Types of Electrophilic Carcinogenic Intermediates

Transcription

1 Types of Electrophilic Carcinogenic Intermediates Procarcinogens Reactive intermediates Enzymes involved in control Aromatic hydrocarbons Epoxides Cytochromes P450 Glutathione S-transferases Epoxide hydrolases Aromatic amines Reactive esters Cytochromes P450 Sulfotransferases Acetyltransferases UDP-Glucuronosyltransferases Glutathione S-transferases Dialkylnitrosamines Carbonium ions/ electron-deficient alkyl groups Cytochromes P450 Vicinal dihaloalkanes Episulfonium ions Glutathione S-transferases

2 The Active Site of Aspergillus niger meh

3 Extended Enzymatic Mechanism of Microsomal Epoxide Hydrolase Tyr Tyr H H H R R R R H Asp nucleophilic attack H + Asp H H hydrolysis H + Asp + R H R N N H His Glu

4 3D Structure of Bacterial Limonene Epoxide Hydrolase Asp 101 Arg 99 Asp 132 Tyr 53 Asn 55

5 Enzymatic Mechanism of Limonene Epoxide Hydrolase D 101 D 101 R 99 H N H 2 N H NH 2 H H H H 2 N Hydrolysis R 99 NH H 2 N NH 2 H H H H H 2 N N 55 N 55 D 132 D 132 Y 53 Y 53

6 Kinetic Analysis of meh Mutant Glu 404 Asp ,10-epoxystearic acid 1/v [nmol -1 x mg meh x min] = wild type meh = meh Glu 404 Asp K M [µm] V max [nmol/mg/min] Wild type meh /s [µm -1 ] meh Glu 404 Asp

7 Relationship between K M, K D and Rate Constants of the meh Enzymatic Mechanism K D k 1 k 2 E + S E S E S E + P K M = K D k 2 k 1 + k 2 k 2 k 1 << k 2 => 1 => K M = K D ; V max ~ k 1 k 1 + k 2 k 1 >> k 2 => k 2 k 1 + k 2 k 2 => K M k 2 = ; V max ~ k 2 k 1 K D k 1 k 1 = k 2 => k 2 k 1 + k 2 = 1 2 => 1 K M = K D ; V max ~ k 1, k 2 2

8 Kinetics of Enzymatic Epoxide Hydrolysis 5 4,5 H 4 3,5 H H E + S ES E-S E + P 3 2,5 2 1,5 1 0,5 S ES E-S P 0 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1 time [s]

9 Threshold of the Genotoxic Effect of Styrene 7,8-oxide in hmeh-transgenic V79 Chinese Hamster Lung Fibroblasts 0.15 elution rate [h -1 ] 0.1 mock transfected 0.05 meh transfected E J styrene 7,8-oxide [µm]

10 Conclusions Xenobiotic-metabolizing EHs are α/β hydrolase fold enzymes working with a catalytic triad The first step of enzymatic epoxide hydrolysis is optimized for speed, allowing efficient detoxification with broad substrate specificity Experimental verification showed that this high speed detoxication introduces a practical threshold of genotoxicity, at least for the epoxides investigated

11 Alwyn Jones Sherry Mowbray Terese Bergfors Martin Hallberg Jinyu Zou Roland Furstoss Jacques Baratti Christophe Morisseau Michael Arand Annette Cronin Heike Dürk Karen Hänel Jan Georg Hengstler Maria Elena Herrero Shirli Homburg Michael Knehr Matthias Lohmann Astrid Mecky Frank Müller Heike Nagel Bruce Hammock Jeff Beetham David Grant Christophe Morisseau Richard Armstrong Conny Cassidy Dick Janssen Manfred Reetz Ari Hirvonen Marek Jakubowski Dominique Lison Vincent Haufroid Pavel Vodicka & Colleagues EU Network of Excellence ECNIS

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13 Threshold of the Genotoxic Effect of Styrene 7,8-oxide in hmeh-transgenic V79 Chinese Hamster Lung Fibroblasts 0.15 elution rate [h -1 ] 0.1 mock transfected 0.05 meh transfected E J styrene 7,8-oxide [µm]

