Search

Recently, we have demonstrated that mice, cultured embryos in α-minimum essential medium (αMEM) and subsequent fed a high-fat, high-sugar diet, developed steatohepatitis. In this study, we investigated using these samples whether the expression of lipid droplet formation genes in the liver is higher in MEM mice, whether these expressions are regulated by histone acetylation, writers/readers of histone acetylation, and the transcriptional factors of endoplasmic reticulum stress. Mice were produced by two-cell embryos in αMEM or standard potassium simplex-optimized medium (control) in vitro for 48 h, and implanted into an oviduct for spontaneous delivery. MEM and control-mice were fed a high-fat, high-sugar diet for 18 wk, and then liver samples were collected and analyzed by histology, qRT-PCR, and chromatin immunoprecipitation assay. Gene expression of Cidea, Cidec, and Plin4 were higher in MEM mice and histone H3K9 acetylation, BRD4, and CBP were higher in MEM mice than in control mice around those genes. However, the binding of endoplasmic reticulum stress-related transcription factors (ATF4, CHOP and C/EBPα) around those genes in the liver, was not clearly differed between MEM mice and control mice. The increased expression of Cidea, Cidec and Plin4 in the liver, accompanied by the development of steatohepatitis in mice induced is positively associated with increased histone H3K9 acetylation and CBP and BRD4 binding around these genes.

Detailed herein is the protocol for synthesis, characterization, and application of POLYseq for cell pooling in single-cell sequencing runs. POLYseq is synthesized through commercially available reagents and is highly tailorable. Synthesis is easily performed in a two-step protocol, utilizing Michael addition only requiring a temperature-stable hot bath capable of holding 90°C. However, care must be taken when mixing reagents for synthesis, as the final product is sensitive to initial mixing ratios of POLYseq reagents. For complete details on the use and execution of this protocol, please refer to Dunn et al. (2021)., Competing Interests: A.D. and T.T. are listed as inventors for a POLY-seq related patent., (© 2021 The Author(s).)

Immune checkpoint inhibitors (ICIs) targeting programmed cell death 1 (PD-1) and cytotoxic T-lymphocyte antigen-4 (CTLA-4) are widely used to treat advanced metastatic cancers. Neutralisation of PD-1 or CTLA-4 by ICIs results in immune-related adverse events (irAEs). The clinicopathological features of twelve patients with hepatic irAEs were evaluated and compared to those of ten patients with autoimmune hepatitis (AIH) or graft-versus-host disease (GVHD). No significant difference was seen in serum levels of transaminases, whereas serum levels of IgG and anti-nuclear antibody were higher in patients with AIH than in those with GVHD or hepatic irAEs. Inflammation was limited to the liver lobes in patients with GVHD or hepatic irAEs, whereas patients with AIH exhibited both portal and lobular inflammation. Immunohistochemical analyses revealed a predominant infiltration of CD8 + T cells and defective accumulation of regulatory T cells (Tregs) expressing forkhead box p3 (FOXP3) in the lobular areas of patients with hepatic irAEs and GVHD. In contrast, periportal lesions of patients with AIH were characterised by an infiltration of CD4 + T cells, CD8 + T cells, CD20 + B cells, and FOXP3 + Tregs. Overall, the activation of CD8 + T cells in the absence of activation of Tregs potentially underlies the immunopathogenesis of hepatic irAEs.

Background & Aims: Preclinical identification of compounds at risk of causing drug induced liver injury (DILI) remains a significant challenge in drug development, highlighting a need for a predictive human system to study complicated DILI mechanism and susceptibility to individual drug. Here, we established a human liver organoid (HLO)-based screening model for analyzing DILI pathology at organoid resolution., Methods: We first developed a reproducible method to generate HLO from storable foregut progenitors from pluripotent stem cell (PSC) lines with reproducible bile transport function. The qRT-PCR and single cell RNA-seq determined hepatocyte transcriptomic state in cells of HLO relative to primary hepatocytes. Histological and ultrastructural analyses were performed to evaluate micro-anatomical architecture. HLO based drug-induced liver injury assays were transformed into a 384 well based high-speed live imaging platform., Results: HLO, generated from 10 different pluripotent stem cell lines, contain polarized immature hepatocytes with bile canaliculi-like architecture, establishing the unidirectional bile acid transport pathway. Single cell RNA-seq profiling identified diverse and zonal hepatocytic populations that in part emulate primary adult hepatocytes. The accumulation of fluorescent bile acid into organoid was impaired by CRISPR-Cas9-based gene editing and transporter inhibitor treatment with BSEP. Furthermore, we successfully developed an organoid based assay with multiplexed readouts measuring viability, cholestatic and/or mitochondrial toxicity with high predictive values for 238 marketed drugs at 4 different concentrations (Sensitivity: 88.7%, Specificity: 88.9%). LoT positively predicts genomic predisposition (CYP2C9∗2) for Bosentan-induced cholestasis., Conclusions: Liver organoid-based Toxicity screen (LoT) is a potential assay system for liver toxicology studies, facilitating compound optimization, mechanistic study, and precision medicine as well as drug screening applications., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Two-thirds partial hepatectomy (PHx) was performed in rats, and the differences in effects between S-allylcysteine (SAC) and other sulfur-containing compounds on regeneration of the remaining liver and restoration of the injury were examined. Three days after two-thirds PHx, rats treated with 300 mg/kg/d, per os (p.o.) SAC showed a 1.2-fold increase in liver weight per 100 g body weight compared with saline-treated controls. In contrast, S-methylcysteine (SMC) (300 mg/kg/d, p.o.) or cysteine (Cys) (300 mg/kg/d, p.o.) did not have a regeneration-promoting effect. In the comparison with control rats, the regenerating liver of SAC-treated rats showed a significantly higher 5-bromo-2'-deoxyuridine labeling index on day 1. In contrast, serum alanine aminotransferase activity, which increases following PHx, was significantly inhibited by SAC and SMC (but not Cys) on day 1 after two-thirds PHx. In addition, SAC induced increases in insulin-like growth factor (IGF)-1 and its receptor mRNA expressions at 1 h after two-thirds PHx, and it increased phosphorylation of extracellular signal-regulated kinase (ERK)2 and Akt at 3 h after two-thirds PHx without affecting serum growth hormone levels. These results demonstrate that SAC is a mitogenic effector of normal remnant liver and promotes recuperation of liver function after two-thirds PHx. Moreover, SAC-induced proliferative effects are mediated via increased mRNA expressions of IGF-1 and its receptor and subsequent phosphorylation of ERK2 and Akt.

Organogenesis is a complex and interconnected process that is orchestrated by multiple boundary tissue interactions 1-7 . However, it remains unclear how individual, neighbouring components coordinate to establish an integral multi-organ structure. Here we report the continuous patterning and dynamic morphogenesis of hepatic, biliary and pancreatic structures, invaginating from a three-dimensional culture of human pluripotent stem cells. The boundary interactions between anterior and posterior gut spheroids differentiated from human pluripotent stem cells enables retinoic acid-dependent emergence of hepato-biliary-pancreatic organ domains specified at the foregut-midgut boundary organoids in the absence of extrinsic factors. Whereas transplant-derived tissues are dominated by midgut derivatives, long-term-cultured microdissected hepato-biliary-pancreatic organoids develop into segregated multi-organ anlages, which then recapitulate early morphogenetic events including the invagination and branching of three different and interconnected organ structures, reminiscent of tissues derived from mouse explanted foregut-midgut culture. Mis-segregation of multi-organ domains caused by a genetic mutation in HES1 abolishes the biliary specification potential in culture, as seen in vivo 8,9 . In sum, we demonstrate that the experimental multi-organ integrated model can be established by the juxtapositioning of foregut and midgut tissues, and potentially serves as a tractable, manipulatable and easily accessible model for the study of complex human endoderm organogenesis.

