Chem.-Biol. Interactions, 27 (1979) 323--334 © Elsevier/North-Holland Scientific Publishers Ltd.

323

CHARACTERISTICS OF ARYL HYDROCARBON HYDROXYLASE ACTIVITY IN RAT MAMMARY EPrrHLIAL CELLS GROWN IN PRIMARY CULTURE *

J.W. GREINER, L.B. MALAN-SHIBLEY and D.H. JANSS Carcinogenesis Program, Frederick Cancer Research Center, Frederick, MD 21701 (U.S.A.) (Received March 26th, 1979) (Revision received May 25th, 1979) (Accepted May 28th, 1979)

SUMMARY

Rat mammary epithelial cells grown in primary culture contain the microsomal enzyme, aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH), which catalyses the oxidative conversion of polycyclic aromatic hydrocarbons (PAH) to more polar derivatives. Constitutive AHH activity, measured with an established fluorometric method, was 46 pmol/mg protein/h in homogenates of rat mammary epithelial cells after 5 days in culture. The addition of dimethylbenz[a]anthracene (DMBA), benz[a]anthracene (BA), or 3-methylcholanthrene (3-MC) to the cell culture medium increased AHH activity 5.3-, 4.7- and 2.4-fold, respectively. Kinetic studies revealed that maximal hydroxylase induction occurred 16 h after 1 #M DMBA was added to the culture medium. The decay of the DMBA-induced hydroxylase was biphasic: one component had a t½ of 15--30 rain and another a t½ of 4 h. Norepinephrine, 1 7 ~ s t r a d i o l and 5,6-benzoflavone also increased AHH activity in mammary epithelial cells in vitro, however, sodium phenobarbital had no effect. Fetal bovine serum (FBS), previously shown to be a potent in vitro inducer of AHH activity, had no effect on either constitutive or DMBAinduced mammary epithelial hydroxylase activities following treatment with 1% activated charcoal. Metyrapone and 7,8-benzoflavone, inhibitors of microsomal mixed function oxidase activity, reduced both constitutive and DMBA* This work was supported by Contract No. N01-CO-75380, with the National Cancer Institute, NIH, Bethesda, MD 20014. Presented in part at the 69th Annual Meeting of the American Association of Cancer Research, Washington, D.C., May, 1978. Abbreviations: AHH, aryl hydrocarbon (benzo[a]pyrene) hydroxylase; BA, benz[a]anthracene; DMBA, dimethylbenz[a]anthracene; DMSO, dimethylsulfoxide; FBS, fetal bovine serum; FBSadj, adsorbed fetal bovine serum; 3-MC, 3-methylcholanthrene; a-NF, 7,8-benzoflavone; ~-ND, 5,6-benzoflavone; PAH, polycyclic aromatic hydrocarbons; PBS, phosphate buffered saline.

324 induced AHH activities when added to homogenates of untreated and DMBAtreated mammary epithelial cells. The addition of 7,8-benzoflavone reduced both constitutive and DMBA-induced hydroxylase activities by approx. 80%, whereas metyrapone addition inhibited these activities by 20%. The study demonstrates several in vitro factors which alter AHH activity in primary cultures of rat mammary epithelial cells.

INTRODUCTION AHH is a microsomal, NADPH-requiring mixed function oxidase(s) which catalyses the conversion of PAH to epoxide intermediates [ 1,2]. The epoxide can rearrange spontaneously, forming phenol and quinone derivatives, or be further metabolized to water soluble and/or more polar compounds. Subsequent metabolism of the epoxide derivatives had been shown to result in the formation of highly reactive compounds (i.e., diol~poxides) which are considered to be ultimate chemical carcinogens [3--5]. Moreover, the modulation of AHH activity has been shown to alter PAH-induced tumorigenesis [6,7]. Therefore, the microsomal aryl hydroxylase functions as an important regulatory step in the carcinogenesis process. AHH has been detected in virtually every tissue investigated, as well as in organ and cell cultures [8--11]. Our interest in the microsomal hydroxylase concerns its probable role in tumor initiation in the rat mammary gland. Huggins et al. [12] reported that a single intragastric dose of DMBA to virgin, Sprague--Dawiey rats resulted in a 100% incidence of mammary adenocarcinomas. However, little is yet known about AHH activity in rat mammary tissue. Recently, we measured AHH activity in rat mammary epithelial cells which were isolated on a Ficoll gradient [13] and have described a hormonesupplemented culture medium whereby these isolated cells can be grown in primary culture [14]. The present study establishes the presence of the substrate-inducible AHH activity in rat mammary epithelial cells grown in primary culture. MATERIALS AND METHODS

