Skin Chemical Carcinogenesis
Hasan Mukhtar, PhD,’ Hans F. Merk, MD.7 and Mohammad Athar, PhD‘
From the *Departments of Dermatology, Case Western Reserve University, School of Medicine, Cleveland, Ohio, and t University of Cologne, Federal Republic of Germany.
The purpose of this review is to update clinical dermatologists about current concepts of chemical carcinogenesis in skin. The incidence of skin cancer is increasing steadily and it has been estimated that of all new cancers diagnosed annually in the United States, almost one third originate in the skin.’ Although solar radiation is the major cause of skin cancers, it is beyond doubt that chemicals also contribute substantially to the growing incidences of cutaneous neoplasms in humans.2 The most common benign tumors induced by chemicals in experimental animals are papillomas and keratoacanthomas, whereas common malignancies are squamous cell carcinomas, basal cell carcinomas and melanomas.3 Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants and represent one of the few clearly defined classes of chemicals responsible for the development of skin cancers.3 Humans are constantly exposed to PAHs through polluted air, cigarette smoke, automobile exhaust, and other air-borne pollutants. During the last four decades, it became clear that chemical carcinogenesis in murine skin, and possibly in human skin, is a stepwise process comprising at least three distinct stages: initiation, promotion, and malignant conversion. This is represented diagramatically in Figure l-l. The initiation stage is essentially an irreversible step in which genetic changes occur possibly in gene(s) controlling differentiation. The promotion stage, on the other hand, leads to the formation of visible lesions through epigenetic mechanisms. This leads to the development of premalignant lesions (palillomas) which can then progress to malignant tumors (carcinomas).3T4 Concept
There is an increasing awareness that the induction of cancer is a multistep process requiring both initiating and promoting substances for the development of a malignant tumor. In murine skin, tumors are generally induced by a single topical application of the initiating agent followed by repeated topical applications of the promoting agent. This is known as the two-stage carcinogenesis protocol. The concept of initiation-promotion leading to papilloma and is shown in Figure 1-2. 7,12-dimethylbenz(a)anthracene Supported in part by carcinoma agent employed in the USPHS grants ES-l 900 (DMBA), a synthetic PAH, is a conventional majority of studies on murine skin carcinogenesis. The initiation phase and CA-38028. of cutaneous chemical carcinogenesis in a two stage carcinogenesis protocol can be achieved by a single topical application of subthreshold 1
FIG. l-l. Multistage carclnogenesis showing initiation-promotion leading to papilioma development and subsequent progression of papilloma to carcinoma.
doses of a carcinogen such as DMBA. This is essentially an irreversible step. This subthreshold dose of carcinogen does not produce tumors over the life span of mice. Promotion with multiple applications of croton oil or a phorbol ester such as 12-O-tetradecanoylphorbol-13-acetate (TPA), however, leads to the development of benign papillomas after a short latency period. The majority of the benign papillomas thus formed regress, whereas only a few of them develop to carcinoma. A lapse of up to 1 year between application of initiator and the beginning of promotor treatment produces a tumor response similar to that observed when promotion is begun only 1 week after initiation
Clinics in Dermatology
Absence of response Number of I+) under the head paptlloma or carcmoma represents relative number 01 tumors wtthln the group
FIG. l-2. Development of papilloma and carcinoma in murine skin following single and multiple topical applications of initiating (DMBA) and/or promoting (TPA) agents.
with the carcinogen.4 Unlike initiation, the promotion phase is initially reversible but becomes irreversible during later stages. Tumor promoters are usually nonmutagenic and noncarcinogenic agents and application of them alone to normal mouse skin does not produce any tumors. A single topical application of a large dose of carcinogen on mouse skin can also result in cutaneous tumor induction. The latency period, however, becomes longer and the number of papillomas produced is lower compared with that produced in initiation-promotion protocol. The repeated applications of subthreshold doses of a carcinogen also induces cutaneous tumors but the number of carcinomas developed with this protocol is much higher than that developed when DMBA is used as initiating agent and TPA is employed as a promoting agent.475
Chemical Aaents as Skin Carcinoaens A large number of chemicals have been identified as initiating agents for cutaneous carcinogenesis in animal test systems. The list of agents summarized in Table l-l is limited to known human exposure. The initiating agents for murine skin include PAHs, nitrosamines, nitrosamide, aromatic amines, and various other alkylating agents.4-fi The promoting agents, which include a wide variety of compounds, are listed in Table 1-2. These compounds include various plant products, PAHs. tobacco products, surface active agents, anthrones, organic peroxides and hydroperoxides, long chain fatty acids and their esters, and phenolic compounds.fi Humans are exposed to some of these agents through environment, occupation, drugs, or diet. Although these agents might act in a cooperative manner with initiators to accelerate the response to the occurrence of skin carcinogenesis, their contribution, if any, to the human skin cancer progression rate is not clear.
