Gut, 1991,32,408-412

Biotransformation enzymes in human intestine:critical low levels in the colon?

W H M Peters, L Kock, F M Nagengast, P G Kremers

AbstractBiotransformation or drug-metabolisingenzymes have an important function in thedetoxication of ingested toxic, carcinogenic,or tumour promoting compounds. Enzymeactivity and isoenzyme composition of threebiotransformation systems: glutathione S-transferase, uridine diphosphate-glucurono-syltransferase, and cytochrome P-450 werestudied in normal small and large intestinalmucosa from three kidney donors. The activityof most drug-metabolising enzymes decreasesslightly from proximal to distal small intestine,whereas in the mucosa of the large intestine asharp fall in activity was observed. The iso-enzyme composition for each of the threebiotransformation systems changed from thesmall to the large intestine. Class Alpha gluta-thione S-transferases were not expressed in thecolon, in contrast to the small intestine whereboth Alpha and Pi class isoenzymes arepresent. In addition, with monoclonal anti-bodies fewer protein bands for UDP-glucu-ronosyltransferases and cytochrome P-450were detected in the colon. In the small intes-tine both isoforms P-4504 and P-450, werepresent, whereas in the colon only reducedamounts of cytochrome P-4504 could be visual-ised. For UDP-glucuronosyltransferase, 53and 54 kDa proteins could be detected in thesmall intestine, but in the colon there was onlyweak staining ofthe 54 kDa band. In the normalhuman colon enzymes are less active andthere are fewer isoenzymes present in themucosa than in the small intestine. Thisimplies a lower level of the detoxifying poten-tial in the colon, which might be important inregard to the high rates ofcarcinogenesis in thecolon.

Division ofGastrointestinal andLiver Diseases, StRadboud Hospital,Nijmegen, TheNetherlandsW H M PetersL KockF M Nagengast

Laboratoire de ChimieMedicale, Institut dePathologie, Universite deLiege, BelgiumP G KremersCorrespondence to:Dr W H M Peters, Division ofGastrointestinal and LiverDiseases, St RadboudHospital, PO Box 9101, 6500HB Nijmegen, TheNetherlands.Accepted for publication26 June 1990

Biotransformation is the sum of all chemicalreactions that alter the nature, water solubility,and distribution of non-nutritive compoundsthat are potentially toxic or carcinogenic. Suchcompounds may enter the body as food com-ponents, food additives, or drugs. The gastro-intestinal tract often is the route of entry ofsuch harmful molecules and the gastrointestinalmucosa can be very active in biotransformation.Data on human intestinal biotransformationenzymes are, however, relatively scarce.'2 Thepresence of both phase IP- and phase II51"6biotransformation enzymes has been shown.Compared with the liver, however, the specificactivity of these enzymes in small intestinalmucosa may be lower, apart from glutathioneS-transferase.'"From the small to the large intestine the

activity of glutathione S-transferase seems to

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Figure 1: Longitudinal distribution of intestinalbiotransformation activities. Activities for cytosolicglutathione S-transferase (GST) microsomal 4-nitrophenoluridine diphosphate-glucuronosyltransferase (UDPGT), andbilirubin UDPGT were determined as indicated underMethods. Enzyme assays were performed in mucosa obtainedfrom segments ofthe proximal (duodenum), mid (jejunum),and distal (ileum) small intestine. In addition, three colonicsegmentsfrom proximal (caecum), mid (transverse colon),and distal (sigmoid colon) large intestine were investigated.Activities were determined in triplicatefor each subject andare given as means (SEM)for three subjects. Small intestinalvalues arefrom four subjects since some data from Peterset al' are included. nd=not detectable.

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Biotransformation enzymes in human intestine: Critical low levels in the colon?

