Carcinogenesis vol.8 no. 10 pp.

1365-1373, 1987

COMMENTARY

Peroxyl free radicals: potential mediators of tumor initiation and promotion

Lawrence J.Marnett
Department of Chemistry, Wayne State University, Detroit, MI 48202, USA

Table I. Oxygen-centered radicals in biological systems Oxygen reduction Lipid peroxidation

In recent years, there has been an explosion of interest in the involvement of free radicals in carcinogenesis (1 - 4 ) . This seems a natural outgrowth of the long overdue realization that free radicals are formed in biological systems and that they contribute significantly to physiological and pathological processes. Much of the emphasis of research linking free radicals and cancer has focused on intermediates of oxygen reduction such as superoxide anion and hydroxyl radical (2). There is another family of oxygencentered free radicals that have received less attention but may be just as important biologically as reduced oxygen intermediates. This Commentary is intended to familiarize the reader with some of the properties of peroxyl radicals and to explain how these species participate in reactions that are relevant to tumor initiation and promotion. Experimental data are presented that are intended to be provocative rather than definitive because this area of investigation is just beginning to evolve. Properties of oxygen-centered free radicals Table I shows the major types of oxygen-centered free radicals in biological systems, the principal pathways of their generation, and their approximate half-lives (5). One electron reduction of O2 (e.g. by redox cycling or by polymorphonuclear leukocytes) produces superoxide anion (O2~), a radical anion that exists in equilibrium with a protonated form, perhydroxyl radical (6). The pKa of the perhydroxyl radical is 4.88 (7), which means that at neutral pH the ratio of (superoxide)/(perhydroxyl radical) is -130:1. It is difficult to estimate the half-life of O2~ because it decomposes by disproportionation (dismutation), the rate of which is a function of the O2~ concentration. Dismutation of two molecules of O2~ generates H2O2, which can be reduced by metals to hydroxyl radical (HO-) (8,9). Hydroxyl radical, which is also a product of the action of ionizing radiation on water, is the most reactive of the oxygen-centered radicals and exhibits a half-life of 10~9 s (10). It only survives a few molecular encounters and cannot penetrate cells or diffuse far from the site of its generation. Because of its importance as a mediator of radiation damage to cells a good deal is known about the reactions of hydroxyl radical with DNA (10). Evidence is accumulating for the production of hydroxyl radical adducts to DNA during the oxygen burst of phagocytic cells in response to particulate or soluble stimuli (11,12). The analog of the perhydroxyl radical in which the H atom is replaced by an organic group is called a peroxyl radical (ROO ) (13). Peroxyl radicals do not dissociate to O2~ because of the
Abbreviations: PGH, prostaglandin H; PGG2, hydroperoxy-endoperoxide; PGH 2 , hydroxy-endoperoxide; BP, benzo[a]pyrene; BP-7,8-diol, 7,8-dihydroxy-7,8-dihydrobenz[a]pyrene; BA-3,4-diol, 3,4-dihydroxy3,4-dihydrobenz[a]anthracene; AHH, aryl hydrocarbon hydroxylase; DMBA, 7,12-dimethylbenz[a]anthracene. IRL Press Limited, Oxford, England

pATa = 4.88

O-OH-O-

-O-O7s
l<T 9 s

R-O 10"6s

Structures of the predominant radical species produced during O2 reduction and lipid peroxidation. These are the two major pathways of oxygen radical formation in biochemical systems. The lifetimes are taken from ref. (5).

stability of the carbon-oxygen bond. The principal pathway of peroxyl radical formation in biological systems is autoxidation (e.g. lipid peroxidation) (14). Compared with other oxygencentered free radicals, peroxyl radicals are stable species capable of diffusing to remote cellular locations. Pryor has estimated that the half-lives of peroxyl radicals are of the order of seconds (5). Changing the organic group attached to the oxygen does not significantly alter the reactivity of peroxyl radicals but it can have a dramatic effect on the physical properties of the radicals. Compared with peroxyl radicals the organic analogs of the hydroxyl radical, called alkoxyl radicals (RO-), are quite reactive (15). They exhibit half-lives of the order of 10~6 s. The principal route of their formation is metal-catalyzed one-electron reduction of organic hydroperoxides. Lipid hydroperoxides, the initial products of lipid peroxidation, are the major organic hydroperoxides in animal tissue (16).
OH(R| HOH(R)

Equation 1
/

C=C

-OH(R)

c-c
/'OH\ (R)

