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[CANCER RESEARCH 41. 1460-1465. April 1981]0008-5472/81 /0041-OOOOS02.00
Influence of Dietary Fatty Acids on the Incidence of Mammary Tumorsin the C3H Mouse1
Ian J. Tinsley,2 John A. Schmitz, and Donald A. Pierce
Department of Agricultural Chemistry ¡I.J.T.], School of Veterinary Medicine ¡J.A.S.], and Department of Statistics [O.A.P.], Oregon State University,' Corvallis,
It is quite clear that both the level and composition of fat inthe diet can influence the incidence and development of sometumor systems. This is particularly true with mammary tumorsin rats and mice where the effect of fat has been observed withspontaneous tumors (25) and with tumors induced by dimeth-ylbenz(«)anthracene (3, 11), A/-nitrosomethylurea (7, 8), anddiethylstilbestrol (9). Epidemiological studies have also associated the incidence of mammary tumors in humans with thelevel of fat in the diet (4).
In general, experiments designed to study the effect of fatcomposition indicate that oils containing polyunsaturated fattyacids tend to enhance tumorigenesis (3). The response torapeseed oil, an oil very low in saturated fatty acids, is somewhat atypical, being comparable to that produced by the moresaturated fats (3).
Some evidence is accumulating, primarily from experimentswith tumor transplants and tissue culture systems, that linoleateis required for the development of mammary tumors (10, 14).A minimal requirement for linoleate has also been suggested
1This study was supported by USPHS Grants CA20998 and CA 27532 from
the National Cancer Institute. Technical Paper 5508, Oregon Agricultural Experiment Station.
2 To whom requests for reprints should be addressed.
Received May 15, 1980; accepted January 12, 1981.
for the development of mammary tumors induced in rats bydimethylbenz(a)anthracene (11 ). Whether any other fatty acidshave specific effects on the incidence and development ofmammary tumors is not known. Although there are numerouspossibilities (22), the mechanism(s) by which fatty acids influence mammary tumorigenesis are not understood.
Statistical methods have been used in this study to furtherisolate the effects of individual fatty acids on the incidence anddevelopment of mammary tumors in the C3H mouse. Thecorrelation between levels of different fatty acids in the dietshas been reduced by using, in addition to selected natural fatsand oils, mixtures prepared from these components. Regression techniques have been used to explore the contributions ofindividual fatty acids on different aspects of tumorigenesis.
MATERIALS AND METHODS
The experimental design was based on that used by Casteref al. (5) to study the effect of dietary fat on the composition oftissue lipid. Eleven natural fats and oils and mixtures of thesefats and oils were used to provide a total of 20 different fats(Table 1) such that the correlation between levels of individualfatty acids was a minimum. In 2 cases, monoglycerides wereused, and oil extracted from alyssum seeds provided an additional source of eicosanoic acid (20:1 ; this designation identifies fatty acids by the number of carbon atoms in the chainfollowed by the number of double bonds). The fatty acid composition, derived from 6 to 8 diet samples taken over thefeeding period, is also given (Table 1). Correlation coefficientsfor the combinations of the 9 major fatty acids are given inTable 2.
The composition of the semisynthetic diet is outlined in Table3, the fat content being held constant at 10% by weight. Afterreviewing the observations of Carroll and Khor (4) and Silver-stone and Tannenbaum (23), 10% fat was selected as a levelwhich should influence tumorigenesis without overwhelmingdifferences due to composition. Diets were mixed regularly,stored in a freezer, and replaced in the animal cages every 2
Dietary Fatty Acids and Incidence of Mammary Tumors
Table 1Fatty acid composition
Each analysis represents the average of at least 8 diet samples. Standard deviations are omitted in the interest of clarity but are less than 10% in most cases.
% of dietary fat by weight in following fatty acids
to 3 days to minimize any untoward effects from rancidity.Peroxide values were determined for all oils prior to use, andpreliminary studies indicated no appreciable increase duringfrozen storage up to 5 weeks. Vitamin E levels were adequateeven for those diets containing high levels of polyunsaturatedfatty acids.
The diet vvc;sanalyzed for zinc and found to contain 5 to 6mg/kg. An exact estimate of the optimum dietary level of zinchas not been established for the mouse, although deficiencysymptoms have been observed in mice fed diets containing 3mg of zinc per kg, and good growth and reproduction havebeen observed in mice fed diets with 30 mg of zinc per kg.
(17). In an ancillary study, no noticeable improvement in performance was obtained by increasing the zinc content of thediet.
The proportion of dietary calories contributed by linoleate inrations containing tallow or butter as the source of fat was 0.3and 0.5%, respectively. Again, an exact requirement of essential fatty acids has not been established for the mouse; however, these intake levels could be considered marginal inreference to those for the rat where an intake of 0.5% ofcalories as linoleate has been determined for females (19).
