Mutagenesis vol. 20 no. 1 pp. 15--22, 2005
1093/mutage/gei001
Effects of a-naphthyl isothiocyanate and a heterocyclic amine, PhIP, on
cytochrome P-450, mutagenic activation of various carcinogens and
glucuronidation in rat liver
Yukio Mori
, Akihiro Koide, Kenjiro Tatematsu,
Shigeyuki Sugie1 and Hideki Mori2
Laboratory of Radiochemistry, Gifu Pharmaceutical University, 6-1
Mitahora-higashi 5-chome, Gifu 502-8585, Japan, 1Department of Pathology,
Kanazawa Medical University, 1-1 Daigaku, Uchinada, Ishikawa 920-0293,
Japan and 2Department of Tumor Pathology, Graduate School of Medicine,
Gifu University, 1-1 Yanagido, Gifu 501-1194, Japan
To elucidate the mechanism underlying suppression by
a-naphthyl isothiocyanate (ANIT) of mammary carcino-
genesis induced by 2-amino-1-methyl-6-phenylimidazo
[4,5-b]pyridine (PhIP), we evaluated hepatic levels of cyto-
chrome P-450 (CYP) enzymes, mutagenic activation of
environmental carcinogens and UDP-glucuronyltransfer-
ase (UDPGT) activities in female Sprague--Dawley rats fed
a high fat diet. Immunoblot analyses revealed induction of
CYP1A1, newly found 51 and 53 kDa proteins and consti-
tutive CYP1A2 and 2B2 by intragastric treatment with
85 mg/kg PhIP eight times for 11 days. Although the extents
of induction were not as high as in the case of PhIP, 3 weeks
feeding of 400 p.p.m. ANIT induced CYP1A1 and the 51
and 53 kDa proteins. CP1A2 level was decreased by the
feeding of ANIT. The mutagenicity in strain TA98 of PhIP,
four other heterocyclic amines (HCAs) and benzo[a]pyrene
was greatly enhanced in the presence of liver S9 mix pre-
pared from rats pretreated with PhIP but not with ANIT.
The mutagenicities of these five HCAs were significantly
decreased in the presence of liver S9 from rats pretreated
with a combination of PhIP and ANIT as compared with
that pretreated with PhIP alone. The level of hepatic
CYP1A2, which is known to be involved in the metabolic
activation of PhIP, was consistently decreased in liver
microsomes from rats administered PhIP plus ANIT as
compared with that from rats administered PhIP alone.
On the other hand, UDPGT activity towards 4-nitrophenol
(4-NP) was enhanced using liver microsomes prepared
from rats pretreated with a combination of PhIP and
ANIT as compared with those pretreated with PhIP or
ANIT alone. These results show that chemoprevention
by ANIT against PhIP-induced rat mammary carcinogen-
esis can be explained by a dual action mechanism, i.e. a
reduction in metabolic activation by hepatic CYP1A2 and
an enhancement of detoxification by 4-NP UDPGT. The
role of the newly found 51 and 53 kDa proteins in activation
of HCAs is also discussed.
Introduction
Lifestyle factors are considered major risk factors in human
cancers and diet contributes to about 35% of the disease (Doll
and Peto, 1981). The adoption of Westernized dietary patterns
in Japan is most likely an important contributing factor to rising


