(CANCER RESEARCH 48. 2492-2497, May I, 1988]

P-450 Enzyme Induction by 5-Ethyl-5-phenylhydantoin and 5,5-Diethylhydantoin,

Analogues of Barbiturate Tumor Promoters Phénobarbitaland Barbital, andPromotion of Liver and Thyroid Carcinogenesis Initiated by7V-Nitrosodiethylamine in Rats1

Bhalchandra A. Diwan,2 Jerry M. Rice, Raymond W. Nims, Ronald A. Lubet, Henry Hu,3 and Jerrold M. Ward

Program Resources, Inc., Frederick Cancer Research Facility, Frederick, Maryland 21701 [B. A. D.J, and Laboratory of Comparatile Carcinogenesis, National CancerInstitute, Frederick, Maryland 21701 [J. M. R., R. W. N., R. A. L., J. M. W.]

ABSTRACT

Male F344/NCr rats, 6 wk old, were fed 500 ppm of phénobarbital(PB) or equimolar doses of either 5-ethyl-5-phenylhydantoin (EPH) or5,5-diethylhydantoin (EEH) in diet for 2 wk and hepatic cytochrome P-450-mediated alkoxyresorufm 0-dealkylase and aminopyrine A'-demeth-ylase activities were determined. Both PB and EPH greatly increased P-450-mediated enzyme activities in rat liver while EEH was ineffective.To evaluate the hydantoins as tumor promoters, 5-wk-old male F344 ratswere given a single i.p. injection of 75 mg /V-nitrosodiethylamine/kg bodyweight. Beginning 2 wk later, they were placed either on normal diet ordiet containing 500 ppm of PB or equimolar doses of EPH or EEH forthe remaining experimental period. Control groups received an i.p. injection of saline followed by each of the test diets. Animals were sacrificedat either 52 or 78 wk. PB and EPH significantly enhanced the development of hepatocellular foci and hepatocellular adenomas at 52 wk andhepatocellular carcinomas at 78 wk in /V-nitrosodiethylamine-initiatedrats. Neither the incidence of hepatocellular neoplasms nor the numberand size of hepatocellular foci was significantly increased by EEH. At 78wk, both PB and EPH enhanced the development of thyroid follicularcell neoplasms in /V-nitrosodiethylamine-initiated rats while no suchenhancement was observed with EEH. Thus, EPH, a long-acting sedative/anticom ulsant, like the structurally similar PB, promoted hepatocellularand thyroid follicular cell carcinogenesis and induced the PB-inducibleform(s) of cytochrome P-450 (P-450,,) in rats. In contrast, EEH unlikebarbital failed to promote hepatocellular and thyroid follicular cell carcinogenesis and also failed to induce PB-inducible form(s) of cytochromeP-450 in rats.

INTRODUCTION

An increasing number of studies during the past 15 yr hasshown that hepatocarcinogenesis is a multistep process, inwhich initiation and promotion stages have been identified inlaboratory rats and mice (1-5). Since the original discovery (1)that hepatocarcinogenesis in rats could be promoted by PB,4 a

variety of other drugs, insecticides, and some endogenous compounds such as steroid hormones have been shown to acceleratehepatocellular tumorigenesis in that species (6-10). The phenomenon of multistage hepatocarcinogenesis has been the subject of several recent reviews (11-13).

Our laboratory has been engaged in studies on structure-promoting activity relationships among sedative, anxiolytic,and anticonvulsant agents prescribed either singly or in combination for the treatment of neurological disorders. Becausethese drugs are often taken in relatively large doses on a daily

Received 10/21/87; revised 1/29/88; accepted 2/4/88.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1Supported in part by the National Cancer Institute under Contract NO1-CO-

23910 with Program Resources, Inc.2To whom requests for reprints should be addressed.3 Present address: Johns Hopkins Applied Physics Laboratory, Laurel, MD

20707.4The abbreviations used are: PB, phénobarbital:EPH, 5-ethyl-5-phenylhydan-

toin; EEH. 5,5-diethylhydantoin; DEN, iV-nitrosodiethylamine.

basis for prolonged periods, they are realistic candidates fortumor-promoting agents. In recent studies (14-16) we haveshown that certain of these compounds promote carcinogenesisin epithelia other than hepatocytes, including those of thethyroid follicle, renal tubule, and urothelium, and that livertumor-promoting activity within a series of barbiturate derivatives varies greatly with molecular structure, persistence intissues as measured by duration of sedative action, and efficiency of induction of specific monooxygenase enzyme activities.

