Morphology and morphometric investigation of hepatocellular preneoplastic lesions and neoplasms in connexin32-deficient mice

Matthias Evert1, Thomas Ott2, Achim Temme2, Klaus Willecke2 and Frank Dombrowski1,3

1 Institut für Pathologie, Otto-von-Guericke-Universität, Magdeburg, Germany and
2 Institut für Genetik, Rheinische Friedrich-Wilhelms-Universität, Bonn, Germany


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Gap junctions are composed of protein subunits, called connexins, and provide a pathway for the exchange of ions and small molecules between contacting cells. This transfer of molecules is thought to be an important pathway for direct cell communication, and is involved in tissue homeostasis, growth control and embryonic development. Impairment of gap junctional intercellular communication (GJIC) via different mechanisms may therefore contribute to dysregulated cellular proliferation and subsequent tumor development. We investigated the effect of Connexin32-deficiency on liver histology and the formation of preneoplastic foci and hepatocellular neoplasms in transgenic knockout mice, as Connexin32 (Cx32) is the major gap junction protein in the liver. Loss of Cx32 does not alter the morphology of extrafocal liver tissue. However, after administration of a single dose of diethylnitrosamine (DEN), given 2 weeks after birth, the number and volume fraction of preneoplastic foci showed a 3.3-fold to 12.8-fold increase in the Cx32-deficient mice as compared with the corresponding wildtype groups, regardless of sex and age of the animals. Number and volume fraction of hepatocellular adenomas and carcinomas also increased significantly in these animals. The experimental groups did not differ in the morphology of the different types of preneoplastic foci and neoplasms. On the other hand, Cx32-deficiency without DEN treatment did not lead to an increase in the spontaneous development of any type of preneoplastic hepatic foci or hepatocellular neoplasms in up to 18-month-old Cx32-deficient mice as compared with wildtype controls. In conclusion, our results indicate that impairment of GJIC in mouse liver due to deletion of the Cx32 coding DNA clearly promotes the carcinogenic effect of DEN administration and results in a higher susceptibility to hepatocellular neoplasms, but does not appear to initiate hepatic tumor development.

Abbreviations: AmCF, amphophilic cell foci; BCF, basophilic cell foci; CCF, clear cell foci; DEN, diethylnitrosamine; FAX, foci of altered hepatocytes; GJIC, gap junctional intercellular communication; HCA, hepatocellular adenomas; HCC, hepatocellular carcinomas; MCF, mixed cell foci; PAS, periodic acid Schiff; PBS, phosphate buffered saline; TCF, tigroid cell foci.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Gap junctions are transmembrane channels between neighboring cells. They consist of two juxtaposed hemichannels, called connexons, each composed of six protein units, called connexins. The connexins are members of a multigene family mapped on different chromosomes, and are expressed in a tissue-specific manner with some overlap. Connexin 32 (Cx32), for example, is the major connexin protein expressed in the liver of rodents and humans (13).

The diameter of gap junctional channels is about 1.5 nm, allowing small hydrophilic molecules up to 1000 Da to pass between such connected cells, for example anorganic ions (N+, K+, Ca2+) or second messengers (cAMP, IP3) (1,2,4,5). In this way, transmission of molecules via gap junction channels enables adjacent cells to communicate, and gap junctional intercellular communication (GJIC) is believed to be an important mechanism in tissue homeostasis, growth and development (48).

Cell culture models have demonstrated that growth of neoplastic cells can be inhibited by coculture with non-neoplastic cells of the same type, and that at least part of this effect depends on an intact, so-called heterologous GJIC between neoplastic and non-neoplastic cells (9). In addition, it has been shown that the expression of gap junction proteins is reduced or their function is impaired in many tumor entities (8,10,11). In addition, one of the most common features of tumor-promoting agents is their ability to inhibit GJIC (1215). Dysfunction of GJIC is therefore considered to be a tumor-promoting condition, and one may conclude that genes coding for connexins represent tumor suppressor genes (16).

