Disposition of toxic compounds and Its Metabolic reaction

1. Disposition of toxic compounds and Its Metabolic reaction Toksikologi Lab. Biokimia Nutrisi

2. Terminology Toksikologi Lab. Biokimia Nutrisi TOXIC SYMPTOM It is any feeling or sign indicating the presence of a poison in the system. TOXIC EFFECTS It refers to the health effects that occur due to exposure to a toxic substance. TOXICITY The adverse effects that a chemical may produce.

3. Terminology Toksikologi Lab. Biokimia Nutrisi Cumulative Effects Over a period of time, the material is only partially excreted and the remaining quantities are gradually collected. The retained toxic compound accumulates and becomes great enough to cause adverse effect

4. Various interactions of chemical substances Additive Combined effect of two chemicals is equal to the sum of the effects of each (2+3=5). Synergistic Combined effect of two chemicals are much greater than the sum (2+2=20). Potentiation One substance does not have a toxic effect on a certain organ or system but when added to another chemical is already toxic. Antagonism Two chemicals interfere with each others in actions.

5. Chemical substances need to achieve an adequate concentration in their target tissues. If the concentration of the toxic chemical remains low in the target organ of toxicity, little or no toxicity will results, whereas if high concentrations are attained, toxicity will result. The two fundamental processes that determine the concentration of a chemical substance at any moment and in any region of the body are: – translocation of chemical molecules, – biotransformation by metabolism of chemical substance and other processes involved in elimination of chemical substance. Toksikologi Lab. Biokimia Nutrisi

6. Absoption Distribution Metabolism Excretion There are 4 phases are interrelated : Absorption Distribution Metabolism Excretion Toksikologi Lab. Biokimia Nutrisi

7. Absorption • is the process of entry of chemical substance from site of exposure into systemic circulation. • It is the first step in the toxicokinetics of a chemical substance. • If the fraction of the chemical substance absorbed in low or the rate of absoption is low, then only a low concentration of the chemical in the target organ may be obtained and thus no toxicity. • The skin, lung, GIT are the main barriers that separate humans or animals from toxic substances. • Chemical substances must cross one of these barriers to exert adverse effect on the body and then pass through various cell membrane Toksikologi Lab. Biokimia Nutrisi

8. Routes of Absorption Toxicants access the body through many absorption routes, spilling into lymphatic and vascular compartments The intravenous route allows complete and immediate penetration, whereas other routes allow delayed and incomplete absorption. Different routes of exposure (inhalation, cutaneous, ingestion) may result in different amounts of toxicants circulating in the bloodstream, as well as various levels of toxicity. Injection > Inhalation > Contact on mucosa > Ingestion Toksikologi Lab. Biokimia Nutrisi

9. Transport of toxicant Toksikologi Lab. Biokimia Nutrisi

10. Transport systems : • Filtration through pores • small molecules may pass through membrane pores • occur down concentration gradient • eg : ethanol, urea • Passive diffusion through the membrane phospholipid • concentration gradient • lipid soluble and non-ionized compound • Active transport • require specific membrane carrier and energy • inhibited by metabolic poisons • saturated at high substrate concentration • specific for endogenous and nutrient substances, eg : fluorouracil Toksikologi Lab. Biokimia Nutrisi

11. Transport systems (continued) • Facilitated diffusion • require specific membrane carrier, but no energy • a concentration gradient • saturated by high substrate concentration • transport of glucose from the cells of the intestine into bloodstream • Phago/pinocytosis • Invagination of the membrane to enclose a particle or droplet • Insoluble substances eg : uranium and asbestos are absorbed into the lungs Toksikologi Lab. Biokimia Nutrisi

12. Distribution • The two Circulatory Systems are by far the most powerful distributors of toxicants throughout the body are • Lymphatic Circulation • Red Blood Cells trapped in fibrin clot. Toksikologi Lab. Biokimia Nutrisi

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14. Different major steps in the overall process leading to a toxic response Toksikologi Lab. Biokimia Nutrisi