1 2 3 4 5 6:.1..I..: 1 3.. 4 5Figure 2: Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) ofpurified intestinal glutathioneS-transferases. Glutathione-agarose purified glutathione S-transferases were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (10% acrylamide, wlv) and subsequently stained with Coomassie brilliant blue. Purifiedintestinal glutathione S-transferases from patient 1 (5-10 ig protein) are shown in panel A, slots 1-6. Purified transferasesfrom patient 2 (8-12 Rgprotein) are shown in panel A, slots 7-12, and thosefrom patient 3 (3-10 Rg protein) are shown inpanel B, slots 1-4. In panelB slot 5 purified hepatic glutathione S-transferases (5 tg protein) from patient 3 were applied.Preparations shown arefrom duodenum (A, I and 7; B, 1), jejunum (A, 2 and 8), ileum (A, 3 and 9; B, 2), caecum (A, 4and 10; B, 3), transverse colon (A, 5 and 11), and sigmoid colon (A, 6 and 12; B, 4). Glutathione S-transferase Pi subunitsare indicated by arrow.

decrease."" The isoenzyme composition fromcytochromes P-45047 as well as glutathioneS-transferases'4 may also be different in largebowel mucosa. Most available data, however,come from analysing separate specimens fromdifferent subjects. Therefore, the possibility can-not be excluded that some of the differences aredue solely to interindividual variations.We have investigated the longitudinal distri-

bution and isoenzyme composition of phase I(cytochrome P-450) and phase II drug-metabol-isilng enzymes (uridine diphosphate-glucurono-syltransferases, glutathione S-transferases) innormal small and large intestinal mucosa fromthree kidney donors. Significant differences inisoenzyme composition and activity were foundbetween the two parts of the intestine.

Methods

TISSUEHuman intestines were obtained from threekidney donors. Donor 1 was a female aged 3-5

months, who died after a head injury (traumacapitis). Donor 2 was an 18 year old man whodied from cerebral damage after a subdural/subarachnoid haemorrhage. Donor 3 was a 49year old man who died after a head injury. Dataon small intestinal biotransformation activitiesobtained from another male kidney donor (an 18year old5) are also incorporated in Figures 1and 4. None of the donors had gastrointestinaldisorders.

After removal, intestine was stored on iceduring transport to the laboratory. The intestinewas opened and cleaned by repeated washingswith ice cold 0-9% NaCl. Tissue was cut intosegments, frozen in liquid nitrogen, and stored at-80°C until use.From segments (length 20-40 cm; area 70-

200 cm2), taken at intervals of 60-120 cm,mucosa was isolated. Mucosa was homogenisedand cytosolic" and microsomal fractions weremade as described previously.41'The investigations were approved by the

local ethical committee on human experimenta-tion.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 1516 1718 19 1. 2 3 4 5Figure 3: Immunodetection ofintestinal glutathione S-transferase-Pi. Human intestinal cytosol (75 RLg protein) was subjectedto sodium dodecyl sulphate-polyacrylamide gel electrophoresis and subsequent Western blotting to nitrocellulose. GST-Pi wasimmunadetected with a monoclonal antibody against GST-Pifrom human placenta.2 Cytosols from proximal to distal smallintestine (panel A, slots 1-6, patient 1; A, 10-15, patient 2; B, 2 and 3, patient 3) andfrom proximal to distal large intestine(A, 7-9, patient 1; A, 16-18, patient 2; B, 4 and 5, patient 3) are shown respectively. In panelA slot 19 and panelB slot Ipurified glutathione S-transferase Pifrom human placenta (0.5 ptg protein) was applied to the gels. The lowest band visible inpanelB slot 2 may be an additional acidic glutathione S-transferase-Pi isoform.

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Peters, Kock, Nagengast, Kremers

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Figure 4: Immunodetection of intestinal UDP-glucuronosyltransferases. Intestinalmicrosomes from patients 2 and 3 were subjected to sodium dodecyl sulphate-polyacrylamidegel electrophoresis (7% acrylamide; w/v) and subsequent Western blotting. The blot wasincubated with a monoclonal antibody against human UDP-glucuronosyltransferases. InpanelA small intestinal microsomesfrom patient 2 (20 pg protein) originating at distances of0, 100, 200, 340, 460, and 610 cmfrom the pylorus were applied in slots 1-6, respectively. Inslots 7-9 large intestinal microsomes (40 [g protein) from the caecum and transverse andsigmoid colon are shown. Slot 10 contains 4 kg hepatic microsomesfrom another patient. InpanelB microsomesfrom liver (15 ig protein), duodenum (50 Rg), ileum (50 Rg), caecum (100Rg), and sigmoid colon (100 kg)from patient 3 are shown in the slots 1-5, respectively.

