poly(ADP-ribose) Polymerase Overactivation Research Articles

The potent inhibitor of poly(ADP‐ribose) polymerase‐1, 5‐aminoisoquinolinone, promotes autophagy and reduces neural tissue damage and locomotor impairment after spinal cord injury in mice (663.4)

Evidence indicate that Poly (ADP‐ribose) Polymerase (PARP) plays an important role in cerebral ischemia/reperfusion, stroke and neurotrauma. Overactivation of PARP consumes NAD+ and ATP culminating in cell dysfunction and necrosis. PARP has also been shown to associate with and regulate the function of several transcription factors. PARP is involved in the up‐regulation of numerous pro‐inflammatory genes that play a pathogenetic role in the later stage of neurotrauma. The aim of this study was to investigate the effects of a potent PARP‐1 inhibitor, 5‐aminoisoquinolinone (5‐AIQ), on secondary inflammatory damage associated with an experimental model of spinal cord injury (SCI). SCI was induced in mice through a spinal cord compression by the application of vascular clips (force of 24 g) to the dura via a four‐level T5 to T8 laminectomy, and 5‐AIQ (3 mg/kg, intraperitoneally, 1 and 6 hours after SCI) was injected into mice and once daily thereafter for 10 days. Our results demonstrated that the administration of 5‐AIQ significantly decreased the phosphorylation of the p70S6K protein and led to higher expression levels of LC3 and Beclin 1 in the injured spinal cord. In addition, neuronal loss and cell death in the injured spinal cord were significantly reduced in the 5‐AIQ‐treated mice compared to the vehicle‐treated mice. Furthermore, the 5‐AIQ‐treated mice showed significantly higher locomotor function in Basso Mouse Scale scores than did the vehicle‐treated mice. These results indicate that 5‐AIQ promoted autophagy, and reduced neural tissue damage and locomotor impairment after SCI.

Parthanatos: mitochondrial‐linked mechanisms and therapeutic opportunities

Cells die by a variety of mechanisms. Terminally differentiated cells such as neurones die in a variety of disorders, in part, via parthanatos, a process dependent on the activity of poly (ADP-ribose)-polymerase (PARP). Parthanatos does not require the mediation of caspases for its execution, but is clearly mechanistically dependent on the nuclear translocation of the mitochondrial-associated apoptosis-inducing factor (AIF). The nuclear translocation of this otherwise beneficial mitochondrial protein, occasioned by poly (ADP-ribose) (PAR) produced through PARP overactivation, causes large-scale DNA fragmentation and chromatin condensation, leading to cell death. This review describes the multistep course of parthanatos and its dependence on PAR signalling and nuclear AIF translocation. The review also discusses potential targets in the parthanatos cascade as promising avenues for the development of novel, disease-modifying, therapeutic agents.

Perinatal asphyxia: CNS development and deficits with delayed onset.

Perinatal asphyxia constitutes a prototype of obstetric complications occurring when pulmonary oxygenation is delayed or interrupted. The primary insult relates to the duration of the period lacking oxygenation, leading to death if not re-established. Re-oxygenation leads to a secondary insult, related to a cascade of biochemical events required for restoring proper function. Perinatal asphyxia interferes with neonatal development, resulting in long-term deficits associated to mental and neurological diseases with delayed clinical onset, by mechanisms not yet clarified. In the experimental scenario, the effects observed long after perinatal asphyxia have been explained by overexpression of sentinel proteins, such as poly(ADP-ribose) polymerase-1 (PARP-1), competing for NAD+ during re-oxygenation, leading to the idea that sentinel protein inhibition constitutes a suitable therapeutic strategy. Asphyxia induces transcriptional activation of pro-inflammatory factors, in tandem with PARP-1 overactivation, and pharmacologically induced PARP-1 inhibition also down-regulates the expression of proinflammatory cytokines. Nicotinamide has been proposed as a suitable PARP-1 inhibitor. Its effect has been studied in an experimental model of global hypoxia in rats. In that model, the insult is induced by immersing rat fetus into a water bath for various periods of time. Following asphyxia, the pups are delivered, treated, and nursed by surrogate dams, pending further experiments. Nicotinamide rapidly distributes into the brain following systemic administration, reaching steady state concentrations sufficient to inhibit PARP-1 activity for several hours, preventing several of the long-term consequences of perinatal asphyxia, supporting the idea that nicotinamide constitutes a lead for exploring compounds with similar or better pharmacological profiles.

