Decreased gap junctional intercellular communication in hexachlorobenzene-induced gender-specific hepatic tumor formation in the rat
Isabelle Plante,
Michel Charbonneau,2 and
Daniel G. Cyr,1,2
Human Health Research Center, INRS-Institut Armand-Frappier, 245 Hymus Boulevard, Université du Québec, Pointe-Claire, QC, H9R 1G6, Canada
 |
Abstract
|
---|
Hexachlorobenzene (HCB), an epigenetic carcinogen, HCB induces the formation of liver tumors in female rats, whereas only a small percentage of males are responsive. Intercellular communication via gap junctions is decreased in carcinogenesis. Gap junctions are composed of proteins termed connexins (Cxs). The objectives of this study were (i) to determine if HCB-induced tumor development is associated with a loss of gap junctional communication; (ii) to assess if HCB causes a gender-specific decrease in the expression of Cx32 and Cx26; and (iii) to establish if these effects result from gender differences in the constitutive expression of these Cxs. Rats were given HCB by gavage for five consecutive days. In the first experiment, control and HCB-treated female rats were sampled on day 100. Intercellular communication was significantly decreased in HCB-treated females compared to controls. To investigate if changes in Cx levels occur prior to day 100, experiments done using male and female rats sampled on day 50. Hepatic mRNA levels for Cx26 and Cx32 were significantly lower only in HCB-treated females as compared to controls. Cx26 mRNA levels were 3-fold higher and Cx32 mRNA levels were 8-fold lower in females compared with males. In a third experiment, ovariectomy abolished any differences between male and female controls for both Cxs, while estradiol had a partial role in the regulation of Cx32. This suggests that the sexual dimorphism in hepatic Cx levels is determined by the ovarian hormones. However, the HCB-induced decrease in Cx32 and Cx26 mRNA levels was maintained in ovariectomized rats, suggesting that the HCB effects are not mediated via an ovary-dependent pathway. Overall results show that HCB exposure induces gender-specific long-term alterations in intercellular gap junctional communication in female rat liver. This effect appears to be a critical mechanism of HCB-induced liver carcinogenesis and tumor promotion.
Abbreviations: Cxs, connexins; Cx26, connexin 26; Cx32, connexin 32; DEN, diethylnitrosamine; DTT, dichlorodiphenyltrichloroethylene; E2, estradiol; HCB, hexachlorobenzene; LY, Lucifer Yellow; OV, ovariectomized; PBB, polybrominated biphenyls; PCB, polychlorinated biphenyls; RhD, RhodamineDextran; TCDD, tetrachlorodibenzo-p-dioxin
 |
Introduction
|
---|
Hexachlorobenzene (HCB) is a widespread environmental contaminant, which has been used as a fungicide and is a byproduct of industrial processes. While the use of HCB as a fungicide has been banned in most industrialized countries, it is a persistent contaminant and is still present in the environment (13). Exposure to HCB has been linked to the development of porphyria and hepatic cancer (38).
Previous studies in rodents have shown that HCB is an epigenetic carcinogen. Studies from our laboratory have shown that short-term exposure (5 days) to HCB renders females more susceptible than males to the development of porphyria, which begins
40 days after the end of the HCB treatment (9). Furthermore, the administration of the tumor initiator diethylnitrosamine (DEN) 95 days after the end of treatment results in the development of hepatic tumors in females, with few males exhibiting tumors. This gender-specific effect of HCB in the liver has also been reported by others (1013). This observation not only allows us to use this exposure model to study the mechanisms of action of HCB, but also provides a unique model to address gender-specific tumor formation.
Gap junctions form intercellular channels between adjacent cells which permit bidirectional communication between cells by selectively allowing the passage of small molecules (<1 kDa) including secondary messengers (e.g. IP3, Ca2+, cAMP). Gap junctions are formed by a family of integral transmembrane proteins termed connexins (Cxs). Cx subunits oligomerize in the trans-Golgi network to form hemichannels or connexons, which consist of six Cxs arranged radially around a central pore. In adult rat hepatocytes there are two main Cxs which modulate intercellular communication: Cx32 and Cx26.
Several studies have shown that gap junctional communication is decreased in carcinogenesis (14). While the reason for this loss of intercellular communication has not been completely elucidated, it has been suggested that the loss of apoptotic control from adjacent cells may be essential for tumor development (15). Furthermore, the loss of cellcell interactions between adjacent cells may be necessary for the clonal expansion of tumors.
The objectives of this study were to determine (i) if HCB-induced tumor development is associated with a loss of gap junctional communication; (ii) whether or not HCB causes a gender-specific decrease in the expression of Cx32 and Cx26; and (iii) if these effects result from a gender difference in the constitutive expression of these Cxs.
 |
Materials and methods
|
---|
Animals and experimental protocol
Animals.
