PROTECTION AGAINST ULTRAVIOLET B RADIATION-INDUCED EFFECTS IN THE SKIN OF SKH-1 HAIRLESS MICE BY A POLYPHENOLIC FRACTION ISOLATED FROM GREEN TEA

Photochemistry and Photobiology, Vol. 58 , No. 5 , pp. 695-700, 1993 Printed in the United States. All rights reserved

003 1-8655/93 $05.00+0.00 @ 1993 American Society for Photobiology

PROTECTION AGAINST ULTRAVIOLET B RADIATION-INDUCED EFFECTS IN THE SKIN OF SKH-1 HAIRLESS MICE BY

A POLYPHENOLIC FRACTION ISOLATED FROM GREEN TEA RAJESH AGARWAL*, SANTOSH K. KATIYAR, SIKANDAR G. KHAN and HASAN MUKHTAR

Department of Dermatology, University Hospitals of Cleveland, Skin Diseases Research Center, Case Western Reserve University, and

Department of Veterans Affairs Medical Center, Cleveland, OH 44106, USA

(Received 1 1 March 1993; accepted 7 June 1993)

Abstract-In prior studies we and others have shown that oral feeding of a polyphenolic fraction isolated from green tea (GTP) or water extract of green tea affords protection against ultraviolet B (UVB) radiation-induced carcinogenesis in SKH-I hairless mice (Wang et al., Carcinogenesis 12, 1527-1 530, 1991). It is known that exposure of murine skin to UVB radiation results in cutaneous edema, depletion of the antioxidant-defense system and induction of ornithine decarboxylase (ODC) and cyclooxygenase activities. In this study we assessed the protective effect of GTP on these UVB radiation-caused changes in murine skin. Oral feeding of 0.2% GTP (wtlvol) as the sole source of drinking water for 30 days to SKH-I hairless mice followed by irradiation with UVB (900 mJ/cm2) resulted in significant protection against UVB radiation-caused cutaneous edema (P < 0.0005) and depletion of the antioxidant-defense system in epidermis ( P < 0.01-0.02). The oral feeding of GTP also resulted in significant protection against UVB radiation-caused induction of epidermal ODC ( P < 0.005-0.01) and cyclooxygenase activities (P < 0.0001) in a time-dependent manner. Our data indicate that the inhibition of UVB radiation-caused changes in these markers of tumor promotion in murine skin by GTP may be one ofthe possible mechanisms ofchemopreventive effectsassociated with green tea against UVB-induced tumorigenesis. The results of this study suggest that green tea, specifically polyphenols present therein, may be useful against inflammatory responses associated with the exposure of skin to solar radiation.

INTRODUCTION

The treatment of cancer is often ineffective once the tumor is diagnosed. Considerable emphasis, therefore, has been placed in identifying cancer chemopreventive agents that inhibit, alter or retard the induction of cancer.) Several studies have shown that naturally occurring polyphenols possess significant cancer chemopreventive effects possibly because of their antioxidant properties.’ In recent years, we and others have shown that polyphenols present in green tea, more specifically epicatechin derivatives, possess cancer chemopreventive effects when evaluated in skin and other animal tumor bioassay systems.j Studies from our laboratory have shown that oral feeding in drinking water or topical application of a polyphenolic fraction isolated from green tea (GTP)? or (-)-epigallocatechin-3-gallate (EGCG) affords protection against chemical carcinogen-induced tumor initiation and complete carcinogenesis in murine skin.44 In recent studies Huang et aL7 and Katiyar et have also shown that topical application of G T P protects against 12- 0-tetradecanoylphorbol- 1 3-acetate-caused tumor promotion in 7,12-dimethylbenz(a)anthracene-initiated murine skin. The mechanism of anti-tumor-promoting effects of green tea appears to involve the inhibition of tumor promoter-caused induction of epidermal ornithine decarboxylase (ODC), cy-

clooxygenase and lipoxygenase activities, edema and hyper- pla~ia.’-~

We have also shown that oral feeding or topical application of G T P affords significant protection against ultraviolet B (UVB) radiation-induced tumorigenicity in SKH- 1 hairless mice.’0 Similar results were also reported from the laboratory of Conney showing cancer chemopreventive effects of water extract of green tea (WEGT) against UVB radiation-induced sunburn lesion formation, tumor initiation and tumor promotion in S M - 1 hairless mice.)’

