* Department of Pathology, Medical College of Ohio, 3055 Arlington Ave., Toledo, Ohio 43614
Received June 25, 2002; accepted October 29, 2002
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ABSTRACT |
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Key Words: gap junctions; hepatocyte; cytochrome P450; tumor promotion; nongenotoxic carcinogens; phenobarbital; biomarkers.
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INTRODUCTION |
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Nongenotoxic carcinogens are difficult to identify. There are no widely accepted short-term tests or bioassays for nongenotoxic carcinogens; this contrasts with genotoxic carcinogens for which many short-term tests and biomarkers exist (Butterworth and Bogdanffy, 1999; Trosko et al., 1998
). Presently, nongenotoxic carcinogens are identified in chronic animal bioassays. This is problematic for regulatory agencies, because humans are exposed to many nongenotoxic agents and most of these are not tested in chronic bioassays because of high costs. Thus, it would be beneficial to develop mechanistically relevant, short-term tests or biomarkers that identify nongenotoxic carcinogens that can be applied across species.
The inhibition of gap junctional intercellular communication (GJIC) is a mechanistically relevant effect common to most nongenotoxic carcinogens and might be useful as a biomarker for these agents (Budunova and Williams, 1994; Klaunig and Ruch, 1990
; Rosenkranz et al., 2000
; Trosko and Chang, 1988
). Gap junctions are a type of cellcell junction that consists of clusters of channels directly linking the interiors of adjacent cells and permitting the flow between cells of ions and molecules less than 1 kDa in diameter (Bruzzone et al., 1996
). This exchange is known as gap junctional intercellular communication (GJIC), which is involved in cellular homeostasis, growth regulation, apoptosis, differentiation, and other functions (Ruch, 2000
). The inhibition of GJIC by nongenotoxic carcinogens is, therefore, a likely factor in their ability to induce cell proliferation and inhibit apoptosis and terminal differentiation.
Strong associations have been observed among many chemicals between the inhibition of GJIC in cell cultures and nongenotoxic carcinogenic activity (Klaunig and Ruch, 1990; Budunova and Williams, 1994
; Trosko and Chang, 1988
). Some studies have used in vivo models and have also reported a similar correlation (Chaumontet et al., 1996
; Ito et al., 1998
; Kolaja et al., 2000
; Sai et al., 2000
; Smith et al., 2000
), but there is less data for in vivo than for in vitro systems. In the present study, we hypothesized that the nongenotoxic rodent hepatocarcinogen phenobarbital (PB) would inhibit GJIC in hepatocytes of a mouse strain (B6C3F1) that was highly sensitive to PB hepatocarcinogenicity, and would have little effect on hepatocyte GJIC in a much less sensitive strain (C57BL/6) (Diwan et al., 1986
; Weghorst et al., 1989
). We previously reported that PB inhibited B6C3F1, but not C57BL/6 mouse hepatocyte GJIC in vitro, following limited exposure durations of less than 24 h (Klaunig and Ruch, 1987
; Ruch and Klaunig, 1988
). But we did not investigate whether this correlation occurred in vivo or following longer exposures in vitro. To address this, we have used fluorescent dye "cut-loading" to assess hepatocyte GJIC in vivo and fluorescent dye microinjection of mouse hepatocyte/rat liver epithelial cell cocultures to address the in vitro effects of PB.
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MATERIALS AND METHODS |
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Hepatocyte isolation, culture, and treatment with PB.
Hepatocytes were isolated from adult male mice by two-stage perfusion of the liver with collagenase D (Boehringer Mannheim, Indianapolis, IN) (Ruch and Klaunig, 1988). Isolated hepatocytes were >90% viable and were plated (1 x 105 cells/cm2) onto 50% confluent cultures of WB-F344 rat liver epithelial cells (Ren and Ruch, 1996
) in standard plastic tissue culture dishes. The culture medium was Richters minimal essential medium (Irvine Scientific Co., Santa Ana, CA) supplemented with 5% FBS, dexamethasone (1 µM), and gentamicin (Life Technologies, Gaithersburg, MD; 40 mg/ml). The cocultures were refed 2 and 24 h after plating of hepatocytes and were treated 48 h later with PB dissolved in DMSO or with DMSO (1 µl/ml; controls). The cocultures were refed and retreated every 3 days.
Assessment of cultured hepatocyte GJIC by fluorescent dye microinjection.
