440 Hasan Mukhtar et a/.

44.

45.

46.

Stanfield and L. K. Roberts (1995) Minimum doses of ultravi- olet radiation required to induce murine skin edema and im- munosuppression are different and depend on the ultraviolet emission spectrum of the source. Photochem. Phutobiol. 62, 106C1075. Fan, P. M. and B. L. Diffey (1985) How reliable are sunscreen protection factors? Br. J . Dermatol. 112, 113-1 18. Katiyar, S. K., C. A. Elmets, R. Agarwal and H. Mukhtar (1995) Protection against ultraviolet-B radiation-induced local and sys- temic suppression of contact hypersensitivity and inflammatory responses in C3WHeN mice by green tea polyphenols. Photo- chem. Photobiol. 62, 855-861. Strickland, F. M., R. P. Pelley and M. L. Kripke (1994) Pre- vention of ultraviolet radiation-induced suppression of contact and delayed hypersensitivity by Aloe barbadensis gel extract. J. Invest. Dermatol. 102, 197-204.

Chemoprevention of Photocarcinogenesis

Rajesh Agarwal and Hasan Mukhtar

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

Disease prevention is an old concept where a 17th century physician Thomas Adams once said “hee is a better physi- cian that keepes disease off us, than hee that cures them being on us. Prevention is so much better than healing, be- cause it saves us the labor of being sick.” Because UV ra- diation from sun exposure is a major cause of nonmelanoma human skin cancers (1-4), significant efforts have been made to devise methods that could protect against the adverse con- sequences of sun exposure. In addition to the prevention strategies like avoiding excessive sun exposure by limiting outdoor activities and wearing protective clothing while out- side in the sun, the one that is receiving increasing attention is the use of “sunscreens” on the body surface to be ex- posed to sun. In more recent times, the approach of topical and/or dietary use of chemical agents is also receiving in- creasing attention as a possibility to protect humans against solar radiation-caused induction of nonmelanoma skin can- cers; both basal cell carcinomas (BCC)@$§ and squamous cell carcinomas (SCC). This in general terms is called chem- oprevention. An attempt has been made in this article to summarize some of these studies. However, greater empha- sis has been placed on two agents, namely a polyphenolic fraction isolated from green tea (GTP) and silymarin. In our published studies and ongoing work, we have shown that these two agents exhibit highly significant protective effects against photocarcinogenesis in the mouse skin model sys- tem.

§§$$Abbreviations: BCC, basal cell carcinomas; DMBA, 7,12-di- methylbenz(a)anthracene; EGCG, ( -)-epigallocatechin-3-gallate; GTP, a polyphenolic fraction isolated from green tea; ODC, or- nithine decarboxylase; SCC, squamous cell carcinomas; TPA, 12- 0-tetradecanoylphorbol- 13-acetate; WEGT, water extract of green tea.

Chemoprevention and an ideal agent

Chemoprevention of cancer is a means of cancer control where the occurrence of the disease, as a consequence of exposure to carcinogenic agents (UV radiation in the case of photocarcinogenesis), can be entirely prevented, slowed or reversed by the administration of one or more chemical agents. It also includes the chemotherapy of precancerous lesions, which in the case of photocarcinogenesis is actinic keratosis. The chemopreventive agents are also known as anticarcinogens, and an ideal agent for human use should have (a) little or no untoward or toxic effects, (b) high ef- ficacy against multiple sites, (c) capability of oral adminis- tration, (d) a known mechanism of action, (e) low cost and (0 the acceptability by the population at risk. While a wide range of laboratory studies and limited epidemiological stud- ies in humans have identified many compounds including several polyphenols, as chemopreventive agents against var- ious cancer cites (3, except the sunscreen as a protection strategy against photocarcinogenesis, only limited studies have assessed the protective effects of chemical agents against photocarcinogenesis (6,7).

Multistage photocarcinogenesis model and associated events

Almost all the studies, whether assessing UV radiatiodsolar radiation as an etiologic factor in the induction of carcino- genesis or delineating different cellular, biochemical and molecular events associated with exposure to UV radiation or those assessing the chemopreventive efficacy of various chemical agents against photocarcinogenesis, have been con- ducted employing a mouse skin model (8-10). Together these studies have been extremely valuable in identifying chemopreventive agents against photocarcinogenesis and have proven extremely useful in defining the mechanism of photocarcinogenesis and identifying the targets associated with photocarcinogenesis, some of which could be modulat- ed to achieve the prevention of cancer due to UV exposure.

