[CANCER RESEARCH 41. 1460-1465.
0008-5472/81
/0041-OOOOS02.00
April 1981]
Influence of Dietary Fatty Acids on the Incidence of Mammary Tumors
in 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,
Oregon 97331
ABSTRACT
Statistical techniques have been used to establish the extent
to which the incidence of spontaneous mammary tumors in
C3H mice could be associated with the levels of individual fatty
acids in their diets. Eleven different fats and oils and nine
mixtures of these fats and oils were selected so that the levels
of the nine major fatty acids varied over a reasonable range
and were not highly correlated with one another. Tumor inci
dence was observed in mice raised on diets containing 10% of
these different fats. Multiple regressions have been calculated,
expressing tumor incidence or time to tumor as a function of
the levels of nine fatty acids, four saturated and five unsaturated, of the dietary lipids. Increased tumor incidence and
decreased time to tumor were observed when increasing levels
of linoleate (18:2) replaced the eight other fatty acids in the
diet while the other polyunsaturated fatty acid, linolenate (18:
3), had little effect on tumor incidence. Four saturated fatty
acids, laurate (12:0), myristate (14:0), palmitate (16:0), and
stéarate(18:0), were studied, with only the latter showing a
significant effect. Increasing levels of stéaratewere associated
with decreased tumor incidence and increased time to tumor.
There was also a suggestion that erucic acid (22:1) reduced
tumor incidence, but oleic acid (18:1) produced no significant
effect.
INTRODUCTION
It is quite clear that both the level and composition of fat in
the diet can influence the incidence and development of some
tumor systems. This is particularly true with mammary tumors
in rats and mice where the effect of fat has been observed with
spontaneous tumors (25) and with tumors induced by dimethylbenz(«)anthracene (3, 11), A/-nitrosomethylurea (7, 8), and
diethylstilbestrol (9). Epidemiological studies have also asso
ciated the incidence of mammary tumors in humans with the
level of fat in the diet (4).
In general, experiments designed to study the effect of fat
composition indicate that oils containing polyunsaturated fatty
acids tend to enhance tumorigenesis (3). The response to
rapeseed oil, an oil very low in saturated fatty acids, is some
what atypical, being comparable to that produced by the more
saturated fats (3).
Some evidence is accumulating, primarily from experiments
with tumor transplants and tissue culture systems, that linoleate
is 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
iment Station.
2 To whom requests for reprints should be addressed.
Received May 15, 1980; accepted January 12, 1981.
1460
Exper
for the development of mammary tumors induced in rats by
dimethylbenz(a)anthracene
(11 ). Whether any other fatty acids
have specific effects on the incidence and development of
mammary tumors is not known. Although there are numerous
possibilities (22), the mechanism(s) by which fatty acids influ
ence mammary tumorigenesis are not understood.
It is not possible to identify the effects of individual fatty
acids, saturated and unsaturated, when comparisons are made
simply among diets, each of which contains a single natural fat
or oil as the fat source. When the fat content is held constant,
there are significant negative correlations between the levels
of different fatty acids; for example, if the level of linoleate is
decreased by substituting tallow for corn oil, one would obtain
a corresponding increase in stéarate.Consequently, one can
not conclude that the difference in response is due to an
increase in the level of one fatty acid or a decrease in the other
or both. Multiple regression methods of analysis do, to some
extent, separate these effects, but the standard errors of indi
vidual regression coefficients are large when the independent
variables are highly correlated.
Statistical methods have been used in this study to further
isolate the effects of individual fatty acids on the incidence and
development of mammary tumors in the C3H mouse. The
correlation between levels of different fatty acids in the diets
has been reduced by using, in addition to selected natural fats
and oils, mixtures prepared from these components. Regres
sion techniques have been used to explore the contributions of
individual fatty acids on different aspects of tumorigenesis.
