A Comparison of Lymphocyte Micro Nuclei and Plasma Micro Nutrients in Vegetarians and...

8/3/2019 A Comparison of Lymphocyte Micro Nuclei and Plasma Micro Nutrients in Vegetarians and Non-Vegetarians

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Carcinogenesis vol.16 no.2 pp.223-23O, 1995

A comparison of lymphocyte micronuclei and plasmamicronutrients in vegetarians and non-vegetarians

Michael Fenech1 and Josephine RinaldiCSIRO Division of Human Nutrition, PO Box 10041, Gouger Street,Adelaide, SA 5000, Australia'To whom correspondence should be addressedWe performed a biochemical and cytogenetic epidemiolog-ical study to establish if there are significant differencesbetween vegetarians (V) and non-vegetarians (NV) in theirperipheral blood lymphocyte micronucleus (MN) index,which is a measure of chromosome damage rate. The levelsof plasma vitamin C (VIT-C), vitamin E (VIT-E), vitaminB 12 (B12) and folic acid were also analysed to assess ifdifferences in chromosome damage rates were associatedwith these potentially antim utagenic micronutrients. Volun-teers were classified as either 'vegetarian' if they hadabstained from eating any flesh foods for at least 3 yearsprior to the study or 'non-vegetarian' if they consumedmeat or meat products at least 5 days/week for at least 3years before participation in the study. The volunteers inthe study consisted of 47 male and 79 female V and 66male and 72 female NV, all of whom were non-smokers forat least 3 years prior to the study. The age of the volunteersvaried between 20 and 89 years. There was no significantdifference in the slope of the age-related increase in MNindex of V and NV of either sex. However, the MN indexwas significantly lower in NV males in the age group 20-40 years and significantly lower for V males in the 41-60years age group. No difference between the MN index ofolder males was detectable and there also was no differencein the MN index of V and NV females across all age groups.V were generally found to h ave significantly higher plasmalevels of VIT-C and folic acid, significantly lower levels ofB12, and similar levels of VIT-E when compared with NV.VIT-C correlated positively w ith MN ind ex in young males,but the reverse was true for B12. In young females folateand B12 appeared to correlate negatively with MN index.VIT-E had no apparent impact on MN index. These datasuggest that the level of folate and B12 may be moreimportant than VIT-C or VIT-E in minimizing chromosomedamage rates in human lymphocytes. Overall, the datafrom this study do not support the hypothesis that V havea lower genetic damage rate than NV.

IntroductionSeveral studies have established that the development of cancerinvolves the accumulation of several m utations at critical gen esstarting at initiation throug h to progression and m etastasis (1 -5) . The rate at which cancer develops is dependent on germlineAbbreviations: SCE, sister chromatid exchange; V, vegetarian; NV, non-vegetarian; CBMN assay, cytokinesis-block micronucleus assay; MN, micro-nucleus; VIT-C, vitamin C; VIT-E, vitamin E; B12, vitamin B l2 ; PHA,phytohaemagglutinin; BN, binucleated cell; MNi, micronuclei; CHOL, choles-terol; Mf, MN frequency in females; Mm, MN frequency in males. Oxford University Press

mutations that are inherited, as well as spontaneous mutationsoccurring as a result of endogenous or exogenous genotoxicagents. It is logical to assume that a decline in genetic damagerate may postpone the onset of cancer and it is, therefore,interesting to determine if genetic damage rates are reducedin those populations that are likely to experience a lowercancer rate due to genetic or lifestyle factors. It has beenestablished that in cancer-prone syndromes, such as ataxiatelangiectasia and Bloom's syndrome, the spontaneous chromo-some damage rates are abnormally elevated (6-8). Conversely,one could expect that in populations, such as the ovo-lactovegetarian Seventh Day Adventists, for which there is adocumented lowered cancer rate for specific cancers whencompared with omnivores (9,10), one might expect that muta-tion rates are relatively low. In view of the emerging interestin vegetarianism (11), it is important to establish if such alifestyle is in fact linked with lowered genetic damage rates.

