6 xenobiotics


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6 xenobiotics

  1. 1. Biotransformation of xenobiotics Department of Biochemistry (J.D.) 2011 1
  2. 2. Greek word ξένος [xenos] means strange• xenobiotics do not occur in the body• they enter body mainly with food or as medications• Chemical industry – produces synthetic compounds which do not occur in nature (plastics, pesticides, dyes, additives…)• Pharmaceutical industry – produces substances of synthetic and natural (plant) origin – do not occur in the body 2
  3. 3. Entry of xenobiotics into body• three principal entries: intestine, lungs, skin• epithelium barrier between blood (ECF) and tissues (ICF) – phospholipid bilayer• penetration of xenobiotic depends on its physical and chemical properties• hydrophobicity facilitates the transport through cell membrane 3
  4. 4. Entry of xenobiotic into cells• Simple diffusion – lipophilic substances, depends on concetration gradient (liver – freely permeable, big pores in cell membrane, the most affected in poisoning)• Facilitated diffusion – transporters• Active transport – primary, secondary• Endocytosis xenobiotics structurally similar with physiological substrates can utilize all available transport systems 4
  5. 5. Biotransformation of xenobiotics in cells• mostly in liver• I. Phase – predominantly hydroxylations, product may be still biologically active• II. Phase – conjugation, product usually inactive• products of biotransformations are more polar - they can be excreted from the body by urine and/or bile 5
  6. 6. Excretion of xenobiotics from cell• primary active transport – needs energy: ATP hydrolysis• special ATP-ases called ABC (ATP binding cassettes)• localized in cell membranes, export xenobiotics from cells into ECF• MRP (multidrug resistence proteins) – in increased expresion, they cause the resistance towards medicines 6
  7. 7. ABC = ATP binding cassettes• superfamily of transmembrane proteins, they have ATP-binding domain(s), substrate binding domain, and transmembrane domain(s)• after ATP binding, ABC can translocate a chemical species across membrane• ABC are located in cell membranes as well as in intracellular membranes• lipids, cholesterol, peptides, drugs, toxins etc. 7
  8. 8. Excretion of xenobiotics from body• chemically modified (more polar) xenobiotics are excreted by urine, bile  stool, or sweat• volatile substance by lungs• intestinal deconjugation and resorption sometimes occur - enterohepatic circulation• excretion into human milk 8
  9. 9. I. phase of biotransformation: examples of reactionsReaction Xenobiotic (example)Hydroxylation (P-450) (hetero)aromatic compounds (Ar-H  Ar-OH)Sulfooxidation dialkylsulfide (R-S-R)  sulfoxide (R-SO-R)Dehydrogenation alcohol / aldehyde hydrate  aldehyde / acidReduction nitrocompounds (R-NO2)  amines (R-NH2)Hydrolysis ester  acid + alcohol Reactions occur mainly in ER, some in cytosol Enzymes of I. phase are rather non-specific – advantage !! 9
  10. 10. Cytochrome P450 (CYP)• superfamily of heme enzymes (many isoforms)• can catalyze different reaction types, mainly hydroxylation• wide substrate specifity - advantage• can be induced and inhibited• occur in most tissues (except of muscles and erythrocytes)• the highest amount in the liver (ER)• exhibit genetic polymorphism ( atypical biotransformations) Abbreviation: P = pigment, 450 = wave length (nm) of a absorption peak after binding CO 10
  11. 11. Contributions of CYP isoforms to drug metabolism 4 % 1A2 1 % other 11 % 2C 52 % 3A4 30 % 2D6 2 % 2E1 11