Male and female SpragueDawley rats (180200 g) were purchased from Charles River Canada (St. Constant, QC). Rats were maintained under a constant photoperiod of 12 h light:12 h dark and received food and water ad libitum. All animal protocols used in this study were approved by the University Animal Care Committee.
Experiment 1.
In previous studies, DEN administration 95 days after a 5-day exposure to HCB induced liver tumors in female rats (9). In order to determine if there is an alteration in hepatic gap junctional communication at day 100, an experiment was done using two experimental groups of female rats: control and HCB-treated. Rats were administered HCB (100 mg/kg) or vehicle (corn oil, controls) by gavage for 5 consecutive days. Rats were sampled 95 days after the end of the HCB treatment and the livers were used to assess junctional communication by dye transfer.
Experiment 2.
To determine if HCB alters mRNA levels of hepatic Cxs, female and male rats were divided into four experimental groups: control females, control males, HCB-treated females and HCB-treated males. Each group consisted of six rats, except for HCB-treated females (n = 7). Rats were administered HCB (100 mg/kg) by gavage every day for 5 consecutive days. Controls received vehicle only (corn oil). Rats were sampled 45 days after the last HCB treatment (day 50 of the experiment) in what has been reported as the early phase of porphyria (9). Rats were anesthetized with xylazine/ketamine (50:10 mg/kg), the livers removed, frozen in liquid nitrogen and stored at 86°C. Northern blot analysis was done to determine the effects of HCB on hepatic Cx32 and Cx26 mRNA levels, while Cx protein levels were determined by western blot analysis.
Experiment 3.
To assess whether or not differences in the constitutive expression of Cx32 and Cx26 mRNA levels in rat hepatocytes were due to ovarian estrogens, an experiment was done using three experimental groups of female rats: intact controls, ovariectomized (OV), and ovariectomized with a silastic implant of estradiol (OV + E2). Each group consisted of 10 individuals. Estradiol-filled polydimethylsiloxane capsules were prepared according to the methods outlined by Stratton et al. (16) and have well characterized steroid release rates (17). Ovariectomized rats were implanted with either an empty capsule (1.6 cm) or a capsule filled with 17ß-estradiol (1.6 cm; Sigma Chemicals, Missassauga, ON). The latter mimics serum estradiol levels. Capsules were maintained in a solution of 2% bovine serum albumin for 3 days prior to the start of the experiment, ensuring that the newly made capsules had a constant estradiol release rate. Rats were killed 10 days after surgery, the livers were removed, frozen in liquid nitrogen and stored at 86°C.
Experiment 4.
To assess whether or not ovarian hormones modulate the HCB response, an experiment was done using four groups of female rats: intact controls, HCB-treated, ovariectomized as well as an ovariectomized and HCB-treated group. There were six animals in each experimental group. Rats were ovariectomized (control were sham operated) and HCB treatment was initiated 3 days after surgery. Rats were given HCB (100 mg/kg) by gavage for 5 consecutive days; intact controls and OV rats received vehicle alone (corn oil). Rats were sampled 40 days after the end of the HCB treatment. The liver from each rat was removed, frozen in liquid nitrogen and stored at 86°C. Northern blot analysis was done to determine hepatic Cx32 and Cx26 mRNA levels.
Incision loading dye transfer
At the time of sampling, livers were excised and a transverse incision was made on the left lobe of the liver of each rat according to the methods of Sai et al. (18). A 100 µl aliquot of a dye mixture [0.5% Lucifer Yellow (Sigma Chemicals); 0.5% RhodamineDextran (Sigma Chemicals) in PBS] was placed in the incision. Following the addition of the dye mixture, three longitudinal incisions were made across the initial incision and an additional 100 µl aliquot of dye mixture was added to incisions. Samples were incubated for 3 min at room temperature and subsequently washed in PBS. A sample of liver was then placed in buffered formalin (10%) overnight, dehydrated and embedded in paraffin. Lucifer Yellow (LY) and RhodamineDextran (RhD) transfer were determined by fluorescence microscopy. The RhD migration was used to assess the non-specific transfer while the LY was used to determine gap junction-specific transfer (18). The extent of the dye migration was determined using digital images that were analyzed using the ImagePro Plus computer program (Media Cybernetics, Silver Spring, MD). Two to three sites per incision were randomly evaluated for dye migration. Means of each animal were used to evaluate differences between groups.
Northern blot analysis
Northern blot analyses were done on total cellular RNA isolated from the liver. RNA was isolated using the guanidinium isothiocyanate method (19). Aliquots of total RNA (10 mg) were separated by electrophoresis in a 1.2% agaroseformaldehyde gel and transferred onto a charged nylon membrane (Genescreen plus, Dupont Chemicals, Missassauga, ON) as previously described (20).
