Introduction

Trichloroethylene (TCE) is a chlorinated hydrocarbon widely used as a solvent in industrial applications. Because of inappropriate usage and transportation, TCE is now a major pollutant in soil and ground water (Wu and Schaum 2000). TCE exposure can cause severe toxic effects to humans, and the EPA (US Environmental Protection Agency) characterized TCE as carcinogenic in 2011 (Chiu et al. 2013). Exposure to TCE is therefore a human health risk in China and other regions of the world (Kamijima et al. 2007).

Liver is the major organ where TCE is typically metabolized into oxidative metabolites by cytochrome P450-dependent oxidation pathways. These metabolites play important roles in TCE-induced hepatotoxicity and carcinogenesis (Allemand et al. 1978; Lash et al. 2000). However, the precise mechanisms underlying TCE hepatotoxicity are unclear.

TCE appears to induce toxic responses in human hepatocytes (L-02 line) by dysregulating the expression of genes encoding CYP1A2 and BAX (BAX is associated with apoptosis) (Xu et al. 2012). Repeated administration of TCE to mice by oral gavage can produce hepatocellular proliferation, mitochondrial dysfunction and glycogen depletion (Sano et al. 2009). Proteome profiles of TCE-induced protein dysregulation in cultured hepatocytes indicate that specific proteins are related to TCE toxicity in human hepatocytes (Liu et al. 2007; Huang et al. 2009). Although there have been numerous studies on TCE-induced liver damage in either mouse in vivo or hepatocytes in vitro, few studies of TCE-induced toxicity in rat liver have been reported.

We studied TCE-induced liver toxicity in rats by exposing rats to TCE by gastric gavage and evaluating its effects on the liver. We then performed a comparative proteomics analysis, focusing on the profiles of liver proteins in TCE-treated SD rats. These studies were followed by analysis of the potential roles of the alterations in protein expression in TCE-induced liver toxicity.

Materials and methods

Animals and treatment

Male Sprague–Dawley (SD) rats, aged 7 weeks with body weights of 103–119 g, were purchased from the Experimental Animal Center of Guangdong Province (Guangzhou, China) and housed under specific pathogen-free (SPF) conditions. The animal experiments were approved by the Committee of Ethnics in the Shenzhen Center for Disease Control and Prevention. Animals were acclimatized to the laboratory conditions for 4 days prior to being randomly divided into four groups with ten rats per group. TCE was purchased from Sigma-Aldrich (Shanghai, China). The animals were fasted for 12 h before each administration, and food was provided 2 h after dosing. Tap water was available ad libitum. TCE was dissolved in corn oil at different doses (0, 250, 500 and 1000 mg/kg per day), with dosage being based on a previous report (Khan et al. 2009). TCE was administered by gastric gavage for 14 consecutive days. The body weight of each rat was recorded every other day during, and at the end of, the study. Animal care and treatments were performed in accordance with the Institutional Animal Care and the NIH Guide for the Care and Use of Laboratory Animals. At the end of the experiment (14 days), blood samples were collected and allowed to coagulate. The serum samples were then obtained and used for analyzing biochemical parameters. During necropsy, samples of liver tissue were taken, weighed and snap-frozen in liquid nitrogen to minimize proteolysis before storing at − 80 °C for analyses.

Serum biochemistry profile

The activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP), and the concentrations of total protein (TP) and albumin (ALB) in serum were analyzed using standard methodology (Bergmeyer et al. 1978), with commercially available kits obtained from Shanghai KEHUA bio-engineering (Shanghai, China). All analyses were performed on an Olympus AU 400 autoanalyzer (Hamburg, Germany).

Protein extraction and CyDye minimal labeling

Frozen liver samples were ground in liquid nitrogen and homogenized in a lysis buffer containing urea (7 M), thiourea (2 M), CHAPS [2% (w/v)], Tris (30 mM) and protease inhibitor cocktail (Thermo Scientific, Rockford, IL, USA). Homogenized samples were sonicated for 30 s, incubated on ice for 30 min and centrifuged at 20,000g for 30 min at 4 °C. Supernatants were precipitated with three volumes of ice-cold acetone at − 20 °C for 2 h, followed by centrifugation at 10,000g for 10 min at 4 °C. The resulting pellets were re-suspended in lysis buffer and centrifuged at 10,000g for 5 min. Protein concentrations of the supernatants were determined with the 2-D Quant kit (GE Healthcare, Pittsburgh, PA, USA) according to manufacturer instructions.

