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• W2022052312 abstract "Drug-induced hepatotoxicity is mainly caused by hepatic glutathione (GSH) depletion. In general, the activity of rodent glutathione S-transferase is 10 to 20 times higher than that of humans, which could make the prediction of drug-induced hepatotoxicity in human more difficult. γ-Glutamylcysteine synthetase (γ-GCS) mainly regulates de novo synthesis of GSH in mammalian cells and plays a central role in the antioxidant capacity of cells. In this study, we constructed a GSH-depletion experimental rat model for the prediction of human hepatotoxicity. An adenovirus vector with short hairpin RNA against rat γ-GCS heavy chain subunit (GCSh) (AdGCSh-shRNA) was constructed and used to knock down the GCSh. In in vitro study in H4IIE cells, a rat hepatoma cell line, GCSh mRNA and protein were significantly decreased by 80% and GSH was significantly decreased by 50% 3 days after AdGCSh-shRNA infection. In the in vivo study in rat, the hepatic GSH level was decreased by 80% 14 days after a single dose of AdGCSh-shRNA (2 × 1011 pfu/ml/body), and this depletion continued for at least 2 weeks. Using this GSH knockdown rat model, acetaminophen-induced hepatotoxicity was shown to be significantly potentiated compared with normal rats. This is the first report of a GSH knockdown rat model, which could be useful for highly sensitive tests of acute and subacute toxicity for drug candidates in preclinical drug development. Drug-induced hepatotoxicity is mainly caused by hepatic glutathione (GSH) depletion. In general, the activity of rodent glutathione S-transferase is 10 to 20 times higher than that of humans, which could make the prediction of drug-induced hepatotoxicity in human more difficult. γ-Glutamylcysteine synthetase (γ-GCS) mainly regulates de novo synthesis of GSH in mammalian cells and plays a central role in the antioxidant capacity of cells. In this study, we constructed a GSH-depletion experimental rat model for the prediction of human hepatotoxicity. An adenovirus vector with short hairpin RNA against rat γ-GCS heavy chain subunit (GCSh) (AdGCSh-shRNA) was constructed and used to knock down the GCSh. In in vitro study in H4IIE cells, a rat hepatoma cell line, GCSh mRNA and protein were significantly decreased by 80% and GSH was significantly decreased by 50% 3 days after AdGCSh-shRNA infection. In the in vivo study in rat, the hepatic GSH level was decreased by 80% 14 days after a single dose of AdGCSh-shRNA (2 × 1011 pfu/ml/body), and this depletion continued for at least 2 weeks. Using this GSH knockdown rat model, acetaminophen-induced hepatotoxicity was shown to be significantly potentiated compared with normal rats. This is the first report of a GSH knockdown rat model, which could be useful for highly sensitive tests of acute and subacute toxicity for drug candidates in preclinical drug development. Glutathione (5-l-glutamyl-l-cysteinylglycine, GSH) 2The abbreviations used are: GSH, glutathione; GCS, γ-glutamylcysteine synthetase; GCSh, GCS heavy chain; shRNA, short hairpin RNA; APAP, acetaminophen; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; m.o.i., multiplicity of infection; PBS, phosphate-buffered saline; CYP, cytochrome P450; pfu, plaque-forming unit; GST, glutathione S-transferase; AST, aspartate aminotransferase; ALT, alanine aminotransferase. 2The abbreviations used are: GSH, glutathione; GCS, γ-glutamylcysteine synthetase; GCSh, GCS heavy chain; shRNA, short hairpin RNA; APAP, acetaminophen; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; m.o.i., multiplicity of infection; PBS, phosphate-buffered saline; CYP, cytochrome P450; pfu, plaque-forming unit; GST, glutathione S-transferase; AST, aspartate aminotransferase; ALT, alanine aminotransferase. is one of the most abundant tripeptides, consisting of glycine, glutamic acid, and cysteine. It serves an important function in protecting tissues against the degenerating effects of oxidative damage by scavenging free radicals from endogenous or exogenous compounds (1Reed D.J. Biochem. Pharmacol. 