Download Effects of Dietary Fats and Butylated

(CANCER RESEARCH 45, 558-560,
February 1985]
Effects of Dietary Fats and Butylated Hydroxytoluene on Mutagen Activation in
Rats1
Debra L. Ponder2 and Nancy R. Green
Department of Nutrition and Food Science, Florida State University, Tallahassee, Florida 32306
of membrane bound enzymes (6). Of concern is the membranebound microsomal drug-metabolizing enzymes responsible for
ABSTRACT
The effects of hydrogenated fats and butylated hydroxytoluene (BHT) in the diets of rats on the hepatic activation of
benzo(a)pyrene, 2-acetylaminofluorene (AAF), and 2-aminofluorene by liver homogenates (S-9 fraction) were evaluated. The
Sa/mone//a/microsomal mutagenicity assay (Strain TA 98) was
utilized to determine the mutagenic potential of the activated
compounds. The S-9 fraction was obtained from animals fed a
15% fat diet consisting of hydrogenated fats (43% frans-fatty
acids) or unsaturated fats (0% frans-fatty acids). BHT was ad
ministered orally (0.5%) 6 days prior to sacrifice in both groups.
The incorporation of BHT in the diet of rats enhanced the
mutagenic potential of AAF and 2-aminofluorene but not of
benzo(a)pyrene. This effect was independent of the lipid com
position of the diet. The most significant increment in the pro
duction of mutagenic metabolites was observed with AAF when
BHT and hydrogenated fats were included in the diet of rats.
Dietary hydrogenated fats appeared to potentiate the effects of
BHT on AAF mutagenicity. Further studies to elucidate the
mechanisms by which BHT and hydrogenated fats enhance AAF
mutagenicity are warranted.
the activation and/or deactivation of xenobiotic substances.
frans-Fatty acids, when incorporated into membrane phospho
lipids, may function as cocarcinogens by altering the activity of
these enzymes.
The purpose of this study was to evaluate the effects of the
incorporation of frans-fatty acids in the diet of rats on the
mutagenic potential of BP,3 AAF, and AF. The frans-fatty acids
were delivered in the form of hydrogenated fat added to the diets
of rats. The testing system consisted of the Ames Salmonella/
microsome mutagenicity assay incorporating liver cell homoge
nates (S-9) from rats fed the frans-fatty acid or control diets.
BHT has been shown to be an effective inducer of microsomal
drug-metabolizing enzymes (11). BHT was chosen as the en
zyme inducer in the study due to its presence in commercially
produced vegetable oils, margarine, and shortenings (7). The
compounds AAF and AF are representative of the aromatic
amines, and BP is representative of the polycyclic aromatic
hydrocarbons. These compounds were chosen because their
metabolism involves the membrane-bound cytochrome P-450
system and their active and inactive metabolites are known.
INTRODUCTION
MATERIALS AND METHODS
The total quantity and type of fat (polyunsaturated, saturated,
hydrogenated) have been associated with the occurrence of
different types of cancer (12, 13). The presence of frans-fatty
acids is an additional possible contributory factor.
The biophysical effects of the tissue incorporation of fransfatty acids, geometric isomers of the naturally occurring c/s-fatty
acids, have not been sufficiently determined. frans-Fatty acids
result from the hydrogénation of vegetable oils or fats and
comprise 16 to 36% of the fatty acids in commercially produced
margarines and shortenings (10). Calculations suggest the per
capita consumption of frans-fatty acids in the US to be 8% of
the total daily fat intake (9).
Animal feeding studies have shown the preferential accumu
lation of frans-fatty acids 18:1 in liver phospholipids (1). Sgoutas
and Kummerow (14) reported that frans-fatty acids caused monomolecular films to be condensed in vitro. Vigo ef al. (16) suc
cessfully hydrogenated model membranes, which resulted in
reduced fluidity of the lipid bilayer.
Alterations in fluidity of cell membranes may alter the activity
1This work was supported by the Developing Scholar Grant (Florida State
University), the Whitney Fund (Florida State University), Sigma Xi Grants-in-Aid of
Research, and Best Foods, Inc.
2 Present address: Division of Nutrition, Department of Community Health,
Emory University School of Medicine, 69 Butler Street, S.E., Atlanta, GA 30303.
To whom requests for reprints should be addressed.
This work represents a portion of the research for a Ph.D. dissertation presented
to the Department of Nutrition and Food Science at Florida State University.
Received January 30, 1984; accepted November 1, 1984.
