1. Introduction

In vitro assays that use biomarkers associated withthe carcinogenic process are currently used by theNational Cancer Institute to screen potential cancerchemopreventive agents. Early markers associatedwith the neoplastic process in rodent and human cellsare associated with defects in the regulation ofcellular differentiation and proliferation [1–3].Human keratinocytes have been used in a number ofstudies to explore the relationships of differentiation,cell proliferation, and oncogenic transformationincluding the impact of various chemical agents onthese processes [4–7]. Studies in our laboratorieshave permitted the development of a cancer chemo-preventive agent assay that uses apparently normalhuman keratinocytes. Although normal human ker-atinocytes do not reproducibly produce carcinogen-induced cell foci in vitro, repeated exposure tothe carcinogen, propane sultone, enhances cellulargrowth and reduces involucrin expression comparedwith that observed in untreated control cultures[8, 9]. These changes require repeated exposure andsufficient culture time or cellular doubling to allowthe production and/or accumulation of carcinogen-altered cells. The changes suggest that propanesultone exposure may either inhibit normal differen-

tiation or enhance the growth properties of cells thathave not differentiated.

Continuous exposure to retinoic acid and othercancer chemopreventive agents can inhibit theenhanced growth and induce involucrin expressionwhen compared with the cultures exposed to propanesultone alone (

Note: Involucrin is a keratin precursorthat is found just inside the cell membrane boundary[5, 7]). In this screening assay, cellular growth, eval-uated using methylene blue staining of cultures, andinvolucrin expression, evaluated using antibodystaining, are used as biomarkers to screen chemicalagents for potential chemoprevention activity.

2. Materials

A. Medium and chemicals01. Keratinocyte growth medium (KGM), No.

CC3001.1

02. Trypsin/EDTA, No. 15400-021.2

03. Fetal bovine serum (FBS), No.16000.010.2

04. Dulbecco’s phosphate buffered saline (PBS),No. 14287-023.2

05. Methanol, No. M 3641.7

06. Methylene blue, No. MB1.7

07. Absolute ethanol, 200 proof, No. 0290.14

Methods in Cell Science 19: 13–18 (March 1997) 1997 Kluwer Academic Publishers. Printed in the Netherlands.

The human epidermal cell assay for chemopreventive agents

Eugene Elmore1,3, Chi Sun2, Hong-R Li2, Gail P. Wyatt4, Julie A. Buchmeier1,3,Vernon E. Steele5 & J. Leslie Redpath2,3

1 Department of Medicine, 2 Department of Radiation Oncology, and 3 Cancer Center, Univerity of California Irvine,Irvine, California, USA; 4 ManTech Environmental Technology Inc., Research Triangle Park, North Carolina, USA;5 Chemoprevention Branch, Division of Cancer Prevention and Control, National Cancer Institute, Bethesda,Maryland, USA

Accepted in revised form 9 October 1996

Abstract. Methods are described for a humankeratinocyte assay for screening cancer preventiveagents. Normal human keratinocytes at the first orsecond subculture are seeded into multi-well dishes.Cultures are repeatedly exposed to the carcinogen,propane sultone, and nontoxic concentrations ofpotential chemopreventive agents. At the end of the

Key words: Carcinogen biomarker, Chemoprevention, Growth inhibition, Human keratinocytes, In vitrotoxicity

Abbreviations: AEC = amino-ethyl carbazole; KGM = keratinocyte growth medium; FBS = fetal bovineserum; EDTA = ethylenediamine tetraacetic acid; DMSO = dimethyl sulfoxide; RA = all-trans-retinoic acid;PS = propane sultone; PBS = Dulbecco’s phosphate buffered saline; T-25 = 25 cm2 culture flask; T-75 =75 cm2 culture flask

exposure period, the cultures are evaluated forgrowth and involucrin expression. Data are analyzedto determine the potential for test agents to inhibitpropane sultone induced growth or to induce involu-crin expression relative to that in propane sultonetreated controls.