1986; 35: 7-13Crossref PubMed Scopus (203) Google Scholar, 2Lu S.C. FASEB J. 1999; 13: 1169-1183Crossref PubMed Scopus (750) Google Scholar). GSH is synthesized from its precursor amino acids in two steps of enzymatic reactions. γ-Glutamylcysteine synthetase (γ-GCS) (3Meister A. Anderson M.E. Annu. Rev. Biochem. 1983; 52: 711-760Crossref PubMed Scopus (5925) Google Scholar) catalyzes the formation of γ-glutamylcysteine from glutamic acid and cysteine. GSH synthetase couples glycine to γ-glutamylcysteine to form GSH. γ-GCS is a rate-limiting step in GSH biosynthesis, and GSH is a feedback inhibitor of γ-GCS activity. γ-GCS is a heterodimeric enzyme composed of a catalytic subunit (heavy chain, 73 kDa) (4Huang C.S. Chang L.S. Anderson M.E. Meister A. J. Biol. Chem. 1993; 268: 19675-19680Abstract Full Text PDF PubMed Google Scholar) and a modulatory subunit (light chain, 27.7 kDa) (5Huang C.S. Anderson M.E. Meister A. J. Biol. Chem. 1993; 268: 20578-20583Abstract Full Text PDF PubMed Google Scholar). Studies performed by purified γ-GCS suggested that the active site exists in the catalytic subunit, whereas the modulatory subunit increases the affinity of the catalytic subunit for glutamic acid and decreases the sensitivity to feedback inhibition by GSH (4Huang C.S. Chang L.S. Anderson M.E. Meister A. J. Biol. Chem. 1993; 268: 19675-19680Abstract Full Text PDF PubMed Google Scholar). In mice, embryos homozygous for the γ-GCS heavy chain (GCSh) mutation fail to gastrulate and die (6Shi Z.Z. Osei-Frimpong J. Kala G. Kala S.V. Barrios R.J. Habib G.M. Lukin D.J. Danney C.M. Matzuk M.M. Lieberman M.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5101-5106Crossref PubMed Scopus (230) Google Scholar). In contrast, homozygous knock-out mice with targeted disruption of the γ-GCS light chain are viable and fertile although the GSH level is decreased by 87% in the liver, and thus this model could be used as a GSH depletion mouse model in vivo (7Yang Y. Dieter M.Z. Chen Y. Shertzer H.G. Nebert D.W. Dalton T.P. J. Biol. Chem. 2002; 277: 49446-49452Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar).Rat is the most frequently used experimental animal for pharmacological and toxicological studies in the drug development process because of their body weight and ease of sampling blood or urine. A standard technique of gene knock out in rat has not been established yet. Recently, a nuclear transfer method has been established and this method may be able to produce conditional knock out and gene replacement in the future (8Zhou Q. Renard J.P. Le Friec G. Brochard V. Beaujean N. Cherifi Y. Fraichard A. Cozzi J. Science. 2003; 302: 1179Crossref PubMed Scopus (335) Google Scholar), but this method is very difficult and not available for general use. Recently, recombinant adenovirus methods are being developed and used for the purpose of clinical therapy or gene delivery in vivo (9Peng Z.C. Hum. Gene Ther. 2005; 16: 1016-1027Crossref PubMed Scopus (361) Google Scholar, 10Chu R.L. Post D.E. Khuri F.R. Van Meir E.G. Clin. Cancer Res. 2004; 10: 5299-5312Crossref PubMed Scopus (135) Google Scholar, 11Kruyt F.A. Curiel D.T. Hum. Gene Ther. 2002; 13: 485-495Crossref PubMed Scopus (89) Google Scholar). Furthermore, a small interfering RNA strategy, which has been proven to be more specific and efficient than the full-length antisense cDNA strategy, has been established (12Meister G. Tuschl T. Nature. 2004; 431: 343-349Crossref PubMed Scopus (1948) Google Scholar). In addition, an adenovirus-mediated short hairpin RNA (shRNA) knockdown approach could reduce the target gene specifically in the liver in mice, resulting in the expected phenotype (13Xu H. Wilcox D. Nguyen P. Voorbach M. Suhar T. Morgan S.J. An W.F. Ge L. Green J. Wu Z. Biochem. Biophys. Res. Commun. 