| Sulfur mustard(SM), a chemical warfare vesicant agent, has been used in several military conflicts, including World Wars I and II and the Iran-Iraq War. Because of its devastating toxicity, its use during the World War I earned it the sobriquet king of the battle gasses. After World War II, SM has also been one of the most predominant agents found in the chemical weapons abandoned in China, causing harm to civilians. Due to its simple and cheap chemical synthesis that makes SM readily available for terrorist use. Bioterrorism preparedness plan proposed that terrorists might use chemical warfare agents and general toxic industrial chemicals as weapon but which they are most likely to use is still neural agents, vesicant agents and cyanide agents. The organs most affected by SM are the skin, eyes and respiratory tract, which are the tissues that come in direct contact with the toxic liquid or vapor. When SM injury became serious and systemic, bone marrow, blood, and immune system are the main target organ.SM is a bifunctional alkylating compound that targets DNA, RNA, proteins, carbohydrates and lipids. The DNA damage induced by SM is considered to be most important triggers of cell death in SM injury. In response to DNA damage, several key biochemical pathways that are important in protecting cells against injury and initiating repair processes are activated. PARP-1, an NAD+-dependent nuclear enzyme implicated in multiple DNA repair pathways(including single strand breaks, double strand breaks and base excision repair), has been shown to be activated upon treatment with SM. PARP-1 uses nicotine adenine dinucleotide(NAD+) as a substrate to catalyze covalent binding of poly(ADP-ribose)(p ADPr) to PARP-1 itself and other nuclear proteins involved in DNA repair. Although PARP activation had been considered to have a positive effect on cells survival by promoting DNA repair, over activation of PARP-1 initiated by severe SM-induced DNA damage and the consequent depletion of NAD+ exacerbates the cell death instead. It is indicated that the activation of PARP-1 is very important in SM injury.In recent years, some protective effects of PARP inhibitors in SM injury in vivo and in vitro have been reported. In the battle drug-screening program initiated by the US Army Medical Research Institute of Chemical Defense, three PARP inhibitors were found to reduce histopathological changes by >50% in the mouse ear model for skin injury among nearly 400 tested compounds. While the cellular NAD+ depletion induced by SM injury could indeed be preserved by treatment with PARP inhibitors, the effect on total cell survival could not be observed. Due to the limited effect of PARP inhibitors in SM injury, it is worth noting that, theoretically, PARP inhibition can block DNA repair and increase cell death induced by alkylating agents. Early studies showed that PARP inhibitors indeed slowed the DNA repair rate in SM injury. Therefore, whether PARP inhibitor could attenuate the cellular damage induced by SM is not sure, and the practical value of PARP inhibitor is controversial.We synthesized some PARP-1 inhibitors which may be under research by U.S. army in treating SM injury. On the other hand, there is a new generation of PARP inhibitors in cancer chemotherapy available, which is much more potent than the classical ones. Whether these more specific PARP inhibitors may also be beneficial in treating SM injured patients remains to be shown. Therefore, in this study we evaluated the PARP-1 inhibitory ability of these compound and the protective effect of these compound in SM injury. Then we study the mechanism of PARP-1 inhibitor in SM injury. 1. The activity evaluation of PARP-1 inhibitors and the protective effect of PARP-1 inhibitor in SM injuryPARP inhibitory activity of eight-five compounds are evaluated by PARP inhibition assay kit. The therapeutic effect of PARP inhibitors on SM injury are evaluated in mouse ear vesicant model(MEVM) and SM-induced Ha Ca T cells. 1.1 The activity evaluation of PARP-1 inhibitors in vitroTo optimize the in vitro screening system based on high sensitive fluorescence for identification of PARP inhibitors, screen potential PARP inhibitors. The results showed that the PARP inhibitory activity of S001 and S003 and its analogues were high, but the PARP inhibitory activity of BPUBA, S002 and its analogues were low, suggesting that future research about the protective effects of PARP inhibitor in SM injury can focus on S001, S003 and its analogues. 