(R)

Equation 2

The two general types of reaction that oxygen-centered free radicals participate in are H abstraction and addition to double bonds (eqns 1 and 2). Both reactions produce carbon-centered radicals as intermediates. The predominant fate of carboncentered radicals is coupling with O2 to produce peroxyl radicals. The fact that O2 is itself a triplet diradical greatly facilitates coupling, which occurs at a diffusion-controlled rate (17). The implication of the sequence of reactions in eqns 1 and 2 is that formation of oxygen-centered free radicals in a cell, either by O2 reduction or autoxidation, eventually results in the generation of peroxyl radicals. It is likely that peroxyl radicals are the major oxygen-centered free radicals generated as a result of oxygen activation and their stability indicates that they can dif1365

L.J.Marnett

(CH2)4CH3 HO2C(CH2)7 HO2C(CH2)7

(CH 2 ) 4 CH 3

(CH2)4CH3 HO2C(CH2)

(CH 2 ) 4 CH 3 HO2C(CH2)7 HO2C(CH2)7

Fig. 1. Mechanism of unsaturated fatty acid metabolism by hematin. Hematin reduces unsaturated fatty acid hydroperoxides by one electron to form alkoxyl radicals that cyclize to the adjacent double bonds generating an epoxy allylic radical. This radical either couples to the hydroxyl group bound to the heme or couples to O 2 to form a peroxyl radical. The peroxyl radical is the oxidant that epoxidizes BP-7,8-diol. This mechanism is consistent with experimental evidence presented in detail in ref. (34).

fuse to cellular loci remote from the site of their generation. Thus, they should be considered prime mediators of radical-induced pathology. One possibility relevant to carcinogenesis is that peroxyl radicals react with DNA. Although a good deal is known about the reaction of hydroxyl radical with DNA, the kinetics and products of reaction of peroxyl radicals with DNA have not been explored so it is difficult to speculate on the likelihood of this possibility. Because of their stability, peroxyl radicals will be much more selective than hydroxyl radical in their reactions with DNA and other macrbmolecules. An alternative possibility is that peroxyl radicals react with other cellular components converting them to derivatives that react with DNA. Evidence for such an indirect role for peroxyl radicals in carcinogenesis is considered below. Peroxyl radicals as mediators of hydroperoxide-dependent oxidations Our interest in the involvement of peroxyl radicals in carcinogenesis was kindled by our investigations of the mechanisms of cooxidation of polycyclic hydrocarbons during prostaglandin biosynthesis (18). The enzyme prostaglandin H (PGH*) synthase is a membrane-bound enzyme widely distributed in mammalian tissue that oxygenates polyunsaturated fatty acids such as arachidonic acid to bicyclic peroxides, which are subsequently converted to prostaglandins, thromboxanes and prostacyclin (19). In addition to the bis-dioxygenase activity, PGH synthase also exhibits a peroxidase activity that catalyzes reduction of the hydroperoxy endoperoxide (PGG2) to the hydroxy endoperoxide (PGHj) by a reducing agent (DH2) (eqn 3) (20). The peroxidase triggers cooxidation of many molecules and several laboratories have explored the possibility that chemical carcinogens that require metabolic activation are cooxidized to reactive derivatives by this hydroperoxide-dependent reaction (21). For example, polycyclic hydrocarbon dihydrodiols, aflatoxin B, and aromatic amines are cooxidized to mutagenic derivatives during prostaglandin biosynthesis (2225). PGH synthase is structurally and functionally unrelated to xenobiotic-metabolizing cytochromes 1366

P-450 so arachidonic-acid-dependent cooxidation constitutes a distinct and complementary pathway to NADPH-dependent oxidation for carcinogen activation (26).

Arachidonic Acid

PGG2

PGH,

Equation 3

Benzo[a]pyrene (BP) is cooxidized during prostaglandin biosynthesis to a mixture of quinones that are believed to derive from 6-hydroxy-BP (27) (eqn 4). 6-Hydroxy-BP is unique among BP phenols in that it is not formed via the intermediacy of aromatic epoxide intermediates (arene oxides) (28). In contrast, 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-7,8-diol) is cooxidized by PGH synthase to the a/iri-dihydrodiolepoxide (eqn 5) (29,30). The importance of this epoxide as the ultimate carcinogenic form of BP and the fact that BP-7,8-diol is epoxidized, whereas BP is not, prompted us to undertake a detailed study of the mechanism of BP-7,8-diol cooxidation. Our studies were facilitated by the discovery that hematin, the prosthetic group of PGH synthase, catalyzes BP-7,8-diol epoxidation by unsaturated fatty acid hydroperoxides (31). Hematin-dependent epoxidation is greatly accelerated by the presence of detergents above their critical micellar concentrations (31). The characteristics of the hematin-dependent reaction are identical to those of the PGH synthase-dependent reaction so it provides a good model system for mechanistic studies (32).