CANCER RESEARCH
Materials. The following were obtained from Sigma Chemical Co. (St.
Louis, MO): BHT; dimethyl sulfoxide; BP; AF; and AAF.
Animals. Male weanling Sprague-Dawley rats (Charles River Breeding
Laboratories, Inc., Wilmington, MA) were divided into 2 groups of 15 and
fed either the experimental or control diets for a period of 5 to 6 weeks.
The control and experimental diets were identical, except in the dietary
fat composition (Table 1). The experimental diet contained 15% partially
hydrogenated fat, which provided 43% frans-fatty acids. The control diet
contained 15% unsaturated fat but 0% trans feffy-acids. The experimen
tal and control groups were further divided into noninduced and induced
groups. The induced group received 0.5% BHT mixed in the diet the last
6 days of the feeding period.
Preparation of Homogenates. The rats were decapitated, and the
livers were removed from the animals under aseptic conditions. All
subsequent steps were carried out at 0-4° following the procedures
described by Ames ef al. (2). For each S-9 fraction, the livers of 3 animals
were pooled to minimize animal-to-animal variation. The S-9 fractions
were stored no longer than 6 months at -70°.
Salmonella typhimurium Mutagenicity Assay. The mutagenicity tests
were performed using strain TA 98 as described by Ames ef al. (2). Each
mutagen concentration was tested in a minimum of 6 plates. For prepa
ration of mutagen concentrations, 1500 ng BP, AAF, or AF were dis
solved in 15 ml dimethyl sulfoxide and stored in sterile, capped tubes in
the dark. The concentrations of each mutagen tested were selected from
the lower end of the linear portion of the dose-response curve for that
mutagen, and the number of revenant colonies for each concentration
3The abbreviations used are: BP, benzo(a)pyrene; BHT, butylated hydroxytoluene; AAF, 2-acetylaminofluorene; AF, 2-aminofluorene.
VOL. 45 FEBRUARY
1985
558
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MUTAGENIC
EFFECTS OF DIETARY
Table 1
Diet composition
Custom fat-free test diet"
Composition of Dietary Fats
Unsaturated0fat
Fatty acid composition
of BHT to the diet of animals (Table 3). Significant (p < 0.05)
increases in the mutagenic response were observed at all con
centrations of AAF tested with the S-9 fractions from animals
receiving BHT and hydrogenated fats in the diet when compared
to the control animals receiving BHT. Thus, dietary inclusion of
the inducer BHT and hydrogenated fats had an additive effect
on the enhancemnt of the mutagenic potential of AAF. In the S9 assays from noninduced animals receiving hydrogenated fats,
a higher concentration of AAF was required for a mutagenic
response than in the S-9 assays from animals receiving unsatu-
29.0
2.0
64.0
4.0
1.0
Casein
Alphacel
Sucrose
Salt mix USP XIV
Vitamin-diet fortification mixture
Hydrogenated0fat
mixture
mixture
FATS AND BHT
12:014:016:016:118:018:118:218:320:022:024:0Unknown%
rated fats. However, no significant differences were noted be
tween the numbers of revenant colonies resulting from the S-9
assays from animals receiving hydrogenated fats or unsaturated
fats with AAF concentrations of 50 ¿¿g
or higher.
The mutagenic potential of AF was significantly increased at
the 10-/¿gconcentration of S-9 assays from noninduced animals
receiving hydrogenated fats as compared to unsaturated fats
(Table 4). This difference was observed only at the 10-jug con
centration and is not considered significant.
trans-fatty acids0.15.30.22.373.616.60.60.70.30.10.20.00.30.210.90.29.356.820.50.80.50.20.10.242.5
" No. 99999; ICN Nutritional Biochemicals. Cleveland. OH.
6 High oteic safflower oil, percentage composition.
0 Specially prepared hydrogenated soybean oil and Mazólacom oil, percentage
composition.
Table2
Effects of dietary tat and BHT on the mutagenic potential of BP in the Ames'
Salmonella/S-9assay
on the following
Inducer chemical (fig/
plate)BHTBP2
»g10
diet:Control76
acid61
*85±5a±7b90
14"60
±
fig20
±1730
>igBackground"NoninducedBP2/ig5
±458
¿ig10
fig20
±1467
10o71
±
±6"67
±13"33
1177
±
±15"98
±30"54
±1333±964±464±7165
12"66
+
±2332
»igBackground"Revertants/plate
±7trans-Fatty ±8PNSCNS<0.05
Revenant colonies are mean ±S.D. (n = 6).
