Role of inhibiting Chk1-p53 pathway in hepatotoxicity caused by chronic arsenic exposure from coal-burning

Abstract

Arsenic is a naturally occurring environmental toxicant, chronic exposure to arsenic can cause multiorgan damage, except for typical skin lesions, liver damage is the main problem for health concern in population with arsenic poisoning. Abnormal apoptosis is closely related to liver-related diseases, and p53 is one of the important hallmark proteins in apoptosis progression. This study was to investigate whether arsenic poisoning-induced hepatocyte apoptosis and the underlying role of p53 signaling pathway. A rat model of arsenic poisoning was established by feeding corn powder for 90 days, which was baked with high arsenic coal, then were treated with Ginkgo biloba extract (GBE) for 45 days by gavage. The results showed that arsenic induced liver damage, increased hepatocyte apoptosis and elevated the expression level of Chk1 and the ratios of p-p53/p53 and Bax/Bcl-2 in liver tissues, which were significantly attenuated by GBE. Additionally, to further demonstrate the potential apoptosis-associated mechanism, L-02 cells were pre-incubated with p53 inhibitor pifithrin-α (PFTα), ataxia telangiectasia-mutated (ATM)/ataxia telangiectasia-mutated and Rad3-related (ATR) inhibitor (CGK733) or GBE, then treated with sodium arsenite (NaAsO₂) for 24 h. The results showed that GBE, PFTα or CGK733 significantly reduced arsenic-induced Chk1 expression and the ratios of p-p53/p53 and Bax/Bcl-2. In conclusion, Chk1-p53 pathway was involved in arsenic poisoning-induced hepatotoxicity, and inhibiting of Chk1-p53 pathway ameliorated hepatocyte apoptosis caused by coal-burning arsenic poisoning. The study provides a pivotal clue for understanding of the mechanism of arsenic poisoning-induced liver damage, and possible intervention strategies.

Keywords

Arsenic poisoning coal hepatocyte apoptosis checkpoint kinase 1 p53

Introduction

Arsenic is a naturally occurring metalloid element and is a well-known environmental toxicant. Chronic exposure to arsenic is a major environmental public health challenge worldwide.¹ In addition to exposure to arsenic through drinking arsenic-contaminated water, another unique pathway of environmental arsenic exposure is through contact with high levels of arsenic-contaminated food and air in Guizhou Province, China.^2,3 A recent study demonstrated that total arsenic level in food and air included corn (the median value in the year of 1998 was 2.64 mg/kg, with 3.77 times higher than the reference standard), chili peppers (the median value in the year of 1998 was as high as 45.07 mg/kg, which was 64.39 times higher than the reference standard), both indoor and outdoor air (the median value was approximately 0.088 mg/m³ and 0.022 mg/m³, respectively, which was exceeded their relative reference standards).⁴ Consuming arsenic-enriched food and air result from combustion of arsenic-contaminated coal (the median value was 397.20 mg/kg, with 8.83 times higher than the reference standard) in unventilated indoor stoves as the lifestyle, residents living in this region suffer from a distinctive type of arsenic poisoning via skin contact and nutrition, inhalation, which was termed as coal-borne arsenicosis, and was characterized by the pathological and the functional damage of multiple organs such as skin, liver, lung and kidney.^4
–6 In particular, hepatotoxicity as a typical characteristic of coal-borne poisoning, and a high 20% prevalence of hepatomegaly was observed in arsenic-poisoning individuals from the survey in 2002.⁵ Although a 20-year follow-up study found that an obvious reduction in the total arsenic levels in both the external environment medium and uptake of individuals, the liver injury in existing patients still exists.^4,7 Currently, the potential molecular mechanisms of arsenic-induced hepatotoxicity remain unclear.