Barbiturates share common structural features with severalother classes of anticonvulsants including hydantoins, oxazoli-dinediones, and succinimides. Hydantoins, especially, are verysimilar to the barbituric acid derivatives in both chemical structure and pharmacological activities. For example, PB and Nir-vanol (5-ethyl-5-phenylhydantoin, EPH; Fig. 1) have remarkably similar pharmacological properties as judged by theneurological effects produced by these drugs in equal plasmaconcentration (17). In addition, EPH like PB alters metabolismof other drugs by the liver (18). EEH is closest in structure tobarbital, a long-acting sedative barbiturate, (Fig. 1) but has lessmarked sedative/depressant effects. Of various hydantoin derivatives used clinically, only phenytoin (5,5-diphenylhydantoin)has been tested as a possible promoter of hepatocellular carcinogenesis in rats (19). Phenytoin failed to promote hepatocarcinogenesis in this study. Other hydantoin derivatives such asEPH and EEH, the structural analogues of well-establishedrodent liver tumor promoters, PB and barbital (14, 19, 20),have not yet been investigated for tumor-promoting activity.One objective of this study was to determine whether dietaryEPH and EEH could promote tumor development in rat liver.

Several earlier studies have shown that a good correlationexists between tumor-promoting activity of barbiturates andtheir ability to induce liver hypertrophy (14, 16, 19, 21), cytochrome P-450 (19, 22), and ornithine decarboxylase (2, 23) inrats. In our recent studies (16, 24) we have extensively tested aseries of barbiturates for tumor-promoting activity and forinduction of hepatic cytochrome P-450-mediated activities including ethoxy-, pentoxy-, and benzyloxyresorufin O-dealkylaseand aminopyrine /V-demethylase. Activities of different barbiturates as promoters of hepatocarcinogenesis showed an excellent correlation with inducing ability for certain hepatic cyto-chrome(s) P-450-mediated activities (pentoxy- and benzyloxyresorufin O-dealkylase, aminopyrine jV-demethylase, and p-ni-troanisole O-demethylase) in the rat. To establish whether theseassociations are true for other classes of tumor promoters, thehydantoin derivatives EPH and EEH were also analyzed forcapacity to induce specific P-450-mediated oxygenase reactions.

MATERIALS AND METHODS

Chemicals

PB (M, 232,000; purity >99%) was purchased from Sigma ChemicalCompany, St. Louis, MO. EPH (M, 204,000) and EEH (M, 156,000)

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(Fig. 1) were synthesized in our laboratory from the correspondingketones via cyanohydrin intermediates by published procedures (25, 26)and were checked for purity and identity by elemental analysis, meltingpoint, and 'H and "C NMR. The synthesized EPH had mp 196-198°C[literature (27), mp 198°C],and EEH had mp 163-164°C [literature(27), mp 163°Cj.The elemental analyses of the synthesized hydantoins

(Galbraith Laboratories, Inc., Knoxville, TN) were as follows.

EPH (C,,HI2N2O2)

Calculated: C 64.69, H 5.92, N 13.72

Found: C 64.84, H 5.80, N 13.81

EEH (C7H12N202)

Calculated: C 53.83, H 7.74, N 17.94

Found: C 53.88, H 7.82, N 18.04

No extraneous NMR peaks were observed in solutions of either compound. 7-Ethoxy-, 7-pentoxy, and 7-benzyloxyresorufin were purchasedfrom Molecular Probes, Inc., Junction City, OR. Dicumarol, resorufin,and aminopyrine were from Aldrich Chemical Company, Milwaukee,WI, and fluorescamine from Roche Diagnostics, Nutley, NJ. DEN(Sigma) was analyzed by gas-liquid chromatography and its purity wasfound to exceed 99%.

