Accepted Manuscript

Title: Pyrazinamide-induced hepatotoxicity is alleviated by

4-PBA via inhibition of the PERK-eIF2␣-ATF4-CHOP
pathway

Author: <ce:author id="aut0005"

author-id="S0300483X17300033-
956716e302044b18c1307fead1d80abd"> Hong-li
Guo<ce:author id="aut0010"
author-id="S0300483X17300033-
a360f03cefb531a965390c627ddc1b15"> Hozeifa M.
Hassan<ce:author id="aut0015"
author-id="S0300483X17300033-
b02f672df504602c9f7c12bed2421200"> Ping-ping
Ding<ce:author id="aut0020"
author-id="S0300483X17300033-
70b7844513d14d657509e1f9347ee5a5"> Shao-jie
Wang<ce:author id="aut0025"
author-id="S0300483X17300033-
2ef79ac2fd2757577a2ef43970fccaf9"> Xi Chen<ce:author
id="aut0030" author-id="S0300483X17300033-
3b7f20e7469f86404fb4ba1f7a7e78ab"> Tao Wang<ce:author
id="aut0035" author-id="S0300483X17300033-
ef26e4b28aeb204433066f64ededd582"> Li-xin
Sun<ce:author id="aut0040"
author-id="S0300483X17300033-
bb130c3850e1978546430ef48dfd43a8"> Lu-yong
Zhang<ce:author id="aut0045"
author-id="S0300483X17300033-
9ea5dc03d899ac01511a308da0bc80af"> Zhen-zhou
Jiang

PII: S0300-483X(17)30003-3
DOI: http://dx.doi.org/doi:10.1016/j.tox.2017.01.002
Reference: TOX 51805

To appear in: Toxicology

Received date: 29-10-2016

Revised date: 11-12-2016
Accepted date: 3-1-2017
Please cite this article as: Guo, Hong-li, Hassan, Hozeifa M., Ding, Ping-
ping, Wang, Shao-jie, Chen, Xi, Wang, Tao, Sun, Li-xin, Zhang, Lu-yong,
Jiang, Zhen-zhou, Pyrazinamide-induced hepatotoxicity is alleviated by 4-
PBA via inhibition of the PERK-eIF2␣-ATF4-CHOP pathway.Toxicology
http://dx.doi.org/10.1016/j.tox.2017.01.002

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Pyrazinamide-induced hepatotoxicity is alleviated by 4-PBA via

inhibition of the PERK-eIF2α-ATF4-CHOP pathway

Hong-li Guo1,2, Hozeifa M. Hassan2,6 Ping-ping Ding2, Shao-jie Wang2, Xi Chen2,

Tao Wang2,3, Li-xin Sun3,4, Lu-yong Zhang2,3,5*, Zhen-zhou Jiang2,4*

Pyrazinamide induces hepatotoxicity via activating ERS

1 Children’s Hospital of Nanjing Medical University, Nanjing, 210008, China

2 Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University,

Nanjing 210009, China;

3 Key Laboratory of Drug Quality Control and Pharmacovigilance (China

Pharmaceutical University), Ministry of Education, Nanjing 210009, China;

4 Jiangsu Key Laboratory of TCM Evaluation and Translational Research, China

Pharmaceutical University, Nanjing 211198, China.

5 Jiangsu Center for Pharmacodynamics Research and Evaluation, China

Pharmaceutical University, Nanjing 210009, China;

6 Department of Pharmacology, Faculty of Pharmacy, University of Gezira,

Wad-Medani, Sudan

Corresponding author: Lu-yong Zhang, lyzhang@cpu.edu.cn; Zhen-zhou Jiang,

beaglejiang@cpu.edu.cn 

1 
 
Graphical abstract

Highlights

Pyrazinamide induces hepatotoxicity in vitro and in vivo via activating ER stress

Pyrazinamide activates the PERK-eIF2α-ATF4-CHOP pathway

Sodium 4-phenylbutyrate ameliorates the hepatotoxicity of Pyrazinamide

Abstract
Pyrazinamide (PZA)-induced serious liver injury, but the exact mechanism of

PZA-induces hepatotoxicity remains controversial. Endoplasmic reticulum (ER)

stress-caused cell apoptosis plays a critical role in the development of drug-induced

liver injury (DILI). However, the direct connection between PZA toxicity and ER

stress is unknown. In this study, we describe the role of ER stress in PZA induced

hepatotoxicity in vivo and in vitro. We found that PZA induces apoptosis in HepG2

cells, and causes liver damage in rats, characterized by increased serum ALT, AST and

