[CANCER RESEARCH 41, 1407-1412, April 1981]0008-5472/81 /0041-OOOOS02.00

Control by Gastrointestinal Hormones of the Hydroxylation of theCarcinogen Benzo(a)pyrene and Other Xenobiotics in Rat Colon1

Wan Fen Fang and Henry W. Strobel2

Department of Biochemistry and Molecular Biology, The University of Texas Medical School at Houston, Houston, Texas 77025

ABSTRACT

The cytochrome P-450-dependent drug metabolism system

activities of rat colon microsomes are increased by gastrointestinal hormones. Pentagastrin, secretin, and cholecystokininoctapeptide increase the specific content of cytochrome P-450over control and stimulate /V-demethylation, O-dealkylation,

and polycyclic aromatic hydrocarbon hydroxylation to differentdegrees. Increase in hydroxylation activity by pentagastrin isdose dependent with a stimulation of 250% of control occurringat a dose level of 250 fig/kg body weight. The increase inhydroxylation activity is prevented by cycloheximide treatmentshowing dependence on protein synthesis. Pentagastrin andcholecystokinin increase the specific content of colon micro-somal P-450 77%, while secretin increases cytochrome P-450content 138% of the control value. Pentagastrin induces a 4-

fold increase in ethylmorphine demethylation, while the majoreffect by secretin is on p-nitrophenetole dealkylation activity(2-fold increase), and the major effects of cholecystokinin areon benzphetamine and benzo(a)pyrene hydroxylation (2-fold

induction). These hormones induce the hydroxylation of othersubstrates also, but the pattern of effects varies with thehormone used. Liver microsomal hydroxylation activities arealso increased significantly by pretreatment with these hormones, although to a lesser degree than colon hydroxylationactivities. Several tissue substances induce the hydroxylationof specific substrates, although no pattern of inductive effectsis evident. For example, reserpine pretreatment doubles therate of benzo(a)pyrene hydroxylation in liver and colon micro-somes but decreases or has no effect on other hydroxylationactivities in liver microsomes and induces only ethylmorphinedealkylation in colon microsomes. On the other hand, 16,16-dimethylprostaglandin E2 induces benzphetamine hydroxylation in the colon but only benzo(a)pyrene hydroxylation in theliver. The gastrointestinal hormones induce drug metabolismwith an apparent pattern of specificities and an increase incytochrome P-450 specific content not seen in animals pre-

treated with the tissue substances tested.

INTRODUCTION

The metabolism of xenobiotics such as benzo(a)pyrene toproducts of altered pharmacological potency occurs in manyorgans and tissues including the liver (20, 21 ), kidney (6), smallintestine (2, 27, 32, 35), and lung (1, 29). Lu ef al. (20) have

1This investigation was supported by Grant CAI9105 awarded through the

National Large Bowel Cancer Project of the National Cancer Institute, Departmentof Health, Education, and Welfare. A preliminary report of part of this investigationhas been presented (9, 33).

1 Recipient of Research Career Development Award 1-K04-CA00311 from

the National Cancer Institute, Department of Health, Education, and Welfare. Towhom requests for reprints should be addressed.

Received October 7, 1980; accepted January 12, 1981.

shown that the substrate specificity for the drug metabolismsystem rests with the cytochrome component using benzo-(a)pyrene as a test substrate, and several laboratories (12-14,

30) have established that the cytochrome component hasmultiple forms with distinct but overlapping substrate preference. In addition, Kawalak and Lu (19) have shown that analogous cytochromes purified from different species (i.e., cytochrome P-448 isolated from rat and liver) can also have quite

different catalytic activities. For instance, these authors (19)reported that rat liver cytochrome P-448 hydroxylated

benzo(a)pyrene at a rate 10 times higher than that of rabbitliver cytochrome P-448. Using various forms of the cytochrome

from the same animal and organ, Wood ef al. (38) and Wislockief al. (37) have shown that various forms of the cytochromehydroxylate benzo(a)pyrene and that this activation ofbenzo(a)pyrene is associated with a marked increase in carci-

nogenicity and mutagenicity.Furthermore, various tissues within the same species hy

droxylate substrates such as benzo(a)pyrene with differingefficiencies and turnover numbers (2, 19, 27, 32, 35). Fangand Strobel have shown that benzo(a)pyrene is hydroxylatedby the rat colon (9) and, more specifically, that benzo(a)pyreneis activated to mutagenic products by the colon (8). Moreover,the hydroxylation and activation of benzo(a)pyrene by thecolon, like those actions by the liver, are markedly responsiveto exogenous inducers (e.g., /8-naphthoflavone). The present

study examines the regulation of both benzo(a)pyrene hydroxylation and the metabolism of other drugs by endogenoushormones and tissue substances. Specifically, this report presents the first evidence that the peptide gastrointestinal hormones modulate the rate of drug metabolism in the colon.

