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Journal of Environmental Science and Health, Part A: Toxic/Hazardous Substances and Environmental Engineering Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lesa20
Cardiac and renal nitrosative-oxidative stress after acute poisoning by a nerve agent Tabun a
b
a
c
d
Dimo Dimov , Radka Hadjiolova , Kamen Kanev , Radka Tomova , Anna Michova , Todor e
d
f
g
Todorov , Rumen Murdjev , Temenujka Boneva & Ivanka Dimova a
Disaster Medicine and Toxicology Deparment, Military Medical Academy, Sofia, Bulgaria
b
Department of Pathophysiology, Faculty of Medicine, Medical University of Sofia, Sofia, Bulgaria
Click for updates
c
Department of Medical Chemistry and Biochemistry, Faculty of Medicine, Medical University of Sofia, Sofia, Bulgaria d
Division of Pathology, Military Medical Academy, Sofia, Bulgaria
e
Department of Pathology, Faculty of Medicine, Medical University of Sofia, Sofia, Bulgaria
f
Division of Clinical Laboratory and Genetics, Military Medical Academy, Sofia, Bulgaria
g
Department of Medical Genetics, Faculty of Medicine, Medical University of Sofia, Sofia, Bulgaria Published online: 01 Jun 2015.
To cite this article: Dimo Dimov, Radka Hadjiolova, Kamen Kanev, Radka Tomova, Anna Michova, Todor Todorov, Rumen Murdjev, Temenujka Boneva & Ivanka Dimova (2015) Cardiac and renal nitrosative-oxidative stress after acute poisoning by a nerve agent Tabun, Journal of Environmental Science and Health, Part A: Toxic/Hazardous Substances and Environmental Engineering, 50:8, 824-829, DOI: 10.1080/10934529.2015.1019801 To link to this article: http://dx.doi.org/10.1080/10934529.2015.1019801
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Journal of Environmental Science and Health, Part A (2015) 50, 824–829 Copyright © Taylor & Francis Group, LLC ISSN: 1093-4529 (Print); 1532-4117 (Online) DOI: 10.1080/10934529.2015.1019801
Cardiac and renal nitrosative-oxidative stress after acute poisoning by a nerve agent Tabun DIMO DIMOV1, RADKA HADJIOLOVA2, KAMEN KANEV1, RADKA TOMOVA3, ANNA MICHOVA4, TODOR TODOROV5, RUMEN MURDJEV4, TEMENUJKA BONEVA6 and IVANKA DIMOVA7 1
Disaster Medicine and Toxicology Deparment, Military Medical Academy, Sofia, Bulgaria Department of Pathophysiology, Faculty of Medicine, Medical University of Sofia, Sofia, Bulgaria 3 Department of Medical Chemistry and Biochemistry, Faculty of Medicine, Medical University of Sofia, Sofia, Bulgaria 4 Division of Pathology, Military Medical Academy, Sofia, Bulgaria 5 Department of Pathology, Faculty of Medicine, Medical University of Sofia, Sofia, Bulgaria 6 Division of Clinical Laboratory and Genetics, Military Medical Academy, Sofia, Bulgaria 7 Department of Medical Genetics, Faculty of Medicine, Medical University of Sofia, Sofia, Bulgaria
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2
We hypothesized that Tabun poisoning, as well as other organophosphorous treatment, cause specific organs’ oxidative changes that have not previously been substantiated investigated. In this regard, a marker for nitrosative-oxidative stress in the main haemodynamic organs (heart and kidney) could reveal the existence of such changes. In this study, for the first time we studied the nitrosative/oxidative stress in heart and kidney after acute Tabun (Ethyl N,N- Dimethylphosphoramidocyanidate) poisoning measuring by immunohistochemistry the expression of 3-nitrotyrosine—a marker for nitrosative-oxidative stress. We investigated nitrotyrozine expression in three different groups of animals (with at least 3 animals in each group): the first group was treated with 0.5 LD50 Tabun and organs were collected after 24 h; the second group received vehicle for the same period; in the third group a highly specific re-activator was applied immediately after Tabun application. Heart and kidney were collected after 24 h. The levels of nitrotyrozine production significantly increased (more than 3 times) in cardiomyocytes after Tabun. The application of reactivator slightly reduced these levels not reaching the basal heart levels. Nitrotyrozine expression in kidney increased more than 2 times after Tabun and application of re-activator did not change it significantly. In conclusion, our study evidently demonstrated that Tabun trigger oxidative-nitrosative stress in heart and kidney and these cellular effects should be protected by an additional anti-oxidant therapy, since acetylcholinesterase re-activator is not efficient in this manner. Keywords: Organophosphorous poisoning, 3-nitrotyrozine, nitrosative-oxidative stress, cardiac and renal damage.
Introduction Organophosphorus compounds (OPC) have been widely used in agriculture for crop protection and pest control. Thousands of these compounds have been synthezied and over one hundred of them have been applied for these purposes.[1] Some have become drugs used in the treatment of myasthenia gravis, e.g., diisopropyl phosphorofluoridate (DPF), tetraethyl pyrophosphate (TEPP), octamethyl pyrophosphotetramide, or they are still used to treat glaucoma (Ecothiopate).[2] Furthermore, the most potent anticholinesterase compounds, including Tabun, Sarin, Address correspondence to Ivanka Dimova, Department of Medical Genetics, Medical University Sofia, 2 Zdrave Str., Sofia 1431, Bulgaria; E-mail: [email protected] Received October 30, 2014. Color versions for one or more of the figures in the article can be found online at www.tandfonline.com/lesa.
