Human skin levels of retinoic acid and cytochrome P 450 derived 4 hydroxyretinoic acid after topical application of retinoic acid in vivo compared to concentrations required to stimulate retinoic acid receptor mediated transcription in vitro

Human skin levels of retinoic acid and

cytochrome P-450-derived 4-hydroxyretinoic

acid after topical application of retinoic acid in

vivo compared to concentrations required to

stimulate retinoic acid receptor-mediated

transcription in vitro.

E A Duell, … , P Chambon, J J Voorhees

J Clin Invest.

1992;

90(4)

:1269-1274.

https://doi.org/10.1172/JCI115990

.

Metabolism of retinoic acid to a less active metabolite, 4-hydroxyretinoic acid, occurs via

cytochrome P-450 isozyme(s). Effect of a pharmacological dose of retinoic acid on the level

of retinoic acid in skin and on cytochrome P-450 activity was investigated. A cream

containing 0.1% retinoic acid or cream alone was applied topically to adult human skin for

four days under occlusion. Treated areas were removed by a keratome and a microsomal

fraction was isolated from each biopsy. In vitro incubation of 3H-retinoic acid with

microsomes from in vivo retinoic acid treated sites resulted in a 4.5-fold increase (P =

0.0001, n = 13) in its transformation to 4-hydroxyretinoic acid in comparison to in vitro

incubations with microsomes from in vivo cream alone treated sites. This cytochrome P-450

mediated activity was oxygen- and NADPH-dependent and was inhibited 68% by 5 microM

ketoconazole (P = 0.0035, n = 8) and 51% by carbon monoxide (P = 0.02, n = 6).

Cotransfection of individual retinoic acid receptors (RARs) or retinoid X receptor-alpha

(RXR-alpha) and a chloramphenicol acetyl transferase (CAT) reporter plasmid containing a

retinoic acid responsive element into CV-1 cells was used to determine the ED50 values for

stimulation of CAT activity by retinoic acid and its metabolites. Levels of all trans and 13-cis

RA in RA-treated tissues were greater than the ED50 values […]

Research Article

Find the latest version:

Human Skin

Levels of Retinoic

Acid

and

Cytochrome P-450-derived 4-Hydroxyretinoic Acid after Topical

Application

of

Retinoic Acid In Vivo Compared

to Concentrations

Required

to

Stimulate

Retinoic

Acid Receptor-mediated Transcription In Vitro

ElizabethA.Duell, Anders Astr6m, ChristopherE.M.Griffiths, PierreChambon,* and John J.Voorhees

Department ofDermatology, University ofMichigan,AnnArbor, Michigan 48109-0528;and*Laboratorie deGenetiqueMoleculaire des Eucaryotesdu Centre National de la RechercheScientifiqueU.184Institut National de la Sante et de la RechercheMedicale,

Faculte de Medecine, Strasbourg,France

Introduction

Metabolism of retinoic acidto a less active

metabolite,

4-hy-droxyretinoic

acid,

occurs via

cytochrome

P450

isozyme(s).

Effect ofapharmacologicaldoseof retinoic acidonthe level of retinoic acid in skin andoncytochrome P-450

activity

was in-vestigated. A cream

containing

0.1% retinoic acid or cream alone wasapplied

topically

toadulthuman skin forfour

days

under occlusion.Treated areas wereremoved

by

a keratome anda microsomal fractionwas isolatedfrom each

biopsy.

In vitro incubation of 3H-retinoic acid with microsomes from in vivo retinoic acid treatedsites resulted ina4.5-fold increase

(P

=

0.0001,

n =

13)

in its

transformation

to

4-hydroxyretinoic

acid in

comparison

to in vitro incubations with microsomes from invivocreamalonetreatedsites. This

cytochrome

P.450 mediated activity was oxygen- and NADPH-dependent and

wasinhibited68% by 5

,M

ketoconazole(P=0.0035,n =

8)

and 51% bycarbonmonoxide

(P

=

0.02,

n =

6).

Cotransfection of individual retinoic acidreceptors

(RARs)

orretinoidXreceptor-a

(RXR-a)

anda

chloramphenicol

ace-tyl transferase

(CAT)

reporter

plasmid containing

aretinoic acid responsive element into CV-1 cellswasusedtodetermine

the

_ED50

valuesfor stimulation of CAT

activity

by retinoic acid andits metabolites. Levels of

al

transand

13-cis

RAin RA-treated tissues were greaterthan the

_ED5,

valuesdetermined for all three RARs with these compounds.

