1.2 mraz

ISBM 2013, September 9-11, 2013, Manchester

Amino acid adducts in urine as a new type of biomarkers of alkylating agents

■■■

Jaroslav Mráz 1, Igor Linhart 2, Šárka Dušková 1, Iveta Hanzlíková 1, Ludmila Dabrowská 1

■■■

1 National Institute of Public Health, Prague: Czech Republic2 Institute of Chemical Technology, Prague, Czech Republic

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Contents

1) Protein adducts compared to other biomarkers

2) Protein adducts: What we know about them

3) Protein adducts: What we don´t know about them

4) Hypothesis

5) Research plan

6) Results

7) Prospectives

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Exposure to chemicals: Types of biomarkers

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� Parent compound in biological material (blood, urine, expired air, saliva, hair, faeces, etc.) B

M of exposure

� Metabolite(s) in biological material (urine)

� Adducts with cellular DNA

BM

of biochemical/

….biological effect

BM

of susceptibility

� DNA adducts in urine

� Adducts with proteins (globin, albumin)

� Altered levels of physiological molecules

� Cytogenetics biomarkers (CA, SCE, MN, etc.)

� Genotype of biotransformation enzymes

3


Exposure to chemicals: Types of biomarkers

4

� Parent compound in biological material (blood, urine, expired air, saliva, hair, faeces, etc.) B

M of exposure

� Metabolite(s) in biological material (urine)

� Adducts with cellular DNA

BM

of biochemical/

….biological effect

BM

of susceptibility

� DNA adducts in urine

� Adducts with proteins (globin, albumin)

� Altered levels of physiological molecules

� Cytogenetics biomarkers (CA, SCE, MN, etc.)

� Genotype of biotransformation enzymes

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Adducts with cellular DNA: � biomarkers of effective dose in target tissue� Invasive sampling (blood)� laborious and tricky analysis

DNA adducts in urine: � correlation with levels of the cellular adducts (?)� non-invasive sampling� Instrumentally demanding analysis

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Why to measure the adducts with globin?

� Surrogate biomarkers instead of the DNA adducts

� Long lifetime in the organism, equal to lifetime oferythrocytes (ca. 125 days in humans)

� Availability of globin in sufficient amounts (140mg/ml blood)

� Analytical procedures available

� Applicable for alkylating agents (e.g., epoxides),arylamines, isocyanates, N,N -dimethylformamide


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Val

COC

H

CH(CH3)2

H2N COC

H

HN

CH2SH

Cys

HN COC

H

(CH2)4NH2

Lys

HN COC

H

CH2

NNH

His

HN COC

H

CH2

OH

Tyr

Nucleophilic sites in globin

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Amino acid sequence of human globin

�� α-chain (141 amino acids)

VLSPADKTNVKAAWGKVGAHAGEYGAEALERMFLSFPT-TKTYFPHFDLSHGSAQVKGHGKKVADALTNAVAHVDDM-PNALSALSDLHAHKLRVDPVNFKLLSHCLLVTLAAHLPAE-FTPAVHASLDKFLASVSTVLTSKYR

�� β-chain (146 amino acids)

VHLTPEEKSAVTALWGKVNVDEVGGEALGRLLVVYPW-TQRFFESFGDLSTPDAVMGNPKVKAHGKKVLGAFSDG-LAHLDNLKGTFATLSELHCDKLHVDPENFRLLGNVLVC-VLAHHFGKEFTPPVQAAYQKVVAGVANALAHKYH

8


Reactive amino acid residues in human globin�� α-chain

VLSPADKTNVKAAWGKVGAHAGEYGAEALERMFLSFPTTKTYFPHFDLSHGSAQVKGHGKKVADALTNAVAHVDDMPNALSALSDLHAHKLRVDPVNFKLLSHCLLVTLAAHLPAEFTPAVHASLDKFLASVSTVLTSKYR