Soman, and VX have been used as “nerve gases” in chemical warfare. Many OPCs, because of their environmental persistence, cause severe environmental pollution. Many people suffer of acute or chronic diseases related to pesticides exposure.[3] The general population might be at risk due to presence of the residues of pesticides, including physical and biological degradation products in air, water and food. This widespread use of OPC has resulted in an increased number of human poisonings. The main mechanism of action is irreversible inhibition of acetylcholine esterase (AChE), resulting in accumulation of toxic levels of acetylcholine (ACh) at the synaptic junctions. Thus muscarinic and nicotinic receptors’ stimulation is induced.[4] However, OPC have many toxicological effects on the body by noncholinergic mechanism.[5] During the last decade basic and clinical research has highlighted the central role of reactive oxygen species
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Cardiac and renal stress after acute Tabun poisoning (ROS) in many diseases, provoked by environmental substances. Accumulation of ROS may be accompanied by the production of reactive nitrogen species (RNS), such as the highly reactive peroxynitrite anion, a strong oxidant formed by the reaction of superoxide anion (.O2¡) and nitric oxide (NO). Under physiological conditions, NO formation is stimulated by flowing blood shear forces acting on the vascular endothelium or by agonist activation of endothelial receptors. Based on this stimulation, Ca2C influx triggers the enzymatic activation of constitutively expressed endothelial membrane-bound nitric oxide synthase (eNOS). Pathophysiological conditions activate the inducible NOS (iNOS), which produces Ca2C-independent NO at a rate 1,000-fold greater than that of eNOS.[6] In the vasculature, iNOS can be induced in infiltrating macrophages and lymphocytes, endothelial cells, smooth muscle cells, or fibroblasts, whereas eNOS is predominantly produced in endothelial cells. Excessive NO binds to cytochrome oxidase, inhibits mitochondrial respiration, increases .O2¡ production, and potentially augments peroxynitrite formation. Peroxynitrite formation has two important biological consequences: loss of bioactive NO and direct cytotoxic effects. Peroxynitrite and its conjugate acid can oxidize a variety of biomolecules, including free thiols (such as cysteine, glutathione, or cysteinyl residues in proteins), lipids, deoxyribose, guanine bases, methionine and phenols. The consequences of these oxidations are protein modification, inhibition of mitochondrial respiration and other enzymes, and lipid peroxidation.[7] The nitration of protein tyrosinyl residues yields 3-nitrotyrosine which is a stable product left by the short-lived peroxynitrite in vivo. The presence of nitrotyrosine suggests peroxynitrite-induced oxidative stress and injury. Oxidative stress has recently been suggested as a factor in the mortality and morbidity induced by OPC poisoning.[8] Many studies have aimed to answer the question whether the toxic effects of OPC can also be followed by oxidative stress and imbalance in antioxidants. Changes in oxidative homeostasis were found to extensively overweigh the inhibition of AChE. Alterations in thiobarbituric acid reactive substances indicate lipid peroxidation as they respond to degradation product of lipids (malondialdehyde) and can be interpreted as a lack of antioxidants for oxidative stress covering. Some organs were more vulnerable to oxidative stress induced by Sarin.[9] So far, studies of oxidative stress after OPC poisoning were mainly focused on the plasma levels of factors of oxidation or antioxidants. Little is known about the specific organs’oxidative changes. The objective of our study was to analyze nitrotyrosine as a marker of NO-induced oxidative stress in heart and kidney after acute OPC poisoning and its treatment. Doing this research, we consider that we will contribute to the elucidation of complex mechanism of OPC
intoxication and assessment for efficacy of antioxidant therapy. We aimed in getting information about nitrotyrozine production in essential human organs (heart and kidney) after Tabun intoxication. It could show oxidative damage, which is of paramount importance for clinical management of the patients.
Materials and methods Animals Twelve Wistar rats were obtained from the vivarium of Military Medical Academy-Sofia (MMA-Sofia). The rats were 6 weeks old at the beginning of the experiment and weighed 180 § 15 g. For the entire experiment, the rats were kept in an air conditioned room at temperature 22 § 2 C, humidity 50 § 10% and light period 12 h per day. Feed and drinking water were provided ad libitum. The entire experiment was approved and supervised by the Ethical Committee of the MMA-Sofia. Compounds and treatments Tabun (Ethyl N,N-Dimethylphosphoramidocyanidate) and cholinesterase reactivators were obtained from the Department of Disaster Medicine and Toxicology, MMASofia. The MMA-Sofia has permission for the manipulation of nerve agents from the government institution. The LD50 was assessed in a separate experiment to be 280 mg kg¡1 (mortality followed up to 24 h). The 12 animals were exposed intramuscularly to Tabun at doses 0 (controls; saline only) and 50% of LD50. Thus three experimental groups were included in the study: the first group was treated by 0.5 LD50 Tabun (140 mg kg¡1) and organs were collected after 24 h; the second group received vehicle for the same period (control group); in the third group a highly specific reactivator HL€ o – 7 (16.7 mg kg¡1) and ¡1 Atropin (10 mg kg ) were applied 1 min after Tabun challenge. After 24 h, animals were sacrificed by carbon dioxide narcosis. Blood, kidney, and heart were collected from the sacrificed animals. Blood was kept in heparinised tubes (Dialab, Prague, Czech Republic) and plasma was collected after blood centrifugation at 3,000 g for 15 min. The collected tissues were fixed by immersion in a freshly prepared solution of 10% (weight/volume) paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) in 0.1 mol L¡1 potassium phosphate buffer, pH 7.4, overnight at 4 C. The samples were then dehydrated through ascending ethanol concentrations and were embedded in paraffin. Immunohistochemical analysis Heart and kidney tissues from rats were fixed in formalin and embedded in paraffin blocks according to standard
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826 procedure then were cut on 4–6 mm tissue sections. Deparaffinization was done in xylene, followed by hydration gradually through graded alcohols and washing in deionized H2O2. Antigen unmasking was performed by heat treatment in 10 mM sodium citrate buffer, pH 6.0 for 5 min at 95 C, slides were cooled in the buffer for additional 20 min. Slides were then incubated for 5 min in 0.5% hydrogen peroxide in deionized H2O2 to quench endogenous peroxidase activity. Specimens were blocked in 1.5% normal blocking serum in phosphate buffer saline (PBS) for 1 h. Incubation with anti-nitrotyrozine primary antibody (sc-32757; Santa Cruz) was done overnight at 4 C. After 3 times of PBS washing, they were incubated for 30 min with biotin-conjugated secondary antibody diluted in PBS with 1.5% normal blocking serum. Detection was done by standard ABC staining system. The sections then were scored for intensity of immunostaining (0 ¡ absent, 1 ¡ faint, 2 ¡ moderate, and 3 ¡ intense) for the antibody and for the proportion of positive cells, and the average value for both parameters was calculated for each animal, analyzing at least 6 sections per animal.