Furthermore,

the

level ofall trans RA was greater than the

_ED5o

for RXR-a whereas the 4-OH RA level was greater than the

_ED5.

for

RAR-,Band

RAR-'y

but lessthanforRAR-aandRXR-a. These

datasuggestthatthereare

sufficient

amountsof retinoic acid in treatedskintoactivategenetranscriptionoverbothRARsand

RXR-a. (J. Clin. Invest. 1992. 90:1269-1274.) Key words:

high performanceliquid chromatography*

P450 inhibitors.

reporter assay.

_ED50

values

This workwaspresented inpart atthe annualmeeting oftheSociety for Investigative Dermatology, Seattle,WA, 1-4 May 1991.

Addressreprintrequests to Dr.ElizabethA.Duell,Departmentof Dermatology,University ofMichigan Medical Center, R6558KresgeI, Box0528,AnnArbor,MI48109-0528.

Receivedforpublication 21 October 1991 and in revisedform 11 February1992.

Retinoidssuchasalltransretinol

(ROL)'

and alltransretinoic acid(RA)areimportantinepithelial proliferationand differ-entiation( 1-3)andrepairofphotodamagedskin(4, 5).ROL is stored in tissuesas

retinyl

esters

(6-8)

orboundtocellular

retinol binding protein (CRBP) (3). LittleornoRA is stored in tissues. Retinoidspresent in human skin areROL,

retinyl

esters,3,4-didehydroretinylesters, and RA(9).RA and

3,4-di-dehydroRA(ddRA)act asimportant morphogensin the chick

wing budsystemeither directly (2)orindirectly (10, 11). The majorretinoic acid receptor(RAR)form expressed in the

epi-dermis is

RAR-'y

( 12,

13). Expressionofretinoid Xreceptor-a (RXR-a)has also been demonstrated in human skin( 14).

Metabolism of RA occurs via a cytochrome P-450(P-450)

mediated enzyme system in tissues such as liver ( 15-17), tra-chea( 15),intestine(16), and rodent skin ( 18 ). Theprimary product formedis4-OH RA which is further transformedto 4-oxo RA. Topical application of RA to skin of 4-d-old rats

increasesRA metabolism fourfold. In vitro ketoconazole in-hibits conversion of RA to 4-OH RA bymicrosomesfromrat

epidermis ( 18 ).Inliver the formation of 4-OH RA via P-450

isozyme(s)isinhibitedboth in vivo and in vitro by ketocona-zole ( 19).

Inthis study a single topical dose of 0.1% RA cream or creamvehicle was applied to adult human skin andkeptunder constantocclusion. After 96 h vehicle- and RA-treated sites weretested for their capacity to metabolize RA and were

ana-lyzedfor RA and RA metabolite content. RA and its

metabo-lites were also tested for theircapacitytoinducea, RAR-(,RAR-'y,orRXR-a-mediatedtranscription ofa reporter gene inacotransfectionsystem.

Methods

Isocitrate, isocitratedehydrogenase, NADPH,NAD, 13-cisRA,and RAwerepurchased fromSigmaChemical Co. (St. Louis, MO).CV-l cells, African Green Monkey kidney cells,were purchasedfrom Ameri-canType Culture Collection (Rockville, MD).3H-RA was obtained from DuPont NEN (Boston, MA)and ['4C]chloramphenicol from AmershamCorp. (Arlington Heights,IL).RA metabolites 4-OH RA, 4-oxo RA, 13-cis 4-oxo RA,and5,6-epoxyRAweregifts fromDrs.

1. Abbreviations used in this paper: CAT, chloramphenicol acetyl transferase; CRBP, cellular retinol binding protein; ddRA, 3,4-didehy-dro retinoic acid; RA, alltransretinoic acid; RAR,RA receptor;ROL, alltransretinol; RXR-a, retinoidX receptor-a.

Abstract

J.Clin. Invest.