1.2 The activity evaluation of PARP-1 inhibitors in Ha Ca T cellThe results above showed that the PARP inhibitory activity of S001 and S003 and its analogues were high, so we evaluated the effect of PARP inhibitors in SM-injuried Ha Ca T cell.We establish the cell model of sulfur mustard injury. With the increase of sulfur mustard exposure dose and time, cell viability decreased gradually. At 6 h post-SM treatment, S001, S003 and its analogues significantly increased cell viability under 1000 μM SM exposure. 1.3 The activity evaluation of PARP-1 inhibitors in vivoIn the mouse ear vesicant model, we found that intraperitoneal injection of PARP inhibitor S003 showed significantly reduced the REW after 0.64mg/ear SM exposure, suggesting that S003 had a protective effect in severe SM injury. However, percutaneous administration of PARP inhibitor S003 showed no protective effect in SM injury. In hairless guinea pig vesicant model, we found that intraperitoneal injection of PARP inhibitor S003 significantly reduced the skin redness and skin blood flow. 2 The study of effect and mechanism of PARP-1 inhibitor in SM injuryIn this part, we selected the most potent PARP inhibitor S003 as example to investigate the effect and mechanism of PARP inhibitor in mouse ear vesicant model(MEVM) and SM-induced Ha Ca T cells. 2.1. The inhibitory effect of PARP-1 inhibitor on SM-induced PARP-1 activationS003 was selected from a screen of 85 compounds synthesized by Beijing institute of Pharmacology and Toxicology and showed outstanding PARP inhibition activity. The IC50 of S003 and the classic PARP inhibitor 3-aminobenzamide(3-AB) was detected, and the results showed that the novel PARP inhibitor S003(IC50=60.22 n M) is over 100 times more potent than 3-AB(IC50=10.86μM, the classical PARP inhibitor).SM is a highly reactive bifunctional alkylating agent that induces DNA damage in cells. DNA damage activates PARP-1, which catalyzes nicotinamide adenine dinucleotide(NAD+) to poly(ADP-ribose)(p ADPr) to PARP-1 itself and to a range of other nuclear proteins. To determine the activation of PARP-1 after SM exposure and to determine the PARP inhibitory activity of S003 in Ha Ca T cells treated with SM, the expression of p ADPr was detected by immunofluorescence and western blot. The increase of p ADPr, which represented the activation of PARP-1, was observed after 6 h of exposure to either 100μM or 1000μM SM. Under exposure to 1000μM SM, the activation of PARP-1 was much greater than under exposure to 100μM SM. Immediate application of S003 after SM exposure could significantly decrease the expression of p ADPr, especially under 1000μM SM exposure. These results suggested that the SM injury could cause PARP-1 activation, which could be marked inhibited by the potent PARP inhibitor S003. 2.2. Effect of PARP-1 inhibitor S003 in SM-induced Ha Ca T cells and the mouse ear vesicant modelIn Ha Ca T cells, after 6 h and 24 h treatment with either 100 μM or 1000 μM doses of SM, a significant decrease in cell viability was observed. At 6 h post-treatment, S003 significantly increased cell viability under exposure to 1000 μM SM, whereas S003 did not protect cell viability under exposure to 100 μM SM. However, the addition of S003 no longer showed a protective effect at 24 h post-treatment for either dose of SM.We obtained similar results for the mouse ear vesicant model, finding that the PARP inhibitor S003 showed a protective effect in severe SM injury.First, the REW and EN were calculated using the formula described in the materials and methods section. The REW and EN scores for the right ear of each animal in the control group were zero. The REW and EN scores for both SM groups were significantly different from those of the control group, and the scores of the group treated with 0.64 mg SM/ear(REW 154.67, EN 2.2) were higher than of the group treated with 0.16 mg SM/ear(REW 204.83, EN 3.9). S003 did not reduce the REW and EN scores in the group exposed to 0.16 mg SM/ear, but S003 reduced the REW(approximately 26%) and EN scores(approximately 40%) in the group exposed to 0.64 mg SM/ear.Then, the structural alterations to the skin of the mouse ears following 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 hours after exposure. Twenty-four hours after exposure to 0.16 mg SM/ear and 0.64 mg SM/ear, the histology evidenced dermis inflammatory cell infiltration and reticular degenerative changes associated with basal cell necrosis of the epidermis, along with hypereosinophilic cytoplasms and nuclear pyknosis. Epidermal cells exposed to 0.16 mg SM/ear showed pyknotic nuclei, but S003 did not show any protective effect. 0.64 mg SM/ear resulted in more pyknotic nuclei then the 0.16 mg SM/ear treatment, and S003 could significantly reduce the number of pyknotic nuclei. 