Levels of Cytochrome P-450-Mediated Aryl Hydrocarbon Hydroxylase (AHH) are Higher in Differentiated than in Germinative Cutaneous Keratinocytes

Jin-feng Guo, M.B., Raya Brown, Ph.D., Charles E. Rothwell, Ph.D., and Isadore A. Bernstein, Ph.D. Dcp:lrtment of Environmental and Industrial Health and Department of Biological Chemistry (lAB), The University of Michigan, Ann Arbor. Michigan, U.S.A.

Induction of microsomal atyl hydrocarbon hydroxylase and cytochrome P-450 was observed in epidermal cells obtained from the skin of newborn rats exposed to benz(a)anthracene by topical exposure and in submerged cultures exposed to the procarcinogen in vitro. The level of aryl hydrocarbon hy­droxylase activity was increased 2.5-fold in vivo and six~ to sevenfold in vitro when the measurements were made on the entire epidermis or the entire culture. respectively. However, separate measurement on genninative (basal) and on differ­entiared cells revealed that AHH was sevenfold higher in differentiated cells as compared with basal cells in the skin of both unexposed animals and animals exposed in vivo. Similar results were obtained in cultured cells exrosed in vitro. Im­munocytochemical staining of sections 0 skin from animals exposed to benz{a)anthracene in vivo with a monoclonal

Manuscript received June 21. 1988; :accepted for publiotion June 19, 1989.

Present :address for Dr. Guo: School of Public Health. Shangh:ai Medical University. Shanghai. People's Republic of China.

Present address for Drs. Rothwell and Bernstein: Parke· Davis Pharma­ceutical Research. 2800 Plymouth Road . Ann Arbor. M148105.

This investigation was supponed by gr1nts £507062 and AM I 5206 from the N:atlonalinstttutes of Health. U. S. Public He:alth Service.

Reprint requesrs to: I. A. Bernstein. 1528 Public Health, The University of Michigan, 109 Observatory Street. Ann Arbor, MI 48109-2029.

Abbrevi:uions: AHH: aryl hydroc:arbon hydroxylase DA: benz(a)anthr.acene BP: benzo(a)pyrene BSA: bovine se rum albumin DMSO: dimethylsulfoxide E.BSS: Earle's balanced salt solution FBS: feta.l bovine serum IgG: immunoglobulin G MEM: minimum essential medium NADH: reduced nicotin:amide adenine dinucleotide NF: naphthoAavone NADPH: reduced nicotinamide 2denine dinucleotide phosphate PAGE: polyacrylamjde gel electrophoresis PAH: polycyclic aromatic hydrocarbons P.450: cytochrome P-4S0 T COD: 2,3.7 .8.tetl'2chlorodibenzo-p-dioxin 3MC: 3-methyl cholanthrene 30H·BP: 3.hydroxy-benzo(a)pyrene

antibody generated against cytochrome P-450c showed a higher binding of the antibody in lower spinous cells than in basal cells in the epidermis. Although more stained cells were observed in exposed cultures than in untreated cultures, the antibody, which inhibits at least 85% of the hydroxylase activity in the skin, inhibited only 6% - \6% of the activity in culture. These observations support the interpretations chat a) differentiated keratinocytes have a higher capacity in the metabolic activation of PAH than do germinative celis, al­though both types of cell are susceptible to induction of cytochrome P-450 by exposure toBA, and b) the cytochrome P-450 induced by exposure of epidermis to benz(a)anthra­cene in vivo exhibits some differences from the one induced upon exposure of keratinocytes to this procarcinogen in vitro. J It,"es' Dermatol 94:86- 93, 1990

The microsomal cytochrome P-4S0 (P-4S0) dependent monooxygenases (mixed function monooxygenases) play an important role in metabolizing drugs. procar­cinogens, pesticides. and other xenobiotics, as well as endogenous compounds such as steroids and fatty

acids [1- 6]. Cytochrome P-450 represents a ubiquitous superfamily of enzymes that includes a number of gene families and subfamilies differing in their amino acid sequences and/or in their substrate specificity (7). One of tlte catalytic activities of these mixed function monooxygenase systems is aryl hydrocarbon hydroxylase (AHH) that catalyzes oxidation of rlyaromatic hydrocarbons (PAH). 3-Methylcholanthrene (3MC , 2,3,7,8-tctrachlorodibcnzo-p-dioxin (TCDD), naphthoflavone (NF), and benz(a)anthracene (BA) can induce cytochrome P-450 in the liver and a variety of extra-hepatic tissues, including skin [8,9 - 12]' both in vivo [2,3,8,9, 13-171 and in vitro [9 - 11,13,18]. In the skin, the site of the largest accumula­tion of these enzymes is the epidermis [91.

