Acetaminophen (APAP) overdose is the most frequent cause of drug-induced acute liver failure. Inhibition of APAP metabolic activation and promotion in APAP disposition are important to protect against APAP-induced liver injury. Tumor suppressor p53 is traditionally recognized as a surveillance molecule to preserve genome integrity. Recent studies have emerged on discovering its functions in metabolic regulation. Our previous study reported that p53 promoted bile acid disposition and alleviated cholestastic syndrome. Here, we examined the effect of doxorubicin (Dox)-mediated p53 activation on APAP-induced hepatotoxicity in mice and revealed a novel role of p53 in regulating APAP metabolism and disposition. Histopathological and biochemical assessments demonstrated that administration of Dox (10 mg/kg/d) before APAP treatment (400 mg/kg) signiﬁcantly alleviated APAP-induced hepatotoxicity. Dox treatment prevented APAP-induced GSH depletion and lipid peroxidation. p53-null mice were more susceptible to APAP-induced liver injury. Further, we found that the expression of drug-metabolizing enzymes and transporters CYPs, SULTs and MRPs was regulated by p53. Dox treatment also promoted Nrf2 activation and increased the expression of Nrf2 target genes including GSTα/μ and NQO1, which contribute to APAP detoxiﬁcation. Overall, this study is the ﬁrst to demonstrate the protective role of p53 in regulating APAP metabolism and disposition, which provides a potential new therapeutic target for APAP-induced liver injury. Introduction metabolites . An overdose of APAP saturates the glu- Acetaminophen (APAP) is a widely used analgesic and curonidation and sulfation pathways, which is further antipyretic drug, which is relatively safe and effective at converted to the reactive intermediate N-acetyl-p-benzo- therapeutic doses. However, APAP in high doses leads to quinone imine (NAPQI) by cytochrome P450 (CYPs) . hepatotoxicity, which is recognized as a major cause of NAPQI is normally detoxiﬁed by conjugation to glu- acute liver failure . Under normal condition, APAP is tathione (GSH), which is further excreted in urine by primarily catalyzed by UDP-glucuronosyltransferases multidrug resistance-associated protein (MRP). However, (UGTs) and sulfotransferases (SULTs) to non-toxic the massive accumulation of NAPQI depletes GSH, which induces oxidative stress and ultimately lead to hepato- cellular damage . Glutathione S-transferases (GSTs) and NAD(P)H Quinone Dehydrogenase 1 (NQO1) are Correspondence: Min Huang (firstname.lastname@example.org) or Huichang Bi (email@example.com) involved in the detoxiﬁcation of APAP by regulating GSH School of Pharmaceutical Sciences, Sun Yat-Sen University, 510006 homeostasis . N-acetylcysteine has been clinically used as Guangzhou, China These authors contributed equally: Jiahong Sun, Yajie Wen, Yanying Zhou. Edited by Y. Wang © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Ofﬁcial journal of the Cell Death Differentiation Association 1234567890():,; 1234567890():,; Sun et al. Cell Death and Disease (2018) 9:536 Page 2 of 12 A. APAP 2 h APAP 6 h APAP 24 h 40 400 40 400 40 400 B. C. ALT level AST level 15000 8000 *** Control *** *** *** Dox APAP APAP + Dox 500 *** 0 0 2 h 6 h 24 h APAP 2 h 6 h 24 h APAP Fig. 1 APAP-induced liver injury is attenuated by activation of p53 in mice. a H&E-stained liver sections were visualized at ×40 and ×400, n = 5. * ** *** Activities of serum ALT (b) and AST (c) after Dox and APAP exposure. Data are presented as the mean ± SEM; n = 5–7. P < 0.05, P < 0.01, P < 0.001 versus APAP group the primary antidote of APAP poisoning by replenishing aimed to explore whether p53 exerts the protective effect GSH, which is only effective for the early stage of APAP on APAP-induced hepatotoxicity and to determine the intoxication . Overall, the current therapeutic strategy for underlying mechanism involved in this hepatoprotection. APAP-induced liver injury is not optimal. Inhibition of The result demonstrated for the ﬁrst time that p53 played APAP metabolic activation and promotion in APAP dis- a vital role in APAP-induced hepatotoxicity by regulating position are an effective strategy to protect against APAP- metabolizing enzymes and transporters related to APAP induced liver injury. metabolism and detoxiﬁcation. Tumor suppressor protein p53 traditionally performs as a transcription factor in controlling genomic stability and Results cell growth . p53 is activated upon a wide variety of sti- APAP-induced liver injury is attenuated by activation of muli, which include but are not limited to DNA damage. p53 in mice Our previous study also reported that p53 signaling To investigate the effect of p53 activation on APAP- pathway was associated with compensatory liver regen- induced liver injury, mice were pretreated with Dox, a eration following APAP-induced liver injury . Recently, speciﬁc activator of p53, at 24 h prior to APAP adminis- accumulating ﬁndings revealed the regulatory role of p53 tration. Histological