2 
 
TBA levels. PZA impairs antioxidant defenses, although this effect did not play an

important role in resulting liver injury. The ER stress related proteins GRP78,

p-PERK, p-eIF2α, ATF4, CHOP and caspase12 were activated after PZA exposure

both in vivo and in vitro. Furthermore, as an ER stress inhibitor, sodium

4-phenylbutyrate (4-PBA) could ameliorate PZA toxicity in HepG2 cells and rat liver.

These results have potential implications for the pathogenesis of PZA-induced

hepatotoxicity in which ER stress especially PERK-eIF2α-ATF4-CHOP pathway

participates in hepatocellular injury.

Key words: Pyrazinamide, Endoplasmic reticulum (ER) stress, hepatotoxicity,

PERK-eIF2α-ATF4-CHOP pathway, sodium 4-phenylbutyrate

1 Introduction:

Pyrazinamide (PZA) remains one of the first-line drug for the therapy of

tuberculosis (Shi et al. 2011) even though its use is associated with severe liver injury

(Kunimoto et al. 2003). Studies have shown that PZA induced a dose-dependent and

sex-related hepatotoxicity. PZA also caused idiosyncratic hepatotoxicity which may

occur at any moment during chemotherapy (Chang et al. 2008). A study confirmed

that the PZA metabolite 5-hydroxypyrazinoic acid (5-OH-PA) was responsible for

PZA-induced liver injury (Shih et al. 2013). However, PZA-induced liver damage

mechanism is unknown and remains controversial.

Endoplasmic reticulum (ER) stress occurs when misfolded and unfolded proteins

accumulated in the ER. ER stress has been implicated in some liver diseases such as

3 
 
viral hepatitis, fatty liver disease, and drug induced hepatotoxicity (Bozaykut et al.

2016). Glucose-regulated protein 78 (Grp78) is the master regulator of ER

homeostasis which interacts with the three stress sensors (Zhu and Lee 2015).

Inositol-requiring protein 1 (IRE1), PKR-like endoplasmic reticulum kinase (PERK)

and activating transcription factor-6 (ATF-6) were the three dominant stress sensors

involved in unfolded protein response (UPR)-signaling pathways, which activation

was shown to mediate ER stress (Liu et al. 2015). PERK phosphorylates eukaryotic

initiation factor 2 alpha (eIF2a) resulting in translation of activating transcription

factor-4 (ATF-4), further to increase recruitment of the transcription factor C/EBP

homologous protein (CHOP) (Joo et al. 2015), which is linked to cell apoptosis. A

growing body of evidence suggests that ER stress is involved in toxicity, especially

chemicals-induced apoptosis as studied with in vivo and/or in vitro models (Huo et al.

2015).

It was considered that ER stress is participated in pathologic pathways including

oxidative stress, peroxisome proliferator-activated receptor (PPAR) signaling and

apoptosis (Hanke et al. 2016; Olivares and Henkel 2015). Our previous study

indicated that PZA caused liver injury was related to these pathways (Zhang et al.

2013). However, the direct connection between ER stress and PZA-induced

hepatotoxicity was unclear. Hence, in this study, several protein markers of ER stress

were used to assess the incidence of ER stress following PZA treatment both in vivo

and in vitro. We revealed that PZA treatment induced ER stress and the ER stress

inhibitor sodium 4-phenylbutyrate (4-PBA) could ameliorate PZA toxicological

4 
 
effects.

2 Materials and methods

2.1 Chemicals and antibodies

PZA (PZA, CAS number 335-67-1, 98% purity), sodium 4-phenylbutyrate

(4-PBA, CAS number 100 1716-12-7, 98% purity), were obtained from

Sigma-Aldrich (St. Louis, MO, USA). Antibodies used in this study are listed in

Supplementary Table S1. Primers for real-time PCR analysis are listed in

Supplementary Table S2. All other chemicals used were of the highest grade

commercially available.

