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REVIEWS OF INFECTIOUS DISEASES • VOL. 12, SUPPLEMENT 2 • JANUARY-FEBRUARY 1990 © 1990 by The University of Chicago. All rights reserved. 0162-0886/90/1201-0069$02.00 The Intestinal Microflora and tbe Colon Cancer Connection Sherwood L. Gorbach and Barry R. Goldin From the Department of Community Health, Tufts University School of Medicine, Boston, Massachusetts The intestinal microflora of humans represents a rich ecosystem composed of metabolically active microorganisms in close proximity to an absorptive mucosal surface. Substrates for bacterial transformation can reach the colonic flora through direct oral ingestion, by biliary secretion into the upper bowel, or by secretion across the mucosa. There. is considerable interest in the possibility that bactenal metabolism of these substrates in the large bowel may play a role in the etiology of cancers that arise in the adjacent epithelium. Metabolic Activities Microorganisms within the flora possess an impressive array of enzymes that can convert exogenous and endogenous compounds into a spectrum of metabolites. In addition to producing constitutive enzymes, these microorganisms can undergo enzyme induction when exposed to high levels of substrate. Table 1 contains a partial list of recognized reactions involving intestinal bacteria. (For further details, see [4, 5].) The Colon Cancer Connection Distribution of the Microflora It is estimated that the intestinal flora of any given person contains more than 400 different species of bacteria [1-3]. Strictly anaerobic bacteria predominate over the facultative forms by a factor of 1,000:1. Among the major anaerobes are Bacteroides, Bifidobacterium, Fusobacterium, Eubacterium, Clostridium, and Peptostreptococcus species. The major bacterial populations are located in the large intestine, where the bacterial concentration is 101 1-1012 cfu/mL of fecal material. The major landmark with respect to bacterial populations is the ileocecal valve; proximal to the valve the upper intestine has small numbers of bacteria and the milieu is rich in oxygen, whereas distal to the valve the bacterial population is dense and a highly anaerobic environment with a low oxidation-reduction potential is found. Thus, the bacterial enzymatic reactions in the colon are characteristically reductive. Please address requests for reprints to Dr. Sherwood L. Gorbach, Department of Community Health, Tufts University School of Medicine, 136 Harrison Avenue, Boston, Massachusetts 02111. S252 Large-bowel cancer is a major disease in Western countries but is rather uncommon in Asia, Africa, and South America [6, 7]. The critical factor that accounts for the fivefold differences in incidence of disease among geographic locations may be the characteristic Western-style diet, which is high in fat and calories and low in dietary fiber [8-10]. By use of food consumption data in various countries, a positive correlation has been found between the consumption of beef, total fat, animal fat, total calories, and animal protein and the incidence of colon cancer. Several studies have shown a protective effect of dietary fiber [11-13], although the relative importance of dietary fat and fiber in the etiology of colon cancer remains controversial. Severalinvestigators have proposed that the effect of diet on cancer development is indirect, consisting primarily of an impact on the ability of the host to metabolize procarcinogens to proximate carcinogens [14-18]. In the case of colon cancer, activation of carcinogens may be mediated by the bacterial flora in the large bowel. Several bacterial enzymes have Downloaded from http://cid.oxfordjournals.org/ at Penn State University (Paterno Lib) on May 12, 2016 Epidemiologic studies and laboratory research have indicated an association between the metabolic activity of the intestinal micro flora and cancer of the large bo.wel. It h~s bee~ suggested that activation of procarcinogens could be mediated enzy~atlcally by l~teStl nal bacteria. The levelsof incriminated colonic bacterial enzymes are Increased by dleta~y fat and inhibited by certain dietary fibers. Organic extracts of feces contain a mu~agem.c substance, presumably derived from bacterial metabolism in the l~rge