Chemical analysis of GTP and WEGT showed that they are mainly constituted of epicatechin derivatives, in general polyphenols.*” Because epicatechin derivatives present in green tea possess significant antioxidant effects, and because in UVB radiation-induced tumorigenicity and tumor promotion, oxidative stress and free radical mechanism leading to cutaneous edema and the induction of ODC and cyclooxygenase activities play a major role,I2 in the present study we assessed whether oral feeding of GTP to SKH- 1 hairless mice protects against UVB radiation-caused ( 1) cutaneous edema, (2) depletion of the antioxidant-defense system in epidermis and (3) induction of epidermal ODC and cyclooxygenase activities, the short-term markers of tumor promotion in cutaneous photo (as well as chemical) carcinogen- e ~ i s . ’ ~

*To whom correspondence should be addressed. tAbbrrvratlons: EGCG, (-)-epigallocatcchin-3-gallate; GSH-r, glu-

tathione reductase; GTP, polyphenolic fraction isolated from green tea, H 2 0 , , hydrogen peroxide; ODC, ornithine decarboxylase; PG, prostaglandin; ROS, reactive oxygen species; TLC, thin-layer chromatography; UVB, ultraviolet B; WEGT, water extract of green tca.

MATERIALS AND METHODS

Chemicals. Hydrogen peroxide (H,02), D,L-omithine, NADPH and oxidized glutathionc were purchased from Sigma Chemical Co. (St. Louis, MO). Arachidonic acid and prostaglandin (PG) metabolites PGE2, PGF2., and PGD2 were purchased from Biomol Research Laboratories Inc. (Plymouth, PA). ~ ,~- [ ‘~C]omi th ine (58 Ci/mmol) was from Amersham Searle (Chicago, IL). [ l-’4C]arachidonic acid

695

696 RAIESH AGARWAL et al.

( 5 2 mCi/mmol) was from New England Nuclear (Boston, MA). All other chemicals were obtained in the purest form commercially available. The GTP from green tea leaves (a product of Korea; dis- tri buted by Hanmi, Inc., Los Angeles, CA) was prepared by a method dewribed earlier.9

..lnimals and protocols. Female SKH- I hairless mice (6 weeks old) obtained from Charles River Laboratories (Wilmington, MA) wcrc used in this study. After amval in our animal facility, the animals were allowed to acclimatize for 15 days before the start of the experiments and were fed purina chow diet and water adlibitum. Throughout the experimental protocols, the mice were maintained at standard conditions: 24 2 2°C temperature, 50 * 10% relative humidity and 12 h light112 h dark cycle. In total, 128 animals were divided into two groups of 64 each and fed with either normal drinking water (control group) or 0.2% GTP (wt/vol) in water as the sole source ofdrinking water (experimental group); this defined feeding regimen was continued up to 30 days. The selection of dose of GTP was based on our previous studies where this dose showed significant cancer chemopreventive effect^.'^.'^ At the end of the feeding regimen, the animals in both control and expenmental groups wcre irradiated only once with UVB (900 mJ/cm2) while housed in spwially designed cages where the animals are held in dividers separated by Plexiglas. The selection of this dose of UVB was based on prior studies where we employed this dose for the induction of tumors on the dorsal skin of SKH-I hairless mice and to assess the protective effects of oral feeding of GTP against UVB radiation- induced tumorigenesis in this mouse model.‘0 The light source uti- lircd was a bank of four Westinghouse FS-40-T-12 fluorescent sun- lamps equipped with a UVB Spectra 305 Dosimeter (Daavlin Co., Bryan, OH). This light source emitted about 80% radiation in the range of 280-340 nm with peak emission at 3 I4 nm as monitored with a SEE 240 photodetector, 103 filter and 1008 diffuser attached to an IL 700 Research Radiometer (International Light, Newbury- port, MA). At the desired time after UVB irradiation, animals were killed by cervical dislocation, and both ears and skin were used for the subsequent studies.

Cutaneous edema. To assess the extent of cutaneous edema viz. weight increase, in UVB-irradiated SKH- 1 hairless mice, and the inhibitory effect of oral feeding of GTP, ear punch weight was used as a measure. A 4 mm-diameter punch of ear skin through the entire ear (two from each ear ofsame mouse) was taken and quickly weighed. The difference in the amount of water gain between unirradiated control and UVB irradiated represented the extent of cutaneous edcma caused by UVB radiation, whereas that between unirradiated control and GTP plus UVB irradiated represented the inhibitory cKcct of GTP against UVB radiation-caused cutaneous edema.

Assays of catalase and glutathione reductase (GSH-r) activities. For biochemical assays, the dorsal UVB-irradiated skin was re- moved and made free of connective tissue and fat. The epidermis was separated from the whole skin by brief heat treatment at 52°C for 30 s, and 100 000 g supernatant, and microsomal fractions were prepared as described Protein concentration was determined by the method of Bradford.’j Catalase activity was determined by following the decomposition of H202 measured as a decrease in absorbance at 240 nm,’o and GSH-r activity was measured as described by Mohandas et al.”

.Issay ofODC activity. Epidermal ODC activity was determined in I00000 g supernatant fraction by measuring the release of I4CO2 from the ~ , ~ - [ ~ ~ C ] o r n i t h i n e by the method of O’Brien et as described by Verma et al.19 The details of the assay procedure are described earlier.y Enzyme activity is expressed as pmol CO, released/h/mg protein.