MATERIALS AND METHODS

Animals. Male Sprague-Dawley rats weighing 150 to 200 g

were obtained from Flow Laboratories, Dublin, Va., for thisstudy. The rats were housed in wire-bottomed cages and

maintained on laboratory chow and water ad libitum throughoutthe course of the study. The gastrointestinal hormones andtissue substances were administered to experimental animalsonce or twice daily for each of 3 consecutive days, and animalswere sacrificed between 8 and 9 a.m. on the fourth day unlessotherwise stated. Animals were fasted overnight before sacrifice in order to aid in the collection of colon microsomes. Theovernight fast did not affect colon microsomal drug metabolismactivities. Each of the hormones was dissolved in 0.9% NaCIsolution for injection, although some of the hormones requiredspecial procedures for preparation of stable solutions. Pentagastrin solutions for injection were prepared fresh each day.To prepare the pentagastrin stock solution, pentagastrin wasbrought into solution in a small amount of 0.9% NaCI solutionby adding 0.1 N NH4OH dropwise until all the pentagastrin

APRIL 1981 1407

Research. on January 13, 2019. © 1981 American Association for Cancercancerres.aacrjournals.org Downloaded from

W. F. Fang and H. W. Strobel

dissolved. The solution was titrated back to pH 7.5 with 0.1 NHCI. The stock solution was further diluted with 0.9% NaCIsolution before i.p. injection. Pentagastrin-treated rats received

one daily injection of 250 fig pentagastrin per kg body weightfor 3 days unless otherwise stated. Secretin was dissolved in0.9% NaCI solution containing cysteine HCI, 1 mg/ml, as areducing agent. Secretin-treated rats received one daily s.c.

injection of 75 units of secretin (27 jug) per kg body weight foreach of 3 days. Cholecystokinin octapeptide was prepared asa concentrated stock solution of 0.5 mg/ml in 0.5 N NaHCO3and diluted into 0.9% NaCI solution before use. Cholecystoki-nin-treated rats received one daily s.c. injection of 20 ftgCholecystokinin per kg body weight for each of 3 days. Cyclo-

heximide was dissolved with 0.9% NaCI solution.In a similar fashion, the tissue substances used in this study

were also dissolved or diluted in 0.9% NaCI solution. 16,16-Dimethylprostaglandin E2 was dissolved in ethanol and dilutedto volume with 0.9% NaCI solution. Prostaglandin-treated ratsreceived 2 daily i.p. injections of 0.4 mg of 16,16-dimethyl-

prostaglandin E2 per kg body weight for each of 3 days.Serotonin (5-hydroxytryptamine) was dissolved in water anddiluted in 0.9% NaCI solution. Serotonin-treated rats received

2 daily i.p. injections of 25 mg of serotonin per kg body weightfor each of 3 days. 5-Hydroxy-L-tryptophan was dissolved in

0.01% ascorbic acid and 0.1 N HCI as a stock solution anddiluted into 0.9% NaCI solution. 5-Hydroxy-L-tryptophan-treated rats received 2 daily i.p. injections of 50 mg of 5-hydroxy-L-tryptophan per kg body weight for each of 3 days.Reserpine, a plant alkaloid, was dissolved with acetic acid anddiluted in 0.9% NaCI solution. Reserpine-treated rats received

one daily i.p. injection of 25 mg of reserpine per kg bodyweight for each of 3 days. All animals were sacrificed on thefourth day, and livers and colons were removed. Microsomeswere prepared from these tissues as described previously (5,9).

Assays. The rate of hydroxylation (N-demethylation) of benz-

phetamine and ethylmorphine by colon microsomes was measured as described previously (21) by formaldehyde liberation,according to the method of Nash (23) as modified by Cochinand Axelrod (3). Reaction mixtures contained 150 /nmol potassium phosphate buffer (pH 7.7), microsomal protein, and waterin a total volume of 1.5 ml. The reaction was initiated by theaddition of 0.15 /imol NADPH, and mixtures were incubated at30°for 10 min. The rate of hydroxylation (O-dealkylation) of p-

nitroanisole and p-nitrophenetole was estimated in similar reaction mixtures as the rate of formation of the product p-

nitrophenol according to the method of Netter and Seidel (25).The hydroxylation of benzo(a)pyrene was measured in 1.0-ml

reaction mixtures containing 100 /umol potassium phosphatebuffer (pH 7.7), 80 nmol benzo(a)pyrene, and 0.15 /¿molNADPH by fluorometric estimation of the 3-hydroxy and other

products according to the methods of Nebert and Gelboin (24),using an excitation wavelength of 386 nm and an emissionwavelength of 515 nm.

Cytochrome P-450-specific content was measured from thedithionite-reduced carbon monoxide difference spectrum ac

cording to the method of Omura and Sato (26), using anextinction coefficient of 91 cm'1 mvT1. NADPH-cytochrome P-

450 (cytochrome c) reducíase activity was measured by amodification (5) of the method of Phillips and Langdon (28) at30°in 1.0-ml reaction mixtures containing 300 /umol potassium

phosphate buffer (pH 7.7) as the rate of reduction of cytochrome c, using an extinction coefficient of 21 cm"1 rtiM"1 at

550 nm (36).Materials. Horse heart cytochrome c, cycloheximide, sero

tonin, 5-hydroxy-i_-tryptophan, and reserpine were purchasedfrom Sigma Chemical Co., St. Louis, Mo. Benzo[a]pyrene, p-nitroanisole, and p-nitrophenetole were obtained from Eastman

Kodak Co., Rochester, N. Y. Benzphetamine hydrochloridewas a gift from Dr. J. W. Hinman of the Upjohn Co., Kalamazoo,Mich. Ethylmorphine was obtained from Merck & Co., Inc.,Rahway, N. J. Pentagastrin was a gift from Ayerst Laboratories,New York, N. Y. Secretin and NADPH were purchased fromBoehringer-Mannheim Biochemicals, Indianapolis, Ind. Chole

cystokinin octapeptide was a gift from Dr. L. Lichtenberger ofDepartment of Physiology, University of Texas Medical Schoolat Houston. 16,16-Dimethylprostaglandin E2 was the gift of Dr.

