- Email: [email protected]
Inhibition of rat hepatic glutathione S-transferase placental form positive foci development by concomitant administration of antioxidants to carcinogen-treated rats
25
Cancer Letters, 55 (1990) 25-29 Elsevier Scientific Publishers Ireland Ltd.
Inhibition of rat hepatic glutathione S-transferase form positive foci development by concomitant administration of antioxidants to carcinogen-treated M. Asamoto,
Y. Shichino,
First
of
Department
Nagoya
467
Pathology,
H. Tsuda* Nagoya
City
placental rats
and N. Ito
University
Medical
School,
1 Kawasumi,
Mizuho-cho,
Mizuho-ku,
(Japan)
(Received 30 August 1989) (Revision received 5 March 1990) (Accepted 11 September 1990)
Summary Inhibition potential
hydroxyanisole luene (BHT),
ofconcomitant
(BHA),
butylated
butylated hydroxyto-
catechol or sodium ascorbate (Na-AsA) administration on development of diethylnitrosamine (DEN) initiated glutathione S-transferase placental form (GST-P) positive foci in rat liver under the influence of Z-acetylaminofluorene (2.AAF) or 3’.methyl-4.dime(3’.Me-DAB) plus thylaminoazobenzene partial hepatectomy (PH) was investigated.
Whereas BHA, BHT and catechol exerted marked inhibitory effects, Na-AsA lacked any The compounds that modifying potential. demonstrated inhibition also induced GST-P in the hepatic periportal areas, suggesting that development of GST-P positive foci is negatively influenced by extra-focal increase in this enzyme form observed with BHA, BHT or
catechol.
Correspondence
ogy,
Nagoya
Miiuho-cho,
to: M. Asamoto, First Department of PatholCity University Medical School. 1 Kawasumi,
Miiuho-ku.
Nagoya 467, Japan. Department of Pathology, School of Medicine, Fujiia-Gakuen Health University, Katsukake-cho, Toyoake, Aichi 470- 11, Japan. *Present address: Second
Keywords: butylated hydroxyanisole; butylated hydroxytoluene; catechol; glutathione Stransferase placental form; rat hepatocarcinogenesis; inhibition Introduction Antioxidants such as BHA, BHT and NaAsA have been widely used as food additives in foods to prevent oxidation of labile lipid components. Another member of this group, catechol, is also present in the environment as an industrial chemical, as well as being a major phenolic component of cigarette smoke. Although Ito and his group [3,4] found that high doses of BHA or catechol can induce stomach neoplasia, Wattenberg et al. [17] have reported that antioxidants, including BHA and BHT, inhibit chemical carcinogenesis in various tissues of many species when administered concomitantly with carcinogens. Concentrating on development of an in vivo medium-term screening system for carcinogens and modifiers, we have established a reliable system for rapid detection of hepatocarcinogens [5] using GST-P as a marker protein for preneoplastic liver cell foci [13]. In addition, it was found that many compounds could act as inhibitory agents in this assay sys-
0 1990 Elsevier Scientific Publishers Ireland Ltd. 0304.3835/90/$03.50 Published and Printed in Ireland
26
tern reducing the induction of GST-P positive foci [5]. Retrospective checking for GST-P staining in slides from rats treated with inhibitors, revealed that strong inhibitors including BHA and catechol, induced GST-P in the periportal areas of treated rat livers. Therefore, in this study, the possible correlation between inhibition of GST-P positive foci development by concomitant administration of antioxidants with carcinogens and the induction of GST-P in the liver portal areas was investigated. Materials and Methods BHA, catechol and Na-AsA were obtained from Wako Pure Chemical Industries Ltd., Osaka, Japan and BHT from the National Institute of Hygienic Science, Tokyo. DEN, 2AAF and 3’-Me-DAB were purchased from Tokyo Chemical Industry Co. A total 215 6-week-old male F344 rats were used. The experimental protocol used was essentially the same as in previous reports [5,15]. Initially, 165 animals were treated i.p. with DEN at a dose of 200 mg/kg body weight, the remaining 50 rats receiving saline
Fig. 1.