To the best of our knowledge there have been only tworelatively small studies comparing sister chromatid exchanges(SCEs*) in vegetarians (V) and non-vegetarians (NV) andboth of these involved Seventh Day Adventists (12,13). Thesestudies showed that SCEs in lymphocytes of children ofSeventh Day Ad ventists were similar to those of children fromthe general population, but the SCE level in adult members(mean age 65 years) of this religious group was significantlylower (by 30%) than that of the general population. Interpreta-tion of these studies was limited by the num bers of subjects andlack of verification of nutrient intake by biochemical analysis.In view of our ongoing research on the impact of diet andother environmental factors on chromosome damage rateassayed using the cytokinesis-block micronucleus (CBMN)assay (14-16), we were interested in: (i) determining if themicronucleus (MN) frequencies in lymphocytes of V and NVare different; (ii) assessing if the levels of micronutrients thatmay be expected to influence chromosome damage rate aredifferent in V and NV; and (iii) establishing if any differencesin MN index could be accounted for by differences in antimuta-genic micronutrients. The micronutrients analysed were theantioxidants ascorbic acid (VIT-C) and a-tocopherol (VIT-E)and the B vitamins folic acid and vitamin B | 2 (B12).Information from such a study should be important because:

(i) it would help establish the extent to which differencesin diet could confound genetic damage studies in humanpopu lations; (ii) micronutrients that could significantly modifychromosome damage rates in vivo may be identified; (iii) itmay help determine if 'normal' micronutrient intakes areoptimal for minimizing chromosome damage rate; and (iv) itmay provide the baseline data required for the appropriatedesign of intervention studies investigating the antimutageniceffects of specific micronutrients.Materials and methodsVolunteer recruitmentVolunteers were recruited by placing an advertisment in local newspapers andby direct contact with various local vegetarian and vegan societies. The223


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M.Fenech and J.Rinaldi

recruited volunteers consisted of 150 females and 114 males aged between20 and 89 years, with a minimum of 15 volunteers/decade for both males andfemales. A total of 126 volunteers were classified as V on the basis that theyhad abstained from eating any flesh foods (red meat, chicken or fish) for atleast 3 years prior to the study and 138 were classified as NV on the basisthat they consumed meat or meat products at least 5 days/week for at least3 years before joining the study. All volunteers who were current smokers,smokers who had ceased smoking less than 3 years before the study, diagnosedwith cancer or had regular complicating usage of medicine were excludedfrom the study.QuestionnairesThe questionnaire for details on lifestyle, occupation and health status wasidentical to the one published by Carrano and Natarajan (17) and the dietaryquestionnaire used was developed at the CSIRO Division of Human Nutrition(18). Detailed analysis of dietary data from these questionnaires and theirrelation to MN frequency data will be reported separately.Blood collection and CBMN assayTo avoid possible confounding by dietary metabolites and diurnal effects, allvolunteers participating in this study were required to fast overnight and todonate a blood sample between 8.00 and 11.00 a.m. before having breakfast,which w as provided. Betwe en 10 and 30 ml blood was collected in heparinizedplastic tubes. Lymphocytes were isolated on Ficoll-Hypaque gradients andthe standard CBMN assay was employed as previously described (14,19).Briefly, lymphocy tes were cultured in plastic round-bottom tubes at a concentra-tion of lX10 6/ml in McCoy's 5A medium, stimulated to divide with phyto-haemagglutinin (PHA) (Wellcome) and 44 h later 4.5 |lg/ml cytochalasin-B(Sigma) were added to accumulate cells that had divided once only asbinucleated cells (B N). Cells were then harvested by cytocentrifugation at72 h post-PHA stimulation, air-dried, fixed in methanol and stained using