It is recognized that carcinogenesis in mouse skin and presumably in human skin and other tissues is a multistage process comprising of initiation, promotion and progression (6,7). In the case of photocarcinogenesis induced in mouse skin, it has been identified that the UVB (290-320 nm) spec- trum of the solar radiation acts as a complete carcinogen as well as a tumor initiator and tumor promoter (6-1 1). As yet no study has clearly shown that UVB (or UVA or UVC) radiation may also be involved in tumor progression. With regard to tumor initiation, it has been shown that UVB ra- diation results in DNA damage to epidermal cells, leading to the formation of cyclobutane dimers and 6-4 photoprod- ucts (12,13). Some of this DNA damage is repaired by en- zymatic pathways catalyzed by endonucleases (12,13), whereas unrepaired lesions ultimately lead to the fixation of mutation in the target gene presumably p53 tumor suppres- sor gene (14-18); no study, however, has shown clearly the fixation of the p.53 gene mutation as the obligatory step in tumor initiation even in the mouse skin photocarcinogenesis protocol. These events ultimately lead to tumor initiation, which is an irreversible process. Further exposure of initiated skin cells, (presumably the stem cells) to UV radiation or other known chemical tumor promoters such as phorbol es-

Photochemistry and Photobiology, 1996, 63(4) 441

Table 14. human skin cancers

Summary of chemopreventive agents against photocarcinogenesis in mouse skin model and in clinical trials of nonmelanoma

Agent Effective against Reference

Studies in mouse skin carcinogenesis model Butylated hydroxytoluene Canthaxanthin Carotenoids a-Difluoromethylornithine Green tea Indomethacin Omega-3 fatty acid sources Retinyl palmitate Silymarin Sunscreens Vitamin E

Human studies: clinical trials Sunscreens Dietary fat Retinoids: all-trans and 13-

cis retinoic acid, etretinate, arotinoid and fenretinide

Indomethacin

UVB in SKH-I hairless UVB in C3WHeN UVB in SKH-1 hairless UVB in BALB/c UVB in SKH-1 hairless UVB in SKH-1 hairless UVB in SKH-1 hairless UVB in C3HMeN UVB in SIU-1 hairless UVB in SKH-1 hairless and C3H UVB in C3HMeN

Actinic keratoses, BCC, SCC Actinic keratoses Actinic keratoses, keratoacanthomas, xeroder-

ma pigmentosum, BCC, SCC, dysplastic nevi, epidermodysplasia vermciformis

Skin cancers in xeroderma pigmentosum pa- tients

( 3 3 ) (46)

(4749)

ter, results in the clonal expansion of initiated cells leading to the formation of many benign papillomas (6,7). This is known as the tumor promotion process, which is reversible at least in early stages, however, becomes irreversible in late stages (6,7). Although the exact mechanism by which UV radiation acts as a tumor promoter is not well defined, it has been shown that UV radiation (a) stimulates DNA synthesis and cell proliferation, (b) causes epidermal hyperplasia and depletion of the antioxidant-defense system, (c) induces or- nithine decarboxylate (ODC) enzyme activity and mRNA expression and prostaglandin synthesis and (d) impairs sig- nal transduction pathways ( 19,20). Because these responses are similar to those induced by many chemical tumor pro- moters like 12-0-tetradecanoylphorbol 13-acetate (TPA) (6,7), it shows some parallelism between skin tumor pro- motion by UV radiation and chemical tumor promoters. In recent years, studies have also shown that UVB radiation results in apoptotic sun burn cell formation and fixation of mutation in p53 gene (14-18,21,22). It is unclear as to what stage in the UVB radiation induced tumorigenesis, these events occur. With regard to tumor progression, it is iden- tified that most of the benign papillomas formed in mouse skin during tumor initiation-promotion regimen of UVB ir- radiation, either remain nonmalignant or regress spontane- ously, whereas some of them convert to malignant carcino- mas (6,7). This is also an irreversible process, and its mech- anism is not yet well defined except that it is assumed that additional genetic changes occur during this stage (6,7). With regard to the role of UV radiation in tumor progression, no study as yet has shown the direct role of UV radiation in tumor progression in mouse skin either in photocarcinogen- esis or chemical carcinogenesis protocols. With respect to multistage photocarcinogenesis, the chemoprevention strat- egy involves identification of agents that can be targeted against each of the these three stages, and therefore chem-

opreventive agents could possess antitumor initiating, anti- tumor promoting and/or antitumor progressing properties.