ENZYME DETERMINATIONSCystosolic glutathione S-transferase activity with1-chloro 2,4-dinitrobenzene as substrate wasmeasured by the method of Habig et al.`7 UDP-glucuronosyltransferase activity with 4-nitro-phenol and bilirubin as substrates was measuredin microsomes and whole homogenate, respec-tively, in the presence of 0-0125% TritonX- 100. 18-20

MISCELLANEOUSGlutathione S-transferases were purified asdescribed previously.'4 Protein was determinedby the method of Lowry et al.2' Sodium dodecylsulphate-polyacrylamide gel electrophoresis wasdone according to Laemmli.22 After staining with

Coomassie brilliant blue, gels were scanned at600 nm with a laser densitometer (LKB 2202Ultrascan, LKB, Bromma Sweden). Immuno-blotting with monoclonal antibodies againstcytochrome P-450,4 UDP-glucuronosyltrans-ferase,'3 and glutathione S-transferases2'24 wasperformed as described previously.'6

ResultsThe intestines from the kidney donors weredivided in segments of approximately 20 cm.Mucosa was isolated from segments taken at theindicated locations (see figure legends). Mucosawas homogenised and subcellular fractionsmade.

In the cytosolic fractions specific activity ofglutathione S-transferase was determined (Fig1). Activities are highest in the proximal smallintestine and show a sharp fall from small to largeintestine.The longitudinal distribution of microsomal

UDP-glucuronosyltransferases with bilirubinand 4-nitrophenol as substrates is also shown inFigure 1. In the small intestine activity of4-nitrophenol UDP-glucuronosyltransferase ismore or less constant, whereas the conjugation ofbilirubin is decreasing. In the three subjectsinvestigated a dramatic fall in activity for 4-nitro-phenol as well as for bilirubin UDP-glucu-ronosyltransferase in the large intestine wasobserved.

Small and large intestinal glutathione S-trans-ferases were purified by affinity chromatographyon glutathione-agarose. These purified prepara-tions are shown in Figure 2. Glutathione Stransferases from small intestine are composed ofboth class Alpha (25 kDa subunits) and class Pi(24 kDa subunits) isoenzymes. In purified gluta-thione S-transferase preparations from colonicmucosa, after Coomassie brilliant blue stainingonly class Pi transferases were present. With amonoclonal antibody developed against gluta-thione S-transferase Pi from human placenta23

1 2 3 4 5 6 7 8 9 101112t 13 14 t 16 17 18 19 1 2 3 4

Figure 5: Immunodetection ofintestinal cytochromes P-45056. Intestinal microsomesfrom patient 1 (panel A, slots 2-10),patient 2 (A, 11-19), and patient 3 (B, 1-4) were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (7%acrylamide, w/v) and Western blotting and subsequently incubated with a monoclonal antibody against cytochromes P450,,.Hepatic microsomes (8 Rg protein) are seen in panelA slot 1, microsomesfrom proximal to distal small intestine (30 pog protein)are in panel A, slots 2-7 and 11-16, panel B, slots 1 and 2. Microsomesfrom proximal to distal large intestine (60 ptg protein)were applied in panel A, slots 8-10 and 17-19, panel B, slots 3 and 4. In slot 13, for unknown reasons, little protein hasmoved into the gel. The 54 kDa band ofcytochrome P450J is indicated by arrow. Other cytochrome P450 related proteinbands seen have molecular masses of52 kDa (liver and intestine) and 56 kDa (liver).

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Biotransformation enzymes in human intestine: Critical low levels in the colon?

