N-(2-Hydroxyethyl)-l-valyl-l-leucine in rat urine as a hydrolytic cleavage product of ethylene oxide adduct with globin
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Ethylene oxide (EO), a genotoxic industrial chemical and sterilant, forms covalent adducts with DNA and also with nucleophilic amino acids in proteins. The adduct with N-terminal valine in globin [N-(2-hydroxyethyl)valine (HEV)] has been used in biomonitoring of cumulative exposures to EO. Here we studied in rats the fate of EO-adducted N-termini of globin after life termination of the erythrocytes. Rat erythrocytes were incubated with EO to produce the HEV levels in globin at 0.4–13.2 µmol/g as determined after acidic hydrolysis. Alternative hydrolysis of the isolated globin with enzyme pronase afforded N-(2-hydroxyethyl)-l-valyl-l-leucine (HEVL) and N-(2-hydroxyethyl)-l-valyl-l-histidine (HEVH), the EO-adducted N-terminal dipeptides of rat globin α- and β-chains, respectively. The ratio of HEVL/HEVH (1:3) reflected higher reactivity of EO with the β-chain. The EO-modified erythrocytes were then given intravenously to the recipient rats. HEVL and HEVH were found to be the ultimate cleavage products excreted in the rat urine. Finally, rats were dosed intraperitoneally with EO, 50 mg/kg. Herein, the initial level of globin-bound HEVL (11.7 ± 1.3 nmol/g) decreased almost linearly over 60 days corresponding to the life span of rat erythrocytes. Daily urinary excretion of HEVL was almost constant for 30–40 days, decreasing faster in the subsequent phase of elimination. Recoveries of the total urinary HEVL from its globin-bound form were 84 ± 6% and 101 ± 17% after administrations of EO and the EO-modified erythrocytes, respectively. In conclusion, urinary HEVL appears to be a promising novel non-invasive biomarker of human exposures to EO.
KeywordsEthylene oxide Globin adducts Hydrolytic cleavage Dipeptidic urinary adducts Biomonitoring
The study was supported by the Grant NT13401-4/2012 from Internal Grant Agency of the Czech Ministry of Health, and by the Czech Ministry of Health, Program DRO (National Institute of Public Health—NIPH, IN 75010330). Purchase of the principal analytical instrument, Orbitrap Q Exactive mass spectrometer, was supported by European Regional Development Fund (ERDF), Integrated Operational Programme Reg. No. CZ.1.06/3.2.01/11.08435. Skilled technical assistance of Hana Chrástecká, Monika Tvrdíková, and Radka Vajtrová (all from NIPH) is gratefully acknowledged.
Compliance with ethical standards
Research involving human and/or animal participants
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. The experiments were approved by the Committee for Animal Protection of the Czech Ministry of Health.
The manuscript does not contain clinical studies or patient data.
Conflict of interest
The authors declare that they have no conflict of interest.
- Altman PL, Dittmer DS (eds) (1974) Biology data book, vol III. Federation of American Societies for Experimental Biology, p 1847Google Scholar
- Basile A, Ferranti P, Pocsfalvi G, Mamone G, Miraglia N, Caira S, Ambrosi L, Soleo L, Sannolo N, Malorni A (2001) A novel approach for identification and measurement of hemoglobin adducts with 1,2,3,4-diepoxybutane by liquid chromatography/electrospray ionisation mass spectrometry and matrix-assisted laser desorption/ionisation tandem mass spectrometry. Rapid Commun Mass Spectrom 15(8):527–540CrossRefGoogle Scholar
- Basile A, Ferranti P, Mamone G, Manco I, Pocsfalvi G, Malorni A, Acampora A, Sannolo N (2002) Structural analysis of styrene oxide/haemoglobin adducts by mass spectrometry: identification of suitable biomarkers for human exposure evaluation. Rapid Commun Mass Spectrom 16(9):871–878CrossRefGoogle Scholar
- Calleman CJ, Ehrenberg L, Jansson B, Osterman-Golkar S, Segerbäck D, Svensson K, Wachtmeister CA (1978) Monitoring and risk assessment by means of alkyl groups in hemoglobin in persons occupationally exposed to ethylene oxide. J Environ Pathol Toxicol 2(2):427–442Google Scholar
- DFG-Deutsche Forschungsgemeinschaft (1991) Analyses of hazardous substances in air, vol 3. Wiley, Hoboken, pp 55–63Google Scholar
- DFG-Deutsche Forschungsgemeinschaft (1997) Analyses of hazardous substances in biological materials, vol 5. Wiley, Hoboken, pp 181–210Google Scholar
- Fennell TR, Sumner SCJ, Walker VE (1992) A model for the formation and removal of hemoglobin adducts. Cancer Epidemiol Biomark Prev 1(3):213–219Google Scholar
- Homolka J (1971) Clinical examination methods, 2nd edn., p 96, Avicenum, Praha (in Czech) Google Scholar
- IARC - International Agency for Research on Cancer (2008) 1,3-butadiene, ethylene oxide and vinyl halides, IARC monographs on the evaluation of carcinogenic risks to humans, vol 97. World Health Organization, GenevaGoogle Scholar
- Lauwerys RR, Hoet P (2001) Industrial chemical exposure. Guidelines for biological monitoring, 3rd edn. Lewis Publishers, Boca Raton (ISBN 1-56670-545-2) Google Scholar
- Mráz J, Hanzlíková I, Moulisová A, Dušková Š, Hejl K, Bednářová A, Linhart I (2016a) Hydrolytic cleavage products of globin adducts in urine as possible biomarkers of cumulative dose. Proof of concept using styrene oxide as a model adduct-forming compound. Chem Res Toxicol 29(4):676–686CrossRefGoogle Scholar
- OSHA-Occupational Safety and Health Administration (2014) Method no. 1010 ethylene oxide. https://www.osha.gov/dts/sltc/methods/mdt/mdt1010/1010.html
- Walker VE, MacNeela JP, Swenberg JA, Turner MJ Jr, Fennell TR (1992) Molecular dosimetry of ethylene oxide: formation and persistence of N-(2-hydroxyethyl)valine in hemoglobin following repeated exposures of rats and mice. Cancer Res 52(16):4320–4327Google Scholar
- Wolfs P, Dutrieux M, Scailteur V, Haxhe JJ, Zumofen M, Lauwerys R (1983) Monitoring of workers exposed to ethylene oxide in a plant distributing sterilizing gases and in units for sterilizing medical equipment. Arch Mal Prof 44(3):321–328 (in French) Google Scholar