Effects of poly (ADP-ribose) polymerase-1 (PARP-1) inhibition on sulfur mustard-induced cutaneous injuries in vitro and in vivo

Chemicals

SM (bis-[2-chloroethyl] sulfide, purity 97.6%) was provided by the Institute of Chemical Defense of CPLA (Chinese People’s Liberation Army). ABT-888 hydrochloride (Tian-Xiang et al., 2013) was synthesized in our institute (purity 99%). The skeletal formula for compound ABT-888 hydrochloride is shown. Other reagents were obtained from Sigma unless otherwise mentioned.

ABT-888 hydrochloride

2-[(S)-2-methylpyrrolidin-2-yl]-1H-benzimidazole-4-carboxamide

Cells

Immortalized epithelial keratinocytes (HaCaT) was a spontaneously transformed aneuploid immortal keratinocyte cell line from adult human skin (Boukamp et al., 1988), which was purchased from the China Center for Type Culture Collection (CCTCC) and cultured in Dulbecco’s Modified Eagle Medium/Ham’s F12 (DMEM/F12).

Detection of PARP-1 enzyme inhibitory activity

The inhibitory activity of the PARP-1 enzyme was detected using the Trevigen’s Homogeneous PARP Inhibition Assay Kit. This assay was performed according to the manufacturer’s instructions.

Animals

Twenty-nine adult male Kunming (KM) mice (18–22 g) were obtained from the Beijing Vital River Laboratory. The animals were acclimatized for one week before being randomly assigned to experimental groups for use in the experimental studies. The mice were housed in an animal house (26 ± 2 °C) and were provided with water and food ad libitum throughout the experiment. Briefly, a total of 29 mice were randomly divided into 5 groups: (i) untreated control (n = 5), (ii) 0.16 mg SM/ear (n = 5), (iii) 0.64 mg SM/ear (n = 7), (iv) 0.16 mg SM/ear + ABT-888 (n = 5), and (v) 0.64 mg SM/ear + ABT-888 (n = 7). The experiments were carried out following protocols approved by the Anima Ethics Committee, Beijing Institute of Pharmacology and Toxicology. The study was conducted according to the Care and Use Guide for Laboratory Animals by the NIH and was approved by the Bioethics Committee of the Beijing Institute of Pharmacology and Toxicology (No. 80-23).

Exposure of HaCaT cells to SM

The exponentially growing HaCaT cells were seeded in either plates or dishes. Before the exposure to SM, the culture medium was discarded and then 100 or 1,000 µM SM were added to the plates. After 30 min, the agent was removed and the cells were washed with phosphate buffered saline (PBS). DMEM/F12 (with 10% fetal calf serum) alone or with ABT-888 was added until cells were analyzed as described.

Cell viability assay

Cell viability was quantified using the Cell Counting Kit-8 (CCK-8) (Dojindo). This assay is based on Dojindo’s highly water-soluble tetrazolium salt. WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] is reduced by dehydrogenases in cells to give an orange, water-soluble formazan dye. The amount of the formazan dye generated by dehydrogenases in cells is directly proportional to the number of living cells. Briefly, exponentially growing HaCaT cells were seeded in 96-well plates at a density of 10,000 cells/well. 6 h or 24 h after exposure to SM and the administration of ABT-888, the CCK-8 reagent was added as recommended by the supplier.

pADPr immunofluorescence

HaCaT cells were seeded in MatTek glass bottom culture dishes and treated with SM and ABT-888. 6 h after exposure to SM and the administration of ABT-888, the cells were washed in PBS and fixed in ice cold 100% methanol for 10 min. The images were obtained by confocal microscopy. The primary antibody used was the anti-pADPr antibody (Abcam) and the secondary antibody was AlexaFluor 488 goat anti-mouse IgG (Molecular Probes). The antibody was dissolved in PBS containing 5% bovine serum albumin (BSA). Images were obtained using a Zeiss LSM 510 META confocal microscope. The mean fluorescence intensity for pADPr was calculated for each individual nucleus using the PI-marked DNA as a nuclear marker. Approximately 30 cells from three different images were analyzed with the ImageJ program.

