CANCER RESEARCH56. 532-537. February I. 19961

ABSTRACT

Epidemiological and laboratory animal model studies suggest that theeffect of dietary fat in colon carcinogenesis depends not only on the

amount but on its fatty acid composition. Animal model studies demonstrated that high dietary corn oil or safflower oil rich in omega-6 fatty

acids increased the colon tumor promotion, whereas diets containing fishoil high in omega-3 fatty acids had no such enhancing effect. One of themechanisms by which high dietary fat enhances colon carcinogenesis maybe through the modulation of colonic mucosal phospholipase A2 (PLA2)and phosphatidylinositol-specific phospholipase C (P1-PLC), which aredominant pathways for arachidonic acid release and formation of ekesanoids. P1-PLC is also responsible for diacylglycerol formation andprotein kinase C-dependent signal transduction and cell proliferation. Inthe present study, we investigated the modulating effect of high fat dietsrich in omega-3 and omega-6 fatty acids on colonic mucosal PLA2, P1-PLCactivities, and eicosanoid (prostaglandins and thromboxane B@)formationfrom arachidonic acid via cyclooxygenase (COX) during different stagesof azoxymethane (AOM)-induced colon carcinogenesis in male F344 rats.At 5 weeks of age, groups of animals were fed the low-fat diet containing5% corn oiL Beginning at 7 weeks ofage, all animals except those intendedfor vehicle treatment received AOM s.c. once weekly for 2 weeks at a doserate of 15 mg/kg body weight. Vehicle-treated groups received an equalvolume of normal saline. One day after the second AOM or vehicletreatment, groups of animals were transferred to experimental diets conmining 23.5% corn oil and 20.5% fish oil + 3% corn oil, whereas onegroup continued on the low-fat diet containing 5% corn oil. Groups ofanimals were then sacrificed at weeks 1, 12, and 36 after the second AOMor saline-treatment. Colonic mucosa harvested at weeks 1, 12, and 36 andcolonic tumors obtained at week 36 were analyzed for PLA2, P1-PLC, andeicosanold formation from arachidonic acid by the action of COX. Theresults demonstrate that colon carcinogen treatment increases the activities of colonic mucosal PLA2 and P1-PLC and the formation of prostaglandins and thromboxane A2 from arachidonic acid through COXthroughout the study period compared to saline-treated animals fed similar diets. The activities of PLA2, P1-PLC, and COX were significantlyhigher in colon tumors compared to colonic mucosa. These results alsodemonstrate that a high-fat diet containing corn oil increases colonicmucosal and tumor PLA2 and P1-PLC and the formation of prostaglan

dins and thromboxane B2 by the action of COX as compared to low

dietary corn oil or a diet high in fish oil. The results of our study offer oneof the mechanisms by which the amount and types ofdietary fat modulatecolon carcinogenesis.

INTRODUCTION

Significant variations in dietary habits among populations of different cultures and life-styles have been associated with a risk for thedevelopment of cancer. Among the dietary habits, dietary fat has

received considerable attention as a risk factor in the etiology of coloncancer (1—5).Several animal model studies have provided additionalevidence that the fatty acid composition of dietary fat is one of thedetermining factors in colon carcinogenesis (6—8).The high levels ofhighly polyunsaturated omega-3 fatty acids such as EPA3 (c20:5, n-3)and DHA (c22:6, n-3) present in marine oils make them uniquedietary fats. Most commonly consumed vegetable oils in the UnitedStates, such as corn oil and safflower oil, contain high levels ofpolyunsaturated fatty acids of the omega-6 type, i.e., LA (c18:2, n-6).Recently, researchers have sought to determine the relative tumorpromoting capabilities of different types of dietary fat, such as fish oiland corn oil. Available data indicated that diets containing highproportions of omega-3 polyunsaturated fatty acids had minimal or nocolon tumor-promoting effect, whereas diets containing increasedlevels of omega-6 polyunsaturated fatty acids such as LA enhancedcolon tumorigenesis in laboratory animal models (9—11). RecentPhase II clinical trials in patients with polyps demonstrate that dietaryfish oil supplements decreased the rectal cell proliferative pattern (12).

