Selective poisoning of Ctnnb1-mutated hepatoma cells in mouse liver tumors by a single application of acetaminophen

Mouse liver tumors that harbor activating mutations in Ctnnb1, encoding β-catenin, express high levels of various cytochromes P450 (CYP), including CYP2E1 and CYP1A2. Acetaminophen (AAP) is metabolized in hepatocytes by these CYPs to the reactive intermediate N-acetyl-p-benzoquinone-imine, which is toxic to hepatocytes at high doses where depletion of glutathione occurs. We have induced liver tumors in mice by treatment with the liver carcinogen N-nitrosodiethylamine followed by chronic treatment with the tumor promoter phenobarbital. Under this protocol, ~80 % of tumors display Ctnnb1 mutations and express high levels of various CYP isoforms. Tumor-bearing animals were given a single intraperitoneal injection of 300 mg/kg body weight AAP, which was well tolerated by the animals. This dose, however, eradicated essentially all larger Ctnnb1-mutated, CYP-positive liver tumors, killing >90 % of hepatoma cells: at 2 days after AAP, large necrotic areas filled with cell debris were observed in tumors. These were infiltrated in the following days by immune cells starting from the outer parts of the damaged areas slowly closing the “wounds” left from the poisoned tumor cells. During this period, there was increased proliferation of normal hepatocytes, but some residual tumor cells were also proliferating. Our results show that a selective poisoning of Ctnnb1-mutated liver tumors by administration of AAP is possible in this experimental system. Application of an adapted protocol could be envisaged for neo-adjuvant treatment of human hepatoblastoma which are frequently mutated in CTNNB1 and often show high expression of CYP2E1.     

Evidence for Cytochrome P450 2E1–Mediated Toxicity of N-Nitrosodimethylamine in Cultured Perivenous Hepatocytes from Ethanol Treated Rats

Abstract: The involvement of cytochrome P450 in the liver toxicity of the potent carcinogen, N-nitrosodimethylamine (NDMA) was investigated in hepatocytes isolated from the periportal or perivenous region by digitonin-collagenase perfusion. Exposure of hepatocytes in culture to NDMA (0.5 or 5 mM) for up to 18 hrs caused little damage, but after 42 hr loss of cell viability became evident, and the extent of cell death was higher in perivenous cells than in periportal cells. Pretreatment of rats with ethanol caused a dramatically enhanced cell damage in perivenous cells (80%) compared to periportal cells (45%). This ethanol pretreatment caused a several-fold induction of cytochrome P450 2E1, as determined both with Western blot and as NDMA demethylase activity, and the effect was observed almost exclusively in perivenous cells. Isoniazid, an inhibitor of cytochrome P450 2E1, completely protected against NDMA toxicity. Glutathione dependent cytoprotective mechanisms and lipid peroxidation did not appear to be critical in NDMA toxicity, as evidence by lack of potentiation of toxicity by buthionine sulfoximine, an inhibitor of glutathione synthesis, and by the absence of increased lipid peroxidation. Instead, the higher expression of cytochrome P450 2E1 in the perivenous cells seems to be the main determinant for the regiospecific toxicity of NDMA, and, consequently, probably also for the associated genotoxicity.     

Zonal differences in DNA synthesis and in transglutaminase activity between perivenous versus periportal regions of regenerating rat liver

We investigated a relationship within zonal differences in DNA synthesis and in transglutaminase (TGase) activity between perivenous versus periportal regions of regenerating rat liver. Using the digitonin/collagenase perfusion technique, hepatocyte subpopulations were isolated from each region at various time points after partial hepatectomy. The amounts of DNA synthesis as well as the levels of TGase mRNA and activity in each subpopulation were measured. Although increased DNA synthesis was observed in both subpopulations with a peak at 24 h after partial hepatectomy, the amount of DNA synthesis in periportal hepatocytes (PPH) was twice as much as that in perivenous hepatocytes (PVH). In PVH, TGase activity peaked at 24 h after partial hepatectomy with a preceding increase in its mRNA expression at 12 h, whereas TGase activity in PPH at 24 h was one-half of that in PVH. As TGase is known to have a growth-arresting activity, our data indicate that relatively higher TGase activity in PVH at 24 h after partial hepatectomy might correlate with relatively lower DNA synthesis in this region compared to periportal region.     

