Biochemical Respond Against Xenobiotic

1. Biochemical Respond Against Xenobiotic

2. Introduction • Xenos: stranger, synonims: biotransformation, drug metabolism • Xenobiotic: is compound that foreign to the body that under normal circumstances is not required by the living body • Examples: pharmaceuticals, pesticides, environmental pollutants (Pb, CO), industrial chemicals, preservatives, colorings and flavorings in food products • Metabolism of xenobiotics are basic to a rational understanding of pharmacology, therapeutics, pharmacy, toxicology, management of cancer or drug addiction.

3. Xenobiotika compounds can enter the body through: • Mouth (food, drugs) • Respiration (cigarette smoke, fumes) • Skin (pesticide poisoning on farmers) • Intra venous • Principal classes of xenobiotics: • Drugs (antibiotics, analgesic, antipyretic, supplements • Carcinogen (food dyes, preservation, alcohol, nitrosamines, artificial sweetenes) • Pollutans/environmental chemical (manufactured environmental chemical)

4. Xenobiotics Absorption Oral Intestine Topical Skin Xenobiotics IV Blood IM, SC, IP Membranes Inhalation Lung

5. Effect of xenobiotics: • the expected effects (the therapeutic effects of drugs / cure or relieve symptoms of disease) • unexpected effects (side effects and toxic effects of drugs) • Through the process of metabolism and excretion processes, the body is able to eliminate all influences that arise after a change in chemical structure • Metabolism xenobiotics holds important meaning in the process of elimination xenobiotics

6. Metabolism of Xenobiotics • Xenobiotics metabolism: • Mechanism of elimination of foreign and undesirable compounds from the body • Control of levels of desirable compounds • Biochemical alteration in the body • Not detoxication reaction, because: • Pro drugs • Pro carcinogenesis • Metabolism increase biologic activity and/or toxicity

7. Divided in 2 phases • Phase 1: hydroxylation catalyzed by members of class of enzymes: monooxygenase or cytovhrome P450s • Phase 2: conjugation the hydroxylated or other compounds produced in phase 1 by spesific enzymes, converted to various polar metabolites by conjugation with glucoronic acid, sulfate, acetate, glutathion or certain amino acids, or by methylation

8. Phase I • Biochemically alter the xenobiotic to change its biologic effect • Location: • Liver (membranes of ER, cytoplasm) • Other tissues (lungs, intestine, skin, kidneys) • Enzymes: • Monooxygenases (hydroxylases, cytochrome P450, Mixed Function Oxidase/MFO • Property of enzymes: • Metabolism of endogenic substances, broad substrate specificity, inducibility

10. Reactions: Hydrolysis, oxidation, reduction RH + O2 + NADPH + H+  R-OH + H2O + NADP lipophilic hydrophilic active inactive inactive active • Results: • Lowering their toxicity • Increasing their toxicity • Bioactivation some xenobiotics (ex. procarcinogen danger of cell, body damage) • Increasing their water solubility

11. Lipophilic Non polar, hydrophobic Hydrophilic Polar • Poorly soluble in water • Need a blood transporter (albumin) • Freely diffuse through membranes • Can be stored in membranes • Slowly eliminated from the body • Water soluble • Difficult transport through membranes • Rapidly eliminated with the urine

12. Cytochrome P450 • Most versatile biocatalyst • Metabolizes > 50% of all drugs and chemical in the body • Membrane-bound protein • Principal component of active site is the heme moiety • NADPH not NADH is involved in the reaction of mechanism

13. Factor affecting metabolism by Cytochrome P450: • Diet and nutrition  different nutrient and elemental deficiencies • Hormonal  different hormones affect metabolism • Age and sex  metabolism slower in neonates and elderly • Genetic  some pathway exhibit genetic variation • Pathological states  diseases involving the liver or kidney lead to alteration in absorption, distribution and excretion of xenobiotics

14. Inducers of cytochrome P450 synthesis: • Drugs: barbiturates, steroid, ethanol, nicotine • Industrial chemical: alcohol, chloroform • Polyaromatic hydrocarbon: benzopyrenes • Inhibitors of cytochrome P450 synthesis: • Competitive binding to and metabolism by cytochrome P450 • Inhibition of synthesis of heme or cytochrome P450 • Inactivation or destruction of cytochrome P450, or destruction of the ER by various agent