vehicle alone. After 2 weeks on basal diet, carcinogen-initiated and control animals were divided into 11 groups (groups l- 11) and 10 groups (groups 12-21)) respectively (see Table 1). Groups l-5 and 12- 16 were given diet containing 0.02% 2-AAF plus 2% BHA (groups 1,12), 1% BHT (groups 2,13), 0.8% catechol (groups 3,14), 5% Na-AsA (groups 4,15) or 2-AAF alone (groups 5,16). Groups 6 -10 and 17-21 were similarly given a diet containing 0.012% 3’-Me-DAB plus 2% BHA (groups 6,17), 1% BHT (groups 7,18), 0.8% catechol (groups 8,19), 5% Na-AsA (groups 9,20) or no addition (groups 10,21). Group 11 received the basal diet without supplement. All animals were subjected to two-thirds partial hepatectomy (PH) at the end of week 3 and killed at week 8. Immunohistochemical staining for GST-P was performed according to previous reports [5,15]. Numbers and areas of GST-P positive foci per cm* were measured using an image analyzer (Olympus VIP-21C) [5,15]. Statistical analyses were performed using Student’s ttest.
GST-P staining in rat liver treated with 3’-Me-DAB + BHA after DEN injection GST-P positive focus and the diffuse GST-P induction in the periportal area.
(group 6). Note the discrete
27 Table I.
Quantitative
values for GST-P positive foci.
Group
Initiation
Promotion
Number (/cm*)
Area (mm2/cmz)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
DEN DEN DEN DEN DEN DEN DEN DEN DEN DEN DEN -
2-AAF + BHA 2-AAF + BHT 2-AAF + Catechol P-AAF + Na-AsA 2-AAF 3’-Me-DAB + BHA 3’-Me-DAB + BHT 3’-Me-DAB + Catechol 3’-Me-DAB + Na-AsA 3’-Me-DAB 2-AAF + BHA 2-AAF + BHT 2-AAF + Catechol 2-AAF + Na-AsA 2-AAF 3’-Me-DAB + BHA 3’-Me-DAB + BHT 3’-Me-DAB + Catechol 3’-Me-DAB + Na-AsA 3’-Me-DAB
46.24 43.52 53.65 37.70 39.77 3.09 6.25 8.18 14.86 19.08 5.02 1.77 2.75 2.75 45.79 40.56 0 0 0 0 0
8.97 10.17 19.85 78.30 79.20 0.32 0.51 0.62 1.46 1.65 0.41 0.10 0.19 0.29 15.13 14.75 0 0 0 0 0
“Significantly bSignificantly Significantly dSignificantly Significantly ‘Significantly gSignificantly
different different different different different different different
compared compared compared compared compared compared compared
to group to group to group to group to group to group to group
f + + f + + + f f f + f f f f +
10.31 13.41 18.54 11.25 12.96 1.49 2.97b 4.w 3.59 5.94 1.56 l.loc 2.55 1.29 3.66 3.22
+ 2.92d f 3.02d f 5.17d f 11.25 f 8.57 f 0.21e + 0.40’ rt: 0.29 f 0.32 f 0.58 + 0.19 f 0.09s + 0.19g f 0.06s f 7.88 +: 2.56
10 at P < 0.001. 10 at P < 0.01. 16 at P< 0.001. 5 at P< 0.001. 10 at P < 0.001. 10 at P< 0.01. 16 at P < 0.001.