MATERIALS
AND METHODS
The experimental design was based on that used by Caster
ef al. (5) to study the effect of dietary fat on the composition of
tissue lipid. Eleven natural fats and oils and mixtures of these
fats and oils were used to provide a total of 20 different fats
(Table 1) such that the correlation between levels of individual
fatty acids was a minimum. In 2 cases, monoglycerides were
used, and oil extracted from alyssum seeds provided an addi
tional source of eicosanoic acid (20:1 ; this designation identi
fies fatty acids by the number of carbon atoms in the chain
followed by the number of double bonds). The fatty acid com
position, derived from 6 to 8 diet samples taken over the
feeding period, is also given (Table 1). Correlation coefficients
for the combinations of the 9 major fatty acids are given in
Table 2.
The composition of the semisynthetic diet is outlined in Table
3, the fat content being held constant at 10% by weight. After
reviewing the observations of Carroll and Khor (4) and Silverstone and Tannenbaum (23), 10% fat was selected as a level
which should influence tumorigenesis without overwhelming
differences due to composition. Diets were mixed regularly,
stored in a freezer, and replaced in the animal cages every 2
CANCER
RESEARCH
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Dietary Fatty Acids and Incidence of Mammary Tumors
Table 1
Fatty 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
22:11.310.1
14:035.5
fatCoconutButterTallowLardOliveCottonseedCornRapeseed
Dietary
17.72.84
12.74.421.300.590.7119.8
51.11.75.54
erucic)SafflowerLinseedSpan
(high
erucic)]Corn
[rapeseed (low
(0.5)Lard
(0.5), rapeseed
(0.25)Coconut
(0.5), olive (0.25). alyssum
(0.5)Linseed
(0.5), safflower
(0.6)Safflower
(0.4), tallow
(0.5)Butter(0.5), olive
(0.4)Tallow
(0.6), cottonseed
(0.2)Linseed
(0.8). span
(0.4),olive
(0.2), glycerol stéarate
(0.4)Glycerol
myristate (0.8), span (0.2)12:0
24.812.2Trace
9.272.241.25
6.523.230.44
Trace
69.116:020.739.731.325.212.914.910.62.016.205.03.96.4116.213.919.79.0427.325.37.354.4918:04.013.819.313.82.
Table 2
Correlation ratios of fatty acid pairs
22:11.00-0.75
12:014:016:018:018:118:218:320:122:112:01.000.230.14-0.13-0.30-0.10-0.15-0.16-0.1214:01.000.04-0.12-0.28-0.26-0.14-0.19-0.1516:01.000.510.20-
1.00
Table 3
Composition of basal diet
ConstituentsCaseinCereloseSalt
mix"Fat
diet200
g565
g50g100g75g10g2
oil)Solfa-flocVitamin
(or
mix*1Vitamin
acetateVitamin
A
0.r>a-Tocopherol
acetateAmount/kg
mg4
mg50
mg
Sait mixture according to Hubbell et al. (13).
6 Amount per kg diet: menadione, 10 mg; thiamine HCI, 10 mg; riboflavin, 10
mg; pyridoxine (vitamin B6), 20 mg; nicotinamide, 50 mg; calcium pantothenate,
30 mg; ascorbic acid, 100 mg; p-aminobenzoic acid, 1.0 g; choline dihydrogen
citrate, 5.0 g; inositol, 1.0g; biotin, 200 mg; folie acid, 1.0 mg; vitamin B,2(0.1%
trituration), 20 mg; and lactose, 2.759 g.
to 3 days to minimize any untoward effects from rancidity.
Peroxide values were determined for all oils prior to use, and
preliminary studies indicated no appreciable increase during
frozen storage up to 5 weeks. Vitamin E levels were adequate
even for those diets containing high levels of polyunsaturated
fatty acids.
The diet vvc;sanalyzed for zinc and found to contain 5 to 6
mg/kg. An exact estimate of the optimum dietary level of zinc
has not been established for the mouse, although deficiency
symptoms have been observed in mice fed diets containing 3
mg of zinc per kg, and good growth and reproduction have
been observed in mice fed diets with 30 mg of zinc per kg.
(17). In an ancillary study, no noticeable improvement in per
formance was obtained by increasing the zinc content of the
diet.
