[CANCER RESEARCH 42, 4284-4288, October 1982]0008-5472/82/0042-OOOOS02.00

Analysis of the Fecal Microflora and Its Enzymatic Activity in

Individuals Genetically Predisposed to Colon Cancer

Jane D. Keathley ' and Cynthia A. Needham

Creighton University, Omaha, Nebraska 681 78 [J. D. K.¡.and Boston University Hospital. Boston. Massachusetts 021181C. A. N.¡

ABSTRACT

The role of the fecal microflora in the induction of coloncancer was investigated in individuals believed to be geneticallypredisposed to colon cancer. Subjects were members of families with increased occurrence of colon and endometrial carcinomas characteristic of the cancer family syndrome. Group1 consisted of 5 cancer family syndrome individuals previouslydiagnosed with colon cancer. Group 2 consisted of 6 cancerfamily syndrome individuals previously diagnosed with endometrial cancer but free of colon cancer. An environmentalcontrol group (Group 3) consisted of 8 spouses of subjects inGroups 1 and 2. Quantitative bacterial cultures and assays of/S-glucuronidase and 7«-dehydroxylase activity were per

formed on fecal samples. No differences in bacterial quantitiesor levels of /i-glucuronidase or 7o-dehydroxylase activity were

found among Groups 1, 2, and 3 or between spouse pairs. Theresults fail to associate quantities or enzymatic activity of theintestinal flora to colon cancer in individuals believed to begenetically predisposed to colon cancer.

INTRODUCTION

It has been hypothesized that the intestinal microflora isresponsible for production of carcinogens and/or cocarcino-

gens in the gut resulting in colon tumor formation (1, 10, 30).Quantitative bacterial cultures of fecal samples from populations at high and low risk for colon cancer have shown somedifferences in fecal bacterial profiles (1, 10, 12); however,these differences have not been confirmed (6, 7, 20).

Analyses of related bacterial metabolism in the gut havecorrelated higher levels of activity of the enzymes /8-glucuron-idase and 7a-dehydroxylase with populations at high risk forcolon cancer (20, 21). /J-Glucuronidase degrades conjugated

products from the liver, releasing toxic substances and possiblycarcinogens or procarcinogens within the bowel (13, 14). 7a-

Dehydroxylase converts primary bile acids to secondary bileacids, which act as colon tumor promoters in animal models(3, 23).

The risk factors in the preceding studies were determined byenvironmental criteria such as geographical location and/ordietary habits. High risk for colon cancer may also be determined genetically, as suggested by a number of syndromes,including inherited adenomatosis of the colon and rectum,Gardner's syndrome, Peutz-Jegher's syndrome, and the CFS2

(15). Investigators have found an increase in excretion ofcholesterol and a decrease in excretion of coprostanol and

1 Present address: Medical Center Hospital of Vermont, Burlington. Vt.2 The abbreviations used are: CFS, cancer family syndrome; PRAS, prere-

duced anaerobically sterilized.Received October 23. 1981; accepted July 8. 1982.

coprostanone among familial polyposis patients compared withrelated and unrelated controls (2, 16, 26). Other fecal constituents including the fecal microflora have not been examined inthese genetically determined high-risk groups.

Members of families fulfilling the criteria for the CFS arebelieved to be genetically predisposed to colon cancer. Thepurpose of this investigation was to study the fecal bacteriaand its associated metabolic activity in CFS family memberswith diagnosed cancer of both the colon and outside the colon.Any differences between the 2 groups might ultimately beassociated with the expression of the genetic predeterminant(s)controlling colon cancer.

MATERIALS AND METHODS

Subjects

CFS has been described by Lynch and Krush (18). Characteristicsof this syndrome are: (a) high incidence of adenocarcinomas of multipleanatomical sites, most frequently of the colon and endometrium; (b)multiple primary malignant neoplasms; and (c) early age of onset. Thesyndrome follows an autosomal dominant mode of inheritance.

Names of subjects for this study and their family and medicalhistories were obtained from the Preventive Medicine Department,Creighton University, Omaha, Nebr. Subjects were selected on thebasis of the following criteria: (a) subjects were members of familiescharacterized by CFS or spouses of CFS family members; (b) subjectsconsumed a high-meat. Western-type diet; (c) subjects had received

no antibiotic therapy for at least 4 weeks prior to specimen collection;and (cO colon cancer subjects had been treated by minor resectiononly.

