This study developed a method to simultaneously quantify the mercapturic acids of acrylonitrile (CEMA) and its genotoxic metabolite cyanoethylene-epoxide (CHEMA) in human urine. The method was applied in a pilot human biomonitoring study with 47 non-smokers and 36 smokers. Smokers had significantly higher levels of both metabolites, with CEMA levels correlated to cigarette consumption. CHEMA was detectable in 90% of smokers and accounted for about 13% of total mercapturic acid levels, indicating oxidative metabolism. The method allows estimation of background and smoking-related acrylonitrile exposure and its genotoxic potential in humans.

1. Quantification of the mercapturic acids of
acrylonitrile and its genotoxic metabolite
cyanoethylene-epoxide in a pilot human
biomonitoring study
T. Schettgen, J. Bertram, T. Kraus
Institute for Occupational and Social Medicine
University Hospital, RWTH Aachen University
www.arbeitsmedizin.rwth-aachen.de

2. Introduction
C
N
H2C
acrylonitrile
possibly carcinogenic to humans (IARC class 2B)
• acrylonitrile-butadiene-styrenepolymers („ABS-plastics“)
• synthetic fibers
3 – 15 µg/cigarette*
indoor air (ETS): 0.5 – 1.2 µg/m3 **
• rubber production
• production Vol.: 1.5 Mio t/year
(USA)
Uniklinik RWTH Aachen – Quantification of the mercapturic acids of acrylonitrile…
ISBM Conference Manchester, 09.09.2013
*Hoffmann
**Nazaroff
D. et al. Chem Res Toxicol 14:767-790 (2001)
and Singer. J Expo Anal Environ Epidemiol 14:S71-S77 (2004)
page 2

3. Metabolism of acrylonitrile in rats*
40 %
30 %
*Sumner
13 %
SCJ, Selvaraj L, Nauhaus SK, Fennell TR. Chem Res Toxicol 10: 1152-1160 (1997)
Uniklinik RWTH Aachen – Quantification of the mercapturic acids of acrylonitrile…
ISBM Conference Manchester, 09.09.2013
page 3

4. Aims and questions
• can we detect CHEMA as a specific metabolite of
genotoxic cyanoethylene-epoxide in human urine?
• what is the portion of oxidative vs. direct metabolism?
• Biomarkers of ACN-exposure?
⇒ development of a method for the simultaneous
determination of CEMA and CHEMA in urine
Uniklinik RWTH Aachen – Quantification of the mercapturic acids of acrylonitrile…
ISBM Conference Manchester, 09.09.2013
page 4

5. Method
Phenomenex Luna C 8(2)
150 x 4.6 mm
column
autosampler
V1
gradient
pump
RAM
pump 2
MercK RAM C 8 (25 µm)
25 x 4 mm
LOQ: 1 µg/L Urin
MS/MS
analytes
Waste
urinary matrix
•use of isotopically labelled internal standards (d3-CEMA, d3-CHEMA )
T. Schettgen, J. Bertram, T. Kraus, Talanta 98: 211-219 (2012)
Uniklinik RWTH Aachen – Quantification of the mercapturic acids of acrylonitrile…
ISBM Conference Manchester, 09.09.2013
page 5