In different animal models, preneoplastic lesions of the liver, initiated by genotoxic agents, may develop to neoplasia after being exposed to tumor-promoting effects, and impairment of the Cx32-mediated GJIC, caused by various agents, e.g. phenobarbital, has been correlated with tumor promotion (17,18). This is in line with the finding that phenobarbital has no tumor-promoting effect on the liver in Cx32-deficient mice (19). In a former study, it has been demonstrated that Cx32-deficient mice show spontaneously more macroscopic visible lesions in the liver and, after treatment with diethylnitrosamine (DEN), a higher number of glucose-6-phosphatase-deficient preneoplastic lesions (20).

To the best of our knowledge, we are the first to investigate the phenotype of Cx32-deficient mice liver at the histological level. In addition, we examined whether transgenic mice deficient for Cx32 show differences to wildtype controls in the development of hepatocellular preneoplastic lesions and tumors, and whether these results can be influenced by administering a single dose of DEN given 2 weeks after birth.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Treatment of the animals
Cx32 deficient mice were used for the present study. They were generated by disrupting the Cx32 coding region in the mouse genome through insertion of a selectable gene that coded for neomycin resistance via homologous recombination (21).

Mice were kept under standard housing conditions with a fixed 12 h/12 h light/dark cycle and food and water ad libitum. Between day 12 and 15 after birth, one experimental group received a single intraperitoneal injection of DEN (Sigma, Heidelberg, Germany; 20 µg/g body weight), diluted with phosphate buffered saline (PBS). DEN-treated and untreated Cx32-deficient males (Cx32Y/-) and females (Cx32-/-) were compared with DEN-treated and untreated C57BL/6/129/Sv-F1 male and female controls, which corresponded to the genetic background of the Cx32-deficient mice.

Experimental design
A total of 284 animals were examined, 121 females (f) and 163 males (m). Of these, 132 (54 f, 67 m) were wildtype controls (Cx32 (+)) and 152 (67 f, 67 m) were Cx32-deficient mice (Cx32 (–)). A total of 110 animals (49 f, 61 m) were treated with DEN (DEN (+)), and 174 (72 f, 73 m) remained untreated (DEN (–)). In this way we obtained eight experimental groups, according to sex (m/f), genetic background (Cx32 (+)/(–)), and DEN-treatment (DEN (+)/(–)). Animals were killed in groups after 3, 6, 9, 12 or 18 months (Table Ia,bGoGo).


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Table Ia. Total number of different types of hepatocellular preneoplastic foci in standard sections of male Cx32-deficient and wildtype mice
 

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Table Ib. Number of different types of hepatocellular preneoplastic foci in standard sections of female Cx32-deficient and wildtype mice
 
Preparation of the livers and histological analysis
The middle lobe of the liver, fixed in PBS-buffered 3% formaldehyde, was cut along its largest diameter, thus obtaining the largest possible area. Samples were dehydrated in graded series of ethanol, embedded in paraffin, and two sections of 3 µm thickness were cut. Sections were dried for 12 h, deparaffinized with xylol, hydrated in a graded series of ethanol and stained with hematoxylin and eosin (HE) as well as with the periodic acid Schiff (PAS) reaction, respectively. The livers of the 18-month-old mice were fixed in 5% formaldehyde and cut into sections of 1 mm thickness. In addition to the standard section of the middle lobe, all visible lesions >2 mm were embedded in paraffin.

Foci of altered hepatocytes were classified histologically into clear cell foci (CCF), mixed cell foci (MCF), basophilic cell foci (BCF), tigroid cell foci (TCF) and amphophilic cell foci (AmCF) (22,23). CCF showed enlarged cell bodies with an extensive glycogen-storage (PAS-positive). BCF were rich in ribosomes. Their basophilic cytoplasm were PAS-negative because of loss of glycogen, and the nuclear/cytoplasmic ratio was increased. TCF were diagnosed when the cells showed a basophilic striped cytoplasm, which was due to highly organized endoplasmic reticulum. Foci of altered hepatocytes (FAH) consisting of cells showing an increase in cytoplasmic basophilia and acidophilia, together with a noticeable homogeneity, are called AmCF. In addition, the nuclei of these altered hepatocytes were enlarged. FAHs which are composed of glycogen-rich and glycogen-poor altered hepatocytes in a close spatial relationship were designated as MCF (2224).

