N-acetylcysteine alleviates PCB52-induced hepatotoxicity by repressing oxidative stress and inflammatory responses

Special chemicals and cell lines

PCB52 (2,2′,5,5′-tetrachlorodiphenyl, purity of 99.4%) was procured from J&K Scientific Company (Beijing, China) and dissolved in dimethylsulfoxide (DMSO) to prepare a 100 mM stock solution. N-acetylcysteine (NAC), and 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) were purchased from Sigma Chemical Co. (St. Louis, MI, USA). A 600 mmol/L stock solution of NAC was prepared by dissolving solid NAC in saline. Normal human hepatocytes, cell line L-02 (HL-7702) and normal rat hepatocytes, cell line Brl-3A were obtained from the Typical Culture Preservation Commission Cell Bank, Chinese Academy of Sciences, Shanghai, China.

Cell culture and treatments

Human L-02 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, Dublin, Ireland) supplemented with 15% fetal bovine serum (FBS) and 100 U/ml penicillin‑streptomycin. Rat Brl-3A cells were cultured in Dulbecco minimum essential medium (DMEM; Gibco, Dublin, Ireland) supplemented with 3% FBS and 100 U/ml penicillin‑streptomycin. Cells were cultured at 37 °C in a thermostatic incubator (Thermo Fisher Scientific Inc., Waltham, MA, USA) infused with carbon dioxide concentration 5%, and 95% saturated atmospheric humidity.

Cells (passage 10) were randomly divided into four groups, including the control, PCB52, NAC, and NAC + PCB52 groups. Following the results of our preliminary experiments (Fig. S1), Brl-3A cells were pretreated with 3 mM NAC for 12 h, while human L-02 cells were pretreated with one mM NAC for 6 h (Gu et al., 2018). The cells were then exposed to 40 μM PCB52 for 48 h and used for subsequent experiments. PCB52 exerted dose- and time-dependent effects (Wang et al., 2018). The concentration of PCB52 and the exposure duration were chosen based on our previous study (Wang et al., 2018) and the changes of cell viabilities in our preliminary experiments.

Determination of cell viability

Cell viability was determined using the cell counting kit-8 (CCK-8; Dojindo, Kumamoto, Japan) according to the manufacturer’s instructions. In brief, cells were first seeded on a 96-well plate. The culture medium was replaced with medium containing PCB52 concentrations of 0, 10, 20, 40 and 80 μmol/L and incubated for 48 h. Then, the PCB media was replaced with a basal medium containing 10 μl of CCK-8 solution and incubated for 3 h at 37 °C. Finally, the absorbance was assessed at 450 nm using a microplate reader (Bio-rad model 680; BIO-RAD, Hercules, CA, USA).

Monitoring ROS levels

The cells, both L-02 (5 × 10⁵ cells/ml) and Brl-3A (2 × 10⁵ cells/ml) were seeded in 25 cm² canted-neck tissue culture flasks (Corning Inc., Corning, NY, USA) and then treated as previously mentioned. Cells were washed twice with PBS, incubated with 10 μmol/L DCFH-DA for 30 min at 37 °C, digested by 0.25% trypsin and washed three times with ice-cold PBS. DCFH-DA fluorescence was analyzed by flow cytometry (BD LSRFortessa™; Becton, Dickinson and Company, Franklin Lakes, NJ, USA).

Examinations of MDA contents

Briefly, the MDA levels were evaluated using an MDA assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), according to the operation manual. Optical densities of the supernatants were read at 532 nm using a microplate reader (Bio-rad model 680; BIO-RAD, Hercules, CA, USA) and the protein concentrations of cells were determined using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). The MDA contents are presented as nmol per mg protein in the bar graph.

