Fd Chem. Tomc Vol 30, No 7, pp 585-588, 1992
0278-6915/92 $5.00+ 0.00 Copyright© 1992Pergamon Press Ltd
Pnnted m Great Bntam All nghts reserved
A COMPARATIVE STUDY OF MICRONUCLEUS FREQUENCY IN PERIPHERAL BLOOD LYMPHOCYTES OF H U M A N SUBJECTS GIVEN DIETARY cis, trans AND SATURATED FAT I. R. RECORD,M. KONSTANTINOPOULOSand P. J. NESTEL CSIRO Division of Human Nutrition, PO Box 10041, Gouger St., Adelaide, S. Australia 5000 (Accepted 16 March 1992)
Abstract--The mlcronucleus test using pertpheral blood lymphocytes has been used to study the clastogenic effects of saturated, cts or trans fatty acids in human volunteers Consumption of diets nch m either saturated fats (palmltic acid), monounsaturated cis fats (ole~c acid) or monounsaturated trans fats (elaidic acid) for 3 wk had no effect on either the number of mlcronuclei or the frequency of micronucleated cells in lymphocytes in culture This study adds to the accumulating body of ewdence to suggest that dietary retake of trans fatty acids Is not especmlly hnked to genetic damage.
INTRODUCTION The relationship between dietary fats and cancer has been the subject of many investigahons over the last few decades. Data from both human and animal studies have linked certain types of cancer, particularly those of the breast, prostate and lower intestine, with elevated dietary fat intake. Dietary fat consumption in the USA for example has increased from 125 g/day in 1909-1913 to 168 g/day in 1979 (Rizek, 1981) and has been linked to mcreases in cancer incidence. In laboratory ammals it has been demonstrated that not only the quantity but also the composition of the fat can have profound effects on tumour development (Cave, 1989). While the strongest relationship in humans has been established between animal fat and cancer, concern was first expressed by Enig et al. (1978) that the increase in cancer incidence was associated with a greater use of chemically modified vegetable oils. Commercially it is considered important to partially hydrogenate vegetable oils in some c~rcumstances to improve their stability and achieve other desired physical qualities (Emken, 1980). Several of these processes not only render the oils more saturated, but also introduce a significant number of trans bonds into the fat, thus producing fatty acids not normally found in nature (Beare-Rogers, 1983). It has been estimated that individuals may consume up to 7 g trams fatty acids per day (Emken, 1980). Because these isomers are relatively uncommon in nature, there has been interest in their possible atherogenic properties (Mensink and Katan, 1990; Nestel et al., 1992a) as well as their carcinogenic potential. Enig et al. (1978 and 1979) have pointed out that in the data from the USA there appears to be a statistical relationship between the increased con-
sumption of trans fatty acids and an increased frequency of some human cancers; however, other authors (Applewhite, 1979; Bailar, 1979; Meyer, 1979) have strongly disputed these conclusions. Animal studies have, in general, failed to demonstrate any significant relationship between trans fatty acid in gestion and carcinogenesis. Awad ( 1981) found that 5% elaidic acad (the trans isomer of oleic acid) m the diet reduced the survival rate of mice bearing Ehrlich's ascites tumour cells. Hunter (1982) pointed out that in this study mice were fed elaidic acid in its triglyceride form and not in its free fatty form and that the fatty acid profiles of the two diets used were different, therefore the conclusions were not necessarily correct. Brown (1981) found no significant difference in the development of spontaneous or induced tumours m groups of mice fed diets high m cis or t r a n s - m o n o e n e fats for 17 months. This negative finding has been confirmed in several other experiments in which trans fatty acids were fed together with known carcinogens (Hogan and Shamsuddin, 1984; Hunter et al., 1985; Reddy et al., 1985; Selenskas et al., 1984; Sugano et al., 1989). Fenech and Morley (1985a) described a rapid, sensitive technique for studymg chromosomal breakage and spindle dysfunction in isolated human peripheral blood lymphocytes. This method involves blocking cellular but not nuclear dwislon with cytochalasin B. Cytogenettc damage can then be quantified by determining the proportion of bmucleated cells containing mmronuclei. The method has been used in a number of situations, such as in the evaluaUon of the genetic effects of age and low-dose X-irradiation (Fenech and Morely, 1985b), benzene inhalation (Hogstedt et al., 1991), marginal folate deficiency (Everson et al., 1988) and cigarette usage (Tomanin et al., 1991). We have used this technique 585