Abnormal apoptosis plays an essential role in liver-related diseases, and p53 is one of the important hallmark proteins in apoptosis-associated biological processes.^8
–10 Evidence has shown that p53-dependent mitochondrial apoptosis was involved in Di-(2-ethylhexyl) phthalate-induced hepatotoxicity via inhibiting anti-apoptotic B cell lymphoma-2 (Bcl-2) and inducing Bax expressions.¹¹ Furthermore, pharmacological inhibition or genetic depletion of p53 can attenuate hepatotoxicity induced by cocaine in mice.¹² Inhibition of p53 apoptosis signaling promoted hepatocyte proliferation, which antagonized acetaminophen-induced hepatotoxicity.¹³ In addition, studies have shown that ginkgo biloba extract (GBE), known as “living fossil,” whose active constituents include flavonol glycosides (24%) and terpene lactones (6%), has the beneficial role such as scavenging free radical, anti-apoptosis.^14,15 However, the impact of p53-mediated apoptosis signaling in coal-burning arsenic poisoning-induced hepatotoxicity has not been investigated. In this study, rats were exposed to different doses of arsenic by feeding corn flour baked with arsenic-contaminated coal from arsenicosis yard in Guizhou Province, China. We firstly verified the rat model of coal-burning arsenic poisoning-induced liver damage by observing poisoning symptoms, liver histopathological examination and liver arsenic content combined with alterations of p53-mediated apoptosis signaling. Additionally, p53 inhibitor pifithrin-α (PFTα), ataxia telangiectasia-mutated (ATM)/ataxia telangiectasia-mutated and Rad3-related (ATR) inhibitor (CGK733) and GBE were used to further investigate the possible role of p53 pathway in L-02 cells by determining expression levels of p53-related proteins.

Materials and methods

Experimental animal groups and administration

This study was performed according to the national and institutional guidelines and approved by the Animal Experimental Ethical Committee of Guizhou Medical University (approval number: 1403059). A total of 48 weaned specific pathogen-free Wistar rats were purchased from the Laboratory Animal Center of the Third Military Medical University [certificate of conformity: SCXK (Chongqing) 2007–0003]. Rats (weight 90 ± 10 g, male and female in half) were housed in sanitized cages at standard relative humidity (60∼70%) for 12-hour light/dark cycle. After 1 week of adaptive feeding, rats were randomly assigned to different groups, including coal-burning arsenic poisoning group and GBE (Yangzi River Pharmaceutical Co., Ltd. in China, approval number Zhunzi Z20027949, China.) treatment group. For coal-burning arsenic poisoning group, 24 rats were divided into the following 4 groups (n = 6/group, female/male = 1) and were fed for 90 days: i) Control group rats were fed with standard feed; ii) low arsenic group rats were fed with corn flour containing 25 mg/kg arsenic; iii) mediate arsenic group rats were fed with corn flour containing 50 mg/kg arsenic; iv) high arsenic group rats were fed with corn flour containing 100 mg/kg arsenic. For GBE treated group, 24 rats were also divided into 4 groups (n = 6/group, female/male = 1) and were fed for 135 days: i) Control group rats were fed with standard feed; ii) single arsenic exposure group rats were fed with standard feed for 45 days after feeding corn flour containing arsenic 100 mg/kg for 90 days; iii) Arsenic+GBE group rats were administered with 25 mg/kg GBE dissolved in deionized water for 45 days once a day after feeding corn flour containing arsenic 100 mg/ kg for 90 days; iv) single GBE group rats were administered with 25 mg/kg GBE once a day after feeding the standard feed for 90 days. Poisoning symptoms such as body weight, food intake, activity, fluffy degree of hair were monitored. Dose design and feed preparation, average arsenic content in feed, average daily feed consumption and actual exposure amount in control, low, medium and high arsenic groups were calculated according to a previous study.¹⁶

Sample collection

At the end of the experiment, the rats were anesthetized by intraperitoneal injection of 0.9% sodium pentobarbital and then euthanized by cervical dislocation. The liver were isolated immediately, rinsed with phosphate buffer saline (PBS), half of the liver were immersed in 4.0% neutral buffered formaldehyde 48 h for histopathological examination. The remaining of liver was used to detect the arsenic content and related protein expression levels, and liver samples were stored at −80 °C until analysis.

Determination of arsenic content in liver

Pre-treatment of samples via wet digestion as follows: moisture of liver were absorbed by filter paper, 0.50 g of liver were added to conical flask, then treated with nitric acid (4 mL) and perchloric acid (1 mL) to digest the tissue on an electric heating plate until the solution gradually became colorless and transparent, and white smoke from acid was evaporated. After the samples were cooled to room temperature, treated with urea (25%, 2.5 mL) and potassium iodide (20%, 1.0 mL), and the volume was made up to a total of 25.0 mL using hydrogen chloride (3 mol/L). Following by filtration, the solution were used to measure arsenic content using a hydride-generation inductive coupled plasma optical emission spectrometer (HG-ICP-OES) (Varian, USA) according to the manufacturer’s instructions. Recovery experiments were performed to verify the feasibility of the method using a standard stock solution of arsenic (1.00 g/L; cat. no. GB08611; National Standard Substance Research Center).