Results Body weight decrease was noted in the BHT and catechol treatment groups and increased mortality after PH was evident in group 7, receiving 3’-Me-DAB plus BHT. There were no statistical differences in food consumption between the groups. The results concerning GST-P positive foci development are summarized in Table I. It was clearly demonstrated that the co-administration of BHA, BHT or catechol in the 2-AAF or 3’-Me-DABcontaining diets brought about an inhibition of GST- P positive foci development in terms of area attained. Also in the case of groups 1216 (withoutDEN injection), administration of BHA, BHT or catechol inhibited the induction
of GST-P positive foci by 2-AAF itself. 3’-MeDAB alone did not induce GST-P positive foci. Na-Asc did not demonstrate any effects on the development of DEN-initiated GST-P positive foci under the influence of 2-AAF or 3’-MeDAB plus PH. Foci number counts in Groups 4 and 5 were not compatible to their area values because of fusion of GST-P positive caused by expansive growth of the lesions. In the livers of rats treated with BHA, BHT, or catechol but not Na-AsA, GST-P binding was found to be strong in periportal areas. Discussion The results clearly showed
of the present investigation that the administration of 2%
BHA, 1% BHT or 0.8% catechol in the diet with 2AAF or 3’-Me-DAB markedly reduced their promoting effects on development of DEN-initiated GST-P positive foci. Countable GST-P positive foci are induced by 2AAF without the DEN initiation group, indicating 2AAF has initiating activity in addition to potent promotion activity. This initiation activity was also inhibited by BHA, BHT or catechol (Groups 12-14). Although 3’-Me-DAB is a liver carcinogen, it did not induce measurable amounts of GST-P positive foci because the dose employed was very low. Therefore it is unclear whether the antioxidants similary inhibited the initating activity of 3’-Me-DAB in this system. Sato et al. [12] earlier reported that the development of DEN-initiated gamma-glutamyl transpeptidase-positive foci and hyperplastic nodules under the influence of 2-AAF was similarly inhibited by diet containing a relatively low dose (0.75%) of BHA and suggested that the induction of specific molecular forms of glutathione S-transferase or UDP-gluconyltransferase might play important roles in this effect. In the light of the present findings the placental form of GST deserves particular consideration in this context, especially since other compounds such as 2-(2furyl)-3(5-nitro-2_furyl)acrylamide, tert-butylhydroquinone, p-methoxyphenol all similarly induce GST-P in the periportal area, while exerting inhibitory effects in a medium term bioassay system [5]. While being a very good enzyme marker for preneoplastic foci and hyperplastic nodules [15], GST-P was also found, in contrast to many other drug-metabolizing enzymes, not to be induced by a large number of carcinogens or tumor promoters such as 2-AAF, 3’-Me-DAB, ethionine, phenobarbital, 3-methylcholanthrene, polychlorinated biphenyls. With regard to mechanisms whereby GST-P could be involved in the inhibition observed in the present experiments, the fact that preneoplastic foci induced by carcinogens are known to be relatively resistant to the toxicity of carcinogens and can respond to regenerative stimuli such as PH, under selection pressure conditions is of interest [3,8,14]. GST-P in foci
may act as an integral part of the defense against cytotoxic substances. Periportal induction of GST-P might endow an equivalent advantage for cell growth on the involved hepatocytes. This would result in a relative decrease in development of GST-P positive foci because of the reported inverse relationship between size attained and proliferation of surrounding hepatocytes [ 161. Phenobarbital is known to be a potent promoter hepatocarcinogenesis when administered after initiation with hepatocarcinogens such as 2-AAF, DEN or azo dyes [6,10,18]. However phenobarbital fed simultaneously with a hepatocarcinogen inhibits hepatic tumorigenesis [9]. Microsomal mixed-function oxygenase inducers, including phenobarbital when administered simultaneously with hepatocarcinogens are thought to accelerate carcinogen metabolism and detoxification by both phase I (cytochrome P-450) and phase II (GSTs) drug metabolizing enzymes [17]. It is known that antioxidants like BHA and BHT also induce such enzymes [ 1,7], including GST-P [ 11,12,17]. Further biochemical or molecular biological studies are clearly required to clarify the relationship between GST-P induction in periportal area, change in other drug metabolizing species, differential proliferating potential and inhibitory effect in rat hepatocarcinogenesis. Acknowledgments The authors would like to express their gratitude to Dr. Kiyomi Sato, Second Department of Biochemistry, Hirosaki University School of Medicine for providing the GST-P antibody and to Dr. Ricardo Cabral for helpful discussion and advice concerning the manuscript. This research was supported in part by Grantsin-aid for Cancer Research from the Ministry of Education, Science and Culture and from the Ministry of Health and Welfare, for a Comprehensive 10 Year Strategy For Cancer Control from the Ministry of Health and Welfare, Japan, the Society for Promotion of Pathology of Nagoya and Experimental Pathological Association.
29
References
10
Benson, A.M., Batzinger, R.P., Ou, S.-Y .L., Bueding, E., Cha, Y.-N. and Talalay, P. (1978) Elevation of hepatic glutathione S-transferase activities and protection against mutagenic metabolites of benzo(a]pyrene by dietary
Peraino, C., Fry, R.J.M. and Staffeldt, E. (1971) Reduction and enhancement by phenobarbital of hepatocarcinogenesis induced in the rat by 2-acetylaminofluorene. Cancer Res., 31, 1506-1512.