C©The American Societyfor Clinical Investigation, Inc. 0021-9738/92/10/1269/06 $2.00

Michael Rosenberger and P. F. Sorter of Hoffmann-LaRoche (Nutley, NJ). Metabolite 3,4-ddRA wasagift from Drs. I. Kompis and M. Klaus ofHoffmann-LaRocheAG (Basel, Switzerland). The 0.1 % RA (Retin-AO)andvehiclecream weresupplied by Ortho Pharmaceutical Corp.(Raritan,NJ). HPLC gradeacetonitrileand methanol were pur-chased fromBurdick& JacksonLaboratoriesInc., adivision ofBaxter Products (Romulus, MI). Spherisorb ODSl column was purchased fromPhaseSeparations Inc. (Norwalk, CT).

RAapplication and biopsy procedure. 0.5 g of RA (0.1% Retin-Ae cream) and 0.5 g of vehicle cream were applied to separately marked 3 x 2 inch areas on the buttock region of adult volunteers and kept oc-cludedfor four dayswithSaran Wrap and secured with surgical tape (20). Atfourdays test areas were evaluated for erythema, washed, and keratomed under 1%lidocainelocal anesthesia (20). In experiments to determine RA and RA metabolite levels, all three RA-treated areas were washed with chlorhexadine soap solution. In addition, two sites were washed with ethanol and one of the ethanol plus soap-washed siteswastape-strippedto"glistening", i.e., forremovalofstratum cor-neum,before keratoming. Epidermalsamples were immediately fro-zen inliquid nitrogen and stored at -80'C until used. Skin punch biopsies (2 mm)were takenfromtape-strippedsites and placed in 10% neutralbuffered formalin.Thesebiopsieswereprocessedfor hematox-ylinandeosin histological analysis ofadequacyofstratum corneum removal bystripping.All subjects gave informed, written consent. The study wasapproved bythe University of Michigan Medical Center Institutional ReviewBoard.

RAmetabolism assay. Microsomal fractions were prepared asprevi-ously described (21 ).RAand RAmetaboliteswerekeptin amber glass in darkenedroomswith yellowlightingwheneverpossible.The rateof RAmetabolismwasdeterminedby incubating 100 gg microsomal pro-tein in 0.01 Mphosphate buffer (pH 7.4) containinganNADPH re-generatingsystem(21),and 3H-RA(1 uM,spact 1 mCi/0.I

_timol).

Sampleswereincubated for 15-45 minat350C.Theaddition of100M1 methanolcontaining 100

,g/ml

ofbutylatedhydroxytoluene cooledto -20°Cterminatedthereaction.Aftercentrifugationat1,000gfor10 min, the supernatantfractionswereanalyzed forRAmetabolitesby reverse-phaseHPLC andliquid scintillationspectrometry.

Ketoconazole,aninhibitorof cytochrome P-450,wasincubatedat 35°C for10 min with all componentsofthe assay present except for 3H-RA whichwasaddedto startthereaction.Forincubationswith CO asinhibitor,all componentsofthe assaywerepresent but tubeswere keptoniceduring 15 minflushingwith CO. Tubesweresealed and transferred to a 35°Cwater bath forformation of RAmetabolites. Control tubeswerehandled in thesame mannerexcept theatmosphere

wasnotequilibrated withCO.

HPLCseparation ofretinoids.AnHPLCsystem(1090M;Hewlett PackardCo.,PaloAlto,CA)containingaSpherisorbODSl column (PhaseSeparations Inc.,25cm X4.6mm),adiode arraydetector,and chemworkstationwasusedtoseparate retinoids. Mobilephasesused forgradientelutionofretinoidswerethoseof Buggeetal.(22).Mobile phase A was acetonitrile:0.02 M ammonium acetate:acetic acid (1:1:0.01) and mobilephaseBwasacetonitrile:0.2 M ammonium ace-tate:aceticacid( 19:1:0.008).At thestartoftherunsolventAwas100% followedbya lineargradientto 30% solvent Bat3 min, ashallow gradientto81%solventB at38 min, and 100% solventB at40 min.The flowratewas0.5ml/minand the total time for theseparationwas45 min. Effluent from the HPLC column floweddirectly into a flow-through scintillation spectrometer (Beta-One; Radiomatic Instru-mentsandChemicalCo., Inc.,Tampa,FL)withacomputerizeddata capture systemtodetermineradioactivityin eachmetabolitepeak.The amountofmetabolite formedwascalculated basedonthespecific activ-ity of added retinoic acid.