Background Micro RNA (miR)-33 targets cholesterol transporter ATP -binding cassette protein A1 and other antiatherogenic targets and contributes to atherogenic progression. Its inhibition or deletion is known to result in the amelioration of atherosclerosis in mice. However, mice lack the other member of the miR-33 family, miR-33b, which exists in humans and other large mammals. Thus, precise evaluation and comparison of the responsibilities of these 2 miRs during the progression of atherosclerosis has not been reported, although they are essential. Methods and Results In this study, we performed a comprehensive analysis of the difference between the function of miR-33a and miR-33b using genetically modified mice. We generated 4 strains with or without miR-33a and miR-33b. Comparison between mice with only miR-33a (wild-type mice) and mice with only miR-33b (miR-33a -/- /miR-33b +/+ ) revealed the dominant expression of miR-33b in the liver. To evaluate the whole body atherogenic potency of miR-33a and miR-33b, we developed apolipoprotein E-deficient/miR-33a +/+ /miR-33b -/- mice and apolipoprotein E-deficient/miR-33a -/- /miR-33b +/+ mice. With a high-fat and high-cholesterol diet, the apolipoprotein E-deficient/miR-33a -/- /miR-33b +/+ mice developed increased atherosclerotic plaque versus apolipoprotein E-deficient/miR-33a +/+ /miR-33b -/- mice, in line with the predominant expression of miR-33b in the liver and worsened serum cholesterol profile. By contrast, a bone marrow transplantation study showed no significant difference, which was consistent with the relevant expression levels of miR-33a and miR-33b in bone marrow cells. Conclusions The miR-33 family exhibits differences in distribution and regulation and particularly in the progression of atherosclerosis; miR-33b would be more potent than miR-33a.

Timely controlled oxygen (O 2 ) delivery is crucial for the developing liver. However, the influence of O 2 on intercellular communication during hepatogenesis is unclear. Using a human induced pluripotent stem cell-derived liver bud (hiPSC-LB) model, we found hypoxia induced with an O 2 -permeable plate promoted hepatic differentiation accompanied by TGFB1 and TGFB3 suppression. Conversely, extensive hypoxia generated with an O 2 -non-permeable plate elevated TGFBs and cholangiocyte marker expression. Single-cell RNA sequencing revealed that TGFB1 and TGFB3 are primarily expressed in the human liver mesenchyme and endothelium similar to in the hiPSC-LBs. Stromal cell-specific RNA interferences indicated the importance of TGFB signaling for hepatocytic differentiation in hiPSC-LB. Consistently, during mouse liver development, the Hif1a-mediated developmental hypoxic response is positively correlated with TGFB1 expression. These data provide insights into the mechanism that hypoxia-stimulated signals in mesenchyme and endothelium, likely through TGFB1, promote hepatoblast differentiation prior to fetal circulation establishment., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Organoid technology provides a revolutionary paradigm toward therapy but has yet to be applied in humans, mainly because of reproducibility and scalability challenges. Here, we overcome these limitations by evolving a scalable organ bud production platform entirely from human induced pluripotent stem cells (iPSC). By conducting massive "reverse" screen experiments, we identified three progenitor populations that can effectively generate liver buds in a highly reproducible manner: hepatic endoderm, endothelium, and septum mesenchyme. Furthermore, we achieved human scalability by developing an omni-well-array culture platform for mass producing homogeneous and miniaturized liver buds on a clinically relevant large scale (>10 8 ). Vascularized and functional liver tissues generated entirely from iPSCs significantly improved subsequent hepatic functionalization potentiated by stage-matched developmental progenitor interactions, enabling functional rescue against acute liver failure via transplantation. Overall, our study provides a stringent manufacturing platform for multicellular organoid supply, thus facilitating clinical and pharmaceutical applications especially for the treatment of liver diseases through multi-industrial collaborations., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Conventional two-dimensional differentiation from pluripotency fails to recapitulate cell interactions occurring during organogenesis. Three-dimensional organoids generate complex organ-like tissues; however, it is unclear how heterotypic interactions affect lineage identity. Here we use single-cell RNA sequencing to reconstruct hepatocyte-like lineage progression from pluripotency in two-dimensional culture. We then derive three-dimensional liver bud organoids by reconstituting hepatic, stromal, and endothelial interactions, and deconstruct heterogeneity during liver bud development. We find that liver bud hepatoblasts diverge from the two-dimensional lineage, and express epithelial migration signatures characteristic of organ budding. We benchmark three-dimensional liver buds against fetal and adult human liver single-cell RNA sequencing data, and find a striking correspondence between the three-dimensional liver bud and fetal liver cells. We use a receptor-ligand pairing analysis and a high-throughput inhibitor assay to interrogate signalling in liver buds, and show that vascular endothelial growth factor (VEGF) crosstalk potentiates endothelial network formation and hepatoblast differentiation. Our molecular dissection reveals interlineage communication regulating organoid development, and illuminates previously inaccessible aspects of human liver development.

The present study aimed to determine the onset time of hepatocarcinogen/hepatocarcinogenic tumor promoter-specific cell proliferation, apoptosis and aberrant cell cycle regulation after post-initiation treatment. Six-week-old rats were treated with the genotoxic hepatocarcinogen, carbadox (CRB), the marginally hepatocarcinogenic leucomalachite green (LMG), the tumor promoter, β-naphthoflavone (BNF) or the non-carcinogenic hepatotoxicant, acetaminophen, for 2, 4 or 6 weeks during the post-initiation phase using a medium-term liver bioassay. Cell proliferation activity, expression of G2 to M phase- and spindle checkpoint-related molecules, and apoptosis were immunohistochemically analyzed at week 2 and 4, and tumor promotion activity was assessed at week 6. At week 2, hepatocarcinogen/tumor promoter-specific aberrant cell cycle regulation was not observed. At week 4, BNF and LMG increased cell proliferation together with hepatotoxicity, while CRB did not. Additionally, BNF and CRB reduced the number of cells expressing phosphorylated-histone H3 in both ubiquitin D (UBD)(+) cells and Ki-67(+) proliferating cells, suggesting development of spindle checkpoint dysfunction, regardless of cell proliferation activity. At week 6, examined hepatocarcinogens/tumor promoters increased preneoplastic hepatic foci expressing glutathione S-transferase placental form. These results suggest that some hepatocarcinogens/tumor promoters increase their toxicity after post-initiation treatment, causing regenerative cell proliferation. In contrast, some genotoxic hepatocarcinogens may disrupt the spindle checkpoint without facilitating cell proliferation at the early stage of tumor promotion. This suggests that facilitation of cell proliferation and disruption of spindle checkpoint function are induced by different mechanisms during hepatocarcinogenesis. Four weeks of post-initiation treatment may be sufficient to induce hepatocarcinogen/tumor promoter-specific cellular responses., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

We have previously reported that a 28-day treatment of carcinogens evoking target cell proliferation activates G1 /S checkpoint function and apoptosis, as well as induction of aberrant ubiquitin D (Ubd) expression, suggesting disruptive spindle checkpoint function, in rats. The present study aimed to determine the onset time of rat liver cells to undergo carcinogen-specific cell cycle aberration and proliferation. Animals were treated orally with a hepatocarcinogenic dose of methyleugenol or thioacetamide for 3, 7 or 28 days. For comparison, some animals were subjected to partial hepatectomy or treated with noncarcinogenic hepatotoxicants (acetaminophen, α-naphthyl isothiocyanate or promethazine). Carcinogen-specific liver cell kinetics appeared at day 28 as evident by increases of cell proliferation, p21(Cip1+) cells, phosphorylated-Mdm2(+) cells and cleaved caspase 3(+) cells, and upregulation of DNA damage-related genes. Hepatocarcinogens also downregulated Rbl2 and upregulated Cdkn1a and Mdm2, and decreased Ubd(+) cells co-expressing phosphorylated-histone H3 (p-Histone H3) and p-Histone H3(+) cell ratio within the Ki-67(+) proliferating population. These results suggest that it takes 28 days to induce hepatocarcinogen-specific early withdrawal of proliferating cells from M phase due to disruptive spindle checkpoint function as evidenced by reduction of Ubd(+) cells staying at M phase. Disruption of G1 /S checkpoint function reflected by downregulation of Rbl2 as well as upregulation of Mdm2 suggestive of sequestration of retinoblastoma protein is responsible for the facilitation of carcinogen-induced cell proliferation at day 28. Accumulation of DNA damage probably in association with facilitation of p53 degradation by activation of Mdm2 may be a prerequisite for aberrant p21(Cip1) activation, which is responsible for apoptosis., (Copyright © 2015 John Wiley & Sons, Ltd.)