bo~el, t~at IS pOSItive in the Ames test. Whether this substance or some other organic chemical IS the putative proximate carcinogen remains speculative, but the evidence continues to point to intestinal bacteria as the metabolic intermediary in colon cancer. Intestinal Microflora and Colon Cancer 5253 Bacterial Enzymes Reaction Glycosidases. Hydrolysis ofglycosidic bonds is one of the best-known examples of bacterial metabolism. Glycosides arc compounds consisting of a nonsugar moiety (aglycone) bound to a sugar byeither an a- or a J3-glycosidin linkage. Glycosides enter the gut from two major sources: the diet and the liver (via the bile). The diet contains a large number of plant glycosides, predominantly flavonoids. Glycosides from the liver include compounds that are glucuronidated and subsequently secreted into the bowel via the bile. The intestinal flora can then hydrolyze the J3-glucuronide bond; hydrolysis leads to the release of biologically active aglycones, some of which are potentially toxic or carcinogenic. The principal glycosidases produced by the intestinal flora are J3-glucosidase, J3-galactosidase, and J3-glucuronidase [21]. Hawksworth et al. [22] concluded on the basis of a study of 50 strains of each of four bacterial species commonly found in the bowel that, on a per-cell basis, Escherichia coli and Clostridium had the highest J3-glucuronidase activity and Lactobacillus and Bifidobacterium the lowest. Bacterial J3-glucuronidase seems to play an important role in the metabolism of colon carcinogens. This substance has a wide substrate specificity and consequently can hydrolyze a large number of different glucuronides. Several studies have shown that intestinal bacterial J3-glucuronidasecan alter or amplify the biologic activity of exogenous and endogenous compounds. For example, toxic aglycones such as methylazoxymethanol from cycasin can be regenerated in situ in the bowel by bacterial J3-glucuronidase. Weisburger et al. have studied the metabolism of the carcinogen N-hydroxyfluorenylacetamide when administered parenterally to conventional and germfree rats [23]. Germfree rats excreted appreciably larger amounts of the glucuronide of N-hydroxyfluorenylacetamide in their feces than did conventional animals. The cecal and fecal metabolites in conventional rats were mostly free unconjugated compounds, whereas the major fraction in germfree animals was conjugated with glucuronic acid or sulfate. Morotomi et al. [24J reported that cell-freeextracts of some strains of intestinal bacteria, including Bacteroidesfragilis, Bacteroides vulgatus, Bacteroides Hydrolysis Glucuronides Glycosides Sulfamates Amides Esters Nitrates Dehydroxylation C-Hydroxy groups N-Hydroxy groups Decarboxylation d-Demethylation Deamination Dehydrogenase Dehalogenation Reduction Nitro groups Double bonds Aw groups Aldehydes Alcohols N-Oxides Nitrosamine formation Aromatization Acetylation Esterification Representative substrate Estradiol 3-glucuronide Cycasin Cyclamate, amygdalin Methotrexate Acetyldigoxin Pentaerythritol trinitrate Bile acids N- Hydroxyfluorenylacetamide Amino acids Biochanin A Amino acids Cholesterol, bile acids DDT (chlorophenothane) p-Nitrobenzoic acid Unsaturated fatty acids Food dyes Benzaldehydes Benzyl alcohols 4-Nitroquinoline l-oxide Dimethylnitrosamine Quinic acid Histamine Gallic acid been implicated in the generation of mutagens, carcinogens, and various tumor promoters: Jl-glucuronidase, J3-glucosidase, J3-galactosidase, nitro reductase, azoreductase, 7·a-steroid dehydrogenase, and 7-ahydroxy-steroid dehydroxylase. The carcinogenic potential of bacterial enzymes in the intestinal micro flora has been illustrated in a series of studies involving experimental colon cancer induced by cycasin. This substance is a naturally occurring J3-glucoside of the methylazoxymethanol extractable from the seeds and roots of cycad plants such as tropical ferns. Laqueur and Spatz discovered that feeding cycasin to infant rats caused hepatomas, renal sarcomas, squamous cell carcinomas of the ear duct, and - in greatest frequency - intestinal adenocarcinomas that were located almost exclusively in the large bowel [19]. The genetic strain of rat had little influence on the carcinogenic effect of cycasin; similar tumors were