Anti-Apoptotic Effect of 3- Aminobenzamide, an Inhibitor of Poly (ADP-Ribose) Polymerase, against Multiple Organ Damage Induced by Gamma Irradiation in Rats

Purpose: This study aimed to investigate the effect of 3-aminobenzamide (3-AB) in doses of 5, 10 and 15 mg/ kg on the inhibition of Poly (ADP-ribose) polymerase (PARP) when combined with ionizing radiation (IR). Material and methods: Rats were treated intraperitonealy, 1 hour prior to irradiation at a dose level of 6 Gray (Gy) and were sacrificed 24 hours after irradiation. Control groups were run concurrently. Results: IR led to an increase of thiobarbituric acid reactive substance (TBARS), nitrite as well as a decrease in total antioxidant capacity associated increase in myeloperoxidase (MPO) with expression of cyclooxygenase-2 (COX-2). Moreover, IR caused an increase in serum lactate dehydrogenase (LDH) activity and cytosolyic Ca+2 associated with an expression of Caspase-3 as well as a decline in complex-I activity and adenosine triphosphate (ATP) level. Pre-treatment with 5 and 10 mg/kg of 3AB guarded against the changes in all the measured parameters, conversely the dose of 15 mg/kg showed no effect on the damage induced by irradiation in the selected tissues. Moreover, 3AB has a dose dependent effect on viability of Vero cells. Conclusion: the selected low doses of 3-AB rather than the higher dose (15mg/kg) protected against radiation induced multiple organ damage.

IR as applied in the radiotherapy clinic induces cell death and DNA damage whereas some processes aim to counteracts that damage and any deficiencies in repair are known to have a large influence on cellular survival after IR. Therefore, modulating the response to IR through the inhibition of DNA repair has gained attention in radiotherapy research (Mo et al. 2015). IR led to an elevation in the oxidative metabolites and a reduction in the antioxidant defense mechanisms in plasma as well as hepatic, renal, lung, and thyroid tissues, resulting in induction of oxidative stress (Kutanis et al. 2016). The hydroxyl radical, produced during oxidative stress or radiation injury, induces a breakage in DNA single strand (Halliwell and Aruoma 1991; Azzam et al. 2012) DNA damage induces cell death (Carante et al. 2015). Consequently, several processes for DNA repair are activated to counteract the DNA strand breaks caused by oxidative stress. One of these processes is the activation of Poly (ADP-ribose) polymerase-1 (PARP-1) which plays a key role in DNA repair and help to maintain DNA integrity. However, the excessive damage of DNA leads to over activation of PARP-1 enzyme causing cell death and apoptosis. Furthermore, PARP-1 interacts with nuclear factor-kappa B (NF-kB) and p53 that have a role in the regulation of apoptosis (Hassa and Hottiger 1999; 2002; Veuger et al. 2009), DNA repair and in immune or inflammatory responses. In addition to PARP activation caused by DNA damage, mitochondrial Ca+2 uptake is another signal leading to cell death.

Pharmacological inhibition of PARP-1 showed a beneficial effect in rodent and large animal models of inflammatory disorders allowing its use as an approach in the treatment of inflammatory diseases (Giansanti et al. 2010) and in cancer therapy (Cerrato et al. 2016).The aim of using PARP inhibitors was to enhance the damage induced in DNA by anti-cancer agents. This was based on the simple principle that if the cytotoxic agents act by damaging the DNA, therefore, the repair of that damage will present a resistance mechanism; thereby; the inhibition of that repair would lead to persistent damage and greater cytotoxicity. New strategies to enhance the efficacy of radiotherapy, used as cancer therapy, focus on the combination with novel targeted agents that might protect against deleterious effects of IR. The combination of 3- Aminobenzamide (3AB), a pharmacological inhibitor of PARP with IR can elicit tumor inhibition with minimal effects on proliferating normal tissue (Albert et al. 2007; Gani et al. 2015). In view of these considerations, the main objective of the present study was twofold, first to assess the role of poly ADP-ribosylation in the regulation of inflammatory and apoptotic processes induced by gamma irradiation. The second aim was to investigate the optimal dose regimen of 3-AB that might largely protect against the multiple organ damage induced by IR in rats.