The proportion of dietary calories contributed by linoleate in
rations containing tallow or butter as the source of fat was 0.3
and 0.5%, respectively. Again, an exact requirement of essen
tial fatty acids has not been established for the mouse; how
ever, these intake levels could be considered marginal in
reference to those for the rat where an intake of 0.5% of
calories as linoleate has been determined for females (19).
Mice were purchased from L. C. Strong Research Founda
tion, San Diego, Calif., as weanling females, with a minimum of
44 animals used for each of the 20 diets. Animals were held in
polycarbonate shoebox cages (3.8 x 19 x 12.7 cm), with 4
mice/cage on corncob bedding, and the room was maintained
at 22 ±1°Cwith a 12-hr lighting cycle. Food consumption for
each cage was measured, and the mice were weighed and
palpated weekly to monitor the development of mammary tu
mors. The size of each tumor was measured with calipers.
Moribund mice were sacrificed by cervical fracture, and each
tumor was weighed, measured, and fixed in 10% buffered
neutral formalin. Any other pathological conditions were noted.
Fixed tissues were imbedded in paraffin, sectioned at 6 to 7
ftm, and stained with hematoxylin and eosin for microscopic
examination.
For each diet, the entire curve P(f) [the percentage of the
population (P), from which the samples were taken, which
would exhibit the first palpable tumor by time f] was estimated
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1461
/. J. Tinsley et al.
tumors (median time to tumor); and (b) Ó = P(65), the proba
bility of a tumor by 65 weeks of age. At times substantially
earlier or later than 65 weeks, there is not enough variation to
tumor incidence to provide useful inferences.
It should be emphasized that both of these aspects are
essentially measuring time to tumor. By the end of the experi
ment, the tumor incidence on all diets was so high, with one
possible exception, as to provide very little evidence of the
differential effects of diets.
for all f values between 0 and 100 weeks of age. The estimate
P(f) of this function was computed by "life-table" methods (1)
to account for mice taken from the experiment for causes other
than tumor development. Although there were no substantial
differences between diets of deaths due to other causes, this
method of estimation of P(f) does adjust for this complication
and also for the effect of the removal of a few mice during early
weeks for tissue analysis.
Statistical analysis consisted of carrying out various multiple
regressions based on models of the form
9
RESULTS
Q = ßo+ I ßK+ e
t=i
where 0 is some selected numerical aspect of the curve P(f),
0 < f < 100, and x,
x9 are the percentages of the
corresponding fatty acids. Since £x,= 100 for all diets, it was
necessary to impose the constraint (£/?= 0) to make the
regression estimates well defined. Consequently, the regres
sion coefficient ß,is essentially the increase in 0 resulting from
a unit increase in x, when all the other x variables are decreased
by equal amounts (1 /8). In other words, ft¡might be conceived
as a "substitution factor," indicating the change in tumorigenic
response produced by increases in the level of a specific fatty
acid as it replaces equal proportions of the other 8, with total
fat remaining constant. Another coefficeint relating response
directly to dietary levels of a fatty acid would differ from ß,by
incorporating both the caloric effect as well as any specific
effect of that fatty acid. One might expect these coefficients to
be positive for all fatty acids given the enhanced tumorigenesis
with increasing levels of total dietary fat. The experimental
design used in this study will not provide an estimate of the
latter parameter.
Some of the Ó aspects considered were: (a) the time (f60)
until 50% of animals have tumors; (b) P(f) at various selected
times (f = 35, 45
95); and finally (c) the age-specific
tumor incidence rates, i.e., the probability of occurrence of a
tumor in various 10-week periods given no tumor up to that
period. It was found that an adequate summary of the effects
is given by the 2 aspects: (a) Q = f50, the time until 50% have
Growth and Food Intakes. There were no marked differ
ences in food intakes or body weights at 7 and 17 weeks with
mice fed these different rations (Table 4). Also, the growth rate
observed in this study was comparable to that reported by
Poiley (18) for this strain. Differences in the average body
weights of the 20 dietary groups are larger at 27 weeks but,
with the increased variability, are not significant. At later stages
of the study, comparisons of body weights become tenuous
with increased variability probably associated with tumor de
velopment and growth. Thus, it would not appear that differ
ences in caloric intake or food efficiency would be factors in
interpreting 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 were
classified 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 focal
or multifocal cardiomyocardiolysis
of variable severity was
present in mice from all dietary groups. The possible associa
tion of these lesions with the dietary variables is being explored
and will be reported elsewhere.
