DETOXIFICATION

DETOXIFICATION

1. Detoxification Gandham.Rajeev Email:[email protected]

2. Detoxification & Biotransformation of Xenobiotics Xenos Xenobiotic Compound that is foreign to body Stranger

3. Introduction As part of normal metabolism, body produces toxins which have to be eliminated. Humans are constantly exposed to exogenous & endogenous toxins. Biotransformation is the process whereby a substance is changed from one chemical to another by a chemical reaction in body.

4. Definition: Detoxification is the process by which toxic substances are converted into more soluble & less toxic substances, which are mainly eliminated through urine & bile. Takes place in the “liver”. The compounds to be detoxified include Drugs – antibiotics, cardiac drugs, steroids, etc.

5. Carcinogens – food dyes, preservatives, artificial sweeteners, alcohols, chemicals & cosmetics etc.

6. Why metabolize? Many endogenous & Xenobiotics are lipophilic . They can easily cross lipid bilayers & transported by lipoproteins. Metabolism of endogenous compounds & xenobiotics allows organisms to convert lipophilic compounds to more water soluble forms which facilitates excretion Many xenobiotic compounds contain aromatic rings & heterocyclic ring structures, that we are unable to degrade or recycle. These structures are hydrophobic

7. Metabolism of foreign compounds occurs as a results phase-I & phase-II reactions. Phase-I reactions are Oxidation Reduction Hydrolysis Phase II is conjugation reactions of phase I compounds. Direct conjugation can also occur.

8. Enzyme systems The cytochrome P450 is involved. Approximately 50% of the drugs are metabolized by isoforms of cytochrome P 450 Use of O 2 that one atom of oxygen enters R-OH and one atom enters water This dual fate of Oxygen accounts for former naming of mono- oxygenases as mixed function oxidases

9. Conjugating agents Conjugating agent Active form Glucuronic acid UDP- glucuronic acid Sulfate PAPS Cysteine Glutathione Acetic acid AcetylCoA

10. Phase I 1.Oxidation 2.Reduction 3.Hydrolysis

11. Phase I vs. Phase II Metabolism Phase I metabolism adds functional group on the molecule & increases water solubility. Phase II metabolism adds an endogenous substrate to produce a water-soluble conjugate & is easily excreted. Phase I activity is located in microsomal fraction of the cell. Phase II activity is located in the cytosolic fraction of the cell.

12. Organs Active in Metabolism Liver Kidneys Gastrointestinal tract Skin Testes Ovaries Adrenals Placenta

13. Phase-I Detoxification by oxidation: Foreign substances - alcohols, aldehydes , amines, anilides , aromatic hydrocarbons & certain drugs are destroyed in the body by oxidation. It occurs predominantly in liver & kidney. CYT P450, ADH, Aldehyde DH & mono amine oxidase are involved.

14. Oxidation Oxidation of Alcohols: These are oxidized to corresponding acids. CH3-CH2-OH Ethyl alcohol NAD + NADH + H + CH3-CHO Acetaldehyde Benzyl alcohol Benzoic acid Ethanol Acetic acid Methyl alcohol Formic acid

15. Oxidation of Aldehyde : Oxidized to corresponding acids. CH3-CHO Acetaldehyde NAD + -CHO -COOH Benzaldehyde Benzoic acid NADH + H + CH3-COOH Acetic acid NAD + NADH + H +

16. Detoxification of Amines: These are oxidized to corresponding acids. Benzylamine Benzoic acid + urea Chloral Trichloroacetic acid Aromatic hydrocarbons: These are oxidized to phenols or phenolic compounds, which are then conjugated with sulphuric & glutamic acids.

17. Anilides : These are oxidized to corresponding phenols Acetanilide Aminophenol Drugs: Chloral is partly oxidized to trichloroacetic acid, which excreted as its salts. Sulfur compounds: Organic sulfur is oxidized to sulfuric acid.

18. Reduction Certain aldehydes e.g., chloral, undergoes reduction to form corresponding alcohol, which is then conjugated with D- glucuronic acid & excreted as corresponding glucuronides . Chloral Trichloroethanol + D- glucuronic acid (excreted as corresponding glucuronides )

19. OH I -NO 2 I NO 2 O 2 N- OH I -NH 2 I NO 2 O 2 N- H 2 O Picric acid Picramic acid Reduction

20. Aromatic nitro compounds, e.g., p- nitrobenzaldehyde is reduced to corresponding amines & excreted after conjugation 3. Hydrolysis The hydrolysis of bonds - ester, glycoside & amide. Usually occurs in liver.

21. Hydrolysis Acetanilide Atropine Procaine Aspirine Salicylic acid + Aspirin acid Aniline + Acetic acid Tropic acid + tropine PABA + Diethylaminoethanol

22. Phase – II Conjugation Reactions Definition: It is a process by which the foreign molecules or its metabolites are coupled with a conjugating agent & converted to soluble, nontoxic derivatives & easily excreted in urine. Features: Conjugating agents are available in the body & some of them are synthesized in the body.

