables were there significant

deviations from normality. However, because of the smallsample size, all of the ANOVAs were repeated with variables

log-transformed and confirmed the results of the untransformedanalyses. The effects of a-tocopherol supplements on adjustedplasma a-tocopherol levels are summarized in Table 2. The

overall test for differences in adjusted plasma a-tocopherol

among dosage groups over time was statistically significant

(repeated measures ANOVA, P = 0.022). At visits 3 and 4,adjusted plasma a-tocopherol levels were significantly in-creased compared to baseline among subjects in the two higher

dosage groups, 60 and 200 mg, but not in the lowest dosage

group, 15 mg. Adjusted plasma a-tocopherol was significantlyincreased by 27 and 38% at visit 4 in the 60- and 200-mg

dosage groups, respectively. The level of increase was similarat visits 3 and 4, which suggested that the increases may stay

relatively constant during the period of 2 weeks between visits3 and 4.

The effects of a-tocopherol supplements on plasma GSHlevels are summarized in Table 3. The mean overall baseline

GSH was 32.9 p.g/ml for all the participants in the study. Theplasma GSH levels were similar in the three dosage groups at

baseline (ANOVA, P = 0.92). The overall test for differencesin GSH among dosage groups over time was statistically sig-

nificant (repeated measures ANOVA, P - 0.024). At visit 4(but not visit 3), GSH was significantly increased (compared tobaseline) in the two higher dosage groups, 60 and 200 mg, butnot in the lowest dosage group, 15 mg. For example, there wasa 67% increase of plasma GSH (mean of the paired difference

within each individual) from 33.1 to 42.2 p.g/ml in the 200-mg

dosage group (paired Wilcoxon test, P < 0.05).To demonstrate the variations in the response of each

individual to a-tocopherol, Figs. 1 and 2 graphically show thewithin-subject augmentation of plasma a-tocopherol and GSHby dosage group. The mean lipid- and water-soluble antioxidant

levels for each dosage group at baseline, visit 3, and visit 4 aresummarized in Table 4. In addition to adjusted a-tocopherol

and GSH, significant dosage effects were also seen for a-car-otene and a-carotene, although the changes in these two van-



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Baseline Visit 3 Visit 4 Baseline Visit 3 Visit .1 Baseline Visit. 3 Visit 4

(A) (B) (C)

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Fig. 1. Within-subject augmenta-

tion of plasma a-tocopherol. Data

points for each subject are presented

as before (baseline) and after (visits 3and 4) a-tocophenol intervention. A,

IS-mg/day dosage group; B, 60-mg/

day dosage group; C. 200-mg/day

dosage group.

Fig. 2. Within-subject augmenta-

tion of plasma GSH. Data points foreach subject are presented as before

(baseline) and after (visits 3 and 4)a-tocopherol intervention. A, l5-mg/

day dosage group; B, 60-mg/day

dosage group; C. 200-mg/day dos-

age group.

0

0.00.0

0

Baseline Visit� 3 Visit 4 Baseline Visit� 3 Visit 4 Baseline Visit 3 Visit 4

ables were not consistent over time. Increases in the lipidprotection ratio were consistent, but of only borderline statis-tical significance (P 0.058).

Data on DNA repair activities are summarized in Table 5separately for each dosage group and time of visit. There were nodose-dependent effects over time on any of the DNA repair mea-

surements by a-tocopherol supplements under our experimentalcondition. To help elucidate the findings in Tables 4 and 5, and fora selection of variables, the mean within-dosage augmentation(compared to baseline) was calculated in the three dosage groups

at visits 3 and 4. For example, at visit 4, subjects in the 15-mgdosage group experienced a O.0l4-�tM mean increase in plasma




5 Unpublished observation.

Table 4 The effects of a-tocopherol supplements on plasma antioxidant defense system and lipid status

Baseline Visit 3 Visit 4Variables

A” B C A B C A B

Repeated measuresS

ANOVA P valueC

Lipid-soluble antioxidants

Unadjusted a-tocopherol (�isi) 24.2 28.9 22.9 26.7 37.0’ 38.3” 30.6’ 35.8” 35.9” 0.062

