Metabolic Activity in Primary Cultures of Fish Hepatocytes

In aquatic toxicology, isolated liver cells from fish can be used as a tool to generate initial information on the hepatic metabolism of xenobiotics, and on the mechanisms of xenobiotic activation or deactivation. This isolation of teleost liver cells is achieved by enzymic dissociation, and monolayer cultures of fish hepatocytes in serum-free medium maintain good viability for 3–8 days. During in vitro culture, fish liver cells express stable levels of phase I and phase II enzymes, such as cytochrome P4501A or glutathione S-transferase, and the cells show an induction of biotransformation enzymes after exposure to xenobiotics. The xenobiotic metabolite pattern produced by fish hepatocytes in vitro is generally similar to that observed in vivo. Limitations to more-intensive application of cultured fish hepatocytes as a screen in aquatic hazard assessment are partly due to the rather limited scope of existing studies, i.e. the focus on one particular species (rainbow trout), and on one particular biotransformation enzyme (cytochrome P4501A), as well as a lack of comparative in vitro/in vivo studies.

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Comparative Proteomic Identification of Protein Disulphide Isomerase A6 Associated with Tert-Butylhydroperoxide-Induced Liver Injury in Rat Hepatocytes

Cellular Physiology and Biochemistry ◽

10.1159/000487968 ◽

2018 ◽

Vol 45 (5) ◽

pp. 1915-1926 ◽

Cited By ~ 2

Author(s):

Chien-Heng Shen ◽

Shui-Yi Tung ◽

Wen-Shih Huang ◽

Kam-Fai Lee ◽

Yung-Yu Hsieh ◽

...

Keyword(s):

Liver Injury ◽

Cell Viability ◽

Er Stress ◽

Primary Cultures ◽

Liver Cells ◽

Cytochrome C Release ◽

Dapi Staining ◽

Tert Butylhydroperoxide

Background/Aims: Oxidants are important human toxicants. They have been implicated in the occurrence and development of liver diseases. Increased intracellular tert-butylhydroperoxide (t-BHP) may be critical for oxidant toxicity, and is commonly used for evaluating mechanisms involving oxidative stress, but the method remains controversial. Methods: Primary cultures of hepatocytes as well as human Hep G2 and mouse FL83B liver cells were obtained. Cell viability was measured by annexin V–FITC/propidium iodide and DAPI staining to determine the effects of t-BHP treatment on acute liver injury. A proteomic assay provided information that was used to identify the differentially expressed proteins following t-BHP treatment; immunohistochemistry and western blotting were performed to detect the expression of PDIA6 activity in apoptotic and endoplasmic reticulum (ER) stress pathways. Results: Our results demonstrate that t-BHP treatment of liver cells increased cell cytotoxicity and the generation of reactive oxygen species. This treatment also increased the level of PDIA6; this was validated in vitro and in vivo based on a comparison of t-BHP-treated and -untreated groups. Treatment of mouse liver FL83B cells with t-BHP activated caspase 3, increased the expression of apoptotic molecules, caused cytochrome c release, and induced Bcl-2, Bax and IRE1α/TRAF2 complex formation. t-BHP-dependent induction of apoptosis was accompanied by sustained phosphorylation of the IRE1α/ASK1/JNK1/2/p38 pathways and PDIA6 expression. Furthermore, t-BHP induced liver FL83B cell viability and apoptosis by upregulating the levels of PDIA6; this process could be involved in the activation of the IRE1α/ASK1/JNK1/2/p38 signalling pathways. Conclusions: We conclude that t-BHP induced an apoptosis cascade and ER stress in hepatocytes by upregulation of PDIA6, providing a new mechanism underlying the effects of t-BHP on liver injury.

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Study of the l-Phenylalanine Ammonia-Lyase Penetration Kinetics and the Efficacy of Phenylalanine Catabolism Correction Using In Vitro Model Systems

Pharmaceutics ◽

10.3390/pharmaceutics13030383 ◽

2021 ◽

Vol 13 (3) ◽

pp. 383

Author(s):

Lyubov Dyshlyuk ◽

Stanislav Sukhikh ◽

Svetlana Noskova ◽

Svetlana Ivanova ◽

Alexander Prosekov ◽

...