Diff-Quik (LAB-A IDS, Australia). Micronuclei (MN i) were scored in 1000 BNaccording to published criteria (14). The same person (J.R.) performed theCBMN assay and scored the slides for the whole of this study to minimizevariation due to operator differences. Furthermore, slides were coded so thatthe operator was not aware of the age, sex and dietary habits of the donorswhen scoring the slides.Analysis o f plasma micronulrientsFresh plasma was obtained from the heparinized blood and snap frozen inliquid nitrogen. Each sample was analysed in duplicate for the four micro-nutrients using the following techniques: B12 and folic acid were measuredsimultaneously using radioimmunoassay (BioRad, Quantaphase II); VIT-Cwas measured spectrophotometrically u sing the dinitrophenylhydrazine m ethodof Roe and Kuether (20); and VIT-E was measured using HPLC by themethod of Hatam and Kayden (21). Because VIT-E is lipid soluble, it is oftenreported as a ratio with cholesterol (CHOL) concentration. We have thereforealso measured CHOL, using a COBAS analyser and an in vitro diagnostic kit(Roche Products) and computed the VIT-E:CHOL ratio.Statistical ana lysis of dataDescriptive statistics, comparison s between groups, correlations and regressionanalysis were performed using the procedures described in Instat (GraphPadSoftware Inc.) and CSS Statistica (StatSoft). Regression analysis of therelationship between MN frequency and age was performed using the leastsquares method; for regression and correlation analysis missing data werecase-wise deleted.

Initial data analysis of plasma micronutrients (16) indicated that there wereweak, but significant, correlations between micronutrient levels and betweenmicronutrient status and age. To avoid the potential confounding effects ofcollinearity when using multiple regression analysis, we first age-adjusted(16) and then log-transformed the age-adjusted MN frequency of each

MALE NON-VEGETARIANS10 0

38ooo

8 0

6 0

r=0.564P


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Micronuclei, micronutrients and vegetarianismindividual and then determined the regression weight of each micronutrientindependently using simple regression analysis.The non-parametric Mann-Whitney U-test was used for comparisonbetween groups using the data for each individual within a group; MN datawere age- and sex-adjusted prior to analysis to minimize the confoundingeffect of these important variables (14,15).ResultsPreliminary analysis of the pooled data showed that MNfrequency in females (Mf) was significantly higher than theMN frequency in males (Mm) in all decades examined (P _ J_ lLJOCO8ocr1, |Q .X

35302520151050

20-40Y

N=15 N=18*

41-60Y 61-90Y

N=16 N=24

V NV V NV V NV(B)

MN FREQUENCY IN VEG. AND NONVEG. FEMALES

o

40353025

8 20a.Ida.

151050

20 -40Y 41-60Y 61-90Y

V NV V NV V NVFig. 2. Comparison of MN frequency in (A) V and NV males and (B) Vand NV females. To facilitate comparison of data between sexes, data forfemales were sex-adjusted to male values using the 1.53 factor described inthe results section. *P = 0.024 (one-tailed test); ** P = 0.027. Significancewas determined using the Mann-Whitney one-tailed test.

influencing the MN index, we analysed the data for femalesand males separately and in each case comparisons for MNiand micronutrients were performed after the individuals weresubdivided into three age groups, namely 20-40, 41-60 and61-90 years. The MN data for each individual in each agegroup were adjusted to the median age of each group (i.e. 30,50 and 75 years respectively) using the slopes of the age-related regression lines illustrated in Figure 1.The MN index in 20-40-year-old males was significantlylower in NV when compared with V, but in the 41-60-year-old males the reverse was true, i.e. the MN index wassignificantly higher in NV males (Figure 2A). In the 61-90-year-old males there were no significant differences betweenV and NV. In females there were no significant differences inthe MN index of V and NV in all of the age groups examined,however, there was a trend for higher MN frequency in NVin the 41-60-year-old group (Figure 2B).Plasma B12 values in V males were on average lower thanthat for NV males (Figure 3A). The differences between Vand NV males were most pronounced in the 20-40-year-oldgroup, in which the mean plasma B12 of V was 160.2 pmol/1and that of NV was 352.4 pmol/1. The extent of this difference

(A)PLASMA B12 IN VEG. AND NONVEG. MALES

500450400350300250200150100500

2 0 - 4 0 y 41-60y 61-90y

V NV V NV V NV

(B)PLASMA B12 IN VEG. A ND NONVEG. F EMA LES400

35 0^ 300o" 250S 200

PLASMA

o

o

500

20-40y*T

n=29|

41-60yn=23

n= 27 ~ ] ~T61-90y

V NV V NV V NVFig. 3. Comparison of plasma B12 in (A) V and NV males, *P = 0.008,** P = 0.018, and (B) V and NV females, *P = 0.013. Significance wasdetermined using the MannWhitney two-tailed test.

