1 First Department of Pathology, Nagoya City University, Medical School, 1-Kawasomi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan,
2 Division of Pathology, National Institute of Health Science, Tokyo, Japan and
3 Division of Toxicology and Metabolism, Nippon Hypox Laboratories Inc., Yamanashi, Japan
Abstract
The aim of this study is to investigate the chemopreventive effects of the synthetic phenolic antioxidant 1-O-hexyl-2,3, 5-trimethylhydroquinone (HTHQ) on 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-associated colon carcinogenesis in rats after initiation with 1,2-dimethylhydrazine (DMH) in male F344 rats. Groups of 2022, 6-week-old male F344 rats were given four subcutaneous injections of 40 mg/kg body wt of DMH during the initial 4 weeks. They were then maintained on powdered basal diet containing 0.03% PhIP alone, PhIP together with 0.5 or 0.125% HTHQ, 0.5 or 0.125% HTHQ alone or basal diet for 32 weeks. A small number (1.1 ± 1.1/rat) of colon tumors were induced by DMH treatment alone. After initiation with DMH, the number of colon tumors was greatly increased to 8.3 ± 5.6 by the administration of PhIP. Additional treatment with HTHQ dose-dependently decreased the multiplicity of colon adenocarcinomas to 4.9 ± 2.8 and 2.6 ± 1.4 with 0.125 and 0.5%, respectively. This treatment similarly reduced atypical hyperplasias of the ventral prostate. Furthermore, HTHQ significantly reduced the multiplicity of duodenal adenocarcinomas induced by DMH + PhIP or DMH alone. Immunohistochemically, HTHQ was revealed to suppress PhIPDNA adduct formation in the epithelial cells of the colon and prostate in a separate 2 weeks experiment. The present results clearly showed that HTHQ has chemopreventive potential for PhIP-associated colon and prostate carcinogenesis. The observed inhibition may largely be due to interference with PhIPDNA adduct formation. In addition, HTHQ has been demonstrated to inhibit duodenal carcinogenesis in the post-initiation stage.
Abbreviations: DEN, N-diethylnitrosamine; Glu-P-1, 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole; HCA, heterocyclic amine; HTHQ, 1-O-hexyl-2, 3,5-trimethylhydroquinone; MeIQx, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine
Introduction
Several heterocyclic amines (HCAs), identified in cooked meats, have been shown to be genotoxic as evaluated by Ames bacterial (1) and in vitro mutation assays using transgenic mice (1,2). Epidemiologically, positive correlations between ingestion of cooked foods and risk of colon cancer have been reported (3,4). In man these HCAs may be excreted in urine after intake of cooked foods (5). In addition, HCADNA adducts have been demonstrated in human tissues (6).
2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), identified as the most abundant mutagenic heterocyclic amine normally generated during cooking (7), exerts carcinogenicity in the mammary gland, colon and prostate of rats (811). In addition to its carcinogenicity, PhIP was shown previously to enhance colon carcinogenesis in a two-stage carcinogenesis model of F344 rats after initiation by five carcinogens including 1,2-dimethylhydrazine (DMH), well known to be a genotoxic colon carcinogen (12). Therefore, HCAs, especially PhIP, may constitute a very important group of environmental carcinogens associated with risk of the cancers that are particularly prevalent in Western countries. It is important to identify chemopreventors of this important environmental carcinogen.
1-O-Hexyl-2,3,5-trimethylhydroquinone (HTHQ), a lipophilic phenolic antioxidant, possesses very strong antioxidant activity, even at low dose levels (13). This compound dose-dependently inhibits 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1) or PhIP-induced mutagenesis with Salmonella TA98 in the presence of S-9 mixture (14).
Previously, we showed that simultaneous treatment with HTHQ potently inhibited PhIP-induced mammary carcinogenesis in female rats without prior initiation treatment (15), and Glu-P-1 or 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx)-induced hepatocarcinogenesis in male rats after initiation with N-diethylnitrosamine (DEN) (16). The inhibitory effect of HTHQ on MeIQx-induced hepatocarcinogenesis were greater than those observed for naturally occurring and synthetic antioxidants such as BHA, BHT, TBHQ and propyl gallate (17). Whether it might also influence colon carcinogenesis induced by PhIP was one question targeted in the present study.
To examine the effect of HTHQ alone or administered together with PhIP in the post-initiation phase of colon carcinogenesis, male rats were treated with these chemicals after initial exposure to DMH and then given HTHQ, separately, or in combination. In addition, to examine the significance of DNA adduct formation for PhIP tumorigenesis and its response to HTHQ, immunohistochemical PhIPDNA adducts in the target tissues were assessed as described previously (18). If this antioxidant exerts chemopreventive effects on PhIP-induced colon carcinogenesis in male rats as well, HTHQ will be one of the most effective candidates for the inhibition of HCA-induced carcinogenesis, and application in the human situation might be proposed after appropriate toxicity testings.