a59-a$9IP-3,6-Ouinono BP-6.12-Ouijione BP-1.6-Ounon8

Equation 4

Peroxyl free radicals

HO "'

P-450 ROO

HO1'"

Equation 5

The epoxide oxygen introduced into BP-7,8-diol by the hematin system derives from O2 rather than hydroperoxide and epoxidation is potently inhibited by antioxidants (31). These observations suggest that epoxidation occurs by a free radical mechanism. Studies with a series of hydroperoxide analogs indicate that fatty acid hydroperoxides with double bonds in proximity to the hydroperoxide group support epoxidation but saturated fatty acid hydroperoxides do not (33). Identification of the products of unsaturated fatty acid hydroperoxide metabolism by hematin and the results of oxygen-labeling experiments provide evidence for a mechanism of hydroperoxide metabolism outlined in Figure 1 (34). Hematin reduces the hydroperoxide by one electron to an alkoxyl radical that cyclizes to the adjacent double bond generating a carbon-centered radical. As discussed above, the carbon-centered radical couples with O2 to form a peroxyl radical, which is the species that epoxides BP-7,8-diol. This mechanism is consistent with all of the experimental observations of the reaction of unsaturated fatty acid hydroperoxides with hematin as well as the epoxidation of BP-7,8-diol. Comparison of the yield of peroxyl radicals with the yield of dihydrodiolepoxide indicates that BP-7,8-diol traps roughly 75% of all the peroxyl radicals generated in the reaction of hematin with unsaturated fatty acid hydroperoxides (33). This is consistent with our findings that the structurally related molecule 7,8-dihydrobenzo[a]pyrene has an extremely high rate coefficient for epoxidation by peroxyl radicals (35).

(+) - anti

o..

P-450

(+)-syn

- BP-7,8-diol

(-) - and Fig. 2. Stereochemistry of epoxidation of BP-7,8-diol by cytochromes P-450 and by peroxyl radicals.

Stereochemistry of peroxyl radical-dependent epoxidation of dihydrodiols The stereoselectivity of epoxidation of BP-7,8-diol by peroxyl radicals is distinct from that exhibited by cytochromes P-450 (36). Oxygen introduction by peroxyl radicals occurs on the same side of the molecule as the 8-hydroxyl group, whereas oxygen introduction by cytochromes P-450 is determined primarily by the orientation of the pyrene moiety with respect to the active site of the enzyme (36,37). Confirmation of the importance of the 8-hydroxyl group to the stereochemistry of epoxidation by peroxyl radicals is provided by the observation that 7,8-dihydrobenzo[a]pyrene is epoxidized to both enantiomers of 9,10-epoxy7,8,9,10-tetrahydrobenzo[a]pyrene to equal extents (38). Figure 2 outlines the stereochemistry of epoxidation of both enantiomers of BP-7,8-diol by peroxyl radicals and cytochromes P-450. ()-BP-7,8-diolthe enantiomer generated in vivo from BP is epoxidized by both pathways to the (-l-)-enantiomer of the antidihydrodiolepoxide. (+)-BP-7,8-diol is epoxidized by peroxyl radicals mainly to the (-)-enantiomer of the anri-diolepoxide but by cytochromes P-450 to the (+)-enantiomer of the iyn-diolepoxide. The distinctive stereochemistry of epoxidation of (+)BP-7,8-diol by peroxyl radicals and cytochromes P-450 provides a useful method for differentiating the two pathways of epoxidation in vitro and potentially in vivo. The reactivity of different peroxyl radicals is similar because the organic group is two atoms removed from the unpaired electron and does not sterically hinder its reaction with other molecules. One expects, therefore, that any system that generates peroxyl radicals would be capable of epoxidizing BP-7,8-diol.

