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. Author manuscript; available in PMC: 2020 Jan 1.
Published in final edited form as: Toxicol Appl Pharmacol. 2018 Oct 10;362:28–34. doi: 10.1016/j.taap.2018.10.012

Contribution of poly(ADP-ribose)polymerase-1 activation and apoptosis in trichloroethene-mediated autoimmunity

Gangduo Wang 1, Huaxian Ma 1, Jianling Wang 1, M Firoze Khan 1,*
PMCID: PMC6286217  NIHMSID: NIHMS1509958  PMID: 30315841

Abstract

Trichloroethene (TCE), a common environmental toxicant and widely used industrial solvent, has been implicated in the development of various autoimmune diseases (ADs). Although oxidative stress has been involved in TCE-mediated autoimmunity, the molecular mechanisms remain to be fully elucidated. These studies were, therefore, aimed to further explore the contribution of oxidative stress to TCE-mediated autoimmune response by specifically assessing the role of oxidative DNA damage, its repair enzyme poly(ADP-ribose)polymerase-1 (PARP-1) and apoptosis. To achieve this, groups of female MRL +/+ mice were treated with TCE, TCE plus N-acetylcysteine (NAC) or NAC alone (TCE, 10 mmol/kg, i.p., every 4th day; NAC, 250 mg/kg/day in drinking water) for 6 weeks. TCE treatment led to significantly higher levels of 8-hydroxy-2’-deoxyguanosine (8-OHdG) in the livers compared to controls, suggesting increased oxidative DNA damage. TCE-induced DNA damage was associated with significant activation of PARP-1 and increases in caspase-3, cleaved caspase-8 and −9, and alterations in Bcl-2 and Bax in the livers. Moreover, the TCE-mediated alterations corresponded with remarkable increases in the serum anti-ssDNA antibodies. Interestingly, NAC supplementation not only attenuated elevated 8-OHdG, PARP-1, caspase-3, cleaved caspase-9, and Bax, but also the TCE-mediated autoimmune response supported by significantly reduced serum anti-ssDNA antibodies. These results suggest that TCE-induced activation of PARP-1 followed by increased apoptosis presents a novel mechanism in TCE-associated autoimmune response and could potentially lead to development of targeted preventive and/or therapeutic strategies.

Keywords: Trichloroethene, oxidative stress, autoimmune disease, poly(ADP-ribose)polymerase-1, apoptosis, N-acetylcysteine

1. Introduction

Autoimmune diseases (ADs), including systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), characterized by autoantibodies mainly directed towards ubiquitous nuclear targets such as DNA and histones, are life-threatening disorders. Such diseases afflict ~50 million Americans and are significant (> $100 billion) US health care cost burden (Blumberg et al., 2012; Caricchio et al., 2003; Walsh and Rau, 2000). In recent years, increasing evidence is presented to provide support that excessive reactive oxygen species (ROS) generation, DNA damage and apoptosis play vital roles in the pathogenesis of ADs (Abdelali et al., 2016; Jog et al., 2009; Ortona et al., 2014; Wang et al., 2016). These studies demonstrate an association among ROS generation, macromolecule (proteins, lipids and DNA) damage, apoptosis, and autoimmunity – strongly supporting the contribution of oxidative stress and apoptosis in the initiation, progression and pathogenesis of ADs both in humans and animals (Abdelali et al., 2016; Frostegard et al., 2005; Grader-Beck et al., 2007; Jog et al., 2009; Ortona et al., 2014; Wang et al., 2010; Wang et al., 2016). Moreover, antioxidants such as N-acetylcysteine (NAC) by decreasing oxidative stress, attenuate autoimmune response and reduce SLE disease activity, further supporting the contribution of oxidative stress in the disease pathogenesis (Lai et al., 2012; López-Pedrera et al., 2016; Ortona et al., 2014; Perl, 2013; Wang et al., 2013).

