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INTRODUCTION
C
HEMOPREVENTION IS DEFINED as reduction of the risk of
cancer development through the use of pharmaceuticals
or micronutrients (33). Cancer preventive strategies are attrac-
tive from the viewpoint of public health. Many studies have ad-
dressed the role of antioxidant micronutrients in vegetables and
fruits in protection against cancers (20). More than two-thirds
of human cancers could be prevented through appropriate life-
style modification. For example, consumption of fruits and
vegetables is associated with a reduced risk of developing can-
cer (99). Numerous phytochemicals derived from edible plants
have been reported to interfere with a specific stage of the car-
cinogenic process (99). Development of dietary compounds as
potential cancer chemopreventive agents is highly desirable,
because of their safety, low toxicity, and general acceptance as
dietary supplements (9).
Phytochemical preparations are marketed as herbal medi-
cines or dietary supplements for a variety of alleged nontoxic
therapeutic effects. However, they have yet to pass controlled
clinical trials for efficacy, and their potential for toxicity is an
understudied field of research (22). Dietary supplementation
of publicly available foods and the ingestion of specific supple-
ments are usually applied for prolonged periods of time. There-
fore, it is essential that the strategy is devoid of risks (103). The
Alpha-Tocopherol Beta Carotene Cancer Prevention Study
Group (1) and the Beta-Carotene and Retinol Efficacy Trial
(76) supplied -carotene and/or vitamin A to smokers and as-
bestos-exposed workers, who were high-risk groups for lung
cancer. After follow-up for several years, higher incidences of
1728
Evaluation for Safety of Antioxidant Chemopreventive Agents
SHOSUKE KAWANISHI, SHINJI OIKAWA, and MARIKO MURATA
ABSTRACT
Antioxidants are considered as the most promising chemopreventive agents against various human cancers.
However, some antioxidants play paradoxical roles, acting as “double-edged sword.” A primary property of
effective and acceptable chemopreventive agents should be freedom from toxic effects in healthy population.
Miscarriage of the intervention by -carotene made us realize the necessity for evaluation of safety before rec-
ommending use of antioxidant supplements for chemoprevention. We have evaluated the safety of antioxi-
dants on the basis of reactivity with DNA. Our results revealed that phytic acid, luteolin, and retinoic acid did
not cause DNA damage under the experimental condition. Furthermore, phytic acid inhibited the formation
of 8-oxo-7,8-dihydro-2-deoxyguanosine, an indicator of oxidative DNA damage, in cultured cells treated with
a H
2
O
2
-generating system. Thus, it is expected that these chemopreventive agents can safely protect humans
against cancer. On the other hand, some chemopreventive agents with prooxidant properties (-tocopherol,
quercetin, catechins, isothiocyanates, N-acetylcysteine) caused DNA damage via generation of reactive oxygen
species in the presence of metal ions and endogenous reductants under some circumstances. Furthermore,
other chemopreventive agents (-carotene, genistein, daidzein, propyl gallate, curcumin) exerted prooxidant
properties after metabolic activation. Therefore, further studies on safety should be required when antioxi-
dants are used for cancer prevention. Antioxid. Redox Signal. 7, 1728–1739.
Forum Review
ANTIOXIDANTS & REDOX SIGNALING
Volume 7, Numbers 11 & 12, 2005
© Mary Ann Liebert, Inc.
Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, Mie, 514-8507, Japan.
14098c31.pgs 11/16/05 3:37 PM Page 1728
lung cancer were observed in the intervention groups than the
placebo groups. We have first demonstrated that -carotene
metabolites have prooxidant properties by the observation of
reactivity with DNA (59). Therefore, we deeply realize the ne-
cessity for evaluation of safety before recommending use of
antioxidant supplements for chemoprevention.
We have evaluated the safety of antioxidants on the basis of
reactivity with DNA. Antioxidant chemopreventive agents with-
out prooxidant properties inhibit the carcinogenic process be-
cause of chelating metals, scavenging reactive oxygen species
(ROS) or interfering with a specific manner (Fig. 1A). On the
other hand, some chemopreventive agents exert prooxidant prop-
erties (Fig. 1B), and some agents exert prooxidant properties
after metabolic activation (Fig. 1C). Details are noted below.
MATERIALS AND METHODS
We have examined the reactivity of antioxidants with
DNA in application to the screening of carcinogenicity for
evaluation of safety. Primary procedures are as follows.