Lack of zonal uptake of estrone sulfate in enriched periportal and perivenous isolated rat hepatocytes.

The zonal uptake of estrone sulfate (E1S; 1 to 400 microM) was investigated in periportal and perivenous rat hepatocytes and cells isolated from whole liver (regular hepatocytes). Transport of E1S by periportal, perivenous, and regular hepatocytes was described by saturable (Kms of 24 to 26 microM and Vmaxs of 1.8 nmol/min/mg protein) and nonsaturable components (2.5 to 3.2 microl/min/mg protein) that were not different among the zonal regions (p >.05, ANOVA). These kinetic constants represented pooled values for the entire complement of transporters for E1S, including two known transporters of E1S: Ntcp, Na+-taurocholate cotransporting polypeptide, and oatp1, the organic anion transporting polypeptide cloned from rat liver. Uptake of E1S was significantly reduced by estradiol 17beta-glucuronide (50 microM) and bumetanide (200 microM), and was inhibited strongly and competitively by pregnenolone sulfate with an inhibition constant of 6.7 microM. Further segregation of the kinetic constants as the sodium-dependent and -independent systems was achieved through simultaneous fitting of data obtained in the presence and absence of sodium from parallel hepatocytic uptake studies. For the periportal, perivenous, and regular hepatocytes, two saturable systems: a sodium-dependent transport system, characterized by similar Vmaxs (1.1 to 1.4 nmol/min/mg protein) and Kms (49 to 55 microM), a sodium-independent transport system of comparable Vmaxs (0.70 to 0.84 nmol/min/mg protein) and Kms (16 to 22 microM), and a linear clearance of 1.7 to 2.7 microl/min/mg protein (ANOVA, p >.05) were obtained. The data suggest that hepatic uptake of E1S involved sodium-dependent and -independent transporter systems. No heterogeneity in transport was observed.     

Uptake of enalapril and expression of organic anion transporting polypeptide 1 in zonal, isolated rat hepatocytes.

Sinusoidal entry is the first obligatory process preceding intracellular drug removal in liver. Transport of the angiotensin converting enzyme inhibitor enalapril (1-750 microM with [(3)H]enalapril), a substrate of Oatp1, the sodium-independent organic anion transporting polypeptide 1 cloned from rat liver, was studied in rat hepatocytes isolated from all zones of the liver (homogeneous) and from enriched periportal (PP) and perivenous (PV) hepatocytes prepared by collagenase perfusion and zone-selective destruction with digitonin, respectively. Uptake was linear over 1 min and was concentration-dependent. Transport by the homogeneous hepatocytes (in the presence and absence of Na(+)) and PP and PV cells was described by single saturable components of similar kinetic constants (K(m) values of 344-461 microM and V(max) values of 9.5-11.6 nmol/min/10(6) cells; P >.05, ANOVA). The K(m) value for enalapril uptake in hepatocytes was of the same order of magnitude compared with that for Oatp1 expressed in HeLa cells transfected with cDNA-Oatp1 and Western blot analysis revealed similar levels of immunoreactive Oatp1 expression in PP and PV hepatocytes. However, enalapril was not taken up by Oatp2 nor by the human OATP expressed in recombinant vaccinia systems.     

