benzpyrene hydroxylase activity in the gastrointestinal tract1...benzpyrene hydroxylase activity in...

Benzpyrene Hydroxylase Activity in

the Gastrointestinal Tract1

LEE W. WATTENBERG, J. LIo@L LEONG, AND PETER J. STRAND

(Department of Pathology, University of Minnesota Medical School, Minneapolis, Minnesota)

SUMMARY

Quantitative and morphologic histochemical studies of the activity of the benzpyrene hydroxylase system in the gastrointestinal tract have been carried out. In normal rats activity is found only in the small intestine. In rats in which an increase inactivity is induced by oral administration of 1,@-benzanthracene, activity is foundthroughout the entire gastrointestinal tract but varies in magnitude from one regionto another. The effects of starvation and of a low-fat diet have been studied and werefound to markedly lower the activity of the enzyme system. In addition to the rat,benzpyrene hydroxylase activity has been found to be present in the duodenum ofmouse, guinea pig, rabbit, hamster, dog, monkey, baboon, and man. The possible roleof this type of system as a protective mechanism against polycycic hydrocarbonswhich might reach the gastrointestinal tract is discussed.

There has been described a group of closelyrelated enzyme systems in liver which are capableof metabolizing compounds not normally presentin the animal body (6â€”8).These enzymes occur inthe microsomes@ and require reduced triphosphopynidine nucleotide (TPNH) and oxygen. An increase in the activities of many of these systemshas been shown to be induced by administration ofa number of polycyclic hydrocarbons (1, 6â€”8).Among the compounds acted upon by these systems are many potent carcinogens including thosebelonging to chemical groups such as the polycyclic hydrocarbons (8), the aminoazo dyes (7),the aromatic amines (5), and fluorenyl derivatives(10, 19). Studies of carcinogenesis induced byaminoazo dyes and @-acetylaininofluorene in therat have shown that an enhanced activity ofthe TPNH-dependent microsomal systems hydnoxylating these compounds protects the animalagainst the carcinogen (17, 18).

In the present communication, studies of benzpyrene hydroxylase will be reported. This TPNHdependent microsomal system oxidizes 3,4-benzpyrene (BP) to hydroxy derivatives and subsequently to quinones (8). In the earlier work its

a This study was supported by research grant C-9599 fromthe National Cancer Institute of the National Institutes ofllealth, U.S. Public Health Service.

Received for publication May 28, 1962.

properties and behavior were studied only in theliver (6, 8). Subsequently a morphologic, histochemical procedure has been developed for demonstrating BP hydroxylase in tissue sections, and application of this procedure has revealed the presence of this system in a number of other tissues(@1). In normal rats it has been found to be presentin the kidney, adrenal, and small intestine as wellas the liver. In rats in which a high activity of BPhydroxylase was induced by intraperitoneal injection of 3-methylcholanthrene, this enzyme systemcould also be found in the thyroid, lung, and testis.In rats in which the skin was painted with 3-methylcholanthrene, activity of this system couldbe demonstrated in the skin (@0).

In the present investigation the distribution ofBP hydroxylase activity has been studied in thegastrointestinal tract in normal animals and under several experimental conditions. This studyseemed important to carry out in view of the increasing evidence of human exposure to environmentalcarcinogens and the possibility that polycydie hydrocarbon-hydroxylating systems, of which

BP hydroxylase can be considered a prototype,may represent a protective mechanism againstingested compounds of this type. Morphologic histochemical as well as conventional homogenatetechnics have been employed so as to give information as to the localization of the reaction as well asquantitative data on its activity.

11@20

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WATTENBERG et al.â€”Benzpyrene Hydroxylase Activity 11@1

MATERIALS AND METHODS

Hi@,tochemicalprocedure.â€”Themorphologic histochemical procedure for the demonstration of BPhydnoxylase activity in tissue sections has beendescribed in detail elsewhere (@1). In this procedure, frozen dried sections are stained with BPand then incubated in a buffered reaction mixturecontaining TPNH. The sections are fixed in formalin and then mounted in an aqueous mediumcontaining 60 per cent glycerol and 1 N NaOH.By means of fluorescence microscopy, sites of BPhydroxylase activity are identified in tissue seetions by the presence of the hydroxy derivatives,which have a bright green fluorescence in contrastto the unaltered BP which maintains a violetfluorescence under these conditions.

