Hepatocytes play a pivotal role in both the synthesis and degradation of numerous endogenous biomolecules, thus maintaining metabolic homeostasis, as well as in the conversion and detoxification of xenobiotic compounds. Based on the location of the blood vessels, the terminal branches of the portal and the hepatic (central) veins and on the direction of the blood flow, hepatocytes of each liver lobule can be divided into two subpopulations, an upstream 'periportal' and a downstream 'perivenous' (pericentral) population. Zonal-specific differences in the metabolic capacities of many enzymes or other proteins, and -to a lesser extent ) of their corresponding messenger RNAs, have been subject to extensive studies throughout the last decades.Many enzymes of intermediary metabolism are not distributed uniformly throughout the liver, but are preferentially expressed in either the periportal or the perivenous hepatocyte subpopulation [1][2][3]. Hence, hepatocytes located in either of the two regions have different, often complementary, functions. Whereas, for example, glycolysis is exclusively active in perivenous hepatocytes, key enzymes of gluconeogenesis, the antagonist pathway, are preferentially expressed in periportal hepatocytes [1]. Zonal-specific expression has also been established for enzymes of amino acid and ammonia metabolism, showing, for example, a higher activity of the urea cycle in periportal cells compared to perivenous hepatocytes [3], whereas glutamine synthesis is exclusively active in the perivenous Hepatocytes located in the periportal and perivenous zones of the liver lobule show remarkable differences in the levels and activities of various enzymes and other proteins. To analyze global gene expression patterns of periportal and perivenous hepatocytes, enriched populations of the two cell types were isolated by combined collagenase ⁄ digitonin perfusion from mouse liver and used for microarray analysis. In total, 198 genes and expressed sequences were identified that demonstrated a ‡ 2-fold difference in expression between hepatocytes from the two different zones of the liver. A subset of 20 genes was additionally analyzed by real-time RT-PCR, validating the results obtained by the microarray analysis. Several of the differentially expressed genes encoded key enzymes of intermediary metabolism, including those involved in glycolysis and gluconeogenesis, fatty acid degradation, cholesterol and bile acid metabolism, amino acid degradation and ammonia utilization. In addition, several enzymes of phase I and phase II of xenobiotic metabolism were differentially expressed in periportal and perivenous hepatocytes. Our results confirm previous findings on metabolic zonation in liver, and extend our knowledge of the regulatory mechanisms at the transcriptional level.Abbreviations GS, glutamine synthetase.

Connexins are subunits of gap junction channels, which mediate the direct transfer of ions, second messenger molecules and other metabolites between contacting cells. Gap junctions are thought to be involved in tissue homeostasis, embryonic development and the control of cell proliferation [1,2]. It has also been suggested that the loss of intercellular communication via gap junctions may contribute to multistage carcinogenesis [3-5]. We have previously shown that transgenic mice that lack connexin32 (Cx32), the major gap junction protein expressed in hepatocytes, express lower levels of a second hepatic gap junction protein, Cx26, suggesting that Cx32 has a stabilizing effect on Cx26 [6]. Here, we report that male and female one-year-old mice deficient for Cx32 had 25-fold more and 8-fold more spontaneous liver tumors than wild-type mice, respectively. Incorporation of bromodeoxyuridine (BrdU) into the liver was higher for Cx32-deficient mice than for wild-type mice, suggesting that their hepatocyte proliferation rate was higher. Furthermore, intraperitoneal injection, two weeks after birth, of the carcinogen diethylnitrosamine (DEN) led, after one year, both to more liver tumors in Cx32-deficient mice than in controls, and to accelerated tumor growth. Loss of Cx32 protein from hepatic gap junctions is therefore likely to cause enhanced clonal survival and expansion of mutated ('initiated') cells, which results in a higher susceptibility to hepatic tumors. Our results demonstrate that functional gap junctions inhibit the development of spontaneous and chemically induced tumors in mouse liver.

