Antioxidants and multistage carcinogenesis in mouse skin

Antioxidants and multistage carcinogenesis in mouse skin

Free Radical Biology & Medicine, Vol. 7, pp. 377-408, 1989 Printed in the USA. All rights reserved. 0891-5849/89 $3.00+ .00 © 1989 PergamonPress plc ...

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Free Radical Biology & Medicine, Vol. 7, pp. 377-408, 1989 Printed in the USA. All rights reserved.

0891-5849/89 $3.00+ .00 © 1989 PergamonPress plc

Review Article ANTIOXIDANTS

AND MULTISTAGE CARCINOGENESIS IN MOUSE SKIN

JEAN-PIERRE PERCHELLET* a n d ELISABETH M . PERCHELLET Anti-Cancer Drug laboratory, Division of Biology, Kansas State University, Ackert Hall, Manhattan, KS 66506, U.S.A. (Received 21 April 1988; Revised and accepted 1 September 1988)

Abstract--The two-step initiation-promotion protocol for the induction of skin tumors in mice is a convenient model to elucidate what molecular events are involved in the multistage process of carcinogenesis and how they can be modulated. The current theories concerning the mechanisms of skin tumor initiation, stages 1 and 2 of tumor promotion, and tumor progression are reviewed. Because chemical carcinogens and tumor promoters may, directly or indirectly, generate reactive oxygen species (ROS) and because various antioxidants inhibit effectively some of the biochemical and biological events linked to tumor initiation, promotion and/or progression, it is conceivable that different sequences and levels of free radical-induced macromolecule damage may contribute to the evolution of the epidermal target cells from the preneoplastic stage to the malignant stage. Keywords--Carcinogenesis, Free radical, Antioxidants, Skin tumors, Initiation, Promotion, Progression

daughter cells. 1-4 Genetic and somatic mutations, as well as epigenetic alterations, may result in neoplastic transformation. The presence of irreversible DNA damage or altered proto-oncogenes increases the probability that the initiated cell might not respond to the regulatory signals for normal growth and differentiation and, after neoplastic transformation and a postulated period of dormancy, might then proliferate into a benign or malignant tumor. Because it is impossible to monitor continually the level of DNA lesions/repair in individual stem cells, it appears difficult to predict if and when such irreversible initiation and transformation of a single precursor cell might occur and, therefore, to determine at the cellular level when carcinogenesis really begins. The early diagnosis of cancer, which is crucial to the efficacy of cancer management, is unfortunately delayed by the extended period of time between the initial interaction of physical, chemical or biologic carcinogens at the cellular level and the appearance of a neoplasm. In most systems studied, there is no evidence of neoplastic growth through much of the latency period. 3 Then, approximately 30 cell doublings are required for most neoplasms to grow exponentially from a single transformed cell to about 109 tumor cells, the estimated limit of clinical or radiologic detection in human, s-7 After only 10 further doublings in volume,

INTRODUCTION

According to the multihit theory for the monoclonal origin of cancer, neoplasia arises when the stem cells of the human tissues that are the random targets of an undetermined number and sequence of inherited, acquired and/or spontaneous cell-damaging events accumulate nonlethal, unrepaired, irreversible, defects that can be transmitted to successive generations of

*Author to whom correspondence should be addressed. Jean-Pierre (PhD, 1974) and Elisabeth (PhD, 1973) Perchellet were trained in Endocrinology under Dr. A. Jost (Laboratory of Comparative Physiology, University Paris VI). After serving in the French Army, Jean-Pierre did postdoctoral work in Biochemistry with Dr. R. K. Sharma (University of Tennessee Center for the Health Sciences, Memphis) and in Oncology with Dr. R. K. Boutwell (McArdle Laboratory for Cancer Research, University of Wisconsin, Madison). He is now Associate Professor at Kansas State University where he directs the Anti-Cancer Drug Laboratory and teaches graduate courses in Human Oncology and Cancer Therapy. His research concerns the biochemistry of mouse skin carcinogenesis and focuses on the molecular mechanisms by which oxidants, antioxidants, and anthracycline antibiotics modulate the multi-stage process of tumor promotion. His wife and collaborator, Elisabeth, held full- and part-time Research Associate positions in Gastroenterology with Dr. J.-J. Bernier (INSERM-U54, St. Lazare Hospital, Paris), in Biochemistry with Dr. R. K. Sharma (University of Tennessee, Memphis) and in Gastroenterology with Dr. W. A. Olsen (Veteran Administration Hospital, University of Wisconsin, Madison) while raising 3 children. Both authors enjoy hiking in National Parks, playing bridge, and visiting art, history, and science museums. 377

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J.-P. PERCHELLETand E. M. PERCHELLET

the tumor burden may become lethal to the host. As illustrated by the current cancer survival statistics, 7 eradicating by combination therapy 100% of the cells resulting from this long preclinical growth of human tumors is a very difficult task. The incidence of cancer at any age seems directly proportional to the number of initiated stem cells accumulated at that a g e ? Since it is impossible to pinpoint the time of tumor cell initiation, there is no way to know how long the progenitor cell remained dormant in the tissue before it started proliferating. Furthermore, it is difficult to extrapolate from doubling times in the clinic to preclinical circumstances in order to estimate the time at which the tumor started growing in the patient before it was detected.

THE NEED FOR EXPERIMENTAL MODELS

OF CARCINOGENESIS A skin papilloma (Pa) becomes visible when the clonal expansion of the initiated epidermal cell reaches a size of 105-106 cells. In classic animal experiments where the complete carcinogenesis or the initiationpromotion treatments are applied directly to mouse epidermis, the first skin tumors appear after 5-7 (benign) and 16-18 (malignant) weeks, s These time intervals represent about 5 and 12% of a mouse's life. If we can extrapolate this in terms of a human life lasting 75 years, these lag periods become 4 and 9 years. Because the stem cells of the human tissues are unlikely to be exposed to such drastic carcinogenic treatments, and for other reasons, the whole process of carcinogenesis in humans may take much longer, it should be noted that a small percentage of the mice whose skins are exposed to these potent carcinogenic treatments never develop any visible neoplasms. It is postulated that many more skin cells are initiated than finally transformed into neoplastic cells and that populations of initiated and dormant tumor cells may exist in organisms for a lifetime without ever expressing their potential for neoplastic transformation and exponential growth? The dormancy of malignant neoplastic cells has clearly been demonstrated, indicating that further stimuli are required to trigger their proliferation. 9 Epidemiological and tumor cell kinetics studies sugest that populations of initiated or dormant tumor cells may exist in the human tissues for several decades before they are, respectively, transformed or triggered into a hyperplastic behavior leading to the diagnosis of a neoplasm? However, most human tumors have a mean cell cycle time of about 2 days but a mean volume doubling time of about 50 days because of the small proportion of proliferating tumor cells (growth frac-

tion) and the high rate of tumor cell death. 3'5'6"~°'~LThe growth of most neoplasms is regarded as an exponential function limited by an exponential retardation. Since mass and doubling time are increasing concomitantly, the rate of tumor growth decellerates because the growth fraction decreases, the cell loss factor increases and sometimes the duration of the cell cycle lengthens. These tumor growth kinetics suggest that it should take only a few months or years for the selected transformed cell to proliferate into a tumor reaching the critical mass of clinical detection. The process of tumorigenesis in humans, therefore, may be characterized by a relatively long period of latency, which is postulated to comprise an undetermined period of dormancy preceding the preclinical portion of the theoretic Gompertzian growth curve (Fig. 1). The hypothetical model in Figure 1 suggests that the cells that are sequentially subjected to genetic mutation and phenotypic transformation by years of exposure to carcinogenic hazards, including tumor initiators and promoters, may remain dormant, that is, maintain their normal rate of self-renewal, for a part of the latency period of the carcinogenic process until one of them finally acquires a proliferative advantage over its neighbor stem cells. In addition to the unknown stimuli triggering proliferation, the dormant neoplastic cells may have to overcome immunologic and other defense mechanisms of the host and induce sufficient vascularization to support their exponential expansion. Thus, after going through occult carcinogenic changes at the molecular level for several months or years, a transformed human cell may all of a sudden speed up its rate of renewal with the result that, within a few more months or years, both patient and physician become finally aware of the presence of a neoplasm. By using experimental tumor models, the long-term objective of fundamental cancer research is to elucidate the molecular events specifically involved in the preclinical portion of the neoplastic process (Fig. 1). Earlier detection and modulation of such events should provide valuable information to design novel cancer therapies applicable in clinical oncology.

THE MOUSE SKIN SYSTEM FOR

MULTISTAGE CARCINOGENESIS Experimental carcinogenesis in mouse skin was pioneered by the works of Mottram 12and Berenblum and Shubik,~3 and studied extensively by Boutwell.~4 The initiating and promoting elements in skin tumor production have been characterized. 14•15 The demonstration of the multistep nature of chemical carcinogenesis in mouse skin was greatly facilitated by the synthesis

Antioxidants and skin carcinogenesis

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of pure polycyclic aromatic hydrocarbon (PAH) carcinogens ~6such as 7,12-dimethylbenz[a]anthracene (DMBA) and the isolation of the potent tumor-promoting agent from croton oil, T M 12-0-tetradecanoylphorbol-13-acetate (TPA). Moreover, the breeding of promotion-sensitive SENCAR mice has provided an excellent model system to pursue studies on the mechanisms of multistage skin carcinogenesis. '4,19 The ex-

perimental induction of tumors in mouse skin is believed to comprise a sequence of initiation, promotion (conversion + propagation), and progression (Table 1). The various treatments "painted" to the shaved backs of the mice during the initiation-promotion protocols 14'15,20and the complete carcinogenesis processesS.2~.22 commonly used for the induction of skin Pa and carcinomas (Ca) are depicted in Table 1.

Table 1. Protocols for Multistage Carcinogenesis in Mouse Skin Description of Treatments (Duration)* Number Initiation

1. DMBA (0.1/~mol; ix) Initiation

2. DMBA (0.1 ~tmol; ix) Initiation

3. DMBA (0.1/lmol; Ix) 4. DMBA (0.1 pmol; ix) 5. DMBA

Agents Administered (Dose/Applications; Total Number of Applications)f Complete Promotion (40 wk)

TPA (8.5 nmol; 80x) Two-Stage Promotion (40 wk) Stage 1 (2 wk) Stage 2 (38 wk)

TPA (8.5 nmol; 4x) Promotion (20 wk) TPA (8.5 nmol; 40x) TPA (8.5 nmol; 40x) TPA

(0.1/lmol; ix) (8.5 nmol; 40x) Complete Carcinogenesis (40 wk)

MEZ (8.5 nmol; 76x) Progression (20 wk) BPx (20 mg, 40x) ; then TPA ENU (8.5 nmol; 36x) (10 ,umol; 4x) ; then TPA MNNG (8.5 nmol; 36x) (1/~mol; 4x)

6. DMBA (3.6/tmol; ix) 7. DMBA (0.1/tmol; 80x) *All treatments are applied topically in 0.2 ml of acetone to the same shaved dorsal area of the skin. tDosages vary with strain. Data are for Charles River CD-1 mice; the frequency of application for repeated treatments is 2x/wk.

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J.-P. PERCHELLETand E. M. PERCIqELLET