2006; 349: 439-448Crossref PubMed Scopus (53) Google Scholar). However, to our knowledge, there is no report that adenovirus-mediated shRNA knock down was successfully applied in rats in vivo. In the present study, we constructed a recombinant adenovirus (AdGCSh-shRNA) that could knock down rat GCSh mRNA efficiently in vitro and in vivo. We established the GSH-depleted rats and this rat model, when treated with acetaminophen (APAP), which is known to be biotransformed to quinoneimine (14Dahlin D.C. Miwa G.T. Lu A.Y. Nelson S.D. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 327-331Crossref PubMed Google Scholar), or some other radical species (15Moldeus P. Andersson B. Rahimtula A. Berggren M. Biochem. Pharmacol. 1982; 31: 1363-1368Crossref PubMed Scopus (72) Google Scholar), demonstrated hepatotoxicity with high sensitivity compared with normal rats.The activity of rodent glutathione S-transferase (GST) is about 10 to 20 times higher than that in human (16Grover P.L. Sims P. Biochem. J. 1964; 90: 603-606Crossref PubMed Scopus (70) Google Scholar). Therefore, active metabolites produced in vivo in rat would be immediately detoxified by GSH conjugation, which would make the prediction of drug-induced hepatotoxicity in human more difficult. From this perspective, the AdGCSh-shRNA-mediated GSH depletion rat model could be useful for predicting the hepatotoxicity caused by unknown active metabolites of drug candidates produced by Phase I enzymes.EXPERIMENTAL PROCEDURESMaterials—APAP and GSH were obtained from Wako Pure Chemical Industries (Osaka, Japan). β-NADPH and glutathione reductase were from Oriental Yeast (Tokyo, Japan). ISOGEN was from Nippon Gene (Tokyo, Japan). ReverTra Ace (Moloney Murine Leukemia Virus Reverse Transcriptase RNaseH Minus) was from Toyobo (Tokyo, Japan). The Adenovirus Expression Vector kit (Dual Version), random hexamer and SYBR Premix Ex Taq were from Takara (Osaka, Japan). The QuickTiter Adenovirus Titer Immunoassay kit was from Cell Biolabs (Tokyo, Japan). Lipofectamine 2000 and minimum essential α medium were from Invitrogen. The GeneSilencer shRNA Vector kit was from Gene Therapy Systems (San Diego, CA). Dulbecco’s modified Eagle’s medium and Ham’s F12 medium were from Nissui Pharmaceutical (Tokyo, Japan). All primers and oligonucleotides for shRNA were commercially synthesized at Hokkaido System Sciences (Sapporo, Japan). Standard metabolites of APAP, such as APAP-glucuronide, APAP-sulfate, APAP-mercapturate, APAP-cysteine, and APAP-GSH, were kindly provided by McNeil Consumer Products (Washington, PA). Other chemicals were of analytical grade or the highest commercially available.Animals—Male Fisher 344 rats (7 weeks old, 130–150 g) were obtained from SLC Japan (Hamamatsu, Japan). Animals were housed in a controlled environment (temperature 25 ± 1 °C, humidity 50 ± 10%, and 12-h light/12-h dark cycle) in the institutional animal facility with access to food and water ad libitum. Animals were acclimatized for a week before use for the experiments. Animal maintenance and treatment were conducted in accordance with the National Institutes of Health Guide for Animal Welfare of Japan, as approved by the Institutional Animal Care and Use Committee of Kanazawa University, Japan.Design of Short Hairpin RNA—Rat GCSh (Gene Bank™ accession code J05181 Gene bank) knock down was achieved by RNA interference using an adenovirus vector-based shRNA approach. The sequences of shRNA-targeted GCSh cDNA were designed by B-Bridge (Mountain View, CA). The sequences of GCSh-shRNA are: top strand, 5′-gatccGTGTGAATGTCCAGAGTTAgaagcttgTAACTCTGGACATTCACACttttttggaagc-3′, and bottom strand, 5′-ggccgcttccaaaaaaGTGTGAATGTCCAGAGTTAcaagcttcTAACTCTGGACATTCACACg-3′. As a negative control, the oligonucleotide sequences of the shRNA target for luciferase from a GeneSilencer shRNA Vector kit were used.Recombinant