Organ dependent enhancement of rat 3,2'-dimethyl-4-aminobiphenyl (DMAB) carcinogenesis by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP): positive effects on the intestine but not the prostate
Katsumi Imaida1,,
Masashi Sano,
Seiko Tamano,
Makoto Asamoto,
Kumiko Ogawa,
Mitsuru Futakuchi and
Tomoyuki Shirai
First Department of Pathology, Nagoya City University Medical School, 1-Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan
 |
Abstract
|
---|
In order to evaluate tumor enhancing effects of the heterocyclic carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), doses of 100 and 300 p.p.m. PhIP were given for 40 weeks to male F344 rats, which initially received 3,2'-dimethyl-4-aminobiphenyl (DMAB). DMAB shows a similar carcinogenic organ spectrum to that of PhIP, including the prostate and colon. PhIP alone at a dose of 300 p.p.m. resulted in the development of prostate and intestine cancers. Furthermore, among the DMAB-treated group, enhancement of intestinal carcinogenesis by 300 p.p.m. PhIP was observed. However, no prostate enhancement was demonstrated in the DMAB + PhIP group. Since PhIPDNA adduct formation in the prostate epithelial cells in a satellite experiment was not affected by pre-treatment with DMAB, it is speculated that the contradictory findings between the intestine and prostate may be due to the specific biological effects of PhIP. Taking into account previous data, that PhIP clearly enhanced rat 1,2-dimethylhydrazine-initiated colon tumorigenesis, the potential of PhIP to enhance colon carcinogenesis may be initiator dependent.
Abbreviations: ACF, aberrant crypt foci; DMAB, 3,2'-dimethyl-4-aminobiphenyl; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; PIN, prostatic intraepithelial neoplasia.
 |
Introduction
|
---|
2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), one of the predominant heterocyclic amines produced in cooked meat and fish (13), is also found in cigarette smoke condensate (4), beer and wine (5). Regarding evidence that human beings are exposed on a daily basis, PhIP has been detected in the urine of healthy volunteers eating a normal diet, but not of in-patients receiving parenteral alimentation (6,7). Experimental evidence of the carcinogenic potential of PhIP in rat colon, mammary glands and prostate (813) suggests that it may play an important role in cancer development in man as a food derived carcinogen, particularly in Western countries in which both consumption of meat and frequencies of colon, breast and prostate cancers are high.
Carcinogenic substances can be theoretically divided into three groups on the basis of their potential to initiate and promote carcinogenesis (14,15). Carcinogens that possess (i) only initiation activity, (ii) both initiation and promotion potential and (iii) only promotion. In general, chemicals that effect both initiation and promotion are named complete carcinogens (16,17). It is likely that promotion, but not initiation, is organ specific. Formation of DNA adducts with chemical carcinogens, including PhIP occurs in a wide variety of organs (1820), and adduct levels do not necessarily correlate with outcome in terms of the carcinogenic responses (20). Tumors can be produced in specific organs by application of certain promoters after treatment with a broad spectrum initiator (21). Thus, from the mechanistic viewpoint, clarification of the promotion potential of PhIP is very important in assessing its hazard risk for human beings. PhIP has been shown to enhance rat mammary and colon carcinogenesis initiated with 7,12-dimethylbenz[a]anthracene and 1,2-dimethylhydrazine (22,23), respectively, in accordance with its carcinogenic organ-tropism.
In the present work, we report that PhIP given after treatment with 3,2'-dimethyl-4-aminobiphenyl (DMAB), which is also carcinogenic in rat intestine (24), prostate (25,26) and urinary bladder (27) enhances tumorigenesis in the colon but not the prostate.
 |
Materials and methods
|
---|
Chemicals
PhIP (PhIP hydrochloride) and DMAB were obtained from the NARD Institute (Osaka, Japan) with purities above 99.9 and 98%, respectively.
