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Biomedical Research (Tokyo) 35 (6) 389-395, 2014

Acrolein, a highly toxic generated under in vivo, aggravates the mouse damage after acetaminophen overdose

1 1 2 1 1 Tomoya ARAI *, Ryo KOYAMA *, Makoto YUASA , Daisuke KITAMURA , and Ryushin MIZUTA 1 Research Institute for Biomedical Sciences, 2 Department of Pure and Applied Chemistry, Faculty of Science and Technology, Tokyo University of Science, 2669 Yamazaki, Noda, Chiba 278-0022, Japan (Received 17 September 2014; and accepted 3 October 2014)

ABSTRACT Although acetaminophen-induced liver injury in mice has been extensively studied as a model of human acute drug-induced hepatitis, the mechanism of liver injury remains unclear. Liver injury is believed to be initiated by metabolic conversion of acetaminophen to the highly reactive interme- diate N-acetyl p-benzoquinoneimine, and is aggravated by subsequent oxidative stress via reactive

species (ROS), including (H2O2) and the hydroxyl (•OH). In this study, we found that a highly toxic unsaturated aldehyde , a byproduct of oxidative stress, has a major role in acetaminophen-induced liver injury. Acetaminophen administration in mice resulted in liver damage and increased acrolein- adduct formation. However, both of them were decreased by treatment with N-acetyl-L-cysteine (NAC) or sodium 2-mercaptoethane- sulfonate (), two known acrolein scavengers. The specificity of NAC and MESNA was

confirmed in culture, because acrolein , but not 2H O2 or •OH toxicity, was inhibited by NAC and MESNA. These results suggest that acrolein may be more strongly correlated with acet- aminophen-induced liver injury than ROS, and that acrolein produced by acetaminophen-induced oxidative stress can spread from dying cells at the primary injury site, causing damage to the ad- jacent cells and aggravating liver injury.

