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Role of the Gastrointestinal Mucosa and Microflora in the Bioactivation of Dietary and Environmental Mutagens or Carcinogens a
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Robert W. Chadwick , S. Elizabeth George & Larry D. Claxton
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USEPA Environmental Research Center Health Effects Research Lab Genetic Toxicology Research Division, Genetic Bioassay Branch, MD-68A, Research Triangle Park, North Carolina, 27711 Published online: 08 Jun 2015.
To cite this article: Robert W. Chadwick, S. Elizabeth George & Larry D. Claxton (1992) Role of the Gastrointestinal Mucosa and Microflora in the Bioactivation of Dietary and Environmental Mutagens or Carcinogens, Drug Metabolism Reviews, 24:4, 425-492 To link to this article: http://dx.doi.org/10.3109/03602539208996302
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DRUG METABOLISM REVIEWS, 24(4), 425-492 (1992)
ROLE OF THE GASTROINTESTINAL MUCOSA AND MICROFLORA IN THE BIOACTIVATION OF DIETARY AND ENVIRONMENTAL MUTAGENS OR CARCINOGENS* ROBERT W. CHADWICK, S. ELIZABETH GEORGE, and LARRY D. CLAXTON USEPA Environmental Reseurch Center Health Effects Research Lab Genetic Toxicology Reseurch Division Genetic Bioassay Brunch, MD-68A Research Triangle Purk, North Carolina 2771 I
I.
INTRODUCTION.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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11.
GENOTOXICITY AND BIOACTIVATION IN THE GASTROINTESTINAL TRACT. . . . . . . . . . . . . . . . . . . . . . A. Esophagus.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Stomach.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Small Intestine.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Large Intestine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
427 431 431 435 439
*The views expressed in this paper are those of the authors and not necessarily those of the Environmental Protection Agency. This paper was refereed by Carl E. Cerniglia. Ph.D., Food and Drug Administration, National Center for Toxicological Research, Jefferson, AR 72079; and by Douglas E. Rickert, Ph.D., Glaxo, Inc., Research Triangle Park, NC 27709. 425 Copyright 0 1992 by Marcel Dekkcr, Inc
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E. Effect of p H . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Intestinal Microflora . . . . . . . . . . . . . . . . . . . . . . . . . . .
442 443
Ill.
INTERACTIONS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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IV.
SOURCES OF ENVIRONMENTAL MUTAGENS . . . . . . . . . 459 A. Foods and Food Products. . . . . . . . . . . . . . . . . . . . . . . . 462 B. Nonfood Genotoxicants . . . . . . . . . . . . . . . . . . . . . . . . . 467 C. Genotoxicant Exposure . . . . . . . . . . . . . . . . . . . . . . . . . 467
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CONCLUSIONS.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
47 I 472
I. INTRODUCTION From Metchnikoff's contention, in 1901, that intestinal microflora may pose a hazard to human health [I], through epidemiological studies in the 1960s and '70s [2-91, which uncovered the relationship between diet and cancer, to recent research which has implicated enzymes of both intestinal mucosa and microflora in the bioactivation of genotoxicants [ 10-331, evidence has accumulated which supports a significant role for the intestinal tract and its contents in the oncogenic process. Humans are continually exposed to potentially harmful chemicals that gain entrance to the intestinal tract via ingested food, water, or inspired air. It has been estimated that over 53,000 chemicals are used by society as drugs, pesticides, household cleaning products, and food additives 1341. Some of these chemicals (promutagens/procarcinogens) become genotoxic upon metabolic activation. More than 90% of the known carcinogens require further enzymatic transformation to form the ultimate carcinogen [lo, I I , 341. The liver is usually considered to be the major drug-metabolizing organ; however, to be absorbed, ingested chemicals must pass through the intestine, where mucosal enzymes can metabolize them via various pathways [35, 361. In their recent review of mucosal biotransformation, Laitinen and Watkin have concluded that the intestine plays a major role in the metabolism of xenobiotics by all animals 1341. Moreover, metabolism of foreign compounds and nutrients by the gut microflora is extensive and highly diverse [37]. There have been a number of recent reviews on the separate roles of the intestinal mucosa [38, 391 and microflora [40-451 in the bioactivation of genotoxicants. However. the close symbiotic relationship between the microflora and the host suggests that it is preferable to consider the whole ecosystem of the gas-
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trointestinal (GI) tract when assessing its role in genetic toxicology. For example, there have been reports from several laboratories of cooperative metabolic interactions between enzymes of the gut mucosa and those of the microbial flora that influence the bioactivation of genotoxicants [ 18. 27, 46-49]. Other reports indicate that production of some mutagenic metabolites requires activation by intestinal anaerobes together with further metabolic conversion by microsomal preparations from either the liver or the intestinal mucosa [24, 50-521. The bioactivation of aromatic nitro compounds exemplifies a multistep process involving enterohepatic circulation, intestinal microflora, and hepatic enzymes [30, 53). While microflora affect the turnover rate of mucosal cells [54, 551, the environment of the mammalian gut can markedly alter the expression of microfloral enzymes (561. In addition, host secretions such as bile salts and glucuronides can modify the activity of the microflora 1571, and bacterial metabolites of these secretions are frequently suspect carcinogens [43, 581. Enterohepatic circulation, resulting from the hydrolytic activity of the microbial flora, delays the excretion of potential genotoxicants and increases the chance for their bioactivation [ 5 9 ] . Though the nature and extent of these interactions have not been completely resolved, this review attempts to provide a contemporary summary of the complex role of the gastrointestinal tract in genetic toxicology.
11. GENOTOXICITY AND BIOACTIVATION IN THE GASTROINTESTINAL TRACT The gastrointestinal tract has been aptly described as a tube extending from the lips to the anus [601. The average length of the human GI tract is 435 cm and it is divided into I 1 well-defined regions (611. In mammals, the structure and function of these regions reflect the diet and life-style of a given species. The esophagus. stomach, small intestine, and large intestine have received the most attention in cancer research, and their relative contributions to the bioactivation of genotoxicants are evaluated in this review. Figure I depicts the intestinal tracts of the widely used laboratory rodent, which is a monogastric herbivore; and man, an omnivore. A large number of bacteria are found in the gastrointestinal tract of mammals. In fact, it has been stated that because of the gastrointestinal flora, there are more cells in the gastrointestinal tract than elsewhere in the body [62]. The number of microorganisms per gram of colon contents can exceed 10" microorganisms per gram dry weight (63, 641 and can compose 40% to 55% of the total fecal mass [65, 661. Some of the major factors determining composition are
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Parotid gland Mouth
Pharynx
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Esophagus
Rectum Anus
FIG. 1. The gastrointestinal tract of the rodent (left) and man (right). diet, pH, oxygen tension, and stress [41, 60, 67-69]. There are several excellent current reviews that discuss the population and ecology of the intestinal tract [37, 60,62, 701. With the exception of toxicants that are absorbed in the mouth or rectum and those entering the lymphatics via the lacteals, all xenobiotics are available for intestinal first-pass clearance [34]. First-pass clearance involves the biotransformation and clearance of a chemical from the body before it reaches the systemic circulation. Biotransformation reactions are generally subdivided into phase I and phase I1 reactions (Fig. 2). Phase I reactions generally involve alteration of the structure of the parent chemical while the phase I1 reactions typically involve addition of an endogenous substance to
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Genotoxicity
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Xenobiotic Enters Body
FIG. 2. Phase I and phase I1 reactions in the biotransformation of genotoxicants. enhance the aqueous solubility and ultimate excretion of the metabolites formed. Both phase I and phase I1 enzymes produce metabolites which are less lipid soluble and are more easily excreted. However, some phase I reactions also activate procarcinogens to ultimate carcinogens [38, 391 while the phase I1 reactions, with a few exceptions 171, 721, lead to detoxification and presystemic elimination of the metabolites formed [73]. Figure 2 indicates some of the major pathways involved in the bioactivation and/or detoxification of genotoxicants in mammals. This section of the review includes a brief discussion of the structure, oncogenic susceptibility, and bioactivation capacity of various regions of the GI tract. In addition, a description of the alimentary tract flora serves as a brief review of the major microorganisms found in the human GI tract and their relative population numbers (Fig. 3). Table 1 , which describes the predominant fecal flora from human, swine, and rats, is included for interspecies comparison.