analysis revealed that centrilobular in various metabolic processes, such as glycolysis, oxida- necrosis was induced at 6 h after APAP treatment and 9, 10 tive phosphorylation, and lipid metabolism . Moreover, peaked at 24 h. Much less hepatocellular injury and we recently revealed a novel role of p53 in regulating bile necrosis was observed in Dox/APAP group, indicating the acid metabolism and disposition, which substantially protection of Dox against APAP-induced liver toxicity prevented bile acid metabolic disorders . On the basis of (Fig. 1a). Moreover, starting from 2 h after APAP treat- these data, we hypothesized that p53 regulates APAP ment, a massive hepatic toxicity was induced as revealed metabolism and detoxiﬁcation and further prevents by increased serum levels of ALT and AST. Co-treated APAP-induced hepatotoxicity. Thus, the current study with Dox signiﬁcantly suppressed the elevation of ALT Ofﬁcial journal of the Cell Death Differentiation Association ALT (U/L) APAP Control APAP+Dox AST ( U/L) Sun et al. Cell Death and Disease (2018) 9:536 Page 3 of 12 A. Total GSH level B. Mitochondrial GSH level C. MDA level D. 4-HNE level 150 150 150 ** 100 100 100 200 *** 50 50 50 100 *** 0 0 0 0 APAP - - + + APAP - - + + APAP - - + + APAP - - + + Dox - + - + Dox - + - + Dox - + - + Dox - + - + E. Total GSH level F. Mitochondrial GSH level G. MDA level H. 4-HNE level 150 150 250 ** ### ### *** 100 100 ## *** 50 100 *** 0 0 0 0 APAP - - + + APAP - - + + APAP - - + + APAP - - + + Dox - + - + Dox - + - + Dox - + - + Dox - + - + Fig. 2 Activation of p53 by Dox prevents APAP-induced oxidative damage in liver. Total (a and e) or mitochondrial (b and f) GSH levels, MDA levels (c and g) and a4-HNE levels (d and h) at 2 or 6 h after APAP exposure were measured. Data are presented as the mean ± SEM; n = 5−6. P < ** *** 0.05, P < 0.01, P < 0.001 and AST levels. Compared to control, no signiﬁcant APAP and the severity of liver injury was compared. change of ALT and AST activities was observed in Dox- Histological analysis demonstrated that massive hepatic −/− treated group, indicating that the dosage of Dox used in toxicity was observed in p53 mice at 6 h after APAP +/+ this study did not induce liver toxicity (Fig. 1b, c). exposure. Compared to p53 mice, a more serious liver −/− injury was induced by a 24-h exposure of APAP in p53 Activation of p53 by Dox prevents APAP-induced oxidative mice (Fig. 3a). Although no signiﬁcant difference of ALT −/− +/+ damage in liver level was observed between p53 and p53 mice at 2 −/− APAP-induced liver injury is triggered by GSH depletion, and 6 h after APAP treatment, ALT level in p53 group 12 +/+ which further causes oxidative damage . Thus, the effect was threefold higher than that in p53 group by 24 h of Dox on APAP-mediated oxidative stress in liver was (Fig. 3b). For AST level, no signiﬁcant difference was −/ explored. At 2 h after APAP exposure, both total and observed in these two groups (Fig. 3c). GSH level in p53 − +/+ mitochondrial GSH levels were reduced to 10–20% of mice was signiﬁcantly lower than that in p53 mice at control, which was not preserved by Dox co-treatment 2 and 24 h after APAP exposure, indicating a more serious (Fig. 2a, b). While GSH levels in the Dox/APAP group oxidative stress induced by p53 deﬁciency (Fig. 3d). returned back to normal at 6 h after APAP exposure, Dox Overall, these results indicated that p53-null mice were promotes the synthesis of GSH upon APAP challenge more susceptible to APAP-induced liver injury. (Fig. 2e, f). Lipid peroxidation is a key index of APAP- mediated oxidative damage in liver . Further, the effect of p53 regulates gene expression of drug-metabolizing APAP and Dox on lipid peroxidation was determined by enzymes and transporters measuring MDA levels. No signiﬁcant change was A number of metabolizing enzymes and transporters observed at 2 h after APAP treatment (Fig. 2c). A twofold participated in APAP metabolism and elimination, which increase of MDA level was induced by APAP at 6 h, which were time-dependently measured after Dox and APAP was attenuated by Dox (Fig. 2g).Similarly,APAP induced a treatment. CYP isoforms such as CYP2E1, CYP3A4 signiﬁcant upregulation of 4-HNE level, which was abol- (human ortholog of CYP3A11) and CYP1A2 play the most ished by Dox treatment (Fig. 2d, h). Thus, we concluded important role in APAP bioactivation. Interestingly, we that Dox prevented APAP-induced liver injury by pro- found that gene levels of Cyp1a1/2e1/3a11 were reduced moting GSH recovery and suppressing lipid peroxidation. by APAP and upregulated by Dox treatment (Fig. 4a). The gene expression of Ugt1a1/6 and Sult1a1 was elevated by The severity of APAP-induced liver injury was enhanced in Dox and was not regulated by APAP (Fig. 4b, e). As shown p53 knockout mice in Fig. 4c, the expression of Mrp2/3/4 were also sig- To further explore the role of p53 in APAP-induced niﬁcantly upregulated in both Dox group and Dox/APAP −/− +/+ liver injury, p53 and p53 mice