Chemopreventive agents against photocarcinogenesis: animal studies

As shown in Table 14, limited studies have shown the chem- opreventive potential of both naturally occurring and syn- thetic chemical agents against photocarcinogenesis in an an- imal model (23-34). These agents include butylated hy- droxytoluene (23) , canthaxanthin (24), carotenoids (25), a- difluoromethylornithine (26) , green tea, (27,28) indomethacin (29), omega-3 fatty acid sources (30) retinyl palmitate (24) silymarin (31,32), sunscreens (33) and vita- min E (34). In this article, however, our emphasis is on stud- ies with green tea and silymarin conducted in our laborato- ries.

Protection against photocnrcinogenesis by green ten. Green tea is widely consumed in China, Korea and Japan, to some extent in India and a few countries in North Africa and Middle East (5,35). The term “green tea” is related to its process of manufacturing in which fresh tea leaves are steamed and/or dried at high temperature with the precaution to avoid oxidation of the polyphenolic compounds, which are mainly epicatechin derivatives. These are the agents that have been shown to be associated with the cancer chemo- preventive effects of green tea. The qualitative and quanti- tative analysis of GTP has shown that it contains four epi- catechin derivatives, namely (-)-epigallocatechin, (-)-epi- gallocatechin-3-gallate (EGCG), (- )-epicatechin and (-)- epicatechin-3-gallate, and constitute 60-70% (wt/wt) of total GTP (5,35). Our laboratory has been studying chemopre- vention of cancer by green tea where in a series of studies we have demonstrated that GTP, water extract of green tea (WEGT) and EGCG, the major polyphenolic compound

442 Hasan Mukhtar eta/.

present in green tea, possess significant anticarcinogenic ef- fects in skin and other tumor bioassay systems (535). In recent years several other laboratories have also verified the cancer chemopreventive effects of GTP, WEGT and EGCG (5,35,36). Below, however, we have provided a brief over- view of studies showing the chemopreventive potential of green tea against photocarcinogenesis in mouse model, and also summarized the work delineating the mechanism of such preventive effect.

Studies from our laboratory for the first time reported the preventive effect of GTP against UVB radiation-induced tu- morigenicity in SKH-1 hairless mice (27). In this study we showed that chronic oral feeding of GTP (0.1%, wt/vol) in drinking water to SKH-1 hairless mice during the entire pe- riod of UVB-carcinogenesis protocol results in significantly lower tumor incidence and multiplicity as compared to the animals that did not receive GTP (27). Results of this study also showed that chronic oral feeding of GTP has an ex- tended 50% probability of tumor development time as com- pared to animals receiving normal drinking water (27). In this study, topical application of GTP before UVB irradia- tion also afforded some protection against photocarcinogen- esis, however, it was lower than that observed after oral feeding of GTP in drinking water (27). In another study from the laboratory of Conney (28) it was shown that feeding WEGT (1.25%, wt/vol) as the sole source of drinking water to SKH-1 hairless mice affords protection in a dose-depen- dent manner, against UVB radiation-induced intensity of er- ythema and area of skin lesions, as well as UVB radiation- induced skin tumor initiation (TPA was used as tumor pro- moter) and skin tumor promotion (7,12-dimethyl- benz[~]anthracene [DMBA] was used as tumor initiator). In another study (37), it has been shown that oral feeding of WEGT or intraperitoneal administration of GTP or EGCG results in the inhibition of tumor growth and also causes partial regression of established papillomas in mouse skin. These results, in conjunction with our studies, suggested the possibility that green tea may reduce the risk of skin cancer induction in humans by solar radiation.