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v)
c
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z 3
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Stomach
Jejunum Ileum GASTROINTESTINAL TRACT
Colon
FIG. 3. Distribution of microbial flora in the hunman GI tract.
TABLE 1 Comparison of Major Genera Isolated from Human, Swine, and Rat Feces Human"
Swine','
Rat"
Bacteroides' Bifidobacterium Eubacterium Fusobacterium Clostridium Peptostreptococcus Peptococcus Streptococcus
Streptococcus' Lactobacillus Fusobacterium Eubacterium Bacteroides Peptostreptococcus
Bacteroides' Lactobacillus Streptococcus Fusobacterium Bi fidobacteri um
uDraser and Barrow [60];Gorbach [156]. 'Moore et al. [69]. "Swine in order of prevalence. "Benno et al. [68]. 'The most abundant genus is listed first in each column.
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A. Esophagus The human esophagus is an elastic 40 cm long tube 1611 which carries food past the region of the heart and lungs (Fig. I ) . The upper 1/3 of the esophagus consists of skeletal muscle while the lower 213 is composed of smooth muscle. About 5 cm above its junction with the stomach, the muscle is hypertrophied to form the gastroesophageal constrictor. In rodents, the esophagus enters the stomach at the inner curvature through a fold in the limiting ridge that separates the forestomach from the glandular stomach [74]. The fold of the limiting ridge makes it impossible for the rat to vomit. The incidence of esophageal cancer varies greatly among different regions of the world 175, 761. A belt of high risk runs from the Middle East through central Asia to China. Other regions of high risk occur in the eastern and southern areas of Africa and in Normandy and Brittany in France. The American Cancer Society estimated that 10,100 new cases of esophageal cancer would occur in the United States in 1989, which constituted about I % of the total cancer incidence for the year [77]. Mortality from esophageal cancer in 1989 was estimated to be 9,400 or 1.87% of the total deaths from cancer in the USA 1771. Chemicals that induce esophageal neoplasms in the laboratory rat include: a number of methyl-alkylnitrosomines [78, 791 and N.N-dibutylnitrosamine [ 801, Zinc deficiency [81-83] and alcohol 176, 811 appear to enhance esophageal neoplasms. Esophageal S9 from rats [84] and cultured esophageal cells from both rats and man [85, 861 are able to activate benzo(a)pyrene. Cultured human fetal esophagus cells are also able to convert 2-aminofluorene and several nitrosamines into metabolites capable of binding to DNA within the cells (851. Activation in the esophagus was less than that observed for the stomach in this study. It has also been reported that nitrosamines produce micronuclei in tissue lining the esophagus [87]. Micronuclei are chromatin fragments from the breakage of chromosomes forming aggregates of chromatin or from spindle malfunction. They remain unaltered in the cytoplasm after division and are indicative of non-disjunction. The esophagus metabolized N-nitroso-N-methylanilinevia initial denitrosation followed by oxidative demethylation to aniline. The same metabolic pattern was observed with S9 fractions [881. From these reports, it is evident that esophageal enzymes are capable of bioactivating xenobiotics to electrophilic species which can bind to macromolecules and initiate carcinogenesis.
B. Stomach The human stomach is a pouch or reservoir, 38 cm in length 1611, which regulates the entry of food into the small intestine (Fig. I ) . The upper region
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of the stomach (fundus) serves for short-term storage of food, the middle region (cardiac) is where food mixing takes place, and the lower portion (pyloric) ends at the pyloric valve, a circular muscular sphincter at the junction with the small intestine. The stomach wall contains glands that secrete mucin, hydrochloric acid, and enzymes, which aid in the digestion of food. The stomach of the rodent is divided into two very distinct parts, the nonglandular forestomach and the glandular stomach. The forestomach is not present in primates, including man, and thus chemical induction of tumors in this part of the rodent stomach is difficult to evaluate in relation to human risk assessment and will not be considered in this review. Though the acid stomach contents of man are usually sterile, Draser has reported trace quantities of bacteria from the stomach contents of fasting people 1701. The primary microorganisms associated with the human stomach are in the genus Lactobacillus [60].These organisms are gram-positive, facultative or anaerobic, nonsporing rods that can withstand low pH 1891. They range in number from -4 to -2 log 10 colony-forming units per gram of wet weight (O.OOO1 to 0.01 CFU/g) [70]. Another group found in the stomach are the Streptococci 1701. These organisms are facultatively anaerobic, grampositive cocci that are generally found in pairs or chains [89]. They can range in number from -5 to -2 log 10 CFU/g wet tissue 1701. Bacteroidps spp. and members of the family Enterobacteriaceae have also been isolated [701. Veillonella spp., an anaerobic gram-negative coccus, Bifidobacterium spp.. Clostridium spp., and yeasts also have been found [62]. These microorganisms are not found in all individuals [70]. A recent issue of the Journal ojthe National Cancer Institute (December 4, 1991) presents the fourth epidemiological study linking cancer of the stomach and the presence of Helicobac!er pylori 1901. While it remains unclear whether this bacteria actually causes gastric cancer, patients with gastric cancer were much more likely to have antibodies to H . pylori than healthy volunteers or patients with several other types of cancer [90]. While there is a high incidence rate of stomach cancer in Japan, other parts of Asia, and South America, it is low and decreasing in North America and Europe [76]. Figure 4 presents the 1970-1979 mortality cancer map for the United States 1911. The data indicate regional differences and a higher mortality rate for Whites than for non-Whites. The American Cancer Society estimated 20.000 new cases of stomach cancer in 1989, which was about 2% of the total cancer cases for the year (771. Mortality from stomach cancer in 1989 was estimated to be 13,900, or 2.8% of the deaths from cancer in the USA 1771. Gastric tumor formation has been induced in animals by 3-methylcholanthrene 1921, 7,12-dimethylbenz(a)anthracene [93], 2,7-bis(acetylamino)fluorenylene (94. 951, 4-hydroxyaminoquinoline-Ioxide 1961, aflatoxin [97]. N-methyl-N'-nitro-N-nitrosoguanidine 198. 991,
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oinaprazole [IOO],and loxtidine [loll. While there is no direct proof that N-nitroso compounds play a causal role in the etiology of stomach cancer in humans, results of epidemiological studies suggest that they are likely to increase the incidence of some tumors 1102, 1031. The generation of genotoxicants in the stomach is dependent on the activities of the phase I and phase I I reactions which occur in this region (Fig. 2). In the stomach, the activity of the phase I I detoxification pathways is very low. For example, the stomach of the rat contains less than 6% of the glutathione-S-transferase[ 1041 and about one third the glucuronosyltransferase I1051 activity found in the small intestine. Sulfotransferase is also reduced in the stomach (731. Since these reactions provide a major defense against electrophilic and free radical intermediates, which can initiate cancer, the low activity found in the stomach may account for its high susceptibility to carcinogenesis. The link between phase I reactions, which generally involve structural modifications of the parent compound. and the bioactivation of these chemicals in the stomach is less clear. Acute or chronic pretreatment with benzo(a)pyrene significantly increased aryl hydrocarbon hydroxylase activity in homogenated samples of both the forestomach and the glandular stomach of the rat 11061. However, tumors formed only in the forestomach. Another study reported that stomach S9 fractions from Aroclor-pretreated rats were unable to convert benzo(a)pyrene to mutagens [107]. On the other hand, cultured fetal human stomach activated benzo(a)pyrene, aflatoxin B , , and certain N-nitrosamines to metabolites that bound to cellular DNA 186). In fact, the activity of the cultured stomach exceeded that of cultured esophagus and liver in the same study. In other work, attempts to activate dimethylnitrosamine (DMN) using homogenates of mouse or hamster stomachs failed, despite the presence of measurable aryl hydrocarbon hydroxylase, and its induction by phenobarbital, Aroclor, or 3-methylcholanthrene 1108, 1091. However, there was no correlation between DMN demethylase activity and the mutagenicity of DMN in the presence of other organ homogenates, including the liver. Thus it was suggested that DMN demethylase was not the rate-limiting step in the activation of this genotoxicant [IOS]. Another report suggested that the high susceptibility of the glandular gastric mucosa to nitrosamine carcinogens may be related to the influence of reduced glutathione (GSH) on their macromolecular binding, since GSH levels in this region are exceedingly high compared to other portions of the GI tract [ I I O ] . Fish extracts and nitrite-treated fish, from Japan, induce adenocarcinomas of the glandular stomach in rats [ 11 I ] , and nitrosated fish produce large amounts of direct alkylating (methylating) chemicals [ 1121, suggesting that endogenous nitrosamide formation under gastric conditions contributes to gastric cancer in the rat. Recently bacterial formation of N-nitroso compounds was reported
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from the administration of precursors in the rat stomach after omeprazoleinduced achlorhydria I1131. These results are relevant to clinical studies, which have suggested that patients with chronic atrophic gastritis or those having undergone Billroth I 1 gastrectomy I114-1 171 and those treated with antisecretory drugs for duodenal ulcers [ I 181 frequently have gastric achlorhydria and are at higher risk for stomach cancer than healthy subjects. Since nitrosation-proficient bacteria are capable of increasing the intragastric formation of nitrosamines in the stomachs of rats with induced gastric achlorhydria. they could perform the same function in patients with this condition and thus account for their higher risk of stomach cancer. Administration of three glandular stomach carcinogens, N-methyl-N'-nitro-Nnitrosoguanidine, N-propyl-N'-nitro-N-nitrosoguanidine,and 4-nitro-Nmethylurethane resulted in the appearance of unscheduled DNA synthesis (UDS) in the pyloric mucosa [ 1191. The assay is quite specific since administration of two nongastric carcinogens or two forestomach carcinogens [I201 failed to induce UDS in the pylorus. Though there are apparently some discrepancies, reported research generally supports the induction of genotoxicity in the stomach through the generation of reactive metabolites in the stomach walls. Because deficient phase I1 conjugation reactions do not permit adequate detoxification of these reactive intermediates, macromolecular binding and genotoxicity result.