J. E. Pike of Upjohn. All other chemicals were reagent grade orbetter.

RESULTS

Induction of Colon Microsomal Hydroxylation Activities byPentagastrin. In order to ascertain whether the mixed-function

oxidase system responds to gastrointestinal hormones, weutilized information on structure function relationships of thesehormones and the availability of synthetic polypeptide hormones. The structure, physiological properties, and actions ofthe major gastrointestinal hormones, gastrin, secretin, andCholecystokinin, have recently been reviewed by Grossman(11). Furthermore, Johnson (16) has shown that the syntheticgastrointestinal hormone pentagastrin exerts a trophic effecton the colon mucosa by stimulating colonie mucosa! DNAsynthesis leading to a rapid increase in mucosal DNA contentand, hence, cell number. As shown in Chart 1, pentagastrinpretreatment of rats brings about a marked increase in thehydroxylation rate of the carcinogen benzo(a)pyrene by colon

> 300

IoP 250 -

X00o =200-

u*

III£ 150 H

oN

100-

0 SO 125 250 375 500

PENTAGASTRIN, pg/kg

Chart 1. Effect of increasing concentrations of pentagastrin on benzo-(a)pyrene hydroxylation activity of rat colon microsomes. A minimum of 10 ratswas used for each point. Rats were given injections of the appropriate dose ofpentagastrin for each 3 days and sacrificed the day after the last injection. Colonmicrosomes were prepared and assayed for benzo(a)pyrene hydroxylation activity. Control levels of benzo(a)pyrene hydroxylation activity are shown in Table 2.

1408 CANCER RESEARCH VOL. 41

Research. on January 13, 2019. © 1981 American Association for Cancercancerres.aacrjournals.org Downloaded from

Benzo(a)pyrene Hydroxylation and Gastrointestinal Hormones

mucosal microsomes from treated rats in a dose-dependent

fashion. Maximal stimulation of benzo(a)pyrene hydroxylationactivity (160% above control) was achieved with doses between 250 and 375 /¿gof pentagastrin per kg body weight,which was in good agreement with the amount of pentagastrinreported to stimulate maximal DNA synthesis in the coloniemucosa (18).

As shown in Table 1, the hydroxylation of benzo(a)pyreneand a variety of drugs is stimulated by pentagastrin pretreatment but to different degrees. The rate of hydroxylation ofsome drugs, especially ethylmorphine, is stimulated after onlya single treatment with pentagastrin. Highest activities wereobserved after 3 days of treatment. The hydroxylation rates ofethylmorphine and benzo(a)pyrene were most responsive topentagastrin pretreatment. Colonie cytochrome P-450 contentwas almost doubled and cytochrome P-450 reducíase activitywas increased by pentagastrin pretreatment.

Having shown the dosage and time dependence of the pentagastrin induction of mucosal drug metabolism system activities, it was important to determine whether pentagastrin induction involves the synthesis of new enzyme protein as hasalready been shown for phénobarbital induction of the liverdrug metabolism system (7) and for the emergence of drugmetabolism activities in neonatal hepatocytes (4). The inhibitorof protein synthesis, cycloheximide, was used to examine thispossibility. As shown in Table 2, cycloheximide injected 45 minbefore pentagastrin for 3 days completely prevented the induction of drug metabolism in the colon by pentagastrin as judgedby the elimination of pentagastrin-dependent increases overthe untreated controls in cytochrome P-450 content, cytochrome c reducíaseactivity, or the rate of hydroxylation of anyof a variety of substrates. These data suggest a role for proteinsynthesis in induction of the colonie drug metabolism systemby pentagastrin.

Induction of Hydroxylation Activities by Cholecystokininand Secretin. The data in the experiments described aboveshow that pentagastrin, a pentapeptide hormone containingthe minimum amino acid sequence required for the physiological activities of gastrin (11), markedly increases drug metabolism activities in the colon through a process involving proteinsynthesis. Of the other major gastrointestinal hormones, cho-lecystokinin has an almost identical COOH-terminal sequenceto that of gastrin. The terminal pentapeptide is the same in the2 hormones. Secretin, on the other hand, has more sequence

Table 1

Induction of coton microsomal drug metabolism system activities bypentagastrin

Rats received injections of pentagastrin, (250 /ig/kg/day) for 1. 2, or 3 daysand were sacrificed 16 hr after the last injection. Microsomes were preparedfrom the colonie mucosa and assayed for the catalytic activity using varioussubstrates. The data are reported as percentage of increase in activity above thecontrol treated only with 0.9% NaCI solution. Control levels are given in Table 2.

% of increase over control

Table 2

Effect of cycloheximide on induction of colon microsomal drug metabolismactivities by pentagastrin

Animals received either a single i.p. injection of pentagastrin (250 g/kg) for 3days or 2 i.p. injections of cycloheximide (1 mg/kg) plus one injection ofpentagastrin for 3 days. Control rats received vehicle only. The animals weresacrificed, and the colon microsomes were assayed as described.

