ToxicologyToxicologyfor MS students for MS students

1.1.2015.2015.

Phase I of biotransformationDr. Pálovics Ágnes

Tel.: 309-1027+36 205 36 44 01

e-mail: PalovicsA@nebih.gov.hu

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Toxicology: definition

Characteristics of the Xenobiotic Properties of the living organisms

Phases Exposure, the basic principle by

Paracelsus Toxicokinetics/pharmacoinetics Toxicodynamics/pharmacodynamics

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Routes of exposure

Non invasive routesoralDermalInhalationOther membranes (nose, eyes, genitals …etc)

Invasive routesinjectionInfusionDialysis treatment

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Oral absorption of xenobioticsKey characteristics:

Molecular size Solubility

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Solubility of some compounds and their incorporation in algal cells

compound Pow

(Coctanol/Cwater)

logPow Microgramm/m3

propane 570 2,756 24 000

glycollate 50 1,699 12000

urea 15 1,176 1000

glycerin 7 0,845 210

sucrose 3 0,477 4

ethanol 0,4898 -0,31 No data

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Structure of the small intestineStructure of the small intestine

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Types of diffusion and active transportTypes of diffusion and active transport

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Types of endocytosisTypes of endocytosis

pinocytosis phagocytosis receptor-dependent endocitózis

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Types of membrane transportTypes of membrane transport

Mechanism Driving force examples

Diffusion through the lipid membrane

thermal movement of the molecules, concentration gradient

Small, hydrophobic molecules (ethanol, amidopyrin

Amphetamine, lidocaine

Diffusion through pores

thermal movement of the molecules, concentration gradient

Small, hydrophylic molecules ; anions, cations, acetyl-salycilic acid, antipyrin)

Facilitated diffusion thermal movement of the molecules

Molecules which can be bound to carriers

Aktive transport Molecules which can be bound to carriers

Molecules which can be bound to carriers,

penicillins

Pynocytosis, phagocytosis

Molecules which can be bound to carriers

Larger molecules (rare ; botulintoxin, antibodies

Diffusion through intercellular spaces

Thernal energy from molecules along a hydrostatic or osmotic gradient

All molecules

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Other factors influencing the absorption of molecules

size solubility Spheric characeristics pH of the environment (acids or bases)

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Influence of the pH on the absorption of some molecules Nicotine

pK=8,5 , absorbs from the mouth (pH: 6.2-7.2)k Nitroglycerin

weak base, to be put under the tongue Acetyl salicylic acid

weak acid, absorbs from the stomach

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pH values in the organism blood 7,35-7,45 mouth 6,2-7,2 stomach 1-3 Duodenum 4,8-8,2 Jejunum 6,3-7,3 Ileum 7,6 colon 7,8-8,0 Rectum 7,8 Cerebral fluid 7,3-7,4 Vagine 3,4-4,2 Urine 4,8-7,5 sweat 4,0-6,8 milk 6,6-7,0

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drug molecule

Highly lipophillic lipophilic polar hydrophilic

accumulation(fatty tissues)

phase I

polar

phase II bioinaktivationconjugation

hidrophilic

extracellular mobilisation

circulation

biliary excretion renal excretion

Steps of biotransformationSteps of biotransformation

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drug molecule

Highly lipophyllic lipophilic polar hydrophylic

accumulation(fatty tissues)

phase I

polar

phase II bioinaktivációkonjugáció

hidrophylic

extracellular mobilisation

circulation

biliary excretion renal excretion

Steps of biotransformationSteps of biotransformation

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Phase I reactions, 1Phase I reactions, 1 I.I.type substrate product

A. Oxydation (MFO)

aromatic hydroxilation

aliphatic hydroxilation

epoxidation

N-hydroxilation

O-dealkilation ROH + CH2O

N-dealkilation R-NH2 + CH2O

S-dealkilation R-SH + CH2O

deamination + NH3

S-oxydation

R R OH

R CH3 R CH2 OH

R CH CH R1CH CH

O

R R1

NH2 HOHN

O CH3R

NH CH3R

S CH3R

CH3R

NH2

S R1R

R C CH3

O

R S R1

O

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Phase I reactions, 2Phase I reactions, 2