Tumor Promotion Study

Two hundred forty weanling male F344/NCr rats were obtained fromAnimal Production, National Cancer Institute-Frederick Cancer Research Facility, Frederick, MD. All animals were housed in an American Association for Accreditation of Laboratory Animal Care-accredited facility. They were housed four per cage in autoclavable polycarbonate cages and given food (NIH autoclavable formula 31 modifiedto contain 6% fat) and water ad libitum. The animals were maintainedat a temperature of 68 to 72°Fand a relative humidity of 50 ±5% with

12 changes room air per h. At 5 wk of age, they were randomly dividedinto 8 groups of 30 rats each. Rats in carcinogen-treated groups receiveda single i.p. injection of DEN at a dose of 75 mg (1.0 mmol)/kg bodyweight in 5 ml sterile saline (groups 1-4). Two wk later, animals wereprovided with either normal diet (group I) or diet containing 500 ppmPB (group 2) or equimolar doses of either EPH (440 ppm; group 3) orEEH (336 ppm; group 4) for the remaining experimental period. Ratsin control groups received 5 ml of sterile saline/kg body weight by i.p.injection and 2 wk later were given diet containing 500 ppm PB (group5) or equimolar doses of either EPH (group 6) or EEH (group 7).Group 8 was an untreated control group. Fifteen rats per group werekilled at 52 wk of age and the remaining animals were killed whenmoribund or at 78 wk when the experiment was terminated. Animalswere weighed weekly.

All animals were carefully necropsied. The livers were removed in

5-ethyl-5-phenylbarbituric acid 5-ethyl-5-phenylhydantoinPhénobarbital Nirvanol

PB EPH

5-5-diethylbarbituric acidBarbital

BB

5.5-diethylhydantoin

Fig. 1. Structures of 5.5-disubstituted barbiturates and hydantoins.

loto and weighed. Selected representative portions of all liver lobes (twosections per lobe) and all liver lesions were fixed in 10% bufferedformalin. Other internal organs including lungs, nasal cavity, kidneys,thyroid gland, thymus, and spleen as well as other organs with grosslesions were routinely removed and fixed in formalin. Hollow organsincluding esophagus, stomach, intestine, and urinary bladder wereopened and mucosa! surfaces were carefully examined for abnormalities. All visible lesions and representative samples from each majororgan were embedded in paraffin, sectioned at 6 ¿im,and routinelystained with hematoxylin and eosin for histological evaluations.

For animals killed at 52 wk, hematoxylin and eosin-stained representative liver sections were examined for number/cm2, area/perimeter,

and volume of hepatocellular foci with the use of an automated imageanalyzer (Videoplan; Carl Zeiss, Inc., New York, NY). After 52 wk,foci were generally too numerous to count accurately. Hepatocellularfoci and tumors (adenomas and carcinomas) were classified accordingto established criteria (28).

Biochemical Study

Determination of Hepatic Alkoxyresorufin O-Dealkylase and Aminopyrine /V-Demethylase Activities. Twenty F344/NCr male rats, 6 wkold, were randomized into 4 groups of 5 each. They were given normaldiet (group 1) or diet containing 500 ppm of PB (group 2) or equimolardoses of EPH (group 3) or EEH (group 4) for 15 days. Rats were killedby CO2 and the entire liver was carefully removed, trimmed free ofextraneous tissue, and washed repeatedly in 0.15 M KC1 (4"C) until the

rinse fluid was free of blood. Livers were weighed and then homogenizedin 0.15 M KC1 (3 ml/g net weight). Postmitochondrial (S-9) andmicrosomal subfractions were obtained as described earlier (29). Proteincontent in the S-9 and microsomal samples was measured using fluorescamine (30) with bovine serum albumin as the standard.