Quantitative procedure.â€”Aquantitative adaptation of the histochemical procedure has been developed employing reaction conditions similar tothose described by Conney, Miller, and Miller forquantitating this system by benzpyrene destruction as manifested by a decrease in the violetfluorescence of this compound (8). In the procedure employed in the present work mucosa wasscraped from the bowel wall with a sharp scalpelblade. A @.5per cent homogenate of this materialwas made in cold (0Â°â€”@Â°C.) isotonic KC1 with aPotter-Elvehjem type of homogenizer with a Teflon pestle. Two ml. of homogenate was pipettedinto a cold (4Â°C.) @5-ml.Erlenmeyer flask eontaming 50 big. of BP in 0.1 ml. of acetone. One milliliten of reaction mixture was added last to initiatethe reaction. It contained the following: TPNH(1 mg.) ; DPNH (0.5 mg.) ; 0.06 M nicotinamide;0.05 M KC1; 0.08 M NaH2PO4.Na2HPO4 buffer atpH 7.4. The flasks were incubated aerobically at370 C. in a covered water bath equipped with a

mechanical shaker. The reaction was stopped bythe rapid addition of 3.0 ml. of cold acetone andthe flasks placed in an iced bath. To this mixturewas then added 9.0 ml. of petroleum ether (Skellysolve B, b.p. 66Â°â€”68Â°C.). The flasks were placedin a covered water bath at 37Â°C. for 10 minutes,which facilitates quantitative extraction of themetabolite into the 10.O-ml. organic solvent phase.After storage in the dark at 4Â°C. for @4â€”48hours,an aliquot (0.5â€”1.0 ml.) of the solvent was removed and extracted with 10.0 ml. of 1 N NaOH.The fluorescence of the aqueous extract was determined in a Farrand photoelectric fluorometer,model A, equipped with a primary filter whichtransmits light maximally at 400 mM and a secondany interference filter with a peak wavelength at

@ and a half band width of 14 mi. A quininesulfate solution (0.3 @g/mlof 0.1 N H2S04) was usedas a fluorescent standard. A specimen of pure 8-

hydroxy-3,4-benzpyrene (8-OH-BP) has been usedto standardize the procedure. The fluorescence ofthis compound is linear in the range assayed (0.001

@gâ€”0.0@@g/ml of 1 N NaOH). All samples wereread within 1â€”5minutes after the alkaline aqueousextracts were obtained. This is necessary, since aslow decrease in intensity of the fluorescence oecurs with time. The fluorescence spectra of thepure sample of 8-OH-BP in 1 N NaOH and thealkaline aqueous extract from our procedure applied to the mucosa of the small intestine and liverwere recorded in an Aminco-Bowman spectrophotofluorometer. The fluorescence spectra werealmost identical, all showing a single maximum at5@0 mi. This was true for highly reactive ratduodenum from animals fed 1,@,-benzanthraceneand liver from rats injected intrapenitoneally withmethylcholanthnene as well as less reactive duodenal and hepatic specimens from control rats. Noeffort has been made to identifly the specific hydroxy derivatives produced by the BP-hydroxylase reaction of the duodenum, but instead theyhave been quantitated as a group. Previously reported studies of this reaction in rat liver homogenates have shown that the reaction productsconsist almost entirely of a mixture of hydroxyderivatives of BP of which the most abundant is8-OH-BP. A small amount of quinone is also produced, but the quinone derivatives of BP do notfluoresce (3, 4). Since the fluorescence spectra ofthe reaction products so closely resemble that of8-OH-BP, a unit of BP-hydroxylase activity hasbeen defined as the activity which will result in anincrease in fluorescence at 5@ m@ equivalent tothat of 1 @qig.of 8-hydroxy-BP per mg. wet weightof tissue per minute. No correction is required forunmodified BP, since under the conditions employed the final alkaline extract shows no fluorescence at 5@ m@ifrom this material.

The activity obtained at 5 minutes was routinely employed in making calculations except in thecase of weakly reactive tissues in which 10 and insome instances @0minutes were used. Tissues reported as negative did not show demonstrable activity after a 1-hour incubation. Under the conditions used there was a linear relationship betweentissue concentration and enzyme activity over therange of tissue concentrations tested, which wasbetween [email protected] and 50 mg. wet weight of tissue inthe reaction mixture.