�� β-chain

VHLTPEEKSAVTALWGKVNVDEVGGEALGRLLVVYPW-TQRFFESFGDLSTPDAVMGNPKVKAHGKKVLGAFSDG-LAHLDNLKGTFATLSELHCDKLHVDPENFRLLGNVLVC-VLAHHFGKEFTPPVQAAYQKVVAGVANALAHKYH

V, Val; C, Cys; H, His; K, Lys99


A) Splitting off the xenobioticsX

VLSPADKTNVKAAWGKVGAHAGECGAEA-----.

mild acidic or alkaline hydrolysis

VLSPADKTNVKAAWGKVGAHAGECGAEA----- + X

B) Selective splitting off the xenobiotic-amino aci d adduct

X X X XVLSPADKTNVKAAWGKVGAHAGECGAEA-----

Edman degradation modified Edman degradation

X X XLSPADKTNVKAAWGKVGAHAGECGAEA----- + XV

Globin adducts: Methods of analysis

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C) Total hydrolysis

X X X XVLSPADKTNVKAAWGKVGAHAGECGAEA-----

acidic hydrolysisenzymatic hydrolysis (Pronase)

V AL XV P A W E G

S G DP G V XH A

N T K A A A G A K XC

XK E

Globin adducts: Methods of analysis

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Simulation of globin adduct level following (A) single exposure, (B) continuous exposure (t=120 d)curve 1: chemical instability not includedcurves 2-5: chemical instability increasing

Fennell T.R.et al.:A model for the formation and re moval of hemoglobin adducts, Cancer Epidemiology, Biomarkers & Prevention 1, 213- 219 (1992)


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N-methylcarbamoylvaline level in globin following single percutaneous exposure to DMF in human volunteers

human volunteers

single percut. exposure(Mráz et al. 2002)

rats

single i.p. administration(Mráz et al.,unpublished)

0

5

10

15

20

0 50 100 150

days

MV

H (

nmol

/g g

lobi

n)

N-methylcarbamoylvaline level in globin following exposure to DMF

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Adducts with cellular DNA

Degradation products in urine(limited information)

Adducts with globin

Degradation products in urine(no information)

?

Adducts with globin: What we don ´t know about them?

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Modified amino acids in urine

Biomarkers of biochemical changes (clinical medicine)

� 3-methylhistidine: muscle breakdown product� nitrotyrosine: stress by nitration� o-, m-tyrosine: oxidative stress� prolyl-4-hydroxyproline: regeneration of bone proteins

Biomarkers of exposure to chemicals(preventive medicine, toxicology)

No examples!

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Hypothesis

Following their lifetime, globin adducts undergo proteolytic degradation to produce:

� free amino acids, which enter the common metabolic cycles

� free amino acid adducts, which are excreted with urine

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Research plan - Stage 1Studies on excretion of amino acid adducts in vivo

1) Synthesis of model amino acid adducts

2) Development of analytical procedures

3) Administration of synthetic amino acid adducts in rats

4) Analysis of rat urine for administered compounds and/or their metabolites

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Precursor in vivo

Reactive species for synthesis

Amino acid

Adduct

N,N-dimethyl-formamide

methyl-isocyanate

Val N-methylcarbamoyl-

Lys N ε-methylcarbamoyl-

ethylene oxide ethylene oxide Val N-(2-hydroxyethyl)-

Cys S-(2-hydroxyethyl)-

acrylamide acrylamide Val N-(2-amidoethyl)-

Cys S-(2-amidoethyl)-

styrene styrene oxide Val N-(2-hydroxy-1-phenylethyl)-N-(2-hydroxy-2-phenylethyl)-

Cys N-(2-hydroxy-1-phenylethyl)-N-(2-hydroxy-2-phenylethyl)-

Adducts to be studied

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CH3

N

O

NH2

O

OH

CH3 CH3

NH2

NH2

O

OH

NHO

OH

CH3 CH3

NH

CH3

O

NH

NH2

O

OH

NH

CH3

O

Val

Lys N(5)-methylcarbamoyl-Lys

(MLU)

methylisocyanate

(MIC)

N-methylcarbamoyl-Val

(MVU)

RESULTS Synthesis of amino acid adducts of MIC and d 3-MIC

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RESULTS Synthesis of amino acid adducts of EO and d 4-EO

2-hydroxyethyl-Val

(HEV)

OH

ONH

OH

OH

ONH2

S

OH

2-hydroxyethyl-Cys

(HEC)

O

OH

NH2

OH

ONH2

SH

O

ethylenoxid

(EO)

Val

Cys

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Adductadmin.