Extraction ofretinoidsfrom biopsy samples. Frozen keratome biopsies,eachweighing - 120 mg,werepowderedandimmediately

transferred into 20 vol of chloroform:methanol(2:1 ) containingtracer amounts of3H-RA.Retinoids were extractedwithgentlemixingfor 15 min followed bycentrifugation at 1,000g for 10 min. Supernatant fractionswereremoved and saved. Pelletswerereextracted with 5 vol

of the same solvent. After centrifugation supernatant fractions were savedand pellets were extracted with methanol:water acidified to pH 3.2 (95:5). After centrifugation the three supernatant fractions were combined and were evaporated to dryness under a stream of nitrogen. Samples were resuspended in methanol for HPLC injection. Recovery ofretinoids and assessment of nonenzymaticisomerizationof alltrans RA to 13-cis RA during extraction procedure were based on the amount of3H-RArecovered as3H-alltrans or 3H- 1 3-cis RA in compar-ison to the amount initially added as all trans3H-RA.

Cotransfection ofCV-J cells and reporter assay. Procedures re-ported by Astrom et al. (23) were used to determine

ED50

values for chloramphenicolacetyltransferase (CAT) activity by RA and RA me-tabolites.CV-l cells were cotransfected with RAR-a,

_RAR-f3,

RAR-y,

or RXR-a expression vectors and the reporter plasmid TRE3-tk-CAT. Human RXR-a cDNA (24) was cloned from human skin as described ( 14)and subcloned into the expression vectorpSVL(Pharmacia LKB Biotechnology, Inc., Piscataway, NJ) for use in the transfection assay ( 14). The effect of variousconcentrationsof RA and RA metab olites (10-"-5 x 104 M) on CAT activity was determined as de-scribed (23).

Other methods. Statistical analyses for all assays were performed using a two-tailed paired t test. Protein content was determined by the method of Lowry et al. (25) with BSA as standard.

The concentrations of retinoids in skin were calculated as follows. Thebiopsies were weighed before pulverization. The recovery of unla-beled material was equated to the recovery of added3H-RA. Calibra-tion curves for the retinoids were stored in the HP-1090 HPLC unit. Theabsorption (peak area) from each sample was converted to nano-grams of retinoid based on the calibration curves. The nanonano-grams of retinoiddivided by the percent recovery gave the value for total reti-noid. This value was divided by the wet weight in grams to give micro-grams per gram skin. 1 g wet wt of tissue was equivalent to 1 ml ofwater for purposes of calculating molarity.

Results

Retention times for nonradioactive retinoid standards are

shown in chromatogram depicted in Fig. 1A.RAmetabolites separated by HPLC after in vitro incubation of 3H-RA with microsomal fractions obtained from biopsies treated in vivo withvehiclecream or0. 1% RAcream areshown in chromato-gramsdepicted in Fig. 1 B(vehicle)and Fig. 1 C (0.1% RA).

Incubation of 3H-RA with microsomes fromRA-treated sites showed

increased

formation of4-OH RA, 4-oxo-RA, andmore

polarmetabolites in comparison to metabolites generated by microsomes from vehicle-treated sites.

Formation ofRAmetabolites wastime (10-60min) and

protein concentration (100-400 ,ug) dependent (data not

shown).Datashowing increased P-450mediated RA metabo-lism four days aftera single topical

application

of 0.1% RA creamin comparison to vehiclecreamfor 13 normal

volun-teers aregiven in

Fig.

2. Therewas a4.5-fold (P= _0.0001) increase in formation of4-OH RA (124.4±17.4pg/min per mgmicrosomal protein)bymicrosomes from RA-treated sites in comparison to formation of metabolites by microsomes

from vehicle treated sites (27.7±5.5 pg/min per mg micro-somal

protein).

There was a 2.6-fold increase in 4-oxo RA

formation (P=_{0.0003) and}a4.8-fold increase in formation of

morepolar metabolites (P=_{0.007) in RA-treated}versus

vehi-cle-treatedareas.