Mechanisms of Tumor Initiation The events considered necessary for initiation include exposure to the carcinogen, its transportation to the target cell, activation to
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Skin Chemical Carcinoaenesis
Chemical agents associated with cutaneous neoplasia in humans.
Exposed oil (cutting
Route of Exposure
and jute batching
Coal tar, pitch
Coke oven worker, roofers and tanners, patients with psoriasis under treatment with Goeckerman regimen
the ultimate carcinogenic metabolite (if the agent is a procarcinogen), and DNA damage leading to an inherited change with the appearance of a preneoplastic lesion, the mutated cell. This concept is diagramatically represented in Figure l-3. The potency of an initiator is known to correlate with its ability to covalently bind with DNA, thus forming specific carcinogen-DNA adducts. The majority of chemical carcinogens are inert biologically and require metabolic activation to highly reactive electrophilic metabolites which then bind with DNA to form covalent adducts. Benzo(a)pyrene, a ubiquitous environmental pollutant to which human exposure is inevitable, is the most widely studied PAH. Extensive studies from several laboratories have identified a specific diol-epoxide derivative of this PAH, known as (+)-7/3,&dihydroxy-9a,lOa-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDE-I), as the ultimate carcinogenic metabolite responsible for cancer induction.798 The complete chemical structure and confirmation of the predominant adduct formed in z&o between BPDE-I and nucleic acids of mammalian (including human) cells and tissues is known. In this adduct, the C-10 position of BPDE-I is linked to the 2-amino group of the guanine residues in DNA.7 Metabolism of PAHs to BPDE-I proceeds through sequential reactions catalyzed
Topical Systemic Systemic
by the cytochrome P-450-dependent enzyme known as aryl hydrocarbon hydroxylase and another enzyme known as epoxide hydrolase.8 Of various isozymes of cytochrome P-450, the P-450~ is important in the formation of carcinogenic metabolites. This isozyme is inducible in response to carcinogenic PAHs and is usually present in undetectable amounts in the skin of the normal unexposed animals? A cytosolic protein known as Ah receptor plays a major role in induction of P-450~ by PAHs. This is, in mice, regulated at a specific genetic site called the Ah locus.10 Observations which may be of clinical interest and practical importance are the demonstration that clotrimazole, a topical antifungal agent, and certain plant phenols are potent inhibitors of carcinogen metabolism, DNA modification, and skin tumor formation in mice exposed to PAHs.11-14
Mechanism of Tumor Promotion Tumor promotion, which is considered to be an epigenetic event, is thought to be the result of clonal expansion of initiated cells leading to clinically evident benign tumors.4 The chemicals or the agents which produce a tissue environment conducive to the clonal outgrowth are known as tumor promoters. The most potent skin tumor promotor identified thus far
Clinics in Dermatology
TABLE 1-2. Skin Tumor Promotors.