Tumor initiation

A single subcarcinogenic dose of DMBA [Table 1 (Protocol 1)] produces no tumors during the life-span of the animals but, after metabolic activation, the electrophilic ultimate carcinogen interacts covalently with, and damages, critical macromolecules, presumably epidermal DNA, 23 to initiate skin carcinogenesis. When the DNA template is replicated before it is repaired, the initiating genetic damage becomes irreversible and • ,34 heritable.New evidence suggests that the c - r a s m proto-oncogene is the target of the initiating mutation in mouse skin carcinogenesis: 25 .~2 1) the activated cr a s Ha oncogene has been isolated from skin Pa; 2) the introduction of mutated r a s m oncogene into epidermal cells can substitute for DMBA initiation in two-stage mouse skin carcinogenesis; and 3) the keratinocytes derived from DMBA-initiated skins and/or resistant to Ca2+-induced terminal differentiation contain an activated c - r a s m oncogene and form squamous Pa in skin grafts on athymic nude mice. A single base substitution activates the c - r a s m proto-oncogene. The frequency of this mutation is dependent on the initiating agent. Over 90% of mouse skin tumors initiated with DMBA have a point mutation (specific A---~T transversion) at the 2nd nucleotide in codon 61 of c - r a s m . Other initiators may induce a point mutation (specific G--~A transition) at the 2nd nucleotide in codon 12 of c - r a s m . Although epigenetic theories are also plausible,; tumor initiation is generally regarded as a permanent alteration of the cell genotype with no neoplastic phenotype. ~533 Epidermal cells initiated with the v - r a s H~ oncogene remain dormant within the skin in the absence of tumor promotion and require TPA treatments to form Pa, suggesting that DMBA-induced point mutation and activation of the c - r a s H~ proto-oncogene represents an initiating event, which is insufficient for skin tumorigenesis unless amplification of the mutated oncogene is triggered by tumor promoters. The persistence of latent initiated cells with the potential to give rise to future neoplasms has been demonstrated. 14.34.35The mechanism by which the c - r a s H~ oncogene induces transformation is unknown. Initiated cells are uniformly resistant to Ca2+-induced terminal differentiation. The correlation between tumor initiation and resistance of epidermal cells to signals for terminal differentiation suggests, therefore, that the initiating event in skin carcinogenesis causes a genetic alteration in the program of terminal differentiation 36 3s Complete

tumor promotion

Repetitive applications of the most potent phorbol ester tumor promoter TPA [Table 1 (Protocol 1)] are

required to trigger molecular events leading the immediate progeny of the DMBA-initiated epidermal cells to the formation of growing skin tumors and achieve complete tumor promotion• ~5,~3 It is theorized that TPA stimulates the expression of the abnormal genetic information within the initiated cells which, because of their altered program of differentiation, acquire a neoplastic phenotype and a proliferative advantage over their normal neighbors. 3'~ Promoting agents by definition are neither mutagenic nor carcinogenic and, therefore, incapable of initiation or complete carcinogenesis by themselves. Moreover, they cannot promote tumor formation in the absence of preexisting initiated cells, as indicated by the experiments in which the sequence initiation-promotion is reversed. H The occasional neoplasms resulting from lifetime treatment of mouse skin with large doses of TPA alone are likely attributable to the promotion of initiated or dormant tumor cells of spontaneous origin. ~4.33The experiments in which promotion precedes initiation or the frequency of TPA application is decreased produce no tumors, suggesting that the promoting effects of individual TPA treatments are transient and, to a certain degree, reversible. ~4 Although the events critical for the selective clonal expansion of initiated cells to form a neoplasm are poorly understood, the evidence that the tumor promoters interact with membranes, stimulate and alter genetic expression and eventually increase the rate of cell proliferation has been reviewed. 33~ 44 TPA has been shown to produce a series of pleiotropic cellular effects including the induction of protein kinase C (PKC) activity. 45'46 phospholipid synthesis 47 and prostaglandin (PG) release, 4~ the synthesis and phosphorylation of epidermal histones, ~ the increase in protease activity, 5° the production of ROS, 5t the overexpression of tumor-specific, promotion sensitive, transforming or cellular proto-oncogenes, 252'~ 32.52.53 the induction of the polyamine biosynthetic pathway 54 followed by sequential increases in RNA, protein, and DNA synthesis, 55 with concomitant alterations in cellular morphology,56 mitotic rate, 44 and degrees of metabolic • ~7 cooperationand terminal differentiation. 4 4 ,-5 8 A major problem is to identify the critical responses which mediate specifically the early and late events required for two-stage tumor promotion. H59.~'° Multistage

tumor promotion

One to four applications of TPA are sufficient to trigger the 1st, partially irreversible, stage of promotion called " c o n v e r s i o n " [Table 1 (Protocol 2)]. Multiple applications of the ineffective promoter mezerein (MEZ) are then required to achieve the 2nd stage of

Antioxidants and skin carcinogenesis promotion called "propagation" and complete the promotion process. 6~ Neither treatment alone is sufficient Overall, the tumor response elicited by the initiationtwo stage promotion protocol [Table 1 (Protocol 2)] is significantly lesser than that observed when TPA is used as a complete promoter [Table 1 (Protocol 1)]. The long-lasting effects of TPA that are essential for stage 1 promotion persist for almost 2 months before declining whereas those of MEZ in stage 2 promotion are rapidly reversible and require a certain frequency of application in order to induce tumors. The sequential inductions of ornithine decarboxylase (ODC) activity, macromolecule synthesis and epidermal cell proliferation are undoubtedly involved in the 2nd stage of tumor promotion. 59 For instance, following DMBA initiation and stage 1 promotion with TPA, the 2nd stage of skin tumor promotion can be completed if only 4 applications of the potent ODC inducer MEZ, which by themselves are ineffective in completing the two-stage promotion process, are followed by repetitive treatments with ethyl phenylpropiolate (EPP), a nonpromoting epidermal hyperplastic agent unable to induce ODC activity. 59 Interestingly, only those phorbol esters which induce the sequential transient expression of the proto-oncogenes c - l o s and cm y c and the ODC gene in mouse skin in vivo cause rapid induction of epidermal hyperplasia, inflammation and stage 2 promotion. 62 The reversibility of complete tumor promotion, therefore, is linked to the 2nd rather than the 1st stage and reflects the reversibility of epidermal hyperplastic transformation induced by TPA.63 The converting activity of TPA in the 1st stage is slowly reversible, requires undisturbed DNA synthesis, 64 and is characterized by the early occurrence of dark basal keratinocytes. 56 An observation that may be relevant to the 1st stage is that the hyperplasia of the epidermis, dermis and hair follicles and the dark basal cells are maximal after the 4th TPA treatment. 65 One theory is that limited applications of TPA in the 1st stage might only trigger the expression of the altered genetic information of the DMBA-initiated cells, which would acquire a neoplastic phenotype but remain dormant. Then, the 2nd stage of tumor promotion may simply involve a selection and clonal expansion of neoplastic cells. Interestingly, the presence of initiated cells is not a prerequisite to establish stage 1 promotion since the specific events responsible for conversion by TPA may be " f i x e d " in normal epidermal cells several weeks before their initiation with DMBA. 66-68Thus, both normal and initiated epidermal cells exhibit a long-lasting but not permanent " m e m o r y " for stage 1 promoting effects of TPA that may complement the past or future

381

irreversible alterations of tumor initiation. 66,68It is not known whether the ability of a few TPA treatments to induce DNA damage, chromosomal aberrations and aneuploidy69 st is linked to their stage 1 tumor-promoting activity. In analogy with the two-hit theory for the monoclonal origin of most neoplasms and the mechanism of viral carcinogenesis involving at least 2 mutational events, the cumulative cell-damaging events triggered by the combinations initiation--stage 1 promotion or stage 1 promotion--initiation may shift the epidermis into a state of increased promotability, 67 in agreement with the report that TPA enhances viral transformation.82 When the start of complete tumor promotion by TPA or the anthrone derivative chrysarobin is delayed 10 weeks after DMBA initiation, the development of skin tumors is faster and sometimes greater than when promotion immediately follows initiation. 83,84 Moreover, when the repetitive treatments with the stage 2 promoter MEZ alone are delayed 10 weeks after DMBA initiation, their effectiveness in promoting skin tumors becomes equal to that of the two-stage promotion protocol [Table 1 (Protocol 2 ) ] . 83 These findings suggest that, because tumor initiation provides indirectly the initiated cells with a proliferative advantage over normal surrounding cells on the basis of their resistance v e r s u s commitment to terminal differentiation, more of the initiated cells have had the time in these initiation/delayed promotion experiments to slowly expand their progeny at the expense of the non-initiated cells until their clone size reaches a critical threshold of promotability. 83 This implies that, by stimulating cell proliferation and differentiation, the few applications of TPA in the 1st stage may accelerate the differentiation and migration of the non-initiated cells out of the basal layer, thereby providing space along the basement membrane to favor indirectly the expansion of the initiated cells resistant to signals of terminal differentiation. 83 Such theory is substantiated by the ability of stage 1 promoters to induce the accumulation of dark basal and suprabasal cells resembling fetal-type dedifferentiated keratinocytes. 56 Therefore, the clonal expansion of the initiated basal keratinocytes at the expense of the normal cells still committed to differentiation, maturation and degeneration may occur spontaneously with time after initiation, but much more slowly then after exposure to a 1st stage tumor promoter. 83 Moreover, in addition to favoring initiated cells over normal cells on the basis of their resistance v e r s u s commitment to terminal differentiation, the toxicity of TPA and other promoters may select rapidly growing initiated cells over more slowly growing initiated or normal epidermal cells. 85 Similarly, it has been postulated that SENCAR mice are more respon-

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J.-R PERCHELLET and E. M. PERCHELLET

sive to the various initiation-multistage promotion protocols because populations of constitutively altered epidermal cells develop spontaneously in these animals and their skins contain a greater proportion of promotable initiated cells than the skins of other less sensitive strains. 37 As opposed to modulating the hyperplastic events responsible for the late propagation of the neoplastic epidermal cells in stage 2 promotion, a breakthrough in understanding the key molecular events involved in the early initiation and conversion phases of skin carcinogenesis would appear more likely to stimulate new ideas for the development of novel methods of cancer detection or effective therapies to prevent, block or inhibit the neoplastic process in humans. However, most in vivo studies on experimental skin carcinogenesis are devoted to the analysis of promotion only (over 60%) with surprisingly few concerned with initiation alone (13%). 86 Recent findings indicate that all DMBA-initiated skin Pa possess a point mutation in the 61st codon of one c-ras Ha allele irrespective of whether complete tumor promotion is achieved with benzoyl peroxide (BPx), TPA or the combination stage 1 TPA-stage 2 MEZ. ~7 This mutation is detected uniquely in the skin Pa as early as 9 weeks after starting promotion and not in the other epidermal cells of the dorsal skin that surround the tumors and are also exposed to the promotion treatment. These data reinforce the theory that the point mutation coincides with the initiating event and that any type of promoting regimen can select these mutation-bearing initiated epidermal cells and induce their transformation and clonal expansion into skin tumors.

Tumor progression

After a relatively short induction period, the twostage system of tumorigenesis [Table 1 (Protocol 1)] produces mostly benign skin Pa which may either persist, regress or later develop into invasive Ca. The development of malignant skin Ca from preexisting Pa is a relatively rare (approximately 5-10% of Pa progress to Ca) and late event (20-40 weeks after the start of promotion) 88 and its frequency appears to be promoter-independent. Experiments with Pa-bearing mice demonstrate that this low frequency of malignant conversion of Pa to Ca is unaffected by the continued application of TPA 89,9° but can be significantly increased by treatments with the free radical generator BPx or the tumor initiators ethylnitrosourea (ENU) and l-methyl-3-nitro- 1-nitrosoguanidine (MNNG) [Table 1 (Protocols 3-5)]. 89,91-94 A balance between the promoting and cytotoxic effects of chronic TPA treatments

has been proposed to explain the fact that increasing the dose and duration of promotion with this agent adversely affects the yield of Ca. 20New studies provide strong evidence that the dose of carcinogen used as initiator plays a critical role in determining which initiated cells will progress to malignancy during or after promotion. 95 Tumor progression, therefore, is proposed to involve a number of genetic alterations. It is theorized that subpopulations of epidermal cells may acquire potentially malignant chromosomal changes at any time before or after their initiation, conversion or propagation. 96 Subsequently, those benign neoplastic cells that have accumulated additional sublethal genetic alterations, besides those associated with tumor initiation, may be selected to develop malignant characteristics. Interestingly, BPx is a weak complete promoter of mouse skin tumors but neither a tumor initiator nor a complete carcinogen. 97 The genotoxic effects of this free radical-generating peroxide may not be sufficient to produce critical initiation lesions but may contribute to certain DNA damage or chromosomal aberrations accelerating the progression of benign epidermal tumor cells toward a higher degree of aneuploidy and malignancy. 93'94

Complete carcinogenesis

Skin tumors can be induced by either a single application of a carcinogenic dose [Table 1 (Protocol 6)] or repeated applications of a subcarcinogenic dose [Table 1 (Protocol 7)] of DMBA. The tumors that arise in these protocols are theoretically the result of both the initiating and promoting abilities of DMBA. Thus, the irreversible and cumulative effects of multiple subcarcinogenic doses of DMBA [Table 1 (Protocol 7)] can achieve tumor initiation, conversion, propagation and progression. In contrast to the DMBA-initiation TPA-promotion regimen [Table 1 (Protocol 1)], complete carcinogenesis by DMBA [Table 1 (Protocols 6, 7)] is characterized by the late occurrence of fewer Pa but the Ca develop much earlier. In the DMBA protocols, therefore, the yield of skin tumors is lower but they have a higher frequency of malignancy and progress to Ca more rapidly. ~ The facts that the glutathione (GSH) peroxidase and ODC responses to TPA and MEZ are different from those to DMBA, and that antioxidant and retinoid treatments inhibit ODC induction and skin tumor promotion by TPA and MEZ but not by DMBA, suggest that the nature and mechanism of tumor promotion by the phorbol esters and related diterpenes may be different from those of the promoting component of DMBA carcin-

Antioxidants and skin carcinogenesis ogenesis. 8,gs-l°° The magnitude and time course of in-

duction of epidermal ODC activity by the anthracenederived tumor promoters chrysarobin and anthralin are considerably different from those characterizing the ODC responses to TPA and MEZ but resemble the effects of DMBA on ODC induction, l°l'm2 Moreover, the lower tumor incidence, the longer latency of Pa development and the greater ratio of Ca:Pa observed with chrysarobin and anthralin as opposed to TPA suggest that the mechanism of ODC induction and skin tumor promotion by chrysarobin and anthralin is different from that of TPA and more like the promoting stage which occurs during complete carcinogenesis w i t h D M B A . 84'1°1'1°2 Although it is not clear whether tumor formation accomplished by the complete carcinogenesis process involves a promoting component with a mechanism analogous to TPA, DMBA, and the anthrone promoters may promote the expression of a subclass of Pa with a high probability of progressing to Ca. The overall tumorigenicity of various compounds or protocols may reflect major differences in their ratios of initiating : promoting ability. 103Although distinct underlying mechanisms may be responsible for these different kinetics of tumor development, the process of skin carcinogenesis by either of these protocols is likely to involve the same basic sequence of cellular alterations (Table 1).

