[CANCER RESEARCH 46, 5529-5532, November 1986|

Effects of Differing Purified Cellulose, Pectin, and Hemicellulose Fiber Diets onFecal Enzymes in 1,2-Dimethylhydrazine-induced Rat Colon Carcinogenesis1

Hugh J. Freeman2

Department of Medicine (Gastroenterology), University of British Columbia, and the Health Sciences Centre Hospital, Vancouver, British Columbia, Canada V6TIW5

ABSTRACT

The fecal microflora enzymes, /J-giucuronidase and 0-glucosidase, aswell as fecal bacterial counts, were examined during colon carcinogenesisin rats administered parenteral 1,2-dimethylhydrazine and fed nutritionally equivalent diets free of fiber or containing one of three single sourcesof dietary fiber (cellulose, hemicellulose, and pectin). Whereas pectin-fed animals had increased fecal 0-glucuronidase activities, those fedcellulose and hemicellulose, two fibers protective in dimethylhydrazinecolon neoplasia, had decreased activities. Although fecal bacterial countswere not significantly changed, similar differential changes in fecal /?-glucosidase activity were noted: cellulose but not pectin or hemicellulosefeeding was associated with reduced activity. Although cellulose fibermay cause differing physiological effects resulting in a reduction in colonieneoplasia development in this experimental animal model, decreasedbacterial metabolic enzyme activation of carcinogens or cocarcinogensmay lead to diminished exposure of colonie cells to exogenous or endogenous mutagens.

INTRODUCTION

The intestinal microflora may play a significant role in thepathogenesis of colon cancer (1). Differences in compositionand concentrations of some fecal bacteria were initially reportedin low compared to high risk colon cancer groups (2). Laterstudies, however, did not confirm these observations, in part,due to difficulties in taxonomic classification (3, 4). Morerecently, their metabolic role has become better appreciated,particularly in relation to carcinogenesis (1, 5-8). Chemicallyinduced colon tumors, for example, may result from procarcin-ogen activation (e.g., cycasin) to a carcinogen (e.g., methylaz-oxymethanol), possibly mediated by bacterial enzymes (e.g., ß-glucosidase) (9, 10). Carcinogens (e.g., W-hydroxy-AAl-fluore-nylacetamide and diethylstilbesterol) are excreted in bile asglucuronides and activated in the intestinal tract through enzymatic hydrolysis by /3-glucuronidase (11-13). Alternatively,administration of a /3-glucuronidase inhibitor reduced the incidence of azoxymethane-induced colon cancer in rats (14). Thesebacterial enzymes are detectable in rat feces and may be influenced by dimethylhydrazine, antibiotics, or differing diets (15-17); diet may also alter human fecal enzyme activities (18-19).

The effect of fiber on these enzymes was first observed in ratsfed grain or beef diets; decreased /3-glucuronidase and increased/i-glucosidase activities were seen in the high-fiber grain group( 15). Later, both increased and decreased enzyme activities werenoted (20-22) after feeding diets with various types of fibers;but precise comparisons, however, are difficult because of differences in doses of fiber, other diet components, animal species, and carcinogens. Since studies (23-26) with single purefiber sources showed differential effects on chemically inducedrat colonie neoplasia, this investigation systematically exam-

Received4/15/86;revised7/28/86;accepted7/31/86.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 inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported by a research grant (MA 9428) to Dr. Freeman

from the Medical Research Council of Canada, Ottawa, Ontario, Canada.2To whom requests for reprints should be addressed, at University of British

Columbia Health Sciences Centre Hospital, 2211 Wesbrook Mall, Vancouver,British Columbia, Canada V6T 1W5.

ined their effects on fecal bacterial enzymes in experimentalcarcinogenesis.

MATERIALS AND METHODS

Animals and Diets. Weaned male Wistar rats weighing 50 to 60 gwere housed in suspended cages with wire mesh floors to preventcoprophagia and were isolated in a carcinogen-containment laboratoryequipped with negative pressure double air lock doors and directed-flow external air exhaust through filters. Water was administered adlibitum at all times. Relative humidity (40%) and environmental airtemperature were constant, and a 12 h light, 12-h dark cycle wasimposed using automated switching devices. Rats were randomly divided into 6 groups of 20 animals (i.e., 10 rats to receive carcinogenand 10 rats to receive no carcinogen in each diet group). Rats were fedone of 6 different chemically defined powder diets of similar physicalappearance containing no fiber or one of the following single fibers atthe 4.5 or 9.0% dose level: cellulose, pectin, and hemicellulose. Theratios of calories derived from fat, protein, and carbohydrate mixed ineach diet were equivalent. Diets were provided by courtesy of Dr. G. A.Spii 1er. Palo Alto, CA. The composition of each diet is shown in Table1. Following 8 weeks of diet administration, individual intakes, outputs,and weight gains for each group were recorded during 1 week with eachanimal housed in an individual cage. Stools and urine were collecteddaily to prevent evaporative loss.

