Center for Environmental and Occupational Health, Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, 2018 Breidenthal Building, 3901 Rainbow Boulevard, Kansas City, Kansas 66160-7417
Received April 28, 2000; accepted June 8, 2000
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ABSTRACT |
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Key Words: cadmium (Cd); gap junctional intercellular communication (GJIC); connexins; cytoskeletal actin; necrosis; apoptosis; cellular proliferation.
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INTRODUCTION |
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Liver is the major target organ for Cd toxicity following acute exposure (Dudley et al., 1982; Habeebu et al., 1998
). The liver injury of acute Cd exposure is dominated by apoptosis and necrosis (Habeebu et al., 1998
). The morphologic injury is reflected in the high levels of liver enzymes in serum, namely alanine aminotransferase (ALT), aspartate aminotransferase (AST), and sorbitol dehydrogenase (SDH). We have recently shown that the liver is also an important organ for the toxicity of chronic Cd exposure (Habeebu et al., 2000
). Unlike acute Cd toxicity, chronic Cd toxicity is manifested primarily as granulomatous inflammation, cell proliferation, nodular hyperplasia, and apoptosis.
Gap junctional intercellular communication (GJIC) refers to the regulation of cellular homeostasis by the passage of low-molecular-weight water-soluble molecules such as ions, second messengers (e.g. cAMP and IP3), growth factors, and neurotransmitters through transmembrane channels called gap junctions (Neveu et al., 1994; Trosko and Goodman, 1994
). Gap junctions consist of an assembly of proteins called connexins. GJIC is thought to be important in the regulation of cell proliferation, differentiation and death, embryonic development, and carcinogenesis. Many oncogenes and tumor promoters inhibit GJIC (Hayashi et al., 1998
; Krutovskikh et al., 1995
; Ren et al., 1998
), while enhanced GJIC activity and transfection of connexin genes into tumor cells suppress tumorigenicity (Chen et al., 1995
; Eghbali et al., 1991
; Hossain et al., 1989
; Ruch et al., 1998
; Yamasaki et al., 1999
). This has led to the suggestion that connexin genes are tumor-suppressor genes (Omori et al., 1998
; Yamasaki et al., 1995
, 1999
).
There are over a dozen different types of connexin proteins (Cao et al., 1998; Yamasaki et al., 1999
) and their distribution is different in various tissues. The major connexins of hepatocytes (liver parenchymal cells) are connexin 32 (Cx32) and connexin 26 (Cx26) (Temme et al., 1998
), whereas biliary epithelium cells and non-parenchymal liver cells express mainly Cx43 (Neveu et al., 1995
). Inhibition of GJIC in the liver could occur via a number of mechanisms, including decreased gap junction permeability, decreased number of gap junctions per cell, and decreased expression of Cx32 and/or Cx26. Inhibition of GJIC has been associated with both cell proliferation (Klaunig and Ruch, 1990
; Loewenstein, 1979
) and increased apoptosis (Trosko and Goodman, 1994
).
Gap junctions, like other intercellular junctions (intermediate and tight junctions), are anchored to the cell membrane by microfilaments and microtubules (Martini and Timmons, 1997). The organized network of microfilaments and microtubules in the cell constitute the cytoskeleton. Microfilaments are composed of linear arrays of polymerized actin, also known as filamentous actin or F-actin. Depolymerization (denaturation) of F-actin leads to disruption of the cytoskeleton.
Acute Cd exposure induces apoptosis and necrosis, two modes of cell death, in the liver (Dudley et al., 1982; Habeebu et al., 1998
). Cd also induces cell proliferation in the liver, especially following chronic exposure, leading to nodular hyperplasia (Habeebu et al., 2000
), which is presumed to be preneoplastic. It is, therefore, of paramount interest to investigate the effect of Cd exposure on GJIC and how it relates to Cd-induced cell proliferation and cell death. An in vitro study by one of the authors (S-HJ) suggests that Cd decreases GJIC in WB-F344 rat liver epithelial cells (manuscript in preparation). Earlier reports indicate that Cd either disrupts gap junctions (Niewenhuis et al., 1997
; Prozialeck and Niewenhuis, 1991
) or has no effect on GJIC (Loch-Caruso et al., 1991
; Mikalsen, 1990
), depending on the model system. The present study was designed to investigate in vivo the effect of acute Cd administration on GJIC in mouse liver, in relation to varying doses of Cd and duration of exposure. Phenobarbital was used as a known positive control for the inhibition of GJIC (Mesnil et al., 1988
; Ruch and Klaunig, 1988
).