383

THE ROLE OF OXIDANTS IN MULTISTAGE SKIN CARCINOGENESIS

Cellular prooxidant states appear to play an important role at critical steps of the process of skin carcinogenesis. 104-110Epidermal cells treated with chemical carcinogens and/or tumor promoters may overproduce ROS and be deficient in their ability to destroy them. Increased levels of potentially damaging oxidants are associated with neoplastic cells but it is unclear whether free radical reactions are a major cause of cellular lesions or merely a consequence of them. Increased radical formation may simply result from the release of intracellular, non-protein bound metal catalysts within damaged cells, ll~

Production of ROS Much research linking free radicals and neoplastic transformation has focused on the intermediates of 02 reduction. The sequential formation of the various types of O2-centered free radicals 112is illustrated (Table 2). The reduction of the molecule of ground-state 02 by a single electron produces the superoxide anion radical 02 ~ (reaction a). It should be noted that 02 ~ exists in e q u i l i b r i u m with a protonated form, perhydroxyl radical HO2", which is a more reactive radical then is 02 ~ in aqueous solution. Spontaneous dismutation of

Table 2. Production and Detoxification of Reactive 02 Species Reaction Radical Generation 02 + e ~ 02; 02- + H + ' HO2" 2 02: + 2 H + ~ H202 + 02 H202 + Fe2+ , Fe3+ + -OH + OH H202 + 02 ~ catalyst Fesalt) 02 + -OH + OHLH + .OH , L. + H20 L" + 02 , LO2' LO2' + LH , L" + LOOH 2 LOOH ~complexes LO" + LO2" + H20 LO. + LH ~ L. + LOH Radical Degradation Fe3+-Desferrioxamine complex aTH + L(O)O. ~ aT. + L(O)OH aT. + L(O)O. , Termination products 2 02- + 2H + SOD H202 + 02 2 GSH + H202 CAW ~ 2 H20 + G S S G ~ G + LOOH S~-d~.d¢.tGSHperoxide LOH + H20 + GSSG

(a) (b) (c, Fenton) (d, Haber-Weiss)

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384

J.-P. PERCHELLETand E. M. PERCHELLET

two molecules of O2 ~- in aqueous solution generates H202 (reaction b). The H202 so formed can be reduced by various metal salts present in tissues to form the hydroxyl radical -OH (Fenton reaction c). 02- and H202 may also interact in vivo to form .OH only if the overall equation is catalyzed by traces of transition metal ions (iron-catalyzed Haber-Weiss reaction d). Because of its extreme reactivity, .OH will immediately react with molecules near the site of its generation. ironically, the less reactive 02 ~ and U20/ may be more damaging because they can diffuse away from their site of generation and induce .OH production in remote cellular locations. In contrast to 02 ~ , U202 can even cross cell membranes and damage other cells. Therefore, the biological damage done by 02"- and H202 that is mediated by .OH formation depends largely on the location of the metal ion complexes catalytically active in reactions c and d.~l~ Low concentrations of H202 induce DNA damage in vivo and in vitro, possibly through ferryl radicals generated by the fenton reaction. ~3 The highly reactive .OH may abstract a hydrogen atom from an unsaturated fatty acid LH to form a lipid radical L. (reaction e) and, therefore, initiate a rapid autocatalytic chain reaction leading to the formation of lipid hydroperoxides (LOOH). After rearrangement of the double bonds to form a conjugated diene, the stabilized lipid molecule containing a carbon radical rapidly reacts with molecular 02 to give a peroxyl radical LO 2" (reaction f), which can then abstract a hydrogen atom from an adjacent lipid molecule to produce a LOOH (reaction g) and so continue the chain reaction of lipid peroxidation. ~2 The decomposition of these lipid hydroperoxides in the presence of transition metal ions yields alkoxyl (LO.) and peroxyl (LO2") radicals (reaction h), which may themselves abstract further hydrogen atoms and contribute to the initiation and progapation of new chain reactions of lipid peroxidation (reactions g and i). The small "transit" pool of non-protein bound iron moving between transferrin, cell cytoplasm, mitochondria and ferritin could provide the catalyst required for .OH formation and chain-propagation of lipid peroxidation (reactions c, d, and h). l~t The lipid peroxyl radicals LO2" may be the major O2-centered free radicals generated in a cell as a result of 02 reduction or lipid peroxidation. Compared with other O2-centered free radicals, LO2" are more stable species capable of diffusing to cellular loci distant from the site of their generation. Therefore, their role in interacting directly or indirectly with DNA and/or other macromolecules and producing cellular lesions associated with carcinogenesis may be more important than that of .OH. l J4 However, lipid peroxidation seems to occur at a late stage in the injury process and may

contribute little to it. ~J5Nucleic acids and proteins may also be oxidized and/or converted to free radical forms, which in turn are highly reactive and may create crosslinks. One of the current hypothesis is that such an oxidative challenge might contribute significantly to the induction, promotion and/or progression of skin tumors. For instance, the role of O2-derived free radicals in mediating ionizing radiation-induced DNA damage and cytotoxicity is well established. ~16'~7 The genetic material is undoubtedly the primary target of oxidant attack in v i v o 1°SA°9tlSA18"t19 since the above ROS consistently induce mutations, DNA adducts and strand breaks, and clastogenic effects in cells subjected to oxidative stress. The possibility has been suggested that the initiating and promoting components of carcinogenesis may be triggered by qualitatively and quantitatively different levels of primary and secondary oxidizing agents causing different levels of mutation, DNA damage and chromosomal aberration.~°4

Detoxification of ROS The major intracellular antioxidant defense relies on enzymatically removing O2- and H202 hopefully before they reach the iron catalysts and form "OH and ferryl radicals. Conversely, an experimental means of antioxidant protection is to decrease the availability of metal catalysts required for the damaging free radical reactions c, d, and h. For example, the specific and powerful iron chelator desferrioxamine methanesulfonate (Desferal; C1BA-GEIGY) may protect against •OH generation and lipid peroxidation by preventing complexes of iron salts from participating as catalysts in radical reactions (j). ~ In addition to the detoxifying enzymes superoxide dismutase (SOD), catalase (CAT), and GSH peroxidase, the cells maintain a multi-level protective system against free radical generation and lipid peroxidation including both lipid-soluble membrane scavengers such as cx-tocopherol (o~TH, vitamin E) and water-soluble cytoplasmic antioxidants such as GSH (Table 2). 12° At the membrane level where the concentration of the natural cellular antioxidant GSH is probably minimal, the lipid-soluble free radical scavenger o~TH may play an important role in preventing oxidative processes from taking place. Effective antioxidant protection by oLTHappears to be due to efficient inhibition of lipid oxy-radical propagation in the bilayer rather than to interception of initiating 02 radicals. 121 Concentrations of c~TH increasing above the threshold of 0.2 mole percent (based on phospholipid content of liposomes) decrease the average radical chain length and the ratio of LO2" to LO. in the bilayer and induce

Antioxidants and skin carcinogenesis termination of lipid oxy-radical propagation (reactions k and 1). Other types of antioxidants may spontaneously and/ or enzymatically prevent the formation of the dangerous •OH radicals by reducing U202 before it can react further. The endogenous antioxidant GSH ~22meets this requirement because it is present at relatively high concentrations in the aqueous part of the cells where reactions a-d are likely to occur. Moreover, reduction of oxidative stress is also accomplished by the action of the following enzymes which control the cellular level of H202 (Table 2).~2° Native SOD or SOD-mimicking compounds such as Cu(II)-(3,5-diisopropylsalicylate)2 (CuDIPS) accelerate the removal of 02; radicals (reaction m). In addition to CAT activity, the detoxification of H202 in the cytoplasm is accomplished largely by the GSH peroxidase-glutathione reductase (GSSG-R) couple, a NADPH-consuming system which is extremely specific for GSH and requires adequate concentrations of glucose and Se to work. 122 Unlike CAT, the Se-dependent form of GSH peroxidase containing selenocysteine at its catalytic site is capable of rapidly detoxifying both U202 and lipid hydroperoxides (reactions n and o). In contrast, non-Se dependent GSH peroxidase uses organic hydroperoxides but not H202 as substrates and has been shown to be associated with another family of protective enzymes, the GSH S-transferases (reaction 0). 123 125There are multiple forms of GSH S-transferases located principally in the cytosol.~26 Although conjugation of GSH to certain xenobiotics may be accomplished to some degree by spontaneous non-enzymatic reactions, conjugation with GSH catalyzed by the GSH S-transferases is an important process for the cellular detoxification of hydrophobic agents bearing electrophilic sites. 122.127Thus, the GSH peroxidase activity of the GSH S-transferase (reaction o) may be of paramount importance to protect from lipid hydroperoxide damage the biological systems that have no or very little Se-dependent GSH peroxidase, even though such protection may not be the primary action of this closely related group of enzymes.~28 In mouse epidermis in vivo and in vitro, Na2SeO3-dependent GSH peroxidase activity represents about 65-75% of the total GSH peroxidase activity.129 Free radicals in tumor initiation

Although increased free radical generation can lead to DNA damage and is potentially carcinogenic, the role of ROS in tumor initiation is not well documented. Transition metal ions, such as iron, are involved in the O2~--dependent formation of more reactive radicals and may affect the initiation of neoplasia. ~2 The free rad-

385

ical-generating agents BPx, lauryl peroxide and H202 may be inactive as skin tumor initiators because the doses of peroxides capable of inducing irreversible DNA lesions may be too toxic to permit the survival of a population of initiated epidermal cells large enough to be promoted.~3° The essential step in the process of skin tumor initiation is the metabolic activation of DMBA by NADPH- and cytochrome P448-dependent TM microsomal mixed-function oxidases that are induced in the epidermis 132,~33 and generate highly reactive electrophilic derivatives, which covalently modify DNA and other cellular macromolecules.134 A sequence of three monooxygenase-, epoxide hydratase- and monooxygenase-catalyzed reactions affecting the angular benzo ring adjacent to the " b a y region ''135 converts the "procarcinogenic" PAH parent molecule DMBA into mutagenic oxidation products often called the " p r o x i m a t e " and "ultimate" forms of the carcinogen: the initial non-K-region epoxide is then hydrated to give a dihydrodiol, and finally the oxidation of the double bond adjacent to the dihydrodiol grouping yields the " b a y region" vicinal diol-epoxide that is probably the 3,4-diol 1,2-oxide of DMBA. 136 Thus, the efficiency of tumor initiation in mouse skin is related both to the dose of DMBA applied and to the extent of induction of the epidermal microsomal aryl hydrocarbon hydroxylase (AHH) activities necessary to catalyze the formation of the electrophilic " b a y region" diol epoxide mutagens in the presence of a NADPH-cytochrome P448 electron transport system. 132.133,137142Genetic studies suggest that, in different strains of mice, high AHH inducibility leads to increased susceptibility to PAH-induced tumors.143 In addition to the formation of electrophilic ultimate carcinogens directly inducing DNA lesions, the metabolic activation/degradation of PAHs may proceed through quinone derivatives and free radical intermediates, thereby producing ROS that may indirectly contribute to the persistence of unrepaired DNA damage resulting in initiation of the affected cells.144 14s An O2-dependent metabolic pathway has been described, in which semiquinone intermediates of PAHs participating in redox cycles are oxidized to quinone metabolites with concomitant reductive generation of 027 subsequently triggering H202 and .OH formation. ~49.150Since ionizing radiations are known to damage DNA either directly or indirectly through free radical generation, ~7 it is interesting to note that a single subtumorigenic dose of ionizing radiations mimics the initiating activity of DMBA in the two-stage initiation-TPA promotion protocol in mouse skin. TM Moreover, the increased generation of ROS by leukocytes during skin inflammation is clearly implicated