Class of Agents Plant products and products lower organisms
Polycyclic aromatic their derivatives
Croton oil,” TPA, Euphorbia lattices*’ Dihydroteleocidin p” Citrus oil*’
+++ +++ +-tt +
l2-0-hexadecanoylphorbol-13. acetate, Phorbol 12,13-dibenzoate
7,12 Dimethylbenz(a)anthracene 7-Bromomethylbenz(a)anthracene Benzo(a)pyrene’*
t+ t-t+ ++
Cigarette smoke condensate,” Tobacco tar**
Sodrum lauryl sulphate” Tween 60
Benzoyl peroxide,‘* Cumene hydroperoxide, Lauryl peroxide, Butyl hydroperoxide
Long chain fatty acids & fatty acid methyl esters
Methyl oleate*’ Methyl 12-oxo-trans-10 octadecenoate
+t t+ ++
+,Weak t+,Modest +t+,Strong “Known human contact
is TPA, a diterpene ester present in a variety of plants of the Euphorbiaceae family. The direct human contact with phorbol ester tumor promotor is limited, although the exposure to other tumor promotors is through various sources.6 Still, the most widely studied tumor promotors are phorbol esters. TPA is a strong inflammatogenic and hyperplasiogenic agent;” however, these properties alone are not sufficient for its tumor-promoting action since agents like ethylphenylpropiolate, turpentine, and acetic acid with similar
inflammatogenic and hyperplasiogenic properties are not tumor promoters.4~6 Several other histopathological alterations, which include induction of dark basal keratinocytes and dismal infiltration of polymorphonuclear leukocytes, in addition to inflammation and hyperplasia, are believed to be important events in the process of skin tumor promotion. The most important biochemical response to the brief exposure to TPA is the induction of epidermal ornithine decarboxylase activity which catalyzes the first step in the biosyn-
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thesis of polyamines.5 Intracellular polyamine levels are regulated in response to cell proliferation, differentiation, and transformation. Inducibility of ornithine decarboxylase and mitogenic activity are considered as important characteristics of tumor promotion. The evidence for this comes from the studies with cr-difluoromethyl ornithine, an irreversible inhibitor of ornithine decarboxylase which effectively inhibits TPA-induced skin tumor promotion. Other biochemical responses to tumor promoters include increased synthesis of DNA, RNA, protein, phospholipids, prostaglandins, increased synthesis, and increased phosphorylation of histone, increased activity of protein kinase C, histidine decarboxylase and protease and decrease in the activities of superoxide dismutase and catalase.4*,15 The importance of several of these individual effects is described elsewhere in this review. Tumor promotion in mouse skin can be divided, at least, into two stages: stage I (conversion) and stage II (propagation). Stage I is characterized by the histopathologic manifestations of induction of dark basal keratinocytes, and the agents capable of producing such effects are known as stage I promoters. Stage II promoters are the agents capable of causing biochemical alterations similar to that of TPA but do not have potential to induce dark basal keratinocytes. The best example of stage II promotor is mezerein, which is as potent as TPA in inducing ornithine decarboxylase activity and enhancing DNA synthesis in mouse epidermis, but is a weak complete tumor promotor.4+ Once stage I tumor promotion is achieved by the application of TPA for only four weeks, further applications with mezerein manifest comparable tumor yields and incidence of tumors as are achieved by TPA. Another good example of a potent stage II promotor is an endogenous second messenger, diacylglycerol which is as good a stage II promotor as mezerein.i6 It is similar to TPA with regards to ornithine decarboxylase induction, DNA synthesis, and arachidonic acid release in mouse epidermis, leukemic cell differentiation, blockage of gap junctional communication, and modulation in intracellular pH. Other evidence for the existence of two-
I / I
4 Absorption Distribution >I
Modified DNA i Plasma (Mutated
Pharmacokinetics and metabolism of leading to the formation of a mutated cell. After absorption and distribution of the procarcinogen to the target cell, it is either excreted as such and/or through metabolic inactivation in the extra cellular matrix. Alternatively, the procarcinogen can be metabolically activated to a reactive electrophilic metabolite which may bind to DNA leading to the formation of the mutated cell.
stage tumor promotion comes from the inhibition studies where anti-inflammatory steroid fluocinolone acetonide and the protease inhibitor, tosyl phenylalanine chloromethyl ketone, have been shown to be potent inhibitorsof stage I tumor promotion, while retinoic acid inhibits only stage II tumor promotion. A plysiatoxin, a polyacetate, and a product of blue-green algae and some other skin toxins like teleocidin and lyngbyatoxin (which are the products of some lower organisms) are tumor promotors that also bind and activate protein kinase C and induce hyperplasia, inflammation, and epidermal differentiation in mouse skin, ultimately leading to the induction of papilloma.17 Humans are exposed to these compounds while swimming in pools. Anthralinl* and benzoyl peroxides,19 clinically important compounds, are effective tumor promotors in mouse skin. The mechanism of their action is not well known; however, they induce protein kinase C activity, but do not bind to it.2’JThe clinical significance of these results is not known and is under investigation. Until this becomes clear, their prolonged use should not be encouraged.