Amelioration of diabetes-induced neurobehavioral and neurochemical changes by melatonin and nicotinamide: Implication of oxidative stress–PARP pathway

Diabetes associated hyperglycemia results in generation of reactive oxygen species which induces oxidative stress and initiate massive DNA damage leading to overactivation of poly (ADP-ribose) polymerase (PARP). In this study, we have elucidated the involvement of oxidative stress–PARP pathway using pharmacological interventions (melatonin, as an anti-oxidant and nicotinamide, as a PARP inhibitor) in diabetes-induced neurobehavioral and neurochemical alterations. Sprague–Dawley rats were rendered diabetic by a single intraperitoneal injection of streptozotocin. Behavioral and cognitive deficits were assessed after 8weeks of diabetes induction using a functional observation battery, passive avoidance and rotarod test. Acetylcholinesterase activity was significantly decreased in hippocampus of diabetic rats as compared to control rats. Diabetic animals showed significant increase in malondialdehyde levels and reduction in NAD levels in hippocampus. Glutamate and GABA levels were also altered in hippocampus of the diabetic animals. Two week treatment with melatonin (3 and 10mg/kg) and nicotinamide (300 and 1000mg/kg) alone and in combination significantly improved the neurobehavioral parameters which were altered in diabetes. Neurotransmitter (glutamate and GABA) levels were improved by these interventions. Our results emphasize that simultaneous inhibition of oxidative stress–PARP overactivation cascade can be beneficial in treatment of diabetes associated CNS changes.

Cellular NAD depletion and decline of SIRT1 activity play critical roles in PARP-1-mediated acute epileptic neuronal death in vitro

Intense poly(ADP-ribose) polymerase-1 (PARP-1) activation was implicated as a major cause of caspase-independent cell death in the hippocampal neuronal culture (HNC) model of acute acquired epilepsy (AE). The molecular mechanisms are quite complicated. The linkage among neuronal death, cellular nicotinamide adenine dinucleotide (NAD) levels, apoptosis-inducing factor (AIF) translocation, SIRT1 expression and activity were investigated here. The results showed that PARP-1 over-activation caused by Mg2+-free stimuli led to cellular NAD depletion which could block AIF translocation from mitochondria to nucleus and attenuate neuronal death. Also, SIRT1 deacetylase activity was reduced by Mg2+-free treatment, accompanied by elevated ratio of neuronal death, which could be rescued by NAD repletion. These data demonstrated that cellular NAD depletion and decline of SIRT1 activity play critical roles in PARP-1-mediated epileptic neuronal death in the HNC model of acute AE.

Parthanatos mediates AIMP2-activated age-dependent dopaminergic neuronal loss

The defining pathogenic feature of Parkinson’s disease is the age dependent loss of dopaminergic neurons. Mutations and inactivation of parkin, an ubiquitin E3 ligase, cause Parkinson’s disease through accumulation of pathogenic substrates. Here we show that transgenic overexpression of the parkin substrate, aminoacyl-tRNA synthetase complex interacting multifunctional protein-2 (AIMP2) leads to a selective, age-dependent progressive loss of dopaminergic neurons via activation of poly(ADP-ribose) polymerase-1 (PARP1). AIMP2 accumulation in vitro and in vivo results in PARP1 overactivation and dopaminergic cell toxicity via direct association of these proteins in the nucleus providing a new path to PARP1 activation other than DNA damage. Inhibition of PARP1 through gene deletion or drug inhibition reverses behavioral deficits and protects in vivo against dopamine neuron death in AIMP2 transgenic mice. These data indicate that brain permeable PARP inhibitors could be effective in delaying or preventing disease progression in Parkinson’s disease.