I R. RECORD et a l
586
to ascertain whether differences in fatty acid composition of diets, especially with respect to trans fatty acids, can influence the frequency of mlcronuclei in human subjects. MATERIALS AND M E T H O D S
Subjects. A total of 17 adult (32-63 yr old) males recruited for a dietary crossover study involving lipid metabolism were used in this investigation. Study parameters have been previously described in detail (Nestel et al., 1992a). Briefly, the men were normotensive with mild hypercholesterolaemia (5-7.5 mmol/litre) but otherwise healthy. Exclusion criteria were alcohol consumpUon greater than 40 g per day, consumption of more than 20 cigarettes per day or any other medication or medical treatment affecting hpid metabolism. Experimentalprotocol. Prior to the commencement of the study the subjects were interviewed by a dietician and instructed to minimize fat mtake by means of simplified food tables. This background diet was supplemented w~th margarine, biscuits and potato crisps containing different oils, each bemg provided for a 3-wk period. The study was camed out double blind and in random treatment periods. Composition of the oils was determined by gas chromatography (Nestel et al., 1992a) and is presented in Table 1. The supplemental fat intake was between 65 and 70 g/day. All food constituents of the supplements other than the fatty acids were considered to be identical. Intake of trans fatty acids from the consumption of the palmitic acid blend was minimal. During the treatment with oleic acid, each individual consumed about 4 g trans fat/day, and during the treatment with elaidic acid thts consumption was approximately 18g/day. Together with the minor amounts of trans fatty acids m dairy and meat fats included in the background &et, trans fatty acids provided about 8% energy during the treatment with elaidic acid compared with just under 2% during the treatment with olelc acid, less than 2% during the habitual diet and less than 1% during the treatment
Table 1 Fatty acid composmon of the test blends (%)
Test blends Fatty acid
Palmmc
Ole,c
Major fats Canola Canola Linseed Sunflower Palm 160 18.0 18.1 cts 18:1 trans 18'2 18 3
Polyunsaturates Monounsaturates* Saturates
*Includes elald~e acid.
Elaldlc Canola Linseed Safflower Hardened canola Palmolem 12.0 40 25 0
310 30 38 0
10.0 40 50 0
-17.0
120 16 0
270 21 0
90 26.0 38.0 340
60 22.0 62.0 140
90 30 0 52 0 160
with palmitic acid. Compliance was assessed by supplying the subjects with electronic scales and instructions to record all food and beverage for 3 consecutive days in each study period. Nutrtent intakes were calculated using a computer-based dietary analysis package (Baghurst and Record, 1984). The protocol was approved by the Human Ethics Committee of the Divtsion of Human Nutrition. Mwronucleusfrequency. Blood was obtained at the start of the study (habitual diet) and at the end of each 3-wk trial period. Peripheral blood lymphocytes were cultured by the method described by Fenech and Morley (1985a). Briefly, lymphoeytes were separated using Ficoll-Paque (Pharmacia), cultured m McCoy's 5A medium at a concentration of 1 x 106 cells/ml and stimulated to divide by the addition of phytohaemagglutmm (5 #g/ml). After 44 hr cytochalasin B was added (4.5/zg/ml), and after a further 28 hr the cells were harvested by cytocentrifugation and stained by a modtfied Wnght's stain (Diff-Quick, Harleco). At least 1000 binucleated cells were scored for the presence of micronuclei. Results were evaluated by paired t-test. RESULTS