Characteristics of histopathology in liver

Histological alterations in liver were examined by hematoxylin eosin (HE) staining. Liver immersed in 4.0% formaldehyde were rinsed overnight, and dehydrated in graded ethanol (50–100%), embedded in paraffin for the following preparation of 4 µm tissue sections, then deparaffinized, rehydrated and finally stained with HE. Liver pathological changes were observed under a BX51 optical microscope (Olympus Corporation).

TUNEL assay of liver tissues

Terminal Deoxynucleotidyl Transferase-mediated deoxyuridine triphosphate Nick-End Labeling (TUNEL) reaction kits (Roche, Germany) was used to determine apoptosis. In brief, after dehydration, liver samples were embedded in paraffin, 4 µm tissue thick sections were prepared, then deparaffinized, dehydrated in graded ethanol, incubated with proteinase K for 30 min at 37 °C and washed with PBS, then treated with 3% H₂O₂ for 10 min at 25°C. After washed with PBS again, incubation with DNase Ⅰ reaction solution for 30 min at 37 °C, and then prepared terminal deoxynucleotidyl transferase and labeling buffer were added for a further incubation at 37 °C for 30 min and following by visualization with DAB, then the thick section were observed under BX51 optical microscope after sealed with neutral balsam on slides. Hepatocyte apoptosis rate was calculated in TUNEL-stained cells using Image-Pro Plus software (version 6.0; National Institutes of Health). A total of 10 fields (magnification, ×400) were selected randomly, and ≥1,000 cells were counted for analysis in each slide and the optical density was calibrated.

Cellular assay and experimental groups

The normal human hepatocyte cell line HL-7702 (L-02) was maintained in DMEM medium (Hyclone; GE Healthcare Life Sciences) supplemented with 10% heat-inactivated fetal bovine serum (Gibco, USA), 100 µg/mL streptomycin and 100 U/mL penicillin at 37°C in a humidified atmosphere with 5% CO₂. L-02 cells have grown to a density of 80%, the medium was replaced with sodium arsenite (NaAsO₂) (Sigma-Aldrich, Merck KGaA) for 24 h at a dose of 1.0, 5.0, 25.0, 50.0 and 100.0 μmol/L, then MTT (Sigma-Aldrich, Merck KGaA) assay was applied to determine cell viability by measuring the absorbance at 490 nm according to the manufacturer’s instructions.

Based on the results of MTT assay, the experiments in vitro were divided into the following groups: i) Control group, L-02 cells were cultured in DMEM medium for 24 h; ii) NaAsO₂ group, L-02 cells were exposed to 50 μmol/L NaAsO₂ for 24 h; iii) NaAsO₂+PFTα group, L-02 cells were pre-treated with 10 μmol/L PFTα (cat. no. SC-45050; Santa Cruz Biotechnology, Inc) for 2 h, which was dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich, Merck KGaA), then replaced with 50 μmol/L NaAsO₂ for 24 h; iv) single PFTα group, L-02 cells were pre-incubated with 10 μmol/L PFTα for 2 h, then replaced with DMEM for 24 h; v) NaAsO₂+CGK733 group, L-02 cells were pre-treated with 10 μmol/L CGK733 (cat. no. SC-202964; Santa Cruz Biotechnology, Inc) for 2 h, which was dissolved in DMSO, then replaced with 50 μmol/L NaAsO₂ for 24 h; vi) single CGK733 group, L-02 cells pre-treated with CGK733 for 2 h, then replaced with DMEM for 24 h; vii) NaAsO₂+GBE group, L-02 cells were pre-treated with 80 μg/mL GBE for 12 h, which was dissolved in DMSO, then replaced with 50 μmol/L NaAsO₂ for 24 h; viii) single GBE group, L-02 cells pre-treated with 80 μg/mL GBE for 12 h, then replaced with DMEM for 24 h. At the end of the experiments, morphological characteristics of L-02 cells and cell viability were examined, and cell lysates were harvested for further western blot analysis.