Preparation of
32
P-5-end-labeled
DNA fragments and detection of DNA damage
Exon-containing DNA fragments obtained from the
human tumor-relative genes such as p53 and p16 tumor sup-
pressor genes and c-Ha-ras-1 protooncogene (8, 11, 88). A
5-end-labeled DNA fragment was obtained by phosphoryla-
tion with [-
32
P]ATP and T
4
polynucleotide kinase (111). The
fragment was further digested with restriction enzyme to ob-
tain a singly labeled fragment. A standard reaction mixture
(in a 1.5-ml microtube) contained the test chemicals, metal
ions and other endogenous compounds,
32
P-5-end-labeled
DNA fragment, and calf thymus DNA in sodium phosphate
buffer. After incubation at 37°C, the DNA fragments were
treated with piperidine or formamidopyrimidine-DNA glyco-
sylase (Fpg) protein. Then, DNA was electrophoresed on an
8% polyacrylamide/8 M urea gel. The autoradiogram was ob-
tained by exposing an x-ray film to the gel. The preferred
cleavage sites were determined by direct comparison of the
cleaved oligonucleotides with a standard Maxam-Gilbert se-
quencing reaction (55). The relative amounts of oligonu-
cleotides from the treated DNA fragments were measured with
a laser densitometer.
Measurement of 8-oxo-7,8-dihydro-2-
deoxyguanosine (8-oxodG) in cellular
and isolated DNA
Human cultured cells were treated with test chemicals.
Then, cells were washed three times with cold phosphate-
buffered saline. Under anaerobic conditions, DNA was ex-
tracted using lysis buffer, RNase A, and proteinase K (61).
For using isolated DNA, calf thymus DNA was incubated
with test chemicals, metal ions, and other endogenous com-
pounds at 37°C. After ethanol precipitation, DNA was di-
gested to the nucleosides with nuclease P
1
and alkaline phos-
phatase, and then analyzed by high-performance liquid
chromatography coupled with electrochemical detector (37).
EVALUATION FOR SAFETY OF ANTIOXIDANTS 1729
FIG. 1. Chemopreventive agents with antioxidant and
prooxidant properties: compounds with antioxidant prop-
erties (A), compounds with prooxidant properties (B), and
compounds with prooxidant properties induced by metabo-
lism (C).
14098c31.pgs 11/16/05 3:37 PM Page 1729
EVALUATION OF
ANTIOXIDANTS FOR SAFETY
Vitamins A and E
-Carotene and vitamin A.
Many epidemiological
studies showed that vitamin A intake decreased incidence of
cancers of lung, bladder, upper gastrointestinal tract, and
breast, as reviewed by Willett and MacMahon (110). It has
been expected that the antioxidant potency of vitamin A and
-carotene may protect against cancer occurrence (24). How-
ever, intervention trials with -carotene failed (1, 76). Regard-
less of the miscarriage of the intervention, attempts to use
retinoids and carotenoids for cancer chemoprevention and
therapy are ongoing (10, 63, 85). Therefore, the causal mecha-
nisms should be elucidated to establish safe approaches in
cancer chemoprevention. We examined the reactivity of -
carotene and its metabolites with DNA. It is known that -
carotene is metabolically converted to two molecules of reti-
nal principally by central cleavage. Then, retinal is further
oxidized to retinoic acid or reduced to retinol (Fig. 2). We de-
termined that both retinol and retinal caused oxidative damage
to cellular and isolated DNA (59). Retinoids significantly in-
duced 8-oxodG formation in HL-60 cells, but did not signifi-
cantly increase 8-oxodG in H
2
O
2
-resistant HP100 cells. Elec-
tron spin resonance spectroscopic studies using a trapping
agent -(4-pyridyl1-oxide)-N-tert-butylnitrone have demon-
strated retinol- and retinal-derived radicals with six-line sig-
nals assigned as carbon-centered radicals. Using the cy-
tochrome c reduction method, generation of superoxide (O
2
)
derived from the autooxidation of retinoids was detected. The
generation of O
2
was significantly correlated with 8-oxodG
formation. We confirmed using isolated DNA that retinol and
retinal induced DNA damage including 8-oxodG in the pres-
ence of Cu(II), whereas retinoic acid and -carotene induced
no or little DNA damage. Both retinol and retinal play impor-
tant roles in carcinogenesis in the intervention studies using
excess amounts of -carotene. On the other hand, retinoic acid
has no prooxidant property (59) but has the potential of regu-
lating cell differentiation (28). A recent intervention study
1730 KAWANISHI ET AL.
suggests 9-cis-retinoic acid has potential chemopreventive
properties in former smokers (47).
