Professional Documents
Culture Documents
377-408, 1989
Printed in the USA. All rights reserved.
Review Article
ANTIOXIDANTS
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
INTRODUCTION
378
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-
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. 1415 The demonstration of the multistep nature of chemical carcinogenesis
in mouse skin was greatly facilitated by the synthesis
o~1014 -tl . . . . .
II . . . . . . . . . . . . .
o~
o 1012
. I . . 7. .
~ IOi
Io 8
II . . . . .
379
10 6
},
.~_ 104
Tumor
Initiation
Tumor Promotion
I
I
.
c ~
~ ' ~
Stem Cell
Stem Cell
Initiation
--27
Neoplastic Cell
Transformation Prolif,ercdion /
l
_
"
. Ii .
,
,.
,~ '
,.
1
"6
I
Postulated Period of
Dormancy
/ i
/~,
/I
/
i
'l
i ....
I ,~'~x~L"Y
~
i u~.t=
u,
I
,I ~
',_T_,',e_
, . . . . . . . . . . ,
>
,,P od of t_ rfi
of
'a~
Prior to, Clinical
,
Deteclim
),
Growth
Period of Leten~;y
I<
Pt~iod of ~ 1
)'
Gl~th
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
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.
380
Tumor initiation
,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
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
381
382
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
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-
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, ll'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 . 84'11'12 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
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
(a)
(b)
(c, Fenton)
(d, Haber-Weiss)
(e)
(f)
(g)
(h)
(i)
(J)
(k)
(I)
(m)
(n)
GSSG-R
NADP~
.~NADPH
Glucose-6-Phosphate Dehydrogenase
Glucose-6-Phosphate
:~,,~
6-Phosphoglucolactone
384
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
385
386
240-,
220
.
,
A.
e~6 16c
14C
120
I01
I
:,=
160
120
BI
moo
Ct
so
:E.,-
6o
40
:~0.. , .
-=
2
5
Time (hours)
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-
388
389
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 37C; 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.
390
J.-P. PERCHELLETand E.
M. PERCHELLET
+ Protein Kinase C
Induction .~.,~iGenerotio n
lf 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.
391
392
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
Initiation
{ DMBA 238-24
IvDMBA 239"240
{ 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
Progression
B p----diol,---O---D MB A ~54
~, BP, BP---dioP s4
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)
394
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. 271'31'32 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. 33 Apparently, ellagic
acid can also scavenge the ultimate carcinogenic
form of BP. 34 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"35 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. 36 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
396
397
398
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-
399
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407
ABBREVIATIONS
ROS--Reactive
Pa--Papilloma
Ca--Carcinoma
oxygen species
408
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)