Animals.â€”Most of the animal experimentationwas done on female Sprague-Dawley rats of 10weeks of age. The source of supply of rodents wasas follows : Sprague-Dawley rats, Holtzman Cornpany, Madison, Wisconsin; Syrian hamsters, Lakeview Hamster Colony, Newfield, New Jersey;



Cancer Research Vol. @,October 196@11@

white Swiss mice, Taconic Farms, Germantown,New York; guinea pigs, Gopher State Caviary, St.Paul, Minnesota; albino rabbits, Oak Crest Rabbitry, Glen Lake, Minnesota. A specimen of baboon duodenum was obtained from Dr. ClaudeHitchcock during an operative procedure on thisanimal; a specimen of duodenum from a 1-yearold female rhesus monkey was obtained immediately after sacrifice of this animal by cerebraltrauma. The dogs employed were female mongrelanimals obtained from the Animal Rescue League;the duodenum was removed immediately followingexsanguination. Specimens of human duodenum

DPNH (Pabst), and sesame oil U.S.P. grade(Magnus, Maybee and Reynard) were used without further purification. 1,@-Benzanthracene (Tenra Chemicals, Inc.) was decolorized with activatedcharcoal, then recrystallized from ethanol. Ahighly purified preparation of 8-hydroxybenzpyrene was generously made available to us byDr. Kenneth H. Harper of the Huntingdon Research Center, Huntingdon, England. The fatfree diet employed was obtained from the Nutnitional Biochemicals Corp., Cleveland, Ohio. Allinorganic salts and organic solvents were Mallinckrodt, AR grade.

Sequential Segments of Small Intestine Cecum ColonRight Lefthalf half

is[S@ untreated Rots

(I)

C

>5

>

U

â€˜1,0>5

05-

V

I

a)Ca)>s154Ca)

II Rots fed 1,2 Benzanthrocene

I

@nk@Un@

StomachFore-@

60 port port

50@

40

30

20

I0

I-, I I I I I

Segment of Rat Gastrointestinal Tract

CHART 1.â€”Distribution of benzpynene hydroxylase activity along the gastrointestinal tract of untreated rats and rats given

20 mg. of 1,2-benzanthracene by mouth 36 hours prior to being sacrificed. The small intestine has been divided into seven segmentsso that differences in activity in sequential portions of the structure may be depicted.

and other human tissues were obtained as soon asthese were excised during the course of surgicalprocedures. One specimen of duodenum was removed incident to the excision of an aortic aneurysm in a 57-year-old male with no gastrointestinalpathology. A second specimen was obtained froma 45-year-old male who was undergoing surgery fora recurrent gastric ulcer. The third specimen wasfrom a 7@-year-old male undergoing surgery for aduodenal ulcer. In all instances the duodenal tissueemployed appeared normal on both gross and micnoscopic examination.

Chemicale.â€”3,4-Benzpyrene (Terra ChemicalsInc.) was purified chromatographically followingthe procedure of Miller (16). TPNH (Boehringen),

RESULTSQuantitative studies; BP-hydroxylase activity in

the small intestine of the rat.â€”Quantitative studiesof the BP-hydroxylase activity in the small intestine of the female rat showed that a moderate activity was present throughout much of the lengthof this structure except for the distal portion, inwhich it was extremely low (Chart 1). Activity hasnot been demonstrated elsewhere in the gastrointestinal tract. In rats given 4 ml. of sesame oilcontaining 1,@-benzanthracene (5 mg/nil by stomach tube) and sacrificed 36 hours later, a profoundincrease in activity was found throughout thesmall intestine. Activity could also be demonstrated in the stomach and the large intestine



ExperimentalgroupSexNo. animalsBenzpyrene

hydroxylaseactivity

(units)Untreated

Untreated1,2-Benzanthracenet in

sesame oilSesame oilsFat-free dietStarvationâ€”72 hoursFemale

Male

FemaleFemaleFemaleFemale12

6

666612.0Â±

4.016 .5 Â±2.0

69.5Â±12.110 . 1 Â± 1.00 .7 Â± 0.10 .9 Â± 0.5

WATTENBERG et al.â€”Benzpyrene Hydroxylase Activity 11@S

(Chart 1). In the stomach, in the small intestine,and in the large intestine, activity was notablylower in the distal portion of these structures thanin the proximal portion.