The purified samples were labeled with Cy2, Cy3 or Cy5 fluorescent dyes (200 pmol dyes per 25 μg protein) (GE healthcare, Pittsburgh, PA, USA) according to manufacturer instructions. Briefly, one half of individual samples from the same group were either labeled with Cy3 or Cy5 to avoid any dye-specific staining bias. An internal standard, made by pooling equal aliquots of all experimental samples, was labeled with Cy2. The labeling reaction was conducted on ice in darkness for 30 min and then quenched with 1 μL of 10 mM lysine (Sigma-Aldrich, Shanghai, China) for 10 min on ice shielded from light.

2D-DIGE electrophoresis

The Cy3- and Cy5-labeled samples were mixed together with an aliquot (25 μg) of Cy2-labeled internal standard. An equal volume of 2 × sample buffer containing urea (7 M), thiourea (2 M), CHAPS [4% (w/v)], dithiothreitol [DTT, 2% (w/v)], and IPG buffer [2% (v/v), at pH 3–11] was added to each mixed sample and incubated on ice for 10 min prior to bringing the total sample volume to 450 μL with rehydration buffer (8 M of urea, 2% w/v of CHAPS, 0.28% w/v of DTT, 0.5% v/v of IPG Buffer at pH 3–10, and 0.002% w/v of bromophenol blue). Twelve differently pooled samples were loaded onto individual IPG strips (pH 3–11, nonlinear gradient, 24 cm) (GE Healthcare, Pittsburgh, PA, USA) for isoelectric focusing (IEF) using an Ettan IPGphor 3 system (GE Healthcare, Pittsburgh, PA, USA). The IEF was programmed in the following five steps: 30 V for 12 h, 500 V for 1 h, 1000 V for 1 h, 8000 V gradient for 6 h, and then the unit was maintained at 8000 V until a total of 90,000 V-hours was reached. After IEF, the strips went through two steps of equilibration before applying on 12.5% SDS-PAGE gels. The second-dimension separation was conducted on an Ettan DALT six electrophoresis system (GE Healthcare, Pittsburgh, PA, USA) at 15 °C at 1 W per gel for 1 h, followed by 11 W per gel for 6 h. All electrophoresis procedures were performed in darkness.

Image acquisition and analysis

After electrophoresis, gels were immediately scanned by a Typhoon Trio Variable Mode Imager (GE Healthcare, Pittsburgh, PA, USA) at 100 μm resolution with excitation/emission wavelengths of 488 (Blue)/520, 532 (Green)/580 and 633 (Red)/670 nm for Cy2, Cy3 and Cy5, respectively. The images were processed by DeCyder 2D v6.5 (GE Healthcare, Pittsburgh, PA, USA) for differential analysis. Student’s t test and one-way ANOVA were used to compare the strength of spots between each treatment and the control. Statistically significant spots (p < 0.05) with an average ratio ≥ 1.2 or ≤ − 1.2 were marked for further analysis.

In-gel digestion and protein identification by MALDI-TOF–MS/MS

The significantly different protein spots were manually excised from a 2-DE gel stained with Coomassie brilliant blue G-250 staining solution. Gel plugs were de-stained with 50% acetonitrile in ammonium bicarbonate aqueous solution (25 mM) followed by dehydration in 100% acetonitrile. After removal of acetonitrile, each gel piece was digested overnight at 37 °C with 0.03 μg of sequencing-grade trypsin (Promega, Madison, WI, USA) in 15 μL of ammonium bicarbonate aqueous solution (25 mM). The extracted peptides were stored at − 80 °C prior to mass spectrometry analysis.