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J.-P. PERCHELLETand E. M. PERCHELLET

in the metabolic activation of PAHs in mouse skin. 152 The oxidative challenge produced by the polymorphonuclear (PMN) leukocytes that are recruited and subsequently activated by two pre-treatments of the skin with TPA 16 h before and immediately prior to initiation has been shown to enhance the metabolic activation of the penultimate carcinogen benzo[a]pyrene (BP) 7,8-dihydrodiol to genotoxic and chemiluminescent metabolites through myeloperoxidase-dependent pathways.~53 Therefore, it is conceivable that the oxidative burst induced by the metabolically stimulated leukocytes infiltrating the sites of inflammation in mouse skin could facilitate or enhance either directly or indirectly the tumor-initiating activity of DMBA. The possible link between inflammation and cancer has been reviewed. ~52,~53The free radicals may act as tumor initiators by direct damaging interaction with DNA, indirect formation of clastogenic factors, or induction of cooxygenation in carcinogen metabolism. The relevance of arachidonic acid metabolism to skin tumor initiation is sugested by the finding that PAH dihydrodiols are cooxidized to mutagenic derivatives during PG biosynthesis.~54'~55 PG endoperoxide synthetase contains both fatty acid cyclooxygenase and hydroperoxide activities, which catalyze the biosynthesis of hydroxy endoperoxides from arachidonic acid. ~56 The peroxidative oxidation mediated by the enzyme PG H synthetase in the presence of arachidonic acid may simultaneously trigger the cooxidation of PAH carcinogens. 157 Arachidonic acid treatment prior to DMBA stimulates tumor initiation, an effect which is abolished by the simultaneous administration of the inhibitor of PG synthesis, indomethacin. 154The mechanism of PG endoperoxide synthetase-dependent cooxygenation, therefore, may play an important role in the oxidative activation of carcinogenic metabolites in tissues with low NADPH-dependent mixed-function oxidase activity. ~54 The excellent correlation observed between LOz" and diol-epoxide formation m suggests that the LOz" generated during arachidonic acid metabolism may be the oxidants that epoxidize the penultimate dihydrodiol metabolite of DMBA to the ultimate diol-epoxide form of the carcinogen. 1~4However, recent studies with isolated epidermal cells suggest that the major source of LOz" may be lipid peroxidation rather than the enzymes of arachidonic acid oxygenation. ~4 During metabolic activation of DMBA, the LOz" radicals play no role in the first epoxidation reaction, which is catalyzed solely by inducible microsomal monooxygenases. In contrast, both LOz" and P448-dependent enzymic reactions may contribute to the terminal step of the pathway of metabolic activation of the initiator DMBA. The kinetics of mixedfunction oxidase induction or inhibition, DNA adduct

formation and skin tumor initiation suggest that, in intact epidermal cells, LOz'-dependent epoxidation of dihydrodiols to diolepoxides may be an effective alternative to cytochrome P448-dependent epoxidation since the induction of the latter enzymic pathway is not absolutely required for the metabolic activation of low tumor-initiating doses of DMBA in mouse skin. 114 In conclusion, the catalytic action of microsomal cytochrome P448-dependent monooxygenases is not exclusively involved in tumor initiation. The tumorinitiating diol epoxides may be formed as well by oxidation of the penultimate metabolites of PAHs through LOz" or other free radicals generated during lipid peroxidation ~59 or the oxidative burst accompanying the state of inflammation, ~52~53 or by the peroxidase component of PG synthetase. 154,L55.157,158

Free radicals in tumor promotion

The relationships between cellular prooxidant states and tumor promotion have been reviewed. 5L~°4-~1°'16°.~61 The role of Oz-centered free radicals in tumor promotion is supported indirectly by the obvservations that 1) tumor promoters increase the generation and decrease the degradation of ROS; 2) certain organic peroxides and free radical-generating systems exhibit tumor-promoting activities and mimic or enhance some of the molecular events linked to tumor promotion; and 3) various antioxidants and free radical scavengers inhibit the biochemical and biological effects of the tumor promoters. The latter point is developed in the following section. Since tumor promoters are not mutagenic and do not interact directly with DNA, it is postulated that their reported effects on aneuploidy ,70,71 chromosomal aberration, 69.7z-75 sister chromatid exchange,76 80 DNA strand breakage, 81 and gene amplification 3°'3zSz'53"Sv are indirect and may be mediated through ROS generated in the TPA-treated epidermal cells themselves and/or released by the activated phagocytic cells infiltrating the skin during inflammation. The fact that TPA does not inhibit gamma radiation-induced DNA repair in resting lymphocytes argues against the hypothesis that tumor promotion is the result of impaired repair of potentially mutagenic lesions in DNA.~62 One of the current hypotheses is that an increased generation of free 02 radicals coupled with a defective antioxidant protective system could explain some of the mechanisms of tumor promotion and progression. Although different interpretations have been proposed to explain these observations, ~63 the activities of the detoxifying enzymes SOD and CAT seem to be significantly depressed by the tumor promoters, 164.165

Antioxidants and skin carcinogenesis

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Fig. 2. Time-response curves for the effects of 1 #M TPA on the level of hydroperoxide (A) GSH peroxidase activity (B) and the intracellular GSH:GSSG ratio (C) in suspensions of disrupted (A) or intact (B,C) mouse epidermal cells freshly isolated by trypsin digestion. '72 The total volume of the incubation mixture was 1 ml; 2 x 106 epidermal cells were resuspended in 0.8 ml of modified Eagle's HeLa cell medium containing 25 mM Hepes buffer, pH 7.0, and 10% bovine serum, and the reagents were added in a total volume of 0.2 ml. Dimethyl sulfoxide (DMSO) was used as a vehicle for TPA; its final concentration in the medium was 0.5%. At the end of each incubation period, 1 ml of 20% trichloroacetic acid (TCA) was added to the cell-free systems and the whole acid-soluble supernatant was collected by centrifugation and mixed with 0.6 ml of ferrithiocyanate assay reagents? 73=74The absorption of the red ferrithiocyanate complex formed in the presence of peroxide was measured against a reagent blank at 480 nm in a Shimadzu-160 double-beam spectrophotometer and compared to H202 standards. The soluble epidermal extracts containing GSH peroxidase activity were prepared from the intact cells and total GSH peroxidase activity was determined with 1.5 mM cumene hydroperoxide as described previously. ,29.,79-~82The enzymic determination of the intracellular levels

387

w h e r e a s c e r t a i n c l a s s e s o f r e a c t i v e 02 i n h i b i t o r s inc l u d i n g C A T and v a r i o u s S O D - m i m i c k i n g c o m p o u n d s g e n e r a l l y i n h i b i t s o m e o f the b i o c h e m i c a l and b i o l o g ical e f f e c t s o f the t u m o r p r o m o t e r s . 99'166-169 R e c e n t l y , w e h a v e e s t a b l i s h e d the s e q u e n t i a l r e l a t i o n s h i p s bet w e e n the e f f e c t s o f T P A on h y d r o p e r o x i d e f o r m a t i o n , G S H p e r o x i d a s e a c t i v i t y and the i n t r a c e l l u l a r ratio o f reduced glutathione (GSH): oxidized glutathione (GSSG). O u r c u r r e n t studies i n d i c a t e that T P A rapidly stimulates h y d r o p e r o x i d e p r o d u c t i o n in intact e p i d e r m a l cell and c e i l - f r e e s y s t e m s i n c u b a t e d in the p r e s e n c e or a b s e n c e o f e n z y m i c and n o n e n z y m i c g e n e r a t o r s o f R O S . 170"171 E p i d e r m a l c e l l s (2 x 106 c e l l s / m l ) f r e s h l y i s o l a t e d f r o m m o u s e skin by trypsin d i g e s t i o n 172 w e r e d i s r u p t e d by s o n i c a t i o n and i n c u b a t e d for v a r i o u s periods o f t i m e in the p r e s e n c e or a b s e n c e o f 1 ktM T P A (Fig. 2 A ) . T h e h y d r o p e r o x i d e c o n t e n t s o f the acids o l u b l e s u p e r n a t a n t s c o l l e c t e d at the end o f the incub a t i o n w e r e a s s a y e d at acid p H by a m o d i f i c a t i o n 173 o f the f e r r i t h i o c y a n a t e m e t h o d . 174 In this T P A - s t i m u l a t e d e p i d e r m a l s y s t e m , the s t e a d y - s t a t e l e v e l o f h y d r o p e r o x i d e s i n c r e a s e s r a p i d l y and s t e a d i l y for a b o u t 2 h and r e m a i n s at 2 2 4 % o f the basal l e v e l up to 4.5 h (Fig. 2 A ) . A f t e r i n c u b a t i o n for 5 h in the p r e s e n c e o f 1 # M concentrations of DMBA, TPA, phorbol- 12,13-didecanoate, MEZ, phorbol- 12,13-dibenzoate, 4-0-methyl T P A , p h o r b o i and EPP, the l e v e l s o f h y d r o p e r o x i d e s are, r e s p e c t i v e l y , 2 3 5 , 2 2 4 , 186, 175, 162, 137, 102, and 9 8 % o f the c o n t r o l l e v e l , s u g g e s t i n g that the hydroperoxide-inducing activities of these agents correlate with their t u m o r - p r o m o t i n g or c a r c i n o g e n i c activities. 170.171 A l t h o u g h the t i m e - c o u r s e and m a g n i t u d e o f the h y d r o p e r o x i d e r e s p o n s e to T P A are i d e n t i c a l , the v a l u e s o f the basal and T P A - s t i m u l a t e d l e v e l s o f h y d r o p e r o x i d e s m e a s u r e d in s u s p e n s i o n s o f intact epi d e r m a l cells are about 5 0 % s m a l l e r than t h o s e o b t a i n e d with the e p i d e r m a l c e l l - f r e e s y s t e m . S i m i l a r results w e r e o b t a i n e d , but at a m u c h l o w e r l e v e l , u s i n g the 4a m i n o a n t i p y r i n e / p h e n o l r e a g e n t and h o r s e r a d i s h per-

of GSH and GSSG was performed in samples deproteinized in 5% TCA/0.01 N HCI. ,79-,82Hydroperoxide level was expressed as n mol H202/2 × 106 disrupted cells/ml, GSH peroxidase activity as ,umol of NADPH oxidized in 1 min/ 2 × 106 cells and the levels of GSH and GSSG as ng/2 x 106 cells. Blank reactions with enzyme source, glutathione, or hydroperoxide samples replaced by buffer were substracted from the data. Results are expressed at each point as % of the basal level of H~O2, GSH peroxidase activity or GSH:GSSG ratio in cells incubated in the absence of TPA. Each point in the graph is the mean value ± SD of 6 replications from 2 different experiments. The mean values of the basal levels of hydroperoxides (8.6 +- 0.4 nmol H202/ml; 100 --- 5%), the basal GSH peroxidase activities (0.167 ± 0.014 k~mol of NADPH oxidized/min/2 x 106 cells; 100 --- 8%) and the basal GSH:GSSG ratios (24.3 --- 1.8; 100 --- 7%) at each point in control incubations have been indicated by the hatched areas.

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oxidase, an enzymic assay performed at physiological pH and which is specific for H202 .175 177 In other in vivo experiments, groups of mice received either acetone, 1, or 2 topical applications of 8.5 nmol of TPA at 24 h interval and were killed i 2 h after the last TPA treatment. When incubated in vitro in the presence or absence of 5 mM NAN3, the levels of hydroperoxides in the homogenates prepared from the skins treated once or twice with TPA were 1.5- and 3-fold higher, respectively, than the control levels in the homogenates from the skins treated with acetone only. 171Phagocytic cells may contribute to the H202 responses in the homogenates prepared from skins pretreated with TPA or DMBA in vivo. The fact that similar H202 responses are detected in epidermal cells and homogenates, which are isolated or prepared from non-treated skins and then incubated directly with TPA or DMBA,~7°'~7~ suggests that the increasing levels of H202 are generated largely by the TPA-and DMBA-treated keratinocytes and cannot reflect entirely the oxidative burst of activated leukocytes infiltrating the skin. Indeed, the keratinocytes may be the major source of H202 in the epidermis. ~TX The sequential and dose-dependent relationships between the decreasing effects of TPA on the intracellular G S H : G S S G ratio and GSH peroxidase activity in mouse epidermis in vivo and in vitro suggest that the GSH-dependent detoxifying system cannot fully protect the epidermal cells for an extended period of time against the oxidative challenge caused by the tumor promoter. 1°°'129179-t~3As shown in Figure 2 (B and C), the incubation of intact epidermal cells with TPA leads within about 20 min to a rapid, transient increase in GSH peroxidase activity, which is concomitant with a dramatic fall in the GSH : GSSG ratio. This is followed after 1 h by a prolonged inhibition of GSH peroxidase activity while the G S H : G S S G ratio remains low. Taken together, the time-dependent studies presented in Figure 2 (A, B, and C) suggest that the increased generation of peroxides by TPA initially stimulates the activity of the GSH-dependent detoxifying system. Although an undetermined amount of spontaneous, nonenzymatic GSH oxidation is likely to occur, the early and transient induction of GSH peroxidase activity in the TPA-treated, H202-producing cells may be responsible for the rapid elevation of GSSG and decline in the intracellular GSH : GSSG ratio. The phenomena observed 2-4.5 h after TPA may be crucial in understanding the cause and prevention of some of the molecular events of tumor promotion. At these times, the TPA-treated cells are still accumulating hydroperoxides but their GSH peroxidase activity is now inhibited and their GSH : GSSG ratio remains abnormally low. Whether it is due to the continual challenge caused

