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Hemoglobin adduct levels in rat and mouse treated with 1,2:3,4-diepoxybutane
Stockholm University, Faculty of Science, Department of Environmental Chemistry.
Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK), Environmental Chemistry.
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2004 (English)In: Chemical Research in Toxicology, ISSN 0893-228X, E-ISSN 1520-5010, Vol. 17, no 6, 785-794 p.Article in journal (Refereed) Published
Abstract [en]

For cancer risk assessment of 1,3-butadiene from rodent cancer test data, the in vivo doses of formed 1,2:3,4-diepoxybutane (DEB) should be known. In vivo doses of DEB were measured through a specific reaction product with hemoglobin (Hb), a ring-closed adduct, N,N-(2,3-dihydroxy-1,4-butadiyl)valine (Pyr-Val), to N-terminal valines. An analytical method based on tryptic digestion of Hb and quantification of Pyr-modified heptapeptides by LC-MS/MS has been further developed and applied in vivo to DEB-treated rats. Furthermore, N-(2,3,4-trihydroxybutyl)valine adducts (THB-Val) to the N-terminal valine in Hb were measured in rats and mice treated with DEB and in a complementary experiment with 1,2-epoxy-3,4-butanediol (EBdiol), using a modified Edman degradation method and GC-MS/MS. In vitro reactions of hemolysate with DEB and EBdiol were used to measure reaction rates for adduct formation needed for calculation of doses and rates elimination in vivo. The results showed that the level of the Pyr-Val adduct per administered dose of DEB was approximately the same in rats as had earlier been observed in mice [Kautiainen et al. (2000) Rapid Commun. Mass Spectrom. 14, 1848−1853]. Levels of the THB-Val adduct after DEB treatment were 3−4 times higher in rat than in mouse, probably reflecting an enhanced hydrolysis of DEB to EBdiol catalyzed by epoxide hydrolase. After EBdiol treatment, the THB-Val adduct levels were about the same in rat and mouse. Calculations from in vitro data show that the Pyr-Val adduct is a relevant monitor for the in vivo dose of DEB and that THB-Val primarily reflects doses to EBdiol. The calculated rates of formation of adducts and rates of elimination agree with expectations. Procedures for quantification of Hb adducts as modified peptides as well as preparation and characterization of peptide standards have been evaluated.

Place, publisher, year, edition, pages
2004. Vol. 17, no 6, 785-794 p.
National Category
Environmental Sciences
URN: urn:nbn:se:su:diva-25171DOI: 10.1021/tx034214gOAI: diva2:199059
Part of urn:nbn:se:su:diva-78Available from: 2004-03-25 Created: 2004-03-25 Last updated: 2010-07-30Bibliographically approved
In thesis
1. Methods for measurement of reactive metabolites as a basis for cancer risk assessment: Application to 1,3-butadiene and isoprene
Open this publication in new window or tab >>Methods for measurement of reactive metabolites as a basis for cancer risk assessment: Application to 1,3-butadiene and isoprene
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

1,3-Butadiene is a general air pollutant associated with combustion of organic matter and is also an extensively used monomer in polymer production. The cancer risk estimation of 1,3-butadiene is encumbered with large uncertainties. Extrapolation from tumour frequencies in long-term animal tests has led to a relatively high figure for the risk associated with 1,3-butadiene exposure. This is mainly based on observations of very high tumour incidences in butadiene-exposed mice, which in this respect are about 100 times more sensitive than rats. It has been hypothesized that a high cancer risk from 1,3-butadiene could be associated with its metabolism to the bifunctional 1,2:3,4-diepoxybutane (DEB) which, in comparison with monofunctional epoxides, 1,2-epoxy-3-butene (EB) and 1,2-epoxy-3,4-butanediol (EBdiol), is a highly effective mutagen, i.e. cancer initiator. Measurement of in vivo doses of DEB is therefore essential for the risk assessment of 1,3-butadiene. Reaction products with hemoglobin offer a possibility of measuring reactive metabolites in vivo. Hemoglobin adducts from EBdiol have in this study been measured with available methods, which are, however, not applicable to the bifunctional DEB, and method development was therefore needed.

This work presents a procedure for measurement of a specific, ring-closed adduct, Pyr-Val, formed from the reaction of DEB with N-terminal valines in hemoglobin. It is based on LC-ESI-MS/MS analysis of the Pyr-modified N-terminal peptides enriched after trypsin digestion of globin. Mouse and rat could be compared regarding the metabolism of EB, DEB and EBdiol. From the data it was concluded that, in 1,3-butadiene exposure, about 60 times higher levels of DEB are formed in mice compared to rats. Estimates of in vivo doses in published cancer tests showed that carcinogenesis in mice is mainly due to DEB, whereas in rat, and possibly man, the monofunctional EBdiol is the predominant causative factor. Preliminarily, the cancer risk assessed from these data is compatible with the epidemiology-based risk estimate of US EPA.

Due to the structural similarity with 1,3-butadiene, certain parallel studies of isoprene (2-methyl-1,3-butadiene) metabolism were initiated. Isoprene is the major endogenously produced hydrocarbon in humans and mammals and shows a similar difference in sensitivity between species for tumour development as 1,3-butadiene. In mice treated with the isoprene monoepoxide, 1,2-epoxy-2-methyl-3-butene (IMO), an in vivo formation of the corresponding diepoxide, 1,2:3,4-diepoxy-2-methyl-butane (IDO), was demonstrated. The in vivo dose of IDO formed from IMO was about half of that of DEB formed from EB. In the analysis of bone marrow erythrocytes an increased frequency of micronuclei, induced by treatment with EB or IMO, showed correlation with the in vivo doses of the respective diepoxides.

With the ambition to reduce animal experiments a general procedure has been developed for trapping reactive metabolites in in vitro test systems, with the specific aim to study differences between species in metabolism of 1,3-butadiene. Vitamin B12 in its reduced form [Cbl(I)] has been used for instant trapping of 1,3-butadiene metabolites formed in S9-mixture. LC-ESI-MS/MS is then used for quantification of the formed alkyl-Cbls. The method has been applied to the epoxide metabolites of 1,3-butadiene, which all form specific alkyl-Cbls in the reaction with Cbl(I)

Place, publisher, year, edition, pages
Stockholm: Institutionen för miljökemi, 2004. 84 p.
National Category
Environmental Sciences
urn:nbn:se:su:diva-78 (URN)91-7265-840-1 (ISBN)
Public defence
2004-04-15, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 13:00
Available from: 2004-03-25 Created: 2004-03-25Bibliographically approved

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Athanassiadis, IoannisTörnqvist, Margareta
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