2.Material and Methods
3-aminobenzamide, High-performance liquid chromatography (HPLC) grade chemicals and solvents for adenosine triphosphate (ATP) assay as well as the other chemicals and reagents used in this study were all purchased from Sigma-Aldrich chemical company (Saint Louis, Missouri, USA) The kinetic ultraviolet (UV) kits from BioSystems S.A (Barcelona, Spain)were used for the determination of lactate dehydrogenase (LDH) activity and for determination of total antioxidant capacity from Biodiagnostics®,( Dokki, Giza, Egypt). Monoclonal Antibodies against Caspase-3 and COX-2 were obtained from Dako Corp, (Carpenteria, CA, USA).A total of forty adult male Wistar rats were used, the animals were ranging in weight 150-180 gm and aging 10–12 weeks. Animals were purchased from the animal breeding unit of the National Research Centre, Giza, Egypt. Rats were acclimatized in the animal facility of the National Centre for Radiation Research and Technology (NCRRT) – (Atomic Energy Authority, Cairo, Egypt) for one week before being used. They were fed standard pellet diet obtained from the National Research Centre (Giza, Egypt.) and allowed free access to water ad libitum. The study conducted in accordance with the guidelines set by the European Economic Community (EEC) regulations (Revised Directive 86/609/EEC) and approved by the Ethics Committee at the Faculty of Pharmacy, Cairo University with PT (1304).Irradiation was carried out at the NCRRT using Gamma Cell-40 biological irradiator with Caesium137 source (Atomic Energy of Canada Limited; Sheridan Science and Technology Park, Mississauga, Ontario, Canada). The radiation dose level was 6 Gy with a dose rate of 0.5 Gy/min.

The rats were blindly allocated to several groups, each consisting of eight rats, assigned as follows:(a)Negative control group (normal non-irradiated animals) consisting of 8 rats. (b)Positive control group irradiated with 6 Gy consisting of 8 rats (Khayyal et al. 2014, El- Ghazaly et al. 2015)c) Pre-treated irradiated group ( consisting of 24 rats); this group was further subdivided into three sub-groups, each consisting of 8 rats, treated with either 5, 10 and 15 mg/ kg of 3 AB as a single dose once before whole body Gamma irradiation (6Gy).Rats were sacrificed 24 hr after exposure to radiation. Blood samples were collected for serum separation and organs (brain, liver, and kidney) were rinsed with ice cold saline and rapidly excised. A small section from the dissected tissues was taken for histological examination and immunohistochemistry. While in another section the remaining part of the brain of each animal was weighed and homogenized in ice cold normal saline (20% w/v homogenate), the homogenate was immediately frozen until analysis. As well as samples of tissue from liver and kidney were collected in ice cold normal saline in Petri dishes, flushed thoroughly to get rid of adhering tissues and blood vessels, dried on filter paper, weighed and cut into portions in ice cold saline bath and stored in -70ᵒC till further analysis. This process was done for each individual rat. Tissues of liver and kidney were homogenized in different media according to the parameter to be measured as described below (20% w/v) using Glass-Col1 homogeniser (Terre Haute Indiana, USA).Lipid peroxidation was determined according to the method of Uchiyama and Mihara (1978), using aliquot of brain homogenate was mixed with1.15 % w/v ice-cold potassium chloride (1:2 ratio). Liver and kidney tissues were homogenized in ice-cold 1.15% potassium chloride.

Total nitrate/nitrite (NOx) was measured according to the method described by Miranda et al. (2001). Total antioxidant capacity was determined using commercially available colorimetric kits; and the assay was carried out according to the manufacturer’s instructions. Myeloperoxidase (MPO) activity was determined in liver and kidney tissue homogenates prepared in 50 mM potassium phosphate buffer (pH6.0) containing 0.5% hexadecyltrimethylammonium bromide, and a mixture of brain aliquot with 50 mM potassium phosphate buffer (pH 6.0) containing 0.5% hexadecyltrimethylammonium bromide. The assay method was that described by Bradley et al. (1982).For determination of cellular damage lactate dehydrogenase activity (LDH) and Cytosolic calcium (Ca+2) concentration were measured. Sera of rats were used for the determination of LDH activity using kinetic ultraviolet (UV) kit, and the assay was carried out according to the manufacturer’s instructions. Cytosolic calcium (Ca+2) concentration was measured in tissue homogenized in cold normal saline. The homogenate was then centrifuged at 105,000 × g for 15 min at 4◦C Thermo Sorvall WX80 ultracentrifuge (DuPont Co., Delaware, USA). The separated cytosolic fraction was used for the determination of cytosolic calcium concentration using a Unicam 939 atomic absorption spectrophotometer (Cambridge, UK). For determination of apoptosis, mitochondrial dysfunction was assessed by measuring complex-I activity and ATP content in mitochondrial fraction which were extracted according to the method of Turpeenoja et al. (1988).