C. Small Intestine The human small intestine is a 150 cm long coiled tube [61] consisting of the duodenum, jejunum, and ileum, and is the principal region for the metabolism and absorption of ingested nutrients and xenobiotics (Fig. I ) . The intestinal mucosa, containing glands and covered with villi, is arranged in folds so that the surface area for digestion and absorption is greatly increased. The flora of the proximal region (duodenum and upper jejunum) of the small intestine resembles that of the stomach. The populations and numbers are similar. However, in the distal region (lower jejunum and ileum), bacterial numbers start to increase 1701. The major populations identified include Lactobacillus spp. ( 5 to 7 log 10 CFU/g wet tissue), Bifidobacterium spp. ( 5 to 7 log 10 CFU/g wet tissue), Bacteroides spp. (2.5 to 7 log CFU/g wet tissue), and Streptococcus spp. (6 log 10 CFU/g wet tissue) 160, 701. Also present are members of the family Enterobacteriaceae (3 to 4 log 10 CFU/g wet tissue) [70]. Clostridium spp., Veillonellu spp., and yeasts have also been isolated [62]. The malignant tumors of the small intestine represent approximately 0. I % of all malignant neoplasms in human pathology and about 3% of the
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malignant neoplasms of the gastrointestinal tract 11211. In 1989 the American Cancer Society estimated 2,700 new cancer cases of the small intestinc (0.27% of the total cancer cases for all sites) from the population of the United States 1771. The mortality from this cancer was estimated to be 900. or 0.18% of the deaths from cancer in the USA for 1989 177). The most common malignant tumors in the small intestine include the adenocarcinomas (50%). the carcinoid tumors (40%). and the connective tissue neoplasms (10%). Though the small intestinal mucosa exhibits higher phase I enzyme activity than any other region of the alimentary canal 1341. the incidence of tumors here only constitutes about 3% of the total occurring in the GI tract. One reason for this apparent discrepancy may be the high activities of the phase I 1 reactions in this region 173, 1041. For example, the activity of glucuronosyl transferase, a phase 11 enzyme, was compared in tissue slices from the stomach, small intestine, cecum, and colon of the rat 11051; and the activities of the stomach, cecum and colon were only 34%, 15%, and 30% of that in the small intestine. Similarly glutathione S-transferase activities in the stomach, colon, and rectum were 5.1%. 3.4%. and 3.4% that of the small intestine 11041. Sulfotransferase is also higher at the proximal than at the aboral end of the intestinal tract in most species 135, 122, 1231. A recent report indicated that cytosolic fractions of the ileum, from 6 patients, catalyzed the sulfation of p-nitrophenol and dopamine at a higher rate than cytosolic fractions from colons of the same patients 11231. The small intestine is the major extrahepatic conjugating organ in many species, even exceeding the activity in the liver in some cases 11241. Since these pathways are a major defense against the genotoxic metabolites generated by phase I reactions in the small intestine, they probably play a significant role in the low susceptibility of this tissue to genotoxicity. Other mechanisms for the detoxification of xenobiotics in the small intestine [125-1281 have been suggested. In one study, an intestinal glutathione transport system has been demonstrated that permits GSH and GSH-S conjugates to be directly eliminated across the brush-border membranes or transported into the lumen to the active site of the y-glutamyltranspeptidase, where they are further metabolized and excreted 11251. In other work, pretreatment of rats with the organochlorine insecticide lindane reduced the estimated absorption rate of parathion from the intestinal tract and the excretion of the hydrolysis product, p-nitrophenol [ 1261. Further investigation showed that the lindane pretreatment stimulated the conversion of parathion, I h after its p.0. administration, to aminoparathion, which is 100-fold less toxic than the parent compound 11291. These results led to speculation that lindane had altered either the nitroreductase activity or the population of the intes-
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tinal microflora. Since nitroreductase is involved in the bioactivation of many aromatic nitro compounds to genotoxic metabolites [30, 531, the relationship between lindane pretreatment and nitroreductase activity was further investigated. Next it was demonstrated that daily p.0. administration of 20 mg/kg lindane for 5 weeks significantly increased nitroreductase in the small intestine but not in the cecum [127]. In this study it was also shown that treatment with the antibiotic neomycin significantly inhibited nitroreductase activity in the cecum but not in the small intestine. This suggested the possible presence of nitroreductase in the mucosal epithelium of the small intestine. To confirm this result, another study investigated the effect of lindane pretreatment on five enzymes: nitroreductase, azo reductase, P-glucuronidase. dechlorinase, and dehydrochlorinase [ 1281. Though there was no effect after 2 weeks, there were significant increases in the nitroreductase and azo reductase activities of the small intestine after 5 weeks of treatment. There was still no effect on the cecum enzyme activities after 5 weeks. Coadministration of antibiotics with lindane markedly reduced microbial flora in both the small intestine and the cecum. However, while enzyme activity in the cecum was barely detectable after the antibiotic treatment, that in the small intestine was not significantly reduced. This supported the presence of the five enzymes in the small intestinal mucosa. Recently the activity of the same five enzymes was determined in the small intestine, large intestine, and cecum of conventional and germ-free Fischer 344 rats and similar results were obtained (Fig. 5). In the lindaneparathion work, the nitroreductase of the small intestine appeared to play a role in the detoxification of parathion to aminoparathion. A similar role has been proposed for this enzyme in the biotransformation of the hepatic carcinogen 2,6-dinitrotoluene [ 1301. This work is discussed in the Interactions sect ion. Tumorigenesis in the rat small intestine has been induced by 1,2dimethylhydrazine I131-1341. In addition, various small intestinal tissue preparations have been reported to activate aromatic amines [ 135-138], benzo[a]pyrene [ 1391, aflatoxin B, 11391, cyclophosphamide [ 1391, and tryptophan pyrolysates Trp-P-l and Trp-P-2 [ 140, 1411. More information on the role of the phase I reactions in activating genotoxicants is available in two recent reviews 138, 391. In summary, the low incidence of tumor formation in the small intestine is due to a number of factors including: ( I ) the short transit time for dietary chemicals in this region, (2) high activity of conjugating enzymes in the small intestinal mucosa [35, 99, 1001, (3) relatively high activity of microsomal detoxification pathways [ 142. 1431, (4) the short life span and rapid shedding of mucosal epithelium 1341, and ( 5 ) the activity of the local immune system (341.