Even though oxidative DNA damage has been implicated in various ADs, the mechanisms by which it contributes to disease pathogenesis and progression remains to be fully elucidated. Extensive DNA damage induced by excessive ROS formation can activate enzymes involved in DNA damage sensing and repair. Poly(ADP-ribose) polymerase-1 (PARP-1), the prototypical representative of the PARP family, is an abundant, chromatin-associated nuclear enzyme responsible for a unique post-translational polyADP-ribosylation reaction involved in DNA repair, transcriptional control, genomic stability, inflammatory processes and several forms of cell death (Ba and Garg, 2011; Jog et al., 2009; Kim et al., 2005; Rosado et al., 2013). Excessive activation of PARP-1 has been implicated in the pathogenesis of various diseases including ADs (Abdelali et al., 2016; Ba and Garg, 2011; Grader-Beck et al., 2007; Jog et al., 2009; Ortona et al., 2014; Rosado et al., 2013; Wang et al., 2016). Moreover, PARP-1 is able to trigger apoptosis inducing factor-mediated caspase-independent and caspase-dependent apoptosis (Cuda et al., 2016; Rosado et al., 2013; Wang et al., 2016). Apoptosis has drawn attention in the past decade in dissecting its role in the pathogenesis of ADs (Ba and Garg, 2011; Cuda et al., 2016; Ortona et al., 2014). However, the status and contribution of PARP-1 and apoptosis in trichloroethene (trichloroethylene, TCE)-mediated autoimmunity has not been explored, and is the focus of this investigation.

Exposure to TCE, a ubiquitous environmental contaminant and a commonly used industrial solvent, is associated with the development of ADs such as SLE and systemic sclerosis, evidenced from both human and experimental animal studies (Khan et al., 1995, 2001; Kilburn and Warshaw, 1992; Wang et al., 2007, 2012, 2013). Previous studies suggest the involvement of oxidative stress in TCE-mediated autoimmunity (Khan et al., 2001; Wang et al., 2007, 2012, 2013, 2015). Although the role of oxidative stress, PARP-1 signaling and apoptosis in the pathogenesis of ADs has been the subject of great interest, the association, contribution and molecular mechanisms of these events in TCE-mediated autoimmunity remain largely unknown. The current studies, therefore, were aimed to assess the potential role of oxidative stress, PARP-1 signaling and apoptosis in TCE-induced autoimmune response by examining oxidative DNA damage, PARP-1 activation and apoptosis-related signaling pathways.

2. Materials and Methods

2.1. Animal and treatment

Female MRL/MpJ (MRL+/+) mice (5-week old) were obtained from the Jackson Laboratory (Bar Harbor, ME) and maintained at the animal house facility of the University of Texas Medical Branch (UTMB) under controlled conditions of temperature and humidity with a 12h light/dark cycle. The mice, provided standard lab chow and drinking water ad libitum, were acclimated for 1 week prior to the treatments. The animals were randomly divided into 4 groups of 6 mice each and exposed to TCE, NAC or TCE + NAC (TCE, 10 mmol/kg in corn oil, i.p., every 4th day; NAC, 250 mg/kg/day through drinking water) (Khan et al., 1995, 2001; Suwannaroj et al., 2001; Wang et al., 2009, 2013, 2014, 2015). The control animals received an equal volume of corn oil. MRL+/+ mice were used in this study because they spontaneously develop various autoantibodies after 9 months and SLE late in the second year of life (Khan et al., 1995, 2001; Wang et al., 2012). This slow development of the disease allows evaluation of specific effects of TCE on inducing/exacerbating the disease in relatively young animals. Female mice were chosen for this study due to higher susceptibility and prevalence of ADs in females (Danchenko et al., 2006; Khan et al., 2001; Wang et al., 2010, 2012). Also, the choice of dose and duration of TCE and/or NAC exposure was based on earlier studies (Khan et al., 1995, 2001; Suwannaroj et al., 2001; Wang et al., 2009, 2013, 2014, 2015). After 6 weeks of TCE, NAC or TCE+NAC treatments, the mice were euthanized under ketamine/xylazine anesthesia, and then blood was withdrawn and major organs removed. The sera, obtained following blood clotting and centrifugation, were stored in small aliquots at −80°C until further analysis. Liver portions were snap-frozen in liquid nitrogen and stored at −80°C for further use. The animal protocol was approved by the Institutional Animal Care and Use Committee of UTMB and all experiments were conducted in accordance with the guidelines of the National Institutes of Health (NIH).