Neuropilin-1 modulates vascular endothelial growth factor-induced poly(ADP-ribose)-polymerase leading to reduced cerebrovascular apoptosis

Cerebral ischemia is encompassed by cerebrovascular apoptosis, yet the mechanisms behind apoptosis regulation are not fully understood. We previously demonstrated inhibition of endothelial apoptosis by vascular endothelial growth factor (VEGF) through upregulation of poly(ADP-ribose)-polymerase (PARP) expression. However, PARP overactivation through oxidative stress can lead to necrosis. This study tested the hypothesis that neuropilin-1 (NP-1), an alternative VEGF receptor, regulates the response to cerebral ischemia by modulating PARP expression and, in turn, apoptosis inhibition by VEGF. In endothelial cell culture, NP-1 colocalized with VEGF receptor-2 (VEGFR-2) and acted as its coreceptor. This significantly enhanced VEGF-induced PARP mRNA and protein expression demonstrated by receptor-specific inhibitors and VEGF-A isoforms. NP-1 augmented the inhibitory effect of VEGF/VEGFR-2 interaction on apoptosis induced by adhesion inhibition through the αV-integrin inhibitor cRGDfV. NP-1/VEGFR-2 signal transduction involved JNK and Akt. In rat models of permanent and temporary middle cerebral artery occlusion, the ischemic cerebral hemispheres displayed endothelial and neuronal apoptosis next to increased endothelial NP-1 and VEGFR-2 expression compared to non-ischemic cerebral hemispheres, sham-operated or untreated controls. Increased vascular superoxide dismutase-1 and catalase expression as well as decreased glycogen reserves indicated oxidative stress in the ischemic brain. Of note, protein levels of intact PARP remained stable despite pro-apoptotic conditions through increased PARP mRNA production during cerebral ischemia. In conclusion, NP-1 is upregulated in conditions of imminent cerebrovascular apoptosis to reinforce apoptosis inhibition and modulate VEGF-dependent PARP expression and activation. We propose that NP-1 is a key modulator of VEGF maintaining cerebrovascular integrity during ischemia. Modulating the function of NP-1 to target PARP could help to prevent cellular damage in cerebrovascular disease.

Effect of Nitric Oxide on Naphthoquinone Toxicity in Endothelial Cells: Role of Bioenergetic Dysfunction and Poly(ADP-ribose) Polymerase Activation

When produced at physiological levels, reactive oxygen species (ROS) can act as signaling molecules to regulate normal vascular function. Produced under pathological conditions, ROS can contribute to the oxidative damage of cellular components (e.g., DNA and proteins) and trigger cell death. Moreover, the reaction of superoxide with nitric oxide (NO) produces the strong oxidant peroxynitrite and decreases NO bioavailability, both of which may contribute to activation of cell death pathways. The effects of ROS generated from the 1,4-naphthoquinones alone and in combination with NO on the activation status of poly(ADP-ribose) polymerase (PARP) and cell viability were examined. Treatment with redox cycling quinones activates PARP, and this stimulatory effect is attenuated in the presence of NO. Mitochondria play a central role in cell death signaling pathways and are a target of oxidants. We show that simultaneous exposure of endothelial cells to NO and ROS results in mitochondrial dysfunction, ATP and NAD(+) depletion, and cell death. Alone, NO and ROS have only minor effects on cellular bioenergetics. Further, PARP inhibition does not attenuate reduced cell viability or mitochondrial dysfunction. These results show that concomitant exposure to NO and ROS impairs energy metabolism and triggers PARP-independent cell death. While superoxide-mediated PARP activation is attenuated in the presence of NO, PARP inhibition does not modify the loss of mitochondrial function or adenine and pyridine nucleotide pools and subsequent bioenergetic dysfunction. These findings suggest that the mechanisms by which ROS and NO induce endothelial cell death are closely linked to the maintenance of mitochondrial function and not overactivation of PARP.

Role of poly(ADP-ribose) polymerases in male reproduction

Poly(ADP-ribose) polymerases (PARPs) are a family of enzymes involved in a wide variety of biological processes, including DNA repair and maintenance of genomic stability following genotoxic stress, and regulates the expression of various proteins at the transcriptional level as well as replication and differentiation. However, excessive activation of PARP has been shown to contribute to the pathogenesis of several diseases associated with oxidative stress (OS), which has been known to play a fundamental role in the etiology of male infertility. Based on the degree and type of the stress stimulus, PARP directs cells to specific fates (such as, DNA repair vs. cell death). A large volume of accumulated evidence indicates the presence of PARP and its homologs in testicular germ line cells and its activity may offer a key mechanism for keeping DNA integrity in spermatogenesis. On the other hand, a possible role of PARP overactivation in OS-induced male reproductive disorders and in human sperm is gaining significance in recent years. In this review, we focus on the findings about the importance of PARP-1 and PARP-2 in male reproduction and possible involvement of PARP overactivation in various clinical conditions associated with male infertility.