Distribution of Glutathione S-Transferase Isoforms in Rat Liver after Induction by β-Naphthoflavone or 3-Methylcholanthrene

Abstract: Regional differences in vulnerability to xenobiotic liver damage may relate to the distribution of the detoxication capacity of the glutathione S-transferases (GST). HPLC analysis of cell lysates obtained by digitonin infusion from either the periportal or the perivenous region revealed that the content of all the GST subunits investigated (1, 2, 3, 4 and 8) was higher in the perivenous region. The strongest perivenous dominance was observed for subunit 1 (Ya) and the α class appeared to be more zonated that the μ class. A similar perivenous dominance was observed by analysis of GST activity with either 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloronitrobenzene (DCNB) or trans-4-phenyl-3-buten-2-one (PBO) as substrate. In contrast, with cumene hydroperoxide (CuOOH) or tert-butyl hydroperoxide (tBOOH) as substarate a reciprocal twofold periportal dominance was observed. Induction by pretreatment with β-naphthoflavone reduced or abolished the perivenous dominance of the α subunits 1, 2 and 8. In contrast, after pretreatment with 3-methylcholan-threne, only the acinar gradient of subunits 2 (Yc) was abolished, while the strong perivenous gradient subunit 1 (Ya) was maintained and that of subunit 8 (Yk) increased. CDNB based assays demonstrated that β-naphtoflavone treatment reduced (from 2.1 to 1.4) while 3-methyl cholanthrene enhanced (to 2.6) the perivenous/periportal GST activity ratio. Assays based on CuOOH or tBOOH indicated that neither the Se-dependent nor the Se-independent glutathione peroxidase activity nor its acinar distribution was affected by the inducers. These results demonstrated that although the expression of all investigated members of the alpha and mu classes is higher in the perivenous region, there are marked isozyme differences, the acinar gradient being particularly prominent for subunit 1 (Ya). The distinct difference in the acinar induction pattern of GST Ya between β-naphthoflavone and 3-methylcholanthrene resembles that reported for cytochrome P450 (CYP1A1 and CYP1A2), also members of the aryl hydrocarbon (Ah) receptor genes, suggesting common regionally acting regulatory elements in the expression of these genes in the liver.     

基于生物信息学筛选乙型肝炎差异表达基因及其在预测肝癌预后中的作用

目的:应用生物数据库及生物信息分析技术探索乙型肝炎病毒(HBV)感染肝细胞的差异表达基因,分析其在肝癌预后中的预测价值。方法:登录基因芯片公共数据库(GEO)下载数据并检测基因芯片品质,应用R软件甄选差异基因,富集分析对差异基因进行分类注释,构建蛋白质相互作用网络筛选核心基因,运用基因表达谱数据动态分析(GEPIA)验证核心差异表达基因,分析5年生存率。结果:合计1041个基因在乙型肝炎病毒感染肝细胞中存在表达差异,基因本体(GO)富集分析显示差异基因主要与细胞外间隙、胞外区、胞外外泌体、环氧化酶P450通路、受体结合相关。京都基因和基因组百科全书(KEGG)通路分析显示差异基因主要参与补体及凝血级联反应、糖酵解/糖异生、视黄醇代谢、过氧化物酶体增殖物激活受体(PPAR)信号通路、碳代谢、代谢途径、初级胆汁酸生物合成、癌症中的蛋白聚糖、胆汁分泌等信号通路。蛋白互作网络筛选出10个关键基因。GEPIA数据库验证结果显示其6个基因高表达于肝癌组织,而生存分析验证激肽原基因1(KNG1)高表达组肝癌患者5年生存率更高。结论:生物信息学技术能有效筛选HBV感染肝细胞中的核心差异基因,并具有预测肝癌预后的作用。     

Species differences in the induction of hepatocellular DNA synthesis by diethanolamine.