Mice were purchased from L. C. Strong Research Foundation, San Diego, Calif., as weanling females, with a minimum of44 animals used for each of the 20 diets. Animals were held inpolycarbonate shoebox cages (3.8 x 19 x 12.7 cm), with 4mice/cage on corncob bedding, and the room was maintainedat 22 ±1°Cwith a 12-hr lighting cycle. Food consumption for
each cage was measured, and the mice were weighed andpalpated weekly to monitor the development of mammary tumors. The size of each tumor was measured with calipers.
Moribund mice were sacrificed by cervical fracture, and eachtumor was weighed, measured, and fixed in 10% bufferedneutral formalin. Any other pathological conditions were noted.Fixed tissues were imbedded in paraffin, sectioned at 6 to 7ftm, and stained with hematoxylin and eosin for microscopicexamination.
For each diet, the entire curve P(f) [the percentage of thepopulation (P), from which the samples were taken, whichwould exhibit the first palpable tumor by time f] was estimated
for all f values between 0 and 100 weeks of age. The estimateP(f) of this function was computed by "life-table" methods (1)
to account for mice taken from the experiment for causes otherthan tumor development. Although there were no substantialdifferences between diets of deaths due to other causes, thismethod of estimation of P(f) does adjust for this complicationand also for the effect of the removal of a few mice during earlyweeks for tissue analysis.
Statistical analysis consisted of carrying out various multipleregressions based on models of the form
9
Q = ßo+ I ßK+ et = i
where 0 is some selected numerical aspect of the curve P(f),0 < f < 100, and x, x9 are the percentages of thecorresponding fatty acids. Since £x,= 100 for all diets, it wasnecessary to impose the constraint (£/?= 0) to make the
regression estimates well defined. Consequently, the regression coefficient ß,is essentially the increase in 0 resulting froma unit increase in x, when all the other x variables are decreasedby equal amounts (1 /8). In other words, ft¡might be conceivedas a "substitution factor," indicating the change in tumorigenic
response produced by increases in the level of a specific fattyacid as it replaces equal proportions of the other 8, with totalfat remaining constant. Another coefficeint relating responsedirectly to dietary levels of a fatty acid would differ from ß,byincorporating both the caloric effect as well as any specificeffect of that fatty acid. One might expect these coefficients tobe positive for all fatty acids given the enhanced tumorigenesiswith increasing levels of total dietary fat. The experimentaldesign used in this study will not provide an estimate of thelatter parameter.
Some of the Ó aspects considered were: (a) the time (f60)until 50% of animals have tumors; (b) P(f) at various selectedtimes (f = 35, 45 95); and finally (c) the age-specific
tumor incidence rates, i.e., the probability of occurrence of atumor in various 10-week periods given no tumor up to that
period. It was found that an adequate summary of the effectsis given by the 2 aspects: (a) Q = f50, the time until 50% have
tumors (median time to tumor); and (b) Ó = P(65), the probability of a tumor by 65 weeks of age. At times substantiallyearlier or later than 65 weeks, there is not enough variation totumor incidence to provide useful inferences.
It should be emphasized that both of these aspects areessentially measuring time to tumor. By the end of the experiment, the tumor incidence on all diets was so high, with onepossible exception, as to provide very little evidence of thedifferential effects of diets.
RESULTS
Growth and Food Intakes. There were no marked differences in food intakes or body weights at 7 and 17 weeks withmice fed these different rations (Table 4). Also, the growth rateobserved in this study was comparable to that reported byPoiley (18) for this strain. Differences in the average bodyweights of the 20 dietary groups are larger at 27 weeks but,with the increased variability, are not significant. At later stagesof the study, comparisons of body weights become tenuouswith increased variability probably associated with tumor development and growth. Thus, it would not appear that differences in caloric intake or food efficiency would be factors ininterpreting effects of diet on tumorigenesis.
Histopathology. As might be expected with the virus-in
duced tumor in this strain (24), the majority of the tumors wereclassified as type A adenocarcinomas. Some type B and mixed,type A and type B, adenocarcinomas were also observed.
A high incidence of generalized amyloidosis as well as focalor multifocal cardiomyocardiolysis of variable severity waspresent in mice from all dietary groups. The possible association of these lesions with the dietary variables is being exploredand will be reported elsewhere.
Tumor Incidence. Estimates of i50, median time to tumor (fwhen P(f) = 0.50), along with values of P(f) at selected 10-
week intervals, are summarized in Table 5. An approximatestandard error for each set of estimates is also included.