Fecal Sample Collection and Transport

Complete contents of a single bowel movement were collected fromeach subject. Feces were collected aseptically in clean, nonsterileZiploc plastic storage bags (268 x 279 mm; Dow Chemical Co.,Indianapolis, Ind.), which were opened and placed in a holder attachedto the toilet seat. Immediately following collection, the specimen washomogenized by kneading for 2 to 3 min. Approximately 1 g was addedto each of 2 preweighed tubes containing 9.0 ml glycerol broth withglass beads (4). The tubes and the remainder of the specimen werefrozen within 15 to 30 min on dry ice for transport to the laboratory,where they were stored at -70°. All specimens were collected andprocessed at the subjects' homes.

Bacterial Cultures

Primary Isolation. Quantitative cultures were initiated within 4 weeksof specimen collection and were performed as described by Sutler efal. (25). One tube of the diluted specimen was weighed, placed in ananaerobic chamber (type B; Coy Manufacturing Co., Ann Arbor, Mich.),and allowed to reach room temperature in an atmosphere of 80%oxygen-free nitrogen, 10% carbon dioxide, and 10% hydrogen. Afterthorough mixing of the sample on a Vortex-type mixer, a series of 10-fold dilutions (10~2 to 10~9) was made in PRAS dilution blanks (11).

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Fecal Microflora and Hereditary Colon Cancer

Media used, purpose, and dilutions plated on each are described inTable 1.

Bacterial Identification. Plates were examined for growth after 48hr of incubation. Each colony type was enumerated and identified.Identification of isolates was performed on single, isolated colonies,which were grown in pure culture when necessary.

Group identification of isolates was based on Gram stain reactionand morphology; colonial morphology; antimicrobial susceptibility; cat-

alase, indole, and lecithinase production; spore formation; growth onbile; esculin hydrolysis; and metabolic end-product analysis.

Metabolic end products were analyzed on a Packard gas Chromatograph, 7400 series. Columns were packed with 10% SP-1000-1%H3POi on Chromosorb W-AW 100/120 mesh (Supelco, Inc., Belle-

fonte, Pa.).

Enzyme Assays

Controls. Control specimens were obtained from a laboratorian.After homogenization of the samples, a set of 10~' dilutions was made

by placing 1.0-g aliquots into preweighed PRAS dilution blanks (9.0ml). The tubes were weighed, thoroughly mixed, and stored at —70°.

Specimen Preparation. On the day of assay, the undiluted subjectspecimen and control tubes were removed from the freezer and thawedin a 37°water bath. Each assay was performed on triplicate aliquots

and on the control specimen.The thawed patient sample was moved into the anaerobic chamber

where 1.0-g aliquots were added to preweighed PRAS dilution blanks(9.0 ml). A 1.0-g aliquot was also placed on a preweighed glass slide.

The slide with the sample was weighed and placed in a glass Petri dishcontaining desiccant. The Petri dish was placed in a drying oven at100° for 24 hr, after which the dried sample and slide were again

weighed and the dry weight of the specimen was calculated./i-Glucuronidase Activity. Samples for /i-glucuronidase activity

were prepared according to Reddy and Wynder (21 ). Standards wereprepared by dissolving purified enzyme (Sigma Chemical Co., St.Louis, Mo.) in distilled water (4°)./?-Glucuronidase activity was assayed

as described by Worthington Biochemical Corp. (29) with the followingmodifications. The reaction mixture was incubated for 5 hr and terminated with 1.0 ml of 0.2 M glycine-0.2 M NaCI, pH 10.4. One unit of

activity is defined as the amount of enzyme that will liberate 1.0 ¡igphenolphthalein from phenolphthalein glucuronide per hr.

7a-Dehydroxylase. 7«-Dehydroxylase activity was determined ac

cording to the methods of Mastromarino ef a/. (19), using as substrate[MC]cholic acid (specific activity, 40 to 60 fiCi/mmol; New England

Nuclear, Boston, Mass.). Following extraction from the incubation

mixture, bile acids were separated by thin-layer chromatography. The

chromatography plates were dried, sprayed with 25% H?SO.,, andcharred at 180°for 30 min. Bile acids appeared as yellow bands which

were identified by comparison with the standards. Radioactivity in eachband was determined in a Packard Tri-Carb Model 3320 liquid scintil

lation spectrometer (Packard Instrument Co., Inc., Downers Grove, III.).Activity was calculated as the percentage of cholic acid microbiallyconverted to deoxycholic acid and was expressed as activity/100 mgdry feces.

Statistical Methods

Data from subject groups were compared using one-way analysis ofvariance. The matched-pair / test was used in comparing individuals.