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between V and NV declined with age, mainly due to aprogressively lower plasma B12 in older NV, possibly due toreduced meat intake or reduced absorption of this m icronutrient.Plasma B12 status in females (Figure 3B) was somewhatdifferent, in that plasma B12 in V females was consistentlyhigher than the values observed for their V male counterpartsand, unlike the males, the plasma B12 status showed a positivetrend with age. A significantly lower plasma B12 w as observedin V females relative to NV females only in the 20-40-year-old age group; the difference was comparably smaller thanthat observed between V and NV males of the same age group(i.e. a 44% difference between V and NV females as comparedwith a 120% difference between V and NV males). In the 20-40-year-old group, 16.4% of the V females, 1.3% of the NVfemales, 25.0% of the V males and 9.0% of the NV maleshad plasma B12 values below 110 pmol/1, which is the highestvalue within the deficient range usually recorded in patientsshowing clinical symptoms of pernicious anaemia (22).Plasma folate levels were consistently higher in V malesand females relative to NV males and females in all agegroups, with statistically significant increments in 20-40-year-old V males and females, and 41-60- and 61-90-year-old V

(A)PLASMA FOLATE IN VEG. AND NONVEG. MALES

females (Figure 4A and B). The highest mean plasma folatelevel was 44.5 nmol/1, recorded in 41-60-year-old V females,and the lowest mean level was 21.0 nmol/1, measured in 20-40-year-old NV males. None of the plasma values in all theindividuals studied were below 3.4 nmol/1, which is the highestlevel in the range usually recorded for anaemic patients asdiagnosed by a range of haematological and biochemicaltests (22).Plasma VIT-C levels were consistently higher in V malesand V females relative to their NV counterparts across all agegroups (Figure 5A and B). These differences were statisticallysignificant (P < 0.05) in all groups studied with the exceptionof the 61-90-year-old female group, in which P = 0.065.There were no obvious alterations in plasma VIT-C levels withage in both sexes in both V and NV. A plasma level of 1.5mg/100 ml usually indicates saturation (19); 79 and 75% ofV males and females respectively and 50 and 47% of NVmales and females respectively had plasma levels of VIT-Cabove 1.5 mg/100 ml. Plasma VIT-E:CHOL ratios did notvary significantly between V and NV across all age groups inboth sexes (Figure 6A and B).

(A)PLSMA V IT -C IN MALE VEG. AND NONVEG.

V NV V NV V NVV NV V NV V NV

(B)PLAS MA FOLATE IN VEG. AND NONVEG. FEM ALES

V NV V NV V NVFig. 4. Comparison of plasma folate in (A) V and NV males, *P = 0.035,and (B) V and NV females, *P = 0.034, **P = 0.0001, ***P = 0.022.Significance was determined using the MannWhitney two-tailed test.

3.0

i 25o 2.0V 1.5

1.00 50.0

(B)PL/WA V IT -C IN VEG AND NONVEG. FEMA LES

20-40y

n=29

n=20

SB

41-60yn=27'*

"- I - .

61-90y

n=23Tn=29

V NV V NV V NVFig. 5. Comparison of plasma VIT-C in (A) V and NV males, *P = 0.009,** P = 0.019, ***P = 0.007, and (B) V and NV females, *P = 0.010,** P = 0.0001. Significance was determined using the Ma nn-W hitne ytwo-tailed test.