In analyzing the effects of different fatty acids, one may usestatistical procedures which would be highly focused and tend
to result in formal tests of significance, or the data may beanalyzed in a broader perspective oriented toward searchingout interesting relationships. Given the exploratory nature ofthe study, the latter approach has been used with the hope ofidentifying as many trends as possible.
Although it is not an essential part of the statistical analysis,it is of interest to calculate the extent to which using mixturesof pure fats and oils increases the precision of inferences aboutapparent effects of specific fatty acids. Assuming a linearregression of some aspect of tumorigenesis on fatty acid levelis a reasonable approximation, it is possible to evaluate theeffectiveness of the design (2). One can compute the numberof replications of the first 11 diets (Table 1), pure fats and oils,required to reduce the standard errors of regression coefficients to that level obtained using all 20 diets. This numbershould be approximately 2 if the use of the mixtures was noteffective. The statistical advantage of the design is quite apparent (Table 6); the levels of effectiveness vary with differentfatty acids because of the varying degree of correlations between fatty acids in the first 11 diets.
Regression coefficients for the 9 fatty acids are given inTable 7 for /50 and P(65), the time at which overall tumorincidence was 55.7%. Note that a substantial amount of thevariation in these 2 quantities can be associated with differences in fatty acid level (r2 = 0.65 and 0.66 for the overall
regression) and that the error term is relatively small. Standarderrors vary considerably among coefficients for different fattyacids; hence, precision of these estimates along with the t
Table 6Replications of first 11 diets needed to give precision obtained with 20 diets
values should both be considered in evaluating the effect ofdifferent fatty acids.
Tumor Yield. The average number of tumors per mouse withtumors ranged from 1.05 to 1.38. No statistically significantrelationships were found in multiple regression of yield ondietary variables. Dietary fat appears to influence incidencerather than yield of mammary tumors in mice (12, 23) while, inrats treated with 7,12-dimethylbenz(a)anthracene, the reverse
is true (3).
DISCUSSION
The regression coefficient for linoleic acid (18:2), while notthe largest, is the most significant, with the lowest standarderror and consistently high f values. The decreased r50 andincreased P(65) would be consistent with other studies, suggesting that this fatty acid is required for the development ofmammary tumors (10, 11, 20). Although linolenic acid (18:3)can inhibit the transformation of linoleate to arachidonic acid(20:4), present in significant amounts in mammary tumor lipid(20), these data do not indicate that this fatty acid has anyspecial effect on the incidence and development of this tumorsystem. Parenthetically, it is interesting to note that an inhibi-
Table 7
Regression coefficients from multiple regressions expressing tx and P(65) as afunction of dietary fatty acid
tory effect of eicosa-5,8,11,14-tetraynoic acid on the growth
of a transplanted mammary adenocarcinoma in mice has beenattributed to inhibition of the conversion of linoleate to arachi-donate (21).
Oleic acid (18:1) substitution appears to have little effect.This may be related to the observation that changes in thedietary level of this fatty acid produce minimal changes in thefatty acid composition of tissue lipids (5). Because of the highstandard error in this study, the effect of eicosanoic acid (20:1) cannot be defined. Erucic acid (22:1), on the other hand,gives a negative coefficient for tumor incidence, the magnitudeof which (though not statistically significant) suggests a decrease in tumor incidence as this fatty acid replaces the other8. Rapeseed oil was the only source of erucic acid in this study,and consequently, it is possible that this effect could be due tosome other constituent of the oil. The response to rapeseed oilconfirms earlier observations of Carroll and Khor (3).
It is not surprising that the in vivo response of linolenateand oleate differs from that observed in vitro since, with thepossible exception of adipose tissue, changes in dietary levelsdo not translate into comparable changes in tissue levels ofthese fatty acids. Linolenate is metabolized rapidly to higherhomologs, and consequently, tissue levels of this particularfatty acid are usually low (15). Concentrations of the higherhomologs would increase with increased levels of linolenate inthe diet; however, the action of these components on the cells
may differ from that of the parent acid. Statistical studies havedemonstrated that the fatty acid composition of tissue lipids isnot particularly responsive to the level of oleate in the diet (5).
Thus, in the analysis of the effect of dietary fat on tumorigenesis, it is not sufficient to simply classify fats as polyunsaturatesor saturates. Individual fatty acids may be having differingeffects on the development of tumors, and the isolation of theseeffects will improve the basis for interpreting the effects of faton cancer.
ACKNOWLEDGMENTS
The competent technical assistance of R. Lowry, B. Jones, and GlenWilson in the preparation of the fat samples and of E. May in the management ofthe animals is acknowledged.
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1981;41:1460-1465. Cancer Res Ian J. Tinsley, John A. Schmitz and Donald A. Pierce Tumors in the C3H MouseInfluence of Dietary Fatty Acids on the Incidence of Mammary