Plasma fatty acids measured by gas chromatography, as reported elsewhere (Nestel et al., 1992b), reflected the change m the composition of dietary fat. At the end of the trans fatty acid-rich diet, the elaldic acid concentration in plasma was seven times higher than the habitual background or the concentration in subjects given palmltic acid-enriched diets. The oleic acid-rich diet doubled plasma elaidic acid because of the partial hydrogenation of canola oil. The frequencies of mlcronucleated cells and micronuclei, the distribution of multiple micronuclei and the range of micronucleated cells are presented in Table 2. None of the dietary treatments had any effect on the variables measured. There was a large variation in mtcronucleus score between subjects, the lowest being 7 and the highest 35, that is a ratio of 5. Scores from indivtduals showed fewer fluctuations, with the greatest vanation recorded being from 11 to 30, but the average ratio of highest to lowest score was 1.7. Analysis of the data by paired t-test showed no effect of the different diets on the micronucleus scores of the subjects; therefore an attempt was made to identify some potential sources of variability. Neither age nor alcohol consumpaon were related to the frequency or number of micronuclei. As only one of the subjects smoked ( < 1 0 cigarettes/day), this parameter was also eliminated as a confounding factor. DISCUSSION
The micronucleus assay using cultured human peripheral blood lymphocytes has proved to be an effective technique for assessing the genotoxic poten-
Dietary fats and genotoxlclty
587
Table 2 Mlcronucleus frequency and distribution In peripheral blood lymphocytes of volunteers fed diets of different fatty acid compomUon Distribution of MN
Dmtary fat type Habitual Palmmc Olelc Elmdlc
Micronucleatedcells* MN* per 1000 BN per 1000 BN 1 2 16 7 + 1 7 18 9 + 2 1 256 24 17 1 + I 3 20 2 + I 8 275 35 18 7 + I 5 21 8 + 1 6 266 28 18 9 + 1 6 21 8 + 2 1 242 24 BN = bmucleated cells MN = mlcronuclel *Means _+SEM of 17 mdwlduals
ttal of a n u m b e r of factors such as s m o k i n g habits ( T o m a n i n et al., 1991), r a d i a t i o n exposure (Fenech a n d Morley, 1985b), benzene i n h a l a t i o n (Hogstedt et al., 1991) a n d m a r g i n a l folate deficiency (Everson et al., 1988). T o our knowledge, possible deleterious effects of dietary fat, caused either by the type or q u a n t i t y o f fat, have not been assessed using this technique. Djuric et al. (1991) have, however, n o t e d t h a t high dietary fat levels are associated with increased oxidative d a m a g e to D N A in o r c u l a t m g lymphocytes. In the present study, the subjects c o n s u m e d fats of different c o m p o s m o n for a period of 3 wk prior to the d e t e r m i n a t i o n o f mlcronucleus frequency. It has been s h o w n (Nestel et al., 1992a) t h a t this length of time is sufficient for sigmficant changes to occur m plasma hpld profiles a n d m cellular fatty acid c o m p o s i t i o n (Glatz et al., 1989). W e f o u n d n o evidence o f deleten o u s effects o f trans fatty acids o n c h r o m o s o m a l integrity m lymphocytes; this m t g h t be due to the fact t h a t the subjects did n o t c o n s u m e the dints long e n o u g h to allow clastogenic changes to be observed. Aiternatwely, some further m u t a g e m c msult m i g h t be needed to d e m o n s t r a t e increased genetic sens~tivlty. It should, however, be pointed out t h a t m this trial the retake o f trans fats was over twice t h a t in countries such as the U K (Sanders, 1988) a n d the U S A (Emken, 1980), where c o n s u m p t i o n of semi-hardened oils is high. The degree of variation between individuals observed m this study has been reported by others (Fenech a n d Morley, 1985a,b). These differences can be attributed to a n u m b e r of factors such as age, genetic disposition a n d exposure to e n v i r o n m e n t a l mfluences, a n d highlights the need for a d e q u a t e controls w h e n using the micronucleus technique to evaluate possible genotoxlc stlmuh. A l t h o u g h the present study c a n n o t prove the safety o f trans fatty acids, the results add to the accumulating body o f evidence suggesting t h a t these isomers are n o t especially linked to genetic damage. Acknowledgements--The authors wish to thank Meadow Lea Foods (Australia) for supplying the test blends and Sr R. McArthur and Ms A Stevens for technical assistance. REFERENCES
Applewhlte T. H. (1979) Statistical "correlations" relating trans fats to cancer' a commentary. Federation Proceedmrs 38, 2435.
