СРАВНИТЕЛНО ИЗУЧАВАНЕ НА НЯКОЛКО КЛАСА ОРГАНИЧНИ СЪЕДИНЕНИЯ ЗА ХЕПАТОТОКСИЧНОСТ. 2. ВЛИЯНИЕ НА МЕТАБОЛИТНАТА АКТИВАЦИЯ

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1 УПРАВЛЕНИЕ И ОБРАЗОВАНИЕ MANAGEMENT AND EDUCATION TOM VII (3) 2011 VOL. VII (3) 2011 СРАВНИТЕЛНО ИЗУЧАВАНЕ НА НЯКОЛКО КЛАСА ОРГАНИЧНИ СЪЕДИНЕНИЯ ЗА ХЕПАТОТОКСИЧНОСТ. 2. ВЛИЯНИЕ НА МЕТАБОЛИТНАТА АКТИВАЦИЯ Светлана Георгиева, Стефан Котов, Яна Колева COMPARATIVE STUDY OF SEVERAL CLASSES OF ORGANIC COMPOUNDS FOR HEPATOTOXICITY. 2. EFFECT OF METABOLIC ACTIVATION Svetlana Georgieva, Stefan Kotov, Yana Koleva ABSTRACT: The metabolism of some xenobiotic chemicals is associated with the formation of toxic intermediates that can elicit liver injury and disrupt the normal functions of this target organ. Therefore, a better understanding of the biochemical mechanisms involved in the manifestation of hepatotoxicity effects is important, since it can contribute to the evaluation of chemicals with respect to their chemical toxicology, thus improving the public awareness of potentially harmful toxicants. Enzymes, catalyzing both the metabolic detoxification and activation of xenobiotics are mostly concentrated in liver as the main metabolic factory. It has been widely recognized that, in many cases, hepatotoxicity, induced by xenobiotics can be attributed to the formation of reactive electrophilic metabolites, due to the biocatalytic action of enzymes in liver. Electrophilic metabolites can interact with several types of functionalities such as amino (-NH 2 ), (-OH) and thiol (-SH) groups in the biological macromolecules, mainly proteins and DNA. Therefore, the aim of the present study was to elucidate the effect of the metabolic activation of several classes of organic and the inherently associated chemical mechanisms, causing liver injury by these xenobiotics. Key words: organic, hepatotoxicity, metabolic activation Introduction The covalent binding of reactive metabolites of drugs to proteins may be a key event in the development of many drug-induced toxicity effects. For example, toxicity can be mediated by a reactive metabolite, covalently binding to critical proteins that are important for maintaining cellular function(s) [13, 14]. Certain other toxicities may be mediated via immunological mechanisms, and a critical component may be the development of hypersensitivity against drugprotein adducts [19]. Although covalent binding of reactive metabolites to protein correlates with the development of a number of drug-induced toxicity effects, the actual sequence of events, leading to the development of the toxicities is unknown. Moreover, with many drugs, the reactive metabolite that can covalently bind to protein may also produce reactive oxygen species or oxygen-containing metabolites that may also produce the toxicity. Thus, al investigations are needed for many drugs to determine the role of covalent binding to protein in the toxicity [8]. The adult liver is the main organ, responsible for metabolizing (and, predominantly, detoxifying) a variety of exogenous as well as endogenous substances, rende them more hydrophilic, which often affects their potency and activity. The enzymes responsible for these actions are primarily expressed in hepatocytes and are mainly divided into two groups: Phase I and Phase II xenobiotic-metabolizing enzymes. The phase I enzymes belong predominantly to the CYP450 family of genes, whose general function is to catalyze the incorporation of polar groups, such as groups, to lipophilic molecules thus rende them more hydrophilic [17]. The main function of the phase II enzymes is to covalently attach a highly-polar, water-soluble moiety to the polar group introduced previously by phase I enzymes. Usually such molecules are sugars or peptides, such as glucuronic acid or glutathione. This usually renders the compound less reactive and prone to more facile excretion [3]. Examples of phase II enzymes are glutathione S-transferase and UDP-glucuronosyl transferase. If the phase II reaction is impaired for some reason, or phase I reaction is induced, this may 239