To whom correspondence should be addressed. Tel: 181 58 237 3931; Fax: 181 58 237 5979; Email: ymori@gifu-pu.ac.jp
Mutagenesis vol. 20 no. 1 ß UK Environmental Mutagen Society 2005; all rights reserved.
doi:10.
rates of colorectal, prostate and mammary cancers (Wynder,
1991). Carcinogenic heterocyclic amines (HCAs) occur in
cooked foods (Sugimura, 1985; Wakabayashi et al., 1992),
with 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)
being reported to be the most abundant HCA at ~480 ng/g
cooked food (Felton et al., 2000), being detectable in 10
volunteers living in Tokyo (0.005--0.3 mg/person) (Wakabayashi
et al., 1997) and 3563 individuals in the USA (0.72--
1.11 mg/person) (Layton et al., 1995). PhIP has been demon-
strated to produce colorectal, prostate and mammary cancers in
rats (Ito et al., 1991; Shirai et al., 1997). It was also shown that
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N-hydroxylation of PhIP by cytochrome P-450 (CYP) 1A2
(Wallin et al., 1990) followed by O-acetylation (Ghoshal
et al., 1995) is the metabolic activation pathway, while ring
hydroxylation by CYP1A1 (Wallin et al., 1990) and
glucuronidation of N-hydroxy-PhIP (N-OH-PhIP) by UDP-
glucuronyltransferase (UDPGT) (Kaderlic et al., 1994a, 1994b)
are detoxification pathways.
Epidemiological studies have shown that high cruciferous
vegetable consumption is inversely related to lung (Verhoeven
et al., 1996) and bladder (Michaud et al., 1999) cancer risk.
Several isothiocyanates are known to occur as glucosinolates in
cruciferous vegetables, such as Japanese horseradish (wasabi),
oriental mustard, broccoli, Brussels sprouts, cabbage and cauli-
flower, and to be released upon hydrolysis of glucosinolates
by myrosinase (Zhang and Talalay, 1994). Naturally occurring
isothiocyanates, such as allyl isothiocyanate (AITC), benzyl
isothiocyanate (BITC), phenethyl isothiocyanate (PEITC) and
4-methylsulfinylbutyl isothiocyanate (sulforaphane), and
synthetic ones have been shown to inhibit chemically induced
skin, mammary gland, lung, forestomach, esophagus, liver and
bladder tumorigenesis (Hecht, 1999). a-Naphthyl isothio-
cyanate (ANIT) is the first isothiocyanate the chemopreventive
effect of which has been examined in rats and is reported
to inhibit liver tumors initiated by 30 -methyl-4-(N,N-
dimethylamino)azobenzene (Sasaki, 1963), ethionine or
N-2-fluorenylacetamide (Sidransky et al., 1966) and bladder
tumors initiated by N-butyl-N-(4-hydroxybutyl)nitrosamine
(Ito et al., 1974).
One of the mechanisms underlying the chemopreventive
effects of these isothiocyanates is related to the ability to
attenuate DNA alkylation by 4-(methylnitrosamino)-1-(3-
pyridyl)-1-butanone (Morse et al., 1989), N-nitrosomethyl-
benzylamine (Wilkinson et al., 1995) or N-nitrosobis
(2-oxopropyl)amine (Nishikawa et al., 1997). It has been
hypothesized that the decrease in DNA alkylation produced
by isothiocyanates may be attributed to inhibition of metabolic
activation reactions and/or induction of detoxifying enzymes.
However, different results for the effects of isothiocyanates on
hepatic CYP have been reported: both increases and decreases
in total CYP content, levels of CYP species and metabolic
activities specific to each CYP species have been observed
15
Y.Mori et al.
(Leonard et al., 1981; Guo et al., 1992, 1993; Manson et al.,
1997; Nishikawa et al., 1997). The activities of quinone reduc-
tase and glutathione S-transferase (GST) in the cytosol of cells
of several organs and hepatocytes from rodents are induced by
BITC, PEITC, AITC, sulforaphane (Zhang and Talalay, 1994)
and 6-methylsulfinylhexyl isothiocyanate (Hou et al., 2000;
Morimitsu et al., 2002). PEITC also enhances UDPGT activity
towards 4-nitrophenol (4-NP) in rat liver microsomes (Guo
et al., 1992), but BITC exhibits no significant effect on
UDPGT activities towards 4-methylumbelliferone and
chloramphenicol (Kassie et al., 2002).
It has been reported that four natural isothiocyanates,
including BITC, and three synthetic norbornyl isothiocyanates
structurally related to sulforaphane inhibit rat mammary
tumorigenesis induced by dimethylbenz[a]anthracene
(DMBA) (Wattenberg, 1977; Zhang and Talalay, 1994),
whereas that initiated by PhIP is not suppressed by BITC (Ino
et al., 1996). Nevertheless, the influence of other isothiocya-
nates on PhIP-induced mammary tumorigenesis remains to be
elucidated. Recently we found that ANIT markedly suppresses
mammary carcinogenesis when given in the initiation phase in
Sprague--Dawley rats induced by PhIP (Sugie et al., 2004). It
was shown that ANIT causes an increase in GST and quinone
reductase activities (Sugie et al., 2004) and a decrease in
total CYP content and the activities of ethoxycoumarin
O-deethylase, benzphetamine N-demethylase (Leonard et al.,
1981), aminopyrine demethylase and aniline hydroxylase
(Drew and Priestry, 1976) in rat liver. However, to our knowl-
edge no data have been provided on the effect of ANIT on
hepatic levels of CYP species, mutagenicity of typical carcino-
gens and UDPGT activities in any animal species.
In order to elucidate the mechanism(s) underlying suppres-
sion of PhIP-induced mammary carcinogenesis by ANIT,
hepatic levels of microsomal CYP enzymes known to activate
typical environmental carcinogens, mutagenic activation of
these carcinogens and two kinds of UDPGT activities were
assayed in female Sprague--Dawley rats treated with ANIT
and/or PhIP.
Materials and methods
Chemicals
Hydrochlorides of 2-amino-6-methyldipyrido[1,2-a:30 ,20 -d ]imidazole
(Glu-P-1) and PhIP, acetates of 3-amino-1-methyl-5H-pyrido[4,3-b]indole
(Trp-P-2) and 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAaC) and
2-amino-3-methylimidazo[4,5-f ]quinoline (IQ), benzo[a]pyrene (BP), N-
nitrosodimethylamine (DMN), 3-methylcholanthrene (MC), phenobarbital
(PB), 7,8-benzoflavone (7,8-BF), 4-NP, bilirubin and UDP-glucuronic acid
were purchased from Wako Pure Chemicals (Osaka, Japan). PhIP was also
purchased from the Nard Institute (Osaka, Japan). Glucose 6-phosphate (G6P),
G6P dehydrogenase (G6PDH), NADP 1, NADPH, NADH and ATP were
obtained from Oriental Yeast Co. (Tokyo, Japan) and furafylline and
7-pentoxyresorufin were from Sigma-Aldrich (Milwaukee, WI). Aflatoxin B1
(AFB1 ) was purchased from Makor Chemicals (Jerusalem, Israel) and ANIT
was from Nacalai Tesque (Kyoto, Japan). All other commercial products were
of the purest grade available. N-nitrosobis(2-hydroxypropyl)amine (BHP) was
synthesized in our laboratory as described previously (Mori et al., 1985).
Animal treatment and tissue preparation
Female 4- or 6-week-old Sprague--Dawley rats purchased from Japan SLC
(Hamamatsu, Japan) were housed in wire cages (2 or 3 rats/cage) and main-
tained under standard laboratory conditions. Twenty female rats, 6 weeks old,
were divided into four groups consisting of five animals. They were fed a
modified AIN-76A high fat diet containing 23.5% corn oil (Clea Japan, Tokyo,
Japan) throughout the experiment (Sugie et al., 2005). As shown in Figure 1,
rats in Groups 2 and 4 were given 400 p.p.m. ANIT in the diet starting at
6 weeks of age. Ten days after initiation of the respective diets Group 1 and 2
rats were given eight doses of corn oil via an intragastric tube for 11 days and
16
Group Treatment
1 vehicle
2 ANIT
3 PhIP
4 PhIP+ANIT
0 7 14 21(days)
Fig. 1. Experimental protocol. Clear, high fat diet; cross-hatched, high fat diet
with 400 p.p.m. ANIT; triangle, intragastric treatment with corn oil (2.5 ml/kg);
closed triangle, intragastric treatment with 85 mg/kg PhIP in corn oil.
Group 3 and 4 rats received 85 mg/kg PhIP in corn oil. All the animals were
decapitated 24 h after the last dose of the vehicle or PhIP. Alternatively,
18 female rats, 7 weeks old, were divided into six groups and given 85 mg/kg
PhIP, 30 mg/kg ANIT dissolved in corn oil or both orally as a single dose and
then were killed either 24 or 48 h after injection. Male Wistar rats were i.p.
injected once a day with PB (80 mg/kg) or MC (20 mg/kg) for 3 days and the
three animals each starved for 24 h after the last dose. Livers were perfused
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in situ with ice-cold sterile 1.15% KCl and 25% homogenates in 1.15% KCl
were prepared. Liver S9 and microsomes were prepared using established
procedures (Mori et al., 2002).
Western blots
Goat anti-rat polyclonal antibodies against CYP1A1, CYP1A2, CYP2B1,
CYP2B2, CYP2E1 and CYP3A2 (Daiichi Pure Chemicals Co., Tokyo, Japan)
were used as primary antibodies. Gel electrophoresis and blot analyses were
performed as described in detail previously (Koide et al., 1999) according
to the established methods of Laemmli (1970) and Towbin et al. (1979),
respectively.
Mutation assay
All tests were carried out using the Ames preincubation assay (Yahagi et al.,
1977). Two N-nitrosamines were dissolved in 100 ml of water and all the other
carcinogens in 50 ml of dimethyl sulfoxide. The mutagenicities of Trp-P-2,
Glu-P-1 and IQ (0.03 mg/plate), MeAaC (10 mg/plate), PhIP and BP (5 mg/
plate), AFB1 (1 mg/plate) and BHP and DMN (10 mg/plate) were checked in
the presence of liver S9 mix, using established procedures (Mori et al., 2001,
2002). The amount of liver S9 fraction was 10 ml/plate for the HCA, 50 ml for
BP and 150 ml for the N-nitrosamines and AFB1 . Salmonella typhimurium
tester strains TA100 and TA98 were employed for the two N-nitrosamines and
the other carcinogens, respectively. The S9 mix contained 4 mM NADPH,
4 mM NADH, 0.5 U G6PDH, 5 mM G6P and 5 mM ATP, except for the
N-nitrosamines, for which 4 mM NADP 1 and 5 mM G6P were used. In the
experiments on CYP inhibition 200 mM 7,8-BF and furafylline were
preincubated with carcinogen substrate and S9 mix (Mori et al., 2001).
Assay for pentoxyresorufin O-depentylase (PROD) activity
PROD activity in liver microsomes was assayed according to the method of
Burke et al. (1985).
Assay of UDPGT activity
UDPGT activities towards bilirubin and 4-NP in liver microsomes were
assayed according to the methods of Heirwegh et al. (1972) and Isselbacher
et al. (1962), respectively.
Results
Figure 2 shows immunoblots and levels (pmol/mg protein) of
microsomal CYP proteins in female Sprague--Dawley rats
treated with ANIT and PhIP for up to 3 weeks. Hepatic CYP1A2
was constitutively detected with an antibody against male rat
CYP1A1 and CYP1A2 in the vehicle group (Group 1). In
Group 2 rats fed 400 p.p.m. ANIT for 3 weeks four bands
corresponding CYP1A1 and CYP1A2 and 53 and 51 kDa
proteins were clearly found, but the CYP1A2 level was
decreased by 34% (P 5 0.05) relative to Group 1 rats. In
contrast, the constitutive CYP1A2 level was 3.8-fold higher in
Group 3 rats (85 mg/kg PhIP) than in Group 1 rats, and