were treated with group. A 24-h treatment of APAP also increased Mrp4 Ofﬁcial journal of the Cell Death Differentiation Association APAP 6 h APAP 2 h % of control % of control % of control % of control % of control % of control % of control % of control Sun et al. Cell Death and Disease (2018) 9:536 Page 4 of 12 A. APAP 6 h APAP 24 h -/- -/- +/+ +/+ p53 p53 p53 p53 B. C. D. ALT level AST level GSH level +/+ +/+ +/+ p53 p53 2500 p53 150 ** -/- -/- -/- p53 p53 p53 ** 2000 50 500 ** 0 0 0 2 h 6 h 24 h APAP APAP 0 h 2 h 6 h 24 h APAP 2 h 6 h 24 h Fig. 3 The severity of APAP-induced liver injury was enhanced in p53 knockout mice. a H&E-stained liver sections after 6 or 24 h after APAP +/+ −/− exposure in p53 and p53 mice. Activities of serum ALT (b), AST (c) and GSH (d) after APAP exposure. Data are presented as the mean ± SEM; ** +/+ n = 5–6. P < 0.01 versus p53 group mRNA level. There was a more than 20-fold increase of measured the protein expression of CYP2E1 after p53 Gstα/μ and Nqo1 mRNA levels in Dox and Dox/APAP activation and depletion. Since mice with a 24-h pre- groups. Notably, a mild upregulation of Gstα/μ and Nqo1 treatment of Dox were collected at 2, 6, and 24 h after expression was also induced by APAP at 24 h (Fig. 4d). APAP exposure, we parallelly measured the expression of To further conﬁrm the regulation of p53 on APAP Nrf2 at 26, 30, and 48 h after Dox treatment alone. As metabolism and elimination, we compared the gene shown in Fig. 6a, Dox treatment alone induced a sig- expression of these metabolizing enzymes and transpor- niﬁcant downregulation of CYP2E1 level at 26 and 30 h. −/− +/+ ters between p53 and p53 mice. Compared to No signiﬁcant difference of CYP2E1 expression was +/+ +/+ −/− p53 mice, mRNA levels of metabolizing enzymes observed between p53 and p53 mice (Fig. 6a). −/− Cyp1a2/3a11 and Ugt1a9 were lower in p53 mice. No Among all the drug-metabolizing enzymes and trans- signiﬁcant difference of Cyp2e1 and Ugt1a1/6 expression porters regulated by p53, the change of Gstα/μ and Nqo1 −/− +/+ was observed between p53 and p53 mice (Fig. 5a, mRNA expression was the most signiﬁcant, which are −/− 5, 13 b). The mRNA level of Sult1a1 in p53 mice was higher well-known Nrf2 downstream target genes .We ﬁrst +/+ than that in p53 mice, which was reduced by APAP at investigated the regulation of p53 on Nrf2 signaling 2 h (Fig. 5e). There was no change of Mrp2/3/4 expression pathway. Nrf2 mRNA and protein levels were elevated by −/− in p53 mice (Fig. 5c). The expression of Gstα/μ and Dox treatment, which were reduced by p53 depletion −/− Nqo1 was signiﬁcantly decreased in p53 mice, which (Fig. 6b and Supplementary Fig. 1). Also, a 24-h exposure was opposite with the change after Dox treatment. of Dox promoted the phosphorylation of Nrf2 (Fig. 6c). As Interestingly, an upregulation of Mrp4, GSTα, and Nqo1 shown in Fig. 6d, Dox alone induced the translocation of levels was induced by APAP regardless of p53 status, Nrf2 into nucleus, which further conﬁrmed the activation indicating that APAP itself also regulates expression of of Nrf2 signaling pathway. We further measured the these drug-metabolizing enzymes and transporters protein expression of GSTα/μ and NQO1 after p53 acti- (Fig. 5c−e). vation or inhibition. At 24 h after APAP exposure, GSTα/ μ and NQO1 protein levels were signiﬁcantly elevated in p53 regulates protein expression of enzymes related to Dox and Dox/APAP groups (Fig. 6e). The protein −/− APAP metabolism and detoxiﬁcation expression of GSTα/μ and NQO1 in p53 mice was +/+ The metabolic activation of APAP is initiated by CYP- lower than that in p53 mice (Fig. 6e). However, a slight mediated conversion to NAPQI, which is the most upregulation of GSTα/μ and NQO1 protein expression proximal event in the toxicity mechanism. CYP2E1 plays was also observed in APAP group, which was consistent the most important role in APAP bioactivation. Here we with the change of mRNA levels (Fig. 6e). Ofﬁcial journal of the Cell Death Differentiation Association Serum ALT U / L Serum AST U / L GSH level (% of control) Sun et al. Cell Death and Disease (2018) 9:536 Page 5 of 12 A. 4 4 4 Cyp1a2 Cyp2e1 Cyp3a11 ### 3 3 3 ** *** *** ** 2 2 2 1 *** 1 ** 1 0 0 0 APAP 2 h 6 h 24 h APAP 2 h 6 h 24 h APAP 2 h 6 h 24 h B. 4 4 4 Ugt1a1 Ugt1a6 Ugt1a9 *** ### ** 3 3 3 *** ### ### *** *** *** ## 2 2 1 1 1 0 0 0 APAP 2 h 6 h 24 h APAP 2h 6h 24 h APAP 2 h 6 h 24 h C. 4 4 5 Mrp2 Mrp3 Mrp4 *** ### ### ** *** *** ** 4 *** 3 3 ### *** ### ** *** 2 2 *** ## ** 1 1 0 0 0 APAP 2h 6h 24 h APAP 2 h 6 h 24 h APAP 2 h 6 h 24 h D. GSTα GSTμ GSTπ ### 50 50 *** *** ### 40 *** 40 ** *** *** ### * *** 30 30 *** ### ## 2 *** ** ** 20 20 *** 10 10 ## 0 0 0 APAP 2 h 6 h 24 h APAP 2 h 6 h 24 h APAP 2 h 6 h 24 h E. 4 Sult1a1 Nqo1 *** ### ** Control ** ### *** ** Dox ** APAP ** APAP + Dox 0 0 APAP 2h 6h 24 h APAP 2 h 6 h 24 h Fig. 4 Dox regulates gene expression of drug-metabolizing enzymes and transporters. a−e mRNA expression of Cyp1a1/2e1/3a11, Ugt1a1/6/9, * ** *** Mrp2/3/4, Gstα/μ/π, Sult1a1, and