2.2. Extraction of genomic DNA and DNA digestion

Genomic DNA was extracted from liver tissues by using a DNA extraction kit (Easy-DNA Kit, Invitrogen, Carlsbad, CA) as per the manufacturer’s instructions. The purity of DNA preparations was assessed by A260/A280 ratio. One hundred μl (~100 xg) of individual genomic DNA samples was digested with nuclease P1 (20 μg nuclease P1 dissolved in 20 mM sodium acetate buffer, pH 4.8) by incubating at 37 °C for 30 min followed by treatment with alkaline phosphatase (AP) (1.3 U or 100 μg AP in 1 M Tris–HCl, pH 7.4) and incubation at 37 °C for 1 h (Ma et al., 2008).

2.3. Determination of 8-OHdG in DNA digests

8-OHdG in DNA digests was quantitated using an ELISA kit (Ma et al., 2008) essentially as described by the manufacturer (BIOXYTECH 8-OHdG-EIA kit, Oxis, Portland, OR). Briefly, 50 μl of the standard or DNA digests from control and various treatment groups (in duplicate) were added to ELISA plate wells precoated with 8-OHdG, followed by addition of 50 μl 8-OHdG-specific antibody (monoclonal) and incubated at 37 °C for 1 h with gentle mixing. After incubation, the plate was washed thoroughly with the wash buffer and 100 μl of the secondary antibody (goat IgG conjugated to horseradish peroxidase) was added and incubated for an additional hour at 37 °C. After washing with wash buffer, 100 μl chromogen substrate (3′,3′,5′,5′-tetramethylbenzidine) was added to each well. The plate was then incubated at room temperature in the dark for 15 min with continuous shaking followed by addition of 100 μl stop solution (1 M H3PO4) to terminate the reaction. The absorbance was measured at 450 nm after 3 min.

2.4. Preparation of nuclear extracts (NEs) for PARP-1 activity

The nuclear protein extracts (NEs) were prepared according to the method in our previous studies (Ma et al., 2008, 2011), with minor modifications. Liver tissues (from all 4-group mice) were cut into smaller pieces, homogenized briefly with a loose glass pestle in cold hypotonic buffer [10 mM HEPES-KOH, 10 mM KCl, 100 μM EDTA, 100 μM EGTA, 1 mM DTT, 0.5 mM PMSF, 2 μg/ml pepstatin, and a complete protease inhibitor cocktail (Roche, Germany)], and incubated on ice for 30 min. Tissues were then homogenized with a tight pestle and centrifuged at 800 g for 5 min to obtain nuclear pellets. Pellets were gently washed two times with homogenizing buffer. Nuclear proteins were extracted in a high salt buffer (20 mM HEPESKOH, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, 2 μg/ml pepstatin, and protease inhibitor cocktail) by incubating for 50 min on ice with reverse mixing at intervals of 10 min. The NEs were cleared by centrifugation (16,000 g, 10 min) and adjusted to 15% with glycerol and stored at −80 °C until further analysis.

2.5. PARP-1 activity assay

PARP-1 activities were measured in the NEs with PARP ELISA assay kit, essentially as described by the manufacturer (Trevigen, Gaithersburg, MD). Briefly, 50 μl of PARP buffer was added to plate wells to rehydrate the histones and incubated for 30 min at room temperature (RT). Twenty five μl standard samples or NEs from all four group mice (in duplicate) were added, followed by addition of 25 μl PARP substrate cocktail and incubated for 30 min at RT. After washing, 50 μl of PAR specific antibody (1:1000) was added and incubated for 60 min at RT. Following washing, 50 μl of PARP 2nd antibody (1:5000) was added and then incubated for 30 min at RT. Following another washing, 100 μl of PeroxyGlow A and B (1:1) was added and chemiluminescence was read immediately by using GloMax 96 Microplate Luminometer (Promega, Madison, WI).