Animals. Female, virgin Sprague--Dawley rats (50--65 days old) were purchased from Charles River Breeding Laboratories, Inc. (Wilmington, MA) and maintained on a diet of Wayne Lab-Blox (Allied Mills Inc., Chicago, IL) and water ad libitum under standardized conditions of light (06:00--18:00 h) and temperature (24.0 -+ 1.5°C). Isolation o f mammary epithelial cells. The procedure for mammary epithelial cell isolation has been reported [14]. Briefly, the rats were sacrificed by CO2 asphyxiation, the abdominal and inguinal mammary fat pads excised, the lymph nodes removed and the tissue minced with a razor blade. The tissue was added tc~-a dissociation medium containing 0.45% collagenase (CLS, Type II, Worthington Biochemical, Freehold, NJ) and incubated at

325 37°C for 60 min. The dissociated tissue was filtered through nylon mesh and the lipocytes removed by centrifugation. The mammary epithelial cells, recognizable as aggregates of alveolar (ductular) and ductal cells, were separated from fibroblasts by centrifugation on a 2--8% Ficoll gradient. The epithelial nature of the cells isolated by this procedure has been confirmed by electron microscopy [15].

Mammary epithelial cell culture conditions. The isolated rat mammary epithelial cells were examined for viability by trypan blue exclusion, counted and added to a hormonally defined medium [14]. The medium contained gentamicin (50 ~g), penicillin (100 units), streptomycin (100 /~g), insulin (50 ng), aldosterone (50 ng), prolactin (50 ng), 17fl~stradiol (0.03 ng), progesterone (3 ng) and 5% charcoal adsorbed FBS [15] in each ml Medium 199-Earle's salts. The mammary epithelial cells were added to 100 mm Falcon petri dishes at a concentration of 0.25 × 106 cell aggregates/dish. Cell cultures were incubated at 37°C in a humidified atmosphere of 95% air : 5% CO2 and the medium changed every 48 h. The day of seeding was designated day 0 and all experiments were done on day 5 when the cells were in the latter stages of logarithmic growth and the monolayers had attained a confluency of 70--85%. Activated charcoal (Mallinckrodt., St. Louis, MO) was added to FBS at a final concentration of 1% (w/v). The mixture was stirred for 15 min at 37°C and the charcoal removed by centrifugation at 10 000 Xg for 15 min at 4°C. The procedure was repeated. After the final centrifugation the adsorbed serum (FBSads) was sterilized by Millipore filtration (0.22 ~m) and stored at -20°C. In vitro induction o f A H H activity. Potential inducers of mammary epithelial AHH activity were added to the culture medium in dimethylsulfoxide (DMSO) or water at a concentration which did not exceed 0.1%. An equal volume of solvent, which was shown not to alter AHH activity, was added to the medium of uninduced mammary epithelial cells. Inspection of the cells by light microscopy revealed no morphological changes following the addition of inducer and/or solvent. Following the induction period, the culture medium was decanted and the cells washed twice with 10 ml Dulbecco's phosphate buffered saline (PBS). The mammary epithelial cells were removed by gentle scrapping with a rubber policeman in 10 ml PBS. Each dish was rinsed with 10 ml PBS and the fractions combined and centrifuged at 500 ×g in a Beckman RC-3 preparative centrifuge for 15 rain at 37°C. The cell pellet was suspended in a glass-glass Potter-Elvehjem homogenizer. Mammary epithelial cells from 4 petri dishes were pooled to provide sufficient protein for triplicate AHH determinations. Determination o f A H H activity. AHH activity in mammary epithelial cell homogenates was determined by the fluormetric procedure of Nebert and Gelboin [16] in an incubation mixture containing 80 ~mol Tris--Cl buffer (pH 7.5), 3 umol MgC12, 0.36 gmol NADPH, 0.66 umol glucose 6-phosphate, 0.15 units glucose-6-phosphate dehydrogenase, 80 wmol benzo[a]pyrene

326 (BP) and 0.8--2.1 mg cell homogenate protein in a final volume of 1 ml. Incubations were carried o u t in a D u b n o f f Metabolic Incubator at 37°C for 60 min. The reaction was stopped b y the addition of I ml acetone, the phenolic metabolites were extracted into 3.5 ml n-hexane incubated at 37°C for 10 min. A 2.5 ml aliquot was extracted with an equal volume of 1 N NaOH and the fluorescence of the alkaline fraction measured immediately using a Turner Model 420 spectrofluorometer with the excitationemission wavelengths set as 390 nm and 522 nm, respectively. One unit of A H H activity was defined as the a m o u n t of enzyme which catalyses the formation of hydroxylated products and results in the fluorescence equivalent of 1 pmol 3-hydroxy-BP in 60 min at 37°C. All samples were read against 3-hydroxy-BP which was generously provided b y the NCI Chemical Repository. Protein concentrations of mammary epithelial cell homogenates were determined spectrophotometrically using bovine serum albumin as the standard [ 17 ]. RESULTS