Nqo1 were measured after Dox and APAP treatment. Data are the mean ± SEM; n = 5. P < 0.05, P < 0.01, P < 0.001 # ## ### versus control group; P < 0.05, P < 0.01, P < 0.001 versus APAP group Ofﬁcial journal of the Cell Death Differentiation Association Fold change of mRNA level Fold change of mRNA level Fold change of mRNA level Fold change of RNA level Fm old change of RNA level Fold change of mRNA level Fold change of mRNA level Fold change of mRNA level Fold change of mRNA level Fold change of mRNA level Fold change of mRNA level Fold change of mRNA level Fold change of mRNA level Fold change of mRNA level Sun et al. Cell Death and Disease (2018) 9:536 Page 6 of 12 A. Cyp1a2 Cyp2e1 Cyp3a11 1.5 2.5 5 2.0 4 *** 1.0 1.5 3 * ** 1.0 2 0.5 ** 0.5 1 * 0.0 0.0 0 APAP 0 h 2 h 6 h 24 h APAP 0 h 2 h 6 h 24 h APAP 0 h 2 h 6 h 24 h B. Ugt1a1 Ugt1a6 Ugt1a9 1.5 1.5 2.5 2.0 1.0 1.0 ** 1.5 1.0 0.5 0.5 0.5 0.0 0.0 0.0 APAP 0 h 2 h 6 h 24 h APAP 0 h 2 h 6 h 24 h APAP 0 h 2 h 6 h 24 h C. Mrp2 Mrp3 Mrp4 2.5 2.0 15 2.0 1.5 1.5 1.0 1.0 0.5 0.5 0.0 0.0 0 APAP 0 h 2 h 6 h 24 h APAP 0 h 2 h 6 h 24 h APAP 0 h 2 h 6 h 24 h D. GSTα GSTμ GSTπ 2.5 40 8 2.0 1.5 1.0 ** 0.5 ** ** * 0.0 0 0 APAP 0 h 2 h 6 h 24 h APAP 0 h 2 h 6 h 24 h APAP 0 h 2 h 6 h 24 h E. Sult1a1 Nqo1 *** +/+ p53 -/- p53 1 *** APAP 0 h 2 h 6 h 24 h APAP 0 h 2 h 6 h 24 h +/+ −/− Fig. 5 Gene expression of drug-metabolizing enzymes and transporters after APAP exposure in p53 and p53 mice. a−e mRNA −/− +/+ expression of Cyp1a1/2e1/3a11, Ugt1a1/6/9, Mrp2/3/4, Gstα/μ/π, Sult1a1, and Nqo1 were measured after APAP treatment in p53 and p53 mice. * ** *** +/+ Data are the mean ± SEM; n = 5. P < 0.05, P < 0.01, P < 0.001 versus p53 group Ofﬁcial journal of the Cell Death Differentiation Association Fold change of RNA level Fold change of mRNA level Fold change of mRNA level Fold change of mRNA level Fold change of mRNA level Fold change of ,RNA level Fold change of mRNA level Fold change of mRNA level Fold change of RNA level Fold change of mRNA level Fold change of mRNA level Fold change ofRmNA level Fold change of mRNA level Fold change of mRNA level Sun et al. Cell Death and Disease (2018) 9:536 Page 7 of 12 Dox 0 h 26 h 30 h 48 h p53 p53 A. CYP2E1 (57 kDa) CYP2E1 (57 kDa) GAPDH (37 kDa) GAPDH (37 kDa) 1.5 1.5 CYP2E1 CYP2E1 1.0 1.0 ** ** 0.5 0.5 0.0 0 p53 Dox 0h 26 h 30 h 48 h p53 Dox 0 h 26 h 30 h 48 h B. C. Nrf2 (97 kDa) Control Dox GAPDH (37 kDa) p-Nrf2 (90 kDa) P53 P53 Nrf2 (97 kDa) β-Acn (43 kDa) GAPDH (37 kDa) p-Nrf2 Nrf2 Nrf2 2.0 1.5 1.5 *** ** ** 1.5 1.0 1.0 *** 1.0 0.5 0.5 0.5 0.0 0 0 Dox 0 h 26 h 30 h 48 h P53 P53 Control Dox D. Control Dox 26 h Dox 30 h Dox 48 h Control Dox APAP Dox + APAP p53 p53 E. GSTα (26 kDa) GSTα (26 kDa) β-Acn (43 kDa) GAPDH (37 kDa) GSTμ (26 kDa) GSTμ (26 kDa) β-Acn (43 kDa) GAPDH (37kDa) NQO1 (31 kDa) NQO1 (31 kDa) β-Acn (43 kDa) GAPDH (37 kDa) GSTα GSTμ NQO1 GSTμ NQO1 1.5 GSTα 1.5 1.5 3 1.5 ** * ** 3 ** 1.0 2 1.0 1.0 1.0 ** 0.5 0.5 1 0.5 0.5 0 0 0 0 0 p53 p53 p53 p53 p53 p53 APAP - - + + APAP - - + + APAP - - + + Dox - + - + Dox - + - + Dox - + - + +/+ −/− Fig. 6 Protein expression of CYP2E1, Nrf2, GSTα/μ, and NQO1 after APAP/Dox exposure in p53 and p53 mice. a, b Liver samples were −/− +/+ subjected to immunoblot with antibodies speciﬁc for CYP2E1 and Nrf2 after Dox treatment alone and in p53 or p53 mice. c Liver samples were subjected to immunoblot with antibodies speciﬁc for phosphorylation of Nrf2 after Dox exposure. d Immunohistochemical staining of Nrf2 in liver section after Dox exposure. e Liver samples were subjected to immunoblot with antibodies speciﬁc for GSTα/μ and NQO1 after Dox and APAP −/− +/+ exposure and in p53 or p53 mice. Quantitation of CYP2E1, GSTα/μ, and NQO1 was normalized to GAPDH or β-actin. Bars represent normalized * ** +/+ # relative densities plotted as mean ± SEM, P < 0.05, P < 0.01 versus control or p53 group; P < 0.05 versus APAP group Ofﬁcial journal of the Cell Death Differentiation Association Fold change of protein level Fold change of protein level Fold change of protein level Fold change of protein level Fold change of protein level Fold change of protein level Fold change of protein level Fold change of protein level Fold change of protein level Fold change of protein level Fold change of protein level Sun et al. Cell Death and Disease (2018) 9:536 Page 8 of 12 A. Mrp2 Mrp3 Mrp4 2.0 1.5 2.0 *** *** 1.5 1.5 1.0 1.0 1.0 0.5 0.5 0.5 0.0 0.0 0.0 Dox (nM) 0 125 250 500 Dox (nM) 0 125 250 500 Dox (nM) 0 125 250 500 Nqo1 Gstα Gstμ 2.5 3 2.0 *** ** *** 2.0 *** 1.5 2 ** 1.5 1.0 1.0 0.5 0.5 0.0 0.0 0 Dox (nM)0 125 250 500 Dox (nM) 0 125 250 500 Dox (nM)0 125 250 500 Control Dox 125 nM Dox 250 nM Dox 500 nM B. Nrf2 DAPI Overlay C. D. Nrf2 ROS level 2.0 *** *** 1.5 *** ### ## 1.0 0.5 0.0 0 Dox (nM) 0 0 125 250 500 0 125 250 500 Dox (nM) 0 125 250 500 APAP (mM) 0 2.5 2.5 2.5 2.5 5.0 5.0 5.0 5.0 Fig. 7 Activation of p53 prevents APAP-induced toxicity in vitro by promoting Nrf2 activation and its downstream target gene expression. a