Diethanolamine increased the incidence and multiplicity of liver tumors in the mouse following chronic exposure. Diethanolamine is known to inhibit cellular choline uptake. Since choline deficiency produces tumors in rodents, diethanolamine, through choline depletion, may result in tumor development in rodents. The potential for diethanolamine to function through this mode of action in humans is not known. The present studies examined the effect of diethanolamine (0-500 mug/ml) and choline depletion on DNA synthesis and changes in expression of genes involved in cell growth pathways in primary cultures of mouse, rat, and human hepatocytes. In mouse and rat hepatocytes DNA synthesis was increased following treatment with 10 mug/ml diethanolamine and higher (3- to 4-fold over control). In contrast, diethanolamine failed to increase DNA synthesis in human hepatocytes. Incubation of hepatocytes in medium containing reduced choline (1/10 to 1/100 of normal medium; 0.898 to 0.0898 mg/l vs. 8.98 mg/l) increased DNA synthesis (1.6- and 1.8-fold of control in mouse and rat hepatocytes, respectively); however, choline depletion did not induce DNA synthesis in human hepatocytes. Mouse and rat hepatocytes incubated in medium supplemented with 2- to 50-fold excess choline reduced diethanolamine-induced DNA synthesis to control levels or below. Gene expression analysis of mouse and rat hepatocytes following diethanolamine treatment showed increases in genes associated with cell growth and decreases in expression of genes involved in apoptotic pathways. These results support the hypothesis that choline depletion is central to the mode of action for the induction of rodent hepatic neoplasia by diethanolamine. Furthermore, since diethanolamine treatment or choline depletion failed to induce DNA synthesis in human hepatocytes, these results suggest that humans may not be at risk from the carcinogenic effects of diethanolamine.     

Regulation of expression of drug-metabolizing enzymes by oncogenic signaling pathways in liver tumors: a review

Mutations in genes encoding key players in oncogenic signaling pathways trigger specific downstream gene expression profiles in the respective tumor cell populations. While regulation of genes related to cell growth, survival, and death has been extensively studied, much less is known on the regulation of drug-metabolizing enzymes (DMEs) by oncogenic signaling. Here, a comprehensive review of the available literature is presented summarizing the impact of the most relevant genetic alterations in human and rodent liver tumors on the expression of DMEs with a focus on phases I and II of xenobiotic metabolism. Comparably few data are available with respect to DME regulation by p53-dependent signaling, telomerase expression or altered chromatin remodeling. By contrast, DME regulation by constitutive activation of oncogenic signaling via the RAS/RAF/mitogen-activated protein kinase (MAPK) cascade or via the canonical WNT/β-catenin signaling pathway has been analyzed in greater depth, demonstrating mostly positive-regulatory effects of WNT/β-catenin signaling and negative-regulatory effects of MAPK signaling. Mechanistic studies have revealed molecular interactions between oncogenic signaling and nuclear xeno-sensing receptors which underlie the observed alterations in DME expression in liver tumors. Observations of altered DME expression and inducibility in liver tumors with a specific gene expression profile may impact pharmacological treatment options.     

Comparison of bidirectional collagenase perfusion with one way perfusion in the isolation of periportal or perivenous hepatocytes in rats]

To selectively isolate hepatocytes from the periportal (PP) and perivenous (PV) regions of rat liver acinus, we compared two different perfusion methods with collagenase: the bidirectional perfusion method (2-P) and the one way perfusion method (1-P). We determined the optimal conditions for each method on the basis of the zonal selectivity of isolated hepatocytes with a hematoxylin-eosin stained liver specimen. By both methods, hepatocytes were selectively isolated from the PP and PV regions. Comparing cell yield and cell viability after the two perfusion methods, 1-P was found to be better than 2-P. Density gradient centrifugation with Percoll was found to be an effective procedure for removing the damaged hepatocytes. We concluded that 1-P could isolate viable PP and PV hepatocytes with a normal glucagon-cyclic AMP response and ultrafine structure in high yield from rat liver.     