Assay ofcyclooxygenase activity. Epidermal microsomal cyclooxygcnase activity was determined by the method of Lysz et ~ 1 . ~ ~ with slight modifications as described recently by Katiyar et aL8 In brief, I50 pL reaction mixture contained I2 pM [14C]arachidonic acid (= 400 000 dpm), 1 m M epinephrine, I mM glutathione and = 10 pg epidermal microsomal protein in 50 mM potassium phosphate buffer, pH 7.4. After incubation at 37°C for 15 min, the reaction was terminated by the addition of 50 pL of 0.2 M HCI. The metabolites of nrachidonic acid were extracted from the incubation mixture with 0.5 mL ofethyl acetate three times. The combined extract was evap- orated to dryness, redissolved in chloroform/methanol/acetic acid/ water ( I 131 10/1.25/1 .O, vol/vol/vol/vol), and subjected to thin-layer

P I L I T I

.c I - I/ T

,& /y GTP + UVB -* 1 I I I

0 12 24 36 48 60 72

Time (hrs) Figure 1. Time-dependent inhibitory effect of GTP against UVB radiation-caused edema in the ear skin of SKH-I hairless mice. Details of the experimental protocols are described in the Materials and Methods. In total, four punch biopsies (4 mm diameter), two from each ear, were pooled. In unirradiated control mice, ear punch weight of one biopsy was 4.5 2 0.3 mg. The data shown as increase in ear punch weight (mg) are those obtained after the subtraction of control values and represent the mean * SE ofeight values obtained from eight mice. The protective effect of GTP was statistically significant (Student’s t test) at 6 h (P < O.OOl), 12 h (P < 0.001), 24 h

( P < O.OOOS), 48 h (P < 0.0005) and 72 h (P < 0.0005).

chromatography (TLC) on a precoated TLC plastic sheet Silica Gel 60 (20 x 20 cm, layer thickness 0.2 mm, MC/B Manufacturing Chemists, Inc., Cincinnati). Thin-layer chromatography plates were developed with the same solvent system and exposed in an iodine chamber for 5 min to visualize the prostaglandin standards. The metabolites of [14C]arachidonic acid corresponding to PGE2, PGF2;, and PGDz were detected by their comigration with authentic standards. The areas in TLC plates corresponding to metabolites were cut, and the radioactivity was determined by counting the samples in a Packard Tri Carb 460 CD liquid scintillation counter equipped with automatic external standardization. The enzyme activity is expressed as pmol PGE2, PGF2,, and PGD2 metabolite formatiodl5 min/mg protein.

Statistical analysis. The statistical significance of difference between UVB and GTP plus UVB groups was evaluated by the Stu- dent’s t test.

RESULTS

Protective efect of GTP against UVB radiation-caused cutaneous edema

To examine whether oral feeding of GTP affords protection against UVB radiation-caused cutaneous edema in SKH- 1 hairless mice, at specific time intervals the ear punch weights were determined in normal water-fed control, UVB alone- irradiated, and GTP-fed and UVB-irradiated groups of mice. As shown in Fig. 1, compared to the normal water-fed untreated control group, irradiation of mice by UVB resulted in an increase in the ear punch weight (edema) in a time- dependent manner. While such an increase in ear punch weight was evident as early as 1 h after UVB irradiation, maximum increase was evident at 48 h post-UVB irradiation (Fig. 1). At this time point, compared to control mice, 1 15% (P < 0.0005) increase in ear punch weight was observed.

Protection by tea 697

Table I . Effect of oral feeding of GTP (0.2%, wt/vol) in drinking water on epidermal catalase and GSH-r activities in UVB-irradiated

SKH- 1 hairless mice*

Treatments? Catalase activity$ GSH-r activity8

Untreated control 461 f 21 71 k 6 G T P fed 487 _+ 26)) 76 5 711

UVB alone 12 h

GTP t UVB 12 h

UVB alone 24 h

GTP t UVB 24 h

postirrad. 363 5 19(79) 50 t_ 4 (70)

postirrad. 490 * 20 (106)li 76 f 7 (107)#

postirrad. 315 -t 12(68) 42 2 4(59)

postirrad. 459 f 22 (99)ll 69 i 7 (97)#

*Data represent mean f SE of four values obtained by assaying each in duplicate from pooled tissues of two animals. For statistical significance, Student’s t test was used between normal water-fed and UVB-irradiated group versus GTP-fed and UVB-irradiated group of mice. Values in parentheses show the percentage of untreated control in each case.

tSKH-1 hairless mice were divided into two groups of 64 animals each, and one group was maintained on normal drinking water, whereas the other was fed with 0.2% GTP (wt/vol) as sole source of drinking water. After 30 days of this treatment regimen, animals in both groups were irradiated to 900 mJ/cm2 dose of UVB radiation. At desired time points, animals were sacrificed, epidermal cytosols prepared, and catalase and GSH-r activities determined as described in Materials and Methods. Data shown here include those obtained without UVB irradiation, and 12 and 24 h after UVB irradiation in the case of both non-GTP-fed and GTP-fed groups of mice.