2.6. Western blot analysis

The protein expression of cleaved PARP-1, cleaved caspase-8/−9, Bcl-2 and Bax was analyzed by using Western blot as described earlier (Ma et al., 2008, 2011; Wang et al., 2013). Briefly, the liver lysates (50 μg of protein) were separated on 12% SDS-PAGE gels and then transferred to polyvinylidene fluoride (PVDF) membranes (Amersham, Arlington Heights, IL). The membranes, following blocking, were probed with specific antibodies (1:5000, Cell Signaling Technology, Danvers, MA) for the above mentioned proteins to detect their expression. β-actin antibody (Sigma) was used to confirm even loading. The quantification of blot signals was performed by densitometry and normalized using the β-actin expression (Ma et al., 2008, 2011; Wang et al., 2013).

2.7. PARP-1 mRNA quantification by real-time (RT) PCR

Total liver RNAs were isolated from controls, TCE- or TCE+NAC-treated mice by using TRIzol reagent following the manufacturer’s instruction (Invitrogen, Carlsbad CA). Reverse transcription was performed prior to RT PCR analysis as reported earlier (Ma et al., 2008, 2011). The RT PCR performance and relative PARP-1 mRNA expression calculations were done as described previously (Ma et al., 2008, 2011). The primer sequences were as follows: forward primer, 5’-ACACCACAAAACCTCAGCCA-3’, reverse primer, 5’-ACAAACCACAAACAACCGGC-3’.

2.8. Anti-single-stranded DNA antibodies (anti-ssDNA) in the serum

The anti-ssDNA antibodies in the sera were analyzed by using a mouse specific ELISA kit (Alpha Diagnostic Int’l, San Antonio, TX) (Khan et al., 1995; Wang et al., 2007), one hundred μl of 1:200 diluted serum samples was used for the analysis.

2.9. Statistical analysis

Unless indicated, all the data are expressed as means ± SD of six mice in each group. Significant differences among mean values were assessed by analysis of variance (one way ANOVA), followed by Tukey-Kramer multiple comparisons test (GraphPad Instat 3 software, La Jolla, CA). A p value of < 0.05 was considered to be statistically significant.

3. Results

3.1. Effect of TCE exposure on oxidative DNA damage in MRL+/+ mice

8-OHdG is one of the most widely recognized biomarkers of oxidative DNA damage (Cheah et al., 2017; Di Minno et al., 2016; Tatsch et al., 2015; Wu et al., 2005; Zhang et al., 2017). Previous studies have shown that TCE exposure enhances ROS generation and promotes oxidative stress, evidenced both in vivo and in vitro (Channel et al., 1998; Khan et al., 2001; Khan and Wang, 2018; Ogino et al., 1991; Wang et al., 2007, 2009, 2012; Zhu et al., 2005). To further evaluate the contribution of TCE-mediated oxidative stress in the induction/exacerbation of an autoimmune response, 8-OHdG formation in the livers of MRL+/+ mice exposed to TCE, NAC or TCE+NAC was quantitated. As shown in Fig. 1, TCE treatment resulted in greatly increased 8-OHdG formation compared to the controls (65% increase, p<0.05). No significant change in 8-OHdG formation was observed in NAC alone treated mice compared to the controls. Remarkably, NAC supplementation clearly protected the TCE-mediated DNA damage, evidenced by significantly reduced level of 8-OHdG in TCE+NAC-treated mice in comparison to TCE alone group (p<0.05).

Fig.1.

Fig.1.

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Levels of 8-OHdG in the livers of MRL+/+ mice after a 6-week exposure to TCE or TCE+NAC. The data are presented as means ± SD (n=6 in each group). *p < 0.05 vs. control group; #p < 0.05 vs. TCE-exposed mice. Note: CON = Controls; TCE = TCE-treated group; T+N =TCE + NAC-treated mice; NAC = NAC-treated mice.