Carcinogenesis. Animals receiving carcinogen were each given s.c.injections of 1,2-dimethylhydrazine dihydrochloride (Aldrich ChemicalCo., Inc., Milwaukee, WI) once weekly for 14 weeks at a dosage of 25rng/kg prepared as a 0.5% solution of 1 inM EDTA (MallinckrodtChemical Works, St. Louis, MO) adjusted to pH 6.5 with sodiumbicarbonate. Controls received an equivalent amount of EDTA atidentical pH. Previous studies had established this as an effectiveregimen in our laboratory for colon tumor induction (27, 28). Ratswere sacrificed 4 weeks after the final injection of 1,2-dimethylhydrazine or the control solution.

Fecal Bacterial Counts. After 8 weeks, fresh fecal pellets were obtained from control and carcinogen-treated animals in the 6 diet groups.Samples were placed into preweighed tubes containing 0.9% sodiumchloride solution at 1:20 (w/v). Tubes were then reweighed to determinewet fecal weight. Each sample was sonicated for 15 s and 100 u\ ofsonicated sample were diluted with 9.9 ml of 0.9% sodium chloridesolution. After vortexing, 10 M' of sample were placed on a 1.0-cm2area of a glass slide using the previously validated method of Holde-mann and Moore (29). Each slide was air dried. Gram stained, andexamined under oil (at x 100) with a light microscope. For each slide,organisms were counted in 2 edge fields and 8 center fields; chains orpairs of bacteria equated to a single clump of organisms. The averagenumber of organisms per field was then calculated. Direct uncorrectedmicroscopic clump counts (DMCC) were defined by the formula (29):

DMCC = no. of bacteria/field x fields/cm2 x 10*.

To determine the corrected DMCC/g weight of feces, corrections fordilution were done.

Fecal Enzyme Activities and Protein. After 8 weeks, fresh fecal pelletswere also collected for protein and enzyme activities; these were placedinto preweighed tubes containing 0. l M phosphate-buffered saline, pH7.0. Samples were sonicated for 30 s, centrifuged at 2000 x g for 5min, and aliquots of supernatant were used immediately. At sacrifice,additional aliquots of fecal material from cecum-proximal colon anddistal colon from each group were collected and processed in an

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FECAL MICROBIAL ENZYMES AND COLON CARCINOGENESIS

Table 1 Rat diet composition

IngredientCasein0

Egg white powderSafflower oilMineral mix*Vitamin mix'

Corn starchCellulose''Pectin'Hemicellulose ^Fiber

free6.95

6.958.004.801.00

72.300004.5%

cellulose6.66

6.667.604.500.95

69.134.50009.0%

cellulose6.34

6.347.24

4.290.91

65.879.0000Diet4.5%

pectin6.66

6.667.604.500.95

69.130

4.5009.0%

pectin6.34

6.347.244.290.91

65.870

9.0004.5%

hemicellulose6.66

6.667.604.500.95

69.1300

4.50* Casein (HCI); edible, extra-grade hydrochloric acid casein, 30 to 40 mesh (Milk Products Ltd., Sydney, Australia).* Mineral mix USP XVII (Teklad; ARS/Sprague-Dawley Corp., Madison, WI) contained per 100 g of mix: sodium chloride, 13.93 g; potassium biphosphate,

38.90 g; anhydrous magnesium sulfate, 5.73 g; calcium carbonate, 38.14 g; ferrous sulfate, 2.70 g; manganese sulfate, 0.401 g; potassium iodide, 0.079 g; zinc sulfate,0.0548 g: cuprícsulfate, 0.0477 g; and cobaltous chloride, 0.0023 g. Selenium was not present in any diet.