HydroxylationactivitiesBenzphetamineEthylmorphinep-Nitroanisolep-NitrophenetoleBenzo(a)pyreneSystem

componentsCytochromecreducíaseCytochromeP-450 contentDay

102860050Day21313436138220Day3172786522832977

ActivitiesBenzphetamine"Ethylmorphine3p-Nitroanisolep-Nitrophenetole6Benzo(a)pyrenecSystem

componentsCytochromecreducíaseCytochromeP-4506Control0.480.351.421.043.5026.713Cyclo

heximide0.470.520.080.0318.6Pentagastrin0.561.322.341.276.4034.423Cyclo

heximide+penta

gastrin0.270.260.720.622.2021.76

nmol formaldehyde liberated per min per mg.' nmol p-nitrophenol liberated per min per mg.' pmol 3-hydroxybenzo(a)pyrene produced per min per mg.

^ nmol cytochrome c reduced per min per mg.' pmol cytochrome P-450 per mg.

homology with pancreatic glucagon than with gastrin. Thus, itwas important to compare the actions of these gastrointestinalhormones with those of pentagastrin. As shown in Table 3,both secretin and Cholecystokinin octapeptide have markedeffects on the drug metabolism activities of the colon. Cytochrome P-450-specific content was increased 2.5-fold by secretin and 1.5-fold by Cholecystokinin. With the exception ofits effects on the rates of O-dealkylation, Cholecystokinin affects the colon drug metabolism activities in the same directionas does pentagastrin. Cholecystokinin stimulates benzpheta-mine hydroxylation a little more and ethylmorphine hydroxylation a little less than does pentagastrin. The main point ofdifference, however, between these highly similar polypeptidesis the marked lowering of O-dealkylation activity after treatment

with Cholecystokinin, whereas pentagastrin pretreatment elicitsa modest increase in O-dealkylation of p-nitroanisole and p-

nitrophenetole. Secretin, which is known to inhibit the physiological effects of gastrin (31), stimulates the colon microsomalhydroxylation of most substrates, with the exception of benz-phetamine and ethylmorphine. Moreover, the stimulatory effectof secretin on O-dealkylation is greater than that of pentagas

trin.The effect of the gastrointestinal hormones on hepatic drug

metabolism system activities is shown in Table 4. In general,some of the effects of these hormones on the liver systemparallel their effects on the colon system, while others do not.O-Dealkylation activity in the liver is not significantly affected

by any gastrointestinal hormone. Pentagastrin does not significantly alter ethylmorphine metabolism in liver, and Cholecystokinin does not alter benzphetamine metabolism. Quantitatively, however, the liver responds to these hormones less wellin terms of the percentage of increase than does the colon.Nonetheless, the control liver activities are higher than theinduced colon activities.

Effects of Tissue Substances on Colon and Liver Microsomal Drug Metabolism System Activities. Pentagastrin, secretin, and Cholecystokinin show some pronounced specificityin the hydroxylation activities that they preferentially induceinter se. They also seem to affect liver and colon systems

APRIL 1981 1409

Research. on January 13, 2019. © 1981 American Association for Cancercancerres.aacrjournals.org Downloaded from

W. F. Fang and H. W. Strobel

differentially. In order to examine further this possible specificity, we tested several tissue substances for their effects ondrug metabolism activity. (Tissue substances, like hormones,have a broad range of effects but are produced by severaltissues.) We chose 5-hydroxytryptamine (serotonin) and itsprecursor 5-hydroxytryptophan, 16,16-dimethylprostaglandin

E2 (a stable prostaglandin derivative), and reserpine (a plantalkaloid) for this study and determined the appropriate dosages. The effects of these tissue substances on colon drug

metabolism activity are shown in Table 5, and the effects onliver drug metabolism activity are shown in Table 6. None ofthe tissue substances induces O-dealkylation in either the liver

or colon systems. In fact, the lowering of activity in pretreatedanimals is statistically significant in most cases. Reserpinepretreatment significantly stimulates colonie ethylmorphine de-methylation. 16,16-Dimethylprostaglandin E2, on the otherhand, markedly stimulates colonie benzphetamine hydroxyl-ation (2.5-fold) but inhibits liver benzphetamine hydroxylation.

Table 3

Induction of colon drug metabolism activities by gastrointestinal hormones

Animals received injections of a single dose of secretin (27 ^g/kg). cholecystokinin octapeptide (20 /ig/kg), or pentagastrin (250 fig/kg) for 3 days as described in Table 1. Microsomal preparations of the liverand colon were assayed for various drug metabolism activities.

HydroxylationactivitiesBenzphetamine3Ethylmorphine3p-Nitroanisolep-NitropheneloledBenzo(a)pyreneSystem

componentsCytochromeP-450content9Cytochromec reductasehControl0.48

±0.02o0.35

±0.031.42±0.101.04±0.173.5±0.713

±2.026.6±2.8Pentagastrin0.56

±0.031.32±0.15C2.34

±0.421.27±0.166.4±0.50C23.0

±2C34.4

±2.2Secretin0.19

±0.04C0.32

±0.021.94±0.282.26±0.03°6.0±0.4C31.0

±3°33.8

±1.9Cholecystokinin0.74

±0.04°0.45

±0.050.39±0.03C0.54±0.05C7.0±0.4e21

±2C28.6

±1.2

nmol formaldehyde liberated per min per mg." Average ±S.D. of at least 3 separate determinations of duplicate assay mixture.c Significantly changed from control group by unpaired i test for at least 3 separate determinations at p

< 0.05." nmol p-nitrophenol liberated per min per mg.8 pmol 3-hydroxybenzo(a)pyrene produced per min per mg.

pmol/mg of colon microsomal protein; nmol/mg of liver microsomal protein.9 nmol Cytochrome c reduced per min per mg.