3.2. Liver PARP-1 activity, cleaved PARP-1 protein and PARP-1 mRNA expression in the MRL+/+ mice

PARP-1 is a nuclear protein activated in response to DNA damage, and ROS induces DNA damage which activates enzymes involved in DNA damage sensing and repair (Ba and Garg, 2011; Grader-Beck et al., 2007; Ivana Scovassi and Diederich, 2004; Jog et al., 2009). PARP-1 activity, cleaved PARP-1 protein and PARP-1 mRNA expression in the livers of control, TCE and TCE+NAC-treated MRL+/+ mice were determined since our data showed that TCE induced oxidative DNA damage. Fig. 2 shows that TCE exposure resulted in remarkable increases in PARP-1 activity (174% increase, p<0.05), cleaved PARP-1 protein expression (207% increase, p<0.05) and PARP-1 mRNA expression (911% increase, p<0.05) in the livers of mice compared to the controls, which also corresponded to the oxidative DNA damage (8-OHdG levels) in the mice following TCE exposure. The increases in the activity, cleaved PARP-1 protein and PARP-1 mRNA expression were also attenuated by NAC supplementation as evident from significantly decreased PARP-1 activity, cleaved PARP-1 protein and PARP-1 mRNA expression in the livers of TCE+NAC-treated mice compared to that in TCE-treated mice (p<0.05), suggesting an association between TCE-induced oxidative DNA damage and PARP-1 activation.

Fig. 2.

Fig. 2.

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PARP-1 activity (A), cleaved PARP-1 protein expression (B) and PARP-1 mRNA expression (C) in MRL+/+ mice. The data are presented as means ± SD (n=6 for ELISA data; n=3 for Western blot and RT PCR data). *p < 0.05 vs. control mice; #p < 0.05 vs. TCE-exposed mice. Note: CON or C = Controls; TCE or T = TCE-treated mice; T+N or TN = TCE plus NAC-treated mice; NAC = NAC-treated mice.

3.3. Caspase 3 activation and cleaved caspase 8 and 9 expression in the livers of MRL+/+ mice

Oxidative stress and consequent apoptosis have been involved in the pathogenesis of ADs (Ba and Garg, 2011; Jog et al., 2009; Ortona et al., 2014). Since TCE exposure led to increased oxidative DNA damage and activation of PARP-1 in the livers, it was of interest to assess whether TCE exposure also induces apoptosis. To determine the apoptotic response, its potential role in TCE-induced autoimmunity and potential protective role of NAC in TCE-mediated apoptosis, the activity/expression of caspases 3, 8 and 9, the key molecular players and markers in apoptosis (Cuda et al., 2016; Galluzzi et al., 2016; Tsapras and Nezis, 2017) were evaluated. TCE treatment resulted in increased caspase 3 activity (91% increase), cleaved caspase 8 (136% increase) and caspase 9 (210% increase) expression in the livers compared to controls (p<0.05); and TCE-induced increases in caspase 3 activity and cleaved caspase 9 expression were attenuated by NAC supplementation (Figs. 3 and 4), suggesting that TCE leads to increased apoptosis and NAC provides protection against TCE-induced apoptosis.

Fig. 3.

Fig. 3.

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Liver caspase 3 activity in MRL+/+ mice exposed to TCE or TCE+NAC for 6 weeks. The data are presented as means ± SD (n=6). *p < 0.05 vs. control mice; #p < 0.05 vs. TCE-exposed mice. Note: CON = Controls; TCE = TCE-treated mice; T+N = TCE plus NAC-treated mice; NAC = NAC-treated mice.

Fig. 4.

Fig. 4.

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Cleaved caspases 8 and 9 protein expression in the livers of MRL+/+ mice exposed to TCE or TCE+NAC for 6 weeks. The data are presented as means ± SD (n=3). *p < 0.05 vs. control group; #p < 0.05 vs. TCE-exposed mice. Note: CON or C = Controls; TCE or T = TCE-treated mice; T+N or TN = TCE plus NAC-treated mice; Casp-8 = cleaved caspase 8; Casp-9 = cleaved caspase 9.