Because exposure of mouse skin to UVB radiation is known to result in cutaneous edema, depletion of the anti- oxidant-defense system and induction of ODC and cycloox- ygenase activities (19), we assessed whether GTP affords protection against these UVB radiation-caused changes in mouse skin (38). In these studies, oral feeding of GTP (0.2%, wt/vol) as the sole source of drinking water for 30 days to SKH-I hairless mice followed by UVB (900 mJ/ cm2) irradiation resulted in significant protection against UVB radiation-caused cutaneous edema and depletion of the antioxidant-defense system in epidermis (38). The oral feed- ing of GTP also resulted in significant protection against UVB radiation-induced epidermal ODC and cyclooxygenase activities (38). The results of this study provided evidence that the inhibition of UVB radiation-caused biochemical changes in mouse skin by GTP may be one of the possible mechanisms of the chemopreventive effect of green tea against photocarcinogenesis in mouse model. Because ex- posure of skin to UV radiation also results in the induction of immunosuppression and cutaneous inflammatory re- sponses (39), we also investigated whether GTP could pro- tect against these UVB-induced effects (40). Immunosup-

pression was assessed in C3H mice by contact sensitization with 2,4-dinitrofluorobenzene applied to UVB-irradiated skin (local suppression) or to a distant site (systemic sup- pression), while double fold skin swelling was used as the measure of UVB-induced inflammation. Topical application of GTP 30 min prior to or after exposure to a single dose of UVB resulted in significant protection against local and systemic suppression of contact hypersensitivity and inflam- mation in mouse dorsal skin in a dose-dependent manner (40). Among the epicatechin derivatives present in GTP, EGCG was found to be the most effective in affording pro- tection against UVB-caused contact hypersensitivity and in- flammatory responses (40). Taken together, the results of these two studies suggested that green tea in general and the epicatechin derivative present therein in particular may be useful agents against inflammatory dermatoses and immu- nosuppression associated with the exposure of the skin to solar radiation.

Protection against photocarcinogenesis by silymarin. Si- lymarin, a flavonoid isolated from the milk thistle of Silybum marianum (L.) Gaertn. (artichoke) (41), is composed of sil- ybin, silydianin and silichristin isomers; the major constitu- ent (>98%) is silybin. For the past two decades silymarin has been used clinically, at least in Europe, for the treatment of alcoholic liver diseases (41,42). In laboratory studies, si- lymarin has been shown to protect against hepatotoxicity induced by allylalcohol, carbon tetrachloride, galactosamine, phalloidin, thioacetamide and microcystin-LR (41,42). The studies with hepatocyte and liver microsomes showed that silymarin affords protection against lipid peroxidation in- duced by several xenobiotic agents (43). In addition, the mechanistic studies have also shown that silymarin is a strong antioxidant capable of scavenging free radicals (44). Because several studies have shown that many antioxidants afford protection against tumor promotion by inhibiting ox- idative stress induced by tumor promoters (6,7,19), and be- cause an increase in the expression of ODC has been sug- gested to be a prerequisite, though not obligatory, for tumor promotion (26,7) in recently published studies we reported the inhibitory effect of topical application of silymarin on TPA and other known skin tumor promoter-induced epider- mal ODC activity and ODC mRNA levels in SENCAR mice (45). The results of these studies suggested the possibility that silymarin could be a useful antitumor-promoting agent capable of ameliorating the tumor-promoting effects of a wide range of tumor promoters including UV radiation. Long-term tumor studies, therefore, were conducted employ- ing mouse skin photocarcinogenesis model to evaluate the cancer chemopreventive effects of silymarin.