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Micronuclei, micronutrients and vegetarianismA separate analysis of this data set, in which data from allage groups were grouped together, suggested that plasma VIT-C w as the only m icronutrient showing a significant correlationwith MN frequency; the correlation was positive and occurredin males, but not in females (16; Figure 7) . We therefore decidedto examine whether the statistically significant differences inthe MN frequencies observed in V and NV males could beexplained by their plasma micronutrient status. We examinedthe correlation between each plasma micronutrient and MN

frequency in the 20-40-year-old group and the 41-60-year-old group after combining the data for V and NV (Table I) . Thisanalysis showed that: (i) there was no significant correlationbetween plasma micronutrient status and MN frequency in the41-60-year-old group; and (ii) that the MN frequency in the20-40-year-old group was, however, correlated positively withplasma folate (r = 0.453), plasma VIT-E:CHOL (r = 0.366)and plasma VIT-C (r = 0.388) and correlated negatively withplasma B12 (r = -0 .20 3) . The interpretation of the lattercorrelations is confounded by relatively strong correlationsbetween plasma folate and plasma VIT-E:CHOL (r = 0.465,P < 0.011) and plasma folate and plasma VIT-C (r =0.526, P < 0.003). The apparent increasing impact of plasma

(A )PLASMA VIT-E/CHOL. RATIO IN VEG. AND N0NVEG. MALES

a.o

ioV NV V NV V NV

(B)PL ASMA V IT -E /CHOL ESTEROL RATIO IN FEMAL E VEG

AND NONVEG.

1 6crd 4

3 -2 r

1

-

20-40yn=20~rn=29 :-;it

41-60y

T

61-90yn=231~ n=29

|

ggg:

V NV V NV V NV

micronutrients on MN frequency with decreasing age is alsoemphasized by a separate analysis of the 20-30-year-old malesin this population sample, which shows a stronger positivecorrelation between plasma VIT-C and MN frequency (r =0.823) and a strong negative correlation between plasma B12and MN frequency (r = 0.799); interpretation of these resultsis also confounded by the strong negative correlation betweenplasma B12 and plasma VIT-C (r = -0 .7 72 , P < 0.002).Analysis of data for females did not reveal any significantcorrelations between plasma m icronutrients and MN frequencyin the specific age groups for which MN frequency differenceswere examined, however, separate data analysis for 20-30-year-old females in this population sample revealed a significantnegative correlation between MN frequency and combinedplasma folate and plasma B12 (r = - 0 . 4 6 3 , P < 0.030) (16).DiscussionSome epidemiological studies have shown that the incidenceof specific cancers and overall mortality rate is reduced inpopulations w ho abstain from or minimize their intake of fleshfoods (9,23,24). How ever, to date there is no clear explanationfor such an observation, although several hypotheses havebeen suggested, such as reduced caloric intake (25), higherintake of antioxidant vitamins such as VIT-C and p-carotene(26) and reduced intake of potent mutagens found in cookedmeat (27).The somatic mutation theory of cancer suggests that geneticdamage rates should be reduced in those individuals or groupswho have a lower risk for cancer. In view of the possibilitythat vegetarianism may indeed be associated with a loweredgenetic damage rate (12) we decided to perform a moredetailed study using the MN index in lymphocytes, whichis an established biomarker for chromosome breakage andchromosome loss. The CBMN assay used in this study is nowwidely adopted for population monitoring due to its relativeease of implementation (14). For example, it was practical forone laboratory to screen the MN index of up to 900 Japanese

REGRESSION WEIGHTS OF PLASMA MICRONUTRIENTS INRELATION TO L0G lo [AGE -AD JUS TED MN FREQUENCY]

i FEMALE

oLJ

a.ouiCL VIT-E/CHOL VIT-C

Fig. 6. Comparison of plasma VIT-E:CH0L ratio in (A) V and NV malesand (B) V and NV females.