Animals
A total of 110 male F344 rats (5- or 6-weeks-old) were purchased from Charles River Japan (Atsugi, Japan) and housed three to a plastic cage on hard-wood chips, in an air-conditioned room at 22 ± 2°C and 50% humidity with a 12 h/12 h lightdark cycle. They were given commercial pellets (Oriental MF; Oriental Yeast, Tokyo) and tap water ad libitum.
DMAB and PhIP treatment
The animals were divided into six groups as shown in Figure 1
(20, 25, 25, 15, 15 and 10 rats for groups 16, respectively). The animals in groups 13 were given DMAB subcutaneously at a dose of 50 mg/kg body weight 10 times at 2 week intervals, as previously reported (21,26). After administration of DMAB, animals in groups 2 and 3 received PhIP incorporated into the diet at doses of 100 and 300 p.p.m., respectively, for 40 weeks. Groups 46 were control groups corresponding to groups 13 and were not given DMAB but received the post-initiation treatment. The experiment was terminated 60 weeks after commencement and all surviving animals were killed.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 1. Experimental protocol. Animals were male F344 rats 5/6-weeks-old at commencement. Closed box, DMAB at a dose of 50 mg/kg body weight, s.c. injections at 2 week intervals. Hatched box, 100 and 300 p.p.m. PhIP for groups 2 and 3, respectively, administered in the diet.
|
|
All rats underwent complete autopsy at the end of the experiment. Accessory sex organs were examined for gross abnormalities and fixed in 10% buffered formalin. For tissue preparation, two sagittal slices of the ventral prostate, three sagittal samples of the dorso-lateral prostate, including the urethra and four transverse samples from each seminal vesicle, including the anterior prostate, were embedded in paraffin. The intestines were inflated with the formalin fixative for about 10 min and then cut along their length and the colons were placed between filter paper in the same fixative for 3 days. All macroscopic intestinal tumors were sampled and embedded in paraffin for histological evaluation. Formalin-fixed colons were stained with methylene blue and the colon mucosal surface of the distal colon was scanned under a light microscope for aberrant crypt foci (ACF). The frequency and crypt multiplicity of foci were recorded (28). Paraffin embedded sections were cut and stained with hematoxylin and eosin for histopathological examination.
Analysis of PhIPDNA adduct formation
In order to determine the influence of DMAB pre-treatment on PhIP metabolism, PhIPDNA adduct formation was investigated. A total of 20 male F344 rats were divided into three groups. (i) DMAB + PhIP group (12 rats): three subcutaneous injections of DMAB at 2 week intervals at a dose of 50 mg/kg body wt for the first 6 weeks and then given PhIP at a dose of 300 p.p.m. in the diet for 4 weeks. (ii) PhIP group (4 rats): PhIP was given for 4 weeks without DMAB pre-treatment. (iii) DMAB group (4 rats): DMAB pre-treatment without PhIP. Animals in the DMAB + PhIP group were killed sequentially and those in the other groups were killed at the end of the experiment. Prostates were fixed in cold acetone and paraffin-embedded ventral prostate sections were prepared for immunohistochemical detection of PhIPDNA adduct formation, as previously described (18).
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, Osaka, Japan) (19). Binary digitized images of ventral prostate sections were obtained automatically by a programmed segmentation procedure. Microscopic images were assessed at a magnification of x600 (at least 10 fields per section). At least 1000 nuclei were counted and labeling indices, expressed as the average of positive cells per total nuclei in the field, were obtained. The color density of the immunostained sections was measured as the optical density, which was automatically measured in obtaining nuclear images. Optical densities were expressed as percent average values.