BP-7,8-diol is epoxidized by the following biochemical systems that generate peroxyl radicalsascorbic acid- and NADPHdependent lipid peroxidation (39), cumene hydroperoxide plus rat liver microsomes (40), phenylbutazone peroxidation by peroxidases (41) and sulfite peroxidation or autoxidation (42). In each case, the major epoxidation product is the anri-dihydrodiolepoxide. Dihydrodiol metabolites of benz[a]anthracene and chrysene are also oxidized to mutagenic products by peroxyl radicals and in the case of 3,4-dihydroxy-3,4-dihydrobenz[a]anthracene (BA-3,4-diol) the principal oxidation product is the a/iri-dihydrodiolepoxide (23,24). The extent of epoxidation of BP-7,8-diol is approximately three times greater than that of BA-3,4-diol (43). The high reactivity of BP-7,8-diol to peroxyl radicals and the unique stereochemistry of epoxidation suggests that BP-7,8-diol and other dihydrodiol metabolites of polycyclic hydrocarbons are excellent diagnostic probes for the detection of peroxyl radicals in chemical or biological systems. Peroxyl radicals and tumor initiation It is important to determine if peroxyl-radical-dependent epoxidation plays a role in metabolic activation of BP or its dihydrodiol metabolites. This requires a characterization of the pathways of metabolism and DNA binding in target tissues for polycyclic hydrocarbon carcinogenesis. A collaborative effort between Thomas Eling's laboratory at the NIEHS and my laboratory at Wayne State University approached this problem using freshly isolated epidermal cells from newborn mouse skin, a target tissue for BP carcinogenesis (44). Freshly isolated cells were employed in order to assess the pathways of metabolism present in the intact tissue and to minimize artifacts such as loss of cytochrome P-450 1367

L.JwMarnett

UNTREATED
ROO- (moderate) inducer P-450 (low)

INDUCED
ROO (moderate)

P-450c (high)

Fig. 4. Pathways of metabolic activation in skin of uninduced and Ahinduced mice.

20

40

60

Retention

Time

(min)

Fig. 3. H.p.l.c. profiles of oxidation products formed by incubation of BP-7,8-diol with freshly isolated epidermal cells from control or /3naphthoflavone-pretreated animals: control (left) and induced (right). Details are provided in ref. (44). TA and CA represent the trans and cis tetraol hydrolysis products of the a/iri-dihydrodiolepoxide. TS and CS represent the trans and cis tetraol hydrolysis products of the yyn-dihydrodiolepoxide.

frequently observed as a result of establishment of primary cultures (45). In addition, epidermal cells from newborn hairless mice contain a very active prostaglandin biosynthetic capacity, which enhanced the probability of detecting arachidonic aciddependent cooxidation (46). Cells were isolated by a trypsin flotation method and immediately incubated in a Hepes-PBS solution containing 5 mM glucose. The cell preparation was a mixture of ~20% basal cells and 80% keratinocytes. Cell viability was initially >82%; no decrease in viability, judged by vital dye exclusion and lactate dehydrogenase release, was observed during the time course of incubation (up to 90 min). Figure 3 displays the profiles of radioactive metabolites eluting from reverse-phase h.p.l.c. columns following incubation of (+)-BP-7,8-diol with 7 X 107 cells prepared from animals pretreated with acetone or j3-naphthoflavone (44). Cells from acetone-treated animals exhibited the same pattern of metabolism as those from untreated animals and were used as the control preparation. Cells from /3-naphthoflavone-treated animals represented a preparation with high levels of the isoenzyme of cytochrome P-450 that exhibits the highest specific activity of polycyclic hydrocarbon oxidation of any of the cytochromes P-450 (47,48). A striking contrast exists between the profiles of oxidation products of (+)-BP-7,8-diol by the control and induced cell preparations. The major oxidation product detected in control cells was the ann-dihydrodiolepoxide whereas the sy/i-dihydrodiolepoxide was the major product in j3-naphthoflavone-induced cells. Oxidation by control cells increased linearly up to 90 min whereas oxidation by j3-naphthoflavone-induced cells plateaued after 30 min. The phenolic antioxidant BHA was a potent inhibitor of (+)-BP-7,8-diol epoxidation by control cells (/50 = 1 fiM) but had no effect on epoxidation by /3-naphthoflavoneinduced cells. All the observations suggest that two distinct pathways of epoxidation operate in the two cell preparations and that they can be modulated independently (Figure 4). Peroxyl free radicals appear to carry out epoxidation in control cells whereas cytochrome P-450 effects epoxidation in /3-naphthoflavoneinduced cells. The capacity for epoxidation appears greater in 1368