Vitamin E. -Tocopherol is widely recognized as being
the most important biological antioxidant of the lipid phase
(35). There is considerable interest in the possibility that vita-
min E (-tocopherol) may be protective against cancer and
cardiovascular diseases (106). On the other hand, several stud-
ies have demonstrated that the vitamin can act as a carcinogen,
at both the initiation and promotion stages (58, 67). We have
indicated that -tocopherol in the presence of Cu(II) can in-
duce extensive DNA damage, including base modification and
strand breakage (113). The predominant DNA cleavage sites
were thymine and cytosine residues. Inhibitory effects of cata-
lase and bathocuproine on DNA damage suggest that H
2
O
2
and Cu(I) are required for the DNA damage. An electron spin
resonance spin-trapping study showed the formation of hy-
droxyl radical (·OH) generated from -tocopherol and Cu(II).
This suggests that ·OH, generated via the reaction of Cu(I)
with H
2
O
2
, is responsible for the induction of DNA damage.
Cu(II) ions are known to bind tightly to DNA, where they can
interact with reducing agents (ascorbic acid, glutathione, phe-
nolics, or NADH) and H
2
O
2
, resulting in oxidative damage to
the nucleic acid, including base modification and strand
breakage (7, 41). Our experiments have shown that -toco-
pherol incorporated into liposomes is able to induce DNA
damage in the presence of Cu(II). It is reasonably considered
that vitamin E may have not only anticarcinogenic effects but
also carcinogenic potentials.
Polyphenols
Isoflavones.
Epidemiological and experimental studies
have shown that soy products can reduce the risk of cancer and
provide other benefits, including lowering cholesterol and
blood pressure and preventing cardiovascular diseases and os-
teoporosis (40, 44, 48, 65, 84, 94). The soy isoflavones, genis-
tein (5,7,4-trihydroxyisoflavone) and daidzein (7,4-dihydrox-
yisoflavone), are representative phytoestrogens (95), and act as
chemopreventive agents against cancers, cardiovascular dis-
ease, and osteoporosis. Because of these health benefits, the
consumption of soy food and the use of isoflavone supplements
have been increasing (56). However, recent studies revealed
that genistein and/or daidzein induced cancers of reproductive
organs in rodents, such as the uterus (66) and vulva (105).
These reports led us to consider that soy isoflavones may have
a carcinogenic effect on female reproductive organs. We
showed that genistein and daidzein exerted cell proliferative
activity on estrogen-sensitive MCF-7 cells (62), as reported
previously, while their metabolites had little or no activity. In
accordance with the data on cell proliferation, the surface plas-
mon resonance sensor showed that genistein and daidzein in-
duced higher affinity binding of estrogen receptor (ER) to es-
trogen response element (ERE), while the metabolites had little
or no binding activity. Isoflavones such as genistein and
daidzein may induce cell proliferation through ER-ERE bind-
ing. On the other hand, the isoflavone metabolites orobol
(5,7,3,4-tetrahydroxyisoflavone) and 7,3,4-trihydroxy-
isoflavone significantly induced 8-oxodG formation in MCF-
CHO
CH
2
OH COOH
β-carotene;
DNA damage (-)
retinal;
DNA damage (++)
retinol (vitamin A);
DNA damage (+)
retinoic acid;
DNA damage (-)
oxidationreduction
FIG. 2. Metabolic conversion of -carotene and the poten-
tial of DNA damage by -carotene and its metabolites.
14098c31.pgs 11/16/05 3:37 PM Page 1730
10A cells (Fig. 3). Interestingly, Fpg treatment revealed that
orobol induced significant cleavage of the guanine residue of
the ACG sequence complementary to codon 273, a well-known
hotspot (49) in the p53 gene (Fig. 4A). Piperidine treatment
cleaved cytosine and guanine residues at the ACG (Fig. 4B).
Similar results were obtained with 7,3,4-trihydroxy-
isoflavone. Oxidative DNA damage by isoflavone metabolites
plays a role in tumor initiation, and cell proliferation by
isoflavones via ER-ERE binding induces tumor promotion
and/or progression, resulting in cancer of estrogen-sensitive or-
gans. Our study raises the possibility that genistein and
daidzein are carcinogenic in estrogen-sensitive organs, even
though isoflavones are generally regarded as chemopreventive
agents.