First, the studies were carried out to assess the preventive effect of silymarin against UVB radiation-caused tumor ini- tiation, tumor promotion and complete carcinogenesis in SKH-1 hairless in mouse skin (31,32). In all these studies, the dose of silymarin used was 6 mg per mouse per appli- cation in 0.2 mL acetone. In an antitumor initiation protocol, UVB as tumor initiator and TPA as the tumor promoting agent, topical application of silymarin for 7 days prior to UVB exposure, afforded 66% protection in terms of tumor multiplicity ( 3 1,32). In the antitumor promotion protocol, DMBA as tumor initiator and UVB as the tumor-promoting agent, the application of silymarin prior to each UVB irra-

Photochemistry and Photobiology, 1996, 63(4) 443

diation resulted in highly significant protection against UVB- caused tumor promotion. The protective effects of silymarin were evident in terms of delay in the latency period on the onset of first tumor, tumor incidence, tumor multiplicity as well as tumor volume (3 1,32). In the anticomplete carcino- genesis protocol employing UVB as carcinogen, the appli- cation of silymarin prior to each UVB irradiation resulted in highly significant protection against UVB-caused tumorige- nicity as evident by prolonged delay in the latency period on the onset of first tumor and highly reduced tumor inci- dence, tumor multiplicity as well as tumor volume (3 132). It was of interest that the protective effect of silymarin was most profound in the complete carcinogenesis protocol of UVB radiation-induced photocarcinogenesis. Taken togeth- er, these studies clearly demonstrated that silymarin pos- sesses a strong chemopreventive potential against photocar- cinogenesis.

We also conducted studies to define the mechanism as- sociated with the preventive effect of silymarin against UVB-caused tumorigenesis (32). In these studies, the appli- cation of silymarin to mouse skin 30 min prior to that of UVB irradiation resulted in significant protection against UVB-caused (a) skin edema, (b) induction of epidermal ODC and cyclooxygenase activities and (c) induction of epi- dermal ODC mRNA expression (32). Detailed studies are needed to define the exact mechanism of the chemopreven- tive effect of silymarin against photocarcinogenesis.

Chemopreventive agents against photocarcinogenesis: human studies

With regard to chemoprevention of photocarcinogenesis in humans, because of the difficulties associated with such kinds of studies, only a limited class of agents are known to protect against nonmelanoma human skin cancers. As listed in Table 14, these include sunscreens (33), dietary fat (46), retinoids (4749) (specifically 13-cis retinoic acid) and in- domethacin (SO). In the “real definition” of chemopreven- tion of nonmelanoma human skin cancers, “sunscreens” are the only class of agents with reasonably proven efficacy at the present time. Several studies have shown the preventive effect of sunscreens against nonmelanoma human skin can- cers (33) , and this is comprehensively reviewed separately in this special issue.

With regard to dietary fat, studies conducted in the mouse model have shown that a high fat diet increases the proba- bility of skin cancer by UV radiation, and conversely, chang- ing to a low fat diet regimen after UV exposure can reduce the incidence of skin cancer (30). Recently Black et al. (46) conducted a study to extrapolate these findings in an animal model to nonmelanoma human skin cancer risk. It was found that the consumption of a low fat diet leads to a significantly lower incidence of actinic keratoses in a population with a history of nonmelanoma human skin cancer (46); actinic ker- atoses is a precancerous condition and appears as red or brown rough scaly patches on the human skin. These results could be of greater significance because human skin actinic keratoses has recently been shown to carry mutations in the pS3 tumor suppressor gene and is implicated in the etiology of some forms of human skin nonmelanoma cancers, both BCC and SCC (22). Regarding the chemoprevention of hu-

man skin cancers by retinoids, as reviewed recently by Chen and De Luca (47), numerous studies have evaluated the ef- ficacy of retinoids in preventing the recurrence of certain human skin cancers, as well as in their treatment (regres- sion). The preneoplastic and neoplastic lesions in human skin that are treated with retinoids include actinic keratosis, keratoacanthomas, xeroderma pigmentosum, BCC and SCC, dysplastic nevi, melanoma and epidermodysplasia verruci- formis (4749). The retinoids employed against these skin lesions include retinoic acid, 13-cis retinoic acid (isotreti- noin), etretinate, arotinoid and fenretinide (4749). With re- gard to indomethacin, in a clinical study it has been shown that indomethacin alleviates skin cancer induction in xero- derma pigmentosum patients (50). To the best of our knowl- edge, no followup work with this agent at least in clinical trials of human skin cancers is available.

Acknowledgements-The original studies with green tea were sup- ported by the American Institute for Cancer Research grants 92B35 and 93A30, and United States Public Health Scrvice grants ES-1900 and P-30-AR-39750. The studies with silymarin were supported by United States Public Health Service grant CA 64514.