2.2 Animal treatment

Female Wistar rats (170-190 g) were obtained from Beijing Vital River

Experimental Animals Centre (Beijing, China). First, rats were randomly divided into

control (n=6) and treatment groups (n=36) by oral gavage with 0.5% CMC-Na or

dose of PZA (2.0 g/kg/d) for 6 consecutive days to investigate whether PZA can

induce hepatotoxicity and liver ER stress. 2 h after each administration, 6 rats in

PZA-treated group were sacrificed under diethyl ether anesthesia to collect blood

samples and liver tissue (marked as control, 2 h, 26 h, 50 h, 74 h, 98 h and 122 h

group). The dose of 2.0 g/kg selected in this study has been referenced in previous

literature (Guo et al. 2016; Zhang et al. 2013), and exceeds the human therapeutic

dose by 11-fold according to body surface area conversion, based on species

sensitivity guidelines from the FDA (Shih et al. 2013). From the initial study results,

5 
 
we chose an appropriate time in which PZA-induced obvious hepatotoxicity and ER

stress to conduct the second experiment. Rats were randomly divided into four groups;

control, 4-PBA (100 mg/kg), PZA (2.0 g/kg/d) and PZA+4-PBA. The 4-PBA solution

used in the animal experiments was prepared by titrating equimolecular amounts of

4-phenylbutyric acid and sodium hydroxide to pH 7.4 (Ricobaraza et al. 2009), which

was administered intraperitoneally 30 min prior to PZA treatment. All animal

treatments were approved by the Committee on the Ethics of Animal Experiments

from the China Pharmaceutical University, and in compliance with the Guiding

Principles in the Use of Animals in Toxicology, which were adopted by the Society of

Toxicology in 1989.

2.3 Serum biochemical assay

Blood was collected in anticoagulant-free tubes and serum was separated by

centrifugation at 3,500 g for 10 min at 4°C. Hepatic injury was assessed by measuring

serum levels of alanine transaminase (ALT), aspartate transaminase (AST) and total

bile acids (TBA) using an automatic clinical analyzer (7080, HITACHI Ltd., Tokyo,

Japan).

2.4 Histopathology

Liver slices from individual rats (n=6 each group) were prepared for histological

examination, stained with hematoxylin and eosin (H&E) and examined by light

microscopy (200 x). Histological assessment was graded using the histological

activity index (HAI) according to the criteria of Knodell et al.22

6 
 
2.5 Cell culture and treatments

The human hepatocarcinoma cell line HepG2 (American Type Culture Collection,

Manassas, VA, USA) was cultured in Dulbecco’s modified Eagle’s medium (DMEM)

containing 10% fetal bovine serum (FBS) on 75-cm2 tissue culture flasks at 37°C in a

humidified atmosphere composed of 95% air and 5% CO2。Drugs were dissolved in

DMEM containing 10% FBS and filter sterilized (0.22 μm Millipore filter, Millipore,

USA). Cells were seeded to appropriate density on tissue culture plates. Cell culture

medium was replaced with media containing PZA (0, 10, 25, 50, 75, 100 mM) or

4-PBA (0.5 mM) following overnight incubation. PZA concentrations chosen in this

study were obtained from previous studies (Tostmann et al. 2010).

2.6 Determination of cell viability and cell apoptosis

Cell viability was measured by the CCK-8 method. Cytotoxicity was measured

by the LDH activity in the supernatant using the LDH Cytotoxicity Assay Kit

(Beyotime Institute of Biotechnology, Nanjing, China) according to the

manufacturer’s instructions. Cell apoptosis was measured using Annexin V-FITC

Apoptosis Detection Kit (BD Pharmingen, USA) according to the manufacturer’s

instructions on the FACSCalibur flow cytometer (BD Biosciences, CA).

2.7 Determination of cellular reactive oxygen species (ROS) and mitochondrial

transmembrane potential (MTP)

Cellular ROS contents and MTP levels were measured by flow cytometry. Briefly,

5 × 105 cells were plated on 60-mm dishes, allowed to attach overnight, and exposed

7 
 
to different concentrations of PZA for 24 h. Cells were stained with 10 μM DCFH-DA

or 10 μM JC-1 (Beyotime Institute of Biotechnology, Nanjing, China) at 37°C for 30

min. Cells were collected and the fluorescence was analyzed using a FACSCalibur

flow cytometer (BD Biosciences, CA).