11
antioxidants. Cancer Res., 38,4486-4495. Farber, E., (1984) Precancerous steps in carcinogenesis. Their physiological adaptive nature. Biochim. Biophys. Acta, 738, 171-180.
Prochaska, H.J. and Talalay, P. (1988) Regulatory mechanisms of monofunctional and bifunctional anticarcinogenie enzyme inducers in murine liver. Cancer Res.. 48. 4776-4782.
12
Sato, K., Kitahara, A., Yin, Z., Waragai, F.. Nishimura, K., Hatayama, I., Ebina, T.. Yamazaki. T., Tsuda, H. and Ito, N. (1984) Induction by butylated hydroxyanisole of specific molecular forms of glutathione S-transferase and UDP-glutamyltransferase and inhibition of development of &C-glutamyl transpeptidase-positive foci in rat liver. Carcinogenesis, 5,473-477. Satoh, K., Kitahara, A., Soma. Y., Inaba. Y.. Hatayama. I. and Sato, K. (1985) Purification, induction and distribution of placental glutathione transferase: a new marker enzyme for preneoplastic cells in the rat chemical carcinogenesis. Proc. Natl. Acad. Sci. USA, 82,3964-3968.
Hirose, M., Kurata, Y., Tsuda, H., Fukushima, S. and Ito, N. (1987) Catechol strongly enhances rat stomach carcinogenesis: a possible new environmental stomach carcinogen. Jpn. J. Cancer Res. (Gann), 78, 1144-1149. Ito, N., Hagiwara, A., Shibata, M., Ogiso, T. and Fukushima, S. (1982) Induction of squamous cell carcinoma in the forestomach of F344 rats treated with butylated hydroxyanisole. Gann, 73.332-334. ho, N., Tsuda, H., Tatematsu, M., lnoue, T., Tagawa, Y., Imaida, K., Fukushima, S. and Asamoto M. (1988) Enhancing effect of various hepatocarcinogens on induction of preneoplastic glutathione S-transferase placental form positive foci in rat - an approach for a new mediumterm bioassay system. Carcinogenesis, 9,387-394. Kitagawa, K., Pitot, H.C., Miller, E.C. and Miller, J.A. (1979) Promotion by dietary phenobarbital of hepatocarcinogenesis by 2-methyl-N,N-dimethyl-4-aminoazobenxene in the rat. Cancer Res., 39, 112- 115. Lam, L.K.T., Sparnins, V.L., Hochalter, J.B. and Wattenberg, L.W. (1981) Effect of 2- and 3-ten-butyl-4hydroxyanisole on glutathione S-transferase and epoxide hydrolase activities and sulfhydryl levels in liver and forestomach of mice. Cancer Res., 41,3940-3943. Moore, M.A., Nakagawa, K. and Ishikawa, T. (1988) Selection pressure and altered hepatocellular islands after a single injection of aflatoxin Bl. Jpn. J. Cancer Res. (Gann), 79,187-194. Narita, T., Watanabe, R and Kitagawa, T. (1980) Mechanisms of inhibition by simultaneously administrated pheno3’-methyl-4-(dimethylamino)azobenzenebarbital of induced hepatocarcinogenesis the rat. Gann. 71, 755758
13
14
Solt, D.B., Medline, A. and Farber, E. (1977) Rapid emergence of carcinogen-induced hyperplastic lesions in a new model for the sequential analysis of liver carcinogenesis. Am. J. Pathol., 88.595-618.
15
Tatematsu, M., Tsuda, H., Shirai. T., Masui, T. and ho. N. (1987) Placental glutathione S-transferase (GST-P) as a new marker for hepatocarcinogenesis: in vivo short-term screening for hepatocarcinogens. Toxicol. Pathol., 15. 60 -68. Tatematsu. M., Aoki, T., Kagawa, M.. Mera. Y. and Ito, N. (1988) Reciprocal relationship between development of glutathione S-transferase positive liver foci and proliferation of surrounding hepatocytes in rats. Carcinogenesis, 9, 221-225. Wattenberg, L.W. (1985) Chemoprevention of cancer. Cancer Res., 45, l-8. Weisburger, J.H., Madison, R.M., Ward, J.M., Viguera. C. and Weisburger E.K. (1975) Modification of diethylnitrosamine liver carcinogenesis with phenobarbital but not with immunosuppression. J. Natl. Cancer Inst., 54. 1185-1188.
