Mutagenesis Advance Access originally published online on December 14, 2004
Mutagenesis 2005 20(1):15-22; doi:10.1093/mutage/gei001
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Mutagenesis vol. 20 no. 1 © UK Environmental Mutagen Society 2005; all rights reserved.
Effects of 
-naphthyl isothiocyanate and a heterocyclic amine, PhIP, on cytochrome P-450, mutagenic activation of various carcinogens and glucuronidation in rat liver
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
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Abstract |
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To elucidate the mechanism underlying suppression by
-naphthyl isothiocyanate (ANIT) of mammary carcinogenesis induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), we evaluated hepatic levels of cytochrome P-450 (CYP) enzymes, mutagenic activation of environmental carcinogens and UDP-glucuronyltransferase (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 constitutive 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 prepared 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 carcinogenesis 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 |
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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 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 µg/person) (Wakabayashi et al., 1997) and 3563 individuals in the USA (0.72–1.11 µg/person) (Layton et al., 1995). PhIP has been demonstrated to produce colorectal, prostate and mammary cancers in rats (Ito et al., 1991; Shirai et al., 1997). It was also shown that 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 cauliflower, 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). -Naphthyl isothiocyanate (ANIT) is the first isothiocyanate the chemopreventive effect of which has been examined in rats and is reported to inhibit liver tumors initiated by 3'-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-nitrosomethylbenzylamine (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 (Leonard et al., 1981; Guo et al., 1992, 1993; Manson et al., 1997; Nishikawa et al., 1997). The activities of quinone reductase 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 isothiocyanates 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 knowledge no data have been provided on the effect of ANIT on hepatic levels of CYP species, mutagenicity of typical carcinogens and UDPGT activities in any animal species.
In order to elucidate the mechanism(s) underlying suppression 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.
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Materials and methods |
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Chemicals
Hydrochlorides of 2-amino-6-methyldipyrido[1,2-a:3',2'-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 (MeA
C) 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+, 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 maintained 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 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 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).
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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 µl of water and all the other carcinogens in 50 µl of dimethyl sulfoxide. The mutagenicities of Trp-P-2, Glu-P-1 and IQ (0.03 µg/plate), MeAC (10 µg/plate), PhIP and BP (5 µg/plate), AFB1 (1 µg/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 µl/plate for the HCA, 50 µl for BP and 150 µl 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+ and 5 mM G6P were used. In the experiments on CYP inhibition 200 µM 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.
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Results |
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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 < 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 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 < 0.01), respectively, compared with Group 3 rats, while this treatment produced no significant decrease in CYP1A1 expression. CYP2B2 level was not increased in Group 2, but was 4.0-fold higher (P < 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 ANIT+PhIP (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.
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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 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 differences 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 ANIT+PhIP was decreased by 66% relative to the PhIP-treated rats.
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To confirm the mutagenic activation induction characteristics 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 activities 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 numbers of revertant colonies/plate after subtraction of spontaneous rates (TA98, 20; TA100, 153) with liver S9 mix from Group 1 rats were 25 ± 6 (mean ± SD) for Glu-P-1, 68 ± 18 for IQ, 117 ± 23 for PhIP, 46 ± 7 for Trp-P-2, 53 ± 7 for MeA
C, 58 ± 5 for BP and 2630 ± 99 for AFB1 in strain TA98 and 109 ± 20 for BHP and 238 ± 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 < 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 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 activities of MeAC and BP were slightly but significantly decreased (20%, P < 0.05). The combined treatment produced no significant changes in mutagenicity of AFB1, BHP and DMN.
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Because induction of mutagenic activation of BHP and DMN by PhIP and suppression by ANIT, which are known to be catalyzed by CYP2B1 and CYP2B2, was not demonstrated, PROD activity was assayed in liver microsomes from female rats after a single or repeated treatments with PhIP. As shown in Table I, PROD activity in Group 3 rats was increased to 4.7-fold above that in Group 1 rats (P < 0.01). The activity in rats 48 h after PhIP treatment as a single dose was almost equal to that in Group 3 rats and that after combined treatment with ANIT was decreased by 64% relative to PhIP-treated rats. Induction of PROD activity by PB was confirmed in male rats, an increase to 57.2-fold (612 ± 13.2 pmol/min/mg protein) above control.
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In an attempt to obtain more information about CYP1A1 and CYP1A2, responsible for PhIP activation, typical CYP inducers and selective inhibitors were used in mutagenic activation assays. Figure 5 shows the effects of MC, PB and two inhibitors specific to the CYP1A subfamily on the mutagenic activity of PhIP in the presence of liver S9 fraction from either Wistar or Sprague–Dawley rats. Treatment of male Wistar rats with MC caused a 14.5-fold increase in their activity, while PB treatment produced no induction. Furafylline and 7,8-BF inhibited mutagenic activity in the presence of liver S9 mix from male rats treated with MC by 82 and 98%, respectively. In the presence of liver S9 mix from female rats of Group 3, in which the activity of PhIP was increased 5.1-fold (Figure 4) above the vehicle control, furafylline and 7,8-BF caused 64 and 85% inhibition, respectively.
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Table II summarizes the effects of PhIP and ANIT treatment for up to 3 weeks on UDPGT activity towards bilirubin and 4-NP in liver microsomes (Groups 1–4). There were no significant differences in UDPGT activity towards bilirubin among the four groups. In contrast, UDPGT activity towards 4-NP in Group 2 and 3 rats was increased to 1.6- and 2.2-fold above the vehicle control (Group 1), respectively, and the combined treatment (Group 4) showed much higher induction (4.7-fold).
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Discussion |
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Marked induction by MC and dramatic inhibition by furafylline and 7,8-BF of the mutagenic activity of PhIP indicate the involvement of CYP1A1 and CYP1A2 (predominantly CYP1A2) in metabolic activation of PhIP by liver S9 fraction from male and female rats. Selective enhancement by PhIP of the mutagenicities of five HCAs in the presence of liver S9 mix reflects selective induction of hepatic CYP1A1 and CYP1A2, especially CYP1A2. These results are in agreement with previous 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 MeAC 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.
We reported that PB increases the mutagenic activity of BHP to 4.4-fold (Mori et al., 1985), hepatic CYP2B2 to 18.3-fold above control and CYP2B1 to 1.9-fold above the induced level of CYP2B2 (Mori et al., 2001) in male rats. PB increased 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 repeatedly 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 demonstration 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 induction 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 hamsters with 2.5 mmol/kg PEITC had no effects on CYP1A2, CYP2B, CYP2E1 and CYP3A (Nishikawa et al., 1997). However, to our knowledge, there are no reports on either suppression 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 produced 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 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 suppressive 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 isothiocyanate 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 applications, a conventional or high fat diet), age and/or sex of the animals, metabolic substrates, etc. However, our results support 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 MeAC 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 carcinogens which can induce CYP1A1 and CYP1A2 and are selectively 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 conjugated 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), indicating that N-glucuronidation of PhIP is an important detoxification 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, consistent 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 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 attributed to a dual action mechanism: a decrease in metabolic activation 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 sulforaphane block the increase in cell proliferation induced by N-nitrosobis(2-oxopropyl)amine in its target organs in hamsters (Nishikawa et al., 1997) and induce apoptosis (Huang et al., 1998; Misiewicz et al., 2003), it is suggested that isothiocyanates are expected to affect chemically induced carcinogenesis through multiple mechanisms.
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* To whom correspondence should be addressed. Tel: +81 58 237 3931; Fax: +81 58 237 5979; Email: ymori{at}gifu-pu.ac.jp
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References |
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Received on January 20, 2004; accepted on October 22, 2004.
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