Effects of clofibric acid on mRNA expression profiles in primary cultures of rat,mouse and human hepatocytes

The mRNA expression profile in control and clofibric acid (CLO)-treated mouse, rat, and human hepatocytes was analyzed using species-specific oligonucleotide DNA microarrays (Affymetrix). A statistical empirical Bayes procedure was applied in order to select the significantly differentially expressed genes. Treatment with the peroxisome proliferator CLO induced up-regulation of genes involved in peroxisome proliferation and in cell proliferation as well as down-regulation of genes involved in apoptosis in hepatocytes of rodent but not of human origin. CLO treatment induced up-regulation of microsomal cytochrome P450 4a genes in rodent hepatocytes and in two of six human hepatocyte cultures. In addition, genes encoding phenobarbital-inducible cytochrome P450s were also up-regulated by CLO in rodent and human hepatocyte cultures. Up-regulation of phenobarbital-inducible UDP-glucuronosyl-transferase genes by CLO was observed in both rat and human but not in mouse hepatocytes. CLO treatment induced up-regulation of L-fatty acid binding protein (L-FABP) gene in hepatocytes of both rodent and human origin. However, while genes of the cytosolic, microsomal, and mitochondrial pathways involved in fatty acid transport and metabolism were up-regulated by CLO in both rodent and human hepatocyte cultures, genes of the peroxisomal pathway of lipid metabolism were up-regulated in rodents only. An up-regulation of hepatocyte nuclear factor 1alpha (HNF1alpha) by CLO was observed only in human hepatocyte cultures, suggesting that this trans-activating factor may play a key role in the regulation of fatty acid metabolism in human liver as well as in the nonresponsiveness of human liver to CLO-induced regulation of cell proliferation and apoptosis.     

Properties of the mitochondrial membrane and carnitine palmitoyltransferase in the periportal and the perivenous zone of the liver. Effects of chronic ethanol feeding

Rats were fed for 35 days a high-fat diet containing either 36% of total calories as ethanol (ethanol group) or an isocaloric amount of carbohydrate (control group). Then, mitochondria were isolated from the periportal and the perivenous zone of the liver in order to study the acinar heterogeneity of the effects of prolonged ethanol administration upon the properties of carnitine palmitoyltransferase I (CPT-I) and its membrane environment. Chronic ethanol ingestion selectively decreased CPT-I activity in periportal hepatocytes but equally increased enzyme sensitivity to malonyl-CoA and enzyme energy of activation in the two zones of the liver. In control animals, mitochondrial membrane showed higher fluidity and lower degree of saturation of phospholipid fatty acyl moieties in periportal than in perivenous hepatocytes. Prolonged ethanol feeding (i) decreased mitochondrial membrane fluidity; (ii) increased the proportion of palmitic acid and decreased that of arachidonic acid in mitochondrial phosphatidylcholine and phosphatidylethanolamine, whereas it drastically reduced the content of linoleic acid and concomitantly increased that of saturated and monoenoic fatty acids in cardiolipin; (iii) suppressed the disordering effects of the addition of ethanol to mitochondrial suspensions. All these ethanol-induced alterations of membrane fluidity and fatty acyl composition were not significantly different between periportal and perivenous mitochondria. In conclusion, chronic ethanol feeding changes the activity of CPT-I in a zone-selective manner but modifies both the regulatory properties of the enzyme and the properties of its lipid environment in a non-zone-selective manner. Hence factors in addition to the properties of the mitochondrial membrane seem to be involved in the ethanol-induced alterations of the CPT-I enzyme.     

Acinar distribution of rat liver arylamine N-acetyltransferase: effect of chronic ethanol and endotoxin exposure

The expression of most drug-metabolising enzymes is highest in the perivenous region of the liver, where drug-induced damage is commonly initiated. Arylamine N-acetyltransferase plays an important role in activation or detoxification of many drugs, carcinogens, pesticides and other xenobiotics, but its acinar distribution is unknown. In this study we have analysed the activity of N-acetyltransferase in cell lysates obtained from the periportal or perivenous region by digitonin treatment during in situ liver perfusion. Livers from control animals were compared with rats chronically exposed either to ethanol by liquid diet or to lipopolysaccharide (endotoxin) by intravenous administration. The activity of N-acetyltransferase in the perivenous region was slightly (+ 20%) higher than in the periportal region. Although chronic ethanol exposure did not change total activity, the acinar distribution was reversed to a higher activity in the periportal region. In contrast, chronic endotoxin significantly increased N-acetyltransferase activity, but did not affect the acinar distribution. This increase was counteracted by simultaneous ethanol treatment. N-Acetyltransferase activity in perivenous lysates was significantly reduced after the co-administration of ethanol and endotoxin compared to that after endotoxin alone. Thus, the perivenous zonation of liver N-acetyltransferase is moderate compared to other transferases or P450 isozymes, and the cellular capacity for N-acetylation in the perivenous region, where xenobiotic activation to reactive intermediates dominates, may be insufficient.     