Acetaminophen-induced liver injury in mice has conversion to the reactive intermediate N-acetyl p- been used as a model of human acute drug-induced benzoquinoneimine (NAPQI) by cytochrome P-450 hepatitis and an in vivo model of necrosis (5, 6). (14). NAPQI covalently binds essential hepatocellu- Acetaminophen is a widely prescribed lar , and is eliminated by . As the drug, and is also sold in numerous preparations as a primary endogenous non-protein sulfhydryl, gluta- single compound or in combination with other med- thione is a major scavenger of free radicals. High ications. Although it is generally safe, an overdose doses of acetaminophen deplete glutathione, thereby of acetaminophen can cause severe liver failure with increasing the formation of significant mortality. Despite substantial efforts in (ROS), such as (H2O2) and the the past, the mechanism of acetaminophen-induced (•OH). ROS and NAPQI are free liver injury remains unclear. Acetaminophen-induced to bind critical cellular proteins, which may result in liver injury is thought to be initiated by its metabolic the reduction of , enhancement of lipid peroxidation, and induction of hepatic ne- crosis (14). Address correspondence to: Dr. Ryushin Mizuta, Re- search Institute for Biomedical Sciences, Tokyo Univer- Acrolein (CH2=CHCHO), a highly reactive α, sity of Science, 2669 Yamazaki, Noda, Chiba 278-0022, β-unsaturated aldehyde, is a common pollutant found Japan Tel: +81-4-7121-4072, Fax: +81-4-7121-4079 E-mail: [email protected] *These authors contributed equally to this work. 390 T. Arai et al. in the environment. Notably, endogenous acrolein is the Tokyo University of Science. generated during oxidative stress (10). Acrolein can be formed in various tissues via lipid peroxidation Acetaminophen-induced liver necrosis. Mice were (19), oxidation (16) and via fasted 14 ~ 16 h before acetaminophen injection but of drugs, such as the anticancer drug cyclophospha- were allowed access to . Acetaminophen was mide (7). Acrolein is primarily metabolized via rap- suspended in 50% Mili-Q water and 50% propylene id reaction with the sulfhydryl groups of glutathione. glycol and was intraperitoneally (ip) injected into Thus, acrolein contributes directly to cellular oxida- mice (600 mg/kg body weight). NAC (1000 mg/kg) tive stress via loss of glutathione (7). Acrolein can and MESNA (1000 mg/kg) were injected ip immedi- form Michael-type addition adducts with cellular ately after acetaminophen injection. Mice were sacri- components, particularly proteins and DNA, result- ficed at 12 h after acetaminophen injection to collect ing in cell toxicity. Elevated levels of acrolein and serum and tissues. Serum alanine aminotransferase acrolein-adducts have been found in ex vivo biologi- (ALT) levels were determined with an automated cal samples from patients, including the plasma of analyzer (Hitachi 7140; Hitachi Instruments Service patients with renal failure (13) and the brains of pa- Co., Tokyo, Japan). A piece of the liver from each tients with neurologic disorders (21). Acrolein can mouse was fixed with formalin and embedded in easily move across cell membranes and tissues due paraffin for hematoxylin- (H&E) staining or to its in water and alcohol; thus, high con- immunohistochemistry. Other pieces of the liver centrations of acrolein produced by lipid peroxida- were frozen in and were stocked in a tion or polyamine oxidation can spread from dying −80°C freezer. cells at the primary site of injury, resulting in dam- age and/or death in adjacent cells (10). Extraction of total RNA and reverse transcription Sodium 2-mercaptoethanesulfonate (MESNA), a polymerase chain reaction. Total RNA was extract- compound, has been shown to scavenge acrole- ed from the removed liver tissues using TRIzol in both experimentally and clinically. MESNA inter- (Sigma-Aldrich, St. Louis, USA) according to the acts with acrolein and forms an inactivate compound, manufacturer’s instructions. Ten micrograms of total which is detectable using mass spectrometry (18). RNA were reverse-transcribed into cDNA using Su- As mentioned before, acrolein is metabolized from perscript with oligo (dT) primer (Invitrogen, Carls- the anticancer drug , and is known bad, USA). For examining the expression of heme to cause hemorrhagic cystitis. To prevent this side oxigenase-1 (Ho-1) gene and 3-phos- effect during chemotherapy, MESNA has phate dehydrogenase (Gapdh) gene, quantitative been routinely given to patients, and has successive- polymerase chain reaction (q-PCR) was conducted ly decreased the incidence of hemorrhagic cystitis using THUNDERBIRD SYBR qPCR Mix (Toyobo, (18). Another thiol compound N-acetyl-L-cysteine Osaka, Japan) and the following primers according (NAC) is known as a glutathione pro-drug that is to the manufacturer’s instructions. Ho-1 Forward: approved for clinical use and is routinely used to 5’-TGGGTCCTCACTCTCAGCTT-3’, Ho-1 Reverse: treat acetaminophen-induced liver injury (11). Re- 5’-GTCGTGGTCAGAACATGG-3’, Gapdh Forward: cently, it was also shown that NAC directly inter- 5’-GGAGAAACCTGCCAAGTATGA-3’, Gapdh Re- acts with acrolein and forms an inactive compounds verse: 5’-CCCTGTTGCTGTAGCCGTATT-3’. (22). Here we show that acrolein-adducts are produced Histopathology. Excised liver was fixed in 10% for- in the of acetaminophen-treated mice, and that malin and embedded in paraffin. Four micron serial MESNA and NAC ameliorate acetaminophen-mediat- sections were used for H&E and TdT-mediated ed liver injury by neutralizing acrolein toxicity, which dUTP Nick-End Labeling (TUNEL) staining. For suggests that acrolein plays a critical role in the the TUNEL assay, the DeadEnd Fluorometric pathogenesis of acetaminophen-induced liver injury. TUNEL System (Promega, Madison, USA) was used according to the manufacturer’s protocol. For immunohistochemical analysis to detect acrolein, MATERIAL AND METHODS deparaffinized sections were immersed in 0.3% 2H O2 Animal treatment. C57BL/6 mice were purchased in to eliminate endogenous ac- from Sankyo-Lab Service (Tsukuba, Japan). All ex- tivity, followed by rinsing with a phosphate buffered perimental procedures using mice were approved by saline (PBS). Acrolein readily reacts with lysine res- the Institutional Animal Care and Use Committee at idues of proteins to form Nε-(3-formyl-3,4-de­ Acrolein-induced liver damage 391 hydropiperidino)lysine (FDP-lysine) (19). Sections NAC that is a glutathione pro-drug and is routinely were then incubated with mouse anti-FDP-lysine an- used to treat acetaminophen-induced liver injury tibody (NOF CORPORATION, Tokyo, Japan) and, (11). Histopathological analysis was conducted to following rinsing with PBS, sections were incubated directly examine hepatic injury (Fig. 1A–D). H&E with peroxidase-conjugated anti-mouse immuno- staining of acetaminophen-treated liver sections re- globulin (Histofine Simple Stain Mouse stain kit) vealed hepatocyte necrosis in the area surrounding (Nichirei Co., Tokyo, Japan). Peroxidase activity on the central veins (Fig. 1B, top, arrows). DNA dam- the sections was developed with 3,3’-diaminobenzi- age in the liver sections was assessed by TUNEL. dine tetrahydrochloride (Nichirei Co.) as the sub- The TUNEL assay was initially developed to deter- strate and then the sections were counterstained with mine apoptosis, but recently we reported that even metyleneblue. The samples were examined with a in necrosis TUNEL-positive signals can be detected microscope (KEYENCE BZ-9000; KEYENCE, (9). Actually, we detected TUNEL-positive signals in Osaka, Japan). the central necrotic regions (Fig. 1B, bottom). In con- trast, NAC- and MESNA-treated livers did not show analysis. Whole liver homogenates any signs of necrosis in both H&E and TUNEL were prepared with BioMasher (nippi, Tokyo, Ja- staining (Fig. 1C and D). pan), and these samples were lysed by modified It is well known that hepatic damage is closely sample buffer [50 mM Tris-HCl (pH 6.8), 2% sodi- correlated with serum ALT levels. Although the ALT um dodecyl (SDS), 1 mg/mL bromophenol levels were increased in acetaminophen-treated mice, blue, 10 mM dithiothreitol, 10% ]. These NAC and MESNA treatment strongly suppressed samples were separated on SDS-12% polyacryl- ALT (Fig. 1E; compare lanes 2 and 3, or lanes 2 amide gel and then electrophoretically transferred and 4, P < 0.05 each). These data together indicate onto nitrocellulose membrane. Membrane immobi- that administration of MESNA prevented acetamino- lized proteins were probed with mouse anti-FDP- phen-induced liver injury as effectively as NAC. lysine monoclonal antibody (NOF CORPORATION) Oxidative stress is hypothesized to be a main or mouse anti-Gapdh monoclonal antibody (Ambion, cause of acetaminophen-induced liver injury. Thus, Austin, USA) and visualized with per- we evaluated the gene expression of Ho-1, an oxi- (HRP)-conjugated rabbit anti-mouse IgG dative stress marker, in the acetaminophen-induced (ZYMED, South San Francisco, USA). liver (17). qPCR analysis showed that Ho-1 expres- sion increased after acetaminophen treatment, and Cell culture. -inactivated fetal calf serum (FCS) that this enhancement was repressed by concomitant was dialyzed against PBS at 4°C. Human Burkitt’s treatment with NAC or MESNA (Fig. 1F, P < 0.05 lymphoma cell line Ramos cells (5 × 104 cells/mL) each). Thus, acetaminophen-induced oxidative stress were cultured in RPMI1640 medium (Nissui Phar- was suppressed by NAC or MESNA treatment. maceutical Co., Tokyo, Japan) supplemented with 50 U/mL streptomycin, 100 U/mL penicillin G, and Reduced acrolein-adduct formation with NAC or 5% dialyzed FCS at 37°C in an of 5% MESNA treatments