Adjusted cs-tocopheroC 0.10 0.10 0.1 1 0.09 0.13’ 0.16” 0.1 1 0.13’ 0.l5� 0.022

a-carotene (�.sM) 0.16 0.33 0.13 0.20 0.25’ 0.19” 023d 0.24 0.20 0.006

p-carotene (�zM) 0.46 0.76 0.36 0.50 0.58’ 0.47 0.64’ 0.63’ 0.50 0.008

Lycopene (�.sM) 0.66 1.10 0.84 0.76 1.04 0.93 0.81 1.06 0.95 0.598

Lipid protection ratio� 5.79 6.39 5.23 5.47 9.15’ 8.77” 6.82 8.00’ 8.14’ 0.058

Water-soluble antioxidants

Vitamin C (;.LM) 44.9 42.0 47.6 53.1 37.0 61.0 45.1 48.8 51.4 0.078

Uric acid QzM) 270 282 267 285 277 318 278 282 281 0.633

GSH (�g/ml) 31.7 33.7 33.1 29.2 35.5 37.3 29.5 40.4’ 42.2’ 0.024

“ Dosage groups: A, 15 mg/day; B, 60 mg/day; C. 200 mg/day.

/‘ For testing dose effects over time.

‘ p < 0.05, compared to baseline, one-sample Wilcoxon test.

,i p < 0.01 , compared to baseline, one-sample Wilcoxon test.,. a-tocopherol concentration divided by plasma triglycerides and cholesterol.

I Ratio of total micromolar concentrations of all lipid antioxidants divided by the concentration of cholesterol (SM).

Table 5 Effects of a-tocopherol supplements on DNA repair measurements

Baseline Visit 3 Visit 4Variables

A” B C A B C A B

Repeated measuresb

ANOVA P valueC

ADPRT (dpml0.5 x 10” cells)

0’/e plasma 20342 20532 18421 24865 23072 13064’ 21742 18774 20633 0.332

1% plasma 20080 19317 15053 21410 17282 11881 18399 17058 15827 0.381

20’T plasma 3613 4133 2671 4416 3795 2163 3719 3616 4027 0.566

UDS (dpmll X l0�’ cells)

MNNGinduced 2034 2611 2411 2007 2366 2473 1564 2910 2938 0.796

UVC induced 2363 2020 2460 2014 2109 1399 1959 2103 2741 0.793

“ Dosage groups: A, 15 mg/day; B, 60 mg/day; C, 200 mg/day.

I, For testing dose effects over time.

‘ P < 0.05. compared to baseline, one-sample Wilcoxon test.

adjusted a-tocopherol, compared to 0.026 p.M in the 60-mg groupand 0.037 �.LM in the 200-mg group.

Discussion

Reactive oxygen species, which can damage DNA, proteins,carbohydrates, and lipids, are constantly generated in cells;

a-tocopherol can eliminate pro-oxidants, decrease DNA dam-age, and function as part of an antioxidant defense system. Thepotential role of micronutrients and related compounds in thecellular antioxidant defense system and their interrelationshipwere previously proposed (33). In this study, we demonstrateda dose-dependent increase of plasma a-tocopherol and GSH

after short-term supplementation of a-tocopherol. Two mech-anisms are proposed: a) a-tocopherol has a “sparing effect” onGSH levels as well as other antioxidants; and b) a-tocopherolmay affect the regulation of enzymes that are involved in thesynthesis and/or metabolism of GSH. Data from this studysuggest that different components of the antioxidant defensesystem interact with each other and provide an overall protec-

tion against reactive oxygen species.