Tumor Incidence. Estimates of i50, median time to tumor (f
when P(f) = 0.50), along with values of P(f) at selected 10week intervals, are summarized in Table 5. An approximate
standard error for each set of estimates is also included.
In analyzing the effects of different fatty acids, one may use
statistical procedures which would be highly focused and tend
Table 4
Body weight and food intake
Body wt (g) at following wk
17
Dietary fat
CoconutButterTallowLardOliveCottonseedCornRapeseed
erucic)SafflowerLinseedSpan
(high
erucic)]Corn
[rapeseed (low
(0.5)Lard
(0.5). rapeseed
(0.25)Coconut
(0.5), olive (0.25), alyssum
(0.5)Linseed
(0.5), safflower
(0.6)Safflower
(0.4), tallow
(0.5)Butter (0.5), olive
(0.4)Tallow
(0.6), cottonseed
(0.2)Linseed
(0.8), span
(0.4)Myristate
(0.2), stéarate(0.4), olive
(0.8), span (0.2)19.3
' Mean ±S.D.
1462
1.1a19.6
±
1.119.2
±
0.919.5
±
1.119.3
±
1.118.8
±
1.318.6
±
1.219.2
±
1.318.6
±
1.219.2
±
1.419.0
±
1.718.9
±
1.319.2
±
1.518.4
±
1.218.5
±
1.318.9
±
1.318.8
±
0.919.3
±
1.718.5
±
1.019.1
±
±1.124.8
1.225.2
±
0.824.2
±
1.225.1
±
1.725.3
±
1.024.8
±
1.424.9
±
0.924.3
±
2.425.1
±
1.225.1
±
0.925.8
±
1.124.9
±
2.424.6±
1.624.1
±
1.324.5
±
.424.9±
.325.3±
.125.5±
.224.8±
.425.1±
±0.830.8
.932.1 ±1
2.029.9±
2.132.3
±
1.831.4
±
1.729.4
±
3.330.7
±
2.430.6
±
1.531.6
±
2.530.9±
2.031.9
±
1.530.8
±
2.430.5±
2.530.3±
2.129.8
±
.931.7 ±1
2.531.6±
3.331.4±
2.031.1
±
1.831.3±
±2.134.2
27
2.835.2±
2.831.9±
2.936.3±
3.234.6
±
2.533.6
±
2.934.6±
3.234.6±
3.035.6
±
3.934.0
±
3.034.8
±
2.834.5±
4.134.6±
3.433.4
±
3.333.3
±
.935.4±1
2.435.1
±
2.735.4±
2.334.6
±
2.335.4±
±2.84.0
CANCER
Av. daily food intake
(9)
0.53.9
±
0.34.4
±
0.33.7
±
0.43.8
±
0.23.8
±
0.33.7
±
0.33.7
±
0.33.7
±
0.33.6
±
0.53.6
±
0.33.8
±
0.23.9±
0.33.9
±
0.33.8
±
0.23.8
±
0.53.9
±
0.34.1
±
0.54.1
±
0.43.8
±
±0.4
RESEARCH
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VOL. 41
Dietary Fatty Acids and Incidence of Mammary Tumors
Table 5
Effect of dietary fat on tumor incidence
wkDietary
variableCottonseedButter
% of mice with palpable tumor at following
(wk)56.3056.3056.6059.0059.5059.9060.2061.3061.3061.4062.0062.0063.0064.0066.0066.4067.4067.5068.5070.1
(0.4)SafflowerSafflower
(0.6), cottonseed
(0.5)SpanCornCoconutCoconut
(0.5), olive
(0.5)OliveTallow
(0.5), safflower
(0.2)Lard
(0.8), span
(0.25)ButterLinseed
(0.5), olive (0.25), alyssum
(0.6)LinseedMyristate
(0.4), tallow
(0.2)Com (0.8), span
(0.5)Linseed
(0.5), rapeseed
(0.4)RapeseedTallowLardApproximate
(0.2), stéarate(0.4), olive
S.E.fx
to result in formal tests of significance, or the data may be
analyzed in a broader perspective oriented toward searching
out interesting relationships. Given the exploratory nature of
the study, the latter approach has been used with the hope of
identifying 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 mixtures
of pure fats and oils increases the precision of inferences about
apparent effects of specific fatty acids. Assuming a linear
regression of some aspect of tumorigenesis on fatty acid level
is a reasonable approximation, it is possible to evaluate the
effectiveness of the design (2). One can compute the number
of replications of the first 11 diets (Table 1), pure fats and oils,
required to reduce the standard errors of regression coeffi
cients to that level obtained using all 20 diets. This number
should be approximately 2 if the use of the mixtures was not
effective. The statistical advantage of the design is quite ap
parent (Table 6); the levels of effectiveness vary with different
fatty acids because of the varying degree of correlations be
tween fatty acids in the first 11 diets.
