(CXC Motif) Ligand 2 by Trovafloxacin Acyl-Glucuronide

1604 Biol. Pharm. Bull. 39, 1604–1610 (2016) Vol. 39, No. 10 Regular Article Acyl-glucuronide as a Possible Cause of Trovafloxacin-Induced Liver Toxicity: Induction of Chemokine (C-X-C Motif) Ligand 2 by Trovafloxacin Acyl-glucuronide Ryo Mitsugi,a Kyohei Sumida,a Yoshiko Fujie, a Robert H. Tukey,b Tomoo Itoh, a and Ryoichi Fujiwara*,a a Department of Pharmaceutics, School of Pharmacy, Kitasato University; 5–9–1 Shirokane, Minato-ku, Tokyo 108–8641, Japan: and b Laboratory of Environmental Toxicology, Departments of Chemistry & Biochemistry and Pharmacology, University of California at San Diego; La Jolla, CA 92023, U.S.A. Received February 27, 2016; accepted July 11, 2016 Trovafloxacin is an antibiotic that was withdrawn from the market relatively soon after its release due to the risk of hepatotoxicity. Trovafloxacin is mainly metabolized to its acyl-glucuronide by uridine 5-diphosphate (UDP)–glucuronosyltransferase (UGT) 1A1. In this study, we examined whether the acyl-glucuronide is involved in the development of hepatotoxicity. A UGT1A1-induced cell model was developed and the toxicity of trovafloxacin acyl-glucuronide was evaluated. The UGT1A1-induced cell model was developed by treating HepG2 cells with chrysin for 48 h. Chemokine (C-X-C motif) ligand 2, a cytokine involved in drug-induced liver injury, was uniquely induced by trovafloxacin in the UGT1A1-induced HepG2 cells. Induction of UGT1A1 resulted in a decrease in cell viability. An in vivo animal study further demonstrated the importance of UGT1A1 in the trovafloxacin-induced liver toxicity. Although the complete mechanism of trovafloxacin-induced liver injury is still unknown, trovafloxacin acyl-glucuronide can be involved in the development of toxic reactions in vitro and in vivo. Key words trovafloxacin; drug-induced liver injury; acyl-glucuronide; chrysin; HepG2 cell Uridine 5′-diphosphate (UDP)–glucuronosyltransferases cyto- and genotoxicity.12) Microarray expression analysis is a (UGTs; EC 2.4.1.17) are a family of membrane-bound en- promising tool to identify genes associated with a drug treat- zymes that catalyze glucuronidation of endogenous and exog- ment. To identify genes that were associated with the trova- enous compounds by transferring the glucuronic acid moiety floxacin-induced liver toxicity, several research groups carried of UDP-glucuronic acid to the substrates.1) Human UGTs are out the microarray expression analysis in human hepatocytes, mainly divided into two distinct families, UGT1 and UGT2, mice, and rats.13–15) The group of genes that were specifically on the basis of evolutionary divergence and homology.2) The induced by the trovafloxacin treatment included topoisomerase UGT1 gene is located on chromosome 2q37 and produces nine I (TOP1), B-cell leukemia/lymphoma 2 (BCL-2)-associated functional enzymes, UGT1A1, UGT1A3, UGT1A4, UGT1A5, transcription factor 1 (BCLAF), Mitofusin1 (MFN1), Metallo- UGT1A6, UGT1A7, UGT1A8, UGT1A9, and UGT1A10, by thionein (MT) 2A, MT1H, and MT1X.13) Although these genes exon sharing.3) The unique first exons encode N-terminal do- might have been induced by the acyl-glucuronide in the hepa- main and the common exons 2 to 5 encode C-terminal domain tocytes, there still was a possibility that the parent compound of UGT1A proteins. Since all of UGTs recognize and utilize itself was involved in the induction of the genes. This was UDP-glucuronic acid as a co-substrate, it has been suggested because a certain amount of trovafloxacin still remained in the that the C-terminal domain is responsible for the co-substrate body even 24 h after the oral and intravenous administration binding. In contrast, UGT1A proteins exhibit overlapping but of trovafloxacin.16–18) distinct substrate specificities, suggesting that the N-terminal Previously, we determined UGT1A1 as the main UGT iso- domain is responsible for the substrate binding. While the form responsible for trovafloxacin acyl-glucuronidation.19) To liver is the most contributing tissue to the metabolism,4) recent investigate whether trovafloxacin acyl-glucuronide is involved findings suggest that extrahepatic tissues such as small intes- in trovafloxacin-induced liver injury, in the present study, a tine play an important role in glucuronidation of endogenous UGT1A1-induced cell model was developed and the toxicity and exogenous compounds.5,6) of trovafloxacin acyl-glucuronide was evaluated. We further Trovafloxacin is an antibiotic that was released on the mar- employed Ugt1-knockout mice to examine the importance of ket in 1998.7) This promising agent was withdrawn from the trovafloxacin acyl-glucuronide in the trovafloxacin-induced market relatively soon after its release due to the risk of hepa- liver injury in vivo. totoxicity including acute liver failure. Trovafloxacin is mainly metabolized by UGTs to its acyl-glucuronide in humans.8) MATERIALS AND METHODS While glucuronides are usually pharmacologically inactive, certain types of glucuronide, especially acyl-glucuronide, Chemicals and