Although the mechanism(s) of colon tumor-promoting effect ofhigh dietary corn oil and lack of tumor promotion by high dietary fishoil is not completely understood, the tumor-promoting effect of highdietary fat has been associated with increased concentrations of colonic lumenal secondary bile acids, i.e. deoxycholic acid and lithocholic acid (4). Laboratory animal model studies demonstrated thatthese secondary bile acids increase colonic mucosal ornithine decarboxylase (a rate-limiting enzyme in polyamine biosynthesis) activity,colonic epithelial polyamine levels, and cell proliferation and act ascolon tumor promoters (13—15).Our recent study demonstrated thatdietary fish oil suppressed colonic mucosal ornithine decarboxylaseand tyrosine-specific protein kinase activities as compared to dietarycorn oil (16). Elevated levels of these enzymes have been associatedwith increased tumor promotion (17, 18). Another related mechanismby which high dietary fat can modulate colon carcinogenesis isthrough alteration of membrane phospholipid turnover and prostaglandin synthesis as shown in Fig. 1 (19, 20). Several previous studieshave established that AA metabolites may modulate the pathogenesisof several immunological and inflammatory diseases (21, 22). Activities of PLA2 and P1-PLC, which are dominant pathways for the AArelease, play a significant role in the outcome of formation of AAmetabolites in colonic mucosa and tumors. It is noteworthy thatelevated levels of PLA2 and P1-PLC and arachidonate metaboliteswere observed in human colon tumors compared to normal colonicmucosa, indicating that increased levels or expression of these enzymes may play a role in colon carcinogenesis (23—26).A recent

3 The abbreviations used are: EPA, eicosapentaenoic acid; DHA, docosahexaenoic

acid; DAG, diacylglycerol; AOM, azoxymethane; AA, arachidonic acid; LA, linoleicacid; COX, cyclooxygenase; LFCO, low-fat diet containing 5% corn oil; HFCO, high-fatdiet containing 23.5% corn oil; HFFO, high-fat diet containing 20.5% fish oil and 3% cornoil; P1-PLC, phosphatidylinositol-specific phospholipase C; PG. prostaglandin; TXB2,thromboxane B2; PAPC, L-a-l-palmitoyl-2-arachidonyl phosphatidylcholine; PIP2, L-3-phosphatidylinositol 4,5-biphosphate; PKC, protein kinase C; HPLC. high-performanceliquid chromatography; PLA2, phospholipase A2.

Received 9/1/95; accepted 11/30/95.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

I Supported by USPHS Grants CA 17613 and CA 37663 from the National Cancer

Institute.2 To whom requests for reprints should be addressed, at Division of Nutritional

Carcinogenesis, American Health Foundation, Valhalla, NY 10595.

532

Modulating Effect of Amount and Types of Dietary Fat on Colonic MucosalPhospholipase A2, Phosphatidylinositol-specific Phospholipase C Activities,and Cyclooxygenase Metabolite Formation during Different Stages ofColon Tumor Promotion in Male F344 Rats1

Chinthalapally V. Rao, Barbara Simi, Tin-Tm Wynn, Kathy Garr, and Bandaru S. Reddy2

Divisions of Nutritional Carcinogenesis (C. V. R.. B. S.. T-T. W., B. S. RI, Research Animal Facility (K. G.J, American Health Foundation, Valhalla, New York /0595

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J@*_]COLON TUMOR PROMOTION BY TYPES AND AMOUNT OF FAT

DIetary Fat

Omega

Omega-3

PL@4 PI@PLC @DAG @PKC

@@ I DAG Ilpas.

EPA AA-'- MAGIlpa.. MAG

@@cloox@gen@e ‘I,

Peroxidase

(CELL PROLIFERATION2)

/Fig. I. Possible interrelationships between the typesand amount of dietary fat and PLA2, P1-PLC, and COXactivities. MAG, monoacylglycerol.

PGE2

@,PGE3

Biologically PGF3 PGF@aBIologicallyl@s cdvs PGD3 PGD2 very active

PGI2 .Icosanoldselcosanolds PGI3

TxA, TxA2

study demonstrated that deoxycholic acid, which is implicated as apromoter of colon carcinogenesis, is a potent activator of P1-PLC inhuman colon mucosa and tumors (23). One of the pathways leading tothe generation of AA involves a direct action of PLA2 on a phospholipid that could include l,2-diacylglycerol or l-O-alkyl-2-acyl-phosphatidylinositol, phosphatidylethylamine, or phosphatidylcholine.The second pathway, mediated by P1-PLC, involves the degradationof phosphatidylinositol 4,5-biphosphate via a sequence of reactionsbeginning with P1-PLC, followed by diglyceride lipase and monoacylglycerol lipase (Fig. 1; Refs. 27 and 28). Also, P1-PLC hydrolyzesphosphatidylinositol 4,5-biphosphate to DAG, which activates PKCthat upon activation can phosphorylate proteins and regulate cellularevents, including cell proliferation and differentiation (27). Only afterliberation from phospholipid is arachidonate available as a substratefor further enzymatic modification by COX, lipoxygenase, and cytochrome P450 (22). COX converts arachidonate to PGs and thromboxane (22). In this connection, it is interesting that administration ofnonsteroidal antiinfiammatory drugs, such as indomethacin, piroxicam, sulindac, and aspirin, which are inhibitors of COX activity,inhibited chemically induced colon tumor development in rats (29—32). Thus, these findings clearly suggest the importance of COX

activity and formation of increased levels of eicosanoids in colontumor promotion.