Peptides released from digestion were analyzed by an UltrafleXtreme MALDI-TOF/TOF mass spectrometer (Bruker Daltonics Inc., Billerica, MA, USA). Briefly, 2 µL of extracted peptide mixtures were spotted onto a polished steel MTP 384 target plate (Bruker Daltonics Inc., Billerica, MA, USA), air dried and covered with a 1 µL CHCA matrix (5 mg/mL in 50% ACN and 0.1% TFA). External calibration was performed using standard peptide calibration mixtures covering peaks between ~ 700 and 4000 Da (Bruker Daltonics Inc., Billerica, MA, USA). The peptide mass fingerprint (PMF) spectra were acquired in the positive reflection mode and the strongest seven peptides per spot were selected automatically for MS/MS analysis in LIFT mode. Protein identification by PMF and MS/MS spectra was performed using the Mascot search engine 2.2 (Matrix Science, Boston, MA, USA) and BioTools software (Bruker Daltonics Inc., Billerica, MA, USA). Confident matches in SwissProt database were defined by the Mascot score, the sequence coverage by matching peptides and statistical significance (p < 0.05).

Bioinformatics analysis

To determine the potential mechanisms mediating hepatotoxicity of TCE, PANTHER (http://pantherdb.org/) was used to classify protein families according to their cellular components, molecular functions and biological processes. STRING (http://string-db.org/, version 9.1) was used to identify protein networks of known and enrich proteins may be modulated in response to TCE exposure. Pathways with p values lower than 0.05 were considered to be significant (Franceschini et al. 2013).

Immunoblotting

To confirm the 2D-DIGE results, proteins with absolute values of average ratio > 1.2 and t values < 0.05 were selected for Western-blot analysis. Liver lysates were prepared with lysis buffer (Beyotime, Shanghai, China), homogenized, and centrifuged at 15,000g for 30 min. Extracts were mixed with protein loading buffer purchased from Thermo Scientific (Rockford, IL, USA) and heated for 5 min at 95 °C. The protein samples were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, Darmstadt, Germany) in a semi-dry transfer unit (GE Healthcare, Pittsburgh, PA, USA). The blotted membranes were subsequently blocked in 5% (w/v) nonfat dry milk in TBS-T buffer (20 mM Tris–HCl, 150 mM NaCl, 0.2% Tween-20, pH 7.5) for 60 min. After being washed in TBS-T, the membranes were incubated with a mixture of several primary antibodies, including anti-GSTM1, anti-PBE, anti-β-Actin, anti-ALDH2, anti-CPS1, anti-ENO1, and anti-DDT antibodies (all from Boster Immunoleader, Pleasanton, CA, USA). After rewashing in TBS-T, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit IgG (Thermo Scientific, Rockford, IL, USA), and anti-goat IgG (Santa Cruz Biotechnology, CA, USA) secondary antibodies. The membranes were then developed using SuperSignal West Dura Substrate (Thermo Scientific, Rockford, IL, USA), and scanned with an Image Quant RT ECL scanner (GE Healthcare, Pittsburgh, PA, USA) for visualization of the proteins bound to the primary antibodies. The relative level of each protein was calculated as a ratio between the grey value of the target band and that of the reference (β-actin).

Statistical analysis

The level of each observed parameter was expressed as mean ± standard deviation (n = 10) and analyzed by SPSS statistical software (version 20.0, Chicago, IL, USA). Differences between multiple groups were calculated using one-way ANOVA followed by a Dunnett’s (two-sided) test. A p < 0.05 value was considered as statistically significant. Figures were generated by GraphPad Prism (version 7.0, GraphPad Software Inc., San Diego, CA, USA).

Results

TCE induces liver injury in rats

At terminal killing, the body weights of test animals in the TCE-treated groups and the control were similar (p > 0.05). However, the liver index significantly increased with the increase in TCE level (dose-dependence) (Table 1; Fig. 1). Additionally, the activities of the liver damage marker enzymes (ALT, AST and ALP) in the serum of TCE-treated rats increased significantly (p < 0.001, compared to the control), in a dose-dependent manner (Fig. 2). The total protein and albumin levels in serum in TCE-treated rats were both significantly (p < 0.001) lowered, compared with their levels in the control (Fig. 2).