Fig. 7. Regression weights of plasma micronutrients in relation tolog-transformed, age-adjusted MN frequency. Regression weights weredetermined by analysing the effect of each plasma micronutrientindependently of other micronutrients. The MN frequency of each subjectwas adjusted to their expected value if they were 55 years old using theequation MN55y = (55 - A) S + M, where MN55y is MN frequencyadjusted to the expected value if subject was 55 years old, A is actual age inyears, M is actual MN frequency and 5 is the slope of the regression linefor MN frequency in relation to age. ** P = 0.014,227


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Table I. CorrelationAge group (years)

20 -3020-4041-60

between MNn

132940

frequency and plasma micronutrientsCorrelation coefficientsFO L0.392 (P --0.453 (P =0.185 (P =

= 0.185)= 0.013)= 0.254)

in males

B1 2-0 .799 (P-0 .203 (P0.174 (P

= 0.001)= 0.291)= 0.284)

VIT-E:CHOL-0 .184 (P =0.366 (P =0.100 (P =

ratio0.547)0.050)0.537)

VIT-C0.823 (P0.388 (P0.128 (P

= 0.001)= 0.037)= 0.430)

atomic bomb survivors to establish if there is a link betweenradiosensitivity and survival (28). Because the MN assay hasan important role as an effective biological dosimeter ofexposure to ionizing radiation (14,29) and exposure to chemicalgenotoxins (30), it is important to ascertain which lifestyleand dietary factors may have a significant imp act on this indexin order to be able to correctly interpret differences observedbetween individuals and/or populations. To date we haveidentified age and sex as being the most important variablesaffecting the MN index and this has been confirmed by otherlaboratories (14,15,28). MN frequency is -1.5 times higher infemales relative to males, which could be due to the loss ofthe X chromosome. The molecular changes affecting lossof X chromosomes are not well understood and althoughundercondensation of the inactive X chromosome has beenimplicated as being the most probable mechanism (31), it isstill not clear if altered nutritional status (e.g. methyl donordeficiency) could alter the rate of X chromosome loss infemales. It is, therefore, probable that the comparison of MNfrequencies between V and NV females may have beenconfounded by the random loss of X chromosomes, whichwould have made the detection of small differences in geneticdamage between groups more difficult to achieve.

This study also identifies variation in the MN index thatcould be attributable to dietary differences. These dietarydifferences w ere not only ascertained by dietary questionnaires,but also by analysis of several micronutrients in plasma thatcould be expected to differ, to varying extents, in V and NVvolunteers. The most important point to emerge concerningthe MN frequencies is that the overall difference between Vand NV subjects, in both males and females, was not remark-able; when differences were apparent (i.e. males aged 20-40and 41-60 years) they did not invariably suggest a lowergenetic damage rate in V subjects. One possible reason whyno remarkable differences were observed in this study is thelikelihood that our NV volunteers included a large proportionof individuals who led a healthy lifestyle and who ate abalanc ed and nutritious diet that effectively only differed fromthat of the V volunteers by the extent of meat intake as aprotein source; this possibility is being explored by ongoingdetailed analysis of the dietary questionairres submitted by thevolunteers. Nevertheless, there were clear and statisticallysignificant differences in plasma micronutrients between Vand NV that were characterized by relatively low B12 andrelatively high folate and VIT-C in V relative to NV. Thesedifferences go in the expected directions and indicate aproportionately higher fruit and vegetable intake in V relativeto NV (32,33).

Both folate and B12 play an important role in the mainten-ance of genetic integrity and gene expression by their centralroles in the synthesis of deoxythymidine monophosphateand the maintenance of methylation of 5-methylcytosine;deficiencies in these micronutrients are known to elevatechromosome breakage rate and MN frequency (34-44). While22 8

considerable focus has been placed on the importance of folatesupplementation as a means of protection against cancer (45-47) and the incidence of neural tube defects (48,49), littleemphasis has been made of the role of B12, even thoughdeficiency in this micronutrient is common, especially in Vindividuals. Recent evidence suggests that: (i) plasma folateand B12 are independent risk factors for neural tube defects;and (ii) that increased risk occurs at levels of folate and B12that are not associated with clinical manifestation of deficiency(50). Together, these findings indicate that both folate and B 12intake should be optimal to minimize pathological effects.Clearly, neither the strictly vegetarian nor the om nivorous d ietwith inadequate fruit and vegetable intake are optimal in thisregard. B12 and folate were the only micronutrients in thisstudy that showed any significant negative correlation withMN frequency, suggesting that the accepted normal values forplasma may not be optimal for minimizing genetic damagerates.