Table 4

Induction of liver drug metabolism activities by gastrointestinal hormones

Experimental details are described in Table 3. All activities are expressed in units described in Table 3 except for theCytochrome P-450 content which is reported as nmol/mg.

HydroxylationactivitiesBenzphetamine

Ethylmorphinep-Nitroanisolep-NitrophenetoleBenzo(a)pyreneSystem

componentsCytochrome P-450 contentCytochrome c reducíaseControl5.28

± 0.25a

6.34 ± 0.563.50 ± 0.663.36 ± 0.21

130.0 ±10.00.76

± 0.05153.4 ±22.9Pentagastrin6.49

± 0.41fl

8.61 ± 0.772.05 ± 0.163.87 ± 0.46

320.0 ±40.0°0.91

± 0.04276.6 ±24.6oSecretin5.06

± 0.1169.66 ± 0.84°

2.96 ± 0.133.37 ± 0.27

248.02 ±21.83d0.97

± 0.03154.6 ± 5.3Cholecystokinin5.51

± 0.42a

7.36 ± 0.821.89± 0.23d

4.0 ± 0.82370.0 ±30.Oc0.83

± 0.06236.3 ±21

" Average ±S.D. as in Table 3.b Averages of 2 determinations of 5 animals each ±variation from the average.c Significant at p < 0.01.'' Significantly changed from control group by unpaired f test for at least 3 separate determinations at p < 0.05.

Table 5

Effects of tissue substances on colon drug metabolism systemAnimals were pretreated with 16,16-dimethylprostaglandin E2,serotonin, 5-hydroxy-L-tryptophan, or reserpine, and the colon microsomes

were assayed for drug metabolism activities. Experimental details are described in Table 1. All activities are expressed in units described forthe colon system in Table 3.

HydroxylationactivitiesBenzphetamine

Ethylmorphinep-Nitroanisolep-NitrophenetoleBenzo(a)pyreneSystem

componentsCytochrome P-450 contentCytochrome c reducíaseControl0.48

±0.02s

0.35 ±0.031.42 ±0.101.04 ±0.173.50 ±0.7013

±225.7 ±2.8Dimethyl-prosta-

glandinE.1.18±0.166

0.37 ±0.060.24 ±0.05C

0.66 ±0.065.0 ±0.8017

±234.8 ±2.2Serotonin0.39

±0.030.44 ±0.0760.27 ±0.17C0.08 ±0.01C

4.40 ±0.8011

±124.8 ±1.55-Hydroxy-L-tryp-

tophan0.57±0.02to

0.62 ±0.0760.286 ±0.02C0.28 ±0.03b

5.0 ±0.4010

±131.7 ±2.6Reserpine0.51

±0.030.65 ±0.0660.26 ±0.03C0.54 ±0.08*7.3 ±0.50615

±215.7 ±1.16

a Average ±S.D. as in Table 3.0 Significantly changed from conlrol group by unpaired f test for at least 3 separate determinalions al p < 0.05.0 Significan! at p < 0.01.

1410 CANCER RESEARCH VOL. 41

Research. on January 13, 2019. © 1981 American Association for Cancercancerres.aacrjournals.org Downloaded from

Benzo(a)pyrene Hydroxylation and Gastrointestinal Hormones

Table 6

Effect of tissue substances on liver drug metabolism systemExperimental procedures are described in Table 4. All activities are expressed in units described in Table 3 except for the cytochrome P-

450 content which is reported as nmol/mg.

HydroxylationactivitiesBenzphetamineEthylmorphinep-Nitroanisolep-NitrophenetoleBenzo(a)pyreneSystem

componentsCytochromeP-450contentCytochromec reducíaseControl5.286.343.503.36130.00.76153.4±±±±±±±0.25a0.560.660.210.100.0522.9Dimethyl-prosta-

glandinE.3.98

±6.27±0.90±1.73

±210.0±0.89

±111.5±0.1

7b0.440.07*0.15°20.0"0.027.3Serotonin4.736.941.772.64130.00.75188.8±0.07±

0.31±0.10"±

0.4±6.0±

0.05±6.95-Hydroxy-L-tryp-

tophan6.438.161.382.52120.00.80185.6±

0.63±0.93±0.28"±0.206±

6.0±

0.02±7.7Reserpine3.514.071.572.70310.00.52198±

0.09C±

0.53±0.156±

0.28±17.0C±

0.036±

23.2* Average ±S.D. as in Table 3.6 Significantly changed from control group by unpaired ( test for at least 3 separate determinations at p < 0.05.c Significant at p < 0.01.

5-Hydroxytryptamine (serotonin) and 5-hydroxytryptophan

have only modest effects on colon and liver drug metabolismactivities. Both substances stimulate ethylmorphine hydroxyl-

ation in the colon to a slight but statistically significant extent.Benzo(a)pyrene hydroxylation is stimulated 2.5-fold in the liverand the colon by reserpine pretreatment and almost 2-fold inthe liver by 16,16-dimethylprostaglandin E2. In general, theaction of each of these tissue substances on the liver or colongives a more diffuse pattern of stimulation than is seen with thegastrointestinal hormones.