In view of the potential significance of secondary bile acids andactivities of PLA2, P1-PLC, and COX in colon tumorigenesis and ofdifferences in tumor-promoting effects of high dietary corn oil andfish oil, we assessed the modulating effect of diets containing LFCO,HFCO, and HFFO on colonic mucosal and tumor PLA2, P1-PLCactivities, and formation of eicosanoids such as PGE2, PGF2a, PGD2,6-keto PGF1a, and TXB2 from AA through COX activity to understand the biochemical mechanisms of the effect of types and amountof dietary fat during the promotional stage of colon carcinogenesis inmale F344 rats.

533

MATERIALS AND METHODS

Materials

AOM (CAS:25843-45-2) was purchased from Ash Stevens (Detroit, MI).[‘4C]AAand PAPC were purchased from NEN-Dupont (Boston, MA). PIP2was obtained from Amersham (Arlington Heights, IL). AA, PGE@, PGF2a,

6-Keto PGF1a,PGD2,AND TXB2and fatty acid standards were procured fromthe Cayman Chemical Company (Ann Arbor, MI). The reverse-phase HPLC@Bondpak C18column was from Waters Associates (Milford, MA). PrecoatedSilica-G plastic TLC plates were from Fisher Scientific Co. (Springfield, NJ).

Animals and Diets

Weanling male F344 rats were purchased from Charles River BreedingLaboratories (Kingston, NY). Fish oil was donated by Menhaden Oil Refinery

ofZapata Protein (USA), Inc. (Reedville, VA). The ingredients of semipurifieddiets were purchased from Dyets, Inc. (Bethlehem, PA). A total of 216 maleF344 rats received at weaning were quarantined for 7 days and then randomlyassigned to one of three dietary groups of LFCO, HFCO, and HFFO. Each

dietary group (72 animals) was then divided into AOM-treated and vehicletreated subgroups. They were housed three each in a plastic cage with filter

tops and maintained under controlled conditions of 21°C at 50% humidity and

a 12-h light/dark cycle. All animals were fed ad libitum. The food cups werereplenished every day.

The composition of experimental diets was based on modified AIN-76Adiet (9) and is shown in Table 1. HFFO diet was formulated to contain 3% corn

oil to alleviate any essential fatty acid deficiency. The percentage compositionof all experimental diets was adjusted so that the animals in all dietary groupswould consume the same amount of calories, protein, vitamins, minerals, andfiber (33). All diets were prepared in our laboratory three times weekly andstored in air-tight containers filled with nitrogen in a cold room at 4°C under

dark. Aliquots of experimental diets were analyzed for their fatty acid cornposition.

Fatty Acid Analysis

The methods for the extraction and separation of lipids from diet sampleswere as described by Bligh and Dyer (34) using chloroform:methanol (2:1)

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Table IPercentage composition of experimentaldiets―Diet

ingredientsCorn

oil dietsFish oil diet

Highfat―Lowfat― Highfat―Casein20.0

23.5023.50DL-Methione0.3

0.350.35Corn

starch52.032.932.9Dextrose13.0

8.328.32Alphacel5.0

5.95.9Corn

oil5.023.53.0Fish

oil0020.5Mineral

mix3.54.114.11Vitaminmix.0

1.181.18Choline

bitartorate0.2 0.240.24

COLON TUMOR PROMOTION BY TYPES AND AMOUNT OF FAT

adding 40 ,xM of PAPC [10 @xCi1@xmol,adjusted with cold substrate], and thereaction mixture was incubated at 37°Cin a shaking water bath for 30 mm.Reaction was terminated by adding 300 pAchloroform:methanol (3:2, v/v). Anadditional 200 j.d of chloroform were added to each sample and mixed

thoroughly. The samples were then centrifuged, and the chloroform layer wasseparated and evaporated to dryness under N7. Five @.tgof AA were added tothe dried extract and redissolved in chloroform. An aliquot of the chloroformextract was then subjected to chromatography on precoated plastic TLC plates(Silica-G). The TLC plates were developed with a solvent system containing

chloroform:methanol:acetic acid:water (90: 12:2: 1, v/v/v/v) and exposed in an

iodide chamber for 5 mm for visualization of AA. The area of each [‘4C]AAmetabolite was determined with a Bioscan System 200 image-scanning counter(Bioscan, Inc., Washington, DC) equipped with a @3-detector.Protein contentwas determined by the Bio-Rad method. Results are expressed as pmoles[‘4C]AAreleased/mg protein/mm.