.~~~~~~~~ ~ ~ ~ ~ ~ ~ ~~~~~~~~~~~~.3ta

Figure 6: Immunodetection of intestinal cytochrome P-450. Intestinal microsomes from patient 1 (panel A, slots 1-9), patient2 (A, 10-18), and patient 3 (B, 2-5) were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (7%acrylamide, w/v) and subsequent Western blotting. Cytochrome P.4505 was detected with a monoclonal antibody. Hepaticmicrosomesfrom patient 3 (4 Rg protein) are seen in panelB slot 1; microsomesfrom proximal to distal small intestine (20 tgprotein) are seen in panel A, slots 1-6 and 10-15, panel B, slots 2 and 3. Microsomes from proximal to distal large intestine(40 [tg protein) were applied in panel A, slots 7-9 and 16-18, panel B, slots 4 and 5. The band representing cytochromeP450 has a molecular mass of52 kDa.

this isoenzyme was indeed readily shown incytosolic fractions from both small and largeintestine (Fig 3).By immunodetection, using a monoclonal

antibody against class Mu glutathione S-trans-ferases,24 small amounts of this isoform weredetected in all intestinal segments from twopatients (not shown). In the intestine frompatient 1 this isoenzyme was absent. Hepaticglutathione S-transferases from patient 3 are alsoshown in Figure 2. Here only class Alphaisoforms and no class Pi subunits are present.On a Western blot, small intestinal micro-

somes from patients 2 and 3, incubated with amonoclonal antibody against UDP-glucuronosyltransferase, show two very closebands. In the corresponding colonic micro-somes, even when twice as much protein isapplied to the gel, only a very weak protein bandis visible (Fig 4). Intestinal microsomes frompatient 1 gave only very weak bands in the smallintestine and no staining at all in the colon (notshown).A Western blot of small intestinal microsomes,

treated with a monoclonal antibody against cyto-chromes P-4504,5,6, shows two bands of 52 and 54kDa (Fig 5). In the proximal small intestine frompatient 1, the 54 kDa band is very weak (Fig 5,panel A). In colonic microsomes the 52 kDa bandis not detectable. Figure 6 shows that immuno-detection of cytochrome P-4505 is possible onlyin small intestinal, but not in colonic, micro-somes from all subjects. It should be noted thathere again for the large intestinal samples twice asmuch protein was applied to the gels.

DiscussionLongitudinal distribution of the biotransforma-tion enzyme activities measured show a decline inactivity, with a sharp fall from the small to thelarge intestine. For glutathione S-transferases,such a large decrease in activity in colonic mucosawas noted before in specimens obtained frompatients with carcinoma of the colon and rec-tum'3 i4 and in rodents.225 The intestines investi-gated here were from patients with no intestinaldisease. Thus the relatively low colonic gluta-thione S-transferase activities are not restricted

to patients with malignancies of the largebowel.23

Glutathione S-transferase subunit compositionis different in small and large intestinal mucosa.The small intestine contains class Alpha, Mu,and Pi isoenzymes, whereas in the colon predomi-nantly class Pi subunits can be detected. ClassMu subunits are present in very small amountsand c4n hardly be seen after Coomassie brilliantblue staining of the sodium dodecyl sulphate gelshown in Figure 2. Thus in contrast to otherreports2627 we found class Pi subunits almostexclusively, both in normal and neoplastic23colonic tissue. Our results confirm the recentimmunohistochemical findings of Hayes et a128giving a similar isoenzyme distribution in smalland large intestine.The presence of bilirubin UDP-glucuronosyl-

transferase activity in the small intestines from allsubjects investigated once again confirms thatbilirubin metabolism is not restricted to theliver.5 12 29 30 Moreover, specific activities of hepa-tic and small intestinal bilirubin UDP-glucu-ronosyltransferase do not differ dramatically(mean (SEM) 704 (42) and 343 (22) nmol/gtissue/h, respectively; data are from patient 3).Similar data obtained from another patient havebeen reported before.'Immunoblot studies with an antibody against

UDP-glucuronosyltransferase show two closebands in the small intestine, whereas in colonicmucosa both bands were hardly visible. The twosmall intestinal bands (53 and 54 kDa) maycorrespond to activities for bilirubin (53 kDa)and 4-nitrophenol (54 kDa).5 Both activities aredrastically reduced in the colon, and here theamount of UDP-glucuronosyltransferase proteinmay be below the detection limit for immuno-blots.For isoenzymes of the cytochrome P-450 bio-

transformation system a similar case exists.Isoform P-450, is detectable in the small intestinebut not in the colon. Another isoenzyme, pro-bably P-4504, is present in both colonic and smallintestinal mucosa. Since more large intestinalmiciosomes were loaded onto the gel it may beconcluded from the immunoblot in Figure 5 thatthe levels of P-4504 in the colon of all patients arelower compared with those in the small intestine.