induced in OsborneMendel, Sprague-Dawley, Fischer, and Wistar rats. The intestinal flora was required for the carcinogenic activity of cycasin: the compound was completely inactive when given orally to germfree rats [20]. thetaiotaomicron, Eubacterium lentum, Peptostrep- Downloaded from http://cid.oxfordjournals.org/ at Penn State University (Paterno Lib) on May 12, 2016 Table 1. Biochemical reactions of intestinal bacteria. Gorbach and Go/din 8254 is mutagenic in the Ames assay, is also carcinogenic [31]. Ponceau 3R, another biologic stain, is reduced in vitro by Fusobacterium to a 2, 4, 5-trimethylaniline that has been determined to be mutagenic [32]. Incubation of methyl orange or methyl yellow with intestinal anaerobes and testing with Salmonella TA1538 in the presenceof a microsomal activating system have produced a positive result for mutagenicity. Both dyes are reductively cleaved to the mutagen N,N-dimethylp-phenylene diamine [33]. Other azo dyes that have been shown to undergo bacterial reduction to mutagenic or carcinogenic products are direct black 38, direct red 2, and direct blue 15. These dyes are converted to benzidine, 3,3dimethylbenzidine, and 3,3-dimethoxybenzidine, respectively. Congo red is also reduced by rat cecal bacteria to benzidine [34]. In the absence of a bacterial reductase system, congo red is not mutagenic for Salmonella TA1538in the presence of a liver activating system; however, preincubation of congo red with cecal bacteria results in a positive mutagenic response. Nitroreductase. The reduction of nitro groups by intestinal bacteria is another source of aromatic amines. Nitroreductase causes the formation of reactive nitroso and N-hydroxy intermediates in the course of converting aromatic nitro compounds to the aromatic amines. The precursor aromatic nitro compounds are commonly found in factory effluents as industrial chemical pollutants. Wheeler et al. [35] studied a similar reaction, the reduction of p-nitrobenzoic acid, in conventional and germfree rats. Conventional animals rapidly converted p-nitrobenzoic acid, whereas germfree rats reduced little of the nitrocompound. A nitrated polynuclear aromatic hydrocarbon, 1nitropyrene, is readily formed by reaction of nitrogen oxides with the combustion product pyrene. Its presence in diesel engine exhaust represents a potential health hazard because of its high mutagenicity in bacterial test systems and its carcinogenicity in rats. When l-nitropyrene was administered orally to conventional rats, 5070-6070 of the dose was detected in the feces as I-aminopyrene [36]. When a similar experiment was performed in germfree rats, l-aminopyrene was not found in the feces. Since reduction of l-nitropyrene to l-aminopyrene is an activation process, the results indicate that the intestinal microflora is important in the metabolic activation of 1nitropyrene, Downloaded from http://cid.oxfordjournals.org/ at Penn State University (Paterno Lib) on May 12, 2016 tococcus species, and E. coli, enhanced the mutagenicity of bile from rats given l-nitropyrene via stomach tube. These bacterial cell-freeextracts hydrolyzed the synthetic l3-n-glucuronides of phenolphthalein and/or p-nitrophenol. Cell-free extracts of bacteria not capable of increasing mutagenicity did not hydrolyze the glucuronides. These data indicate that the glucuronides of I-nitropyrene metabolites secreted into the bile can be hydrolyzed in the intestine by bacterial B-glucuronidases to potent mutagenic aglycones. Azoreductase. Most of the artificial coloring additives used in the food, printing, and textile industries are dyes that contain a single azo bond (monoazo dyes). The water-soluble dyes are not absorbed well from the intestine and are subject to bacterial action in the large bowel [25]. A number of studies have shown that the bacterial flora can reductively hydrolyze the azo bond by the action of azoreductase (an enzyme confined largely to intestinal bacteria); this reaction results in the formation of substituted aromatic amines [26], a class of bacterially generated compounds including a number of wellestablished carcinogens [27]. Large-bowel cancer occurs more commonly in Western countries than elsewhere, and the extent of the use of azo dyes is related to the degree of