Acumen

HaCaT cells were seeded in 96-well plates and treated with SM. After 6 h of exposure to SM, the cells were washed with PBS and fixed with 4% paraformaldehyde for 15 min and permeabilized in 100% pre-cooled methanol for 5 min. The cells were then blocked in 5% BSA and incubated with the anti-pADPr antibody (Abcam) for 1 h followed by labeling with AlexaFluor 488 goat anti-mouse IgG (Molecular Probes) for 1 h. Then, the cells were stained with 0.3 μ g/well Hoechst 33342 (Sigma) in PBS for 30 min. The plates were scanned on an Acumen eX3 laser scanning cytometer (TTP LabTech, Melbourne, UK), and the pADPr/nuclear Total Fluorescence Intensity was calculated using the Acumen eX3 software.

Western blot

Briefly, the cells were washed with cold PBS and lysed on ice for 30 min in a lysis buffer containing 1× protease inhibitor cocktail (Roche). The cell lysates were centrifuged at 12,000× g for 30 min, and the supernatants were collected. The proteins were separated by SDS-PAGE and transferred to NC membranes (Bio-Rad), which were blocked for 2 h in PBS with 5% BSA and 0.1% Triton X-100. Subsequently, the membranes were incubated with primary antibodies overnight at 4 °C followed by incubation with the appropriate horseradish peroxidase (HRP)-labeled secondary antibodies for 2 h at room temperature. The immunoreactive bands were detected using SuperSignal West Pico Chemiluminescent detection reagents (Thermo SCIENTIFIC). The rabbit anti-PARP-1 antibodies (Cell Signaling Technology), rabbit anti-Actin antibody (Abcam), mouse anti-pADPr antibody (Abcam) and mouse anti-GAPDH antibody (Cell Signaling Technology) were used as primary antibodies.

Detection of γ-H2AX

HaCaT cells were seeded in 6-well plates. Six hours or 24 h after SM exposure and ABT-888 administration, the cells were harvested, permeabilized and fixed. The primary antibody was the anti-gamma H2A.X (phospho S139) antibody (Abcam) and the secondary antibody was anti-rabbit IgG-FITC (Santa Cruz Biotechnology). A FACSCalibur flow cytometer (Becton, Dickinson and Company, BD) was used to detect the fluorescence intensity.

NAD⁺ quantification

Intracellular NAD⁺ levels were measured using the NADH/NAD Quantification kit (Sigma) according to the manufacturer’s instructions. Briefly, HaCaT cells were seeded and harvested 6 h or 24 h after treatment. Next, cells were extracted with NADH/NAD Extraction Buffer. Total NAD⁺ was detected in a 96-well plate following the protocol instructions. The color reaction was read with a 450 nm filter (Enspire 2300, PERKINELMER, PE).

Determination of intracellular ATP

The concentration of intracellular ATP was determined using a bioluminescence ATP assay kit (Beyotime) (Sigma) according to the manufacturer’s instructions. Briefly, HaCaT cells were seeded and harvested 6 h or 24 h after treatment. The cells were extracted with lysis buffer and an ATP detection working solution was added to each well of a black 96-well plate, followed by incubation for 3 min at room temperature. Then, the cell lysate samples were added to the wells and the luminescence was measured immediately (Enspire 2300, PE).

Measurement of caspase 3/7 activity

Caspase 3/7 activities were measured using the Caspase-Glo 3/7 assay Kit (Promega, Corp.) following manufacturer’s instruction. 6 h or 24 h after treatment, the HaCaT cells were lysed, and the substrate cleavage by caspase 3/7 was measured from the generated luminescent signal (Enspire 2300, PE).

Quantification of apoptotic and necrotic cell death

Apoptosis and necrosis were measured by annexin/propidium iodide (PI) double staining. Six hours or 24 h after treatment, HaCaT cells were harvested and washed with PBS. Then, the cells were re-suspended in 500 µl binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4) and stained with 5 µl annexin V-FITC and 5 µl PI (Invitrogen). The cells were incubated in a dark at room temperature for 15 min according to the manufacturer’s instructions and subjected to flow cytometry.

