1 drug molecule Highly lipophyllic lipophilic polar hydrophylic accumulation (fatty tissues) phase I polar phase IIbioinactivation conjugation hydrophylic extracellular mobilisation circulation excetion with bile excretion with urine Steps of biotransformation

Phase II reactions Conjugation reaction Functional group on the xenobiotic or its metabolite Cofactor (endogenous partner) EnzymePlace of the reaction Glucuronic acid conjugation -OH, -COOH, - NH 2, -NH, -SH, -CH UDPGAGlucuronyl transferase (GT) Smooth endoplasmic reticulum Sulphate conjugation Aromatic OH/NH 2 -COOH PAPSSulfotransferase (ST) Cytosol Glycine conjugation aromatc-NH 2, - COOH CoenzymeA Glycine Amino acid aciltranszferase Mitochondria Acetylationaromatic/aliphatic- NH 2, hidrazinok, -SO 2, NH 2 Acetil coenzyme AN-, O-acetil transferase Cytosol ( membranes) Methylationaromatic-OH, NH 2, NH -SH Adenosyl-S- metionin MetiltransferaseCytosol ( membranes Glutathione conjugation epoxide, organic haloids Reduced glutathione Glutathione transferase Cytosol ( membranes II. Non conjugation reaction enzymes: epoxide hydrolases, glyoxalases, carboxylesterases

-OH, -COOH, -NH 2, -NH, -SH, -CH UDPGAGlucuronic acid conjugation Smooth endoplasmic reticulum

Synthesis of UDP glucuronic acid

Glucuronidation of phenol

Characteristics of glucuronide- conjugation It is fast (coupled to phase I reactions in the endoplasmic reticulum), It is common, It can not be saturated, The same xenobiotic molecule can conjugate with several glucuronides, The enzyme is polymorphic, The effect is mostly inactivation, but activation can also happen.

cofactorsxenobiotic Mixed Function Oxidase system in the smooth endoplasmic reticulum fp1, fp2: flavoproteins b5:ciytochrome b 5

Glucuronidation can produce a more active or a less active molecule Glucuronids of morphine 6-glucuronide- morphine is much more effective than the parent molecule 3-glucuronide morphine is totally uneffective

Sulphate conjugatio n Aromatic OH/NH 2 -COOH PAPSSulphotransferaseCytosol

Sulphatation of phenol sulphurilase SO ATPAPS + PPi (pyrophosphate) APS-Phosphokinase APS + ATPPAPS + ADP

The characteristics of sulphate conjugation There is some substrate specificity It can be saturated It is the second most freguent phase II reaction It takes place in the cytosol The enzyme sulphotransferase is polymorphic The effect is mostly inactivation, but activation can also happen.

Glycine conjugation (amino acid conjugaton) aromatic-NH 2, -COOH CoenzymeA Glycine (or other amino acid) Aminoacid-acyltransferase Mitochondria

Acetylation aromatic/aliphatic-NH 2, hydrazins, -SO 2, NH 2 Acetyl coenzyme A N-, O-acetyl transferase Cytosol ( also in membranes)

Methylation aromatic-OH, NH 2, NH -SH Adenosyl-S- methionin Methyl-transferase Cytosol ( also in membranes)

Glutathione conjugation epoxides, organic haloids Reduced glutathione Glutathione transferase Cytosol (also in membranes)

Glutathione conjugation of some compounds:

Formation of mercapturic acid after glutathione conjugation

Non conjugation reactions of phase II Reactions catalysed by: Epoxide hydrolases Glyoxalases Carboxylesterases

Detoxication of epoxides by hydratation The reaction is catalysed by an epoxide hydrolase enzyme. From bromobenzene 3,4-oxide bromobenzene 3,4-dihydrodiol is formed. It is assumed that the first step is that the enzyme deprotonates water rather than activating the epoxide ring.

Phase II reactions mean bioinactiovation in most of the cases. There are some exceptions, where they result in bioactivation.

Formation of carbonium és nitrenium ions from sulphate conjugates of benzyl alcohols and hydroxamic acids

Reversion of inactivation in the urinary bladder Reversion of inactivation in the urinary bladder

Characteristics of the biotransformation reactions 1.No strict substrate specificity, 2.Induction and inhibition 3.Not only xenobiotics but endogenous substrates

Metabolism studies In vitro studies in vivo studies with labelled xenobiotics (different doses, single and repeated treatment)

Metabolic pathways of the pesticide dimethachlor (18 metabolites identified)

Factors influencing biotransformation of a xenobiotic Species Intra-species genetic variations Age Physiological status Other xenobiotics

Ratio of glucuronidation and sulphatation in some species glucuronidation (%) Sulfatation (%) cat087 human2371 rat2568 rabbit4645 pig1000

Biotransformation of Amphetamine in rabbits, rats, guinea pigs and dogs

Genetic polymorphism of biotransformation enzymes Differences in the base sequence of the DNAin the base sequence of the DNA In the amino acide squence of an enzymeIn the amino acide squence of an enzyme In the reaction kineticsIn the reaction kinetics % of fast acetilation in some human populations% of fast acetilation in some human populations Europeans40%Europeans40% Asiatics80%Asiatics80% Inuits96%Inuits96%

Consequences of acetylation kinetics: different side effects Isoniazid (drug against tuberculosis)Isoniazid (drug against tuberculosis) slow acetilation: neurotoxic effects fast acetilation: liver problems Hidralazin (drug against high blood pressure) Hidralazin (drug against high blood pressure) slow acetilation: Lupus eritomatosus fast acetilation: no specific side effect

Differences between sexes Hormones influence the lipid environment of enzymes. Sex-differences in the activity of CYP P450 enzymes. (The hypothalamus is releasing a feminizing factor, which results in „feminine liver” having somewhat lower metabolising capacity in general than the liver of males. :

The role of age in biotransformation

The influence of the physiological status of the individual: Diseases,Fasting. The influence of other xenobiotics Enzyme induction by PAHs, Enzyme inhibition by heavy metals, Dietary factors, Smoking, consumption of alcohols, illegal drugs…

Effects of exposure before birth Thalidomide 1953 synthesis, Chemie Grünenthal putting the drug on the market

Thalidomide babies

Thalidomide S: sedative effect R: teratogenic

Cause of the tragedy: thalidomide was tested on adult mice only…. Differences in species: mouse: superoxide glutathione- conjugation human: Superoxide glutathion conjugation Differences of age: foetuses have a limited (or missing) metabolising capacity