We aimed to clarify the hepatocarcinogen-specific disruption of cell cycle checkpoint functions and its time course after repeated administration of hepatocarcinogens. Thus, rats were repeatedly administered with hepatocarcinogens (methapyrilene, carbadox and thioacetamide), a marginal hepatocarcinogen (leucomalachite green), hepatocarcinogenic promoters (oxfendazole and β-naphthoflavone) or non-carcinogenic hepatotoxicants (promethazine and acetaminophen) for 7, 28 or 90 days, and the temporal changes in cell proliferation, expression of G1/S and spindle checkpoint-related molecules, and apoptosis were examined using immunohistochemistry and/or real-time RT-PCR analysis. Hepatocarcinogens facilitating cell proliferation at day 28 of administration also facilitated cell proliferation and apoptosis at day 90. Hepatocarcinogen- or hepatocarcinogenic promoter-specific cellular responses were not detected by immunohistochemical single molecule analysis even after 90 days. Expression of Cdkn1a, Mad2l1, Chek1 and Rbl2 mRNA also lacked specificity to hepatocarcinogens or hepatocarcinogenic promoters. In contrast, all hepatocarcinogens and the marginally hepatocarcinogenic leucomalachite green induced Mdm2 upregulation or increase in the number of phosphorylated MDM2(+) cells from day 28, irrespective of the lack of cell proliferation facilitation by some compounds. However, different Tp53 expression levels suggest different mechanisms of induction or activation of MDM2 among hepatocarcinogens. On the other hand, hepatocarcinogenic methapyrilene and carbadox downregulated the number of both ubiquitin D(+) cells and proliferating cells remaining in M phase at day 28 and/or day 90, irrespective of the lack of cell proliferation facilitation in the latter. These results suggest that hepatocarcinogens disrupt spindle checkpoint function after 28 or 90 days of administration, which may be induced ahead of cell proliferation facilitation.

Background: Recent study has demonstrated the important role of endothelial-mesenchymal interactions in 3-dimensional self-organization of immature progenitor populations with the use of mimicking of organogenesis. Here, we show that the same principle can be applicable to adult mature cells, ie, human adult hepatocytes (hAHs)., Methods: We cultivated hAHs with fluorescence-labeled human mesenchymal cells (hMSCs) and human umbilical vein endothelial cells (HUVECs) in micro-well culture plates and observed them for 9 days. Fluorescence microscopy imaging analyses were performed to evaluate the internal structures of generated 3-dimensional tissues. Maintenance of in vitro protein production capacity was examined with the use of enzyme-linked immunosorbent assay (ELISA)., Results: hAHs started to self-organize into 3-dimensional tissue with the use of coculturing with hMSCs and HUVECs. Live imaging analyses showed that endothelial cells started sprouting inside the generated tissues after 2 days of culture. ELISA showed that human albumin production capacity was improved with the use of coculture compared with hAHs-only culture after 9 days., Conclusions: We demonstrated that 3-dimensional vascularized hepatic tissue could be generated from hAHs by reconstituting endothelial-mesenchymal interactions. Future studies are needed to evaluate the therapeutic potential of vascularized hepatic tissue transplantation, and this may pave a new way to establish a new transplantation modality as an alternative to hepatocyte transplantation., (Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.)

Background: Liver ischemia/reperfusion (I/R) injury is a high risk factor in liver transplantation and it influences graft survival. One of the major events during I/R injury is the generation of cytotoxic oxygen radicals. Recently, hydrogen gas has been reported to have antioxidant properties and protective effects against organ dysfunction induced by I/R injury. The aim of this study is to investigate effects of hydrogen on porcine liver reperfusion injury., Materials and Methods: Six outbred pigs weighing 20 kg were used for the experiment. Under general anesthesia, the venous bypass between the left femoral vein and the splenic vein to the left jugular vein was made using a centrifugal pump. Then, we used a total vascular exclusion clamp (all in- and out-flow to the liver was clamped) for 60 minutes. Hydrogen (5 ppm) saturated with lactate Ringer's solution was prepared. This solution was infused through the portal vein just before reperfusion (hydrogen group)., Results: Aspartate aminotransferase levels in the control versus hydrogen group in 30, 60, and 120 minutes after reperfusion were 1560.3, 1925.3, and 2342.5 versus 175.3, 200.7, and 661.00 IU/L, respectively. Lactate dehydrogenase (LDH) levels in the control versus hydrogen groups in 30, 60, and 120 minutes after reperfusion were 23,235.0, 3496.7, and 4793.5 versus 663.3, 802.0, and 983.7 IU/L, respectively. The hydrogen gas level in liver tissue increased to 954.6 ppm immediately after reperfusion; however, it disappeared within 30 minutes., Conclusion: The solution containing hydrogen gas was safe and had remarkably protective effects on the porcine during liver I/R and may be applied in the clinical setting., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Generation of functional and vascularized organs from human induced pluripotent stem cells (iPSCs) will facilitate our understanding of human developmental biology and disease modeling, hopefully offering a drug-screening platform and providing novel therapies against end-stage organ failure. Here we describe a protocol for the in vitro generation of a 3D liver bud from human iPSC cultures and the monitoring of further hepatic maturation after transplantation at various ectopic sites. iPSC-derived specified hepatic cells are dissociated and suspended with endothelial cells and mesenchymal stem cells. These mixed cells are then plated onto a presolidified matrix, and they form a 3D spherical tissue mass termed a liver bud (iPSC-LB) in 1-2 d. To facilitate additional maturation, 4-d-old iPSC-LBs are transplanted in the immunodeficient mouse. Live imaging has identified functional blood perfusion into the preformed human vascular networks. Functional analyses show the appearance of multiple hepatic functions in a chronological manner in vivo.

Disruptive epigenetic gene control has been shown to be involved in carcinogenesis. To identify key molecules in piperonyl butoxide (PBO)-induced hepatocarcinogenesis, we searched hypermethylated genes using CpG island (CGI) microarrays in non-neoplastic liver cells as a source of proliferative lesions at 25 weeks after tumor promotion with PBO using mice. We further performed methylation-specific polymerase chain reaction (PCR), real-time reverse transcription PCR, and immunohistochemical analysis in PBO-promoted liver tissues. Ebp4.1, Wdr6 and Cmtm6 increased methylation levels in the promoter region by PBO promotion, although Cmtm6 levels were statistically non-significant. These results suggest that PBO promotion may cause altered epigenetic gene regulation in non-neoplastic liver cells surrounding proliferative lesions to allow the facilitation of hepatocarcinogenesis. Both Wdr6 and Cmtm6 showed decreased expression in non-neoplastic liver cells in contrast to positive immunoreactivity in the majority of proliferative lesions produced by PBO promotion. These results suggest that both Wdr6 and Cmtm6 were spared from epigenetic gene modification in proliferative lesions by PBO promotion in contrast to the hypermethylation-mediated downregulation in surrounding liver cells. Considering the effective detection of proliferative lesions, these molecules could be used as detection markers of hepatocellular proliferative lesions and played an important role in hepatocarcinogenesis., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

A critical shortage of donor organs for treating end-stage organ failure highlights the urgent need for generating organs from human induced pluripotent stem cells (iPSCs). Despite many reports describing functional cell differentiation, no studies have succeeded in generating a three-dimensional vascularized organ such as liver. Here we show the generation of vascularized and functional human liver from human iPSCs by transplantation of liver buds created in vitro (iPSC-LBs). Specified hepatic cells (immature endodermal cells destined to track the hepatic cell fate) self-organized into three-dimensional iPSC-LBs by recapitulating organogenetic interactions between endothelial and mesenchymal cells. Immunostaining and gene-expression analyses revealed a resemblance between in vitro grown iPSC-LBs and in vivo liver buds. Human vasculatures in iPSC-LB transplants became functional by connecting to the host vessels within 48 hours. The formation of functional vasculatures stimulated the maturation of iPSC-LBs into tissue resembling the adult liver. Highly metabolic iPSC-derived tissue performed liver-specific functions such as protein production and human-specific drug metabolism without recipient liver replacement. Furthermore, mesenteric transplantation of iPSC-LBs rescued the drug-induced lethal liver failure model. To our knowledge, this is the first report demonstrating the generation of a functional human organ from pluripotent stem cells. Although efforts must ensue to translate these techniques to treatments for patients, this proof-of-concept demonstration of organ-bud transplantation provides a promising new approach to study regenerative medicine.