2.8 Determination of T-SOD, MDA, T-AOC and GSH activities

The total superoxide dismutase (T-SOD, U in mg protein) and malondialdehyde

(MDA, nmol in mg protein) activity in HepG2 cells and rat liver was analyzed using a

SOD assay kit and a MDA assay kit (Beyotime Institute of Biotechnology, Nanjing,

China) respectively.

The total antioxidant capacity (T-AOC, unit in mg protein) and glutathione (GSH,

mg in g protein) activity in rat liver was analyzed using a T-AOC assay kit and a GSH

assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) respectively.

2.9 Real-time PCR analysis

Total RNA was extracted with TRIzol (Vazyme Biotech, Nanjing, China) and was

reverse-transcribed to obtain cDNA. Quantitative reverse transcription-PCR was

performed using the SYRBGreen PCR Master Mix (Vazyme Biotech, Nanjing, China).

Gene expression was evaluated by the ΔΔCT method, using GAPDH as a reference

gene.

2.10 Western blotting

Protein was extracted from the liver tissue or cells in RIPA buffer together with

phosphatase and protease inhibitors. Cell lysates were mixed with 4X Laemmlisample
8 
 
buffer, boiled and separated by SDS-PAGE. Proteins were transferred to a PVDF

membrane (Bio-Rad, Hercules, CA). After non-specific blocking with skim milk or

BSA for 1 hour, the membranes were probed with primary antibody (1:500–1000) in 5

ml blocking buffer overnight at 4 °C. Next, the membranes were washed 4 times with

Tris-buffered saline with 0.1% Tween 20 (TBST) and incubated with an appropriate

HRP-conjugated secondary antibody. Membranes were further washed 4 times with

TBST, incubated with an ECL solution (Millipore, USA) and digitally imaged with a

CCD camera.

2.11 Statistical analysis

Values were reported as mean ± SD. Student's t-test was used, where applicable.

ANOVA analysis was applied to assess interactions between groups and differences

between means. p<0.05 was accepted as statistically significant. All represented data

from in vitro experiments were assessed from at least three independent experiments.

3 Results

3.1 PZA treatment induced liver injury both in vitro and in vivo

HepG2 cells were used to test the toxicity of PZA in vitro. The CCK-8 assay

showed that HepG2 cells exposure to different concentrations of PZA for 24 hours

had reduced cell viability with an IC50 of 87 mM (Fig. 1A). As in Fig. 1B, PZA

significantly increased LDH levels in cell supernatant at 10 mM (2-fold, p < 0.05), 25

mM (2.1-fold, p < 0.05), 50 mM (2.5-fold, p < 0.01), 75 mM (5-fold, p < 0.001) and

100 mM (4-fold, p < 0.01) respectively, which was an indicator of cellular membrane

9 
 
damage (Fotakis and Timbrell 2006). To evaluate the apoptotic effects of PZA, the

apoptotic cells were detected by Annexin V-FITC/PI double staining and

cleaved-caspase 3 protein levels were detected by western blot. As shown in Fig. 1C

and D, PZA treatment for 24 h significantly increased apoptotic cells by about 9% (p

< 0.05), 20% (p < 0.01) and 28% (p < 0.001) at the concentrations of 25 mM, 50 mM,

75 mM respectively. The cleaved-caspase 3 protein levels were consistent with

apoptosis (Fig. 1 E).

In vivo, we found PZA significantly increased serum ALT levels 2-fold (p < 0.01)

and 3-fold (p < 0.001) in the 98 h and 122 h groups respectively (Fig. 2A). Serum

AST levels were increased 1.8-fold (p < 0.05) in the 122 h group (Fig. 2B). Serum

TBA levels were increased 3-fold (p < 0.05) and 5-fold (p < 0.01) in the 98 h and 122

h groups respectively (Fig. 2C). Hepatic histopathological examination revealed a

predominantly macrovesicular steatosis (Fig. 2D-b) in the 98 h group, and mild

necrosis and inflammation (Fig. 2D-c) in the 122 h group. The toxicity score was

shown in Fig. 2E.

The above results illustrate PZA induced liver injury both in HepG2 cells and rat

liver.