Western blot analysis

Liver tissues were homogenized with PBS using the Potter-Elvehjem Tissue Homogenizer (Shanghai Shuoguang Electronics Co., Ltd.), then centrifuged for 5 min at 4°C, 700 g using a Z36HK centrifuge (Hermle Labortechnik GmbH) to obtain homogenates. Total protein was extracted from homogenized liver tissues or L-02 cells lysates by adding RIPA lysis buffer [150 mM NaCl, 1% Triton X-100, 50 mM Tris-HCl (pH 7.4), 0.1% SDS, 1% NP-40, 1% sodium deoxycholate, 1 mM PMSF and proteinase inhibitor]. Subsequently, protein samples were centrifuged at 1,200 g, 4°C for 5 min, and the supernatants were obtained for determining protein concentration using a BCA detection kit (cat. no. P0010; Beyotime Institute of Biotechnology). Western blot analysis of Chk1, phosphorylated-p53 (p-p53), p53, Bax and Bcl-2 was performed as follows: 20 µg protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were transferred onto PVDF membranes (EMD Millipore, USA). Non-specific binding sites were blocked with 5% non-fat milk diluted with Tris buffered saline with Tween (TBST), and membranes were incubated at 4°C overnight with following primary antibodies: Rabbit polyclonal anti-Chk1 antibody (cat. no. 10362-1-AP; Proteintech Group, Inc), rabbit polyclonal anti-p53 antibody (cat. no. sc-6243; Santa Cruz Biotechnology, Inc), rabbit polyclonal anti-p-p53 antibody (phosphorylated serine (Ser) 20; cat. no. sc-21872-R; Santa Cruz Biotechnology, Inc), rabbit polyclonal anti-Bax antibody (cat. no. sc-493; Santa Cruz Biotechnology, Inc) and rabbit polyclonal anti-Bcl-2 antibody (cat. no. BAO412; Beijing Dingguo Changsheng Biotechnology Co., Ltd), then the membranes were washed with TBST, and a further incubation for 2 h with horseradish peroxidase-conjugated secondary antibody (cat. no. IH-0011; Beijing Dingguo Changsheng Biotechnology Co., Ltd) at room temperature. After washed with TBST again, the chemiluminescence signals were visualized with enhanced chemiluminescence reagent (Beyotime Institute of Biotechnology) using a Bio-Rad Imaging System (Bio-Rad Laboratories, Inc). The density of the bands was quantified using Image Lab software (version 2.0; Bio-Rad Laboratories, Inc), GAPDH (cat. no. BM1623; Beijing Dingguo Changsheng Biotechnology Co., Ltd) was used as internal control in equal samples, and p53 protein level was used as control for evaluating the phosphorylation level of p53 in Ser 20. 6 rat samples or 3 independent cellular samples were detected for statistical analysis.

Statistical analysis

SPSS software (version 21.0; IBM Corp.) was used for data analysis. Experimental data in vivo and in vitro were collected and were expressed as mean ± standard deviation. Analysis of variance between groups was performed by one-way analysis of variance (ANOVA), and Student-Newman-Keuls (SNK) test was used for further multiple comparisons. Alternatively, Kruskal Wallis followed by Mann-Whitney U test with Bonferroni correction was used under the condition of non-normally distributed data. The level of statistical significance was set at P < 0.05.

Results

Coal-burning arsenic poisoning-induced liver damage in rats was successfully established

Coal-burning arsenic poisoning rat model was successfully established as previously described.¹⁶ To validate liver damage caused by arsenic poisoning in rats, arsenic content and characteristics of histopathology in liver were determined. As presented in Table 1, after 90 days of low arsenic, mediate arsenic or high arsenic exposure, arsenic content in liver showed increased levels of 1.7, 2.3 and 4.3 fold compared to that of control group, respectively (P < 0.05), and arsenic content in high arsenic group was higher than that of low arsenic group (P < 0.05). Furthermore, liver histopathology showed the features of vacuolation, cytoplasmic loosening and congestion of hepatic sinuses in low arsenic group, and mediate arsenic group showed vacuolation, cytoplasmic loosening, congestion of hepatic sinuses, inflammatory infiltration and punctate necrosis. Congestion of hepatic sinuses, inflammatory infiltration, punctate necrosis, and fibroblast proliferation were observed in high arsenic group (Figure 1). These results indicated that coal-burning arsenic poisoning induced liver damage.

Table 1.

Arsenic accumulation in liver tissues and hepatotoxicity induced by arsenic exposure.