RESULTS

Subjects. Subjects were divided into 3 groups for examination. Group 1 consisted of 5 CFS members who had beendiagnosed and treated for colon cancer. Three females, 64,64, and 77 years old, respectively, and 2 males, 63 and 54years old, respectively, made up this group. Their respectiveages at the time of colon cancer diagnosis were 48, 47, 69,47, and 47 years. Group 2 consisted of 6 CFS family memberswith no evidence of colon cancer but in whom either endome-

trial or breast cancer had occurred. This group consistedentirely of females, 66, 64, 72, 56, 38, and 55 years old,respectively, whose ages at the time of endometrial or breastcancer diagnosis were 49, 46, 39, 45, 29, and 34 years old,respectively. Group 3 consisted of 8 spouses of the individualsin Groups 1 and 2. The individuals in the latter group had noknown genetic risk for colon cancer but environmental anddietary exposure similar to that of Groups 1 and 2. Group 3consisted of 6 males and 2 females. Ages were not availablefor 3 subjects in this group, the remaining subjects were 50,59, 65, 69, and 70 years old at the time of specimen collection.

Subjects were requested to complete dietary questionnaires.It was determined that all consumed meats other than poultryand fish at least 4 times/week. Subjects indicated that mealswith no meat were generally consumed at breakfast. Foursubjects in Group 1 consumed diets unaffected by surgerywhile 2 subjects reported a limited intake of fresh fruits anddairy products.

Table 1

Media lor quantitative cultures

MediumDilutions to be plated" Purpose

AerobicincubationMannitolsaltagarStreptococcus

KfagarSheepbloodagarMacConkey's

agarSabouraud'sagario-2,io-2.io-2.IO'2,IO'2,10—,io-,10

-,10-,10-,10

6,1Q-6,io-e.io-6,io-6,io-8io-8io-8io-8io-6StapnylococciEnterococciPredominant

aerobicfloraAerobicand facultatively anaerobic Gram-negativebacilliYeast

AnaerobicincubationRifampinagarBifidobacteriumagarKanamycin-vancomycin

laked-bloodagarNeomycin-vancomycinLactobacillus

selectiveagarSheepbloodagarRich

agarPhenylethyl alcohol agar with vitaminKNeomycin

agarCycloserinemannoseagarEgg

yolk agar10-2,1CT2,io-2.IO'2,10-',IO"3.io-3.io-3.io-3,IO'3.10-',10

4,104,10-,10-,IO'3,io-5.io-5.io-5.io-5.IO'5,io-3.10

«,io-6.IO'6.io-6,10~5,IO"7,io-',IO'7,io-'.io-'.io-5.1010-10-10-10-10-10-10-10-10-10-Fusobacteriumsp. and Clostridiumsp.Bifidobacteriumsp.Bacteroidessp.Fusobacterium

sp. and Veionellasp.Lactobacillussp.Total

counts nd predominantfloraFastidiousorganismsGram-positiveorganismsAnaerobic

cocci and Clostridiumsp.Lecithinase-negativeClostridiumsp.Clostridium

sp.8 0.1-ml portion of each dilution is plated on each medium used (25).

Dilutions to be plated on egg yolk agar were heated in an 80°water bath for 10 min prior to inoculation in order to detect spore formers.

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J. D. Keathley and C. A. Needham

Table 2Mean viable culture counts of fecal bacteria isolated from Groups 1, 2, and 3

OrganismAerobic

and facultatively anaerobicGNBEnterococciStaphylococciYeastBacillus

sp.StreptococciTotal

aerobesAnaerobic

GPCLactobaci/lusBifidobacteriumLecithinase-negative

clostridiaLecithinase-positiveclostridiaEubactehumUnidentified

GPBPropionibacteriumAnaerobic

GNCBacteroidesfragilisBacteroidesmelaninogenicusBacteroidessp.FusobacteriumUnidentified

GNBTotalanaerobesTotal

culture countsGroup

1(5)"9.269.02±±8.31

±8.87'8.768.12'(4)(4)(4)Nl"5.497.849.509.906.709.8910.103.209.029.688.6210.548.208.38NI'8.1410.8410.93±±±±±±±±±±±±±r±5.437.799.706.629.7210.033.158.729.498.578.578.158.338.09(4)(2)(4)(4)(5)(5)(1)(4)(3)(1)(1)(1)(1)(1)Group

2(6)8.529.176.193.586.129.869.969.527.799.128.768.099.419.468.818.81±±±±±±±±±±±±±±±8.27