Transient transfection of PARP-1shRNA and selection of stable transfectants

The RNAi lentivirus that was used to specifically interfere with the PARP-1 gene was designated PARP-1 shRNA (5′-CCGGCGACCTGATCTGGAACATCAACTCGAGTTGATG TTCCAGATCAGGTCGTTTTT-3′), and the control shRNA, which was used as the negative control, was designated NC. To establish stable cell lines with knockdown of PARP-1, Lv-shPARP-1 was transfected into HaCaT cells. At 24 h after transfection, the cells were portioned into new plates and subjected to selection with 3 µg/ml Puromycin (Invitrogen) for 10 days. Independent colonies were isolated and confirmed by western blotting and PCR. Control colonies stably transfected with Lv-shCon were also generated in parallel. Lv-shPARP-1 and Lv-shCon were constructed by BioWit Technologies Co., Ltd (Shenzhen, China).

RNA isolation and RT-PCR

Total RNA from cultured cells was extracted with Trizol reagent (Invitrogen) according to the manufacturer’s instructions. RNA (1 µg) was converted to cDNA using the PrimeScript RT reagent kit (Takara Biotechnology). Two sets of primers were used for PCR: GAPDH forward, 5′-AGGTGAAGGTCGGAGTCAAC-3′ and reverse, 5′-CGCTCCTGGAA GATGGTGAT-3′; and PARP-1 forward, 5′-GAGCATCCCCAAGGACTCG-3′ and reverse, 5′-CCGCTGTCTTCTTGACTTTC-3′. The mRNA expression levels were determined using the SYBR Premix Ex Taq II kit (Takara Biotechnology). The relative mRNA expression of PARP-1 was calculated using the 2-ΔΔCt method.

Luminex assay

At 6 h or 24 h after SM exposure, HaCaT cells were lysed. The cell suspension was transferred into a centrifuge tube and gently rocked for 10–15 min at 4 °C. Then, the particulate matter was removed by filtration using EMD Millipore filters. The Phospho-JNK (Thr183/Tyr185), Phospho-p53 (ser46), Active Caspase 9 (Asp315), Active Caspase 8 (Asp384), c-PARP (p89), Phospho-AKT (Thr308) and Phospho-mTOR (Ser2448) protein levels were determined with the Luminex assays using a MILLIPLEX MAP (Millipore Corp) according to the manufacturer’s protocols. The data were collected using a Luminex 200 analyzer (Luminex Corp).

Mouse ear vesicant model

In the mouse ear vesicant model (MEVM), five microliters (32 mg/ml and 128 mg/ml) of SM in propylene glycol was applied to the medial surface of the right ears of KM mice. Propylene glycol was applied to the medial surface of the right ear of KM mice in the control group. ABT-888 was administered (i.p.) at dose of 200 mg/kg once 30 min before or 10 min after the SM exposure. The injury was measured from the edema response. The ear edema for each animal was initially expressed as a percentage of the increase in relative ear weight (REW) to normal and was calculated from the difference in ear weights between the right and left ears using the following formula: ${\displaystyle \text{\%}\mathrm{REW}=\frac{\mathrm{SM}\left(\mathrm{or}\mathrm{ABT\; -\; 888}+\mathrm{SM}\right)\text{exposed ear weight}\left(\text{right ear}\right)-\text{Vehicle control ear weight}\left(\text{left ear}\right)}{\text{Vehicle control ear weight}\left(\text{left ear}\right)}\times 100.}$ The effect of the drug (modulation of edema) was expressed as the percentage in reduction from the positive control (SM-exposed only) and was calculated from the difference in the mean % REW between the ABT-888 + SM group and the SM only group (positive control) using the following formula (Babin et al., 2000): ${\displaystyle \text{\%}\text{Reduction from model}=\frac{\left(\mathrm{Mean}\text{\%}\mathrm{REW\; ABT\; -\; 888}+\mathrm{SM}\right)-\left(\mathrm{Mean}\text{\%}\mathrm{REW}\mathrm{SM}\right)}{\mathrm{Mean}\text{\%}\mathrm{REW}\mathrm{SM}}\times 100.}$

Histopathology

Immediately after obtaining the tissue weight, skin punches were fixed in 10% neutral buffered formalin (NBF). All NBF specimens were embedded in paraffin, sectioned and stained with hematoxylin and eosin (H& E) for evaluation.