08. Dimethyl sulfoxide (DMSO), No. D5879. 7

09. Chlorine bleach.10. Rabbit anti-human involucrin antibody, No.

BT 601. 8

11. Amino-ethyl carbazole, No. A5754. 7

12. Goat anti-rabbit antibody, No. A102PN. 9

13. Tryptocase soy broth, No. T8907. 3

14. Thioglycollate medium, No. T9032. 7

15. Paraformaldehyde, No. P6148. 7

16. Propane sultone, No. P8643. 7

17. Acetone, No. P4206. 7

18. All-trans-retinoic acid, No. 2625. 7

19. Keratinocytes, No. CC2503. 1

20. Glacial acetic acid, No. 412-1. 4

21. Hydrochloric acid, No. SA48-500. 4

22. Sodium hydroxide, No. SS266-1. 4

23. Hydrogen peroxide, No. H325-100. 4

B. Glassware and plastics01. Culture flasks, 25 cm 2 (T-25): No. 3325; 75

cm2 (T-75): No. 3375.3

02. Multi-well culture dishes, 24 well: No. 3524;96 well: No. 3596.3

03. Plastic pipets, 1 ml: No. 430952; 5 ml: No.430954; 10 ml: No. 430955.3

04. Centrifuge tubes, 15 ml: No. 43630; 50 ml:No. 430522.3

05. Pipet tips, 1000

µl: No. 21-236-2A; 200 µl:No. 21-236-1; 20 µl: No. 21-236-4.4

06. Biohazard bags, No. 01-830D.4

07. Gloves-latex, No. 11-394-4C.4

08. Tyvek lab coats, No. 01-361-51B.4

09. Nunc freezer vials, No. T2850-2C.5

10. Nitrile N-Dx gloves, No. 11-395-19B.4

C. Equipment01. Humidified, CO2 tissue culture incubator, No.

3200 (NAPCO)4 or equivalent.02. Pipettors, 1–10 µl: No. P20; 10–100 µl: No.

P100; 100–1000 µl: No. P1000.6

03. Pipetaid with filter, No. 13-681-15C.4

04. Tissue culture hood, with exhaust ducting,No. 1162,12 or equivalent.

05. Water bath, 13-874-77F.4

06. Centrifuge, No. 05-101-5.4

07. Microscope, Inverted, No. IMT-2,10 or equiv-alent.

08. Multi-well plate reader, No. 450.11

09. Computer with spreadsheet program.10. Spreadsheet program (Quattro Pro 6.0,13 or

similar)11. Multi-channel pipettor, No. 21-377-122.6

3. Procedure

Note: The procedures described for this assayinclude using a potent carcinogen, propanesultone and a weak carcinogen, amino-ethylcarbazole (AEC). The user is hereby cautionedthat health and safety guidelines must be devel-

oped to permit the safe handling of carcinogensand procedures must be in place for the safedisposal of the liquid and dry waste from theassay exposures. Also, since human cell sourcesare used, care should also be taken to protect thelaboratory staff and others from potentialexposure to viruses that may be associated withthe cells. Even if the cultures have been tested forhepatitis and HIV, they may still have undetectedvirus present.

A. Preparation of solutions1. Cell culture and treatment solutions0 The culture medium, keratinocyte growth

medium (KGM), is supplied as completemedium except for the addition of pituitaryextract at the time of use. Sterile Dulbecco’sphosphate buffered saline can be purchased orprepared and autoclaved. Trypsin/EDTA ispurchased as a 10× stock and diluted withsterile PBS and refrozen in single use aliquotsin 15 ml centrifuge tubes. Trypsin neutralizingsolution is prepared by adding 2% fetal bovineserum to complete KGM. Propane sultone isprepared as a 7.5 mg/ml stock in acetone. Thestock is divided into single use (400 µl)aliquots, the vials wrapped in foil, and storedin a –70 °C freezer. A vial of the propanesultone stock is placed on ice and allowed towarm to approximately 0 °C at the time ofuse. Retinoic acid is prepared by adding coldethanol to a tube containing a preweighedamount (~20 mg) of all-trans-retinoic acidto obtain a final concentration of 2 mg/ml.Once dissolved, the solution is distributed incryovials in 500 µl aliquoits. Care is taken tokeep the tubes cold during the procedure,which will reduce the possibility of evapora-tion of the ethanol. At the time of use, onecryovial is removed and diluted to 0.2 mg/mlwith cold ethanol. To prepare the treatmentmedium, 2.5 µl of this stock is added per mlof acetone/KGM or PS/KGM.Unless the solubility is already known, eachtest agent must be tested to determine theappropriate solvent. The solvent should permitaddition of the maximum test agent concen-tration to the test medium while maintainingthe solubility of the test agent in the finalsolution. The preferred solvents are culturemedium, water, DMSO, ethanol, or acetoneand the final solvent concentration permittedin the test medium is 0.25%.