Results In addition to AChE blocking, OPC could have damaging effect on different essential organs. We aimed to investigate nitrotyrozine production in heart and kidney after Tabun poisoning, since NO responds very fast to any change in physiological homeostasis of the organism and its excessive production could lead to formation of substances, related to oxidative stress of the tissue, such as nitrotyrozine. NO-production is tightly connected to hemodynamics and thus it could influence mainly the most important hemodynamic organs – heart and kidney. We investigated nitrotyrozine expression in three different groups of animals (with at least 3 animals in each group): the first group was treated by 0.5 LD50 Tabun and organs were collected after 24 h; the second group received vehicle for the same period; in the third group a highly specific re-activator was applied immediately after Tabun application. Hematoxilin and eosin staining of heart and kidney tissues did not show any morphological changes for the investigated period of 24 h. Heart nitrotyrozine expression 24 h after Tabun treatment was dramatically increased in comparison with its levels without treatment (Figs. 1A and B). More than 70% of cardiac cells were positive with a high intensity; positivity was observed in endothelial cells as well (Fig. 1D). In control animals less than 25% of cells showed some positivity; mainly endothelial cells were slightly nitrotyrozinepositive (Fig. 1D). After application of AChE re-activator, we observed decrease in nitrotyrozine levels of about 10% compared to Tabun treatment, but still far from the normal levels in non-treated heart (Figs. 1C, D). Most of the cells demonstrated still high expression.
Dimov et al. In kidney samples, the increase of nitrotyrozine expression was less prominent than in the heart, but still there was evidently higher positivity in Tabun treated animals (Fig. 2A-B, G), compared to controls –22.8% of cells on average (Tabun) versus 9.6% of cells on average in controls (Fig. 2C-D, G). The most remarkable difference was in glomerular capillaries, whereas distal tubules showed some consistent expression even without treatment. The application of AChE re-activator did not change considerably the expression of nitrotyrozine in glomerules (Fig. 2E-F, G) –19% of cells on average were positive.
Discussion OPC have a common mechanism of pharmacological or toxicological action, ultimately modifying cholinergic signaling through disruption of acetylcholine degradation. However, many studies indicate that a number of cholinesterase inhibitors have additional sites of action that may have pharmacological or toxicological relevance. These effects can occur at concentrations below those affecting cholinergic transmission. Knowledge of selective additional targets may play a key role in the optimization of strategies for poisoning therapy and in the further elucidation of toxicity mechanisms for this class of compounds. That is why it is important to investigate the alterations in NO-production and oxidative status and find biomarkers for NO-induced oxidative stress in essential organs like heart and kidney after acute Tabun intoxication. In this study for the first time we studied the nitrosative/oxidative stress in heart and kidney after acute Tabun poisoning measuring the expression of 3-nitrotyrosine by immunohistochemistry. The oxidative stress is induced by ROS which include partially reduced forms of molecular oxygen-hydroxyl radical (.OH), superoxide anion (.O2¡), hydrogen peroxide (H2O2), lipid peroxides, and hypochlorous acid (HClO). Nitric oxide is produced by a variety of mammalian cells, including vascular endothelium, neurons, smooth muscle cells, macrophages, neutrophils, platelets, and pulmonary epithelium. The physiological actions of NO range from modulating the vascular system (blood flow, inhibition of platelet aggregation, angiogenesis), to regulating the immune system (cellular immunity by macrophage, neutrophil killing of pathogens, non-specific host defense) and controlling neuronal functions (neurotransmission, synaptic plasticity in the central nervous system, oscillatory behavior of neuronal networks. NO influences on several aspects of cardiomyocyte contractility, from the fine regulation of excitation-contraction coupling to modulation of (presynaptic and postsynaptic) autonomic signaling and mitochondrial respiration. This multifaceted involvement of NO in cardiac physiology is supported by a tight molecular regulation of the three NO synthases (NOS), from cellular spatial confinement to
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Cardiac and renal stress after acute Tabun poisoning
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Fig. 1. Nitrotyrozine expression in the heart after treatment by Tabun (A), vehicle (B), Tabun C AChE re-activator (C). D represents the percentage of nitrotyrozine-positive cells.