Differences in body and organ weights were analyzed by means of the Student's t-test in combination with the F-test for equal variance. Incidences of tumors and other histopathological lesions were analyzed by the Fisher's exact probability test (two tailed).
 |
Results
|
---|
DMAB and PhIP treatment
In this experiment, survival rates at the end of the experiment in the DMAB-treated groups were <50%, because of development of tumors in the subcutaneous tissue and Zymbal glands. Furthermore, a dose of 300 p.p.m. PhIP showed a significant suppressive effect on body weight gain in both DMAB-initiated and non-initiated groups (Figure 2
; Table I
). Therefore, experimentation of the groups given DMAB was terminated at week 56. As shown in Table I
, prostate and liver weights of rats given 300 p.p.m. PhIP were lower than the corresponding control values.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 2. Growth curves of animals given 50 mg/kg body weight DMAB at 2 week intervals followed by either 100 or 300 p.p.m. PhIP. All surviving animals given DMAB were killed at week 56.
|
|
Prostate and seminal vesicle lesions, the incidences of which are shown in Table II
, included prostatic intraepithelial neoplasia (PIN) and adenocarcinomas, as well as dysplasia of the seminal vesicle. DMAB alone induced a low incidence of adenocarcinomas of the ventral prostate and high incidences of PIN in the ventral, dorso-lateral and anterior prostate and dysplasia of the seminal vesicles. PhIP itself also induced PIN and adenocarcinomas of the ventral prostate in a dose-related fashion. However, in groups receiving the combined treatment of DMAB and PhIP, the incidences of these lesions were decreased rather than increased. The appearance of ACF of the colon, on the other hand, was clearly enhanced by 300 p.p.m. PhIP (Table III
). Numbers were greater than the sum of those in rats given the individual chemicals, suggesting a synergistic action. The incidences of adenoma and/or adenocarcinoma of the small intestine and colon were also increased by the subsequent administration of PhIP but a statistical significance was only seen for the small intestine (Table IV
). The multiplicity of small intestinal and colon tumors showed the same tendency as seen for the incidences.
Analysis of PhIPDNA adduct formation
Analysis of PhIPDNA adduct formation in immunohistochemically stained sections did not reveal any differences in either optical density or labeling indices between groups of DMAB and DMAB followed by PhIP at weeks 2 or 4 (Table V
). No adduct formation was detected in rats given DMAB alone.
 |
Discussion
|
---|
The data from the present experiment confirmed that PhIP is carcinogenic to the prostate and intestine of rats. The reason why the incidences of prostate cancer and intestinal tumors were low as compared with previous data (8) is presumably due to the shorter duration of treatment and the lower dose used. In spite of the tumorigenic response in the prostate and seminal vesicle with the 40 week administration of PhIP, no enhancement of tumor development was demonstrable after DMAB initiation. Tumorigenic response in the small intestine, on the other hand, was clearly enhanced by PhIP and that in the colon, at the ACF level, was also strongly promoted. However, the increase in development of adenomas and/or carcinomas of the colon was not statistically significant. There was no clear difference in the PhIPDNA adduct formation in prostate epithelial cells between the DMABPhIP and PhIP groups, precluding the possibility that the lack of enhancement was the result of reduced PhIP activation because of prior DMAB exposure. Another possible mechanism of this observation may be related to apoptosis, which could be induced by PhIP treatment (29,30), and prior to the treatment of DMAB possibly affects these changes differently at each target organ.
Our previous data (31,32) demonstrated that the incidence of ventral prostate carcinomas in rats given PhIP at a dose of 400 p.p.m. for the initial 20 weeks of a 60 week experiment was not very different from that in rats given PhIP at a dose of 400 p.p.m. for 52 weeks (67 versus 55%). The incidence of colon carcinomas was 52% in rats given PhIP for 52 weeks as opposed to only 20% in rats given PhIP for the initial 20 weeks. These findings indicate that the tumorigenic response of the prostate and colon differs greatly regarding treatment period. This fact suggests that PhIP possesses initiation activity but lacks, or has only weak, enhancing effects for rat prostate and seminal vesicles, while both initiation and promotion are exerted in the intestine. Further evidence in support of this hypothesis is provided by the finding that a dose of 100 p.p.m. PhIP for 104 weeks induced colon cancers in rats but not prostate neoplasms (12). In other words, continuous long-term exposure to PhIP is very effective in promoting colon tumor development but not for prostate tumorigenesis. This is completely the reverse of the situation with DMAB-induced colon and prostate carcinogenesis (24), i.e. shorter treatments with a higher dose being very effective for induction of colon tumors, while longer treatments with a lower dose being more effective for prostate tumor production. Therefore, chemical carcinogens possess may different degrees of initiation and promotion activity, which is dependent on the target organ.