Fig. 2. Immunoblots and densitometric determination of expression of CYP
protein in liver microsomes from female Sprague--Dawley rats treated with
ANIT, PhIP or both. Liver microsomes were pooled from five rats each
treated with vehicle (lane 1) or PhIP (lane 3) eight times for 11 days, ANIT
(lane 2) for 3 weeks or PhIP1ANIT (lane 4). Lanes 5 contains CYP standards
from male Sprague--Dawley rats treated with MC (A), PB (B and C) or
acetone (D). (A) and (B) contain 1.0 mg and (C) and (D) contain 0.4 mg
microsomal protein. The values represent means of pmol/mg microsomal
protein obtained from 4--8 experiments. *P 5 0.05 and **P 5 0.01,
compared with the vehicle group (lane 1); #P 5 0.01, compared with the
PhIP-treated group (lane 3) (Student’s t-test). n.d., not detected.
the same level of high induction of the 51 kDa protein and a
lesser induction of CYP1A1 and the 53 kDa protein were
observed. Combined treatment with ANIT (Group 4) decreased
CYP1A2 and the 53 and 51 kDa proteins by 63, 29 and 52% (P
5 0.01), respectively, compared with Group 3 rats, while this
treatment produced no significant decrease in CYP1A1 expres-
sion. CYP2B2 level was not increased in Group 2, but was 4.0-
fold higher (P 5 0.01) in Group 3 than in Group 1, and the
induced level of CYP2B2 was significantly decreased to almost
the constitutive level by combined treatment with ANIT1PhIP
(Group 4). CYP2B1 was not constitutively expressed and was
not induced in any group of rats and there were no significant
differences in either CYP3A2 or CYP2E1 level among the four
groups.
In order to confirm the new evidence for CYP induction and
suppression, the potency of ANIT and PhIP in modifying
CYP1A and CYP2B expression was further checked in liver
microsomes from female rats orally treated with 30 mg/kg
ANIT, corresponding to the daily intake in the diet, and 85
mg/kg PhIP as a single dose. As shown in Figure 3, CYP1A1
and the 53 and 51 kDa proteins were clearly induced in rats 24
h after ANIT treatment to the same or higher levels as Group 2
rats, while no significant alteration in the level of CYP1A2 was
observed. Forty-eight hours after ANIT treatment the levels of
CYP1A1 and the 53 kDa protein were markedly decreased and
Chemopreventive effects of a-naphthyl isothiocyanate
Fig. 3. Immunoblots and densitometric determination of expression of four
CYP1A proteins (A) and CYP2B1 and 2B2 (B) in liver microsomes from
female Sprague--Dawley rats orally treated with 30 mg/kg ANIT, 85 mg/kg
PhIP or both as a single dose. Liver microsomes were pooled from three rats
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each 24 and 48 h after treatment. Lanes MC and PB contain CYP standards
and the values represent the means as described in the legend to Figure 2.
*P 5 0.01, compared with the vehicle group; #P 5 0.01, compared with the
PhIP-treated group (Student’s t-test). n.d., not detected.
those of CYP1A2 and the 51 kDa protein were also decreased
to the same levels as in Group 2 rats. Similar inductions of the
four CYPs as in Group 3 rats were seen in rats 24 and 48 h after
PhIP treatment; CYP1A2 level was increased to 3.9-fold above
control, with the same level of the 51 kDa protein and a lesser
induction of CYP1A1 and the 53 kDa protein. Combined
treatment with ANIT decreased the induced levels of three
CYP1A-related proteins by 31--49%, compared with those in
rats 24 h after PhIP treatment. In rats 48 h after combined
treatment CYP1A1 was almost negligible and the other three
proteins were 54--62% lower compared with those after PhIP
treatment. On the other hand, there were no significant differ-
ences in CYP2B2 levels among the four groups of rats 24 h
after treatment, while that in rats 48 h after ANIT treatment was
decreased by 66% relative to the vehicle group. CYP2B2 in rats
48 h after PhIP treatment was 3.8-fold above control and that
in rats 48 h after combined treatment with ANIT1PhIP was
decreased by 66% relative to the PhIP-treated rats.
To confirm the mutagenic activation induction characteris-
tics of ANIT and PhIP, 9 carcinogens which are known to be
metabolically activated by CYP1A1, CYP1A2, CYP2B1,
CYP2B2, CYP2E1 and CYP3A2 were assayed in Salmonella
strains TA98 and TA100. Figure 4 shows the mutagenic activ-
ities of five HCAs including PhIP, BP, AFB1 , BHP and DMN
in the presence of liver S9 mix from female rats treated with
ANIT and/or PhIP for up to 3 weeks (Groups 2--4). The num-
bers of revertant colonies/plate after subtraction of spontaneous
rates (TA98, 20; TA100, 153) with liver S9 mix from Group 1 rats
were 25 6 6 (mean 6 SD) for Glu-P-1, 68 6 18 for IQ, 117 6
23 for PhIP, 46 6 7 for Trp-P-2, 53 6 7 for MeAaC, 58 6 5
for BP and 2630 6 99 for AFB1 in strain TA98 and 109 6 20
for BHP and 238 6 8 for DMN in strain TA100. No enhancing
effects on mutagenicity were observed with all the carcinogens
tested in Group 2 rats. On the other hand, mutagenic activities of
five HCAs and BP were increased by PhIP treatment (Group 3),
by 2.8- to 12.9-fold (P 5 0.01) relative to those in Group 1
rats, but no significant alterations in mutagenicity were
observed with AFB1 , BHP and DMN. In Group 4 rats
17
Y.Mori et al.