6. Chromatogram
XIC of -MRM (5 pairs): 231.0/83.9 amu from Sample 15 (Qreal_15)
Quantifying transition
9.29
4500
processed urine sample of a smoker (creatinine: 1.05 g/L, Cotinine: 1360 µg/L)
CHEMA
4000
23.4 µg/L
3500
Intensity, cps
XIC of -MRM (5 pairs): 214.9/161.8 amu from Sample 15 (Qreal_15)
3000
2500
12.34
2000
1.6e4
CEMA
1500
1000
15.72
500
1.2e4
1
2
3
4
5
6
7
8 9 10 11 12 13 14 15 16 17
Time, min
XIC of -MRM (5 pairs): 231.0/101.9 amu from Sample 15 (Qreal_15)
4000
9.29
Qualifying transition
CHEMA
3600
Intensity, cps
0
188 µg/L
1.4e4
3200
1.0e4
8000
6000
4000
2800
Intensity, cps
2000
2400
2000
0
1600
1
2
3
4
5
6
7
1200
XIC of -MRM (5 pairs): 218.0/164.9 amu from Sample 15 (Qreal_15)
800
400
0
8 9 10 11 12 13 14 15 16 17
Time, min
1
2
3
4
5
6
8 9 10 11 12 13 14 15 16 17
Time, min
XIC of -MRM (5 pairs): 234.0/83.9 amu from Sample 15 (Qreal_15)
1.2e4
1.6e4
1.4e4
Intensity, cps
1.2e4
1.0e4
8000
Intensity, cps
D3-CHEMA
1.8e4
D3-CEMA
1.4e4
9.27
2.0e4
12.30
1.6e4
7
1.0e4
8000
6000
4000
6000
2000
4000
13.19
2000
0
0
1
2
3
4
5
6
7
8 9 10 11 12 13 14 15 16 17
Time, min
Uniklinik RWTH Aachen – Quantification of the mercapturic acids of acrylonitrile…
ISBM Conference Manchester, 09.09.2013
1
2
3
4
5
6
7
8 9 10 11 12 13 14 15 16 17
Time, min
page 6

7. Precision and accuracy
Analyte
Qlow
Qmedium
Qhigh
Qreal
Within
Between
Within
Between
Within
Between
Within
Between
series
series
series
series
series
series
series
series
(n=6)
(n=10)
(n=6)
(n=10)
(n=6)
(n=10)
(n=6)
(n=10)
c (µg/L urine)
CHEMA
std. dev.(%)
8.3
3.9
42.0
8.0
2.6
85.7
5.9
2.9
3.2
accuracy (%)
98
100
15.1
71.8
3.5
4.7
---
94
c (µg/L urine)
CEMA
23.2
---
142.9
std. dev.(%)
accuracy (%)
8.8
11.2
2.2
101
Uniklinik RWTH Aachen – Quantification of the mercapturic acids of acrylonitrile…
ISBM Conference Manchester, 09.09.2013
5.6
96
2.4
196.4
6.4
95
3.1
14.5
---
---
page 7

8. Pilot human biomonitoring study
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./day
cotinine (U): 56 – 4940 µg/L urine
approval of the ethics committee of the RWTH Aachen University for collection of urine samples (EK 206/09)
Uniklinik RWTH Aachen – Quantification of the mercapturic acids of acrylonitrile…
ISBM Conference Manchester, 09.09.2013
page 8

9. Results
CEMA
µg/L
n > LOD
Non-smokers median
CHEMA
µg/g Creatinine
µg/L
37 (79 %)
µg/g Creatinine
4 (8.5 %)
1.5
<1
<1
95th perc.
6.0
5.9
1.1
0.8
max. value
(n=47)
1.9
16.4
7.8
3.0
1.8
n > LOD
36 (100 %)
34 (94 %)
smokers
median
184
148
29.3
23.1
(n=36)
95th perc.
412
393
91
74
max. value
907
485
147
98
Uniklinik RWTH Aachen – Quantification of the mercapturic acids of acrylonitrile…
ISBM Conference Manchester, 09.09.2013
page 9

10. Relationship CHEMA:CEMA
Mean portion of CHEMA relative to CEMA ~ 13 %
Uniklinik RWTH Aachen – Quantification of the mercapturic acids of acrylonitrile…
ISBM Conference Manchester, 09.09.2013
page 10

11. Smoking and CEMA
800
700
y = 11.4 * x + 29.3
r = 0.446
p = 0.006
CEMA (µg/L urine)
600
500
400
300
200
100
0
0
5
10
15
20
25
30
35
40
45
cigarettes/day
Uniklinik RWTH Aachen – Quantification of the mercapturic acids of acrylonitrile…
ISBM Conference Manchester, 09.09.2013
page 11