Groups	Arsenic content in liver (μg/g)	Hepatocytes apoptosis (%)	Chk1	p53
Control group	1.05 ± 0.31	9.00 ± 0.59	0.26 ± 0.05	0.36 ± 0.19
Low arsenic group	2.87 ± 1.32^*	9.27 ± 0.36	0.41 ± 0.08^*	1.06 ± 0.36^*
Mediate arsenic group	3.53 ± 1.39^*	16.49 ± 2.06^*#	0.45 ± 0.04^*	1.15 ± 0.17^*
High arsenic group	5.56 ± 2.46^*#	18.83 ± 1.28^*#	0.55 ± 0.07^*	0.74 ± 0.27^*
Levels of linear trend in ANOVA	13.37^☆	54.50^☆	2.14^☆	4.03^☆

Figure 1.

Coal-burning arsenic poisoning induced liver damage in rats. Representative HE staining images (magnification, × 400) and histopathological feature alterations in liver tissues. (A) control group; (B) low arsenic group; (C) mediate arsenic group; (D) high arsenic group; the arrows of red, purple, yellow, green, black and brown represent vacuolation, cytoplasmic loosening, congestion of hepatic sinuses, inflammatory infiltration, punctate necrosis and fibroblast proliferation, respectively.

The data are expressed as the mean ± standard deviation; n = 6. Arsenic content in liver (μg/g) was determined by HG-ICP-OES; Hepatocyte apoptosis is presented as a percentage. Chk1, p53 protein levels were evaluated by relative optical density, GAPDH as internal control. *P < 0.05, ^# P < 0.05 indicate a significant difference compared with control group and low arsenic group, respectively. The linear trends were obtained with ANOVA test among different arsenic exposure groups, ^☆ P < 0.05 indicates a linear trend with increasing doses of arsenic.

Arsenic induces hepatocyte apoptosis via activation of Chk1-53 pathway in rats

As presented in Figure 2, TUNEL results showed a dose-dependent increase in hepatocyte apoptosis rate after exposure to low, mediate and high doses of arsenic for 90 days (P < 0.05). Also, hepatocytes showed the features of chromatin condensation, which was attached to the periphery of the nuclear membrane and was shaped with crescent- or ring-like with a color variation from yellow to dark brown. Furthermore, a significant elevation of Chk1 and p53 levels in liver tissues was observed after exposure to arsenic (Table 1), suggesting that Chk1-p53 pathway was activated in arsenic-induced hepatotoxicity.

Figure 2.

Coal-burning arsenic poisoning induced hepatocyte apoptosis in rats. Representative TUNEL staining images (magnification, × 400) and apoptosis alterations in liver tissues. (A) control group; (B) low arsenic group; (C) mediate arsenic group; (D) high arsenic group. Black arrows represent the apoptosis of hepatocytes. (E) the bar graph represents apoptosis rate of hepatocytes. Data are presented as mean ± standard deviation; n = 6. *P < 0.05 vs. control; ^# P < 0.05 vs. low arsenic group.

Inhibition of Chk1-p53 pathway ameliorated arsenic-induced hepatotoxicity via reduction of hepatocyte apoptosis in rats

As presented in Figure 3, the control group and GBE treated group displayed normal radiation disposition of hepatic cords, and no obvious pathological changes were observed. High arsenic group showed vacuolation, cytoplasmic loosening, punctate necrosis. GBE could effectively decrease the above vacuolation formation. As shown in Figure 4, a significant reduction of hepatocyte apoptosis rate was also observed after GBE treatment. Moreover, as shown in Figure 5, after treated with GBE, the level of Chk1 and the ratios of p-p53/p53 and Bax/Bcl-2 showed a significant downregulation compared with single arsenic exposure group (P < 0.05) (Table S1). This indicated that GBE exerted protective effect against arsenic-induced hepatotoxicity via inhibition of Chk1-p53 signaling.

Figure 3.

GBE ameliorated coal-burning arsenic poisoning-induced liver damage in rats. Representative images of HE staining (magnification, × 400) and histopathological features alteration in liver tissues. (A) control group; (B) high arsenic group; (C) high arsenic + GBE group; (D) GBE group. The arrows of red, purple, black represent vacuolation, cytoplasmic loosening and punctate necrosis, respectively.

Figure 4.