Reagents UDP-glucuronic acid can exhibit an increased reactivity compared to the parent (UDPGA), alamethicin, chrysin, estradiol, and estradiol 3-O- compounds.9) Acyl-glucuronide-associated toxicity has been glucuronide were purchased from Sigma-Aldrich (St. Louis, reported in vivo and in vitro10,11); however, there are also re- MO, U.S.A.). Trovafloxacin was purchased from Wako Pure ports showing that acyl-glucuronidation did not induce the Chemical Industries, Ltd. (Osaka, Japan). Recombinant human * To whom correspondence should be addressed. e-mail: [email protected] © 2016 The Pharmaceutical Society of Japan Vol. 39, No. 10 (2016) Biol. Pharm. Bull. 1605 tumor necrosis factor α (TNF-α) was purchased from Roche for 24 h. Forty-eight hours after changing the culture medium (Mannheim, Germany). Primers were commercially synthe- to a normal or a chrysin-containing DMEM medium, the cells sized at Life Technologies (Carlsbad, CA, U.S.A.). All other were co-treated with trovafloxacin (50 µM) and TNF-α (4 ng/ chemicals and solvents were of analytical grade or the highest mL) for 24 h. Cell viability was measured using a 3-(4,5-di- grade commercially available. methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) Cell Culture and Chemical Treatments The human Cell Counting Kit (Nacalai Tesque, Kyoto, Japan) according to hepatoma HepG2 cells were obtained from DS Pharma Bio- the manufacturer’s protocol. medical Co., Ltd. (Osaka, Japan). HepG2 cells were grown Animals Heterozygous Ugt1 (Ugt1+/−) mice23) and in Dulbecco’s modified Eagle’s medium (DMEM) containing C57BL/6NCrSlc mice were used to obtain wild type (Ugt1+/+), 100 U/mL penicillin, 100 µg/mL streptomycin, and 10% fetal Ugt1+/−, and UGT1 knockout (Ugt1−/−) mice. Genomic DNA bovine serum (FBS) and were maintained at 37°C in a humid- was isolated from tail biopsies and was used as a template for 24) ified atmosphere containing 5% of CO2. Before the treatment, genotyping PCR. All animals received food and water ad li- HepG2 cells were seeded into six-well plates at 5×105 cells/ bitum, and mouse handling and experimental procedures were well. After 24 h, the culture medium was changed to a normal conducted in accordance with our animal care protocol, which or a chrysin-containing DMEM medium and subsequently was previously approved by Kitasato University. cells were maintained for 48 h until harvesting. RNA was iso- Two-day-old mice were subcutaneously treated with tro- lated from the cells and was used for the quantitative-reverse- vafloxacin (150 mg/kg) or canola oil. Three hours after the transcription PCR (Q-PCR) analysis. The microsomal fraction injections, mice were subcutaneously treated with lipopolysac- was also obtained from the cells. Control and the UGT1A1- charide (LPS) (5 mg/kg) or saline. Nine hours after the second induced HepG2 cells were further treated with trovafloxacin treatment, blood was obtained from the submandibular vein (50 µM) for 24 h. and serum was prepared. Serum alanine aminotransferase Q-PCR Analysis cDNA was synthesized from total RNA (ALT) levels were determined using a Transaminase CII-test using ReverTra Ace qPCR RT Master Mix (Toyobo, Tokyo, Wako kit (Wako Pure Chemical Industries, Ltd.) according to Japan) according to the manufacturer’s protocol. Q-PCR was the manufacturer’s protocol. performed with THUNDERBIRD SYBR qPCR Mix (Toyobo), Statistical Analysis Significant differences of UGT1A1 and the reactions were run in a CFX96 Real-Time PCR Detec- expression in HepG2 cells were analyzed by Dunnett’s test. tion System (Bio-Rad, Hercules, CA, U.S.A.). Expression of The Tukey–Kramer test was used to determine the signifi- glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA cance in the gene expression studies. The unpaired t-test was was used as an internal control for the cDNA quantity and used to determine the significance in the enzyme and cell vi- quality. Primer pairs that can detect UGT1A1 and GAPDH ability assays. p<0.05 was considered as significant. were reported previously.20,21) Other primers used were newly established with a program, Primer Blast (National Institutes RESULTS of Health). We confirmed that all of the primer sets produced specific bands and that bands were not detected when the PCR Development of the UGT1A1-Induced Cell Model reaction was conducted without cDNA. After an initial dena- Chrysin, phenytoin, carbamazepine, and phenobarbital can in- turation at 95°C for 30 s, the amplification was performed by duce UGT1A1 in vitro and in vivo.25–27) To determine the most denaturation at 95°C for 5 s, annealing at an appropriate tem- potent UGT1A1 inducer in HepG2 cells, we preliminarily perature for 30 s, and extension at 72°C for 30 s for 45 cycles. conducted a cell-based induction assay.28) HepG2 cells were Enzyme Assay in Vitro and in Cells Microsomes were treated with a lower (10 µM) and a higher (50 µM) concentra- prepared as described before.22) Estradiol 3-O- and trova- tion of chrysin, phenytoin, carbamazepine, and phenobarbital.

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