An enhancing effect of PhIP on DMAB-initiated intestinal tumorigenesis was observed, an effect clear in the case of the small intestine but not in the colon. Our previous study demonstrated that PhIP exerted the enhancing effect in 1,2-dimethylhydrazine-initiated colon carcinogenesis (22,23). For example, 300 p.p.m. PhIP administered in the diet for 36 weeks enhanced the incidence of colon tumors (adenomas/carcinomas) from 75 to 95% and their multiplicity from 1.3 to 9.1 (23). These findings suggest that enhancement of intestinal tumorigenesis by PhIP may rely on the initiator. A similar initiator-dependent tumor enhancing potential of given chemicals was previously observed for a liver promoter (33).
In conclusion, although PhIP is carcinogenic to both rat intestine and prostate, the present data indicate that it only exerts a significant tumor enhancing potential in the small intestine after DMAB initiation. Furthermore, the exertion of colon tumor enhancement by PhIP may be dependent on the initiating agent.
 |
Notes
|
---|
1 To whom correspondence should be addressedEmail: imaida{at}med.nagoya-cu.ac.jp 
 |
Acknowledgments
|
---|
This research was supported in part by a Grant-in-Aid from the Ministry of Health and Welfare for the Second Term Comprehensive 10 Year Strategy for Cancer Control, Japan, Grants-in-Aid for cancer research from the Ministry of Education, Science, Sports and Culture and the Ministry of Health and Welfare of Japan, and grants from the Society for Promotion of Toxicologic Pathology of Nagoya, Japan and the Experimental Pathology Research Association, Japan.
 |
References
|
---|
-
Felton,J.S., Knize,M.G., Shen,N.H., Lewis,P.R., Andresen,B.D., Happe,J. and Hatch,F.T. (1986) The isolation and identification of a new mutagen from fried ground beef: 2-amino-1 methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Carcinogenesis, 7, 10811086.[Abstract]
-
Sugimura,T. and Wakabayashi,K. (1991) Heterocyclic amines: new mutagens and carcinogens in cooked foods. Adv. Exp. Med. Biol., 283, 569578.[Medline]
-
Gross,G.A., Turesky,R.J., Fay,L.B., Stillwell,W.G., Skipper,P.L. and Tannenbaum,S.R. (1993) Heterocyclic aromatic amine formation in grilled bacon, beef and fish and grill scrapings. Carcinogenesis, 14, 23132318.[Abstract]
-
Manabe,S., Yohyama,O., Wada,A. and Aramaki,T. (1991) Detection of a carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), in cigarette condensate. Carcinogenesis, 12, 19451947.[Abstract]
-
Manabe,S., Suzuki,H., Wada,O. and Ueki,A. (1993) Detection of the carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in beer and wine. Carcinogenesis, 14, 899901.[Abstract]
-
Wakabayashi,K., Ushijima,H., Takahashi,M., Nukaya,H., Kim,S.-B., Hirose,M., Ochiai,M., Sugimura,T. and Nagao,M. (1993) Exposure to heterocyclic amines. Environ. Health Perspect., 99, 129133.[ISI][Medline]
-
Ushiyama,H., Wakabayashi,K., Hirose,M., Itoh,H., Sugimura,T. and Nagao,M. (1991) Presence of carcinogenic heterocyclic amines in urine of healthy volunteers eating normal diet, but not in patients receiving parenteral alimentation. Carcinogenesis, 12, 14171422.[Abstract]
-
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, 15031506.[Abstract]
-