CO2 (22). When factors that reduce cell toxicity of We next examined whether acrolein levels are in- acrolein or ROS were investigated, they were added creased in the liver following acetaminophen treat- after the addition of acrolein or ROS in the culture. ment. Acrolein readily reacts with lysine residues in The viable cell number was counted under micro- proteins to form FDP-lysine (19). This reaction is de- scope in the presence of 0.25% trypan blue. Acrole- tectable by western blot analysis with an anti-FDP- in was purchased from KANTO CHEMICAL Co. lysine antibody. As shown in Fig. 2, acrolein-adducts (Tokyo, Japan). NAC, MESNA, vitamin C, and cat- were increased after acetaminophen treatment, alase were purchased from Sigma-Aldrich. whereas treatment with NAC or MESNA suppressed the adduct formation (Fig. 2). This was also demon- strated by an immunohistochemical analysis of FDP- RESULTS lysine (Fig. 3). Necrotic regions surrounding the Protective role of NAC and MESNA in acetamino- central veins, as shown with H&E staining (Fig. 3B, phen-induced hepatic injury top), were stained with the anti-FDP-lysine antibody To investigate the role of acrolein in acetaminophen- in the livers of mice treated with acetaminophen induced liver injury, we administered MESNA to (Fig. 3B, bottom). FDP-lysine positive staining was acetaminophen-treated mice. As a control, we used not evident in those mice co-treated with NAC or 392 T. Arai et al.