23. D- glucuronic acid Certain amino acids as glycine , cysteine . Conjugation occurs mainly in liver & some extent in kidney Conjugation produces less toxic, more soluble compounds which are excreted. . Conjugation occurs independently or it can follow oxidation, reduction or hydroxylation

24. Types of Conjugation 1.Methylation 2.Acetylation 3.Conjugation with Sulfuric acid 4.Conjugation with D- glucuronic acid 5.Conjugation with Amino acids 6.Conjugation with Glutamine 7.Conjugation with Glutathione

25. Methylation Detoxification by methylation is limited Usual methyl donor is SAM (active methionine ) Methylation of heterocyclic N- atom compounds of the pyrimidine & Quinoline types. e.g , Nicotin amide -CONH2 -CONH2 CH3 N Nicotinamide N Methyl Nicotinamide SAM

26. Acetylation Acetyl coA , takes part in conjugation reactions Conjugation with acetic acid occurs only with aromatic amino groups Sulpha drugs are conjugated by acetylation & excreted as acetylated derivatives. Sulfanilamide Acetyl CoA PABA Acetyl CoA Acetylated sulfanilamide Acetyl derivative of PABA Acetylation is catalyzed by acetyl transferase .

27. Conjugation with sulfuric acid PAPS is the sulfate group donor Sulfuric acid is used for detoxification of phenolic & hydroxyl groups Substances like phenol, cresol, indole are formed in the gut by the action of intestinal bacteria are absorbed & transported to liver, where they are conjugated with sulfate to form Ethereal sulfates, which are excreted in urine, less toxic & more acidic.

28. Phenol Active sulfate OH I O II O-S-O I II O Phenyl sulfuric acid Conjugation with sulfuric acid

29. Conjugation with D- glucuronic acid It is most important & commonest detoxification process. D- glucuronic acid is participated in this reaction, as its active form UDP- glucoronic acid which is formed in uronic acid pathway

30. Compounds conjugated with glucuronic acid Bilirubin to form bilirubin diglucuronide Aromatic acids, e.g. benzoic acid Phenols & other secondary & tertiary aliphatic alcohols. Certain drugs like morphine, menthol, pyramidon , acetalinide , sulfa pyridine, etc. Antibiotics like chloramphenicol

31. Hormones: Thyroid hormones & derivatives of steroids. Glucuronic acid form two types of linkages An ether linkage, e.g. in phenyl glucuronide An ester linkage, e.g. in benzoyl glucuronide Formation of glucuronides play an important role in detoxification of exogenous & endogenous compound & their excretion as corresponding glucuronides .

32. Conjugation with Amino acids Glycine combines with harmful substances - aromatic carboxylic acids to form harmless derivatives which are excreted in urine. Ex. Benzyl Co A is conjugated with glycine to form hippuric acid. Benzyl CoA + Glycine Hippuric acid Nicotinic acid + Glycine Nicotinuric acid

33. L- Cysteine : Aromatic compounds are conjugated with L- cysteine in the presence of acetic acid to form mercapturic acids. Bromobenzene + L- cysteine + acetic acid Bromophenyl mercapturic acid Bromobenzene + L- cysteine + acetic acid Naphthyl mercapturic acid

34. Conjugation with Glutamine Glutamine conjugates phenyl acetic acid to form phenylacetyl glutamine & excreted in urine Accounts for mousy odour of urine in PKU. Phenyl acetic acid + Glutamine Phenyl acetyl Glutamine

35. Conjugation with Glutathione It is a tripeptide . Potentially toxic electrophilic xenobiotics (carcinogens) are conjugated to the nucleophilic GSH Glutathione S- transferase is involved. Present in liver cytosol & other tissues. Exhibit different substrate specifities

36. P 450 = absorption peak at 450 nm The cytochrome P450 contain large group of enzymes. CYPs are major enzymes involved in drug metabolism. The most common reaction catalyzed by cytochrome P450 is a monooxygenase reaction (Oxidation), e.g. insertion of one atom of oxygen into an organic substrate (RH) while the other oxygen atom is reduced to water. RH + O 2 + 2H + + 2e – → ROH + H 2 O Role of cytochrome P-450 enzymes

37. Cytochrome P450 Activity Requires Oxygen NADPH Embedded in lipid bilayer next to cytochrome P450 oxido-reductase . Monooxygenase is also called as mixed function oxidase , is associated with microsomes.

38. About 150 isoforms are present. CYP denotes a cytochrome P450 CYP1A1 CYP=CytochromoP450 1=Family A=Subfamily 1=is the first individual member of that subfamily. Isoforms of cytochrome P450

39. Properties of human cytochrome P450 Involved in phase I of the metabolism. Involved in the metabolism of many endogenous compounds (e.g. steroids). All are hemoproteins . Exhibit broad substrate specificity. Extremely versatile catalysts, catalyzing about 60 types of reactions. They catalyze reactions involving introduction of one atom of oxygen into the substrate & one into water.

40. Their hydroxylated products are more water soluble & facilitating their excretion. Present in liver & also in intestine, brain & lungs. Located in smooth endoplasmic reticulum or in mitochondria In some cases their products are mutagenic or carcinogenic Many have a molecular mass of about 55kDa. Their activities may be altered in diseased tissues (e.g. cirrhosis), affecting drug metabolism.

41. References Harper’s Biochemistry – 25 th edition Medical Biochemistry – AR Aroor Text book of Biochemistry – DM Vasudevan

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