2 leave the organism with an excess of reactive molecules derived from phase I reaction, which can be detrimental. This can be among the principal reasons for the drug-induced hepatotoxicity as reactive metabolites of the parent compound are formed, which subsequently negatively affect cellular functions [17]. The aim of the present study was to elucidate the effect of the metabolic activation of several classes of organic and the inherently associated chemical mechanisms, causing liver injury by these xenobiotic organic chemicals. Materials and Methods Compounds. The metabolic activation in liver for different classes of organic was investigated (Table 1). OECD (Q)SAR Application Toolbox. (Quantitative) Structure-Activity Relationships [(Q)SARs] are methods for estimating properties of a chemical from its molecular structure and have the potential to provide information on the hazards of chemicals, while reducing time, monetary costs and animal testing currently needed. To facilitate practical application of (Q)SAR approaches in regulatory contexts by governments and industry and to improve their regulatory acceptance, the OECD (Q)SAR project has developed various outcomes such as the principles for the validation of (Q)SAR models, guidance documents as well as the QSAR Toolbox [16]. Metabolic pathways documented for 200 organic chemicals in different mammals are stored in a database format that allows easy computeraided access to the metabolism information. The collection includes chemicals of different classes, with variety of functionalities such aliphatic hydrocarbons, alicyclic s, furans, halogenated hydrocarbons, aromatic hydrocarbons and haloaromatics, amines, nitro-derivatives, and multifunctional. In vivo and in vitro (predominantly, with liver microsomes as experimental systems) studies were used to analyze the metabolic fate of chemicals. Different sources, including monographs, scientific articles and public websites were used to compile the database [10, 16]. Results and Discussion Many chemically-induced toxicity effects are believed to be mediated by the metabolic activation to electrophilic derivatives. Metabolism of xenobiotics is usually catalyzed by the drugmetabolizing enzyme system, and the reactive metabolites formed can covalently bind not only to sites in proteins, but, also, to other sites such as those on DNA, RNA purine and pyrimidine bases, or react with smaller molecular weight endogenous substances such as glutathione tripeptide. Two general concepts have been advanced to explain the mechanisms, by which covalent binding may produce toxicity. One possible mechanism is (a): covalent interaction or alte the protein(s) and/or DNA in such a way that normal functioning cannot be maintained, e. g., by covalently binding to a critical enzyme, necessary for energy production in the cell or to enzymes, involved in ion homeostasis; or causing genotoxicity and mutagenicity. Another mechanism is (b): binding to protein(s) and thus causing the chemical-protein adduct to serve an immunogen. Subsequent exposure causes an immune reaction that results in a cytotoxic response [8]. The concept of metabolic activation and its importance in chemical toxicology has been derived from an analysis of the formation of protein adducts from toxic chemicals [11]. Electrophilic intermediates/metabolites and parent chemicals can react with many different sites in cells. The role of in initiating the carcinogenic process has been extensively investigated [1, 2, 7, 12]. However, electrophilic metabolites may not only react with sites in DNA but may also bind to proteins, RNA, and to endogenous substances of lower molecular weight such as glutathione [8]. The complexity of the reaction of electrophilic metabolites with the various sites within cells and the reasons why different electrophilic reagents react at different sites have been interpreted on the basis of the concepts of hard and soft electrophiles/nucleophiles (hard and soft acids/bases) [5, 9, 18]. Some examples of belonging to different organic classes were elucidated for their probable metabolic activation in liver (observed and predicted) and, respectively, protein and (Table 1). 240

3 Nitroaromatic Table 1. Probable metabolic activation of different classes of organic by (Q)SAR Application Toolbox Class of organic compound Name of compound Structure of compound Metabolic activa tion [ref] Observed liver metabolism by Toolbox (Protein and ) Nitrobenzene [6, 20] 8 metabolites; Aromatic amines, Aromatic amines and nitro 4-nitroaniline [6, 20] 4 metabolites; DNA binding - Aromatic amines, Aromatic amines and nitro 7 metabolites; nitroso DNA binding - Aromatic nitro Halonitrobenzenes 2-chloronitrobenzene 7 metabolites; Michaeltype - Aromatic nitroso 3-chloronitrobenzene [6, 20] 3 metabolites; aromatic amines and nitro [6, 20] 3 metabolites; Liver Metabolism Simulator by Toolbox (Protein and ) 6 metabolites; Michaeltype - Aromatic Aromatic amines 8 metabolites; - Michael- 241

4 Dinitroben zenes Halobenzene type - Aromatic amines and nitroso 5 metabolites; nitroso; - Aromatic nitroso com- 4-chloronitrobenzene pounds [6, 20] No metabolites 16 metabolites; - Michaeltype, nitroso DNA binding - Aromatic amines, nitro 1-chloro-2,4- dinitrobenzen e Bromoben zene aromatic amines [6, 20] 6 metabolites; aromatic amines and nitro [6, 20] 7 metabolites; Michael-type, Nucleophilic cyclo to diketones and Nucleophilic DNA binding 8 metabolites; - Nucleophilic 242

5 α,βunsaturated aldehydes p-benzoqui none 1,2-dibromobenzene [6, 20] No metabolites 6 metabolites; - Michaeltype and 1,3-dibromobenzene [6, 20] No metabolites 5 metabolites; - Acrolein [4,15, 21] No metabolites 5 metabolites; Schiff base formation; No Citral [4,15, 21] No metabolites 1 metabolite; No DNA binding 2,5-dichlorobenzoqui none [4,15, 21] No metabolites No metabolites p-benzoqui none [4,15, 21] No metabolites No metabolites Acrylates Methylacry late [4,15, 21] No metabolites 4 metabolites; - Methacrylates methyl [4,15, 21] No metabolites 7 metabolites; 243