3.4. Bcl-2 and Bax expression in the livers of MRL+/+ mice

The process of apoptosis has emerged as possible source of autoantigens which could play a vital role in pathogenesis of ADs (Cuda et al., 2016; Grader-Beck et al., 2007; Ko et al., 2016). To further evaluate apoptotic pathways, Bcl-2 family, a group of apoptotic regulation proteins (Cuda et al., 2016; Kale et al., 2018; Ortona et al., 2014), was determined in various treatment groups. The results revealed that the Bcl-2 (anti-apoptotic gene) expression was significantly decreased (54% decrease) whereas Bax (pro-apoptotic gene) expression was remarkably increased (130% increase) in livers of TCE-exposed mice in comparison to control mice (p<0.05). Furthermore, the TCE-mediated changes were attenuated by NCA supplementation (Fig. 5). These findings suggest a dysregulation of pro- and anti-apoptotic pathways caused by TCE treatment and protection provided by NAC from TCE-mediated apoptosis.

Fig. 5.

Fig. 5.

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Bcl-2 and Bax protein expression in the livers of MRL+/+ mice exposed to TCE or TCE+NAC for 6 weeks. The data are presented as means ± SD (n=3). *p < 0.05 vs. control group; #p < 0.05 vs. TCE-exposed mice. Note: CON or C = Controls; TCE or T = TCE-treated mice; T+N or TN = TCE plus NAC-treated mice.

3.5. Anti-ssDNA autoantibodies in mouse sera

Anti-nuclear target antibodies are most responsive and characteristic of ADs, and represent key biomarkers in the evaluation of ADs (Arriens et al., 2017; Ippolito et al., 2011; Pisetsky, 2017). Previous studies have shown that TCE exposure induces an autoimmune response in experimental animals evidenced by increased autoantibodies including anti-nuclear antibodies (ANA) and anti-dsDNA antibodies (Griffin et al., 2000; Khan et al., 1995; Wang et al., 2013, 2015). To further evaluate TCE-mediated autoimmune response and possible association with oxidative stress and cell apoptosis, serum anti-ssDNA antibodies in TCE, NAC or TCE+NAC exposed MRL+/+ mice were determined by using a mouse specific ELISA kit (Fig. 6). Compared to controls, TCE exposure led to remarkable increases in serum anti-ssDNA antibodies (69% increase, p<0.05), which were attenuated following NAC supplementation, as evident from their decreased levels in mice treated with TCE+NAC (p<0.05), suggesting that NAC provides protection from TCE-mediated autoimmune response in these mice.

Fig. 6.

Fig. 6.

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Anti-ssDNA antibodies in the sera of MRL+/+ mice exposed to TCE or TCE+NAC for 6 weeks. The data are presented as means ± SD (n=6). *p < 0.05 vs. control group; #p < 0.05 vs. TCE-exposed mice. Note: CON = Controls; TCE = TCE-treated mice; T+N = TCE plus NAC-treated mice; NAC = NAC-treated mice.

4. Discussion

Exposure to TCE, a widely used industrial agent and a common environmental toxicant, is associated with the development of autoimmunity, evidenced from studies in both humans and animals (Griffin et al., 2000; Cooper et al., 2009; Khan et al., 1995, 2001; Kilburn and Warshaw, 1992; Wang et al., 2007, 2009, 2012, 2013). Increasing evidence has suggested free radical-mediated reactions as the potential mechanism in the pathogenesis of ADs (Khan et al., 2001; Kurien and Scofield, 2008; Oates, 2010; Wang et al., 2010, 2016). NAC, a precursor of GSH, which also reduces oxidative stress by directly scavenging ROS and modulating pro-inflammatory NF-κB signaling pathway, has shown beneficial effects, including protection against autoimmunity in both human and animal studies ((Lai et al., 2012; López-Pedrera et al., 2016; Perl, 2013; Wang et al., 2013). Previous studies in MRL+/+ mice suggest that excessive ROS generation following TCE exposure may play a key role in TCE-mediated autoimmunity through lipid peroxidation and protein modifications (Khan et al., 2001; Khan and Wang, 2018; Wang et al., 2007, 2009, 2012, 2013). To further investigate the potential of oxidative stress-mediated events particularly DNA damage and provide novel mechanistic evidence for the role of ROS-associated pathways in the pathogenesis of ADs, we conducted studies in female mice treated with TCE or TCE along with NAC and evaluated the biomarkers of oxidative DNA damage and apoptosis, with a major focus on PARP-1 activation for their contribution to autoimmunity.