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. .
FIG. 5. Enzyme activity of germ-free (top) and conventional Fischer 344 rats (bottom). The enzyme data are mean micrograms of metabolite per gram tissue per hour t SEM from 6 rats. The asterisk represents a response significantly different from conventional animals at p < 0.05.
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D. Large Intestine The human large intestine, consisting of the cecum, ascending colon, transverse colon, rectum, and anus, is the section of the gastrointestinal tract where absorption is completed (Fig. I ) . Some cellulose is partly digested by bacteria and undigested residues are expelled in the feces. The large bowel of humans is about 175 cm long [61], has a surface area of 1300 cm’, and holds an average 220 g of contents 11441. The greatest and most diverse microbial population is found in the large intestine. In people consuming Western diets, with high fat and protein content, bacteria are a major component and constitute about 50% of the fecal solids (661. Of primary importance are the obligately anaerobic, non-spore-forming. gramnegative rods. Members of this group include Bucteroides spp. and Fusobucterium spp. 141, 60, 1451. Numbers of Bacteroides spp. can reach 7.9 log 10 CFU/g contents 1601. Fecal numbers can range from 10.0 to 1 I . 3 log 10 CFU/g 160, 1451. Another major group found in the human colon are the obligately anaerobic, gram-positive non-spore-forming rods. These include Luctobucillus spp. (6.4 log 10 CFU/g contents) and Bijidobucterium spp. 141, 60, 621. Clostridium spp., which are gram-positive, sporeforming rods, compose another predominant group in the large intestine (41, 601. Members of the genus Eubucterium spp.. which are gram-positive anaerobic non-spore-forming rods, are also found in relatively high numbers ( 10.7 CFU/g feces) [145]. Several different facultative and obligately anaerobic gram-positive cocci are found in the large intestine. These include Streptococcus jaeculis and other related Streptococcus spp. [60, 621. Members of the obligately anaerobic family Peptococcaecae include Peptococcus spp., Peptostreptococ.ci~.~ spp.. and Ruminococcus spp. 141. 60, 621. The Enterobacteriaceae are present in relatively low numbers in the large intestine. These microorganisms are characterized by their facultative nature and are gram-negative non-spore-forming rods. Escherichia cofi (6.5 log 10 CFU/g contents) is found in the large intestine 1601. Other genera isolated include Proteus, Klebsiella, and Sulmonellu [60]. The incidence of colon cancer is higher among North American and Western Europeans than among residents of Africa, Asia, and South America 16, 7, 146, 1471. In tact, colon cancer is the second most frequent cancer in the United States. I t has been stated that over 6% of Americans living today are expected to develop colon cancer at some time during their lives and about one half of these people will die from it 11481. Figure 6 presents the 1970-1979 mortality rate from colon cancer in the United States 1911. The data indicated a higher mortality rate for Whites than for non-Whites. Moreover, for unknown reasons, significant death rate from colon cancer in non-Whites seemed to be confined to the eastern half of the
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country. There did not appear to be a sex difference. The American Cancer Society estimated 151,000 new cases of colon-rectum cancer in 1989, which was about 15% of the total cancer cases predicted for the year (771. Mortality from colon-rectum cancer in 1989 was estimated to be 61,300, or 12.2% of the deaths from cancer in the USA (771. Because of the high incidence of colon cancer, considerable attention has been given to its etiology, including a variety of end points to demonstrate the metabolic activation of genotoxicants in the colon: ( I ) covalent binding to DNA in siru, (2) chromosomal abberations in situ, (3) Micronucleus test in siru, (4) sister chromatid exchanges in siru, and ( 5 ) nuclear anomalies in situ [38]. These assays have been described and evaluated in a recent review by Combes (381. Thies and Siegers [39] have succinctly summarized experimental evidence suggesting a role of mucosal enzymes in the activation of intestinal carcinogens as follows: ( I ) Intestinal mucosa is capable of activating procarcinogens to mutagenic metabolites, as evidenced by an increase in the number of revertants in the Salmonella mutagenicity assay, by cell transformation in tissue culture, and by alterations of DNA. Induction of microsoma1 enzymes enhances these effects. (2) Induction of intestinal microsomal enzymes significantly alters the rate of tumor formation in experimental animals. (3) Genetic variations, concerning the rate of tumor formation between strains of the same species, are correlated with different activities of activating and detoxifying enzymes in the target organs from these animals. The activation of genotoxicants such as benzo[a]pyrene, 2-aminoanthracene, 2-aminofluorine, dimethylnitrosamine, I ,2-dimethylhydrazine, N-nitrosobis 3-methylcholanthrene, (2-oxopropyl)amine, 3,2-dimethyl-4-aminobiphenyl, N-nitroso-N-methylurea, bromodeoxyuridine, 2-amino-3,4-dimethylimidazole, and 2-nitro-p-phenylenediaminein colon tissue preparations has been recently reviewed 138). Often the colon tissue preparations were effective only after their cytochrome P-450/448 activity had been induced by (3naphthoflavone or Aroclor. Phenobarbital had no effect. A recent report indicates that (3-naphthoflavone induced cytochrome P4501AI in both the colon and small intestine of rats while phenobarbital induce cytochrome P450IIBI in the small intestine only (1491. Differences in the P-450 isozyme composition can affect the metabolism of potential carcinogenic chemicals and may play a role in the bioactivation of xenobiotics in the colon, a tumor-susceptible tissue. The high incidence of colon cancer in the US population may also be related to the low concentration and reduced diversity of the biotransformation enzymes in this region (149, 1501. Animal studies have demonstrated that the activities of the major phase I1 enzymes in the colon are significantly lower than that in the small intestine. For example, the colon of the rat contains about 3% of the glutathioneS-transferase [ 1041 and 30% of the UDP-glucuronosyltransferase activity
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[IOO]found in the small intestine. More details are provided in recent reviews [73, 1511. Recently low levels and reduced diversity of both phase 1 and phase I1 enzymes have been reported in mucosa from human colons when compared to that in the small intestines of the same individuals [ 1501. A decline in the oral-aboral distribution of glutathione-S-transferase activity in the human intestinal tract was observed, with a sharp fall from the small to the large intestine [ISO]. In the small intestine, class alpha, mu, and pi isoenzymes of glutathione-S-transferasewere detected whereas in the colon, class mu predominated. Similarly, the UDP-glucuronosyltransferase activity of the human colon was barely visible in immunoblot studies with an antibody against UDP-glucuronosyltransferase, whereas two bands were clearly visible in the small intestine. The level of cytochrome P-450 in the colons of all patients was lower than that in the small intestines of the same individuals. Two isozymes of cytochrome P-450 were detected in the small intestine while only one of these was present in the colon. The lower activity and reduced isoenzyme composition of these biotransformation enzymes contribute to a decreased level of detoxification in the colon [ISO]. On the other hand, there is a gradual increase in P-glucuronidase activity toward the lower end of the intestinal tract, with high activity found in the colon [152]. P-Glucuronidase releases many genotoxic metabolites from their nontoxic glucuronides. In summary, low phase I1 detoxification activity, high P-glucuronidase activity, and the reduced isoenzyme composition of the biotransformation enzymes may account for the greater susceptibility of the colon to cancer. E. Effect of pH Gastrointestinal enzyme activities are influenced by pH [37]. In the human, pH increases from
Drug Metabolism Reviews Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/idmr20
Role of the Gastrointestinal Mucosa and Microflora in the Bioactivation of Dietary and Environmental Mutagens or Carcinogens a
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Robert W. Chadwick , S. Elizabeth George & Larry D. Claxton
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USEPA Environmental Research Center Health Effects Research Lab Genetic Toxicology Research Division, Genetic Bioassay Branch, MD-68A, Research Triangle Park, North Carolina, 27711 Published online: 08 Jun 2015.