Kinetics o f A H H induction. The dose-response curves for BA, DMBA and 3-MC-induced A H H activities in mammary epithelial cells grown in primary culture are summarized in Fig. 1. A H H activity increased linearly as increasing amounts of BA, DMBA or 3-MC were added to the culture medium until maximal induction for each PAH was reached at 1 #M. At that con-

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[PAH]-~M Fig. 1. E f f e c t s o f various P A H o n A H H a c t i v i t y in r a t m a m m a r y epithelial cells g r o w n in p r i m a r y c u l t u r e . M a m m a r y epithelial cells were isolated f r o m 5 0 - - 6 5 ~ i a y ~ l d S p r a g u e - D a w l e y rats a n d g r o w n in p r i m a r y c u l t u r e as d e s c r i b e d in Materials a n d M e t h o d s . D M B A , B A a n d 3-MC were a d d e d t o t h e c u l t u r e m e d i u m f o r 16 h a t t h e c o n c e n t r a t i o n s listed. T h e c o n t r o l cells received a n e q u a l v o l u m e ( 1 0 ,1) o f t h e s o l v e n t , DMSO. E a c h d a t a p o i n t r e p r e s e n t s t h e m e a n o f triplicate d e t e r m i n a t i o n s w h o s e s t a n d a r d errors did n o t vary m o r e t h a n 10%.

327

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Hours Fig. 2. Time course of DMBA induction of AHH activity. Rat mammary epithelial cells were cultured for 5 days. The plates were divided into two groups: DMBA-treated (; ; ) and control (c o ). DMBA (1 #M) was added in 10 #1DMSO and the control cells received the same volume of DMSO. At the times indicated, the cells from four 100 mm petri dishes were pooled and AHH activities determined on both groups. Each point represents the mean +- S.E. of three separate experiments.

centration, BA, DMBA and 3-MC increased mammary epithelial A H H activity 5.3-, 4.7- and 2.3-fold, respectively, over the control activity. The addition of 10 or 100 ~M BA or DMBA to the mammary epithelial cell culture medium resulted in submaximal increases in A H H activity. Moreover, 3-MC added at those concentrations had no effect on constitutive A H H activity. The time course of DMBA-induced A H H activity in primary cultures of m a m m a r y epithelial cells is shown in Fig. 2. Mammary A H H activity increased up to 16 h following the addition of 1 ~M DMBA. The level of hydroxylase induction declined between 16 and 30 h, after which a plateau of A H H activity (approx. 100 units/mg protein) was maintained to 44 h. The in vitro addition of cycloheximide (3.5/~M) has been shown to inhibit protein synthesis and result in a time~lependent decay of A H H activity [18,19]. Fig. 3 represents the biphasic decay of the DMBA-induced h y d r o x y lase in mammary epithelial cells in vitro. Following cycloheximide addition to the culture medium of DMBA-treated mammary epithelial cells, the decay o f A H H activity consisted of a rapid c o m p o n e n t with a tl/2 of 15--30 min and a slow c o m p o n e n t with a tl/2 of 4 h. A H H activity in DMBA-treated mammary epithelial cells also declined following DMBA removal from the culture medium. The decay was biphasic with tl/2 of 15--30 min and 9.7 h, respectively. Other inducers of A H H activity. The addition o f naphthalene (1/~M) to the m a m m a r y epithelial cell culture medium reduced constitutive AHHactivity b y 50%, whereas 5,6-benzoflavone (5-NF) (1 ~M) increased hydroxylase activity 2.1-fold (Table I), The addition of 10 and 100/~M norepinephrine increased A H H activity 2.1- and 3.2-fold, respectively,,while sodium phenobarbital had no effect. O p t i m u m g r o w t h of rat m a m m a r y epithelial

328 TABLE I OTHER IN VITRO INDUCERS OF MAMMARY EPITHELIAL AHH ACTIVITY Mammary epithelial cells were isolated from Sprague--Dawley rats and grown in primary culture for 5 days (confluency = 80--85%). Naphthalene, ~-NF, 17~-estradiol or DMBA were added in a final volume of 0.1% DMSO. Sodium phenobarbital and norepinephrine were added i n t h e same volume of 0.9% NaC1. After 16 h, the culture medium was decanted and AHH activity determined fluorometrically using mammary epithelial cell homogenates. Values r e p r e s e n t t h e mean + S.E.; 4--6 determinations for each group. Inducer