2. Methylene blue staining solution is preparedby adding 0.4 g of methylene blue to 30 mlof methanol and once the dye is dissolvedadding 70 ml of deionized water.

3. Preparation of reagents for involucrin stainingA 2%-paraformaldehyde solution is preparedby dissolving 2 g of paraformaldehyde in

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100 ml of PBS. A few drops of phenol redare added and enough sodium hydroxide(1N) added to turn the solution pink. Thesolution is then heated until the paraformalde-hyde is in solution [caution: use low heatonly to prevent the release of toxic fumes].Hydrochloric acid (1N) is then added to returnthe solution to orange (about pH 7).The primary antibody is prepared by adding0.1 ml goat serum, 0.1 ml of 1% albumin inPBS, 10 ml of PBS and using this solutionto dilute the rabbit anti-human involucrinantibody 1:1. The secondary antibody solutionis prepared by adding 0.1 ml of 1% albuminin PBS to 9 ml of PBS and 1 ml of the sec-ondary antibody, goat anti-rabbit.

4. The amino-ethyl carbazole (AEC) solution isprepared by first preparing a 10× stock ofAEC by adding 60 ml of DMSO to 10.02grams of AEC. Just prior to use, dilute asample of the stock 1:10 with DMSO. TheAEC working solution should be used imme-diately after preparation due to potential pre-cipitation. To prepare the working solution,50 ml of 0.02 M sodium acetate in distilledwater is mixed with 6 ml of the diluted AEC.This solution is then filtered to remove anyprecipitate and 0.4 ml of 0.3% H2O2 added.The sodium acetate solution can be preparedas a 50× stock and diluted with distilled water.

B. Culture preparation1. Cultures of keratinocytes are used after the

first or second subculture. If cryopreservedcultures are used, they must be frozen at thefirst subculture and used at the second. Theculture should demonstrate good growth qual-ities over the duration of the experiment.Cultures are cryopreserved in 10% FBS and10% DMSO. For thawing, a vial is removedfrom the nitrogen freezer and placed in a37 °C water bath to thaw rapidly. Care is takennot to get water on the sealing rim of the vial.The thawed vial is then swabbed with analcohol wipe or a sterile gauze pad soakedwith 70% ethanol and the vial allowed to airdry. The contents of each thawed vial aretransferred to a 15 ml conical centrifuge tube,10 ml of complete KGM added and the tubecentrifuged at 20 °C for 5 min at 1000 rpm.The pellet is resuspended is KGM and trans-ferred to T-75 culture flasks at 5 × 105 cellsper flask in 10 to 12 ml of culture mediumper flask. The flasks are then incubated at37 °C.

2. To subculture, cells are washed once with0.25% trypsin/EDTA, and 1 ml of trypsin/EDTA added to the flask, and the cells incu-bated for 3 to 6 min. The trypsin associatedwith the detached cells is neutralized by

adding 3 ml of complete KGM + 2% fetalbovine serum (FBS) then centrifuged at 1000rpm at 20 °C for 5 min. The cells are thenresuspended in KGM and seeded at an appro-priate plating density and medium volume forthe experimental procedure.

C. Human epidermal cell cytotoxicity (range-finding) assay1. To choose a concentration range for the assay,

a preliminary cytotoxicity assay is performed.To do this, approximately 5 × 103 viable cells(in equal aliquots) are plated in 1 ± 0.5 ml ofculture medium into wells of a 24-well tissueculture dish. The cell culture medium iscomplete KGM. The cells are allowed toattach overnight and then exposed to the testagent at various concentrations after one day(Day 1). The highest test agent concentrationshould be

< 1 mg/ml in culture medium or thehighest soluble concentration. If a solvent isused other than culture medium or water, thehighest concentration should be one in whichthe solvent does not exceed 0.25% in the treat-ment medium.