16

Ratio to mutagenic activities obtained with Group1

14
∗
∗
12

10 ∗

8
## ##
6 ## ∗
∗
4
## ∗
##
#
2

0
Glu-P-1 IQ PhIP Trp-P-2 MeAαC BP AFB1 BHP DMN

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Fig. 4. Mutagenic activities of various carcinogens in the TA98 strain (HCAs, BP and AFB1 ) and TA100 strain (BHP and DMN) in the presence of liver S9
from female Sprague--Dawley rats treated with ANIT, PhIP or both. Liver S9 was pooled from five rats each treated with ANIT (Group 2) (lightly stippled),
PhIP (Group 3) (heavily stippled) or PhIP1ANIT (Group 4) (cross-hatched). Each test was carried out in duplicate (4--8 plates) and the activities represent
the means 6 SD of the ratio to the mutagenic activity obtained with the vehicle group (Group 1). *P 5 0.01, compared with the vehicle group; #P 5 0.05 and
##
P 5 0.01, compared with the PhIP-treated group (Group 3) (Student’s t-test).

the induced activities of Glu-P-1, IQ, PhIP and Trp-P-2 were

decreased to almost half or less those in Group 3 rats, while the Table I. PROD activity in liver microsomes from female Sprague--Dawley
rats treated with 85 mg/kg PhIP eight times for 11 days or 85 mg/kg PhIP and
activities of MeAaC and BP were slightly but significantly 30 mg/kg ANIT as a single dose
decreased (20%, P 5 0.05). The combined treatment produced
no significant changes in mutagenicity of AFB1 , BHP and Treatment PROD activity
DMN. (pmol/min/mg protein)
Because induction of mutagenic activation of BHP and Repeated treatment
DMN by PhIP and suppression by ANIT, which are known to Vehicle (Group 1) 10.1 6 0.2
be catalyzed by CYP2B1 and CYP2B2, was not demonstrated, PhIP (Group 3) 47.3 6 6.7a (4.7)
PROD activity was assayed in liver microsomes from female Single treatment
Vehicle 10.7 6 1.9
rats after a single or repeated treatments with PhIP. As shown PhIP 55.4 6 9.3a (5.2)
in Table I, PROD activity in Group 3 rats was increased to 4.7- PhIP1ANIT 20.2 6 1.7a;b (1.9)
fold above that in Group 1 rats (P 5 0.01). The activity in rats
48 h after PhIP treatment as a single dose was almost equal The results are expressed as means 6 SD of 3--7 experiments with liver
to that in Group 3 rats and that after combined treatment with microsomes from five rats 24 h after repeated treatment and from three rats
48 h after single treatment. Values in parentheses show the ratio to the
ANIT was decreased by 64% relative to PhIP-treated rats. activity obtained with each vehicle group.
Induction of PROD activity by PB was confirmed in male a
P 5 0.01, compared with each vehicle group (Student’s t-test).
b
rats, an increase to 57.2-fold (612 6 13.2 pmol/min/mg P 5 0.01, compared with PhIP group (Student’s t-test).
protein) above control.
In an attempt to obtain more information about CYP1A1 and 4-NP in liver microsomes (Groups 1--4). There were no sig-
CYP1A2, responsible for PhIP activation, typical CYP nificant differences in UDPGT activity towards bilirubin
inducers and selective inhibitors were used in mutagenic acti- among the four groups. In contrast, UDPGT activity towards
vation assays. Figure 5 shows the effects of MC, PB and two 4-NP in Group 2 and 3 rats was increased to 1.6- and 2.2-fold
inhibitors specific to the CYP1A subfamily on the mutagenic above the vehicle control (Group 1), respectively, and the
activity of PhIP in the presence of liver S9 fraction from either combined treatment (Group 4) showed much higher induction
Wistar or Sprague--Dawley rats. Treatment of male Wistar rats (4.7-fold).
with MC caused a 14.5-fold increase in their activity, while PB
treatment produced no induction. Furafylline and 7,8-BF inhib-
Discussion
ited mutagenic activity in the presence of liver S9 mix from
male rats treated with MC by 82 and 98%, respectively. In the Marked induction by MC and dramatic inhibition by furafyl-
presence of liver S9 mix from female rats of Group 3, in which line and 7,8-BF of the mutagenic activity of PhIP indicate
the activity of PhIP was increased 5.1-fold (Figure 4) above the involvement of CYP1A1 and CYP1A2 (predominantly
the vehicle control, furafylline and 7,8-BF caused 64 and 85% CYP1A2) in metabolic activation of PhIP by liver S9 fraction
inhibition, respectively. from male and female rats. Selective enhancement by PhIP of
Table II summarizes the effects of PhIP and ANIT treatment the mutagenicities of five HCAs in the presence of liver S9 mix
for up to 3 weeks on UDPGT activity towards bilirubin and reflects selective induction of hepatic CYP1A1 and CYP1A2,

18
 Chemopreventive effects of a-naphthyl isothiocyanate

3000 100
A B
furafylline
2500
80 7,8-benzoflavone

TA 98 revertants/plate
2000

% of control
60
1500
40
1000

20
500

0 0
none MC PB MC PhIP
Wistar ( ) Wistar ( ) SD ( )

Fig. 5. Effects of CYP inducer (A) and inhibitor (B) on the mutagenic activity of PhIP with liver S9 from Wistar or Sprague--Dawley rats. Each bar represents
the mean 6 SD (4--8 plates). The mutagenic activities in (B) were compared with incubation in the absence of CYP inhibitor as shown in (A) for male rats
and with the activitiy (565 6 94 revertants/plate) obtained with female rats treated with PhIP in Group 3.