'Vitamin mix AOAC (Teklad; ARS/Sprague-Dawley Corp.) contained per 100 g of mix: vitamin A (dry, stabilized), 200,000 IU; vitamin D (dry, stabilized),20,000 IU; vitamin E (dry, stabilized), 1,000 IU; menadione, 0.05 g; choline, 20.0 g; p-aminobenzoic acid, 1.0 g; inositol, 1.0 g; niacin, 0.4 g; calciuim rf-pantothenate,0.4 g; nhollmm, 0.08 g; thiamin HCI, 0.05 g; pyridoxine HCI, 0.05 g; folie acid, 0.02 g; biotin, 0.004 g; vitamin B,2, 0.0003 g.

'Avfcel, PH-105 cellulose (FMC Corp., Philadelphia, PA).' High methoxyl pectin (Sunkist Growers, Ontario, CA).'Highly refined hemicellulose (Staley Co. Decatur, IL).

identical fashion. Supernatant from rats at sacrifice was frozen at -20*C

for enzyme assays and protein determination.For j3-glucuronidase activity (15,18,30), incubations were performed

in triplicate at 37°Cfor 15 min in a shaking water bath. The final

concentrations in 1.0 ml of the incubation mixture included 0.02 Mphosphate-buffered saline, pH 7.0; 0.1 mM EDTA, 1 mM phenolphthal-ein-/3-D-glucuronide; and distilled water with the enzyme supernatant.The reaction was stopped with 4.0 ml of 0.2 M glycine buffer, pH 10.4,in 0.2 M sodium chloride and read at 540 nm with a Bausch & LombModel 710 dual-beam spectrophotometer (Bausch & Lomb, Rochester,NY). All reactions were linear with respect to concentration and incubation time to 45 min.

For /3-glucosidase activity (15, 31), incubations were performed intriplicate at 37'C for 60 min in a shaking water bath. The final

concentrations in 1.0 ml of the incubation mixture included 0.02 Mphosphate-buffered saline, pH 7.4; 1 mM p-nitrophenol-/3-o-glucoside;and distilled water with the enzyme supernatant. The reaction wasstopped with 5.0 ml of 0.01 M sodium hydroxide and read at 450 nm.All reactions were linear with respect to concentration and incubationtime to 60 min.

For fecal protein, determinations were done in triplicate based onthe method of Lowry et al. (32) using bovine serum albumin as thestandard.

Statistical Analysis. Collected data were analyzed statistically usingthe unpaired Student's r test.

RESULTS

Weight and Carcinogenesis Studies. Water and food intakeswere similar for all groups. Only mean stool weights weresignificantly greater in the two (4.5 and 9.0%) cellulose groupsand the 9.0% pectin group compared to fiber-free group (all, P< 0.05). In addition, each of these diet groups had significantlyhigher mean stool weights than did the 4.5% pectin group (all,P < 0.05). Although cellulose-fed rats tended to show greaterweight gain compared to other diet groups, no statistical difference was observed. In addition, no statistical differences inmean total body weight or mean weekly weight gain wereobserved.

The small and large bowel mucosa of control rats in eachdiet group were normal; in particular, no inflammatory changeswere seen in contrast to a previous report suggesting thatcellulose caused increased numbers of neutrophils in smallbowel mucosa (33). In carcinogen-treated rats, colon tumorswere seen in all groups, especially in distal colon. Table 2 showsthe mean ±SE number of tumors at this site per rat for each

Table 2 Fecalbacterialcountsand colontumors

DietgroupFiber

free4.5% cellulose9.0% cellulose4.5% pectin9.0% pectin4.5% hemicelluloseColony

count"13.81

3.332.355.09

12.703.59Tumors/rat*1.20

±0.300.53 ±0.22C0.33 ±0.17'

1.20 ±0.241.33 ±0.310.27 ±0.15'

' Fecal bacterial counts, DMCC x 10'°/gfeces.6 Mean ±SE number of distal colon tumors per rat.' P (t test versus control) < 0.05.

Table3Diet

groupFiber

free4.5% cellulose9.0% cellulose4.5% pectin9.0% pectin4.5% hemicelluloseFecal

0-glucuronidaseactivitiesControl

rats23.56±2.36"

3 1.58 ±4.6816.32 ±1.12*

147.07 ±13.33*102.66 ±8.54*

15.41 ±0.83*Carcinogen-treated

rats8.93

±1.267.77 ±0.84

11.42 ±1.1115.61 ±2.13*42.71 ±3.94*

12.48 ±0.60" Mean ±SE (/<mol phenolphthalein-0-D-glucuronide substrate hydrolyzed per

min per mg fecal protein); n = 10 control or carcinogen-treated (8 weeks) rats/group.