To cite this article: Robert W. Chadwick, S. Elizabeth George & Larry D. Claxton (1992) Role of the Gastrointestinal Mucosa and Microflora in the Bioactivation of Dietary and Environmental Mutagens or Carcinogens, Drug Metabolism Reviews, 24:4, 425-492 To link to this article: http://dx.doi.org/10.3109/03602539208996302
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DRUG METABOLISM REVIEWS, 24(4), 425-492 (1992)
ROLE OF THE GASTROINTESTINAL MUCOSA AND MICROFLORA IN THE BIOACTIVATION OF DIETARY AND ENVIRONMENTAL MUTAGENS OR CARCINOGENS* ROBERT W. CHADWICK, S. ELIZABETH GEORGE, and LARRY D. CLAXTON USEPA Environmental Reseurch Center Health Effects Research Lab Genetic Toxicology Reseurch Division Genetic Bioassay Brunch, MD-68A Research Triangle Purk, North Carolina 2771 I
I.
INTRODUCTION.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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11.
GENOTOXICITY AND BIOACTIVATION IN THE GASTROINTESTINAL TRACT. . . . . . . . . . . . . . . . . . . . . . A. Esophagus.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Stomach.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Small Intestine.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Large Intestine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
427 431 431 435 439
*The views expressed in this paper are those of the authors and not necessarily those of the Environmental Protection Agency. This paper was refereed by Carl E. Cerniglia. Ph.D., Food and Drug Administration, National Center for Toxicological Research, Jefferson, AR 72079; and by Douglas E. Rickert, Ph.D., Glaxo, Inc., Research Triangle Park, NC 27709. 425 Copyright 0 1992 by Marcel Dekkcr, Inc
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E. Effect of p H . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Intestinal Microflora . . . . . . . . . . . . . . . . . . . . . . . . . . .
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INTERACTIONS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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IV.
SOURCES OF ENVIRONMENTAL MUTAGENS . . . . . . . . . 459 A. Foods and Food Products. . . . . . . . . . . . . . . . . . . . . . . . 462 B. Nonfood Genotoxicants . . . . . . . . . . . . . . . . . . . . . . . . . 467 C. Genotoxicant Exposure . . . . . . . . . . . . . . . . . . . . . . . . . 467
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CONCLUSIONS.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
47 I 472
I. INTRODUCTION From Metchnikoff's contention, in 1901, that intestinal microflora may pose a hazard to human health [I], through epidemiological studies in the 1960s and '70s [2-91, which uncovered the relationship between diet and cancer, to recent research which has implicated enzymes of both intestinal mucosa and microflora in the bioactivation of genotoxicants [ 10-331, evidence has accumulated which supports a significant role for the intestinal tract and its contents in the oncogenic process. Humans are continually exposed to potentially harmful chemicals that gain entrance to the intestinal tract via ingested food, water, or inspired air. It has been estimated that over 53,000 chemicals are used by society as drugs, pesticides, household cleaning products, and food additives 1341. Some of these chemicals (promutagens/procarcinogens) become genotoxic upon metabolic activation. More than 90% of the known carcinogens require further enzymatic transformation to form the ultimate carcinogen [lo, I I , 341. The liver is usually considered to be the major drug-metabolizing organ; however, to be absorbed, ingested chemicals must pass through the intestine, where mucosal enzymes can metabolize them via various pathways [35, 361. In their recent review of mucosal biotransformation, Laitinen and Watkin have concluded that the intestine plays a major role in the metabolism of xenobiotics by all animals 1341. Moreover, metabolism of foreign compounds and nutrients by the gut microflora is extensive and highly diverse [37]. There have been a number of recent reviews on the separate roles of the intestinal mucosa [38, 391 and microflora [40-451 in the bioactivation of genotoxicants. However. the close symbiotic relationship between the microflora and the host suggests that it is preferable to consider the whole ecosystem of the gas-
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trointestinal (GI) tract when assessing its role in genetic toxicology. For example, there have been reports from several laboratories of cooperative metabolic interactions between enzymes of the gut mucosa and those of the microbial flora that influence the bioactivation of genotoxicants [ 18. 27, 46-49]. Other reports indicate that production of some mutagenic metabolites requires activation by intestinal anaerobes together with further metabolic conversion by microsomal preparations from either the liver or the intestinal mucosa [24, 50-521. The bioactivation of aromatic nitro compounds exemplifies a multistep process involving enterohepatic circulation, intestinal microflora, and hepatic enzymes [30, 53). While microflora affect the turnover rate of mucosal cells [54, 551, the environment of the mammalian gut can markedly alter the expression of microfloral enzymes (561. In addition, host secretions such as bile salts and glucuronides can modify the activity of the microflora 1571, and bacterial metabolites of these secretions are frequently suspect carcinogens [43, 581. Enterohepatic circulation, resulting from the hydrolytic activity of the microbial flora, delays the excretion of potential genotoxicants and increases the chance for their bioactivation [ 5 9 ] . Though the nature and extent of these interactions have not been completely resolved, this review attempts to provide a contemporary summary of the complex role of the gastrointestinal tract in genetic toxicology.
11. GENOTOXICITY AND BIOACTIVATION IN THE GASTROINTESTINAL TRACT The gastrointestinal tract has been aptly described as a tube extending from the lips to the anus [601. The average length of the human GI tract is 435 cm and it is divided into I 1 well-defined regions (611. In mammals, the structure and function of these regions reflect the diet and life-style of a given species. The esophagus. stomach, small intestine, and large intestine have received the most attention in cancer research, and their relative contributions to the bioactivation of genotoxicants are evaluated in this review. Figure I depicts the intestinal tracts of the widely used laboratory rodent, which is a monogastric herbivore; and man, an omnivore. A large number of bacteria are found in the gastrointestinal tract of mammals. In fact, it has been stated that because of the gastrointestinal flora, there are more cells in the gastrointestinal tract than elsewhere in the body [62]. The number of microorganisms per gram of colon contents can exceed 10" microorganisms per gram dry weight (63, 641 and can compose 40% to 55% of the total fecal mass [65, 661. Some of the major factors determining composition are
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Parotid gland Mouth
Pharynx
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Rectum Anus
FIG. 1. The gastrointestinal tract of the rodent (left) and man (right). diet, pH, oxygen tension, and stress [41, 60, 67-69]. There are several excellent current reviews that discuss the population and ecology of the intestinal tract [37, 60,62, 701. With the exception of toxicants that are absorbed in the mouth or rectum and those entering the lymphatics via the lacteals, all xenobiotics are available for intestinal first-pass clearance [34]. First-pass clearance involves the biotransformation and clearance of a chemical from the body before it reaches the systemic circulation. Biotransformation reactions are generally subdivided into phase I and phase I1 reactions (Fig. 2). Phase I reactions generally involve alteration of the structure of the parent chemical while the phase I1 reactions typically involve addition of an endogenous substance to
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Genotoxicity
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Xenobiotic Enters Body
FIG. 2. Phase I and phase I1 reactions in the biotransformation of genotoxicants. enhance the aqueous solubility and ultimate excretion of the metabolites formed. Both phase I and phase I1 enzymes produce metabolites which are less lipid soluble and are more easily excreted. However, some phase I reactions also activate procarcinogens to ultimate carcinogens [38, 391 while the phase I1 reactions, with a few exceptions 171, 721, lead to detoxification and presystemic elimination of the metabolites formed [73]. Figure 2 indicates some of the major pathways involved in the bioactivation and/or detoxification of genotoxicants in mammals. This section of the review includes a brief discussion of the structure, oncogenic susceptibility, and bioactivation capacity of various regions of the GI tract. In addition, a description of the alimentary tract flora serves as a brief review of the major microorganisms found in the human GI tract and their relative population numbers (Fig. 3). Table 1 , which describes the predominant fecal flora from human, swine, and rats, is included for interspecies comparison.
