Immunohistochemical Demonstration of the Gap Junctional Protein Connexin 32 and Proliferating Cell Nuclear Antigen in Glutathione S-Transferase Placental Form-Negative Lesions of Rat Liver Induced by Diethylnitrosamine and Clofibrate

Abstract

The distributions of a gap junctional protein, connexin 32 (cx 32), and proliferating cell nuclear antigen (PCNA) were examined immunohistochemically in glutathione S-transferase placental form (GST-P)-negative foci, induced in rat liver by initiation with diethylnitrosamine (DEN, 200 mg/kg) followed by promotion with clofibrate (1% in diet) in an in vivo medium-term assay system for hepatocarcinogenesis. The results were compared to those in GST-P-positive foci induced by DEN alone. The treatment with clofibrate caused the appearance of GST-P-negative foci, increased in size as compared to GST-P-positive foci in the same liver or induced by the DEN alone. The proportion of PCNA-positive hepatocytes in GST-P-negative foci was significantly higher than in the surrounding parenchyma, indicating increased cell proliferation. The numbers of cx 32-positive spots per hepatocyte in GST-P-negative foci were clearly decreased, reaching 65.4% at week 20 and 51.8% at week 30 of values for surrounding normal hepatocytes. In GST-P-positive foci induced by DEN, only a slight decrease (80%) was observed at week 8. These findings show that a positive association between the sustained inhibition of gap junctional intercellular communication and increased cell proliferation of GST-P-negative foci in Fischer-344 male rats induced with DEN and promoted with clofibrate.

Keywords

Gap junction peroxisome proliferator cell proliferation hepatocarcinogenesis

References

Ashby J. , Brady A. , Elcombe CR , Elliott BM , Ishael J. , Tugwood JD , Kettle S. , and Purchase IFH (1994). V. The role of cell proliferation in peroxisome proliferator mediated-hepatocarcinogenesis. In: Mechanistically-Based Human Hazard Assessment of Peroxisome Proliferator-Induced Hepatocarcinogenesis . Human Exp. Toxicol. 13(suppl. 2): S36-S40.

Cattley RC , Marsman D. , Conolly RB , and Popp JA (1992). Cell proliferation and promotion in peroxisome proliferator-induced rodent hepatocarcinogenesis. CIIT Activities 12: 1-5.

Cattley RC , Marsman D. , and Popp JA (1991). Age-related susceptibility to the carcinogenic effect of the peroxisome proliferator WY-14,643. Carcinogenesis 12: 469-473.

Hosokawa S. , Tatematsu M. , Aoki T. , Nakanowatari J. , Igarashi T. , and Ito N. (1989). Modulation of diethylnitrosamine-initiated placental gultathione S-transferase positive preneoplastic and neoplastic lesions by clofibrate, a hepatic peroxisome proliferator. Carcinogenesis 10: 2237-2241.

Ito N. , Tatematsu M. , Hasegawa R. , and Tsuda H. (1989). Medium-term bioassay system for detection of carcinogens and modifiers of hepatocarcinogenesis utilizing the GST-P positive liver cell focus as an endpoint marker. Toxicol. Pathol. 17: 630-641.

Ito S. , Watanabe T. , Yoshioka K. , Tateno C. , Okuno Y. , Yanaihara N. , and Yositake A. (1993). Immunohistochemical demonstration of c-myc oncogene product and GST-P in rat hepatic preneoplastic lesions induced by diethylnitrosamine and clofibrate. Biomed. Res. 14: 345-351.

Krutovskikh VA , Oyamada M. , and Yamasaki H. (1991). Sequential changes of gap-junctional intercellular communications during multistage rat liver carcinogenesis: Direct measurement of communication in vivo. Carcinogenesis 12: 1701-1706.

Mensnil M. , Fitzgerald DJ , and Yamasaki H. (1988). Phenobarbital specifically reduces gap junction protein mRNA level in rat liver. Mol. Carcinog. 1: 79-81.

Milks LC , Kumar NM , Haughten R. , Unwin N. , and Gilula NB (1988). Topology of the 32 kd gap junction protein determined by site-directed antibody localizations. EMBO J. 7: 2967-2975.

10.

Neveu MJ , Hully JR , Paul DL , and Pitot HC (1990). Reversible alteration in the expression of the gap junctional protein connexin 32 during tumor promotion in rat liver and its role during cell proliferation. Cancer Commun. 2: 21-31.

11.

Ogiso T. , Tatematsu M. , Tamano S. , Hasegawa R. , and Ito N. (1990). Correlation between medium-term liver bioassay system data and results of long-term testing in rats. Carcinogenesis 11: 561-566.

12.

Rao MS , Tatematsu M. , Subbarao V. , Ito N. , and Reddy K. (1986). Analysis of peroxisome proliferator-induced preneoplastic and neoplastic lesions of rat liver for placental form of glutathione S-transferase and γ-glutamyltranspeptidase. Cancer Res. 46: 5287-5290.

13.

Reddy JK and Lalwai ND (1983). Carcinogenesis by hepatic peroxisome proliferators: Evaluation of the risk of hypolipidemic drugs and industrial plasticizers to humans. CRC Crit. Rev. Toxicol. 12: 1-58.

14.

Reddy JK and Qureshi SA (1973). Tumorigenicity of the hypolipidaemic peroxisome proliferator ethyl-α-p-chloro-phenoxyisobutyrate (clofibrate) in rats. Br. J. Cancer 40: 476-482.

15.

Satoh K. , Kitahara A. , Soma 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 hepatocarcinogenesis. Proc. Natl. Acad. Sci. USA 82: 3964-3968.

16.

Stott WT (1988). Chemically induced proliferation of peroxisomes: Implications for risk assessment. Reg. Toxicol. Pharmacol . 8: 125-159.

17.

Sugie S. , Mori H. and Takahashi M. (1987). Effect of in vivo exposure to the liver tumor promoters phenobarbital or DDT on gap junctions of rat hepatocytes: A quantitative freeze-fracture analysis. Carcinogenesis 8: 45-51.

18.

Tanaka K. , Smith PF , Stromberg PC , Eydelloth RS Herold EG , Grassman SJ , Frank JD , Hertzog PR Soper KA , and Keenan KP (1992). Studies of early hepatocellular proliferation and peroxisomal proliferation in Sprague-Dawley rats treated with tumorigenic doses of clofibrate. Toxicol. Appl. Pharmacol. 116: 71-77.

19.

Yamasaki H. (1990). Gap junctional intercellular communication and carcinogenesis. Carcinogenesis 11: 1051-1058.

20.

Zeilmarker MJ and Yamasaki H. (1986). Inhibition of junctional intercellular communication as a possible short-term test to detect tumor-promoting agents: Results with nine chemicals tested by dye transfer assay in Chinese hamster V79 cells. Cancer Res. 46: 6180-6186.