Effects of various experimental regimens on theBP-hydroxylase activity of the rat duodenum.â€”Starvation for 7@hours markedly reduced the BPhydroxylase activity of the duodenum, as can beseen in Table 1. A second procedure which hasbeen found to markedly decrease the BP-hydroxylase activity of the duodenum is feeding of a fatfree diet. After 8 days on such a diet very littleBP-hydroxylase activity remained (Table 1). Theenzyme system in the duodenum seems particularly sensitive to these effects, since studies of otherorgans in which BP-hydroxylase activity occurs,such as liver, kidney, and adrenal, show no significant alteration under these conditions. Data onthe magnitude of the increase in BP-hydroxylaseactivity following administration of 1,@-benzanthracene are also presented in Table 1, along withthose for control animals fed sesame oil, which wasused as the vehicle for this polycyclic hydnocarbon. The level of activity in the duodenum of thenormal male rat was similar to that of the female,Table 1.

BP-hydroxylase activity in species other than therat.â€”Studies have been carried out to determinewhether the duodenum of species other than therat have BP-hydroxylase activity. All the speciesincluded showed activity, but the magnitude varied quite widely (Table @).Of all the specimensstudied, those for man had the lowest activity. Information as to the extent to which the preoperative and operative procedures as well as the diseased states of these individuals result in an alteration of activity from a normal level is not availableat present. In man, the stomach and colon havealso been tested for BP-hydroxylase activity, andnone has been found.

Morphologic hi@tochemicd studies of the distribution ofBP-hydroxylase in the gastrointestinal tract ofthe rat.â€”In the normal rat a positive reaction canbe observed in the small intestine, where it is foundin the epithelial cells covering the viii. Cells in theglandular erypts, the stroma, and muscle are negative. The intensity of the activity is not uniformthroughout the length of the small intestine, butcan be perceived only in approximately the proximal half of this structure. Even in the proximal portion of the small intestine, the reaction isnot intense, and in some specimens it cannot beseen at all. Activity has not been observed in theesophagus, stomach, cecum, or the large intestine.

In rats given 1,@-benzanthracene by mouth andsacrificed 36 hours later, a high activity of the BP

hydroxylase activity in the proximal portion of thesmall intestine was seen. In addition, activity became perceptible in portions of the gastrointestinaltract in which a positive reaction could not befound in normal animals. A positive reaction couldbe observed in the forestomach. The activity waslimited to the squamous epithelium and was uniform throughout the forestomach except in itsmost distal portion, where the reaction becameweaker. In the squamous epithelium immediatelyadjacent to the glandular portion of the stomachno activity was apparent. The entire glandularportion of the stomach gave a negative reaction.The duodenal mucosa exhibited an intense reac

TABLE 1

EFFECT OF DIETARY FACTORS AND 1,2-BENZANTHRACENEON THE BENZPYRENE HYDROXYLASE AcTivITY

OF THE DUODENASL MUCOSA OF THE RAT

a One unit is equivalent to the fluorescence of 1 @g.of8-hydroxy.BP formed/mg wet weight of tissue/minute. MeanÂ±standarddeviation.

t 4ml.ofsesameoilcontaining1,2-benzanthracene(5mg/ml) given via stomach tube 56 hours prior to sacrifice.

@ 4 ml. of sesame oil given via stomach tube 36 hours priorto sacrifice.

tion (Fig. 1). As in the normal animal, the positivereaction was confined to the epithelium coveringthe villi. This intense activity persisted throughoutmost of the length of the small intestine but diminished progressively in the distal portion of theileum. In the cecum and right half of the colonweak activity could be seen in the surface epithehum. No activity was observed in the glandularcnypts. In the more distal portions of the large intestine a positive reaction has not been observed.

A comparison of the results of the morphologichistochemical procedure for demonstrating BPhydnoxylase activity with the quantitative oneclearly showed that the quantitative method wasmore sensitive. This was evident from the fact thatstructures such as the glandular stomach and lefthalf of the colon of rats fed 1,@-benzanthracenegave a negative reaction with the histochemicalprocedure but did have definite activity whichcould be quantitated.



AnimalBenzpyrene hydroxylase activity(units*)Mouse

GuineapigRabbitHamsterDogMonkeyBaboonMan2.0,5.0

1.5,2.52.0,1.31.7,1.32.3,4.013.43.00.3,0.3,0.2

Cancer Research11f24 Vol. @,October 196@2

Histochemical procedures in which perylenewas used as a substrate (@1) gave very similar results to those with BP. With both BP and peryleneas substrates, enhanced activity in the small intestine and stomach has been obtained in experiments in which 3-methylcholanthrene, 3,4-benzpyrene, or 1,@,5,6-dibenzanthracene have been administered by stomach tube as well as those inwhich 1,@-benzanthracene was given. 1,@-Benzanthracene has been employed to induce increasesin BP-hydroxylase activity in most of the work,since this compound is effective and also presentsa minimum carcinogenic hazard.