2.3. Effect of PARP-1 inhibitor S003 in SM-induced NAD+/ATP depletion and apoptosis/necrosisSM has been shown to activate the enzymatic activity of PARP-1 by decreasing the NAD+ concentration in either human skin grafts or keratinocytes ex vivo. 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 S003, Ha Ca T cells were exposed to SM and the concentrations of intracellular NAD+ and ATP were measured 6 h later. SM treatment induced a dose-dependent decrease in NAD+ and ATP levels. Immediate application of S003 after SM exposure could prevent the reduction in NAD+ and ATP.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 Ha Ca T cells doubly stained for annexin V and PI to observe the SM-induced apoptosis and necrosis and to assess the effect of S003. The results showed that only 1000 μM SM could cause Ha Ca T cell apoptosis and necrosis at 6 h after SM exposure. S003 significantly decreased the percentage of apoptotic and necrotic cells, but showed no significant effect in cells exposed to 100 μM SM. However, S003 seemed to insignificantly increase apoptosis.The annexin V/PI double staining results showed that SM caused apoptotic and necrotic cell death at 6 h in Ha Ca T cells and that S003 could reduce the SM-induced damage of SM. 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 Ha Ca T cells with 100 or 1000 μM SM for 6h. After 6 h of exposure to SM, a dose-dependent increase in caspase-3/7 activity was observed. At 6 h after SM exposure, S003 decreased caspase 3/7 activity in cells exposed to 1000 μM SM but increased caspase 3/7 activity in cells exposed to 100μM SM. When apoptosis occurs, PARP-1 is cleaved by activated caspase 3/7 into p89 and p24 fragments. To observe the effects of S003 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 1000 μM SM exposure, S003 decreased the expression of c-PARP. However, S003 showed no significant effect on c-PARP levels in cells exposed to 100 μM SM.These results showed that the PARP inhibitor S003 significantly increased the cell viability at 6 h after exposure to 1000 μM SM, but showed no effect after exposure to 100 μM SM. The cell protective effect of the PARP inhibitors in severe SM injury is mainly due to the protective effect of NAD+ and ATP depletion and the reduction in apoptosis and necrosis. Under conditions of mild SM injury, the intracellular NAD+ and ATP were not depleted and apoptosis and necrosis were very mild. This might be the part of the reason that the PARP inhibitor had no significant protective effect on mild SM induced cell death.Although the PARP inhibitor S003 showed some protective effects at 6 h after SM exposure, S003 had no significant protective effects at 24 h after SM exposure. Consistent with the findings for cell survival, the protective effects of S003 with respect to NAD+ and ATP depletion were no longer significant at 24 h after SM exposure. In addition, the effects of S003 in reducing apoptosis and necrosis were also significantly decreased. At 24 h after exposure to 1000 μM SM, S003 showed no significant effects on ANNEXIN V/PI staining or the expression of c-PARP. We expected to notice that S003 could still reduce PI staining in the cells exposed to 100μM SM and decrease the caspase 3/7 activity in cells exposed to 100 μM or 1000 μM SM. However, the protective effects of the PARP inhibitor S003 in SM injury were overall less obvious at 24 h than at 6 h after SM-induced injury. 2.4. Effect of PARP-1 inhibitor S003 in SM-induced DNA damageH2AX is a sensitive marker for DNA double-stranded breaks(DSB). When DSB occurs, H2 AX is phosphorylated at Ser139 in the nucleosomes surrounding the break point. To determine the effect of SM on DNA damage and the potential effect of S003 on preventing SM injury, the phosphorylation of H2AX(S139) was detected in Ha Ca T cells. Ha Ca T cells were exposed to 100μM or 1000μM SM before DMEM/F12(with 10% fetal calf serum) alone or with 10μM S003 was added. The cells were then analyzed by flow cytometry. The results showed that 100μM and 1000μM SM increased the phosphorylation of H2 AX in Ha Ca T cells at both 6 h and 24 h after SM exposure. S003 enhanced the phosphorylation of H2 AX caused by SM exposure at 24 h. This result indicated that S003 might aggravate SM-induced DSB in Ha Ca T cells by increasing the phosphorylation of H2 AX, indicating that the PARP inhibitor not only failed to protect the cell against SM injury, but actually increased the DNA damage. 3 Effect of PARP-1 knockdown in SM-treated Ha Ca T cellsTo further investigate the potential effect of PARP-1 on the toxicity of SM, we used RNA interference to knock down the endogenous PARP-1 in Ha Ca T cells. 