3.2 PZA disturbed the antioxidant defense system both in vitro and in vivo

Oxidative stress is related to both apoptosis and drug induced liver injury. We

analyzed parameters involved in oxidative stress in vitro and in vivo to investigate

whether PZA treatment caused oxidative stress. PZA treatment for 24 h significantly

decreased SOD activity 27% (p < 0.05), 28% (p < 0.05), 50% (p < 0.01) and 66% (p <
10 
 
0.001) at the concentrations of 10 mM, 25 mM, 50 mM and 75 mM respectively in

the cytoplasm of HepG2 cells (Fig. 3A). Furthermore, PZA treatment for 24 h

significantly reduced mitochondrial transmembrane potential at the concentrations of

10 mM (4.1-fold, p < 0.01), 25 mM (5.2-fold, p < 0.001), 50 mM (5.8-fold, p < 0.001)

and 75 mM (7.3-fold, p < 0.001) (Fig. 3B and C). However, interestingly, there was

no obvious influence on ROS levels after PZA treatment in HepG2 cells (Fig. 3D). As

mitochondrial pathway-induced apoptosis is one route for apoptosis, we determined

marked protein levels (Bcl-2, Bax and caspase 9) of this pathway, and there were no

significant changes (Fig. 3E).

Next, the activities of antioxidant enzymes in rat liver including T-AOC and

T-SOD were determined. As results showed, PZA treatment increased T-AOC activity

(1.4-fold, p < 0.05 and 2.2-fold, p < 0.01 respectively) but reduced T-SOD activity

(19%, p < 0.05 and 26%, p < 0.05 respectively) in the 98 h and 122 h groups (Fig. 4 A

and B). MDA, a lipid peroxidation marker, was increased after PZA exposure in all

groups with no significant differences except the 50 h group (2.1-fold, p < 0.05)(Fig.

4C). GSH is one of the major endogenous antioxidant, which contents were increased

in rat livers treated with PZA in the 122 h group (2.9-fold, p < 0.01) (Fig. 4D). These

results suggest that PZA disturbs the antioxidant defense system in rat livers after 6

days treatment.

3.3 PZA induces ER stress and activated PERK-eIF2α-ATF4-CHOP pathway in

vitro and in vivo

To investigate ER stress occurrence after PZA exposure, the ER stress markers

11 
 
GRP78, IRE-1α, XBP1s, ATF4, ATF6 and CHOP were analyzed for mRNA

expression in HepG2 cells and rat livers. PCR analysis (Fig. 5A) showed a significant

up-regulation in the mRNA expression of GRP78, IRE-1α, XBP1s, ATF4, CHOP and

ATF6 in HepG2 cells treated with PZA, especially at high exposure concentrations

(50 mM and 75 mM). Meanwhile, in PZA-treated rats, ER stress markers were

elevated to different degrees (Fig. 5B). At the mRNA level, GRP78, IRE-1α, XBP1s,

ATF4, CHOP and ATF6 expressions was continually increased following the first

administration of PZA with significant differences starting in the 50 h and/or 74 h

groups, and maximized in the 98 h and/or 122 h groups, while compared with the 98 h

group, GRP78 and CHOP were slightly decreased in the 122 h group (Fig. 5B).

Considering the key role of CHOP in cell apoptosis, we next analyzed the

PERK-eIF2α-ATF4-CHOP pathway by western blot. As Fig. 5C shown, PZA

treatment increased the protein expression of GRP78 , p-PERK, p-eIF2α, ATF4 and

CHOP. These results indicate that activation of PERK-eIF2α-ATF4-CHOP pathway

may contribute to PZA-induced apoptosis in HepG2 cells. In rat livers, protein levels

of these markers were up-regulated but their elevation started following the third

dosage (Fig. 5C). GRP 78 was increased in the 50 h group and declined in the 122 h

group with p-eIF2α significantly increased in the 74 h group. ATF4 increased

throughout PZA administration. Concomitant with an increase in p-eIF2α levels, there

was a pronounced elevation in CHOP, an ER stress terminal marker. The protein level

of p-PERK was increased starting in the 26 h group. These results indicate that PZA

activates ER stress and PERK-eIF2α-ATF4-CHOP pathway, which maybe an

12 
 
important reason of PZA-induced liver injury.

3.4 Sodium 4-phenylbutyrate ameliorates the hepatotoxicity of PZA in vitro

Considering the role of ER stress in PZA-induced hepatotoxicity, we used the ER

stress inhibitor 4-PBA to test whether it could ameliorate the toxic effects of PZA in

vitro. We selected 4-PBA at a concentration of 0.5 mM that has no significant

cytotoxicity (Tung et al. 2015). Our results showed that ER stress related proteins

levels including GRP78, p-PERK, p-eIF2α, ATF4 and CHOP were increased after

PZA treatment and this increase was prevent in the presence of 4-PBA (Fig. 6A).