DISCUSSION

The regulation of the rate of drug metabolism by altering theamount of component enzymes present is a central issue instudies of carcinogenesis from the standpoint of activation ofenvironmental or dietary precarcinogens, in studies of chemo-

therapeutic management of tumors from the standpoint ofremoval of activated anticancer drugs, and in the activation ofsome anticancer drugs (e.g., cyclophosphamide). The demonstration of drug metabolism systems in many extrahepatictissues (22) makes the regulation of drug metabolism evenmore complicated by virtue of different tissue sensitivities toregulatory effectors. Our data provide the first evidence showing that the gastrointestinal hormones significantly affect therate of drug metabolism in the colon and, to a lesser degree,the liver. The physiological action of gastrin on the stomach,increased acid output, was reported to have a peak effect ofdoubling the basal level after 7 days of treatment (31 ). Johnson(15, 16) reported that 6 pentagastrin injections within 48 hrsignificantly increased DMA synthesis in the colonie mucosa aswell as in the oxyntic gland area, duodenum, ileum, and pancreas. The dose-response curve presented by Johnson (Ref.15, Fig. 3) for pentagastrin-stimulated DMA synthesis in colonie

mucosa is essentially identical to that shown in Chart 1 in thispaper, showing the increase in benzo(a)pyrene hydroxylationspecific activity after pretreatment with increasing dosages ofpentagastrin. This pentagastrin-induced increase in the spe

cific enzyme activity is dependent on new protein synthesisand, presumably, DNA synthesis as indicated by the sensitivityof induction to cycloheximide treatment. Thus, our resultsconfirm the trophic effects of pentagastrin on colonie mucosa(15, 16) and extend those results by showing specific effectson enzyme activities.

Furthermore, both secretin and cholecystokinin octapeptide

have related, but not identical, inductive effects on colon drugmetabolism activities. This is somewhat unexpected, since ithas been shown that, unlike pentagastrin, secretin does notstimulate colonie mucosal DNA synthesis and, when given incombination with pentagastrin, actually inhibits pentagastrin-stimulated DNA synthesis (18). Similarly, cholecystokinin octapeptide has been shown, at best, to have a weak trophicaction at moderate doses in the duodenum but not in thestomach (17). Nonetheless, these hormones stimulate drugmetabolism and cytochrome P-450 content in the colon andthe liver. Each of these hormones has an individual pattern ofeffects. For instance, secretin increases p-nitrophenetole,whereas cholecystokinin increases benzphetamine metabolismin the colon (Table 3). Secretin actually appears to decreasebenzphetamine metabolism. The basis of this apparent specificity may lie in the discrete pattern of P-450 cytochromesinduced by hormone pretreatment. Although the content ofcolonie mucosal cytochrome P-450 rises after hormone pre

treatment, we cannot yet specify the form or forms of cytochrome P-450 accounting for that rise in cytochrome content.Identification of specific hormone-responsive forms of cytochrome P-450 could aid us in understanding the discrete

effects of these hormones on drug metabolism. Induction ofcolon microsomal cytochrome P-450-specific content by gastrointestinal hormones ranges from 1.6-fold for cholecystokininand 1.8-fold for pentagastrin to 2.8-fold for secretin, comparingfavorably with the 3.8-fold increase elicited by phénobarbitalpretreatment (9). The 8-fold induction of cytochrome P-448content by /8-naphthoflavone, however, is the largest inductionof colonie cytochrome P-450 (P-448) yet reported (9).

The gastrointestinal hormones also affect hepatic drug metabolism, although the changes are not as pronounced as thosein the colon. That this group of gastrointestinal hormonesaffects the liver as well as the colon makes effects on drugmetabolism a more generalized phenomenon. This does notrule out specificity as a tissue characteristic as is seen in thelack of any stimulation of hepatic O-dealkylation activity inresponse to any of the hormones.

The effects of the tissue substances on drug metabolismactivities in the colon and in the liver are fewer and more diffusethan are the effects of the gastrointestinal hormones. Nonetheless, the stimulatory effects on enzyme activities where presentare dramatic. For instance, reserpine, which depletes stores ofthe biogenic amines, increases benzo(a)pyrene hydroxylationmore than 2-fold in the liver and colon. Such effects are of

APRIL 1981 1411

Research. on January 13, 2019. © 1981 American Association for Cancercancerres.aacrjournals.org Downloaded from

W. F. Fang and H. W. Strobel

particular significance in light of the recent demonstration ofinhibition of malignant growth of colon tumors by 5,6-dihy-droxytryptamine, a cytotoxic congener of serotonin (34). Serotonin, on the other hand, apparently acts to stimulate cellproliferation in experimentally induced colonie carcinomas(34).

The relationship between the trophic effects of tissue substances and gastrointestinal hormones and the effects of thesecompounds on drug metabolism remains to be defined. Inany event, the gastrointestinal hormones and tissue substancesdo affect the growth rate and drug metabolism activity of thecolon, and now it appears that at least serotonin and relatedcompounds exert cytotoxic effects on colonie tumors. Sincethe gastrointestinal hormones do increase cytochrome P-450content and hydroxylation of benzo(a)pyrene, it is very likelythat the activation of other carcinogens is also favored byinduction of the drug metabolism system by one or more of thehormones, as we have shown previously for phénobarbital orß-naphthoflavone (8).