Keyword(s):

Phenylalanine Ammonia Lyase ◽

In Vitro Model ◽

High Efficiency ◽

Tumor Regression ◽

Liver Cells ◽

Model Systems ◽

In Vivo Studies ◽

Vitro Model

The kinetics of l-phenylalanine ammonia-lyase (PAL) penetration into the monolayer of liver cells after its release from capsules was studied. The studies showed the absence of the effect of the capsule shell based on plant hydrocolloids on the absorption of l-phenylalanine ammonia-lyase in systems simulating the liver surface. After 120 min of incubation, in all variants of the experiment, from 87.0 to 96.8% of the enzyme penetrates the monolayer of liver cells. The combined analysis of the results concludes that the developed encapsulated form of l-phenylalanine ammonia-lyase is characterized by high efficiency in correcting the disturbed catabolism of phenylalanine in phenylketonuria, which is confirmed by the results of experiments carried out on in vitro model systems. PAL is approved for the treatment of adult patients with phenylketonuria. The encapsulated l-phenylalanine ammonia-lyase form can find therapeutic application in the phenylketonuria treatment after additional in vitro and in vivo studies, in particular, the study of preparation safety indicators. Furthermore, it demonstrated high efficacy in tumor regression and the treatment of tyrosine-related metabolic disorders such as tyrosinemia. Several therapeutically valuable metabolites biosynthesized by PAL via its catalytic action are included in food supplements, antimicrobial peptides, drugs, amino acids, and their derivatives. PAL, with improved pharmacodynamic and pharmacokinetic properties, is a highly effective medical drug.

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Cytochrome P4501A-activity in rainbow trout (Oncorhynchus mykiss) hepatocytes after exposure to PCB#126 and astaxanthin: In vitro and in vivo studies

Marine Environmental Research ◽

10.1016/s0141-1136(97)00101-3 ◽

1998 ◽

Vol 46 (1-5) ◽

pp. 1-5 ◽

Cited By ~ 7

Author(s):

Patric Amcoff ◽

Hans Börjeson ◽

Leif Norrgren ◽

Maija Pesonen

Keyword(s):

Rainbow Trout ◽

Oncorhynchus Mykiss ◽

In Vivo Studies ◽

Rainbow Trout Oncorhynchus Mykiss ◽

Cytochrome P4501a ◽

Pcb 126

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Hydroxyapatite nanoparticle-induced mitochondrial energy metabolism impairment in liver cells: in vitro and in vivo studies

Journal of Applied Toxicology ◽

10.1002/jat.3450 ◽

2017 ◽

Vol 37 (8) ◽

pp. 1004-1016 ◽

Cited By ~ 7

Author(s):

Yang Xue ◽

Qingqing Chen ◽

Jiao Sun

Keyword(s):

Energy Metabolism ◽

Liver Cells ◽

In Vivo Studies ◽

Mitochondrial Energy Metabolism

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Transfer of newly made triglyceride from hepatocytes to preexisting extracellular very low density lipoproteins

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y87-058 ◽

1987 ◽

Vol 65 (3) ◽

pp. 337-343

Author(s):

Gen Yoshino ◽

George Steiner

Keyword(s):

Low Density Lipoprotein ◽

Density Lipoprotein ◽

Liver Cells ◽

Low Density Lipoproteins ◽

In Vivo Studies ◽

Low Density ◽

Very Low Density Lipoproteins ◽

Apoprotein B

Previous in vivo studies suggested a new model to describe the metabolism of very low density lipoproteins (VLDL). It was hypothesized that some of the lipoprotein triglyceride was transferred directly from hepatocytes and intestinal mucosal cells into preexisting extracellular VLDL particles. These studies employ an in vitro system to test this hypothesis. Isolated rat liver cells containing newly made radioactive triglyceride were prepared. These cells were incubated in medium to which exogenous VLDL had or had not been added. The presence of extracellular VLDL (rat or human) stimulated the transfer of labeled triglyceride out of the liver cells. This triglyceride was recovered in the medium's VLDL (as determined by its density and its precipitability by MnCl2–heparin or by anti-apoprotein B). Although these studies focussed on VLDL, preliminary data showed that similar triglyceride transfer occurred in the presence of the other apoprotein B containing lipoprotein, low density lipoprotein (LDL). However, in the presence of equivalent amounts of LDL, this triglyceride transfer was less than that seen in the presence of exogenous VLDL. Furthermore, the increased triglyceride released in the presence of LDL occurred entirely in the d < 1.006 fraction of the medium. That released in the presence of VLDL was recovered in the d > 1.006 fraction. Hence, we conclude that the transfer of the newly made triglyceride was from the cell to the extracellular lipoprotein that had been added to the medium. The transfer of triglyceride to VLDL did not depend on the synthesis and release of new VLDL particles because it was not accompanied by a change in the production of [14C]leucine VLDL protein, it was not blocked by chloroquine, and the LDL induced triglyceride release occurred into the d > 1.006 fraction. This transfer did not depend on the previously described triglyceride-transfer factor. The present in vitro studies support the model suggested by our earlier in vivo studies. The VLDL particle does not appear to be metabolized as a complete intact unit. Rather, some of its major lipid component, triglyceride, can move directly into and out of already existing extracellular lipoproteins.