6 Mutagenic response, revenant colonies per plate, show a 2-fold increaseover
background revenants.
0 NS, not significant.
d Background data consist of the mean ±S.D. of the number of revenant
colonies resulting from the incorporation of 100 /il TA 98 culture, 20 »/I
dimethyl
sulfoxide, and 500 n\ of the S-9 mix.
was averaged. The mean values for the control and mutagen concentra
tions were reported. For statistical analysis of data, Student's t test was
DISCUSSION
These experiments substantiate the usefulness of the Ames'
mutagenicity testing system in detecting the influence of dietary
fat on the hepatic activation of mutagens. This finding is in
agreement with the conclusions of Castro ef al. (5), as well as
those of Black and Gerguis (3). The current study provides a
qualitative, not quantitative, assessment of the effects of dietary
fat and the antioxidant BHT on the metabolic activation of BP,
AAF, and AF.
The incorporation of BHT in the diet of rats receiving hydro
genated or unsaturated fat enhanced the mutagenic potential of
AAF and AF but not of BP. This effect was independent of the
lipÃ-dcomposition of the diet. Weak inhibitory properties of BHT
toward the mutagenicity of BP were not observed in this study
as they were in the study by Calle and Sullivan (4).
The most significant increment of the production of mutagenic
metabolites occurred when BHT and hydrogenated fat were
added to the animals' diets. The inclusion of BHT and hydrogenTable3
Effects of dietary fat and BHT on the mutagenicpotential of 2-MF in the Ames'
Salmonella/S-9 assay
Inducer
chemical
(/^g/plate)BHTAAF20
diet:Control261on the following
acid487
±152"-"559
956446
±
±111"30±4224
t¡gBackground0NoninducedAAF20
ng50
ttg100
used.
±117"744±1516844
±227*33±950±10175
RESULTS
BHT is not an effective inducer of BP mutagenicity, as evi
denced in this study (Table 2). No significant differences were
observed in the mutagenic potential of BP in the microsomal
assays from BHT-induced and noninduced animals. In addition,
the presence of hydrogenated fats in the diets of rats with or
without the inclusion of BHT had no notable effects on BP
mutagenicity.
The mutagenic potential of AAF was enhanced by the addition
CANCER
RESEARCH
jig50
94"226
±
141"228
±
±159"33
nglOOngBackground0Revertants/plate
±109"271
138"32
±
±7trans-Fatty
±8P<0.05<0.05<0.05NS"NS
* Revenant colonies are mean ±S.D. (n = 6).
6 Mutagenic response, revenant colonies per plate, show a 2-fold increaseover
background revenants.
c Background data consist of the mean value ±S.D. of the number of revenant
colonies resulting from the incorporation of 100 (<iTA 98 culture, 20 pi dimethyl
sulfoxide, and 500 «¿I
of the S-9 mix.
" NS, not significant.
VOL. 45 FEBRUARY
1985
559
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MUTAGENIC
EFFECTS OF DIETARY FATS AND BHT
Table 4
Effects of dietary fat and BHT on the mutagenic potential of 2-AF in the Ames'
Salmonella/S-9assay
Inducer
chemical
kg/plate)BHTAF2
f<g10
figBackground0
diet:Control983
on the following
acid860
±148a'6
1585 ±150"
1652
±512"30
±4466
±226"
1458±2886
2191
±537633
NS
NSNSNSNS
±9565
the usefulness of the in vitro mutagenicity test in assessing the
effects of dietary factors on mutagenesis. More specifically,
(a) dietary BHT enhanced the activation of AAF and AF, and (b)
the incorporation of hydrogenated fats potentiated the effects of
BHT on AAF mutagenicity. Further studies to elucidate the
mechanisms by which BHT and hydrogenated fats enhance AAF
mutagenicity are warranted. In addition, the effects of BHT and
hydrogenated fats on tumorigenesis in animals following AAF
administration are worthy of investigation.
NoninducedAF2
REFERENCES
±245"
±311"
1150±2616
869 ±409"
[Ã-Q10
5
1432 ±410"
900 ±417"
fig
<0.05
Background''Revertants/plate
33 ±7frans-Fatty 32 ±8PNSC
* Revertant colonies are mean ±S.D. (n = 6).
Mutagenic response, revenant colonies per plate, show a 2-fold increaseover
background revenants
c NS, not significant.
"Background data consist of the mean ±S.D. of the number of revenant
1. Alfin-Slater, R. B., and Aftergood, L. Nutritional role of hydrogenatedfats. In:
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3. Black, H. S., and Gerguis,J. Use of the Ames test in assessingthe relation of
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Pathol. Toxicol., 4: 131-138, 1980.