Furthermore, PZA exposure induced cell apoptosis (24%, p < 0.01 vs. control) which

was weakened by 4-PBA (18%, p < 0.01 vs. control; p < 0.05 vs. PZA) at the

concentration of 50 mM (Fig. 6B and C). At the concentration of 75 mM, PZA

treatment induced cell apoptosis (29%, p < 0.001 vs. control) which was weakened by

4-PBA (21%, p < 0.01 vs. control; p < 0.05 vs. PZA) (Fig. 6B and C). These results

suggest that PZA-induced HepG2 cell apoptosis is partly mediated through the ER

stress, at least.

3.5 In vivo amelioration of PZA toxicity by 4-PBA

According to the in vitro results, we next investigated the effect of 4-PBA in vivo.

From our previous results, we chose the time (122 h) in which PZA induced obvious

hepatotoxicity and ER stress. The GRP78, p-PERK, p-eIF2α, ATF4 and CHOP

proteins were significantly reduced in PZA and 4-PBA co-treated rat livers (Fig. 7A).

Interestingly, PZA significantly increased serum ALT, AST and TBA levels, but

13 
 
4-PBA treatment caused substantial and significant reductions in serum levels of ALT

(15%, p < 0.05 vs. PZA), AST (28%, p < 0.05 vs. PZA) and TBA (50%, p < 0.05 vs.

PZA)(Fig. 7B). Hepatic histopathological examination revealed coagulative necrosis

with inflammatory cell infiltration in hepatocytes from the PZA group (Fig. 7C-c).

Only mild macrovesicular steatosis in hepatocytes was seen in the PZA and 4-PBA

co-treatment group (Fig. 7C-d), which illustrated that 4-PBA ameliorates PZA

toxicity. The score of toxicity was shown in Fig. 7D.

4 Discussion

The severe hepatotoxicity associated with PZA has limited its use in clinic for the

treatment of TB (Singh et al. 1995). Moreover, when used in combination therapy,

PZA has been found to increase isoniazid and rifampicin hepatotoxicity (Centers for

Disease and Prevention 2001). However, the toxic mechanism of PZA remained

unknown. Therefore, we investigated the PZA-induced liver injury effect both in vivo

and in vitro, and elucidated the molecular mechanism involved in this hepatotoxicity.

Our previous study suggested that PZA destroyed rat liver antioxidant defenses

and caused lipid peroxidation (Zhang et al. 2013), hence, we first illustrated the

involvement of oxidative stress in PZA caused hepatotoxicity. Increased ROS

generation, usually related to oxidative stress, has been shown to induce liver injury

and to adversely alter lipids, proteins and DNA (Serviddio et al. 2013). In the present

study, results showed that PZA treatment had no influence on ROS generation. In

addition, the key mitochondrial apoptotic protein levels including Bcl-2, Bax and

caspase 9 were not significantly altered following PZA exposure. Singh et al. (Singh
14 
 
et al. 2012) showed that NAC, as an antioxidant, failed to reverse PZA-induced

cytotoxicity in HepG2 cells. Further results indicated that PZA treatment decreased

total SOD activity and increased MDA levels, resulting in antioxidant defense system

imbalance in vivo. GSH is a major endogenous substance involved in detoxification of

drugs metabolized in the liver (Yoshida et al. 2015). Increased MDA production is

usually related to a decrease in GSH. However, our data indicate that hepatic GSH

contents were increased following PZA exposure, suggesting that elevation of GSH

may be induced to ameliorate the toxicity of PZA in the liver. Zhang’s study indicated

that PZA increased MDA contents but decreased both total GSH and T-AOC levels in

rat livers after treatment for 28 d (Zhang et al. 2013). In this study, rats were treated

with PZA for only 5 d. The different phenomena from the earlier study might be

attributed to different treatment periods. Hence, we also speculated that GSH and

T-AOC, as non-enzymatic antioxidants, which play an important protective role in

intracellular antioxidant defense in the body, would be compensatory increased when

PZA stimulated the liver as a toxic substance in response to ameliorate toxicity. With

the prolongation to 28 days, liver compensation ability couldn’t be enough to resist

the toxic effect of PZA, because elevation of GSH could also be a sign for the induced

oxidative stress, that have been resisted by the GSH level, which with more drug-liver

contact time, this compensatory effect will be abolished. Although we showed that

PZA impaired rat liver antioxidant defense system, the involvement of oxidative stress

in this effect needed further investigations.