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MATERIALS AND METHODS |
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Animals.
Adult male mice (CF1, 2530 g body weight) were housed in animal facilities certified by the American Association for the Accreditation of Laboratory Animal Care, at 70 ± 2°F with a 12-h light/dark cycle. They were provided food (Purina Laboratory Mouse Chow, St. Louis, MO) and tap water ad libitum. Cd solutions were prepared by dissolving CdCl2 (99% pure, certified ACS grade, Fisher Chemical Co.) in 0.9% saline. Radioactive Cd, 0.01 µCi 109Cd/kg (109CdCl2, NEN Research Products, Boston, MA) was added to the solutions to enable determination of the amount of Cd in the liver. Mice (n = 5) were injected ip with 30 µmol CdCl2/kg. One group was administered phenobarbital, 800 ppm in drinking water, for 72 h. Blood was collected and livers were removed at 1.5, 3, 6, 9, 14, 24, and 48 h later. For dose-response studies, mice (n = 5) were injected with CdCl2 ip at 5, 10, 20, 30, 40, and 60 µmol/kg. Blood and liver specimens were collected 9 h later. A portion of the left lobe of the liver was excised and used for analysis of gap junctional intercellular communication (GJIC) by measuring ex vivo fluorescent dye transfer (see below). Another portion of the liver was placed in 10% neutral buffered formalin for histopathological analysis. Additional portions of liver were immediately frozen in liquid nitrogen and stored at 80°C for immunohistochemical analysis of connexin proteins and cytoskeletal actin.
Tissue and blood analysis.
At the end of the exposure to Cd, animals were anesthetized, decapitated to collect blood, and necropsied. A portion of liver was fixed in 10% neutral buffered formalin, and Cd content was determined by 109Cd analysis. Liver sections were processed by standard histological techniques, and stained with hematoxylin and eosin (H&E) for examination by light microscopy. Serum was analyzed for alanine aminotransferase and sorbitol dehydrogenase activities using commercially available kits from Sigma (St. Louis, MO).
GJIC analysis by ex vivo fluorescent dye transfer.
Strips of fresh liver were used to analyze GJIC as previously described (Krutovskikh et al., 1991; Krutovskikh et al., 1995
). Briefly, thin strips of liver were washed in ILDT buffer (incision-loading dye transfer buffer), and then placed in fresh ILDT buffer at 37°C, containing 1 mg/ml Lucifer Yellow-CH (Molecular Probes, Eugene, OR). Small incisions were made along one edge of the liver strips using a very sharp, clean blade. The strips were left to incubate in the Lucifer Yellow medium for 5 min. Next, the strips were washed in fresh ILDT buffer and fixed in 10% neutral buffered formalin for 24 h. They were then processed by standard histological techniques, and mounted on glass slides for immunofluorescence microscopy. Digital images were captured to disk, using a fluorescence microscope (Nikon Optiphot-2, Nikon, Japan, equipped with filters for FITC and rhodamine fluorescence) connected via an image grabber to an Apple Macintosh computer. The computer was equipped with NIH Image 1.60, image-capture and analysis software, available free on the Internet from the National Institutes of Health.
Immunohistochemistry.
Immunohistochemical localization and analysis of connexins 26 and 32 (Cx26 and Cx32) in the liver was performed on cryosections of the liver as described previously (Green et al., 1996). Liver cryosections, 5 µm thick, were placed on polylysine-coated microscope slides, fixed in acetone for 10 min at 20°C, and air-dried. Sections were blocked for 30 min in goat serum diluted 1:100 in PBS buffer, and primary antibody to Cx26 or Cx32 (Zymed Laboratories, Inc., San Francisco) was applied at 1:100 dilution for 2 h at room temperature. FITC-labeled secondary antibody (Zymed) was applied at 1:100 dilution for 1 h at room temperature. Slides were mounted in Slow-Fade antifade solution (Molecular Probes, Eugene, OR) and analyzed under immunofluorescence confocal microscopy. Images were captured on a Meridian Insight Plus Laser Scanning Confocal Microscope (Meridian Instruments, Inc., Okenos, MI), equipped with a dual laser system consisting of independent argon- and krypton-ion lasers and Meridian Insight-IQ Image Processing and Quantitation Computer System. The images were subsequently analyzed quantitatively using NIH Image 1.60. The number of signals of Cx26 and Cx32 was counted in 10 randomly selected high-power fields, and divided by the number of hepatocytes in the fields, to obtain the number of Cx26 or Cx32 signals per hepatocyte.