Gene expression in hepatocytes within the liver lobule is differentially regulated along the portal to central axis; however, the mechanisms governing the processes of zonation within the lobule are unknown. A model for zonal heterogeneity in normal liver is proposed, based on observations of differential expression of genes in liver tumors from mice that harbor activating mutations in either Catnb (which codes for ␤-catenin) or Ha-ras. According to the model, the regulatory control consists of two opposing signals, one delivered by endothelial cells of the central veins activating a ␤-catenin-dependent pathway (retrograde signal), the other by blood-borne molecules activating Ras-dependent downstream cascades (anterograde signal). In conclusion, gradients of opposing signaling molecules along the portocentral axis determine the pattern of enzymes and other proteins expressed in hepatocytes of the periportal and pericentral domains of the liver lobule. (HEPATOLOGY 2006;43:407-414.) E ven though the architecture of the liver tissue appears rather uniform, a closer view shows the existence of repetitive small structural and functional units called lobules that compose the liver parenchyma. Hepatocytes within the liver lobule differ in their enzyme content and subcellular structure according to their location relative to the afferent and efferent blood vessels, the terminal branches of the portal and the hepatic (central) veins, respectively (for reviews see 1,2 ). Based on the location of the blood vessels and the direction of the blood flow, the individual liver lobule can therefore be subdivided into an upstream "periportal" and a downstream "perivenous" (pericentral) region. Hepatocytes located in either of the two regions have different, often complementary functions, as indicated by differences in the content and activity of key enzymes of the intermediary and xenobiotic metabolism.Two types of zonal patterns of gene expression have been described: one group of genes is stably expressed (non-inducible) within only one or a few layers of hepatocytes of the liver lobule, either pericentral or periportal, whereas a second group of genes displays a more dynamic (inducible) expression that may gradually diminish along the axis of the lobule. 3,4 One of the best-known examples for an enzyme encoded by a member of the first group of genes is glutamine synthetase (GS), which plays a key role in ammonia metabolism. GS is stably expressed at very high levels within only one to two layers of hepatocytes surrounding the central veins, and the number of hepatocytes expressing the enzyme is hardly affected by external stimuli under physiological conditions. 5 Other enzymes, exemplified by key enzymes of carbohydrate metabolism, are also zonally expressed, but in a more gradual pattern. In addition, these latter enzymes undergo dynamic changes in expression as an adaptive response to changes in the hormonal or nutritional status. Xenobiotic metabolizing enzymes, including various cytochromes P450 (CYPs), represent an int...

Tumor promoters are non-mutagenic chemicals which increase the probability of cancer by accelerating the clonal expansion of cells transformed during tumor initiation. Phenobarbital (PB) is an antiepileptic drug which promotes hepatocarcinogenesis in rodents when administered subsequent to an initiating carcinogen like diethylnitrosamine (DEN). Here we have investigated the prevalence and patterns of mutations in two genes, Haras and b-catenin, both known mutational targets in mouse hepatocarcinogenesis. Liver tumors were generated by a single administration of DEN to 6 week old mice followed by feeding of PB (0.05%) containing or control diet for 39 weeks. Mutations at Ha-ras codon 61 were screened by allele-speci®c oligonucleotide hybridization; b-catenin mutations were detected by direct sequencing of PCR products spanning exon 2. In tumors from mice treated with DEN alone, the prevalence of Ha-ras mutations was *30% (6/20), while no b-catenin mutations (0/13) were detectable in tumors of this treatment group. By contrast, Ha-ras mutations were undetectable in tumors from mice treated with DEN/PB (0/32), while *80% (37/46) of tumors from this group showed b-catenin mutations. These results demonstrate that PB strongly aects the prevalence of mutations in the two cancer-related genes, presumably by positive and negative selection for cells harboring the respective mutation. Oncogene (2001) 20, 7812 ± 7816

A number of mouse skin tumors initiated by the carcinogens N-methyl-N'-nitro-N-nitrsoguanidine (MNNG), methylnitrosourea (MNU), , and 7,12-dimethylbenz[alanthracene (DMBA) have been shown to contain activated Ha-ras genes. In each case, the point mutations responsible for activation have been characterized. Results presented demonstrate the carcinogen-specific nature of these ras mutations. For each initiating agent, a distinct spectrum of mutations is observed. Most importantly, the distribution of ras gene mutations is found to differ between benign papillomas and carcinomas, suggesting that molecular events occurring at the time of initiation influence the probability with which papillomas progress to malignancy. This study provides molecular evidence in support of the existence of subsets of papillomas with differing progression frequencies. Thus, the alkylating agents MNNG and MNU induced exclusively G -* A transitions at codon 12, with this mutation being found predominantly in papillomas. MCA initiation produced both codon 13 G -> T and codon 61 A --T transversions in papillomas; only the G -> T mutation, however, was found in carcinomas. These findings provide strong evidence that the mutational activation of Ha-ras occurs as a result of the initiation process and that the nature of the initiating event can affect the probability of progression to malignancy.The carcinogenic nature of many chemicals has long been associated with their ability to bind to DNA and to cause somatic mutations (1). These lesions, if they occur at certain critical locations in the genome, are thought to effect key functions in initiating or furthering neoplastic development. Increasing evidence suggests that the activation of cellular protooncogenes to their oncogenic forms plays a central role in the development of neoplasia and thus identifies protooncogenes as candidate targets for chemical carcinogens (2-4). The most frequently detected oncogenes, both in human tumors and in many animal tumor model systems, are members of the ras gene family-Ha-, Ki-, and N-ras (2-4). These oncogenes differ from their normal cellular counterparts by having acquired a single point mutation at codon 12, 13, 61, or 117 (2). Studies on several experimental model systems have identified, for a number of chemically induced tumors, the particular activating mutations involved (5-7). From these has developed a correlation between carcinogen and ras mutation, suggesting a causal relationship between carcinogen treatment, ras activation, and, in these tumor systems at least, the initiation of tumorigenesis.