3 6 3 5 4
Range of
>3 0 I 3 3
mlcronucleated cells 7-30 8-28 8-27 10-35
Awad A B. (1981) Trans fatty acids in tumor development and host survival Journal of the National Cancer Institute 67, 189-192 Baghurst K I. and Record S. J (1984) A computensed d~etary analysis system for use w~th diaries or food frequency questionnaires. Community Health Studies $, 11-18 Ballar J C (1979) Dietary fat and cancer trends--a further cntlque. Federation Proceedings 38, 2435-2436. Beare-Rogers J L (1983) Trans and positional isomers of common fatty acids Advances m Nutrmonal Research 5, 171-200 Brown R R (1981) Effects of dietary fat on incidence of spontaneous and induced cancer in mice. Cancer Research 41, 3741-3742 Cave W T. (1989) Differential effects of specific types of dietary lipid on mammary tumor development In Carcmogenests and Dietary Fat Edited by S. Abraham. pp 85-98 Kluwer, Boston, MA Djunc Z , Hellbrun L. K., Reading B A., Boomer A., Valenote F A and Martmo S (1991) Effects of a low fat diet on levels of oxidative damage to DNA in human peripheral nucleated blood cells Journal of the National Cancer Institute 83, 766-760. Emken E A. (1980) Nutritional aspects of soybean oll utlhzatlon. In Worm Soybean Research Conference 1 l. Proceedmgs Edited by F T Corbln pp 667~79 Westvlew Press, Boulder, CO. Enlg M G., Munn R. J and Keeney M (1978) Dietary fat and cancer trends--a critique Federation Proceedings 37, 2215-2220 Enlg M G , Munn R. J. and Keeney M. (1979) Response to crmclsms of "Dietary fat and cancer trends--a critique" Federation Proceedmgs 38, 2437-2439 Everson R. B, Wehr C M , Erexson G L. and MacGregor J T (1988) Association of marginal folate depletion with Increased human chromosomal damage in vivo demonstration by analysis of mlcronucleated erythrocytes Journal o f the National Cancer Institute 80, 525-529 Fenech M and Morley A A (1985a) Measurement of mtcronuclel m human lymphocytes. Mutation Research 147, 29 36 Fenech M. and Morley A A. (1985b) Cytoklnesls-block mlcronucleus method In human lymphocytes effect of in wvo aging and low dose X-irradiation Mutation Research 161, 193-198 Glatz J F C., Soffers A. and Katan M B (1989) Fatty acid composmon of serum cholesterol esters and erythrocyte membranes as indicators of hnoleic aod intake in man. Amerwan Journal of Chntcal Nutrmon 49, 269-276. Hogan M L. and Shamsuddln A. M (1984) Large intestinal carcinogenesis 1" promotional effect of dmtary fatty acid isomers in the rat model Journal o f the National Cancer Institute 73, 1293-1296 Hogstedt B , Holmen A , Karlsson A., Ralhle G., Nlllius K and Vestlund K (1991) Gasoline pump mechanics had increased frequencies and sizes of mlcronuclel in lymphocytes stimulated by pokeweed mltogen. Mutanon Research 263, 51-55.
588
I.R. RECORD et al.
Hunter J. E , Ip C. and HoUenbach E. J. (1985) Isomeric fatty acids and tumongenem: a commentary on recent work. Nutrition and Cancer 7, 199-209. Mensink R P. and Katan M. B. (1990) Effect of &etary trans fatty acids on high density and low denmy hpoprotein levels in health subjects. New England Journal o f Medicine 323, 439-445. Meyer W, H (1979) Further comments on "Dietary fats and cancer trends--a critique". Federation Proceedings 38, 2436-2437. Nestel P J., Noakes M., Belling G , McArthur R , Chfton P. M. and Abbey M. (1992a) Plasma cholesterol lowering potential of edible oll blends suitable for commercial use. Amerwan Journal o f Climcal Nutrition 55, 46-50. Nestel P. J., Noakes M., Belling G. B., McArthur R , Clifton P. M and Abbey M (1992b) Trans fatty acids m the Australian diet' effect on plasma hpid and lipoprotein concentraUons Proceedings o f the 3rd Internatwnal Congress on Essential Fatty AcMs and Eicosanoids. In Press. Reddy B. S., Tanaka T. and Siml B. (1985) Effect of &fferent levels of &etary trans fat or corn oll on
azoxymethane-mduced colon carcinogenesis m F344 rats. Journal o f the National Cancer Insutute 75, 791-798 Rlzek R. L. (1981) Food supply studies and consumption survey statistics on fat m United States diets. Cancer Research 43, 2477s-2484s. Sanders T A. B (1988) Essential and trans fatty acids in nutrition. Nutrltwn Research Reviews 1, 57-78. Selenskas S L., Ip M. M. and Ip C (1984) Similarity between trans fat and saturated fat in the modification of rat mammary carcinogenesis. Cancer Research 44, 1321-1326. Sugano M., Watanabe M , Yoshlda K , Tomioka M , Miyamoto M and Kritchevsky D (1989) Influence of &etary cis and trans fats on DMH-induced colon tumors, steroid excretion, and ¢lcosanoid production m rats prone to colon cancer. Nutr~twn and Cancer 12, 177-187. Tomanm R., Ballann C,, Nardmi B., Mastrangelo G. and Sarto F (1991) Influence of smoking habR on the frequency of micronuclel m human lymphocytes by the cytokinesls block method Mutagenesis 6, 123-126
0278-6915/92 $5.00+ 0.00 Copyright© 1992Pergamon Press Ltd
Pnnted m Great Bntam All nghts reserved
A COMPARATIVE STUDY OF MICRONUCLEUS FREQUENCY IN PERIPHERAL BLOOD LYMPHOCYTES OF H U M A N SUBJECTS GIVEN DIETARY cis, trans AND SATURATED FAT I. R. RECORD,M. KONSTANTINOPOULOSand P. J. NESTEL CSIRO Division of Human Nutrition, PO Box 10041, Gouger St., Adelaide, S. Australia 5000 (Accepted 16 March 1992)
Abstract--The mlcronucleus test using pertpheral blood lymphocytes has been used to study the clastogenic effects of saturated, cts or trans fatty acids in human volunteers Consumption of diets nch m either saturated fats (palmltic acid), monounsaturated cis fats (ole~c acid) or monounsaturated trans fats (elaidic acid) for 3 wk had no effect on either the number of mlcronuclei or the frequency of micronucleated cells in lymphocytes in culture This study adds to the accumulating body of ewdence to suggest that dietary retake of trans fatty acids Is not especmlly hnked to genetic damage.