FIG. 1-4. A scheme depicting signal transuction systems that generate various second messenger molecules leading to unscheduled signals manifesting tumor promotion in response to tumor promotor. TPA. Phospholipase C (PLC), which may beactivated In response to an external stimulus by a receptor (R) or a nonreceptor mediated pathway, catalyzes the breakdown of phosphotidylinositol 43 diphosphate (PIP,) leading to the generation of two separate arms of second messengers, i.e., diacylglycerol (DAG) that activates protein kinase C (PKC) and inositol 1,4.5 triphosphate that mobilizes Ca** from internal stores leading to the activation of calmodulin/ Ca*‘-dependent enzymes and perhaps PKC as well. TPA binds to PKC at the same site where DAG binds to it leading to its activation and translocation from
cytoplasm to plasma membrane. Membrane bound PKC phosphorylates various growth factor receptors (GFR) which then mediate unscheduled signals for cellular proliferation. PKC is also believed to increase ornithine decarboxylase (ODC) activity, a prerequisite for cellular proliferation. This leads to the elevated levels of polyamines (PA), the precursors for the synthesis of nucleotides and eventually of DNA. The unscheduled signals from plasma membrane to nucleus may also activate oncogenes, eg, ras oncogene, the product of which is a 21 KD membrane bound protein (P”). P*l also appears to play a role In the activation of PLC.
Molecular Basis of Tumor Promotion The molecular tumor promotion
mechanism(s) are complex
involved in and not well
Clinics in Dermatology
understood. They appear to be different for different types of tumor promotors. The biologic response observed after exposure to phorbol esters include changes in membrane functions and modulation in gene expression, differentiation, and growth. Hormone-like action of these agents is indicated by the requirement of specific structure for optimum activity through a specific cellular receptor and pleotropic cellular responses to brief exposures of their very small concentrations. Some evidence indicates that protein kinase C is a receptor for phorbol esters, and tumor promotion by these agents is mediated by their receptor binding. The sequences of molecular events occurring in this process are shown in Figure l-4. Protein kinase C is activated by TPA in viva in the presence of Ca2’ and phospholipids forming a part of the signal transducing system. Several lines of recent evidences have clearly established that activation of phospholipase C by an external stimuli generates two separate arms of second messengers, diacylglycerol, and inositol 1,4,5 triphosphate.6121 Diacylglycerol is an endogenous activator of protein kinase C. TPA binds to protein kinase C at the same site where diacylglycerol binds to it. Inositol 1,4,5 triphosphate mobilizes Ca2’ from internal stores leading to the activation of calmodulin/ Ca”‘-dependent enzymes which may, in turn, lead to protein kinase C activation via indirect mechanisms. The activation of protein kinase C phosphorylates various growth factor receptors which then receive unscheduled signals leading to the enhanced synthesis of DNA along with the amplified expression of oncogenes,” the detailed description of which is given separately in this review. The product of rasHa oncogene (p”) may also play a role in the modulation of activity of phospholipase C.” Physiological inducers of epidermal differentiation stimulate endogenous phosphotidyl inositol metabolism and generate diacylglycerol. This has led to the belief that the phosphotidylinositol-protein kinase C second messenger system may regulate epidermal differentiation. In this regard, the action of TPA mimics the action of the endogenous
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Skin Chemical Carcinogenesis
second messenger system. In response to TPA, all epidermal cells do not differentiate, but only immature keratinocytes are stimulated for proliferation.22 This heterogenous action is responsible for the regenerative hyperplasia in TPA-treated murine skin which is similar to a hyperplastic response caused by repeated wounding/abrasion, i.e., physical tumorpromoting agents.6 Thus initiated, papilloma and carcinoma cells behave like immature epidermal cells and proliferate in response to exposure with TPA.22 Since initiated cells have a constitutive defect of proliferating in superficial strata, they have a growth advantage and clonally expand to manifest a visible tumor,4p6+1 The molecular mechanism leading to tumor promotion by macrocyclic terpenoids (including phorbol esters, mezerein, and toleocidin) involves phospholipase C-dependent activation of protein kinase C. This receptor for these promoters is implicated in the transduction of altered and unscheduled signals for cellular proliferation (Fig. l-4) as discussed previously. Unlike