posttranslational allosteric modulation by specific interacting proteins, acting in concert to restrict the influence of NO to a particular intracellular target in a stimulus-specific manner. Loss of this specificity, such as produced on excessive NO delivery (oxidative stress) may result in cellular disturbances leading to heart failure. Nitrotyrosine is widely used as a marker of nitroxidative stress, associated with cardiac injury in hypertension, ischemia, cardiomyopathy. It was factor of measure for cardiovascular complications in metabolic syndrome, associated with impaired redox signaling, mitochondrial activity, and dysregulation of cellular metabolism in the heart.[10] Levels of nitrotyrosine, going together with chronic oxidative-nitrosative stress in metabolic syndrome, decreased markedly by treatment with tempol, a membrane-permeable radical scavenger. Type 1 diabetes triggered cardiac oxidative stress and hyperglycemia up-regulated cardiac 3-nitrotyrosine expression, which was significantly attenuated by coenzyme Q10 supplementation. Here we present the results, showing significantly increased levels of nitrotyrosine production (more than 3 times) in cardiomyocytes after treatment by Tabun, suggesting an additional mechanism of cardiac cellular injury for the OPC. The application of reactivator slightly reduced these levels not reaching the basal normal heart levels. OPC induce acute poisoning with myocardial necrosis. Creatinine kinase and lactate dehydrogenase levels are increased after OPC poisoning. Cardiac manifestations are sinus tachycardia, sinus bradycardia, hypertension, hypotension, impaired heart rate and force contraction, ECG changes, prolonged QTc interval, ST
segment elevation, low amplitude T waves, extrasystoles and prolonged PR interval.[11] In normal kidneys, eNOS is located on vascular endothelial cells. iNOS is expressed in some distal tubular segments in normal kidney, but after stimulation, iNOS can also be expressed in glomerular mesangial cells, smooth muscle cells, proximal tubular cells and inflammatory cells and plays a role in processes of oxidative stress.[12] Although many studies report a protective role for endothelial-derived NO, high production of NO by iNOS is damaging. Extensive production of NO may lead to oxidative damage by interacting with reactive oxygen species such as superoxide anion (.O2¡), produced by macrophages, mononuclear cells, mesangial cells and endothelial cells.[13] Interaction between .O2¡ and NO will lead to the formation of peroxynitrite (ONOO¡), a cytotoxic molecule capable of initiating lipid peroxidation, and nitration of tyrosine residues (nitrotyrosine), resulting in loss of protein structure and function.[14] It has been shown that in pathological conditions NO production in the presence of . O2¡ lead to oxidative damage of tubular epithelium. A sustained glomerular reduction in eNOS expression combined with a marked increase in interstitial and glomerular iNOS expression is critically involved in the pathogenesis of renal damage. Nitrotyrosine was elevated in the kidney of high fat-fed mice. This study indicated that magnolia extract BL153 administration may be a novel approach for renoprotection in obese individuals by anti-inflammation and antioxidative stress activity. Nitrotyrosine abnormalities were ameliorated by RTA dh404 administration in chronic kidney disease, which is largely driven by systemic oxidative
Dimov et al.
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Fig. 2. Nitrotyrozine production in kidney samples after treatment by Tabun (A-B), vehicle (C-D), Tabun C AChE re-activator (E-F). The objects in rectangles are showed in higher magnification. G represents the percentage of nitrotyrozine-positive cells.
stress and inflammation. It is shown that the nephroprotective activity of berberine against cysplatin-induced renal injury, which could be also attributed to the inhibition of oxidative/nitrosative stress.[15] NO-induced oxidative stress, evaluated by nitrotyrozine expression in kidney, increased by more than 2 times after acute OPC poisoning according to our results and application of re-activator did not change it significantly.
Many studies reviewed by the Ontario College show positive associations between pesticide exposure and solid tumors, including kidney cancer. In children constantly exposed to low levels of pesticides in their food and environment, an elevated risk of kidney cancer was observed. The chronic exposure to pesticides leads to kidney failure. The mechanism is still to be elucidated. OPC poisoning is associated with enhanced lipid peroxidation, reduced
Cardiac and renal stress after acute Tabun poisoning glutathione levels and elevated antioxidant status, and increased oxidative stress.[16] Recently, increasing attention has been put to vascular endothelial dysfunction after OPC intoxication.[17] Current state of knowledge regarding pesticides and oxidative stress is that stimulation of free radical production, induction of lipid peroxidation, and disturbance of the total antioxidant capability of the body are mechanisms of toxicity in most pesticides, including organophosphates. The activation of RhoA/Rhokinase/MEK1/ERK1/2/iNOS pathway is associated with oxidative/nitrosative stress leading to distant and target organ injury.
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Conclusion Our study evidently demonstrated that acute Tabun poisoning trigger oxidative-nitrosative stress in heart and kidney, and these cellular effects should be protected by an additional anti-oxidant therapy, since AChE reactivator is not efficient in this manner.
Funding This work was supported by Counsel of Medical Sciences, Medical University Sofia, Contract No. 40/2014.
References [1] Mogda, M.; El-Kashoury, A.; Rashed, M.; Koretem K. Oxidative and biochemical alterations induced by profenofos insecticide in rats. Nature Sci. 2009, 7(2), 1–15. [2] Kumar S.; Fareedullah M.; Sudhakar, Y.; Venkateswarlu, B.; Kumar, E. Current review on organophosphorus poisoning. Arch. Appl. Sci. Res. 2010, 2(4), 199–215. [3] Alavanja, M.; Ross, M.; Bonner, M. Increased cancer burden among pesticide applicators and others due to pesticide exposure. Cancer J. Clin. 2013, 63(2), 120–142. [4] Moshiri, M.; Darchini-Maragheh, E.; Balali-Mood M. Advances in toxicology and medical treatment of chemical warfare nerve agents. DARU J. Pharm. Sci. 2012, 20, 1–24.
