[CANCER RESEARCH54, 3145—3152, June 15, 19941 ABSTRACT Direct intercellular signal transduction Is achieved by the passage of small molecules through gap junctions (Gi). Previous studies in our labentory showed that the liver tumor promoter phenobarbital (PB) reversibly decreases the abundance of the GJ protein connexln32 (Cx32) In both preneoplastic-altered hepatic foci and centrolobular hepatocytes (M. J. Neveu et al., Cancer Commun., 2: 21—31, 1990). Because the inhibitory effects of PB on GJ intercellular communication are prevented by the nonspecific cytochrome P-450 Inhibiter SKF-525A (J. E. Klaunig, et al., Toxicol. AppL Pharmacol., 102: 533-563, 1990), we investigated whether alterations In Cx32 are coincident with changes In the m*jor PB-Inducible cytochrome P-450, termed b/c or IIB1/2. Immunostaining of liver cryosections from rats fed dietary PB demon strated that centrolobular hepatocytes that exhibit reduced Cx32 express enhanced cytochromeP4SOIIB1/2protein. In contrast, no change In the periportal distribution of connexin26 immunoreactivity was found in PB-treated rats. In addition, rats were treated with the structurally re lated barbiturates pentobarbital, amobarbital, barbital, and barbituric acid. We found that the extent ofthe hepatic lobule occupied by coincident ceutrolobular alterations in Cx32 and P.450 staining correlates with the ability of the compounds to promote liver oncogenesis. To determine the molecular mechanisms responsible for the modifica don in Cx32 staining, we examined the mRNA and protein levels of Cx32 and P4SOILB1/2in total-tissue homogenates from PB-treated rats. North era blotting demonstrated that dietary PB dramatically induced P-45011B1mP.NA,but the sameRNAsamplesfailedto showalterationsIn Cx32 steady-state transcripts. Consistent with these findings, the level of Cx32 protein in total liver homogenates did not change in rats chronically fed PB. Ei*minatlon of Cx32 solubifity in 20 mat NaOH demonstrated that PB treatment results in the generation of a NaOH-soluble form of Cx32 (Le., 47 kDa). In addition, trypsinized paraffin-embedded liver sections from PB-treated rats exhibited difñise cytoplasmic Cx32 staining that was restricted to centrolobular cells. Our results show that PB and related barbiturate tumor promoters reversibly down-regulate punctate Cx32 staining in centrolobular hepatocytes posttranslatlonaliy, possibly through modification(s) in the transport, assembly, and/or turnover of GiL INTRODUCTION GJs4 are aggregates of transmembrane pores that mediate intercel lular signal transduction by regulating the intercellular transfer of ions, metabolites, second messengers, and other molecules less than Received 6/23/93; accepted 4/13/94. Thecostsof publicationof thisarticleweredefrayedinpartbythepaymentof page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 The studies described in this investigation were supported by grants from the National Cancer Institute (CA-07175 and CA-45700), USPHS. M. J. N. was a predoctoral traineein Environmental Toxicologyof theNationalInstituteof Environmental Health Sciences (ESO-7015). 2 Present address: Pfizer Central Research, Eastem Point Rd., Groton, CT 06340. 3 To whom requests for reprints should be addressed, at McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, 1400 University Ave., Madison, WI 53706.1599. 4 The abbreviations used are: GJ, gap junction; GJIC, gap junctional intercellular communication; Cx32, connexln32; Cx26, connexin26; Cx43, connexin43; PB, pheno barbital; DDT, 1,1,1-trichloro-2,2-bis(p.chlorophcnyl)cthane; RT, room temperature; SDS,sodiumdodecylsulfate;PAGE, polyacrylamide gel electrophoresis. 