mRNA expression of Mrp2/3/4, Gstα/μ, and Nqo1 was measured after Dox treatment in AML12 cells. b Immunocytochemistry for Nrf2 (Green) and DAPI (blue). Images were captured at 24 h of exposure to Dox by confocal microscopy. c mRNA expression of Nrf2 was measured after a 24-h Dox treatment. d ROS levels were detected by H DCF, and ﬂuorescence was measured by plate reader at 48 h after APAP and Dox exposure. Data are the * ** *** ## ### mean ± SEM; n = 5. P < 0.05, P < 0.01, P < 0.001 versus control group; P < 0.01, P < 0.001 versus APAP group Ofﬁcial journal of the Cell Death Differentiation Association Fold change of mRNA level Fold change of mRNA level Fold change of mRNA level Fold change of mRNA level Fold change of mRNA level Fold change of ROS level Fold change of mRNA level Fold change of mRNA level Sun et al. Cell Death and Disease (2018) 9:536 Page 9 of 12 To characterize the role of Nrf2 in Dox-mediated pro- A. Dox (μM) 0 0 250 250 500 500 tection against APAP toxicity, AML12 cells were trans- Nrf2 siRNA - + - + - + fected with siRNA targeting Nrf2. Nrf2 mRNA expression Nrf2 (97 kDa) was reduced by 50% at 48 h and by 70% at 72 h after transfection (Supplementary Fig. 2). Moreover, GSTα (26 kDa) Nrf2 siRNA transfection signiﬁcantly reduced NRF2 protein expression. Dox treatment induced an upregula- GAPDH (37 kDa) tion of NRF2 protein level in scrambled siRNA- transfected cells. Dox increased protein levels of NQO1 GSTμ (26kDa) and GSTα/μ in scrambled siRNA-transfected cells, which NQO1 (31 kDa) was abolished by Nrf2 knockdown (Fig. 8a). Further, we investigated the protection efﬁcacy of Dox in Nrf2- GAPDH (37 kDa) knockdown AML12 cells. After 48 h of Nrf2 siRNA transfection, cells were treated with APAP and Dox for 24 B. ROS level h and intracellular ROS levels were measured. Compared to normal AML12 cells, the inhibition of APAP-induced ROS accumulation by Dox was attenuated by 150 Nrf2 silencing (Fig. 8b). Discussion The present study demonstrated the protective effect of p53 on APAP-induced liver toxicity by regulating the Nrf2 siRNA + + + + expression of drug-metabolizing enzymes and transpor- APAP (mM) 0 2.5 2.5 2.5 ters, which enhanced APAP metabolism and suppressed Dox (nM) 0 0 250 500 oxidative damage. p53-null mice were more susceptible to Fig. 8 The protective effect of p53 against APAP toxicity is Nrf2- APAP-induced liver injury. Moreover, p53 promoted Nrf2 dependent. a AML12 cells were transfected with scrambled and activation and the transcription of its downstream target Nrf2 siRNA for 36 h and further treated with Dox for 48 h. Cell samples were subjected to immunoblot with antibodies speciﬁc for GSTα/μ genes related to APAP detoxiﬁcation. and NQO1. b AML12 cells were transfected with Nrf2 siRNA for 48 h Under the catalysis of UGTs and SULTs, APAP is pri- and further treated with APAP and Dox for 24 h. Intracellular ROS were marily converted to pharmacologically inactive glucuronide measured by H DCF. Data are the mean ± SEM; n = 5. *P < 0.05 versus and sulfate conjugates, which is further excreted into the control group urine or bile by MRPs . An increase of UGTs, SULTs, and MRPs expression was observed after p53 activation, indi- Activation of p53 prevents APAP-induced toxicity in vitro cating that Dox promotes the conversion of APAP to non- by promoting Nrf2 activation and its downstream target toxic metabolites as well as the elimination of APAP gene expression metabolites. The metabolic activation of APAP is initiated To further elucidate the protective mechanism of p53 by CYP-mediated conversion to NAPQI, which is the most against APAP toxicity, we treated AML12 cells with Dox proximal event in the toxicity mechanism . NAPQI causes (125–500 nM) and also found a dose-dependent upregu- oxidative stress and binds covalently to liver proteins, lation of mRNA levels of Mrp2/3/4, Nqo1, and GSTα/μ which is normally detoxiﬁed by GSH both none- (Fig. 7a). Next, we investigated if the protective effect of nzymatically and enzymatically in a reaction catalyzed by 15, 16 p53 worked through the activation of Nrf2. There was a GSTs . In this study, we found that a slight increase of clear translocation of Nrf2 (Fig. 7b, green) into the these CYPs mRNA expression was induced by Dox. p53 nucleus (Fig. 7b, blue) at 24 h after Dox exposure, which is deﬁciency did not induce a signiﬁcant change of CYP2E1 necessary for Nrf2 activation. Moreover, the mRNA protein expression, which was consistent with previously expression of Nrf2 was elevated by Dox treatment, indi- published study . While Dox treatment suppressed the cating that p53 enhanced the activity of Nrf2 signaling CYP2E1 level, activation of p53 might inhibit APAP pathway (Fig. 7c). Further, AML12 cells was exposed with bioactivation. There was also a profound increase of GSTs APAP (2.5–5 mM) and Dox, and determined whether expression after Dox exposure. Moreover, NQO1, which Dox inhibits APAP-induced intracellular ROS accumu- controls redox homeostasis and facilitates adaptation