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412 Peters, Kock, Nagengast, Kremers

The above experimental data on biotransfor-mation enzyme activities in the intestines ofhealthy kidney donors show a constant ordecreasing activity along the small intestine and asignificant fall in activity in the colon. For allthree enzyme systems investigated here thenumber of isoenzymes in the colon is reduced.This pattern of both reduced enzyme activitiesand isoenzyme composition undoubtedly willresult in a lower level ofdetoxication in the colon.Activities of biotransformation enzymes in thecolon may further decrease with increasingage.'4 3' In addition, levels of j3-glucuronidase arehighest in the colon.32 This could result in lesstoxic glucuronides becoming toxic again bydeconjugation. Indeed, high concentrations ofmutagens have been detected in the humancolon.33 In conclusion, the detoxicating capacityin the human colon could be at a critical low levelwhich may contribute to the high rate of coloniccancer.

1 Laitinen M, Watkins JB. Mucosal biotransformation. In:Rozman K, Hanninen 0, eds. Gastrointestinal toxicology.Amsterdam: Elsevier, 1986: 169-92.

2 Intestinal metabolism of xenobiotics. Koster AS, Richter E,Lauterbach F, Hartmann F, eds. Progress in Pharmacologyand Clinical Pharmacology 1989; 7(2).

3 Hoensch HP, Steinhardt HJ, Weiss G, Haug D, Maier A,Malchow H. Effects of semisynthetic diets on xenobioticmetabolizing enzyme activity and morphology of smallintestinal mucosa in humans. Gastroenterology 1984; 86:1519-30.

4 Peters WHM, Kremers PG. Cytochromes P-450 in the intes-tinal mucosa of man. Biochem Pharmacol 1989; 38: 1535-8.

5 Peters WHM, Nagengast FM, van Tongeren JHM. Gluta-thione S-transferase, cytochrome P-450 and UDP-glucu-ronosyltransferase in human small intestine and liver.Gastroenterology 1989; %: 783-9.

6 Watkins PB, Wrighton SA, Schuetz EG, Molowa DT,Guzelian PS. Identification of glucocorticoid-inducible cyto-chromes P-450 in the intestinal mucosa of rats and man.J Clin Invest 1987; 80: 1029-36.

7 Strobel HW, Newaz SN, Fang WF, et al. Evidence for thepresence and reactivity of multiple forms of cytochromeP-450 in colonic microsomes from rats and humans. In:Rydstrom J, Montelius J, Bengtsson M, eds. Extrahepaticdrug metabolism and chemical carcinogenesis. Amsterdam:Elsevier Science, 1983: 57-66.

8 Autrup H, Harris CC, Trump BF, Jeffrey AM. Metabolism ofbenzo(a)pyrene and identification of the major benzo(a)-pyrene-DNA adducts in cultured human colon. Cancer Res1978; 28: 3689-96.

9 Mayhew JW, Lombardi PE, Fawaz K, et al. Increasedoxidation of a chemical carcinogen, benzo(a)pyrene, bycolon tissue biopsy specimens from patients with ulcerativecolitis. Gastroenterology 1983; 85: 328-34.

10 Mekhail-Ishak K, Hudson N, Tsao MS, Batist G. Implicationsfor therapy of drug-metabolizing enzymes in human coloncancer. CancerRes 1989; 49: 4866-9.

11 Peters WHM. Isolation and partial characterization of humanintestinal glutathione S-transferases. Biochem Pharmacol1988; 37: 2288-91.

12 Hartmann F and Bissell DM. Metabolism of heme and

bilirubin in rat and human small intestinal mucosa. J ClinInvest 1982; 70: 23-7.

13 Siegers CP, Bose-Younes H, Thies E, Hoppenkamps R,Younes M. Glutathione and GSH-dependent enzymes intumorous and nontumorous mucosa of the human colon andrectum. J Cancer Res Clin Oncol 1984; 107: 238-41.