16
17 18
Cancer Letters, 55 (1990) 25-29 Elsevier Scientific Publishers Ireland Ltd.
Inhibition of rat hepatic glutathione S-transferase form positive foci development by concomitant administration of antioxidants to carcinogen-treated M. Asamoto,
Y. Shichino,
First
of
Department
Nagoya
467
Pathology,
H. Tsuda* Nagoya
City
placental rats
and N. Ito
University
Medical
School,
1 Kawasumi,
Mizuho-cho,
Mizuho-ku,
(Japan)
(Received 30 August 1989) (Revision received 5 March 1990) (Accepted 11 September 1990)
Summary Inhibition potential
hydroxyanisole luene (BHT),
ofconcomitant
(BHA),
butylated
butylated hydroxyto-
catechol or sodium ascorbate (Na-AsA) administration on development of diethylnitrosamine (DEN) initiated glutathione S-transferase placental form (GST-P) positive foci in rat liver under the influence of Z-acetylaminofluorene (2.AAF) or 3’.methyl-4.dime(3’.Me-DAB) plus thylaminoazobenzene partial hepatectomy (PH) was investigated.
Whereas BHA, BHT and catechol exerted marked inhibitory effects, Na-AsA lacked any The compounds that modifying potential. demonstrated inhibition also induced GST-P in the hepatic periportal areas, suggesting that development of GST-P positive foci is negatively influenced by extra-focal increase in this enzyme form observed with BHA, BHT or
catechol.
Correspondence
ogy,
Nagoya
Miiuho-cho,
to: M. Asamoto, First Department of PatholCity University Medical School. 1 Kawasumi,
Miiuho-ku.
Nagoya 467, Japan. Department of Pathology, School of Medicine, Fujiia-Gakuen Health University, Katsukake-cho, Toyoake, Aichi 470- 11, Japan. *Present address: Second
Keywords: butylated hydroxyanisole; butylated hydroxytoluene; catechol; glutathione Stransferase placental form; rat hepatocarcinogenesis; inhibition Introduction Antioxidants such as BHA, BHT and NaAsA have been widely used as food additives in foods to prevent oxidation of labile lipid components. Another member of this group, catechol, is also present in the environment as an industrial chemical, as well as being a major phenolic component of cigarette smoke. Although Ito and his group [3,4] found that high doses of BHA or catechol can induce stomach neoplasia, Wattenberg et al. [17] have reported that antioxidants, including BHA and BHT, inhibit chemical carcinogenesis in various tissues of many species when administered concomitantly with carcinogens. Concentrating on development of an in vivo medium-term screening system for carcinogens and modifiers, we have established a reliable system for rapid detection of hepatocarcinogens [5] using GST-P as a marker protein for preneoplastic liver cell foci [13]. In addition, it was found that many compounds could act as inhibitory agents in this assay sys-
0 1990 Elsevier Scientific Publishers Ireland Ltd. 0304.3835/90/$03.50 Published and Printed in Ireland
26
tern reducing the induction of GST-P positive foci [5]. Retrospective checking for GST-P staining in slides from rats treated with inhibitors, revealed that strong inhibitors including BHA and catechol, induced GST-P in the periportal areas of treated rat livers. Therefore, in this study, the possible correlation between inhibition of GST-P positive foci development by concomitant administration of antioxidants with carcinogens and the induction of GST-P in the liver portal areas was investigated. Materials and Methods BHA, catechol and Na-AsA were obtained from Wako Pure Chemical Industries Ltd., Osaka, Japan and BHT from the National Institute of Hygienic Science, Tokyo. DEN, 2AAF and 3’-Me-DAB were purchased from Tokyo Chemical Industry Co. A total 215 6-week-old male F344 rats were used. The experimental protocol used was essentially the same as in previous reports [5,15]. Initially, 165 animals were treated i.p. with DEN at a dose of 200 mg/kg body weight, the remaining 50 rats receiving saline
Fig. 1.