Mitochondrial complex-I activity was determined according to the method of Whitfield et al. (1981) based on the reaction catalyzed by mitochondrial complex-I by following the decrease in absorbance due to the oxidation of NADH coupled with the reduction of ubiquinone-1 (Coenzyme-Q1) to ubiquinol-1 at 340 nm. Mitochondrial ATP extraction was carried out as described before by Glick et al.(1993). ATP was quantified using HPLC according to a previously published method reported by Harmsen et al. (1982). Briefly, the HPLC system with UV detector (Chromatec) was set according to the condition of separation: C18 column (Hypersil), flow rate: 0.5 mL/min, temperature: 25˚ C, injector volume: 20 mL, run time: 10 min, mobile phase 0.2M KH2PO4: acetonitrile: methanol (9.6:0.3:0.1). The retention time of separated peak was determined at wavelength 254 nm.Tissue samples were collected from the brain, liver and kidneys of rats in different groups and fixed in 10% buffer neutral formalin. Washing was carried out using tap water. Serial dilutions of alcohols (methyl, 70 % ethyl, and absolute ethyl alcohol) were used for dehydration. Specimens were cleared in xylene and embedded in paraffin at 56ᵒ C in hot air oven for 24 hr. Paraffin/bees wax tissue blocks were prepared for sectioning at 4 ml thicknesses by a sledge microtome. The obtained tissue sections were collected on glass slides, deparaffinized, and stained by hematoxylin& eosin stain for routine examination using a light microscope (Bancroft et al. 1996; Bancroft and Gamble 2010).

Immunohistochemical studies were carried out for detection of Caspase-3 and COX-2 expression on paraffin tissue sections prepared and stained according to method of Dakoimmunohistochemical staining methods described by Hsu et al. (1981).Cryopreserved Vero (Green African monkey kidney) cells were obtained from the Cell Bank of VACSERA (Cairo, Egypt). Cells were cultured in a humidified atmosphere (5%CO2, 37oC) with RPMI-1640 Medium supplemented with 2% fetal bovine serum (FBS) for maintenance of cell growth. Every five days, cells were passaged after trypsinization with pre-warmed trypsin– EDTA solution. In one experiment Vero cells were irradiated with 6Gy. In another one, they were pretreated with different doses of 3AB for 1 hr before irradiation.Cell viability was determined by a quantitative colorimetric assay with 3-(4,5 dimethylthiazol- 2-yl)-2,5-diphenyltetrazoli-umbro-mide (MTT) as previously described by Lakatos et al.(2013). 96-well plates were inoculated with 1 X 105 cells/ml (100 µl / well) and incubated at 37°C for 24hr. Cells were pretreated with various L-TH concentrations for different time points, and then exposed to CIS (60μM/ml) for 24hr. After treatment, 20 µl of MTT solution (5mg/ml in PBS, BIO BASIC CANADA INC) was added to the wells and incubated at 37°C/3h. Afterward, 200μl of DMSO was added to each well, and the plates were agitated at 150 rpm for 5 min. Finally, the absorbance was measured at 560 nm.Quantitative data were expressed as mean ± standard error (SEM) and analyzed by one-way analysis of variance (ANOVA) followed by Tukey-Kramer multiple comparison test. Graph pad software instant (version 7) was used to carry out these statistical tests. The level of statistical significance was taken at P ≤0.05.