of lation. Similarly, intracellular ROS level was elevated by cells to oxidative stress, was also upregulated by p53 acti- fourfold after a 48-h exposure of APAP, which was vation. The higher levels of these enzymes that are ameliorated by the simultaneous treatment of Dox responsible for APAP detoxiﬁcation account for the more (Fig. 7d). effective protection by intracellular antioxidants to APAP Ofﬁcial journal of the Cell Death Differentiation Association Fold change of ROS level Sun et al. Cell Death and Disease (2018) 9:536 Page 10 of 12 challenge. Therefore, we concluded that activation of p53 protective effect of Dox is achieved by promoting Nrf2 effectively prevents APAP accumulation by promoting activation and the transcription of its downstream target APAP metabolism and elimination. It is important to note genes related to APAP detoxiﬁcation. It is very important to that the expression of several enzymes was upregulated by note that the Nrf2 signaling pathway may not be the only −/− Dox, which was not signiﬁcantly changed in p53 mice. factor contributing to the protection of Dox against APAP- This observation was consistent with our previously pub- induced liver injury. Our previous study demonstrated that p53 directly regulates metabolizing enzymes and transpor- lished results . We found that Dox induced the upregu- lation of MRP2/3/4 protein expression, which was ters such as CYP2B6 and MRP3, which are related to APAP abolished by p53 deﬁciency, indicating the p53-dependent metabolism and detoxiﬁcation . Published study also regulation of these enzyme expressions by Dox. Only reported that p53 directly activated CYP3A4 transcription . MRP4 expression was reduced by p53 deﬁciency. There In the current study, CYPs related to APAP bioactivation was no signiﬁcant difference of MRP2/3 levels between was also found to be regulated by p53. Therefore, p53 might +/+ −/− p53 and p53 mice. We speculated that many sig- also directly regulate the transcription of CYPs related to naling pathways (Nrf2, PI3K/Akt, and MEK/ERK signaling APAP metabolism, which needs to be addressed in future pathways) also have been shown to involve in the regula- studies. tion of MRPs expression, which induced a compensatory p53 is a tumor suppressor that plays an important role regulation of these enzymes in response to the depletion of in regulating cell growth, DNA repair, and apoptosis. 18, 19 p53 . Under severe DNA damage by APAP overdose, p53 is An elevation of ALT level was induced at 2 h after activated to inhibit cell proliferation or trigger cell apop- APAP exposure, which was prevented by Dox. GSH tosis . Moreover, our previous study also reported that depletion induced by APAP was not reversed by Dox. dynamic and coordinated regulation of Nrf2 and Also, no signiﬁcant change of lipid peroxidation was p53 signaling pathways was associated with compensatory observed after APAP and Dox treatment. In contrast, both liver regeneration after APAP-induced acute liver injury . ALT and AST levels were dramatically reduced by Dox at As shown in Supplementary Fig. 3, the expression of core −/ 6 h after APAP exposure. Moreover, Dox prevented cell cycle protein CDK4 and cyclin D1 was lower in p53 − +/+ APAP-induced GSH depletion and accumulation of lipid mice than that in p53 mice. Therefore, suppression peroxidation, indicating that the maximum protective of p53 activity might prevent cell apoptosis and promote effect of Dox was achieved at 6 h after APAP exposure. compensatory liver regeneration, which might contribute Starting from 2 h after APAP exposure, a more than to a relatively mild liver injury at early time points of tenfold increase of GSTs and NQO1 levels was induced by APAP exposure . Consistent with published study, APAP Dox, which led us to investigate whether these detoxifying induced a more severe damage in liver at 24 h after APAP enzymes play a major role in the defense against APAP exposure . We speculated that suppression of p53 fails to toxicity. enhance APAP metabolism and detoxication regulated by Nrf2 regulates the expression of a battery of cytoprotective metabolizing enzymes and transporters, which was posi- genes encoding intracellular detoxifying enzymes, including tively correlated with this exacerbated liver damage. MRPs,NQO1,and GSTs,which areresponsible forAPAP Overall, this experiment was done in an effort to com- elimination and detoxiﬁcation . It has been known that prehensively investigate the regulatory role of p53 on activation of Nrf2 confers potent resistance against acute APAP metabolism. This study is the ﬁrst to demonstrate drug toxicity . Our results showed that Dox increased Nrf2 that p53 prevents against APAP-induced liver injury by expression and promoted the activation and translocation of regulating the metabolizing enzymes and transporters Nrf2 into the nucleus, indicating that p53 enhanced the related to APAP detoxiﬁcation and elimination. These activity of Nrf2 signaling pathway. Previous published study