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v)
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Stomach
Jejunum Ileum GASTROINTESTINAL TRACT
Colon
FIG. 3. Distribution of microbial flora in the hunman GI tract.
TABLE 1 Comparison of Major Genera Isolated from Human, Swine, and Rat Feces Human"
Swine','
Rat"
Bacteroides' Bifidobacterium Eubacterium Fusobacterium Clostridium Peptostreptococcus Peptococcus Streptococcus
Streptococcus' Lactobacillus Fusobacterium Eubacterium Bacteroides Peptostreptococcus
Bacteroides' Lactobacillus Streptococcus Fusobacterium Bi fidobacteri um
uDraser and Barrow [60];Gorbach [156]. 'Moore et al. [69]. "Swine in order of prevalence. "Benno et al. [68]. 'The most abundant genus is listed first in each column.
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A. Esophagus The human esophagus is an elastic 40 cm long tube 1611 which carries food past the region of the heart and lungs (Fig. I ) . The upper 1/3 of the esophagus consists of skeletal muscle while the lower 213 is composed of smooth muscle. About 5 cm above its junction with the stomach, the muscle is hypertrophied to form the gastroesophageal constrictor. In rodents, the esophagus enters the stomach at the inner curvature through a fold in the limiting ridge that separates the forestomach from the glandular stomach [74]. The fold of the limiting ridge makes it impossible for the rat to vomit. The incidence of esophageal cancer varies greatly among different regions of the world 175, 761. A belt of high risk runs from the Middle East through central Asia to China. Other regions of high risk occur in the eastern and southern areas of Africa and in Normandy and Brittany in France. The American Cancer Society estimated that 10,100 new cases of esophageal cancer would occur in the United States in 1989, which constituted about I % of the total cancer incidence for the year [77]. Mortality from esophageal cancer in 1989 was estimated to be 9,400 or 1.87% of the total deaths from cancer in the USA 1771. Chemicals that induce esophageal neoplasms in the laboratory rat include: a number of methyl-alkylnitrosomines [78, 791 and N.N-dibutylnitrosamine [ 801, Zinc deficiency [81-83] and alcohol 176, 811 appear to enhance esophageal neoplasms. Esophageal S9 from rats [84] and cultured esophageal cells from both rats and man [85, 861 are able to activate benzo(a)pyrene. Cultured human fetal esophagus cells are also able to convert 2-aminofluorene and several nitrosamines into metabolites capable of binding to DNA within the cells (851. Activation in the esophagus was less than that observed for the stomach in this study. It has also been reported that nitrosamines produce micronuclei in tissue lining the esophagus [87]. Micronuclei are chromatin fragments from the breakage of chromosomes forming aggregates of chromatin or from spindle malfunction. They remain unaltered in the cytoplasm after division and are indicative of non-disjunction. The esophagus metabolized N-nitroso-N-methylanilinevia initial denitrosation followed by oxidative demethylation to aniline. The same metabolic pattern was observed with S9 fractions [881. From these reports, it is evident that esophageal enzymes are capable of bioactivating xenobiotics to electrophilic species which can bind to macromolecules and initiate carcinogenesis.
B. Stomach The human stomach is a pouch or reservoir, 38 cm in length 1611, which regulates the entry of food into the small intestine (Fig. I ) . The upper region
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of the stomach (fundus) serves for short-term storage of food, the middle region (cardiac) is where food mixing takes place, and the lower portion (pyloric) ends at the pyloric valve, a circular muscular sphincter at the junction with the small intestine. The stomach wall contains glands that secrete mucin, hydrochloric acid, and enzymes, which aid in the digestion of food. The stomach of the rodent is divided into two very distinct parts, the nonglandular forestomach and the glandular stomach. The forestomach is not present in primates, including man, and thus chemical induction of tumors in this part of the rodent stomach is difficult to evaluate in relation to human risk assessment and will not be considered in this review. Though the acid stomach contents of man are usually sterile, Draser has reported trace quantities of bacteria from the stomach contents of fasting people 1701. The primary microorganisms associated with the human stomach are in the genus Lactobacillus [60].These organisms are gram-positive, facultative or anaerobic, nonsporing rods that can withstand low pH 1891. They range in number from -4 to -2 log 10 colony-forming units per gram of wet weight (O.OOO1 to 0.01 CFU/g) [70]. Another group found in the stomach are the Streptococci 1701. These organisms are facultatively anaerobic, grampositive cocci that are generally found in pairs or chains [89]. They can range in number from -5 to -2 log 10 CFU/g wet tissue 1701. Bacteroidps spp. and members of the family Enterobacteriaceae have also been isolated [701. Veillonella spp., an anaerobic gram-negative coccus, Bifidobacterium spp.. Clostridium spp., and yeasts also have been found [62]. These microorganisms are not found in all individuals [70]. A recent issue of the Journal ojthe National Cancer Institute (December 4, 1991) presents the fourth epidemiological study linking cancer of the stomach and the presence of Helicobac!er pylori 1901. While it remains unclear whether this bacteria actually causes gastric cancer, patients with gastric cancer were much more likely to have antibodies to H . pylori than healthy volunteers or patients with several other types of cancer [90]. While there is a high incidence rate of stomach cancer in Japan, other parts of Asia, and South America, it is low and decreasing in North America and Europe [76]. Figure 4 presents the 1970-1979 mortality cancer map for the United States 1911. The data indicate regional differences and a higher mortality rate for Whites than for non-Whites. The American Cancer Society estimated 20.000 new cases of stomach cancer in 1989, which was about 2% of the total cancer cases for the year (771. Mortality from stomach cancer in 1989 was estimated to be 13,900, or 2.8% of the deaths from cancer in the USA 1771. Gastric tumor formation has been induced in animals by 3-methylcholanthrene 1921, 7,12-dimethylbenz(a)anthracene [93], 2,7-bis(acetylamino)fluorenylene (94. 951, 4-hydroxyaminoquinoline-Ioxide 1961, aflatoxin [97]. N-methyl-N'-nitro-N-nitrosoguanidine 198. 991,
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oinaprazole [IOO],and loxtidine [loll. While there is no direct proof that N-nitroso compounds play a causal role in the etiology of stomach cancer in humans, results of epidemiological studies suggest that they are likely to increase the incidence of some tumors 1102, 1031. The generation of genotoxicants in the stomach is dependent on the activities of the phase I and phase I I reactions which occur in this region (Fig. 2). In the stomach, the activity of the phase I I detoxification pathways is very low. For example, the stomach of the rat contains less than 6% of the glutathione-S-transferase[ 1041 and about one third the glucuronosyltransferase I1051 activity found in the small intestine. Sulfotransferase is also reduced in the stomach (731. Since these reactions provide a major defense against electrophilic and free radical intermediates, which can initiate cancer, the low activity found in the stomach may account for its high susceptibility to carcinogenesis. The link between phase I reactions, which generally involve structural modifications of the parent compound. and the bioactivation of these chemicals in the stomach is less clear. Acute or chronic pretreatment with benzo(a)pyrene significantly increased aryl hydrocarbon hydroxylase activity in homogenated samples of both the forestomach and the glandular stomach of the rat 11061. However, tumors formed only in the forestomach. Another study reported that stomach S9 fractions from Aroclor-pretreated rats were unable to convert benzo(a)pyrene to mutagens [107]. On the other hand, cultured fetal human stomach activated benzo(a)pyrene, aflatoxin B , , and certain N-nitrosamines to metabolites that bound to cellular DNA 186). In fact, the activity of the cultured stomach exceeded that of cultured esophagus and liver in the same study. In other work, attempts to activate dimethylnitrosamine (DMN) using homogenates of mouse or hamster stomachs failed, despite the presence of measurable aryl hydrocarbon hydroxylase, and its induction by phenobarbital, Aroclor, or 3-methylcholanthrene 1108, 1091. However, there was no correlation between DMN demethylase activity and the mutagenicity of DMN in the presence of other organ homogenates, including the liver. Thus it was suggested that DMN demethylase was not the rate-limiting step in the activation of this genotoxicant [IOS]. Another report suggested that the high susceptibility of the glandular gastric mucosa to nitrosamine carcinogens may be related to the influence of reduced glutathione (GSH) on their macromolecular binding, since GSH levels in this region are exceedingly high compared to other portions of the GI tract [ I I O ] . Fish extracts and nitrite-treated fish, from Japan, induce adenocarcinomas of the glandular stomach in rats [ 11 I ] , and nitrosated fish produce large amounts of direct alkylating (methylating) chemicals [ 1121, suggesting that endogenous nitrosamide formation under gastric conditions contributes to gastric cancer in the rat. Recently bacterial formation of N-nitroso compounds was reported
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from the administration of precursors in the rat stomach after omeprazoleinduced achlorhydria I1131. These results are relevant to clinical studies, which have suggested that patients with chronic atrophic gastritis or those having undergone Billroth I 1 gastrectomy I114-1 171 and those treated with antisecretory drugs for duodenal ulcers [ I 181 frequently have gastric achlorhydria and are at higher risk for stomach cancer than healthy subjects. Since nitrosation-proficient bacteria are capable of increasing the intragastric formation of nitrosamines in the stomachs of rats with induced gastric achlorhydria. they could perform the same function in patients with this condition and thus account for their higher risk of stomach cancer. Administration of three glandular stomach carcinogens, N-methyl-N'-nitro-Nnitrosoguanidine, N-propyl-N'-nitro-N-nitrosoguanidine,and 4-nitro-Nmethylurethane resulted in the appearance of unscheduled DNA synthesis (UDS) in the pyloric mucosa [ 1191. The assay is quite specific since administration of two nongastric carcinogens or two forestomach carcinogens [I201 failed to induce UDS in the pylorus. Though there are apparently some discrepancies, reported research generally supports the induction of genotoxicity in the stomach through the generation of reactive metabolites in the stomach walls. Because deficient phase I1 conjugation reactions do not permit adequate detoxification of these reactive intermediates, macromolecular binding and genotoxicity result.