Exposure to gamma irradiation led to variable changes in the different parameters of oxidative stress measured in the brain, liver and kidney. Total antioxidant capacity was markedly reduced from normal by 38%, 56.9% and 47% in tissues of brain, liver and kidney respectively after exposure to 6 Gy (Fig.1.І). The reduction in total antioxidant capacity was associated with an increase in level of the other oxidative stress parameters, namely TBARS and nitrite. The former was raised from normal by three folds in brain and liver tissues but in kidney tissues by 2 folds (Fig.1.ІІ). The latter was raised by 107%, 108% and 111% in brain, liver and kidney respectively (Fig.1.ІІІ). Pre-treatment with doses of 5, 10 and 15mg/Kg of 3 AB almost completely protected against the changes in TBARS (Fig.1.ІІ) and total antioxidant capacity induced by irradiation (Fig.1.І). Conversely the dose of 15mg/kg was not as effective in preventing the changes induced by 6 Gy in nitrite levels (Fig.1.ІІІ). Neutrophils accumulation is indicated by the release and activation of MPO, exposure to whole body gamma-radiation (6Gy) showed a marked increase in brain tissue levels of MPO by 4 folds while increased in liver by 2 folds and in kidney by 170%(Fig.1.ІХ).Pretreatment with the doses of 5 and 10 mg/kg of 3 AB showed a marked decline in the MPO levels .While the dose of 15 mg/Kg showed a marked increase by three folds from normal in brain tissues, while an increase in liver and kidney tissues by 265% and 144% respectively(Fig.1.ІХ).Insert Fig (1)On the other hand, irradiation led to a dramatic elevation in serum LDH activity increased by 5 folds (Fig.2.І). In parallel irradiation with 6 Gy led to a marked increase in the cytosolic calcium level by approximately 3 folds in brain and kidney and 186 % in liver tissues (Fig.2.ІІ).

This increase in Ca+2 was associated with depletion of the mitochondrial contents of complex-I activity, decreased by 55%, 47% and 53% in brain, liver and kidney respectively (Fig.2.ІІІ) and consumption of mitochondrial ATP, which decreased by 40%, 37%, 33% in brain, liver and kidney respectively (Fig.2.ІХ), these results revealed significant apoptotic changes and a high degree of cellular injury. While pretreatment with 5 and 10 mg/kg of 3 AB cause almost complete protection against these changes, conversely pretreatment with 15 mg/kg showed significant changes in apoptotic and cellular damage parameters.Insert Fig (2)In accordance with the biochemical assessment, the irradiated animal tissues showed inflammatory and apoptotic changes which were indicated by a positive immunohistochemicalreaction for COX-2 and Caspase-3. It was found that sections of Brain, liver and kidney of control rats were negatively stained for both COX-2 (Figs 3) and Caspase-3 expression (Figs 4) in examined tissues. While sections of brain, liver and kidney of irradiated rats showed variable degrees of brown positively stained apoptotic cells for both COX-2 (Figs 3) and Caspase-3 expression (Fig.4). Regarding tissue sections of irradiated rats that were pretreated with 3 AB 5mg/kg and 10mg/kg showed negative staining for both COX-2 and Caspase-3. However, tissue sections of irradiated rats that were treated with a dose of 15mg/kg 3AB showed positive expression of COX-2 (Figs. 3) and Caspase-3. (Figs.4)Insert Fig (3),(4)On the same line, histological examination indicated that microscopical examination of different sections taken form specimens of Brain, Liver and Kidney of control rats revealed normal histological pictures. While, microscopical examination of different sections of irradiated rats revealed congestion with mild degree of neuronal degeneration and necrosis was observed in few cases, as some neurons appeared with pyknotic nuclei or without nuclear structure.

Regarding the examined liver sections of gamma irradiated rats it revealed; marked hepatic congestion with disorganization of hepatocytes. Kupffer cell activation and marked hepatocellular necrobiotic changes were observed. Histopathological examination of renal tissues of irradiated rats revealed distinctive pattern of renal lesions. Congestion of the glomerular capillaries and intertubular blood vessels were an evident finding. In addition, renal tubular epithelium showed variable degrees of necrobiotic changes specially those of the proximal convoluted tubules with scattered necrotic cells. On the other hand, examination of tissue sections taken from irradiated rats that pretreated with 5,10 and 15mg/kg of 3AB, it revealed that; the use of 5mg/kg was the best in protecting the tissue against the harmful effects of radiation. Rats pretreated with 5mg/kg before radiation exposure showed only mild changes in their examined tissue specimens. While rats pretreated with 10mg/kg showed moderate necrobiotic changes in their tissues, rats retreated with 15mg/kg showed severe changes in their tissues.Insert Fig (5)Vero cells could tolerate gamma irradiation up to dose level of 6Gy. Interestingly, Pretreatment with high doses of 3AB showed a radio-sensitizing effect toward IR, indicated by loss of Vero cells viability. (Fig. 6).