industrialization of the country. A connection may exist between the number of cases of cancer and the use of azo dyes [28]. The reduction of azocompounds by azoreductase is believed to be mediated through a free-radical mechanism, which produces intermediates that react with proteins and amino acids. Azoreductase also can reduce food dyes, releasing phenyl- and naphthylsubstituted amines. These compounds have been implicated as chemical carcinogens [29]. The amines generated in the bowel via the azoreductase reaction are probably further oxidized to proximal carcinogens by microsomal enzymes in the intestinal mucosa. The role of bacteria in the generation of mutagens from a number of azo dyes is noteworthy. There is a 90070 correlation between carcinogenicityand mutagenicity for aromatic amines and azo dyes tested with the salmonella/microsomal mutagenicity test. The transformation of azo dyes by intestinal bacteria may be a necessary prerequisite of carcinogenicity.Trypan blue is widely used as a biologic stain and is not mutagenic, but reduction by a cell-freeextract of Fusobacterium produces a mutagen. O-Toluidine [30], which Intestinal Microflora and Colon Cancer Miller and Miller [37] and Weisburger and Weisburger [29], after reviewing the evidence, have suggested that the products of these reactions are extremely important in chemical carcinogenesis. Diet and Bacterial Enzymes Bile Acids Bile acids have been studied extensively as candidate carcinogens because of their structural similarity to the carcinogenic polycyclic aromatic hydrocarbons [42]. The concentration of fecal bile acids is increased in people eating a meat (high-fat) diet. High concentrations of fecal bile acids cause colonic bacteria to produce larger amounts of 7-a-dehydroxylase, the enzyme involved in conversion of primary to secondary bile acids [43]. This finding was confirmed by the addition of chenodeoxycholic acid to a growing culture of Eubacterium species, resulting in a striking increase in 7-a-dehydroxylase activity [44]. Salvioli and co-workers found increased fecal concentrations of cholic and chenodeoxycholic acid and decreased fecal concentrations of secondary bile acids after daily administration 'of lyophilized Streptococcus jaecalis to healthy volunteers [45]. Demographic studies have demonstrated a correlation between high fecal concentrations of secondary bile acids and the Western high-beef diet [46]. Hill et al. noted that the fecal microflora of North Americans and Western Europeans contained more bacterial strains capable of 7-a-dehydroxylation than did the fecal microflora of Ugandans or Indians [47]. In studies by Mowar et al. involving Japanese living in Akita, Japan, and Japanese living in Hawaii and consuming a Western-style diet, the latter group had higher levels of fecal deoxycholic acid; however, little difference was noted in the other fecal bile acids [48]. These authors also found that fecal concentrations of coprostanol and coprostanone, degradation products of cholesterol, were higher in people eating a Western-style diet [48]. Mastromarino and coworkers studied patients with colon cancer and found elevated levels of both 7-a-dehydroxylase and cholesterol dehydrogenase compared with those in healthycontrols [49J. Elevations of both enzymes also were noted in patients with nonhereditary colonic polyps [17J. MacDonald et al. [50] measured the activity of fecal NAD- and NADP-dependent 7-a-hydroxysteroid dehydrogenase, which converts hydroxy-bile acids to keto-bile acids, in vegetarian Seventh Day Adventists and subjects consuming a mixed Western diet. The activity was lower in the vegetarian group. The authors suggested that increased fecal NAD- and NADP-dependent 7-a-hydroxysteroid dehydrogenase is associated with a risk of large-bowel cancer. Downloaded from http://cid.oxfordjournals.org/ at Penn State University (Paterno Lib) on May 12, 2016 The effect of diet on fecal bacterial B-glucuronidase, nitroreductase, and azoreductase activities has been studied in rats [14, 38]. Rats initially maintained on a grain diet and then shifted after several weeks to a meat diet showed a twofold rise in fecal l3-glucuronidase and nitroreductase activitieson the meat diet. This increase started within 6 days, although the