Adenovirus—To generate the recombinant adenovirus vector expressing GCSh-shRNA, pGSU6-GFP plasmids were recombined into the pAxcwit using the cosmide-terminal protein complex (COS-TPC) method according to the manufacturer’s instruction. In brief, double strand oligo DNA for shRNA of GCSh and luciferase were inserted into the BamHI and NotI sites of the pGSU6-GFP vector. This product was digested by HincII and inserted into the SwaI site of the pAxcwit vector. This pAxcwit vector and the parental adenovirus DNA terminal protein complex were co-transfected into 293 cells by Lipofectamine 2000. The recombinant adenovirus was isolated and propagated into the 293 cells. An adenovirus containing shRNA of GCSh (AdGCSh-shRNA) and one containing shRNA of luciferase (AdLuc-shRNA) were constructed. The titer was determined by a QuickTiter Adenovirus Titer Immunoassay kit. The titers of AdGCSh- or AdLuc-shRNA were 4.95 × 1011 pfu/ml and 2.98 × 1011 pfu/ml, respectively. The viral stock solution was concentrated with the Amicon Ultra-15 filtration system (Millipore, Billerica, MA) for the in vivo study.Cell Culture—The 293 cell line and rat hepatoma cell lines BRL3A and H4IIE were obtained from American Type Culture Collection (Manassas, VA). The human hepatoma cell line HLE was obtained from the Japanese Collection of Research Biosources (Tokyo, Japan). The mouse hepatoma cell line Hepa1–6 was kindly provided by Dr. S. Kaneko (Kanazawa University, Japan). The 293 cell line was maintained in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (BioWhitaker, Walkersville, MD), 3% glutamine, 16% sodium bicarbonate, and 0.1 mm nonessential amino acids (Invitrogen) in a 5% CO2 atmosphere at 37 °C. BRL3A cells were maintained in Ham’s F12, HLE and Hepa1–6 cells were maintained in Dulbecco’s modified Eagle’s medium, and H4IIE cells were maintained in α-minimal essential medium. All cell lines were infected by the adenovirus in medium containing 5% fetal bovine serum.Real-time Reverse Transcription PCR Analysis—RNA from the hepatoma cells or from liver specimens was isolated using ISOGEN. Rat GCSh and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were quantified by real-time reverse transcription PCR. Primer sequences used in this study were as follows: rat GCSh, 5′-ATG CAGTATTCTGAACTACC-3′ and 5′-ACAAACTCAGATTCACCTAC-3′; mouse GCSh, 5′-TCTAACAAGAAACATCCGGCA-3′ and 5′-GGTCAGGTCGATGTCATTGTA-3′; human GCSh, 5′-ATTAGAAGAAAATCAGGCTC-3′ and 5′-GTAGCCAACTGATCATAAAG-3′; rat GAPDH, 5′-GTTACCAGGGCTGCCTTCTC-3′ and 5′-GGGTTTCCCGTTGATGACC-3′; mouse GAPDH, 5′-TCACCAGGGCTGCCATTTG-3′ and 5′-CTCACCCCATTTGATGTTAGT; human GAPDH, 5′-CCAGGGCTGCTTTTAACTC-3′ and 5′-GCTCCCCCCTGCAAATGA-3′. For the reverse transcription process, total RNA (2 μg) and 150 ng of random hexamer were mixed and incubated at 70 °C for 10 min. RNA solution was added to a reaction mixture containing 100 units of ReverTra Ace, reaction buffer, and 0.5 mm dNTPs in a final volume of 40 μl. The reaction mixture was incubated at 30 °C for 10 min, 42 °C for 1 h, and heated at 98 °C for 10 min to inactivate the enzyme. The real-time PCR was performed using the Smart Cycler (Cepheid, Sunnyvale, CA). PCR mixture contained 1 μl of template cDNA, SYBR Premix Ex Taq solution, and 10 pmol of sense and antisense primers. The PCR condition for GAPDH and GCSh were as follows. After an initial denaturation at 95 °C for 30 s, the amplification was performed by denaturation at 94 °C for 4 s, annealing and extension at 64 °C for 20 s for 45 cycles. Amplified products were monitored directly by measuring the increase of the dye intensity of the SYBR Green I (Molecular Probes, Eugene, OR) that binds to double strand DNA amplified by PCR.Western Blot Analysis—The H4IIE cell lysates, 1.5 μg, were separated on 10% SDS-polyacrylamide gels and transferred onto polyvinylidene difluoride membrane (Immobilon-P; Millipore). The specific proteins were detected