induced cells because the yield of jy/i-dihydrodiolepoxide in j3naphthoflavone-induced cells is 10 times higher than the yield of a/jtt-dihydrodiolepoxide in control cells. We attempted to investigate the source of peroxyl radicals in the epidermal cells. The high prostaglandin biosynthetic capacity of epidermal cells from hairless mice makes arachidonic acid metabolism a likely pathway. However, the prostaglandin H synthase inhibitor indomethacin has no effect on BP-7,8-diol epoxidation by cells (44). PGH synthase activity in these epidermal cells is 45 times higher than lipoxygenase activity so it seems that enzymes of arachidonic acid oxygenation are not responsible for a significant amount of peroxyl radical generation. Measurement of malondialdehyde formation by the cells indicated a close correlation between the time course of lipid peroxidation and anri-diolepoxide formation. Lipid peroxidation is an efficient source of peroxyl radicals and has been shown in other tissues to epoxidize BP-7,8-diol (39). Cellular lipid peroxidation is often associated with toxicity but as noted above there appears to be no increase in cell death during the course of the incubations of the epidermal cells. If these results can be extrapolated to mouse skin in vivo they suggest that peroxyl free radicals play an important role in oxidative activation of carcinogens. Peroxyl radicals will be more important for activation of molecules that do not induce cytochrome P-450 (assuming that the molecule in question is oxidized by peroxyl radicals). One such molecule appears to be BP-7,8diol. It is a potent tumor initiator when applied topically to SENCAR or CD-I mice, followed by phorbol ester tumor promotion [100% tumor incidence at 150 nmol ()-BP-7,8diol/animal] (49). Topical application of this dose of BP7,8-diol to mice induces only a very slight increase in AHH activity (~ 30%) whereas the same dose of/?-naphthoflavone increases AHH activity 10-fold (44). This means that the pathways responsible for metabolic activation of BP-7,8-diol in mouse skin are those operative at the time of its administration and not any pathways induced as a result of the treatment. The relative contribution of peroxyl radicals to BP-7,8- diol epoxidation appears to be greater than the contribution of cytochrome P-450 in untreated animals. What is the relevance of these observations on the oxidation of BP-7,8-diol to metabolic activation of BP and other polycyclic hydrocarbons in skin? The major DNA adducts formed after topical administration of BP or 7,12-dimethylbenz[a]anthracene (DMBA) are derivatives of bay-region dihydrodiolepoxides (50, 51). This means that metabolic activation has to occur by pathways that generate the 7,8-oxide of BP or the 3,4-oxide of DMBA, hydrate the arene oxides to dihydrodiols and finally epoxidize the isolated double bond of the dihydrodiols. The first oxidation in this sequence has to occur via a cytochrome-P-450dependent reaction because no other mammalian enzymes epoxidize aromatic rings of polycyclic hydrocarbons. This reaction is not effected by peroxyl radicals; in fact, peroxyl radicals oxidize BP to quinones (27). The identity of the cytochrome P-450