Prevention of Acute/Severe Hypoglycemia-Induced Neuron Death by Lactate Administration

Hypoglycemia-induced cerebral neuropathy can occur in patients with diabetes who attempt tight control of blood glucose and may lead to cognitive dysfunction. Accumulating evidence from animal models suggests that hypoglycemia-induced neuronal death is not a simple result of glucose deprivation, but is instead the end result of a multifactorial process. In particular, the excessive activation of poly (ADP-ribose) polymerase-1 (PARP-1) consumes cytosolic nicotinamide adenine dinucleotide (NAD(+)), resulting in energy failure. In this study, we investigate whether lactate administration in the absence of cytosolic NAD(+) affords neuroprotection against hypoglycemia-induced neuronal death. Intraperitoneal injection of sodium L-lactate corrected arterial blood pH and blood lactate concentration after hypoglycemia. Lactate administered without glucose was not sufficient to promote electroencephalogram recovery from an isoelectric state during hypoglycemia. However, supplementation of glucose with lactate reduced neuronal death by ∼80% in the hippocampus. Hypoglycemia-induced superoxide production and microglia activation was also substantially reduced by administration of lactate. Taken together, these results suggest an intriguing possibility: that increasing brain lactate following hypoglycemia offsets the decrease in NAD(+) due to overactivation of PARP-1 by acting as an alternative energy substrate that can effectively bypass glycolysis and be fed directly to the citric acid cycle to maintain cellular ATP levels.

Further Studies on the Hypothesis of PARP-1 Inhibition as a Strategy for Lessening the Long-Term Effects Produced by Perinatal Asphyxia: Effects of Nicotinamide and Theophylline on PARP-1 Activity in Brain and Peripheral Tissue

Oxygen interruption leads to death when re-oxygenation is not promptly re-established. Re-oxygenation triggers a cascade of biochemical events for restoring function at the cost of improper homeostasis. The effects observed long after perinatal asphyxia (PA) have been explained by over-expression of sentinel proteins, such as poly(ADP-ribose) polymerase-1 (PARP-1), competing for NAD(+) during re-oxygenation, leading to the idea that sentinel protein inhibition constitutes a therapeutic strategy. We studied the effects of nicotinamide and theophylline on PARP-1 activity assayed in brain and peripheral (heart) rat tissue 1-24h after birth, as well as on changes in behaviour and monoamine neurotransmission in adult rats. PA was induced by immersing rat foetuses into a water bath for 0 or 21min. After resuscitation, the pups were treated with nicotinamide (0.8mmol/kg, i.p.), theophylline (0.14mmol/kg, i.p.) or saline (0.9% NaCl) and nurtured by surrogate dams, pending behavioural and microdialysis experiments, or euthanised after birth for assaying PARP-1 activity. To estimate the in vivo distribution of a single dose of nicotinamide or theophylline into brain and peripheral compartment, a series of animals were implanted with microdialysis probes, one into the brain and other subcutaneously, 1h after birth, assaying the drugs with a HPLC-UV system. Nicotinamide, but not theophylline prevented the long-term effects induced by PA. Only nicotinamide produced a consistent decrease in PARP-1 activity in brain and heart, whether assayed in control or asphyxia-exposed pups. The present results support the idea that the long-term effects induced by PA imply PARP-1 over-activation.

