[CANCER RESEARCH 48, 2519-2523, May 1, 1988]

Kinetics of Phénobarbital Inhibition of Intercellular Communication in Mouse

HepatocytesRandall J. Ruch and James E. Klaunig1

Department of Pathology, Medical College of Ohio, Toledo, Ohio 43699

ABSTRACT

Gap junction-mediated intercellular communication in untreated andphenobarbital-treated C57BL/6 x C3H FI mouse hepatocytes was eval

uated by microinjection of fluorescent Lucifer Yellow CH dye. Intercellular communication (dye coupling) was detected in untreated hepatocytesafter 0.5 h in culture, reached a maximum level in 24- and 48-h-old

cultures (85.2%), and then decreased over the next 72 h. Phénobarbital(20-500 Mg/ml) decreased dye coupling in a dose-related manner when

added to freshly plated cultures. This inhibitory effect was evident during0.5-12 h of treatment but was not seen in cultures treated for 24 h.

Phénobarbitalalso decreased dye coupling within 30 min when added toestablished (24-h-old) hepatocyte cultures. This effect was maximal after

2 h treatment. In these cultures, dye coupling recovered within 15 minafter removal of the promoter. Hepatocytes, pretreated with phénobarbital for 24 h, did not show inhibition of dye coupling after reapplicationof phénobarbital. Thus, phénobarbital inhibited mouse hepatocyte dyecoupling rapidly and reversibly, and the cells became refractory to theinhibitory effect after prolonged treatment.

INTRODUCTION

Cell growth may be regulated by the cell-to-cell exchange ofsmall molecules and ions through gap junctions (i.e., intercellular communication) (1). The loss of gap junctions or a decrease in cell-to-cell communication may predispose cells toenhanced growth (1). Gap junctions are decreased or absent inregenerating liver (2, 3), and intercellular communication canbe decreased by growth factors (4) and tumor promoters (5-8).Several types of neoplastic cells have also been shown to havea reduced or a complete loss of intercellular communication(D-

The loss or inhibition of intercellular communication is alsothought to be a mechanism involved in tumor promotion (8).Tumor promoters often have mitogenic activity in their targettissue, thus permitting the expansion of the initiated cell population (9, 10). This stimulated cell growth may increase thelikelihood of additional genetic events required for completeneoplastic transformation to occur in an initiated cell (10). Themechanism by which tumor promoters exert their mitogeniceffect remains unclear. However, it is now known that nearlyall tumor promoters inhibit intercellular communication bothi/i vivo (11-13) and in vitro (8). This inhibitory effect appearsto be characteristic of tumor promoters, not genotoxic carcinogens or cytotoxic agents (14). Therefore, one possible mechanism by which tumor promoters enhance initiated cell growthmay be through their ability to inhibit intercellular communication.

Work in our laboratory has focused on defining the mechanisms by which tumor promoters inhibit hepatocyte intercellular communication (7, 14-16). We have previously assessedhepatocyte intercellular communication by gap-junctional passage of [3H]uridine nucleotides from prelabeled "donor" hepatocytes to nonlabeled "recipient" hepatocytes by autoradiogra-

Received 9/22/87; revised 1/12/88; accepted 2/8/88.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1To whom requests for reprints should be addressed.

phy (7). With this method, we have shown that several livertumor promoters inhibited hepatocyte intercellular communication, whereas genotoxic carcinogens and hepatocytotoxinsdid not (7, 14). In addition, the inhibitory effect of liver tumorpromoters on rodent hepatocyte intercellular communicationcorrelated with in vivo strain and species sensitivities to thehepatocarcinogenic activity of the promoters (16).