terpenoid tumor promoters, free radical generating tumor promoters act through different mechanisms even though they can also activate protein kinase C but do not bind to it.23 Oxidant promoters, by oxidizing membrane lipids, activate phospholipase AZ, result in the release of free arachidonic acid which leads to enhanced synthesis of prostaglandins. T-Butyl hydroperoxide, superoxide anions and hydrogen peroxide have been shown to stimulate prostaglandin synthesis in pig aorta epithelial cells and lung fibroblasts, and perhaps similar a mechanism exists in epidermal cells.23 Prostaglandins and other products of the lipoxygenase pathway are formed from the me&stable hydroperoxy precursors, hydroperoxy arachidonic acid, prostaglandin G2, and 15-hydroperoxy prostaglandin E2. It has been suggested that these intermediates, when present in low concentrations, may participate in a feedback loop which results in further amplification of eicosanoid metabolism. Some of the metabolites of arachidonic acid such as prostaglandin E2 and prostaglandin Fzcu are important for the induction of hyperproliferation and its maintenance in
mouse skin.23p24 Since hydroperoxides are known to increase lipid peroxidation leading to membrane damage, this may result in an efflux of Ca2+ from the intracellular stores in the endoplasmic reticulum and mitochondria. Based on this, it has been suggested that the increase in the levels of prostaglandin Ez and prostaglandin Fzcr, along with elevated levels of cytosolic Ca2’ by organic hydroperoxides, might be responsible for tumor promotion in mouse skin by these compounds.24
Gap Junctional Intercellular Communication and Tumor Promotion Intercellular communication is considered an important cellular mechanism for regulating cellular growth and differentiation.25 This is carried out through the exchange of ions and molecules which act as messenger signals. TPA has been shown to block gap junctional intercellular communication.26 Precise biochemical mechanisms involved in the regulation of gap junction are not clearly understood; however, the down regulation of intercellular communication was found to be associated with an increase in protein kinase C activity,25y26 suggesting the role of a second messenger system in this process. The gap junction intercellular communication may be an important event in the development of pathology during tumor promotion. The initiated cells which are scattered randomly in the population of normal cells remain dormant for long periods of time under the growth-regulating influences of their normal neighbor cells. The blockage of gap junction intercellular communication results in the modulation of repressor effects of normal cells on the adjacent initiated cells leading to their expansion into miniclones. The continuous repeated exposure of tumor-promoting hyperplasiogenic agents results in further expansion of miniclones into tumor mass.
Tumor Progression Conversion of benign papilloma into carcinoma is known as malignant transformation.
Clinics in Dermatology
It has been suggested that the clinical and biological events of tumor progression represent the result of sequential selection of variant subpopulations within this clone. It has also been hypothesized that such clonal evolution might result from enhanced genetic instability in tumor cells. This may increase the probability of further genetic alterations and their subsequent selection. Extensive information regarding the alterations in cellular behavior and morphology during this process can be found elsewhere.28 Important characteristics of this stage of neoplasm are the tendency to grow with time at a faster rate, and the switchoff mechanism for the growth control is poorly operated. (Usually, it is not the result of shortening in the cell cycle time but represents an increase in the growth function.) Under certain circumstances, the loss of command for growth regulation may be related to the loss of specific receptors in the subpopulation of cells for circulating hormones and other growth factors. The gradual increase in malignancy leads to the disappearance or decrease in the organelles and metabolic functions necessary for specialized activities in the cell.** The mechanism of tumor progression is poorly understood; however, some possible factors may include single gene mutation that could destabilize the genome, gross chromosomal abnormalities, and cellular heterogeneity within the neoplastic clone itself. In addition to these acquired mechanisms, the increased genetic instability in neoplastic cells of a small segment of the human population is the result of inherited gene defects present in all cells of the body.28 Support for this hypothesis also comes from the reports that the individuals with genetic disorders like Bloom’s syndrome, Fanconi’s anemia, ataxia telangiectasia, and xeroderma pigmentosum, have an inherited defect in DNA repair or in some other aspect of DNA housekeeping and show an increased incidence of cutaneous malignancy.29 In addition to these mechanisms, some extracellular factors like oncogenic virus, continued presence of long lived carcinogenic chemical or radioisotope, and repeated exposure to clastogenic materials may also destabilize host cell genome.z*-3”