total effect required 12-17days. Fecal azoreductase also increased by approximately twofold when rats were shifted to the meat diet. An increase in specific activity of this enzyme was noted 4-10 days after the dietary change. Similar increases in fecal bacterial enzymes were noted when the grain diet was supplemented with 30070 beef fat without other beef products [39]. Variations in diet influence the activity of fecal bacterial enzymes in humans in a manner similar to that reported in rats. Fecal bacterial enzymes were studied in omnivorous individuals who ate a mixed Western diet, lactovegetarians who consumed a diet that excluded all animal foods except milk products, and strict vegetarians [40]. Omnivores had considerably higher levelsof fecal B-glucuronidase, nitroreductase, and 7-a-dehydroxylase than did lactovegetarians or strict vegetarians. Azoreductase levels were significantly lower among the strict vegetarians than among omnivores. Elimination of red meat from the diet of omnivores and fiber supplementation of the mixed Western diet produced no significant change in any enzyme activity except that of 7-a-dehydroxylase. In both cases levels of this enzyme fell significantly during the period of dietary adjustment. In another study [41], rats on either grain or beef diets were fed Lactobacillus acidophi/us. The activities of fecaljl-glucuronidase, azoreductase, and nitroreductase were significantly reduced in the beef-fed rats, but no changes were seen in the grain-fed animals, whose fecal enzyme activities were already low. The results cited above indicate that diet and other factors can alter levels of bacterial enzymes in the gastrointestinal tract, perhaps affecting the formation of carcinogens and the concentration of tumorpromoting substances. 8255 8256 High-Fat Diets Dietary Fiber A protective effect against colon cancer has been ascribed by some authors to consumption of dietary fiber [11]. The authors of a recent study found the risk of colon cancer to be relatively low in people who ate larger amounts of crude dietary fiber, fruits, and vegetables, although intake of grains did not appear to be protective [53]. The effect of fiber on fecal bacterial enzymes was studied in animals fed either a grain or a beef diet; the grain diet was associated with lower activity of ~glucuronidase, nitroreductase, and azoreductase [14]. The specific components of fiber have been studied more intensively in an effort to discern their effects on fecal enzymes. Fecal bacterial glycosidase activity was lower in animals fed a high-eellulose diet than in those fed a standard laboratory diet [54]. In another study three single sources of dietary fibercellulose, hemicellulose, and Pectin- were fed to lab- oratory animals, and the effects on fecal bacterial enzymes, fecal bacterial counts, and dimethylhydrazine-induced colon cancer were observed [55]. Cellulose and hemicellulose decreased the activity of fecal enzymes, whereas pectin increased that activity. Fecal bacterial counts were not altered by any of the three fiber sources. Protection against chemically induced colon cancer was related to bacterial enzyme levels in that celluloseand hemicellulose protected the animals, whereas pectin had no effect. Protection Against Experimental Colon Cancer by Manipulation of the Intestinal Microflora The evidence presented above implicating the microflora in colon cancer is circumstantial- either from in vitro laboratory studies or from metabolic epidemiology. More direct evidence implicating bacteria in colon cancer is derived from studies in animal models involving chemical induction of large-bowel cancer. Dimethylhydrazine, an experimental carcinogen that is metabolized by the liverto methylazoxymethanol, is similar to cycasin in its structure and carcinogenic. effects [56]. Rats fed a high-beef diet were more susceptible to the carcinogenic effects of dimethylhydrazine than were rats fed a grain diet. When dimethylhydrazine was fed to rats on high-beef and high-grain diets, the rates of tumor production were 83070 and 31070, respectively [16]. In studies in which germfree and conventional rats werechallenged with 3,2-dimethyl-4-aminobiphenyl (DMAB), there were significantly fewer colonic tumors among germfree animals [57]. When