INTRODUCTION The relationship between dietary fats and cancer has been the subject of many investigahons over the last few decades. Data from both human and animal studies have linked certain types of cancer, particularly those of the breast, prostate and lower intestine, with elevated dietary fat intake. Dietary fat consumption in the USA for example has increased from 125 g/day in 1909-1913 to 168 g/day in 1979 (Rizek, 1981) and has been linked to mcreases in cancer incidence. In laboratory ammals it has been demonstrated that not only the quantity but also the composition of the fat can have profound effects on tumour development (Cave, 1989). While the strongest relationship in humans has been established between animal fat and cancer, concern was first expressed by Enig et al. (1978) that the increase in cancer incidence was associated with a greater use of chemically modified vegetable oils. Commercially it is considered important to partially hydrogenate vegetable oils in some c~rcumstances to improve their stability and achieve other desired physical qualities (Emken, 1980). Several of these processes not only render the oils more saturated, but also introduce a significant number of trans bonds into the fat, thus producing fatty acids not normally found in nature (Beare-Rogers, 1983). It has been estimated that individuals may consume up to 7 g trams fatty acids per day (Emken, 1980). Because these isomers are relatively uncommon in nature, there has been interest in their possible atherogenic properties (Mensink and Katan, 1990; Nestel et al., 1992a) as well as their carcinogenic potential. Enig et al. (1978 and 1979) have pointed out that in the data from the USA there appears to be a statistical relationship between the increased con-
sumption of trans fatty acids and an increased frequency of some human cancers; however, other authors (Applewhite, 1979; Bailar, 1979; Meyer, 1979) have strongly disputed these conclusions. Animal studies have, in general, failed to demonstrate any significant relationship between trans fatty acid in gestion and carcinogenesis. Awad ( 1981) found that 5% elaidic acad (the trans isomer of oleic acid) m the diet reduced the survival rate of mice bearing Ehrlich's ascites tumour cells. Hunter (1982) pointed out that in this study mice were fed elaidic acid in its triglyceride form and not in its free fatty form and that the fatty acid profiles of the two diets used were different, therefore the conclusions were not necessarily correct. Brown (1981) found no significant difference in the development of spontaneous or induced tumours m groups of mice fed diets high m cis or t r a n s - m o n o e n e fats for 17 months. This negative finding has been confirmed in several other experiments in which trans fatty acids were fed together with known carcinogens (Hogan and Shamsuddin, 1984; Hunter et al., 1985; Reddy et al., 1985; Selenskas et al., 1984; Sugano et al., 1989). Fenech and Morley (1985a) described a rapid, sensitive technique for studymg chromosomal breakage and spindle dysfunction in isolated human peripheral blood lymphocytes. This method involves blocking cellular but not nuclear dwislon with cytochalasin B. Cytogenettc damage can then be quantified by determining the proportion of bmucleated cells containing mmronuclei. The method has been used in a number of situations, such as in the evaluaUon of the genetic effects of age and low-dose X-irradiation (Fenech and Morely, 1985b), benzene inhalation (Hogstedt et al., 1991), marginal folate deficiency (Everson et al., 1988) and cigarette usage (Tomanin et al., 1991). We have used this technique 585
I R. RECORD et a l
586
to ascertain whether differences in fatty acid composition of diets, especially with respect to trans fatty acids, can influence the frequency of mlcronuclei in human subjects. MATERIALS AND M E T H O D S
