The Late-Stage Protective Effect of Mito-TEMPO against Acetaminophen-Induced Hepatotoxicity in Mouse and Three-Dimensional Cell Culture Models

An overdose of acetaminophen (APAP), the most common cause of acute liver injury, induces oxidative stress that subsequently causes mitochondrial impairment and hepatic necroptosis. N-acetyl-L-cysteine (NAC), the only recognized drug against APAP hepatotoxicity, is less effective the later it is administered. This study evaluated the protective effect of mitochondria-specific Mito-TEMPO (Mito-T) on APAP-induced acute liver injury in C57BL/6J male mice, and a three dimensional (3D)-cell culture model containing the human hepatoblastoma cell line HepG2. The administration of Mito-T (20 mg/kg, i.p.) 1 h after APAP (400 mg/kg, i.p.) injection markedly attenuated the APAP-induced elevated serum transaminase activity and hepatic necrosis. However, Mito-T treatment did not affect key factors in the development of APAP liver injury including the activation of c-jun N-terminal kinases (JNK), and expression of the transcription factor C/EBP homologous protein (CHOP) in the liver. However, Mito-T significantly reduced the APAP-induced increase in the hepatic oxidative stress marker, nitrotyrosine, and DNA fragmentation. Mito-T markedly attenuated cytotoxicity induced by APAP in the HepG2 3D-cell culture model. Moreover, liver regeneration after APAP hepatotoxicity was not affected by Mito-T, demonstrated by no changes in proliferating cell nuclear antigen formation. Therefore, Mito-T was hepatoprotective at the late-stage of APAP overdose in mice.


FS2. Chop induction in APAP liver injury and the effect of JNK inhibitor.
JNK activation is the early consequence of APAP-induced mitochondrial oxidative or nitrosative stress. To confirm the involvement of JNK, SP600125 a JNK inhibitor (30 mg/kg, i.p.) was administered 1h before APAP (400 mg/kg, i.p.) injection. After 8h of the APAP administration, the blood and tissue samples were collected. Serum ALT level was estimated and Chop mRNA level was analyzed by quantitative RT-PCR. SP600125 significantly attenuated the APAP liver injury (Fig. S2A) and Chop mRNA expression in C57BL/6J mice. (Fig. S2B).
A B Figure S2. Effects of a JNK inhibitor SP600125 on APAP-induced serum ALT elevation (A), and hepatic Chop mRNA expression in mice (B). The mice were treated with APAP (400 mg/kg, i.p.) and 1h before SP600125 (30 mg/kg, i.p.) or saline was administered. SP600125 was dissolved in HS-15 solution, and HS-15 solution used as a vehicle. Blood samples were collected 8h after APAP injection from mouse and the serum ALT activity measured. Each value represents the mean ± SEM. (n = 5). **p<0.01 vs. vehicle group and #p<0.05 vs. APAP group.
Following the overdose of APAP, excessive unstable reactive metabolite NAPQI was generated and bound to mitochondrial and cellular proteins. In the mitochondria, oxidative and nitrosative stresses were generated by APAP-induced activated JNK. Therefore, the effect of Mito-T (20 mg/kg, i.p.) on mitochondrial oxidative stress was estimated using a mitochondrial-specific ROS detection fluorescent probe, Mito-SOX ( Figure S3A) and a CM-H2DCFDA probe ( Figure S3B) following APAP (400 mg/kg, i.p.) injection. A single dose of Mito-T markedly attenuated the APAP-induced increased ROS in mouse livers and its efficacy on mitochondrial ROS scavenging was confirmed.
A B Figure S3. Effect of Mito-T on mitochondrial ROS generation in mouse livers. Mouse liver samples were collected 4 h after APAP (400 mg/kg, i.p.) injection. Mito-T (20 mg/kg, i.p.) was added 1 h after APAP administration. Mitochondria were isolated from mouse livers and mitochondrial ROS generation was estimated using Mito-SOX and CM-H2DCFDA probes. The fluorescence was measured 10 min after Mito-SOX (A) and CM-H2DCFDA (B) addition. Each value represents the mean ± SEM. (n = 3-4). **p < 0.01 vs vehicle group, and #p < 0.05 vs APAP group. Figure S4. Schematic representation of APAP pathophysiology and action point of Mito-T.

S 1. Mitochondrial ROS determination
Mitochondrial oxidative stress was measured using MitoSOX and CM-H2DCFDA. For this study, mitochondria were isolated from mouse livers using a commercially available Mitochondria Isolation Kit for Tissue (Thermo Fisher Scientific, Waltham, MA, USA) according to the supplier's protocol. For MitoSOX, isolated mitochondria were suspended in suspension buffer (125 mM KCl, 2 mM K2HPO4, 20 mM HEPES, 5 mM malic acid, 5 mM pyruvic acid, 4 mM MgCl2, 3 mM ATP, 50 μM EGTA) and protein was quantitated by the BCA method. The amount of mitochondrial protein was adjusted to 200 μg, and then MitoSOX was added and incubated at 37°C for 5 min. The fluorescence (485 nm for excitation and 590 nm for emission) was measured by a microplate reader (Tecan Co., Ltd., Männedorf, Switzerland). For H2-DCFDA, isolated mitochondria were suspended in suspension buffer (125 mM sucrose, 150 mM KCl, 10 mM HEPES-KOH, 2.5 μM rotenone, 5 mM KH2PO4, 5 mM succinate). The amount of mitochondrial protein was adjusted to 250 μg by the BCA method. Then, CM-H2DCFDA was added and incubated at 37°C for 5 min. The fluorescence (485 nm for excitation and 530 nm for emission) was measured by a microplate reader.