6.2 schettgen

Quantification of the mercapturic acids ofacrylonitrile and its genotoxic metabolitecyanoethylene-epoxide in a pilot human

biomonitoring study

T. Schettgen, J. Bertram, T. Kraus

Institute for Occupational and Social MedicineUniversity Hospital, RWTH Aachen University

www.arbeitsmedizin.rwth-aachen.de

Introduction

Uniklinik RWTH Aachen – Quantification of the mercapturic acids of acrylonitrile…

ISBM Conference Manchester, 09.09.2013page 2

CH2

C N

acrylonitrile

possibly carcinogenic to humans (IARC class 2B)

• acrylonitrile-butadiene-styrene-polymers („ABS-plastics“)

• synthetic fibers• rubber production• production Vol.: 1.5 Mio t/year

(USA)

3 – 15 µg/cigarette*

indoor air (ETS): 0.5 – 1.2 µg/m3 **

*Hoffmann D. et al. Chem Res Toxicol 14:767-790 (2001)**Nazaroff and Singer. J Expo Anal Environ Epidemiol 14:S71-S77 (2004)

Metabolism of acrylonitrile in rats*

*Sumner SCJ, Selvaraj L, Nauhaus SK, Fennell TR. Chem Res Toxicol 10: 1152-1160 (1997)


ISBM Conference Manchester, 09.09.2013

40 %

30 % 13 %



Aims and questions

• can we detect CHEMA as a specific metabolite ofgenotoxic cyanoethylene-epoxide in human urine?

• what is the portion of oxidative vs. direct metabolism?

• Biomarkers of ACN-exposure?

⇒development of a method for the simultaneousdetermination of CEMA and CHEMA in urine



autosamplerV1

RAM

Waste

pump 2

column

gradientpump

MS/MS

analytes

urinary matrix

Phenomenex Luna C 8(2)150 x 4.6 mm

MercK RAM C 8 (25 µm)25 x 4 mm

LOQ: 1 µg/L Urin•use of isotopically labelled internal standards (d3-CEMA, d3-CHEMA )

T. Schettgen, J. Bertram, T. Kraus, Talanta 98: 211-219 (2012)

Method

ChromatogramXIC of -MRM (5 pairs): 231.0/83.9 amu from Sample 15 (Qreal_15)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17Time, min

0

500

1000

1500

2000

2500

3000

3500

4000

4500

Intensity, cps

9.29

15.72

XIC of -MRM (5 pairs): 234.0/83.9 amu from Sample 15 (Qreal_15)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17Time, min

0

2000

4000

6000

8000

1.0e4

1.2e4

1.4e4

1.6e4

1.8e4

2.0e4

Intensity, cps

9.27

13.19

CHEMA

D3-CHEMA

Quantifying transition


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17Time, min

0

400

800

1200

1600

2000

2400

2800

3200

3600

4000

Intensity, cps

9.29CHEMA

Qualifying transition


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17Time, min

0

500

1000

1500

2000

2500

3000

3500

4000

4500

Intensity, cps

9.29

15.72


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17Time, min

0

2000

4000

6000

8000

1.0e4

1.2e4

1.4e4

1.6e4

1.8e4

2.0e4

Intensity, cps

9.27

13.19

CHEMA

D3-CHEMA

Quantifying transition


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17Time, min

0

400

800

1200

1600

2000

2400

2800

3200

3600

4000

Intensity, cps

9.29CHEMA

Qualifying transition


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17Time, min

0

2000

4000

6000

8000

1.0e4

1.2e4

1.4e4

1.6e4

Intensity, cps

12.34


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17Time, min

0

2000

4000

6000

8000

1.0e4

1.2e4

1.4e4

1.6e4

Intensity, cps

12.30

CEMA

D3-CEMA


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17Time, min

0

2000

4000

6000

8000

1.0e4

1.2e4

1.4e4

1.6e4

Intensity, cps

12.34


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17Time, min

0

2000

4000

6000

8000

1.0e4

1.2e4

1.4e4

1.6e4

Intensity, cps

12.30

CEMA

D3-CEMA

processed urine sample of a smoker (creatinine: 1.05 g/L, Cotinine: 1360 µg/L)

23.4 µg/L

188 µg/L



Precision and accuracy

Analyte Qlow Qmedium Qhigh Qreal

Within

series

(n=6)

Between

series

(n=10)

Within

series

(n=6)

Between

series

(n=10)

Within

series

(n=6)

Between

series

(n=10)

Within

series

(n=6)

Between

series

(n=10)

CHEMA

c (µg/L urine) 8.3 42.0 85.7 23.2

std. dev.(%) 3.9 8.0 2.6 5.9 2.9 3.2 3.5 4.7

accuracy (%) 98 100 94 --- ---

CEMA

c (µg/L urine) 15.1 71.8 142.9 196.4

std. dev.(%) 8.8 11.2 2.2 5.6 2.4 6.4 3.1 14.5

accuracy (%) 101 96 95 --- ---





Non-smokers: n = 47 (44 m/ 3 f)age: 21 – 77 years (median: 48 years)cotinine (U): < 1 – 34 µg/L urine

smokers: n = 36 (35 m/ 1 f)age: 27 – 66 years (median: 45 years)cigarette consumption: 2 – 35 cig./daycotinine (U): 56 – 4940 µg/L urine