2. The chemopreventive exposure consists ofthat highest concentration and four or morelog dilutions of that concentration. The testagent treatment is continuous until the cellsare either subcultured for a second exposureor fixed and stained for evaluation. At leastfour wells per exposure group are used. Thenegative control is exposure to KGM medium(or the highest solvent concentration, whenused). After 6 to 7 days (depending on theculture growth) two of the wells for eachexposure group are subcultured into two addi-tional wells using identical subculture ratios.The solvent control culture density is used todecide the subculture ratio. These cultures areallowed to attach overnight and the culturemedium replaced with the same test agentconcentration used in the first exposure. Theremaining two culture wells from the firstexposure are fixed, stained, and the relativegrowth determined.

3. The concentration of test substance thatreduces the relative growth by approximately20 to 50% is estimated from the exposure dataand is used as the highest concentration (HC)for the human epidermal cell assay.

D. Human epidermal cell assay1. The concentration of test agents used in

human epidermal cell assay consist of thehighest test substance concentration (HC fromcytotoxicity assay above), and four half-logdilutions of that concentration. Multi-welldishes are seeded with normal, early passagehuman keratinocyte cells in complete KGMand treated on day 1 or 2. All test dishes,

15

excluding the medium and/or solvent controls,are then exposed to 7.5 µg/ml of propanesultone (PS). The chemopreventive test agentis added to the appropriate groups on the sameday and at each media change thereafter. Themedium is changed twice weekly during theexperiment. The positive control group is PS+ all-trans-retinoic acid (RA). The RA (0.03µg/ml) is added at each media change.

2. When the test agent is soluble in media, thecontrols consist of an acetone control(exposed to 0.1% acetone) and an acetone +RA control (exposed to RA continuously). IfDMSO or other solvent is required, the appro-priate solvent control is added.

3. After the start of the exposures, the chemo-preventive and PS groups are exposed to PSfor three to four days each week and CP isadded with each media change. The multi-wellplate, into which the cells will be initiallyplated, is labeled ‘P0’ (Passage 0). Just priorto or at confluence, the cells from one wellof each treatment group are trypsinized andreseeded at approximately 4–5 × 103 cells/welland the plate labeled ‘P1’. The day after thesubculture, the cultures are retreated with theappropriate test agent or control solutions asdone for the first treatment. The cultures arerefed as described for ‘P0’. This process isrepeated until the cells reached ‘P3’.

4. When the ‘P2’ plates are confluent, one wellfrom each treatment condition is trypsinizedand plated at 1 × 103/well in two 96-welldishes. Three wells per group are seeded intoeach dish. One dish is used to measure growthinhibition and the other used to measureinvolucrin induction. If the number of cellsin a treatment dish is less than 6000, thenthat treatment is considered as toxic and willnot be evaluated for either endpoint. The‘P3’ plates are incubated for 6 to 7 daysprior to evaluating for growth or involucrinexpression.

5. Growth inhibitionAfter 6 to 7 days, the cells in one of the96 well dishes are stained using methyleneblue. The stained cultures are rinsed withtap water and allowed to dry. The dye isthen extracted in 100 µl of methanol/glacialacetic acid/water (100:1:99) solution andabsorbance at 595 nm determined using amicro-plate reader. The average reading of5 to 6 blank wells is used to correct theabsorbance in each well for the plate (back-ground) absorbance.

6. Involucrin inductionAt the time of methylene blue staining, thesecond 96 well dish is evaluated for involu-crin expression. Standard immunoperoxidase

methodology is used for this purpose. Culturesare washed twice with PBS, fixed with 2%paraformaldehyde for 20 min, washed threetimes with PBS, treated with H2O2 for 5 min(100 µl/well), washed three times with PBSleaving the second wash on the cells for 30min, exposed with primary rabbit-anti-humanantibody for involucrin (20 min) (50 µl/well),wash three times with PBS, then exposed tothe secondary perioxidase labeled goat anti-rabbit antibody (20 min) (50 µl/well). Theantibody treated cells are then rinsed threetimes with PBS and exposed for 10 min to100 µl/well of the chromogenic substrateAEC, which is oxidized to a brown precipi-tate on cells with bound antibody. The treatedcultures are then rinsed twice with tap waterand 100 µl of DMSO added to each well todissolve the brown precipitate. The opticaldensities at 450 nm are read using a platereader. The average reading of five to sixblank wells is used to correct the absorbancein each well for the plate (background)absorbance.

