Involvement of diclofenac acyl-β-<scp>d</scp>-glucuronide in diclofenac-induced cytotoxicity in glutathione-depleted isolated murine hepatocytes co-cultured with peritoneal macrophages

Kawase, Atsushi; Kaneto, Ayaka; Ishibashi, Mao; Kobayashi, Akihiro; Shimada, Hiroaki; Iwaki, Masaya

doi:10.1080/15376516.2018.1544384

Cited by 6 publications

(5 citation statements)

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“…HepG2, M0, M1, or M2 alone at 1 × 10 5 cells/cm 2 were treated with DIC at doses of 100, 200, 300, 500, 800, and 1000 µM. After 24 h, the LDH released into the medium was measured with the LDH Cytotoxicity Detection Kit [ 6 ]. LDH release in direct co-cultures of HepG2 cells with M0, M1, and M2 (1:0, 1:0.1, 1:0.4, and 1:1) or in indirect co-cultures (1:0 and 1:0.4) was measured 24 h after DIC treatments (500 µM).…”

Section: Methodsmentioning

confidence: 99%

“…Total RNA was extracted from HepG2 cells, M0, M1, and M2 3 h and 24 h after DIC or vehicle treatment using Sepasol RNA I Super G, and then reverse-transcribed into complementary DNA using ReverTra Ace qPCR RT Master Mix. Time points of 3 h and 24 h after DIC treatment were chosen because the relatively high concentrations of DIC-AG were detected 3 h after DIC treatment and the protein changes and cytotoxicity were observed 24 h after DIC treatment in the preliminary experiments and report [ 6 ]. The PCR mixtures were incubated at 95 °C for 10 s, and then amplified for 40 cycles of 95 °C for 5 s, 59 °C for 20 s, and 72 °C for 40 s using the Fast SYBR Green Master Mix [ 30 ].…”

Section: Methodsmentioning

confidence: 99%

“…NSAIDs-AG is formed from NSAIDs catalyzed by UDP-glucuronosyltransferases (UGTs) such as UGT1A9 and UGT2B7 and is excreted into the bile via multidrug resistance-associated protein 2 (MRP2). Our group previously showed the involvement of NSAIDs-AG in inducing DILI [ 5 ] and established an in vitro system to evaluate DILI using isolated murine hepatocytes co-cultured with peritoneal macrophages [ 6 ]. Another group demonstrated that a test using monocyte-derived hepatocyte-like cells was useful to identify DILI-associated drugs [ 7 ].…”

Section: Introductionmentioning

confidence: 99%

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Diclofenac-Induced Cytotoxicity in Direct and Indirect Co-Culture of HepG2 Cells with Differentiated THP-1 Cells

Kawase

Takashima

Tanaka

et al. 2022

IJMS

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Non-steroidal anti-inflammatory drugs (NSAIDs) such as diclofenac (DIC) frequently induce drug-induced liver injury (DILI). It is unclear whether macrophages such as M1 and M2 participate in NSAID-associated DILI; elucidating this relationship could lead to a better understanding of the detailed mechanism of DILI. We co-cultured human hepatoma HepG2 cells with M1 or M2 derived from human monocytic leukemia THP-1 cells to examine the roles of M1 and M2 in DIC-induced cytotoxicity. DIC was added to the direct or indirect co-cultures of HepG2 cells with M1 or M2 (HepG2/M1 or HepG2/M2, respectively) at cell ratios of (1:0, 1:0.1, 1:0.4, and 1:1). In both direct and indirect HepG2/M2 co-cultures (1:0.4), there was lower lactate dehydrogenase release compared with HepG2/M1 co-cultures. Other NSAIDs as well as DIC showed similar protective effects of DIC-induced cytotoxicity. There were only slight differences in mRNA levels of apoptosis- and endoplasmic reticulum stress-associated factors between M1 and M2 after DIC treatment, suggesting that other factors determined the protective effects of M2 on DIC-induced cytotoxicity. Levels of high mobility group box 1 (HMGB1) in the medium and the mRNA expression levels of HMGB1 receptors were different between M1 and M2 after DIC treatment. Increased HMGB1 concentrations and expression of toll-like receptor 2 mRNA in M1 were observed compared with M2 after DIC treatment. In conclusion, these results suggested that the HMGB1/TLR2 signaling axis can be suppressed in M2 but not M1, leading to the different roles of M1 and M2 in NSAID-induced cytotoxicity.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Diclofenac-Induced Cytotoxicity in Direct and Indirect Co-Culture of HepG2 Cells with Differentiated THP-1 Cells

Kawase

Takashima

Tanaka

et al. 2022

IJMS

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“…after PB treatments (100 mg/kg, i.p., saline) to WT and D mice were determined by LC-MS/MS according to modification method (Kawase, Kaneto, et al, 2018). Briefly, a 5-μL plasma was deproteinated by adding 50 μL methanol followed by centrifugation at 2000 × g for 10 min.…”

Section: Quantitation Of Plasma Pb Concentrations By Lc-ms/ms the Plasma Pb Concentrations 24 Hrmentioning

confidence: 99%

Protein Kinase N Family Negatively Regulates Constitutive Androstane Receptor-Mediated Transcriptional Induction of Cytochrome P450 2b10 in the Livers of Mice