by rabbit anti-human GCSh polyclonal antibody, cross-reacting to rat GCSh (sc-22755; Santa Cruz Biotechnology, Santa Cruz, CA) at a dilution of 1:200. The protein bands were developed by biotinylated second antibody-peroxidase reaction. The quantitative analysis of protein expression was performed using ImageQuant TL Image Analysis software (Amersham Biosciences).GSH Level—Cell lysates were mixed in 5% (w/v) metaphosphoric acid and incubated on ice for 10 min. After the addition of 0.125 m sodium phosphate buffer containing 6.3 mm EDTA, pH 7.5, the cell lysates were centrifuged at 13,000 × g at 4 °C for 5 min. Livers (100 mg) were homogenized with ice-cold 5% sulfosalicylic acid and centrifuged at 8,000 × g at 4 °C for 10 min. The GSH concentration in the supernatant was measured as described previously (17Tietze F. Anal. Biochem. 1969; 27: 502-522Crossref PubMed Scopus (5502) Google Scholar).Adenovirus Infection and APAP Administration in Rats—Fourteen days after one intravenous injection of AdGCSh-shRNA or AdLuc-shRNA at 2 × 1011 pfu/ml/body, the rats were orally administered APAP suspended in 0.5% carboxymethylcellulose (0, 300, 1000 mg/kg body weight). Blood samples were collected at 0, 30, 60, 120, and 180 min after the APAP treatment. Twenty-four hours after the administration of APAP, serum samples were collected for assessment of transaminase levels and for APAP metabolite analysis. The liver was fixed in buffered neutral 10% formalin. The fixed samples were embedded in paraffin and sectioned at a thickness of 2 μm and stained with hematoxylin-eosin for microscopic examination. Rat liver cytosol and microsomes were prepared as described previously (18Guengerich F.P. Shimada T. Yun C.H. Yamazaki H. Raney K.D. Their R. Coles B. Harris T.M. Environ. Health Perspect. 1994; 102 ((Suppl.): 49-53Crossref PubMed Scopus (77) Google Scholar). In all experiments, the rats were not treated by fasting prior to the APAP treatment or sacrifice.GST Activity and Cytochrome P450 (CYP) Content—The cytosolic GST activity was determined using 1-chloro-2,4-dinitrobenzene as a substrate according to the method of Habig et al. (19Habig W.H. Pabst M.J. Jakoby W.B. J. Biol. Chem. 1974; 249: 7130-7139Abstract Full Text PDF PubMed Google Scholar). The microsomal cytochrome P450 content was determined by the method of Omura and Sato (20Omura T. Sato R. J. Biol. Chem. 1964; 239: 2370-2378Abstract Full Text PDF PubMed Google Scholar).Determination of Plasma Concentrations of APAP and Its Metabolites—The plasma concentrations of APAP and its metabolites were measured using a high performance liquid chromatography method described previously (21Kim Y.C. Lee S.J. Toxicology. 1998; 128: 53-61Crossref PubMed Scopus (39) Google Scholar). In brief, plasma was mixed with an aliquot of acetonitrile containing theophylline as an internal standard. After extraction and centrifugation, the resulting supernatant was evaporated under nitrogen. The residue was diluted with distilled water as necessary before being injected into high performance liquid chromatography. APAP and its metabolites, APAP-GSH, APAP-cysteine, APAP-mercapturate, APAP-glucuronide, and APAP-sulfate, were separated in a Mightysil RP-18 column (4.6 × 150 mm; 5 μm; Kanto Chemical, Tokyo, Japan). APAP and the metabolites, eluted with 1.8% aqueous acetic acid-methanol-H2O (66:9:100) at a flow rate of 1.0 ml/min, were monitored at 248 nm.Statistical Analysis—Statistical analyses were performed with the GraphPad Instat version 2.0 computer program (GraphPad Software, San Diego, CA) by Student t-test, Dunnett’s post hoc test, or Bonferroni test.RESULTSChanges of GCSh mRNA Expression and GSH Level in Various Hepatoma Cell Lines—To investigate the knockdown effect on the cells, various hepatoma cells were infected with AdGCSh-shRNA or AdLuc-shRNA (negative control adenovirus) at a multiplicity of infection (m.o.i.) of 20 for 3 days. Real-time