* P (t test versus fiber-free group) < 0.05 for either control or carcinogen-

treated rats. See text.

group. A significant reduction in tumor number was observedfor both cellulose and hemicellulose groups compared to thefiber-free or either pectin group (all, P < 0.05).

Fecal Bacterial Counts. Fecal bacterial counts (i.e., DMCC x10'°/gfeces) for each diet were: fiber free, 8.71; 4.5% cellulose,

6.47; 9.0% cellulose, 3.44; 4.5% pectin, 4.72; 9.0% pectin, 7.95;and 4.5% hemicellulose, 4.78. Although fiber-fed animals hadlower bacterial counts compared to the fiber-free diet, theresults were not statistically significant. Table 2 shows resultsfor carcinogen-treated rats. Although reductions in these carcinogen rats tended to be more for the cellulose and hemicellulose groups, the results were not statistically significant.

Fecal /3-Glucuronidase Activity. Table 3 shows /S-glucuroni-dase activities in fresh fecal specimens following 8 weeks offiber administration. In control animals, significant decreasesin mean activities were seen for the cellulose or hemicellulosediets, whereas increases occurred with both pectin diets compared to the fiber-free diets (all, P< 0.05). In addition, increasedactivities were seen with both pectin diets compared to bothcellulose or hemicellulose diets (all, P < 0.05). In this study,parenteral dimethylhydrazine per se reduced ß-glucuronidaseactivities in carcinogen-treated compared to non-carcinogen-

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FECAL MICROBIAL ENZYMES AND COLON CARCINOGENESIS

treated rats in each diet group (all, P < 0.05). Even in carcinogen-treated animals, however, mean ß-glucuronidase activitieswere increased for both pectin groups (P < 0.05).

Fecal /3-Glucosidase Activity. Table 4 shows results for ß-glucosidase activities in fresh fecal specimens following 8 weeksof fiber administration. In control rats, significant increases inmean 0-glucosidase activities were seen in both pectin andhemicellulose groups compared to the fiber-free group (all, P <0.05); cellulose had no significant effect. In carcinogen-treatedrats, however, mean activities were significantly decreased forboth of the cellulose, but not the pectin or hemicellulose groupscompared to the fiber-free group (both, P < 0.05).

Colonie Distribution of Fecal Enzymes. Ten weeks after theseinitial fecal studies, 0-glucuronidase and /3-glucosidase activitieswere measured in fecal specimens following sacrifice in proximal and distal colon for each diet group. Results are shown inTable 5. No differences in mean enzyme activities were detectedbetween the proximal and distal colon for any diet group.

For 0-glucuronidase, mean fecal activity was decreased inboth colon sites for cellulose (i.e., 9.0%) compared to the fiber-free group (P < 0.05). In contrast, activities were increased forboth pectin diets (both, P < 0.05).

For 0-glucosidase, mean fecal activity was increased in bothcolon sites compared to the fiber-free group (P < 0.05). Exceptfor the 4.5% cellulose group in the proximal colon (P < 0.05),there were no other differences.

DISCUSSION

The results in this study indicate that specific fibers, if derivedfrom a single source, have differing effects on fecal microbialenzyme activities during chemically induced colon carcinogen-esis. Pectin, but not cellulose or hemicellulose, increased fecal0-glucuronidase activity during carcinogenesis in both the control and carcinogen-treated animals. In contrast, cellulose andhemicellulose, two fiber sources that are protective in di-methylhydrazine-induced colonie neoplasia (23-25), decreasedenzyme activity. After sacrifice, similar differential changes infecal 0-glucuronidase activity were observed despite freezing

Table 4 Fecal fi-glucosidase activities

DietgroupFiber

free4.5% cellulose9.0% cellulose4.5% pectin9.0% pectin4.5% hemicelluloseControl

rats2.09±0.19"

1.86 ±0.162.02 ±0.183.16 ±0.43*2.86 ±0.25*4.13 ±0.11*Carcinogen-treated

rats1.51

±0.330.50 ±0.08*0.81 ±0.08*1.42 + 0.140.87 ±0.051.00 ±0.13

" Mean ±SE (//MID]p-nitrophenol-fi-D-glucosidc substrate hydrolyzed per minper mg fecal protein); n = 10 control or carcinogen-treated (8 weeks) rat/group.

b P (t test versus fiber-free group) < 0.05 for either control or carcinogen-

treated rats. See text.