  12. 12. Mechanism of CYP hydroxylation• the formation of hydroxyl group• monooxygenase: one O atom from O2 molecule is incorporated into substrate between C and H (R-H  R-OH )• the second O atom + 2H from NADPH+H+ give water R-H + O2 + NADPH + H+  R-OH + H2O + NADP+ 2 e- + 2 H+ 12
  13. 13. Components of cytochrome P450 + 2H ++ + FeNADPH + H FAD hem RH O2 + +++ NADP FADH 2 Fe hem R OH H 2O cyt. reduktasa cyt P-450cytochrome P450 contains three cofactors and two enzymes:• NADPH+H+, FAD, heme• NADPH:CYP reductase (separates 2 H  2 e- + 2 H+) ER• cytochrome P-450 (hydroxylase) 13
  14. 14. Detailed scheme shows reductive activation of O2 cyt P-450 A H substrate A-H substrát A H 3+ Fe e cyt P-450 cyt P-450 A H NADPH + H 3+ 2+ Fe Fe NADP O2 e 2H cyt P-450 A H 2+ Fe O2 cyt P-450 A H 2+ Fe O2 A OH hydroxylovanýhydroxylated product substrát H2 O 14
  15. 15. Hydroxylation by CYP450 occurs in endogenous and exogenous substrates• Endoplasmic reticulum: squalene, cholesterol, bile acids, calciol, FA desaturation, prostaglandins, xenobiotics• Mitochondria: steroidal hormones 15
  16. 16. Compare various hydroxylationsSubstrate Product Reagent Coreductant Other comp.Phenylalanine tyrosine O2 BH4 * -Xenobiotic xen-OH O2 NADPH+H+ FAD, hemeProline 4-OH-Pro O2 2-oxoglutarate Fe2+, ascorbateDopamine noradrenaline O2 ascorbate Cu2+* tetrahydrobiopterine 16
  17. 17. Main isoforms of human cytochrome P450 Various isoforms prefer different substrates, have different inducers and inhibitorsCYP Substratea Inducera InhibitoraCYP1A2 theophylline tobacco smoke erythromycinCYP2A6 methoxyflurane phenobarbital methoxsalemCYP2C9 ibuprofen phenobarbital sulfaphenazoleCYP2C19 omeprazole phenobarbital teniposideCYP2D6 codeine rifampicine quinidineCYP2E1 halothane alcohol disulfiramCYP3A4 diazepam phenobarbital grapefruita Examples from many possible compounds. 17
  18. 18. Inducers and inhibitors of CYP450• some xenobiotics induce the synthesis of CYP – the metabolic capacity of CYP is enhanced• if administered inducer + drug, both metabolized by the same CYP isoform  drug is metabolized faster  drug is less effective• some xenobiotics inhibit CYP• the most common isoform CYP3A4 metabolizes more than 120 different pharmaceutical drugs• inhibitors of CYP3A4 are e.g. macrolide antibiotics, grapefruit (furanocoumarins), ketoconazole• if administered inhibitor + drug  increased drug level  overdosing  side effects 18
  19. 19. Genetic polymorphism of CYP450 usual drug dose (ultra)rapidpoor metabolizer extensive metabolizer metabolizer most of populationhigher drug level normal response no / insufficient effect side effects clinical effect of drug intoxication 19
  20. 20. Example I. Phase of biotransformation of benzene hydroxylation hydroxylace (CYP 450) H O H 20
  21. 21. Example Biotransformation of polycyclic aromatic hydrocarbons (PAH) H2O HO O epoxid reactive epoxide OH dihydrodiol benzo[a]pyrene O interactions with DNA, mutations vazba na DNA, mutace tumours (kůže, lungs) nádory (skin, plíce) HO OH 21
  22. 22. PAH in environment• Industrial sources: combustion of fossil fuels (coal, petroleum oil, etc.), production of coke, asphalt ...• Non-industrial sources: forest fires, combustion of household rubbish, cigarette smoke …• Foods: fried, grilled, smoked, roasted foods, overheated fats and oils, burnt (singed) bread, pastry … 22
  23. 23. II. Phase of biotransformation• conjugation – catalyzed by transferases• synthesis = endergonic reaction, one of the reactants must be activated• xenobiotic after I. phase reacts with endogenous conjugation reagent• conjugate is more polar, less active, easily excreted by urine and/or bile (stool) 23
  24. 24. Conjugation reactions and reagentsReaction Reagent Group in substrateGlucuronidation UDP-glucuronate -OH, -COOH, -NH2Sulfation PAPS -OH, -NH2, -SHMethylation SAM -OH, -NH2Acetylation acetyl-CoA -OH, -NH2Sulfide formation glutathione Ar-halogen, Ar-epoxideAmide formation glycine, taurine -COOH 24