14 Typical meh Substrates Cl Cl Cl Cl N benzo[a]pyrene 4,5-oxide Cl Cl HEM NH 2 carbamazepine 10,11-ox H cis-9,10-epoxystearic acid CH 3 CH 3 cis-stilbene oxide styrene 7,8-oxide androstene oxide octane-1,2-epoxid

15 Mouse seh Structure

16 EH from Agrobacterium at 2.1 Å seh from mouse at 2.8 Å EH from Aspergillus at 1.7 Å Nardini et al., J.Biol.Chem Argiriadi et al., PNAS 1999 Zou et al., Structure 2000 Leukotriene A 4 hydrolase from human at 1.95 Å EH from Rhodococcus at 1.2 Å Thunnissen et al., Nature Struct. Biol Arand et al., EMB J., in press

17 EH from Aspergillus

18 EH from Agrobacterium

19 seh from mouse (only the C-terminus displayed)

20 LTA4 hydrolase

21 seh from mouse (complete dimer)

22 EH from Rhodococcus

23 EH from Rhodococcus

24 EH from Aspergillus

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26 Structural Relationship between Mammalian Epoxide Hydrolases and other Enzymes Related by Sequence Similarity Catalytic Triad Mammalian meh? Haloalkane dehalogenase Mammalian seh

27 Enzymatic Mechanism of Epoxide Hydrolysis R H δ + δ δ + - Asp/Glu R - H H - Asp/Glu - Asp H H N NH His - Asp HN + NH His Step 1 Nucleophilic Attack R - H - Asp/Glu Step 2 Hydrolysis Asp H H N NH His seh meh Asp His Asp/Glu

28 Expected Effect of the meh Concentration on the Epoxide Steady State Concentration epoxide concentration [µm] 0,8 0,15 0,1 no meh 25 µm meh 0,05 50 µm meh time [s]

29 Steady State Kinetics of Epoxide Detoxification 1000 Epoxide steady state concentration [µm] Rate of epoxide formation [µm x s -1 ]

30 2-Pheny lethanol H H 1-Phenyl ethanol Correlation between Styrene- and Styrene xide-exposure and Biomarker Blood Levels of Healthy Non-Smokers (Data taken from Rappaport et al., Cancer Res. (1996) 56, pp ) Styrene Biomarker Styrene (n = 21) Styrene oxide (n = 8) CYP H 4-Hydroxystyrene Exhaled Styrene 0.948*** S-Albumin (α) * S-Albumin (β) S-DNA (1) Styrene-7,8-oxide GST Glutathion conjugates S-DNA (2) 0.434* EH SCEs * Average exposure level: Styrene = 50.5 mg/m 3 ; Styrene oxide = 129 µg/m 3 H H * p < 0.05; ** p < 0.01; *** p < Phenyl glycol UGT Glucuronide H ADH H H H N H Phenyl glyoxylic acid Mandelic acid Hippuric acid

31 Epoxide Hydrolase

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33 Genotoxins Microsomal epoxide hydrolase (meh) Soluble epoxide hydrolase (seh) Signal molecules Epoxides Limonene epoxide hydrolase (LEH) Carbon source

34 EH from Agrobacterium at 2.1 Å seh from mouse at 2.8 Å EH from Aspergillus at 1.7 Å Nardini et al., J.Biol.Chem Argiriadi et al., PNAS 1999 Zou et al., Structure 2000 Leukotriene A 4 hydrolase from human at 1.95 Å EH from Rhodococcus at 1.2 Å Thunnissen et al., Nature Struct. Biol Arand et al., EMB J., in press

35 EH from Aspergillus

36 EH from Aspergillus

37 Evaluation of the Detoxification Efficacy of meh concentration [µm] 5 4,5 4 S real S ermittelt deducedb Pfrom P 3,5 3 2,5 2 1,5 1 0, ,2 0,4 0,6 0,8 1 time Zeit [s]