6 methacrylate Conclusion Many chemically-induced hepatotoxicity effects are believed to be mediated by the metabolism (bioactivation) to electrophilic intermediates. The hepatotoxic effects can be mediated by covalent binding of a reactive metabolite to proteins. Covalent binding to proteins in many cases correlates with the incidence and severity of the hepatotoxicity. References 1. Beland, F.A., F.F. Kadlubar Formation and persistence of arylamine DNA adducts in vivo. Environmental Health Perspectives, 62, pp Belinsky, S.A., C.M. White, T.R. Devereux, M.W. Anderson DNA adducts as a dosimeter for risk estimation. Environmental Health Perspectives, 76, pp Board, P., A. Blackburn, et al Polymorphism of phase II enzymes: identification of new enzymes and polymorphic variants by database analysis. Toxicology Letters, , pp Chan, K. and P.J. O Brien Structure activity relationships for hepatocyte toxicity and electrophilic reactivity of α,βunsaturated esters, acrylates and methacrylates. Journal of Applied Toxicology, 28, pp Coles, B Effects of modifying structure on electrophilic reactions with biological nucleophiles. Drug Metab.Rev, 15, pp Hakimelahi, G.H. and G.A. Khodarahmi The identification of toxicophores for the prediction of mutagenicity, hepatotoxicity and - opening, Michael-type and Schiff base formation; cardiotoxicity. Journal of the Iranian Chemical Society, 2(4), pp Harris, C.C., A. Weston, J.C. Willey, G.E. Trivers, D.L. Mann Biochemical and molecular epidemiology of humanc ancer: indicators of carcinogen exposure, DNA damage, and genetic predisposition. Environmental Health Perspectives, 75, pp Hinson, J.A., D.W. Roberts Role of covalent and noncovalent interactions in cell toxicity: Effects on proteins. Annual Review Pharmacology and Toxicology, 32, pp Ketterer, B Detoxication reactions of glutathione and glutathione transferases. Xenobiotica, 16, pp Mekenyan, O.G., S.D. Dimitrov, T.S. Pavlov and G.D. Veith A systematic approach to simulating metabolism in computational toxicology. I. The TIMES heuristic modeling framework. Current Pharmaceutical Design, 10, pp Miller, E.C., J.A. Miller The presencea nd significance of bound aminoazo dyes in the livers of rats fed p- dimethylaminoazobenzene. Cancer Research, 7, pp Miller, E.C., J.A. Miller Mechanisms of chemical carcinogenesis: nature of proximate carcinogens and interactions with macromolecule. Pharmacological Reviews, 18, pp Mitchell, J.R., D.J. Jollow Metabolic activation of drugs to toxic substances. Gastroenterology, 68, pp Nelson, S.D., P.G. Pearson Covalenat nd noncovalenitn teractions in acute lethal cell injury caused by chemicals. Annual Review Pharmacology and Toxicology, 30, pp

7 15. O Brien, P.J, A.G. Siraki and N. Shangari, Aldehyde sources, metabolism, molecular toxicity mechanisms, and possible effects on human health. Critical Reviews in Toxicology, 35, pp OECD (Q)SARs Application Toolbox: 3,en_2649_34379_ _1_1_1_1,0 0.html 17. Park, B.K., M. Pirmohamed, et al The role of cytochrome P450 enzymes in hepatic and extrahepatic human drug toxicity. Pharmacology & Therapeutics, 68, pp Pearson, R.G., J. Songstad Application of the principle of hard and soft acids and bases to organic chemistry. Journal of American Chemical Society, 89, pp Pohl, L.R., H. Satoh, D.D. Christ The immunologic and metabolic basis of drug hypersensitivities. Annual Review Pharmacology and Toxicology, 28, pp Rathi, M.A., L. Thirumoorthi, M. Sunitha, P. Meenakshi, D. Gurukumar and V.K. Gopalakrishnan Hepatoprotective activity of Spermacoce hispida linn. extract against nitrobenzene induced hepatotoxicity in rats. Journal of Herbal Medicine and Toxicology, 4, pp Rikans, LE The oxidation of acroleine by rat liver aldehyde dehydrogenases. Relation to allyl alcohol hepatotoxicity, Drug metabolism and disposition, 15, pp Светлана Фоткова Георгиева, асистент Университет Проф. д-р Асен Златаров Медицински колеж Катедра Фармация Бул. Ст. Стамболов Бургас, България fotkova@abv.bg Яна Колева Колева, гл. ас. д-р Университет Проф. д-р Асен Златаров Катедра Органична химия бул. Проф. Якимов Бургас, България ykoleva@btu.bg Стефан Котов, доц. д-р Университет Проф. д-р Асен Златаров Катедра Органична химия бул. Проф. Якимов Бургас, България skotov@btu.bg 245