Dechlorination CCl4 [CCl3.] CHCl3

oxidatíve desulfuration

P

O

S O

O

R3

R2R1

P

O

O O

O

R3

R2R1

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Phase I reactions, 3Phase I reactions, 3

type substrate product

A.Oxydation ( non MFO)

II. Amino oxydation R – CHO + NH3

III. Dehydrogenation CH3CHO + CH3COOH

B. Reduction

Azoreduction R – N = N – R1 R – NH2 + R1 – NH2

nitroreduction R – NO2 R – NH2

carbonil reduction

C. Hydrolysis

esthers +

amides R – CONH2 R – COOH + NH3

R CH2 NH2

CH3 CH2 OH

R C R1

O

R CH R1

OH

R C O

O R1

R C OH

O

R1 OH

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Cl CH2 C NH2

CH3

CH3

Cl CH2 C NHOH

CH3

CH3

Biotransformation of chlorfentermine and amphetamineBiotransformation of chlorfentermine and amphetamine

CH2 CH NH2

CH3

CH2 C O

CH3

+ NH3

chlorphentermine

amphetamine

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cofactors xenobiotic

MFO: Mixed Function OxydaseMFO: Mixed Function Oxydaseenzyme complex enzyme complex

fp1, fp2: flavoproteinsb5: cytochrome b5

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Carbon monoxyde difference spectrum Carbon monoxyde difference spectrum of cytochrome P-450of cytochrome P-450

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Structure of HemStructure of Hem

Cys (of the P-450 molecule)

víz

N

N

N

N

CH3

CH2

CH2CH3

CH3

CH3

OH

O

OH

O

Fe

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Cytochrome P–450Cytochrome P–450 mono mono--oxoxyygengenase activityase activity

2+, 3+: oxydation state of irone: electroneSH: substrate

SH + NADPH + H+ + O2 SOH + NADP + H2O

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Difference spectrum of type I and type II interactionDifference spectrum of type I and type II interaction

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Bioinactivation or bioactivationBioactivation during oxydation of benzeneBioactivation during oxydation of benzene

OH

HC+

H

O-

H

OHH

OH

benzol epoxid NIH-SHIFT

fenol

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Highly reactive intermediatesHighly reactive intermediates

compound

name structure intermediate Type of toxic effect

Bromo-benzene Necrosis in the liver

Vynil chloride Liver tumour

aniline methemoglobinaemia

dimethyl-nitrose amine

H3C+ carcinogenesis

Carbon tetrachloride

CCl4.CCl3

Necrosis in the liver

chloroform CHCl3 Necrosis in the kidney

Br Br

OH

H Cl

H H

H Cl

HO

NH2 NH

OH

N N

CH3

CH3 O

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Some cytochrome enzymesSome cytochrome enzymes

CYP 1 A-1 (gut) • Inducible: PAH, tobacco smoke, de novo protein synthesis

•Substrates.: theophylline, coffein, activation of precarcinogens, inactivation of carcinogens• activity inhibited by flavonoids

CYP 1 A-2 (liver)

CYP 2 A-6 • small quantities• Substrates: coumarine, nicotine

CYP B-6 •Important in rodents• Substrate: cyclophosphamide prodrug, transformed into an anti-tumour agent

CYP 2 C-9 • weakly inducible (phenobarbital, rifampicin)• Substrates: tolbutamide, warfarin

CYP 2 C-19 • Polymorphic enzyme• Substrates: mefenitoin, thalidomide

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CYP 2 D-6 • Polymorphic• Non inducible• Substrate: propranolol•

CYP 2 E-1 • Inducible• Substrates: small molecules, solvents

CYP 3 A • most abundant, main enzyme in most drug metabolism• Inducers: phenobarbital, dexametazon, rifampicin• Very active in the gut

CYP 4 A • active in rodents, inducer: clofibrate,

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Biotransformation in other parts of the body

Biotransformation can be active in all cells, especially:

In the gastrointestinal tract In the respiratory system and in the skin.

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General characteristics of CYP 450 enzymes

products of 4 gene families, number of iso-forms in the human liver >40

Number of reactions catalysed >60 Weak substrate specificity

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Non MFO catalysed reactions in phase I

Non- MFO catalysed oxidationaminooxidationdehydrogenation

ReductionAzo-nitro-karbonil-compounds

Hydrolysis: esthers, amides