The O-dealkylation of the alkoxy resorufins by hepatic S-9 fractionswas measured using a modification (29) of assays previously describedfor measuring these activities in microsomes (31-33). The O-dealkyla

tion of all 3 alkoxyresorufin (ethoxy, benzyloxy, pentoxy) substrateswas determined under the same conditions. The final substrate concentration used was 5.0 ^M, the S-9 concentration used was between 100and 200 u^ nil. and reaction rates were determined during the linearportion of the reaction. These conditions resulted in somewhat higheractivity rates, particularly in control animals, than were routinelyachieved in prior studies (24, 32).

For measurement of aminopyrine /V-demethylase activity, 5.0-mlincubation mixtures in 0.1 M potassium phosphate buffer, pH 7.4,contained 5 mM MgCI2, 10 mM semicarbazide hydrochloride, 6.5 ITIMglucose-6-phosphate, 0.65 mM NADP, and 5 mM aminopyrine. Thereaction was started by addition of 1-2 mg microsomal protein andstopped after incubation at 37°Cwith shaking for 10-20 min by

addition of 3.0 ml 5% ZnSO4 and 3 ml of 4.5% Ba(OH)2. Aftercentrifugation the content of formaldehyde was determined by themethod of Nash (34).

Statistical Analysis

The significance of differences in body weights, liver weights, thenumber of foci/cm2, and their volume among different treatment groupswere evaluated with Student's t test. The significance of differences in

the incidence of tumors among different groups was evaluated by theX2test. Alkoxyresorufin O-dealkylation and aminopyrine /V-demethyl-

ase data were analyzed for statistical significance between varioustreatment groups and the controls using the 2-sample Kruskal-Wallisnonparametric rank test (35).

RESULTS

Tumor Promotion Study

Body and Liver Weight. No significant difference in dailymean food intake (g/kg body weight/day) was observed indifferent groups. No differences in body weight gain were seenbetween rats in different groups until 60 wk of age. A slight

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decrease in body weight gain in older rats (78 wk) receivingeither PB (409 ±85 g, mean ±SD) or EPH (415 ±68 g)following DEN treatment was related to higher incidence ofmalignant hepatocellular neoplasms as compared to those thatreceived DEN alone (425 ±43 g). A significant increase in theliver to body weight ratio was observed at 52 and 78 wk of agein rats that received either PB or EPH following DEN administration, in comparison with rats that had been given DENalone. This increase in liver weight in PB- and EPH-treatedgroups was clearly due to the growth of neoplasms in theseanimals. No significant increase in liver weights or liverrbodyweight ratio was observed in rats fed EEH following exposureto DEN (group 4).

Survival. All rats in every treatment group were alive at 52wk of age. Between 52 and 78 wk survival of DEN-EEH (group4) rats was better than that of DEN.EPH (group 3) or DEN.PB(group 2) rats (refer to Fig. 2). In the DEN-EPH group 8 of 15rats (53%) and in the DEN-PB group 9 of 15 rats (60%)survived until 78 wk. All of the rats that died between 52 and78 wk had hepatocellular lesions. All rats given hydantoins orPB but no DEN or left untreated (groups 5-8) survived for the

duration of this study.Development of Hepatocellular Foci. At 52 wk, foci of altered

hepatocytes of clear, eosinophilic, mixed, and basophilic celltypes were seen in animals treated with either DEN alone(group 1) or DEN followed by the test chemicals. Mixed andbasophilic foci were more commonly observed in animalstreated with DEN alone whereas eosinophilic and clear cell fociwere especially evident in DEN-PB (group 2) or DEN-EPH(group 3) animals.

As shown in Fig. 3, animals that were initiated with DENand then given either PB (group 2) or EPH (group 3) developedsignificantly more foci/cm2 than did animals treated with DEN

alone (group 1). Although the number of DEN-initiated foci(group 1) was slightly increased after EEH treatment (group 4),this difference was not significantly different. No foci wereobserved at this time (52 wk) in control animals including thosegiven only PB, EPH, or EEH (groups 5-8).