Climbing STAIRs towards clinical trials with a novel PARP-1 inhibitor for the treatment of ischemic stroke

Climbing STAIRs towards clinical trials with a novel PARP-1 inhibitor for the treatment of ischemic stroke

Poly(ADP-Ribose) Polymerase-1: A Novel Therapeutic Target in Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is the most common gastrointestinal disease of infancy, afflicting 11% of infants born 22-28 wk GA. Both inflammation and oxidation may be involved in NEC pathogenesis through reactive nitrogen species production, protein oxidation, and DNA damage. Poly(ADP-ribose) polymerase-1 (PARP-1) is a critical enzyme activated to facilitate DNA repair using nicotinamide adenine dinucleotide (NAD+) as a substrate. However, in the presence of severe oxidative stress and DNA damage, PARP-1 overactivation may ensue, depleting cells of NAD+ and ATP, killing them by metabolic catastrophe. Here, we tested the hypothesis that NO dysregulation in intestinal epithelial cells during NEC leads to marked PARP-1 expression and that administration of a PARP-1 inhibitor (nicotinamide) attenuates intestinal injury in a newborn rat model of NEC. In this model, 56% of control pups developed NEC (any stage) versus 14% of pups receiving nicotinamide. Forty-four percent of control pups developed high-grade NEC (grades 3-4), whereas only 7% of pups receiving nicotinamide developed high-grade NEC. Nicotinamide treatment protects pups against intestinal injury incurred in the newborn rat NEC model. We speculate that PARP-1 overactivation in NEC may drive mucosal cell death in this disease and that PARP-1 may be a novel therapeutic target in NEC.

Inhibition of poly(ADP-ribose) polymerase-1 attenuates the toxicity of carbon tetrachloride

Carbon tetrachloride (CCl4) is routinely used as a model compound for eliciting centrilobular hepatotoxicity. It can be bioactivated to the trichloromethyl radical, which causes extensive lipid peroxidation and ultimately cell death by necrosis. Overactivation of poly(ADP-ribose) polymerase-1 (PARP-1) can rapidly reduce the levels of β-nicotinamide adenine dinucleotide and adenosine triphosphate and ultimately promote necrosis. The aim of this study was to determine whether inhibition of PARP-1 could decrease CCl4-induced hepatotoxicity, as measured by degree of poly(ADP-ribosyl)ation, serum levels of lactate dehydrogenase (LDH), lipid peroxidation, and oxidative DNA damage. For this purpose, male ICR mice were administered intraperitoneally a hepatotoxic dose of CCl4 with or without 6(5H)-phenanthridinone, a potent inhibitor of PARP-1. Animals treated with CCl4 exhibited extensive poly(ADP-ribosyl)ation in centrilobular hepatocytes, elevated serum levels of LDH, and increased lipid peroxidation. In contrast, animals treated concomitantly with CCl4 and 6(5H)-phenanthridinone showed significantly lower levels of poly(ADP-ribosyl)ation, serum LDH, and lipid peroxidation. No changes were observed in the levels of oxidative DNA damage regardless of treatment. These results demonstrated that the hepatotoxicity of CCl4 is dependent on the overactivation of PARP-1 and that inhibition of this enzyme attenuates the hepatotoxicity of CCl4.

Ex vivo supplementation with nicotinic acid enhances cellular poly(ADP-ribosyl)ation and improves cell viability in human peripheral blood mononuclear cells

Poly(ADP-ribosyl)ation is a posttranslational modification of proteins, which is mainly catalyzed by poly(ADP-ribose) polymerase-1 (PARP-1) by using NAD+ as substrate and is directly triggered by DNA strand breaks. Under mild genotoxic stress poly(ADP-ribose) (PAR) formation plays an important role in DNA repair whereas severe genotoxic stress and the ensuing overactivation of PARP-1 induce cellular NAD+ depletion, energy failure and ultimately cell death. We are interested in studying the consequences of moderately enhanced enzymatic activity under conditions of DNA damage. Here we chose supplementation of cells with the NAD+ precursor nicotinic acid (NA) as a strategy. In order to reliably assess PAR accumulation in living cells we first developed a novel, sensitive flow-cytometric method for the rapid analysis of poly(ADP-ribose) accumulation (RAPARA). Our data showed that ex vivo supplementation of human peripheral blood mononuclear cells (PBMC) with low concentrations of NA significantly raised cellular NAD+ levels by 2.1-fold. Upon X-irradiation or exposure to hydrogen peroxide or N-methyl-N′-nitro-N-nitrosoguanidine, PAR accumulation was significantly increased and sustained in NA-supplemented cells. Furthermore, NA-supplemented PBMC displayed significantly higher cell viability due to a lower rate of necrotic cell death. In summary, ex vivo supplementation of human PBMC with NA increases cellular NAD+ levels, boosts the cellular poly(ADP-ribosyl)ation response to genotoxic treatment, and protects from DNA-damage-induced cell death.