  25. 25. Biosynthesis of UDP-glucuronate P O CH2 HO CH2 HO CH2 O O UTP O OH OH OH HO OH HO O P HO O UDP OH OH OH glukosa-1-P UDP-glukosa UDP-glucose glukosa-6-P glucose 6-P glucose 1-P + O O NAD C H2O + O NAD OH glukosiduronáty(bis)glucosiduronates HO O UDP OH UDP-glukuronát UDP-glucuronate 25
  26. 26. UDP-glucuronate COO O OH OHO O O NH HO P O O O P N O O O O-glycoside bond O N-glycoside bond of ester type OH OH 26
  27. 27. Glucuronates are the most common conjugates• O-glycosides ether type (Ar-O-glucuronate, R-O-glucuronate) ester type (Ar-COO-glucuronate)• N-, S-glycosides• Substrates: aromatic amines, amphetamines, (acetyl)salicylic acid, drugs, flavonoids ...• Endogenous substrates: bilirubin, steroids 27
  28. 28. bilirubin bisglucosiduronate 28
  29. 29. Example Biotransformation of amphetamine amphetamine Phase I reaction Phase II reaction 4-hydroxyamphetamine 4-hydroxyamphetamine 4-O-glucosiduronate Phase I reaction Phase II reaction 4-hydroxynorephedrine 4-hydroxynorephedrine 4-O-glucosiduronate 29
  30. 30. PAPS phosphoadenosyl phosphosulfate NH 2Physiological sulfations: N O N OGlycosaminoglycanes S O O N Nheparine, dermatane sulfate, O Pkeratane sulfate, O O Ochondroitine sulfate etc.Sulfoglycosphingolipids O O OH(acidic glycolipids, sulfatides) P O O 30
  31. 31. Example Biotransformation of phenol hydroxylation hydroxylace (CYP 450) H OH conjugation konjugace O glucuronate glukuronát O SO3H sulfát 31
  32. 32. Glutathione – three functions -glutamyl-cysteinyl-glycine• Reductant = antioxidant (glutathione peroxidase)• Conjugation agent (glutathione transferase) endogenous substrates – leukotrienes• AA Transport into cells (-glutamyltransferase, GMT) 32
  33. 33. Glutathione (GSH) NH2 H O  N HOOC  N COOH  O CH2 H SHelectrophilic site R-X + GSH  R-SG + XH (R-X epoxides, halogenalkanes) nucleophilic group 33
  34. 34. Example R-SG sulfide is converted to mercapturic acids and excreted CoA-SH acetyl-S-CoA GSH Glu + Gly epoxide N-acetyl-S-substituted cysteine (mercapturic acid) 34
  35. 35. Methylations are involved in the inactivation of catecholamines MAO monoamine oxidase, COMT catechol-O-methyltransferase MAO O HO CH2 CH2 NH2 HO CH2 C - NH3 H dopamine HO HO dihydroxyphenylacetaldehyde O O COMT HO CH 2 C HO CH 2 C OH SAM OH O HO CH 3 homovanillic acid dihydroxyphenylacetic acidInactivation can proceed in the reverse order: first COMT, then MAO, product is the same. 35
  36. 36. Conjugation with amino acids (amide formation)• glycine, taurine• xenobiotics with -COOH groups• amide bond formation• endogenous example: conjugated bile acids 36
  37. 37. Toluene biotransformation CH3 CH2OH COOHtoluene benzylalcohol benzoic acid O glycine glycin O C C O OH NH CH2 C OH benzoic acid benzoová kys. hippurová kyselina hippuric acid (activated by CoA) (N-benzoylglycin) (N-benzoylglycine) 37
  38. 38. 38
  39. 39. Biotransformation of ethanol in liver (cytosol) H H alkoholdehydrogenasa alcohol dehydrogenase (AD) O H3C C O + NAD H3C C + NADH+ H H H acetaldehyd acetaldehyde aldehyddehydrogenasa acetaldehyde dehydrogenase (AcD) H H H NAD OH3C C O + H2O H3C C O H3C C - 2H  NADH + H+ OH OH aldehyd-hydrát acetaldehyde hydrate acetic acid octová kyselina 39
  40. 40. • Alcohol dehydrogenase (AD) – metalloenzyme (Zn), more isoforms, in liver, lungs, kidneys, intestine, and other tissues• some isoforms are less active in females• Acetaldehyde dehydogenase (AcD) – more isoforms, liver, cytosol and mitochondria 40