Imaida,K., Hagiwara,A., Yada,H., Masui,T., Hasegawa,R., Hirose,M., Sugimura,T., Ito,N. and Shirai,T. (1996) Dose-dependent induction of mammary carcinomas in female SpragueDawley rats with 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. Jpn. J. Cancer Res., 87, 11161120.[ISI][Medline]
-
Hagiwara,A., Murai,T., Yoshino,H., Goshima,H., Mori,S., Takashima,A., Shirai,T. and Fukushima,S. (1999) Hepatocarcinogenic activity of N-butyl-N-(4-hydroxybutyl)nitrosamine in rats is not modified by sodium L-ascorbate. Teratog. Carcinog. Mutagen., 19, 3342.[ISI][Medline]
-
Hagiwara,A., Boonyaphiphat,P., Tanaka,H., Kawabe,M., Tamano,S., Kaneko,H., Matsui,M., Hirose,M., Ito,N. and Shirai,T. (1999) Organ-dependent modifying effects of caffeine and two naturally occurring antioxidants a-tocopherol and n-tritriacontane-16,18-dione, on 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced mammary and colonic carcinogenesis in female F344 rats. Jpn. J. Cancer Res., 90, 399405.[ISI][Medline]
-
Hasegawa,R., Sano,M., Tamano,S., Imaida,K., Shirai,T., Nagao,M., Sugimura,T. and Ito,N. (1993) Dose-dependence of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) carcinogenesis in rats. Carcino- genesis, 14, 25532557.[Abstract]
-
Shirai,T., Cui,L., Takahashi,S., Futakuchi,M., Asamoto,M., Kato,K. and Ito,N. (1999) Carcinogenicity of 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) in the rat prostate and induction of invasive carcinomas by subsequent treatment with testosterone propionate. Cancer Lett., 143, 217221.[ISI][Medline]
-
Ito,N., Tsuda,H., Hasegawa,R. and Imaida,K. (1982) Sequential observation of pathomorphologic alterations in preneoplastic lesions during the promoting stage of hepatocarcinogenesis and the development of short-term test system for hepatopromoters and hepatocarcinogens. Toxicol. Pathol., 10, 3749.
-
Ashby,J., Elliott,B.M., Lefevre,P.A., Styles,J. and Longstaff,E. (1983) Initiation/promotion versus complete carcinogenicity in the rodent liver. Environ. Health Perspect., 50, 339346.[ISI][Medline]
-
Ito,N., Hasegawa,R., Imaida,K., Hirose,M., Shirai,T., Tamano,S. and Hagiwara,A. (1997) Medium-term rat liver bioassay for rapid detection of hepatocarcinogenic substances. J. Toxicol. Pathol., 10, 111.
-
Kunze,E., Woltjen,H.H. and Albrecht,H. (1983) Absence of a complete carcinogenic effect of phenacetin on the quiescent and proliferating urothelium stimulated by partial cystectomy. A 2-year feeding study in rats. Urol. Int., 38, 223228.[ISI][Medline]
-
Takahashi,S., Tamano,S., Hirose,M., Kimoto,N., Ikeda,Y., Sakakibara,M., Tada,M., Kadlubar,F.F., Ito,N. and Shirai,T. (1998) Immunohistochemical demonstration of carcinogenDNA adducts in tissues of rats given 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP): Detection in paraffin-embedded sections and tissue distribution. Cancer Res., 58, 43074313.[Abstract]
-
Shirai,T., Nakamura,A., Fukushima,S., Tada,M., Morita,T. and Ito,N. (1990) Immunohistochemical demonstration of carcinogenDNA adducts in target and non-target tissues of rats given a prostate carcinogen, 3,2'-dimethyl-4-aminobiphenyl. Carcinogenesis, 11, 653657.[Abstract]
-
Shirai,T., Takahashi,S., Cui,L., Yamada,Y., Tada,M., Kadlubar,F.F. and Ito,N. (1998) Use of polyclonal antibodies against carcinogenDNA adducts in analysis of carcinogenesis. Toxicol. Lett., 102/103, 441446.