vehicle alone. After 2 weeks on basal diet, carcinogen-initiated and control animals were divided into 11 groups (groups l- 11) and 10 groups (groups 12-21)) respectively (see Table 1). Groups l-5 and 12- 16 were given diet containing 0.02% 2-AAF plus 2% BHA (groups 1,12), 1% BHT (groups 2,13), 0.8% catechol (groups 3,14), 5% Na-AsA (groups 4,15) or 2-AAF alone (groups 5,16). Groups 6 -10 and 17-21 were similarly given a diet containing 0.012% 3’-Me-DAB plus 2% BHA (groups 6,17), 1% BHT (groups 7,18), 0.8% catechol (groups 8,19), 5% Na-AsA (groups 9,20) or no addition (groups 10,21). Group 11 received the basal diet without supplement. All animals were subjected to two-thirds partial hepatectomy (PH) at the end of week 3 and killed at week 8. Immunohistochemical staining for GST-P was performed according to previous reports [5,15]. Numbers and areas of GST-P positive foci per cm* were measured using an image analyzer (Olympus VIP-21C) [5,15]. Statistical analyses were performed using Student’s ttest.
GST-P staining in rat liver treated with 3’-Me-DAB + BHA after DEN injection GST-P positive focus and the diffuse GST-P induction in the periportal area.
(group 6). Note the discrete
27 Table I.
Quantitative
values for GST-P positive foci.
Group
Initiation
Promotion
Number (/cm*)
Area (mm2/cmz)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
DEN DEN DEN DEN DEN DEN DEN DEN DEN DEN DEN -
2-AAF + BHA 2-AAF + BHT 2-AAF + Catechol P-AAF + Na-AsA 2-AAF 3’-Me-DAB + BHA 3’-Me-DAB + BHT 3’-Me-DAB + Catechol 3’-Me-DAB + Na-AsA 3’-Me-DAB 2-AAF + BHA 2-AAF + BHT 2-AAF + Catechol 2-AAF + Na-AsA 2-AAF 3’-Me-DAB + BHA 3’-Me-DAB + BHT 3’-Me-DAB + Catechol 3’-Me-DAB + Na-AsA 3’-Me-DAB
46.24 43.52 53.65 37.70 39.77 3.09 6.25 8.18 14.86 19.08 5.02 1.77 2.75 2.75 45.79 40.56 0 0 0 0 0
8.97 10.17 19.85 78.30 79.20 0.32 0.51 0.62 1.46 1.65 0.41 0.10 0.19 0.29 15.13 14.75 0 0 0 0 0
“Significantly bSignificantly Significantly dSignificantly Significantly ‘Significantly gSignificantly
different different different different different different different
compared compared compared compared compared compared compared
to group to group to group to group to group to group to group
f + + f + + + f f f + f f f f +
10.31 13.41 18.54 11.25 12.96 1.49 2.97b 4.w 3.59 5.94 1.56 l.loc 2.55 1.29 3.66 3.22
+ 2.92d f 3.02d f 5.17d f 11.25 f 8.57 f 0.21e + 0.40’ rt: 0.29 f 0.32 f 0.58 + 0.19 f 0.09s + 0.19g f 0.06s f 7.88 +: 2.56
10 at P < 0.001. 10 at P < 0.01. 16 at P< 0.001. 5 at P< 0.001. 10 at P < 0.001. 10 at P< 0.01. 16 at P < 0.001.