1000 daltons in mass (1, 2). Cloning studies have identified a family of GJ proteins, termed connexins, that are distinguished by their predicted molecular mass (2). Many normal biological processes including tissue homeostasis, embryonic development, glandular se cretion, cellular differentiation, and growth control are modulated by GJIC (2—4). Alterations in the number of GJs or the level of GJIC have been found in preneoplastic and neoplastic cells from several rodent and human tissues. Furthermore, many tumor promoters, oncogenic pro teins, and growth factors modulate OJIC (2—7).In addition, transfec tion of tumor cells with either Cx32 or Cx43 results in reduced growth rates in cell culture and in nude mice (3, 4). In rodent and human liver, the abundance of GJs, the level of GJIC, and the expression of Cx32 are diminished in most preneoplastic altered hepatic foci and carcinomas (4, 6, 7). In addition to these changes in mutated cells, the liver tumor promoter PB decreases the morphological abundance of GJs (8) and the level of punctate Cx32 staining in centrolobular hepatocytes (9). Furthermore, physiological studies demonstrated that PB rapidly inhibits hepatocyte GJIC in primary cultures (7, 10) and liver tissue slices (11). The PB-induced alterations in both GJIC and Cx32 staining are rapidly reversible after withdrawal of PB (9, 10). In this report we examined whether the PB-induced changes in Cx32 staining in centrolobular hepatocytes associate with other previously characterized changes in gene expression. Administration of PB to rodents induces a program of adaptive changes in centrolobular hepatocytes. The modifications include in creased levels of smooth endoplasmic reticulum, cytochrome P45OIIB1/2, epoxide hydrolase, and isozymes of glutathione S-trans ferase (12, 13). A strong association exists between the ability of a subclass of compounds, including barbiturates, to promote hepatocar cinogenesis and the ability to induce cytochrome P45011B1/2 enzy matic activity (14). Relatively minor modifications in the structure of barbiturates can modify these activities (see Fig. 1). Amobarbital, pentobarbital, and barbituric acid are weak inducers of P45OIIB1/2 enzymatic activity and are ineffective promoters of rat hepatocarci nogenesis (15—20).Furthermore, the inducibility of P45011B1/2 ac tivity correlates with the susceptibility of various strains of rodents to PB promotion (14, 22). The present study examines whether alterations in Cx32 and P45OIIB1/2 protein expression occur in livers from rats treated with PB, amobarbital, barbituric acid, pentobarbital, or barbital. Because the inhibitory effect of PB on GJIC in primary hepatocytes is prevented by the cytochrome P-450 inhibitor SKF-525A (23, 24), changes in Cx32 and P45011B1/2 may colocalize. In support of this hypothesis, PB is a weak or ineffective inhibitor of GJIC in fibroblasts (V79) and in liver epithelial cells (WB-F344) that exhibit low levels of microsomal enzyme activity (7). To determine which molecular mechanisms were responsible for centrolobular alterations in Cx32 staining, we examined the expression of Cx32 mRNA and protein after dietary PB. Contrary to a previous report, which suggested that PB reduces the level of Cx32 mRNA (25), our results show that PB induces posttranslational alterations in the 3145 Colocalized Alterations in Connexin32 and Cytochrome P45011B1/2 by Phenobarbital and Related Liver Tumor Promoters' Mark J. Neveu,2 Karlee L. Babcock, Effiot L. Hertzberg, David L. Paul, Bruce J. Nicholson, and Henry C. Pitot@ McArdle Laboratory for Cancer Research and Center for Environmental Toxicology, University of Wisconsin@ Madison, Wisconsin 53706-1599 fM. J. N., K L. B., H. C. P.J; Department of Neuroscience Albert Einstein College of Medicine, Bronx New York 10461 fE L. H.J; Department of Anatomy and Cell Biology, Harvard Medical School, Boston@Massachusetts 02115 [D. L P.]; and Department ofBiological Sciences, SUNY-Buffalo, Buffalo, New York 14260 [B. J. N.J Research. on December 5, 2021. © 1994 American Association for Cancer cancerres.aacrjournals.org Downloaded from