GBE alleviated coal-burning arsenic poisoning-induced hepatocyte apoptosis in rats. Representative images of TUNEL staining (magnification, × 400) of liver tissues in (A) control group; (B) high arsenic group; (C) high arsenic + GBE group; (D) GBE group. Black arrows represent apoptosis of hepatocytes. (E) the bar graph represents apoptosis rate of hepatocytes; Data are presented as mean ± standard deviation; n = 6. *P < 0.05 vs. control; ^# P < 0.05 vs. arsenic exposure group; ^& P < 0.05 vs. GBE group.

Figure 5.

GBE reduced coal-burning arsenic poisoning-activated expression levels of Chk1-p53 pathway proteins in liver tissues of rats. (A) The representative western blot bands of Chk1, p53, p-p53, Bax and Bcl-2 in liver tissues, GAPDH as internal control, and p53 level was used as the control of p-p53 level. (B) The bar graph represents the levels of Chk1, the ratios of p-p53/p53 and Bax/Bcl-2. Data are presented as mean ± standard deviation; n = 6. *P < 0.05 vs. control; ^⋆ P < 0.05 vs. arsenic exposure group; ^& P < 0.05 vs. GBE group.

Inhibition of Chk1-p53 pathway reduced NaAsO₂-induced apoptosis in L-02 cells

The cultured L-02 cells were exposed to 1, 5, 25, 50 or 100 μmol/L NaAsO₂ for 24 h, a significant decrease in cell viability was observed at doses of 25, 50 and 100 μmol/L compared with control. At doses ≥25 μmol/L NaAsO₂, L-02 cells showed the characteristics of apoptosis, including shrinkage, increase in intercellular space, shedding and suspension of cells in culture medium (Figure S1). Also, the level of Chk1 and the ratios of p-p53/p53 and Bax/Bcl-2 showed a significant upregulation in 50 μmol/L NaAsO₂ group compared with those of control. Pre-incubation with GBE and CGK733 significantly reduced NaAsO₂-induced Chk1 level and the ratios of p-p53/p53 and Bax/Bcl-2 (Figure 6). Similarly, the ratio of p-p53/p53 and Bax/Bcl-2 were also obviously decreased after pre-treated with PFTα (Figure 6), more details as referred Figure S2 and Table S2–S4.

Figure 6.

Expression levels of Chk1-p53 pathway proteins induced by NaAsO₂ in L-02 cells were decreased after treated with GBE, CGK733 or PFTα. L-02 cells were pre-incubated with GBE (A), CGK733 (B) or PFTα (C), and then treated with 50 μM NaAsO₂ for 24 h. The bar graph represents the levels of Chk1, the ratios of p-p53/p53 and Bax/Bcl-2. Data are presented as mean ± standard deviation; n = 3. *P < 0.05 vs. control; ^⋆ P < 0.05 vs. NaAsO₂ group; ^△ P < 0.05 vs. single GBE, CGK733 or PFTα group, respectively.

Discussion

Arsenicosis caused by burning arsenic-contaminated coal is one of the major environmental public health concerns in Guizhou Province, China.¹⁷ Approximately 200,000 local residents are at risk of coal-burning arsenic exposure, and about 3,000 local residents have been diagnosed with coal-burning arsenicosis. Furthermore, clinical incidents of skin lesions, liver damage, lung dysfunction, neuropathy and nephrotoxicity are observed among populations exposure to coal-burning arsenic.⁵ In this study, a rat model of coal-burning arsenic poisoning and L-02 cells exposed to NaAsO₂ were used to explore the role of Chk-p53 pathway in arsenic-induced hepatotoxicity. The results suggested that arsenic poisoning-induced a significant increase in hepatocyte apoptosis, and upregulation of Chk1 and p53 levels. Moreover, GBE could effectively alleviate the feature of vacuolation formation and hepatocyte apoptosis, accompanied by a significant decreased in Chk1 level and the ratios of p-p53/p53 and Bax/Bcl-2. In addition, in vitro results demonstrated that GBE, PFTα or CGK733 reduced NaAsO₂-induced apoptosis via inhibiting Chk1-p53 pathway, indicating that Chk1-p53 pathway is closely related to arsenic-induced hepatotoxicity, and inhibition of Chk1-p53 pathway exerts protective roles against arsenic-induced apoptosis.