Actjvation of procarcinogens by cytochrome P-450 is considered to be a key step in the process of chemical carcinogenesis [ef. 7J. After entering the body procarcinogens :lte metabolized by the mixed function monooxygenases. among other enzymes, to be­come potent carcinogens. It is important to understand the detailed role of P-450 in epidermal carcinogenesis because the epidermis is exposed to a variety of genotoxic compounds as a result of the skin's interface with the body's external environment :and because cutane­ous cancer is rhe most common m:alignanr human tumor [19}. As a contribution to such an understanding, this paper reports a) that early differentiated cells of the epidermis are more responsive than the germinative population to induction of AHH activity upon

0022.202X/90/S03.S0 Copyright © 1990 by The Society for Investigative Dennatology,lne.

86

VOL. 94. NO.1 JANUARY 1990

exposure to 3MC and BA and b) that the P-450 isozyme induced in the ep_idermis by topical exposure to these chemicals in vivo appears to differ from the P-450 induced by exposing a submerged culture of keratinocytes to the same PAH.

MATERIALS

Reduced nicotinamide adenine dinucleotide (NADH), reduced nicotinamide adenine dinucleotide phosphate (NADPH), bcnz(a)anthracene (BA). 3-merhylcholanrhrene (3MC). bcnzo(a)pyrene (BP). and bovine serum albumin (BSA) were ob­tained from Sigma Chemical Co. (St. Louis, MO): 3-hydroxy­benzo(a)pyrene (30H-BP) from the National Cancer Institute (Be­rhesda, MD); phenol reagenr and dimerhylsulfoxide (DMSO) from Fisher Scienrific Co. (Fair Lawn, NJ); rhe PAGE reagents and appa­ratus, from Bio-Rad (Richmond, CAl. and protein molecula.r weight standard kit and Percoll, &om Pharmacia, Inc. (Piscataway, NJ). Earle's balanced salt solution (EBSS) and minimum essential medium (MEM) were purchased from Hazelcon Research Products (Denver. PAl: feral bovine serum (FBS) from Gibco Laborarories (Grand Island, NY) and Flow Laborarories (New York, NY). Fluo­rescein isothiocyanate (FITC)-Iabeled sheep anti-mouse IgG was obtained from Cappel Laborarories (Wesr Chesrer, PAl, and OCT embedding medium was obcained from Miles Laboratories Inc. (Naperville, IL) .

The newborn rats that were used in these studies were from a CFN strain (Carworth Farms) and were obtained from a randomly inbred colony maintained by the Toxicology Laboratory at the School of Public Healrh of The University of Michigan.

METHODS

Culture of Cutaneous Keratinocytes from Newborn Rats Keratinocytes were isolated from the cutaneous epidermis of new­born rats (2.d old) as described by Vaughan er al [20J. The basal ce lls were separated from differentiated cells on PercolJ dens ity gradients 12 1). Tile kcrdlinocytcs were suspended in 38% Percoll in EBSS and centrifuged at 30,000 X g for 15 min at 4 °c . The basal cel ls consti­tuted the lowermost band on this Percoll gradient. The basal cell fraction, consisting of greater than 95% small, round cells with a high nuclear to cytoplasmic ratio, was suspended at a density of 4 X 10' cells/ml) in MEM conraining 10% FBS and supplemented with insulin. hydrocortisone. and antibiotics [20]. The calcium COI1-

centration of this medium was 2 mM. Eight miJIiliters of the sus· pension were then seeded into l00-mm Corning tissue culNre dishes and incubated at 37°C in an atmosphere of 5% COfair at 85% humidity. In some experiments, cells were cultured III low calcium medium. The low calcium medium consisted of calcium­free MEM containing 10% Chelex-treated FBS and supplemented as described above. The concentration of the calcium was adjusted to 0.08 mM by the addition of an adequate amount of calcium chloride. The seeding densi ty for the low calcium culture was 1.3 X l OS cells/ml in medium. Five days after seeding, cultures were exposed [0 several concentrations of BA or 3MC. T he PAH were dissolved in DMSO, and the final concentration ofDMSO in the medium was 0.15%.