Membrane-bound P1-PLC activity was measured by the method of Bleasdale et a!. (37) with some modifications using [3H]PIP2 (5 Ci/mmol) as asubstrate (36). P1-PLC activity of membrane proteins (100—200 j.tg) was

determined in a total volume of a 250-pA reaction mixture containing 30 mrsiHEPES-NaOH buffer (pH 7.2), 5 mMDTF, 4 mrvtCaC12,2 mMEGTA, 0.9 mrsiMgSO4, and 50 @.tM[3H]PIP, (50 pCi/mmol). The reaction was initiated byadding substrate to the mixture and incubating at 37°Cfor 20 mm in a shakingwater bath. The reaction was terminated by the addition of 0.2 ml of chloroform:methanol (1 :2, v/v) and then 0.3 ml of 1 M HC1. The incubation mixture

was mixed vigorously and centrifuged to yield two phases. An aliquot of 0.3

ml of the aqueous layer containing [3H]inositol 1,4,5-triphosphate was transferred into a scintillation vial containing 10 ml of scintillation cocktail.

Radioactivity was counted in a Beckman model LD6800 scintillation counter.The activity is expressed as pmoles of [3H]inositol 1,4,5-triphosphate formedfrom [3H]PIP2/mgprotein/15 mm.

COX Activity. Colonic mucosaand tumorsfrom individualanimalswerehomogenized in 1:3 (w/v) volumes of 100 missTris-HC1buffer (pH 7.2), usinga Polytron tissue homogenizer. The samples were then centrifuged at 9000 X gat 4°Cfor 10mm. The supernatant fraction was centrifuged at 100,000 X g for1 h. The resulting microsomal pellet was resuspended in 50 mr@ipotassiumphosphate buffer (pH 7.4) for the assay of COX activity. The COX activity of

colonic mucosa and of tumors was measured by methods published previously

(36). Briefly, a 150-pJ reaction mixture containing 12 p@M[‘4C]AA(420,000dpm), 1 mM epinephnne, 1 mr@tglutathione in 50 mr@iphosphate buffer, and25—35,@.gof mucosal or tumor microsomal protein was incubated at 37°Cfor15 mm. The reaction was then terminated by the addition of 40 p3 of 0.2 MHC1.The COX metabolites of AA were extracted three times with 0.5 ml ofethyl acetate. The combined extracts were evaporated to dryness under N2,redissolved in chloroform, and subjected to TLC using Silica-G plates, whichwere developed in a solvent system containing chloroform:methanol:aceticacid:water (100:15:1.25:1, v/v/v/v). They were then exposed in an iodide

chamber for 5 mm for visualization of the standards. The metabolites of[‘4C]AAcorresponding to PGE2, PGF2@,PGD-,, 6-keto PGF1@,and TXB2were detected by their comigration (R@values) with authentic standards. Thearea of each metabolite was determined with the Bioscan System 200 image

scanning counter equipped with a n-detector.

Statistical Analysis

Differences in body weights and biochemical parameters between thegroups were analyzed by Student's t test and ANOVA. Differences wereconsidered statistically significant at P < 0.05.

RESULTS

As expected, corn oil diet contained LA as major fatty acid,whereas fish oil diet contained primarily DHA and EPA (Table 2).HFFO diet, which was formulated to contain 3% corn oil, showedreduced levels of LA relative to HFCO diet. The body weights ofanimals treated with vehicle or AOM and fed the control and experimental diets were comparable throughout the study (data not shown).However, animals fed LFCO diet gained about 10% lesser bodyweight when compared to those fed HFCO and HFFO diets in both

a Diet was formulated on the basis of the American Institute of Nutrition standard

reference diet with the modification of varying sources of carbohydrate (9).b Additional corn oil and fish oil were added at the expense of starch. The composition

of high-fat diets was adjusted so that all animals in various dietary groups would consumeapproximately the same amount of protein. minerals, vitamins, fiber, and calories (9).

containing butylated hydroxytoluene (0.005%). Fatty acid phenacyl estersfrom lipids were prepared according to the method of Borch (35). These fattyacid derivatives were analyzed by a HPLC method using a Waters HPLCsystem driven by model 5 10 system controller (Waters Associates, Milford,

MA). Briefly, the fatty acid derivatives were separated on a Waters Bond pakC,8 column (30 cm length X 4 mm diameter) with a step-wise gradient ofacetonitrile and water at a flow rate of 1.5 ml/min. Elutions of fatty acidphenacyl esters were monitored at 240 nm wavelength in a Waters 990photodiode array detector. Identification of individual fatty acids was madewith authentic standards derivatized and analyzed under the same conditions.