14 Peters WHM, Roelofs HMJ, Nagengast FM, van TongerenJHM. Human intestinal glutathione S-transferases. BiochemJ 1989; 257: 471-6.

15 Peters WHM, Allebes WA, Jansen PLM, Poels LG, CapelPJA. Characterization and tissue specificity of a monoclonalantibody against human UDP-glucuronosyltransferase.Gastroenterology 1987; 93: 162-9.

16 Peters WHM, Jansen PLM. Immunocharacterization of UDPglucuronyltransferase isoenzymes in human liver, intestineand kidney. BiochemPharnacol 1988; 37: 564-7.

17 Habig WH, Pabst MJ, Jakoby WB. Glutathione S-trans-ferases. The first enzymatic step in mercapturic acid forma-tion.I Biol Chem 1974; 249: 7130-6.

18 Peters WHM, Jansen PLM, Nauta H. The molecular weightsof UDP-glucuronosyltransferase determined with radiation-inactivation analysis. A molecular model of bilirubin UDP-glucuronosyltransferase. J Biol Chem 1984; 259: 11701-5.

19 Peters WHM, Jansen PLM, Cuypers HTM, de Abreu RA,Nauta H. Deconjugation ofglucuronides catalyzed by UDP-glucuronyltransferase. Biochim Biophys Acta 1986; 873:252-9.

20 Peters WHM, Jansen PLM. Microsomal UDP-glucuro-nyltransferase-catalyzed bilirubin diglucuronide formationin human liver.JHepatol 1986; 2: 182-94.

21 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Proteinmeasurement with the Folin Phenol reagent. J Biol Chem1951; 193:265-75.

22 Laemmli UK. Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature 1970; 227:680-5.

23 Peters WHM, Nagengast FM, Wobbes T. GlutathioneS-transferases in normal and cancerous human colon tissue.Carcinogenesis 1989; 10: 2371-4.

24 Peters WHM, Kock L, Nagengast FM, Roelofs HMJ.Immunodetection with a monoclonal antibody of gluta-thione S-transferase Mu in patients with and withoutcarcinomas. Biochem Pharmacol 1990; 39: 591-7.

25 Siegers CP, Riemann D, Thies E, Younes M. Glutathione andGSH-dependent enzymes in the gastrointestinal mucosa ofthe rat. CancerLett 1988; 40: 71-6.

26 Kodate C, Fukushi A, Narita T, Kudo H, Soma Y, Sato K.Human placental form of glutathione S-transferase a newimmunohistochemical marker for human colonic carcinoma.JpnJ Cancer Res 1986; 77: 226-9.

27 Clapper ML, Hoffman SJ, Tew KD. Characterization ofglutathione S-transferases in human colon carcinomas. Pro-ceedings ofthe American Association for Cancer Research 1988;29: 290.

28 Hayes PC, Harrison DJ, Bouchier IAD, McLellan LI, HayesJD. Cytosolic and microsomal glutathione S-transferaseisoenzymes in normal human liver and intestinal epithelium.Gut 1989; 30:854-9.

29 Anand BS, Narang APS, Dilawari JB, Koshy A, Datta DV.Bilirubin glucuronyltransferase activity in the human gastricand duodenal mucosa. IndianJ Med Res 1980; 71: 109-11.

30 Chowdhury RJ, Wolkoff AW, Arias IM. Heme and bilepigment metabolism. In: Arias IM, Popper H, Schachter D,Schafritz DA, eds. The liver, biology and pathobiology. NewYork: Raven Press, 1982: 309-32.

31 McMahon TF, Beierschmitt WP, Weiner M. Changes in phaseI and phase II biotransformation with age in male Fisher 344rat colon: relationship to colon carcinogenesis. Cancer Let1987; 36:273-82.

32 Koster AS, Frankhuyzen-Sierevogel AC, Noordhoek J. Distri-bution of glucuronidation capacity (1-naphtol andmorphine) along the rat intestine. Biochem Pharnacol 1985;34: 3527-32.

33 Ichinotsubo D, Rice S, Abe C, et al. Mutagens formed inmucosal tissue of the human gastrointestinal tract. In: StichHF, ed. Carcinogens and mutagens in the environment. Vol II.Boca Raton FL: CRC Press, 1983: 27-40.

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