Hepatocyte GJIC was evaluated by microinjection of fluorescent Lucifer yellow CH dye (Sigma) (Ruch and Klaunig, 1988). Three dishes per treatment condition were sampled and 10 hepatocytes per dish were microinjected and filled with dye. All hepatocytes directly adjacent to microinjected ones were evaluated for dye uptake (dye-coupling) 5 min after microinjection. The data from three replicate cultures were pooled, and the percentage of dye-coupled cells for all injections was determined and is presented in the figures. Treatment groups were compared using the Chi-square test (Gad and Weill, 1986
). GJIC in WB-F344 cells was also evaluated by dye microinjection.
Hepatocyte viability in cocultures.
Cocultured hepatocytes were counted to determine whether PB affected hepatocyte survival. Coculture medium was removed and replaced with trypan blue dye. Fifteen min later, the dye was removed and the cells were washed with Ca+2-Mg+2free phosphate-buffered saline (PBS). Trypan blue dye-excluding (viable) hepatocytes in nine randomly selected fields on each dish were counted at x400 magnification. The total number of viable hepatocytes per dish was determined by multiplying the mean number of viable hepatocytes per field by the surface area of the 35 mm dish (962 mm2) and dividing that number by the area of the microscopic field (0.076 mm2). Three replicate dishes were evaluated per treatment group. Groups were compared statistically by Students t-test.
Treatment of mice with PB.
Mice were randomly divided into eight groups of 46 animals each (Table 1). They were administered PB by a single intraperitoneal injection (0.1 mg/kg in sterile 0.9% saline; groups 2 and 6) or given in drinking water (500 ppm, groups 4 and 8). Control mice were injected with saline (groups 1 and 5) or were given deionized drinking water (groups 3 and 7). Mice injected with PB or saline were euthanized 24 h after injection. Mice given PB in the drinking water were euthanized after 14 days of administration.
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The sections were examined microscopically using a Nikon fluorescence microscope equipped with a fluorescein filter set. Dye-loaded hepatocytes along the cut edges were identified easily by their bright yellow fluorescence. GJIC was quantified by counting the number of dye-stained hepatocytes perpendicular to the cut edge. Dye-loaded cells along the cut edge were not counted. Four randomly selected points along each cut edge were evaluated. For each mouse, three liver pieces were "cut-loaded," sectioned, and evaluated in this way for a total of 12 counts per animal. The mean number of dye-coupled cells for each animal was determined from these 12 evaluations. From these means, treatment group means and standard deviations were determined, then compared statistically using Students t-test (Gad and Weill, 1986).
Analyses of Cx26 and Cx32 expression and localization.
Northern and Western blot assays and fluorescent immunohistochemistry were performed as described (Ren and Ruch, 1996) to investigate the effects of PB on mouse liver Cx26 and Cx32 mRNA and protein expression. For Northern analyses, total RNA was isolated from cocultures or from frozen liver pieces (
0.5 g). Frozen liver was first pulverized under liquid nitrogen. Total RNA was isolated from cultures or pulverized tissue using 1 ml of ice-cold Tri-Reagent (Molecular Research Center, Inc., Cincinnati, OH) according to the manufacturers protocol. The purified RNA was quantified spectrophotometrically, separated by electrophoresis in 1% agarose gels (10 µg total RNA per sample), transferred to Hybond N+ nylon membranes (Amersham-Pharmacia Biotech, Piscataway, NJ) by vacuum blotting, and cross-linked to the membranes by UV light exposure. The membranes were hybridized with Cx32, Cx26, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) radiolabeled probes prepared from full-length cDNAs. Membranes were exposed to X-ray film (Kodak Biomax MR; Fisher Scientific) with intensifying screens for 13 days. Films were developed in an automatic X-ray film developer.
For Western blot analyses of Cx32 and Cx26 protein, membrane-enriched extracts were prepared from cell cultures or pulverized frozen liver tissue (0.5 g per sample). Cells were harvested from culture dishes by scraping into ice-cold PBS, then pelleted by centrifugation. One ml of ice-cold hypotonic lysis buffer (10 mM Tris, pH 7.5; 1 mM iodoacatamide; 2 mM phenylmethylsulfonylfluoride) was added to each cell pellet or tissue sample and these were disrupted by sonication on ice. The cell fragments were alkalinized by the addition of 1.5 µl of 40 mM NaOH, then pelleted by centrifugation (13,000 x g for 30 min). The pellets were dissolved in 2% sodium dodecylsulfate (SDS) and protein concentrations were determined using the Bio-Rad DC Protein Assay (Bio-Rad Corp., Richmond, CA). Proteins (40 µg per sample) were separated by SDSPAGE on 12% polyacrylamide gels under reducing conditions. Proteins were transferred to Immobilon-P membranes and Cx26 and Cx32 were detected using mouse monoclonal anti-Cx26 and anti-Cx32 antibodies (Zymed, South San Francisco, CA).