by the presence of TPA in the incubation medium or to the now depleted intracellular level of GSH, it is postulated that, a couple of hours after TPA treatment, the endogenous GSH antioxidant system of the epidermal cells cannot reduce the excessive level of peroxidation possibly triggering macromolecule-damaging events linked to the tumor-promoting activity of TPA. Increased peroxidation is easier to detect when hydroperoxide decomposition is inhibited. ~v4'~84For example, whole skin homogenates incubated for 5 h in the presence of NaN3 contain 4 times more hydroperoxides than in its absence (Table 3). The increased accumulation of peroxides is also observed in epidermal systems incubated in the presence of 3-amino1,2,4-triazole, diethyl maleate (DEM), l-chloro-2,4dinitrobenzene (CDNB) and L-buthionine-SR-sulfoximine, the compounds that inhibit CAT activity or decrease the availability of GSH. 171 The enzymic and nonenzymic generation of hydroperoxides in mouse skin homogenates incubated for 5 h at 37°C in 50 mM potassium phosphate buffer, pH 7.4, containing 118 mM NaCI, 5.36 mM KCI, 1 mM CaCI2, 0.85 mM MgSO4 and 5 mM dextrose under a 95% 02-5% CO2 atmosphere is reported in Table 3. TPA is equally effective in stimulating hydroperoxide production in control and NaN3-treated homogenates. TPA enhances the hydroperoxide responses to various enzymic systems including NADPH, glucose (G)/glucose oxidase (GO) and xanthine (X)/xanthine oxidase (XO). Moreover, TPA can also stimulate the nonenzymic, NADPH-independent production of peroxides promoted by ascorbic acid (ASC, vitamin C) or the FeCI~/FeSO4/ EDTA complex. These results suggest that TPA may increase the levels of peroxidation and cellular damage in mouse epidermis through the stimulation of the prooxidant activities of various endogenous enzymic and nonenzymic sources of ROS. Our results are consistent with the observations that TPA stimulates the generation of ROS in leukocytes and provokes a chemiluminescence (CL) response that peaks and persists after 5 - 3 0 min. ~5 As early as 1976, DeChatelet et al. ~86have shown that TPA rapidly increases the cellular production of H202. The cytotoxic potential of activated macrophages and granulocytes has also been demonstrated to correlate with their ability to release H202 in response to TPA. I87 Several phorbol ester derivatives with various tumorpromoting activities have been shown to stimulate the generation of H202 by macrophages during a 60-rain incubation period. ~88Moreover, TPA has already been reported to stimulate H202 production in mouse skin in vivo after 30 min.~89 In murine epidermis, TPA induces the specific activity of XO, ~9° a O2~-generating

Antioxidants and skin carcinogenesis

389

Table 3. Enzymic and NonenzymicGeneration of Hydroperoxidesin Mouse Skin (epidermis + dermis) HomogenatesTreated with TPA Hydroperoxide Formationt

Incubation System* Control (no addition) +NAN3 +NAN3 + N A D P H +NAN3 + glucose/glucose oxidase +NAN3 + xanthine/xanthine oxidase +NAN3 + ascorbate +NAN3 + FeCIflFeSO4/EDTA TPA (1 pM) +NAN3 +NAN3 + N A D P H +NAN3 + glucose/glucose oxidase +NAN3 + xanthine/xanthine oxidase +NAN3 + ascorbate + NaN3 + FeC13/FeSOJEDTA

nmol/mg Protein/5 h r : ~ 10.6 -+ 0.4 46.0 _+ 1.9 100.5 +- 4.6 128.3 _+ 5.8 149.4 _+ 7.1 84.6 - 3.2 130.3--- 5.2 23.0 +- 0.9 94.7 _+ 3.7 188.5 -+ 8.3 293.5 -+ 15.3 255.3-+ 12.3 114.1 _+ 4.6 216.7 _+ 10.0

% of Control

% of Respective Incubate Without TPA

100 434 948 1210 1410 798 1229 217 893 1778 2769 2408 1076 2044

217 206 188 229 171 135 166

*Thc volume of each incubate was 1 ml with a protein concentration of 1.5 mg/ml. The concentrations used were: 2.5 mM NAN3; 1 mM NADPH; 2.5 mM glucose/0.5 munit glucose oxidase; 1.0 mM xanthine/0.5 munit xanthine oxidase; 0.2 mM ascorbate; 0.1 mM FeClfl0.1 mM FeSOd0,1 mM EDTA. tDetermined after incubation for 5 h at 37°C; ice-cold blanks containing all the components of the incubation mixtures were subtracted from each value. SMean + SD of 6 replicates in 2 different experiments.

system shown to mimic the effects of TPA on ODC induction TM and promotion of cell transformation.192 The tumor-promoting activity of TPA may be related to its peroxisome proliferative activity.193 In analogy with TPA, the peroxisome proliferators are not mutagenic and do not covalently interact with DNA but may modulate gene expression and function as tumor promoters in a two-stage carcinogenesis model. 194,195Peroxisome proliferators stimulate the production of H202 and other ROS and decrease the enzymic defense against peroxidative damage.196 The resultant oxidant stress may cause DNA damage either directly or by initiating lipid peroxidation. Since ROS, when present in excess, trigger lipid peroxidation of membranes and cellular damage, it would be logical to assume that the early imbalance between hydroperoxide production and degradation observed after TPA in our studies is accompanied by epidermal lipid peroxidation. However, using the thiobarbituric acid assay, a decline in epidermal lipid peroxidation has been described 4-22 h after TPA treatment in v i v o . 197 In this study, lipid peroxidation increased whereas GSH peroxidase activity decreased with the aging of mouse epidermis. However, it might be critical to re-assess the effects of TPA on microsomal lipid peroxidation at shorter time intervals and after multiple TPA treatments using the diene conjugation procedure. The level of lipid peroxidation might not necessarily parallel the H202 response to TPA. In liver homogenate in vitro prepared from mice treated

in vivo for 14 days with peroxisome proliferators, the steady-state level of H202 is increased whereas both GSH peroxidase activity and lipid peroxidation are decreased.198 Interestingly, the tissue level of GSH peroxidase appears to be sensitive to the level of lipid peroxides. Because the cells maintain a multi-level defense against lipid peroxidation, the sequential transient increase and prolonged decrease in epidermal GSH peroxidase activity that we observed after TPA might be linked more directly to the fluctuation of lipid peroxides than to that of H202. Therefore, a decrease in lipid peroxidation ~97 and GSH peroxidase activity 100.129,179-183several hours after TPA might not necessarily indicate a decrease in oxidative stress to the cell. Although the levels of H202 and lipid peroxides might evolve differently at later times because of different levels of detoxification, both H202 generation and lipid peroxidation might be implicated in the early oxidative challenge altering the GSH-dependent antioxidant system during TPA treatment. The increased level of peroxidation and the decreased efficacy of the GSH protective system may be characteristic of the tumor growth process. 199 However, it is not known whether increased peroxidation is indicative of the benign or malignant state of the skin during carcinogenesis. Inflammatory macrophages from promotion-sensitive SENCAR mice secrete 4 times more H202 than the corresponding cells from promotion-resistant C57BL/6 mice. 2°° Theoretically, if increased peroxidation mediates to some ex-

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tent tumor promotion, the skins of these mice should elicit peroxidative responses to TPA related to their different sensitivities to tumor promotion by TPA. ROS mediate nonenzymic, NADPH-supported, and XO-catalyzed lipid peroxidation in microsomal preparations from whole skin, dermis or epidermisfl °~ NADPH-dependent H20 2 formation in microsomes is mainly due to NADPH oxidase while about 1/3 may arise from the autoxidation of cytochrome P450.174'j84 Skin microsomes incubated with NADPH, FeCI3/ADP or low concentrations of ASC form lipid peroxides. Moreover, the nonenzymic catalysts FeCI3 and ADP enhance remarkably the enzymic generation of lipid peroxides in epidermal microsomes. 2°~ The nonenzymic FeSO4/ ADP-supported rate of lipid peroxidation has been studied in epidermal homogenates prepared after the very heat treatment used in our laboratory. 2°2 The tumor-promoting activities of the phorbol esters in mouse skin correlate well with their abilities to induce ODC activity and DNA synthesis in epidermal cells. 2°3 Since the levels of peroxidation in control and TPA-treated epidermal samples may be several folds greater in the presence of NAN3, NADPH, G/GO, X/XO, ASC and FeC13/FeSOg/EDTA than in their absence (Table 3), one would expect these enzymic and nonenzymic stimulators of lipid peroxidation to enhance as well the sequential effects of TPA on GSH metabolism, ODC induction and macromolecule synthesis. By magnifying the effects of TPA, these enzymic and nonenzymic oxidant-generating systems might provide valuable tools to elucidate the role of peroxidation in the molecular mechanism of tumor promotion. Indeed, our preliminary studies (unpublished data) indicate that concentrations of NaN3 (1-10 pM), CDNB (1-5/~M) and/or NADPH (0.01-0.1 mM) and G / G O (0.01-0.25 munit/ml) several folds lower than those shown to inc r e a s e hydroperoxide accumulation in skin homoge-

M. PERCHELLET

nates (Table 3) can enhance the ODC-inducing activity of TPA in suspensions of intact epidermal cells ~72 or in cultures of intact skin explants, z°4 Interestingly, it has been suggested that mouse skin sensitivity or resistance to TPA as a hyperplasiogen and as a tumor promoter is likely to be explained on the basis of oxidant generation and 8-1ipoxygenase induction rather than ODC induction. 2°5 The significance of increased production of ROS and elevated peroxidation to the multistage process of skin tumor promotion is purely speculative. Some of the major issues to be addressed have been depicted (Fig. 3). The relevance of PKC induction to the level of peroxidation/GSH-dependent detoxification during tumor promotion remains to be determined. The potential role of PKC in stimulus-response coupling has been reviewed. 45'46,2°6 PKC is probably a prime target of several chemically unrelated tumor promoters which mimic the membrane perturbations caused by diacylglycerol. There is sufficient evidence to suggest that PKC induction mediates, at least partially, the molecular mechanism by which tumor promoters alter gene expression. PKC induction has been implicated in the elaboration of ROS by neutrophils, leukocytes and inflammatory cells. 2°7-218 Moreover, exogenous diacylglycerols stimulate O2 ~ production, suggesting that endogenous diacylglycerols may function as messengers for this biological response. 2°8,2~°,2~,2j~ However, the primary involvement of this enzymic activity in modulating the production and/or detoxification of peroxides in epidermal systems treated with TPA has yet to be demonstrated. In intact cells, phorbol esters activate NADPH oxidase, the en'zyme system responsible for the oxidative burst. The phorbol ester promoters similarly activate the oxidase in a reconstituted system which includes a membrane fraction, ATP, phospholipids, NADPH and either a cytosolic

+ Protein Kinase C Induction .~.,~iGenerotio n l°f Reactive ._~ Resulting --'] Agent with ~ ~. 102 Sl~cies 7 Free Radical[ oae o Tumor-Promoting ~/ Challenge[ Dam_._ t_ Activity and I ? Mocromo~cules, ? + l Detoxification[ Increased [ • ~ Membranes, DNA,"~-~ ~ ] o f Reactive ] Level of l Chromosomes _ Natural Antioxidon f t [ Oz Species [ Peroxidation_.J Protective Systems :;~ +

Multistage _ lumor. Promohon and Progression

Chemotherapy ? Fig. 3. Postulatedrole of free radicals and epidermal peroxidationin the process of skin tumor promotion and/or progression.

Antioxidants and skin carcinogenesis fraction or purified P K C . 46 Incidentally, the non-TPA type tumor promoters palytoxin and thapsigargin, which do not bind to phorbol ester receptors or activate PKC, also stimulate O2 ~-formation in neutrophits, suggesting that these compounds activate the NADPH oxidase system of the cells by different signal transduction mechanisms. 219 TPA stimulates xanthine dehydrogenase (XD) synthesis and the conversion of existing and newly synthesized XD to XO. 19° Several antibiotics that suppress skin inflammation prevent H202 production by inhibiting the cell systems generating ROS.22° Moreover, the ability of TPA to induce peroxisomal enzyme activities and H202 generation is inhibited by cycloheximide, suggesting that these effects of TPA may be largely the result of de novo protein synthesis. ~93 New findings suggest that the induction of XO by TPA is a consequence of the hyperplasia induced by the tumor promoter rather than the cause of it. 22~ In contrast to non-tumor promoters and purely hyperplastic agents, TPA stimulates the accumulation of hydroperoxides in epidermal cells and cell-free systems in vitro (Fig. 2, Table 3) 170'171 but the significance of H202 production, lipid peroxidation and GSH-dependent detoxification to the modulation of gene expression during skin tumor promotion by TPA is not known. In relation with their weak skin tumor-promoting activities, 97 the peroxides mimic some of the effects of TPA on PKC induction, 222 cell transformation, 223 single strand break in DNA, 224GSH peroxidase inhibition and ODC induction, 1°° but at doses considerably higher than those of TPA. However, the peroxides enhance the progression phase of carcinogenesis. 93'94 In accord with our findings on GSH m e t a b o l i s m, 1°°'18°'~8Hs3 structure function studies indicate that the ability of different phorbol esters to stimulate the generation of ROS and inhibit their degradation, and damage DNA and chromosomes correlates with their potency as tumor promoters. 72"1°5"1°8"164'188"19° However, the relevance of these events to a specific stage of tumor promotion is not clear: different promoters produce single strand breaks in DNA of mouse epidermal cells in relation with their complete and stage 2 tumor-promoting activities 224 but they stimulate H202 formation and DNA base modification in relation with their potency as stage 1 tumor promoters. 225 It has been postulated that H202-induced damage might be responsible for the " m e m o r y " effect induced by stage 1 tumor prorooters. 225 On the other hand, PKC induction and generation of 02; do not appear to be of critical importance for stage 1 conversion. 66,169In agreement with the identical effects of TPA and MEZ on GSH metabolism and ODC induction observed in our studies, l°°.18°,181.ls3the