Considerable controversy exists on the potential role of VIT-C in protection against cancer. Clearly a high intake of freshfruit and vegetables is associated with a high VIT-C and folicacid intake, but it is not clear if these micronutrients are, infact, the main cause of the associated lower cancer rates.While some studies show protection of VIT-C against cancer(51-55), others suggest that high VIT-C aggravates cancer riskin chemically induced animal cancers (56,57) and there isgood evidence for its clastogenic action in vitro (55,58,59)and in vivo (60). Our studies indicate a clear positive correlationbetween MN frequency and plasma VIT-C in males, but donot prove that VIT-C itself is the direct cause. This could onlybe ascertained by intervention studies with high doses of VIT-C. Of all the micronutrients examined, VIT-E appears to havethe least influence on the MN index, suggesting either thatlipid peroxidation is unlikely to have important secondaryeffects on DNA integrity or that the range of VIT-E values inthis study was too narrow to discern any dose effects. Somerecent epidemiological studies have suggested that protectionagainst cancer and coronary heart disease by VIT-E was onlyevident in those individuals who supplemented their normaldietary intake with VIT-E capsules (6163).

In analysing the relationship of plasma micronutrients andMN frequency in males it became evident that the influenceof micronutrient status was increasingly pronounced withdecreasing age, to the extent that the plasma micronutrientsexamined had no obvious impact on MN frequency in agegroups above 40 years, while significant and progressivelyhigher correlations could be detected in age groups below40 years. The correlations observed have been somewhatconfounded by other significant correlations between theplasma levels of the micronutrients examined. These findingssuggest that the reduced MN frequency in V males in the 4 1 -60 year age group could not be explained by the differencesin micronutrient status between V and NV, but the reversewas true with regard to the MN frequency reduction in NV


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Micronuclei, micronutrients and vegetarianismmales in the 20-30 and 20-40 year age groups. These dataare indicative of a possible role for VIT-C in aggravating D NAdamage rates in young males and a possible beneficial effectassociated with elevated B12 status. The data for females inthe 20-30 year age group also show a dose-related reductionin MN frequency in relation to the combined folate and B12plasma levels, thus reinforcing the notion that genetic damagerates in lymphocytes may be minimized by optimizing folateand B12 intake beyond the currently accepted normal plasmavalues. Our observations do not appear to support the viewthat lowered cancer rates in vegetarians could be explained bytheir higher VIT-C intake nor do they suggest that VIT-Csupplementation would be a successful approach to minimizegenetic damage rates in lymphocytes.

Becaus e MN frequency appeared to be effectively influencedby micronutrient status only in the younger age groups, itseems that dietary interventions investigating the relationshipbetween diet and genetic damage would be most successful ifrestricted to individuals aged less than 30 years. This appearsto make sense, because it would be expected that age-related biological changes, such as reduced absorption ofmicronutrients and increased genetic damage rate, are likelyto overwhelm any doseresponse effects produced by normalintake of miconutrients in the older categories; this effectivelyalso implies that supplementation over and above normaldietary intake may be required to observe any effects ongenetic damage rates in the elderly.

In conclusion, more attention needs to be given to theinfluence of diet on baseline genetic damage rates because: (i)it helps to identify those micronutrients and those age groupswith which positive effects are most likely to be obtained; and(ii) it enables a more refined interpretation of observed geneticdamage rates when the MN assay is used as a biomarker ofexposure to physical or chemical genotoxins. The lack ofan exceptional difference in spontaneous genetic damage inlymphocytes of V and NV does not exclude the possibilitythat mutation rates in other tissues may be different.AcknowledgementsWe would like to acknowledge the contribution of the numerous volunteerswho participated in this study, as well as the help of Dr Peter Clifton, SrRosemary McArthur and Sr Maria Nugent, who assisted us in the collectionof blood. In particular we would also like to acknowledge the supportingtechnical assistance provided by Clare Aitken at various critical points of theproject and Dr Ivor Dreosti for reviewing the manuscript. This research waspartially funded by financial grants from the Anti-Cancer Foundation of SouthAustralia and the Australian Meat Research Corporation.References

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