12. Relationship CEMA:Cotinine
Uniklinik RWTH Aachen – Quantification of the mercapturic acids of acrylonitrile…
ISBM Conference Manchester, 09.09.2013
page 12

13. Kinetics after smoking cessation
Smoker (m, 48 years, 1 pack/day) ceased smoking for 33 h and collected urine samples
220 start of smoking cessation
CEMA
CHEMA
Mercapturic acid (µg/g creatinine)
200
180
160
140
120
end of smoking cessation
100
80
60
40
20
0
0
10
20
30
40
time (h)
t½ ~ 10-12 h
Uniklinik RWTH Aachen – Quantification of the mercapturic acids of acrylonitrile…
ISBM Conference Manchester, 09.09.2013
page 13

14. Overview
Literature overview: metabolites of acrylonitrile in urine of smoking individuals of the gen. population
Study
This Study,
smokers (n=36)
Schettgen et al. 2008
smokers (n=14)
Eckert et al. 2012
smokers (n=40)
Alwis et al. 2012
smokers (n=347)
Calafat et al.1999
smokers (n=170)
CEMA
(µg/L)
median
max. value
median
max. value
median
max. value
mean
CHEMA
(µg/L)
HEMA
(µg/L)
184
907
29
147
n.b.
n.b.
n.b.
n.b.
n.b.
187 ± 181
n.b.
n.b.
n.b.
median
95th perc.
4.0
6.0
4.3
49.4
1.9 ± 3.7
2.8
15.4
CHEMA (not HEMA) is the main mercapturate of cyanoethylene-epoxide in humans (≠ rats)!
Uniklinik RWTH Aachen – Quantification of the mercapturic acids of acrylonitrile…
ISBM Conference Manchester, 09.09.2013
page 14

15. Summary and Conclusions
fully automated quantification of mercapturates of acrylonitrile (ACN) and genotoxic
cyanoethylene‐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 and
allows a closer estimation of its genotoxic potential
Uniklinik RWTH Aachen – Quantification of the mercapturic acids of acrylonitrile…
ISBM Conference Manchester, 09.09.2013
page 15

16. Quantification of the mercapturic acids of acrylonitrile and its genotoxic
metabolite cyanoethylene-epoxide in a pilot human biomonitoring study
T. Schettgen, J. Bertram, T. Kraus
Thank you for your attention!
Institute for Occupational and Social Medicine
University Hospital, RWTH Aachen University
www.arbeitsmedizin.rwth-aachen.de

17. Comparison to acrylamide mercapturates
M.I. Böttcher, T. Schettgen, B. Kütting, M. Pischetsrieder, J. Angerer: Mercapturic acids of acrylamide and glycidamide
as biomarkers of the internal exposure to acrylamide in the general population. Mutat Res 580 (1-2): 167-176 (2005)
Similar relationship between CHEMA and CEMA: ~ 0.13
=> Similar oxidative metabolism for acrylamide and acrylonitrile in humans
Uniklinik RWTH Aachen – Quantification of the mercapturic acids of acrylonitrile…
ISBM Conference Manchester, 09.09.2013
page 17

18. Influence of GSTT1 ?
relationship urinary CHEMA/CEMA
0.6
No visible relationship!
0.5
Slight tendency towards higher oxidative
metabolism at low GSTT1-activity in
accordance with literature…
0.4
0.3
0.2
0.1
0.0
0
50
100
150
200
250
300
350
400
hGSTT1 activity (pmol/mg * min)
Uniklinik RWTH Aachen – Quantification of the mercapturic acids of acrylonitrile…
ISBM Conference Manchester, 09.09.2013
page 18

19. 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….
Uniklinik RWTH Aachen – Quantification of the mercapturic acids of acrylonitrile…
ISBM Conference Manchester, 09.09.2013
page 19