References

1. Elmets, C. A. (1991) Cutaneous photocarcinogenesis. In Phar- macology of the Skin (Edited by H. Mukhtar), pp. 389416. CRC Press, Boca Raton, FL.

2. Ananthaswamy, H. N. and W. E. Pierceall (1990) Molecular mechanisms of ultraviolet radiation carcinogenesis. Photochem. Photobzol. 52, 1 119-1 136.

3 . Marks, R. (1995) An overview of skin cancers, incidence and causation. Cancer (Suppl.) 75, 607-612.

4. Epstein, J. H. (1989) Photocarcinogenesis, skin cancer, and ag- ing. In Aging and the Skin (Edited by A. K. Balin and A. M. Kligman), pp. 307-346. Raven Press, New York, NY.

5. Agarwal, R. and H. Mukhtar (1995) Cancer chemoprevention by polyphenolic compounds present in green tea. Drug News & Perspect. 8, 2 16-225.

6. Agarwal, R. and H. Mukhtar (1991) Cutaneous chemical car- cinogenesis. In Pharmacology of the Skin (Edited by H. Mu- khtar), pp. 371-387. CRC Press, Boca Raton, FL.

7. DiGiovanni, J. (1 992) Multistage carcinogenesis in mouse skin. Pharmacol. Ther. 54, 63-128.

8. Kripke, M. L. (1977) Latency, histology, and antigenicity of tumors induced by ultraviolet light in three inbred mouse strains. Cancer Rex 37, 1395-1400.

9. Epstein, J. H. (1978) Photocarcinogenesis: a review. Nutl. Can- cer Inst. Monogr. 50, 13-25.

10. Forbes, P. D. (1981) Photocarcinogenesis: an overview. J . In- vest. Dernuitol. 77, 139-1 43.

1 I . Yuspa, S. H. And A. A. Dlugosz (1991) Cutaneous carcinogen- esis: natural and experimental. In Biochemistry and Physiology of the Skin, 2nd ed. (Edited by L. A. Goldsmith), pp. 1365- 1402. Oxford University Press, New York.

12. Lippke, J. A,, K. Gordon, D. E. Brash and W. A. Haseltine (1981) Distribution of UV-light-induced damage in a defined sequence of human DNA: detection of alkaline-sensitive lesions at pyrimidine nucleotide-cytidine sequences. P roc. Natl. Acud. Sci. USA 78, 3388-3392.

3. Rosenstein, B. S. and D. L. Mitchell (1987) Action spectra for the induction of pyrimidine (6-4) pyrimidine photoproducts and cyclobutane dimers in normal human skin fibroblasts. Photo- chern. Photohiol. 45, 775-78 1.

4. Nataraj, A. J., J. C. Trent, I1 and H. N. Ananthaswamy (1995) p53 gene mutations and photocarcinogenesis. Photochem. Pho- tohiol. 62, 21 8-230.

5. Brash, D. E., J. A. Rudolph, J. A. Simon, A. Lin, G. J. Mc- Kenna, H. P. Baden, A. J . Halperin and J . Ponten (1991) A role for sunlight in skin cancer: UV-induced p53 mutations in squa-

444 Hasan Mukhtar et at.

mous cell carcinoma. Proc. Natl. Acad. Sci. USA 88, 10124- 10128.

16. Pierceall, W. E., T. Mukhopadhyay, L. H. Goldberg and H. N. Ananthaswamy (1991) Mutations in the p53 tumor suppressor gene in human cutaneous squamous cell carcinomas. Mol. Car- cinogenesis 4, 445449.

17. Ziegler, A., D. J. Leffell, S. Kunala, H. W. Sharma, M. Gailani, J. A. Simon, A. J. Halperin, H. P. Baden, P. E. Shapiro, A. E. Bale and D. E. Brash (1993) Mutation hotspots due to sunlight in the p53 gene of nonmelanoma skin cancers. Proc. Natl. Acad. Sci. USA 90, 42164220.