PISA, A novel pharmacodynamic assay for assessing poly(ADP-ribose) polymerase (PARP) activity in situ

Introduction Poly ADP-ribose polymerase (PARP) maintains genomic integrity by repairing DNA strand breaks, however over-activation of PARP following neural tissue injury is hypothesized to cause neuronal death. Therefore, PARP inhibitors have potential for limiting neural injury under certain conditions. A reliable method for assessing PARP activity in brain is critical for development of novel inhibitors with CNS activity. We developed the PARP In Situ Activity (PISA) assay to provide a direct, quantitative assessment of CNS PARP activity in vitro or in vivo. Methods The assay utilized brain sections from rats with striatal kainic acid (KA) lesions and 3H- or biotinylated NAD + as the substrate to assess PARP activity. Following optimization of the assay, it was used to assess in vitro and in vivo efficacy of known and novel PARP inhibitors. The assay also was used to assess PARP activity in male and female gonad-intact and ovariectomized rats. Results Using 3H-NAD + as the substrate, PARP activity was greater ( p < 0.01) in tissue from KA-lesioned vs. non-lesioned rats. Using biotinylated NAD + it was revealed that PARP activity was present ipsilateral to the KA injection site, and labeling was blocked by incubation with excess unlabeled NAD + or PARP inhibitors. The PARP inhibitor, 3-aminobenzamide and several novel inhibitors reduced ( p < 0.01) polymerase activity in vitro. Furthermore, the inhibitor MRLSD303 reduced ( p < 0.001) PARP activity in vivo in both male and female rats. Finally, administration of the novel PARP inhibitor MRLIT115 dose-dependently reduced ( p < 0.001) polymerase activity in vivo. Discussion The PISA assay provides a direct, quantitative method for assessing PARP activity in vitro and provides critical information on factors underlying in vivo efficacy of chemical inhibitors including brain penetration and target engagement. These findings support use of the PISA assay as a screening tool for testing efficacy of PARP inhibitors in brain.

Postnatal Age Influences Hypoglycemia-Induced Poly(ADP-ribose) Polymerase-1 Activation in the Brain Regions of Rats

Poly(ADP-ribose) polymerase-1 (PARP-1) overactivation plays a significant role in hypoglycemia-induced brain injury in adult rats. To determine the influence of postnatal age on PARP-1 activation, developing and adult male rats were subjected to acute hypoglycemia of equivalent severity and duration. The expression of PARP-1 and its downstream effectors, apoptosis-inducing factor (Aifm1), caspase 3 (Casp3), NF-kappaB (Nfkb1) and bcl-2 (Bcl2), and cellular poly(ADP-ribose) (PAR) polymer expression were assessed in the cerebral cortex, hippocampus, striatum, and hypothalamus at 0 h and 24 h posthypoglycemia. Compared with the control group, PARP-1 expression increased in the cerebral cortex of adult rats 24 h posthypoglycemia, but not at 0 h, and it was accompanied by increased number of PAR-positive cells. The expression was not altered in other brain regions. Aifm1, Nfkb1, Casp3, and Bcl2 expressions also increased in the cerebral cortex of adult rats 24 h posthypoglycemia. Conversely, hypoglycemia did not alter PARP-1 expression and its downstream effectors in any brain region in developing rats. These data parallel the previously demonstrated pattern of hypoglycemia-induced brain injury and suggest that PARP-1 overactivation may determine age- and region-specific vulnerability during hypoglycemia.