Table 1 The influence of TCE on liver index in rats
Full size table
Fig. 1
figure 1

Influence of TCE on the liver index of rats. Data are means and SD of ten replicates; **p < 0.01, compared with control; ***p < 0.001, compared with control

Full size image
Fig. 2
figure 2

Alterations of serum indicators of liver injury in SD rats exposed to TCE. See legend of Fig. 1; ***p < 0.001, compared with the control

Full size image

Differentially expressed proteins in the livers of TCE-exposed rats

To study the mechanism underlining TCE-induced hepatotoxicity, we evaluated the changes in the expression of hepatic proteins using 2D-DIGE. A total of 2000 protein spots were detected. Among these, 66 proteins were differentially expressed in TCE-treated rats (by one-way ANOVA, p < 0.05 combined with more than a 1.2-fold intensity difference) as identified by MALDI-TOF/TOF mass spectrometry (Fig. 3). The presence of different spots corresponding to the same protein was related to the occurrence of different isoforms, mainly due to post-translational modifications. Among the differentially expressed protein spots, 50 (belonging to 39 different proteins) were up-regulated, while 33 spots (27 different proteins) were down-regulated. The results of protein identification are summarized in Table 2.

Fig. 3
figure 3

Representative 2D-DIGE merged image of protein expression in the livers of SD rats untreated and treated with TCE. Proteins extracted from rats in the control and those in the TCE (500 mg/kg) treatment were differentially labeled with Cy3 in green and Cy5 in red (a merged spot would appear yellow)

Full size image
Table 2 Statistical data and MALDI-TOF MS/MS identification results of differentially expressed proteins
Full size table

Protein classification and functional analysis

PANTHER was used for protein classification analysis. Most of the differentially expressed proteins were mainly localized in the cytoplasm (63%) and mitochondrion (26%). The majority of proteins were associated with catalytic (68%) and binding activities (12%). Altered proteins were mainly involved in metabolic processes (55%) (Fig. 4).

Fig. 4
figure 4

Functional classification of TCE-induced differential proteomic profiles according to their cellular localization (a), molecular function (b) and biological processes (c). The classification was conducted using the PANTHER classification system

Full size image

Protein–protein interactions were analyzed by STRING (Franceschini et al. 2013). The data suggested a combined effect of these proteins in TCE-induced hepatotoxicity (Fig. 5). We categorized these proteins by their involvement in KEGG pathways (p < 0.05) using the “Enrichment” module integrated within STRING. As shown in Table 2, the pathways perturbed by TCE included fatty acid metabolism, PPAR signaling pathway, glycolysis/gluconeogenesis, and glutathione metabolism.

Fig. 5
figure 5

Protein interaction networks of TCE-induced differential proteomic profiles. The networks were generated with STRING database for direct physical and indirect functional protein interaction analysis (PBE is also named Ehhadh)

Full size image

Verification of PBE as differentially expressed in TCE-induced hepatic injury

Six metabolic enzymes (aldehyde dehydrogenase (Aldh2), alpha-enolase (Eno1), d-dopachrome decarboxylase (Ddt), carbamoyl-phosphate synthase (Cps1), glutathione S-transferase Mu 1 (Gstm1) and peroxisomal bifunctional enzyme (PBE) appeared to be associated with TCE-induced hepatotoxicity. Their levels were evaluated by Western-blot analysis. Aldh2 was down-regulated in the livers of the rats exposed to TCE for 14 days at doses of 250 and 1000 mg/kg (Fig. 6), while Gstm1 was up-regulated at doses of 500 and 1000 mg/kg. PBE was significantly up-regulated by TCE at doses of 500 and 1000 mg/kg. PBE was the only protein that demonstrated TCE dose-dependent enhancement of expression in the livers of rats.