Table 5 Fecal enzymes in carcinogen-treated rats

/3-GlucuronidaseDiet

groupFiber

free4.5% cellulose9.0% cellulose4.5% pectin9.0% pectin4.5% hemicel

luloseProximal

colon12.14±1.23°

11.09± 1.746.65 ±0.64*

42.53 ±10.44*27.42 ±7.33*

13.18 ±2.21Distal

colon13.93±

1.5910.48 ±1.616.92 ±0.89*

43.62 ±9.49*24.27 ±3.13*

14.04 ±4.24/3-GlucosidaseProximal

colon0.79

±0.092.12 ±0.31*

0.92 ±0.101.16 ±0.231.07 ±0.072.37 ±0.21*Distal

colon1.49

±0.131.86 ±0.261.30 + 0.141.73 ±0.221.25 + 0.102.03 ±0.22*

" Mean ±SE; n = 10 carcinogen-treated (14 weeks) rats/group.*jP (t test versus fiber-free group) < 0.05 for either /3-glucuronidase or ß-

glucosidase activities. See text.

and storage of fecal specimens from proximal and distal colon.These studies are consistent with previous reports on fecal ß-glucuronidase in this chemically induced animal model of coloncancer. Bauer et al. (20) found an increased incidence of di-methylhydrazine-induced colorectal tumors and increased fecalß-glucuronidase activity in male Sprague-Dawley rats fed a6.5% pectin, but not a 20% wheat bran or carrot fiber diet; thisenzyme was postulated to play a role in carcinogen activation;however, separate animals not treated with carcinogen wereused for fecal enzyme measurements. This may not be validsince the present report as well as previous studies (16) indicatethat dimethylhydrazine administration per se may alter fecal ß-glucuronidase activity. In a later study (21), the same investigators reported that 5% pectin and 5% guar caused differentialeffects on colon tumor incidence and fecal 0-glucuronidaseactivities, but their earlier findings for pectin-fed animals werenot confirmed. London et al. (34) also observed enzyme activitydifferences in male BALB/c mice for several different highfiber-containing bran diets in dimethylhydrazine-induced coloncarcinogenesis. However, the precise effects of single sourcesof pure fiber per se, as examined in the present report, couldnot be defined since bran, in particular, contains multiplediffering fiber (i.e., cellulose, hemicellulose, and lignin) andnon-fiber components (i.e., starch, simple sugars, fat, and protein (35).

Similar differential effects were observed in the present studyfor /3-glucosidase activity in the different diet groups. Pectinand hemicellulose, but not cellulose, increased fecal activity incontrol rats, whereas cellulose, but not pectin or hemicellulose,decreased fecal activity during the carcinogen protocol. Theseresults are consistent with a previous report (22) showingdecreased fecal glycosidase activities in dimethylhydrazine-treated male Wistar rats administered cellulose-added diets; inthat report, the intriguing suggestion was made that luminalmutagens derived from dietary ß-glycosidesmight be reducedby cellulose feeding, possibly explaining the reduced colontumor incidence with this fiber. The present study confirmsthese earlier observations (22) of reduced 0-glucosidase activitywith a high cellulose diet using two different doses of purecellulose compared to a fiber-free diet. Furthermore, this studysuggests that this effect is specific for cellulose compared toother non-cellulosic polysaccharide components, at least if administered in identical fiber concentrations and in identicaldiets. The precise mechanism for this differential effect ofcellulose fiber on the intestinal microflora enzymes duringcarcinogenesis still requires elucidation. However, as previouslysuggested (1, 15, 18, 22), fiber-induced alterations in bacterialenzymes present in the colonie lumen may serve to shed lighton the protection offered by cellulose fiber diets in experimentalcolon carcinogenesis (23, 24). In a recent report (14), inhibitionof bacterial /3-glucuronidase caused a reduction in the appearance of carcinogen-induced colon tumors. Although cellulosefiber might cause differing physiological effects to reduce coloncancer incidence in this animal model, decreased bacterial metabolic activation of carcinogens or cocarcinogens might resultin diminished exposure of colonie cells to either exogenous orendogenous mutagens.

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1986;46:5529-5532. Cancer Res   Hugh J. Freeman  Rat Colon CarcinogenesisFiber Diets on Fecal Enzymes in 1,2-Dimethylhydrazine-induced Effects of Differing Purified Cellulose, Pectin, and Hemicellulose

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