Hepatocellular adenomas (HCA) were diagnosed when the lesions were sharply limited and compressed the surrounding liver parenchyma. Hepatocellular carcinomas (HCC) were diagnosed when the lesion exhibited trabeculae thicker than three cell layers in at least two separate areas and showed a higher number of mitotic figures (2325).

The size of the liver sections was determined using a square point lattice system, each point representing 1 mm2. The morphometrical analysis of preneoplastic lesions and neoplasms was done at a magnification of x100. The volume density of altered hepatocellular foci and tumors was determined by the point-counting stereological technique with an ocular grid described by Weibel (26). At least 600 points were counted per section.

Statistical analysis
We investigated the experimental groups for possible differences in the total number and volume fraction by statistical analysis with Wilcoxon–Mann–Whitney test. The chi-square test was applied to determine the relative amount of animals bearing at least one neoplasm. P < 0.05 was considered statistically significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Morphology
Regarding the extrafocal liver parenchyma, the experimental groups did not differ in their morphology, particularly regarding the architecture of the liver acini, shape and size of portal tracts, number and morphology of bile ducts, shape and width of hepatocyte trabeculae and sinusoids. Hepatocytic nuclei were typically shaped and showed no hyperchromasia or polymorphism; the chromatin pattern was regular. The cytoplasm contained a normal amount of glycogen and no increased basophilia. The number, distribution, shape and size of the nonparenchymal cells as well as the amount and composition of the extracellular matrix appeared normal. No inflammatory reaction was found in the extrafocal liver tissue.

Different types of FAH emerged in all experimental groups (Table Ia,bGoGo, Figure 1a–dGo). Male DEN-treated Cx32-deficient mice were the first to show preneoplastic lesions. They were already visible at an age of 3 months and could be assigned to several types of foci (Table IaGo). At an age of 6 and particularly 9 months, however, nearly all visible foci were of the mixed-cell type. This predominant existence of MCF was seen in the DEN (+)-groups, particularly with increasing age. The Cx32-deficient and wildtype animals did not differ in the morphology of preneoplastic lesions.



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Fig. 1. Clear cell foci (a) were among the rare foci which emerged in DEN (–) wildtype and Cx32-deficient liver. The clear cell focus in (a) was found in a male DEN (–) Cx32-deficient animal at 12 months (Table I). The surrounding extrafocal hepatocytes do not show morphological alterations. In (b–d), a liver section from a 9-month-old DEN (+) male Cx32-deficient mouse is shown. Multiple foci of altered hepatocytes are easily visible at low magnification ((b) PAS stain). Most of these foci are of the mixed cell type ((c and d) compare Table IGoGo). (c) and (d) represent the same mixed cell focus which fills a complete liver acinus. Staining with hematoxylin and eosin (c) shows the increase in basophilia, and the PAS stain (d) demonstrates differences in glycogen storage of the cells in this focus. While in the foci of altered hepatocytes the acinar architecture is conserved, the adenomas show a loss of acinar structure and an expansive growth, the latter is clearly visible in (e). Three additional FAH are shown in (e) (female Cx32-deficient, DEN (+), 12 months). The HCC shown in (f) (female, Cx32-deficient, DEN (+), 12 months) is representative for all HCCs seen of this study. The tumor is highly differentiated, exhibits a trabecular growth pattern, an increase in cytoplasmic basophilia, and mitotic figures (arrows). Magnifications: long (horizontal) edges of the photomicrographs represent in (a) and (f) 1.4 mm, in (b) 14.4 mm, in (c) and (d) 1.8 mm, and in (e) 11.5 mm.

 
The first HCA appeared simultaneously at an age of 6 months in male and female DEN-treated Cx32-deficient mice as well as in male DEN-treated wildtype animals (see Table IIGo). Female wildtype control animals did not exhibit the first spontaneous HCAs until they reached the age of 12 months. HCAs were sharply demarcated to the surrounding parenchyma (Figure 1eGo). They usually showed a trabecular pattern built by one to two layers of glycogen-rich hepatocytes.