Fig. 4 depicts the average size of foci in different groups ofrats at 52 wk of age. The mean volume of foci was significantlyhigher (P < 0.05) in groups given PB (group 2) or EPH (group3) following DEN initiation than in rats given only DEN (group1). The mean volume of foci in the rats given EEH after DEN(0.13 ±0.17, group 4) was significantly lower (P < 0.05) thanin those given either PB (group 2) or EPH (group 3) after DENadministration.

XI

65WCÕKS

16

14

12

u. 10

3 8

Si 4

DEN DEN DEN DENPB EPH EEH

Fig. 3. Mean number of hepatocellular foci/cm in different groups of rats at52 wk; mean ±SE. *. significantly different from group 1 (DEN) at P < 0.05.

0.6

0.5

0.4

0.3

0.0DEN.PB DEN.EPH DEN.EEH

Fig. 2. Cumulative percentage of rats dying with tumors between 50 and 78wk. A, DEN; O, DEN/PB; •DEN/EPH; A, DEN/EEH.

Fig. 4. Mean focus volume (mm3) in different groups of rats at 52 wk; mean±SE. *, significantly different from group 1 (DEN) at P < 0.05.

Development of Hepatocellular Neoplasms. The incidences ofhepatocellular tumors observed in different experimentalgroups at 52 wk are shown in Table 1. In rats initiated withDEN, both PB and EPH enhanced the incidence of hepatocellular adenomas. EPH was as effective as PB in promotinghepatocarcinogenesis initiated by DEN. Also, histologically,the liver architecture (i.e., enlargement of centrilobular hepatocytes) of rats treated with EPH did not differ from hepaticmorphology in rats treated with PB. Rats of both groups hadhepatocytomegaly. The incidence of hepatocellular carcinomaswas 10% in DEN-PB animals and 20% in DEN-EPH animals.EEH treatment subsequent to DEN administration (group 4)did not significantly modify the incidence of hepatocellularneoplasms and the livers of EEH-fed rats were not significantlydifferent than those of rats given only DEN. Results presentedin Table 1 clearly show that group 2 animals treated with PBsubsequent to DEN and group 3 animals receiving EPH following DEN had significantly enhanced hepatocellular adenomayields compared to rats treated with DEN alone (P < 0.05).

By 78 wk, almost all of the DEN-treated rats (groups 1-4)had developed hepatocellular adenomas although the multiplicity of such lesions was significantly greater (P < 0.05) in group2 (DEN-PB) and group 3 (DEN-EPH) rats as compared togroup 1 animals (DEN alone; Table 1). The incidence of hepatocellular carcinomas (Table 1) was also significantly higher (P< 0.05) in rats given either PB (group 2, 70%) or EPH (group3, 60%) subsequent to DEN. Multiple hepatocellular carcino-

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Table l Effect of PB, EPH, and EEH on hepatocellular carcinogenesis initiated by DEN in male F344INCr rats

Hepatocellular tumors observedat52wk°No.

of animals with hepatocellulartumorsTreatment

groupsDEN

DEN PBDEN- EPHDEN EEHSaline -PBSaline EPHSaline- EEHSalineAdenomas(%)3(20)

14 (93)*13(87)*

2(13)0

1(7)00Carcinomas

(%)01(7)

4(27)1(7)

0000Tumors

per rat withtumorsAdenomas

(mean ±SE)1.7

±0.73.4 ±0.6*2.7 ±0.5*

1.5 ±0.50

1.0 ±000Carcinomas

(mean ±SE)0

1.0 ±01.3 + 0.31.0 ±0

0000Between

52 and 78wk°No.

of animals with hepatocellulartumorsAdenomas

(%)10

(67)14(93)15 (100)9(60)

0000Carcinomas(%)M7)

11 (73)12(80)2(13)

0000Tumors

per rat withtumorsAdenomas

(mean ±SE)1.4

±0.37.4 ±1.2*

10.5 ±1.2*

3.3 ±1.20000Carcinomas

(mean ±SE)1.0

±02.4±0.5

1.7 ±0.21.0 ±0

0000

°There were 15 rats in each group.* Statistically significant compared to group (DEN); P< 0.05.