reverse transcription PCR analysis and GSH assay were performed to examine the GCSh mRNA expression and GSH suppression (Fig. 1). The expression level of GCSh mRNA was significantly reduced to 20–30% in BRL3A, H4IIE, and Hepa1–6 cells. In contrast, AdGCSh-shRNA was less potent, reducing GCSh mRNA to 45% in HLE cells. The GSH level was suppressed only in H4IIE cells by 50%, despite the efficient GCSh mRNA knock down in BRL3A and Hepa1–6 cells. Based on these results, H4IIE cells were used in the next experiments.Time-dependent Knockdown Effect of AdGCSh-shRNA in H4IIE Cells—To investigate the most efficient condition for infection, H4IIE cells were infected with AdGCSh-shRNA at m.o.i. 10 or 20 for 1, 2, 3, and 5 days. GCSh mRNA was reduced after 24 h of infection, and an 80% decrease of GCSh mRNA was achieved after 2 days of infection (Fig. 2A). The decrease of GCSh mRNA was accompanied by a decrease in GCSh protein (Fig. 2B). The GSH level was significantly reduced by 50% after 3 days of AdGCSh-shRNA m.o.i. 20 infection (Fig. 2C). There was no difference between 3 and 5 days of infection. These results suggested that a m.o.i. of 20 and 3 days of infection could be an appropriate condition for cytotoxicity experiments.FIGURE 2Time-dependent knockdown effect in AdGCSh-shRNA-infected H4IIE cells. GCSh mRNA (A), GCSh protein (B), and GSH content (C) were determined in H4IIE cells infected with AdLuc-shRNA or AdGCSh-shRNA. GCSh protein was quantified by immunoblotting as described under “Experimental Procedures.” Data represent the mean ± S.D. (n = 3). *, p < 0.05, **, p < 0.01, and ***, p < 0.001 compared with AdLuc-shRNA-infected cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Effect of APAP Treatment in H4IIE Cells Infected with AdGCSh-shRNA—To investigate the effect of GSH depression on the cytotoxicity of APAP, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed. H4IIE cells were infected with AdGCSh-shRNA at m.o.i. 20 or with AdLuc-shRNA in the same conditions as a negative control. After 3 days of infection, H4IIE cells were exposed to various concentrations of APAP of 0, 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5 mm. After 24 h of APAP treatment, AdGCSh-shRNA-infected cells showed no toxic effects compared with control (data not shown).Infection of AdGCSh-shRNA to Rat—To further investigate in vivo in rat, a single tail vein injection to Fisher 344 rats was made to deliver AdGCSh-shRNA. The effects of GCSh knock down were examined 14 days after infection. Control animals were treated with PBS or AdLuc-shRNA. The hepatic GCSh mRNA expression was remarkably decreased dose dependently (Fig. 3A). At the dose of 2 × 1011 pfu/ml/body, GCSh mRNA was significantly decreased by 90%. Consistent with the decrease of GCSh mRNA, the hepatic total GSH level was also reduced to 20% at doses above 2 × 1011 pfu/ml/body (Fig. 3B). The hepatic GSH level in AdLuc-shRNA-infected rat was slightly increased compared with the PBS-treated rats (Fig. 3B).FIGURE 3Effects of adenovirus infection on hepatic GCSh mRNA (A), GSH level (B), ALT and AST (C) in rats. All experiments were performed 14 days after AdGCSh-shRNA or AdLuc-shRNA infection. Data represent the mean ± S.D. (n = 4 or 5). *, p < 0.05 and **, p < 0.01 compared with PBS-treated rats or AdGCSh-shRNA-infected rats.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To examine the hepatotoxic effect of the adenovirus infection, serum AST and ALT were measured 14 days after infection. As shown in Fig. 3C, AST was significantly elevated in the 4.0 and 8.0 × 1011 pfu/ml/body infection by 1.6- and 4.1-fold, respectively, compared with the control. ALT was significantly elevated in only the 8.0 × 1011 pfu/ml/body infection by 2.2-fold compared with the control. The dose of 2.0 × 1011 pfu/ml/body did not affect the AST and ALT, and thus this condition was adopted in the next experiments. The cytochrome P450 