391

stage 2 tumor promoter MEZ is at least as potent as TPA in increasing the generation and decreasing the detoxification of ROS. 105,108,164A88A90However, the hydroperoxide response to TPA is greater than that induced by MEZ in epidermal cell and cell-free systems. 170.171In general, purely inflammatory or hyperplastic agents and compounds with no or only stage 1 tumor-promoting activity produce none of these effects. Our findings that peroxides, complete and stage 2 tumor promoters concomitantly deplete the level of GSH, inhibit GSH peroxidase activity and induce polyamine biosynthesis suggest that the accumulation of nondetoxified ROS in the epidermis may be one of the components of the later stages of skin tumor promotion and progression. Other evidence suggests that an oxidative challenge is involved in the 2rid rather than the 1st stage of skin tumor promotion: treatments with butylated hydroxyanisole (BHA), etTH + GSH, or a SOD analogue inhibit both the ODC-inducing and complete tumor-promoting activities of TPA but fail to inhibit skin tumor promotion when applied in combination with TPA in the 1st stage. 1°°,1°8,169In mouse skin, the free radical-generating peroxides have been shown to induce dark basal cells, a characteristic of complete and stage 1 tumor promoters. 56,13° In SENCAR mice, retinoic acid (RA), which triggers CL and the development of dark cells, elicits stage 1 and complete tumor-promoting activities. 226 Since the luminescence response is believed to be due to 02 free radical generation, the data suggest that the ability of RA to promote tumors and induce dark basal keratinocytes may be due to initial oxidative reaction at the cell membrane. It is not known whether the increased level of peroxidation in mouse epidermis contributes significantly to the induction of DNA and chromosome damage during skin tumor promotion and progression and if it would be possible to prevent TPA-induced genetic lesions by boosting the natural antioxidant protective systems of the epidermal cells. Since tumor promoters are reactive 02 generators whose action appears to be mediated to a certain degree by free radical production, peroxidation and DNA strand break, 71-73.75,81.166.227-23°it is important to establish whether H202-mediated oxidation of macromolecules is accompanied by DNAdamaging events associated largely with certain stages of tumor promotion or progression. Sister chromatid exchanges 76-8° may be implicated in the partially irreversible I st stage of promotion. Moreover, the stage 1 tumor-promoting activity of H202 may be linked to its ability to induce DNA lesions leading to chromosome-type as well as chromatid-type aberrations. TM Therefore, it is fundamental to assess whether the different potencies of various compounds during tumor

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J.-P. PERCHELLETand E. M. PERCHELLET

initiation, stage 1 and 2 promotion, and progression could be explained on the basis of different levels of peroxidation causing different levels of DNA damage (Fig. 3). Free radicals in tumor progression

When applied to the skin of Pa-bearing mice, the free radical generators BPx and H,O2 are more potent than TPA in stimulating the malignant progression of skin tumorsd 3'94 It is not known whether chemically unrelated compounds such as TPA, chrysarobin, DMBA, BPx, MNNG and ENU increase the epidermal levels of peroxidation and genetic lesions in relation with their potency in tumor progression. As pointed out before, DMBA does not mimic the effects of TPA and MEZ on epidermal GSH peroxidase and ODC activities ~°~ and some antioxidant inhibitors of skin tumor promotion by TPA and MEZ fail to inhibit DMBA-induced ODC activity and carcinogenesis by a single large dose or by repeated small doses of DMBA.~.~ t0o.232However, in our preliminary studies, DMBA appeared at least as potent as TPA in inducing hydroperoxide accumulation in the epidermal system. 17°lTj In contrast to TPA, the effects of both BPx and DMBA on mouse skin are characterized by direct production of single strand breaks in DNA, induction of a high Ca:Pa ratio and resistance to inhibition by RA, suggesting that the promoting component of BPx may resemble more the promoting component of DMBA carcinogenesis than that of TPA. 233 Moreover, strains of mice refractory to promotion by phorbol esters in two-stage initiation-promotion protocols respond to promotion by BPx and to complete skin carcinogenesis protocols. The induction of single strand breaks by phorbol esters in vitro may be related to the induction of terminal differentiation. 224Therefore, phorbol ester tumor promoters would induce DNA strand breaks and accelerate terminal differentiation in normal non-initiated basal epidermal cells but not in the initiated progenitor cells resistant to phorbol ester-mediated DNA strand breaks and signals of terminal differentiationf133 To explain the mechanism of tumor promotion by BPx, it has been postulated that this free radical generator might induce extensive DNA strand breaks and cytotoxic effects in normal epidermal cells and, therefore, favor indirectly the clonal expansion of the initiated epidermal cells that are more resistant to BPx-induced genetic lesions and cytotoxicity. Furthermore, the initiated or benign neoplastic keratinocytes sustaining and surviving limited DNA damage due to their relative resistance to the potent effects of BPx, MNNG, or ENU, might accumulate sufficient additional genetic

lesions to progress towards malignancy. The oxidative challenge caused by TPA in the epidermis may not be sufficient to cause such malignant conversion in preneoplastic or neoplastic keratinocytes resistant to TPAinduced DNA strand breaks. THE EFFECTS OF ANTIOXIDANTS ON MULTISTAGE SKIN CARCINOGENESIS

The anticarcinogenic activity of antioxidants in various tumor systems has been reviewed. 234-237 With a few exceptions, most antioxidant treatments tested inhibit the multistage process of skin carcinogenesis (Table 4). The process of anticarcinogenesis by these compounds remains obscure. The mechanisms of antioxidant protection elicited by different classes of inhibitors, such as free radical scavengers, thiol delivery systems, compounds enhancing or mimicking enzymic activities involved in detoxification, retinoids and inhibitors of arachidonic acid metabolism, are varied and likely to alter in a different manner and to a different degree the effects of different categories of skin carcinogens and tumor promoters. Antioxidants in tumor initiation and complete carcinogenesis

The mechanisms by which antioxidants may prevent the generation of carcinogenic compounds, block their interaction with critical target sites in epidermal cells and/or suppress the neoplastic process elicited by carcinogenic agents have not been clearly elucidated. 235 During initiation or complete carcinogenesis by a single large dose or repeated small doses of a carcinogen, antioxidants may alter the enzymic activities involved in metabolic activation, degradation and detoxification of the PAHs and/or scavenge free radical species directly or through the enhancement of the host defense systems. Modifying the metabolism of PAH carcinogens is complicated in that the microsomal monooxygenase system can both activate and detoxify those compounds. It is postulated that antioxidants exert chemoprotective effects against neoplasia in laboratory animals largely through selective induction of phase II detoxification pathways which facilitate the enzymic activation and clearance of activated metabolites through conjugation reactionsf175 In addition to the inhibition of the oxidative metabolic activation of procarcinogens to ultimate electrophiles, the data reviewed in Table 4 suggest that various reducing agents and electron scavengers can inhibit the processes of skin carcinogenesis and tumor initiation by trapping the reactive electrophilic ultimate forms of chemical carcinogens and the free radicals generated during skin irradiation, thereby preventing their mu-

Table 4. Effects of Antioxidants on Multistage Skin Carcinogenesis* Treatments (Mode of Administration)t

Initiation { DMBA 238-24° IvDMBA 239"240

Ellagic acid (top) Quercetin (top) Garlic oil (top) Ajoene (top) Onion oil (top) Propenyl sulfide (top) Indomethacin (top) Flurbiprofen (top)

{ BP, BP-diol-epoxide 2~s

Flurbiprofcn + arachidonic acid (top) Phenidone (top) ETYA (top) ETYA + arachidonic acid (top) Lipoxygenase inhibitor AA861 (top) 3,4,2' ,4'-Tetrahydroxychalcone (top) RA (top; orally)

{ MC244~ ~, BP 245 ~ DMBA 238"24°

{ DMBA 239 { DMBA 239 ~, DMBA247; - - O - - D M B A 99

{ DMBA§

$ BP 2s°

BP, ---O--DMBA TM

{BP,

~,TPA 99,16s ,~TPA 242 { CRO 241 ,LTPA 1~'2'~2 { TPA 100 { TPA 183

~,TPA§; ~, BPx§ ENU/TPA§; $ MNNG/TPA§

~,TPA 249 TPA TM $ TPA z52 { TPA TM ~,TPA 252 { TPA253'254; 1' TPA 255 T TPA2S5

T TP A252 1"TPA 252

$ TPA 48 { TPA 48

~ BP TM { BP TM

{ TPA 256 { TPA z56 ~,TPAg8.257; { CRO 258 Anthralin 259 { TPA98257; { CRO 26° Anthralin 259 { TPAZ61; 1' TPA26111

--4)---DMBA z61 Initiation

Na2SeO3 (i.p.) + aTH (top) CuDIPS (top) GSH (i.p.) + aTH (top) DDTC (i. p. ) Garlic oil (top) Onion oil (top) lndomethacin (top) RA (top) 13-cis-RA (top)

Progression

B p----diol,---O---D MB A ~54 ~, BP, BP---dioP s4

RA analogs (retinoids) (top; orally; i.p.) DMSO (top)

Complete Promotion ~, CRO238'241:~; { TPA nX'-242 TPA 243; - - O - - C R O 238 ~, TPA 243 { CRO TM TPA 2~; --O---TPA 24s ~, CRO 238,241 { CRO23~; ~, TPA lc~/ TPA lc~j { TPA 1°° TPA lc°

aTH (top) ASC (top) ASC analogs (top) aTH + ASC + BHT + GSH (diet) Se (dw) Na2Se (top; diet) Na2SeO3 (diet; i.p.) Na2SeO3 + GSH (i.p.) Na2SeO3 (i.p.) + aTH (top) Na2SeO3 + GSH (i.p.) + aTH (top) BHA (top) BHT (top) CuDIPS (top) Cys (i.p.) Cysteamidc (top) GSH (i.p.) GSH (i.p.) + ctTH (top) DDTC (i.p.)

Two-Stage Promotion Stage 1 T TPAm° --O---TPA 169 - - O - - T P A 1°~ J, TPA 183 { TPA¶ { TPA¶ T TPA48 { TPA262; - - O - - T P A ~69

{ CRO 25~ { CRO 26°

Stage 2 { MEZm° { MEZ I~ ~ MEZ 183 { MEZ¶ { MEZ¶ ~, MEZ 262 ~ TPA 263

Complete Carcinogenesis# aTH (top; orally) ASC (diet) aTH + ASC + BHT + GSH (diet) Na2Se (top; diet) Na2SeO3 (diet; dw) Na2SeO3 (i.p.) + ctTH (top) CuDIPS (top) Cysteamide (top) GSH (i.p.) + aTH (top) ODTC (i.p.) Ellagic acid (top; dw) Garlic oil (top) Onion oil (top; top + dw) RA (top; i.p.) 13-cis-RA (orally) IS-carotene (top) DMSO (top)

MC (rsd)238; $ DMBA (rsd) 2~,265.266.* { UV-light (rsd) 267 { UV-light (rsd) 2°2.26s { MC (rsd); ~ BP (rsd) 23s BP (rsd)238; ,~ UV-light (rsd) 269 ----
*Enhancing effect ( I' ); Inhibitory effect ( $ ); No significant effect (--O---) tTreatments administered by topical application to the skin (top), by intraperitoneal injection (i.p.), in drinking water (dw), orally or in the diet. ~tCRO, croton oil; MC, 3-methylcholanthrene §Perchellet, J. P. unpublished data. IlTopical application to the skin but at a site remote from initiation and TPA treatments. ¶Belman, S.; Perchellet, J. P. unpublished data. #Treatment with either a single large dose (sld) or repeated small dose (rsd) of a carcinogen. **Hamster buccal pouch mucosa carcinogenesis. ttHamster lingual mucosa carcinogenesis.