It is known that after transurethral resection of the prostate (TUR-P) or a bladder tumor (TUR-BT), necrotizing granuloma formation associated with massive eosinophil accumulation can be detected at the site of the scar, revealing marked eosinophilia. This condition is called post-TUR prostatitis or cystitis. In the present study, we noticed a similar phenomenon in five patients who underwent cholecystectomy, of whom four had gallbladder adenocarcinoma and one had metastatic liver cancer originating from the rectum. We detected necrotizing granulomas with massive eosinophil accumulation, associated with marked eosinophilia. To induce these phenomena, the interval between the first operation (i.e., cholecystectomy) and the second operation (i.e., resection of the hepatic bed and extrahepatic bile duct) is very important. If the interval was 1 week, no granuloma formation was detected. On the other hand, if it was more than 2 weeks, the resected hepatic bed contained necrotizing granulomas with substantial eosinophil accumulation combined with an increase in peripheral eosinophilia (up to 34% in one case). Secondary resection was necessary to induce eosinophilia after cholecystectomy. In this sense, malignancies possessed a relationship with delayed eosinophilia. In the granulomas, some foreign body-type multinucleated giant cells were positive for both anti-interleukin (IL)-5 and CD68 antibodies. In sharp contrast, no eosinophilia was detected after cholecystectomy, with or without hepatic resection consequent to severe adhesion. Clinicians as well as pathologists should keep in mind that these peculiar phenomena of eosinophil accumulation surrounding the necrotizing granulomas and peripheral eosinophilia after cholecystectomy could occur.

We performed in vitro, animal, and human studies to clarify the effect of mayonnaise on β-carotene intake and its mechanism. In an artificial gastric juice model, we examined the transfer of β-carotene from grated carrot to mayonnaise or vegetable oil. Mayonnaise was more easily dispersed in artificial gastric juice than vegetable oil. The β-carotene concentration was greater in mayonnaise than vegetable oil. In rats, the postprandial serum β-carotene concentration in the mayonnaise group (β-carotene with mayonnaise) was higher than that in the control (β-carotene only) and vegetable oil (β-carotene with vegetable oil) groups. Continuous feeding of dietary β-carotene (14 d), employing mayonnaise or egg yolk, resulted in an increased accumulation of β-carotene in the liver. In a human study, diets were provided in the form of (1) carrot as a control (CON), (2) carrot juice (JU), (3) carrot with oil (OIL) and (4) carrot with mayonnaise (MS). Following collection of fasting blood samples, nine adult males consumed one of the four diets in random order. Fasting and postprandial changes in serum β-carotene were assessed at 2, 3, 4, 6 and 8 h following ingestion of each diet. The incremental areas under the curves of serum β-carotene concentration were higher following MS than following both CON and JU. In conclusion, we suggest that mayonnaise contributes to raising the serum β-carotene concentration when consumed with carrots rich in β-carotene, and that its mechanism is related to the emulsifying property of the egg yolk contained in mayonnaise.

The elongation of poly(A) tails in cytoplasm is essential for oogenesis and early embryogenesis in Xenopus laevis. mGLD-2 is a mouse homologue of Xenopus cytoplasmic poly(A) polymerase xGLD-2. We found an association of mGLD-2 with cytoplasmic polyadenylation components, CPEB and CPSF described in Xenopus oocytes. To clarify the role of mGLD-2 in mouse, we produced an mGLD-2 disrupted mouse line by homologous recombination. In spite of the ubiquitous expression of mGLD-2, the disrupted mice were apparently normal and healthy. Moreover, it was demonstrated that mGLD-2 disruption did not affect the poly(A) tail elongation in oocytes using reporter RNAs. Coincide with these observations, the maturation of the oocytes was normal and the mice were fertile. Thus mGLD-2 is dispensable for full-term development and oogenesis. Our results also indicate that there is another source of cytoplasmic poly(A) polymerase in mouse.

Aim: To evaluate the effects of combined radiofrequency ablation and transcatheter arterial embolization with iodized oil on ablation time, maximum output, coagulation diameter, and portal angiography in a porcine liver model., Methods: Radiofrequency ablation (RFA) was applied to in vivo livers of 10 normal pigs using a 17-gauge 3.0 cm expandable LeVeen RF needle electrode with or without transcatheter arterial embolization (TAE) with iodized oil (n=5). In each animal, 2 areas in the liver were ablated. Direct portography was performed before and after RFA. Ablation was initiated at an output of 30 W, and continued with an increase of 10 W per minute until roll-off occurred. Ablation time and maximum output until roll-off, and coagulated tissue diameter were compared between the 2 groups. Angiographic changes on portography before and after ablation were also reviewed., Results: For groups with and without TAE with iodized oil, the ablation times until roll-off were 320.6 +/- 30.9 seconds and 445.1 +/- 35.9 seconds, respectively, maximum outputs were 69.0 +/- 7.38 W and 87.0 +/- 4.83 W and maximal diameters of coagulation were 41.7 +/- 3.85 mm and 33.2 +/- 2.28 mm. Significant reductions of ablation time and maximum output, and significantly larger coagulation diameter were obtained with RFA following TAE with iodized oil compared to RFA alone. Portography after RFA following TAE with iodized oil revealed more occlusion of the larger portal branches than with RFA alone., Conclusion: RFA following TAE with iodized oil can increase the volume of coagulation necrosis with lower output and shorter ablation time than RFA alone in normal pig liver tissue.

Curcumin is a plant-derived diferuloylmethane compound extracted from Curcuma longa, possessing antioxidative and anticarcinogenic properties. Antioxidants and oxidative stress are known to induce the expression of certain classes of detoxification enzymes. Since the upregulation of detoxifying enzymes affects the drug metabolism and cell defense system, it is important to understand the gene regulation by such agents. In this study, we demonstrated that curcumin could induce the expression of human glutathione S-transferase P1 (GSTP1). In HepG2 cells treated with 20muM curcumin, the level of GSTP1 mRNA was significantly increased. In luciferase reporter assays, curcumin augmented the promoter activity of a reporter construct carrying 336bp upstream of the 5'-flanking region of the GSTP1 gene. Mutation analyses revealed that the region including antioxidant response element (ARE), which overlaps AP1 in sequence, was essential to the response to curcumin. While the introduction of a wild-type Nrf2 expression construct augmented the promoter activity of the GSTP1 gene, co-expression of a dominant-negative Nrf2 abolished the responsiveness to curcumin. In addition, curcumin activated the expression of the luciferase gene from a reporter construct carrying multiple ARE consensus sequences but not one with multiple AP1 sites. In a gel mobility shift assay with an oligonucleotide with GSTP1 ARE, an increase in the amount of the binding complex was observed in the nuclear extracts of curcumin-treated HepG2 cells. These results suggested that ARE is the primary sequence for the curcumin-induced transactivation of the GSTP1 gene. The induction of GSTP1 may be one of the mechanisms underlying the multiple actions of curcumin.

There is an interesting clinical report indicating that aciclovir, which is mainly excreted into urine, decreases the systemic clearance of theophylline by inhibiting cytochrome P450 (CYP) 1A2-mediated metabolism. In this study, we investigated the effect of aciclovir on the metabolism of theophylline, and on the activity and expression of hepatic CYP1A2 in rats. Theophylline (10 mg/kg) was injected intravenously into rats treated with two different dosages of aciclovir. When theophylline was simultaneously administered with aciclovir (50 mg/kg), the systemic clearance of theophylline and metabolic clearance of its major metabolites, 1-methyluric acid and 1,3-dimethyluric acid, were unchanged. In place of theophylline, when 1-methyl-3-propylxanthine (2.5 mg/kg), which is almost metabolized by CYP1A2 in rats, was coadministered intravenously with aciclovir (50 mg/kg), the pharmacokinetics of 1-methyl-3-propylxanthine was also unchanged. When theophylline was administered to rats pretreated with repeated intraperitoneal injections of aciclovir (25 mg/kg twice daily for 3 d), no significant differences in the systemic clearance of theophylline and its metabolic clearance to 1-methyluric acid and 1,3-dimethyluric acid were observed between the control and aciclovir-treated rats. This dosage of aciclovir did not change the activity of 7-ethoxyresorufin O-dealkylation, which is represented as CYP1A2 activity. In Western blot analysis, no significant change in the protein levels of hepatic CYP1A2 was observed between the control and aciclovir-treated rats. The present study suggests that aciclovir has no effect on the pharmacokinetics and metabolism of theophylline and on the activity and expression of hepatic CYP1A2 in rats.

