BIOTRANSFORMATION

Enzymatic processes in liver and other tissues that modify the chemical structure of xenobiotics, render them more water-soluble, increase their elimination, decrease their half-life Biotransformed metabolites are chemically different from the parent molecule
PHASE I Expose or Add functional groups Xenobiotic Oxidation Reduction Hydrolysis Primary products Excretion PHASE II Biosynthetic conjugation Secondary products

Lipophilic (e.g. barbital vs hexobarbital) t1/2= 55h vs t1/2 of months, real t1/2= 5hr

Hydrophilic (Ionizable)

PHASE I
Add or expose functional groups: -OH, -SH, -NH2, -COOH, (small increase in hydrophilicity)

Phase II
Conjugation with endogenous molecules: glucuronic acid, sulfate, glutathione, ... covalent bonds formed (large increase in hydrophilicity)
Table 6.1 C&D

Major organ of biotransformation Secondary organs of biotransformation kidney (proximal tubule) LIVER (hepatocytes) importance of liver physiology/anatomy lungs (type II cells) first-pass elimination testes (Sertoli cells) skin (epithelial cells) intestines (microbial flora)
Preparation of Phase I enzymes (e.g. liver) endoplasmic reticulum (microsomes) and cytosol Liver homogenate Centrifuge at 9 000g (separation of mitochondria, lysosomes, nuclei, broken cells) Supernatant at 105 000g Precipitate (Phase I) microsomes Supernatant (Phase II) cytosol

Biotransformation

vs metabolism vs toxicity (bioactivation) vs target organ vulnerability

PHASE I Hydrolysis
Carboxylesterases, cholinesterases, organophosphotases e.g. hydrolysis of procaine (used as local anaesthetic) Peptidases Epoxide Hydrolase (EH) -detoxifying enzyme for epoxides (aromatic, unstable and reactive molecules) -formation of diols (accessible to Phase II) -EH present in many tissues

Fig. 6.4 C&D, Hydrolyation of epoxides by EH

Role of EH in the biotransformation of benzo[a]pyrene B[a]P

Inactivation of benzo[a]pyrene 4,5-oxide Conversion of B[a]P to tumorigenic diolepoxide

Fig. 6.6 C&D

REDUCTION
e.g Azo- and Nitro-Reductions Azo-reduction N=N Prontosil Fig. 6.6 C&D [4H] -NH2 1,2,4-triaminobenzene -NH2 Sulfanilamide

Azo- and Nitro-reductions can be catalyzed by enzymes of intestinal flora (especially in large intestine) by cytochrome P450 (usually oxidizing enzyme), has the capacity to reduce xenobiotics under low oxygen or anaerobic conditions interactions with reducing agents (reduced forms of glutathione, NADP,)

REDUCTION Role of intestinal microbial flora in biotransformation

reduction enhanced in anaerobic environment important for nitroaromatic compounds Oxidation by cytochrome P450 Conjugation with glucuronic acid by UDP-GT (uridine diphosphate glucuronyl transferase) Excretion of glucuronide in bile Reduction by microbial flora under anaerobic conditions Reabsorption from intestine (enterohepatic cycling) Hydroxylation of amine group Conjugation with sulfate or acetate Reactive metabolites Tumorigenic

Fig. 6.9 C&D

OXIDATION
Monoamine oxidase (MAO), Multiple function oxidase (MFO) Flavin Monooxygenases (nicotine, cocaine,) Alcohol dehydrogenase (liver ADH, gastric ADH) oxidation of alcohols to aldehydes low ADH links to high blood alcohol levels lower gastric ADH in women/men ADH activity lower after fasting clinical alcoholics have lower gastric ADH racial differences in gastric ADH Aldehyde dehydrogenase (ALDH) oxidation of aldehydes to carboxylic acid racial differences in ALDH face flushing syndrome associated with low ALDH
ADH R-CH2OH
NAD+

ALDH R-C=O H R-C=O OH

NADH + H +

NADH + H+

NAD+ + H2O

Fig. 6.19 C&D

Biotransformation of alcohol

Fig. 6.20C&D

CYTOCHROME P450
most versatile oxidative enzyme heme-containing protein ferric Fe2+ reduced to ferrous Fe3+ reduced cytP450 can bind oxygen

Table 6.6 C&D

6.35 C&D

Nomenclature of cytochrome P450 use of recombinant DNA CYP Amino acid sequence known for many cytP450s <40% a.a. homology, assigned to different genes (CYP1, CYP2, CYP3,..) 40-55% a.a. homology, different subfamily (CYP2A, CYP2B, CYP2C,) >55% a.a. homology, different members of same subfamily (CYP2A1, CYP2A2,) Use of Knockout Mice Inhibition of cytochrome P450 (aminobenzotriazole inhibits synthesis of cytP450) detergents, CO,

Fig. 6.35 C&D

Cytochrome P450
-can oxidize a substrate (e.g. a xenobiotic) -can transport electrons -many isoforms of cytP450 isolated (in human, rat, others) -importance of NADPH-cytochrome P450 reductase (embedded in ER membrane with cytP450) -catalytic cycle -inducible by specific substrates (e.g. phenobarbital, benzo[a]pyrene, methylcholanthrene) Simplified reaction: Substrate (RH) + O2 + NADPH + H+ Product (ROH) + H2O + NADP+ How can we demonstrate a role for cytP450 in biotransformation of a XB? -cytP450 activity increases after exposure to XB -enzymatic inhibitors of cytP450 (e.g. aminobenzotriazole) have an effect -absorption spectrum of cytP450 is modified in presence of XB -reaction can be reconstituted with components of the catalytical cycle (e.g. NADPH)

Hydroxylation of substrates by cytochrome P450

Fig. 6.36 C&D

Deactivation (detoxication)

Role of cytochrome P450 and peroxidase in activation of benzene in bone marrow

Fig. 6.29 C&D

PHS prostaglandin H synthase

Halothane hepatitis and activation by oxidation and reduction

Fig. 6.16 C&D

Reactions catalyzed by cytochrome P450

Fig. 6.43 C&D

Biotransformation of acetaminophen the good, the bad and the ugly

Nephrotoxicity & Hepatotoxicity at excess doses

Fig.6.28 C&D