Regression coefficients for the 9 fatty acids are given in
Table 7 for /50 and P(65), the time at which overall tumor
incidence was 55.7%. Note that a substantial amount of the
variation in these 2 quantities can be associated with differ
ences in fatty acid level (r2 = 0.65 and 0.66 for the overall
regression) and that the error term is relatively small. Standard
errors vary considerably among coefficients for different fatty
acids; hence, precision of these estimates along with the t
values should both be considered in evaluating the effect of
different fatty acids.
Tumor Yield. The average number of tumors per mouse with
tumors ranged from 1.05 to 1.38. No statistically significant
relationships were found in multiple regression of yield on
dietary variables. Dietary fat appears to influence incidence
rather than yield of mammary tumors in mice (12, 23) while, in
rats treated with 7,12-dimethylbenz(a)anthracene,
the reverse
is true (3).
DISCUSSION
The regression coefficient for linoleic acid (18:2), while not
the largest, is the most significant, with the lowest standard
error and consistently high f values. The decreased r50 and
increased P(65) would be consistent with other studies, sug
gesting that this fatty acid is required for the development of
mammary 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 any
special effect on the incidence and development of this tumor
system. Parenthetically, it is interesting to note that an inhibiTable 7
Regression coefficients from multiple regressions expressing tx and P(65) as a
function of dietary fatty acid
Fatty
acid12:014:016:018:018:118:218:320:122:1r»VRMSC-0.060.02-0.080.19-0.02-0.10-0.01-0
Table 6
Replications of first 11 diets needed to give precision obtained with 20 diets
Fatty
acid12:014:016:018:018:118:218:320:122:1Replications142152232132295533
0.51±0.18(1.12)(-0.84)(0.45)(-1.61
Mean ±S.E.
Numbers in parentheses, f value testing ß,= 0.
c Residual mean square.
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tory effect of eicosa-5,8,11,14-tetraynoic
acid on the growth
of a transplanted mammary adenocarcinoma in mice has been
attributed to inhibition of the conversion of linoleate to arachidonate (21).
Oleic acid (18:1) substitution appears to have little effect.
This may be related to the observation that changes in the
dietary level of this fatty acid produce minimal changes in the
fatty acid composition of tissue lipids (5). Because of the high
standard 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 magnitude
of which (though not statistically significant) suggests a de
crease in tumor incidence as this fatty acid replaces the other
8. Rapeseed oil was the only source of erucic acid in this study,
and consequently, it is possible that this effect could be due to
some other constituent of the oil. The response to rapeseed oil
confirms earlier observations of Carroll and Khor (3).
The largest regression coefficients, though not the most
precise, are observed in stéarate(18:0). Increasing levels of
this fatty acid in the diet are associated with higher values of
fso and lower tumor incidence at 65 weeks. These data suggest
that it is unlikely that palmitate (16:0) has an effect comparable
to stéarate, if anything, it would be the reverse. The effects
associated with varying levels of myristate (14:0) and laurate
(12:0) appear to be small, although there is a suggestion that
lauric acid may have a positive effect on tumor incidence at 65
weeks. The enhanced tumor yield with coconut oil compared
to tallow in rats treated with dimethylbenz(a)anthracene
(11 )
may reflect an effect of lauric acid.