Quercetin and luteolin. Flavonoids, particularly
flavonol and flavone, are commonly found in many vegetables
and herbs. Quercetin is the most widely distributed flavonol,
and luteolin is one of the most widely distributed flavones in
the plant kingdom. Onions are rich in quercetin, which has per-
ceived benefits to human health, including anticarcinogenic
properties (26). On the other hand, quercetin has been reported
to be carcinogenic (13, 70, 77). Considerable evidence sug-
gests that quercetin has prooxidant activity (6, 86), and DNA-
damaging ability in cultured cells (18, 23). Previously, we
showed that quercetin induced DNA damage by prooxidative
effects, but luteolin did not, in a simplified in vitro model
(114). Some flavonoids, including quercetin and luteolin, are
topoisomerase II (topo II) inhibitors and act as anti-tumor
agents (4, 115). Topo II inhibitors can induce apoptosis by
means of DNA-replication-associated damage involving
formation of a stable drug–topo II–DNA ternary complex,
called a cleavable complex (64). We showed that both quercetin
and luteolin induced DNA cleavage to 1–2-Mb and less than
200-kb DNA fragments, followed by DNA ladder formation in
EVALUATION FOR SAFETY OF ANTIOXIDANTS 1731
HL-60 cells (112). The significant increase in 8-oxodG forma-
tion in HL-60 cells and no increase in their H
2
O
2
-resistant
clone HP 100 cells were observed after treatment with
quercetin. It indicated that quercetin induced oxidative DNA
damage and that H
2
O
2
was the main mediator of such damage.
Luteolin inhibited topo II activity more strongly than quercetin
in crude nuclear extract. Inhibition of topo II activity by lute-
olin was associated with DNA cleavage by cleavable complex
formation. Luteolin-induced DNA cleavage and DNA ladder
formation in HP 100 cells were similar to those in HL-60 cells.
These results suggest that H
2
O
2
-mediated DNA damage is the
0
0.05
0.1
0.15
0.2
0.25
8-oxodG/10
5
dG
Control
Genistein
Orobol
Daidzein
7,3',4'-OH-IF
6,7,4'-OH-IF
**
*
FIG. 3. Intracellular 8-oxodG formation by isoflavone
metabolites in MCF-10A cells. Human mammary epithelial
MCF-10A cells were treated with 10 µM isoflavones or their
metabolites in the experimental medium at 37°C for 1 h. Re-
sults are expressed as means ± SE of values obtained from
three independent experiments. OH-IF, trihydroxyisoflavone.
*p < 0.05, **p < 0.01, significantly different compared with
the control by Student’s t test.
T
G
C
G
G
A
G
A
T
T
C
T
C
T
T
C
C
T
C
T
G
T
G
C
G
C
C
G
G
C
T
T
C
T
C
C
C
A
G
G
A
C
A
G
GC
C
A
A
A
A
C
A
C
G
C
A
C
T
C
C
A
A
A
G
C
T
G
T
G
C
G
G
A
G
A
T
T
CT
C
T
T
C
C
T
C
T
G
T
G
C
G
C
C
G
G
T
C
T
C
T
C
C
C
A
G
G
A
C
A
G
G
C
A
C
A
A
A
C
A
C
G
C
A
C
C
T
C
A
A
A
G
C
T
G
Complementary to
Codon 273
(3')
(5')
2.0
1.0
Absorbance
(A) Piperidine treatment
3.0
2.0
1.0
14540 14530 14520 14510 14500 14490 14480
Nucleotide number of
p53
tumor suppressor gene
Absorbance
(5')
(3')
(B) Fpg treatment
FIG. 4. Site specificity of DNA cleavage induced by
isoflavone metabolites in the presence of Cu(II) and
NADH. The reaction mixture contained the
32
P-5-end-
labeled 443-bp DNA fragment (ApaI 14,179–EcoRI 14,621*),
20 µM per base of calf thymus DNA, 5 µM orobol, 20 µM
CuCl
2
, and 200 µM NADH in 10 mM phosphate buffer (pH
7.8) (*
32
P label). After incubation for 1 h at 37°C, the DNA
fragments were treated with piperidine (A) or Fpg protein (B)
and electrophoresed by the Maxam-Gilbert sequencing reac-
tion (55). The relative amounts of DNA fragments were mea-
sured by scanning the autoradiogram with a laser densitometer.
The horizontal axis shows the nucleotide number of the human
p53 tumor suppressor gene, and underscoring shows the com-
plementary sequence to codon 273 (nucleotide numbers
14,486–14,488).
14098c31.pgs 11/16/05 3:37 PM Page 1731
main pathway in quercetin-induced apoptosis, and topo II-
mediated DNA cleavage is that in luteolin-induced apoptosis.
Several papers indicated that luteolin does not have mutagenic-
ity and carcinogenicity (13, 70, 77). Luteolin can be expected
as a comparatively safe chemopreventive agent.