A patient presented with hematemesis due to gastric variceal bleeding with an intratumoral arterioportal shunt. Contrast-enhanced CT revealed gastric varices and hepatocellular carcinoma with tumor thrombi in the right portal vein. Angiography and angio-CT revealed a marked intratumoral arterioportal shunt accompanied with reflux into the main portal vein and gastric varices. Balloon-occluded retrograde venography from the gastro-renal shunt showed no visualization of gastric varices due to rapid blood flow through the intratumoral arterioportal shunt. The hepatic artery was temporarily occluded with a balloon catheter to reduce the blood flow through the arterioportal shunt, and then concurrent balloon-occluded retrograde transvenous obliteration (BRTO) was achieved. Vital signs stabilized immediately thereafter, and contrast-enhanced CT revealed thrombosed gastric varices. Worsening of hepatic function was not recognized. BRTO combined with temporary occlusion of the hepatic artery is a feasible interventional procedure for ruptured high flow gastric varices with an intratumoral arterioportal shunt.

In a 42-year-old Japanese woman with esophageal varices, abdominal ultrasound (US) demonstrated a hyperechoic lesion 3 cm in diameter in segment 4 (S4). This nodular lesion had high intensity on T1-weighted magnetic resonance imaging (MRI), low intensity on T2, and very high intensity on superparamagnetic iron oxide (SPIO) enhanced MRI. Angiography showed sparse distribution of arterial branches and dense distribution of portal branches in S4. Meandering, thin arteries were seen in the peripheral area of the right lobe. The second branches of the portal vein were hardly visualized anywhere in the liver. Computed tomography arterioportography (CTAP) revealed portal blood flow dominance in this nodular lesion. There was no evidence of ischemic liver damage, such as thromboembolic episodes, laboratory data of liver damage, coagulation abnormalities etc. Therefore this abnormality was more likely to be caused by anomalous changes than thrombotic changes. Needle biopsy revealed no atypical cells. Two years later, the nodule size was reduced to 1.9 cm, showing its benign nature. Based on these findings, this lesion was classified as a new type of large regenerative nodule (LRN) associated with anomalies in the portal veins and arteries. This is the first report of an LRN of this size in which portal vein perfusion was dominant. Moreover, this lesion was difficult to differentiate from hepatocellular carcinoma (HCC) by imaging. Analysis of the images and pathological features of this case would contribute to a better understanding of the pathogenesis of nodular lesions of the liver.

We present three infants who showed hypertransaminasemia without extrahepatic symptoms, which improved by eliminating cows' milk formulas. The levels of milk protein-specific lymphocyte proliferation were elevated in all three patients. These cases indicate the importance of food allergy in the diagnosis of infantile hepatic dysfunction., ((c) 2005 Blackwell Munksgaard)

Cathodoluminescence (CL) is the light that is emitted from a material irradiated by an electron beam. The present study was undertaken to show the applicability to biological studies of a scanning electron microscope (SEM) equipped with a high-sensitive cathodoluminescence detection system. For this purpose, we injected inorganic fluorescent powders (P43) suspended in phosphate buffered saline into rat blood circulation, fixed the animals with glutaraldehyde within a day, and observed the hepatic tissues with a SEM. Our instrument enabled the simultaneous collection of both secondary electron (SE) and CL images of these tissues. Backscattered electron (BSE) images of the same portion were also able to be obtained with this microscope. SE and BSE images clearly showed the three-dimensional structure of the hepatic tissues including hepatocytes, Kupffer cells, Ito cells, and sinusoidal epithelial cells, while CL images visualized cathodoluminescence signals emitted from P43 as bright spots. We observed non-coated tissues under a low-vacuum condition and metal-coated tissues under a high-vacuum condition, and found that the high-vacuum observation of metal-coated tissues provided high quality CL images of P43 in the Kupffer cells. The superimposition of the CL images onto the corresponding SE or BSE images revealed that bright spots in the CL images were produced by the fluorescent powders uptaken by Kupffer cells. These findings indicate that the detection of CL as well as SE or BSE signals by SEM all provide us with useful information on the distribution of fluorescent tracers in tissues and cells in three-dimensional images.

Background: Antibodies to cytochrome P4502D6 (CYP2D6) were measured and their prevalence compared with that of antibodies to liver-specific arginase in patients with autoimmune hepatitis (AIH)., Methods: Anti-CYP2D6 antibodies were measured by sensitive radioligand assay and enzyme-linked immunosorbent assay (ELISA), and anti-arginase antibodies were measured by ELISA in 132 patients (definite AIH 11, probable AIH 36, hepatitis C 20, hepatitis B 23, other autoimmune diseases 42) and 50 healthy controls., Results: CYP2D6 index (radioligand assay) was significantly higher in all groups of patients than those in healthy controls. A higher index than the cut-off value (mean+3 S.D. in healthy controls) was found in 36.4%, 44.4%, 25.0%, 17.4% and 28.6% of patients with definite AIH, probable AIH, hepatitis C, hepatitis B and other autoimmune diseases, respectively. CYP2D6 index was not related to serum IgG, anti-nuclear antibody or AIH scores, and was weakly correlated with anti-arginase antibody activity. When CYP2D6 index and anti-arginase antibodies were combined, 55.3% of AIH patients were positive for either one or both antibodies., Conclusions: Anti-CYP2D6 antibodies by radioligand assay were frequently present in patients with AIH. Combined tests for anti-CYP2D6 radioligand assay and anti-arginase antibodies resulted in detection of 55% of AIH patients.

We investigated the effects of prostaglandin (EP) receptor subtype agonists on DNA synthesis and proliferation in primary cultures of adult rat hepatocytes to elucidate their mechanisms of action. Maintained in short-term cultures (i.e. 3.5 h) in a serum-free, defined medium, hepatocyte parenchymal cells underwent DNA synthesis and proliferation in the presence of sulprostone (10(-6) M), PGE(2) (10(-6) M), and 17-phenyl-trinor-PGE(2) (10(-9) M) in a time- and dose-dependent manner. PGE(2) was less potent than 17-phenyl-trinor-PGE(2) in stimulating hepatocyte mitogenesis. Sulprostone (10(-6) M) and 11-deoxy-PGE(1) (10(-6) M) showed weak and insignificant stimulation, respectively, for hepatocyte mitogenesis. These effects of PGE(2), 17-phenyl-trinor-PGE(2), and sulprostone were abolished by treatment with a specific EP(1) receptor antagonist, SC-51322, or the PLC inhibitor U-73122. The effects of these EP(1) receptor agonists were potentiated by ionomycin and blocked by verapamil. Hepatocyte mitogenesis was almost completely blocked by specific inhibitors of growth-related signal transducers, such as genistein, wortmannin, PD98059, and rapamycin. A monoclonal antibody against TGF-alpha dose-dependently inhibited PGE(2)- and 17-phenyl-trinor-PGE(2)-induced hepatocyte mitogenesis. Treatment with the EP(1) receptor agonists significantly increased the secretion of TGF-alpha, reaching a maximum within 5 min. The increase in TGF-alpha secretion was blocked by SC-51322, U-73122, somatostatin, and verapamil and potentiated by ionomycin. These results indicate that the proliferative mechanisms of action of EP(1) receptor agonists are mediated through an increase in the autocrine secretion of TGF-alpha, which is dependent on the EP(1) receptor/G-protein involved in PLC regulation/PLC/Ca(2+) system. The locally secreted TGF-alpha, in turn, acts as a complete mitogen that stimulates the tyrosine kinase/MAPK pathway in these cells.