Full-length Cx26 and Cx32 cDNA probes were obtained as generous gifts from Drs B.Nicholson (SUNY, Buffalo, NY) and D.Paul (Harvard University, Cambridge, MA). The cDNA probes were labeled by random priming with [32P]dCTP (Oligonucleotide Labelling Kit, Pharmacia-Amersham Biotech, Baie D'Urfé, QC; 21). Each northern blot was standardized for RNA loading by hybridizing the membranes with an end-labeled oligonucleotide probe recognizing the 18S rRNA (21). Hybridizations of the cDNA probes and 18S rRNA probe were done as previously described (21). The resulting unsaturated phosphorimages were scanned using a Molecular Dynamics Phosphorimager (PhosphorImager SITM Molecular Dynamics) and the integrated area under the curve for each signal was standardized against the signal for the 18S rRNA to determine the relative levels of either Cx32 or Cx26 mRNA.
Western blot analyses
Frozen liver samples were homogenized in buffer (0.25 mM sucrose; 10 mM TrisHCl pH 7.5; 50 mg/ml leupeptin; 50 mg/ml aprotinin; 25 mg/ml pepstatin; 50 mg/ml antipain; 2.5 g/ml phenylmethylsulfonyl fluoride) and centrifuged at 10 000 g for 10 min at 4°C. The resulting supernatant containing the gap junctions was removed and its protein content determined using the Bio-Rad Protein Assay (Bio-Rad, Missassauga, ON).
Protein samples (50 mg) were diluted in loading buffer (Laemmli buffer), and loaded onto a 12% polyacrylamide gel with a 5% stacking gel (22). Electrophoresis was done at 120 V for 1.5 h until the dye front reached the end of the gel.
The gel was removed from the glass plates, and the proteins transferred onto a nitrocellulose membrane using a Bio-Rad Transblot apparatus at 100 V for 45 min in transfer buffer (25 mM TrisBase, 0.195 M glycine, 0.1% SDS, 20% methanol and 2 mM CaCl2). At the end of the transfer, the membrane was removed. The transfer of proteins was determined by staining the membrane with a solution of Ponceau Red S (0.5% Ponceau, 1% acetic acid). Nitrocellulose membranes containing the transferred proteins were washed and then blocked overnight with Tris-buffered saline (TBS: 100 mM Tris, 54 mM NaCl, pH 7.5) containing 0.1% Tween and 5% Carnation instant milk. Membranes were then incubated for 1.5 h at room temperature with primary antisera (1.0 µg/ml rabbit anti-mouse Cx26 or 1.0 µg/ml mouse anti-rabbit Cx32; Chemicon International, Temecula, CA). The membranes were washed three times with TBS containing 0.1% Tween and subsequently incubated for 45 min at room temperature with secondary antibody (0.4 µg/ml anti-rabbit-IgG conjugated to an alkaline phosphatase; Santa Cruz Biotechnology, Santa Cruz, CA; or 4.8 µg/ml anti-mouse-IgG conjugated to horseradish peroxidase; Sigma Chemicals). Signal detection was done according the manufacturer's recommendations. To standardize for protein loading, the membranes were probed for actin. Membranes were incubated for 1 h with monoclonal mouse anti-actin (1.0 µg/ml; Sigma Chemicals). The membranes were washed three times with TBS containing 0.1% Tween and subsequently incubated for 1 h at room temperature with secondary antibody (3.6 µg/ml anti-mouse-IgG conjugated to peroxidase; Sigma Chemicals). Signal detection was done according to the manufacturer's instructions.
Statistics
To determine differences in Cx mRNA and protein between experimental groups, the data were tested for normality using the KolmogorovSmirov test while the Levine median test was done for equal variance. Statistical differences between groups were determined by ANOVA followed a posteriori by a StudentNewmanKeuls test for multiple comparisons between experimental groups. Significance was established at P < 0.05. All analyses were done using the SigmaStat computer software (Jandel Scientific Software, San Rafael, CA).
 |
Results
|
---|
Experiment 1
The functionality of gap junctional intercellular communication was determined at day 100 using the incision-loading/dye transfer method as previously described (18). This method evaluates intercellular communication in vivo by tracking RhD as an indicator of non-specific dye migration (Figure 1Ai
) and LY as an indicator of gap junctional communication (Figure 1Aii
). Gap junctional communication is measured by overlaying the micrographs for each dye and determining the difference in migration between the LY and the RhD (Figure 1Aiii
). Results indicate that in HCB-treated females (Figure 1Bii
) gap junctional communication was significantly decreased by
36% (Figure 1C
) compared with control females (Figure 1Bi
).