Culture of Hepatoma Cells Rat hepatoma cell line. ATCC H4EII , was grown in MEM containing 15% FBS ilnd supplemented as described above in Corning tissue culture flasks T -75 (15 mJ per flask). For subculruring, rhe cells were derached from rhe flask by tre:ament with 0.25% trypsin solution containing 0.02% EDTA. The dC[3ched cells were sedimcoted, resuspended at a density of 4 X lOS cells/ml in the medium, and 8 ml of the suspension was seeded in a 100 mm Corning tissue culture dish. Exposure to BA and 3MC was carried out as described for keratinocytes.

Preparation of Micro somes Preparation of microsomal suspen­sions, from either keratinocytes or hepatoma cells in culrure, fol­lowed rhe procedures described by J in er al [1 Ij. Briefly, 24 or 48 h after the addition of the PAH, cultures were washed th ree times with ice-cold Dulbecco's phosphate-buffered saline. and the cells were scraped off the cul ture dish with a rubber policeman. The cells

AHH IN BASAL AND DIFFERENTIATED KERATINOCYTES 87

were then resuspended in 100 m.M potassium phosyhace buffer, pH 7.4, rharcontained 10 mM EDT A and 20% glycero . All subsequent steps were carried out at 4 ·C. The cell suspension was then soni­cated in a Bronson sonicator with six bursts of 10 sec each. After each burst the samples were cooled by dipping the container in ice for 30 sec. The homogenate was centrifuged at 800 X g for 20 min; rhe resulting supernatant solution was then centrifuged at 9,000 X g for 20 min. and that supernate was centrifuged at t 05,000 X g for 60 min to sediment the microsomes. The microsomal pellet was resuspended in the potassium phosphate buffer and centrifuged for a second period of 60 min ar 105,000 X g. The final peller was ho­mogenized in the same buffer using a teflon pestle homogenizer and was stored at -70·C until used.

E xposure of Rat Skin in Vivo Newborn rats (3-d old) were used in these experiments. One-tenth of a mill iliter of BA solution in DMSO (10 mg/ ml) was spread wirh a glass rod on rhe skin of rhe back of each animal. After a period 0(24 h, the skin was excised and the keratinocytes were isolated in the same manner as described above. Basal cells were scparatcd from the diffe rentiated cells by density gradient sedimentation at 30,000 X g for t 5 min at 4 "C in 38% isotonic Percoll and microsomes were obtained from each population as described above.

Assay o f AHH The microsomal AHH activity was measured by the fluorometric method of Nerhert and Gelboin [10], as modified by Bickers er al [9J and Jin er al [1 Ij. Samples of 10- 100 Jl I of rhe microsomal suspension that contained 20 - 150 ,ug of protein were added ro a solurion of 67.5 JIM KH,PO .. pH 7.0 (adjusred wirh dilute KOH). containing 4.2,uM MgCI2 and 25,uM nicotinamide to make a final volume of900 JlI. In experiments that involved the use of antibodies to inhibit the reaction, a suitable volume of ascites fluid or purified IgG, obrained as described by Rorhwell [22]. was included in this solution. Then 100 JlI of a second solution contain­ing 0.7 mg ofNADPH, 0.7 mg ofNAD H, and 0.7 mg ofBSA was added. The enzyme reaction was initiated by adding 40 nmols ofBP in 20,u1 of acetone, and the reaction mixture was incubated in the dark with continuous shaking for 30 min at 37·C. The reaction was terminated by adding 1 ml of ice-cold acetOne and 3 ml of ice-cold hexane and mixing on a Vortex mixer. The aqueous phase was separated from the hexane phase either by centrifugation at 400 X g for 4 min or by letting the mixture stand for 20 min. Two-and-a­half milliliter aliquots of the hexane phase were mixed with 1 ml of 1 N KOH in wa[er, and, after the separation, the aqueous phase was analyzed for fluorescence in a Perkin-Elmer spectrophotofluoro­meter using an excitation wavelength of 396 nm and an emission wavelength of522 nm with a slit width of 10 nm. The experiments were carried out under semi-dark conditions.

--200

~

- _ . ~oma Cells - Kerallnotyles

.. 11

"" 4

I ~----

/ /

120 , / E /

I f ' I .. 811

I

~ I

I I

1 ,

'" I

/'

f • • 2 • • . I. ~OB'"

Fig ure 1. AH H 2ctiviry in kc=ratinocytes 2nd hCP2t0l112 cdls after exposure to diffcrcni concentrations of BA. Por experimental details see Mnhods.

88 GUO IT AL

0.05

I 0.0<

~ 0.03

.... '20 ...

CulM*' Keratinocytes ---Control

-E' .....

... ... 500 om

Figure 2. Reduced CO-complex spectrum of the microsomal homogenates from kcratinocytes grown in medium containing 2 mM Ca++. Confrol curvt represents absorbance in the presence of dithionite alone. For experimentaJ details see Mtlhods.

