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UNC-Chapel Hill ENVR 442 - Protective Systems Against Toxicity

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TXCL: TOXC 442, Syllabus, Protective SystemsProtective Systems Against ToxicityToxicology 442, Biochemistry 442Environmental Sciences 442Biochemical ToxicologyDavid J. [email protected] Bioinformatics Bldg966-4685Concurrent AssignmentGlutathione1.** In: Hodgson E, Smart RC. INTRODUCTION TO BIOCHEMICAL TOXICOLOGY, 3rd ed., 2001 pp. 126-130, pp. 130-131, pp. 239-248, pp. 217-218, pp. 230-235, pp. 239-2522.** Parkinson A. Biotransformation of Xenobiotics. In: Klaassen CD, CASARETT AND DOULL'S TOXICOLOGY, 2001, 6th ed., pp.213-2183. Lu SC. Regulation of hepatic glutathione synthesis: current concepts and controversies. FASEB Journal, 13: 1169-1183 (1999) [available at: http://www.nlm.nih.gov, then PubMed, then "Lu SC 1999"Metallothionein4. In: Hodgson E, Smart RC. INTRODUCTION TO BIOCHEMICAL TOXICOLOGY, 3rd ed. (2001). pp. 337-3405. Miles AT, Hawksworth GM, Beattie JH, Rodilla V. Induction, Regulation, Degradation, and Biological Significance of Mammalian Metallothioneins. Crit. Rev. in Biochemistry and Molecular Biology, 35: 35-70 (2000)OptionalMarzullo L. An update of N-acetylcysteine treatment for acute acetaminophen toxicity in children. Current Opinion in Pediatrics, 17: 239-245 (2005). This first part of the review is a good presentation of the background for the role of glutathione. Available at: http://www.nlm.nih.gov, then PubMed, then "Marzullo L 2005", then Lippincott Williams & Wilkins, then Ovid GatewayExamination questions fromLecture materialAssigned readingsTypes of examination questions:Short discussion questions1. Glutathione and Sulfo- or S-Transferases1.1. Reactions of GSHOne of the cellular systems which constitutes a protective function against toxicants centers around glutathione.Properties which contribute to this protective role:(a) –SH is a very reactive nucleophilic site(b) tissue concn generally is relatively high – the normal concn of GSH in liver of various animals is 4-10 mM(c) multiple forms of glutathione S-transferaseswhich catalyze the reaction of various electrophilic compounds with GSH, thereby neutralizing their electrophilic sites and rendering the products more water-soluble in a (generally) overall detoxication process. 8 transferases can be chromatographically separated from rat liver.Substrates of S-transferases: Although each of the S-transferases has some preferencefor certain substrates, each of the transferases often have overlapping substrate specificities and not rigid specificities.(d) GSH “can non-enzymatically reduce a number of substances, such as peroxides and free radicals” and a later example of carbonyl compounds with reactive double bonds1.2 At least 2 separate cellular GSH pools:CytosolLarger poolRapid changes in concentrationMitochondriaDo not synthesize their own GSH—take up GSH actively from the cytosol (and also actively extrude GSH)Concn usually changes slowly10 nmolesGSH/mg protein in rat liver homogenate3.5-4 nmoles GSH/mg of protein in rat liver mitochondria1.3 Further Metabolism of GSH ConjugatesTypical pattern for further metabolism:Acetylation of free NH2 of cysteinyl residue yields a mercapturic acid [S-alkylated derivative of N-acetylcysteine]Treatment of animal with toxicant = excretion of the metabolite as a mercapturic acidIn contrast to the amides formed by conjugation of xenobiotics to other amino acids, glutathione conjugates are thioethers, which form by nucleophilic attack of glutathione thiolate anion (GS-)Glutathione S transferases are present in most tissues, with very high concentrations in liver.Conjugation reactions can be divided into two types:• (Michael) addition reactions = glutathione is added to an activated double bond or strained ring system• Displacement reactions = glutathione displaces an electron withdrawing group (esp. halides)Cellular DistributionCytosolic (>95% of total) Glutathione S-transferases: 7 classes (with multiple subunits within each class)• alpha (i.e., GSTA). Humans 4 subunits, rats at least 5 subunits• mu GSTM. Humans 6 subunits, rats 6 subunits• pi GSTP• Theta• Kappa•Sigma•ZetaMicrosomal (<5% of total): distinct from cytosolic transferasesdiethyl fumarate2 5web.indstate.edu/thcme/mwking/glutathione.gifγ- glutamylcysteine synthetase(glutamate cysteine ligase)Glutathione biosynthesisglutathione synthetase(b.2) Decrease tissue GSH concn. by inhibition of GSH synthesisglutamate + cysteine yield glutamylcysteine (catalyzed by gamma-glutamylcysteinesynthetase) Inhibitor: BSO=Buthionine sulfoximine (also by Methionine sulfoximine)Inhibitors of glutathione biosynthesisL-2-oxothiazolidine-4-carboxylate (“2-oxo”)2-Oxo readily enters cells and is converted intracellularly to cysteineEffects of Altered GSH Concn on Hepatic Toxicity:Gillette and coworkersTwo examples have been extensively studied:AcetaminophenBromobenzene (metabolized via arene oxide intermediate)Both compounds exert their cellular toxicity – liver – by conversion by the cytochrome P.450 dependent mixed function oxidase todifferent reactive metabolites which then react covalently with tissue macromolecules.For the same toxicant – namely acetaminophen – what effect does the tissue concnof GSH have on the extent of covalent reaction of the metabolite to hepatic proteins.Two types of experiments:(measurements at 120 min. after acetaminophen dosage in mice)*Threshold – normal cell has excess GSH1 hour after administration of single dosage level (750 mg/kg mice) of acetaminophenAt any dosage, 2-15% of acetaminophen administered to animals is transformed by the cytochrome P-450 dependent MFO to a chemically reactive metabolite.At low – or therapeutic – dosages, virtually all of the reactive metabolite is converted to a glutathione conjugate that is ultimately excreted as a mercapturic acid derivative. But at high dosages of the drug, the GSH in liver is decreased to such an extent that the reactive metabolite can no longer be completely inactivated by GSH and a portion of the metabolite becomes covalently bound to liver proteins.25%15%31%22%47%39%James et al. Toxicol Appl Pharmacol. 1993 Feb;118(2):159-68Control 34±4James et al. Toxicol Appl Pharmacol. 1993


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