View larger version (48K):
[in this window]
[in a new window]
|
Fig. 1. Effects of HCB on intercellular communication in female rat liver. Rats were administered HCB (100 mg/kg) by gavage every day for 5 days and sampled on day 100 of the experiment. Livers were excised and subject to incision-loading/dye-transfer analysis using RhD and LY. For all images, RhD (A, panels i and iii) and LY (A, panels ii and iii; B, panels i and ii) fluorescence is shown by white arrows (100x). The distance of migration is indicated by a white line (A and B). Section B represents LY migration in a control (panel i) and in a HCB-treated female liver (panel ii). Hepatic gap junctional intercellular communication in an intact female and HCB-treated groups are shown (C). The data are expressed as the mean ± SEM (n = 6). The `a' in section C indicates that the mean is significantly different from controls (P < 0.05).
|
|
Experiment 2
Female rats were treated with HCB and sampled 45 days after the end of treatment (day 50 of the experiment). HCB administered to rats using this model does not induce liver injury as determined by the absence of histological changes and normal plasma levels of alanine aminotransferase (ALT) (data not shown). HCB-treated females had significantly lower levels of Cx32 (Figure 2A
) and Cx26 (Figure 2B
) mRNA levels compared with females given vehicle alone (controls). Cx26 mRNA levels were
40% lower in treated females compared with controls, whereas Cx32 mRNA levels were
30% lower. There were no significant differences in either Cx32 (Figure 2C
) or Cx26 (Figure 2D
) mRNA levels between control and HCB-treated males. This is consistent with the fact that only female rats are sensitized to HCB tumor promotion.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 2. Effects of HCB on hepatic Cx32 and Cx26 mRNA levels in male and female rats. Rats were administered HCB (100 mg/kg) by gavage every day for 5 days and sampled on day 50 of the experiment. Total cellular RNA was isolated and subjected to northern blot analysis using specific Cx32 and Cx26 cDNA probes. Data were standardized for loading using an 18S rRNA probe. Hepatic Cx32 (A, C) and Cx26 (B, D) mRNA levels in female (A, B) and male (C, D) rats are shown. The data are expressed as the mean ± SEM (n = 6 for all groups except HCB-treated females, n = 7). The `a' indicates that the mean is significantly different from controls (P < 0.05).
|
|
While Cx26 and Cx32 mRNA levels were not altered in male rats, it is interesting to note that there are important differences in the basal levels of expression for these two Cxs between males and females. Cx26 mRNA levels were almost 3-fold higher in female rats compared with males, whereas Cx32 mRNA levels were 8-fold higher in male rats compared with females (see control levels in Figure 2A to D
).
To establish whether or not HCB decreases in Cx26 and Cx32 mRNA also occurred at the protein level, liver samples of control and HCB-treated female rats were subjected to western blot analysis. The results indicate that at the protein level, the effects of HCB are in fact much more pronounced than at the mRNA level. Cx26 protein levels were
63% lower in HCB-treated rats (Figure 3B
), whereas Cx32 levels were decreased by 90% relative to female control rats (Figure 3A
).

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 3. Effects of HCB on Cx32 and Cx26 protein levels in female rat liver. Rats were administered HCB (100 mg/kg) by gavage every day for 5 days and sampled on day 50 of the experiment. Livers were homogenized and a 50 µg aliquot of protein from each rat was subjected to western blot analysis using Cx26 (A) and Cx32 (B) antisera. Data were standardized for loading using an actin antisera. The data are expressed as the mean ± SEM (n = 6). The `a' indicates a significant difference from controls (P < 0.05).
|
|
Experiment 3
The differences in Cx32 and Cx26 mRNA levels between control males and females suggest that ovarian hormones may be important regulators of hepatic Cx mRNA levels. To establish whether or not this was the case, we compared Cx26 and Cx32 mRNA levels in the livers of intact control females with those from ovariectomized rats (OV) and ovariectomized rats given an estradiol implant (OV + E2).
Northern blot analysis indicates that hepatic Cx26 mRNA levels in OV females were
3-fold lower than levels in intact controls (Figure 4A
), while Cx32 mRNA levels were 6-fold higher in OV females (Figure 4B
) compared with controls. The differences between intact controls and OV rats are almost identical to the differences observed in the basal mRNA expression of these two Cxs between males and females. Interestingly, estradiol maintenance did not alter Cx26 mRNA levels, as these were comparable to levels in OV rats. This suggests that the differences in Cx26 mRNA levels are not the result of a decrease in circulating estradiol levels in OV rats. Unlike Cx26 mRNA levels, Cx32 mRNA levels appear to be partially regulated by estradiol, as levels were significantly lower in OV + E2 rats than levels found in OV rats. However, Cx32 mRNA levels remained significantly elevated compared with intact controls, suggesting that other ovarian factors may contribute to the regulation of steady-state hepatic Cx32 mRNA levels (Figure 4
).