Standard curves were obtained by using several dilutions of 30H­BP in water, [0 which I ml ice-cold acetone and 3 ml hexane were added. The standard solutions were then subjected to the same steps as the experimental samples. Results were expressed as pmol 30H­BP / min/ mg protein. Protein was determined by the method of Lowry et 201 [23] using crystalline BSA as standard.

Determination ofp~4S0 The P-450 in microsomal suspensions prepared from epidermal cultures grown in medium with 2 mM Ca++ was mcasured using the merhod of Omura and Saro [24]. The samples. solubilized with Tergitol NP- t 0 at a detergent concentra­tion of 1.1 % and a protein concentration of about 4 mg/ml. were analyzed by CO-difference spectroscopy in a Varian Cary Model 219 spectrophotometer. The concentration of P-450 was deter­mined using the absorbance difference between 450 and 490 nm with an extinction coefficient of91 mM- 1 cm- I [cf24J.

lndirect Immunofluorescent Techniques

Ski" SectiOtiS Pieces of skin from the backs of treated and untreated newborn rats were frozen in a drop of OCT on a cryostat holder that was partially immersed in liquid nitrogen. Fresh frolen sections, 4 -6 Jim thick. were mounted on coverslips, air dried. rinsed with 0.01 M phosphatc buffeted saline at pH 7.2-7.4 (PBS), and stained.

Cell Cljltures Basal cel ls were plared on glass coverslips (0.2 ml of the 0.15% suspension per 22 X 11 mm coverslip) and grown and exposed as described above. At the end of the exposure period (24 h) the slides were rinsed in PBS and stained.

Staitji"g proud life Fresh frozen sections and unfixed cultures 011

coverslips were incubated for t h ar room temperature with the monoclonal anti P-450c, 7B11, [22] diluted 1: 10,1 :50, or 1: 100 in PBS containing 1 % BSA. At the end of the incubation period the coverslips were rinsed for 20 min in three changes of PBS. The slides were rhen further incubated for 1 h at room temperature with FlTC-labcled sheep anti-mouse IgG diluted 1: 50 in PBS contain­ing 1 % BSA, rinsed for 20 min in three changes of PBS. mounted in glycerin buffered to pH 9 with 0.1 M Na,PO" 6.xed in 4% formal­dehyde in 0.1 M phosphate. pH 7.0, for 5 min at room remperature. and rinsed for 20 min in PBS.

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Table L AH H Activity in Microsomes from Basal and Differentiated Keratinocytes After Topical Exposure to BA

for 24 h In Vivo

Cells

Basal Differentiated

AHH Activity" (pmols 30H-BP formed/mg

microsomal protein/min)

Control'

1.35 ± 0.59 9.40 ± 0.47

Expos<d

3.78 ± 0.46 23.7±3.5

·Values are shown ±st;;andud dcvi.uion. N - 3. Analyses wcrc donc in trlplicatc for cach dal;l pomt in an t"-X~rim("nt.

·Control group rcccivcd only DMSO. Separarion and cnzym.nic an.1lytieal tech­niques are described In M~thoJJ.

Stained slides were cxamined with a Zeiss photomicroscope II. using transmitced light with a 490-nm excitation fi1cer and a 530-11111 barrier filtcr.

Preparation and Characterization of Monoclonal Antibodies 7BlI and 6C9.4 The monoclonal anti-P-450c, 7B 11 , was gener­ated in rills laboratory [22] by the hybridoma technique [cf25-26] against P-450c, isolated. and partially purified by the method of Ryan et al [27J from the livers of rats exposed to 3MC. Monoclonal antibody 7Bt 1 binds to rabbit her-atic P-450 isozyme 4 but nor [0

isozyme 2, 3A, 3B, 3C t or 6 [22 . It inhibits the activation of (-)­trans-7 .8-dihydrobenzo(a}pyrene-7 .8-diol~HJ (Midwest Research Institute. Kansas City. MO) for binding to DNA in the procedure of Belvedere et al {28] and the AHH activity in the hepatic microsomes of 3-MC-induced rats [22]. The rabbit cytochrome P-450 used in this srudy were purified in the laboratory of M. J. Coon [291 who generously provided samples of these proteins.

The monoclonal anti-P-450b. 6C9.4. was generated against cy~ tochrome P-4S0b :1.S described by Rothwell et :1I [301. In contrast to

7B 11 , 6C9.4 does not inhibit the AHH or DNA~binding activity catalyzed by the preparation of cytochrome P-4S0c used as antigen in thc generation of 7B 11 [22].