Results Body weight decrease was noted in the BHT and catechol treatment groups and increased mortality after PH was evident in group 7, receiving 3’-Me-DAB plus BHT. There were no statistical differences in food consumption between the groups. The results concerning GST-P positive foci development are summarized in Table I. It was clearly demonstrated that the co-administration of BHA, BHT or catechol in the 2-AAF or 3’-Me-DABcontaining diets brought about an inhibition of GST- P positive foci development in terms of area attained. Also in the case of groups 1216 (withoutDEN injection), administration of BHA, BHT or catechol inhibited the induction
of GST-P positive foci by 2-AAF itself. 3’-MeDAB alone did not induce GST-P positive foci. Na-Asc did not demonstrate any effects on the development of DEN-initiated GST-P positive foci under the influence of 2-AAF or 3’-MeDAB plus PH. Foci number counts in Groups 4 and 5 were not compatible to their area values because of fusion of GST-P positive caused by expansive growth of the lesions. In the livers of rats treated with BHA, BHT, or catechol but not Na-AsA, GST-P binding was found to be strong in periportal areas. Discussion The results clearly showed
of the present investigation that the administration of 2%
BHA, 1% BHT or 0.8% catechol in the diet with 2AAF or 3’-Me-DAB markedly reduced their promoting effects on development of DEN-initiated GST-P positive foci. Countable GST-P positive foci are induced by 2AAF without the DEN initiation group, indicating 2AAF has initiating activity in addition to potent promotion activity. This initiation activity was also inhibited by BHA, BHT or catechol (Groups 12-14). Although 3’-Me-DAB is a liver carcinogen, it did not induce measurable amounts of GST-P positive foci because the dose employed was very low. Therefore it is unclear whether the antioxidants similary inhibited the initating activity of 3’-Me-DAB in this system. Sato et al. [12] earlier reported that the development of DEN-initiated gamma-glutamyl transpeptidase-positive foci and hyperplastic nodules under the influence of 2-AAF was similarly inhibited by diet containing a relatively low dose (0.75%) of BHA and suggested that the induction of specific molecular forms of glutathione S-transferase or UDP-gluconyltransferase might play important roles in this effect. In the light of the present findings the placental form of GST deserves particular consideration in this context, especially since other compounds such as 2-(2furyl)-3(5-nitro-2_furyl)acrylamide, tert-butylhydroquinone, p-methoxyphenol all similarly induce GST-P in the periportal area, while exerting inhibitory effects in a medium term bioassay system [5]. While being a very good enzyme marker for preneoplastic foci and hyperplastic nodules [15], GST-P was also found, in contrast to many other drug-metabolizing enzymes, not to be induced by a large number of carcinogens or tumor promoters such as 2-AAF, 3’-Me-DAB, ethionine, phenobarbital, 3-methylcholanthrene, polychlorinated biphenyls. With regard to mechanisms whereby GST-P could be involved in the inhibition observed in the present experiments, the fact that preneoplastic foci induced by carcinogens are known to be relatively resistant to the toxicity of carcinogens and can respond to regenerative stimuli such as PH, under selection pressure conditions is of interest [3,8,14]. GST-P in foci
may act as an integral part of the defense against cytotoxic substances. Periportal induction of GST-P might endow an equivalent advantage for cell growth on the involved hepatocytes. This would result in a relative decrease in development of GST-P positive foci because of the reported inverse relationship between size attained and proliferation of surrounding hepatocytes [ 161. Phenobarbital is known to be a potent promoter hepatocarcinogenesis when administered after initiation with hepatocarcinogens such as 2-AAF, DEN or azo dyes [6,10,18]. However phenobarbital fed simultaneously with a hepatocarcinogen inhibits hepatic tumorigenesis [9]. Microsomal mixed-function oxygenase inducers, including phenobarbital when administered simultaneously with hepatocarcinogens are thought to accelerate carcinogen metabolism and detoxification by both phase I (cytochrome P-450) and phase II (GSTs) drug metabolizing enzymes [17]. It is known that antioxidants like BHA and BHT also induce such enzymes [ 1,7], including GST-P [ 11,12,17]. Further biochemical or molecular biological studies are clearly required to clarify the relationship between GST-P induction in periportal area, change in other drug metabolizing species, differential proliferating potential and inhibitory effect in rat hepatocarcinogenesis. Acknowledgments The authors would like to express their gratitude to Dr. Kiyomi Sato, Second Department of Biochemistry, Hirosaki University School of Medicine for providing the GST-P antibody and to Dr. Ricardo Cabral for helpful discussion and advice concerning the manuscript. This research was supported in part by Grantsin-aid for Cancer Research from the Ministry of Education, Science and Culture and from the Ministry of Health and Welfare, for a Comprehensive 10 Year Strategy For Cancer Control from the Ministry of Health and Welfare, Japan, the Society for Promotion of Pathology of Nagoya and Experimental Pathological Association.
29
References
10
Benson, A.M., Batzinger, R.P., Ou, S.-Y .L., Bueding, E., Cha, Y.-N. and Talalay, P. (1978) Elevation of hepatic glutathione S-transferase activities and protection against mutagenic metabolites of benzo(a]pyrene by dietary
Peraino, C., Fry, R.J.M. and Staffeldt, E. (1971) Reduction and enhancement by phenobarbital of hepatocarcinogenesis induced in the rat by 2-acetylaminofluorene. Cancer Res., 31, 1506-1512.