TABLE 2

BENZPYRENE IIYDROXYLASE ACTIVITY

IN THE DIJODENAL MUC0SA OF

V@uuousANIMALSPECIES

tion it is barely detectable; most of the small intestine has a very high activity except for its distalsegment, and the cecum and proximal half of thelarge intestine has a moderate activity while theleft half of this structure including the rectum hasa low activity.

The presence of polycyclic hydrocarbon-hydroxylating systems in the gastrointestinal tractmay represent a mechanism whereby the animalis protected against the carcinogenic effects ofingested polycyclic hydrocarbons, since it has beenshown for BP that its hydroxy derivatives are considerably less carcinogenic than the parent compound (4, 9). Such a mechanism would protect notonly the gastrointestinal tract, but along withliver and other organs which have BP-hydroxylaseactivity would serve in having a more general protective effect, since it has been shown that ingested polycyclie hydrocarbon can produce malignant neoplasms at distant sites such as the breast(13). A protective mechanism of this type in the

gastrointestinal tract might be particularly important for man, since there is substantial evidencethat the human may ingest benzpyrene as well asother polycyclic hydrocarbons by virtue of theirformation during the cooking or smoking of foods(@,11, 1@2,14, 15). In addition to ingested material,some polycyclic hydrocarbon which is inhaledprobably reaches the gastrointestinal tract in swallowed respiratory secretions which contain thesecompounds.

The results of the present study indicate thatthe level of BP-hydroxylase activity in gastrointestinal mucosa may be varied by diet. Starvationor a low-fat diet decreases the activity, whereasingestion of polycyclic hydrocarbons induces anincrease in activity. If the excess of unaltered carcinogenic polycyclic hydrocarbon over that whichis hydroxylated represents the main carcinogenichazard, the amount of unaltered material woulddepend not only on the dose of these compoundstaken into the gastrointestinal tract but also thehydroxylase activities of the sequential segmentsof the gastrointestinal tract. This latter in turnwould depend on the intrinsic ability of the panticular segment to form this enzyme and the vanous dietary stimuli which modify this intrinsic

a Each value represents an individualanimal.

DISCUSSION

The results of the present study have shownthat in normal rats BP-hydroxylase activity ispresent in the small intestine and is either absentor of extremely low magnitude in other segmentsof the gastrointestinal tract. In animals whichhave received 1,92-benzanthracene by stomachtube, activity can be demonstrated throughoutthe entire gastrointestinal tract but shows considerable variation from one region to another. Inaddition, in each of the anatomic subdivisions ofthe gastrointestinal tract the activity is markedlyhigher in the proximal part than in the distal one.Thus the squamous portion of the stomach shows amoderate activity, whereas in the glandular por

FIG. 1.â€”Duodenum from a rat fed 1,2-benzanthracene.

Benzpyrene hydroxylase procedure. A positive reaction ispresent in the cytoplasm of epithelial cells covering the viffiand appears as a bright fluorescence. Other histologic elementsin the section are visualized by virtue of a weaker fluorescence

of unaltered benzpyrene. X150.



S

5@

I



WATTENBERG et al.â€”Benzpyrene Hydroxylase Activity l1@5

level. In considering the types of hazard frompolycyclic hydrocarbons in terms of the ability ofthe gastrointestinal tract to detoxify these compounds, the maximum one would be sporadic highdoses of carcinogen with a dietary regimen givingminimum stimulation to the induction of polycyclic hydrocarbon hydroxylases. In contrast, aconsiderably more favorable situation would existwhere frequent lower doses are taken and wherethe diet stimulates a high hydroxylase formation.Under these conditions there would be present notonly a relatively high level of activity to beginwith, but this would be further enhanced by theadditional inducing effect of the low doses of polycyclic hydrocarbon.

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1962;22:1120-1125. Cancer Res Lee W. Wattenberg, J. Lionel Leong and Peter J. Strand Benzpyrene Hydroxylase Activity in the Gastrointestinal Tract

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