15. Cell Membrane Toxicity • When toxic substances released in extracellular fluids rapidly meet cell membranes. • Alterations to the membranes come from altering the state of membrane receptors (neurotransmission, hormones, cytokines), • i.e. • dissolving or altering the lipid matrix, or from binding or cross linking membrane protein. • inhibits the cell membrane’s Na+/K+ pump (ATPase enzyme), eliminating its electrical polarization. • expand lipid membranes and increase their fluidity and permeability. • Oxidation of membrane lipids • Disturbing cytoplasm Ca that leading toloss of cell functions and cell death Toksikologi Lab. Biokimia Nutrisi

16. Cell Metabolism Toxicity (1) blocking one pathway usually leaves alternatives for cells to compensate with, and (2) cells usually have some reserve which allows them to pull through brief episodes of intoxication Toksikologi Lab. Biokimia Nutrisi

17. Storage • Storage is a type of bioaccumulation. Tissues and animals may bio-accumulate toxic halogenated defenses from compounds they synthesize or from external sources such as their diet • Site of storage • Bone • Fat : Body fat is a storage compartment that varies in size, depending on the individual. In dieting, autointoxication can occur as toxic substances are released into the blood. • Resulting Distributions for Lead and Dioxin body tissue distributions may vary substantially between toxicants. Hair and nails have very high concentrations of lead, and are often used to document lead intoxication. Toksikologi Lab. Biokimia Nutrisi

18. METABOLIC REACTIONS OF TOXIN AND TOXICANT COMPOUNDS (biotransformation) Toksikologi Lab. Biokimia Nutrisi

19. Metabolism (biotransformation) refers to the processes by which foreign chemicals are structurally altered by enzymatic reactions. • Theliverhas generally the highest capacity for metabolism of many types of foreign chemicals and most of the enzymatic reactions which convert lipophilic substances to hydrophilic conjugates. • Lipophilic substances entering the body would accumulate and remain there for a long time. Toksikologi Lab. Biokimia Nutrisi

20. Foreign compound metabolism may occur in most tissues of the body, with lung, kidney and intestines usually having intermediate capacities and skin, gonads, placenta and adrenals having fairly low capacities. • Many of the detoxifications reactions occur within the endoplasmic reticulum of individual cells. • most proteins are actually enzymes agents that facilitate the transformation of one chemical into another. Toksikologi Lab. Biokimia Nutrisi

21. Biochemical Transformations Systemic poisons in the body undergo : • biochemical processes that increase or reduce their toxicities, or change toxicants to forms that are readily eliminated from the body. Biotransformationrefers to changes in xenobiotic compounds as a result of enzyme action. Detoxication: the body metabolizes xenobiotic compounds in ways that usually reduce toxicity and facilitate removal of the substance from the body. Toxication or activation: nontoxic substances are metabolized to toxic ones or by which toxicities are increased by biochemical reactions. Toksikologi Lab. Biokimia Nutrisi

22. Pathways of xenobiotic species prior to their undergoing any biochemical interactions that could lead to toxic effects Toksikologi Lab. Biokimia Nutrisi

23. Compounds with a high degree of polarity, such as relatively ionizable carboxylic acids, are less likely to enter the body system and, when they do, tend to be quickly excreted. • Volatile compounds, such as dichloromethane or diethylether, are expelled so quickly from the lungs that enzymatic metabolism is less • nonpolar lipophilic compounds, (relatively less soluble in aqueous biological fluids and more attracted to lipid species, are resistant to enzymatic attack. For instan polychlorinated biphenyls (PCBs) tend to bioaccumlate in lipid tissue. Toksikologi Lab. Biokimia Nutrisi

24. Process biotransformation pathways Toksikologi Lab. Biokimia Nutrisi

25. Phase-I Biotransformation • catabolic (breakdown) enzymatic reactions • Increase polarity or water solubility by insertion, addition or exposure of reactive groups, initiating the conversion of the molecule: a polar group is either unmasked or added • Reaction typical are oxidation, reduction and hydrolysis. • The reactions are carried out by enzymes in microsomes and in the endoplasmic reticulum. • The major system is cytochrome P-450, an array of (7700 known distinct sequences) mono-oxygenase enzymes in the smooth endoplasmic reticulum. Fe++ acts as a source of electrons and oxygen. The “450” (nm) refers to the bluish color of the enzymes. Toksikologi Lab. Biokimia Nutrisi