RESULTS

Induction of AHH Activity and ofp.4S0 In Vitro Treatment with llA induced AHH activity in both hepatoma cells and keratin­ocytes in culture (Fig I). The keratinocyces were grown in 2 ruM calcium ion for 5 d and. thercfore. consisted of differentiated as well as basal cells. The microsomal fraction from the induced heparoma cells showed 2.5-4.0 times higher AHH activity than the micro­somes from keratinocytes.

P-450 was not detected by CO-difference spectroscopy in micro­somal suspensions from either uninduced kerarinocytes or hepa­toma cultures. A value of 9.70 pmoles P-450/mg protein was de­tected in microsomal suspensions of keratinocytes exposed to BA in culture (Fig 2). Under comparable conditions of exposure and assay. 40.0 pmoles P-450/ mg protein were derected in microsomal sus­pensions of hepatoma cells. These are minimal values for (he cy­tochrome P-450 because there was a significant amount of absorb­ance at 420 nm, probably resulting from the alteration of P-450 during the isolation procedure.

Table 0_ AHH Activity in Monolayer and Stratificd Cultures of Rat Keratinocytes Exposed to 2.5,uM BA In Vitro

AHH Activity" (pmols 30H-BP fonned/mg microsomal protein/min)

Culture'

Low calcium Normal calcium

24-h Exposure Connol

0.60 ± 0.17 9.97 ± 0.47

Exposed

4.4 1 ± 0.42 63.6 ± 5.83

Control

0.59 ± 0.25 10.5 ± 0.57

·Valucs are shown ± srand..ud devimon. N - 3. Analyses for each dan poinl 10 c.:lch experimcnt wcrr dOllc in ITIpllcatC'.

Expo~d

3.59 ± 0.79 57.2 ± 15.3

VOL. 94, NO. I JANUARY 1990

Comparison of Microsomal Levels of AHH in Germinative (Ba.al) and Diff .. entiated Cell. Table I shows that the activity of AHH measured in the microsomal fraction of cU[aneOU$ epi­dermis from newborn rats topically exposed [0 BA in vivo was about 2.5 times greater than the activity in microsomes from unexposed animals. The data in T able I further demonstnte that the AHH activity in both induced ce ll s and comro l cdls were about 7 times greater in microsomes isolated from differentiated cells than in mi­crosomes from basal cells.

Because the animals were exposed to BA by (opical application. it was possible that the higher activity of AHH in differentiated cells was a function of an earlier and greater exposure to BA as the chemical made its way down into the tissue fro m the surface. To tcst thi s possibiliry, a compari son was made berween microsomcs de­rived from basal cells and microsomes from differentiated ce lls that were exposed to SA in vitro. The induction of AHH activity hy exposure (Q BA in basal cel ls was tested in a culture ofkerarinocytes grown in low calcium medium. When grown in mediulll comain­ing a concentration of about 0.1 mM Ca++. rat keratinocytes grow as a monolayer 131] . At a concentration of 0.08 mM Ca++ in rhe medium, the cui cure consiscs of about 60% germinative baul ce ll s and 40% non-germinative early differentiated cell s [321 . The in­duction of AHH in differentiated cells was studied in keratinocytes grown in medium with a nonnal level (about 2 mM) of ca lcium. When grown under these condi tions. a strdtified culture that con­tained mostly differentiated ce ll s was obtained ref 33 - 34]. Data presented in Table Il show that exposure to BA (or 24 h increased the activity of AH H 6 - 7 times greater than control in both types of culture.

AHH activity was about 15 times higher in the microsomes fro m the induced cultures having the higher percentage of differentiated cells as compared with the microsomes from the induced cultures consisting predominantly of basal ce lls . The data in Table II also show rhat an exposure of 48 h did not produce a significantly greater level of AI 11-1 than was present after an exposure of24 h . A compari­son of the data in Tables I and II demonstrates that the degree of induction achieved in both ce ll types was greater in vitro than in vivo.

The localiza tion of the induced AHH activity in differentiated cells was confi rmed by immunostaining of sk.in sections from ani­mals induced by topical exposure to BA. Monoclonal antibody 78 II, which inhibits AHH activity in microsomes isolated from such treated animals [22]. was used. Figure 3 shows that cells o f the spinous layer were indeed highly stained. while cells from other strata of the skin were either stained slightly or nOt stained at all.