Differing response to the 4 saturated fatty acids is of interest
and would substantiate the observations of Caster et al. (6)
who demonstrated that saturated fatty acids also differed in
their effects on a number of physiological parameters. Of
particular interest was a highly significant effect of stéarateon
liver lipid, cholesterol content of liver lipid, and plasma choles
terol. These workers also defined 2 groups of saturates, C4,8,12,16 and C-6,10,14,18, based on effects on food intake
and growth. In this study, responses to the 4 saturated fatty
acids would tend to confirm this classification and would sup
port the contention of Caster ef a/, that "saturated fatty acids
should not be considered just as a group of non-essential,
similar energy sources, but as a group of nutrients each of
which is biochemically and physiologically significant in its own
right."
Conclusions of this study are confirmed in part by recent
observations of the effects of fatty acids on the growth of
normal and neoplastic rat mammary epithelial cells (26). In
both cell systems, linoleate enhanced and stéarateinhibited
growth. However, in contrast to observations reported here,
both oleate and linolenate enhanced growth, the former being
more active with neoplastic cells and the latter with normal
cells.
It is not surprising that the in vivo response of linolenate
and oleate differs from that observed in vitro since, with the
possible exception of adipose tissue, changes in dietary levels
do not translate into comparable changes in tissue levels of
these fatty acids. Linolenate is metabolized rapidly to higher
homologs, and consequently, tissue levels of this particular
fatty acid are usually low (15). Concentrations of the higher
homologs would increase with increased levels of linolenate in
the diet; however, the action of these components on the cells
1464
may differ from that of the parent acid. Statistical studies have
demonstrated that the fatty acid composition of tissue lipids is
not particularly responsive to the level of oleate in the diet (5).
The use of median tumor incidence or incidence over some
specified time interval is one approach to the analysis of the
effect of the dietary variables. A more sophisticated treatment
would be required for the comprehensive analysis of the ob
vious differences in the time course of tumor incidence (Table
4). Of particular interest in this regard is the response of mice
raised on the ration containing 10% tallow. The time course of
tumor incidence through 65 weeks is not markedly different
from that of the other 19 dietary groups; however, in the next
30 weeks, further incidence of tumors was much less. This
decreased tumor incidence cannot be attributed to increased
mortality. It is possible that, over the prolonged feeding period,
a low-order deficiency in essential fatty acids is achieved which
could inhibit tumorigenesis. Fatty acid analysis of tissues (to
be reported elsewhere) would be indicative of such a possibil
ity; however, no gross deficiency symptoms were observed.
Mice raised on rations containing 10% butterfat might be
expected to show a similar response; however, this was not
the case. The higher level of stéaratein tallow may also be a
factor in the differences in response of the 2 dietary groups.
Thus, in the analysis of the effect of dietary fat on tumorigen
esis, it is not sufficient to simply classify fats as polyunsaturates
or saturates. Individual fatty acids may be having differing
effects on the development of tumors, and the isolation of these
effects will improve the basis for interpreting the effects of fat
on cancer.
ACKNOWLEDGMENTS
The competent technical assistance of R. Lowry, B. Jones, and Glen
Wilson in the preparation of the fat samples and of E. May in the management of
the animals is acknowledged.
REFERENCES
1. Armitage, P. Statistical Methods in Medical Research. New York: John Wiley
& Sons, 1971.
2. Brownlee. K. A. Statistical Theory and Methodology in Science and Engi
neering. New York: John Wiley & Sons. 1965.
3. Carroll, K. K.. and Khor, H. T. Effects of level and type of dietary fat on
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1465
Influence of Dietary Fatty Acids on the Incidence of Mammary
Tumors in the C3H Mouse
Ian J. Tinsley, John A. Schmitz and Donald A. Pierce
Cancer Res 1981;41:1460-1465.
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