the role of dietary fat in promoting colonic carcinogenesis was studied during challenge with DMAB, more animals fed a high-fat diet than animals fed a lowfat diet had colonic tumors. Dietary fat had no effect on the incidence of tumors when germfree animals were studied; this observation further emphasizes the role of the microflora in this process. The administration of oral antibiotics influences the metabolic activity of the intestinal flora and the induction of cancer. Three groups of beef-fed rats were studied: a control group, a group receiving tetracycline, and a group receivingerythromycin [58]. There was a striking reduction in the incidence of intestinal tumors in the antibiotic-treated groups after challenge with dimethylhydrazine: colonic tumors developed in 74070 of control animals but in only 20070 of the tetracycline group and 22070 of the erythro- Downloaded from http://cid.oxfordjournals.org/ at Penn State University (Paterno Lib) on May 12, 2016 Several studies have implicated dietary fat as the nutrient most closely associated with the high incidence of colon cancer in Western populations [7-10]. Various theories have been advanced to explain the role of dietary fat in colonic carcinogenesis [8]. A highfat diet causes increased bile flow and higher fecal levels of bile acids; as noted above, bile acids have been implicated in colon cancer, possibly as a cocarcinogen. It has been suggested that dietary fat can increase mucosal enzyme systems, an effect that may promote activation of carcinogens [51]. A high-fat diet may also increase bacterial enzyme activities in the large bowel. Temple and EI-Khatib [52] studied the effects of a high-fat diet in mice, measuring both intestinal mucosal and fecal enzymes. An increase was noted in mucosal levels of 5'-nucleotidase but not in mucosal levels of P-glucuronidase. There was an increase in fecal bacterial reductase in Swiss mice fed the high-fat diet but not in C57BL/l mice. These bacterial enzyme changes correlated with susceptibility of the strain of animal to dimethylhydrazine-induced colon cancer: Swiss mice, which had increased levelsof bacterial enzymes,werehighly susceptible to tumor formation, whereas C57BL/l mice were resistant to cancer induction. As noted above, beef fat, when added to a grain diet, caused increases in the activities of several bacterial enzymes [39]. Gorbach and Goldin Intestinal Microflora and Colon Cancer Fecal Mutagens Distributions in different populations. Bruce et al. [60] first reported that organic extracts of human feces contained material positive in the bacterial mutagenic test developed by Ames et al. [61]. Using a number of separation techniques, Bruce and associates found that mutagenic activity was associated with a single compound. Several groups investigated the occurrence and levels of fecal mutagens in populations at different risk for colon cancer. In a study conducted in South Africa, fecal specimens were obtained from low-risk rural blacks and from high-risk whites in Johannesburg [62]. The frequency of mutagenic substrates found in the feces differed significantly between the two populations. Only 20/0 of the fecal specimens obtained from the rural black population contained mutagens; in contrast, 15% of those from the white population tested positive. Reddy et al. [63] measured fecal mutagenic activity in three populations: residents of New York City who consumed a typical Western diet (considered at high risk for colon cancer), Seventh Day Adventists, and residents of the rural Finnish town of Kuopio (both considered at low risk for colon cancer). Fecal specimens collected from the three groups were freeze-dried, extracted with ether, partially purified by silica gel chromatography, and assayed with Salmonella test strains TA9S and TAloo, with and without a liver microsomal activating system. Of the fecal specimens from the high-risk population, 22070 were directly mutagenic on strain TA9S, 11 070 were directly mutagenic on strain TAloo, and 6070 were' mutagenic with S-9 activation on strain TAloo. Of the Kuopio samples, 13070 were mutagenic on strain TA9S, with microsomal activation required in all cases; no mutagenicity was observed on strain TA100. None of the samples from Seventh Day Adventists