To verify that the immunocytochemical staining and the mea­suremcnr of AHH activity in the microsonul fraction resulted from the same form of cytochrome P-450, the ability of7Bl1 to inhibit the AHH activity induced by BA was studied. The data in Table III demonstrate that 7611 had a profound effect (87% inhibition) on the activity of AHH in microsomes obtained from animals exposed to BA in vivo. Surprisingly, although marc 7Bll-positive cells were observed in treated cultures of keratinocytes than untreated cultures (Fig 4) , rhe AHH activity in microsomes prepared from cells exposed to BA in culture was inhibited only 5% - 16% by the antibody (Table III). The monoclonal antibody to cytochrome P-450b, 6C9.4, did not inhibit the AHH activity in the microsomal fraction from epidennis induced in vivo or keratinocytes induced in viero (Table III).

The tech no logy involved in isolating and assaying AHH activity in cultures per se was not responsible for the failure: of 7811 co inhibit the AHH induced in keratinocytes in vitro. Using the same procedures for AHH induction. iso lation and assay. 7 B11 inhibited [he AHH activity in cel ls of the hepatoma line. H4EII, by 83% and 84%, respectively, in two experiments (Table III ). The inhibitory effect frol11 7 611 on AHH induced in vitro in keratinocytes was only about 16%. Furthermore. when va rious mixtures made from microsomal AHH activity derived from keratinocytes induced in vitro and from epidermis induced in vivo were assayed for inhibi~ tion by 7 B 11. the inhibitions show n by each mixture corresponded

AHH IN BASAL AND DIFFERENTIATED K£RATINOCYTES 89

_.- ... .... -.... .... .... , '--

.... ... ~:.;.~ -''b.-:''" M .j --- ...... -... ~ ..... -; '-:'-." .

,: { · · I' .ltt .. " II , • , I ' , " , .... ~ • ;

" ...... ",' " ... .,: I, I!J 'I C, T ,

• , , • : .~, !tI ' :.' ' .• "1, . , ...... • , I , t ,-- I

I , ., Ii' ~!l(pW • I .' ., _ ' I ,~ .. "

"

Figu re 3. I3l1lding of thC' monoclonal anti P-4S0c. 7 1} II , fO sect ions of skin from the rat. Sections were 1011llunostamed :IS described in M~/hods. Cel ls frollllhe lower ~pmous layer. but not from the basal (b) and granular layers or dlC' sir-uum corneum (SC) of the epidermiS. are sUlIled in the skin from treated anll111ls (d dermis; h hair root; s: spmou$ layer). A: Section of skin from tre;ued anllual ( IOOX); B. Elongated cells from the lower spinous layer arc mongly stallled (BOO X); C: Section of sk in from umre:tted animal. No H311l1ng is observed (800 X).

90 GUO IT AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Table m. Effect of Monoclonal Antibodies, 7Bl1 and 6C9.4, on Microsomal AHH Activities in Kerarinocytes Induced In Vivo and In Vitro and Hepatoma Cells Induced In Vitro

AHH Activiry" (pmols 30HMBA formed/mg of protein/ min)

Antibody'

7811 6C9.4 (pg IgG) 8A In vivo

o 1.25 2.5 5

10

o

1.25 2.5 5

10 (PI ascires fluid)

o 0 I 5

10 25

5 5

4.92'> 1.77 1.11 0.87 0.62 5.02 5.36 5.17 5.31

4.9 0.98 0.49 0.40 0.40

Kc:rarinocytc:s'

'The difference between duphc:ue samples for each cUu POint wu less than S%.. 'Thr source of the anubody was either punfled IgG or hybridoma ascHn fluid.

BA In virro

8.08 7.54 8.82 6.87 6.80

8.1

7.5 8.5

SA In vitro

162 90.7 69.7 50.2 25.9

Hepatoma Cells'

3MC In vitro

231 110 75.5 54.2 38.7

'The source of enzyme' used In Ihls txpenment wu a 9,000 gsupcmat:anl fraction which had a lowc:r AHH content than 2. microsomal prt'J»ration. For the in VIYO group. animau were p:untcd With t rug o£SA 011 th" slun orche ~ck. :md the Jkln wu processed 24 h l;a!er. For the culrur~ group. Ic.er.mnocyta were exposed to 2.5J1M BA. H~PlItolTU cdls w~r~ uposc-d (0 dth~r 1O,uM BA or 30 JIM 3MC. For oth~r eJ[penm~nf;a1 dct;alls see Methods.

to the sum of the inhibitions that would have occurred if each component had been assayed separately (Fig 5).

DISCUSSION

The studies of Bickers and colleagues [cf9,3S-37] have shown that AHH activity ilnd the isozyme of P-4S0 that is a component of AHH can be induced by 3MC. BA. and polychlorinated biphenyl in the epidennis of the rat in vivo, as well as in keratinocytes from the mouse, in culture.