A growing of body evidence illustrates the connection between ER stress and

15 
 
liver injury (Koga et al. 2015), but the relationship between ER stress and

PZA-induced hepatotoxicity is still not well elucidated. Our results demonstrate that

PZA exposure induces ER stress both in vitro and in vivo. PERK-eIF2α-ATF4-CHOP

pathway is one of the major ER stress pathways. Lin’s study suggested that PERK

signaling activation was largely sustained with unmitigated ER stress (Lin et al. 2007)

and sustained PERK signaling impaired cell proliferation and promoted apoptosis

(Lin et al. 2009). By phosphorylating eIF2α and activating ATF4, PERK induced

CHOP expression. CHOP was reported to mediate cell death by inducing

pro-apoptotic genes (Puthalakath et al. 2007) and repressing anti-apoptotic genes

(McCullough et al. 2001). Following PZA exposure, the expression of p-PERK,

p-eIF2α, ATF4 and CHOP were up-regulated in both HepG2 cells and rat liver,

suggesting that PZA activating PERK-eIF2α-ATF4-CHOP pathway resulting in

apoptosis and liver damage. Interestingly, the expressions of CHOP decreased in the

122 h-treated group compared to those of the 98 h-treated group, while the toxicity

was more severe. These findings imply that other potentially, unidentified pathways

are involved in PZA-induced liver injury.

As a chemical chaperone, 4-PBA is shown to inhibit ER stress through reducing

the accumulated misfolded proteins in the ER (Kolb et al. 2015). In this study, 4-PBA

ameliorates PZA-induced toxicity both in cells and rat livers, indicating the key role

of ER stress in PZA hepatotoxic action. 4-PBA also reduced the apoptotic rate in

HepG2 cells after PZA treatment, which may be due to the inhibition of CHOP, the

ER stress-mediated apoptosis pathway (Marciniak et al. 2004). As an ER stress

16 
 
inhibitor, 4-PBA has been found to inhibit the occurrence of ER stress in rat liver and

to alleviate the hepatotoxicity of PZA by improving hepatic function in rat liver,

especially via the reduction of serum TBA levels. Previous investigations suggested

that bile acids induce ER stress resulting in hepatocellular injury (Adachi et al. 2014;

Bochkis et al. 2008), and CHOP deficiency reduced liver fibrosis induced by

cholestasis (Tamaki et al. 2008). In addition, PZA can interact with anion transporters

such as uric acid transporter (Sato et al. 2011), which may also be with bile acid

transporters. Indeed, we found that PZA changes expression of genes participated in

bile acid biosynthesis and transport in the early study (Guo et al. 2016), and that this

expression was repressed by 4-PBA (Fig. S1). Although the changes were modest, we

consider that this effect may be due to the 4-PBA hepatoprotection properties which

were mediated through ER stress inhibition. Furthermore, our previous study found

that taurine-conjugated ursodeoxycholic acid (TUDCA) alleviated PZA-induced

cholestatic liver injury while the mechanism was not fully understood (Guo et al.

2016). Interestingly, TUDCA was an inhibitor of ER stress, so part of its

hepatoprotection mechanism might be due to ER stress inhibition. To clarify the

mechanism by which 4-PBA or TUDCA ameliorates PZA-induced toxicity still

needed further studies.

Conclusion

In summary, our findings indicate that short-term PZA treatment activated

PERK-eIF2α-ATF4-CHOP pathway and induced ER stress in rat liver and HepG2

cells resulting in hepatotoxicity and the inhibitor of ER stress 4-PBA can ameliorate

17 
 
these toxic effects. These results have potential implications for the pathogenesis of

PZA-induced liver toxicity in which ER stress and PERK-eIF2α-ATF4-CHOP

pathway play an important role.

Funding Sources

This study was supported by the National Natural Science Foundation of China

(No.81273604, 81320108029, 81173651), Natural Science Foundation of Jiangsu

Province (BK20151439), the Priority Academic Program Development of Jiangsu

Higher Education Institutions (PAPD), and this study was partially supported by the

111 Project (111-2-07).

Conflict of interest

The authors declare that no conflicts of interest.