Materials and methods
Animals
Five-week-old male F344 rats were obtained from Charles River, Japan Inc. (Atsugi, Japan). They were randomly divided into groups of three animals per plastic cage with hard wood chips as bedding in an air-conditioned room at 22 ± 2°C and 55 ± 5% humidity with a 12 h light/dark cycle. Food (Oriental MF, Oriental Yeast Co., Tokyo, Japan) and tap water were available ad libitum.
Chemicals
DMH was obtained from Tokyo Kasei Kogyo Co. Ltd (Tokyo, Japan), PhIP from Toronto Research Chemicals, Inc. (Ontario, Canada) and HTHQ from Nippon Hypox Laboratories Inc. (Yamanashi, Japan). Anti-PhIPDNA adduct antibodies established in our laboratory were used as reported previously (18,19).
Treatment
Experiment 1: chemopreventive effects of HTHQ on PhIP-associated carcinogenesis. Groups of 2022, 6-week-old male F344 rats were given four subcutaneous injections of DMH at a dose of 40 mg/kg body wt during the initial 4 week period. They were then maintained without further treatment or on powdered basal diet containing 0.03% PhIP alone, PhIP together with 0.5 or 0.125% HTHQ, or 0.5 or 0.125% HTHQ alone for 32 weeks and then killed for histopathological examination. The diets were prepared every 2 weeks with incorporation of 2% corn oil and stored in the dark at 4°C before use. They were replaced twice a week throughout the experimental period in conjunction with measurement of food consumption. Body weights were recorded every week during the experiment.
Experiment 2: immunohistochemical evaluation of PhIPDNA adduct formation. Groups of six or seven 6-week-old rats were maintained on powdered basal diet containing 0.03% PhIP alone or PhIP together with 0.5 or 0.125% HTHQ, prepared as for experiment 1, for 2 weeks. The control group was given basal diet without supplement. Tissues were fixed in acetone and then processed routinely for anti-PhIPDNA adduct immunohistochemical staining as well as histopathological examination.
Immunohistochemistry of PhIPDNA adducts
The methods for immunohistochemical evaluation of PhIPDNA adducts were as described previously (18). Briefly, sections were incubated with diluted anti-PhIPDNA adduct antibody (1:10 000) overnight at 4°C and then sequentially exposed to biotin-labeled goat anti rabbit IgG and ABC (Vectastain ABC elite kit, Vector Laboratories). For quantification of PhIPDNA adduct positive cells, immunostained sections were examined with a light microscope connected to an Image Processor for Analytical Pathology (IPAP, Sumika Technos Corp., Osaka, Japan) (20). Binary digitized images of colon, small intestine (jejunum and ileum), duodenum, prostate and pancreas sections were obtained automatically by a programmed segmentation procedure. All microscopic images were measured at a magnification of 600 (at least 20 fields/one section) for one rat. At least 3000 nuclei were counted and labeling indices expressed as the average positive cells per total nuclei in the field obtained.
Statistical analysis
Statistical analysis of differences between means and incidence was carried out using analysis of variance (ANOVA), and KruskalWallis test, respectively. When positive, post-hoc multiple comparison tests were applied to evaluate statistical significance between any treatment subgroups.
Results
Experiment 1: chemopreventive effects of HTHQ on PhIP-associated carcinogenesis
The average final body weights of all groups treated with PhIP were lower than those without PhIP. However, additional HTHQ treatment did not affect body weight gain. No significant differences in liver, kidney and prostate weights were observed between the HTHQ treatment and control groups (data not shown). In the colon, the average numbers of both adenomas and carcinomas per rat were greatly increased by the administration of PhIP after DMH initiation (Table I). Although the incidences of colon adenocarcinomas were not significantly reduced by the additional treatment with HTHQ, the multiplicity of adenocarcinomas was dose-dependently decreased to 4.9 ± 2.8 and 2.6 ± 1.4 by 0.125 and 0.5% HTHQ, respectively, as compared with 8.3 ± 5.6 for the PhIP alone group. A tendency for reduction in both incidence and multiplicity was also observed with HTHQ 0.5% after DMH treatment.
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We have reported previously colon carcinogenicity of PhIP in F344 rats administered the compound in the diet at a dose of 400 p.p.m. for 52 weeks; the incidence of colon carcinoma in males was 55% (16 of 29 rats), and the average number of colon carcinoma was 0.65/rat (10). Administration of 100 p.p.m. PhIP for 104 weeks induced colon carcinomas in 43% of rats, although 25 p.p.m. for 104 weeks induced no colon tumors (11). Moreover, treatment with 300 p.p.m. PhIP for 24 weeks enhanced colon carcinogenesis in F344 rats after treatment with five carcinogens including DMH (12). However, no malignant tumors were observed after PhIP alone treatment, probably because the experimental period (only 24 weeks of treatment) was too short to induce tumors without a prior initiation procedure. In the present study, DMH alone treatment induced a small number of colon carcinomas and tumor development was enhanced by subsequent administration of 300 p.p.m. PhIP for 32 weeks. These results are in line with the previous report (12).