Unfortunately, the [3H]uridine method to detect hepatocyte

intercellular communication is limited in that the kinetics ofthe inhibitory effect of a promoter cannot easily be studied.This is because there is a delay between gap-junctional passageof [3H]uridine nucleotides from donor to recipient cells and

incorporation into recipient RNA (7). In hepatocytes, this delaylimits the earliest time of detection of intercellular communication to 4 h after establishment of donor-recipient cultures (7).Also, agents that inhibit recipient cell RNA synthesis mightartifactually result in observed decreases in intercellular communication. To circumvent these limitations of the nucleotidetransfer method, we have in this investigation evaluated hepatocyte intercellular communication by microinjection of fluorescent dye (Lucifer Yellow CH) and observing spread of dyeinto adjacent cells (dye coupling). Lucifer Yellow CH is plasmamembrane impermeable (17) but small enough (M, 440) to passthrough liver cell gap junctions (molecular weight exclusionlimit of about 1000; Ref. 18). Thus, detection of hepatocyteintercellular communication using Lucifer Yellow CH dye coupling is nearly instantaneous and dependent only on the existence of permeable gap junctions, not on tracer incorporation.In the present study, we have utilized Lucifer Yellow CH dyecoupling to evaluate the time course of the inhibitory effect ofphénobarbital,a liver tumor promoter (19), and recovery fromthe inhibitory action of phénobarbital on male C57BL/6 xC3H F, (hereafter called B6C3F,) mouse hepatocyte intercellular communication.

MATERIALS AND METHODS

Animals. Male B6C3F,/CNIBR mice, 4-6 months old, were purchased from Charles River Laboratories, Inc. (Wilmington, MA), andused exclusively in this study. Mice were housed in polycarbonate cagesand fed Purina Lab Chow Blox (Ralston Purina Co., St. Louis, MO)and water ad libitum.

Chemicals. Phénobarbitaland Lucifer Yellow CH were purchasedfrom Sigma Chemical Co. (St. Louis, MO). All other reagents andtissue culture supplies were purchased from sources previously indicated(7).

Hepatocyte Isolation and Culture. Hepatocytes were isolated by two-stage collagenase perfusion through the portal vein (20) and plated outat 1 x 10* viable cells per 60-mm plastic dish in 3 ml medium. Initial

viability of the isolated cells, determined by trypan blue dye exclusion,was always above 90%. The cells were cultured in Leibovitz's L-15

medium supplemented with glucose (1 mg/ml), dexamethasone (1 MM),gentamicin sulfate (50 Mg/ml), and fetal bovine serum (10%, v/v;Hyclone Laboratories, Logan, UT) at 37'C (21). The cultures were

refed with 3 ml medium/dish after a 2-h attachment period.Detection of Hepatocyte Intercellular Communication by Lucifer Yel

low Dye Injection. Microelectrodes were pulled from 1.5-mm-diametersingle barrel glass Kwik-Fil capillaries (World Precision Instruments,

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Inc., New Haven, CT) using a Narishige model PE-2 vertical micro-electrode puller (Narishige Scientific Instrument Laboratory, Tokyo,Japan). Microelectrode tip diameters measured approximately 1 Aim.Microelectrode tips were backfilled with 5% (w/v) Lucifer Yellow CHin 0.1 M LiCl, and microelectrode barrels were backfilled with 0.1 MLiCl. Hepatocyte cultures (2-120 h old) were observed under a NikonOptiphot UFX-II epifluorescence microscope (Nikon, Inc., GardenCity, NJ) at xlOO at room temperature. "Donor" hepatocytes were

impaled with the microelectrode under phase contrast microscopy anddye was iontophoretically ejected using continuous 3 nA current for 1min. Five min after cessation of dye injection, hepatocytes in directcontact with donor hepatocytes (i.e., recipient hepatocytes) were evaluated under epifluorescence for evidence of dye accumulation (dyecoupling). All recipients in contact with injected donors were evaluatedfor evidence of dye coupling. The percentage of dye-coupled recipientswas determined for each treatment and sampling time. Differences inthe number of dye-coupled and non-dye-coupled recipients betweentreatment groups were statistically evaluated by 2 x 2 x2 analysis (22).Dye-coupled cells were photographed by epifluorescence-phase contrastmicroscopy with Tri-Pan film (ASA 400) (Eastman Kodak Co., Rochester, NY).