Acinar heterogeneity in hepatic transport of dibromosulfophthalein and ouabain studied by autoradiography, normal and retrograde perfusions and computer simulation

This study is aimed to investigate the relative involvement of periportal (zone 1) and perivenous (zone 3) hepatocytes in the uptake and biliary excretion of the organic anion dibromosulfophthalein (DBSP) and the uncharged cardiac-glycoside ouabain. The localization in the acinus of [35S]BSP (sulfobromophthalein, the tetra-bromo-analogue of DBSP) and [3H]ouabain administered to livers perfused with normal and retrograde flow, was detected by autoradiography. The plasma disappearance and biliary excretion rates of DBSP and [3H]ouabain were determined in normal and retrograde perfusions. In addition, computer simulations were performed to predict the effect of reversal of the perfusate flow on the plasma disappearance and biliary excretion rate curves and on the concentration of label in zones 1 and 3. Autoradiography showed that 2 and 10 min after injection of [35S]BSP to normally and retrogradely perfused livers, the label was uniformly distributed in the liver acinus. The same results were found 30 sec and 10 min after injection of [3H]ouabain to normally and retrogradely perfused livers. The plasma disappearance and biliary excretion rate of DBSP were slightly faster in retrograde perfusions compared to normal perfusions both with and without a basal bile salt infusion of 15 mumole/hr. This could not be explained by an acinar heterogeneity with respect to any of the DBSP transport steps (plasma to liver, liver to plasma, liver to bile) as was shown by computer simulations. The plasma disappearance and biliary excretion rate of ouabain were similar in normal and retrograde perfusions. It is concluded that periportal and perivenous hepatocytes are equally involved in the uptake of (D)BSP and ouabain from the medium. However, due to the particular distribution patterns no conclusions can be drawn from normal and retrograde perfusions about the relative involvement of these cells in biliary excretion, as was shown by computer simulation. The unaffected kinetic behaviour of the retrogradely perfused livers indicated that no liver damage occurs during retrograde perfusion with respect to transport function.     

Acinar Distribution of Rat Liver Arylamine N‐Acetyltransferase: Effect of Chronic Ethanol and Endotoxin Exposure

Abstract: The expression of most drug‐metabolising enzymes is highest in the perivenous region of the liver, where drug‐induced damage is commonly initiated. Arylamine N‐acetyltransferase plays an important role in activation or detoxification of many drugs, carcinogens, pesticides and other xenobiotics, but its acinar distribution is unknown. In this study we have analysed the activity of N‐acetyltransferase in cell lysates obtained from the periportal or perivenous region by digitonin treatment during in situ liver perfusion. Livers from control animals were compared with rats chronically exposed either to ethanol by liquid diet or to lipopolysaccharide (endotoxin) by intravenous administration. The activity of N‐acetyltransferase in the perivenous region was slightly (+ 20%) higher than in the periportal region. Although chronic ethanol exposure did not change total activity, the acinar distribution was reversed to a higher activity in the periportal region. In contrast, chronic endotoxin significantly increased N‐acetyltransferase activity, but did not affect the acinar distribution. This increase was counteracted by simultaneous ethanol treatment. N‐Acetyltransferase activity in perivenous lysates was significantly reduced after the co‐administration of ethanol and endotoxin compared to that after endotoxin alone. Thus, the perivenous zonation of liver N‐acetyltransferase is moderate compared to other transferases or P450 isozymes, and the cellular capacity for N‐acetylation in the perivenous region, where xenobiotic activation to reactive intermediates dominates, may be insufficient.