26. Phase-I Biotransformation • The role of the mono-oxygenases is the oxidation or reduction of existing groups present on the molecule or the insertion of molecular oxygen into aromatic rings to form oxepin or benzene oxide • Another group of Phase I enzymes includes the hydrolases (esterases, proteases, lipases), their function being to hydrolyze peptide and ester bonds of a wide variety of chemicals to expose polar carboxyl, and hydroxyl or amino groups. • These enzymes sometimes transform aromatic compounds into epoxides (R-O-R), compounds significantly more toxic (mutagenic) than their parents. Toksikologi Lab. Biokimia Nutrisi

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28. Phase-II Biotransformation • binding of a substrate produces a conjugation productthat normally is less toxic than the parent xenobiotic compound or its phase I metabolite and more readily excreted from the body. • enzymatic reactions also occur in microsomes and mitochondria, but mostly the enzymes are cytosolic enzymes • The reaction are transfer covalently normal body constituents (sulfate, glutathione, acetyl/glycine, methyl, glucuronyl, amino acids as well as ornithine) to reactive groups of xenobiotic. Toksikologi Lab. Biokimia Nutrisi

29. PHASE I REACTIONS • adds a functional group to a hydrocarbon chain or ring or modifies one that is already present. • Oxidation of C, N, S, and P is most important • Reduction may occur on reducible functionalities by addition of H or removal of O. • Hydrolysis processes require the xenobiotic compound have a hydrolyzable group. Toksikologi Lab. Biokimia Nutrisi

30. I.1. Oxidation Reactions • Monooxidationsoccur with O2 as the oxidizing agent, one atom of which is incorporated into the substrate, and the other going to form water. • The key enzymes of the system are the cytochrome P-450 enzymes, it found most abundantly in the livers of vertebrates, reflecting the liver’s role as the body’s primary defender against systemic poisons, and in the kidney, ovaries, testes, and blood. The presence of this enzyme in the lungs, skin, and gastrointestinal tract may reflect their defensive roles against toxicants. Toksikologi Lab. Biokimia Nutrisi

31. Epoxidationconsists of adding an oxygen atom between two C atoms in an unsaturated system. Both of the epoxidation reactions shown below have the effect of increasing the toxicities of the parent compounds, a process called intoxication. Toksikologi Lab. Biokimia Nutrisi

32. I.2. Hydroxylation • The attachment of –OH groups to hydrocarbon chains or rings. • Hydroxylation can follow epoxidation, as shown by the following rearrangement reaction for benzene epoxide: Toksikologi Lab. Biokimia Nutrisi

33. I.3. Epoxide Hydration • The addition of H2O to epoxide rings, • Formation of a dihydrodiol by hydration of epoxide groups can be an important detoxication process in that the product is often much less reactive to potential receptors than is the epoxide. Toksikologi Lab. Biokimia Nutrisi

34. Epoxidation and hydroxylation of benzo(a)pyrene (left) to form carcinogenic benzo(a)pyrene 7,8-diol-9,10-epoxide (RALAT) Toksikologi Lab. Biokimia Nutrisi

35. I.4. Oxidation of Noncarbon Elements • The oxidation of nitrogen, sulfur, and phosphorus is an important type of metabolic reaction in xenobiotic compounds. It can be an important intoxication mechanism by which compounds are made more toxic. Toksikologi Lab. Biokimia Nutrisi

36. I.5. Alcohol Dehydrogenation • Produce aldehydes from primary alcohols that have the –OH group on an end carbon and produce ketones from secondary alcohols that have the –OH group on a middle carbon. • This is an important detoxication process because aldehydes are lipid soluble and relatively toxic, whereas carboxylic acids are more water soluble and undergo phase II reactions leading to their elimination. Toksikologi Lab. Biokimia Nutrisi