ROS has the potential to initiate cellular damage of proteins, lipids and DNA (Barrera et al., 2015; Finkel, 2011; Kurien and Scofield, 2008). Earlier studies have demonstrated that TCE exposure induced oxidative damage of protein and lipids (Channel et al., 1998; Khan et al., 2001; Wang et al., 2012, 2013, 2014, 2015). However, oxidative DNA damage and its potential role in TCE-mediated pathogenesis of ADs is not clearly understood. Therefore, to assess the effect of TCE exposure on DNA oxidation and its involvement in TCE-mediated autoimmunity, 8-OHdG, one of the most widely used biomarkers of oxidative damage of DNA (Cheah et al., 2017; Di Minno et al., 2016; Tatsch et al., 2015; Zhang et al., 2017), was determined following TCE exposure with or without NAC. TCE treatment in MRL+/+ mice in this study resulted in increased liver 8-OHdG, suggesting the potential of TCE in inducing oxidative NDA damage. Remarkably, antioxidant NAC (a cell-permeable precursor of glutathione) supplementation attenuated the TCE-induced 8-OHdG formation, supporting the contribution of oxidative stress in TCE-associated DNA damage, and also suggesting the potential of NAC to attenuate or avert the TCE-mediated oxidative DNA damage.

PARP-1, a sensor of DNA damage, is a nuclear protein selectively activated in response to DNA damage (Abdelali et al., 2016; Ba and Garg, 2011; Ivana Scovassi and Diederich, 2004; Jog et al., 2009). TCE-induced oxidative DNA damage observed in this study led us to assess the activation of PARP-1. Our results show that TCE exposure significantly increased cleaved PARP-1 expression and PARP-1 activation, and NAC supplementation remarkably attenuated the increases in both cleaved PARP-1 expression and PARP-1 activation. These findings provide, for the first time, a clear evidence for the increased activation of PARP-1 following TCE exposure through oxidative stress-mediated signaling pathways. Our findings are consistent with other reports that PARP-1 activation and subsequent cell death could have important roles in the pathogenesis of diseases, and PARP-1 inhibition and consequent cell death inhibition, could be a novel therapeutic target for ADs (Ba and Garg, 2011; Grader-Beck et al., 2007; Jog et al., 2009; Ortona et al., 2014; Rosado et al., 2013). Thus, our findings not only suggest a potential role of PARP-1 activation in TCE-mediated autoimmunity, but also provide a clear rationale for evaluating apoptotic pathways leading to autoimmune responses.

One of major outcomes resulting from PARP-1 activation is apoptosis leading to cell death (Abdelali et al., 2016; Ba and Garg, 2011; Ivana Scovassi and Diederich, 2004; Wang et al., 2016). Excessive ROS generation can induce cell apoptosis by triggering multiple pathways including the functions of caspase proteins (Ortona et al., 2014; Rosado et al., 2013; Trachootham et al., 2008). Since TCE exposure caused oxidative DNA damage and also led to activation of PARP-1, it was of interest to investigate the consequent effects on apoptotic pathways. Bcl-2 family of proteins, which is divided into three groups (1) anti-apoptotic proteins such as Bcl-2, Bcl-X, Bcl-W; (2) pro-apoptotic proteins such as Bax, Bak, Bok; and (3) proapoptotic BH3-only proteins such as Bad, Bid, Bim, play key role in regulating activation of caspase cascade leading to apoptosis (Cuda et al., 2016; Kale et al., 2018; Ortona et al., 2014). Caspase family, which is classified based on their primary apoptotic role as initiators (caspase-2, −8, −9, and −10), executioners (caspase-3, −6, and −7), and inflammatory caspases (caspase-1, −4, and −5), has been well characterized as the key mediators of both extrinsic and intrinsic apoptosis (Galluzzi et al., 2016; Kale et al., 2018; Tsapras and Nezis, 2017). Even though the molecular mechanisms by which apoptosis causes ADs is not fully understood, the role of the excessive apoptosis in the modification of endogenous targets leading to autoantigens, breaking self-tolerance and regulating T cell functions are some of the subjects of great interest (Ba and Garg, 2011; Cuda et al., 2016; Ortona et al., 2014; Stuart and Hughes, 2002). The present study demonstrated that TCE exposure not only resulted in caspase 3 activation, increased cleaved caspase 8 and 9, but also showed reduced expression of Bcl-2 (anti-apoptotic member) and greatly enhanced Bcl-2 member Bax (pro-apoptotic member), suggesting TCE exposure results in an imbalance in pro- and anti-apoptosis pathways to contribute to an autoimmune response. Moreover, NAC supplementation clearly attenuated these alterations, further supporting that TCE exposure mediated apoptosis via oxidative stress-mediated mechanisms.