Table 2 Effects of hydantoins administered in the diet on cytochrome(s) P-450activities in the rat

1 :'3.'1,'.f?Vo*o- •. *> . •- .-»'* •*î ,

L, ' 'V-,-; '••Ó. ,'< •£• ». jfk

%m¿':^.--'*'; = ••.i'-'.'. ••¿•jVí':-ï.-;--:^--;:V:"Fig. 5. Hepatocellular carcinoma initiated by DEN and promoted by EPH in

a 68-wk-old rat. x 250.

kr^r'.v-Tiv/L'.•;>•>tv«x;...í'f%.f „--*•\-- •"'-•í ,,i*k rf*-*r>-,;'.^ .

... >.••..•-"•:»;'v'Sífiolr,"; -.•

.-••Ai-1'-*'••'-^•'•v.. ' •'<•";.'•<',<>*.?.á.•••. *AI , ¿í-^- -a,v.;/?.J;v 'J.'">/0>-rrvWià&ìri. ;JM&'ÃŒ̂í•;i-.*.*^,'<r •». ./ ,;•

,:5rí¿>'•H X. -*'.^ ..,-?-•-..lí

••. .

;Fig. 6. A portion of lung showing metastasis from the hepatocellular carci

noma illustrated in Fig. 5. x 100.

Treatment"Control

PBEPHEEH0-Dealkylation

of phenoxazoneethers*Ethoxy-56.3

±9.2122.0+ 12.7''123.0± 13.6"

87.0 ±18.0Pentoxy-31.9

±5.7947.0+ 15.0"

1062.0 ±43.0''8 1.0 ±6.8''Benzyloxy-49.2

±6.41549.0 ±36.0''1490.0 ±57.0"

94.2 ±7.8''Aminopyrine

W-demeth-ylationc12.3+

1.941.5 + 6.0"39.9 ±2.6"19.6+ l.l"

" F344 rats received control diet or diet containing either 500 ppm phénobar

bital or equimolar doses of ethylphenylhydantoin or diethylhydantoin for 15 days.Enzyme assays were done in duplicate; values given are mean ±SD for five malerats per treatment.

* Values are in units of picomoles resorufin formed/min/mg S-9 protein at28-C.

c Values are in units of nanomoles formaldehyde formed/min/mg microsomalprotein at 37*C.

"Significantly different from control group, P < 0.05 (2-sample Kruskal-

Wallis nonparametric rank test).

mas were commonly observed in rats of groups 2 and 3 whilefew such lesions were seen in animals of groups 1 and 4 (Table1). Furthermore, two rats in group 2 (DEN-PB) and three ratsin group 3 (DEN-EPH) had lung métastases(Figs. 5 and 6)while none was found in groups exposed to DEN alone (group1) or DEN followed by EEH (group 4).

Development of Nonhepatic Tumors. The most common typesof nonhepatic tumors observed in this study included those ofthe nasal cavity, thyroid, pituitary gland, testes, and lympho-reticular system (Table 2). Nasal cavity tumors were observedonly in DEN-treated rats and their incidence was not affectedby the administration of test compounds. The incidences oftesticular tumors (Leydig cell tumor), pituitary tumors, andlarge granular lymphocyte leukemias were predictably high inolder rats (78 wk) of both control and experimental groups andwere unaffected by treatment.

Thyroid tumors were most frequently observed in rats ofgroup 2 (DEN-PB) and group 3 (DEN-EPH). Thyroid tumorsof follicular cell origin were seen in 5 of 15 rats (33%) in group2 and 4 of 15 rats (27%) in group 3 while no such tumors wereobserved in other groups. Histologically, these tumors wereclassified as follicular cell adenomas (Fig. 7) and adenocarci-nomas. The historical incidence of thyroid tumors in rats givenDEN alone in our two last studies (14, 16) was 2 tumors in 35animals. Thus, PB and EPH but not EEH have a significant (P< 0.05) enhancing effect on the development of thyroid tumorsin DEN-initiated animals. Although follicular cell hyperplasiaswere seen in PB only or EPH only animals no goitrogenicchanges were observed.