Radiotherapy is used at some stage in the treatment of around 50% of cancer patients. IR causes damage to normal tissues as well as inducing DNA breaks that are considered the most cytotoxic event. The exposure to IR causes that damage primarily via the generation of reactive oxygen species (ROS) (Virag and Szabo 2002; Azzam et al. 2012). Such destructive effect was found to stimulate the DNA nick-sensor enzyme, poly (ADP-ribose) polymerase (PARP) (Kiang et al 2012). This process is accompanied by much consumption of PARP-1-substrate, NAD+, and consequently mitochondrial ATP to facilitate DNA repair and maintain the genomic integrity (Virag and Szabo 2002; Ba and Garg 2011). Benzamides, particularly the 3-aminobenzamide (3AB), inhibit PARP by interfering with the binding of NAD+ to the enzyme’s active site. In addition, 3-AB deactivates the PARP by binding to DNA and thereby prevents the recognition of DNA breaks by the enzyme (Southan and Szabo 2003).The present study showed an increase in the level of TBARS (end product of lipid peroxidation) in the selected tissues of irradiated rats which were accompanied with a decrease in the total antioxidant capacity. These findings are consistent with previous researchers who found that IR induced toxic metabolite production and caused endogenous antioxidant enzyme degradation (Kutanis et al. 2016). The elevated level of free radicals can eventually lead to DNA damage resulting in the over activation of PARP enzyme (Areti et al. 2014). Moreover, PARP activation might not only result from oxidative damage but it also has a role in the generation of more free radicals (Obrosova et al. 2005). The current study showed that pretreatment with 3AB at the three selected doses showed a free radical scavenging effect via guarding against the elevation in the level of TBARS and the reduction of total antioxidant capacity, and hence decreasing the state of oxidative stress induced by IR in the three examined tissues. Thus, PARP inhibitors with anti-oxidative potency, like 3AB, could indirectly contribute to the decrease in PARP activity by decreasing the free radical that evoked oxidative DNA damage.

PARP is known to be a key-regulator enzyme of nuclear factor κB (NF-κB); which plays a central role in the expression of different cytokines and other inflammatory mediators (Jog et al. 2009; Ba et al. 2010). Exposure to IR resulted in an increase in the expression of COX-2 and a rise in the stable end product of NO synthesis, nitrite in the brain, liver and kidney tissues due to the increased activity of PARP enzyme induced by irradiation. This could be explained on the ground that PARP recruits the inducible nitric oxides synthase (iNOS) and cyclo-oxygenase-2 (COX-2); enzymes that were mainly involved in inflammatory process (Jog et al. 2009; Park et al. 2004; Laube et al. 2016). Therefore, pre-treatment with 3-AB at dose level of 5 and 10 mg/kg showed capability of lowering the expression of COX-2 as well as the level of nitrite in the three selected irradiated organs. This was achieved by inhibiting PARP enzyme to a certain extent without reaching complete blockage as seen with the high dose of 3AB (15 mg/kg) which retained the state of inflammation. Multiple evidences indicated attenuation of nitric oxide production in different pharmacological models upon PARP inhibition, for example: renal ischemia/reperfusion induced in rats (Oztas et al. 2009) and gamma irradiated wound healing (El-Hamoly et al. 2015).The recruitment of neutrophils could have a role in causing tissue necrosis and dysfunction as they represent a major source of reactive oxygen radicals (Grisham 1994; Mittal et al. 2014). The present study showed an increase in MPO activity (an index of tissue-associated neutrophil infiltration) in the irradiated tissues, which is in accordance with previously reports (Hepgül et al.2010; Khayyal et al. 2014; El-Hamoly et al. 2015). The inhibition of PARP by 3AB in the small dose levels decreased MPO contents, reflecting a reduced neutrophil infiltration. In parallel to this outcome, PAPR-1 inhibition or even gene-inactivation has been shown to protect against neutrophil infiltration and increase in MPO activity (Koh et al. 2005; Baiet al.2009; Yu et al. 2016).

As mentioned before, the link between PARP activation and cell death, apoptosis and necrosis, would be according to the enhanced use of NAD+ followed by ATP depletion, a situation which can occur when DNA is extensively damaged (Schraufstatter et al. 1986; Fouquerel and Sobol 2014). Therefore, a decrease in level of ATP was observed in tissues of irradiated rats which might be attributed to its consumption by PARP enzyme in repairing the DNA damage induced by radiation. However, when pre-treated with 3AB in either dose of 5 or 10 mg/kg, the ATP level was restored providing that PARP activation is the main metabolic pathway consuming ATP during oxidant insult. Unlikely, complete blockage of PARP enzyme with the higher dose of 3AB (15 mg/kg) inversely led to much consumption of ATP contents as compared to lower doses. This can possibly occur by allowing the activated PARP to bind DNA breaks again and use more NAD+ to continue the cycle. The NAD+ content is replenished by a nicotinic acid mononucleotide adenylyl transferase-1 enzyme (Belenky et al. 2007). If not replenished or excessively used by hyperactive PARP, the depletion of NAD+ and the exhaustion of ATP result in impaired energy metabolism and, consequently, caused cell necrosis (Alano et al. 2010). Excessive activation of PARP-1 may deplete NAD+ pools and secondary ATP in efforts to resynthesize NAD+. All together these findings have driven us to recognize that the observed depletion in the ATP content in the examined tissues after irradiation could be due to the oxidative DNA damage leading to PARP over-activation and progression of inflammatory cascades.