signiﬁcant data may provide a clinically relevant argument also reported that p21, a p53 target gene stabilizes Nrf2 by for using p53 against APAP-induced acute liver injury. binding to Keap1 and interfering with its ability to promote Nrf2 ubiquitylation and proteasomal degradation .Inlight Methods of the role of Nrf2 in the protection of p53 against APAP- Animal experiments induced liver damage, Nrf2 was depleted using siRNA Male C57BL/6 mice (6–8 weeks old) were obtained transfection to study whether lack of this transcription factor from Laboratory Animal Center of Sun Yat-Sen Uni- renders hepatocytes more vulnerable to APAP toxicity after versity (Guangzhou, China). p53 knockout and paired p53 activation. Our results illustrated that Dox suppressed wild-type mice (19–20 g) were established by NIFDC and APAP-induced ROS accumulation, which was abolished by Beijing Biocytogen Co., Ltd and purchased from the Nrf2 silencing. Moreover, Dox failed to upregulate the National Center of Laboratory Rodents. All animals were expression of NQO1 and GSTs in Nrf2-knockdown hepa- maintained under controlled conditions (22–24 °C, tocytes. Overall, our study allows us to speculate that the 55–60% humidity, and 12-h light/dark cycle) with free Ofﬁcial journal of the Cell Death Differentiation Association Sun et al. Cell Death and Disease (2018) 9:536 Page 11 of 12 access to standard food and water. All procedures fol- GSH measurement lowed the Regulations of Experimental Animal Adminis- GSH levels of total or mitochondria extracts in the tration issued by the Ministry of Science and Technology liver were measured using a GSH assay kit (Nanjing of the People’s Republic of China. Animal protocols were Jiancheng Bioengineering Institute, Nanjing, China). approved by the Ethics Committee on the Care and Use of Liver mitochondria were isolated by differential cen- Laboratory Animals of Sun Yat-Sen University. All ani- trifugation following the assay kit’s instruction (Sangon mals were fasted 3 h or overnight before APAP adminis- Tech). tration, since the severity of APAP-induced could be regulated by fasting period . A relative mild liver injury qRT-PCR analysis +/+ −/− model was used in p53 and p53 mice by reducing qRT-PCR analysis for the expression of target genes in the fasting time to 3 h. Dox (Aladdin Company, Shanghai, mouse livers and AML12 hepatocytes was performed as China) at 10 mg/kg or saline was administered to WT described previously . The sequences of gene-speciﬁc mice by intraperitoneal injections at 24 h before APAP primers were listed in Supplementary Table 1. administration. APAP (Sigma-Aldrich, St. Louis, MO) solution was made fresh in 0.9% saline at 40 mg/ml, and Western blot analysis mice were administered a single dose of 400 mg/kg APAP Proteins extracted from mice liver tissue or AML12 by intraperitoneal injection. Mice were killed at 0, 2, 6, hepatocytes were prepared using radioimmunoprecipitation and 24 h after APAP treatment. Serum samples and liver assay lysis buffer (Biocolor BioScience and Technology, tissues were harvested. Tissues were ﬂash frozen in liquid Shanghai, China) and were determined by the BCA Pro- nitrogen and stored at –80 °C for further use. tein Assay (Thermo Scientiﬁc, Rockford, IL). Protein extract was separated in 8–12% SDS-PAGE and electro- Cell culture and treatment phoretically transferred onto polyvinylidene ﬂuoride AML-12 cells, a nontumorigenic mouse hepatocyte cell membranes. After blocking with 5% non-fat dry milk in line, were obtained from the American Type Culture Tris-buffered saline, membranes were incubated over- Collection (ATCC, Manassas, VA) and cultured in night with primary antibodies, including NRF2 (Cell Sig- DMEM/F12 containing 0.005 mg/ml insulin, 0.005 mg/ml naling Technology, Shanghai), phosphate NRF2 (Abcam), transferrin, 5 ng/ml selenium, 40 ng/ml dexamethasone CYP2E1 (Cell Signaling Technology, Shanghai), NQO1 and 10% FBS at standard cell culture conditions (5% CO , (Sangon Biotech, Shanghai), GSTα (Sangon Biotech, Shanghai), GSTμ (Sangon Biotech, Shanghai), CDK4 95% air). 100 nM negative small interference RNA (siRNA) or siRNA targeting at Nrf2 (RiboBio, Guangzhou, (Sangon Biotech, Shanghai), Cyclin D1 (Cell Signaling China) were transiently transfected using lipofectamine Technology, Shanghai), β-actin (Santa Cruz Biotechnol- 2000 siRNA Transfection Reagent (Roche diagnostics, ogy, Dallas), and GAPDH (Santa Cruz Biotechnology, Mannheim, Germany). Cells were treated with Dox (125 Dallas, TX). A secondary horseradish peroxidase- −500 nM) or APAP (2.5−5 mM) at 48 h after siRNA conjugated anti-rabbit or anti-mouse IgG antibody transfection and incubated for 24−48 h. (Santa Cruz Biotechnology, Dallas) was subsequently applied, and then speciﬁc bands were visualized using an Histologic evaluation enhanced chemiluminescence detection kit (Engreen Tissues were immediately ﬁxed in formaldehyde, Biosystem, Beijing, China). embedded in parafﬁn, sectioned, and stained with hema- toxylin and eosin