content and GST activity slightly increased in AdGCSh-shRNA-treated rats (data not shown).APAP-induced Hepatotoxicity in AdGCSh-shRNA-infected Rat—To determine whether APAP-induced hepatotoxicity was potentiated by the suppression of hepatic GSH, rats were tail vein-injected once with 2.0 × 1011 pfu/ml/body AdGCSh-shRNA or AdLuc-shRNA. After 14 days, APAP was orally administered without previous fasting. The serum AST and ALT levels are shown in Figs. 4, A and B. Twenty-four hours after APAP administration, 300 mg/kg treatment did not result in hepatotoxicity. In contrast, the AdGCSh-shRNA-infected rats treated with 1000 mg/kg APAP demonstrated a significant increase of AST (2159 ± 1156 units/liter) and ALT (924 ± 667 units/liter) compared with AdLuc-shRNA infected rats. Without fasting treatment, the AdLuc-shRNA and normal rats administered 1000 mg/kg APAP did not show hepatotoxicity. The results of the histological examination in 1000 mg/kg APAP-administered rats are shown in Fig. 4C. Remarkable hepatic necrosis, especially around the central vein, was observed in AdGCSh-shRNA-treated rats given 1000 mg/kg APAP, consistent with the elevation of AST and ALT. There was no histological change in the other groups.FIGURE 4Hepatotoxic effect of APAP in AdGCSh-shRNA-infected rats. APAP was orally administered without previous fasting. After 24 h, serum AST (A) and ALT (B) were measured, and hematoxylin-eosin staining (C) was performed in sections of rat liver. Hepatic necrosis was observed only in APAP-administered rats infected with AdGCSh-shRNA. Arrow indicates areas of hepatic necrosis caused by 1000 mg/kg APAP treatment. Data represent the mean ± S.D. (n = 4 or 5). ***, p < 0.001 compared with each AdLuc-shRNA-infected group.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Metabolism of APAP in Rats Infected with Adenovirus—Changes in the plasma concentration of APAP and its metabolites are shown in Fig. 5. For APAP, APAP-glucuronide, and APAP-sulfate, the maximum plasma concentration was observed 30 min or 1 h after APAP administration. The concentration of APAP-glucuronide was significantly elevated in rats infected with AdGCSh-shRNA compared with AdLuc-shRNA-infected rats (Fig. 5B). On the other hand, APAP-sulfate, a major detoxification product in rats generated directly from APAP, was decreased (Fig. 5C). For APAP-GSH, APAP-cysteine, and APAP-mercapturate, the maximum plasma concentration was observed 1 h after APAP administration in rats infected with AdGCSh-shRNA. As for the rats infected with AdLuc-shRNA, the concentration of APAP-GSH was gradually decreased, whereas the concentrations of APAP-cysteine and APAP-mercapturate were slightly increased.FIGURE 5Changes of the plasma concentrations of APAP and its metabolites in rats infected with the adenovirus. Rats were administered APAP (1000 mg/kg, p.o.). Data represent the mean ± S.D. (n = 3). *, p < 0.05, **, p < 0.01, and ***, p < 0.001 compared with the AdLuc-shRNA-infected group.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Continuation of the Depletion of GSH Level—To examine the continuation of the hepatic GSH depletion, rats were tail vein-injected once with 2 × 1011 pfu/ml/body AdGCSh-shRNA. After 2, 3, 4, and 5 weeks, the hepatic GSH level was measured (Fig. 6). Hepatic GSH was significantly decreased by 80% at 2 to 3 weeks after infection. The hepatic GSH was reduced by 66 and 45% at 4 and 5 weeks after infection, respectively. In addition, at 7 and 10 days after infection of AdGCSh-shRNA, the hepatic GSH was decreased by 20 and 50%, respectively (data not shown). The effects of the circadian rhythm were also examined 2 weeks after infection with AdGCSh-shRNA (data not shown). The hepatic GSH level was lower than those from PBS-treated rats at all the time points examined, and no effect of the circadian rhythm on the GSH level was observed in rats infected with AdGCSh-shRNA.FIGURE 6Time-dependent changes of hepatic GSH level in rats infected with AdGCSh-shRNA. Rat liver was excised at 2, 3, 4, and 5 weeks after infection with AdGCSh-shRNA. Data represent the mean ± S.D. (n = 3 to 5). ***, p < 0.001 compared with the PBS-treated group.