We are unable as yet to define which of the multiple forms ofcytochrome P-450 is(are) increased by gastrointestinal hor

monal pretreatment. It has, however, already been shown thatvarious forms of cytochrome P-450 hydroxylate benzo-(a)pyrene to different products, and the activation ofbenzo(a)pyrene is associated with an increase in carcinogenic-ity and mutagenicity (37, 38). The effects of gastrointestinalhormones on the metabolic profile of benzo(a)pyrene catalyzedby rat colon is now under study as a model system. Suchapproaches applied to human systems, also under study, arenecessary before a relationship between colonie metabolismof benzo(a)pyrene or other dietary and environmental carcinogens and human colon cancer can be demonstrated. Theresults reported in this paper, however, are suggestive of thatpossibility.

REFERENCES

1. Arine. E., and Philpot, R. M. Preparation and properties of partially purifiedpulmonary cytochrome P-450 from rabbits. J. Biol. Chem., 251: 3213-

3220, 1976.2. Chhabra, R. S.. Pohl. R. J., and Fouts, J. R. A comparative study of

xenobiotic-metabolizing enzymes in liver and intestine of various animalspecies. Drug Metab. Dispos. 2:443-447. 1974.

3. Cochin, J.. and Axelrod. J. Biochemical and pharmacological changes inthe rat following chronic administration of morphine, nalorphine, and nor-morphine. J. Pharmacol. Exp. Ther, 725: 105-110, 1959.

4. Dallner, G.. Siekevitz, P., and Palade, G. E. Biogenesis of endoplasmicreticulum membranes. II. Synthesis of constitutive microsomal enzymes indeveloping rat hepatocyte. J. Cell Biol., 30. 97-117, 1966.

5. Dignam, J. D., and Strobel. H. W. NADPH-cytochrome P-450 reducíasefrom rat liver: purification by affinity chromatography and characterization.Biochemistry, 16: 1116-1123, 1977.

6. Ellin, A., Jacobson, S. B., Schenkman, J. B., and Orrenius, S. CytochromeP-450k of rat kidney cortex microsomes: its involvement in fatty acid u- and(w-1) hydroxylation. Arch. Biochem. Biophys., 150: 64-71. 1972.

7. Ernster. L., and Orrenius. S. Substrate-induced synthesis of the hydroxyl-ating enzyme system of liver microsomes. Fed. Proc.. 24: 1190-1199,

1965.8. Fang, W. F., and Strobel, H. W. Activation of carcinogens and mutagens by

rat colon mucosa. Cancer Res.. 38: 2939-2944. 1978.9. Fang, W. F., and Strobel. H. W. The drug and carcinogen metabolism system

of rat colon microsomes. Arch. Biochem. Biophys.. 186: 128-138, 1978.10. Fang, W. F., and Strobel. H. W. Effects of gastrointestinal hormones on drug

metabolism in rat colon mucosa microsomes. Fed. Proc., 30: 365, 1979.11. Grossman. M. I. Gastrointestinal hormones. In: J. A. Parsons (ed.). Peptide

Hormones, pp. 105-116. London: University Park Press, 1976.12. Guengerich, F. P. Separation and purification of multiple forms of microsomal

cytochrome P-450: activities of different forms of cytochrome P-450 towardsseveral compounds of environmental interest. J. Biol. Chem., 252: 3970-

3979, 1977.13. Guengerich, F. P. Separation and purification of multiple forms of microsomal

cytochrome P-450: partial characterization of three apparently homogeneous cytochromes P-450 prepared from livers of phénobarbital- and 3-meth-ylcholanthrene-treated rats. J. Biol. Chem., 253: 7931-7939, 1978.

14. Haugen, D. A., van der Hoeven, T. A., and Coon, M. J. Purified livermicrosomal cytochrome P-450: separation and characterization of multipleforms. J. Biol. Chem., 250: 3567-3570, 1975.

15. Johnson, L. R. Trophic action of gastrointestinal hormones. In: J. C. Thompson (ed.). Symposium on the Gastrointestinal Hormones, pp. 215-230.Austin, Texas: The University of Texas Press. 1975.

16. Johnson, L. R. New aspects of the trophic action of gastrointestinal hormones. Gastroenterology, 72. 788-792, 1977.

17. Johnson, L. R.. and Guthrie, P. D. Effect of cholecystokinin and 16,16-dimethylprostaglandin E2 on RNA and DMA of gastric and duodenal mucosa.Gastroenterology, 70: 59-65. 1976.

18. Johnson, L. R., and Guthrie, P. D. Effect of secretin on colonie DMAsynthesis. Proc. Soc. Exp. Biol. Med., 158: 521-523, 1978.

19. Kawalak, J. C., and Lu, A. Y. H. Reconstituted liver microsomal enzymesystem that hydroxylates drugs, other foreign compounds, and endogenoussubstrates. VIM. Different catalytic activities of rabbit and rat cytochromes P-448. Mol. Pharmacol., 11: 201-210, 1975.