Experimental Procedure

Beginning at 5 weeks of age, all animals were fed the modified AIN-76A

(LFCO) diet. At 7 weeks of age, animals allotted to carcinogen treatment weregiven AOM s.c. once weekly for 2 weeks at a dose rate of 15 mg/kg body

weight, whereas the animals intended for the vehicle treatment were givennormal saline. One day after the second AOM or saline treatment, groups ofanimals treated with AOM or normal saline were transferred to HFCO and

HFFO diets, whereas additional groups were continued on LFCO diet. Allanimals were weighed twice monthly until the experiment was terminated. Six

animals from each AOM- and vehicle-treated group fed the control and

experimental diets were sacrificed by CO2 euthanasia at weeks I, 12, and 36after the last AOM or saline injection. The colons were rapidly removed andrinsed in ice-cold normal saline. They were slit open longitudinally, freed fromall contents, and cleaned with ice-cold normal saline. They were laid flat on a

glass plate, and the mucosa were scraped with a microscopic glass slide. The

mucosal samples were quickly frozen in liquid nitrogen and stored at —80°Cuntil analyses. In addition, colon tumors were harvested at week 36, snapfrozen in liquid nitrogen, and then stored at —80°Cuntil analyses.

Biochemical Analysis

PLA2 and P1-PLC Activities. Samples of colonic mucosa collected from

individual rats at weeks 1, 12, and 36 and colon tumors harvested at week 36

were homogenized in 1:3 (w/v) volumes of homogenizing buffer containing 30mM Tris-HCI (pH 7.4), 140 m@iNaCl, 5 mM KC1, 20 @.tMEDTA, 10 @tg/mlleupeptin, 50 j.tg/ml trypsin inhibitor, and 1 m'vi phenylmethylsulfonyl fluoride, and the homogenates were centrifuged at 100,000 X g at 4°Cfor 1h. Theresulting supernatant fraction was used for cytosolic PLA2 activity, and thepellet fraction was redissolved in 30 mM HEPES-NaOH buffer (pH 7.2)

containing 0.2% Triton X-100 and used for the analysis of membrane-bound

P1-PLC activity.Cytosolic PLA, activity was measured by our method published previously

using [‘4C]PAPC (40—60 mCilmmol) as substrate (36). PLA2 activity of

cytosolic protein was carried out in a total volume of a l00-@.d reaction mixture

containing 50 mM sodium HEPES (pH 7.3), 0.8 mrvi CaCl2, 0.02% Triton

X-lOO, and 20—30@xgof cytosolic protein. The reaction was initiated by

534

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Table 2 Fatty acidcomposition ofhigh-fat―Fatty

acidHigh-fat

dietsCorn

oil (%)Fish oil(%)c

12:00.08NDc

14:00.436.88c16:011.2018.24c16:ln-70.207.16c18:02.012.74cl8:ln-924.3115.85c

l8:2n-658.827.12c

l8:3n-31.210.90c20:4n-601.18c

20:5n-3017.41c22:6n-3013.14Total

suturated fatty acids1 3.7127.86Totaln-3 fattyacids1.2130.85Totaln-6 fattyacids58.828.30Totaln-9 fattyacids24.3115.85a

See Table I for diet composition.