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J.-P. PERCHELLETand E. M. PERCHELLET

tagenic interaction with epidermal DNA. The reducing agents, therefore, may act as nucleophiles competing with nucleophilic sites on DNA for the created radicals. As demonstrated during the photocarcinogenic response of mouse skin, free radical reactions, specifically chain propagation of epidermal lipid peroxidation, may play a major role in carcinogenesis. 2°2 Modulation of the level of epidermal lipid peroxidation and skin carcinogenesis appears possible through treatments that alter the polyunsaturated lipid:antioxidant ratio. A major function of oLTHis to inhibit both enzymic and nonenzymic lipid peroxidation and prevent the spread of damaging lipid radicals through membranes, o~TH may also prevent the formation of proximal carcinogens.239 In the standard model for hamster buccal pouch carcinogenesis, repeated applications of I 0 mg of aTH on alternate days to those when 0.5% DMBA solutions are painted thrice a week can not only inhibit DMBA carcinogenesis but may entirely prevent tumor development when less potent 0.1% DMBA solutions a r e u s e d . 266 The important intracellular reducing agent ASC has been suggested to be a protective agent against development of cancer. However, the controversy surrounding the efficacy of this agent on cancer chemotherapy comes from the fact that, under certain conditions, the free radicals generated during the oxidation of ASC may mediate DNA strand scissions and potentiate the mutagenic effects of carcinogens. 276 In vitro, ASC exerts opposite effects at different concentrations: at concentrations < 1 raM, ASC has a prooxidant effect on lipid peroxidation, whereas at concentrations >1 mM it fails to promote peroxidation and generally functions as an antioxidant. 277 The pronounced chemoprotective role, as well as the adverse effects, of the synthetic phenolic antioxidants butylated hydroxytoluene (BHT) and BHA commonly used in the food industry to scavenge 02containing radicals and prevent the oxidative deterioration of many food products has been reviewed thoroughly. 237 Electron spin resonance studies in vitro show that BHT scavenges the .OH and .MNNG formed by the interaction of H202 with MNNG by reacting with these labile free radicals and forming a stable phenoxyl free radical of BHT. 27~The scavenging effect of BHT on .OH, therefore, may be related to its effect in lowering carcinogenesis. The chemoprotective action of BHA, BHT and ethoxyquin requires intact cells to be operational and has been associated with an indirect effect on the epidermal metabolizing system which leads to a decrease in covalent binding of carcinogens to D N A . 239'279 The protective effects of these antioxidants may be accounted for, at least in part, by their ability to elevate the GSH S-transferases. 28° Preliminary results indicating that the inhibitors of PG

endoperoxide synthetase inhibit mouse skin initiation by BP and BP-7,8-diol but not by DMBA suggest the possible involvement of PG biosynthesis in BP and BP-7,8-diol tumor initiation, perhaps by a cooxygenation mechanism.154 However, BHA, an inhibitor of LO2"-dependent metabolism, but not the modifiers of PG H synthetase activity, significantly inhibits the LO2.-dependent epoxidation, which is the major pathway of BP-7,8-diol oxidation in skin cells prepared from animals that are not treated with inducers of mixed-function oxidase. ~s~ Since the SOD biomimetic agent CuDIPS is a potent inhibitor of initiation of SENCAR skins 247 and inhibits complete carcinogenesis in CD- 1 mice by a single large dose or multiple small doses of DMBA, 99 it is possible that, in contrast to SENCAR mice, the requirement for a high dose of DMBA for initiation of CD- 1 skins and the lower CuDIPS : DMBA ratio applied on CD- 1 skins might have masked the anti-initiating activity of CuDIPS in these mice. 99 The covalent binding of 3HDMBA to epidermal DNA is remarkably reduced by CuDIPS pretreatment, suggesting that the anti-carcinogenic properties of this compound may reflect a perturbation in O2--dependent metabolic activation of DMBA. 99 As an alternative explanation, copper complexes could interact with the microsomal electron transport system and inhibit mixed-function oxidation reactions through mechanisms independent of their SOD-like chemical reactivity. This hypothesis is substantiated by the observation that CuDIPS and CuSO4, but not bovine liver SOD, inhibit BP-dependent mutagenesis and cytochrome P450 reductase activity. The in vitro antimutagenic activity of CuDIPS as scored in the Ames assay, therefore, seems independent of its salicylate structure and is mediated through a copperdependent but non-SOD-associated inhibition of P450 reductase activity. 2~ Thiols, sulfides and synthetic sulfur-rich chemicals have been shown to inhibit successfully the effects of several carcinogens, presumably by blocking their metabolic activation and/or by inducing enzyme systems known to aid in their detoxification. The inhibitory effects of L-cysteine (Cys) on the mutagenic activities of several carcinogens have been described. 282 In vitro treatments with GSH alone and GSH + cytosol or purified GSH S-transferases are active in inhibiting DNA binding of reactive metabolites of BP z83 as well as the cytotoxicity and mutagenicity of these carcinogens in mammalian cells. 284 The dithiolthiones may inhibit tumor development by increasing cellular thiol levels and inducing enzymes concerned with the maintenance of GSH pools as well as enzymes important to electrophile detoxification. 279 Dially sulfide, the thioether that occurs naturally in garlic, can modify

Antioxidants and skin carcinogenesis hepatic mixed-function oxidase and stimulate hepatic sulfate conjugation reactions leading to eventual detoxification and elimination of the electrophile mutagenic forms of the carcinogens. 285 In addition, the endogenous thioredoxin/thioredoxin reductase system, a thioenzyme which reduces radicals at the surface of the skin, may play an important role in preventing free radical-induced cell damage during skin carcinogenesis.286 Moreover, various intracellular thiols may protect DNA-modifying enzymes such as DNA methylase from carcinogen damage. 287 The antioxidant effects of disulfiram and its reduction product, diethyldithiocarbamate (DDTC), have been described. 234.288Both compounds inhibit chemical carcinogenesis in the gastrointestinal tract. Because of the chelating properties of its sulfhydryl moieties, DDTC is a rather nonspecific inhibitor of several metal-containing enzymes, including SOD. 28~ Although the inhibition of P450-dependent microsomal enzyme activities may be involved, the GSH peroxidase-like and free radical-quenching activities of DDTC are likely to explain some of the remarkable inhibitory effects elicited by this compound during complete and multistage skin carcinogenesis. ~82j83 It has been suggested that CS2, a metabolite of disulfiram, may be the chemical species responsible for the carcinogen-inhibiting effects brought about by disulfiram and DDTC. TM A recent study indicates that DDTC protects mouse tissues from hyperoxic injury. 29° The data suggest that the mechanism of this antioxidative property is indirect and involves the delayed increase in the GSH enzyme levels following DDTC injection. 29~ It has been postulated that DDTC is effective in protecting against lipid peroxidation of microsomal membranes in the nM range because of its hydrophobicity, whereas the effects of other hydrophilic thiol-containing molecules such as GSH and cysteamine are observed in the mM range only. 288 Mixed results have been published concerning the potential anticarcinogenicity and/or mutagenicity of G S H . 292-294 A model of an indirect mechanism of GSH mutagenesis involving ROS is described in which cleavage of GSH by 2t-glutamyltranspeptidase, followed by auto-oxidation of the resulting cysteinylglycine may produce free radicals, which lead to the penultimate mutagen H202 .295 The role of Se in tumorigenesis has been extensively reviewed 296 and its possible action on both the initiating and promoting components of chemical carcinogenesis has been disc u s s e d . 297 The anticarcinogenic properties of Se may be attributed, at least partially, to its ability to maintain the integrity of the Se-dependent enzyme GSH peroxidase, which detoxifies peroxides. The synergistic effects of o~TH and Se in the chemoprevention of car-

395

cinogenesis have been described. 298,299Our recent finding that Na2SeO3 + a T H and GSH + aTH, the combined treatments that inhibit complete and stage 2 tumor promotion in the multistage protocols, fail to inhibit the carcinogenicity of a single large dose of DMBA and even enhance the induction of skin tumors by repeated applications of subcarcinogenic doses of DMBA is intriguing. ~°° The discrepancies between some of the effects of aTH, Se and GSH on skin carcinogenesis presented in Table 4 have been discussed previously. 100 The antineoplastic activity of ellagic acid has been reviewed. 3°° The inhibition of skin tumor initiation and carcinogenesis by ellagic acid 248,27°,271may result from decreased metabolic activation and increased conjugation reactions, as indicated by the findings that ellagic acid inhibits epidermal AHH activity, induces epidermal GSH S-transferase activities, and decreases the formation and covalent binding of ultimate carcinogens to epidermal DNA. 271'3°1'3°2 Ellagic acid binds to DNA and it has been suggested that this binding may be a mechanism by which ellagic acid inhibits mutagenesis and carcinogenesis. 3°3 Apparently, ellagic acid can also scavenge the ultimate carcinogenic form of BP. 3°4 Surprisingly, the antitumor-promoting activity of this interesting antioxidant has not been studied. Vitamin A is required for the maintenance and function of differentiation of epithelial cells. Vitamin A and its analogs (retinoids) are capable of inhibiting the development of Ca induced by chemical carcinogens in various organs. The role of retinoids in the modification of multistage skin carcinogenesis is well documented, 8"9~'23z'25~'262'263"3°5 The RA-binding protein may be involved in the expression of biological and antitumorigenic activities of the retinoids. Since the retinoids have been demonstrated to stimulate cellmediated immunity, there is some suggestion that retinoids may also retard carcinogenesis by serving to enhance the organism's immune response. 3°6 The discrepancy between some of the effects of the retinoids on complete skin carcinogenesis may be related to their variable chemical reactivity toward free radicals. Incubation of 13-cis-RA with peroxidase and hydroperoxides or with LOz--generating systems leads to rapid 02 uptake, retinoid oxidation and formation of oxygenated products. ~J4The conflicting reports concerning the effects of retinoids on complete and multistage skin carcinogenesis, therefore, may be explained by the fact that the retinoids are not only efficient at trapping reactive oxidants, thereby lowering the steady-state oxidant concentration, but that, under certain conditions, they can actually enhance LO2" and LO. formation. 114

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J.-P. PERCHELLETand E. M PERCHELLET

The fact that our understanding of the mechanism of tumor promotion is itself still evolving increases the difficulty of trying to explain how antioxidants may be affecting this process. Reviewing the effects of the numerous modifiers of tumor promotion is a monumental task but a survey of the literature reveals clearly the relevance of ROS to the mechanism of tumor promotion. Basically, these findings can be classified in three categories.

various cell systems. 169,310,314,316.317For example, retinal has been shown to decrease PKC induction and H202 formation in TPA-treated neutrophils. 2~3 Several adrenal steroids shown to inhibit skin tumor promotion by TPA also inhibit the ability of TPA to stimulate 02- production in leukocytes. 3~8.3~9 Furthermore, the Ca 2+ channel and calmodulin antagonists shown to inhibit PKC induction by TPA are also capable of inhibiting the CL response in epidermal cells 3t° and the production of 02- and H202 in leukocytes stimulated by TPA. 21~'-~16'217

Antioxidative effects of anti-tumor promoters. Various agents which decrease the biological and biochemical events linked to skin tumor promotion also inhibit the ability of TPA to induce the generation of ROS. For instance, a protease inhibitor such as tosyl phenylalanine chloromethyl ketone is effective against complete and stage 1 skin tumor promotion 59'3°7 and a number of protease inhibitors from various sources, specially those active against chymotrypsin, block the production of 02 ~ and H202 and the CL response by TPA-activated PMN leukocytes. 3°~ 311 However, the SOD-inhibitory activity of the protease inhibitors may be independent from their anti-proteolygic activity. 3~2 Not surprisingly, CuDIPS inhibits the TPA-induced CL response in mouse epidermal cells 313 and the ability of TPA-stimulated PMN leukocytes to generate oxidants ~69 and enhance the metabolic activation of PAH carcinogens. ~52 The O j response and toxicity of TPA-stimulated leukocytes or the CL observed in TPAtreated epidermal cells are inhibited to various degrees by SOD, 314 quercetin, J69 7,8-benzoflavone and ASC. 31° Although they do not react directly with 02- the phenolic antioxidant BHA 3~° and several of its analogs, 3~5 which inhibit TPA-stimulated O2- formation and CL in PMN leukocytes or epidermal cells, also inhibit ASC-initiated lipid peroxidation in relation with their ability to inhibit TPA-induced ODC activity in mouse epidermis. The results imply that BHA and its lipophilic analogs may scavenge free-radical derivatives of 02: more directly involved in the promotional stage of carcinogenesis. 3~5 In addition, the specific lipoxygenase inhibitors and agents with dual cyclooxygenaselipoxygenase inhibitory activities shown to inhibit skin tumor promotion in different strains of mice also block the TPA-induced 02 ~ release from leukocytes ~6~ and CL response in mouse epidermal cells, 313 whereas the specific cyclooxygenase inhibitors are ineffective. These data suggest that at least a major part of the TPA-induced CL response is due to the increased metabolism of arachidonic acid, most probably by the lipoxygenases. 3~3 Moreover, another class of anti-promoting agents, the retinoids, also inhibits the oxidative challenge caused by TPA, MEZ, or teleocidin B in