Conc. (~M)

AHH activity

Induced/control

(units/mg protein) None Naphthalene

~-NF Sodium Phenobarbital Norepinephrine

17~-Estradiol DMBA 17~-F~tradiol + DMBA

--

1.0 1.0 10.0 100.0 10.0 100.0 10.0 1.0 10.0 1.0

45 24 93 36 44 96 143

-+ 7 ±6 ± 5a ±5 ±4 ± 12 a ± 8a

--

0.5 2.1 0.8 1.0 2.1 3.2

156 ± 8 a 250 ± 16 a

3.6 5.3

240 ± 8 a

5.3

a p < 0.05 (vs. control AHH activity)

cells requires t h e a d d i t i o n o f 0.03 ng 1 7 ~ s t r a d i o l t o t h e c u l t u r e m e d i u m . T h e a d d i t i o n o f 10 ~ M 1 7 ~ s t r a d i o l t o t h e c u l t u r e m e d i u m f o r 16 h increased A H H a c t i v i t y 3.6-fold. E i t h e r D M B A (1/~M) a l o n e or in c o m b i n a t i o n w i t h 10 /~M 1 7 ~ s t r a d i o l s t i m u l a t e d m a m m a r y epithelial A H H a c t i v i t y 5.3-fold ( T a b l e I). Serum effects on A H H activity. C o n s t i t u t i v e m a m m a r y epithelial A H H activities r e m a i n e d u n c h a n g e d in cells c u l t u r e d in t h e p r e s e n c e o f 5%, 10%, 15% a n d 20% c h a r c o a l FBSads (Table II). In c o n t r a s t , A H H a c t i v i t y was significantly higher in cells c u l t u r e d w i t h u n t r e a t e d F B 8 a n d , m o r e o v e r , h y d r o x y l a s e a c t i v i t y increased c o n c o m i t a n t l y in cells g r o w n in 5%, 10% a n d 15% u n t r e a t e d FBS. T h e m a g n i t u d e s o f D M B A - i n d u c e d A H H a c t i v i t y in cells c u l t u r e d in e i t h e r 5% or 10% FBSa& w e r e similar. In m a m m a r y epithelial cells c u l t u r e d w i t h 5% a n d 10% u n t r e a t e d FBS, h o w e v e r , t h e levels o f D M B A i n d u c t i o n w e r e 8.6- a n d 9.8-fold, r e s p e c t i v e l y . Inhibition o f A H H activity. T a b l e I I I s u m m a r i z e s t h e e f f e c t s o f 7 , 8 - b e n z o f l a v o n e ( a - N F ) [3,8] a n d m e t y r a p o n e [ 2 0 ] , t w o i n h i b i t o r s o f m i x e d f u n c t i o n o x i d a s e a c t i v i t y , o n c o n s t i t u t i v e a n d D M B A - i n d u c e d A H H activities in r a t m a m m a r y epithelial cells in p r i m a r y c u l t u r e . Prior a d d i t i o n o f 0.1 m M a - N F t o m a m m a r y epithelial cell h o m o g e n a t e s i n h i b i t e d c o n s t i t u t i v e a n d D M B A - i n d u c e d A H H activities b y 80% a n d 84%, respectively. In c o n t r a s t , m e t y r a p o n e (0.1 raM) a d d i t i o n r e d u c e d t h e c o n s t i t u t i v e a n d i n d u c e d h y d r o xylases b y 18% a n d 28%, r e s p e c t i v e l y (Table III).

329 T A B L E II SERUM EFFECTS ON CONSTITUTIVE AND DMBA-INDUCED AHH ACTIVITY Rat m a m m a r y epithelial cells were seeded in c o m p l e t e m e d i u m containing 5% adsorbed FBS. The m e d i u m was r e m o v e d 24 h later and replaced with m e d i u m containing t h e indicated s e r u m concentrations. M e d i u m was changed every 48 h and D M B A (1 ~M) was added o n day 4 w h e n t h e m o n o l a y e r s were 70--75% c o n f l u e n t . U n i n d u c e d epithelial cells received an e q u a l aliquol (10 ~1) DMSO and A H H activities were d e t e r m i n e d as described in Materials and Methods. Values represent m e a n ± S.E.; 4--6 d e t e r m i n a t i o n s for each group. Mammary epithelial cells

Serum cone.

(%)

Unindueed

5 10 15 20

DMBA-induced

5 10

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