Fig. 1 Protective role of NAC and MESNA in acetaminophen-induced hepatic injury. (A–D) Serial sections of acetamino- phen-treated mouse livers were stained with H&E (top) or TUNEL (bottom), and microscopically examined. Arrows: necrotic areas. Scale bar: 500 μm. (E) Serum ALT levels of mice treated with only (lane 1, n = 4), acetaminophen (APAP) alone (lane 2, n = 5), APAP and NAC (lane 3, n = 5), and APAP and MESNA (lane 4, n = 5). Asterisk (*): P < 0.05. (F) q- PCR analysis for Ho-1 gene expression in the liver of mice treated with vehicle only (lane 1, n = 2), APAP alone (lane 2, n = 3), APAP and NAC (lane 3, n = 3), and APAP and MESNA (lane 4, n = 3). Asterisk (*): P < 0.05. (Student’s t-test).

MESNA (Fig. 3C and 3D). These data indicate that acrolein is generated in the acetaminophen-treated liver, and can be inactivated by MESNA and NAC.

Specificity of NAC and MESNA against acrolein Next, we examined whether NAC and MESNA could specifically neutralize acrolein toxicity. ROS,

such as H2O2 and •OH, are toxic compound generat- ed under oxidative stress. According to a protocol by Yoshida et al. (22), we compared the neutraliza- tion activity of NAC or MESNA against acrolein,

H2O2, and •OH (H2O2 with vitamin C). The toxicity was evaluated using Ramos Burkitt’s lymphoma cells. As shown in Fig. 4A, cell toxicity caused by 20 μM acrolein was prevented by either 50 μM Fig. 2 Suppression of acetaminophen-induced acrolein- adduct formation by NAC or MESNA treatments. Acrolein- NAC or 50 μM MESNA, but not by 30 U/mL cata- adducts (FDP-lysine) in liver extracts were detected with lase. The results suggest that the sulfhydryl com- western blot analysis using anti-FDP-lysine antibody. Liver pounds rapidly inactivated acrolein and protected was excised from mice treated with vehicle only (lane 1), the cells from acrolein-mediated toxicity. Cell toxic- acetaminophen (APAP) alone (lanes 2 and 3), APAP and NAC (lanes 4 and 5), and APAP and MESNA (lanes 6 and ity caused by 100 μM H2O2 was prevented by 30 U/ 7). Gapdh protein level was shown as loading control. +/−: mL , but not by 1 mM NAC or 1 mM treatment with/without indicated reagents. MESNA (Fig. 4B). We also tested whether the com- Acrolein-induced liver damage 393