Table 3Effect of types and amountof dietary fat on colonicmucosal and tumor PL42 and P1-PLC activitiesin male F344ratsWeeks

on experimentaldiets following carcinogenDietary

regimenAOM-treatedSaline-treatedLow-fat

cornHigh-fatHigh-fat fish Law-fat cornHigh-fatHigh-fatfishorvehicle treatmentoilcorn oiloil oilcornoiloilPLA,―Colon

mucosaI27.9±35c.d.e38.4 ±4.825 ±4.0―@ 12.3 ±1.715.6 ±2.510.9±1221.1±33@@e27.9 ±3.818.7 ±5.0―@ 9.8 ±l.4―@13.9 ±1.77.9 ±12d,e3622.6±36d.e29.3 ±4.418.3 ±24df 11.6 ±17d.e16.0 ±1.910.5±Tumor55.5±7Ø@.e75.8 ±8.650.0 ±3.9@P1-PLC―Colon

mucosa22.0 ±25df32.5 ±4.321.4 ±3.4@31.8

±46dJ3.828.7 ±44d,g34.2±41d.e42.4 ±4.030.8 ±37df

COLON TUMOR PROMOTION BY TYPES AND AMOUNT OF FAT

depend predominantly on the direct action of PLA2 (28). These resultssuggest that production and availability of AA for the synthesis ofeicosanoids may be limited in animals fed HFFO diet due to adecrease in colonic mucosal and tumor PLA2.

P1-PLC Activity. As summarizedin Table3, carcinogentreatmentsignificantly (P < 0.05) increased the colonic P1-PLC activity at alltime points. P1-PLC activity was also found to be increased(P < 0.001) in colonic tumors compared to mucosa. Dietary LFCOand HFFO resulted in decreased colonic mucosal and tumor P1-PLC

activity as compared to HFCO diet (P < 0.05—0.001) in both salineand AOM-treated groups. Since P1-PLC is involved in the generationof AA from phospholipids through the modulation of DAG andmonoacylglycerol lipase (Fig. 1), decreased levels of colonic mucosaland tumor P1-PLC observed in animals fed HFFO as compared tothose fed HFCO may suggest that the availability of AA for eicosanoid synthesis may be reduced in animals fed HFFO diet.

COX Activity. COX activitywas analyzedby quantitatingthe rateof formation of PGs and TXB2 from [‘4C]AA.The levels of formationof PGE2, PGF2, PGD2, 6-keto PGFJ , and TXB2 in colonic mucosa andtumor tissues are summarized in Table 4. The results indicate thatthere was a 3—6-foldsurge in the formation of all COX metabolitesduring the first week after AOM administration in all dietary groupsas compared to their respective saline-treated groups. These levelswere reduced to about 75% at weeks 12 and 36. Carcinogen-treatedanimals exhibited significantly (P < 0.05—0.001) enhanced colonicmucosal COX activity, as indicated by the formation of PGs andTXB2 from AA as compared to those treated with saline in all dietarygroups. Colonic PGE2, PGF2, PGD2, and TXB2 levels were significantly (P < 0.05—0.001)higher in animals treated with AOM and fedHFCO diet compared to those fed LFCO or HFFO diets at all timepoints; somewhat similar trends were observed in the saline-treatedgroups. In general, the formation of colonic mucosal 6-keto PGF1 wasnot affected (P > 0.05) by types and amount dietary fat. The COXactivity was 3—4-fold higher in colonic tumors than in mucosa.Animals fed HFFO diet showed significantly (P < 0.05—0.001)lowerCOX activity in the tumors as compared to those fed HFCO diet. Theresults of this study indicate that the formation of eicosanoids fromAA via COX can be influenced by the types and amount of dietary fat.Dietary fat rich in omega-3 fatty acids decreased the formation ofcolonic mucosal and tumor PGs and TXB2, which are productsderived from AA via COX when compared to a diet high in omega-6

AOM- and saline-treated groups. Since the aim of this study was tounderstand the mechanisms of types and amount of dietary fat incolon tumor promotion, the tumors harvested at week 36 were used toassay the biochemical parameters that are involved in colon tumorigenesis and not subjected to histopathology. However, the gross colontumor incidences (% animals with tumors) in animals fed the experimental diets were as follow: LFCO, 48%; HFCO, 82%; and HFFO,

52%.PLA2 Activity. The activity of PLA2analyzed in colonic mucosa

at weeks 1, 12, and 36 and in tumors at week 36 is summarized inTable 3. Carcinogen administration significantly elevated the activities of colonic mucosal PLA, as compared to saline treatment, irrespective of dietary regimen throughout the period. It is noteworthy

that there was a 2—3-foldincrease in the PLA2 activity of the colontumors when compared to surrounding colonic mucosa. The activityof PLA2 in the colonic mucosa was higher in AOM-treated animalsfed the HFCO diet when compared to those fed the LFCO and HFFOdiets. In saline-treated animals, dietary HFCO produced a significantincrease (P < 0.05) in colonic mucosal PLA2 activity compared to