829 [5] Terry, J. Functional consequences of repeated organophosphate exposure: potential non-cholinergic mechanisms. Pharmacol. Ther. 2012, 134, 355–365. [6] Soski, S.; Dobutovic, B.; Sudar, E.; Obradovic, M.; Nikolic, D.; Djordjevic, J.; Radak, D.; Dimitri P.; Mikhailidis, D.; Isenovic, E. Regulation of inducible nitric oxide synthase (iNOS) and its potential role in insulin resistance, diabetes and heart failure. Open Cardiovasc. Med. J. 2011, 5, 153–163. [7] Miric, D.; Kisic, B.; Stolic, R.; Miric, B.; Mitic, R.; JanicijevicHudomal, S. The role of xanthine oxidase in hemodialysis-induced oxidative injury: Relationship with nutritional status. Oxidat. Med. Cell. Longev. 2013, 2013, 245–253. [8] Nurulain, S.; Szegi, P.; Tekes, K. Antioxidants in organophosphorus compounds poisoning. Arch. Industr. Hyg. Toxicol. 2013, 64(1), 169–177. [9] Pohanka, M.; Rom anek, J.; Pikula, J. Acute poisoning with sarin causes alteration in oxidative homeostasis and biochemical markers in Wistar rats. J. Appl. Biomed. 2012, 10, 187–193. [10] Li, Y.; Sarkar, O.; Brochu, M.; Anand-Srivastava, M.B. Natriuretic Peptide receptor-C attenuates hypertension in spontaneously hypertensive rats: role of nitroxidative stress and gi proteins. Hypertension 2014, 63(4), 846–855. [11] Thomaz, J.; Martins, N.; Monteiro, D.; Rantin, F.; Kalinin, A. Cardio-respiratory function and oxidative stress biomarkers in Nile-tilapia exposed to the organophosphate insecticide trichlorfon. Ecotoxicol. Environ. Saf. 2009, 72, 1413–1424. [12] Du, C.; Guan, Q.; Diao, H.; Yin, Z.; Jevnikar, A. Nitric oxide induces apoptosis in renal tubular epithelial cells through activation of caspase-8, AJP - Renal Physiol. 2006, 290(5), 1044–1054. [13] Shah, S.; Baliga, R.; Rajapurkar, M.; Fonseca, V. Oxidants in chronic kidney disease. J. Amer. Soc. Nephrol. 2007, 18(1), 16–28. [14] Luthra, A.; Gupta, N.; Kaufman, P.; Weinreb, R.; Y€ ucel, Y. Oxidative injury by peroxynitrite in neural and vascular tissue of the lateral geniculate nucleus in experimental glaucoma. Exper. Eye Res. 2005, 80(1), 43–49. [15] Domitrovic, R.; Cvijanovic, O.; Pernjak-Pugel, E.; Skoda, M.; Mikelic, L.; Crncevic-Orlic, Z. Berberine exerts nephroprotective effect against cisplatin-induced kidney damage through inhibition of oxidative/nitrosative stress, inflammation, autophagy and apoptosis. Food Chem. Toxicol. 2013, 62, 397–406. [16] Rastogi, S.K.; Satyanarayan, P.; Ravishankar, D.; Tripathi, S. A study on oxidative stress and antioxidant status of agricultural workers exposed to organophosphorus insecticides during spraying. Ind. J. Occup. Environ. Med. 2009, 13, 131–134. [17] Xiong, X.M.; Dai, W.; LI, P.; Wu, S.; Hu, M. Subchronic toxicity organophosphate insecticideinduced damages on endothelial function of vessels in rabbits by inhibiting antioxidases. Progr. Biochem. 2010, 37(11), 1232–1239.
Journal of Environmental Science and Health, Part A: Toxic/Hazardous Substances and Environmental Engineering Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lesa20
Cardiac and renal nitrosative-oxidative stress after acute poisoning by a nerve agent Tabun a
b
a
c
d
Dimo Dimov , Radka Hadjiolova , Kamen Kanev , Radka Tomova , Anna Michova , Todor e
d
f
g
Todorov , Rumen Murdjev , Temenujka Boneva & Ivanka Dimova a
Disaster Medicine and Toxicology Deparment, Military Medical Academy, Sofia, Bulgaria
b
Department of Pathophysiology, Faculty of Medicine, Medical University of Sofia, Sofia, Bulgaria
Click for updates
c
Department of Medical Chemistry and Biochemistry, Faculty of Medicine, Medical University of Sofia, Sofia, Bulgaria d
Division of Pathology, Military Medical Academy, Sofia, Bulgaria
e
Department of Pathology, Faculty of Medicine, Medical University of Sofia, Sofia, Bulgaria
f
Division of Clinical Laboratory and Genetics, Military Medical Academy, Sofia, Bulgaria
g
Department of Medical Genetics, Faculty of Medicine, Medical University of Sofia, Sofia, Bulgaria Published online: 01 Jun 2015.
To cite this article: Dimo Dimov, Radka Hadjiolova, Kamen Kanev, Radka Tomova, Anna Michova, Todor Todorov, Rumen Murdjev, Temenujka Boneva & Ivanka Dimova (2015) Cardiac and renal nitrosative-oxidative stress after acute poisoning by a nerve agent Tabun, Journal of Environmental Science and Health, Part A: Toxic/Hazardous Substances and Environmental Engineering, 50:8, 824-829, DOI: 10.1080/10934529.2015.1019801 To link to this article: http://dx.doi.org/10.1080/10934529.2015.1019801
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Journal of Environmental Science and Health, Part A (2015) 50, 824–829 Copyright © Taylor & Francis Group, LLC ISSN: 1093-4529 (Print); 1532-4117 (Online) DOI: 10.1080/10934529.2015.1019801
Cardiac and renal nitrosative-oxidative stress after acute poisoning by a nerve agent Tabun DIMO DIMOV1, RADKA HADJIOLOVA2, KAMEN KANEV1, RADKA TOMOVA3, ANNA MICHOVA4, TODOR TODOROV5, RUMEN MURDJEV4, TEMENUJKA BONEVA6 and IVANKA DIMOVA7 1
Disaster Medicine and Toxicology Deparment, Military Medical Academy, Sofia, Bulgaria Department of Pathophysiology, Faculty of Medicine, Medical University of Sofia, Sofia, Bulgaria 3 Department of Medical Chemistry and Biochemistry, Faculty of Medicine, Medical University of Sofia, Sofia, Bulgaria 4 Division of Pathology, Military Medical Academy, Sofia, Bulgaria 5 Department of Pathology, Faculty of Medicine, Medical University of Sofia, Sofia, Bulgaria 6 Division of Clinical Laboratory and Genetics, Military Medical Academy, Sofia, Bulgaria 7 Department of Medical Genetics, Faculty of Medicine, Medical University of Sofia, Sofia, Bulgaria
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We hypothesized that Tabun poisoning, as well as other organophosphorous treatment, cause specific organs’ oxidative changes that have not previously been substantiated investigated. In this regard, a marker for nitrosative-oxidative stress in the main haemodynamic organs (heart and kidney) could reveal the existence of such changes. In this study, for the first time we studied the nitrosative/oxidative stress in heart and kidney after acute Tabun (Ethyl N,N- Dimethylphosphoramidocyanidate) poisoning measuring by immunohistochemistry the expression of 3-nitrotyrosine—a marker for nitrosative-oxidative stress. We investigated nitrotyrozine expression in three different groups of animals (with at least 3 animals in each group): the first group was treated with 0.5 LD50 Tabun and organs were collected after 24 h; the second group received vehicle for the same period; in the third group a highly specific re-activator was applied immediately after Tabun application. Heart and kidney were collected after 24 h. The levels of nitrotyrozine production significantly increased (more than 3 times) in cardiomyocytes after Tabun. The application of reactivator slightly reduced these levels not reaching the basal heart levels. Nitrotyrozine expression in kidney increased more than 2 times after Tabun and application of re-activator did not change it significantly. In conclusion, our study evidently demonstrated that Tabun trigger oxidative-nitrosative stress in heart and kidney and these cellular effects should be protected by an additional anti-oxidant therapy, since acetylcholinesterase re-activator is not efficient in this manner. Keywords: Organophosphorous poisoning, 3-nitrotyrozine, nitrosative-oxidative stress, cardiac and renal damage.