Compounds determined / yield in urine (%) 1)

Parent compound Metabolic product

HEV HEV 29; 33

HEC HEC <1 %HEMA,CMMA

n.m.2)

MVU MVU 58; 105

MLU MLU 0,5; 0,5 MLU-Ac 46; 70

1) Individual values, n=2; 2) n.m. not measured

RESULTSYields of the adducts and their metabolites in rat urine (0-24 h) after i.p. administration, 10 mg/kgAnalysis: HPLC/MS of diluted urine

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OH

O

NH2

NHNHCH3

O

OH

O

NH

NHNHCH3

O

CH3

OMLU

MLU-Ac

OH

O

NH2

S

OH

OH

O

NH

S

CH3

O

OH O

OH

O

NH

S

OH

CH3

O

HEC

CMMAHEMA

RESULTS Synthesis of metabolites of amino acid adducts

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RESULTS Analytical procedures for amino acid adducts in uri ne

Solid phase extraction (SPE) + HPLC/MS/MS(= standard strategy)

fully developed for MLU -Ac and HEV, partially for MVU, HEMA, CMMA

Single-step derivatization and extraction with reactive alkylchloroformates + HPLC/MS/MS(= experimental strategy)

product identification and initial assessment of reaction yields: rather unpredictable

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Research plan - Stage 2Studies on the fate of native globin adducts follow ing

their physiological removal from circulation

1) Incubation of erythrocytes from donor rats with t he adducting agent

2) Washing the erythrocytes to remove all unbound fo rms of the adducting agent

3) Transfusion of the washed erythrocytes to accepto r rats

4) Determination of globin adduct level in the washe d adducted erythocytes and in blood of acceptor rats following the transfusion

5) Determination of globin adduct level in blood of acceptor rats at regular intervals during whole lif etime of the erythrocytes

6) Analysis of rat urine for the amino acid adducts and possibly other products (at regular intervals as in 5) 24


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RESULTSAnalysis for adducts after administration of N -methyl-formamide (NMF) in rats (1000 mg/kg)

■ Analysis of globin for MVU and MLUhydrolysis with pronase + HPLC/MS/MS

MVU and MLU found as expected, also found several adducts with dipeptides (e.g.,N -methylcarbamoyl-Val-His)

■ Analysis of urine for MVU and MLU -AcSPE + HPLC/MS/MS


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RESULTSAnalysis for adducts after in vitro incubation of r at erythrocytes with EO, 100 mM

■ Analysis of globin for HEV and HEC hydrolysis with pronase + HPLC/MS/MS

X XVLSPAD---- VLSPAD----

expected found

XV L A XVL S A

S P D P D

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RESULTSAnalysis for adducts after i.p. administration of E O in rats (50 mg/kg)

■ Analysis of urine for N -2-hydroxyethyl-Val-LeuSPE + HPLC/MS/MS

detected !

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Prospectives

If physiological degradation globin adducts would indeed result in urinary excretion of amino acid adducts and/or related products, these would constitute a new category of biomarkers, combining three major advantages:

� Specificity for the parent compound

� Monitoring of long-term cumulative exposures

� Non-invasive sampling

Possible extension to other proteins

Possible aplications in toxicology, clinical medici ne, pharmacology

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Acknowledgements

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1.2 mraz

Technology

proteins globin

manchestersimulation

biological material

biomarkers prevention

surrogate biomarkers

humans availability

biological effectadducts

single exposure