15. Induction of cytochrome P450  important clinical implication  biochemical mechanism of drug interaction, example: Patient is taking warfarin (metabolized by CYP2C9) and phenobarbital (an inducer P450) • Level of CYP2C9 will be elevated 3-4 fold after 5 days • Warfarin will be metabolized much more quickly  dosage become inadequate  dose must be increased to be terapeutically effective

16. Example of a reactions catalyzed by cyt P450 The figure is from: Color Atlas of Biochemistry / J. Koolman, K.H.Röhm. Thieme 1996. ISBN 0-86577-584-2

19. Polymorphism: Natural variations in a gene, DNA sequence, or chromosome that have no adverse effects on the individual and occur with fairly high frequency in the general population Structure of human cytochrome P450 CYP2C9

20. Some important drug reactions due to mutant or polymorphic form of enzims or protein

22. Human CYP families and their main functions. Data adapted from (Gonzalez 1992, Nelson et al. 1996, White et al. 1997, Nelson 1999, Lund et al. 1999)

24. Phase II • Phase I derivates are made more polar (water soluble) through conjugation reaction • Location: • Liver (intestine mucosa, skin): ER, cytoplasm • Properties: • Need of an endogenic substance • Synthetic reactions • Energy consumption

25. Results: • Highly polar conjugates (increasing water solubility) • Decreased toxicity • Important reactions: • Enzymes: transferase

26. Conjugation Reactions • Glucuronidation • Endogen reactant: UDP glucuronic acid • Enzyme: UDP glucuronyl transferase • Location: microsome • Reactive site: OH, COOH, NH2, SH, C-C • Type of substrates: phenol, alcohols, carboxylic acids, hydroxylamines, sulfonamides • Examples: acetaminophen, nitrophenol

27. Sulfate conjugation • Endogen reactant: phosoadenosylphosphosulfate • Enzyme: sulfotransferase • Location: cytosol • Reactive site: NH2, OH • Type of substrates: phenol, alcohols, aromatic amines • Examples: aniline, phenol, acetaminophen • Acetylation • Endogen reactant: acetyl-CoA • Enzyme: n-Acetyltransferase • Location: cytosol • Reactive site: NH2, SO2NH2, OH • Type of substrates: amines • Examples: isoniazid, sulfonamides

28. Glutathione conjugation • Endogen reactant: glutathione • Enzyme: glutathione S-transferase • Location: cytosol • Reactive site: epoxides, organic halides, organic nitro compounds, unsaturated compounds • Type of substrates: epoxides, arene oxides, nitro groups, hydroxylamines • Examples: bromobenzene • Methylation • Endogen reactant: s-adenosyl-methionine • Enzyme: transmethylase • Location: cytosol • Reactive site: NH2, SH, OH • Type of substrates: phenols, amines, catecholamines • Examples: pyridine, histamine, epinephrine

31. Xenobiotics Drug Metabolism • Drugs that are active → phase 1 metabolism xenobiotik to change the active drug into inactive • Drugs that have not been active → phase 1 metabolism xenobiotik convert an inactive drug becomes active.

32. Alcohol METHANOL (CH3OH) • lower narcotic effect than ethanol • slower excretion from the body • metabolized by the same enzymes as ethanol • causes harder sickness (formaldehyde) • serious intoxication: 5 – 10 ml (lethal dose  30 ml) • no symptoms immediately after drunkenness (6 – 30 hours) • headache, pain in back, loss of sight • metabolic acidosis • therapy: ethanolemia  1 ‰ (1 - 2 days), liquids

33. Ethanol • A small molecule; both lipid and water soluble • Readily absorbed from the intestine by passive diffusion • Small percentage of ingested ethanol (0-5%) enters the mucosal cells of the upper GI tract (tongue, mouth, esophagus, and stomach) • The remainder enters the blood, of which 85 to 98% is metabolized in the liver, and only 2 to 10% is excreted through the lungs or kidneys

34. Distribution of ethanol in the body: • the equilibrium concentration of ethanol in a tissue depends on the relative water content of that tissue • ethanol is practically insoluble in fats and oils, although like water, it can readily pass through biological membranes • No plasma protein binding ethanol • Factors affecting ethanol absorption: • concentration of ethanol, passive diffusion (higher concentrationgreater absorption), blood flow at site of absorption(efficient blood flow greater absorption), rate of ingestion, food (presence of food in stomach retards gastric emptying, reduces absorption of ethanol)