Biochemical Study

Effects of PB, EPH, and EEH on Body and Liver Weights.Animals in all treatment groups showed normal and compar-

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Fig. 7. Follicular cell adenoma (papular) type) of the thyroid gland in a 78-wk-old rat exposed to DEN followed by EPH. x 250.

able weight gains. Ratios of liver to body weights following 15days dietary administration were significantly higher (P < 0.05)in rats fed EPH (7.09 ±0.40, mean ±SD) and PB (7.53 ±0.29) as compared to those fed EEH (5.86 ±0.46) or controldiet (5.65 ±0.23). Absolute weights were also significantlyincreased in EPH- and PB-treated rats compared to the con

trols.Effects of PB, EPH, and EEH on Ethoxy-, Pentoxy-, and

Benzyloxyresorufin O-Dealkylase Activities. As shown in Table2, both PB and EPH dramatically (~30-fold) increased pentoxy-and benzyloxyresorufm O-dealkylase activities in rat liver after15 days of feeding. On the other hand, only a slight (2- to 3-fold) increase in activity of these enzymes was evident in liversof rats fed EEH. Ethoxyresorufin O-dealkylase activity wasinduced about 2- to 3-fold over control values by PB and EPHwhile no such increase in the activity of this enzyme was seenfollowing EEH treatment.

Effects of PB, EPH, and EEH on Aminopyrine /V-DemethylaseActivity. A significant increase of aminopyrine yV-demethylaseactivity over control values was observed in rats fed all testcompounds (Table 2). However, the increase was much higherin rats fed either EPH or PB (3- to 4-fold) than in those fedEEH (1.5-fold).

DISCUSSION

EPH is a hepatic metabolite of the anticonvulsant drug 3-methyl-5-phenyl-5-ethylhydantoin (17) and has been consideredto contribute to both the therapeutic effect and the toxicity ofthe parent drug. At equal plasma concentrations, EPH and PBproduce similar anticonvulsive effects (17). Under the tradename Nirvanol, EPH was used clinically for the treatment ofchorea in children in the 1920s but caused a high incidence oftoxic side effects.

From this study it is clear that EPH is also comparable toPB in both the specificity and the intensity of its inducing effecton hepatic O-dealkylase activities toward the phenoxazone substrates pentoxy- and benzyloxyresorufin, both of which arespecifically mediated in rats by the PB-inducible form of cyto-chrome P-450, P-450b (32). Both PB and EPH cause increases

in these activities in rats of approximately 30- to 35-fold. Incomparison with the 2- to 4-fold increases in aminopyrine N-demethylase also induced by these compounds, the alkoxyre-sorufin O-dealkylase reactions clearly provide an extremelysensitive assay for P-450h induction.

This study clearly showed that EPH, given after the initiatingdose of DEN, like its barbiturate analogue PB, also had anenhancing effect on the development of hepatocellular foci andhepatocellular neoplasms. There was a significant increase (P< 0.05) in the number of focus transections/cm2 in livers of

rats exposed to EPH as compared to the number in livers ofrats exposed to DEN alone (Fig. 3). EPH also enhanced thedevelopment of hepatocellular neoplasms resulting in a significant increase in the percentage of rats bearing liver tumors andin the number of tumors per liver (Table 1). At equimolar doses,both PB and EPH exhibited approximately equal potency inenhancing liver growth and promoting preneoplastic and neo-plastic liver lesions in DEN-pretreated rats. Moreover, a pro

moting effect on the incidence of follicular tumors of thethyroid, comparable in magnitude to that of PB (14), wasobserved in rats that received EPH following DEN initiation.EPH thus appears fully comparable to PB in regard to both theepithelia on which it has promotion effects and the dosagerequired for promotion in these tissues.