Kawase

Mukai

Tateishi

et al. 2021

The Journal of Pharmacology and Experimental Therapeutics

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In receptor-type transcription factors-mediated cytochrome P450 (P450)s induction, few studies have attempted to clarify the roles of protein kinase N (PKN) in the transcriptional regulation of P450s. This study aimed to examine the involvement of PKN in the transcriptional regulation of P450s by receptor-type transcription factors, including the aryl hydrocarbon receptor, constitutive androstane receptor (CAR), and pregnane X receptor. The mRNA and protein levels, and metabolic activity, of P450s in the livers of wild-type (WT) and double-mutant (D) mice harboring both PKN1 kinase-negative knock-in and PKN3 knockout mutations [PKN1 T778A/T778A ; PKN3 −/− ] were determined following treatment with activators for receptor-type transcription factors. mRNA and protein levels, and metabolic activity, of CYP2B10 were significantly higher in D mice treated with the CAR activator phenobarbital (PB), but not with 1,4-bis((3,5-dichloropyridin-2-yl)oxy)benzene, compared with WT mice. We examined the CAR-dependent pathway regulated by PKN following PB treatment, because the extent of CYP2B10 induction in WT and D mice was notably different in response to treatment with different CAR activators. The mRNA levels of Cyp2b10 in primary hepatocytes from WT and D mice treated with PB alone or in combination with SKI-1 (a Src inhibitor), or U0126 (a This article has not been copyedited and formatted. The final version may differ from this version.

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“…The protein expression levels of PIP5K1A, PIP5K1B and PIP5K1C were determined by targeted proteomics as described previously (32,33). Briefly, cell lysates (50 g) diluted in 25 l phase transfer surfactant buffer were treated with 10 mM dithiothreitol and 42 mM iodoacetamide and digested by 1 g trypsin at 37°C for 18 h. Peptides were separated on a reversed-phase column (AdvanceBio Peptide Map C18, 2.1 × 150 mm, 2.7 m; Agilent Technologies, Richardson, TX, USA) and detected on a LC-MS/MS apparatus consisting of an LC system (UltiMate 3000 series, Thermo Fisher Scientific, Waltham, MA, USA) and a TSQ Endura Triple Quadrupole Mass Spectrometer with electrospray ionization (Thermo Fisher Scientific).…”

Section: Determination Of Expression Of Pip5k1a Pip5k1b and Pip5k1c mentioning

confidence: 99%

Decrease in Multidrug Resistance-associated Protein 2 Activities by Knockdown of Phosphatidylinositol 4-phosphate 5-kinase in Hepatocytes and Cancer Cells

et al. 2019

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Purpose: The plasma membrane localization and transport activity of multidrug resistance-associated protein 2 (MRP2/ABCC2) and P-glycoprotein (P-gp/ABCB1) efflux transporters are governed by transporter-associated proteins. Phosphatidylinositol 4,5-bisphosphate (PIP2) formed by phosphatidylinositol 4-phosphate 5-kinase type 1 (PIP5K1) activates the linker function of radixin for efflux transporters. Radixin is involved in the plasma membrane localization of efflux transporters. We examined whether PIP5K1 could be a target for the modulation of transporter activities in hepatocytes and cancer cells. Methods: The effects of PIP5K1 depletion by siRNA in mouse primary hepatocytes, PANC1 human pancreatic carcinoma cells, and HepG2 human hepatocellular carcinoma cells on the intracellular accumulation of MRP2 and P-gp substrates were examined. Results: PIP5K1A depletion resulted in increased intracellular accumulation of carboxydichlorofluorescein, a MRP2 fluorescent substrate, in mouse primary hepatocytes, PANC1 cells, and HepG2 cells. In PANC1 and HepG2 cells, the transport activities of MRP2 were significantly decreased by PIP5K1C depletion. However, the transport activities of P-gp were unchanged by PIP5K1 depletion. PIP2 levels were unchanged between control and PIP5K1A- or PIP5K1C-depleted HepG2 cells. MRP2 mRNA levels showed few changes in HepG2 cells following PIP5K1A or PIP5K1C depletion. The expression of phosphorylated radixin was decreased by PIP5K1A and PIP5K1C depletion, although total radixin levels were unchanged. Conclusions: These data suggest that PIP5K1A and PIP5K1C could be target proteins for modulating MRP2 function, partly because of the resulting changes of the linker function of radixin.

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Involvement of diclofenac acyl-β-d-glucuronide in diclofenac-induced cytotoxicity in glutathione-depleted isolated murine hepatocytes co-cultured with peritoneal macrophages

Cited by 6 publications

References 45 publications

Diclofenac-Induced Cytotoxicity in Direct and Indirect Co-Culture of HepG2 Cells with Differentiated THP-1 Cells

Diclofenac-Induced Cytotoxicity in Direct and Indirect Co-Culture of HepG2 Cells with Differentiated THP-1 Cells

Protein Kinase N Family Negatively Regulates Constitutive Androstane Receptor-Mediated Transcriptional Induction of Cytochrome P450 2b10 in the Livers of Mice

Decrease in Multidrug Resistance-associated Protein 2 Activities by Knockdown of Phosphatidylinositol 4-phosphate 5-kinase in Hepatocytes and Cancer Cells

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