35. Factors that determine the rate and route of ethanol oxidation in individuals include: • genotype  polymorphic forms of ADH and acetaldehyde dehydrogenase can greatly affect the rate of ethanol oxidation and the accumulation • Drinking history  the level of gastric ADH decreases and CYP2E1 increase with the progression from a naïve, to a moderate, a heavy and chronic consumer of alcohol • Gender  blood levels of ethanol after consuming a drink normally higher for women than men, because of lower levels of gastric ADH activity

36. Factors that determine the rate and route of ethanol oxidation in individuals include: • Quantity—The amount of ethanol an individual consumes over a small amount of time determines its metabolic route.

37. Metabolism of Ethanol • Metabolism occurs by two pathways • The first pathway comprises two steps • The first step, catalyzed by the enzyme alcohol dehydrogenase, takes place in the cytoplasm: • The second step, catalyzed by aldehyde dehydrogenase, takes place in mitochondria:

38. First Pathway • Ethanol consumption leads to an accumulation of NADH • High concentration of NADH inhibits gluconeogenesis by preventing the oxidation of lactate to pyruvate will cause the reverse reaction to predominate, and lactate will accumulate  the consequences may be hypoglycemia and lactic acidosis • The NADH glut also inhibits fatty acid oxidation, the excess NADH signals that conditions are right for fatty acid synthesis  triacylglycerols accumulate in the liver, leading to a condition known as "fatty liver"

39. Second Pathways • The ethanolinduciblemicrosomal ethanol-oxidizing system (MEOS) • Cytochrome P450-dependent pathway generates acetaldehyde and subsequently acetate while oxidizing biosynthetic reducing power, NADPH, to NADP+ • Because it uses oxygen, this pathway generates free radicals  damage tissues • Because the system consumes NADPH, the antioxidant glutathione cannot be regenerated  exacerbating the oxidative stress • Approximately 10 to 20% of ingested ethanol is oxidized through a microsomal oxidizing system (MEOS), comprising cytochrome P450 enzymes in the endoplasmic reticulum (especially CYP2E1)

40. Figure 1 Oxidative pathways of alcohol metabolism. The enzymes alcohol dehydrogenase (ADH), cytochrome P450 2E1 (CYP2E1), and catalase all contribute to oxidative metabolism of alcohol. ADH, present in the fluid of the cell (i.e., cytosol), converts alcohol (i.e., ethanol) to acetaldehyde. This reaction involves an intermediate carrier of electrons, nicotinamide adenine dinucleotide (NAD+), which is reduced by two electrons to form NADH. Catalase, located in cell bodies called peroxisomes, requires hydrogen peroxide (H2O2) to oxidize alcohol.

41. Liver mitochondria can convert acetate into acetyl CoA in a reaction requiring ATP • The accumulation of acetyl CoA has several consequences: • ketone bodies will form and be released into the blood, exacerbating the acidic condition already resulting from the high lactate concentration • If ethanol is consistently consumed at high levels, the acetaldehyde can significantly damage the liver  leading to cell death

42. Liver damage from excessive ethanol consumption occurs in three stages • the aforementioned development of fatty liver • alcoholic hepatitis groups of cells die and inflammation results • cirrhosis fibrous structure and scar tissue are produced around the dead cells • The cirrhotic liver is unable to convert ammonia into urea, and blood levels of ammonia rise  toxic to the nervous system  cause coma and death • Cirrhosis of the liver arises in about 25% of alcoholics, and about 75% of all cases of liver cirrhosis are the result of alcoholism

43. Alcoholic Liver Disease (ALD) is a term used to describe the spectrum of liver injury associated with acute and chronic alcoholism • The 3 stages of Alcoholic Liver Disease are: • Hepatic steatosis (fatty change) • Alcoholic hepatitis • Alcoholic cirrhosis • Interrelationships among stages of Alcoholic Liver Disease:

44. Alcohol, biochemistry and metabolism, Ghassan Hemased, MD

45. Alcohol, biochemistry and metabolism, Ghassan Hemased, MD

50. Potential alcohol-medication interactions involving cytochrome P450 enzymes in the liver (Alcohol Research and Health, 1999)