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Table II. Total number of hepatocellular tumors in standard sections of Cx32-deficient and wildtype mice
 
All HCCs were well differentiated and larger than the HCAs. The trabeculae consisted mostly of three or more layers of enlarged hepatocytes with moderate cellular and nuclear pleomorphism (Figure 1fGo). Mitoses and apoptotic bodies were more frequently seen than in HCAs. The first HCC was found in male DEN-treated Cx32-deficient mice at an age of 9 months. All other HCCs did not occur until they reached the age of 12 months. Regarding the morphology of the neoplasms, there were no differences between the experimental groups.

No HCCs were visible in the middle lobes of the livers of 37 male and 41 female DEN (–) Cx32-deficient animals during the first 12 months of life. Two of 21 male DEN (–) Cx32-deficient animals exhibited a HCC when the entire livers were investigated.

Quantitative analysis
Cx32-deficient versus wildtype.
DEN (–): Preneoplastic lesions and neoplasms were rare and did not appear until 12 months of age (with one exception, Tables I–IIIGoGoGoGo). There was no statistically significant difference between Cx32-deficient and wildtype animals. Of 29 male DEN (–) mice (19 Cx32-deficient and 11 wildtype) at an age of 18 months, the Cx32-deficient animals showed four clear-cell foci and four amphophilic cell foci (mean number 0.3 ± 1.1; mean volume fraction: 0.1 ± 0.2%), whereas the wildtype animals did not show any FAH at all (Table IaGo). Six neoplasms (four HCAs, two HCCs) were found in the Cx32-deficient animals (mean number): 0.2 ± 0.4; mean volume fraction: 6.2 ± 12.9%), a single HCA occurred in the wildtype group (mean number: 0.1 ± 0.4; mean volume fraction: 3.5 ± 11.6%; Table IIGo). All differences between the Cx32-deficient and wildtype groups were not statistically significant.


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Table III. Mean number and volume fraction of hepatocellular preneoplastic foci and tumors in Cx32-deficient and wildtype mice
 
DEN (+): After DEN-treatment, the first preneoplastic lesions appeared after 6 months in wildtype animals and after 3 months in Cx32-deficient animals. In male Cx32-deficient mice, the number (volume fraction) of preneoplastic foci was increased 5.0-fold (5.9-fold) in the 6 months group, and 3.3-fold (4.6-fold) in the 9 months group; in female animals 4.8-fold (12.0-fold) in the 6 months group, 3.4-fold (8.2-fold) in the 9 months group and 7.6-fold (12.8-fold) in the 12 months group as compared with the wildtype groups (Table IIIGo). After 9 and 12 months, Cx32-deficient mice showed a higher number and volume fraction of neoplasms (mainly adenomas and few carcinomas) than their wildtype controls (Table IIIGo). In addition, the portion of female knockout mice developing neoplasms was significantly higher in the 9 months and 12 months group as compared with wildtype controls (4/6 versus 0/6 at an age of 9 months and 6/6 versus 2/6 at an age of 12 months, P < 0.05).

Thus, without DEN-treatment, Cx32-deficient male and female mice did not exhibit more spontaneous preneoplastic lesions or tumors than their wildtype controls. After DEN-treatment, however, the number and volume fraction of DEN-induced preneoplastic foci and neoplasms were significantly increased in the Cx 32-deficient groups.

DEN (+) versus DEN (–).
Without DEN-treatment, only 4.8% of the animals showed one to 6 spontaneous preneoplastic lesions per cm2 liver area. After DEN-treatment, however, 70% of the animals exhibited numerous (up to 37) preneoplastic lesions in the same area. The increase in volume fraction was similar (Table IIIGo).

Without DEN-treatment, only 1.4% of the mice (two females of the 12 month group) exhibited a total of three adenomas, whereas after DEN administration, 27% developed neoplasms, mainly adenomas. This increase in number was accompanied by a corresponding increase in volume fraction (Table IIIGo).

As expected, DEN-treatment caused a highly significant increase in the number and volume fraction of preneoplastic lesions and neoplasms in all experimental groups (Table IIIGo).