In the present experiment, HTHQ alone did not inhibit colon carcinogenesis in the post-initiation period of DMH initiation. However, it showed strong inhibition when PhIP was additionally administered. The development of pre-neoplastic prostatic atypical hyperplasias and regenerative pancreas basophilic foci, which are considered to be associated with PhIP itself, was also inhibited by HTHQ. In previous studies, simultaneous treatment with 0.5% HTHQ exhibited strong inhibitory effects on mammary carcinogenesis induced by PhIP without prior treatment (15), and liver carcinogenesis induced by MeIQx, Glu-P-1 (16) or a HCA mixture after initiation treatment with DEN (21). Therefore, present results have indicated that HTHQ inhibited carcinogenicity of PhIP in all its target organs.
PhIP is metabolized to the N-hydroxy form in large part by hepatic CYP1A2 and further metabolism to O-acetylated or O-sulfated hydroxy-PhIP is thought to be responsible for reactive forms, which can bind to macromolecules such as DNA in the cells (22,23). A relationship between DNA-adduct levels and carcinogenicity has been well documented for many carcinogens (2429). DNA modification through covalent binding of carcinogen(s) is thought to be an essential step for the activity of genotoxic carcinogens but not in itself enough to initiate carcinogenesis. In addition to possible formation of DNA adduct types that are non-relevant to carcinogenesis, a high efficiency of DNA repair and, therefore inadequate fixation of heritable DNA lesions, also have been suggested as complicating factors. Therefore, it has been shown that DNA-adduct formation is a prerequisite but itself not sufficient for carcinogenicity, and DNA adduct levels may correlate with carcinogenic response in only target organs (29,30). In fact, HCADNA adducts have been demonstrated in non-target tissues of carcinogenicity (18,23).
HTHQ was demonstrated to strongly (60100%) reduce mutagenicities of many carcinogenic HCA such as PhIP, Glu-P-1, 3-amino-1-methyl-5-H-pyrido[4,3-b]indole (Trp-P-2), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) or MeIQx in the Ames assay (14,31). Furthermore, our previous data demonstrated that HTHQ could interfere with the activity of methoxyresorfin O-demethylase (MROD), which is a marker enzyme for CYP1A2 in vitro (32). HTHQ inhibition of PhIPDNA adduct formation in the present experiment could be due to inhibition of metabolic activation via the cytochrome P450 system, and the mechanism whereby HTHQ inhibited PhIP carcinogenicity is largely due to inhibition of initiation process.
Our previous study demonstrated that the strong antioxidant activity of HTHQ might not be responsible for the inhibition of MeIQx-induced hepatocarcinogenesis (17). However, recently the N-hydroxy derivative of MeIQx was found to cause Cu(II)-mediated DNA damage, including 8-OH-dG formation in calf thymus DNA, particularly in the presence of NADH (33). A highly purified cytochrome P-450 reductase and NADPH system was shown to generate superoxide radicals from HCAs as identified by the electron spin trapping method (34). Therefore, a contribution of antioxidative activity of HTHQ cannot necessarily be ruled out. Whether the observed inhibition of prostate carcinogenesis and regenerative basophilic foci development in the pancreas by HTHQ involves the same mechanisms as for colon carcinogenesis remains to be determined.
PhIP also enhanced small intestinal carcinogenesis. This fact has already been reported in rats pre-treated with five carcinogens including DMH (12), or with 3,2'-dimethyl-4-aminobiphenyl (35). However, as the carcinogenicity of PhIP in the small intestine was not evident in rodents in long-term carcinogenicity studies (9,11,36), the present findings suggest that PhIP possesses weak tumor promotion potential in the small intestine after appropriate initiation. The increase in PhIPDNA adduct levels in the small intestine after PhIP treatment further indicates possible weak carcinogenicity of PhIP in the small intestine at higher doses.
In the duodenum, almost the same incidence and the multiplicity of carcinomas were induced by DMH following treatment with or without PhIP. Although DNA adduct levels were increased, PhIP did not influence duodenum carcinogenesis in line with earlier reports that adducts do not necessarily correlate with carcinogenesis. On the other hand, HTHQ significantly lowered the multiplicity of duodenal tumors with or without PhIP administration. The result clearly indicates that HTHQ possesses chemopreventive activity on duodenal carcinogenesis in the post-initiation stage.
In conclusion, the present results clearly showed an enhancing effect of PhIP on DMH-induced colon and small intestinal carcinogenesis, and that HTHQ has chemopreventive potential for PhIP-associated colon and prostate carcinogenesis, and for duodenal carcinogenesis in the post-initiation period. The observed inhibition may largely be due to interference with PhIPDNA adduct formation. From the present experiment and our previous studies, it can be concluded that HTHQ is a promising chemopreventive agent for protection against heterocyclic amine-related carcinogenesis.
Notes
4 To whom correspondence should be addressed Email: futakuch{at}med.nagoya-cu.ac.jp
Acknowledgments
This work was supported in part by Grants-Aid for Cancer Research from the Ministry of Education, Sports, Science and Culture, and the Ministry of Health and Welfare of Japan, a Grant-in-aid from the Ministry of Health and Welfare for the Second Term Comprehensive 10-Year Strategy for Cancer Control, Japan, and a grant from the Society for Promotion of Toxicologic Pathology of Nagoya, Japan.
References