Oncogenes in Cutaneous Carcinogenesis Oncogenes are genes that all normal cells contain and in their natural states are known as protooncogenes. Their expression is, sometimes, a part of the normal cell activity. Protooncogenes are activated by mutation or translocation that may ultimately result in tumor growth or tumorigenesis.31 The role of oncogenes was first demonstrated by Robert A. Weinberg who showed the transformation of normal cells into cancerous cells by transfecting normal cells with the DNA from tumor cells. Since then, about 40 specific oncogenes have been identified and isolated.32 The oncogene that has been demonstrated in many chemically-induced skin tumors is mutated Harvey ras gene (rasHa). This gene is a member of a multigene family, present in the genome of all multicellular organisms. All the members of this gene family code for a closely-related membrane-bound 21KD protein called Pzl. This protein binds to guanosine triphosphatase and has an intrinsic guanosine triphosphatase activity. The activation of rasHa in skin appears to be mediated by a point mutation in the cellular oncogene. The product of mutated oncogene P2i is altered by one amino acid in which guanosine triphosphate activity is reduced.32p33 In other studies, similar results have been obtained by mutating cloned normal rasHa by the exposure to a skin carcinogen.34 The mutation of rasHa seems to be an early initiating event, but absence of this activated oncogene in all papillomas and conversion of such a papilloma into carcinoma by the incorporation of cloned activated rasHa oncogene, suggests its role in malignant transformation.32-34 Balmain and his group demonstrated that the introduction of activated Harvey murine sarcoma virus ras genes into epidermal cells of mouse skin in z&o followed by treatment with TPA-induced benign papillomas. Some of these benign papillomas progressed to invasive carcinoma.35 This is quite similar to that of the two-stage initiation promotion protocol. The event of activation of rasHa does not, however, seem to be sufficient for oncogenic action unless it is coupled with other important events which are not clearly defined.
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dioctanoylglycerol: A potent stage II mouse skin tumor promotor. Cancer Res. 1988;48:1736-1739.
1. Scott0 J, Fears T, Lisiecki E, et al. Incidence of nonmelanoma skin cancer in the United States, 1977 8: Preliminary report, DHEW Publication No. (NIH) 80-2154. Bethesda, MD, 1980, National Cancer Institute.
17. Arcoleo JP, Weinstein IB. Activation of protein kinase C by tumor promoting phorbol esters, teleocidin and aplysiamxin in the absence of added calcium. Carcinogenesis. 1985;6:213-218.
2. Adams RM. Occupational skin cancer. In: Adams RM, ed. Occupational Skin Disease. New York: Grune and Stratton, 198382-98.
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4. Slaga TJ. Mechanisms involved in two-stage carcinogenesis in mouse skin. In: Slaga TJ, ed. Mechanisms of tumor promotion. Vol. 2. Florida: CRC Press. 1984:116.
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21. Argyris TS. Regeneration and the mechanism of epidermal tumor promotion. CRC Crit Rev Toxicol. 1985;14: 211-258.
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7. Harris CC. Future directions in the use of DNA adducts as internal dosimeters for monitoring human exposure to environmental mutagens and carcinogens. Environ Health Persp. 1985;185-191. 8. Conney AH. Induction of microsomal enzymes by foreign chemicals and carcinogenesis by polycyclic aromatic hydrocarbons. GHA Clowes Memorial Lecture. Cancer Res. 1982;42:4875-4917. 9. Mukhtar H, Bickers DR. Drug metabolism in skin: Comparative activity of the mixed-function oxidases, epoxide hydratase and glutathione-S-transferase in liver and skin of the neonatal rat. Drug Metab Dispos. 1981;9:311-314.
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12. Mukhtar H, Das M, Khan WA, et al. Exceptional activity of tannic acid among naturally occurring plant against 7,12phenols in protecting dimethylbenz(a)anthracene-, benzo (a)pyrene-, 3methvlcholanthreneand N-methvl-N-nitrosoureainduced skin tumorigenesis in mice. Cancer Res. 1988;48:2361-2365.
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Clinics in Dermatology
Address for correspondence: Hasan Muhktar, PhD, Department Administration Medical Center, 10701 East Boulevard, Cleveland,
of Dermatology, OH 44106.