Snmol H 2 0 2 consumed/min/mg protein. Pnmol NADPH oxidized/min/mg protein. IIStatistically not significant. 1IP < 0.01. #P < 0.02.

Even at 72 h post-UVB irradiation, a significant increase in ear punch weight, though less than that observed at 48 h, was evident (Fig. 1). Oral feeding of GTP for 30 days prior to UVB irradiation, however, resulted in significant protection against the UVB radiation-caused cutaneous edema at each time point examined (Fig. 1). Compared to UVB alone, GTP-fed and UVB-irradiated animals did not show any increase in ear punch weight at 1 and 3 h and negligible increase at 6, 12 and 72 h post-UVB irradiation. At peak time point of cutaneous edema in UVB alone-irradiated animals viz. 48 h, oral feeding of GTP showed highly significant protection (P -: 0.0005) as evident by only 28% increase in ear punch weight compared to untreated control animals where 1 15% increase occurred (Fig. 1). The GTP alone had no effect on increase in ear punch weight, suggesting that there was no spontaneous edema due to GTP feeding (data not shown).

Protective efect of GTP against UVB radiation-caused depletion of epidermal catalase and GSH-r activities

At specific time intervals, epidermal catalase and GSH-r activities were also determined in normal water-fed controls, GTP-fed controls, UVB alone-irradiated, and GTP-fed and UVB-irradiated groups of mice. As shown by data in Table 1 , oral feeding of GTP as a sole source of drinking water did not result in any measurable change in epidermal catalase

and GSH-r activities in SKH- 1 hairless mice. At all the time points studied, irradiation of SKH- 1 hairless mice to UVB radiation resulted in significant decrease in epidermal catalase and GSH-r activities only at 12 and 24 h post-UVB irradiation; the decrease was more pronounced in the case of GSH-r activity compared to that in catalase activity at both time points (Table 1) . No measurable change in the activities of these two enzymes was observed at other time points studied post-UVB irradiation (data not shown). Oral feeding of GTP for 30 days prior to UVB irradiation resulted in protection against the UVB radiation-caused decrease in the activities of these enzymes at both time points (Table 1). The protection was significant ( P < 0.014.02) to an extent that it reversed the depleted enzyme activity levels compa- rable to those observed in the case of the normal water-fed control group of mice (Table 1).

Protective effect of GTP against U VB radiation-caused induction of epidermal ODC activity

In further studies, we assessed the protective effect of oral feeding of GTP on UVB radiation-induced epidermal ODC activity as a function of time after UVB irradiation. As shown in Fig. 2, irradiation of SKH-1 hairless mice to a 900 mJ/ cmz dose of UVB radiation resulted in a significant increase in epidermal ODC activity at all time points studied. The increase in enzyme activity was maximum at 24 h post-UVB irradiation; however, two peaks of induced enzyme activity were observed at 3 and 24 h post-UVB irradiation (Fig. 2). Even at the last time point studied viz. 72 h, the enzyme activity was about 6.4-fold higher than that in the untreated control group of mice. Oral feeding of GTP for 30 days prior to UVB irradiation resulted in significant protection (P < 0.005-0.0 1) against UVB radiation-caused induction of epidermal ODC activity (Fig. 2). While the protective effects of GTP were evident at all time points studied, the pattern was similar to the curve obtained with UVB alone-induced epidermal ODC activity (Fig. 2).

Protective effect of GTP against UVB radiation-caused induction of epidermal cyclooxygenase activity

The effect of oral feeding of GTP as a sole source of drinking water on UVB radiation-caused induction of epidermal cyclooxygenase activity is shown in Fig. 3. As quantitated by the formation of PG metabolites from arachidonic acid in the presence of epidermal microsomes from UVB-irradiated animals, a significant increase in enzyme activity was observed in a time-dependent manner. Collaborative to the results obtained for UVB radiation-caused cutaneous edema, while a significant increase in enzyme activity was evident at each time point studied, maximum increase was observed at 48 h post-UVB irradiation. When these data were considered in terms of PGE2, PGF,, and PGD, metabolite formation, compared to the untreated control group of mice, a six- to eight-fold ( P < 0.0005) increase in the formation of these metabolites was observed at the 48 h time point in the UVB alone-irradiated group of mice (Fig. 3). Oral feeding of GTP afforded significant protection (P < 0.000 1) against the UVB radiation-caused induction of epidermal cyclooxygenase activity also in a time-dependent manner (Fig. 3). In terms of PG metabolite formation, at the peak time viz. 48

698 RAJFSH AGARWAL et al.

250 4 T

I I I I I I I

0 12 24 36 48 60 72

Time (hrs) Figure 2. Time-dependent inhibitory effect of GTP against UVB radiation-caused induction of epidermal ODC activity in SKH- 1 hairless mice. Details of the experimental protocols are described in the Materials and Methods. Each data point represents the mean _+

SE of four individual values; epidermis from two animals was pooled for cach determination. The protective effect ofGTP was statistically significant (Student’s 1 test) at all time points studied; P < 0.01 (at

I , 3, 12, 24 and 48 h) and <0.001 (at 6 and 72 h).

h, oral feeding of GTP resulted in 62, 72 and 73% inhibition of PGE2, PGF,,, and PGDz formation, respectively, induced by UVB irradiation (Fig. 3).

