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Molecular and Cell Biology Section, Faculty of Nutrition [M. Y. H., J. R. L., R. J. C., L. A. D., R. S. C.], Department of Statistics [J. S. M., N. W., R. J. C.], and Center for Environmental and Rural Health [J. R. L., N. W., R. J. C., R. S. C.], Texas A&M University, College Station, Texas 77843-2471, and Paterson Institute for Cancer Research, Manchester, M20 4BX United Kingdom [R. H. E.]
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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With respect to putative mechanisms of action, select dietary polyunsaturated fatty acids may modulate carcinogen activation (2, 6) and DNA repair enzymes (7, 8) . It has been demonstrated recently that dietary fish oil, containing 20:5n-3 and 22:6n-3, reduces the incidence of activating G-to-A point mutations of K-ras in colonic mucosa of AOM3 -treated rats (9, 10) . This chemopreventive effect is consistent with the induction of MGMT (EC 2.1.1.63), an alkyl group acceptor that rapidly removes AOM-induced promutagenic O6-methylguanine DNA adducts, thereby preventing oncogenic G-to-A mutations (11, 12) . This is noteworthy because there is substantial evidence that select mutagenic and lethal lesions involve the O6 position of guanine (13) . Therefore, dietary fish oil may protect against colon carcinogenesis by either decreasing DNA adduct formation and/or enhancing DNA adduct removal (DNA repair).
Another mechanism to protect against colonic DNA damage is the induction of apoptosis (14, 15) . Although apoptosis generally occurs with low frequency in the colon, i.e., less than one apoptotic cell per crypt (15, 16) , there is compelling evidence that it plays a central role in the regulation of cell number and the eradication of harmful cells (15–18) . We have shown that dietary fish oil confers protection against experimental tumorigenesis during the promotion phase, in part by enhancing the deletion of cells through activation of apoptosis rather than decreasing cell proliferation (4, 15) . However, the effect of diet on damage-induced cell suicide (targeted apoptosis) in the colon during tumor initiation has not been examined to date. Therefore, in this study, we determined the ability of fish oil feeding to simultaneously modulate O6-methylguanine DNA adduct formation (DNA damage), removal (DNA repair), and deletion (apoptosis) during the initiation stage of colonic malignant transformation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animals.
The animal use protocol was approved by the University Animal
Care Committee of Texas A&M University and conformed to the NIH
guidelines. Thirty male weanling Sprague Dawley rats (Harlan, Houston,
TX) were provided with diets differing only in the type of fat (corn
oil or fish oil). The rats were acclimated for 1 week before receiving
the defined diets, and then stratified by body weight so that mean
initial body weights did not differ between groups. Animals were
provided with the defined diets for 2 weeks before AOM injection and
throughout the entire duration of the study, and had free access to
food and water at all times. Forty-eight-h food intakes were measured
after 1 week of receiving the diets. Body weights were recorded weekly
throughout the study.
Diets.
The two defined diets (Table 1
) differed only in the type of fat (corn oil or fish oil) as previously
described (20)
. The major differences between the fatty
acid composition of the two lipid sources were significantly higher
amounts of 14:0; 16:1n-7; 20:5n-3; and 22:6n-3 in the fish oil compared
with the corn oil diet, and higher amounts of 18:2n-6 and 18:1n-9 in
the corn oil diet. Dietary fat was provided at 15 g/100 g of diet. The
fish oil diet contained 3.5 g of corn oil/100 g of diet to ensure
that essential fatty acid requirements were met. Animals were provided
with fresh diet daily. The fish oil also contained 1 g/kg of
-tocopherol and 1.5 g/kg of 
-tocopherol and 0.025 g/100 g of
tertiary butylhydroquinone as antioxidants. Corn oil was supplemented
with - and -tocopherol and tertiary butylhydroquinone to
obtain antioxidant levels equivalent to that in fish oil. All diets
contained pectin, a fermentable fiber, at 6 g/100 g of diet. These
dietary ingredients were selected based on our previous studies in
which dietary fish oil protected against AOM-induced colon
tumorigenesis compared with corn oil (15)
. Pectin was
selected because the protective effect of fish oil is enhanced when a
highly fermentable fiber source is in the diet (15)
.
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In Vivo Measurement of
O6-Methylguanine.
Rats were euthanized by CO2 exposure, the
entire colon was removed and rinsed with PBS, and the distal colon was
subsequently isolated. Distal colonic sections were fixed in ethanol
for an in vivo measurement of
O6-methylguanine adducts as previously
described (21, 22)
using mouse monoclonal
anti-O6-methylguanine. The specificity
of this monoclonal antibody has been previously demonstrated
(21–23)
. Liver
O6-methylguanine DNA adducts in
AOM-injected animals were used as a positive control (24)
.