EEH is structurally similar to barbital. However, unlike barbital, EEH is pharmacologically inactive as either a hypnoticor an anticonvulsant (36). Barbital has been shown to promotehepatocarcinogenesis (14, 20) and thyroid and renal carcino-genesis (14, 15) in rodents, and is also known to induce hepaticcytochrome P-450 (14), PB-specific cytochrome(s) P-450 activities (24), and aminopyrine ¿Y-demethylaseactivity (14) in rats.In contrast, EEH failed to increase liver growth or promotedevelopment of either preneoplastic or neoplastic hepatocellular lesions and caused minimal increase in cytochrome P-450-dependent drug-metabolizing enzymes.

Several recent reports have shown a strong correlation between the liver tumor-promoting activity of several distinctclasses of compounds and their ability to induce liver hypertrophy and cytochrome(s) P-450-dependent drug-metabolizing enzymes (8, 19, 24). The metabolism of many substances ismediated by specific forms of cytochrome P-450. Thus, thedealkylation of pentoxy- and benzyloxyresorufin is mediated bythe major PB-inducible forms of cytochrome(s) P-450 in therat (29, 32, 33). Within the class of barbiturates, the ability topromote hepatocarcinogenesis is closely associated with theability of each compound to increase liver growth and inducedealkylase activities mediated by the major PB-specific cytochrome P-450 (pentoxy- and benzyloxyresorufin O-dealkylase)in male rats (16, 24). The correlation between induction ofalkoxyresorufin dealkylase activity and capacity to promotehepatocarcinogenesis previously established for barbituratesclearly extends to their hydantoin analogues. Thus, the induction of liver growth associated with functional increases incertain cytochrome P-450 species suggests tumor-promotingpotential. The findings of this study that the hydantoin derivatives EPH and EEH display a strong relationship between livertumor-promoting activity and induction of PB-specific cyto-chrome(s) P-450 are consistent with this view. Interestingly, inour recent study, we found that the liver tumor-promotingchlorinated hydrocarbon pesticides (e.g., DDT, lindane) arealso potent inducers of pentoxy- and benzyloxyresorufin O-dealkylase activities in rats.5 Thus, the ability to induce PB-

' R. A. Lubet. et al., manuscript in preparation.

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specific cytochrome(s) P-450 in the liver appears to warrantfurther evaluation as a molecular marker for screening chemicals for ability to promote hepatocarcinogenesis.

The commonly used anticonvulsant 5,5-diphenylhydantoin

(phenytoin; Dilantin) was found by Peraino et al. (19) to beineffective in promoting hepatocellular carcinogenesis in Spra-gue-Dawley rats when fed mixed in diet at 500 ppm. Theseauthors also observed no increase in liver weight or liver DNAsynthesis in diphenylhydantoin-treated animals in their acuteexposure study. Phenytoin has, however, been shown to inducehepatic DNA synthesis and hyperplasia in the rat in an earlierstudy (37). This drug is known to induce microsomal enzymeactivity in several rodent species (38, 39) and in man (40, 41).We have found6 this drug to be effective in inducing O-dealkyl-ation of pentoxy- and benzyloxyresorufin in F344 rats, althoughthe magnitude of the response is approximately one-half of thatachieved with EPH or PB. Thus, based on our results and thoseof others (37, 38, 41), we consider that further experiments areneeded on the possible effects of diphenylhydantoin on hepatocarcinogenesis in rats.

The mechanisms underlying the promoting action of EPH orof the barbiturates in the rat liver are not yet known. However,the present study clearly showed that EPH exerted biochemicaleffects quite similar to those of PB, a well-established rodentliver tumor promoter. Thus, in consideration of similarities inthe biochemical effects between PB and EPH, it seems probablethat the action of EPH in liver tumor promotion is quite similarto that of PB. However, the present data do not rule out thepossibility that tumor promotion by PB and by EPH proceedsby different mechanisms.

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1988;48:2492-2497. Cancer Res   Bhalchandra A. Diwan, Jerry M. Rice, Raymond W. Nims, et al.   in Rats

-NitrosodiethylamineNand Thyroid Carcinogenesis Initiated by Promoters Phenobarbital and Barbital, and Promotion of Liver5,5-Diethylhydantoin, Analogues of Barbiturate Tumor P-450 Enzyme Induction by 5-Ethyl-5-phenylhydantoin and

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