Datashowing inhibition ofP-450mediated

activity

by

ke-toconazolearegiveninFig.3.Withbiopsies fromRA-treated

sites assourceof microsomestherewas a68% decrease in4-OH

RAformation

(P

=

0.0035,

n=

8)

inincubations

containing

5

micro-( ₆₁

E

41

21

% 6,00

a

CM

a

4,00

U

a

.2

2,00

6,00 U IL

a

>.' 4,00

.U

.2

2,00

la

0-O\

-!o

-0O

0-0'

TIME (MIN)

I I

10 20

TIME (MIN)

z W 200

I-0

2150

2 1

*t5

30

I0

-C

10

10

0-,.

A

10 20

TIME (MIN)

30

Figure 1. Reverse-phaseHPLCseparation of retinoidsasdetailed in Methods.(A)Chromatogramshowsretention times for nonradioac-tiveretinoidstandards.(B)Depictsthechromatogramobtained after invitro incubationof3H-RA with microsomal fractions from vehi-cle-treatedsites. (C) Depictschromatogramobtained after in vitro incubation of3H-RAwithmicrosomal fraction fromRA-treatedsites.

T

T

IL-w

UR

umE

Figure3. Ketoconazoleinhibits retinoic acid-induced cytochrome P-450-mediated transformation of retinoic acid into 4-OHretinoic acid. Conditionsfor in vitroassayofthemicrosomal fraction from RA-treatedsitesandanalytical proceduresaredetailed inMethods. Comparisonof3H-RAversus3H-RA+ 5 uMketoconazolegave the followingpercent decreasesand P values:4-OHRA(68%; 0.0035), 4-oxo RA(82%; 0.003),andpolarmetabolites (53%; 0.029).n =8. *,3H-RA- 1

_AM;

o, 3H-RA+KETO-5MM.

somal protein) in comparison to 3H-RA alone (154.1±26.2 pg/min per mgmicrosomal protein). There weresignificant

decreases in formation of 4-oxo RA (82%, P = 0.003, n = 8)as well as more polar metabolites (53%, P = 0.029, n = 7) in

incubations containing ketoconazoleand 3H-RA in compari-son to3H-RA alone. As shown inFig. 4,COsignificantly de-creased by 51%(P=0.02,n = 6)formation of 4-OH RA.

Concentrations of retinoids required to obtain half-maxi-mal stimulation

(ED50)

of CAT. activity by RAR-a,

_RAR-fl,

RAR-y,

or RXR-a in cotransfected CV-1 cells aregiven in Table I. Data for RAR-,y, the major receptorexpressedin epi-dermal cells, show that RA and 3,4 ddRA have an

_EDm

of 1-2 nM while 4-oxo RA requires a 10-fold higher concentration (13 nM) and 4-OH RA a 20-fold higher concentration (30

nM). The 5,6epoxy RA ED50for

RAR-Ey

was49nM. To determinewhetherconcentrations of RA or its metabo-litesreachedsufficient levelstopotentially activate

transcrip-tionby RARs or RXR-a after topicalapplicationofRA,levels of RA and its metabolites present in viableepidermisat four

daysweredetermined. Fig. 5 shows that washing the sites with

soap and ethanol was notsufficienttoremove RAremaining

onthesurface of skin. After tapestripping theamountofRA

measured decreased 80% whereas 4-OH RA levels remained

z W 125

I. 100'

75

8

25'

E 0'

4-OHRA 4-OXO-RA POLAR METABOLITES

Figure2.Retinoic acidtreatmentof human skin in vivocauses

cy-tochromeP-450 mediated increased retinoic acid metabolism.In vi-troassayconditions and analytical proceduresaredetailed in

Meth--ods.Comparison of RA-treatedversusvehicle-treated sitesgavethe

followingpercentincreases and P values:4-OH RA(349%; 0.000 ), 4-oxo RA(162%; 0.0003),andpolarmetabolites (381%;0.007).n

= 13.m,VEH;o,RA.

7r

4-OH-RA 4-OXO-RA

Figure 4. COinhibits cytochrome P-450-mediated retinoic acid

me-tabolism into 4-OH retinoic acid. Microsomal fractions from sites treatedinvivo with 0.1%RAcream wereusedin in vitroincubations.

Assayconditions and analytical proceduresaredetailed in Methods.