Introduction Organophosphorus compounds (OPC) have been widely used in agriculture for crop protection and pest control. Thousands of these compounds have been synthezied and over one hundred of them have been applied for these purposes.[1] Some have become drugs used in the treatment of myasthenia gravis, e.g., diisopropyl phosphorofluoridate (DPF), tetraethyl pyrophosphate (TEPP), octamethyl pyrophosphotetramide, or they are still used to treat glaucoma (Ecothiopate).[2] Furthermore, the most potent anticholinesterase compounds, including Tabun, Sarin, Address correspondence to Ivanka Dimova, Department of Medical Genetics, Medical University Sofia, 2 Zdrave Str., Sofia 1431, Bulgaria; E-mail: [email protected] Received October 30, 2014. Color versions for one or more of the figures in the article can be found online at www.tandfonline.com/lesa.
Soman, and VX have been used as “nerve gases” in chemical warfare. Many OPCs, because of their environmental persistence, cause severe environmental pollution. Many people suffer of acute or chronic diseases related to pesticides exposure.[3] The general population might be at risk due to presence of the residues of pesticides, including physical and biological degradation products in air, water and food. This widespread use of OPC has resulted in an increased number of human poisonings. The main mechanism of action is irreversible inhibition of acetylcholine esterase (AChE), resulting in accumulation of toxic levels of acetylcholine (ACh) at the synaptic junctions. Thus muscarinic and nicotinic receptors’ stimulation is induced.[4] However, OPC have many toxicological effects on the body by noncholinergic mechanism.[5] During the last decade basic and clinical research has highlighted the central role of reactive oxygen species
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Cardiac and renal stress after acute Tabun poisoning (ROS) in many diseases, provoked by environmental substances. Accumulation of ROS may be accompanied by the production of reactive nitrogen species (RNS), such as the highly reactive peroxynitrite anion, a strong oxidant formed by the reaction of superoxide anion (.O2¡) and nitric oxide (NO). Under physiological conditions, NO formation is stimulated by flowing blood shear forces acting on the vascular endothelium or by agonist activation of endothelial receptors. Based on this stimulation, Ca2C influx triggers the enzymatic activation of constitutively expressed endothelial membrane-bound nitric oxide synthase (eNOS). Pathophysiological conditions activate the inducible NOS (iNOS), which produces Ca2C-independent NO at a rate 1,000-fold greater than that of eNOS.[6] In the vasculature, iNOS can be induced in infiltrating macrophages and lymphocytes, endothelial cells, smooth muscle cells, or fibroblasts, whereas eNOS is predominantly produced in endothelial cells. Excessive NO binds to cytochrome oxidase, inhibits mitochondrial respiration, increases .O2¡ production, and potentially augments peroxynitrite formation. Peroxynitrite formation has two important biological consequences: loss of bioactive NO and direct cytotoxic effects. Peroxynitrite and its conjugate acid can oxidize a variety of biomolecules, including free thiols (such as cysteine, glutathione, or cysteinyl residues in proteins), lipids, deoxyribose, guanine bases, methionine and phenols. The consequences of these oxidations are protein modification, inhibition of mitochondrial respiration and other enzymes, and lipid peroxidation.[7] The nitration of protein tyrosinyl residues yields 3-nitrotyrosine which is a stable product left by the short-lived peroxynitrite in vivo. The presence of nitrotyrosine suggests peroxynitrite-induced oxidative stress and injury. Oxidative stress has recently been suggested as a factor in the mortality and morbidity induced by OPC poisoning.[8] Many studies have aimed to answer the question whether the toxic effects of OPC can also be followed by oxidative stress and imbalance in antioxidants. Changes in oxidative homeostasis were found to extensively overweigh the inhibition of AChE. Alterations in thiobarbituric acid reactive substances indicate lipid peroxidation as they respond to degradation product of lipids (malondialdehyde) and can be interpreted as a lack of antioxidants for oxidative stress covering. Some organs were more vulnerable to oxidative stress induced by Sarin.[9] So far, studies of oxidative stress after OPC poisoning were mainly focused on the plasma levels of factors of oxidation or antioxidants. Little is known about the specific organs’oxidative changes. The objective of our study was to analyze nitrotyrosine as a marker of NO-induced oxidative stress in heart and kidney after acute OPC poisoning and its treatment. Doing this research, we consider that we will contribute to the elucidation of complex mechanism of OPC
intoxication and assessment for efficacy of antioxidant therapy. We aimed in getting information about nitrotyrozine production in essential human organs (heart and kidney) after Tabun intoxication. It could show oxidative damage, which is of paramount importance for clinical management of the patients.