  41. 41. Alternative pathways for alcohol metabolismER:MEOS (microsomal ethanol oxidizing system, CYP2E1)CH3-CH2-OH + O2 + NADPH+H+  CH3-CH=O + 2 H2O + NADP+It is activated on higher alcohol levels (> 0.5 ‰)  increasedproduction of acetaldehydePeroxisomes: oxidation of ethanol by hydrogen peroxide, catalaseCH3-CH2-OH + H2O2  CH3-CH=O + 2 H2O 41
  42. 42. Metabolic consequences of EtOH biotransformation Ethanol AD, AcD AD MEOS part. soluble acetaldehyde excess of NADH in cytosol in membrane PL (hangover) reoxidation by pyruvate adducts with acetate proteins excess of lactate  acidosis nucleic acids toxic effects amines lack of pyruvate  hypoglycaemia on CNS acetyl-CoA various products FA/TAG synth. liver steatosis 42
  43. 43. Acetaldehyde reacts with biogenic aminesto tetrahydroisoquinoline derivatives (animal alkaloids) HO HO NH2 N HO - H2O HO H dopamine CH 3 H O C salsolinol CH3 6,7-dihydroxy-1-methyl-1,2,3,4- acetaldehyde tetrahydroisoquinoline Neurotoxin ? 43
  44. 44. Tests for detection of ethanol intakeLiver enzymes: GTM, AST, ALT, GMD, CHSFatty acids ethyl esters (FAEE) appear in the blood in 12 – 18 h after drinking and can bedetected even 24 h after alcohol in blood is no more increased. However, traces of FAEEs aredeposited in hair for months and may serve as a measure of alcohol intake.Ethyl glucosiduronate (EtG) increases in the blood synchronously with the decrease ofblood ethanol and can be detected (in the urine, too) after few days, even up to 5 days.Phosphatidyl ethanol (PEth) is present in the blood of individuals, who have been drinkingmoderate ethanol doses daily, in even 3 weeks after the last drink.Carbohydrate-deficient transferrin (CDT). In the saccharidic component of eachtransferrin molecules, there are 4 – 6 molecules of sialic acid. Drinking to excess disturbes theprocess of transferrin glycosylation so that less sialylated forms of transferrin (with only twoor less sialyl residues per molecule, CDT) are detected in blood during approximately 4weeks after substantial alcohol intake. 44
  45. 45. Per milles of alcohol in blood ‰ = per mille = 1/1000 malcohol (g)alcohol in blood (‰) = mbody (kg)  f Biological feature Males Females 0.67 (males) Total body water 60 – 67 % 50 - 55 % 0.55 (females) Total body fat 10 – 20 % 20 – 30 % 45
  46. 46. Oxidation of ethylene glycol proceeds stepwise with a number of intermediates CHO oxid. oxid. CH2OH CHO CHO COOH COOH oxid. oxid. glyoxal CH2OH CH2OH oxid. oxid. CHO COOH ethylenglykolethylene glykolaldehyd glycolaldehyde glyoxalic glyoxalová štavelová oxalic acid COOH kyselina kyselina acidglycol CH2OH glykolová kyselina glycolic acid in kidneys  calcium oxalate stones  renal failure 46
  47. 47. TobaccoSubstances nicotine, the products of incomplete combustioninvolved euphoria, psychical relaxation, increase of pulse rate, vasoconstriction, stimulates adrenaline release (silent stress),Effects increases salivary and gastric secretion, stimulates intestinal peristalsis (defecating effect of the first morning cigarette)Symptoms typical smell, yellow fingers and teethof abuse lung diseases (COPD*, cancer), heart attack,Risks erectile dysfunctions, premature wrinkles * chronic obstructive pulmonary disease 47
  48. 48. Nicotine is the principal alkaloid of tobacco 2 3 more basic 1 N pKB = 6,16 2 CH3 N 1less basicpKB = 10,96 3-(1-methylpyrrolidine-2-yl)pyridine 48
  49. 49. What happens during cigarette burning?• temperature about 900 C• dried tobacco undergoes incomplete combustion• very complicated mixture of products• nicotine partly passes to smoke, partly decomposes Cigarette box Nicotine: 0.9 mg/cig. Tar: 11 mg/cig. 49