Occurrence of Dye Coupling in Nontreated Mouse Hepatocytes duringthe First 120 h of Culture. To determine the extent of intercellularcommunication (dye coupling) in nontreated hepatocytes over a prolonged culture period (120 h), nontreated cultures were sampled at 0.5,1, 2, 4, 6, 8, 12, 24, 48, 72, 96, and 120 h after initial plating andevaluated for dye coupling. The cultures were refed just prior to the2-, 24-, 48-, 72-, 96-, and 120-h sampling times.

Effects of Phénobarbitalon Dye Coupling in Mouse HepatocyteCultures. Initial experiments were performed to determine if phénobarbital could inhibit intercellular communication (dye coupling) betweennewly cultured cells and if the effect was sustained during the first 48h of culture. Immediately after plating, cultured hepatocytes weretreated with phénobarbital(20, 100, or 500 Mg/ml) in DMSO2 or with

DMSO (0.2%). After 2 h attachment, the cultures were refed andretreated with phénobarbitalor DMSO only. Dye coupling in phénobarbital- and DMSO-treated cultures was determined at 0.5, 1, 2, 4, 6,8, 12, 24, and 48 h after plating. The cultures were refed and retreatedwith phénobarbitalprior to the 2-h sampling time but not at 24 and 48h.

To determine if phénobarbitalcould inhibit intercellular communication in established cultures (with preexisting gap junctions) and toassess the minimum duration of treatment necessary for an inhibitoryeffect to be seen, 24-h-old cultures were treated with phénobarbital(500Mg/ml) or DMSO (0.2%) only and evaluated for dye coupling 0.25, 0.5,1, 2, 3, and 4 h after treatment.

Studies were also performed to assess how rapidly hepatocyte dyecoupling recovered following removal of phénobarbital.Twenty-four-h-old cultures were treated with phénobarbital(500 jig/ml) or DMSO(0.2%) for 2 h and then evaluated for dye coupling. Similar cultureswere treated with phénobarbital(500 fig/ml) or DMSO (0.2%) for 2 hand then washed 3 times with culture medium and refed with 3 ml ofculture medium. At 0, 0.25, 0.5, and 1 h after refeeding, dye couplingwas determined in the phénobarbital-and DMSO-pretreated cultures.

Experiments were also performed to determine if the hepatocytesbecame refractory to the inhibitory effect of phénobarbitalon dyecoupling after prolonged exposure (0-24 h in culture). Hepatocytecultures were treated with phénobarbital(500 Mg/ml) or DMSO (0.2%)at plating (0 h) and after attachment and refeeding (2 h) and thenassayed for dye coupling after 8 and 24 h. Additional phénobarbitaland DMSO-treated cultures were refed after 24 h treatment and secondarily treated with either phénobarbital (500 ¿ig/ml)or DMSO(0.2%). Dye coupling was determined in these cultures 2 h later.

RESULTS

Fig. 1 depicts phase contrast-fluorescence photomicrographsof dye-coupled mouse hepatocytes after 2 h (Fig. la) or 24 h

2The abbreviation used is: DMSO, dimethyl sulfoxide.

R

Fig. 1. Phase contrast-fluorescence photomicrographs of fluorescent dye coupling between donor mouse hepatocytes (D) microinjected with Lucifer YellowCH and adjacent recipient hepatocytes (R) in (a) 2 li old cultures and (/>)24-h-old cultures, x 250.

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Fig. 2. Dye coupling levels in untreated mouse hepatocytes in primary culture.