Acomparison of incubation withRA+COversusRAalonegavethe

followingpercentdecreases and Pvalues:4-OH RA(51 %;0.02)and 4-oxoRA(53%; 0.07).n=6. .,3H-RA- I MM;o,3H-RA+CO

- MM.

z i 150 E 125'

IL -i

R 100' 0 75'

I 50

25 o5

TableI. Concentrations of Retinoids RequiredtoObtain Half-Maximal Stimulation

(ED5,)

of

TRE3-tk-CAT

Transcription by Retinoic Acid Receptorsand RXR-aCotransfectedintoCV-1 Cells

ED50

Compounds RAR-a RAR-,B RAR-Dy RXR-a nM

RetinoicAcid 24* 4* 1.3* 236

dd-Retinoic Acid 34 13 1.8 245

4-oxo-Retinoic Acid 79 28 13 377

4-OHRetinoic Acid >500 75 30 >500 5,6-EpoxyRetinoic Acid 237 56 49 >500

Data representtheaverage valuefortwo tothreetitrations from101 to5X

l-4

Mretinoid concentrations. * These datapoints forRA werepreviously published (23)andareincludedtofacilitate compar-isontometabolitedata.

the sameregardless ofthe method of tissue preparation. All transand 13-cisRA were present in almost equal amounts.

The 13- cisRA present intissuewasnotduetononenzymatic

isomerization

during extraction since 91.2%±1.7% of the

'H-RAaddedastracer todeterminerecovery of materialremained

asalltransand 8.8%±1.1%was 13-cis RA. As shown in Table

II,

all

trans RA was present as824 ng/gmwetwt or- _2.75

_,uM

concentration

(three

log units higher

than the

_ED50

for

RAR-y),

13 cis-RAas745 ng/gm wet wt or - 2.5

tiM

(60X

ED"o

value

for

RAR-y),

and4-OHRA as93 ng/gmwet wt or - 0.3

,M

concentration

(10X

_ED50

value for

RAR-'y).

4-Oxo RA wasnotdetected in RA-treated

biopsies

even

though

epidermal

microsomes from similar

biopsies

showed conversiontoboth 4-OH and 4-oxo RA.

Discussion

The

effects of

daily application

ofRA without occlusion to

photodamaged skin

havebeen

investigated

in clinicalstudies (4,5). The

improvement

seen

is

variable and dose

dependent

(4,5). Pilot studies with 0.1%RAcreamwithorwithout

occlu-sion

used

topically

for various

periods

of time showed that RA

under occlusion for four

days

gavea

highly

consistent

erythem-atous

reaction

as well as characteristic

histological

(20)

re-7

5

0

WASH WASH+EtOH WASH

EtH

+STRIPPED

Figure 5. Retinoidcontentinareastreated fourdayswith0.1%RA creamfollowedbyremovalof surface RAbytheindicated methods. Extractionproceduredetailed in Methods. Concentrations instripped

tissuesampleswerealltransRA( 2.7

_AM),

13 cis-RA

(nu

2.5

MAM),

and 4-OH RA(-- 0.3

MM).

n= 5foreachmethod ofsurfaceRA removal.o, all trans RA; m, 13-cisRA;

.,

4-OHRA.

Table I. Comparison ofTissueLevelsafterTopical Application

ofAllTrans RetinoicAcidto HumanSkinIn Vivowith

ED50

ValuesforTranscriptionalActivation of

TRE3-tk-CA

T in a

CotransfectionAssay

alltransRA 13-cis RA 4-OH RA

ng/g wet wt 824±281 745±24 93±36

nM 2750 n~2480 310

Retinoid(nM)

ED50RAR-a 24* 190* > 500

EDORAR-#

4* 43* 75

ED50RAR-y

1.3* 140* 30

ED,1RXR-a 236 ND > 500

*_{These datapointswerepreviously published (23) and are included}

forfacilitatingcomparisons.

sponsein 9

of

10patients. Becauseof thisconsistent retinoid response,thefourdayoccludedpatch was used in the current

study.

Routine

nonoccluded clinical use ofRA cream

pro-ducedaninconsistent retinoidresponse too variable for

experi-mentation.

Inthepresent study asingletopical application of RA with

occlusioncontinuously for four daystoadult human skin

re-sulted in

sufficient

retinoic acid penetration into viablelayers

of the

epidermis

toinduceRAmetabolism.Our data and that

of

others (

18)

suggestthat

this

increased metabolism is

proba-bly dueto

increased

P-450

activity.