Identification of metallothionein (MT) isoforms on capillary zone electrophoresis (CZE) analysis was studied using a linear polyacrylamide-coated capillary at pH 7.4 and EDTA. The CZE system was able to separate standard (purified and commercially available) MT specimens into their isoforms within 10 min. The peaks of MT-1 and MT-2 isoforms disappeared on addition of EDTA to the specimen, and the disappearance was shown to be time-dependent and dose-dependent, although the reason why the peaks decreased is still unclear. A heat-treated cytosol fraction prepared from Zn-injected mouse liver showed many major and minor peaks on CZE analysis. Two major peaks were identified to be MT-1 and MT-2, respectively, by co-injection with the purified MT isoforms. When EDTA was added to the cytosol fraction, the two major peaks, MT-1 and MT-2, and three other minor peaks disappeared time-dependently. Therefore, each MT isoform in the cytosol fraction can be identified by the addition of EDTA, also the peaks are identified by the corresponding migration times of purified MTs. Unknown substances like MT sub-isoforms may also be detected, although this question warrants clarification. From these results, it was concluded that the addition of EDTA is useful for identification of MT isoforms in cytosol fractions on CZE analysis.

The aim of this study was to identify metallothionein (MT) isoforms in mouse liver by using capillary zone electrophoresis (CZE). Purified MT-1 and MT-2 isoforms were completely separated by CZE using a polyacrylamide-coated tube at physiologic pH. There were two peaks in the cytosol fraction prepared from zinc-injected mouse liver, in which the migration times corresponded with those of purified MT-1 and MT-2 isoforms. When anti-MT monoclonal antibody was added with the purified MT-1 or MT-2 solution, the peaks decreased. Furthermore, the two peaks in the cytosol prepared from Zn-injected mouse liver decreased in a time-dependent manner from the electropherogram after the addition of the antibody. Therefore, those peaks were identified as MT-1 and MT-2 isoforms, respectively. In conclusion, the addition of anti-MT monoclonal antibody to the cytosol fraction of tissues is an effective method for identification of MT isoforms after separation using CZE.

We investigated the mechanisms of transforming growth factor-beta1 (TGF-beta1) inhibition on transforming growth factor-alpha (TGF-alpha)-induced DNA synthesis and proliferation in primary cultures of adult rat hepatocytes. TGF-alpha (1.0 ng/ml) produced a 4.2-fold elevation of DNA synthesis during 3 h of culture and a 1. 2-fold increase in nucleus number (proliferation) during 4 h of culture. TGF-beta1 dose dependently inhibited the TGF-alpha-induced hepatocyte DNA synthesis and proliferation: half-maximal inhibition occurred at a TGF-beta1 concentration of 0.08 ng/ml. The inhibitory effects of 1.0 ng/ml TGF-beta1 were almost completely reversed by adenylate cyclase inhibitors, 2,4-dideoxyadenosine (10(-6) M), and somatostatin (3 x 10(-7) M), or by a specific inhibitor of protein kinase A, H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride; 10(-7) M). In addition, while TGF-alpha did not affect the basal cellular adenosine 3',5'-monophosphate (cAMP) levels, TGF-beta1 was found to produce dose-dependent increases in cellular cAMP levels. The cAMP-elevating effects of TGF-beta1 were also reversed by 2,4-dideoxyadenosine (10(-6) M), and somatostatin (3 x 10(-7) M), but not by H-89 (10(-7) M). The present results suggest that the specific mechanisms involved in the growth inhibitory effect of TGF-beta1 on TGF-alpha-induced hepatocyte DNA synthesis and proliferation are via stimulation of adenylate cyclase, which increases intracellular cAMP and subsequently activates protein kinase A.

Cytochrome P450 isoforms involved in the benzo[a]pyrene metabolism in the Chinese hamster liver were characterized. The activity of benzo[a]pyrene hydroxylase in male hamster livers increased markedly by treatment with 3-methylcholanthrene (25 mg/kg per day, i.p., 3 days) and moderately with phenobarbital (60 mg/kg per day) and dexamethasone (100 mg/kg per day). In contrast, the ability for the mutagenic activation of benzo[a]pyrene determined by the mutagenicity test was increased most markedly by treatment with phenobarbital and significantly with 3-methylcholanthrene, but not with dexamethasone. These observations are similar to those in the rat rather than in the Syrian hamster. Western blot analysis and assay of the enzymes associated with cytochrome P450 isoforms showed that the 3-methylcholanthrene treatment elevated markedly the level of CYP1A2, but not that of CYP1A1, while the phenobarbital treatment elevated markedly the level of CYP2A and CYP3A, but not that of CYP2B. Further, immunoinhibition study demonstrated that, in Chinese hamster livers, CYP2A and CYP1A2 were mainly involved in the mutagenic activation of benzo[a]pyrene and CYP3A in the benzo[a]pyrene hydroxylase activity, respectively.

We investigated the effects of transforming growth factor alpha (TGF-alpha) on DNA synthesis and proliferation in primary cultures of adult rat hepatocytes and examined the influence of alpha and beta adrenoceptor agonists on the TGF-alpha-induced responses. TGF-alpha (1.0 ng/ml) produced a 4.1-fold elevation of DNA synthesis during 3 h of culture and a 1.2-fold increase in the nucleus number (proliferation) during 4 h of culture at a cell density of 3.3 x 10(4) cells/cm(2). The TGF-alpha-induced hepatocyte DNA synthesis and proliferation were dose-dependent at EC(50) values of 0.36 ng/ml and 0.45 ng/ml, respectively. Hepatocyte DNA synthesis and proliferation induced by 1.0 ng/ml TGF-alpha did not reduce even at higher initial plating densities (5.0 x 10(4) and 1.0 x 10(5) cells/cm(2)). Increasing concentrations of the beta(2) adrenoceptor agonist metaproterenol (10(-7)-10(-6) M) markedly reduced the proliferative effects of TGF-alpha, whereas those of the alpha(2) adrenoceptor agonist 5-bromo-6-[2-imidazolin-2-yl-amino]-quinoxaline (UK-14304; 10(-6)-10(-5) M) and the alpha(1) adrenoceptor agonist phenylephrine (10(-7)-10(-6) M) significantly potentiated the TGF-alpha action. The proliferative effects of TGF-alpha (1.0 ng/ml) were not affected significantly by a monoclonal antiepidermal growth factor receptor antibody (1-100 ng/ml) and were almost completely blocked by specific inhibitors of signal transducers such as genistein (10(-5) M), 1-6[[17beta-3methoxyestra-1,3, 5(10)-trien-17-yl]amino]hexyl]-1H-pyrrol2,5-dione (U-73122; 0(-5) M), wortmannin (5 x 10(-7) M), sphingosine (5 x 10(-6) M), 2'-amino-3'-methoxyflavone (PD98059; 5 x 10(-5) M), and rapamycin (10 ng/ml). These results suggest that among the elements that link signals of cell surface receptor to the nucleus, the proliferative action of TGF-alpha is mediated, at least, by tyrosine kinase, phospholipase C, phosphatidylinositol 3-kinase, protein kinase C, mitogen-activated protein kinase kinase, and ribosomal protein p70 S6 kinase.

We compared the effects of insulin-like growth factor I (IGF-I) and II (IGF-II) on DNA synthesis and proliferation and investigated various signal transduction mechanisms involved in insulin-like growth factor-induced mitogenesis in primary cultures of adult rat hepatocytes. IGF-I stimulated hepatocyte DNA synthesis and proliferation with an EC50 of 75 ng/ml within 4 h of culture. These effects were sensitive to the IGF-I concentration and cell density. Hepatocyte proliferation induced by IGF-I was potentiated by metaproterenol (10(-6) M) as well as by 8-bromo-cAMP, phorbol 12-myristate 13-acetate (PMA; 10(-8) M) and was inhibited by U-73122 (1-(-[[17beta-3-methoxyestra-1,3,5(10)-triene-17-yl]amino]hexyl]-+ ++1Hpyrrol-2,5-dione)), genistein, wortmannin, PD98059 (2'-amino-3'-methoxyflavone) and rapamycin. The IGF-I effect was independent of pertussis toxin (100 ng/ml). IGF-II also dose dependently stimulated hepatocyte DNA synthesis and proliferation with an EC50 of 0.75 ng/ml within 4 h of culture. However, these effects were not dependent on the initial plating density. The stimulatory effects of IGF-II were potentiated by UK-14304 (5-bromo-6-[2-imidazolin-2-ylamino]-quinoxaline) (10(-5) M) and inhibited by phenylephrine, PMA, metaproterenol, 8-bromo-cAMP, PD98059, rapamycin, and pertussis toxin. The IGF-II effects were not affected by genistein, U-73122, and wortmannin. These results suggest that IGF-I and IGF-II rapidly stimulate the DNA synthesis and proliferation of adult rat hepatocytes by separate mechanisms.