Omission of primary antibody and preadsorption with ligand were used as
negative controls (22)
. At least 20 crypt columns/animal
that met architectural criteria were chosen for analysis. The staining
intensity was assessed by cell position within the crypt as previously
described (22, 25)
. Images of colonic crypts were captured
on a MICROSTAR IV Reichert microscope networked to a Sony DXC-970 MD
3CCD camera and a Power Macintosh computer. Images were processed using
NIH Image, version 1.61. Captured images were adjusted using offset and
gain to optimize the image brightness and contrast so that the greatest
difference between light and dark-stained pixels was achieved. Offset
and gain were determined by pre-analysis of multiple darkly and lightly
stained tissues to expand the scale in the necessary region to enhance
detection. Once established, the settings remained constant for all
samples. Representative photomicrographs have been published
(22)
.
In Situ Apoptosis Measurement.
This method is based on the terminal deoxynucleotidyl
transferase-mediated dUTP-biotin nick end labeling method
(26)
using a kit from Oncor (Gaithersburg, MD).
Paraformaldehyde-fixed, paraffin-embedded distal colon sections were
prepared (15)
. Positive control slides were treated with
DNase I (Ambion, Austin, TX) at 37°C. Negative control slides were
incubated without terminal deoxynucleotidyl transferase enzyme. The
antibody-antigen complex was visualized by incubation with
diaminobenzidine tetrahydrochloride (Sigma, St. Louis, MO; Refs.
15
and 22
). Apoptotic cells were identified
based on a combination of positive staining and morphological criteria
as described by Kerr et al. (27)
. Crypt height
in number of cells and the number and location of apoptotic cells were
recorded, with 20 crypts analyzed per animal. The apoptotic index was
100 times the mean of the number of apoptotic cells per crypt column
divided by the mean of the total number of cells per crypt column.
In Vivo Measurement of Repair Enzyme.
Colonic MGMT expression was determined using rabbit anti-rat
alkyltransferase antibody and a tyramide signal amplification system
(NEN Life Science Products, Boston, MA) as we have previously described
(22)
. The specificity of this antibody has been documented
(26)
. Endogenous peroxidase activity was quenched by
immersing tissue sections in 3%
H2O2 in methanol. Antigen
was retrieved by microwave treatment with 0.1 M sodium
citrate solution (pH 6.0). To block nonspecific background staining,
tissue sections were incubated with avidin, biotin, and TNB buffer
[0.1 M Tris-HCl (pH 7.5), 0.15 M NaCl, and
0.5% blocking reagent from the NEN kit]. Omission of primary antibody
was used as a negative control. At least 20 crypt columns/animal were
randomly chosen. The staining intensity was assessed by cell position
within the crypt using an image analysis system (NIH Image, version
1.61; Ref. 22
). Epithelial cells on the left side
of the crypt were selected, the image was digitized, and the staining
intensity was plotted. Background staining intensity was determined on
10 randomly obtained images on each slide outside of the tissue section
and subtracted from the staining intensity of collected data.
Statistical Analyses.
DNA adduct levels, apoptosis, and DNA repair enzyme expression were
analyzed using two-way ANOVA to determine the effect of fat, time, and
fat x time interaction. When Ps for the interactions
were <0.05, means of all diet groups were separated using the
Student-Newman-Keuls multiple range test. When Ps were
<0.05 for the effects of fat, time, or carcinogen but not for the
interactions, overall means for fat, time, or carcinogen treatment
groups were separated by using the Student-Newman-Keuls multiple range
test. Data on DNA adducts were also analyzed using PROC MIXED in SAS
(SAS Institute Inc.). This mixed model approach simultaneously accounts
for the between animal, within animal between crypts, and within crypt
variation. Statistical inferences based on properly modeled variation
were then used to evaluate the relationship between cell position
within the crypt and adduct level and to determine the main effect of
fat, time, carcinogen, and their interactions. The effects of diet on
the regression of apoptotic index on adduct level, repair enzyme on
adduct level, and repair enzyme on apoptosis were tested using linear
regression. Differences between fish and corn oil within crypt tertiles
were determined by one-way ANOVA. Comparisons among the different crypt
compartments (bottom, middle, and upper tertiles) were made using
paired t tests.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Overall, corn oil-fed animals had more (P < 0.05)
DNA adducts compared with fish oil-fed animals. These differences were
detected at 6 h (P = 0.027) and were greatest at
12 h (P = 0.002; Fig. 2
). Statistical likelihood ratio tests indicated that the relationship
between DNA adduct levels and cell position within the crypts was
parabolic in nature. Specifically, at each diet/time point, roughly
1800 data points (3 rats x 20 crypts x 30 cell positions)
contributed to the parabolic fit (Fig. 3
). These plots demonstrate the shape and nature of the distribution of
the DNA adduct level measurements within the crypts, and they indicate
a consistent suppressive effect of fish oil on adduct level throughout
the crypt, regardless of the time after injection or cell position
within the crypt.