Catechins. Catechins, including catechin, epicatechin,
and epigallocatechin gallate (EGCG), are a class of flavonoids
with potent antioxidant and cancer chemopreventive proper-
ties. Catechins are believed to be an active constituent of green
tea. Several epidemiological studies suggest that green tea con-
sumption is associated with a reduced risk of several forms of
cancer in human populations (34, 46). On the other hand, sev-
eral human cohort and case-control studies have indicated sig-
nificant positive relationships between green tea consumption
and cancers of various organs (50, 102, 116). In addition, green
tea catechins have been reported to enhance colon carcinogen-
esis in rats (31). We showed that the content of 8-oxodG of
DNA in HL-60 cells treated with 1 mM catechin was signifi-
cantly increased in comparison to untreated cells, whereas cat-
echin did not cause a significant increase in the amount of 8-
oxodG in HP100 cells (74). Addition of bathocuproine
significantly decreased the amounts of 8-oxodG induced by 1
mM catechin, suggesting the possible role of endogenous cel-
lular copper in the activation of catechin to a DNA damaging
species. Epicatechin appears to be more potent than catechin in
the induction of 8-oxodG in cells. To clarify underlying mecha-
nisms, we examined DNA damage by catechins by using iso-
lated DNA. In the presence of Cu(II), the amount of 8-oxodG
increased with increasing concentration of catechins. The addi-
tion of 100 µM NADH enhanced Cu(II)-mediated 8-oxodG
formation induced by catechin and epicatechin. NADH is a re-
ductant existing at high concentrations (100–200 µM) in cells
(53). Amounts of 8-oxodG formed by epicatechin were larger
than those by catechin in the presence of NADH. The calcula-
tion has indicated that both the highest occupied molecular or-
bital of these catechins and the lowest unoccupied molecular
orbital of their corresponding quinones are localized on their B
rings. The calculated highest occupied molecular orbital en-
ergy of epicatechin (8.08 eV) is lower than that of catechin
(8.12 eV), suggesting that epicatechin is more easily oxidized
than catechin. The lowest unoccupied molecular orbital of the
quinone form of epicatechin is larger (0.65 eV) than that of
the quinone form of catechin (0.67 eV), suggesting that the
quinone form of epicatechin is easily reduced by NADH than
that of catechin. These differences between catechin and epi-
catechin can be explained by a steric effect of the OH group at
the 3-position of their C rings.
Catechins, especially EGCG, can ameliorate free radical
damage to DNA, under certain conditions (2, 3). To assess the
safety, we compared the intensity of DNA damage in the
presence of metal ions by four catechins: catechin, epigallo-
catechin (EGC), epicatechin gallate (ECG), and EGCG (21).
DNA fragments treated with various concentrations of cate-
chins in the presence of metal ions were detected by autoradi-
ography. Oligonucleotides were detected as a result of DNA
damage. In the presence of an Fe(III) complex such as
Fe(III)EDTA and Fe(III)ADP, EGC and EGCG induced DNA
1732 KAWANISHI ET AL.
damage. In addition, ECG also caused mild DNA damage.
Catechin induced only slight DNA damage. The order of
DNA damaging ability was EGCG ≈ EGC > ECG >> cate-
chin. In the presence of Cu(II), all four catechins induced
DNA damage; the order of DNA damaging ability was EGC >
catechin > EGCG > ECG. Piperidine treatment revealed that
catechins induced not only direct breakage of the deoxyribose
phosphate backbone but also base modification in the pres-
ence of metal ions.
At the present time, human trials for green tea catechins
are already in progress some countries (82, 109). Catechins
may have the dual function of carcinogenic and anticarcino-
genic potentials. These findings require further studies on
safety and risk assessment of catechins.
Propyl gallate (PG). PG is widely used as an impor-
tant synthetic antioxidant in the food industry. In contrast, the
National Toxicology Program (68) reported that PG induced
preputial gland tumors, islet-cell tumors of the pancreas, and
pheochromocytomas of the adrenal glands in male rats. PG
also induced malignant lymphoma in male mice. Accumulation
of PG may contribute to carcinogenesis. However, the mecha-
nism leading to carcinogenesis has not yet been clarified. We
demonstrated that PG significantly increased 8-oxodG forma-
tion in HL-60 cells (45). However, PG itself did not increase
the level of 8-oxodG in isolated calf thymus DNA in the pres-
ence of metal ions. PG is hydrolyzed enzymatically to gallic
acid (GA) by cellular carboxylesterase. We demonstrated that
GA increased the amounts of 8-oxodG in the presence of
Cu(II), Fe(III)EDTA, and Fe(III)ADP. From these results, it is
considered that GA, produced from PG by esterase, may be in-
volved in oxidative DNA damage in cultured human cells.