Overlapping cDNAs encoding dihydropyrimidine dehydrogenase (DPD), the rate-limiting enzyme of pyrimidine degradation, were isolated from a rat liver cDNA library, and a 4,353 bp cDNA sequence with a 3,075 bp open reading frame encoding a polypeptide of 1,025 residues with a molecular mass of 111,458 Da was determined. Western blot analysis of COS-7 cells that were transfected with an RT-PCR-derived DPD cDNA showed that the product from the cDNA binds to an anti-serum for DPD. Northern blot analysis was performed by using a partial fragment of the cloned cDNA as a probe. The mRNA level, the protein level, and the activity of DPD in the rat liver increased to the adult level by 3 mo of age. The levels of DPD in the liver of adrenalectomized rats increased 48 h after glucocorticoid administration. The increase of DPD after birth may be caused by the increase in glucocorticoid level.

Magnetic resonance imaging (MRI) of hepatocellular carcinoma (HCC) in cirrhotic and noncirrhotic livers was reviewed in 26 Japanese patients. Thirteen of the 15 HCC from noncirrhotic livers were iso- to low intense to surrounding parenchyma on T1-weighted spin-echo (SE) images. In contrast, seven of the 11 HCC in cirrhotic livers were high intense (p < 0.05). Signal intensity ratio and contrast-to-noise ratios of HCC in cirrhotic livers on T1-weighted images were higher (p < 0.05 and p < 0.01, respectively) than those of HCC in noncirrhotic livers.

We investigated whether or not proliferation of adult rat hepatocytes induced by platelet-derived growth factor (PDGF) is affected by alpha1-adrenoceptor agonists such as phenylephrine during the early and late phases of primary culture. Adult rat hepatocytes underwent significant DNA synthesis after culture with 10 ng/ml of PDGF for 2 hr at a low cell density (3.3 x 10(4) cells/cm2). Under these culture conditions, the number of nuclei increased significantly during the 3.5-hr culture period. Hepatocyte DNA synthesis and proliferation induced by 10 ng/ml of PDGF decreased slightly as a result of increasing the initial plating density. An alpha1-adrenoceptor agonist, phenylephrine (10(-6) and 10(-5) M), alone did not affect hepatocyte DNA synthesis and proliferation, but markedly potentiated PDGF-induced hepatocyte DNA synthesis and proliferation. The phenylephrine effect was mimicked by phorbol myristate acetate (10(-7) M), but not by ionomycin (10(-5) M). The mitogenic effects of PDGF were almost completely blocked by treating hepatocytes with genistein (5 x 10(-6) M), U-73122 (3 x 10(-6) M), sphingosine (10(-5) M), wortmannin (10(-7) M) and rapamycin (10 ng/ml). These results demonstrate that PDGF can induce the proliferation of adult rat hepatocytes rapidly in primary culture, regardless of the initial plating density. The present results also suggest that following stimulation with PDGF, activation of tyrosine kinase, phospholipase C, phosphatidylinositol 3-kinase, protein kinase C (PKC) and p70 ribosomal protein S6 kinase is essential for the proliferation of adult rat hepatocytes. The co-mitogenic effects of phenylephrine may involve PKC activation.

The aim of this study was to evaluate the correlation of development of the collateral circulation to the liver during hepatic arterial infusion chemotherapy (HAIC) with the presence of hepatic tumours adjacent to the hepatic surface, and with pretreatment occlusion of aberrant hepatic arteries. In 102 patients with unresectable malignant hepatic tumours treated with HAIC using an implantable port system, development of collaterals to the liver was assessed with CT arteriography using the implantable port and pre- and postoperative angiography. Aberrant hepatic arteries, if present, were occluded prior to treatment for hepatic arterial redistribution. Collaterals to the liver were seen in 29 patients, who had 35 areas with collateral perfusion: 22 areas were in the right posterosuperior area, 6 in the left peripheral area and 7 in the right or left lobar area. Collaterals were revealed more frequently in patients with hepatic tumours adjacent to the hepatic surface than in those without hepatic tumours in peripheral areas in the liver (p < 0.0001). In addition, collaterals developed more frequently in patients with an aberrant hepatic arterial anatomy compared with those with conventional anatomy (p = 0.0007). Our results indicated that patients with hepatic tumours adjacent to the hepatic surface and with pretreatment occlusion of aberrant hepatic arteries had the potential to develop collaterals to the liver during HAIC.

We investigated the effects of hepatocyte growth factor (HGF) and platelet-derived growth factor (PDGF) on the expression of alpha2- and beta2-adrenergic responses in primary cultures of adult rat hepatocytes. HGF (1 and 5 ng/ml) rapidly stimulated the expression of beta2-adrenergic responses and significantly increased alpha2-adrenergic responses with a maximal response at 21 h. The stimulatory effects of HGF were reduced dependent on the initial plating density and were completely blocked by cycloheximide (5 microM). On the other hand, PDGF (10 ng/ml) significantly increased the beta2-adrenergic response, but only slightly increased the alpha2-adrenergic responses. Expression of the alpha2- and beta2-adrenergic responses by PDGF was independent of the initial plating density. The expression of these responses was blocked by cycloheximide (5 microM). Northern blot analysis of the hepatocyte mRNA showed increased expression induced by the HGF and PDGF of the both alpha2- and beta2-adrenergic receptor mRNA, and this expression was inhibited by actinomycin D (5 ng/ml). These results indicate that the expression of alpha2- and beta2-adrenergic responses produced by these two growth factors is regulated differently by the cell density of the primary cultures. The results suggest that the expression of alpha2- and beta2-adrenergic responses is mediated through increased synthesis of these receptor proteins.

We investigated whether or not beta and alpha adrenergic agonists could affect proliferation of adult rat hepatocytes induced by hepatocyte growth factor (HGF) during the early and late phases of primary culture. Adult rat hepatocytes underwent significant DNA synthesis after culture with 5 ng/ml HGF for 3 h at a low cell density (3.3 x 10(4) cells/cm2). Under these culture conditions, the number of nuclei increased significantly during a subsequent 4-h culture period. Hepatocyte DNA synthesis and proliferation induced by 5 ng/ml HGF was reduced at high cell densities near confluence. A beta adrenergic agonist, metaproterenol (10(-7) M), and dibutyryl cAMP significantly potentiated hepatocyte DNA synthesis and proliferation at a concentration as low as 10(-7) M when cultured in combination with 5 ng/ml HGF. Similarly, an alpha-1 adrenergic agonist, phenylephrine (10(-6)-10(-4) M) markedly potentiated HGF-induced hepatocyte DNA synthesis and proliferation. The phenylephrine effect was mimicked by a phorbol ester (10(-6) M), but not by ionomycin (10(-6) M). The mitogenic effects of HGF were almost completely blocked by simultaneous treatment of hepatocytes with genistein (5 x 10(-6) M), U-73122 (10(-6) M), wortmannin (10(-7) M), sphingosine (3 x 10(-6) M) and rapamycin (10 ng/ml). These results demonstrate that HGF can rapidly induce proliferation of adult rat hepatocytes in primary culture. However, this effect is dependent on the initial plating density. The co-mitogenic effects of metaproterenol and phenylephrine may involve both protein kinase A and protein kinase C activation, respectively. The results also suggest that following stimulation with HGF, activation of tyrosine kinase, phosphatidylinositol 3-kinase, phospholipase C and p70 ribosomal protein S6 kinase is essential for hepatocyte proliferation.