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). Maximum apoptosis was achieved by 6–9 h after AOM injection. Most of
the apoptosis was located toward the base of the crypt (Fig. 5
) where the stem cells are located. In addition, there was a strong
position and time effect (Fig. 5)
, indicating that cell death was
dramatically increased (P < 0.05) in the base of the
crypt at 6 h and continued through 12 h. Although there was
no overall effect of lipid source on apoptosis (Fig. 4)
, fish oil
feeding resulted in a significantly higher (P = 0.035)
level of apoptosis in the upper one-third (upper tertile) of the crypt
compared with corn oil treatment (Fig. 6
).
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shows that there was no main effect of either time or diet
(P > 0.05) on alkyltransferase expression. Similar to
previous reports (22, 34)
, expression was detected
predominantly in the nuclei; however, some cytoplasmic staining was
also observed. The expression of MGMT was localized primarily to the
upper portion of the crypt. Interestingly, there was a strong diet x position interaction effect (P = 0.004) within the
colonic crypt at 12 h after AOM injection (Fig. 8
). Specifically, the steady-state level of MGMT for the fish oil fed
animals was 4-fold higher (P < 0.001) in the upper
versus lower region of the crypt, whereas there was no such
effect for corn oil-fed animals.
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). The slopes were not significantly different for the lower or middle
tertiles (P > 0.05). These data indicate that fish oil
treatment selectively enhances the targeted removal of DNA adducts by
increasing apoptosis in the top one-third of the crypt. Because polyps
and tumors eventually develop from loss of growth control and retention
of cells at the top of the crypt, the significant difference in fish
oil versus corn oil on apoptosis targeted to this region may
account, in part, for the observed protective effect of fish oil
against experimentally induced colon tumorigenesis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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100 mg/day) of 20:5n-3 and 22:6n-3 in the form of fish
compared with 1000–3000 mg/day by the Japanese (39)
. In
comparison, diets rich in n-6 polyunsaturated fatty acids, found in
plant oils such as corn oil, enhance the development of colon tumors
(2, 38, 40) . This is particularly relevant because
Americans consume >10 times the amount of this lipid source needed to
meet minimal essential fatty acid requirements (41, 42)
.
With respect to the putative mechanism(s) of action, we have
demonstrated that the balance between colonic epithelial cell
proliferation, differentiation, and apoptosis can be favorably
modulated by dietary fish oil, conferring protection against
carcinogenic agents (15)
. This is of significance because
markers of apoptosis are better predictors of colon tumor outcome in
tumorigenesis studies where diet is an experimental variable
(15)
. In addition, it is now clearly established that the
malignant transformation of colonic epithelium is associated with a
progressive inhibition of apoptosis (16, 18, 43)
. These
in vivo findings support our postulate that dietary n-3
polyunsaturated fatty acids act to facilitate the apoptotic removal of
carcinogen adducted cells. In a continuation of our efforts to
elucidate the mechanisms by which dietary 20:5n-3 and 22:6n-3 reduce
colon tumor incidence, we determined how fish oil modulates
methylation-induced DNA adduct levels in the colon during the initial
stages of malignant transformation in the rat AOM carcinogenesis model. AOM is metabolized to methylazoxymethane by p450-dependent multifunction oxidases in the liver. Subsequently, methylazoxymethane is converted to methylazoxyformaldehyde by alcohol dehydrogenase and eventually converted to methyldiazonium, the ultimate carcinogen (44, 45) . The carcinogenicity of AOM can be inhibited using metabolic inhibitors (46) . We have shown that colonic O6-methylguanine adduct levels increase at a much faster rate in rats fed corn oil compared with fish oil. Although adduct levels were not different at the 0- and 3-h time points, the protective effect may be explained by the modulation of enzymology related to carcinogen activation, thereby altering the amounts and activities of oxidative (Phase 1) and conjugative (Phase 2) xenobiotic metabolizing enzymes (46, 47) . Additional experiments are needed to address this hypothesis. It is unlikely that the protective effect of fish oil supplementation is related to an effect on MGMT-mediated DNA repair because overall, fish oil feeding had no significant effect on the induction of MGMT in the distal colonic epithelium during the time frame of this experiment. MGMT serves as the stoichiometric acceptor protein for O6-methylguanine adducts, transferring the methyl group from DNA to the protein (12) . This transfer inactivates MGMT and is irreversible. Therefore, a colonocyte’s ability to withstand damage is in part related to the number of MGMT molecules it expresses and to the rate of de novo synthesis. Although not the focus of this study, it is possible that "back up" systems, such as DNA mismatch repair, which also recognize O6-methylguanine adducts, may have been influenced by dietary lipid composition (48) .