Materials and methods Animals Twelve Wistar rats were obtained from the vivarium of Military Medical Academy-Sofia (MMA-Sofia). The rats were 6 weeks old at the beginning of the experiment and weighed 180 § 15 g. For the entire experiment, the rats were kept in an air conditioned room at temperature 22 § 2 C, humidity 50 § 10% and light period 12 h per day. Feed and drinking water were provided ad libitum. The entire experiment was approved and supervised by the Ethical Committee of the MMA-Sofia. Compounds and treatments Tabun (Ethyl N,N-Dimethylphosphoramidocyanidate) and cholinesterase reactivators were obtained from the Department of Disaster Medicine and Toxicology, MMASofia. The MMA-Sofia has permission for the manipulation of nerve agents from the government institution. The LD50 was assessed in a separate experiment to be 280 mg kg¡1 (mortality followed up to 24 h). The 12 animals were exposed intramuscularly to Tabun at doses 0 (controls; saline only) and 50% of LD50. Thus three experimental groups were included in the study: the first group was treated by 0.5 LD50 Tabun (140 mg kg¡1) and organs were collected after 24 h; the second group received vehicle for the same period (control group); in the third group a highly specific reactivator HL€ o – 7 (16.7 mg kg¡1) and ¡1 Atropin (10 mg kg ) were applied 1 min after Tabun challenge. After 24 h, animals were sacrificed by carbon dioxide narcosis. Blood, kidney, and heart were collected from the sacrificed animals. Blood was kept in heparinised tubes (Dialab, Prague, Czech Republic) and plasma was collected after blood centrifugation at 3,000 g for 15 min. The collected tissues were fixed by immersion in a freshly prepared solution of 10% (weight/volume) paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) in 0.1 mol L¡1 potassium phosphate buffer, pH 7.4, overnight at 4 C. The samples were then dehydrated through ascending ethanol concentrations and were embedded in paraffin. Immunohistochemical analysis Heart and kidney tissues from rats were fixed in formalin and embedded in paraffin blocks according to standard
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826 procedure then were cut on 4–6 mm tissue sections. Deparaffinization was done in xylene, followed by hydration gradually through graded alcohols and washing in deionized H2O2. Antigen unmasking was performed by heat treatment in 10 mM sodium citrate buffer, pH 6.0 for 5 min at 95 C, slides were cooled in the buffer for additional 20 min. Slides were then incubated for 5 min in 0.5% hydrogen peroxide in deionized H2O2 to quench endogenous peroxidase activity. Specimens were blocked in 1.5% normal blocking serum in phosphate buffer saline (PBS) for 1 h. Incubation with anti-nitrotyrozine primary antibody (sc-32757; Santa Cruz) was done overnight at 4 C. After 3 times of PBS washing, they were incubated for 30 min with biotin-conjugated secondary antibody diluted in PBS with 1.5% normal blocking serum. Detection was done by standard ABC staining system. The sections then were scored for intensity of immunostaining (0 ¡ absent, 1 ¡ faint, 2 ¡ moderate, and 3 ¡ intense) for the antibody and for the proportion of positive cells, and the average value for both parameters was calculated for each animal, analyzing at least 6 sections per animal.
Results In addition to AChE blocking, OPC could have damaging effect on different essential organs. We aimed to investigate nitrotyrozine production in heart and kidney after Tabun poisoning, since NO responds very fast to any change in physiological homeostasis of the organism and its excessive production could lead to formation of substances, related to oxidative stress of the tissue, such as nitrotyrozine. NO-production is tightly connected to hemodynamics and thus it could influence mainly the most important hemodynamic organs – heart and kidney. We investigated nitrotyrozine expression in three different groups of animals (with at least 3 animals in each group): the first group was treated by 0.5 LD50 Tabun and organs were collected after 24 h; the second group received vehicle for the same period; in the third group a highly specific re-activator was applied immediately after Tabun application. Hematoxilin and eosin staining of heart and kidney tissues did not show any morphological changes for the investigated period of 24 h. Heart nitrotyrozine expression 24 h after Tabun treatment was dramatically increased in comparison with its levels without treatment (Figs. 1A and B). More than 70% of cardiac cells were positive with a high intensity; positivity was observed in endothelial cells as well (Fig. 1D). In control animals less than 25% of cells showed some positivity; mainly endothelial cells were slightly nitrotyrozinepositive (Fig. 1D). After application of AChE re-activator, we observed decrease in nitrotyrozine levels of about 10% compared to Tabun treatment, but still far from the normal levels in non-treated heart (Figs. 1C, D). Most of the cells demonstrated still high expression.
Dimov et al. In kidney samples, the increase of nitrotyrozine expression was less prominent than in the heart, but still there was evidently higher positivity in Tabun treated animals (Fig. 2A-B, G), compared to controls –22.8% of cells on average (Tabun) versus 9.6% of cells on average in controls (Fig. 2C-D, G). The most remarkable difference was in glomerular capillaries, whereas distal tubules showed some consistent expression even without treatment. The application of AChE re-activator did not change considerably the expression of nitrotyrozine in glomerules (Fig. 2E-F, G) –19% of cells on average were positive.
Discussion OPC have a common mechanism of pharmacological or toxicological action, ultimately modifying cholinergic signaling through disruption of acetylcholine degradation. However, many studies indicate that a number of cholinesterase inhibitors have additional sites of action that may have pharmacological or toxicological relevance. These effects can occur at concentrations below those affecting cholinergic transmission. Knowledge of selective additional targets may play a key role in the optimization of strategies for poisoning therapy and in the further elucidation of toxicity mechanisms for this class of compounds. That is why it is important to investigate the alterations in NO-production and oxidative status and find biomarkers for NO-induced oxidative stress in essential organs like heart and kidney after acute Tabun intoxication. In this study for the first time we studied the nitrosative/oxidative stress in heart and kidney after acute Tabun poisoning measuring the expression of 3-nitrotyrosine by immunohistochemistry. The oxidative stress is induced by ROS which include partially reduced forms of molecular oxygen-hydroxyl radical (.OH), superoxide anion (.O2¡), hydrogen peroxide (H2O2), lipid peroxides, and hypochlorous acid (HClO). Nitric oxide is produced by a variety of mammalian cells, including vascular endothelium, neurons, smooth muscle cells, macrophages, neutrophils, platelets, and pulmonary epithelium. The physiological actions of NO range from modulating the vascular system (blood flow, inhibition of platelet aggregation, angiogenesis), to regulating the immune system (cellular immunity by macrophage, neutrophil killing of pathogens, non-specific host defense) and controlling neuronal functions (neurotransmission, synaptic plasticity in the central nervous system, oscillatory behavior of neuronal networks. NO influences on several aspects of cardiomyocyte contractility, from the fine regulation of excitation-contraction coupling to modulation of (presynaptic and postsynaptic) autonomic signaling and mitochondrial respiration. This multifaceted involvement of NO in cardiac physiology is supported by a tight molecular regulation of the three NO synthases (NOS), from cellular spatial confinement to
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Cardiac and renal stress after acute Tabun poisoning
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Fig. 1. Nitrotyrozine expression in the heart after treatment by Tabun (A), vehicle (B), Tabun C AChE re-activator (C). D represents the percentage of nitrotyrozine-positive cells.