4. Calle,L. M., and Sullivan,P. D. Screeningof antioxidants and other compounds
for antimutagenic properties toward benzo(a)pyrene-inducedmutagenicity in
Strain TA 98 of Salmonellatyphimurium. Mutât.Res., 707: 99-114,1982.
5. Castro, C. E., Felkner, I. C., and Yang, S. P. Dietary lipid-dependentactivation
of the carcinogen W-2-fluorenylacetamidein rats as monitored by Salmonella
typhimurium. Cancer Res., 38: 2836-2841,1978.
6. Coleman, R. Membrane-bound enzymes and membrane ultrastructure.
Biochim. Biophys. Acta, 300:1-30, 1973.
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Food Cosmetic Toxicol., 8: 409-412, 1970.
8. Cramer,J. W., Miller, J. A., and Miller, E. C. N-Hydroxylation: a new metabolic
reaction observed in the rat with the carcinogen2-acetylaminofluorene.J Biol
Chem., 235: 885-888, 1960.
9. Enig, M. C., Munn, R. J., and Kenney, M. Dietary fat and cancer trends: A
critique. Fed. Proc., 37: 2215-2220,1978.
10. Enig, M. G., Pallansch, L. A., Sampugna, J., and Kenney, M. Fatty acid
composition of the fat in selected food items with emphasis on trans-compo
nents. J. Am. Oil Chem. Soc., 60:1788-1795, 1983.
11. Gilbert, D., and Goldberg, L. A liver microsomal enzyme induced by the
treatment of fats with butylated hydroxytoluene. Food Cosmet. Toxicol., 5:
481-490, 1967.
12. Hopkins, G. M., and West, C. G. Minireview: possible roles of dietary fats in
carcinogenesis.Life Sci., 79:1103-1116,1976.
13. Pearce, M. L., and Dayton, S. Incidence of cancer in men on a diet high in
polyunsaturatedfat. Lancet, 7: 464-467,1971.
14. Sgoutas, D., and Kummerow, F. Incorporation of frans-fatty acids into tissue
lipids. Am. J. Clin. Nutr., 23: 1111-1119,1970.
15. Shut, H. A. J., Wirth, P. J., and Thorgeirsson, S. S. Mutagenic activation of Whydroxy-2-acetylaminofluorene in the Salmonella test system: the role of
deacetylation by liver and kidney fractions from mouse and rat Mol PharmaCOl.,74. 682-692, 1978.
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Arylhydroxamic acid acyltransferase in the mutagenicity of N-hydroxy-N-fluorenylacetamidein Salmonellatyphimurium. Cancer Res., 38: 613-618,1978.
uQ
colonies resulting from the incorporation of 100 ni TA 98 culture, 20 ¡A
dimethyl
sulfoxide, and 500 ^l of the S-9 mix.
ated fat in the diet resulted in an aproxÃ-mate 2-fold increase in
the mutagenic potential of AAF. However, the incorporation of
hydrogenated fat without BHT did not significantly influence the
mutagenicity of AAF. Thus, it appears that hydrogenated fat and
BHT have a synergistic effect on AAF mutagenicity. In compari
son, the mutagenicity of AF was enhanced but not significantly
increased by the inclusion of hydrogenated fats and BHT.
The effect of dietary BHT and lipid composition on the hepatic
activation of premutagenic compounds appears not to be related
to the class of the mutagenic compound. In the present study,
the modifications enhancing mutagenicity of the aromatic amine
AAF did not significantly affect the mutagenic potential of the
aromatic amine AF. Reportedly, the most important metabolic
pathway in the mutagenic activation of AAF in the Salmonella
test system is the deacetylation of W-hydroxy-AAF to the hydroxylamine /V-OH-AF (15, 17). Both AAF and AF must first be
hydroxylated by the mixed-function oxidase system in the microsomal fraction of the cell (8). Since the mutagenic potential of
AAF, not AF, is significantly affected by BHT and dietary fat, it
is likely that components of the deacetylase reaction, not the
hydroxylation reaction, are affected.
In conclusion, evidence has been presented which illustrates
CANCER
RESEARCH
VOL. 45 FEBRUARY
1985
560
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1985 American Association for Cancer Research.
Effects of Dietary Fats and Butylated Hydroxytoluene on
Mutagen Activation in Rats
Debra L. Ponder and Nancy R. Green
Cancer Res 1985;45:558-560.
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