37. I.6. Metabolic Reductions • Reductions are carried out by reductase enzymes; for example, nitroreductase enzyme catalyzes the reduction of the nitro group. • Reductase enzymes are found largely in the liver and to a certain extent in other organs, such as the kidneys and lungs. • Most reductions of xenobiotic compounds are mediated by bacteria in the intestines, the gut flora. • Intestinal flora are known to mediate the reduction of organic xenobiotic sulfones and sulfoxides to sulfides: Toksikologi Lab. Biokimia Nutrisi

38. Functional Groups That Undergo Metabolic Reduction Toksikologi Lab. Biokimia Nutrisi

39. I.7. Metabolic Hydrolysis Reactions • Hydrolysisinvolves the addition of H2O to a molecule accompanied by cleavage of the molecule into two species. • Hydrolysis is a very important aspect of many xenobiotic compounds, such as pesticides (esters, amides, or organophosphate esters). Toksikologi Lab. Biokimia Nutrisi

40. I.8. Metabolic Dealkylation • Many xenobiotics contain alkyl groups, such as the methyl (–CH3) group, attached to atoms of O, N, and S. An important step in the metabolism of many of these compounds is replacement of alkyl groups by H, • These reactions are carried out by mixed-function oxidase enzyme systems. • Reaction for the plant systemic insecticide demeton: Toksikologi Lab. Biokimia Nutrisi

41. Metabolic dealkylation reactions shown for the removal of CH3 from N, O, and S atoms in organiccompounds. Toksikologi Lab. Biokimia Nutrisi

42. I.9. Removal of Halogen • Many xenobiotic compounds that contain covalently bound halogens (F, Cl, Br, I) is the removal of halogen atoms (dehalogenation). • This may occur by reductive dehalogenation,in which the halogen atom is replaced by hydrogen,or two atoms are lost from adjacent carbon atoms, leaving a carbon–carbon double bond. Toksikologi Lab. Biokimia Nutrisi

43. Oxidative dehalogenationoccurs when oxygen is added in place of a halogen atom, Toksikologi Lab. Biokimia Nutrisi

44. PHASE II REACTIONS OF TOXICANTS • Also known as conjugation reactionsbecause they involve the joining together of a substrate compound with another species that occurs normally in (is endogenous to) the organism. • This can occur with unmodified xenobiotic compounds, xenobiotic compounds that have undergone phase I reactions, and compounds that are not xenobiotic species. Toksikologi Lab. Biokimia Nutrisi

45. The substance that binds to these species is called an endogenous(present in and produced by the body) conjugating agent. • The conjugation product is usually less lipid soluble, more water soluble, less toxic, and more easily eliminated than the parent compound. Toksikologi Lab. Biokimia Nutrisi

46. Overall process of conjugation that occurs in phase II reactions. Toksikologi Lab. Biokimia Nutrisi

47. II. 1. Conjugation by Glucuronides • Glucuronidesare the most common endogenous conjugating agents in the body. They react with xenobiotics through the action of uridine diphosphate glucuronic acid (UDPGA). • This transfer is mediated by glucuronyl transferase enzymes. These enzymes occur in the endoplasmic reticulum, where hydroxylated phase I metabolites of lipophilic xenobiotic compounds are produced. Toksikologi Lab. Biokimia Nutrisi

48. A generalized conjugation reaction of UDPGA with a xenobiotic compound can be represented as the following: Toksikologi Lab. Biokimia Nutrisi

49. II.2. Conjugation by Glutathione (GSH) • The importance of glutathione in reducing levels of toxic substances can be understood by considering that loss of H+ from –SH on glutathione leaves an electron-rich –S– group (nucleophile) that is highly attractive to electrophiles. • Electrophiles are important toxic substances because of their tendencies to bind to nucleophilic biomolecules, including nucleic acids and proteins. Such binding can cause mutations (potentially cancer) and result in cell damage. Toksikologi Lab. Biokimia Nutrisi

50. Glutathione conjugate of a xenobiotic species (HX–R), followed by formation of glutathione and cysteine conjugate intermediates (both of which may be excreted in bile) and acetylation to form readily excreted mercapturic acid conjugate. Toksikologi Lab. Biokimia Nutrisi