C. Small Intestine The human small intestine is a 150 cm long coiled tube [61] consisting of the duodenum, jejunum, and ileum, and is the principal region for the metabolism and absorption of ingested nutrients and xenobiotics (Fig. I ) . The intestinal mucosa, containing glands and covered with villi, is arranged in folds so that the surface area for digestion and absorption is greatly increased. The flora of the proximal region (duodenum and upper jejunum) of the small intestine resembles that of the stomach. The populations and numbers are similar. However, in the distal region (lower jejunum and ileum), bacterial numbers start to increase 1701. The major populations identified include Lactobacillus spp. ( 5 to 7 log 10 CFU/g wet tissue), Bifidobacterium spp. ( 5 to 7 log 10 CFU/g wet tissue), Bacteroides spp. (2.5 to 7 log CFU/g wet tissue), and Streptococcus spp. (6 log 10 CFU/g wet tissue) 160, 701. Also present are members of the family Enterobacteriaceae (3 to 4 log 10 CFU/g wet tissue) [70]. Clostridium spp., Veillonellu spp., and yeasts have also been isolated [62]. The malignant tumors of the small intestine represent approximately 0. I % of all malignant neoplasms in human pathology and about 3% of the
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malignant neoplasms of the gastrointestinal tract 11211. In 1989 the American Cancer Society estimated 2,700 new cancer cases of the small intestinc (0.27% of the total cancer cases for all sites) from the population of the United States 1771. The mortality from this cancer was estimated to be 900. or 0.18% of the deaths from cancer in the USA for 1989 177). The most common malignant tumors in the small intestine include the adenocarcinomas (50%). the carcinoid tumors (40%). and the connective tissue neoplasms (10%). Though the small intestinal mucosa exhibits higher phase I enzyme activity than any other region of the alimentary canal 1341. the incidence of tumors here only constitutes about 3% of the total occurring in the GI tract. One reason for this apparent discrepancy may be the high activities of the phase I 1 reactions in this region 173, 1041. For example, the activity of glucuronosyl transferase, a phase 11 enzyme, was compared in tissue slices from the stomach, small intestine, cecum, and colon of the rat 11051; and the activities of the stomach, cecum and colon were only 34%, 15%, and 30% of that in the small intestine. Similarly glutathione S-transferase activities in the stomach, colon, and rectum were 5.1%. 3.4%. and 3.4% that of the small intestine 11041. Sulfotransferase is also higher at the proximal than at the aboral end of the intestinal tract in most species 135, 122, 1231. A recent report indicated that cytosolic fractions of the ileum, from 6 patients, catalyzed the sulfation of p-nitrophenol and dopamine at a higher rate than cytosolic fractions from colons of the same patients 11231. The small intestine is the major extrahepatic conjugating organ in many species, even exceeding the activity in the liver in some cases 11241. Since these pathways are a major defense against the genotoxic metabolites generated by phase I reactions in the small intestine, they probably play a significant role in the low susceptibility of this tissue to genotoxicity. Other mechanisms for the detoxification of xenobiotics in the small intestine [125-1281 have been suggested. In one study, an intestinal glutathione transport system has been demonstrated that permits GSH and GSH-S conjugates to be directly eliminated across the brush-border membranes or transported into the lumen to the active site of the y-glutamyltranspeptidase, where they are further metabolized and excreted 11251. In other work, pretreatment of rats with the organochlorine insecticide lindane reduced the estimated absorption rate of parathion from the intestinal tract and the excretion of the hydrolysis product, p-nitrophenol [ 1261. Further investigation showed that the lindane pretreatment stimulated the conversion of parathion, I h after its p.0. administration, to aminoparathion, which is 100-fold less toxic than the parent compound 11291. These results led to speculation that lindane had altered either the nitroreductase activity or the population of the intes-
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tinal microflora. Since nitroreductase is involved in the bioactivation of many aromatic nitro compounds to genotoxic metabolites [30, 531, the relationship between lindane pretreatment and nitroreductase activity was further investigated. Next it was demonstrated that daily p.0. administration of 20 mg/kg lindane for 5 weeks significantly increased nitroreductase in the small intestine but not in the cecum [127]. In this study it was also shown that treatment with the antibiotic neomycin significantly inhibited nitroreductase activity in the cecum but not in the small intestine. This suggested the possible presence of nitroreductase in the mucosal epithelium of the small intestine. To confirm this result, another study investigated the effect of lindane pretreatment on five enzymes: nitroreductase, azo reductase, P-glucuronidase. dechlorinase, and dehydrochlorinase [ 1281. Though there was no effect after 2 weeks, there were significant increases in the nitroreductase and azo reductase activities of the small intestine after 5 weeks of treatment. There was still no effect on the cecum enzyme activities after 5 weeks. Coadministration of antibiotics with lindane markedly reduced microbial flora in both the small intestine and the cecum. However, while enzyme activity in the cecum was barely detectable after the antibiotic treatment, that in the small intestine was not significantly reduced. This supported the presence of the five enzymes in the small intestinal mucosa. Recently the activity of the same five enzymes was determined in the small intestine, large intestine, and cecum of conventional and germ-free Fischer 344 rats and similar results were obtained (Fig. 5). In the lindaneparathion work, the nitroreductase of the small intestine appeared to play a role in the detoxification of parathion to aminoparathion. A similar role has been proposed for this enzyme in the biotransformation of the hepatic carcinogen 2,6-dinitrotoluene [ 1301. This work is discussed in the Interactions sect ion. Tumorigenesis in the rat small intestine has been induced by 1,2dimethylhydrazine I131-1341. In addition, various small intestinal tissue preparations have been reported to activate aromatic amines [ 135-138], benzo[a]pyrene [ 1391, aflatoxin B, 11391, cyclophosphamide [ 1391, and tryptophan pyrolysates Trp-P-l and Trp-P-2 [ 140, 1411. More information on the role of the phase I reactions in activating genotoxicants is available in two recent reviews 138, 391. In summary, the low incidence of tumor formation in the small intestine is due to a number of factors including: ( I ) the short transit time for dietary chemicals in this region, (2) high activity of conjugating enzymes in the small intestinal mucosa [35, 99, 1001, (3) relatively high activity of microsomal detoxification pathways [ 142. 1431, (4) the short life span and rapid shedding of mucosal epithelium 1341, and ( 5 ) the activity of the local immune system (341.