p53 is involved in the process of pathogenesis and progression due to excessive cellular stress, and is closely associated with apoptosis and inflammatory in response to environmental toxicants, such as arsenic, cadmium and chromium.^18
–20 p53 protein modification by phosphorylation, acetylation or ubiquitination may be a landmark event in response to multiple organ toxicity.^21,22 Especially, phosphorylation modification has been proposed to be essential for p53 stabilization, activation of p53 target genes, and interaction with transcriptional co-activators.²³ Phosphorylation of different sites have different physiological functions, literature has shown that p53 Ser 20 is critical site for disruption p53-MDM2 interaction and resulting in dissociation of p53 from MDM2 and finally enhancing stabilization of p53.^24,25 Our results showed that p53 Ser 20 phosphorylation in response to the toxic stimulation of arsenic in vivo and in vitro, which provide evidence for further exploring whether arsenic-induced phosphorylation of p53 Ser 20 weakened the interaction between p53 and MDM2. Multiple serine/threonine kinases have been implicated in the upstream signaling leading to p53 phosphorylation, among of that, Chk1 has been closely associated with phosphorylation of p53 in genotoxic stress-induced DNA damage.^26
–28 Lines of evidences revealed that DNA damage is one of the important pathogenic mechanisms in arsenic poisoning.^29,30 In our study, we found that the arsenic increased the Chk1 levels in vivo and in vitro. Furthermore, CGK733 and GBE ameliorated NaAsO₂-induced Chk1 level and p-p53 (Ser 20) in vitro. Studies have shown that phosphorylation of p53 in Ser 20 is causally related to p53-mediated apoptosis in human gliomas, and mitochondrial apoptosis pathway is also linked to p53 level in arsenite-mediated hepatotoxicity in mice.^31,32 The Bcl-2 protein family including the canonical anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax is not only associated with p53, both are transcriptional targets of p53, and the ratio of Bax/Bcl-2 is one of significant factors of mitochondrial apoptosis pathway.^33,34 Our results demonstrated that arsenic elevated the ratio of Bax/Bcl-2 in vivo and in vitro, and PFTα, CGK733 or GBE led to a significant reduction in the ratio of that. Moreover, downregulation of p53 and Bax levels and the ratio of Bax/Bcl-2 by GBE were also confirmed in other cells.^35,36 As well known, transcriptional activity of p53 and/or its selectivity toward different subset genes and/or subcellular localization and/or stability trigged by phosphorylation at specific residues determined fate of cellular in response to stress.³⁷ Evidence have reported that p53 nuclear import was the dominant consequence in high dose of arsenic-induced toxicity in human embryo lung fibroblast (HELF) cells and human keratinocytes (HaCaT) cells.^38,39 Moreover, published articles have suggested that cytoplasmic accumulation of p53 in response to low levels of arsenic exposure in MCF-10A cells.⁴⁰ And mitochondrial localization of the low level p53 protein was observed in proliferative mouse sarcoma (L929), rat immortalized fibroblasts (FR3T3), rat pheochromocytoma (PC12), human primary fibroblasts (HF) and human lung carcinoma (A549), but p53 was translocated into nuclear under etoposide treatment.⁴¹ All these findings provide a scientific basis for a further understanding of the mechanisms of p53 in arsenic-induced hepatotoxicity. On the other hand, p53 is associated with immune-related inflammatory signaling network, which plays an important role in arsenic-induced toxicological effects.¹⁸ Existing study suggested that mouse embryonic fibroblasts (MEFs) with p53 knock-down (p53^−/−) resulted in a decrease of pro-inflammatory cytokines such as interleukin 6 (IL-6), nuclear factor kappa-B (NF-κB), and upregulation of anti-inflammatory cytokines, including interleukin-10 (IL-10) when exposure to arsenic.⁴² Also, studies demonstrated that arsenic induced p53-medicated apoptosis and NF-κB pathway in liver cells could be reversed by pomegranate or carnosic acid.^11,32 These studies indicate that p53 is closely related to apoptosis and inflammatory signaling in arsenic-induced cytotoxicity.