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Table II. UDPGT activities in liver microsomes from female
Sprague--Dawley rats repeatedly treated with PhIP, ANIT or both for up to 3 weeks
Group Treatment UDPGT activity (nmol/min/mg protein)
Bilirubin
1 Vehicle 0.20 6 0.03
2 ANIT 0.18 6 0.02 (0.9)
3 PhIP 0.17 6 0.01 (0.9)
4 PhIP1ANIT 0.22 6 0.02 (1.1)
Each test was carried out with liver microsomes pooled from five rats. The
results are expressed as means 6 SD of 3--5 experiments. Values in
parentheses show the ratio to the activity obtained with the vehicle group
(Group 1).
a
P 5 0.05, compared with Group 1 (Student’s t-test).
b
P 5 0.01, compared with Group 1 (Student’s t-test).
especially CYP1A2. These results are in agreement with pre-
vious findings that hepatic CYP1A1 and CYP1A2 are involved
in metabolic activation of a number of HCAs (Degawa et al.,
1988) and that several HCAs, including PhIP, induce hepatic
CYP1A subfamily members, especially CYP1A2, in male
F344 rats (Degawa et al., 1989; Mori et al., 2003). It has
been reported that the 51 kDa protein, in addition to CYP1A1
and CYP1A2, is induced by PhIP in liver microsomes from
male F344 rats (Adachi et al., 1991), but not by MeIQx (Mori
et al., 2003), seven other HCAs (Degawa et al., 1989) and
4-aminoazobenzene derivatives (Degawa et al., 1986). In this
study it has been demonstrated that PhIP can induce the 53 kDa
protein in addition to these three CYP1A-related proteins in
liver microsomes from female Sprague--Dawley rats. The 51
kDa protein induced by PhIP has been reported to contribute
to mutagenic activation of Glu-P-1 and Trp-P-2 (Adachi et al.,
1991). Recently we found that MeIQx enhances the mutagenic
activities of several HCAs, but not MeAaC and BP, which are
predominantly activated by rat CYP1A1 (Degawa et al., 1988),
in spite of induction of rat hepatic CYP1A1 (one-fifth of the
induced CYP1A2 level) (Mori et al., 2003). In contrast, PhIP
caused a significant increase in the mutagenic activities of both
carcinogens, although the hepatic CYP1A1 level in female
Sprague--Dawley rats treated with PhIP is almost equal to that
in male F344 rats treated with MeIQx. Accordingly, these
findings suggest that the 53 and 51 kDa proteins may contribute
to the activation of HCAs and BP.
4-Nitrophenol
We reported that PB increases the mutagenic activity of BHP
to 4.4-fold (Mori et al., 1985), hepatic CYP2B2 to 18.3-fold
12.9 6 2.2 above control and CYP2B1 to 1.9-fold above the induced level
20.5 6 4.7a (1.6) of CYP2B2 (Mori et al., 2001) in male rats. PB increased
28.5 6 0.6 (2.2)
60.1 6 6.5b (4.7)
PROD activity to 57.2-fold above the vehicle control in male
rats, while PhIP induced hepatic CYP2B2, but not CYP2B1,
to ~4-fold and increased PROD activity to ~5-fold above the
respective controls in female rats. Therefore, it is reasonable
that liver S9 fraction from female rats treated with PhIP repeat-
edly or as a single dose could not enhance the mutagenicity
of BHP, indicating insufficient induction of CYP2B2 and
CYP2B1 for activation. Nevertheless, this is the first demon-
stration that HCA can induce hepatic CYP2B2 and PROD
activity in rats. DMN is mutagenetically activated by rat
CYP2E1, CYP2B1 and CYP2B2 (Yang et al., 1987; Mori
et al., 2001) and AFB1 by CYP2B1 and CYP3A2 (Mori et al.,
2001). Thus, it seems reasonable that PhIP has no effect on the
mutagenic activation of two carcinogens, reflecting no induc-
tion of hepatic CYP2B1, CYP2E1 and CYP3A2 in female rats.
It has been reported that feeding of 0.1% PEITC for 2 weeks
induces CYP1A1, CYP1A2, CYP2B1 and CYP2B2, but not
CYP2E1 and CYP3A2, in male Fisher rats (Manson et al.,
1997). Intragastric treatment of male F344 rats with 1 mmol/kg
PEITC also induces CYP2B1, but not CYP2B2 and CYP2E1
(Guo et al., 1992), while intragastric treatment of female ham-
sters with 2.5 mmol/kg PEITC had no effects on CYP1A2,
CYP2B, CYP2E1 and CYP3A (Nishikawa et al., 1997). How-
ever, to our knowledge, there are no reports on either suppres-
sion or induction of CYP isoforms by other isothiocyanates in
any animal species. Feeding a 400 p.p.m. ANIT-containing diet
for 3 weeks and intragastric treatment as a single dose pro-
duced a decrease in hepatic CYP1A2 but a clear increase in
three other CYP1A-related proteins; these did not affect the
mutagenic activities of five HCAs and BP in ANIT-treated rats,
indicating that these changes may be insufficient for mutagenic
activation. On the other hand, no significant change was