Furthermore, inductionof

P-450

activity

in response to RAhasbeen reported in other

tissues ( 15-19).Inaddition, 5,6epoxy RA can beformed

ei-therbya

lipid peroxidase

(26) or aP-450-mediated activity (27). However, the P-450 isozyme(s) induced inhumanskin inresponse to

topical

RAis unknown.

P-450enzymespurified from rabbit liverhave been

evalu-ated for

their

capacity

to metabolize RA. IA2 and IIB4

iso-zymes were mosteffective in metabolizing RA to 4-OH RA

while

IIC3,IIE 1,IG2, and IIE2wererelatively ineffective,and

isozymes

IA 1 andIIIA6wereineffective (28).

Cultured human

keratinocytes

have beenshowntocontain the mRNA for P-450

isozyme

IA 1 (29). However, the IAI

isozyme

was oneoftwoforms

from

rabbit liver that didnot

metabolize

RA. An

immunohistochemical

study of cultured human

epidermal

foreskin keratinocytes showed binding of monoclonal antibodiesto

isozymes

belongingto

families

IIand

III

(30).

Inneonatalrats

topical application of

3-methylcholan-threnewas more

effective

than

topical application of

RA in inducing RA metabolism by P-450 isozyme(s)(18).

Pheno-barbitolwastheleasteffective inducer ofRAmetabolism ( 18).

In hamsters intraperitoneal injection of methylcholanthrene increased RA metabolism in liver twofold while orallygiven

RA increased RA metabolism eightfold in liver ( 16). Thus

metabolism ofRA in response to inducers of P-450 activity

may vary as a result ofroute of administration, species in-volved, ortissuestudied.

Cytochrome

P-450mediated metabolism ofRA may bea

significant mechanism for

regulation

of

pharmacological

RA levels invivo.Infacttheconversion ofRAtolessactive

attenuatesphysiologically relevant, endogenous RA levelscan notbe addressed with the data presented here. However, our

results do demonstrate that topical application ofpharmacolog-ical dosesof RA in humans induce a 4.5-fold increase in

metab-olism

ofRAto4-OH RA andmorepolar metabolites. Keto-conazole,atopical antifungal agent currentlyin routine

clini-cal use, is an inhibitor ofsome P-450 mediated activities.

Ketoconazoleatconcentrations of 7X

10-7

Mto5 X 10-6M

inhibits

invitroRAmetabolism

by

rat

epidermal

microsomes. Similarconcentrationsarerequiredtosuppresscertain P-450 dependent reactions in steroid synthesis (19). One possible side effect of ketoconazoleuse is increased RAlevels dueto

decreasedRAmetabolism. Thismaybe of clinical importance inas muchas simultaneoususe oftopical ketoconazole and

RAcould decreasetheamountof RA requiredtoobtain clini-calefficacy.

Todetermine thepotency

of

ddRA and RA

metabolites

in

comparison

to RA and 13-cis RA in RAR- and RXR-me-diated

transcriptional activation,

a reporter gene (TRE3-tk-CAT)wascotransfected together withreceptorexpression vec-torsinto

CV-l

cellsandconcentrations

required

toobtain half-maximal induction

of

CAT

activity

(ED50)weredetermined.

Similar

reporter assay systemshave beenestablished with cell lines other than CV-1cells. The

ED50s

observedforRAin these

systems varywith RARreceptor typerather than the cell line used

for

cotransfection

procedure (23, 24).

The CV-l cells used in thereporter assay are

epithelial

cells but the

appropriate-ness

of

anyin

vitro

system to in vivo occurrences is always

open to

question.

Of

the three known

RARs,

RAR-'y

is the

major expressed

form in human skin (

12, 13)

and inthereporter assay system

used in these

experiments

RAR-,y has the lowest ED50

for all

trans RA(1.3 nM)

(23),

alltransddRA(1.8 nM),

all

trans

4-oxo-RA (13 nM), and

all

trans4-OH RA (30 nM). The

ED50valuesare two- tofourfold higher forRAR-,B than those

determined for

RAR-y

except for

all

trans ddRA which is

seventimes

higher.