Data reported in the present communication show that exposure to BA in vivo or in vitro results in an increase in AHH activity in che microsomal fraction of rat epidermal cdls. Topical exposure to BA in vivo resulted in a 2.S-fold increase in microsomal AHH activity (Table I). Assay of AHH activity in basal (germinative) and differ­entiated (non-dividing) popubtions, separately, showed thac the level in the differentiated cells was 7 times higher than in the basal cells. A comparison of the normal and induced microsomal activities derived from cells in culture (Table II) ind.icated that an exposure of 24 h co BA resulted in a six- [0 sevenfold increase in the levd of microsomal AHH. The reason for the greater percentage increase in the AHH activity of the cells rhat were induced in vitro as compared with those induced in vivo is nOt obvious but may be a function of the formation of a differem isozyme of P-450 as discussed below. The explanation for the lower level of AHH activity in the unin­duced low calcium culture (60% basal cells) as compared with the basal cells isolated from the untreated epidermis is also not known. Perhaps the newborn animals utilized in these experiments were slightly induced as a resuh of environmental exposure to a PAH during gestation. However, the level of AHH activity in uninduced cultures grown in normal calcium (mostly differentiated cells) was not strikingly different from that in the differentiated population of uninduced epidermis.

Studies were not done to ascertain that the observed increases in the AHH activity involved Jt nOllo protein synthesis. but it is likely that an induction ofp-4S0 did occur. Monoclonal antibody 7B11, which was shown to bind to purified P-450 isozyme 4 (beta­naphthoflavone-induced) but not to isozyme 2 (phcnobarbital-

induced) [221. inhibited 85% of the AHH activity in epidermal microsomes trom rats exposed topically to BA (Table III).

Monoclonal antibody 6C9.4, which inhibits the phenobarbital­induced form ofP-450. did nOt inhibit the AHH activity (Table III) in either the in vivo or in vitro induced epidermal preparations, indicating that cytochrome P-4S0b makes little, if any, contribution to the AHH response under the conditions of these experiments. In fact , a slight increase in the activity of AHH was seen when 6C9.4 was addcd to the reaction mixture during the assay for AHH . Possi­bly. othcr P-4S0 isozymes in the preparation might havc depressed the AHH activity by diverting substrate through another metabolic pathway.

The higher level of AHH activity in differentiated keratinocytes as compared with the germinative population of cells appears to be at variance with data presented by Wiebel et al [39J, who showed in a series of cell lines derived from rat hepatoma line H41lE that the P-450 species found in the less differentiated or "dedifferentiated" derivatives were the types induced by PAH, while the species found in the more differentiated derivatives were of the types induced by phenobarbital. The differentiative state was defined by the secretion of albumin and the presence of the hepatic isozymes of alcohol dehydrogenase and aldolase in the derivative. However, Cudas and Hankinson [40] provided data that indicated that the failure of the more differentiated hepatoma derivatives to express induction of AHH was a funcrion of a deficiency in Ah (aromatic hydrocarbon) receptor activity.

Knutson and Poland [41] have suggested that the Ah recepto r plays a role in the regulation of cell proliferation and differentiation. Although the data in the present paper cannot be used to confirm a regulatory role fo r the Ah receptor ill differentiation, they do sup­port a relationship between differentiation and induction of P-4S0, the protein that is induced when the Ah receptor is activated by a PAH. It is possible that the higher activities of AHH and P-4S0 in the differentiated epidermal cells are a function of an increase in the density of Ah receptors as the cell differentiates.

The activation of procarcinogens such as PAH for binding co DNA is considered to be a step in the initiation of a cell for carcino­genesis [2]. In this context, it appears odd that differentiated cells should have a higher level of AHH than basal cel ls. Mitosis of

VOL. 94. NO. 1 JANUARY 1990

Figure 4. Binding of [he monoclonal anti P-450, 7B II , to differentiating culture of epidermal keratinocytes. Cultures were grown 011 coversl ips. treated, and srcained as described in Methods. More stained cells :ueobserved in treated cultures (A) than in unrreated culcures (B) (800X).

initiated cells is obviously necessary for the development of a carci­noma. Possibly, initiation of differentiated epidennal cells results in a reactivation of rhe cell's ability to double DNA and undergo mitos is. Vaughan et al [42] have shown that differentiated cells retain the ability to replicate their DNA. although they normally do not do so.