High-performance liquid chromatography analysis of the prod-
ucts generated from PG incubated with esterase revealed that
PG converted into GA. On the basis of these results, the possi-
ble mechanisms of metal-mediated DNA damage induced by
GA, a metabolite of PG, are proposed as follows: Metal-
mediated autooxidation of GA generates the semiquinone radi-
cal. In the presence of metal ion (M
n
), H
2
O
2
is generated by
O
2
dismutation with concomitant reduction of M
n
to M
n
1
. In
the presence of Cu(II), GA induces DNA damage by the inter-
action of Cu(I) and H
2
O
2
to form a Cu(I)–hydroperoxo com-
plex such as Cu(I)OOH. Fe(III)EDTA-mediated DNA damage
resulting from exposure to GA is caused by ·OH generated
from the Fenton reaction. The ·OH is extremely short-lived and
travels a very short distance in water (72, 101). This can be one
of the reasons that Cu(II)-mediated DNA damage caused by
GA is stronger than Fe(III)EDTA-mediated damage, although
autooxidation of GA mediated by Fe(III)EDTA is faster than
that by Cu(II). It is concluded that GA plays an important role
in PG carcinogenicity.
Miscellaneous
Curcumin.
Curcumin is the major yellow pigment in
turmeric, curry, and mustard, and has also been widely used in
cosmetics and drugs (17). A major source of human consump-
tion of curcumin is turmeric, which is used as a coloring agent
14098c31.pgs 11/16/05 3:37 PM Page 1732
and spice in many foods (27). Studies on the chemopreventive
efficacy of curcumin have shown that it possesses both anti-
initiating and antipromoting activities in several experimental
systems (17). Animal studies have demonstrated that curcumin
inhibits carcinogenesis in various tissues, including skin (32),
colorectal (83), oral (100), forestomach (92), and mammary
(93) cancers. In contrast, the National Toxicology Program
study (71) showed that dietary administration of turmeric oleo-
resin with a high curcumin content (79–85%) induced clitoral
gland adenomas in female rats. There is also evidence for car-
cinogenic activity of the turmeric oleoresin in mice based on an
increased incidence of hepatocellular adenoma. We demon-
strated the prooxidant property of curcumin with metabolic ac-
tivation by CYP enzymes (CYP 2D6, 1A1, 1A2, 2E1) (87).
Figure 5 shows mechanisms of curcumin-induced anticancer
and carcinogenic effects. In a metabolic pathway, curcumin is
converted by hydrogenation to tetrahydrocurcumin, the more
promising chemopreventive agent (75). In contrast, curcumin
undergoes O-demethylation catalyzed by certain CYPs to O-
demethyl curcumin (36). O-Demethyl curcumin is then autoxi-
dized into the O-demethyl curcumin radical, leading to the pro-
duction of the corresponding o-quinone form. Several studies
indicate that NAD(P)H may non-enzymatically reduce o-
quinones to catechols through two-electron reduction (29). The
formation of the NAD(P)H-dependent redox cycle results in
enhanced O
2
generation, leading to enhancement of oxidative
DNA damage. Thus, the NADH-dependent redox cycle of O-
EVALUATION FOR SAFETY OF ANTIOXIDANTS 1733
demethyl curcumin may continuously generate ROS and medi-
ate oxidative DNA damage. The anticarcinogenic effect of cur-
cumin is associated with its influence on metabolizing en-
zymes (96, 104). High CYP activity may yield much
O-demethyl curcumin, leading to a carcinogenic effect.
Isothiocyanates (ITCs). Organic ITCs (R-N=C=S),
also known as mustard oils, are widely distributed in plants,
many of which are consumed by humans. Vegetables belonging
to the family Cruciferae and genus Brassica (e.g., broccoli and
cauliflower) contain substantial quantities of ITCs, mostly in
the form of their glucosinolate precursors (117). Extracts of broc-
coli sprouts were effective in reducing tumors in carcinogen-
treated rats (19). The National Toxicology Program has evalu-
ated that allyl ITC (AITC) is carcinogenic to rats (69). It has
been reported that benzyl ITC (BITC) and phenethyl ITC
(PEITC) exhibit promotion potential during the post-initiation
stage (30, 51). Figure 6A shows carcinogenic potential and the
intensity of DNA damage by ITCs in our system (60). The
highly electrophilic central carbon atom of the -N=C=S group
(117) should be hydrolyzed to give rise to the SH group. Figure
6B shows a proposed mechanism of DNA damage induced by
ITCs (60). Autooxidation of the SH group is coupled with gen-
eration of O
2
from O
2
. The generation of H
2
O
2
by O
2
dismu-
tation and O
2
-mediated reduction of Cu(II) to Cu(I) occur.