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 4. Effects of ovariectomy on hepatic Cx32 and Cx26 mRNA levels in female rats 10 days after surgery. Rats were ovariectomized (OV) and given an estradiol implant (OV + E2) (control were sham-operated). Rats were sampled 10 days after surgery. Total cellular RNA was isolated from livers and subjected to northern blot analysis using specific Cx32 and Cx26 cDNA probes. Data were standardized for loading using an 18S rRNA probe. Hepatic Cx26 (A) and Cx32 (B) mRNA levels in female rats are shown. The data are expressed as the mean ± SEM (n = 10). The `a' indicates that the mean is significantly different from controls (P < 0.05) and `b' indicates that the mean is significantly different from the OV group.
|
|
Experiment 4
The results from the first two experiments indicate that the ovary and ovarian hormones may be important regulators of hepatic Cx26 and Cx32 mRNA levels. In this experiment we wanted to assess whether or not the ovary could influence the HCB response with respect to hepatic Cx26 and Cx32 mRNA levels. To address this objective an experiment was designed using four experimental groups of female rats: a control group, an HCB-treated group, an OV group, and an HCB-treated OV group (OV-HCB). Rats were sampled on day 45 of the experiment.
Both Cx26 and Cx32 mRNA levels were lower in the HCB-treated group compared with control female rats (Figure 5
). Ovariectomy resulted in a significant decrease in Cx26 mRNA levels by almost 75% (Figure 5A
). Interestingly, Cx32 mRNA levels were not significantly different from controls (Figure 5B
), suggesting that the increased levels observed 10 days after ovariectomy may be transient. Furthermore, there were also decreases in both Cx32 and Cx26 mRNA levels in the OV-HCB group compared with the OV group (Figure 5
). This suggests that HCB-induced lower hepatic Cx32 and Cx26 mRNA levels are not the result of an ovarian-mediated pathway.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 5. Effects of HCB on hepatic Cx32 and Cx26 mRNA levels in female rats and ovariectomized female rats 45 days after the first day of HCB exposition. Rats were ovariectomized (OV) (control were sham-operated) and administered HCB (100 mg/kg) by gavage every day for 5 days, beginning 3 days after the surgery. The livers were sampled on day 45 of the experiment. Total cellular mRNA was isolated and subjected to northern blot analysis using specific Cx32 and Cx26 cDNA probes. Data were standardized for loading using an 18S rRNA probe. Hepatic Cx26 (A) and Cx32 (B) mRNA levels in females are shown. The data are expressed as the mean ± SEM (n = 6). The `a' indicates that the mean is significantly different from controls (P < 0.05) and `b' denotes a significant difference from OV (P < 0.05).
|
|
 |
Discussion
|
---|
HCB promotes gender-specific tumor formation in female rat livers (9,10). The mechanism responsible for this gender-specific effect is unknown. It has been shown that the formation of tumors is associated with a loss of intercellular communication (2326). In the present study we have demonstrated that HCB administration resulted in significantly lower levels of both Cx32 and Cx26 mRNA and protein levels in the livers of female rats sampled 45 days after the last administration of HCB (Figures 2 and 3
). Interestingly, Cx32 and Cx26 mRNA levels in the livers of male rats which received an identical exposure to HCB were not significantly different from vehicle-exposed controls. Since female rats develop hepatic tumors, our results suggest that decreased Cx32 and Cx26 expression, as well as decreased gap junctional intercellular communication, are associated specifically with carcinogenesis.
It has been suggested that the majority of malignant tumors have altered intercellular communication. While it has not been clearly established why there is aberrant gap junctional intercellular communication in tumor cells, transfecting malignant cells with certain Cxs, such as Cx26, can decrease tumor progression, thereby suggesting that Cxs and gap junctional communication may act as tumor suppressors (27). What is particularly interesting with the present observations is that HCB decreases gap junctional communication prior to the formation of hepatic tumors in female rats. This suggests that the decreased expression of cellular Cxs and consequent decrease in gap junctional communication are early events in the process of chemically-induced liver carcinogenesis. In fact, Cx32 may serve as an early marker of liver tumors. This is similar to the loss of Cx43 expression, which has been suggested as a marker of breast tumors (28). Furthermore, since HCB is an epigenetic carcinogen, it is unlikely that HCB exposure results in mutations to either the Cx32 or Cx26 genes, but rather that their regulation is altered via other intracellular pathways.
While other organochlorines such as tetrachlorodibenzo-p-dioxin (TCDD) (29), pentachlorophenol (18), polychlorinated biphenyls (PCB) (30,31), clofibrate (31), dichlorodiphenyltrichloroethylene (DDT) (32,33) and the metal cadmium (34) have all been shown to decrease gap junctional communication, HCB is the only chemical to our knowledge which causes gender-specific decreased gap junctional communication. Environmental carcinogens such as PCBs, TCDD and polybrominated biphenyls (PBBs) have also been suggested to cause female-specific hepatic tumors, however, it is not known whether or not these chemicals act via similar mechanisms as HCB.
Our present data suggest that there exists a sexual dimorphism in Cx32 and Cx26 mRNA levels of control rats. This dimorphism may be responsible for rendering female rats more susceptible to the formation of hepatic tumors than their male counterparts. Our data indicate that the ovary appears to be an important regulator of hepatic Cx26 mRNA levels, but estradiol does not appear to be responsible for this regulation. While it is difficult to establish which other ovarian factors are responsible for regulating Cx26, other studies have reported that progesterone can regulate Cx mRNA levels in the uterus (35). Further studies will be necessary to establish whether progesterone regulates Cx26 in the liver.