Epidermal AHH activity induced in vivo is strongly inhibited by 78 t I, while the activity induced in keratinocytes in culture is mini­mally inhibited. This observation suggests that one of the catalytic proteins responsible for AHH activity is different in the two cases. In fact. the percentage of inhibition that occurs in the AHH activity derived from keratinocytes induced in vitro appears to correspond to the level of AHH present in uninduced cells, although this point requires further study.

Two observations argue against the possibility that a technical artifact accounts for the difference seen in the inhibitory effects of 7B 11 on epidermal AHH induced in vivo and in vitro. First, the: inhibition observed when 7B 11 was added to mixtures of the micro­somal fractions frol11 cells induced in vivo and in vitro was the sum of the degrees of inhibition expected if each were tested separately.

AHH IN BASAL AND DIFFERENTIATED KERATINOCYTES 91

"\

' 00

300

200

100

• , ,

o IOI . o.mWprt)1eIn 0

101 Mute gtO!~n 100

, , , , \ \ , , , , , \ ~

, , ~

,. 7'

, , , , , , , " • -1'111 , ,

\ .1'1"', , , , , ,

50 50

SouI"Ot of AHH

, , , , , , ., , ,

7, 2'

, , , , , , "

, , , .. 100 o

Figure S. Effect of monoclonal antibody 791 1 on AHH activity in mixed preparations of 9,000 X g supernatant fractions from epidermis exposed to DA ill vivo and keratinocyte cul tures exposed to llA in vitro. Puri6ed 7ll t I IgG (5,u1) was used in each case except controls. For other technical details, see Methods. Each data point represents the average of duplicate values. Each value varied by no more than 5% from the mean.

Second, rhe AHH activity in microsomes from hepatoma ce lls in­duced in vitro by either BA or 3MC. using the same conditions as were applied in the case of keratinocytcs, was inhibited to nearly 85% by 7B1!.

The difference between AHH from keratinocytes induced in vitro and in vivo clearly concerns cytochrome P-450, because 7B l l binds to a purified form of cytochrome P-450 obtained from rabbit liver and inhibits AHH activity in epidermal microsomes from newborn rats exposed to BA (Table III). The observation that 7Bl l decorates many more cells in an induced culture as compared with an uninduced cuhure (Fig 4), while not inhibiting the increased level of microsomal AHH derived from the induced culture, sug­gesrs thar the isozyme of cytochrome P-450 induced in vitro differs from the one induced in vivo in some way. In both situations, 7B l1 appears co bind. but the monoclonal antibody inhibi ts the caralytic activity only of thc molecule induced in vivo. Cytochromc P-450 represents a la rge family of isozymes thar differ in their amino acid sequence and rheir immunologic properties [cf 6.7,381. It appears poss ible that the isozyme of cytochrome P-450c that is induced by SA in cultured keratinocytes is structurally difFerenr in some subtle way from that induced in vivo in the epidermis and in vitro in rhe hepatoma line, such [hat 7B 11 binds to the isozyme bur fails to interfere with its cataly tic activity. The mOst obvious explanation for rhis situation would be that the actual inducing molecule is different in vivo and in vitro. This could be true ifBA is metabolized differently in the two conditions. It is also conceivable that the two

92 GUO ET AL

P-450 molecules have different conformations as a result of their synthesis in cells that are existing in different environments. Both would have the epitope specific for 7811. but the spatial relation­ship between rhe epifope and rhe active center would be different so that the antibody would not inhibit the activity of the P-450 synthe­sized in vitro. It is also possible that in vitro a regulatory gene is affected CO activate a different structural gene for P-450 than is activated in vivo. The biosynthesis of different keratin peptides in vivo and in vitro represents a precedent for this rype of phenomenon [431 · Note Added in Proof A paper by DiGiovanni j, Gill RD, Nctti ­kumara AN, Colby All, and Reiners 11 1 r, entided Effect of Extra­cellular Calcium Concentration on [he Metabolism of Polycyclic Aromatic Hydrocarbons by Cultured Mouse Kcratinocytes. which reports complementary experiments and conclusions. will be pub­lished shorrly in Cancer Research .

Wr IVlsl, ro rxpms our .~ ratlt'4dt to Dr. XI1IXi" Ding. Dt!partmfPlt of Bio lo.~i(al Chemistry. Tnt U,m't.'wty of Mich',~Qn. for his aSSislQnct l4Ii(h (ht sptctropho(()­mrtrie mraSUTl'Pnr"t of ryrochromt P-450. Wtalso wish (0 (hank 0, Minor J Coon

anJ Dr. Dmgf'" ttlltorial as.cista"u

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