The ability to yield an SH group from ITCs may depend on the
NADP
+
NADPH
G-6-P
G-6-PDH
CYPs
OHHO
O
H
3
C
O
CH
3
OO
OHHO
H
3
CO OH
OO
OHHO
H
3
CO O
OO
OHO
H
3
CO O
OO
Cu(II)
Cu(I)
O
2
-
O
2
H
2
O
2
O
2
-
O
2
Cu(I)OOH
Oxidative DNA damage
Glucono-δ-lactone-
6-phosphate
NADPH
NADP
+
Curcumin
o
-Demethyl curcumin
H
2
O
2
o
-quinone form
OHHO
O
H
3
C
O
CH
3
OO
Tetrahydrocurcumin
Cancer chemoprevention
FIG. 5. Proposed mechanisms of curcumin-induced anti-cancer and carcinogenic effects. G-6-P, glucose 6-phosphate;
G-6-PDH, glucose 6-phosphate dehydrogenase.
14098c31.pgs 11/16/05 3:37 PM Page 1733
length of the methylene chain, i.e., ITCs with shorter methy-
lene chains can yield greater amounts of the SH group, result-
ing in stronger DNA damage through O
2
generation. We dem-
onstrated that the order of DNA damaging ability is AITC >
BITC > PEITC. AITC is carcinogenic, while BITC and PEITC
alone have tumor-promoting activities. ITCs with certain
length of methylene chains may have anti-tumor activities
alone.
N-Acetylcysteine. Cancer prevention by N-acetylcys-
teine has been shown to be effective in several animal experi-
ments (5, 14). The oral administration of N-acetylcysteine
completely prevented the induction of DNA alterations of vari-
ous natures in rat lung cells (38). In addition, N-acetylcysteine
prevented the in vivo formation of carcinogen–DNA adducts,
and suppressed the development of tumors in rodents (14).
Thus, numerous studies indicate that N-acetylcysteine can pre-
vent mutation and cancer through a variety of mechanisms (15,
16). EUROSCAN, a randomized trial of a 2-year supplement
of retinyl palmitate and/or N-acetylcysteine in patients with
head and neck cancer or lung cancer, resulted in no benefit
(107). Relevantly, Sprong et al. (97) have reported that low-
dose N-acetylcysteine protects against endotoxin-mediated ox-
idative stress by scavenging H
2
O
2
, while higher doses may
have the opposite effect. We demonstrated that the content of
8-oxodG in HL-60 cells was increased by the N-acetylcysteine
treatment, but not in HP100 cells (73). Therefore, it is consid-
ered that generation of H
2
O
2
plays an important role in N-
acetylcysteine-induced 8-oxodG formation. Numerous studies
have indicated that the formation of 8-oxodG causes misrepli-
cation of DNA that may lead to mutation or cancer (90). The
formation of 8-oxodG in cellular DNA induced by N-
acetylcysteine is noteworthy in relation to the report that 8-
oxodG results in GT transversions. There is growing evidence
1734 KAWANISHI ET AL.
that compounds that are antioxidants at some concentrations
become prooxidants at other concentrations.
Phytic acid. Phytic acid (myo-inositol hexaphosphoric
acid) is present in plants, particularly in cereals, nuts, oil seed,
legumes, pollen, and spores (25, 89). In mammalian cells,
phytic acid and the lower inositol phosphates are present as in-
tracellular molecules (98). It has been reported that phytic acid
is chemopreventive in rodent colon and mammary carcinogen-
esis models, and in transplanted fibrosarcoma models (39, 91).
Phytic acid has been used as an antioxidant and could conceiv-
ably be a protective agent in the human diet (78). Our previous
study (57) revealed that phytic acid efficiently inhibited oxida-
tive DNA damage. Phytic acid decreased the formation of 8-
oxodG in cultured cells treated with the H
2
O
2
-generating sys-
tem using glucose oxidase, whereas phytic acid did not
influence the accumulation of H
2
O
2
. The formation of 8-oxodG
in calf thymus DNA by H
2
O
2
and Cu(II) was decreased by
phytic acid. Experiments using
32
P-labeled isolated DNA dem-
onstrated that phytic acid inhibited DNA damage by H
2
O
2
and
metals, although myo-inositol did not inhibit oxidative DNA
damage. Phytic acid and its derivatives bind metal ions such as
Cu(II), Zn(II), and Cd(II) in vitro (54). The present study has
revealed that the molar ratio between Cu(II) and phytic acid on
inhibition of oxidative DNA damage is about 3. This result is
supported by the work of Vohra et al. (108), who found that the
binding ratio of the moles of metal/moles of phytic acid was
about 3.5 at pH 7.5. These findings have led us to consider that
phytic acid containing six phosphates functions as a metal
chelator to inhibit the generation of highly reactive species
such as ·OH and Cu(I)–hydroperoxo complex from H
2
O
2
.