The effects of PB on Cx26 and Cx32 localization were determined by immunohistochemistry. Frozen liver sections (56 µ) were prepared using a cryostat, mounted onto microscope slides coated with aminopropyl triethoxysilane (Sigma), fixed in 100% acetone, and stored at 80°C until use. For immunostaining, the sections were warmed to room temperature, rehydrated in PBS, and incubated sequentially in 3% normal goat serum (Jackson ImmunoResearch Laboratories, West Grove, PA) in blocking buffer (4% milk, 10% bovine serum albumin, and 0.3% Triton X-100) for 1 h, rabbit polyclonal anti-Cx26 or anti-Cx32 antibody (1:100 in PBS; Zymed) for 3 h, and fluorescein isothiocyanate-conjugated goat antirabbit antibody (1:100 in PBS; Jackson Immunoresearch) for 2 h. Sections were washed extensively in PBS after antibody incubations and mounted with Anti-Fade Solution (Molecular Probes, Eugene, OR).
Northern blot analyses of Cyp2b1 expression.
Northern blot analyses were performed as described above to determine the effects of PB on Cyp2b1 mRNA expression in the mouse liver. The probe was a single-stranded DNA oligonucleotide specific for Cyp2b1, 5'-GGTTGGTAGCCGGTGTGA-3' (Omiecinski et al., 1985) (Life Technologies) and was radiolabeled with [
32P]ATP (Amersham-Pharmacia Biotech) using T4 polynucleotide kinase (Life Technologies).
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RESULTS |
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DISCUSSION |
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Many groups have reported that PB and other nongenotoxic carcinogens impair GJIC and have observed a correlation between carcinogenicity and the impairment of GJIC (Budunova and Williams, 1994; Klaunig and Ruch, 1990
; Trosko and Chang, 1988
). In fact, the reduction of GJIC appears to be one of the most common effects of nongenotoxic (and some genotoxic) carcinogens described to date. This correlation has been observed across organs and species (including monkey and human) and correlative structure-activity relationships also have been observed (Baker et al., 1995
; Pugh et al., 2000
; Ren and Ruch, 1996
; Upham et al., 1998
). In fact, when a variety of in vitro tests for carcinogenicity were considered, the inhibition of GJIC correlated best with carcinogenicity (Rosenkranz et al., 2000
).
These relationships suggest that the inhibition of GJIC is a biomarker for nongenotoxic carcinogens and might be exploited as a short-term bioassay for such agents. Presently, there are no satisfactory short-term tests that identify nongenotoxic carcinogens and two-year rodent bioassays are necessary to do so. Two-year carcinogenicity bioassays are time-consuming, expensive, and consequently not performed for most nongenotoxic agents to which humans are exposed. The development and validation of GJIC assays using a panel of primary cultured cell types or tissues, including those of human origin, might be highly sensitive and predictive of nongenotoxic carcinogenicity and would benefit risk assessment.
Although many groups have reported that PB inhibits hepatocyte GJIC, the mechanism remains unclear. We saw no changes in connexin gene expression at the mRNA or protein levels and did not observe changes in gap junction localization in sensitive B6C3F1 mouse hepatocytes. In rat liver, PB has been reported to alter connexin expression and gap junction localization, although the data are not consistent. Administration of PB for 4 and 11 weeks decreased Cx32 mRNA content in rat liver in one study (Neveu et al., 1994), but did not decrease Cx32 mRNA or protein levels in rat liver in other studies (Chaumontet et al., 1996
; Krutovskikh et al., 1995
). However, decreased dye coupling and Cx32 immunostaining of membrane gap junctions were seen. The decreased immunostaining was correlated with increased expression of CYP2B1 (Krutovskikh et al., 1995
) and cytoplasmic localization of Cx32-containing gap junctions (Chaumontet et al., 1996
). However, we saw no change in Cx32 or Cx26 immunostaining in mouse liver after PB administration (Fig. 7
). These inconsistent results might reflect species or protocol differences.