Antitumor-promoting effects of antioxidants. The direct quenching reactivity of cellular antioxidants against specific free radicals has been reviewed. 32° The various antioxidant treatments, which decrease the oxidative challenge caused by TPA, also inhibit the other biochemical events linked to the skin tumor-promoting activity of this compound. While cupric sulfate and diisopropylsalycylic acid are ineffective, t6~ SOD, CuDIPS, or other copper complexes with potent SOD mimetic activities inhibit the ability of TPA to induce ODC activityfl ~'t~7~6~ DNA synthesis and mitosisfl 2~ sister chromatid exchanges, chromosomal aberration and polyploidization 72,79 and the promotion of neoplastic transformation in vitro 322 324 and to decrease the 13-adrenergic responsiveness of lymphocytes. 325In contrast, CuD1PS does not inhibit DMBA-induced ODC activity, suggesting that the mechanism by which DMBA induces this enzymic marker of stage 2 tumor promotion is different from that of TPA. 99 Most of the effects of TPA described above are also inhibited by CAT and various antioxidants and free radical scavengers, including BHA, BHT, mannitol, a T H , disulfiram, selenous acid and the synthetic lipophilic derivative of ASC, ascorbyl palmitate, j67,24:~'321-329 in addition, a T H inhibits TPA-stimulated deacylation of cellular lipids, PG production and altered cell morphology in vitro. 33° Interestingly, the inhibitor of tumor promotion quercetin inhibits epidermal lipoxygenase activity and the induction of ODC activity but not the stimulatory effect of TPA on DNA synthesis. 24~ The complex effects of the inhibitors of arachidonic acid metabolism on PG synthesis, ODC induction, DNA synthesis, and skin tumor promotion in different strains of mice treated with TPA have been excellently reviewed. 4~'253 Both the cyclooxygenase/endoperoxidase pathway of PG synthesis and the lipoxygenase pathway of hydroperoxy (HPETE)- and hydroxy-eicosatetraenoic (HETE) acid formation play a mediatory role in TPA-induced inflammation, ODC activity, DNA synthesis, cell differentiation, in vitro promotion of neoplastic transformation and skin tumor formation. 254"255"323"329"331"332 Specific lipoxygenase inhibitors

Antioxidants in tumor promotion

Antioxidants and skin carcinogenesis

and agents with dual cyclooxygenase-lipoxygenase inhibitory activities consistently antagonize the tumorpromoting effects of TPA. In contrast, the antitumorpromoting activities of specific cyclooxygenase inhibitors are variable. 254.255 In certain strains of mice and under certain conditions, the cyclooxygenase inhibitors indomethacin and flurbiprofen may shunt arachidonic acid metabolism into the alternate lipoxygenase pathway and enhance the tumor-promoting activity of TPA, suggesting that the HPETEs and other products of the lipoxygenase pathway play an important role in the mechanism of ODC induction by TPA and may be more intimately involved in promotion than the PGs. 48.332 The generation of ROS and the induction of the lipoxygenase pathway producing reactive oxidized metabolites of arachidonic acid, therefore, appears to be essential to the molecular mechanism of skin tumor promotion by TPA. Specific lipoxygenase inhibitors lacking PKC inhibitory effects strongly suppress TPAinduced ODC activity and skin tumor formation. 256 The effectiveness of RA and 2 1 retinoids as inhibitors of TPA -3°5328or anthralin-induced ODC activity 259 in mouse epidermis in vivo has been demonstrated. In vitro, RA or its analogs also inhibit the enhancing effects of TPA on DNA synthesis, mitosis, and the transformation of 3-methylcholanthrene(MC)-treated c e l l s . 321'326 Cutaneous RA pretreatments inhibit by almost 100% TPA-increased epidermal GSSG-R activity, an event postulated to decontrol the availability of deoxyribonucleotides for DNA biosynthesis. 333 However, the potent inhibitor of ODC induction and skin tumor promotion by TPA, RA, fails to inhibit and, under some experimental conditions, significantly enhances ODC induction and tumor formation by DMBA. 87-Bromomethylbenz[a] anthracene (BrMBA), a structural analogue of DMBA, is a weak mouse skin tumor-initiating agent and a weak complete carcinogen but a good tumor promoter when applied repeatedly to DMBA-initiated mouse skin.l°3 The doses of RA and dexamethasone which inhibit skin tumor promotion by TPA, also inhibit epidermal ODC induction and skin tumor promotion by BrMBA. 232 In contrast, 7,8-benzoflavone, which does not inhibit TPA-induced ODC activity, inhibits the ODC and tumor responses to DMBA but neither ODC induction nor tumor promotion by BrMBA. 232The discrepancy between the effects of these various compounds on the induction of skin tumors by different protocols might result from different levels of genetic lesions and hyperplastic, cytostatic or cytotoxic activities in the TPA-and DMBAtreated epidermis. In some cases, the inhibitory effects that some agents exert on the propagating activities of TPA or MEZ could be masked by equal or greater stimulatory effects on the other stages theoretically

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involved in complete carcinogenesis by DMBA. For instance, RA inhibits the overall TPA-induced responses but has the ability to act as either a weak 1st stage or complete tumor promoter in the initiationpromotion system for skin carcinogenesis in SENCAR m i c e . 226

Our results indicate that the inhibitory effects of Cys, GSH, and o~TH on ODC induction are proportional to their abilities to prevent the decrease in the GSH :GSSG ratio caused by TPA and inhibit the incidence of skin tumors in the initiation-promotion prot o c o l . 242 In mouse epidermis in vivo and in vitro, the inhibitory effects of combined treatments with Cys or GSH, Na2SeO3 or selenocystamine and ~TH on TPAdecreased GSH peroxidase activity are additive, in relation with their additive inhibitory effects on TPAinduced ODC activity, t°°,~29 A number of thiol-containing molecules that are readily transported and/or enzymatically converted to GSH intracellularly are more effective than GSH treatment in increasing the intracellular thiol level of various tissues. In mouse epidermis in vivo and in vitro, the monoethyl and monomethyl esters of GSH, N-acetyl-L-cysteine and L-2oxothiazolidine-4-carboxylate are all significantly more effective than GSH in inhibiting the sharp decline in the intracellular G S H : G S S G ratio, the prolonged decrease in GSH peroxidase activity and the induction of ODC activity caused by TPA. ~2"~s3 Moreover, DDTC, which prevents totally the initial drop in the G S H : G S S G ratio of TPA-treated epidermal cells and is more potent that 16 other antioxidants in inhibiting TPA-decreased GSH peroxidase activity, is also the most potent thiol delivery agent to inhibit TPA-induced epidermal ODC activity in vitro and in v i v o . 182'183 In addition, garlic oil, onion oil and dipropenyl sulfide, a constituent of onion oil, all inhibit to diverse degrees the effects of TPA on the GSH-dependent detoxifying system in relation with their abilities to inhibit TPAinduced ODC activity in mouse epidermis in vitro and in vivo. ls~'z52 The GSH peroxidase-like activity of garlic oil has been shown. It should be noted that these inhibitors of skin tumor promotion also inhibit soybean and mouse epidermal lipoxygenase activities. 251'252'334 Although DMSO may scavenge free radicals, it is difficult to find a common explanation for the range of effects of DMSO on experimental tumorigenesis. 261 When DMSO is used as the solvent instead of acetone, it enhances complete skin carcinogenesis by BP, does not alter initiation by DMBA, and inhibits skin tumor promotion by TPA. Interestingly, DMSO applied topically to the skin before each TPA treatment inhibits skin tumor promotion when it is administered to the initiated back of the mouse but increases skin tumor promotion when it is applied on the non-initiated ab-

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domen. 26t Without modifying the initiating event, DMSO could enhance complete skin carcinogenesis by increasing the penetration, metabolism and DNA-damaging effects of carcinogenic doses of PAHs. Perhaps, DMSO alters specifically the promoting components of carcinogenesis. That the effects of DMSO on skin tumor promotion depend on the site of its administration in relation to TPA treatment suggests that the intact DMSO molecules affecting the initiated epidermal cells following direct penetration through the skin and the DMSO metabolites affecting the same cells indirectly through the circulation exert opposite effects on the mechanism of TPA promotion. 26~

Tumor-promoting effects of pro-oxidants. The treatments that worsen the oxidative challenge caused by TPA enhance the events linked to skin tumor promotion by TPA. For instance, the promotional activity of methyl ethyl ketone peroxide, a potent lipid-peroxidizing agent, is potentiated by DEM which is known to deplete intracellular GSH, suggesting that lipid peroxidation may be important in skin tumor promotion. 335 Chronic treatment of skin Pa with the GSH-depleting agent DEM also enhances markedly their malignant conversion to C a . 336 Similarly, the treatments with NaN3 plus NADPH, G / G O or X/XO that enhance the hydroperoxide response to TPA in our recent experiments 17°.j7~ also increase, although to a lesser degree, TPA-induced epidermal ODC activity in mouse skin explants. Moreover, the H2Oz-generating enzyme GO mimics the inhibitory effects of several liver tumor promoters on gap junctional intercellular communication, an event which is inhibited by several antioxidants including SOD and otTH. 337 In mouse epidermis in vivo, topical adriamycin (ADR, doxorubicin) treatments enhance ODC induction and skin tumor promotion by TPA. In epidermal cells incubated in the presence of ADR, the tumor promoter causes a greater sequential rapid increase and prolonged decrease in GSH peroxidase activity accompanied by a greater decrease in the GSH :GSSG ratio. 179 Since free radical generation and lipid peroxidation may be associated with ADR toxicity, these data suggest that the enhancement of the ODC-inducing and tumor-promoting activities of TPA by ADR may be the result of an increased oxidative challenge altering to a greater degree than TPA alone the GSH-dependent antioxidant protective system of the epidermal cells.

CONCLUSION

The review of the inhibitory effects of the antioxidants in the mouse skin tumor system confirms the important role of 02 metabolites in the multistage pro-

cess of carcinogenesis. Although the anti-carcinogenic and/or antitumor-promoting activities of some of these compounds may be caused to a certain degree by mechanisms independent from their antioxidant properties, it is likely that their effectiveness as inhibitors of tumor formation is due largely to their ability to scavenge, reduce and detoxify, directly or indirectly and through spontaneous or enzyme-catalyzed reactions, the various unstable electrophile intermediates affecting the epidermal cells during the sequential phases of skin carcinogenesis. The results of these animal tumor experiments suggest that, if increased levels of peroxidation coupled with decreased antioxidant protection play a major role during the long and multistage process of neoplasia, the antineoplastic activity of the natural SOD, CAT and GSH-dependent detoxifying systems might be preserved and ameliorated through chemotherapeutic intervention and the use of antioxidant systems that complement each other. For instance, the mechanisms of antioxidant protection by the GSHdependent detoxifying system and ~xTH are independent, complementary, and protect each other. Treatments or diets that would enhance on a life-long basis the natural antioxidant protection of the organism, therefore, might be valuable in cancer chemoprevention by decreasing the risk, increasing the threshold and delaying the advent of tumor initiation. Our research suggests that free radical scavengers, GSH level-raising agents, Se-containing compounds and thiol delivery systems that prevent the decline in the GSH : GSSG ratio and maintain GSH peroxidase activity stimulated for extended periods of time may facilitate a more complete destruction of peroxides during TPA treatment, in relation with their effectiveness as inhibitors of ODC induction, hyperplasia and skin tumor promotion and progression. The ultimate goal is to determine whether the protective effects of these antioxidant treatments are sufficient to block the growth or induce the regression of skin tumors that have already developed. This information is of importance to assess the potential use of antioxidants in the management of certain human neoplasms resistant to conventional therapy. Acknowledgements--The authors gratefully acknowledge the financial support of the NIH (Grant CA-40083 awarded by the National Cancer Institute, Department of Health and Human Services), the American Cancer Society (Grant BC-600), the Wesley Foundation of Wichita (Wesley Scholar Program: Molecular Biology and Cell Growth Regulation), and the Center for Basic Cancer Research, Kansas State University REFERENCES 1. Moolgavkar, S. H.; Knudson, A. G. Jr. Mutation and cancer: a model for human carcinogenesis. J. Natl. Cancer Inst. 66:1037-1052; 1981.

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ABBREVIATIONS ROS--Reactive Pa--Papilloma Ca--Carcinoma

oxygen species

PAH--Polycyclic aromatic hydrocarbon DMBA--7,12-Dimethylbenz[a]anthracene TPA-- 12-0-Tetradecanoylphorbol- ! 3-acetate MEZ--Mezerein PKC--Protein kinase C BPx--Benzoyi peroxide PG--Prostaglandin ODC--Ornithine decarboxylase EPP--Ethyl phenylpropiolate

408

J.-P. PERCHELLETand E. M. PERCHELLET

MNNG--Methyl-3-nitro- 1-nitrosoguanidine ENU--Ethylnitrosourea GSH--Reduced glutathione GSSG--Oxidized glutathione GSSG-R--Glutathione reductase RA--Retinoic acid (vitamin A) BrMBA--7-Bromomethylbenz[a]anthracene SOD--Superoxide dismutase CAT--Catalase oLTH--c~-Tocopherol (vitamin E) CuDIPS--Cu(II)-(3,5-diisopropylsalicylate)2 AHH--Aryl hydrocarbon hydroxylase PMN--Polymorphonuclear BP--Benzo[a]pyrene MC--Methylcholanthrene

DMSO--Dimethyl sulfoxide DEM--Diethyl maleate CL--Chemiluminescence CDNB--1-Chloro-2,4-dinitrobenzene G/GO/Glucose/glucose oxidase X / XO--Xanthine / xanthine oxidase ASC--Ascorbic acid (vitamin C) XD--Xanthine dehydrogenase BHA--Butylated hydroxyanisole BHT--Butylated hydroxytoluene Cys--L-cysteine DDTC--Diethyldithiocarbamate HPETE/HETE--Hydroperoxy- / hydroxy -eicosatetraenoic acid ADR--Adriamycin (doxorubicin)