Histochemistry.
Cytoskeletal actin was localized using rhodamine-labeled phalloidin (Molecular Probes, Eugene, OR). Five-µm-thick cryosections of liver were placed on polylysine-coated microscope slides, fixed in acetone for 10 min at 20°C, and air-dried. Sections were permeabilized with 0.2% Triton X-100 for 10 min. A 1:40 dilute solution of rhodamine-labeled phalloidin was applied for 20 min. Slides were mounted in Slow-Fade antifade solution (Molecular Probes) and analyzed under immunofluorescence confocal microscopy. Images were captured to disk as described above.
Statistics.
Data are expressed as means ± standard errors of mean. Comparisons between control and treated groups of mice were performed with one-way analysis of variance (ANOVA), followed by Duncan's multiple comparison test. Statistical significance was set at p < 0.05.
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RESULTS |
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Gap junctional intercellular communication was analyzed by measuring the distance of ex vivo fluorescent dye transfer from a clean-cut surface of the liver. Ten randomly selected sites were measured per mouse liver, giving 50 measurements per treatment group. Cd induced a time-dependent decrease in gap junctional intercellular communication (Fig. 1, left column). In control liver the fluorescent dye Lucifer Yellow was transferred ex vivo a distance of 132.8 ± 5.24 µm in 5 min. Following ip administration of 30 µmol Cd/kg, the ex vivo dye transfer decreased rapidly to 73.20 ± 3.42 µm (44.9% inhibition when compared to control value; Fig. 2A
) by 6 h. The ex vivo dye transfer was at its nadir from 6 to 14 hr, after which there was a gradual but mild recovery. Phenobarbital (800 ppm PB in water for 72 h), used as a known positive control (Mesnil et al., 1988
; Ruch and Klaunig, 1988
), reduced the dye transfer to 79.52 ± 3.44 µm (40.1% inhibition).
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The effects of Cd exposure on the major connexins of liver, Cx26, and Cx32 were studied by fluorescence immunohistochemistry and confocal microscopy. In control mice, the connexins were visualized as a series of bright fluorescent signals along the hepatocyte plasma membrane (Fig. 3A). Cd administration caused a decrease in the number, size, and intensity (brilliance) of the connexin signals (Fig. 3B
). The decrease was time- and dose-dependent. In control mice there were 9.01 ± 0.2 Cx26 signals per cell. Following Cd administration (30 µmol/kg), the number of signals per cell decreased rapidly, falling to 4.93 ± 0.17 (45.3% decrease compared to control value) by 6 h (Fig. 4
, upper panel). The number was then essentially maintained at this level, falling only slightly to 4.22 ± 0.45 (53.2% decrease) by 48 hr. Phenobarbital (800 ppm in water for 72 h) decreased Cx26 signals to 5.43 ± 0.48 (39.7% decrease; Fig. 4
, upper panel).
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The number of connexin signals in relation to dose of Cd was also analyzed. There was a marked decrease in the number of signals per cell for both Cx26 and Cx32 following administration of 5 µmol Cd/kg (Fig. 5). With increasing dose of Cd above 5 µmol/kg, the decrease in the signal number per cell became gradual for both connexins. At 30 µmol Cd/kg the signal number for Cx26 was 4.31 ± 0.37 (52.2% decrease), and at 60 µmol Cd/kg it was 2.85±0.39 (68.4% decrease; Fig. 5
, upper panel). The dose-dependent decrease in Cx32 signals was less marked than for Cx26 signals. At 30 µmol Cd/kg the signal number for Cx32 was 4.76±0.20 (42.7% decrease), and at 60 µmol Cd/kg it was 3.19±0.20 (61.6% decrease; Fig. 5
, lower panel).