The difference we observed between mouse strains, however, is clearly not the result of treatment protocol, but due to genetic differences between the B6C3F1 and C57BL/6 mice. One possibility is strain differences in PB metabolism and detoxification. PB is a potent inducer of Cyp2b1 in rodent liver and the inducible phenotype has been correlated with barbiturate liver carcinogenicity (Rice et al., 1994). The C57BL/6 mouse, however, is an exception. Diwan et al. (1986)
reported that PB induced Cyp2b1 in C57BL/6 mice, and we observed that induction of this cytochrome and hepatomegaly were similar in this strain and B6C3F1 mice. Thus, induction of the cytochrome per se does not appear to account for the strain-specific differences in sensitivity of GJIC to PB. However, cytochrome P450 activity appears to be important in the inhibition of hepatocyte GJIC by PB. Inhibitors of cytochrome P450 activity (e.g., SKF-525A) prevented the blockage of hepatocyte GJIC by PB (Guppy et al., 1994
). Induction of cytochrome P450 by PB also increased the production of reactive oxygen species due to uncoupling of the cytochrome P450 cycle (Venditti et al., 1998
) and hepatocyte GJIC is sensitive to free radicals (Ruch and Klaunig, 1986
). Therefore, the strain differences might be related to relative antioxidant capacity and ROS detoxification in the face of increased cytochrome P450 activity. Mouse strain susceptibility to liver tumor promotion by nongenotoxic agents has been related to oxidant stress and alterations in hepatic antioxidant function (Ahotupa et al., 1993
; Stevenson et al., 1995
). Thus, the inhibition of hepatocyte GJIC by PB could involve ROS that have direct or indirect effects on gap junctions. In fact, antioxidants prevented the inhibition of hepatocyte GJIC by PB (Ruch and Klaunig, 1986
). Differences between B6C3F1 and C57BL/6 hepatocytes might be related to the extent of ROS generation and detoxification.
We also observed that the inhibition of B6C3F1 hepatocyte GJIC in vitro was transient, whereas that in vivo was not, at least after 14-day treatment. The transient inhibition in vitro by PB was reported earlier and was not due to depletion or degradation of the compound, because culture medium from refractory cells retained inhibitory activity towards naïve hepatocytes and treatment of refractory cells with fresh PB had no effect on GJIC (Ruch and Klaunig, 1988). Thus, the hepatocytes were truly refractory to PB. The mechanism is unclear but is not due to upregulated connexin expression. The refractory effect is reminiscent of the transient inhibition of GJIC induced by TPA that is due to the downregulation of protein kinase C (PKC) (Ren et al., 1998
). It is unlikely, however, that PKC is involved in the PB effect, because potent inhibitors of the kinase and pretreatment of hepatocytes for 24 h with TPA did not prevent the reduction of hepatocyte GJIC by PB (Ren et al., 1998
).
It is also interesting that refractoriness was only observed in vitro. This could be related to the much higher concentrations and more rapid onset of exposure of PB in cultured hepatocytes. These cells were treated instantaneously with 0.255.0 mM PB, whereas hepatocytes in vivo were exposed more gradually to the drug as the animals drank the PB-treated water and steady-state serum levels were achieved. These levels are much lower than the in vitro treatments. In male mice administered 500 ppm PB in the drinking water for 14 days, serum PB levels were approximately 1025 mg/ml (0.040.1 mM) (Diwan et al., 1986). Thus, the rapid-onset exposure of hepatocytes in vitro to high concentrations of PB might activate a protective mechanism or cause the downregulation of the inhibitory mechanism. The lack of a refractory effect in vivo might be due to the gradual exposure and lower serum concentration of PB or another mechanism.
The maximal inhibition of GJIC by PB was approximately 50% in vivo and in vitro. This suggests some hepatocytes are resistant to PB. This might be related to the lobular heterogeneity of hepatocytes, although we did not investigate lobular position differences in the cut-loading studies. Position-dependent differences in hepatocyte drug sensitivity are due to nutrient, oxygen, and drug concentration gradients across the lobule; differences in Phase I and Phase II metabolic capacity; and hepatocyte age and ploidy (Moslen, 1996). Presumably, lobular position affects hepatocyte sensitivity to GJIC inhibitors like PB. Accordingly, Neveu et al. (1994)
reported the induction of cytochrome P450 and reduction of Cx32 immunostaining was most prominent in centrilobular hepatocytes of rats fed dietary PB.
In summary, PB decreased B6C3F1 mouse hepatocyte GJIC in vivo and in vitro and had no effect on C57BL/6 mouse hepatocyte GJIC. The decrease in GJIC was not due to altered expression or abnormal localization of Cx26 and Cx32, but to a post-translational mechanism. The inhibition of GJIC correlated with strain-specific sensitivities to PB hepatocarcinogenicity and suggests that the inhibition is involved in the carcinogenic mechanism of PB. These data and those from other groups (Chaumontet et al., 1996; Ito et al., 1998
; Kolaja et al., 2000
; Sai et al., 2000
; Smith et al., 2000
) also suggest that in vivo and/or in vitro GJIC assays are mechanism-based biomarkers for nongenotoxic carcinogenicity.
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NOTES |
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