(6)9.13(5)5.96

(2)3.38(3)6.07(3)9.82(4)9.06

(6)7.47(5)9.06(5)8.58(5)7.86(4)8.89(6)9.02(5)8.77(1)8.77(1)Group

3(7)9.495.725.143.645.429.039.629.177.709.1310.399.689.249.17NI110.24±±±±±±±±±±±±±i±9.45

(7)5.48(5)4.79(4)3.59

(2)5.37(4)8.99

(4)8.86

(6)7.51(6)8.69

(7)10.23(6)9.46(4)8.81

(7)8.80(5)9.79(7)Ni"

Ni"9.81Nl"8.53±±9.62

(3)8.44

(3)10.638.67Ni'8.4710.6710.60±i±8.30

(4)8.26

(2)10.68

a Numbers in parentheses, number of subjects represented.6 GNB, Gram-negative bacilli; GPC. Gram-positive cocci; GPB, Gram-negative bacilli; GNC. Gram-negative cocci.c Log,o mean number of organisms/g dry feces ±S.E.d Not isolated.

Table 3

Mean values of enzymatic activity in Groups 1, 2, and 3

7a-Dehydroxylase (%converison to deoxy-

/3-Glucuronidase(units/mg cholic acid/100 mg drydry feces) feces)

Group 1Group 2Group 3189.8

±47.4a

256.4 ±72.4177.1 ±32.935.4

±10.149.7 ±10.742.1 ±10.4

Mean ±S.E.

Surgical therapy of the subjects in Group 1 included thefollowing: right colectomy (2 subjects), removal of a 22-cmsection of cecum (one subject), removal of a 22-cm section of

the descending colon (one subject), and resection of the rightcolon and distal ileum (one subject).

Quantitative Cultures. Storage of aliquots of a control specimen in glycerol broth at —70°for up to 4 weeks did not

decrease quantities of bacteria recovered by culture. Meanvalues of each group of bacteria found in Groups 1, 2, and 3are listed in Table 2. No significant differences were noted inquantities of any bacterial group between the subject groups.Comparisons between spouse pairs showed no statisticallysignificant differences between CFS family members and theirspouses in quantities of any bacteria.

/i-Glucuronidase Activity. The mean values of total /S-glu-

curonidase activity in each group are shown in Table 3. Themean difference in total activity between spouse pairs was39.9 units/mg dry feces, and the standard deviation of thedifference was 93.1 units/mg. No statistically significant differences were noted in either comparison. The mean value ofthe yß-glucuronidase control sample was 176.4 ±34.7 (S.D.),with a coefficient of variation of 20%.

7a-Dehydroxylase Activity. Mean values of percentage of

conversion to deoxycholic acid in each group are shown in

Table 3. The mean difference in conversion of cholic acid todeoxycholic acid between spouse pairs was 5%, and thestandard deviation of the difference was 36%. No significantdifferences were noted in either comparison. The mean valueof the 7a-dehydroxylase control sample was 72.0 ±3.1, with

a coefficient of variation of 4%.

DISCUSSION

Cancer of the large intestine represents 15% of all malignantneoplasms in the United States (5). The incidence is highest incountries of relatively advanced socioeconomic status, andcorrelations have been found between colon cancer incidenceand certain dietary patterns. These patterns include high levelsof dietary animal fats and proteins and low levels of dietaryfiber and unrefined carbohydrates. Based on these correlations, it has been suggested that cancers of the digestive tractmight be caused by the ingestion of carcinogens or by dietarycomponents that are metabolized (possibly by the intestinalmicroflora) within the digestive tract to carcinogenic compounds.

A unique population of CFS family members was available tous to investigate the proposed relationship of fecal microflorato colon cancer. Three groups of individuals were selectedfrom this population. Subjects in Group 1 had expressed theirgenetic predisposition for cancer in the form of large bowelcarcinoma. Members of Group 2 had expressed their geneticpredisposition in the form of endometrial or breast cancer. Thepurpose of this investigation was to detect any differences inthe intestinal microflora between these 2 groups that might becorrelated with the development of colon cancer in one groupbut not in the other. The control group (Group 3) was includedto establish the influence of dietary or environmental differences.

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Fecal Microflora and Hereditary Colon Cancer

Dietary information obtained from subjects in this study wasnot extensive but did indicate the consumption of a typicalWestern diet in all cases. Differences that were reported werefelt to be minor.