Peroxyl free radicals

isoenzyme in mouse skin that catalyzes arene oxide formation is not known, nor is it clear whether the isoenzyme is present in epidermal tissue prior to hydrocarbon administration or is induced as a result of treatment (vide infra). The terminal step in the metabolic activation pathwayoxygenation of dihydrodiols to dihydrodiolepoxidesis effected by peroxyl radicals or by cytochrome P-450. The relative contribution that either of the oxidizing agents make to this step probably depends on the state of induction of the epidermal cells. If little induction has occurred, peroxyl radicals may make a major contribution to oxidation but if significant induction has occurred, cytochrome P-450 should predominate (vide supra). These findings raise some provocative questions about the pathways of metabolic activation in mouse skinhistorically the most important target tissue for polycyclic hydrocarbons. Early studies of the metabolism of DMBA in mouse skin established the kinetics of disappearance of the hydrocarbon, the kinetics of induction of AHH activity, the time-dependence of inhibition of metabolism by flavones and other polycyclic hydrocarbons, and the time-dependence of inhibition of tumor-initiating activity by flavonoids (52-55). Total binding to skin macromolecules is high at 6 h followed by a slight increase at 12 h (53,54). This parallels the time course for formation of deoxynucleoside adducts in epidermal DNA (51). The time course for binding is inversely related to the time course for inhibition of tumor initiation by the mixed-function oxidase inhibitor a-naphthoflavone (53). Maximal inhibition ( 80% below control) is observed when a-naphthoflavone is applied simultaneously with DMBA; less inhibition is observed when a-naphthoflavone is applied 6 h after DMBA, and no inhibition is detected 12 h after DMBA. If a-naphthoflavone inhibits tumorigenesis by inhibiting DMBA metabolism, these experiments indicate that the crucial metabolic events occur early after DMBA is applied. This fits well with the rapid time course for covalent binding of DMBA to skin macromolecules. The time course for induction of AHH activity in skin has also been determined (53-55). Slight to moderate induction is detected 4 h after treatment with 100 nmol DMBA, a dose that is 10 times the tumor-initiating dose. Maximal induction is observed 8-16 h after treatment. Considering the time course for inhibition of tumorigenesis by a-naphthoflavone ( 0 - 6 h), it seems that metabolic activation is nearly complete before any significant induction of cytochrome P-450 occurs. This is especially true at low tumor-initiating doses of hydrocarbons. These considerations imply that, although cytochrome P-450 catalyzes the oxidation of DMBA to dihydrodiols, induction of the enzyme is not required for metabolic activation in skin. Extension to metabolic activation of BP is complicated by the fact that a-naphthoflavone does not inhibit tumor initiation by BP, an observation that is still not understood (46). Even discounting the importance of induction to metabolic activation of polycyclic hydrocarbons, the chemical arguments advanced above and the a-naphthoflavone inhibition studies with DMBA indicate that cytochrome P-450 is responsible for the oxidation of BP, at least to the 7,8-oxide. Further oxidation of the dihydrodiol to a dihydrodiolepoxide can be effected by a cytochrome P-450 or by peroxyl radicals. The extent of the contribution either pathway makes to this terminal activation step in vivo is unknown. Our studies in intact epidermal cells suggest that peroxyl-radical-dependent epoxidation may be more important than cytochrome-P-450-dependent epoxidation in the skin of untreated mice. Curiously, we find little evidence that cytochrome P-450 oxidizes BP-7,8-diol at all in cells from uninduced animals (44). This makes one wonder how oxidation of BP to BP-7,8-diol

(a cytochrome P-450-requiring reaction) occurs in the absence of induction. Perhaps BP is a much better substrate for the trace amounts of cytochrome P-450 in uninduced epidermal cells. Rapid cytochrome-P-450-dependent metabolism of 5-methylchrysene has been observed in mouse skin in vivo (56) which may indicate that unoxidized polycyclic hydrocarbons are good cytochrome P-450 substrates. A direct comparison of the rate of oxidation of BP and BP-7,8-diol by epidermal enzymes has not been performed. Perhaps only trace amounts of metabolism, an amount that can be supplied by the balance of oxidizing enzymes present in uninduced skin, is required to initiate tumorigenesis. Immunochemical experiments indicate that rat skin contains detectable cytochrome P-450c only following induction and then exclusively in follicular cells (57). Skin from uninduced animals exhibits primarily cytochrome P-450),, again only in follicular cells (57). It is important to realize that the deoxynucleoside adducts quantitated in mouse skin following administration of BP or DMBA represent a very small percentage of the total dose applied. Calculations from several literature reports provide a range of DNA binding of 0.0002-0.009% of the applied dose or 2 - 9 0 p.p.m. (50,51,58-64). Whether this low percentage binding is due to low rates of metabolism to dihydrodiolepoxides or inefficient binding of dihydrodiolepoxides to DNA is uncertain [the range of DNA-binding reported following adminstration of racemic syn- and anft-dihydrodiolepoxides corresponds to 0.002 0.010% (20-100 p.p.m.) (65)] but it suggests that quantitatively minor pathways of metabolism could be of major importance biologically. The findings that BP-7,8-diol is a very weak inducer of epidermal cytochrome P-450 and is a poor substrate for the trace amounts of cytochrome P-450 that exist in uninduced animals have implications for carcinogenicity testing on skin. A priori one might expect that BP-7,8-diol would exhibit much higher tumor-initiating activity than BP because it is a proximate carcinogen. This assumes that BP and BP-7,8-diol are equally reactive toward metabolizing enzymes in mouse skin. In fact, ()-BP-7,8-diol is only equipotent to BP as a tumor initiator and (+)-BP-7,8-diol is - 7 0 % less potent (49). Our studies imply that significant differences exist between the pathways responsible for dihydrodiolepoxide formation from BP and BP-7,8-diol in mouse skin. These differences may account for the lower than expected tumor-initiating activity of BP-7,8-diol. This discussion is admittedly speculative but it is so, in part, because little is known about the in vivo metabolism of polycyclic hydrocarbons in mouse skin. Tests of the hypothesis that peroxyl radicals play a role in BP-7,8-diol epoxidation seem to have highlighted deficiencies in our understanding of target tissue metabolic activation as much as they have clarified it. Quantitative studies of the oxidation of indirect-acting carcinogens in skin seem to be a high priority. Such studies may not only identify non-cytochrome-P-450-dependent mechanisms of activation but may also explain anomalous tumor-initiating activities of certain polycyclic hydrocarbon derivatives and the lack of activity of compounds that are potent carcinogens in non-epidermal tissues (e.g. nitrosamines). Peroxyl radicals and tumor promotion The tumor-promoting activity of peroxides that are chemical sources of free radicals and the ability of antioxidants to inhibit promotion constitute some of the major supportive evidence for the hypothesis that free radicals are involved in tumor promotion (66,67). The half-lives of oxygen-centered free radicals shown 1369