Fig. 3 Acrolein-adduct formation in the damaged area of the liver after acetaminophen treatment. Serial sections of livers of acetaminophen-treated mice were stained with H&E (top) or anti-FDP-lysine antibody (bottom), and microscopically exam- ined. The liver was excised from mice treated with vehicle only (A), acetaminophen alone (B), acetaminophen and NAC (C), and acetaminophen and MESNA (D). Necrotic areas surrounding central veins were shown by arrows (B). Scale bar: 500 μm. pounds could prevent the toxicity of radical •OH, injury (Fig. 1), acrolein-adduct formation (Fig. 2 and which can be generated from H2O2 by adding vita- 3), and acrolein-mediated cell toxicity (Fig. 4). min C (22). H2O2 (10 μM) alone was not sufficient These data strongly suggested that acetaminophen- to induce cell death (Fig. 4C), but the addition of induced liver injury is primarily mediated by acrole-

1 mM vitamin C rendered the same amount of H2O2 in-induced cytotoxicity through adduct formation. fully toxic (Fig. 4D). Cell toxicity caused by the Acrolein is likely generated from lipid peroxidation radical •OH was prevented by 30 U/mL catalase, but and polyamine oxidation via oxidative stress in the not by 1 mM NAC or 1 mM MESNA (Fig. 4D). initially affected hepatocytes following acetamino- These results suggest that NAC and MESNA specif- phen treatment. ically neutralize the cell toxicity of acrolein, but not Recently, acrolein has been implicated as a factor of H2O2 and •OH. in several diseases, including spinal cord injury (4) and brain infarction (12). The pathophysiology of spinal cord injury is characterized by an initial, pri- DISCUSSION mary injury, followed by secondary injury process- Acetaminophen-induced liver injury is hypothesized to es, in which oxidative stress is a critical component. be mediated by the metabolite NAPQI and ROS. In Further, acrolein has been implicated as the media- this study, however, we identified acrolein as anoth- tor of the secondary injury (4). Using a mouse mod- er factor that can exacerbate acetaminophen-induced el of photochemical-induced thrombosis, Saiki et al. liver injury. We found that acrolein-adduct forma- compared the relative importance of acrolein and tion was increased in the necrotic area surrounding ROS in cell damage during brain infarction. They the central vein in the liver after acetaminophen found that acrolein is more strongly involved in cell treatment, and that liver necrosis and acrolein-adduct damage than ROS during brain infarction (12). Our formation were markedly suppressed by treatment results in the acetaminophen-induced liver injury with NAC or MESNA. model also supported this conclusion. Due to its sol- NAC is a known precursor of glutathione, and ubility in water, acrolein can easily move across cell has been clinically used to prevent acetaminophen- membranes. Thus, high concentrations of acrolein induced liver injury. Furthermore, NAC is thought are produced by oxidative stress, and can spread to react directly with •OH and H2O2, thereby reduc- from the dying cells of the primary injury to dam- ing their toxicity (1). Nevertheless, Yoshida et al. age and kill adjacent cells and tissues (10), though recently showed that NAC is a strong scavenger of the way to distinguish primary injury from second- acrolein and directly neutralizes its toxicity (22). ary injury has not been established. Although it has MESNA is also a known scavenger of acrolein. Fol- been widely hypothesized that the major factor re- lowing interaction with acrolein, MESNA forms an sponsible for cell damage under oxidative stress inactivate compound that is detectable by mass conditions is ROS, it now appears that acrolein, a spectrometry (18). Using NAC and MESNA, we de- byproduct of oxidative stress, is more toxic than tected a reduction in acetaminophen-induced liver ROS and promotes secondary injury in various tis- 394 T. Arai et al.

ness of NAC and MESNA in preventing aggravation of these diseases. In conclusion, we found that acrolein is more strongly correlated than ROS with cell damage in acetaminophen-induced liver injury, and propose that acrolein produced under oxidative stress after acetaminophen-treatment can spread from the dying cells of primary injury to damage adjacent cells and aggravate the liver injury.

Acknowledgments This work was supported by Grants-in Aid for Sci- entific Research (C) 21590542 from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan (to RM).

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