(Fig. Ib) in culture. Intercellular communication (dye coupling)in nontreated mouse hepatocyte cultures over the first 0.5-120h of culture is shown in Fig. 2. Dye coupling increased rapidlyduring the first hours of culture to a value of 56.4% dye-coupled

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MOUSE HEPATOCYTE INTERCELLULAR COMMUNICATION

recipient cells at 2 h. This level of dye coupling displayed amore gradual increase during the next 22 h in culture to amaximal level at the 24-h sampling time of 85.2%. Dye couplingremained stable in 48-h cultures but then decreased markedlyover the next 72 h in culture to 39.2% dye-coupled recipients.Refeeding of cultures with fresh medium had no effect on dyecoupling when compared to nonrefed cultures (data not shown).This suggests that factors in the medium did not contribute tothe decline in dye coupling over the 120-h culture period.

When phénobarbital(20-500 ng/m\) was added to the newlyplated cultures and dye coupling was assessed over the next0.5-48 h, a dose-responsive inhibition of dye coupling wasobserved in the cultures sampled up to 12 h (Fig. 3). However,no statistically significant difference in dye coupling was observed in the cultures sampled at 24 and 48 h, indicating thatthe hepatocytes had recovered from the phénobarbitalinhibitory effect.

Phénobarbitalalso inhibited dye coupling when administeredto established (24-h-old) hepatocyte cultures (Fig. 4). The initialdye coupling level in the 24-h-old cultures was 84.0%, and thislevel remained similar over the next 240 min in DMSO-treatedcontrol cultures. When phénobarbital(500 ug/ml) was addedto the cultures, a statistically significant inhibition of dye cou-

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CULTURE DURATION (h)Fig. 3. Dose-related inhibition of dye coupling in mouse hepatocyte cultures

treated with phénobarbital(PB) over 48 h culture duration (*/> < 0.05 versus

DMSO control group).

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TREATMENT DURATION (min)Fig. 4. Inhibition of dye coupling by phénobarbital(/'/() in established (24-h-

old) mouse hepatocyte cultures (*P < 0.05 versus DMSO control group).

pling was evident after 30 min exposure. Dye coupling levelsthen decreased further with increasing duration of phénobarbital exposure to a minimum of 31.9% after 2 h exposure. Thislevel of inhibition remained similar in the 3- and 4-h-exposedcultures.

The recovery from phenobarbital-mediated inhibition of dyecoupling in the 24- and 48-h-treated cultures (Fig. 3) suggestedeither that the hepatocytes had become unresponsive to theinhibitory effect of phénobarbitalafter prolonged exposure orthat phénobarbitalhad been altered or removed from the media.To test these two hypotheses, two experiments were performed.First, the hepatocytes were preexposed to phénobarbital(500Mg/ml) for 24 h and then evaluated for their dye couplingresponse to a second administration of phénobarbital(500 /ig/ml) (Table 1). In cultures that were treated with phénobarbitalat plating (0 h) and refeeding (2 h), dye coupling was significantly decreased after 8 h (Table 1). However, after 24 htreatment with phénobarbital, dye coupling returned to thecontrol level (Table 1). These cultures were then refed at 24 hand treated with a second application of phénobarbital(500 /¿g/ml) or DMSO (0.2%) for 2 h. In DMSO-preexposed cultures,secondary application of phénobarbitalresulted in a significantinhibition of dye coupling, while in hepatocyte cultures preexposed to phénobarbital,secondary application of phénobarbitalhad no effect on dye coupling (Table 1). In a second experiment,hepatocyte cultures were treated with phénobarbital(20, 100,or 500 Mg/ml) or DMSO (0.2%) for 24 h. After the 24-htreatment period, "conditioned" media from the treated cul

tures were transferred to nontreated, 24-h-old hepatocyte cultures. After 2 h incubation in the "conditioned" media, dye

coupling was assayed. There was a dose-related inhibition ofdye coupling by phénobarbitalin these cultures (Table 2) indicating that phénobarbitalwas still present in an active form in"conditioned" media. Thus, these two experiments indicate that

the recovery from the inhibitory effect of phénobarbital onhepatocyte dye coupling in 24-h-treated cultures was not dueto loss or alteration of phénobarbital,but instead a change inthe hepatocyte response to the promoter.