Binding of metals to phytic acid could facilitate the elimination
of potentially toxic heavy metals from the organism. Phytic
acid is expected to effectively inhibit oxidative DNA damage
Allyl isothiocyanate (AITC)
Benzyl isothiocyanate (BITC)
Phenethyl isothiocyanate (PEITC)
carcinogenic potential
H
2
H
2
C
=
C
–
C
-
N
=
C
=
S
H
H
2
C
-
N
=
C
=
S
C
–
C
-
N
=
C
=
S
H
2
H
2
initiation promotion
DNA damage
R–N=C=S
H
2O
+++
+++
++
+
+
R–N–C–SH
H
O
Cu(II)
Cu(I)
O2
-
O2
O2
H2
R–N–C–S•
H
O
Cu(I)OOH
DNA damage
A
B
FIG. 6. Carcinogenic potential and DNA damage induced by ITCs (A and B).
14098c31.pgs 11/16/05 3:37 PM Page 1734
by chelating Cu(II) as well as Fe(II) and Fe(III). We have con-
cluded that phytic acid may inhibit the generation of highly re-
active species from H
2
O
2
by chelating transition-metal ions, re-
sulting in prevention against cancer.
CONCLUSION
We summarize the conception about carcinogenesis and
chemoprevention on the basis of our results and literatures (Fig.
7). Most of the carcinogens yield ROS in the presence of en-
dogenous substances such as metal ions, NADH, and photosen-
sitizers (42, 43). In addition, infection and inflammation induce
several cancers via nitrative DNA damage with generation of
reactive nitrogen species (52, 79–81). Relevantly, chemopre-
vention with aspirin and other anti-inflammatory agents should
only be considered established (33), via inhibition of cyclooxy-
genase-2 and inducible nitric oxide synthase (12). ROS and re-
active nitrogen species induce DNA damage, resulting in muta-
tion and carcinogenesis, if DNA damage is not repaired.
Antioxidant chemopreventive agents without a prooxidant
property inhibit the carcinogenic process (Fig. 1A). Phytic acid
can protect DNA from ROS by chelating metals. Luteolin and
retinoic acid exert anticarcinogenic potency by scavenging ROS
and/or by specific manners. From the point of view of safety,
compounds lacking a prooxidant property are suitable for
chemoprevention, even if the antioxidant efficacy is mild. How-
ever, some chemopreventive agents exert prooxidant properties
after metabolic activation (-carotene, curcumin, etc.) or with-
out metabolic activation (-tocopherol, quercetin, catechins,
etc.). Trials to establish chemopreventive activity by antioxi-
dants have been inconclusive (33). Furthermore, dietary supple-
mentation of publicly available foods and the ingestion of spe-
cific supplements are usually applied for prolonged periods of
time. Therefore, we would like to emphasize the importance of
assessment for safety of chemopreventive agents.
EVALUATION FOR SAFETY OF ANTIOXIDANTS 1735
ACKNOWLEDGMENTS
This work was supported by the Japan Health Foundation,
the Health Research Foundation, and Grants-in-Aid for Sci-
entific Research from the Ministry of Education, Science,
Sports and Culture of Japan.
ABBREVIATIONS
AITC, allyl isothiocyanate; BITC, benzyl isothiocyanate;
ECG, epicatechin gallate; EGC, epigallocatechin; EGCG, epi-
gallocatechin gallate; ER, estrogen receptor; ERE, estrogen re-
sponse element; Fpg, formamidopyrimidine-DNA glycosylase;
GA, gallic acid; ITC, isothiocyanate; O
2
, superoxide; ·OH, hy-
droxyl radical; 8-oxodG, 8-oxo-7,8-dihydro-2-deoxyguano-
sine; PEITC, phenethyl isothiocyanate; PG, propyl gallate;
ROS, reactive oxygen species; topo II, topoisomerase II.
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Address reprint requests to:
Shosuke Kawanishi
Department of Environmental and Molecular Medicine
Mie University Graduate School of Medicine
2-174 Edobashi, Tsu, Mie, 514–8507, Japan
E-mail: kawanisi@doc.medic.mie-u.ac.jp
Received for publication May 19, 2005; accepted June 27,
2005.
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