The histopathological endpoints described in epidermal necrosis (EN; inner surface of the SM treated ear) were given the following severity scores as previously described (Babin et al., 2000): (0) no lesion or change; (1) change in <5% of the entire tissue section; (2) change in 10–40% of the entire tissue section; (3) change in 50–80% of the entire tissue section; (4) change in >90% of the entire tissue section. For example, a severity score of 2 (change in <10%–40% of the entire tissue section) indicates 10%–40% of the epidermal cells are necrotic cells in a 400× field. Histopathology was evaluated in a blind experiment and the scores were reported as the mean and standard deviation of each group.

Statistical analysis

All values were expressed as the means ± SEM. For the evaluation of statistical significance, a two-tailed Student’s t-test was performed. Bonferroni test was used for post hoc comparison. P < 0.05 was considered statistically significant.

PARP-1 activation increased in HaCaT cell following SM treatment, which could be prevented by ABT-888

SM is a highly reactive bifunctional alkylating agent that induces DNA damage. DNA damage activates PARP-1, which catalyzes the transfer of ADP-ribose units from nicotinamide adenine dinucleotide (NAD⁺) to PARP-1 itself and a range of other nuclear proteins. To determine the activation of PARP-1 and the PARP inhibitory activity of ABT-888 in SM treated HaCaT cells, the expression of pADPr was detected by immunofluorescence and western blotting. The increase of pADPr, which represented the activation of PARP-1, was observed after 6 h of exposure to SM at concentrations of either 100 µM or 1,000 µM. The activation of PARP-1 under 1,000 µM SM exposure was much greater than under 100 µM SM exposure. Immediate application of ABT-888 after SM exposure could significantly decrease the expression of pADPr, especially under 1,000 µM SM exposure. In addition, ABT-888 alone group and control group did not show any significant difference in the expression of pADPr (Figs. 1A–1E). These results suggested that the SM injury could cause PARP-1 activation, which could be marked inhibited by the potent PARP inhibitor ABT-888. The PARP-1 inhibitory effect of ABT-888 was also confirmed using the Trevigen’s Homogeneous PARP Inhibition Assay Kit (Fig. 1F).

PARP inhibitor ABT-888 had a protective effect in HaCaT cells after severe SM injury

In HaCaT cells, at 6 h post-treatment by ABT-888, cell viability was significantly increased under 1,000 µM SM exposure (Fig. 2A), whereas ABT-888 did not protect cell viability under 100 µM SM exposure. Moreover, the addition of ABT-888 no longer showed the protective effect at 24 h post SM exposure (Fig. 2B). ABT-888 alone group and control group did not show any significant difference in cell viability.

PARP inhibitor ABT-888 had a protective effect in mouse ear vesicant model after severe SM injury

In the SM-induced mouse ear vesicant model, we also observed protective effect of ABT-888. ABT-888 showed a protective effect in severe SM injury. The structural alterations of the skin in the mouse ears post SM exposure were observed. Exposure to 0.16 mg SM/ear and 0.64 mg SM/ear resulted in moderate to severe edema and epidermal necrosis at 24 h after SM exposure. Twenty-four hours post-exposure to 0.16 mg SM/ear and 0.64 mg SM/ear, the dermis inflammatory cell infiltration and reticular degenerative changes associated with basal cell necrosis of the epidermis were observed, along with hypereosinophilic cytoplasms and nuclear pyknosis. Epidermal cells exposed to 0.16 mg SM/ear showed pyknotic nuclei, but ABT-888 did not show any protective effect. More pyknotic nuclei of epidermal cells were observed in the mouse ear skin exposed to 0.64 mg SM/ear than that exposed to 0.16 mg SM/ear and ABT-888 could significantly reduce the number of pyknotic nuclei (Fig. 3A).