-
Shirai,T., Tamano,S., Kato,T., Iwasaki,S., Takahashi,S. and Ito,N. (1991) Induction of invasive carcinomas in the accessory sex organs other than the ventral prostate of rats given 3,2'-dimethyl-4-aminobiphenyl and testosterone propionate. Cancer Res., 51, 12641269.[Abstract]
-
Hasegawa,R., Miyata,E., Futakuchi,M., Hagiwara,A., Nagao,M., Sugimura,T. and Ito,N. (1994) Synergistic enhancement of hepatic foci development by combined treatment of rats with 10 heterocyclic amines at low doses. Carcinogenesis, 15, 10371041.[Abstract]
-
Hirose,M., Takahashi,S., Ogawa,K., Futakuchi,M., Shirai,T., Shibutani,M., Uneyama,C., Toyoda,K. and Iwata,H. (1999) Chemoprevention of heterocyclic amine-induced carcinogenesis by phenolic compounds in rats. Cancer Lett., 143, 173178.[ISI][Medline]
-
Ito,N., Shirai,T., Tagawa,Y., Nakamura,A. and Fukushima,S. (1988) Variation in tumor yield in the prostate and other target organs of the rat in response to varied dosage and duration of administration of 3,2'-dimethyl-4-aminobiphenyl. Cancer Res., 48, 46294632.[Abstract]
-
Shirai,T., Sakata,T., Fukushima,S., Ikawa,E. and Ito,N. (1985) Rat prostate as one of the target organs for 3,2'-dimethyl-4-aminobiphenyl-induced carcinogenesis: effects of dietary ethinyl estradiol and methyltestosterone. Jpn. J. Cancer Res. (Gann), 76, 803808.[ISI][Medline]
-
Shirai,T., Iwasaki,S., Masui,T., Mori,T., Kato,T. and Ito,N. (1993) Enhancing effect of cadmium on rat ventral prostate carcinogenesis induced by 3,2'-dimethyl-4-aminobiphenyl. Jpn. J. Cancer Res., 84, 10231030.[ISI][Medline]
-
Shirai,T., Fukushima,S., Kawabe,M., Shibata,M.-A., Iwasaki,S., Tada,M. and Ito,N. (1991) Selective induction of rat urinary bladder tumors by simultaneous administration of 3,2'-dimethyl-4-aminobiphenyl (DMAB) and butylated hydroxyanisole or butylated hydroxytoluene is associated with increased DMABDNA adduct formation. Carcinogenesis, 12, 13351339.[Abstract]
-
Bird,R.P. (1987) Observation and quantification of aberrant crypts in the murine colon treated with a colon carcinogen: preliminary findings. Cancer Lett., 37, 147151.[ISI][Medline]
-
Zhu,H., Boobis,A.R. and Gooderham,N.J. (2000) The food-derived carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine activates S-phase checkpoint and apoptosis and induces gene mutation in human lymphoblastoid TK6 cells. Cancer Res., 60, 12831289.[Abstract/Free Full Text]
-
Venugopal,M., Callaway,A. and Snyderwine,E.G. (1999) 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) retards mammary gland involutin in lactating SpragueDawley rats. Carcinogenesis, 20, 13091314.[Abstract/Free Full Text]
-
Shirai,T., Sano,M., Tamano,S., Takahashi,S., Hirose,M., Futakuchi,M., Hasegawa,R., Imaida,K., Matsumoto,K., Wakabayashi,K., Sugimura,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, 195198.[Abstract]
-
Shirai,T., Cui,L., Takahashi,S., Futakuchi,M., Asamoto,M., Kato,K. and Ito,N. (1999) Carcinogenicity of 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) in the rat prostate and induction of invasive carcinomas by subsequent treatment with testosterone propionate. Cancer Lett., 143, 217221.[ISI][Medline]
-
Shirai,T., Imaida,K., Ohshima,M., Fukushima,S., Lee,M.-S., King,C.M. and Ito,N. (1985) Different responses to phenobarbital promotion in the development of
-glutamyltranspeptidase-positive foci in the liver of rats initiated with diethylnitrosamine, N-hydroxy-2-acetylaminofluorene and aflatoxin B1. Jpn. J. Cancer Res. (Gann), 76, 1619.[ISI][Medline]
Received December 29, 2000;
revised April 25, 2001;
accepted April 26, 2001.