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DISCUSSIONIn this study, a recombinant adenovirus vector expressing an shRNA-directed rat GCSh was generated (AdGCSh-shRNA). The GSH level was efficiently decreased by 50% only in H4IIE cells infected with AdGCSh-shRNA (Fig. 1). The target sequence of the rat GCSh is the same as mouse, but it differs from that of human. This would probably affect the mRNA knockdown efficiency. The lack of a decrease of GSH in BRL3A cells may be due to differences in the expression levels of coxsackie and adenovirus receptor (22Huang K.C. Altinoz M. Wosik K. Larochelle N. Koty Z. Zhu L. Holland P.C. Nalbantoglu J. Int. J. Cancer. 2005; 113: 738-745Crossref PubMed Scopus (41) Google Scholar).In the cytotoxicity study, APAP treatment did not show a toxic effect in AdGCSh-shRNA-infected cells compared with AdLuc-shRNA-infected cells. APAP is mainly metabolized by UDP-glucuronosyltransferases and sulfotransferases, partly by CYP enzymes (23Howie D. Adriaenssens P. Prescott L.F. J. Pharm. Pharmacol. 1977; 29: 235-237Crossref PubMed Scopus (203) Google Scholar, 24Tone Y. Kawamata K. Murakami T. Higashi Y. Yata N. J. Pharmacobio-dyn. 1990; 13: 327-335Crossref PubMed Scopus (22) Google Scholar). APAP toxicity is highly dependent upon bioactivation by CYP enzymes to the reactive intermediate N-acetyl-p-benzoquino" @default.
• W2022052312 created "2016-06-24" @default.
• W2022052312 creator A5001851785 @default.
• W2022052312 creator A5019715351 @default.
• W2022052312 creator A5051993790 @default.
• W2022052312 creator A5065037081 @default.
• W2022052312 creator A5065428536 @default.
• W2022052312 creator A5066645932 @default.
• W2022052312 creator A5089297113 @default.
• W2022052312 date "2007-08-01" @default.
• W2022052312 modified "2023-10-14" @default.
• W2022052312 title "Knock Down of γ-Glutamylcysteine Synthetase in Rat Causes Acetaminophen-induced Hepatotoxicity" @default.
• W2022052312 cites W1538128477 @default.
• W2022052312 cites W154126403 @default.
• W2022052312 cites W1570767231 @default.
• W2022052312 cites W1581414347 @default.
• W2022052312 cites W1631043697 @default.
• W2022052312 cites W1951469402 @default.
• W2022052312 cites W1964953888 @default.
• W2022052312 cites W1967325600 @default.
• W2022052312 cites W1967684747 @default.
• W2022052312 cites W1970360497 @default.
• W2022052312 cites W1971296064 @default.
• W2022052312 cites W1976339214 @default.
• W2022052312 cites W1988466756 @default.
• W2022052312 cites W1989069505 @default.
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• W2022052312 cites W2007064404 @default.
• W2022052312 cites W2014780970 @default.
• W2022052312 cites W2018419931 @default.
• W2022052312 cites W2021633756 @default.
• W2022052312 cites W2028627047 @default.
• W2022052312 cites W2034005931 @default.
• W2022052312 cites W2038568679 @default.
• W2022052312 cites W2041030326 @default.
• W2022052312 cites W2043952117 @default.
• W2022052312 cites W2045904650 @default.
• W2022052312 cites W2048168782 @default.
• W2022052312 cites W2050525622 @default.
• W2022052312 cites W2069769399 @default.
• W2022052312 cites W2079043903 @default.
• W2022052312 cites W2079749916 @default.
• W2022052312 cites W2080543048 @default.
• W2022052312 cites W2086975308 @default.
• W2022052312 cites W2089552291 @default.
• W2022052312 cites W2101804080 @default.
• W2022052312 cites W2117354924 @default.
• W2022052312 cites W2133833958 @default.
• W2022052312 cites W2136954900 @default.
• W2022052312 cites W2143542298 @default.
• W2022052312 cites W2157294014 @default.
• W2022052312 cites W4245957869 @default.
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