3.1 The knockdown efficiency of PARP-1 and the effect of PARP-1 knockdown in SM-treated Ha Ca T cellsTo examine the knockdown efficiency of PARP-1 in Ha Ca T cells, we detected the m RNA and protein level of PARP-1. The results showed that PARP-1 sh RNA significantly decreased the levels of PARP-1 m RNA by 90% compared to the level in the control. The level of PARP-1 protein was also significantly decreased by RNA interference.To determine whether PARP-1 is involved in the toxicity of SM, we examined the cell viability in PARP-1-knock-down and control Ha Ca T cells exposed to SM. The results showed that the downregulation of PARP-1 significantly increased cell viability compared with that of the control SM-treated cells, suggesting that PARP-1 knockdown suppressed the toxicity of SM at the level of cell viability. 3.2 The effect of PARP-1 knockdown in SM-treated Ha Ca T cells on apoptosis pathwaySM-induced DNA injury has been reported to cause PARP-1 activation, which may lead to apoptosis. To test this possibility, we examined the apoptosis checkpoint signals using Luminex assays, the Caspase-Glo 3/7 assay and Annexin V-APC staining assay to clarify the relationship between PARP-1 activation and SM-induced apoptosis. The results suggested that the silencing of PARP-1 suppressed the SM-induced apoptosis checkpoint signals in the Ha Ca T cells. 3.3 The effect of PARP-1 knockdown in SM-treated Ha Ca T cells on Akt/m TORpathwayPreviously, it was reported that PARP-1 is involved in autophagy induced by DNA damage through the activation of the key autophagy regulator m TOR. To investigate whether SM exposure activated m TOR and how PARP-1 participated in this process, the phosphorylation of Akt and m TOR was evaluated in PARP-1-knockdown and NC Ha Ca T cells. The results showed that in the Ha Ca T cells treated with NC sh RNA, the phosphorylation of Akt and m TOR was suppressed in a dose-dependent manner in response to SM stimulation. PARP-1 knockdown significantly increased the phosphorylation of Akt and m TOR. The results suggested that PARP-1 knockdown reversed the SM-induced suppression of the Akt/m TOR pathway, which might affect autophagy following SM-induced injury.The results showed that knockdown of PARP-1 protected cell viability and downregulated the apoptosis checkpoints, including p-JNK, p-p53, Caspase 9, Caspase 8, c-PARP and Caspase 3, following SM-induced injury. Furthermore, knockdown of PARP-1 also reversed the SM-induced suppression of the Akt/m TOR pathway. Taken together, our study reveals a novel relationship between PARP-1 and the activation of the apoptosis/autophagy pathway in SM-induced injury and indicates that the protective effects of PARP inhibitors in SM injury might be due to the regulation of the apoptosis/autophagy pathway.Conclusions:1. S003 is a new generation of PARP-1 inhibitor which is over 100 times more potent than the classical PARP-1 inhibitor 3-AB.2. PARP-1 inhibitor S003 has a therapeutic effect in mouse ear vesicant model(MEVM), hairless guinea pig vesicant model and SM-induced Ha Ca T cellsafter severe SM injury.3. PARP-1 inhibitor S003 could reduce SM-induced NAD+/ATP depletion, apoptosis/necrosis in SM-induced Ha Ca T cells, which might be the reason why PARP-1 inhibitor exerts a protective effect.4. PARP-1 knockdown could reduce SM injury by regulating apoptosis and autophagy pathway in SM-induced Ha Ca T cells.5. PARP-1 inhibitor further enhanced the DNA damage after SM exposure.6. S003 could develop into a new drug for the prevention and treatment of SM exposure in the future.In conclusion, in this study we used a new generation of specific and potent PARP-1 inhibitor S003 to investigate the effect and mechanism of PARP-1 inhibitor in SM injury, the results showed that PARP-1 inhibitor S003 has a therapeutic effect in mouse ear vesicant model(MEVM), hairless guinea pig vesicant model and SM-induced Ha Ca T cells after severe SM injury. PARP-1 downregulation could reduce SM-induced NAD+/ATP depletion, apoptosis/necrosis and autophagy in SM-induced Ha Ca T cells, which might be the part of the reason why PARP-1 inhibitor exerts a protective effect. However, PARP-1 inhibitor S003 further enhanced the DNA damage after SM exposure, which indicated that we should be very careful in the application of PARP-1 inhibitors in SM injury treatment. In addition, the mechanism by which PARP-1 inhibition is protective against SM-induced injury is mainly focused on the mitigation of the NAD+ depletion. However, based on the evidence provided in this article, we have reason to believe that the effect of PARP-1 in the apoptosis and autophagy pathways in sulfur mustard-induced injury cannot be ignored. This study provided a new theoretical basis for PARP-1 inhibition in the treatment of sulfur mustard-induced injuries. |
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