The ED50 valuesforRAR-a werethe

high-est

obtained with

any

of

the three RARs. The

ED50

valuesfor

all

trans RA(23) and

all

transddRA

with

RAR-awassimilar

tothe valueobtained with 4-OHRAand

RAR-'y

(30 nM). The

ED50of 4-OH RA for RAR-a wassimilarto thatofRXR-a

(> 500nM).RXR-ahas beenshowntobeexpressed inskin in vivo (14)but demonstrates higher ED50 values for all

com-pounds tested comparedtoRARs.TheED50sinthe presenceof

RXR-a are20times higher foreach compound in comparison tovalues

for

RAR-y.

The lowest

_{ED_0}

for 13-cis RA is 43 nM

withRAR-,B receptor (2

3).

The value for 1 3-cis RA with RAR--yis 140nM(23) (TableII).

Todetermine whether metabolites mightplay a significant

role inretinoid effectsobservedfollowingtopical application of

RA, treated siteswere analyzedfor presence of RA and RA

metabolites.

Washing withsoap and ethanol was not sufficient to remove RAstillpresent on the surface and within stratum corneumof skinasevidenced

by

4

_jig

RA per gwet wt

(

12

,uM)

in the

specimen.

Removal ofstratumcorneum and

sur-faceRAwithtapestripping reduced these values to 1.5

,tg

RA per g wet wt (5 ,M). The method of preparing the site for

biopsy didnotchange the level of 4-OH RA (93 ng/g wet

wt,

0.3 ,uM).

Thissuggests that the presence of

4-OH

RA indicates

increasedRAcontent and increased metabolism of RA within

living

layers

of

_epidermis

without

_{necessitating}

_tape

_stripping

ofskintodetermineRAmetabolism.

Intape-stripped biopsy samples, 53% ofRA extracted was

alltransRA(2.7

,qM)

and 47%was 13-cis RA(2.5

,M).

This concentration of alltransRA is

2,000

times higher than the

ED50

for

RAR-y

and50-foldhigher thantheED50 forRXR-a

inthe cotransfectionassay.Thehigh levels of extracted13-cis RA aftertopical application of alltransRAandapparentlack ofsignificant nonenzymatic isomerization during retinoid

ex-traction from tissue suggests that

epidermis

may contain an

active RA isomerase. Theamountof 13-cisRAin these

sam-ples

is

10-fold

higher

than the

EDOs

in thereporter assay

of

140

nMwith

RAR-y

and 190nMwithRAR-aand50-fold higher than the

_ED,0

of43nMfor RAR-3

(23).

The4-OHRAlevels

were 10times

higher

than the ED50determined for RAR-,yand

four times

higher

than the ED50 for RAR-gi but less than the

ED50

of> 500 nM with RAR-a and RXR-a in the cotransfec-tionassay.

The above

comparisons

aremadeonthe

assumption

that concentrations

ofretinoids

in skin havefunctional

activity

sim-ilartothatobserved when thesame

retinoids

areaddedtocell cultures

containing genetically engineered

retinoid response systems.

However,

theremaybe

unrecognized

differences

be-tween invivo and in vitro cell

penetration, distribution,

and metabolism.

In

conclusion,

the effects observed

clinically

aftertopical

application

of

pharmacological

doses ofalltrans RA under occlusionfor four

days

may be due in partto

all

trans RAand 13-cis RAaswellas4-OH RA. The large

quantity of all

trans RA as well as lesser but

substantial

amounts

of 13-cis

and 4-OH RA in relation totheconcentration

required

for half-maximal

activation of transcription

in the

cotransfection

assay suggeststhat the

biological

responseof skin

(20)

inresponseto RA under short-term occlusion maybe

mediated

atleast in

part

by

both RARs and RXR-a. Whether molecular

changes

in

skin inresponsetoRA

application

canbemediated by

RAR-and

RXR-a-independent

mechanisms remains unknown.

Acknowledgments

WewishtothankUlrikaPetterssonforexcellenttechnical assistancein the reporter gene assay, Robin Gardnerfor providing biopsy materials, and TedHamiltonfor statisticalanalyses.

Thisstudywassupportedin partbyagrantfromR. W. Johnson PharmaceuticalResearchInstituteand theBabcock Endowment.

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