We investigated whether or not insulin and cAMP-elevating agents induce the proliferation of adult rat hepatocytes during the early and late phases of primary culture. Adult rat hepatocytes synthesized a significant amount of DNA when cultured in the presence of 10(-7) M insulin for 3 h. Under these conditions, the number of nuclei increased within 4 h. Hepatocyte DNA synthesis and proliferation were not essentially affected by the initial plating densities. Other cAMP-elevating agents, such as glucagon, forskolin and dibutyryl cAMP, as well as beta-adrenoceptor agonists (i.e., metaproterenol and isoproterenol) alone had no effect on either hepatocyte DNA synthesis or proliferation in primary culture. In contrast, these agents potentiated both processes at concentrations as low as 10(-7) M when cultured in combination with 10(-7) M insulin. The stimulatory effects of beta-adrenoceptor agonists and other cAMP-elevating agents were significantly blocked by the cAMP-dependent protein kinase inhibitor, H-89 (N-[2-(p-(bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride; 10(-7) M). The mitogenic effect of insulin upon hepatocytes was almost completely suppressed by genistein (5 x 10(-6) M), wortmannin (10(-7) M) and by rapamycin (10 ng/ml). These results show that insulin rapidly induced the proliferation of adult rat hepatocytes in primary culture. The mitogenic effects of insulin were potentiated by beta-adrenoceptor agonists and cAMP-elevating agents. The effects of beta-adrenoceptor agonists and cAMP-elevating agents may be mediated through cAMP-dependent protein kinase. In addition, the activation of receptor tyrosine kinase, phosphoinositide 3-kinase and p70 ribosomal protein S6 kinase may be involved in the insulin signal transduction pathway.

We investigated whether or not epidermal growth factor (EGF) and cAMP-elevating agents induce the proliferation of adult rat hepatocytes during the early (4 h after adding EGF) and late phases (21 h after adding EGF) of primary cultures. Adult rat hepatocytes did not significantly proliferate after culture with 20 ng/ml EGF for 4 h at a density of 1 X 10(5) cells/cm2. In contrast, when the density was decreased by about one-third to 3.3 X 10(4) cells/cm2, the number of nuclei increased about 1.2-fold after culture with 10-20 ng/ml EGF for 4 h. Under these culture conditions, DNA synthesis began within 2-4 h of exposure to 20 ng/ml of EGF, although at the high cell density, DNA was not synthesized during this period. The beta-adrenoceptor agonists, metaproterenol and isoproterenol, and other cAMP-elevating agents, such as glucagon, forskolin, and dibutyryl cAMP, potentiated both hepatocyte DNA synthesis and proliferation about 1.4-fold when cultured in combination with 20 ng/ml EGF. The stimulatory effects of metaproterenol and other cAMP-elevating agents were specifically blocked by the cAMP-dependent protein kinase inhibitor, H-89 (10(-7) M). The effect of EGF was almost completely suppressed by genistein (5 X 10(-6) M) and rapamycin (10 ng/ml), but it was unaffected by wortmannin (10(-7) M). These results demonstrate that mature rat hepatocytes can proliferate very rapidly in low-density cultures with EGF, the effects of which were potentiated by beta-adrenoceptor agonists and cAMP-elevating agents. In addition, the activation of receptor tyrosine kinase and p70 ribosomal protein S6 kinase may be involved in EGF-induced hepatocyte DNA synthesis and proliferation.

Metallothionein (MT) isoforms from various liver tissues were separated with capillary zone electrophoresis (CZE) using a polyacrylamide-coated tube at neutral pH. The electrophoresis was performed on MT-1 and MT-2 purified from mouse, rat, rabbit and human livers. The retention times of mouse and rat MT-1 coincided, while the retention times of rabbit and human MT-1 were longer. The retention times of MT-2 purified from the four sources were the same. MT-1 and MT-2 separated more definitely with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-Tris buffer (25 mM, pH 7.4) than with N-tris(hydroxymethyl)methyl-3-aminopropane sulfonic acid (TAPS)-Tris buffer (25 mM, pH 7.7) or with N-(2-acetamido)iminodiacetic acid (ADA)-Tris buffer (25 mM, pH 7.4). In addition, liver MT isoforms prepared from Zn- or Cd-administered mice could be separated.

Although there is much evidence to suggest that lipopolysaccharide (LPS)-induced elevation of hepatic metallothionein (MT) contents is mediated by cytokines, the presence of MT-inducing activity in the serum of LPS-treated animals has not been examined. It was found that serum from LPS-treated mice stimulated MT induction in a hepatoma cell culture. The MT-inducing activity in serum was highest 2 h after LPS injection. Tumor necrosis factor and interleukin (IL)-6 levels in the serum were highest 1 and 2 h, respectively, after LPS injection. Anti-mouse IL-6 monoclonal antibody neutralized MT-inducing activity in serum obtained from mice 2 h after LPS injection. The MT-inducing activity in serum was blocked by the glucocorticoid antagonist, RU38486. A similar requirement for glucocorticoid was also observed in an IL-6-stimulated culture. These results show that the LPS-induced elevation of hepatic MT is mediated by IL-6, and the expression of the stimulating activity of IL-6 is dependent on the presence of glucocorticoid.

Induction of isozymes of drug-metabolizing enzymes by butylated hydroxytoluene (BHT) was studied in the male ddY mouse and Chinese hamster. In mice given 0.05 and 0.15% BHT in the diet for 14 days cytochrome P-450 contents and the activities of uridine diphosphate-glucuronyl transferase (UDP-GT) and pentoxyresorufin O-dealkylase were markedly increased, while in those fed 0.15% BHT testosterone 6 alpha-, 16 alpha- and 16 beta-hydroxylases were greatly increased, which indicated induction of cytochrome P-450 isozymes of the CYP2B family. Western blot analysis also showed an increased level of the isozyme immunorelated to rat CYP2B2 by BHT feeding. The activities of aryl hydrocarbon hydroxylase, ethoxycoumarin O-deethylase (ECOD), erythromycin N-demethylase and glutathione S-transferase (GST) remained unchanged. In Chinese hamsters given 0.05 and 0.15% BHT in the diet for 14 days activities of ECOD and GST were induced, but cytochrome P-450 contents and the activities of other enzymes were unaffected. Testosterone 15 alpha-hydroxylase was induced in hamsters fed 0.15% BHT. These findings suggested that BHT administration in the hamster induced CYP2A2-type isozyme, which was confirmed by Western blot analysis. BHT treatment enhanced activation of benzo[a] pyrene (B[a]P) as determined by the mutagenicity test, especially in Chinese hamsters. The results suggest that BHT treatment induces specific isozymes of drug-metabolizing enzymes and might modify the expression of toxicities of other chemicals.

To investigate the manganese status in magnesium deficiency, 40 male Wistar rats, 3 wk old, were divided into two groups and fed a magnesium deficient diet or a normal synthetic diet for 2 wk. Dietary magnesium depletion decreased magnesium levels in brain, spinal cord, lung, spleen, kidney, testis, bone, blood, and plasma, while it elevated the magnesium level in liver. In magnesium-depleted rats, calcium concentration was increased in lung, liver, spleen, kidney, and testis, while it was decreased in tibia. In magnesium-depleted rats, manganese concentration was decreased in plasma and all tissues except adrenal glands and blood. Dietary magnesium depletion diminished pyruvate carboxylase (EC 6.4.1.1) activity in the crude mitochondrial fraction of liver. Positive correlation was found between the liver manganese concentration and the pyruvate carboxylase activity. In the magnesium-depleted rats, glucose was decreased while plasma lipids (triglycerides, phospholipids, and total cholesterol) were increased. These results suggest that dietary magnesium deficiency changes manganese metabolism in rats.

To study the metabolism of K Vitamins (VK) in the liver, two types of natural VK, phylloquinone (K1) and menaquinone-4 (MK-4), were administered to male Wistar rats orally (P.O.), intravenously (I.V.) and intraperitoneally (I.P.). Blood and a small portion of the liver (and ascites by I.P.) were collected 8 times up to 72 h (P.O.) or 24 h (I.V. and I.P.). A modified assay procedure followed by high performance liquid chromatography (HPLC) was developed to detect VK from small amounts of liver tissue. After oral administration of both K1 and MK-4 (10 mumol/kg-P.O.), their concentrations in the liver increased from 1 h then reached a maximum at 6 h (10 nmol/g v.s. 0.35 nmol/g). After intravenous or intraperitoneal administration of K1 and MK-4 (0.5 mumol/kg-I.V. and I.P.), MK-4 concentrations in the liver reached a maximum faster than those of K1 (1.2 nmol/g -3 h vs. 1.3 nmol/g -0.5 h I.V. and 0.97 nmol/g -6 h vs. 0.47 nmol/g -1 h I.P.). MK-4 also increased in the liver from 6 h to 12 h (0.11 nmol/g -12 h) after oral administration of K1 (P.O.). These results indicate that K1 stays in plasma and liver longer than MK-4 and orally administered K1 might be transformed partially into MK-4 in the liver.