DISCUSSION

1 Jltraviolet radiation, besides being a potent DNA-dam- aging agent, also induces effectively an acute inflammatory response in the skin subsequently leading to cellular proliferation that results in n e o p l a ~ i a . ~ ’ - ~ ~ Several studies have shown a relation between the production of reactive oxygen species (ROS) and tumor promotion.25-*8 Ultraviolet radiation has also been shown to result in free radical formation in human and murine skin and epidermal k e r a t i n o ~ y t e s . ~ ~ ~ ~ ~ The IJVB-irradiated skin undergoes lipid peroxidation that leads to severe damage to the cellular membranes.j’ Recently, the role of hydroxyl radical in sunlight-induced skin damage via induction of lipid peroxidation has been suggested.32 In the present study, therefore, the protective effect of GTP against UVB radiation-caused cutaneous edema in SKH- I hairless mice suggests that the antioxidant properties of GTP may be responsible a t least for its protective effects against LJVB radiation-caused inflammation in murine skin!,” Be- cause in early stages like edema and inflammation, tumor promotion is r e v e r ~ i b l e , ’ ~ , ~ ~ it can be presumed that antioxidant characteristics of G T P scavenge free radicals either directly or by terminating biological reactions that generate free radicals and thereby inhibit the promotion stage of tumorigenesis.

The cellular concentration of ROS is determined by their rates of production and detoxification. Antioxidant enzymes function cooperatively and any change in one of them may break the equilibrium leading to excessive production of ROS, which may cause cellular damage and other biochemical al-

5000

4000

3000

2000

1000

0 5000

4000

3000

2000

1000

0 I I I I I 1

5000 - C

4000 -

0 12 24 36 48 60 72

Time (hrs) Figure 3. Time-dependent inhibitory effect of GTP against UVB radiation-caused induction of epidermal cyclooxygenase activity in SKH- I hairless mice. Details of the experimental protocols are described in the Materials and Methods. Panels A, B and C represent, respectively, PGE2, PGF,,, and PGDz formation. Each data point reprcsents the mean 2 SE of four individual values; epidermis from two animals was pooled for each determination. The protective effect of GTP was statistically significant (Student’s t test) at all time points studied ( P < 0.000 1 , at 3-72 h in the case of each PG metabolite).

terations such as inflammation, lipid peroxidation, D N A damage and enzyme activation or ina~tivation.’-~--” Such al- terations subsequently have been suggested to play a major role in the multistage process of c a r c i n o g e n e s i ~ . ~ ~ ~ ~ ~ J ~ Ultra- violet B radiation is known t o decrease the activities of cutaneous catalase, GSH-r and glutathione peroxidase, indi- cating that the antioxidant defense system of the skin can be depleted by UVB radiation.3638 Collaborative to that, in the present study a significant decrease in epidermal catalase and GSH-r activities was observed in SKH-1 hairless mice exposed t o UVB radiation. However, the oral feeding of G T P to these mice prior to their exposure to UVB radiation afforded significant protection against the UVB radiation-caused decrease in the activities of these enzymes.

Moreover, the significant decrease in epidermal catalase and GSH-r activities in UVB-irradiated SKH- 1 hairless mice indicates a greater free radical load for these enzymes in the skin resulting from UVB radiation. The ameliorating effects

Protection by tea 699

of GT’P against the UVB radiation-caused decrease in the activilies of these enzymes, therefore, may be due to the scavenging properties of GTP against such a free radical load. However, the increase in the activities of catalase and GSH-r in the skin of GTP-fed and UVB-exposed SKH-1 hairless mice i s not presumably at the transcriptionaVtranslationa1 levels, because oral feeding of G T P to SKH- 1 hairless mice did not result in significant increases in the cutaneous activities of these enzymes (Table l).j4

Induction of ODC, a well-known marker of cellular proliferation has been considered to be a rather specific mani- festation of the tumor promotion process for both chemical as well as photocarcinogene~is.’~~~~ The protective effect of GTP against the UVB radiation-caused induction of epidermal O D C activity in the present study further indicates its role more specifically a t the promotion stage of multistage skin carcinogenesis. The UVB radiation-caused changes in the skin of SKH-1 hairless mice, observed in the present study, further suggest a correlation between the UVB-induced pro-oxidant effect and ODC induction. Additional evidence showing the involvement of pro-oxidant state in the induction of epidermal ODC activity includes a superoxide anion-generating xanthine/xanthine oxidase system, which induces ODC activity in isolated mouse k e r a t i n o c y t e ~ . ~ ~ This assumption is supported by our study showing that nordihydroguaiaretic acid, a known antioxidant, inhibits significantly against a free radical-generating compound benzoyl peroxide-caused induction of epidermal ODC activity and skin tumor p r o m ~ t i o n . ~ ~