18. Kanjilal, S., W. E. Pierceall, K. K. Cummings, M. L. Kripke and H. N. Ananthaswamy (1993) High frequency of p53 mu- tations in ultraviolet radiation-induced murine skin tumors: ev- idence for strand bias and tumor heterogenicity. Cancer Res. 53, 2961 -2964.

19. Agarwal, R. and H. Mukhtar (1993) Oxidative stress in skin chemical carcinogenesis. In Oxidative Stress in Dermatology (Edited by J. Fuchs and L. Packer), pp. 207-241. Marcel Dek- ker, New York.

20. Miller, C. C., P. Hale and A. P. Pentland (1994) Ultraviolet B injury increases prostaglandin synthesis through a tyrosine ki- nase-dependent pathway. Evidence for UVB-induced epidermal growth factor receptor activation. J . B i d . Chem. 269, 3529- 3533.

21. Kane, K. S. and E. V. Maytin (1995) Ultraviolet B-induced apoptosis of keratinocytes in murine skin is reduced by mild local hyperthermia. J . Invest. Dermatol. 104, 62-67.

22. Ziegler, A,, A. S. Jonason, D. J. Leffell, 3. A. Simon, H. W. Sharma, J. Kimmelman, L. Remington, T. Jacks and D. E. Brash (1994) Sunburn and p53 in the onset of skin cancer. Nature 372, 773-776.

23. Black, H. S. and M. M. Mathews-Roth (1991) Protective role of butylated hydroxytoluene and certain carotenoids in photo- carcinogenesis. Photochem. PhotobioL 53, 707-71 6.

24. Gensler, H. L., M. Aickin and Y. M. Peng (1990) Cumulative reduction of primary skin tumor growth in UV-irradiated mice by the combination of retinyl palmitate and canthaxanthin. Can- cer Lett. 53, 27-31.

25. Mathews-Roth, M. M. and N. I. Krinsky (1985) Carotenoid dose level and protection against UV-B induced skin tumors. Pho- tochem. Photobiol. 42, 35-38.

26. Gensler, H. L. (1991) Prevention by alpha-difluoromethylorni- thine of skin carcinogenesis and immunosuppression induced by ultraviolet irradiation. J. Cancer Res. Clin. Oncol. 117, 345- 350.

27. Wang, 2 . Y., R. Agarwal, D. R. Bickers and H. Mukhtar (1991) Protection against ultraviolet B radiation-induced photocarcin- ogenesis in hairless mice by green tea polyphenols. Carcino- genesis 12, 1527-1530.

28. Wang, 2. Y., M.-T. Huang, T. Ferraro, C.-Q. Wong, Y.-R. Lou, M. Iatropoulos, C. S. Yang and 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.

29. Reeve, V. E., M. J. Matheson, M. Bosnic and C. Boehm-Wilcox (1995) The protective effect of indomethacin on photocarcino- genesis in hairless mice. Cancer Lett. 95, 2 13-2 19.

30. Black, H. S., J. I. Thornby, J. Gerguis and W. Lenger (1992) Influence of dietary omega-6, -3 fatty acid sources on the ini- tiation and promotion stages of photocarcinogenesis. Photo- chem. Photobiol. 56, 195-199.

31. Agarwal, R., S. K. Katiyar and H. Mukhtar (1995) Protective effects of silymarin against ultraviolet B radiation-induced tu- morigenesis in SKH- 1 hairless mouse skin. J. Invest. Dermatol. 104, 635.

32. Agarwal, R., S. K. Katiyar and H. Mukhtar (1995) Protection against photocarcinogenesis in SKH-1 hairless mice by sily- marin. Photochem. Photobiol. 61S, 14s.

33. Pathak, M. A. (1991) Sunscreens: principles of photoprotection. In Pharmacology of the Skin (Edited by H. Mukhtar), pp. 229- 248. CRC Press, Boca Raton, FL.

34. Gerrish, K. E. and H. L. Gensler (1993) prevention of photo- carcinogenesis by dietary vitamin E. Nutr. Cancer 19, 125-133.

35. Mukhtar, H., S. K. Katiyar and R. Agarwal (1994) Green tea and skin-anticarcinogenic effects. J. Invest. Dermatol. 102, 3-7.