HFFO diet at weeks 1, 12, and 36. Dietary administration of LFCOinhibited PLA2 activity at weeks 12 and 36 only as compared toHFCO. Colonic tumor PLA2 was significantly reduced in animals fedHFFO and LFCO diets as compared to those fed HFCO diet. Asindicated in Fig. 1, AA generation from membrane phospholipids will

1 37.6 ±39d,e@@ 4.6 39.2 ±4.l@@e12 47.5 ±3•7d.f 60.3 ±7.0 36.1 ±36 56.8 ±45df 67.8 ±7.2 46.4 ±44d.g

Tumor 120 ± 12 135 ±9.5 84 ± l2―@

a PLA, activity is expressed as pmol [ 4CIAA released from I ‘4C]PAPC/mg protein/mm at 37°C.

h P1-PLC activity is expressed a pmol [3H]inositol 1,4,5-triphosphate formed from [3H]PIP2/mg protein/IS mm at 37°C.

‘Mean ± SD (n = 6).

d Significantly different from high-fat corn oil group among AOM-treated or saline-treated groups.

ep < O05•

fp < 0.01.5p < 0.001.

535

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Table 4 Effect of types and amount of dietary fat on the formation of PGE2, PGF2a 6-Keto PGF1 a' PGD2, andTXB2 from AA byCOX in the colonic mucosa andtumorsWeeks

on experimentaldiets following carcinogenDietary

regimenAOM-treatedSaline-treatedHigh-fatHigh-fat

fishLaw-fatcornHigh-fatHigh-fatorvehicle treatmentLaw-fat corn oilcorn oiloiloilcorn oilfishoilPOE2―Colon

mucosa11797± 1ç@b.c.d1977 ± 1361518 ±92c.d313 ±20329 ±29261 ±38@e12353±37C1472 ±49339 ±38'@237 ±30@348 ±38252 ±21'@―36351

±38(e4f,@ ±55295 ±40@215 ±2l@329 ±29192 ±25@Colontumor1 112 ±l22'@1568 ±1241373 ±1fJ9c.dPGF2aColon

mucosa11039± 137@.e1317 ± 106953 ±153@252 ±42286 ±56251 ±3212350

±38@e441 ±58260 ±42@―258 ±49c.d317 ±59240±36339±54c.e467 ±73282 ±39c@'236 ±51'@310 ±38222 ±59c.eColon

tumor809 ±105c.d987 ±177746 ±62ce6-Keto

POF1aColonmucosaI753

± @b.e.d897± 106737 ±83c.e304 ±44320 ±43297 ±4712300

±55341 ±49299 ±42239 ±51c.d278 ±47259 ±3436296

±35312 ±59283 ±24249 ±51254 ±38241 ±51Colontumor1029 ±1551085 ± 187831 ±95c.ePOD2Colon

mucosa1522± 116c.d680 ±73568 ±53121 ±21'@155 ±13103 ±l4@e12228±40@'290 ±31177 ±37c.e122 ±24177 ±241 11 ±2l@36203

±23'@287 ±54127 ±36'@141 ±27158 ±361 19±21c.dColontumor587 ±108642 ±91574 ±58TXB2Colon

mucosa11056± l09@e1298 ±173973 ±l00'@250 ±41c.d302 ±33244 ±39C@@12328±5l'@―376 ±35306 ±30'@'231 ±43c.d287 ±44246 ±4436331±61c.d74296 ±38c.e251 ±34273 ±42275 ±36Colon

tumor801 ±l02@e962 ±96773 ±112'@

COLON TUMOR PROMOTION BY TYPES AND AMOUNT OF FAT

a pmoles of PGF2, PGF2a, 6-Keto PGF1@, POD2, or ‘@‘@2formed from [‘4C]AA/mg protein/iS mm.

b Mean ± SD (n = 6).

C Significantly different from high-fat corn oil group among AOM-treated or saline-treated groups.

dp < o.os.

@p<0.01.fP < 0.001.

fatty acids. In addition, it was also reported that omega-3 fatty acids,i.e., EPA and DHA present in fish oil, inhibit the formation of PGsand TXB2 via COX activity (38, 39). Furthermore, because of additional double bonds within EPA compared to AA, COX enzymemetabolizes EPA to PGE3 and TBA3 instead of PGE2 and TXB2 (Fig.1), suggesting altered biological properties compared with the eicosanoids derived from AA (20, 40).