ADs are characterized by autoantibodies mainly against nuclear targets such as DNA. It is evident from our data that oxidative stress, PARP-1 activation and apoptotic cell death followed by increased anti-ssDNA antibodies are major consequences of TCE exposure. Therefore, it was of special interest to test if NAC, which protected against TCE-mediated oxidative DNA damage, PARP-1 activation and apoptosis, also attenuated and/or averted TCE-mediated autoimmune response. Remarkably, NAC supplementation indeed attenuated the TCE-mediated increases in anti-ssDNA antibodies, which not only support that TCE exposure induces autoimmune response (Griffin et al., 2000; Khan et al., 1995; Wang et al., 2007, 2009, 2012, 2013), but also support the potential role of oxidative DNA damage, PARP-1 activation and apoptosis in TCE-induced autoimmunity.

In conclusion, our data demonstrate that TCE treatment leads to significant increases in 8-OHdG formation in the livers, suggesting increased oxidative DNA damage. TCE-induced oxidative DNA damage was associated with significant increases in the levels of PARP-1, activities of caspase-3, caspase-8 and −9 and alterations in Bcl-2 and Bax levels in the livers. Interestingly, NAC supplementation not only attenuated 8-OHdG and PARP-1, caspase-3, cleaved caspase-9 and Bax levels, but also the TCE-mediated autoimmune response evidenced from reduced serum anti-ssDNA antibodies. The results suggest that TCE-induced oxidative DNA damage along with increased apoptosis may play a significant role in TCE-associated autoimmunity, and excessive activation of PARP-1 presents a novel mechanism of TCE-mediated autoimmune response via increased apoptosis of the cells. The key findings on PARP-1 activation and apoptosis presented in this study have led us to depict the plausible pathways of TCE-mediated autoimmune response and protection provided by antioxidant NAC (Fig. 7). Further studies to unravel the distinct pathways by which TCE induces/accelerates autoimmunity via increasing oxidative stress, especially blocking specific signaling pathways, knocking out/down target genes, and identification of autoantigens will further reveal critical mechanisms in TCE-associated autoimmune response, and could potentially lead to novel preventive/therapeutic strategies.

Fig. 7.

Fig. 7.

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The potential mechanisms of TCE-mediated autoimmunity and protection provided by NAC. TCE-induced ROS formation followed by DNA damage leads to PARP-1 activation and consequent apoptosis. Overproduction of ROS also causes a variety of structural modifications of the endogenous molecules, including DNA, lipid and protein damage, which could be the potential sources of neoantigens. After cell apoptosis and antigen processing, these events could elicit autoimmune responses by breaking immune tolerance and stimulating lymphocytes. NAC supplementation could attenuate TCE-mediated autoimmunity by reducing ROS generation and attenuating PARP-1 activation and DNA, lipid, protein damage.

Highlights:

  1. TCE exposure resulted in increased DNA oxidation and activation of PARP-1

  2. TCE led to increased apoptosis evidenced by changes in caspases and Bcl-2 family

  3. TCE treatment resulted in significant increases in anti-ssDNA antibodies in sera

  4. NAC attenuated changes in 8-OHdG, PARP-1, caspases, and also the autoantibodies

  5. Apoptosis via activation of PARP-1 contributed to TCE-mediated autoimmunity

Acknowledgements

This work was supported by R01Grants ES016302 and ES026887 from the National Institute of Environmental Health Sciences (NIEHS), NIH, and its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIEHS, NIH.

Footnotes

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Conflict of interest

The authors declare no competing financial interests.

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