Cyclooxygenase is known to play an important role in cutaneous inflammation, cell proliferation and skin tumor promotion.28 Arachidonic acid, released predominantly from skin phosphatidylcholine by the activation of skin phospho- lipasc A, due to a variety of mechanical, chemical or hor- monal stimuli, undergoes oxidative metabolism involving the cyclooxygenase pathway resulting in the formation of PG such as PGE,, PGF,,, and PGD, et~.”,~’ Ultraviolet radiation-caused induction of cyclooxygenase activity in cultured cells, as measured by the formation ofPGE2 from arachidonic acid, is also known.44 In the skin, several studies have shown the involvement of PG in inflammatory processes and epidermal growth in response to wound repair and in prolifer- ative skin disease p s ~ r i a s i s . ~ ’ - ~ ~ The UVB radiation-caused significant increase in cyclooxygenase activity in the skin of SKH- I hairless mice, as measured by PGE2, PGF,, and PGD, formation, further suggests its correlation with the UVB- caused pro-oxidant state, and collectively its role in UVB- caused tumor promotion. The inhibitory effects of oral feeding of G T P to mice against UVB radiation-caused induction of cyclooxygenase activity suggest a protective role of GTP against the generation of oxidative stress and inflammation, which play key roles in the promotion stage ofcarcinogenesis. The possibility that GTP might work as a sunscreen to protect against UVB radiation-induced effects in skin, a s observed in the present study, cannot be ruled out. More studies are needed to answer this issue employing individual epicatechin derivatives present in GTP, as GTP itselfdoes not show very strong absorbance in the UVB range (data not shown).

In summary, the results of our study suggest that GTP, in general green tea, protects against UVB radiation-caused tumorigenicity by inhibiting UVB radiation-caused cutaneous

edema, depletion of the epidermal antioxidant-defense system and induction of epidermal ODC and cyclooxygenase activities. These results also suggest that the antineoplastic effects of GTP may be more pronounced a t the promotion stage o f multistage carcinogenesis.

Acknowledgemenrs-This work was supported in part by USPHS grant ES-1900 and P-30-AR-39750, and American Institute for Can- cer Research grant 92B35. R.A. is a recipient ofan Upjohn Company Foundation Career Development Award through the Dermatology Foundation. S.G.K. is supported by a postdoctoral fellowship from NIH under training grant T-AR-07569.

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

1 I .

12.

13.

14.

15.

REFERENCES

Weinstein, I. B. ( 1 99 1) Cancer prevention: recent progress and future opportunities. Cancer Rex 51 (Suppl.), 5080s-5085s. Stich, H. F. (1991) The beneficial and hazardous effects of simple phenolic compounds. Mutar. Res. 259, 307-324. Mukhtar, H., 2. Y. Wang, S. K. Katiyar and R. Aganval ( I 992) Tea components: antimutagenic and anticarcinogenic effects. Prey. Med. 21, 351-360. Wang, Z. Y., W. A. Khan, D. R. Bickers and H. Mukhtar (1 989) Protection against polycyclic aromatic hydrocarbon-induced skin tumor initiation in mice by green tea polyphenols. Carcinogen- esis 10, 411-415. Khan, W. A,, Z . Y. Wang, M. Athar, D. R. Bickers and H. Mukhtar (1988) Inhibition of the skin tumorigenicity of (+)- 7P,8a-dihydroxy-9u, 1 Oa-epoxy-7,8,9, I O-tetrahydroben- zo(a)pyrene by tannic acid, green tea polyphenols and quercetin in SENCAR mice. Cancer Lett. 42, 7-12. Katiyar, S. K., R. Agarwal, Z . Y. Wang, A. K. Bhatia and H. Mukhtar (1 992) (-)-Epigallocatechin-3-gallate in Camellia si- nensis leaves from Himalayan region of Sikkim: inhibitory effects against biochemical events and tumor initiation in SEN- CAR mouse skin. Nutr. Cancer 18, 73-83. Huang, M.-T., C. T. Ho, Z. Y . Wang, T. Ferraro, T. Finnegan- Olive, Y. R. Lou, J . M. Mitchell, J. D. Laskin, H. Newmark, C. S. Yangand A. H. Conney (1992) Inhibitory effect oftopical application of a green tea polyphenol fraction on tumor initiation and promotion in mouse skin. Carcinogenesis 13,947-954. Katiyar, S. K., R. Agarwal, G. S. Wood and H. Mukhtar (1992) Inhibition of 12-0-tetradecanoylphorbol- 13-acetate-caused tumor promotion in 7,12-dimethylbenz[a]anthracene-initiated SENCAR mouse skin by a polyphenolic fraction isolated from green tea. Cancer Rex 52, 6890-6897. Aganval, R., S. K. Katiyar, S. I. A. Zaidi and H. Mukhtar (1992) Inhibition of tumor promoter-caused induction of ornithine decarboxylase activity in SENCAR mice by polyphenolic fraction isolated from green tea and its individual epicatechin derivatives. Cancer Res. 52, 3582-3588. Wang, 2. Y., R. Agarwal, D. R. Bickersand H. Mukhtar (1991) Protection against ultraviolet B radiation-induced photocarcin- ogenesis in hairless mice by green tea polyphenols. Carcinogen- esis 12, 1527-1530. Wang, Z . Y., M. T. Huang, T. Ferraro, C. Q. Wong, Y. R. Lou, M. Iatropoulos, C. S. Yangand A. H. Conney (1992) Inhibitory effect of green tea in the drinking water on tumorigenesis by ultraviolet light and 12-0-tetradecanoylphorbol- 13-acetate in the skin of SKH-1 mice. Cancer Res. 52, 1162-1 170. Hug, D. H. and J. K. Hunter (1991) Photomodulation ofen- zymes. J. Photochem. Photobiol. 10, 3-22. Agarwal, R. and H. Mukhtar (1991) Cutaneous chemical carcinogenesis. In Pharmacology ofthe Skin (Edited by H. Mukh- tar), pp. 371-387. CRC Press, Boca Raton, FL. Khan, S. G., S. K. Katiyar, R. Aganval and H. Mukhtar (1992) Enhancement ofantioxidant and phase I1 enzymes by oral feeding of green tea polyphenols in drinking water to SKH- 1 hairless mice: possible role in cancer chemoprevention. Cancer Res. 52, 4050-405 2. Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Bzochem. 72, 248-254.