Cx32 mRNA levels were significantly higher in OV rats sampled 10 days after surgery. Unlike Cx26, Cx32 mRNA levels were decreased in OV rats given estradiol, although Cx32 mRNA levels were not maintained at control levels. This suggests that estradiol partially regulates Cx32 mRNA levels and that other ovarian factors contribute to the regulation of hepatic Cx32. The ovarian regulation of hepatic Cx32 mRNA levels however, appears to be transitory, since OV rats sampled 45 days after surgery had Cx32 mRNA levels that were not significantly different from intact control rats. Several studies have reported that either cellular levels or the cellular distribution of Cxs in reproductive and endocrine tissues can be influenced by steroid hormones (3641). While in many of these studies estradiol increases Cx levels, our data clearly indicate that in the liver, estradiol down-regulates Cx32 or does not influence Cx26 mRNA levels. Our results indicate that the ovary and, most likely, ovarian hormones are responsible for the sexual dimorphism in hepatic Cx26 and Cx32 mRNA levels of control rats.
In OV rats sampled at day 45, HCB decreased both Cx26 and Cx32 mRNA levels, despite the fact that Cx26 mRNA levels were already depressed as a result of the ovariectomy. This suggests that the effects of HCB are not mediated via a peripheral action of HCB on the ovary, but are most likely the result of a direct effect of HCB on the liver. This is particularly interesting, since HCB did not alter Cx26 and Cx32 mRNA levels in the males (Figure 2
), indicating that the gender-dependent differences in Cx26 and Cx32 mRNA levels induced by HCB can be attributed to non-ovarian factors.
Cx32 is
10 times more abundant than Cx26 in the liver. The importance of both Cxs in promoter-induced tumors may therefore be different. It has been demonstrated using mutated Cx32 transgenic mice that a lack of Cx32 results in a higher incidence of spontaneous and chemically-induced hepatic tumors (42). Omori et al. (43) have recently reported that the occurrence of spontaneous liver tumors in hepatic Cx32 knockout mice was not different from wild-type mice. However, these mice were more susceptible to DEN-induced hepatocarcinomas than wild-type mice. Based on our results, it is tempting to speculate that exposure to HCB leads to a `chemically-induced knock-down' of Cx32 in female rats, leading to hepatic liver tumor formation.
In conclusion, HCB exposure results in gender-specific long-term alterations in intercellular gap junctional communication in female rat liver. This decrease occurs prior to the development of hepatic tumors and may represent an early indicator of chemically-induced carcinogenesis. This effect occurs independently of the presence of the ovaries, thereby suggesting that non-ovarian factors play an important role in HCB-induced decreased intercellular communication and carcinogenesis.
 |
Notes
|
---|
1 To whom correspondence should be addressed E-mail: daniel.cyr{at}inrs-sante.uquebec.ca 
2 Both authors contributed equally to all parts of the work 
 |
Acknowledgments
|
---|
This study was supported by Toxic Substances Research Initiative in the form of a grant to both D.G.Cyr and M.Charbonneau, and by a studentship from the Armand-Frappier Foundation to IP. Drs. B.Nicholson (SUNY, Buffalo, NY) and D.Paul (Harvard University, Cambridge, MA) are thanked for their generous gifts of cDNA probes. Rupert Abdalian, Guylaine Lassonde and Julie Dufresne are thanked for their assistance.
 |
References
|
---|
- Newhook,R. and Meek,M.E. (1994) Hexachlorobenzene: evaluation of risks to health from environmental exposure in Canada. Environ. Carcino. Ecotox. Rev., C12, 345360.[ISI]
- Williams,G.M., Iatropoulos,M.J. and Weisburger,J.H. (1996) Chemical carcinogen mechanisms of action and implications for testing methodology. Exp. Toxicol. Pathol., 48, 101111.[ISI][Medline]
- Government of Canada (1993) Canadian Environmental Protection Act Priority Substances List. Supporting Document, Hexachlorobenzene. National Health and Welfare/ Department of the Environment, Ottawa, Canada, 56 pp.
- Legault,N., Sabik,H., Coopert,S.F. and Charbonneau,M. (1997) Effects of estradiol on the induction of porphyria by hexachlorobenzene in the rat. Biochem. Pharmacol., 54, 1925.