19
Y.Mori et al.
observed in hepatic levels of CYP2B1, CYP2B2, CYP2E1 and
CYP3A2, in accord with the observation of no induction of the
mutagenic activities of BHP, DMN and AFB1 . However, these
results are not consistent with the previous finding that feeding
of 220--1000 p.p.m. ANIT for 2--6 weeks exerts a clear sup-
pressive effect on metabolic activities specific to CYP1A1,
CYP2B1 and CYP2B2 in liver microsomes from male F344
rat (Leonard et al., 1981). Further, PEITC causes a decrease in
metabolic activities specific to CYP1A1, CYP1A2, CYP2B1,
CYP2B2, CYP2E1 and CYP3A2 in male F344 rats 6 h after
treatment, but an increase in those specific to CYP1A1,
CYP1A2, CYP2B1 and CYP2B2 in the same animals 24 h
after treatment (Guo et al., 1993). BITC, phenylbutyl isothio-
cyanate and phenylhexyl isothiocyanate show similar effects
on these metabolic activities, except for the CYP1A1, CYP1A2
and CYP2B1 activities (Guo et al., 1993). The reasons for these
discrepancies are currently unknown, but it is suggested that
the differences might be due to experimental conditions such as
timing of death, treatment regimen (i.e. one or several applica-
tions, a conventional or high fat diet), age and/or sex of the
animals, metabolic substrates, etc. However, our results sup-
port the previous finding that feeding 600 p.p.m. ANIT for 24
weeks produces no significant effect on hepatocarcinogenesis
in male rats initiated with N-nitrosodiethylamine (Makiura
et al., 1973), which is known to be activated by CYP2E1,
CYP2B1 and CYP2B2 (Mori et al., 2002).
The combination of ANIT and PhIP caused a marked
decrease in the PhIP-induced level of hepatic CYP1A2, in
accord with the observation of a marked decrease in the
PhIP-induced mutagenic activities of Glu-P-1, IQ and PhIP.
Although ANIT slightly induced CYP1A1 and the 51 and 53
kDa proteins, the combination with PhIP caused a significant
decrease in hepatic levels of these proteins and the mutagenic
activities of MeAaC and BP compared with those in PhIP-
treated rats. This is, to our knowledge, the first observation of
suppression by an isothiocyanate of highly induced levels of
CYP and mutagenic activation of HCAs in rat liver. Thus,
ANIT may suppress carcinogenesis by other HCAs or carcino-
gens which can induce CYP1A1 and CYP1A2 and are selec-
tively activated by the CYP1A subfamily, including newly
found proteins, through a dramatic inhibition of metabolic
activation in the liver. ANIT also decreased PhIP-induced
levels of CYP2B2 and PROD activity, suggesting the
possibility of suppressing tumorigenesis initiated with
carcinogens which are metabolically activated by CYP2B2.
PhIP--glutathione conjugate is not found in the bile and urine
of rats (Alexander et al., 1991) and N-OH-PhIP is not conju-
gated by hepatic GST (Kaderlik et al., 1994b). N-OH-PhIP N3-
glucuronide and N-OH-PhIP N2 -glucuronide are the major
metabolites of PhIP in bile and urine of rats (Alexander et al.,
1991; Kaderlik et al., 1994a). d-Galactosamine markedly
decreases the formation of the two N-glucuronides of N-OH-
PhIP and increases the formation of DNA adducts and
unscheduled DNA synthesis (Kaderlik et al., 1994b), indicat-
ing that N-glucuronidation of PhIP is an important detoxifica-
tion reaction. The UDPGT1A subfamily is predominantly
involved in the biotransformation of N-OH-PhIP (Nowell
et al., 1999). PhIP clearly enhanced hepatic 4-NP UDPGT
activity in female rats, but not bilirubin UDPGT activity, con-
sistent with previous findings in male rats treated with MeIQx
(Mori et al., 2003), suggesting that UDPGT1A may be
involved in glucuronidation of N-hydroxy-HCAs. Induction
of UDPGT activity towards 4-NP by ANIT is in agreement
20
with that by PEITC (Guo et al., 1992), however, the much
higher induction of 4-NP UDPGT activity in combination with
PhIP implies promotion of detoxification of N-OH-PhIP. BITC
is reported to suppress rat mammary tumors initiated by
DMBA, but not those initiated by PhIP (Ino et al., 1996).
DMBA is detoxified by quinone reductase (Long et al.,
2001), GST and UDPGT (Liu et al., 1994), and BITC clearly
induces hepatic GST (Vos et al., 1988) and quinone reductase
(Guo et al., 1993) activities, but not UDPGT1A (Kassie et al.,
2002). Accordingly, BITC may be unable to inhibit PhIP-
induced rat mammary tumorigenesis due to a lack of ability
to induce hepatic UDPGT1A.
In conclusion, the present study has demonstrated that PhIP
and ANIT have a bifunctional action, with induction of CYP1A
proteins and UDPGT activity and that suppression by ANIT of
PhIP-induced mammary carcinogenesis in rats can be attribu-
ted to a dual action mechanism: a decrease in metabolic activa-
tion of PhIP, predominantly by hepatic CYP1A2, and an
increase in detoxification by 4-NP UDPGT, but not by
CYP1A1. Together with the findings that PEITC and sulfora-
Downloaded from http://mutage.oxfordjournals.org/ by guest on November 15, 2015
phane block the increase in cell proliferation induced by
N-nitrosobis(2-oxopropyl)amine in its target organs in ham-
sters (Nishikawa et al., 1997) and induce apoptosis (Huang
et al., 1998; Misiewicz et al., 2003), it is suggested that iso-
thiocyanates are expected to affect chemically induced carci-
nogenesis through multiple mechanisms.
References
Adachi,H., Degawa,M., Miura,S., Hashimoto,Y., Sugimura,T. and Esumi,H.
(1991) Induction of putative new cytochrome P450 isozyme in rat liver by
2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. Biochem. Biophys. Res.
Commun., 174, 797--803.
Alexander,J., Wallin,H., Rossland,O.J., Solberg,K.E., Holme,J.A., Becher,G.,
Andersson,R. and Grivas,S. (1991) Formation of a glutathione conjugate and
a semistable transportable glucuronide conjugate of N2 -oxidized species of
2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in rat liver.
Carcinogenesis, 12, 2239--2245.
Burke,M.D., Thompson,S., Elcombe,C.R., Halpert,J., Haaparanta,T. and
Mayer,R.T. (1985) Ethoxy-, pentoxy- and benzyloxyphenoxazones and
homologues: a series of substrates to distinguish between different
induced cytochrome P-450. Biochem. Pharmacol., 34, 3337--3345.
Degawa,M., Kojima,M., Sato,Y. and Hashimoto,Y. (1986) Induction of a high
spin form of microsomal cytochrome P-448 in rat liver by 4-aminoazo-
benzene derivatives. Biochem. Pharmacol., 35, 3565--3570.
Degawa,M., Ueno,H., Miura,S., Ohta,A. and Namiki,M. (1988) A simple
method for assessment of rat cytochrome P-448 isozymes responsible for
the mutagenic activation of carcinogenic chemicals. Mutat. Res., 203,
333--338.
Degawa,M., Tanimura,S., Agatsuma,T. and Hashimoto,Y. (1989)
Hepatocarcinogenic heterocyclic aromatic amines that induce cytochrome
P448 isozymes, mainly cytochrome P448H (P-4501A2), responsible for
mutagenic activation of the carcinogens in rat liver. Carcinogenesis, 10,
1119--1122.
Doll,R. and Peto,R. (1981) The causes of cancer: quantitative estimates of
avoidable risks of cancer in the United States today. J. Natl Cancer Inst., 66,
1191--1308.
Drew,R. and Priestry,B.G. (1976) Microsomal drug metabolism during
a-naphthylisothiocyanate induced cholestasis. Toxicol. Appl. Pharmacol.,
35, 491--499.
Felton,J.S., Jagerstad,M., Knize,M.G., Skog,K. and Wakabayashi,K. (2000)
Contents in foods, beverages and tobacco. In Nagao,M. and T. Sugimura
(eds), Food Borne Carcinogens: Heterocyclic Amines. John Wiley & Sons,
Chichester, UK.
Ghoshal,A.G., Davis,C.D., Schut,H.A.J. and Synderwine,E.G. (1995) Possible
mechanisms for PhIP-DNA adduct formation in the mammary gland of
female Sprague--Dawley rats. Carcinogenesis, 16, 2725--2731.
Guo,Z., Smith,T.J., Wang,E., Sadrieh,N., Ma,Q., Thomas,P.E. and Yang,C.S.
(1992) Effects of phenethyl isothiocyanate, a carcinogenesis inhibitor, on