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DISCUSSION |
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In the present study, Cd caused a time-dependent (over a 48-h period) and dose-dependent (560 µmol Cd/kg) inhibition of GJIC. In the time-course study, maximal inhibition of GJIC was attained by 6 h. In comparison, the maximal level of apoptosis is not attained until 9 h following administration of the same dose of Cd (Habeebu et al., 1998). Inhibition of GJIC, thus, precedes the occurrence of Cd-induced apoptosis. This suggests that the inhibition of GJIC may play a regulatory role in the induction of apoptosis by Cd. In the dose-response studies, the inhibition of GJIC is progressive. The level of apoptosis induced by Cd also increases progressively with increasing dose of Cd (Habeebu et al., 1998
). Again, these observations are in agreement with the suggestion that inhibition of GJIC plays a role in Cd-induced apoptosis. The concept that inhibition of GJIC may regulate apoptosis is not new (Schulte-Hermann et al., 1990
; Trosko and Goodman, 1994
). Lifshitz and coworkers compared apoptosis in intact colonic tissue and isolated colonic crypts with apoptosis in isolated colonic cell populations at different stages of development, and concluded that loss of intercellular communication was responsible for the extensive apoptosis they observed in the isolated colonic cells (Lifshitz et al., 1998
).
GJIC is usually studied at the cell population level. In a recent report (Wilson et al., 2000), Trosko's group demonstrated clearly that changes in GJIC at the cell population level do not necessarily mirror changes in GJIC in the individual cells making up the population. Rather, GJIC in a given cell depends on the stage of the cell in the cell cycle (G0, G1, S, G2 or M), the type of cell (hepatocyte, biliary epithelial cell, endothelial cell, or Ito cell), as well as whether or not the cell is undergoing apoptosis or necrosis. Trosko's group subjected cells of the epithelial cell line WB-F344 to serum deprivation and found that the population GJIC and apoptosis increased progressively with increasing duration of serum deprivation. In contrast, GJIC in apoptotic and mitotic cells decreased as apoptosis and mitosis progressed in response to serum deprivation. Their data suggests that GJIC serves primarily to regulate the induction or initiation of apoptosis and mitosis. Trosko's group concluded further that apoptosis and mitosis are regulated initially by similar mechanisms.
Cd induced cell proliferation in the liver in the present study (data not shown) and our earlier studies (Habeebu et al., 1998, 2000
). The rapid decrease in GJIC (30% inhibition by 1.5 h, Fig. 2A
) precedes cell proliferation, thus suggesting that Cd-induced cell proliferation may be mediated by inhibition of GJIC. Most studies of GJIC are focused on its role in cell proliferation and carcinogenesis, resulting in a wide body of evidence suggesting that not only is GJIC essential for normal cell homeostasis (Guthrie and Gilula, 1989
; Holder et al., 1993
; Trosko et al., 1998
), but also that its inhibition or absence is necessary for the development of most tumors (Yamasaki et al., 1991, 1999
). Many lines of evidence have been presented in the literature in support of this hypothesis. Most oncogenes and tumor promoters inhibit GJIC (Azarnia et al., 1989
; de-Feijter et al., 1996
; Hayashi et al., 1998
; Krutovskikh et al., 1995
). GJIC is impaired in most cancer cells (Yamasaki, 1991
). Many tumors show aberrant (intracytoplasmic or nuclear) localization of gap junction proteins (connexins) (Krutovskikh et al., 1994
; de-Feijter et al., 1996
), decreased numbers of gap junctions (Krutovskikh et al., 1991
), or decreased expression of connexins (Cesen et al., 1998
). Transfection of GJIC genes into tumor cell lines restores normal cell growth (Chen et al., 1995
; Mehta et al., 1991
). Finally, it has been shown that knockout mice lacking Cx32, the major connexin of hepatocytes, are characterized by a high incidence of spontaneous and chemically induced tumors in the liver (Moennikes et al., 1999
; Temme et al., 1997
). It has recently been suggested that connexin expression per se, rather than GJIC level, is more closely related to growth control, suggesting that connexins may have a GJIC-independent function (Yamasaki et al., 1999
).
This study of Cd-induced GJIC changes in the liver focused mainly on Cx26 and Cx32. Connexin 32 is the major connexin of hepatocytes, and Cx26 is the next in significance (Krutovskikh et al., 1991; Temme et al., 1998
; Wilgenbus et al., 1992
). Connexin 43 is found mainly in biliary epithelium and mesodermal cells in the liver (Berthoud et al., 1992
; Neveu et al., 1995
).