  50. 50. Cigarette smoke contains• free base of nicotine – binds to receptors in the brain• CO – binds to hemoglobin to give carbonylhemoglobin (tissue ischemia)• nitrogen oxides – may generate reactive radical species• polycyclic aromatic hydrocarbons (PAH) (pyrene, chrysene, benzo[a]pyrene …), main components of tar they can attack and damage DNA, carcinogens• other substances (N2, CO2, HCN, CH4, esters …) 50
  51. 51. How to disclose a smoker?1. saliva test smoker’s saliva contains much higher level of thiocyanate than saliva of non-smoker, thiocyanate is generated from CN- → SCN- reaction with Fe3+ ions give red complex2. nicotine in urine3. minor tobacco alkaloids in urine (cotinine, nornicotine, anatabine, anabasine) 51
  52. 52. Example Biotransformation of nicotine N CH3 N nicotine N N OH H CH3 N N nornicotine 5-hydroxynicotine nicotine-N-glucuronate N O cotinine-N-glucuronate CH3 N cotinine 52
  53. 53. Biotransformations of selected drugsDrug Biotransformation MetaboliteCodeine demethylation morphine (active, another way)Bromhexin hydroxylation + demethylation ambroxol (active, the same)Paracetamol conjugation, oxidation conjugates (mostly inactive)Aspirin hydrolysis, hydroxyl., conjug. conjugates (inactive) 53
  54. 54. Bromohexin is the prodrug of an expectorant ambroxol N-demethylation hydroxylation bromohexin ambroxol (prodrug) (expectorant)Antitussic codeine (3-O-methylmorphine) is transformed slowly into morphine O-demethylation codeine morphine (antitussic) (analgesic, an addictive drug) 54
  55. 55. Acetaminophen (p-acetaminophenol, paracetamol) N-(4-hydroxyphenyl)acetamide prepared in 1893, common analgetic-antipyretic, overt the counter, without a prescription The amide bond is not hydrolyzed! oxidation of only a small part to cyt P450 N-acetyl-p-benzoquinoneimide (NAPQI), unless the conjugating capacity is exhausted~ 3 % excretedunchangedinto the urine if conjugation capacity CONJUGATION is limited, GSH unwanted side effects: – covalent bonding to proteins, – oxidation of –SH groups in enzymes, – depletion of GSH, – hepatotoxicity at 60 % as glucosiduronate overdosing 30 % as sulfate ester mercapturic acid 55
  56. 56. Acetylsalicylic acid (Aspirin) is an analgetic-antipyretic with antiinflammatory effect; over the counter, minute doses inhibit aggregation of blood platelets. acetylation of macromolecules (acetylation of COX inhibits the synthesis of prostaglandins)esterase UDP-glucuronate and O UDP salicyl glucosiduronate salicyloyl glucosiduronate salicylatecyt P450 glycine o-hydroxyhippurate (salicyloylglycine, salicyluric acid) gentisate oxid. 2,5-dihydroxyhippurate quinone (gentisoylglycine, (and products of its glycine gentisuric acid) polymerization) 56
  57. 57. Polypragmasy - application of multiple remedies simultaneously • it is proper to avoid application of too many different remedies together • interactions between different drugs or their metabolites can cause enhancement or inhibition of pharmacological effects • the mixed type hydroxylases (cyt P450) are inducible, their activities may increase many times in several days, so that the remedies are less efficient • if the load of the detoxifying system is high, minor pathways of transformation can be utilized and produce unwanted side-effects due to the formation of toxic metabolites • intensive conjugation with glutathione can result in depletion of this important reductant in the cells 57
  58. 58. Selected biochemical markers of liver damage (in serum)Analyte Reference values ChangeALT 0,1 - 0,8 kat/l GMD 0,1 - 0,7 kat/l GMT 0,1 - 0,7 kat/l Bilirubin 5 - 20 mol/l Ammonia 5 - 50 mol/l Urobilinogens (urine) up to 17 mol/l ------------------------------ --------------------- -------------Pseudocholinesterase 65 - 200 kat/l Urea 3 - 8 mmol/l Albumin 35 - 53 g/l  58