Real-time Quantitative PCR

Total RNA was extracted using TRIzol^® Reagent (Thermo Fisher Scientific, Waltham, MA, USA), and the RNA quality and quantity were measured using a NanoDrop2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Complementary DNA (cDNA) was synthesized from the total RNA using the Prime Script RT Reagent Kit (RR036A; Takara Biotechnology Co., LTD., Shiga, Japan). Primers for tumor necrosis factor (TNF) receptor-associated factor 6 (Traf6), myeloid differentiation factor88 (MyD88), TNF, interleukin (IL)1B, Keap1, Nfe2l2/Nrf2, heme oxygenase (Hmox/HO)-1, NAD(P)H quinone oxidoreductase 1 (NQO1), Sqstm1/P62, and β-actin were designed using the Get Prime software (https://gecftools.epfl.ch/getprime). Real-time Quantitative PCR (RT-qPCR) was conducted using TB Green™ Premix Ex Taq™ GC (RR820A; Takara, Shiga, Japan) on a Light Cycler^® 96 System (Roche Life Science, Penzberg, Germany). Relative quantification of gene expression was performed based on the comparative CT (2^–ΔΔCt) method. The mRNA expression levels of target genes were normalized to that of the β-actin internal standard. Data were presented as fold changes over the controls, which were shown as 1. The sequences of primers used for RT-qPCR are shown in Table S1.

Western blot analysis

Total protein was extracted by lysis using a RIPA buffer (containing 1 mmol/L PMSF), 30 min incubation in ice, and total proteins were collected. Protein concentrations were determined using a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Proteins (20 µg per sample) were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (BIO-RAD, Hercules, CA, USA). The membranes were then blocked with 5% milk-Tris-buffered-Tween solution for 2 h at room temperature, incubated with primary antibodies at 4 °C overnight, followed by incubation in the appropriate secondary antibodies. Finally, blots were incubated in enhanced chemiluminescent (ECL) detection reagent (BIO-RAD Laboratories, Inc., Hercules, CA, USA) for 3 min. Densitometric analysis was performed using the Tanon Gel Image System (version 4.2). Band intensities were normalized using GAPDH as an internal control. Data on relative integrated optical density values of bands are displayed in the form of bar charts, with the control presented as 1. This study used primary antibodies against toll-like receptor 4 (TLR4, dilution 1:5,000, sc293072; Santa Cruz Biotechnology, Dallas, TX, USA), Traf6 (dilution 1:3,000, sc8409; Santa Cruz Biotechnology, Dallas, TX, USA), MyD88 (dilution 1:3,000, sc-74532; Santa Cruz Biotechnology, Dallas, TX, USA), Nrf2 (dilution 1:2,000, sc-13032; Santa Cruz Biotechnology, Dallas, TX, USA), keap1 (dilution 1:3,000, sc-15246; Santa Cruz Biotechnology, Dallas, TX, USA), HO-1 (dilution 1:2,000, sc-390991; Santa Cruz Biotechnology, Dallas, TX, USA), NF-κB p65 (dilution 1:2,000, sc-8008; Santa Cruz Biotechnology, Dallas, TX, USA) and GAPDH (dilution 1:5,000, sc-32233; Santa Cruz Biotechnology, Dallas, TX, USA).

Statistical analysis

Numeric data are presented as mean ± standard error (SE) of at least three independent replicates. Statistical analysis was conducted using the SPSS statistical software (version 16.0; IBM, Armonk, NY, USA). Differences in mean values between the control and PCB52-treated cells were evaluated by the Independent-Samples T-test, while differences between the control, PCB52, NAC, and NAC + PCB52 groups were analyzed by One-Way analysis of variance (ANOVA). A p-value < 0.05 was considered statistically significant.

NAC pretreatment alleviates PCB52-induced hepatotoxicity

Compared to the control group, 48 h exposure of Brl-3A and L-02 cells to PCB52 reduced cell viabilities in a dose-dependent manner (Fig. S1). The most significant reduction was obtained at PCB52 concentrations of 40 and 80 μM. Consistent with our previous study (Wang et al., 2018), a PCB52 concentration of 40 μM was found as the most suitable for further studies.