11
antioxidants. Cancer Res., 38,4486-4495. Farber, E., (1984) Precancerous steps in carcinogenesis. Their physiological adaptive nature. Biochim. Biophys. Acta, 738, 171-180.
Prochaska, H.J. and Talalay, P. (1988) Regulatory mechanisms of monofunctional and bifunctional anticarcinogenie enzyme inducers in murine liver. Cancer Res.. 48. 4776-4782.
12
Sato, K., Kitahara, A., Yin, Z., Waragai, F.. Nishimura, K., Hatayama, I., Ebina, T.. Yamazaki. T., Tsuda, H. and Ito, N. (1984) Induction by butylated hydroxyanisole of specific molecular forms of glutathione S-transferase and UDP-glutamyltransferase and inhibition of development of &C-glutamyl transpeptidase-positive foci in rat liver. Carcinogenesis, 5,473-477. Satoh, K., Kitahara, A., Soma. Y., Inaba. Y.. Hatayama. I. and Sato, K. (1985) Purification, induction and distribution of placental glutathione transferase: a new marker enzyme for preneoplastic cells in the rat chemical carcinogenesis. Proc. Natl. Acad. Sci. USA, 82,3964-3968.
Hirose, M., Kurata, Y., Tsuda, H., Fukushima, S. and Ito, N. (1987) Catechol strongly enhances rat stomach carcinogenesis: a possible new environmental stomach carcinogen. Jpn. J. Cancer Res. (Gann), 78, 1144-1149. Ito, N., Hagiwara, A., Shibata, M., Ogiso, T. and Fukushima, S. (1982) Induction of squamous cell carcinoma in the forestomach of F344 rats treated with butylated hydroxyanisole. Gann, 73.332-334. ho, N., Tsuda, H., Tatematsu, M., lnoue, T., Tagawa, Y., Imaida, K., Fukushima, S. and Asamoto M. (1988) Enhancing effect of various hepatocarcinogens on induction of preneoplastic glutathione S-transferase placental form positive foci in rat - an approach for a new mediumterm bioassay system. Carcinogenesis, 9,387-394. Kitagawa, K., Pitot, H.C., Miller, E.C. and Miller, J.A. (1979) Promotion by dietary phenobarbital of hepatocarcinogenesis by 2-methyl-N,N-dimethyl-4-aminoazobenxene in the rat. Cancer Res., 39, 112- 115. Lam, L.K.T., Sparnins, V.L., Hochalter, J.B. and Wattenberg, L.W. (1981) Effect of 2- and 3-ten-butyl-4hydroxyanisole on glutathione S-transferase and epoxide hydrolase activities and sulfhydryl levels in liver and forestomach of mice. Cancer Res., 41,3940-3943. Moore, M.A., Nakagawa, K. and Ishikawa, T. (1988) Selection pressure and altered hepatocellular islands after a single injection of aflatoxin Bl. Jpn. J. Cancer Res. (Gann), 79,187-194. Narita, T., Watanabe, R and Kitagawa, T. (1980) Mechanisms of inhibition by simultaneously administrated pheno3’-methyl-4-(dimethylamino)azobenzenebarbital of induced hepatocarcinogenesis the rat. Gann. 71, 755758
13
14
Solt, D.B., Medline, A. and Farber, E. (1977) Rapid emergence of carcinogen-induced hyperplastic lesions in a new model for the sequential analysis of liver carcinogenesis. Am. J. Pathol., 88.595-618.
15
Tatematsu, M., Tsuda, H., Shirai. T., Masui, T. and ho. N. (1987) Placental glutathione S-transferase (GST-P) as a new marker for hepatocarcinogenesis: in vivo short-term screening for hepatocarcinogens. Toxicol. Pathol., 15. 60 -68. Tatematsu. M., Aoki, T., Kagawa, M.. Mera. Y. and Ito, N. (1988) Reciprocal relationship between development of glutathione S-transferase positive liver foci and proliferation of surrounding hepatocytes in rats. Carcinogenesis, 9, 221-225. Wattenberg, L.W. (1985) Chemoprevention of cancer. Cancer Res., 45, l-8. Weisburger, J.H., Madison, R.M., Ward, J.M., Viguera. C. and Weisburger E.K. (1975) Modification of diethylnitrosamine liver carcinogenesis with phenobarbital but not with immunosuppression. J. Natl. Cancer Inst., 54. 1185-1188.
16
17 18