showed mutagenicity with any test strain. These data indicate that the incidence of colon cancer may be correlated with the presence of mutagens in the fecal stream. In a subsequent study by Reddy et al. [64], fecal comutagens were measured in two of the three populations discussed above: the New York City residents and the Seventh Day Adventists. Fecal specimens were selected on the basis of having shown no mutagenic activity in the previous study. Fecal extracts were prepared and tested for their ability to enhance the indirect-acting mutagen and carcinogen 2-acetylaminofluorene (2-AAF) and the direct-acting mutagen N-methyl-N -nitro-N-nitroguanide (MNNG). Dose-related enhancement of 2-AAF on strains TA9S and TA100 was observed with fecal extracts from both populations, but extracts from the vegetarian group had significantly lower comutagenic activity on strains TA9S. Comutagenic activity with MNNG was the same for both groups. Kuhnlein et al. [65] observed significant differences in the mutagenicity of water extracts from feces of lactoovovegetarians. The mutagenic activity was assayed with the fluctuation test described by Green and Muriel [66]. Activity was detected in concentrations as low as 1 mg of fecal supernatant/mL, and the level of activity was significantly lower for vegetarians than for nonvegetarians. There was, however, considerable variation among individuals. For example, the subjects with the highest and lowest activities were both from the vegetarian group. Thus the results are difficult to interpret. Varied mutagenic activity of the water extracts toward the test strains indicated the presence of several different mutagens. Ferguson et al. [67] used repair-proficient and repair-deficient strains of E. coli to investigate the DNA-damaging activity of ethanol-soluble fecal extracts. Feces from European patients with colorectal cancer, age-matched controls, Maoris, Samoans, and lactoovovegetarian Seventh Day Adventists were analyzed for DNA-modifying activity. The Europeans had the highest activity regardless of whether or not they had cancer. The rate of positive samples was lower in the Polynesian groups, and there were no unequivocally positive samples in the Seventh Day Adventist group. Downloaded from http://cid.oxfordjournals.org/ at Penn State University (Paterno Lib) on May 12, 2016 mycin group. Fecal J3-glucuronidase activity was also significantly reduced in antibiotic-treated animals. Similarly, administration of a J3-glucuronidase inhibitor decreased the formation of colon cancer in conventional animals treated with the experimental carcinogen azoxymethane, a compound similar to dimethylhydrazine [59]. In another study, beef-fed rats were given dimethylhydrazine to induce colonic tumors; viable Lactobacillus was included in the diet [16]. At the 2Q-week observation time, the rate of colon cancers was significantly lower among beef-fed animals given Lactobacillus than among beef-fed controls; however, no difference was noted at 36 weeks.These findings suggest that Lactobacillus increases the latency or induction time for colon cancers, probably by suppressing the metabolic activity of the colonic flora. 8257 Gorbach and Goldin 8258 icallyat 37°C, mutagen was produced. Studies on pure cultures revealed that five species of Bacteroides (B. fragilis, B. ovatus, B. uniformis, B. thetaiotaomicron, and strain 3452A) produced the mutagen. These strains are commonly found in human feces. The in vitro production of mutagen was inhibited by fermentable carbohydrates such as glucose, starch, and dextran. Some 40 other species of intestinal anaerobes tested for production of the mutagen all proved negative. The facts that Bacteroides species are common in the human colon and that only a relatively small percentage of individuals produce the mutagen indicate that the precursor is the determining factor in its production. This possibility was confirmed by the demonstration that bacteria from feces not containing mutagen were capable of producing it in the presence of the precursor compound. The precursor - which is a product of other bacteria in the colon, a result of the diet, or a metabolite derived from the host - is necessary, in combination with Bacteroides, for mutagen production. It has