The increase in relative ear weight (REW) to normal represented the ear edema. The REW and epidermal necrosis (EN) scores for the right ear of each animal in the control group were zero (Table S3). The REW and EN scores of all SM groups were significantly different from those of the control group, and the scores of the group exposed to 0.64 mg SM/ear (REW 204%, EN 3.9) were higher than those of the group exposed to 0.16 mg SM/ear (REW 154%, EN 2.2). ABT-888 did not reduce the REW and EN scores in the group exposed to 0.16 mg SM/ear, but ABT-888 reduced the REW (approximately 26%)and EN scores (approximately 40%) in the group exposed to 0.64 mg SM/ear (Figs. 3B and 3C). The results were similar when ABT-888 was applied either after or before SM treatment (Table S10). In addition, the ABT-888 alone group and control group did not show any significant difference (Table S10).

The PARP inhibitor ABT-888 could prevent the reduction of NAD⁺ and ATP in HaCaT cell after SM exposure

SM has been shown to activate the enzymatic activity of PARP-1 by decreasing the NAD⁺ concentration in either human skin grafts (Gross et al., 1985) or keratinocytes ex vivo (Rosenthal et al., 1998). Severe SM-induced DNA damage over-activates PARP-1, leading to the depletion of NAD⁺ and ATP. To determine the NAD⁺ and ATP concentration after SM exposure and the effect of ABT-888, HaCaT cells were exposed to SM and the concentrations of intracellular NAD⁺ andATP were measured 6 and 24 h later. SM exposure could decrease NAD⁺ and ATP levels (Figs. 4A–4D). The ABT-888 alone group and control group did not show any significant difference in NAD⁺ and ATP concentration. Immediate application of ABT-888 after SM exposure could prevent the reduction of NAD⁺ and ATP.

The PARP inhibitor ABT-888 suppressed SM-induced apoptosis and necrosis in HaCaT cells

It has been known that SM-induced DNA injury causes PARP activation, which may lead to necrosis or apoptosis. PARP-1 has been shown to participate in apoptotic and necrotic cell death pathways. Therefore, we used flow cytometry to analysis HaCaT cells doubly stained for annexin V and PI to observe the SM-induced apoptosis and necrosis and to assess the effect of ABT-888. The results showed that only 1,000 µM SM caused HaCaT cell apoptosis and necrosis after SM exposure. ABT-888 significantly decreased the percentage of apoptotic and necrotic cells (Figs. 5E and 5F).

Thus, we next assessed whether SM activates the caspase-PARP pathway, which is the main executor of apoptotic processes. Caspase-3/7 is responsible for the cleavage of PARP-1 during apoptosis. Therefore, the caspase-3/7 activity was measured after treatment of HaCaT cells with 100 or 1,000 µM SM for 6 h or 24 h. At 6 h following 100 or 1,000 µM SM exposure, an increase in caspase-3/7 activity was observed. ABT-888 decreased caspase 3/7 activity in cells exposed to 1,000 µM SM but increased caspase 3/7 activity in cells exposed to 100 µM SM (Fig. 5A). At 24 h following SM exposure, ABT-888 decreased the caspase 3/7 activity in HaCaT cells (Fig. 5B).

When apoptosis occurs, PARP-1 is cleaved by activated caspase 3/7 into p89 and p24 fragments. To observe the effects of ABT-888 on apoptosis at different doses and incubation times with SM, a western blot was performed to detect c-PARP (cleavage of PARP-1, an apoptosis-specific p89 fragment). The results showed that SM could significantly increase the expression of c-PARP. At 6 h after 1,000 µM SM exposure, ABT-888 decreased the expression of c-PARP. However, ABT-888 showed no significant effect on c-PARP levels in cells exposed to 100 µM SM (Fig. 5C). However, ABT-888 had no significant protective effects on c-PARP levels at 24 h after SM exposure (Fig. 5D). In addition, ABT-888 alone group and control group did not show any significant difference in caspase-3/7 activity, c-PARP levels, percentage of apoptotic and necrotic cells.

PARP-1 knockdown protected HaCaT cells from SM-induced cell death

To further investigate the mechanism of PARP-1 on the toxicity of SM, we used RNA interference to knock down the endogenous PARP-1 in HaCaT cells. To examine the effect of PARP-1 knockdown on the mRNA level, the total mRNA from control and PARP-knockdown HaCaT cells was isolated and processed for RT-PCR. The relative mRNA expression of PARP-1 was calculated. The results showed that PARP-1 shRNA significantly decreased the levels of PARP-1 mRNA by 90% compared to the level in the control (Fig. 6A). The level of PARP-1 protein was also significantly decreased by RNA interference (Fig. 6B).