DISCUSSION

The results of the present study demonstrate that administration ofAOM enhanced the activities of colonic mucosal PLA2 and P1-PLCand the formation of PGs and TXB2 from AA via COX, and theseincreases were observed throughout the promotion and progressionstage of colon carcinogenesis. Induction of these enzyme activities inthe colon mucosa by carcinogen treatment is in agreement withprevious studies (25, 32, 36, 41). Also, the colonic mucosal PLA2 andP1-PLC activities and formation of eicosanoids were sustained at highlevels throughout the study period. We are not aware of any previousstudies in which the colonic PLA2 and P1-PLC activities and theformation of eicosanoids from AA were compared during differentstages of colon tumor promotion. It is also noteworthy that theactivities of PLA2 and P1-PLC and the formation of PGs and TXB2were higher in colon tumors than in mucosa, suggesting an enhancedrelease of arachidonate from the phospholipids and increased formation of eicosanoids in colon tumors through the activation of COX.

The results of this study demonstrate that high dietary corn oil

increased colonic mucosal and tumor PLA2 and P1-PLC activities ascompared to a low corn oil diet, whereas high dietary fish oil distinctlyinhibited the activities of these enzymes as compared to a high cornoil diet. Although the mechanism of alteration of mucosal PLA2 andP1-PLC activities by the types and amount of dietary fat is not clearlyknown, it is possible that the increased levels of omega-6 fatty acidsor omega-3 fatty acids may exert their enhancing or inhibitory effectby directly acting on the PLA2 and P1-PLC, or alternatively by actingon their regulators. Also, colonic secondary bile acids have beenshown to activate colonic mucosal PLA2 (42) and P1-PLC (43). In thiscontext, it is noteworthy that high dietary corn oil increases thecolonic lumenal (or fecal) secondary bile acids as compared to alow-fat diet containing corn oil (4). Our recent unpublished resultsindicate that the lumenal concentrations of secondary bile acids suchas deoxycholic acid and lithocholic acid in the animals fed the dietscontaining low and high amounts of corn oil and high fish oil were1.56, 3.18, and 1.74 mglg dry sample, respectively, suggesting thathigh dietary fish oil had no enhancing effect on the production ofsecondary bile acids in the lumen of the colon as compared to a highcorn oil diet. Therefore, the feeding of a diet rich in corn oil results inincreased levels of secondary bile acids and omega-6 fatty acids in thelumen of the colon, which activate colonic mucosal PLA2 and P1-PLCactivities (42, 43) for the release of free AA and other products ofphospholipid breakdown. AA is metabolized via the COX pathway toa number of prostanoids. Products of the COX pathway modulate theproliferation rates of a variety of cell types (40). Consistent with these

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COLON TUMOR PROMOTION BY TYPES AND AMOUNT OF FAT

results were several investigations that demonstrated that PG synthesis inhibitors, such as nonsteroidal antiinflammatory agents, inhibitcolon carcinogenesis in laboratory animal models (29—32). Theseinteresting observations suggest the potential importance of AA metabolism and pGs in modifying tumor promotion by omega-6 andomega-3 fatty acids in colon carcinogenesis.

In conclusion, the results of the present study provided evidencethat high-fat diets containing corn oil significantly enhanced thecolonic PLA2 and P1-PLC activities, indicating PLA2 and P1-PLCdependent cellular activities may play an important role in highdietary corn oil-induced colon tumor promotion. The results of ourcurrent and earlier studies from our laboratory and of others demonstrate that a high-fat diet containing corn oil as compared to a low cornoil diet and high dietary fish oil increase the colonic lumenal secondary bile acids that modulate the colonic mucosal P1-PLC and DAG

(Fig. 1). This in turn directly activates the PKC and PKC-dependentcell proliferation and/or is responsible for the increase of cellular AAlevels through the mediation of DAG and monoacylglycerol lipases asproposed in Fig. 1 (43—47).Also, diets rich in corn oil activate colonicmucosal PLA2, thereby increasing phospholipid turnover and therelease of free AA and other products of phospholipid breakdown.The increased levels of AA are metabolized via the COX pathway toa number of prostanoids that enhance tumor promotion.

ACKNOWLEDGMENTS

We thank Laura DiSciorio for preparation of the manuscript, the staff of theResearch Animal Facility and Histopathology Facility for expert technicalassistance, and Zapata Protein (USA), Inc. for kindly providing Menhaden fishoil.

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1996;56:532-537. Cancer Res   Chinthalapally V. Rao, Barbara Simi, Tin-Tin Wynn, et al.   of Colon Tumor Promotion in Male F344 RatsCyclooxygenase Metabolite Formation during Different StagesPhosphatidylinositol-specific Phospholipase C Activities, and

,2Colonic Mucosal Phospholipase AModulating Effect of Amount and Types of Dietary Fat on

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