Fig. 6
figure 6

Verification of TCE-targeted proteins in the rat liver. The levels of identified proteins in the liver samples from three rats in each group were confirmed by Western blot analysis. β-Actin was used as the loading control. a Protein bands of Aldh2, Eno1, Ddt, Cps1, Gstm1, and PBE. b Relative levels of Aldh2, Eno1, Ddt, Cps1, Gstm1, and PBE. Data are means and SD of three replicates; *p < 0.05, **p < 0.01, ***p < 0.001, compared with the control

Full size image

Discussion

Following exposure of SD rats to TCE by gastric gavage (for 14 days), significant increases in the liver index and the activities of liver damage marker enzymes (ALT, AST and ALP) were observed. This indicates that rats, in addition to mice, are vulnerable to TCE-induced liver injury. Furthermore, albumin and total protein in serum were decreased after treatment of TCE. Albumin in serum is mainly produced by the liver. Moreover, liver is also the major organ where TCE is metabolized for hepatotoxicity. The active metabolites of TCE may impair hepatocellular function and decrease albumin synthesis, which contributes to the reduction of albumin in serum (Carvalho and Verdelho Machado 2018; Alves de Mattos 2011). Because albumin is the most abundant protein in serum, its reduction usually leads to a drop of the total protein in serum.

Quantitative analysis by proteomic approaches suggested that the expression levels of 66 individual proteins were altered in the livers of TCE-treated rats. Bioinformatic analysis revealed that many of these proteins are metabolic enzymes mainly participating in glycolysis and the metabolism of lipids and glutathione. This result is consistent with other studies (Lash et al. 2014). The levels of 6 proteins, among 39 different proteins enhanced for expression, were further verified by Western blot analysis. After functional analysis, the six proteins were involved in different biological processes associated with liver injury or hepatic carcinogenesis (Puri 2019; He et al. 2020; Seo et al. 2019; Park et al. 2019; Yu et al. 2018; Gao et al. 2018; Panisello-Rosello 2018; Koen et al. 2016; Kumar et al. 2009). Thus, these proteins were selected for further verification. Of these proteins, the levels of Eno1, Ddt and Cps1 did not change; Aldh2 and Gstm1 were down- and up-regulated, respectively, without a typical dose–response relationship. However, the expression of PBE increased in a dose-dependent manner in the livers of rats exposed to TCE.

Aldh2 is the main enzyme responsible for acetaldehyde oxidation in ethanol metabolism, which implies that its down-regulation in the livers of rats by TCE may increase the oxidative burdens in the body and potentially inhibit ethanol metabolism. This observation is consistent with previous reports demonstrating induction of oxidative stress in the liver of mice by TCE (Atkinson et al. 1993) and decreased expression of Aldh2 and its enzymatic activity (Wang et al. 2010). Gstm1 (a critical enzyme for glutathione (GSH) metabolism) was up-regulated in the livers of rats exposed to TCE. A major function of GSH in mammalian cells is to attenuate oxidative stress, a factor implicated in cancer development and progression (Watanabe and Nagashima 1983). Thus, enhanced expression of Gstm1 might mediate multiple pathologic processes, such as the liver injury induced by TCE.

PBE is a bifunctional enzyme, which has two functional domains, the N-terminal region with enoyl-CoA hydratase activity and the C-terminal region with 3-hydroxyacyl-CoA dehydrogenase activity (Uchida et al. 1992). It is one of the four enzymes in the peroxisomal β-oxidation pathway. PBE is indispensable for the production of medium-chain dicarboxylic acids, which are essential for the coordinated induction of mitochondrial and peroxisomal oxidative pathways during fasting (Houten et al. 2012). Deficiency of this protein can cause peroxisomal disorders such as Zellweger syndrome (Itoh et al. 2000). PBE is also a potential biomarker for hepatocellular carcinoma, as indicated by high-throughput RNA-seq analysis (Gao et al. 2018; Jiang et al. 2019). PBE binds and activates the activation function-1 region of PPARα, a nuclear receptor associated with hepatotoxicity (Juge-Aubry et al. 2001). Interestingly, TCE-induced liver toxicity and carcinogenesis are also mediated partly by activation of PPARα (Mandard et al. 2004; Kyrklund et al. 1983; Klaunig et al. 2003). Therefore, the enhanced expression of PBE in the livers of rats exposed to TCE is likely induced via activation of PPARα by TCE, and PBE may contribute to the hepatotoxicity of TCE.

In summary, the results of this study indicate that TCE induces liver injury in rats. In this model, the expression of 66 liver proteins was altered, among which, PBE, a peroxisomal bifunctional enzyme, can be enhanced for expression, possibly in response to oxidative stress caused by TCE exposure. The role of PBE in TCE-induced liver injury is worth further investigation.