Several studies have suggested that the chemopreventiveproperties of a-tocopherol include reducing micronuclei fre-quencies (6), preventing peroxidative damage to DNA by di-

etary polyunsaturated fat (34), and inhibiting mammary tumorsinduced by 9,lO-dimethyl-l,2-benzanthracene (35). In thisstudy, we evaluated an additional potential mechanism of a-to-

copherol in cancer prevention: modulation of DNA repair ac-tivities. Our earlier studies have shown that DNA repair activ-ities can be modulated by oxidant/antioxidant balance;pretreatment of PMLs with H2O, or buthionine sulfoximine

(depletion of GSH) resulted in a decrease of DNA repairactivity (17, 36). Furthermore, our preliminary results from a

colon cancer case-control study suggested a positive correlationbetween plasma GSH level and UV-induced UDS in PMLs.5 Inthis study, however, we did not detect any consistent effects ofa-tocopherol on three DNA repair measurements, ADPRT andUV- and MNNG-induced UDS.

Data from this study should be interpreted with cautionbecause several factors may affect the study results. It is obvi-

ous that a sample size of 10 in each dosage group with a trialperiod of 4 weeks allows only preliminary conclusions andmust be considered as a pilot project for a larger confirmatorystudy in the future. Furthermore, under our experimental con-

ditions, assay for DNA repair activities by UDS may be influ-enced by the initial DNA damage levels. Two confoundingfactors may affect our ability to detect the effects of a-tocoph-

erol on DNA repair: a) supplementation of a-tocopherol mayprotect cells from DNA damage and result in lower DNA repairsynthesis, which may even out the effects of a-tocopherol onDNA repair; and b) if there is a delayed response to a-tocoph-




erol, a longer trial period may be required to evaluate its effects

on DNA repair. However, it is also possible that the protective

effect of a-tocopherol against carcinogenesis may support amechanism for the protective effect of a-tocopherol against

DNA damage, without affecting DNA repair activities. Wei et

a!. (37) reported that the use of vitamin supplements was not

correlated with DNA repair capacity in T lymphocytes trans-fected with a damaged plasmid. However, plasma antioxidantswere not measured in their study; the relationship betweenantioxidants and DNA repair activity remains to be explored.

In this study, short-term supplements of a-tocopherol in-

crease both plasma a-tocopherol and GSH in a time- and

dosage-dependent fashion, but do not have consistent effects onDNA repair activities. Handelman et a!. (38) raised importantissues concerning the steady state after a change of a-tocoph-

erol intake, which should be addressed in future trials. It wasestimated that more than 2 years are required for the a-tocoph-erol/’y-tocopherol ratio to reach a new steady state after achange in a-tocopherol intake. Also, in a cross-sectional mea-

surement in five subjects who reported long-term use of a-tocopherol supplements (�250 mg/day), and in five other sub-

jects who reported no supplement use, the adipose a-tocoph-erol/y-tocopherol ratio clearly discriminated between the twogroups. In the future, a longer trial period of a-tocopherol maybe required. In addition, the adipose a/’y-tocopherol ratio may

be valuable in ranking individuals according to long-term a-to-copherol intake (38).

The normal intake of vitamin E in United States dietsranges between 4 and 22 lU/day in adults who are not taking

vitamin E supplements (average values, 1 1-13 lU/day). Thisintake maintains an average blood level of approximately 23p�M, very similar to the mean baseline plasma a-tocopherollevel of 27.9 ,.aM among our subjects. Concerning the selection

of appropriate cohort for dietary chemoprevention studies, oneimportant concept is that supplementation of a-tocopherol tosubjects on a nutritionally adequate diet does not always pro-vide additional benefit or protection (39); therefore, supplemen-

tation of micronutrients to subjects on a nutrient-deficient diet

may provide maximal benefit or protection against cancer. In

summary, short-term supplements of a-tocopherol can modu-

late the overall antioxidant defense system but without consis-tent effects on DNA repair activities. On the basis of the resultsfrom this small pilot study, future confirmatory studies should

address import issues related to the selection of study cohort,the optimal dose of a-tocopherol, the duration of trial, and thespecificity of DNA repair assays.

Acknowledgments

The authors thank all the participants in the study; Hoffman LaRoche for pro-

viding the study compound; Dr. Bing L. Ma, Betty Hom, and Elizabeth Hom for

technical assistance: and Dr. C. A. Thomas, Jr. (Pantox Laboratories) for helpful

discussion.

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