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FIG. 5. Enzyme activity of germ-free (top) and conventional Fischer 344 rats (bottom). The enzyme data are mean micrograms of metabolite per gram tissue per hour t SEM from 6 rats. The asterisk represents a response significantly different from conventional animals at p < 0.05.
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D. Large Intestine The human large intestine, consisting of the cecum, ascending colon, transverse colon, rectum, and anus, is the section of the gastrointestinal tract where absorption is completed (Fig. I ) . Some cellulose is partly digested by bacteria and undigested residues are expelled in the feces. The large bowel of humans is about 175 cm long [61], has a surface area of 1300 cm’, and holds an average 220 g of contents 11441. The greatest and most diverse microbial population is found in the large intestine. In people consuming Western diets, with high fat and protein content, bacteria are a major component and constitute about 50% of the fecal solids (661. Of primary importance are the obligately anaerobic, non-spore-forming. gramnegative rods. Members of this group include Bucteroides spp. and Fusobucterium spp. 141, 60, 1451. Numbers of Bacteroides spp. can reach 7.9 log 10 CFU/g contents 1601. Fecal numbers can range from 10.0 to 1 I . 3 log 10 CFU/g 160, 1451. Another major group found in the human colon are the obligately anaerobic, gram-positive non-spore-forming rods. These include Luctobucillus spp. (6.4 log 10 CFU/g contents) and Bijidobucterium spp. 141, 60, 621. Clostridium spp., which are gram-positive, sporeforming rods, compose another predominant group in the large intestine (41, 601. Members of the genus Eubucterium spp.. which are gram-positive anaerobic non-spore-forming rods, are also found in relatively high numbers ( 10.7 CFU/g feces) [145]. Several different facultative and obligately anaerobic gram-positive cocci are found in the large intestine. These include Streptococcus jaeculis and other related Streptococcus spp. [60, 621. Members of the obligately anaerobic family Peptococcaecae include Peptococcus spp., Peptostreptococ.ci~.~ spp.. and Ruminococcus spp. 141. 60, 621. The Enterobacteriaceae are present in relatively low numbers in the large intestine. These microorganisms are characterized by their facultative nature and are gram-negative non-spore-forming rods. Escherichia cofi (6.5 log 10 CFU/g contents) is found in the large intestine 1601. Other genera isolated include Proteus, Klebsiella, and Sulmonellu [60]. The incidence of colon cancer is higher among North American and Western Europeans than among residents of Africa, Asia, and South America 16, 7, 146, 1471. In tact, colon cancer is the second most frequent cancer in the United States. I t has been stated that over 6% of Americans living today are expected to develop colon cancer at some time during their lives and about one half of these people will die from it 11481. Figure 6 presents the 1970-1979 mortality rate from colon cancer in the United States 1911. The data indicated a higher mortality rate for Whites than for non-Whites. Moreover, for unknown reasons, significant death rate from colon cancer in non-Whites seemed to be confined to the eastern half of the
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country. There did not appear to be a sex difference. The American Cancer Society estimated 151,000 new cases of colon-rectum cancer in 1989, which was about 15% of the total cancer cases predicted for the year (771. Mortality from colon-rectum cancer in 1989 was estimated to be 61,300, or 12.2% of the deaths from cancer in the USA (771. Because of the high incidence of colon cancer, considerable attention has been given to its etiology, including a variety of end points to demonstrate the metabolic activation of genotoxicants in the colon: ( I ) covalent binding to DNA in siru, (2) chromosomal abberations in situ, (3) Micronucleus test in siru, (4) sister chromatid exchanges in siru, and ( 5 ) nuclear anomalies in situ [38]. These assays have been described and evaluated in a recent review by Combes (381. Thies and Siegers [39] have succinctly summarized experimental evidence suggesting a role of mucosal enzymes in the activation of intestinal carcinogens as follows: ( I ) Intestinal mucosa is capable of activating procarcinogens to mutagenic metabolites, as evidenced by an increase in the number of revertants in the Salmonella mutagenicity assay, by cell transformation in tissue culture, and by alterations of DNA. Induction of microsoma1 enzymes enhances these effects. (2) Induction of intestinal microsomal enzymes significantly alters the rate of tumor formation in experimental animals. (3) Genetic variations, concerning the rate of tumor formation between strains of the same species, are correlated with different activities of activating and detoxifying enzymes in the target organs from these animals. The activation of genotoxicants such as benzo[a]pyrene, 2-aminoanthracene, 2-aminofluorine, dimethylnitrosamine, I ,2-dimethylhydrazine, N-nitrosobis 3-methylcholanthrene, (2-oxopropyl)amine, 3,2-dimethyl-4-aminobiphenyl, N-nitroso-N-methylurea, bromodeoxyuridine, 2-amino-3,4-dimethylimidazole, and 2-nitro-p-phenylenediaminein colon tissue preparations has been recently reviewed 138). Often the colon tissue preparations were effective only after their cytochrome P-450/448 activity had been induced by (3naphthoflavone or Aroclor. Phenobarbital had no effect. A recent report indicates that (3-naphthoflavone induced cytochrome P4501AI in both the colon and small intestine of rats while phenobarbital induce cytochrome P450IIBI in the small intestine only (1491. Differences in the P-450 isozyme composition can affect the metabolism of potential carcinogenic chemicals and may play a role in the bioactivation of xenobiotics in the colon, a tumor-susceptible tissue. The high incidence of colon cancer in the US population may also be related to the low concentration and reduced diversity of the biotransformation enzymes in this region (149, 1501. Animal studies have demonstrated that the activities of the major phase I1 enzymes in the colon are significantly lower than that in the small intestine. For example, the colon of the rat contains about 3% of the glutathioneS-transferase [ 1041 and 30% of the UDP-glucuronosyltransferase activity
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[IOO]found in the small intestine. More details are provided in recent reviews [73, 1511. Recently low levels and reduced diversity of both phase 1 and phase I1 enzymes have been reported in mucosa from human colons when compared to that in the small intestines of the same individuals [ 1501. A decline in the oral-aboral distribution of glutathione-S-transferase activity in the human intestinal tract was observed, with a sharp fall from the small to the large intestine [ISO]. In the small intestine, class alpha, mu, and pi isoenzymes of glutathione-S-transferasewere detected whereas in the colon, class mu predominated. Similarly, the UDP-glucuronosyltransferase activity of the human colon was barely visible in immunoblot studies with an antibody against UDP-glucuronosyltransferase, whereas two bands were clearly visible in the small intestine. The level of cytochrome P-450 in the colons of all patients was lower than that in the small intestines of the same individuals. Two isozymes of cytochrome P-450 were detected in the small intestine while only one of these was present in the colon. The lower activity and reduced isoenzyme composition of these biotransformation enzymes contribute to a decreased level of detoxification in the colon [ISO]. On the other hand, there is a gradual increase in P-glucuronidase activity toward the lower end of the intestinal tract, with high activity found in the colon [152]. P-Glucuronidase releases many genotoxic metabolites from their nontoxic glucuronides. In summary, low phase I1 detoxification activity, high P-glucuronidase activity, and the reduced isoenzyme composition of the biotransformation enzymes may account for the greater susceptibility of the colon to cancer. E. Effect of pH Gastrointestinal enzyme activities are influenced by pH [37]. In the human, pH increases from