Concurrent targeting of nitrosative stress–PARP pathway corrects functional, behavioral and biochemical deficits in experimental diabetic neuropathy

Peroxynitrite mediated nitrosative stress, an indisputable initiator of DNA damage and overactivation of poly(ADP-ribose) polymerase (PARP), a nuclear enzyme activated after sensing DNA damage, are two crucial pathogenetic mechanisms in diabetic neuropathy. The intent of the present study was to investigate the effect of combination of a peroxynitrite decomposition catalyst (PDC), FeTMPyP and a PARP inhibitor, 4-ANI against diabetic peripheral neuropathy. The end points of evaluation of the study included motor nerve conduction velocity (MNCV) and nerve blood flow (NBF) for evaluating nerve functions; thermal hyperalgesia and mechanical allodynia for assessing nociceptive alterations, malondialdehyde and peroxynitrite levels to detect oxidative stress–nitrosative stress; NAD concentration in sciatic nerve to assess overactivation of PARP. Additionally immunohistochemical studies for nitrotyrosine and Poly(ADP-ribose) (PAR) was also performed. Treatment with the combination of FeTMPyP and 4-ANI led to significant improvement in nerve functions and pain parameters and also attenuated the oxidative–nitrosative stress markers. Further, the combination also reduced the overactivation of PARP as evident from increased NAD levels and decreased PAR immunopositivity in sciatic nerve microsections. Thus, it can be concluded that treatment with the combination of a PDC and PARP inhibitor attenuates alteration in peripheral nerves in diabetic neuropathy (DN).

MLH1 mediates PARP-dependent cell death in response to the methylating agent N-methyl-N-nitrosourea

Background:Methylating agents such as N-methyl-N-nitrosourea (MNU) can cause cell cycle arrest and death either via caspase-dependent apoptosis or via a poly(ADP-ribose) polymerase (PARP)-dependent form of apoptosis. We wished to investigate the possible role of MLH1 in signalling cell death through PARP.Methods:Fibroblasts are particularly dependent on a PARP-mediated cell death response to methylating agents. We used hTERT-immortalised normal human fibroblasts (WT) to generate isogenic MLH1-depleted cells, confirmed by quantitative PCR and western blotting. Drug resistance was measured by clonogenic and cell viability assays and effects on the cell cycle by cell sorting. Damage signalling was additionally investigated using immunostaining.Results:MLH1-depleted cells were more resistant to MNU, as expected. Despite having an intact G2/M checkpoint, the WT cells did not initially undergo cell cycle arrest but instead triggered cell death directly by PARP overactivation and nuclear translocation of apoptosis-inducing factor (AIF). The MLH1-depleted cells showed defects in this pathway, with decreased staining for phosphorylated H2AX, altered PARP activity and reduced AIF translocation. Inhibitors of PARP, but not of caspases, blocked AIF translocation and greatly decreased short-term cell death in both WT and MLH1-depleted cells. This MLH1-dependent response to MNU was not blocked by inhibitors of ATM/ATR or p53.Conclusion:These novel data indicate an important role for MLH1 in signalling PARP-dependent cell death in response to the methylating agent MNU.

PCB-induced endothelial cell dysfunction: Role of poly(ADP-ribose) polymerase

Polychlorinated biphenyls (PCBs) are persistent environmental pollutants implicated in the development of pro-inflammatory events critical in the pathology of atherosclerosis and cardiovascular disease. PCB exposure of endothelial cells results in increased cellular oxidative stress, activation of stress and inflammatory pathways leading to increased expression of cytokines and adhesion molecules and ultimately cell death, all of which can lead to development of atherosclerosis. To date no studies have been performed to examine the direct effects of PCB exposure on the vasculature relaxant response which if impaired may predispose individuals to hypertension, an additional risk factor for atherosclerosis. Overactivation of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP) following oxidative/nitrosative stress in endothelial cells and subsequent depletion of NADPH has been identified as a central mediator of cellular dysfunction. The aim therefore was to investigate whether 2,2′,4,6,6′-pentachlorobiphenyl (PCB 104) directly causes endothelial cell dysfunction via increased oxidative stress and subsequent overactivation of PARP. Exposure of ex vivo rat aortic rings to PCB 104 impaired the acetylcholine-mediated relaxant response, an effect that was dependent on both concentration and exposure time. In vitro exposure of mouse endothelial cells to PCB 104 resulted in increased cellular oxidative stress through activation of the cytochrome p450 enzyme CYP1A1 with subsequent overactivation of PARP and NADPH depletion. Pharmacological inhibition of CYP1A1 or PARP protected against the PCB 104-mediated endothelial cell dysfunction. In conclusion, the environmental contaminants, PCBs, can activate PARP directly impairing endothelial cell function that may predispose exposed individuals to development of hypertension and cardiovascular disease.