Compared with the control, no significant changes in Brl-3A cell viabilities were observed following 12 h NAC pre-treatment at concentrations 3, 5, and 10 mM. Pretreatment with NAC at concentrations 3 and 5 mM significantly ameliorated PCB52-induced cytotoxicity in Brl-3A cells (Fig. S2). Accordingly, 3 mM NAC was chosen to perform subsequent analyses. Similar to the findings of our preliminary experiment, NAC pretreatment significantly repressed PCB52-induced hepatotoxicity in Brl-3A cells (Fig. 1A). In L-02 cells also, cell viability significantly increased in the NAC + PCB52 group compared with the PCB52 group (Fig. 1B), while no change in cell viability was observed in the NAC group compared with the control (Fig. 1).

NAC pretreatment mitigated PCB52-induced ROS accumulation and MDA content

The levels of ROS in PCB52-treated cells were significantly higher than in control groups (Figs. 2A and 2B). Compared with the PCB52 group, NAC pretreatment significantly reduced ROS levels in both Brl-3A (Fig. 2A) and L-02 cells (Fig. 2B). However, no significant changes in ROS levels were observed between the NAC group and the control group.

Moreover, MDA contents in PCB52-treated Brl-3A and L-02 cells were considerably higher than in control groups (Figs. 2C and 2D). Consistently, NAC pretreatment significantly reduced MDA content compared to the PCB52 group. However, no significant differences in MDA content were observed between the NAC group and the control group.

NAC pretreatment reduces PCB52-induced inflammatory response

In Brl 3A cells, compared to the control group, the expression of Traf6, MyD88, and Tnf were significantly increased in PCB52 group (Fig. 3A), indicating activation of inflammatory pathways. Also, the expression of Hmox1, Nqo1, and Sqstm1 was significantly upregulated by exposure to PCB52, while no significant changes were detected in the expression levels of Keap1 and Nfe2l2 (Fig. 3B). NAC pretreatment significantly reduced expression of Traf6, MyD88, and Tnf as compared to the PCB52 group (Fig. 3A), while no significant changes in the expression of Keap1, Nfe2l2, Hmox1, Nqo1, and Sqstm1 were observed (Fig. 3B). Besides, no significant changes in gene expression were observed between the NAC group and the control group. Downregulated expression of Hmox1 in the NAC + PCB52 group could suggest a reduction of a compensatory response to an oxidative and inflammatory reaction. However, no significant changes in the expression of Keap1, Nfe2l2, Nqo1, and Sqstm1 were observed.

In L-02 cells, compared with the control, the expression of MYD88, TNF, and IL1B were significantly upregulated in PCB52 group (Fig. 3C), while no significant changes were detected in the expression levels of TRAF6, KEAP1, NFE2L2, HMOX1, and NQO1 (Fig. 3D). Expression of MYD88, TNF, and IL1B was significantly decreased in the NAC + PCB52 group compared with the PCB52 group (Fig. 3C). However, no significant changes in the expression of TRAF6, KEAP1, NFE2L2, HMOX1, and NQO1 were observed between the two groups. Also, no significant difference was found in the NAC group compared with the control group.

Consistent with the RNA levels, western blot analysis showed that the protein expression of TLR4, MyD88, Traf6, and NF-κB p65 was significantly increased in PCB52-treated Brl-3A cells, as compared with the control (Figs. 4A–4E). However, no significant changes were observed in the protein expression of keap1, Nrf2, and HO-1 (Figs. 4A and 4F–4H). Furthermore, overexpression of TLR4, MyD88, Traf6, and NF-κB p65 proteins was also observed in PCB52-treated L-02 cells (Figs. 4I–4M). Meanwhile, no significant changes in the expression of keap1, Nrf2, and HO-1 proteins were found (Figs. 4I and 4N–4P), which concurs with the observations made in Brl-3A cells. Furthermore, expression of TLR4, MyD88, Traf6, and NF-κB p65 proteins was significantly repressed in the NAC + PCB52 group compared with the PCB52 group in both Brl-3A (Figs. 4A–4E) and L-02 cells (Figs. 4I–4M), suggesting reduced inflammation following NAC pretreatment. However, no significant changes in keap1, Nrf2, and HO-1 proteins were observed.