With regard to the deletion of colonocytes, in the rat distal
colon, epithelial cells are derived from a stem cell population at the
base of the crypt and migrate from a region of active cell
proliferation in the bottom two-thirds of the crypt toward the top of
the crypt, obtaining a differentiated or apoptotic phenotype. Cells at
the lumenal surface are subsequently exfoliated into the fecal stream
(18)
. Generally, apoptosis occurs after terminal
differentiation (16)
. This form of cell deletion is
predominant in the upper region of the crypt, indicating that
"spontaneous" apoptosis is unlikely to effectively regulate stem
cell number (16, 18)
. An important exception to this
sequence of events occurs during the initiation of tumorigenesis when
there is an immediate apoptotic response to DNA adduct formation
(22, 31, 32)
. Previous reports suggest that this form of
damage-induced cell deletion (targeted apoptosis) is primarily
localized to the bottom two-thirds of the crypt, where actively
proliferating cells reside (16, 18, 22) . This is somewhat
puzzling because cells with DNA adducts reside along the entire crypt
axis immediately after carcinogen administration (Fig. 3)
. Our data
also suggest that dietary fish oil enhances targeted apoptosis in the
nonproliferating transit cells, i.e., at higher cell
positions. Specifically, in the upper one-third of the crypt, the
region where polyps and tumors eventually develop, DNA adduct levels
were positively correlated with apoptosis in the fish oil-fed animals
(Figs. 6 and 9)
. In contrast, DNA adduct levels were negatively
correlated with apoptosis after corn oil feeding, indicating the
suppression of this protective response. Thus, targeted apoptosis
should decrease DNA-adducted cells and reduce the possibility for
clonal expansion into the colonic lumen (as a polyp) (49)
.
Although the effect of dietary lipid on apoptosis is significant, it is
numerically small and one might question its role in cancer prevention.
In this regard, there is cogent evidence to indicate that apoptosis is
a central component of cell number regulation in the colonic epithelium
(15, 16, 18, 43)
. In addition, it is becoming increasingly
apparent that small changes in the percentage (<1%) of apoptotic
cells in the crypt can contribute to the development of colon tumors
(4, 15, 16, 18)
.
The confinement of the apoptogenic effect of dietary fish oil to the upper one-third of the colonic crypt may indicate a modifying effect of cell differentiation. Interestingly, we have previously demonstrated that fish oil supplementation increases indices of cell differentiation in the rat colon (4) . Unfortunately, the identity of the signaling pathways responsible for the transition from terminal differentiation to apoptosis remains to be determined. A second hypothesized mechanism by which fish oil may enhance apoptosis may be through its ability to influence cell-matrix contact. Because fatty acids are critical constituents of biological membranes and their composition can be altered by dietary lipids (50) , it is possible that n-3 polyunsaturated fatty acids modulate cell viability by influencing crypt habitat/matrix adhesion (51, 52) . The specific effects of diet on colonocyte matrix proteins and adhesion molecules require further study. Finally, our recent findings indicate that MGMT is also highly expressed toward the top of the crypt (22) . Because the upper crypt region would have more direct exposure to lumenal carcinogens, this would suggest that cells on the lumenal surface are afforded inducible repair and deletion mechanisms.
Maximum levels of apoptosis occurred 9 h after AOM injection
(Fig. 4)
. Although we did not measure colonic cell proliferation in
this study, previous findings indicate that the elevation of
methylation-induced apoptosis is associated with synchronous inhibition
of mitosis in the colon (31, 32)
. This is consistent with
the fact that O6-methylguanine adducts
signal S-phase arrest (49)
. It is generally presumed that
the depression of cell proliferation occurs so that either repair or
apoptosis can ensue.
In conclusion, dietary n-3 polyunsaturated fatty acids found in fish oil confer protection against experimental colon tumorigenesis in part by reducing the level of DNA adducts and by enhancing the deletion of cells through the activation of targeted apoptosis. These data support our hypothesis that 20:5n-3 and 22:6n-3 act as anticarcinogens.
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Acknowledgments |
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Footnotes |
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1 Supported in part by NIH Grants CA57030
(to R. J. C.), CA59034 (to R. S. C.), CA61750 (to J. R. L.), and
CA74552 (to N. W.) and by NIEHS Grant P30-ES09106. 
2 To whom requests for reprints should be
addressed, at 442 Kleberg Biotechnology Center, Texas A&M University,
College Station, TX 77843-2471. Phone: (409) 845-0419; Fax: (409)
862-2662; E-mail: chapkin{at}acs.tamu.edu
3 The abbreviations used are: AOM, azoxymethane;
MGMT,
O6-methylguanine-DNA-methyltransferase.
Received 1/17/00; revised 4/26/00; accepted 5/17/00.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-3 fatty acid diet. Mutat. Res., 378: 23-30, 1997.[Medline]
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