In this study, Cd induced a time- and dose-dependent decrease in the size and intensity of Cx26 and Cx32 signals (Figs. 3A and 3B) and in the number of Cx26 and Cx32 signals per cell (Figs. 4 and 5
, all panels). These findings suggest that Cd induces a decrease in Cx26 and Cx32 proteins as well as in the number of gap junctions per cell, thus explaining the inhibition of GJIC in liver from mice exposed to Cd. The possibility that Cd also inhibits GJIC by altering the phosphoryla+tion state of Cx26 and Cx32 cannot be ruled out. Cd-induced decrease in connexin proteins could be due to decreased mRNA expression (transcription), post-transcriptional factors (e.g., decreased mRNA stability), increased protein degradation, or combinations of the above. Each of these mechanisms has been reported in at least one model system (Baker et al., 1995
; Cesen et al., 1998
; Saez et al., 1998
; Temme et al., 1998
).
Some chemicals affect Cx26 and Cx32 differentially, resulting in alteration in the expression of one connexin in one direction (decrease or increase), while leaving the other connexin unaffected or altering its expression in the opposite direction. TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin), a potent rodent liver tumor promoter, decreases Cx32 mRNA in isolated hepatocytes but has no effect on Cx26 mRNA (Baker et al., 1995). Interleukins 1 and 6 increase Cx26 protein expression in immortalized MHSV12 mouse hepatocytes, while simultaneously decreasing Cx32 protein expression (Temme et al., 1998
). In contrast to these examples, in the present study, Cd appears to have decreased Cx26 and Cx32 protein levels coordinately in the liver (Figs. 4 and 5
). This suggests that Cd probably acts via a mechanism common to both Cx26 and Cx32. One such mechanism is the induction of oxidative stress in hepatocytes. The cellular toxicity of Cd has been attributed in part to induction of oxidative stress (Dong et al., 1998
; Shaikh et al., 1999
). Oxidative stress has been shown to inhibit GJIC (Ruch et al., 1989
; Upham et al., 1997
). More specifically, free radical scavengers, which reduce the level of oxidative stress of a cell, enhance the expression of Cx26 and Cx32 (Kojima et al., 1996
). It is conceivable, therefore, that Cd decreases the expression of Cx26 and Cx32 via Cd-induced oxidative stress.
In the present study, Cd was found to disrupt cytoskeletal actin (Fig. 6). Actin is the major protein that forms the cell's microfilament network (Martini and Timmons, 1997
), and it has been shown to be physically associated with gap junctions (Lo et al., 1996
; Murray et al., 1997
), along with other types of intercellular junctions. Disruption of cytoskeletal actin results in changes in the structure of these intercellular junctions (gap, intermediate, and tight junctions), destruction of the intercellular junctions (Amsterdam et al., 1992
), or internalization of gap junctions (Koike et al., 1993
). In the present study, Cd caused a time- and dose-dependent disruption and loss of cytoskeletal actin. This is consistent with earlier reports, which indicate that Cd disrupts the organization of cytoskeletal actin in vitro (Jeong et al., 1998
; Prozialeck and Niewenhuis, 1991
; Wang et al., 1996
). Others have shown that the effect of Cd on cytoskeletal actin depends on the concentration of Cd; high doses of Cd produce depolymerization (more common) or polymerization (less common) of actin, depending on the cell type, and low doses produce the reverse effect on the same cells (DalleDonne et al., 1997
; Wang and Templeton, 1996
). In the present study, Cd produced depolymerization (disruption and loss) of cytoskeletal actin at all doses tested (560 µmol/kg). The disruption and loss of cytoskeletal actin progressed in a manner that paralleled the Cd-induced progressive inhibition of GJIC and decrease in Cx26 and Cx32 signals per cell. It is probable, therefore, that loss of cytoskeletal actin contributed to the decrease in Cx26 and Cx32 signals and inhibition of GJIC observed in this study. A similar association was observed in a study of three subclones (M17, M6 and M3) from the MGC-803 human stomach carcinoma cell line (Han et al., 1996
). M17, with severe disruption of microfilament (and microtubule) networks, showed severe inhibition of GJIC. In contrast, M6 and M3, with less disruption of the microfilament network, had normal GJIC.
In conclusion, this study demonstrates that Cd inhibits GJIC in mouse liver in a time- and dose-dependent manner, and this inhibition is correlated with parallel decreases in the major connexins of the liver, Cx32 and Cx26. The inhibition of GJIC is also correlated with Cd-induced disruption and loss of cytoskeletal actin. Further studies are needed to pinpoint the mechanisms by which Cd decreases GJIC and the expression of Cx32 and Cx26 in the liver.
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ACKNOWLEDGMENTS |
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NOTES |
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2 To whom correspondence should be addressed. Fax: (913) 588-7501. E-mail: cklaasse{at}kumc.edu.
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