To study the effect of PARP-1 knockdown in the toxicity of SM, we examined the cell viability in PARP-1-knock-down and control HaCaT cells exposed to SM using the CCK-8 kit. The results showed that 100 or 1,000 µM SM exposure decreased the cell viability 6 h and 24 h after SM treatment. The downregulation of PARP-1 significantly increased cell viability compared with that of the control SM-treated cells (Figs. 6C and 6D), suggesting that PARP-1 knockdown suppressed the toxicity of SM at the level of cell viability.

PARP-1 knockdown suppressed the SM-induced apoptosis checkpoint signals in HaCaT cells

In HaCaT cells treated with NC shRNA, the protein levels of phospho-JNK (Thr183/Tyr185), phospho-p53 (ser46), active Caspase 9 (Asp315), active Caspase 8 (Asp384), c-PARP (p89) and Caspase 3/7 activity were enhanced in response to SM stimulation (Figs. 7A–7L). However, 24 h after exposure to 1,000 µM SM, the protein levels of phospho-JNK (Thr183/Tyr185) (Fig. 7B), phospho-p53 (ser46) (Fig. 7D), active Caspase 9 (Asp315) (Fig. 7F), and active Caspase 8 (Asp384) (Fig. 7H) were extremely low, possibly because these early apoptosis signals could not be detected during the late apoptosis. In the HaCaT cells treated with PARP-1 shRNA, all of the apoptosis checkpoint signals mentioned above was downregulated compared to the cells treated with NC shRNA (Figs. 7A–7L). These results suggested that the silencing of PARP-1 suppressed the SM-induced apoptosis checkpoint signals in the HaCaT cells.

PARP-1 knockdown reversed the SM-induced suppression of the Akt/mTOR pathway

Previously, it was reported that PARP-1 is involved in autophagy induced by DNA damage through the activation of the key autophagy regulator mTOR (Munoz-Gamez et al., 2009). To investigate whether SM exposure activated mTOR and how PARP-1 participated in this process, the phosphorylation of Akt and mTOR was evaluated in PARP-1-knockdown and NC HaCaT cells. The results showed that in the HaCaT cells treated with NC shRNA, the phosphorylation of Akt and mTOR was suppressed in response to SM stimulation (Figs. 8B and 8D). At 24 h after exposure of HaCaT cells to SM, PARP-1 knockdown significantly increased the phosphorylation of Akt and mTOR (Figs. 8B and 8D), whereas PARP-1 knockdown had no effect on the phosphorylation of Akt and mTOR at 6 h after exposure to SM (Figs. 8A and 8C). The results suggested that PARP-1 knockdown reversed the SM-induced suppression of the Akt/mTOR pathway, which might affect autophagy following SM-induced injury. Taken together, the results suggest that the silencing of PARP-1 could protect HaCaT cell from SM-induced injury by regulating the apoptosis and autophagy pathways.

PARP inhibitor ABT-888 further enhanced the DNA damage after SM exposure

H2AX is a sensitive marker for DNA double-stranded breaks (DSB) (Fernandez-Capetillo et al., 2004). When DSB occurs, H2AX is phosphorylated at Ser139 in the nucleosomes surrounding the break point (Thiriet & Hayes, 2005). To determine the effect of SM on DNA damage and the potential effect of ABT-888 on preventing SM injury, the phosphorylation of H2AX (S139) was detected in HaCaT cells. HaCaT cells were exposed to 100 µM or 1,000 µM SM before DMEM/F12 (with 10% fetal calf serum) alone or with 10 µM ABT-888 was added. The cells were then analyzed by flow cytometry. The results showed that exposure to 100 µM and 1,000 µM SM increased the phosphorylation of H2AX in HaCaT cells at both 6 h (Fig. 9A) and 24 h (Fig. 9B) following SM exposure. ABT-888 enhanced the phosphorylation of H2AX caused by SM exposure at 24 h (Fig. 9B). This result indicated that PARP inhibitor actually increased the DNA damage after SM exposure.