700 WEsn AGARWAL et al.

16. Claiborne, A. (1985) Catalasc activity. In CRC Handbook of Methods for Oxygen Radical Research (Edited by R. A. Green- wald), pp. 283-284. CRC Press, Boca Raton, FL.

17. Mohandas, J., J. J. Marshall, G. G. Duggin, J. S. Horvath and D. Tiller (1984) Differential distribution of glutathione and glutathione-related enzymes in rabbit kidney: possible impli- cation in analgesic nephropathy. Cancer Res. 44, 5086-509 1.

18. OBricn, T. G., R. C. Simsiman and R. K. Boutwell (1975) Induction of the polyamine-biosynthetic enzymes in mouse epidermis by tumor-promotingagents. Cancer Res. 35, 1662-1 670.

19. Verma, A. K., B. G. Shapas, H. M. Rice and R. K. Boutwell (1979) Correlation of the inhibition by retinoids of tumor promoter-induced mouse epidermal ornithine decarboxylase activity and of skin tumor promotion. Cancer Res. 39, 419-425.

20. Lysz, T. W. and P. Needleman (1982) Evidence for two forms of fatty acid cyclooxygcnase in brain. J. Neurochetn. 38, 1 1 11- I 117.

21. Epstcin, J. H., K. Fukuama and K. Fye ( I 970) Effect of ultraviolet radiation on the mitotic cycle and DNA, RNA and protein synthesis in mammalian epidermis in vivo. Photochem. Photo-

12. Shah, G., R. Ghosh, P. A. Amstad and P. A. Cerutti (1993) Mechanism of induction of c-fos by ultraviolet B (290-320 nm) in mouse JB6 epidermal cells. Cancer Res. 53, 38-45.

23. Ananthaswamy, H. N. and W. E. Pierceall (1990) Molecular mechanisms of ultraviolet radiation carcinogenesis. Photochem. Photobiol. 52, 11 19-1 136.

14. Matsui, M. S. and V. A. DeLeo (1991) Longwave ultraviolet radiation and promotion of skin cancer. Cancer Cells 3, 8-1 1.

25 Slaga, T. J., A. J. P. Klein-Szanto, L. L. Triplett, L. P. Yotti and J . E. Trosko ( 1 98 1) Skin tumor-promoting activity of benzoyl peroxide, a widely used free radical generating compound. Sci- ence 213, 1023-1025.

26. Kensler, T. W. and M. A. Trush (1 984) Role of oxygen radicals in tumor promotion. Environ. Mutagen. 6 , 593-6 16.

27. Pryor, W. A. (1985) Cancer and free radicals. In Antimuta- genesis and Anticarcinogcncsis Mechanisms (Edited by D. M. Shankel, P. E. Hartman, T. Kada and A. Hollaender), pp. 45- 59. Plenum Press, New York.

28. Agarwal, R. and H. Mukhtar (1993) Oxidative stress in skin chemical carcinogenesis. In Oxidative Stress in Dermatology (Edited by L. Packer and J. Fuchs), pp. 207-24 1, Marcel Dekker, New York, NY.

29. Pathak, M. A. and K. Stratton (1968) Free radicals in human skin before and after exposure to light. Arch. Biochem. Biophys. 123, 468-476.

30. Taira, J., K. Mimura, T. Yoneya, A. Hagi, A. Murakami and K. Makino (1992) Hydroxyl radical formation by UV-irradiated epidermal cells. J. Biochem. 11 1, 693-695.

