Download Peroxyl free radicals: potential mediators of tumor initiation and

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
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
Table I. Oxygen-centered radicals in biological systems
Oxygen reduction
Lipid
peroxidation
pATa = 4.88
O-O-
•-O-O7s
R-O
10"6s
H-Ol<T 9 s
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).
HOH(R)
•OH(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
(CH 2 ) 4 CH 3
HO2C(CH2)7
(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 (22—25). 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
PGH,
PGG2
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
Equation 4
BP-6.12-Ouijione
BP-1.6-Ounon8
Peroxyl free radicals
P-450
ROO
1
HO "'
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).
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-diol—the 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.
(+) - 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.
BP-7,8-diol is epoxidized by the following biochemical systems
that generate peroxyl radicals—ascorbic 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
INDUCED
ROO- (moderate)
ROO (moderate)
inducer
P-450 (low)
P-450c (high)
Fig. 4. Pathways of metabolic activation in skin of uninduced and Ahinduced mice.
0
Retention
Time
20
40
60
(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 4—5 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 pathway—oxygenation of dihydrodiols
to dihydrodiolepoxides—is 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 skin—historically 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.
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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