subsequently been demonstrated that cell-free extracts of Bacteroides in the presence of bile can produce the mutagen. The bacterial enzyme system is not oxygen sensitive; however, the mutagen is produced under only anaerobic conditions [69]. Structure offecal mutagen. The structure of the fecal mutagen was first announced in 1982 [72] and was confirmed independently in the following year (73]. The characteristic ultraviolet spectra and shift on oxidation implied that the compound was a pentaene. Chemical ionization, mass spectrometry, and nuclear magnetic resonance analysis elucidated the structure of the compound, called (8)-3-(1,3,5,7,9dodecapentaenyloxy)-1,2-propanediol (figure 1). The compound is a vinyl ether and is a conjugated laurylglyceroI. There are no known mutagens with similar structures. The mutagen structurally resembles a group of ether-linked lipids that are found in anaerobic bacteria and in low concentrations in some mammalian tissues [74]. Conclusion There is no direct evidence that the human intestinal microflora influences the risk of colon cancer. What has emerged from the various studies cited in this review is that the intestinal microflora is capable of engaging in reactions that can generate carcinogens, mutagens, or tumor promoters in the large bowel. Furthermore, various treatments that alter the Downloaded from http://cid.oxfordjournals.org/ at Penn State University (Paterno Lib) on May 12, 2016 Isolation ofether-soluble mutagen from human feces. Bruce et al. [6OJ described the presence of mutagenic activity in an ether extract of freeze-dried feces. The ether extract demonstrated maximal activity when washed with aqueous sodium hydroxide and then neutralized prior to testing. The washed ether extract was subsequently dried, suspended in benzene, placed on a silica gel column, and eluted with benzene and ether. A single fraction contained most of the activity. This was the first evidence that the mutagenic activity in the ether fraction was associated with a single compound or a class of similar compounds. The purification and ultraviolet spectra of the compound were described by two collaborating groups [68, 69]. The compound has a peak in the ultraviolet at 365 nm and a high extinction coefficient. It has a lime-green fluorescence when exposed to long-wavelength ultraviolet light. The high absorbance allowed investigators [69] to devise a rapid high-performance liquid chromatography method based on the area of optimal density in column fractions; this method is 10 times more sensitive than the Ames test. The compound is highly sensitive to oxidation; it is inactivated by liver microsomal preparations normally added to bacterial mutagen assays to activate compounds by oxidation. Oxidation can be inhibited by inclusion of antioxidants such as butylated hydroxytoluene in the organic solvents and can be prevented completely if purification is performed under an atmosphere of argon in solvents containing butylated hydroxytoluene. The mutagen is very stable under strictly anaerobic conditions. When oxidation is allowed to proceed under controlled conditions, the three ultraviolet peaks shift to lower wavelength and the fluorescence disappears. Bacterial production of fecal mutagen. The amount of mutagenic activity increases dramatically if a fecal specimen already containing mutagen is incubated anaerobically at 37°C for several days. Mutagen production is inhibited by exposure of the feces to oxygen, low temperature, autoclaving, or radiation [70]. It was not possible to generate a mutagen in vitro when fecal specimens were suspended in bacteriologic media. However, the addition of bile to the media resulted in the production of mutagen [71]. It was then demonstrated that a precursor was present in feces containing mutagen. When this precursor was added to bacteriologic media containing bile, inoculated with fresh feces, and incubated anaerob- Intestinal Micro/lora and Colon Cancer H2CO(CH-CH)5CH2CH 3 I H-C-OH I CH 20H Figure 1. Structure of fecal mutagen (8)-3-(1,3,5,7,9dodecapentaenyloxy)-1,2-propanediol. References 1. Simon GL, Gorbach SL. The human intestinal microflora. Dig Dis Sci 1986;31:147S-62S 2. Finegold SM, Attebery HR, Sutter VL. 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