Fig. 5 illustrates that the 24-h-old hepatocytes recovered fromthe inhibitory effect of phénobarbitalon intercellular communication in a rapid fashion when fresh medium was added tothe cultures. When 24-h hepatocyte cultures were treated withphénobarbital(500 //g/ml) for 2 h, a significant decrease in dyecoupling was seen compared to DMSO control cultures (39.4%

Table 1 Development of refractoriness to phénobarbitalinhibition ofmouse hepatocyte dye coupling

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and2hDMSO

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44.781.4

82.5" Hepatocyte cultures were treated for 24 h (or 8 h) with either PB (500 >ig/

ml) or DMSO (0.2%). After 8 h, 2 groups of cultures (DMSO and PB treated)were assessed for dye coupling. After 24 h, an additional 2 groups of cultures (PBand DMSO treated) were evaluated for dye coupling. Medium was removed fromthe remaining cultures and replaced with medium containing either PB or DMSOand evaluated for dye coupling after 2 h of treatment.'' PB. phénobarbital.

c P < 0.05; x2 test.

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Table 2 Effects of "conditioned" phenobarbital-containing media on mousehepatocyte dye coupling in 24-h-old cultures

Dye coupling

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Fig. 5. Recovery of dye coupling in established (24-h-old) mouse hepatocytecultures following removal of phénobarbital(PB) after 2 h pretreatment (*P <0.05 versus DMSO-pretreated control group).

versus 87.1%, respectively). This level of inhibition was similarto that seen in cultures treated with phénobarbital(500 //g/ml)for 2 h (Fig. 4). When phénobarbital was removed from thecultures, the levels of dye coupling in phenobarbital-pretreatedcultures rapidly approached control levels (Fig. 5). Immediatelyafter washing and refeeding (O min recovery time) dye couplinghad increased to 58.1 % but was still significantly less than thecontrol level (83.9%). However, after 15 min recovery, dyecoupling in the phenobarbital-pretreated cultures was equivalent to the control level and remained so over the remainingsampling times.

DISCUSSION

Intercellular communication through gap junctions may beone mechanism by which cell growth is regulated (1). Tumorpromoters might enhance tumor formation in vivo by inhibitingintercellular communication (8). In the present study, we evaluated the kinetics of the development of intercellular communication in untreated and phenobarbital-treated culturedB6C3Fi mouse hepatocytes. Previously we have examined intercellular communication between rodent hepatocytes by following the gap junctional passage of [3H]uridine nucleotidesfrom prelabeled donor cells to nonlabeled recipient cells (7, 14-16). The [3H]uridine method cannot be used to evaluate theshort-term effects of tumor promoters on hepatocyte intercellular communication or the kinetics of the inhibitory effect ofthe promoter. Therefore, in the present study, we have utilizedfluorescent dye coupling techniques to study the kinetics of thedevelopment of intercellular communication in nontreated andphenobarbital-treated hepatocyte cultures.

In nontreated, freshly plated mouse hepatocyte cultures, dye

coupling increased during the first 48 h in culture and thendecreased over the next 3 days in culture (Fig. 2). The nonlineardevelopment of intercellular communication suggests that thereis a rate-limiting component or process of hepatocyte gapjunction formation. This may be the availability of gap junctionsubunits [connexons (2)] in the cultured cells, the number ofhepatocyte-to-hepatocyte contacts, and/or de novo synthesis ofconnexon protein subunits. The peak dye coupling level seen inthe nontreated cultures (85.2%) was similar to the level ofintercellular communication detected by the [3H]uridine

method (83.0%) (7). However, intercellular communication wasnot detected with the [3H]uridine assay until 4 h in culture (7),

whereas dye coupling was seen after 0.5 h in culture in thepresent study. This suggests that the sensitivities of the twomethods for detecting peak levels of hepatocyte intercellularcommunication are similar but that the dye injection method isbetter suited to analyzing the rates of development of intercellular communication.