31. Dixit, R., H. Mukhtar and D. R. Bickers (1983) Studies on the role of reactive oxygen species in mediating lipid peroxide formation in epidermal microsome of rat skin. J. Invest. Der- matol. 81, 369-375.

32. Ogura, R., M. Sugiyama, J. Nishi and N. Haramaki (1991) Mechanism of lipid radical formation following exposure of epidermal homogenate to ultraviolet light. J. Invest. Dermatol. 97, 1044-1047.

33. Cerutti, P. A. and B. F. Trump (1991) Inflammation and oxidative stress in carcinogenesis. Cancer Cell 3, 1-7.

bid. 12, 57-65.

34. Sun, Y. (1990) Free radical antioxidant enzymes, and carcinogenesis. Free Radical Biol. Med. 8, 583-599.

35. Perchellet, J.-P. and E. M. Perchellet (1989) Antioxidants and multistage carcinogenesis in mouse skin. Free Radical Biol. Med. 7, 377-408.

36. Connor, M. J. and L. A. Wheeler (1987) Depletion of cutaneous glutathione by ultraviolet radiation. Photochem. Photo- bid. 46. 2 39-245.

37. Fuchs, J., M. E. Huflejt, L. M. Rothfuss, D. S. Wilson, G. Car- camo and L. Packer ( I 989) Impairment of enzymic and nonen- zymic antioxidants in skin by UVB irradiation. .I. Invest. Der- matol. 93, 769-773.

38. Pence, B. C. and M. F. Naylor (1990) Effects of single-dose ultraviolet radiation on skin superoxide dismutase, catalase and xanthine oxidase in hairless mice. J . Invest. Dermatol. 95, 2 13- 216.

39. Hillebrand, G. G., M. S. Winslow, M. J. Benzinger, D. A. Heit- mcyer and D. L. Bissett (1 990) Acute and chronic ultraviolet radiation induction of epidermal ornithine decarboxylase activity in hairless mice. Cancer Res. 50, 1580-1584.

40. Fischer, S . M., G. S. Cameron, J. K. Baldwin, D. W. Jasheway and K. E. Patrick (1988) Reactive oxygen in the tumor promotion stage of skin carcinogenesis. Lipids 23, 592-597.

41. Athar, M., H. Raza, D. R. Bickers and H. Mukhtar (1990) Inhibition of benzoyl peroxide mediated tumor promotion in 7, I2-dimethylbenz(a)anthracene initiated skin of SENCAR mice by antioxidants nordihydroguaiaretic acid and diallyl sulfide. J. Invest. Dermatol. 94, 162-1 65.

42. Furstenberger, G. and F. Marks (1990) The role ofeicosanoids in normal, hyperplastic, and neoplastic growth in mouse skin. In Eicosanoids and the Skin (Edited by T . Ruzicka), pp. 107- 124. CRC Press, Boca Raton, FL.

43. Furstenberger, G., M. Gross and F. Marks (1982) On the role of prostaglandins in the induction of epidermal proliferation, hyperplasia and tumor promotion in mouse skin. In Prosta- glandins and Cancer: First International Conference on Pros- taglandins and Related Lipids, Vol. 2 (Edited by T. J. Powles, R. S. Bochman, K. V. Honn and P. Ramwell), pp. 239-254. Alan R. Liss, New York.

44. Hanson, D. L. and V. A. DeLeo (1989) Long wave ultraviolet radiation stimulates arachidonic acid release and cyclooxygenase activity in mammalian cells in culture. Photochem. Photo- biol. 49, 423-430.

45. Bonta, J. L. and M. J. Parnham (1978) Prostaglandins and chronic inflammation. Biochem. Pharmacol. 27, 161 1-1 623.

46. Samuelsson, B. (1983) Leukotrienes: a new class of mediators of immediate hypersensitivity reactions and inflammation. In Advances in Prostaglandin, Thromboxane and Leukotriene Re- search, Vol. 1 I (Edited by B. Samuelsson, R. Paoletti and P. W. Ramwell), pp. 1-13. Raven Press, New York.

47. Anggard, E. and C. E. Jonsson (1972) Formation of prostaglandins in the skin following a bum injury. In Prostaglandins in Cellular Biology, Vol. 1 (Edited by P. W. Ramwell and B. B. Phams), pp. 269-29 1. Plenum, New York.

48. Kragballe, K., G. J. Fisher and J. J. Voorhees (1990) Eicosa- noids, phosphoinositides, and the pathophysiology of psoriasis. In Eicosanoids and the Skin (Edited by T. Ruzicka), pp. 67-78. CRC Press, Boca Raton, FL.

Documents

PROTECTION AGAINST ULTRAVIOLET B RADIATION-INDUCED EFFECTS IN THE SKIN OF SKH-1 HAIRLESS MICE BY A POLYPHENOLIC FRACTION ISOLATED FROM GREEN TEA