L.J.Marnett
HO2C

HO2C-

Fig. 5. Mechanism of epoxidation of 13-m-retinoic acid by retinoid-derived peroxyl radicals.

in Table I make it clear that one cannot simply weigh out a known amount of free radical and determine its promoting activity on skin as one can do with fully covalent molecules. One also cannot extract skin and identify the free radicals (if any) produced following phorbol ester treatment. So far, it has not been possible to directly determine whether compounds like benzoyl peroxide actually enhance free radical formation in vivo under conditions where they act as promoters. All techniques for studying free radical involvement in promotion are by nature indirect and are based on chemical trapping experiments or spectroscopic experiments to provide evidence for the existence of unstable radical intermediates. For example, Kensler and Taffe have recently reported trapping methyl radicals with the spin trap 5,5-dimethyl1-pyrroline-N-oxide following addition of cumene hydroperoxide to isolated mouse epidermal cells (4). Another implication of the data in Table I is that if oxygencentered radicals are involved in tumor promotion it should be mirrored by an increase in peroxyl radical levels. The discussion in the previous sections suggests that BP-7,8-diol should be extremely useful as a probe for the generation of peroxyl radicals in intact cells or perhaps even in vivo. The high rate and stereoselectivity of the reaction of BP-7,8-diol with peroxyl radicals means that one should be able to quantitate the oxidation of radiolabeled material by cell or tissue preparations (preferably the (+)-enantiomer, which is commercially available) to tetrahydrotetraols that are diagnostic of the intermediacy of peroxyl radicals in the oxidations. If the levels of oxygen-centered free radicals increase following administration of tumor promoters the increases should be reflected in the levels of peroxyl radicals trapped by BP-7,8-diol. Conversely, agents that inhibit free radical formation or scavenge them should decrease the peroxyl-radicalmediated epoxidation of BP-7,8-diol. Thus, the experimental strategy developed to test the role of peroxyl radicals in tumor initiation should be equally useful for testing their involvement in promotion. Just as the decomposition of peroxide promoters to free radicals is assumed but not founded, the anti-promoting activity of antioxidants is hypothesized to result from radical scavenging. Antioxidant compounds usually contain a phenol or aromatic amine functionality that is the radical trapping moiety. However, some compounds that are potent inhibitors of tumor promotion, but are not considered classic antioxidants, also may act by altering the steady-state levels of free radicals in initiated epidermal cells. Retinoids (e.g. ail-trans- or 13-cw-retinoic acid) are potent inhibitors of epidermal promotion by phorbol esters and are generally believed to act by affecting the state of differentiation of the cells 1370