xenobiotic-metabolizing enzymes and nitrosamine metabolism in rats.
Carcinogenesis, 13, 2205--2210.
Guo,Z., Smith,T.J., Wang,E., Eklind,K.L., Chung,F.-L. and Yang,C.S. (1993)
Structure--activity relationships of arylalkyl isothiocyanates for the inhibi-
tion of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone metabolism and
the modulation of xenobiotic-metabolizing enzymes in rats and mice.
Carcinogenesis, 14, 1167--1173.
Hecht,S.S. (1999) Chemoprevention of cancer by isothiocyanates, modifiers of
carcinogen metabolism. J. Nutr., 129, 768S--774S.
Heirwegh,K.P., Van de Vijver,M. and Fevery,J. (1972) Assay and properties
of dititonin-activated bilirubin uridine diphosphate glucuronyltransferase
from rat liver. Biochem. J., 129, 605--618.
Hou,D.X., Fukuda,M., Fujii,M. and Fuke,Y. (2000) Transcriptional regulation
of nicotinamide adenine dinucleotide phosphate: quinone oxidoreductase in
murine hepatoma cells by 6-(methylsufinyl)hexyl isothiocyanate, an active
principle of wasabi (Eutrema wasabi Maxim). Cancer Lett., 161, 195--200.
Huang,C., Ma,W.-Y, Li,J., Hecht,S.S. and Dong,Z. (1998) Essential role of
p53 in phenethyl isothiocyanate-induced apoptosis. Cancer Res., 58,
4102--4106.
Ino,N., Sugie,S., Ohnishi,M. and Mori,H. (1996) Lack of inhibitory effect
of benzyl isothiocyanate on 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyr-
idine (PhIP)-induced mammary carcinogenesis in rats. J. Toxicol. Sci., 21,
189--194.
Isselbacher,K.J., Chrabas,M.F. and Quinn,R.C. (1962) The solubilization and
partial purification of a glucuronyl transferase from rabbit liver microsomes.
J. Biol. Chem., 237, 3033--3036.
Ito,N., Matayoshi,K., Matsumura,K., Denda,A., Kani,T., Arai,M. and
Makiura,S. (1974) Effect of various carcinogenic and non-carcinogenic
substances on development of bladder tumors in rats induced by N-butyl-
N-(4-hydroxybutyl)nitrosamine. Jpn. J. Cancer Res., 65, 123--130.
Ito,N., Hasegawa,R., Sano,M., Tamano,S., Esumi,H., Takayama,S. and
Sugimura,T. (1991) A new colon and mammary carcinogen in cooked
food, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP).
Carcinogenesis, 12, 1503--1506.
Kaderlik,K.R., Minchin,R.F., Mulder,G.J., Ilett,K.F., Daugaard-Jenson,M.,
Teitel,C.H. and Kadlubar,F.F. (1994a) Metabolic activation pathway for
the formation of DNA adducts of the carcinogen 2-amino-1-methyl-6-
phenylimidazo[4,5-b]pyridine (PhIP) in rat extrahepatic tissues.
Carcinogenesis, 15, 1703--1709.
Kaderlik,K.R., Mulder,G.J., Shaddock,J.G., Casciano,D.A., Teitel,C.H. and
Kadlubar,F.F. (1994b) Effect of glutathione depletion and inhibition of
glucuronidation and sulfation on 2-amino-1-methyl-6-phenylimidazo[4,5-b]
pyridine (PhIP) metabolism, PhIP--DNA adduct formation and unsched-
uled DNA synthesis in primary rat hepatocytes. Carcinogenesis, 15,
1711--1716.
Kassie,F., Rabot,S., Uhl,M., Huber,W., Qin,H.M., Helma,C., Schulte-
Hermann,R. and Knasm€uller,S. (2002) Chemopreventive effects of garden
cress (Lepidium sativum) and its constituents toward 2-amino-3-methyl-
imidazo[4,5-f]quinoline (IQ)-induced genotoxic effects and colonic
preneoplastic lesions. Carcinogenesis, 23, 1155--1161.
Koide,A., Fuwa,K., Furukawa,F., Hirose,M., Nishikawa,A. and Mori,Y.
(1999) Effect of cigarette smoke on the mutagenic activation of
environmental carcinogens by rodent liver. Mutat. Res., 428, 165--176.
Laemmli,U.K. (1970) Cleavage of structural proteins during the assembly of
the head of bacteriophage T4. Nature, 227, 680--685.
Layton,D.W., Bogen,K.T., Knize,M.G., Hatch,F.T., Johnson,V.M. and
Felton,J.S. (1995) Cancer risk of heterocyclic amines in cooked foods: an
analysis and implications for research. Carcinogenesis, 16, 39--52.
Leonard,T.B., Popp,J.A., Graichen,M.E. and Dent,J.G. (1981) a-
Naphthylisothiocyanate induced alterations in hepatic drug metabolizing
enzymes and liver morphology: implications concerning anticarcinogenesis.
Carcinogenesis, 2, 473--482.
Liu,J.Z., Zhang,B.Z. and Milner,J.A. (1994) Dietary selenite modifies
glutathione metabolism and 7,12-dimethylbenz(a)anthracene conjugation
in rats. J. Nutr., 124, 172--180.
Long,D.J.,II, Waikel,R.L., Wang,X.J., Roop,D.R. and Jaiswal,A.K. (2001)
NAD(P)H:quinone oxidoreductase 1 deficiency and increased susceptibility
to 7,12-dimethylbenz[a]anthracene-induced carcinogenesis in mouse skin.
J. Natl Cancer Inst., 93, 1166--1170.
Makiura,S., Kamamoto,Y., Sugihara,S., Hirao,K., Hiasa,Y., Arai,M. and Ito,N.
(1973) Effect of 1-naphthylisothiocyanate and 3-methylcholanthrene on
hepatocarcinogenesis in rats treated with diethylnitrosamine. Gann, 64,
101--104.
Manson,M.M., Ball,H.W.L., Barrett,M.C., Clark,H.C., Judah,D.J.,
Williamson,G. and Neal,G.E. (1997) Mechanism of action of dietary
chemoprotective agents in rat liver: induction of phase I and II drug
Chemopreventive effects of a-naphthyl isothiocyanate
metabolizing enzymes and aflatoxin B1 metabolism. Carcinogenesis, 18,
1729--1738.
Michaud,D.S., Spiegelman,D., Clinton,S.K., Rimm,E.B., Willett,W.C. and
Giovannucci,E.L. (1999) Fruit and vegetable intake and incidence of
bladder cancer in a male prospective cohort. J. Natl Cancer Inst., 91,
605--613.
Misiewicz,I., Skupinska,K. and Kasprzycka-Guttman,T. (2003) Sulforaphane
and 2-oxohexyl isothiocyanate induce cell growth arrest and apoptosis
in L-1210 leukemia and ME-18 melanoma cells. Oncol. Rep., 10,
2045--2050.
Mori,Y., Yamazaki,H., Toyoshi,K., Makino,T., Obara,T., Yokose,Y. and
Konishi,Y. (1985) Mutagenic activation of carcinogenic N-nitrosopropyl-
amines by rat liver: evidence for a cytochrome P-450 dependent reaction.
Carcinogenesis, 6, 415--420.
Mori,Y., Koide,A., Fuwa,K. and Kobayashi,Y. (2001) N-benzylimidazole for
preparation of S9 fraction with multi-induction of metabolizing enzymes
in short-term genotoxicity assays. Mutagenesis, 16, 479--486.
Mori,Y., Koide,A., Kobayashi,K., Morimura,K., Kaneko,M. and Fukushima,S.
(2002) Effect of ethanol treatment on metabolic activation and detoxifica-
tion of esophagus carcinogenic N-nitrosamines in rat liver. Mutagenesis, 17,
251--256.
Mori,Y., Koide,A., Kobayashi,Y., Furukawa,F., Hirose,M. and Nishikawa,A.
(2003) Effects of cigarette smoke and a heterocyclic amine, MeIQx on
cytochrome P-450, mutagenic activation of various carcinogens and
Downloaded from http://mutage.oxfordjournals.org/ by guest on November 15, 2015
glucuronidation in rat liver, Mutagenesis, 18, 87--93.
Morimitsu,Y., Nakagawa,Y., Hayashi,K. Fujii,H., Kumagai,T., Nakamura,Y.
Horio,F., Itoh,K., Iida,K., Yamamoto,M. and Uchida,K. (2002)
A sulforaphane analogue that potently activates the Nrf2-dependent
detoxification pathway. J. Biol. Chem., 277, 3456--3463.
Morse,M.A., Amin,S.G., Hecht,S.S. and Chung,F.L. (1989) Effects of
aromatic isothiocyanates on tumorigenicity, O6-methylguanine formation
and metabolism of the tobacco-specific nitrosamine 4-(methylnitrosamino)-
1-(3-pyridyl)-1-butanone in A/J mouse lung. Cancer Res., 49, 2894--2897.
Nishikawa,A., Lee,I.-S., Uneyama,C., Furukawa,F., Kim,H.-C., Kasahara,K.,
Huh,N. and Takahashi,M. (1997) Mechanistic insights into chemopreven-
tive effects of phenethyl isothiocyanate in N-nitrosobis(2-oxopropyl)amine-
treated hamsters. Jpn. J. Cancer Res., 88, 1137--1142.
Nowell,S.A., Massengill,J.S., Williams,S., Radominska-Pandya,A.,
Tephly,T.R., Cheng,Z., Strassburg,C.P., Tukey,R.H., MacLead,S.L.,
Lang,N.P. and Kadlubar,F.F. (1999) Glucuronidation of 2-hydroxyamino-
1-methyl-6-phenylimidazo[4,5-b]pyridine by human microsomal UDP-
glucuronosyltransferases: identification of specific UGT1A family isoforms
involved. Carcinogenesis, 20, 1107--1114.
Sasaki,S. (1963) Inhibitory effects by alpha-naphthylisothiocyanate on
development of hepatoma in rats treated with 30 -methyl-4-
dimethylaminoazobenzene. J. Nara Med. Assoc., 14, 101--115.
Shirai,T., Sano,M., Tamano,S. Takahashi,S., Hirose,M., Futakuchi,M.,
Hasegawa,R., Imaida,K., Matsumoto,K., Wakabayashi,K., Sugiura,T. and
Ito,N. (1997) The prostate: a target for carcinogenicity of 2-amino-1-
methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) derived from cooked foods.
Cancer Res., 57, 195--198.
Sidransky,H., Ito,N. and Verney,E. (1966) Influence of a-naphthylisothio-
cyanate on liver tumorigenesis in rats ingesting ethionine and N-2-
fluorenylacetamide. J. Natl Cancer Inst., 37, 677--686.
Sugie,S., Ohnishi,M., Ushida,J. Wakabayashi,K., Yamamoto,T., Hara,A.,
Koide,A., Mori,Y., Tanaka,T., Kohno,H., Suzuki,R. and Mori,H. (2004)
Effect of a-naphthylisothiocyanate on 2-amino-3-methylimidazo[4,5-b]pyr-
idine (PhIP)-induced mammary carcinogenesis in rats. Int. J. Cancer,
in press.
Sugimura,T. (1985) Carcinogenicity of mutagenic heterocyclic amines formed
during cooking process. Mutat. Res., 150, 33--41.
Towbin,H., Staehelin,T. and Gordon,J. (1979) Electrophoretic transfer of
proteins from polyacrylamide gels to nitrocellulose sheets: procedure and
some applications. Biochemistry, 76, 4350--4354.
Verhoeven,D.T.H., Goldbohm,R.A., van Poppel,G., Verhagen,H. and
Brandt,P.A. (1996) Epidemiological studies on Brassica vegetables and
cancer risk. Cancer Epidemiol. Biomarkers Prev., 5, 733--748.
Vos,R.M., Snoek,M.C., van Berkel,W.J., Muller,F. and van Bladeren,P.J.
(1988) Differential induction of rat hepatic glutathione S-transferase
isoenzymes by hexachlorobenzene and benzyl isothiocyanate. Comparison
with induction by phenobarbital and 3-methylcholanthrene. Biochem.
Pharmacol., 37, 1077--1082.
Wakabayashi,K., Nagao,M., Esumi,H. and Sugimura,T. (1992) Food-derived
mutagens and carcinogens. Cancer Res., 52 (suppl.), 2092s--2098s.
Wakabayashi,K., Totsuka,Y., Fukutome,K., Oguri,A., Ushiyama,H. and
Sugimura,T. (1997) Human exposure to mutagenic/carcinogenic
21
Y.Mori et al.