Studies on apoptotic signaling cascades have shown that mitochondria play a pivotal role in the regulation of the apoptotic process by having a direct effect on energy production, regulating the activation of caspases, participating in intracellular calcium ion homeostasis, and production of reactive oxygen species ( Kowaltowskiet al. 2004; Orrenius 2004; Khayyal et al. 2014). PARP acts on mitochondria and according to the extent of oxidative stress, DNA damage, and PARP activation, different cell death pathways may be activated, inducing caspase-dependent apoptotic cell death (Yu et al. 2006; Modjtahedi et al. 2006). The present findings showed that the development of apoptosis evidenced by the increased expression of caspase in tissues was indeed associated with an increase in cytosolic calcium content. This might be attributed to PARP activation following radiation exposure since PARP protein has been reported to be a suitable substrate of Caspase-3, thus playing a role in the initiation of apoptosis (Nosseri et al. 1994; Morales et al. 2014). Furthermore, the inhibition of mitochondrial respiratory chain complex-I by radiation declared that the bioenergetics failure induced by mitochondrial dysfunction have a role in inducing apoptosis (Wang et al. 2013). This was in agreement with the present findings that showed a mitochondrial involvement in the apoptotic process represented by the decline in complex-I activity, which was evidenced in all tissues following radiation exposure. Calcium has been previously reported to have a role in the apoptotic process (Trump and Berezesky 1995; Kalogeris et al. 2014). By taking into consideration the previous studies which showed that mitochondrial Ca2+ uptake is required for PARP-1 activation, it was suggested that PARP-1 activation is a downstream event of mitochondrial Ca2+ overload (Duan et al. 2007). Evidence suggests that the increase in mitochondrial Ca2+lead to ROS production such as superoxide (O2•−) which react with NO to form peroxynitrite (ONOO−), which is more deleterious for DNA damage (Szaboand Ohshima 1997; Kalogeris et al. 2014), leading consequently for PARP-1 activation. Therefore, it was suggested that PARP inhibitors could have anti-apoptotic effect which was clearly defined in this study by the use of 3AB in low doses since they counteracted the apoptotic effect induced by radiation via decreasing the expression of caspase enzyme and mitochondrial Ca2+ as well as elevating the level complex-I.

Accompanying the previous in vivo results, outcomes extrapolated from MTT Vero cells viability assay revealed a marked dose dependent effect of 3AB on Vero cell viability. One interesting finding that also verified in the current work is that high doses of 3AB could increase the sensitivity of Vero cells (the kidney of African green monkey) toward gamma radiation. Such dose-dependent effect was in line with the findings of Caldini et al. (2011) on HUVAC and aortic endothelial cells, while the angiogenic role of 3AB and hence cells proliferation and viability was accordingly affected by the final concentration.All these consequences of the exposure to IR could lead to the observed histological damage in the rat brain, liver and kidney. Based on these data, we expected that decreasing ROS or increasing antioxidant status may provide protection from IR damage which required PARP activity for repair. The use of 3AB in doses of 5 and 10 mg/kg was capable of guarding against all the damage induced by irradiation whereas the high dose of 3AB (15 mg/kg) failed to decrease the apoptotic as well as the inflammatory response. Thus, complete PARP inhibition by high doses of PARP inhibitors more likely impair or delay DNA repair in examined tissues thus counteracting the minimal protection provided by lower doses.

Pharmacological inhibition of PARP to a certain extent might be accepted as an approach in the treatment of inflammation induced during exposure to IR. The controversy between the anti- apoptotic and pro-apoptotic effect of 3AB due to the change in its doses might rely on the efficacy of PARP inhibitors in potentiating normal tissue damage after 3-Aminobenzamide course of radiotherapy.