Subjects. A total of 17 adult (32-63 yr old) males recruited for a dietary crossover study involving lipid metabolism were used in this investigation. Study parameters have been previously described in detail (Nestel et al., 1992a). Briefly, the men were normotensive with mild hypercholesterolaemia (5-7.5 mmol/litre) but otherwise healthy. Exclusion criteria were alcohol consumpUon greater than 40 g per day, consumption of more than 20 cigarettes per day or any other medication or medical treatment affecting hpid metabolism. Experimentalprotocol. Prior to the commencement of the study the subjects were interviewed by a dietician and instructed to minimize fat mtake by means of simplified food tables. This background diet was supplemented w~th margarine, biscuits and potato crisps containing different oils, each bemg provided for a 3-wk period. The study was camed out double blind and in random treatment periods. Composition of the oils was determined by gas chromatography (Nestel et al., 1992a) and is presented in Table 1. The supplemental fat intake was between 65 and 70 g/day. All food constituents of the supplements other than the fatty acids were considered to be identical. Intake of trans fatty acids from the consumption of the palmitic acid blend was minimal. During the treatment with oleic acid, each individual consumed about 4 g trans fat/day, and during the treatment with elaidic acid thts consumption was approximately 18g/day. Together with the minor amounts of trans fatty acids m dairy and meat fats included in the background &et, trans fatty acids provided about 8% energy during the treatment with elaidic acid compared with just under 2% during the treatment with olelc acid, less than 2% during the habitual diet and less than 1% during the treatment
Table 1 Fatty acid composmon of the test blends (%)
Test blends Fatty acid
Palmmc
Ole,c
Major fats Canola Canola Linseed Sunflower Palm 160 18.0 18.1 cts 18:1 trans 18'2 18 3
Polyunsaturates Monounsaturates* Saturates
*Includes elald~e acid.
Elaldlc Canola Linseed Safflower Hardened canola Palmolem 12.0 40 25 0
310 30 38 0
10.0 40 50 0
-17.0
120 16 0
270 21 0
90 26.0 38.0 340
60 22.0 62.0 140
90 30 0 52 0 160
with palmitic acid. Compliance was assessed by supplying the subjects with electronic scales and instructions to record all food and beverage for 3 consecutive days in each study period. Nutrtent intakes were calculated using a computer-based dietary analysis package (Baghurst and Record, 1984). The protocol was approved by the Human Ethics Committee of the Divtsion of Human Nutrition. Mwronucleusfrequency. Blood was obtained at the start of the study (habitual diet) and at the end of each 3-wk trial period. Peripheral blood lymphocytes were cultured by the method described by Fenech and Morley (1985a). Briefly, lymphoeytes were separated using Ficoll-Paque (Pharmacia), cultured m McCoy's 5A medium at a concentration of 1 x 106 cells/ml and stimulated to divide by the addition of phytohaemagglutmm (5 #g/ml). After 44 hr cytochalasin B was added (4.5/zg/ml), and after a further 28 hr the cells were harvested by cytocentrifugation and stained by a modtfied Wnght's stain (Diff-Quick, Harleco). At least 1000 binucleated cells were scored for the presence of micronuclei. Results were evaluated by paired t-test. RESULTS
Plasma fatty acids measured by gas chromatography, as reported elsewhere (Nestel et al., 1992b), reflected the change m the composition of dietary fat. At the end of the trans fatty acid-rich diet, the elaldic acid concentration in plasma was seven times higher than the habitual background or the concentration in subjects given palmltic acid-enriched diets. The oleic acid-rich diet doubled plasma elaidic acid because of the partial hydrogenation of canola oil. The frequencies of mlcronucleated cells and micronuclei, the distribution of multiple micronuclei and the range of micronucleated cells are presented in Table 2. None of the dietary treatments had any