E. Experimental design1. Rangefinding assay

Chemical treatment Number of wells‘P0’ ‘P1’

Media control or solvent(highest %) 04 02

Highest concentration(HC) of test agent 04 02

0.1 × HC 04 020.01 × HC 04 020.001 × HC 04 020.0001 × HC 04 02

Total 24 12

2. Human epidermal cell assay

Chemical treatment Number of wellsa

Acetone control (0.1%) 037.5 µg PS/ml +

0.03 µg/ml RA 03Acetone + 0.03 µg/ml RA 03Media or solvent (highest

concentration used) 037.5 µg PS/ml 037.5 µg PS/ml + HCb 037.5 µg PS/ml + 0.3 HC 037.5 µg PS/ml + 0.1 HC 037.5 µg PS/ml + 0.03 HC 037.5 µg PS/ml + 0.01 HC 03

Total 30a at least 2 wells.b HC = highest concentration of chemopre-ventive test agent.

16

F. Experimental evaluations and criteria for apositive response1. The percent inhibition of growth and the

percent induction involucrin expression fol-lowing exposure to the test substances aredetermined at various concentrations. Thecalculated values for inhibition or inductionare determined as follows.

2. For determining growth inhibition, the meanvalue for each CP group is subtracted from themean value of the PS group and divided bythe value for the PS group. These numbers aremultiplied by 100 to obtain the percent inhi-bition relative to PS.

3. For determining involucrin induction, theaverage absorbance values for the involu-crin endpoint are divided by the averageabsorbance values obtained in the methyleneblue stained parallel cultures to obtain the nor-malized values for involucrin expression. Thenormalized value for the PS group is sub-tracted from the normalized values of each CPgroup and divided by the normalized PS groupvalue. These numbers are multiplied by 100to obtain the percent involucrin inductionrelative to PS.

4. The raw data may be entered into Quattro Proor other spreadsheet program to facilitate thedata calculations.

5. For an experiment to be considered valid,the propane sultone treated cultures mustdemonstrate at least a 40% increase in growthrelative to the solvent control. Also, thepositive control or the test agent must induceat least a 30% inhibition of growth. At aminimum two data points are required foreach test article concentration.

6. For a test chemical to be considered positive,it should demonstrate inhibition of car-cinogen-induced growth by 30% or more(relative to the propane sultone controls) attwo consecutive nontoxic test chemical con-centrations and/or it should demonstrate anincrease in involucrin expression of 20% ormore (relative to the propane sultone controls)at two consecutive nontoxic test chemicalsconcentrations.

4. Discussion

This procedure has been used to screen chemicals forthe National Cancer Institute’s Division of CancerPrevention and Control. The assay requires consid-erable attention to detail and several weeks to obtaindata. Starting with an actively growing culture atearly passage is important. Also, each laboratory willneed to decide the appropriate subculture ratios toprevent the cultures from becoming confluent prior

to the time of the next subculture. This will dependupon the growth rate of the culture, the quality of theculture medium and culture procedures, and thequality of the incubator environment. While we havebeen able to subculture the cell cultures at ratios of1:10 to 1:18, each decision is based on the cultureappearance at the time of subculture. If cells areallowed to become over confluent, the cells may beforced into differentiation in some culture treatmentor control conditions, thereby creating bias in thedata.

It is important that the split ratios be identical forall wells in an experiment. This internal controlensures that treatment conditions that producetoxicity will be selected against and result in lowercell numbers at latter subcultures. If sufficient cellsare not present in one well of each treatment to seedtwo sets of wells in the 96-well dishes for assessmentof the different endpoints, the individual treatmentcondition is considered as toxic. Also, once data isassessed, if it is apparent that the highest concentra-tion(s) produce considerable inhibition of growth (ingreat excess of the solvent control growth) then thatconcentration is also considered as toxic and the datanot used in the assessment.

The use of dye absorbance as an estimate ofrelative cell number requires that data be obtained ondifferent density cultures. Figure 1 presents theresults of two parallel experiments conducted in ourlaboratories to study this issue. The data demonstratethe reproductivity of staining in parallel culturesand the reasonable linearity with increasing cellnumber.