stain (H&E) according to a standard Immunocytochemistry protocol. H&E-stained liver sections were examined using a AML-12 cells were ﬁxed with 4% paraformaldehyde Leica DM5000B microscope (Leica, Heidelberg, Germany). (Sigma-Aldrich) in PBS for 15 min after respective expo- sures. The cells were permeabilized with 0.25% Triton X- Biochemical evaluation 100 for 10 min and were then incubated in 10% serum APAP-induced liver injury was evaluated by measuring (Sigma-Aldrich) blocking solution containing 0.3 M gly- serum alanine transaminase (ALT) and aspartate transa- cine for 30 min. Cells were exposed to anti-Nrf2 antibody minase (AST) catalytic activities. ALT and AST levels (1:100 in blocking solution; Santa Cruz Biotechnology, were analyzed using commercial kits (Kefang Biotech, Dallas) overnight at 4 °C, followed by incubation with Guangzhou, China) on a Beckman Synchron CX5 Clinical appropriate ﬂuorescence-conjugated secondary anti- System (Beckman Coulter, Brea, CA) according to the bodies (Molecular Probes) for 1 h. Nuclei were counter- manufacturer’s protocol. To assess lipid peroxidation stained with 49,6-diamidino-2-phenylindole (DAPI). induced by APAP, levels of malondialdehyde (MDA) were Images were acquired using a ﬂuorescence confocal also determined using commercially available kits (Nanj- microscope (Zeiss Violet Confocal; Zeiss, Oberkochen, ing Jiancheng Bioengineering Institute, Nanjing, China). Germany) with a ×40 objective. Ofﬁcial journal of the Cell Death Differentiation Association Sun et al. Cell Death and Disease (2018) 9:536 Page 12 of 12 ROS detection 4. McGill, M. R. et al. The mechanism underlying acetaminophen-induced hepatotoxicity in humans and mice involves mitochondrial damage and Changes in intracellular ROS were measured by the ROS- nuclear DNA fragmentation. J. Clin. Invest. 122, 1574–1583 (2012). reactive ﬂuorescent indicator 29,79-dichlorodihydro- 5. Chan, K., Han, X. D. & Kan, Y. W. An important function of Nrf2 in combating ﬂuorescein diacetate (H DCFDA) (Molecular Probes). After oxidative stress: detoxiﬁcation of acetaminophen. Proc. Natl. Acad. Sci. USA 98, 4611–4616 (2001). respective exposures, the medium was removed and the cells 6. Saito, C., Zwingmann, C. & Jaeschke, H. Novel mechanisms of protection were washed once with PBS and then incubated with 10 mM against acetaminophen hepatotoxicity in mice by glutathione and N- acetylcysteine. Hepatology 51,246–254 (2010). H DCFDA for 30 min at 37 °C. Mean ﬂuorescence intensity 7. Goldstein, I. et al. Understanding wild-type and mutant p53 activities in of DCF was measured using the BioTek Synergy H1 Hybrid human cancer: new landmarks on the way to targeted therapies. Cancer Gene plate reader (excitation, 485 nm; emission, 530 nm). DCF Ther. 18,2–11 (2011). ﬂuorescence was standardized based on cell viability. 8. Fan, X. et al. Dynamic and coordinated regulation of KEAP1-NRF2-ARE and p53/p21 signaling pathways is associated with acetaminophen injury responsive liver regeneration. Drug Metab. Dispos. 42,1532–1539 (2014). Statistical analysis 9. Bensaad, K. et al. TIGAR, a p53-inducible regulator of glycolysis and apoptosis. The data are shown as means ± SEM. Statistical analyses Cell 126,107–120 (2006). 10. Goldstein, I. et al. p53, a novel regulator of lipid metabolism pathways. J. were performed using one-way analysis of variance with Hepatol. 56,656–662 (2012). Tukey’s post-hoc test. GraphPad Prism 5.0 (GraphPad 11. Chen, P. et al. p53-mediated regulation of bile acid disposition attenuates Software, Inc., La Jolla, CA) was used for statistical analyses. cholic acid-induced cholestasis in mice. Br.J.Pharmacol. 174,4345–4361 (2017). 12. Du, K., Ramachandran, A. & Jaeschke, H. Oxidative stress during acet- Acknowledgements aminophen hepatotoxicity: sources, pathophysiological role and therapeutic This study was ﬁnancially supported by the Natural Science Foundation of potential. Redox Biol. 10, 148–156 (2016). China (Grant: 81522047, 81703599, 81573489, 81730103, 81320108027), the 111 13. Aleksunes, L. M. et al. Induction of Mrp3 and Mrp4 transporters during acet- project (Grant: B16047), the Key Laboratory Foundation of Guangdong aminophen hepatotoxicity is dependent on Nrf2. Toxicol. Appl. Pharmacol. Province (Grant: 2011A060901014), the Natural Science Foundation of 226,74–83 (2008). Guangdong (Grant: 2015A030313124, 2017A030310330), and the National Key 14. McGill, M. R. & Jaeschke, H. Metabolism and disposition of acetaminophen: Research and Development Program (Grant: 2017YFC0909303). recent advances in relation to hepatotoxicity and diagnosis. Pharm. Res. 30, 2174–2187 (2013). Conﬂict of interest 15. Jaeschke, H.,Xie,Y.& McGill, M. R. Acetaminophen-induced liver Injury: from The authors declare that they have no conﬂict of interest. animal models to humans. J. Clin. Transl. Hepatol. 2,153–161 (2014). 16. Zheng, L. et al. 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Published: May 10, 2018
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