20. Lu, A. Y. H., Kuntzman, R., West, S. B.. Jacobson. M., and Conney, A. H.Reconstituted liver microsomal enzyme system that hydroxylates drugs,other foreign compounds, and endogenous substrates. II. Role of the cytochrome P-450 and P-448 fractions in drug and steroid hydroxylations. J.Biol. Chem., 247: 1727-1734, 1972.

21. Lu, A. Y. H., Strobel, H. W.. and Coon, M. J. Properties of a solubilized formof the cytochrome P-450-containing mixed-function oxidase of liver microsomes. Mol. Pharmacol., 6: 213-220, 1970.

22. Lu, A. Y. H., and West, S. B. Reconstituted mammalian mixed-functionoxidases: requirements, specificities, and other properties. Pharmacol. Ther.A 2. 337-358. 1978.

23. Nash, T. The colorimetrie estimation of formaldehyde by the Hantzschreaction. Biochem. J., 55: 416-421, 1953.

24. Nebert, D. W., and Gelboin, H. V. Substrate-inducible microsomal arylhydroxylase in mammalian cell culture. I. Assay and properties of inducedenzyme. J. Biol. Chem., 243: 6242-6249, 1968.

25. Netter. K. J.. and Seidel, G. An adaptively stimulated O-demethylatingsystem in rat liver microsomes and its kinetic properties. J. Pharmacol. Exp.Ther., 146: 61-65, 1964.

26. Omura, T., and Sato, R. The carbon monoxide-binding pigment of livermicrosomes. I. Evidence for its hemoprotein nature. J. Biol. Chem., 239:2370-2378, 1964.

27. Pantuck, E. J., Hsiao, K. C., Kuntzman, R., and Conney. A. H. Intestinalmetabolism of phenacetin in the rat: effect of charcoal-broiled beef and ratchow. Science (Wash. D. C.), 187: 744-745, 1975.

28. Phillips, A., and Langdon, R. G. Hepatic triphosphopyridine nucleotide-cytochrome c reducíase: isolation, characterization, and kinetic studies. J.Biol. Chem., 237 2652-2660. 1962.

29. Philpot, R. M., Arine, E., and Fouts, J. R. Reconstitution of the rabbitpulmonary microsomal mixed-function oxidase system from solubilized components. Drug Metab. Dispos., 3: 118-126, 1975.

30. Ryan, D., Lu, A. Y. H., West, S. B., and Levin, W. Multiple forms ofcytochrome P-450 in phénobarbital- and 3-methylcholanthrene-treated rats:separation and spectral properties. J. Biol. Chem., 250: 2157-2163, 1975.

31. Stanley, M. D., Coalson, R. E., Grossman, M. I., and Johnson, L. R. Influenceof secretin and pentagastrin on acid secretion and parietal cell number inrats. Gastroenterology, 63: 264-269, 1972.

32. Stohs, S. J., Grafstrom, R. C., Burke, M. D., Moldeus, P. W., and Orrenius,S. The isolation of rat intestinal microsomes with stable cytochrome P-450and their metabolism of benzo(a)pyrene. Arch. Biochem. Biophys., 777:105-116, 1976.

33. Strobel, H. W., Fang, W. F., and Oshinsky, R. J. Role of colonie cytochromeP-450 in large bowel carcinogenesis. Cancer (Phila.), 45: 1060-1065.1980.

34. Tutton, P. J. M., and Barkla, D. H. Cytotoxicity of 5,6-dihydroxytryptaminein dimethylhydrazine-induced carcinomas of rat colon. Cancer Res., 37:1241-1244, 1977.

35. Wattenburg. L. W. Studies of polycyclic hydrocarbon hydroxylases of theintestine possibly related to cancer: effect of diet on benzpyrene hydroxylaseactivity. Cancer (Phila.), 28: 99-102. 1971.

36. Williams, C. H., and Kamin, H. Microsomal triphosphopyridine nucleotide-cytochrome c reductase of liver. J. Biol. Chem., 237: 587-595, 1962.

37. Wislocki, P. G.. Chang, R. L., Wood, A. W., Levin, W., Yagi, H., Hernandez,O., Mäh,H. D., Dansette. P. M., Jerina, D. M., and Conney, A. H. Highcarcinogenicity of 2-hydroxybenzo(a)pyrene on mouse skin. Cancer Res.,37:2608-2611, 1977.

38. Wood, A. W.. Levin. W.. Lu, A. Y. H., Yagi, H., Hernandez, O., Jerina, D. M.,and Conney, A. H. Metabolism of benzo(a)pyrene and benzo(a)pyrenederivatives to mutagenic products by highly purified hepatic microsomalenzymes. J. Biol. Chem., 25Õ: 4882-4890, 1976.

1412 CANCER RESEARCH VOL. 41

Research. on January 13, 2019. © 1981 American Association for Cancercancerres.aacrjournals.org Downloaded from

1981;41:1407-1412. Cancer Res   Wan Fen Fang and Henry W. Strobel  Colon

)pyrene and Other Xenobiotics in Ratathe Carcinogen Benzo(Control by Gastrointestinal Hormones of the Hydroxylation of

  Updated version

  http://cancerres.aacrjournals.org/content/41/4/1407

Access the most recent version of this article at:

   

   

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/41/4/1407To request permission to re-use all or part of this article, use this link

Research. on January 13, 2019. © 1981 American Association for Cancercancerres.aacrjournals.org Downloaded from