- Carpenter,H.M., Williams,D.E. and Buhler,D.R. (1985) Hexachlorobenzene-induced porphyria in Japanese quail: an in vitro study of changes in cytochrome P-450 and monooxygenases. J. Toxicol. Environ. Health, 16, 207217.[ISI][Medline]
- Arnold,D.L., Moodie,C.A., Charbonneau,S.M., Grice,H.C., McGuire,P.F., Bryce,F.R., Collins,B.T., Zawidzka,Z.Z., Krewski,D.R., Nera,E.A. and Munro,I.C. (1985) Long-term toxicity of hexachlorobenzene in the rat and the effect of dietary vitamin A. Food Chem. Toxicol., 23, 779793.[ISI][Medline]
- Cabral,J.R., Mollner,T., Raitano,F. and Shubik,P. (1979) Carcinogenesis of hexachlorobenzene in mice. Int. J. Cancer, 23, 4751.[ISI][Medline]
- Cabral,J.R., Shubik,P., Mollner,T. and Raitano,F. (1977) Carcinogenic activity of hexacholorobenzene in hamsters. Nature, 269, 510511.[ISI][Medline]
- Krishnan,K., Brodeur,J. and Charbonneau,M. (1991) Development of an experimental model for the study of hexachlorobenzene-induced hepatic porphyria in the rat. Fund. Appl. Toxicol., 17, 433441.[ISI][Medline]
- Smith,A.G. and Cabral,J.R. (1980) Liver-cell tumours in rats fed hexachlorobenzene. Cancer Lett., 11, 169172.[ISI][Medline]
- Lambrecht,R.W., Ertürk,E., Grunden,E.E., Headley,D.B., Peters,H.A., Morris,C.R. and Bryan,B.T. (1983) Renal toxicity and tumorigenicity of hexachlorobenzene (HCB) in Syrian golden hamsters (H) after subchronic administration. Proc. Fed. Am. Soc. Exp. Biol., 42, 786.
- Smith,A.G., Francis,J.E., Dinsdale,D., Manson,M.M. and Cabral,J.R. (1985) Hepatocarcinogenicity of hexachlorobenzene in rats and the sex difference in hepatic iron status and development of porphyria. Carcinogenesis, 6, 631636.[Abstract]
- Pereira,M.A., Herren,S.L., Britt,A.L. and Khoury,M.M. (1982) Sex difference in enhancement of GGTase-positive foci by hexachlorobenzene and lindane in rat liver. Cancer Lett., 15, 95101.[ISI][Medline]
- Yamasaki,H., Krutovskikh,V., Mesnil,M., Columbano,A., Tsuda,H. and Ito,N. (1993) Gap junctional intercellular communication and cell proliferation during rat liver carcinogenesis. Environ. Health Persp., 101, 191198.
- DeoCampo,N.D., Wilson,M.R. and Trosko,E.J. (2000) Cooperation of blc-2 and myc in the neoplastic transformation of normal rat liver epithelial cells is related to the down-regulation of gap junction-mediated intercellular communication. Carcinogenesis, 21, 15011506.[Abstract/Free Full Text]
- Stratton,I.G., Ewing,L.L. and Desjardins,C. (1973) Efficacy of testosterone-filled polydimethylsiloxane implants in maintaining plasma testosterone in rabbits. J. Reprod. Fertil., 35, 235244.[Medline]
- Brawer,J.R., Schipper,H. and Robaire,B. (1983) Effects of long term androgen and estradiol exposure on the hypothalamus. Endocrinology, 112, 194199.[ISI][Medline]
- Sai,K., Kanno,J., Hasegawa,R., Trosko,J.E. and Inoue,T. (2000) Prevention of the down-regulation of gap junctional intercellular communication by green tea in the liver of mice fed pentachlorophenol. Carcinogenesis, 21, 16711676.[Abstract/Free Full Text]
- Chomczynski,P. and Sacchi,N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem., 162, 156159.[ISI][Medline]
- Cyr,D.G., Hermo,L., Blaschuck,O.W. and Robaire,B. (1992) Distribution and regulation of epithelial cadherin messenger ribonucleic acid and immunocytochemical localization of epithelial cadherin in the rat epididymis. Endocrinology, 130, 353363.[Abstract]
- Cyr,D.G., Dufresne,J., Pillet,S., Alfieri,T.J. and Hermo,L. (2001) Expression and regulation of metallothioneins in the rat epididymis. J. Androl., 22, 124135.[Abstract/Free Full Text]
- Cyr,D.G., Hermo,L. and Laird,D.W. (1996) Immunocytochemical localization and regulation of connexin43 in the adult rat epididymis. Endocrinology, 137, 14741484.[Abstract]
- Yamasaki,H., Krutovskikh,V., Mesnil,M., Tanaka,T., Zaidan-Dagli,M.L. and Omori,Y. (1999) Role of connexin (gap junction) genes in cell growth control and carcinogenesis. C. R. Acad. Sci. III, 322, 151159.[ISI][Medline]
- Ruch,R.J. (1994) The role of gap junctional intercellular communication in neoplasia. Ann. Clin.. Lab. Sci., 24, 216231.[Abstract]
- Hotz-Wagenblatt,A. and Shalloway,D. (1993) Gap junctional communication and neoplastic transformation. Crit. Rev. Oncog., 4, 541558.[ISI][Medline]
- Kanno,Y. (1985) Modulation of cell communication and carcinogenesis. Jpn J. Physiol., 35, 693707.[ISI][Medline]
- Hircshi,K.K., Xu,C.E., Tsukamoto,T. and Sager,R. (1996) Gap junction genes Cx26 and Cx43 individually suppress the cancer phenotype of human mammary carcinomas cells and restore differentiation potential. Cell Growth Diff., 7, 861870.[Abstract]
- Laird,D.W., Fistouris,P., Batist,G., Alpert,L., Huynh,H.T., Carystinos,G.D. and Alaoui-Jamali,M.A. (1999) Deficiency of connexin43 gap junctions is an independent marker for breast tumors. Cancer Res., 59, 41044110.[Abstract/Free Full Text]
- Warngard,L., Bager,Y., Kato,Y., Kenne,K. and Ahlborg,U.G. (1996) Mechanistical studies of the inhibition of intercellular communication by organochlorine compounds. Arch. Toxicol., 18, 149159.