With data obtained from valid experiments,propane sultone induced growth ranged from 54 to300% above that observed in the solvent controlswith an average of 116%. Retinoic acid inhibitedpropane sultone induced growth by 48 to 82% withand average inhibition of 64%. These data were pre-sented to provide insights into assay performanceexpectations. Each laboratory will need to accumu-late their own data on assay performance.

Figures 2 and 3 present the data obtained withblack tea extract, a potential cancer preventive agent,which produced a positive response for both end-points in the assay. Figure 2 presents data for the

17

Figure 1. Methylene blue dye absorbance as a functionof keratinocyte cell number.

growth inhibition endpoint and Figure 3 presents datafor the involucrin endpoint.

As with any cell culture procedure, careful atten-tion to detail with permit the reproducible perfor-mance of this assay procedure. Controls are necessaryand should be included in each assay. Multiple testagents can be evaluated in a single experiment whichwill allow for more efficient use of time.

Acknowledgments

Supported by the Division of Cancer Prevention andControl, NCI under contract numbers: N01-55503-01; N01-95172-04; N01-2546-01; N01-2546-02; andN01-55138.

Notes on suppliers

01. Clonetics Corportion, San Diego, CA, USA02. Life Technologies, Gaithersburg, MD, USA03. Corning Costar, Cambridge, MA, USA04. Fisher Scientific, Pittsburgh, PA, USA05. Intermountain Scientific, Kaysville, UT, USA06. Rainin Instrument Co., Woburn, MA, USA

07. Sigma Chemical, St Louis, MO, USA08. Biomedical Technologies, Stoughton, MA, USA09. American Qualex, La Mirada, CA, USA10. Olympus America, Lake Success, NY, USA11. Bio-Rad, Cambridge, MA, USA12. Forma Scientific, Marrietta, OH, USA13. Wordperfect, Orem, UT, USA14. Quantum Chemical Company, Tuscola, IL, USA

References

01. Boone CW, Kelloff GJ, Malone WF (1990).Identification of candidate cancer chemopreventiveagents and their evaluation in animal models andhuman clinical trials: A review. Cancer Res 50: 2–9.

02. Steele VE, Stoner GD, Boone CW, Kelloff GJ (1992).Cellular and molecular targets for chemoprevention.Boca Raton: CRC Press, 373 p.

03. Hennings H, Holbrook KA, Yuspa SH (1983). Factorsinfluencing calcium-induced terminal differentiationin cultured mouse epidermal cells. J Cell Physiol 16:265–281.

04. Boyce ST, Ham RG (1983). Calcium-regulated dif-ferentiation of normal human epidermal keratinocytesin chemically defined clonal culture and serum-freeserial culture. J Invest Dermatol 81: 33–40.

05. Okumura H, Matsumto K, Hashimoto K, YoshikawaK (1991). A new method for quantifying keratinocytedifferentiation using immunofluorescent staining ofinvolucrin and cytofluorography. Exp Cell Res 192:647–650.

06. Strickland JE, Jetten AM, Kawamura H, Yuspa SH(1994). Interaction of epidermal growth factor withbasal and differentiating epidermal cells of mice resis-tant and sensitive to carcinogenesis. Carcinogenesis5: 735–740.

07. Wilke MS, Hsu BM, Wille Jr JJ, Pittelkow MR, ScottRE (1988). Biological mechanisms for the regulationof normal human keratinocyte proliferation and dif-ferentiation. Am J Pathol 131: 171–181.

08. Elmore E, Korytynski EA, Wyatt GP (1990).Applications for normal human epithelial cells:Cytotoxicity and cellular differentiation. In VitroCellular Develop Biol 26: 34a.

09. Elmore E, Sun C, Li H-R, Buckmeier JA, Steele VE,Kelloff GJ, Redpath JL (1996). Comparison of growthinhibition and involucrin expression in human epithe-lial cells are endpoints for screening cancer preven-tive agents in vitro. In Vitro Cellular Develop Biol 32:63A.

Address for correspondence: Eugene Elmore, PhD,Radiaton Oncology, Med. Sci I, B 118, Department ofMedicine, University of California at Irvine, Irvine, CA92697, USAPhone: (714) 589 8517; Fax: (714) 824 3566E-mail: eelmore@uci.edu

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Figure 2. Concentration dependent inhibition of growthas with black tea extract.

Figure 3. Concentration dependent induction of involu-crin with black tea extract.