posttranslational allosteric modulation by specific interacting proteins, acting in concert to restrict the influence of NO to a particular intracellular target in a stimulus-specific manner. Loss of this specificity, such as produced on excessive NO delivery (oxidative stress) may result in cellular disturbances leading to heart failure. Nitrotyrosine is widely used as a marker of nitroxidative stress, associated with cardiac injury in hypertension, ischemia, cardiomyopathy. It was factor of measure for cardiovascular complications in metabolic syndrome, associated with impaired redox signaling, mitochondrial activity, and dysregulation of cellular metabolism in the heart.[10] Levels of nitrotyrosine, going together with chronic oxidative-nitrosative stress in metabolic syndrome, decreased markedly by treatment with tempol, a membrane-permeable radical scavenger. Type 1 diabetes triggered cardiac oxidative stress and hyperglycemia up-regulated cardiac 3-nitrotyrosine expression, which was significantly attenuated by coenzyme Q10 supplementation. Here we present the results, showing significantly increased levels of nitrotyrosine production (more than 3 times) in cardiomyocytes after treatment by Tabun, suggesting an additional mechanism of cardiac cellular injury for the OPC. The application of reactivator slightly reduced these levels not reaching the basal normal heart levels. OPC induce acute poisoning with myocardial necrosis. Creatinine kinase and lactate dehydrogenase levels are increased after OPC poisoning. Cardiac manifestations are sinus tachycardia, sinus bradycardia, hypertension, hypotension, impaired heart rate and force contraction, ECG changes, prolonged QTc interval, ST
segment elevation, low amplitude T waves, extrasystoles and prolonged PR interval.[11] In normal kidneys, eNOS is located on vascular endothelial cells. iNOS is expressed in some distal tubular segments in normal kidney, but after stimulation, iNOS can also be expressed in glomerular mesangial cells, smooth muscle cells, proximal tubular cells and inflammatory cells and plays a role in processes of oxidative stress.[12] Although many studies report a protective role for endothelial-derived NO, high production of NO by iNOS is damaging. Extensive production of NO may lead to oxidative damage by interacting with reactive oxygen species such as superoxide anion (.O2¡), produced by macrophages, mononuclear cells, mesangial cells and endothelial cells.[13] Interaction between .O2¡ and NO will lead to the formation of peroxynitrite (ONOO¡), a cytotoxic molecule capable of initiating lipid peroxidation, and nitration of tyrosine residues (nitrotyrosine), resulting in loss of protein structure and function.[14] It has been shown that in pathological conditions NO production in the presence of . O2¡ lead to oxidative damage of tubular epithelium. A sustained glomerular reduction in eNOS expression combined with a marked increase in interstitial and glomerular iNOS expression is critically involved in the pathogenesis of renal damage. Nitrotyrosine was elevated in the kidney of high fat-fed mice. This study indicated that magnolia extract BL153 administration may be a novel approach for renoprotection in obese individuals by anti-inflammation and antioxidative stress activity. Nitrotyrosine abnormalities were ameliorated by RTA dh404 administration in chronic kidney disease, which is largely driven by systemic oxidative
Dimov et al.
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Fig. 2. Nitrotyrozine production in kidney samples after treatment by Tabun (A-B), vehicle (C-D), Tabun C AChE re-activator (E-F). The objects in rectangles are showed in higher magnification. G represents the percentage of nitrotyrozine-positive cells.
stress and inflammation. It is shown that the nephroprotective activity of berberine against cysplatin-induced renal injury, which could be also attributed to the inhibition of oxidative/nitrosative stress.[15] NO-induced oxidative stress, evaluated by nitrotyrozine expression in kidney, increased by more than 2 times after acute OPC poisoning according to our results and application of re-activator did not change it significantly.
Many studies reviewed by the Ontario College show positive associations between pesticide exposure and solid tumors, including kidney cancer. In children constantly exposed to low levels of pesticides in their food and environment, an elevated risk of kidney cancer was observed. The chronic exposure to pesticides leads to kidney failure. The mechanism is still to be elucidated. OPC poisoning is associated with enhanced lipid peroxidation, reduced
Cardiac and renal stress after acute Tabun poisoning glutathione levels and elevated antioxidant status, and increased oxidative stress.[16] Recently, increasing attention has been put to vascular endothelial dysfunction after OPC intoxication.[17] Current state of knowledge regarding pesticides and oxidative stress is that stimulation of free radical production, induction of lipid peroxidation, and disturbance of the total antioxidant capability of the body are mechanisms of toxicity in most pesticides, including organophosphates. The activation of RhoA/Rhokinase/MEK1/ERK1/2/iNOS pathway is associated with oxidative/nitrosative stress leading to distant and target organ injury.
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Conclusion Our study evidently demonstrated that acute Tabun poisoning trigger oxidative-nitrosative stress in heart and kidney, and these cellular effects should be protected by an additional anti-oxidant therapy, since AChE reactivator is not efficient in this manner.
Funding This work was supported by Counsel of Medical Sciences, Medical University Sofia, Contract No. 40/2014.
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