- Bager,Y., Kenne,K., Krutovskikh,V., Mesnil,M., Traub,O. and Wärngard,L. (1994) Alteration in expression of gap junction proteins in rat liver after treatment with the tumour promoter, 3,4,5,3',4'-pentachlorobiphenyl. Carcinogenesis, 15, 24392443.[Abstract]
- Krutovskikh,V.A., Mesnil,M., Mazzoleni,G. and Yamasaki,H. (1995) Inhibition of rat liver gap junction intercellular communication by tumor-promoting agents in vivo association with aberrant localization of connexin proteins. Lab. Invest., 72, 571577.[ISI][Medline]
- Tateno,C., Ito,S., Tanaka,M., Oyamada,M. and Yoshitake,A. (1994) Effect of DDT on hepatic gap junctional intercellular communication in rats. Carcinogenesis, 15, 517521.[Abstract]
- Ruch,R.J., Bonney,W.J., Sigler,K., Guan,X., Matesic,D., Schafer,L.D., Dupont,E. and Trosko,J.E. (1994) Loss of gap junctions from DDT-treated rat liver epithelial cells. Carcinogenesis, 15, 301306.[Abstract]
- Fang,M.Z., Mar,W.C. and Cho,M.H. (2001) Cadmium-induced alterations of connexin expression in the promotion stage of in vitro two-stage transformation. Toxicology, 161, 117127.[ISI][Medline]
- Risek,B., George,K.F., Hahn,D.W. and Gilula,N.B. (1995) Gap junction regulation in the uterus and ovaries of immature rats by estrogen and progesterone. J. Cell Sci,, 108, 10171032.[Abstract/Free Full Text]
- MacKenzie,L.W., Puri,C.P. and Garfield,R.E. (1983) Effect of estradiol-17ß and prostaglandins on rat myometrial gap junctions. Prostaglandins, 26, 925941.[Medline]
- Grümmer,R., Chwalisz,K., Mulholland,J., Traub,O. and Winterhager,E. (1994) Regulation of connexin26 and connexin43 expression in rat endometrium by ovarian steroid hormones. Biol. Reprod., 51, 11091116.[Abstract]
- Grümmer,R., Traub,O. and Winterhager,E. (1999) Gap junction connexin genes Cx26 and Cx43 are differentially regulated by ovarian steroid hormones in rat endometrium. Endocrinology, 140, 25092516.[Abstract/Free Full Text]
- Antoskiewicz,B., Muller,G., Grummer,R. and Winterhager,E. (1996) Induction of connexin32 expression by potential embryonic signals in rabbit uterine epithelium. Early Pregnancy, 2, 253263.[Medline]
- Orsino,A., Taylor,C.V. and Lye,S.J. (1996) Connexin-26 and connexin-43 are differentially expressed and regulated in the rat myometrium throughout late pregnancy and with the onset of labor. Endocrinology, 137, 15451553.[Abstract]
- Shinohara,K., Funabashi,T., Mitushima,D. and Kimura,F. (2000) Effects of estrogen on the expression of connexin32 and connexin43 mRNAs in the suprachiasmatic nucleus of female rats. Neurosci. Lett., 286, 107110.[ISI][Medline]
- Temme,A., Buchmann,A., Gabriel,H.D., Nelles,E., Schwarz,M. and Willecke,K. (1997) High incidence of spontaneous and chemically induced liver tumors in mice deficient for connexin32. Curr. Biol., 7, 713716.[ISI][Medline]
- Omori,Y., Zaidan Dagli,M.L., Yamakage,K. and Yamasaki,H. (2001) Involvement of gap junctions in tumor suppression: analysis of genetically-manipulated mice. Mutat. Res., 477, 191196.[ISI][Medline]
Received January 25, 2002;
revised March 18, 2002;
accepted April 5, 2002.