or interrupting message transduction by the promoter (68-71). Consistent with the latter view, retinoids prevent induction of ornithine decarboxylase in mouse skin following administration of phorbol esters and they inhibit protein kinase C activity in vitro (72,73). The chemical structures of retinoids suggest they should be quite reactive with free radicals at a number of sites in the extended polyene chain. In fact, we have found that incubation of 13-cw-retinoic acid with peroxidase and hydroperoxides or with peroxyl-radical-generating systems leads to rapid oxygen uptake, retinoid oxidation and formation of oxygenated products (74). The major product of reaction of 13-c/s-retinoic acid with peroxyl radicals is 5,6-epoxy-13-c-retinoic acid and the rate of its formation is somewhat higher than the rate of formation of the anft-dihydrodiolepoxide from BP-7,8-diol (V.M.Samokyszyn and L.J.Marnett, unpublished). Reaction with peroxidases and hydroperoxides produces a mixture of 4-hydroxy-13-cw-retinoic acid and 5,6-epoxy-13-cw-retinoic acid (80). Mechanistic studies indicate that peroxidase higher oxidation states remove the H atom from the 4 position generating a carbon-centered radical that couples with O2 to generate a retinoid peroxyl radical (eqn 6). Addition of the peroxyl radical to the 5,6 double bond produces 5,6-epoxy- 13-c-retinoic acid and a retinoid alkoxyl radical that is reduced to 4-hydroxy-13-rij-retinoic acid (Figure 5).

Equation 6

The complex nature of the reaction of retinoids with peroxyl radicals and peroxidases suggests that they are not only efficient at trapping reactive oxidants, thereby lowering the steady-state oxidant concentration, but that under certain conditions they can actually enhance peroxyl and alkoxyl free radical formation. This may be relevant to occasional reports that retinoids enhance rather than inhibit tumor promotion and highlights another set of metabolic experiments that seem worthwhile to perform (75-77). Despite the potent chemopreventive activity of retinoids and related compounds and despite the fact that they possess chemical funtionalities that are extremely reactive toward oxidizing agents, the fate of these molecules on mouse skin has never been determined. This may be related to observations that oxidized retinoic acid derivatives exhibit somewhat lower biological activities in

Peroxyl free radicals

cell culture than do the parent molecules (78). The opinion may have arisen that retinoids do not require metabolic activation and although this may be correct for hormonal activities or even chemopreventive activities in internal organs, it may not be possible to extrapolate this conclusion to mouse skin, a tissue exposed to high oxygen tensions and possessing active oxygenase-dependent metabolic pathways. In fact, 13-c/.r-retinoic acid is the most reactive molecule toward peroxyl radicals that we have worked with. Nearly complete oxidation in vitro is detected at initial retinoid concentrations of 1 /tM, a concentration that approaches the levels attained in tumor promotion experiments. If BP-7,8-diol is epoxidized in mouse skin cells by peroxyl radicals, it is virtually certain that 13-c-retinoic acid will be epoxidized under similar conditions because it is more reactive to peroxyl radicals. The ease of oxidation of certain retinoids by transient oxidants likely to be generated in promotion-sensitive cells may provide a new basis for testing the biochemical mechanisms of their antipromoter or promoter action.

References
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Summation This Commentary is not intended to convince readers that peroxyl radicals play a role in metabolic activation or tumor promotion but to stimulate them to design experiments to test their involvement in a variety of experimental models. A good deal of information is already available in the literature about the chemistry of peroxyl radicals and systems have been described that generate reasonable concentrations of peroxyl radicals albeit rapidly (33, 34). These systems should be useful for evaluating the ability of peroxyl radicals to oxidize compounds of interest. The other purpose of this discussion is to illustrate some of the gaps in our knowledge of the biochemistry of chemical carcinogenesis in skin. Epidermal carcinogenesis is important from a historical and practical point of view (77). It was the first tissue in which chemicals were shown to induce cancer and it is still routinely employed for testing new chemicals as potential carcinogens or for defining structure-activity relationships. It provided the experimental basis for the multi-stage model of carcinogenesis and work in epidermal systems has revealed many of the key biochemical events that follow administration of the initiator or promoter. Included in these advances are many elegant studies that have elucidated the nature of the reaction of the ultimate carcinogen with DNA. Recent years have witnessed a shift in the focus of experimentation in carcinogenesis towards molecular aspects of alteration of gene structure and expression by chemicals. The shift was hastened by development of recombinant DNA techniques that helped identify important target genes and the precise structural changes that chemicals induce in them. It is important to note, though, that this natural migration to the new frontier does not mean that everything is known about the problems heretofore studied. Much needs to be learned about the metabolic events responsible for critical events in skin. The burst of experimentation on the role of free radicals in carcinogenesis and the questions that these studies raise hopefully serve to emphasize this.

Acknowledgements
I am grateful to Donna Pruess-Schwartz and Victor Samokyszyn for critically reading the manuscript. This work was supported by research grants from the American Cancer Society (BC 2441) and the National Institutes of Health (CA 43209 and GM 23642). L.J.M. is a recipient of a Faculty Research Award of the American Cancer Society (FRA 243).

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