effect on the variables measured. There was a large variation in mtcronucleus score between subjects, the lowest being 7 and the highest 35, that is a ratio of 5. Scores from indivtduals showed fewer fluctuations, with the greatest vanation recorded being from 11 to 30, but the average ratio of highest to lowest score was 1.7. Analysis of the data by paired t-test showed no effect of the different diets on the micronucleus scores of the subjects; therefore an attempt was made to identify some potential sources of variability. Neither age nor alcohol consumpaon were related to the frequency or number of micronuclei. As only one of the subjects smoked ( < 1 0 cigarettes/day), this parameter was also eliminated as a confounding factor. DISCUSSION
The micronucleus assay using cultured human peripheral blood lymphocytes has proved to be an effective technique for assessing the genotoxic poten-
Dietary fats and genotoxlclty
587
Table 2 Mlcronucleus frequency and distribution In peripheral blood lymphocytes of volunteers fed diets of different fatty acid compomUon Distribution of MN
Dmtary fat type Habitual Palmmc Olelc Elmdlc
Micronucleatedcells* MN* per 1000 BN per 1000 BN 1 2 16 7 + 1 7 18 9 + 2 1 256 24 17 1 + I 3 20 2 + I 8 275 35 18 7 + I 5 21 8 + 1 6 266 28 18 9 + 1 6 21 8 + 2 1 242 24 BN = bmucleated cells MN = mlcronuclel *Means _+SEM of 17 mdwlduals
ttal of a n u m b e r of factors such as s m o k i n g habits ( T o m a n i n et al., 1991), r a d i a t i o n exposure (Fenech a n d Morley, 1985b), benzene i n h a l a t i o n (Hogstedt et al., 1991) a n d m a r g i n a l folate deficiency (Everson et al., 1988). T o our knowledge, possible deleterious effects of dietary fat, caused either by the type or q u a n t i t y o f fat, have not been assessed using this technique. Djuric et al. (1991) have, however, n o t e d t h a t high dietary fat levels are associated with increased oxidative d a m a g e to D N A in o r c u l a t m g lymphocytes. In the present study, the subjects c o n s u m e d fats of different c o m p o s m o n for a period of 3 wk prior to the d e t e r m i n a t i o n o f mlcronucleus frequency. It has been s h o w n (Nestel et al., 1992a) t h a t this length of time is sufficient for sigmficant changes to occur m plasma hpld profiles a n d m cellular fatty acid c o m p o s i t i o n (Glatz et al., 1989). W e f o u n d n o evidence o f deleten o u s effects o f trans fatty acids o n c h r o m o s o m a l integrity m lymphocytes; this m t g h t be due to the fact t h a t the subjects did n o t c o n s u m e the dints long e n o u g h to allow clastogenic changes to be observed. Aiternatwely, some further m u t a g e m c msult m i g h t be needed to d e m o n s t r a t e increased genetic sens~tivlty. It should, however, be pointed out t h a t m this trial the retake o f trans fats was over twice t h a t in countries such as the U K (Sanders, 1988) a n d the U S A (Emken, 1980), where c o n s u m p t i o n of semi-hardened oils is high. The degree of variation between individuals observed m this study has been reported by others (Fenech a n d Morley, 1985a,b). These differences can be attributed to a n u m b e r of factors such as age, genetic disposition a n d exposure to e n v i r o n m e n t a l mfluences, a n d highlights the need for a d e q u a t e controls w h e n using the micronucleus technique to evaluate possible genotoxlc stlmuh. A l t h o u g h the present study c a n n o t prove the safety o f trans fatty acids, the results add to the accumulating body o f evidence suggesting t h a t these isomers are n o t especially linked to genetic damage. Acknowledgements--The authors wish to thank Meadow Lea Foods (Australia) for supplying the test blends and Sr R. McArthur and Ms A Stevens for technical assistance. REFERENCES
Applewhlte T. H. (1979) Statistical "correlations" relating trans fats to cancer' a commentary. Federation Proceedmrs 38, 2435.