The study showed no significant differences in quantities ofbacterial species either among Groups 1, 2, and 3 or betweenspouse pairs. This failure to demontrate significant differencessuggests that absolute numbers of fecal bacteria may not varyin relation to development of colon cancer in CFS family members, at least not in a manner detectable by current techniques.

Because of the difficulties encountered in collecting specimens from various locations throughout the United States, weelected to have specimens shipped frozen. Crowther (4) hasshown that survival of microorganisms during freezing is enhanced by diluting specimens in glycerol broth and that lossesof most species are insignificant using this method of transport.Our preliminary studies showed no decrease in total culturecounts after storage at —70°.Hedges ef al. (9) further exam

ined this method of shipping fecal specimens and found thatnumbers of colony-forming units on egg yolk lactose sugarmedium were reduced 20- to 50-fold after freezing in glycerolbroth. Lecithinase-positive clostridia were particularly suscep

tible to freezing. Bacterial quantities in this study were low incomparison with results of other investigators. Our total anaerobic counts were approximately 1 log lower than counts fromnonfrozen specimens reported by Mastromarino et al. (20).These differences in bacterial quantities may have been relatedto our shipping method.

Levels of carcinogen-related metabolism were also assessed. /3-Glucuronidase activity did not differ among Groups1, 2, and 3 or between spouse pairs. Fecal 7«-dehydroxylase

activity was also measured, with no significant differencesnoted among Groups 1, 2, and 3, or between spouse pairs.

While it is possible that some enzyme-producing bacteria

may be lost during freezing, our assays were designed tomeasure existent enzymatic activity. /?-Glucuronidase and 7a-

dehydroxylase activities are both stable at frozen temperatures(21, 27). Our control specimens maintained enzymatic activityat —70°throughout the time period of the study. Investigators

have shown that 7a-dehydroxylase activity can be induced in

some species but not in others (24, 28). Maximum inducedactivity occurs during log-phase growth 2 to 3 hr after exposure

to cholic acid. Our incubation period (3 hr) was not long enoughto allow bacteria to reach log-phase growth and produce sig

nificant amounts of enzymatic activity. Thus, we feel that ourresults are valid assessments of enzyme activity present in thespecimen at the time of collection.

Our failure to demonstrate increased enzymatic activity inCFS colon cancer subjects may be explained in several ways:(a) Colon carcinogenesis may involve enzymatic activities otherthan or in addition to the 2 studied here. Other metabolicprocesses that have been theoretically linked with colon cancerinclude cholesterol degradation and reduction of A/-nitrosocompounds in feces, (b) Our population size was small due tothe difficulty of obtaining good samples from a wide range oflocations. Larger numbers of subjects may point out differencesin fecal constituents not noted in this study, (c) Difficulties wereencountered in maintaining the precision of the /J-glucuroni-dase assay. Fresh standards were prepared daily, and standards and samples were maintained at 4°until the incubation

period began. Nevertheless, control values varied widely from

day to day. It is therefore difficult to determine what role ß-glucuronidase might play in the etiology of colon cancer in thestudy population.

(d1) Fecal enzymatic activity may be more a reflection of

dietary practices than a major factor in colon carcinogenesis.Subjects in this study consumed similar diets and exhibitedsimilar levels of fecal enzymatic activity. Increased enzymaticactivity is found in populations at high risk for colon cancer;however, dietary practices also differ between these populations and low-risk populations (8, 21, 22).

(e) Increased fecal enzymatic activity has been reported incolon cancer patients not characterized by a family history ofcolon cancer (8, 19, 21). Lynch (17) states that genetic susceptibility and environmental exposure interact to varying degrees within a population. He attributes the variance in cancerliability in a population to varying proportions of heritability andenvironmental exposure. Our CFS subjects may thus be moreliable to colon cancer than are non-CFS subjects because of

a higher degree of inborn susceptibility of these individuals tolevels of promoting agents that would not affect a non-CFS

subject. Thus, fecal enzymatic activity may contribute to colontumor promotion even though no difference in levels of enzymatic activity are found in different colon cancer populations.

The results presented here point out the need for completeevaluation of genetic influences in colon carcinogenesis, withemphasis on the interaction of genetic and environmental factors in production of disease.

ACKNOWLEDGMENTS

The authors gratefully acknowledge the assistance of Patrick Lynch, J. D.,and the Preventive Medicine Department. Creighton University, Omaha, Nebr.

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1982;42:4284-4288. Cancer Res   Jane D. Keathley and Cynthia A. Needham  Individuals Genetically Predisposed to Colon CancerAnalysis of the Fecal Microflora and Its Enzymatic Activity in

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