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Ptgs2 activation by endotoxin mediates the decrease in Igf1, but not in Igfbp3, gene expression in the liver

Journal of Endocrinology ◽

10.1677/joe-08-0205 ◽

2008 ◽

Vol 198 (2) ◽

pp. 385-394 ◽

Cited By ~ 4

Author(s):

A I Martín ◽

M López-Menduiña ◽

E Castillero ◽

M Granado ◽

M A Villanúa ◽

...

Keyword(s):

Gene Expression ◽

Nitric Oxide ◽

Direct Action ◽

Primary Cultures ◽

Inhibitory Effect ◽

Liver Cells ◽

Male Wistar Rats ◽

Igf Binding

The aim of this work was to analyse the role of cyclooxygenase-2 (Ptgs2) in endotoxin-induced decrease in Igf1 and Igf binding protein-3 (Igfbp3). For this purpose, male Wistar rats were injected with lipolysaccharide (LPS) and/or the Ptgs2 inhibitor meloxicam. LPS induced a significant decrease (P<0.01) in serum concentrations of Igf1 and Igfbp3 and their mRNAs in the liver. Meloxicam administration prevented the inhibitory effect of LPS injection on serum Igf1 and its liver mRNA. By contrast, meloxicam administration was unable to modify the inhibitory effect of LPS on Igfbp3. LPS injection also induced a decrease in GH receptor (Ghr) mRNA in the liver, and meloxicam attenuated this effect. In order to elucidate a direct action of the Ptgs2 inhibitor on the liver cells, the effect of LPS and/or meloxicam was studied in primary cultures of hepatocytes with non-parenchymal cells. LPS decreased Igf1 and Ghr but not Igfbp3 gene expression in liver cells in culture. Meloxicam administration attenuated the inhibitory effect of LPS on Igf1 mRNA, whereas it did not modify the decrease in Ghr mRNA after LPS. The effect of meloxicam on the LPS response does not seem to be mediated by changes in nitric oxide or tumour necrosis factor (Tnf) production, since meloxicam did not modify the stimulatory effect of LPS on nitric oxide or Tnfα gene expression both in vivo and in vitro. All these data suggest that LPS-induced Ptgs2 activation decreases Igf1 gene expression in liver cells.

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Chemopreventive Effect of Cratoxylum formosum (Jack) ssp. pruniflorum on Initial Stage Hepatocarcinogenesis in Rats

Molecules ◽

10.3390/molecules26144235 ◽

2021 ◽

Vol 26 (14) ◽

pp. 4235

Author(s):

Piman Pocasap ◽

Natthida Weerapreeyakul ◽

Rawiwan Wongpoomchai

Keyword(s):

Hepatocellular Carcinoma ◽

Early Stage ◽

In Vivo Studies ◽

Nuclear Antigen ◽

Apoptotic Cells ◽

Glutathione S Transferase ◽

Initial Stage ◽

Indigenous Plant

Cratoxylum formosum ssp. pruniflorum (Kurz) Gogelein (CP) is an indigenous plant found mainly in southeast Asia. Several in vitro studies have confirmed its activity against hepatocellular carcinoma; however, in vivo studies of the effect of CP on liver cancer are needed. This study investigated the effect of CP on early-stage hepatocarcinogenesis in rat liver when using diethylnitrosamine (DEN) as a carcinogen. Immunohistochemistry was used to detect (a) upregulation of glutathione S-transferase placental (GST-P) positive foci, (b) the proliferating cell nuclear antigen PCNA, and (c) apoptotic cells in the liver as indicators of early-stage carcinogenesis. Immunohistochemical parameters were observed in rats given CP orally following DEN injection. Rats given DEN presented overexpression of GST-P positive foci, PCNA, and apoptotic cells, indicating the formation of cancerous tissues, and these effects were diminished by CP treatment. CP thus inhibited hepatocarcinogenic effects in an animal model. These results could help plan further in vivo studies and support the use of CP to prevent processes that promote the pathogenesis of hepatocellular carcinoma in humans.

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