3 6 3 5 4
Range of
>3 0 I 3 3
mlcronucleated cells 7-30 8-28 8-27 10-35
Awad A B. (1981) Trans fatty acids in tumor development and host survival Journal of the National Cancer Institute 67, 189-192 Baghurst K I. and Record S. J (1984) A computensed d~etary analysis system for use w~th diaries or food frequency questionnaires. Community Health Studies $, 11-18 Ballar J C (1979) Dietary fat and cancer trends--a further cntlque. Federation Proceedings 38, 2435-2436. Beare-Rogers J L (1983) Trans and positional isomers of common fatty acids Advances m Nutrmonal Research 5, 171-200 Brown R R (1981) Effects of dietary fat on incidence of spontaneous and induced cancer in mice. Cancer Research 41, 3741-3742 Cave W T. (1989) Differential effects of specific types of dietary lipid on mammary tumor development In Carcmogenests and Dietary Fat Edited by S. Abraham. pp 85-98 Kluwer, Boston, MA Djunc Z , Hellbrun L. K., Reading B A., Boomer A., Valenote F A and Martmo S (1991) Effects of a low fat diet on levels of oxidative damage to DNA in human peripheral nucleated blood cells Journal of the National Cancer Institute 83, 766-760. Emken E A. (1980) Nutritional aspects of soybean oll utlhzatlon. In Worm Soybean Research Conference 1 l. Proceedmgs Edited by F T Corbln pp 667~79 Westvlew Press, Boulder, CO. Enlg M G., Munn R. J and Keeney M (1978) Dietary fat and cancer trends--a critique Federation Proceedings 37, 2215-2220 Enlg M G , Munn R. J. and Keeney M. (1979) Response to crmclsms of "Dietary fat and cancer trends--a critique" Federation Proceedmgs 38, 2437-2439 Everson R. B, Wehr C M , Erexson G L. and MacGregor J T (1988) Association of marginal folate depletion with Increased human chromosomal damage in vivo demonstration by analysis of mlcronucleated erythrocytes Journal o f the National Cancer Institute 80, 525-529 Fenech M and Morley A A (1985a) Measurement of mtcronuclel m human lymphocytes. Mutation Research 147, 29 36 Fenech M. and Morley A A. (1985b) Cytoklnesls-block mlcronucleus method In human lymphocytes effect of in wvo aging and low dose X-irradiation Mutation Research 161, 193-198 Glatz J F C., Soffers A. and Katan M B (1989) Fatty acid composmon of serum cholesterol esters and erythrocyte membranes as indicators of hnoleic aod intake in man. Amerwan Journal of Chntcal Nutrmon 49, 269-276. Hogan M L. and Shamsuddln A. M (1984) Large intestinal carcinogenesis 1" promotional effect of dmtary fatty acid isomers in the rat model Journal o f the National Cancer Institute 73, 1293-1296 Hogstedt B , Holmen A , Karlsson A., Ralhle G., Nlllius K and Vestlund K (1991) Gasoline pump mechanics had increased frequencies and sizes of mlcronuclel in lymphocytes stimulated by pokeweed mltogen. Mutanon Research 263, 51-55.
588
I.R. RECORD et al.
Hunter J. E , Ip C. and HoUenbach E. J. (1985) Isomeric fatty acids and tumongenem: a commentary on recent work. Nutrition and Cancer 7, 199-209. Mensink R P. and Katan M. B. (1990) Effect of &etary trans fatty acids on high density and low denmy hpoprotein levels in health subjects. New England Journal o f Medicine 323, 439-445. Meyer W, H (1979) Further comments on "Dietary fats and cancer trends--a critique". Federation Proceedings 38, 2436-2437. Nestel P J., Noakes M., Belling G , McArthur R , Chfton P. M. and Abbey M. (1992a) Plasma cholesterol lowering potential of edible oll blends suitable for commercial use. Amerwan Journal o f Climcal Nutrition 55, 46-50. Nestel P. J., Noakes M., Belling G. B., McArthur R , Clifton P. M and Abbey M (1992b) Trans fatty acids m the Australian diet' effect on plasma hpid and lipoprotein concentraUons Proceedings o f the 3rd Internatwnal Congress on Essential Fatty AcMs and Eicosanoids. In Press. Reddy B. S., Tanaka T. and Siml B. (1985) Effect of &fferent levels of &etary trans fat or corn oll on
azoxymethane-mduced colon carcinogenesis m F344 rats. Journal o f the National Cancer Insutute 75, 791-798 Rlzek R. L. (1981) Food supply studies and consumption survey statistics on fat m United States diets. Cancer Research 43, 2477s-2484s. Sanders T A. B (1988) Essential and trans fatty acids in nutrition. Nutrltwn Research Reviews 1, 57-78. Selenskas S L., Ip M. M. and Ip C (1984) Similarity between trans fat and saturated fat in the modification of rat mammary carcinogenesis. Cancer Research 44, 1321-1326. Sugano M., Watanabe M , Yoshlda K , Tomioka M , Miyamoto M and Kritchevsky D (1989) Influence of &etary cis and trans fats on DMH-induced colon tumors, steroid excretion, and ¢lcosanoid production m rats prone to colon cancer. Nutr~twn and Cancer 12, 177-187. Tomanm R., Ballann C,, Nardmi B., Mastrangelo G. and Sarto F (1991) Influence of smoking habR on the frequency of micronuclel m human lymphocytes by the cytokinesls block method Mutagenesis 6, 123-126