36. Yang, C. S. and Z. Y. Wang (1993) Tea and cancer. J . Natl. Cancer Inst. 85, 1038-1049.

37. Wang, Z. Y., M.-T. Huang, C.-T. Ho, R. Chang, W. Ma, T. Ferraro, K. R. Reuhl, C. S. Yang and A. H. Conney (1992) Inhibitory effect of green tea on the growth of established skin papillomas in mice. Cancer Res. 52, 6657-6665.

38. Agarwal, R., S. K. Katiyar, S. G. Khan and H. Mukhtar (1993) Protection against ultraviolet B radiation-induced effects in the skin of SKH-1 hairless mice by a polyphenolic fraction isolated from green tea. Photochem. Photobiol. 58, 695-700.

39. Elmets, C. A,, P. R. Bergstresser, R. E. Tigelaar, R. J. Wood and J. W. Streilein (1983) Analysis of the mechanism of unre- sponsiveness produced by haptens painted on skin exposed to low dose ultraviolet radiation. J. Exp. Med. 158, 781-794.

40. Katiyar, S. K., C. A. Elmets, R. Agarwal and H. Mukhtar (1995) Protection against ultraviolet B radiation-induced local and sys- temic suppression of contact hypersensitivity and edema re- sponses in C3WHeN mice by green tea polyphenols. Photo- chem. Photobiol. 62, 855-861.

41. Mereish, K. A,, D. L. Bunner, D. R. Ragland and D. A. Creasia (199 1 ) Protection against microcystin-LR-induced hepatotoxic- ity by silymarin: biochemistry, histopathology, and lethality. Pharmac. Res. 8, 273-277.

42. Letteron, P., G. Labbe, C. Degott, A. Berson, B. Fromenty, M. Delaforge, D. Larrey and D. Pessayre (1990) Mechanism for the protective effects of silymarin against carbon tetrachloride-in- duced lipid peroxidation and hepatotoxicity in mice. Biochem. Pharmacol. 39, 2027-2034.

43. Carini, R., A. Comoglio, E. Albano and G. Poli (1992) Lipid peroxidation and irreversible damage in the rat hepatocyte mod- el. Protection by the silybin-phospholipid complex IdB 1016. Biochem. Pharmacol. 43, 21 11-21 15.

44. Comoglio, A,, G. Leonarduzzi, R. Carini, D. Busolin, H. Bas- aga, E. Albano, A. Tomasi, G. Poli, P. Morazzoni and M. I . Magistretti (1990) Studies on the antioxidant and free radical scavenging properties of IdB 1016 a new flavanolignan complex. Free Radical Res. Commun. 11, 109-115.

45. Agarwal, R., S. K. Katiyar, D. W. Lundgren and H. Mukhtar (1994) Inhibitory effect of silymarin, an anti-hepatotoxic fla- vonoid, on 12-0-tetradecanoylphorbol- 13-acetate-induced epi- dermal ornithine decarboxylase activity and mRNA in SENCAR mice. Carcinogenesis 15, 1099-1 103.

46. Black, H. S., A. Herd, L. H. Goldberg, J. E. Wolf, 3. I. Thornby, T. Rosen, S. Bruce, J. A. Tschen, J. P. Foreyt, L. W. Scott, S. Jaax and K. Andrews (1994) Effect of a low-fat diet on the incidence of actinic keratosis. N. Engl. J. Med. 330, 1272-1275.

47. Chen L .X . and L. M. De Luca (1995) Retinoid effects on skin cancer. In Skin Cancer: Mechanism and Human Relevance (Ed- ited by H. Mukhtar), pp. 401424. CRC Press, Boca Raton, FL.

48. Lippman, S. M., J. F. Kessler and F. L. Meyskens, Jr (1987) Retinoid as preventive and therapeutic anticancer agents (Part 11). Cancer Trear. Rep. 71, 493-515.

49. Lippman, S. M. and F. L. Meyskens, Jr (1989) Results of the use of vitamin A and retinoid in cutaneous malignancies. Phar- macol. Ther. 40, 107-122.

50. Al-Saleem, T., 2. S. Ali and M. Gassab (1980) Skin cancer in xeroderma pigmentosum: response to indomethacin and ster- oids. Lancet 1, 264-265.