Males versus females.
As we noticed only a very limited number of spontaneous preneoplastic lesions and neoplasms in general, there was no statistical significant difference between male and female animals in the non-DEN-treated groups. On the other hand, after DEN-treatment, male animals developed a higher number and larger preneoplastic foci and neoplasms than did females in nearly all experimental groups (Table IIIGo).

Time
In animals exhibiting preneoplastic lesions and neoplasms, we noticed a continuous increase in number and volume fraction in almost all groups (Table IIIGo).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
To the best of our knowledge, we are the first to histomorphologically analyze the phenotype of Cx32-deficient mice liver. The loss of Cx32 containing gap junctions did not cause any congenital architectural disorder and, in contrast to other transgenic mouse models, caused no secondary alterations of the extrafocal tissue, e.g. acute or chronic hepatitis (27,28). The phenotypes of preneoplastic lesions were similar to those seen in many other animal models of hepatocarcinogenesis (22,29). However, since MCF was the predominant lesion phenotype occurring even at younger age, a particular progression sequence from one lesion phenotype to another was not found.

As expected, the Cx32-deficient and wildtype animals exhibited a higher number of preneoplastic lesions and tumors than did their non-treated counterparts, because DEN is a well-known carcinogen that is used in many animal experimental protocols to induce tumor development in rodent liver (17,19,20,30).

The higher number and volume fraction of FAH and neoplasms in male animals is in line with previous observations. These studies revealed that after DEN-administration, female mice of this strain developed fewer tumors than did male mice, because sex hormones exert strain-dependent effects on hepatocarcinogenesis (20,31,32). As expected, there was only a very small number of spontaneous preneoplastic lesions and tumors in wildtype animals, because the mouse strain used in this study is not very susceptible to hepatocarcinogenesis (30,33).

The loss of Cx32 protein did not lead to an increase in the number or volume fraction of spontaneous preneoplastic lesions or tumors during 18 months' observation. This is in contrast to a previous report according to which male and female Cx32-deficient mice developed a considerably higher number of spontaneous liver tumors than did wildtype mice in a 12 months' observation period (20). This difference may be explained by the use of different experimental evaluation methods. In the previous study, visible lesions were counted only on the liver surface and considered as neoplasms, whereas in this study, different types of FAH and neoplasms were classified histomorphologically.

Although Cx32 deficiency alone had no effect on hepatocarcinogenesis in up to 18-month-old mice, Cx32-deficient mice were more susceptible to tumor development than wildtype controls after treatment with DEN. It has previously been demonstrated that Cx32-deficient animals showed a higher proliferation rate of hepatocytes than did the wild type animals, an observation that may be attributed to impaired GJIC (20). Increased proliferation is believed to be a tumor-promoting condition in several models of carcinogenesis (34,35). Therefore, the fact that there was a higher number of tumors in Cx32-deficient animals after DEN administration may at least partially be attributed to increased proliferation of hepatocytes. Nevertheless, it still remains unclear whether increased proliferation per se has tumor-promoting effects. Ledda-Columbano and coworkers have recently shown that feeding of high doses of the strong direct hepatocellular mitogen triiodothyronine (T3) resulted in a significant decrease in hepatocellular preneoplasias and neoplasias in the Solt/Farber model (36,37). The impaired GJIC in the hepatocytes of the Cx32-deficient mice, in addition to increased proliferation, probably exerts additional influences (e.g. metabolic alterations) on DEN-damaged cells. Further studies are necessary to reveal those additional factors that might have a progressive effect of hepatocarcinogenesis in Cx32-deficient mice.


    Notes
 
3 To whom correspondence should be addressed at: Institut für Pathologie der Otto-von-Guericke-Universität Magdeburg, Leipziger Str. 44, D-39120 Magdeburg, Germany Email: Frank.Dombrowski{at}medizin.uni-magdeburg.de Back


    Acknowledgments
 
The authors wish to thank Mrs Inge Heim for her excellent technical assistance. This work has been supported by the Deutsche Forschungsgemeinschaft (DO 622/1-4 to F.D., and SFB 284 project C6 to K.W.) as well as the Deutsche Krebshilfe (to K.W.).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received November 5, 2001; accepted January 28, 2002.