Pilot human biomonitoring study

approval of the ethics committee of the RWTH Aachen University for collection of urine samples (EK 206/09)



CEMA CHEMA

µg/L µg/g Creatinine µg/L µg/g Creatinine

Non-smokers

(n=47)

n > LOD 37 (79 %) 4 (8.5 %)

median 1.9 1.5 < 1 < 1

95th perc. 6.0 5.9 1.1 0.8

max. value 16.4 7.8 3.0 1.8

smokers

(n=36)

n > LOD 36 (100 %) 34 (94 %)

median 184 148 29.3 23.1

95th perc. 412 393 91 74

max. value 907 485 147 98

Results



Relationship CHEMA:CEMA

Mean portion of CHEMA relative to CEMA ~ 13 %

Smoking and CEMA

0 5 10 15 20 25 30 35 40 45

0

100

200

300

400

500

600

700

800

y = 11.4 * x + 29.3r = 0.446p = 0.006

CEM

A (µ

g/L

urin

e)

cigarettes/day



Relationship CEMA:Cotinine



0 10 20 30 40

0

20

40

60

80

100

120

140

160

180

200

220

end of smoking cessation

start of smoking cessation

Mer

capt

uric

aci

d (µ

g/g

crea

tinin

e)

time (h)

CEMA CHEMA



Kinetics after smoking cessation

t½ ~ 10-12 h

Smoker (m, 48 years, 1 pack/day) ceased smoking for 33 h and collected urine samples



Overview

Study CEMA(µg/L)

CHEMA(µg/L)

HEMA(µg/L)

This Study,smokers (n=36)

median 184 29n.b.

max. value 907 147Schettgen et al. 2008smokers (n=14)

mediann.b. n.b.

4.0max. value 6.0

Eckert et al. 2012smokers (n=40)

mediann.b. n.b.

4.3max. value 49.4

Alwis et al. 2012smokers (n=347)

mean 187 ± 181 n.b. 1.9 ± 3.7

Calafat et al.1999smokers (n=170)

mediann.b. n.b.

2.895th perc. 15.4

CHEMA (not HEMA) is the main mercapturate of cyanoethylene-epoxide in humans (≠ rats)!

Literature overview: metabolites of acrylonitrile in urine of smoking individuals of the gen. population



Summary and Conclusionsfully automated quantification of mercapturates of acrylonitrile (ACN) and genotoxiccyanoethylene‐epoxide (CEO) in urine

Background burden to acrylonitrile in the general population

Smoking considerably contributes to the internal burden to acrylonitrile

CHEMA detectable in 90 % of all smokers‘ urine samples

Close relationship between both metabolites

Mean relationship CHEMA/CEMA is about ~ 0.13

Roughly estimated urinary half‐life of ~10 – 12 h

CHEMA (not HEMA) is the main mercapturate of CEO in humans (≠ rats)!

Method is highly suitable for human biomonitoring of exposure to acrylonitrile andallows a closer estimation of its genotoxic potential

Quantification of the mercapturic acids of acrylonitrile and its genotoxicmetabolite cyanoethylene-epoxide in a pilot human biomonitoring study

T. Schettgen, J. Bertram, T. Kraus

Thank you for your attention!

Institute for Occupational and Social MedicineUniversity Hospital, RWTH Aachen University

www.arbeitsmedizin.rwth-aachen.de

Similar relationship between CHEMA and CEMA: ~ 0.13=> Similar oxidative metabolism for acrylamide and acrylonitrile in humans

M.I. Böttcher, T. Schettgen, B. Kütting, M. Pischetsrieder, J. Angerer: Mercapturic acids of acrylamide and glycidamideas biomarkers of the internal exposure to acrylamide in the general population. Mutat Res 580 (1-2): 167-176 (2005)

Comparison to acrylamide mercapturates



Influence of GSTT1 ?

0 50 100 150 200 250 300 350 400

0.0

0.1

0.2

0.3

0.4

0.5

0.6re

latio

nshi

p ur

inar

y C

HEM

A/C

EMA

hGSTT1 activity (pmol/mg * min)

No visible relationship!

Slight tendency towards higher oxidative metabolism at low GSTT1-activity in accordance with literature…



Accidental environmental release of acrylonitrile

On May 4th/5th 2013, a train derailed and caught fire near Wetteren/Belgium, releasing ~ 300 t of acrylonitrile in the environment.Besides fire fighters and police, about 1.500 people from the village were potentially exposed to acrylonitrile….



6.2 schettgen

Health & Medicine

metabolism of acrylonitrile

rwth aachen university

t of acrylonitrile

b acrylonitrilebutadiene

gl urine smokers

gl urineapproval

processed urine sample

c gl urinecema23