heterocyclic amines and comutagenic beta-carbolines. Mutat. Res., 376,

253--259.
Wallin,H., Mikalsen,A., Guengerich,F.P., Ingelman-Sundberg,M.,
Solberg,K.E., Rossland,O.R. and Alexander,J. (1990) Differential rates
of metabolic activation and detoxification of the food mutagen 2-amino-
1-methyl-6-phenylimidazo[4,5-b]pyridine by different P450 enzymes.
Carcinogenesis, 11, 489--492.
Wattenberg,L.W. (1977) Inhibition of carcinogenic effects of polycyclic
hydrocarbons by benzyl isothiocyanate and related compounds. J. Natl
Cancer Inst., 58, 395--398.
Wilkinson,J.T., Morse,M.A., Krestry,L.A. and Stoner,G.D. (1995) Effect
of alkyl chain length on inhibition of N-nitrosomethylbenzylamine-induced
esophageal tumorigenesis and DNA methylation by isothiocyanates.
Carcinogenesis, 16, 1011--1015.
Wynder,E.L., Fujita,Y., Harris,R.E., Hirayama,T. and Hiyama,T. (1991)
Comparative epidemiology of cancer between the United States and
Japan. Cancer, 67, 746--763.
Yahagi,T., Nagao,M., Seino,Y., Matsusima,T., Sugimura,T. and Okada,M.
(1977) Mutagenicities of N-nitrosamines on Salmonella. Mutat. Res., 48,
121--130.
Yang,C.S., Patten,C., Lee,M.J., Li,M., Yoo,J.S., Pan,J. and Hong,J. (1987)
Enzymatic mechanisms in the metabolic activation of N-nitrosodialkyla-
mines. In Bartsch,H., O’Neill,I. and Schulte-Hermann,R. (eds), The
Relevance of N-Nitroso Compounds to Human Cancer, Exposure and

Downloaded from http://mutage.oxfordjournals.org/ by guest on November 15, 2015

Mechanism. IARC Scientific Publication no. 84. IARC, Lyon, pp. 104--108.
Zhang,Y. and Talalay,P. (1994) Anticarcinogenic activities of organic
isothiocyanate: chemistry and mechanisms. Cancer Res., 54, 1976s--1981s.

Received on January 20, 2004; accepted on October 22, 2004

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