Based on the aforementioned studies, speculating that crosstalk between p53-medicated apoptosis and inflammatory pathways might be involved in liver damage caused by arsenic. Although the underlying inflammatory-related mechanism need to be further clarified in coal-burning arsenic poisoning, our present results suggested that inhibition of Chk1-p53 pathway ameliorated arsenic poisoning-induced apoptosis. In addition, it is worthy of note that potential adverse effects of inhibition of p53. A study demonstrated that inhibition of p53 by hypoxia inducible factor-2α (HIF-2α) was a key event in arsenite-induced malignant transformation of human bronchial epithelial cells.⁴³ Therefore, inhibition of p53 may be a double-edged sword: on one hand, inhibiting p53 may attenuate p53-sensitized apoptosis and inflammatory signaling in high dose of arsenic-induced acute toxicity; on the other hand, p53 inactivation may result in the development of immortalized cells with DNA damage and genetic mutations, which may contribute to tumorigenesis in low concentration of arsenic-induced chronic toxicity. Here, two limitations in this study should be pointed out for improvement in our following study. Firstly, the detailed mechanism of p53-mediated apoptosis and inflammatory signaling should be elucidated in subsequent experiments using different doses of arsenic. Secondly, the intersection between p53-mediated apoptosis and inflammatory signaling in multi-organ toxicity induced by arsenic should be investigated.

Conclusion

This study provides some limited evidences that Chk1-p53 pathway is involved in coal-burning arsenic poisoning-induced liver damage. Another major finding is that inhibiting of Chk1-p53 pathway alleviates coal-burning arsenic poisoning-induced hepatotoxicity (Figure 7). Overall, our study provides a clue for deeper understanding of the mechanism of coal-burning arsenic poisoning-induced liver damage, and possible intervention ways.

Figure 7.

Schematic diagram for current study. Chk1-p53 pathway and the downstream balance of Bax/Bcl-2 play an important role in arsenic-induced hepatocyte apoptosis. In combination with inhibitor of Chk1 (CGK733) and p53 (PFTα) validated that GBE ameliorates arsenic-induced hepatocyte apoptosis, and the process is associated with arsenic-induced Chk1-p53 pathway and the imbalance of Bax/Bcl-2 ratio. The study provides evidence for understanding the liver damage mechanism duo to arsenic poisoning, and possible intervention strategies for GBE.

Supplemental material

Supplemental Material, sj-pdf-1-het-10.1177_0960327120988880 - Role of inhibiting Chk1-p53 pathway in hepatotoxicity caused by chronic arsenic exposure from coal-burning

Supplemental Material, sj-pdf-1-het-10.1177_0960327120988880 for Role of inhibiting Chk1-p53 pathway in hepatotoxicity caused by chronic arsenic exposure from coal-burning by Yuan Yang, Chunyan Liu, Tingting Xie, Dapeng Wang, Xiong Chen, Lu Ma and Aihua Zhang in Human & Experimental Toxicology

Footnotes

Authors’ note

Yuan Yang, Chunyan Liu and Tingting Xie contributed equally to this work.

Acknowledgements

The authors gratefully thank Maolin Yao, Chun Yu, Yuyan Xu and Yong Hu for their contributions to the animal experiments.

Author contributions

The experiments were performed by TX and CL, and experimental data was analyzed by YY, DW, XC, and LM. The study design, the review and revision of the final manuscript were performed by AZ.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by grants from the Natural Science Foundations of China (Grant Nos. U1812403, 81430077 and 81872569).

ORCID iD

Aihua Zhang

Supplemental material

Supplemental material for this article is available online.

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Supplementary Material

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Role of inhibiting Chk1-p53 pathway in hepatotoxicity caused by chronic arsenic exposure from coal-burning

Abstract

Keywords

Introduction

Materials and methods

Experimental animal groups and administration

Sample collection

Determination of arsenic content in liver

Characteristics of histopathology in liver

TUNEL assay of liver tissues

Cellular assay and experimental groups

Western blot analysis

Statistical analysis

Results

Coal-burning arsenic poisoning-induced liver damage in rats was successfully established

Arsenic induces hepatocyte apoptosis via activation of Chk1-53 pathway in rats

Inhibition of Chk1-p53 pathway ameliorated arsenic-induced hepatotoxicity via reduction of hepatocyte apoptosis in rats

Inhibition of Chk1-p53 pathway reduced NaAsO2-induced apoptosis in L-02 cells

Discussion

Conclusion

Supplemental material

Supplemental Material, sj-pdf-1-het-10.1177_0960327120988880 - Role of inhibiting Chk1-p53 pathway in hepatotoxicity caused by chronic arsenic exposure from coal-burning

Footnotes

Authors’ note

Acknowledgements

Author contributions

Declaration of conflicting interests

Funding

ORCID iD

Supplemental material

References

Supplementary Material

Inhibition of Chk1-p53 pathway reduced NaAsO₂-induced apoptosis in L-02 cells