Reference

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Figure captions:

Fig. 1 PZA-induced cytotoxicity in HepG2 cells. HepG2 cells were treated with PZA

in different concentrations for 24 h. Cell viability (A) and LDH activity in media (B)

were measured by CCK-8 assay and LDH kit, respectively. HepG2 cells were treated

with PZA in different concentrations for 24 h. Cell apoptosis (C and D) was detected

using flow cytometer and total apoptotic cells (early and late-stage apoptosis) were

represented by the right side of the panel (Annexin V staining alone or together with

PI). Cleaved-caspase3 protein level (E) was detected by western blot. Data were

expressed as mean ± SD of three independent experiments. *P<0.05, **P<0.01,

***P<0.001 versus control group

Fig. 2 PZA increased rat serum ALT, AST and TBA levels. Rats were treated with

PZA (2.0 g/kg) for different hours. (A) The effect of PZA on serum ALT level. (B)

The effect of PZA on serum AST level. (C) The effect of PZA on serum TBA level.

Data were expressed as mean ± SD (n=6). *P<0.05, **P<0.01, ***P<0.001 versus

control group. (D) Hepatic histopathology in PZA-treated rats. There were no

remarkable changes in the control group (a); predominant macrovesicular steatosis in

hepatocytes (dotted arrow) in the 98 h group (b); and coagulation necrosis with

inflammatory cell infiltration in hepatocytes (solid arrow) in the 122 h group (c). (E)

The histologic toxicity score. H&E stain 200 ×

Fig. 3 PZA exposure disturbed the antioxidant defense system in vitro. (A) PZA effect

on SOD activities measured by SOD kit in HepG2 cells. (B and C) The change of

24 
 
mitochondrial membrane potential was detected using JC-1 kit by flow cytometry in

HepG2 cells and the rate was represented by the right lower side of the panel. (D)

Intracellular ROS generation induced by increasing concentration of PZA was

measured in HepG2 cells by staining with DCFH-DA  (10 μM). (E) Protein levels of

Bcl-2, Bax and caspase9 were analyzed by western blot. Data were expressed as mean

± SD of three independent experiments. *P<0.05, **P<0.01 versus control group

Fig. 4 PZA exposure disturbed the antioxidant defense system in vivo. Effects of PZA

on T-AOC, T-SOD, MDA and GSH levels in rat liver, which measured by different

kits. Data were expressed as mean ± SD, n=6 in vivo. *P<0.05, **P<0.01 versus

control group

Fig. 5 PZA exposure induced ER stress in vitro and in vivo. Changes of mRNA

expression involved in ER stress in HepG2 cells (A) and rat livers (B) were

determined by RT-PCR. Changes of protein levels (C) involved in ER stress were

analyzed by western blot.*P<0.05 versus control group

Fig. 6 Chemical chaperone 4-PBA ameliorated PZA toxic effects in vitro. HepG2 cells

were treated with PZA for 24 h in the absence or presence of 4-PBA (0.5 mM). The

protein levels involved in ER stress (A) were detected by western blot. Cell apoptosis

(B and C) was detected using flow cytometry and total apoptotic cells (early and

late-stage apoptosis) were represented by the right side of the panel (Annexin V

staining alone or together with PI). Data were expressed as mean ± SD of three

independent experiments
25 
 
Fig. 7 Chemical chaperone 4-PBA ameliorated PZA toxic effects in vivo. Rats were

treated with PZA for 6 days in the absence or presence of 4-PBA (100 mg/kg). (A)

Total tissue lysates from livers of rats exposed to PZA for 6 days were subjected to

western blotting for proteins involved in ER stress (n=3). (B) Effects of PZA on

serum ALT, AST and TBA levels (n=6). (C) Hepatic histopathology in PZA-treated

rats. There were no remarkable changes in control and 4-PBA groups (a and b);

coagulation necrosis with inflammatory cell infiltration in hepatocytes (solid arrow)

were seen in PZA group (c); mild macrovesicular steatosis in hepatocytes (dotted

arrow) in PZA and 4-PBA co-treatment group (d). (D) The histologic toxicity score.

H&E stain 200 × * (compared with control group) or # (compared with PZA group)

p< 0.05, ** or ## p< 0.01

26 
 
Fig 1

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Fig 4

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