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6.2 schettgen 6.2 schettgen Presentation Transcript

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