Phénobarbitalinhibited dye coupling in freshly plated hepatocyte cultures after 0.5-12 h of treatment (Fig. 3). Intercellularcommunication was decreased at the first sampling time of 0.5h and remained below control levels throughout 12 h of treatment. In addition, phénobarbitalinhibited dye coupling betweenhepatocytes of 24-h-old cultures that had established gap junctions (Fig. 4). The inhibitory effect occurred rapidly, beingevident 30 min after phénobarbitaladdition, and reaching fulleffect after 2 h treatment. Dye coupling in established hepatocyte cultures also recovered rapidly (within 15 min) followingphénobarbitalremoval (Fig. 5). Thus, in both freshly plated andestablished hepatocyte cultures, phénobarbitalinhibited intercellular communication within 30 min after treatment and rapidrecovery from the inhibition occurred following removal of thephénobarbital.

The mechanism by which phénobarbitalinhibited hepatocyteintercellular communication is not likely to be due to gapjunction degradation or decreased synthesis of gap junctionprotein. Rapid recovery from the inhibition as seen in Fig. 5would not be expected if de novo gap junction synthesis had tooccur following phenobarbital-induced gap junction degradation. Instead it is conceivable that phénobarbitalinhibited hepatocyte intercellular communication by either affecting anendogenous intracellular system that controls gap junctionpermeability and/or by altering the structure of the gap junctionitself. Control of hepatocyte gap junction permeability may beup-regulated by cyclic AMP-dependent protein kinases (23) anddown-regulated by protein kinase C activation (24-26) and byincreased intracellular levels of H+ or Ca2+ ions (27). Inhibition

of mouse hepatocyte intercellular communication by phénobarbital may be due to phénobarbitaleffects on hepatocyte cyclicAMP levels (28), oxygen free radical production (15), activationof protein kinase C (29) or effects on hepatocyte Ca2* levels

(30). Recently, Chauhan and Brockerhoff (29) have providedevidence that phénobarbitalcompetes with diacylglycerol forthe protein kinase C receptor site. Activation of protein kinaseC by 12-O-tetradecanoylphorbol-13-acetate or synthetic diacyl-glycerols results in the rapid inhibition of intercellular communication in many types of cells (24-26). In addition, barbiturates are capable of disordering membrane lipids (31) andbinding to membrane proteins (32). These effects, alone or incombination, might also contribute to the mechanism of inhibition of hepatocyte intercellular communication by phénobarbital.

Mouse hepatocytes became refractory to the inhibitory effectof phénobarbitalon intercellular communication (Tables 1 and

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MOUSE HEPATOCYTE INTERCELLULAR COMMUNICATION

2) suggesting that in vivo the inhibition of hepatocyte intercellular communication may be only an initial, transient effect ofphénobarbital treatment during the prolonged tumor promotion process. Sugie et al. (13), in contrast, have shown thatphénobarbitaladministration to rats for 2-8 weeks resulted ina decrease in the size of hepatocyte gap junctions and in thearea of the plasma membrane occupied by the gap junctions. Itis also possible that preneoplastic "initiated" hepatocytes are

more responsive to the inhibitory effects of phénobarbitalonintercellular communication and/or are less capable of developing refractoriness to the inhibitory effect. If intercellularcommunication is a regulatory component of cellular replication (1), then the latter hypothesis may be substantiated by theresults of Schulte-Hermann et al. (33). They demonstrated thatnormal hepatocytes in rat liver cease responding to the hyper-plastic effect of phénobarbital after prolonged treatmentwhereas putatively preneoplastic focal cells continue to proliferate during chronic phénobarbitaladministration.

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