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Methods for measurement of reactive metabolites as a basis for cancer risk assessment: Application to 1,3-butadiene and isoprene
Stockholm University, Faculty of Science, Department of Environmental Chemistry.
Responsible organisation
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
Identifiers
URN: urn:nbn:se:su:diva-78ISBN: 91-7265-840-1 (print)OAI: oai:DiVA.org:su-78DiVA: diva2:199063
Public defence
2004-04-15, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 13:00
Opponent
Supervisors
Available from: 2004-03-25 Created: 2004-03-25Bibliographically approved
List of papers
1. A liquid chromatography tandem mass spectrometric method for in vivo dose monitoring of diepoxybutane, a metabolite of butadiene
Open this publication in new window or tab >>A liquid chromatography tandem mass spectrometric method for in vivo dose monitoring of diepoxybutane, a metabolite of butadiene
2000 In: Rapid Commun. Mass Spectrom., ISSN 0951-4198, Vol. 14, 1848-1853 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:su:diva-25170 (URN)
Note
Part of urn:nbn:se:su:diva-78Available from: 2004-03-25 Created: 2004-03-25Bibliographically approved
2. Hemoglobin adduct levels in rat and mouse treated with 1,2:3,4-diepoxybutane
Open this publication in new window or tab >>Hemoglobin adduct levels in rat and mouse treated with 1,2:3,4-diepoxybutane
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.

National Category
Environmental Sciences
Identifiers
urn:nbn:se:su:diva-25171 (URN)10.1021/tx034214g (DOI)
Note
Part of urn:nbn:se:su:diva-78Available from: 2004-03-25 Created: 2004-03-25 Last updated: 2017-12-13Bibliographically approved
3. Adducts to N-terminal valines in hemoglobin from isoprene di-epoxide, a metabolite of isoprene
Open this publication in new window or tab >>Adducts to N-terminal valines in hemoglobin from isoprene di-epoxide, a metabolite of isoprene
Show others...
2004 (English)In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 18, no 18, 2177-2184 p.Article in journal (Refereed) Published
Abstract [en]

Isoprene (2-methylbuta-1,3-diene) is a multi-site carcinogen in rodents. To evaluate the role of the diepoxide metabolite (1,2:3,4-diepoxy-2-methylbutane) in carcinogenesis, measurements of in vivo doses of the diepoxide are needed. The in vivo dose may be inferred from levels of reaction products with hemoglobin (Hb adducts). This report presents in vitro studies of the adduct formation by the diepoxide of isoprene with valinamide and oligopeptides as model compounds of N-terminal valines in hemoglobin (Hb). In the reaction with valinamide it was shown that isoprene diepoxide forms as the main product a ring-closed adduct, which is a pyrrolidine derivative [N,N-(2,3-dihydroxy-2-methyl-1,4-butadiyl)valinamide, MPyr-Val]. The analysis was performed by gas chromatography/mass spectrometry (GC/MS) (EI and PICI) after acetylation. The ring-closed adduct was also identified by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) as the main product in the reaction between isoprene diepoxide and standard hepta- or (2H8)octapeptides, corresponding to the N-terminal peptides of the α-chains in mouse and rat Hb. These peptides, alkylated with isoprene diepoxide, to be used as internal standards and calibration standards for quantification of MPyr-adduct levels in vitro and in vivo, were analyzed with respect to the degree of MPyr-alkylation by two independent methods, amino acid analysis and HPLC-UV; similar results were obtained using these methods. A method for measurement of Hb adducts as modified peptides, used earlier to measure a similar adduct to N-terminal valines in Hb from the diepoxide of 1,3-butadiene, has in the present work been tested for application to isoprene diepoxide. The method is based on tryptic degradation of globin and LC/ESI-MS analysis of N-terminal Pyr-heptapeptides of the Hb α-chain enriched by HPLC. MPyr-adduct levels in isoprene diepoxide alkylated hemolysate from mouse erythrocytes incubated with different concentrations of isoprene diepoxide (2 and 10 mM) for 1 h were quantified. The adduct level was about 50 nmol/g α-chain Hb per mM × h. From the adduct levels the rate constant of isoprene diepoxide for reaction with N-terminal valine was calculated to be about 1.6 times faster than for diepoxybutane

Place, publisher, year, edition, pages
John Wiley & Sons, Inc, 2004
National Category
Environmental Sciences
Identifiers
urn:nbn:se:su:diva-25172 (URN)10.1002/rcm.1608 (DOI)
Note
Part of urn:nbn:se:su:diva-78Available from: 2004-03-25 Created: 2004-03-25 Last updated: 2017-12-13Bibliographically approved
4. Hemoglobin adducts and micronuclei in rodents after treatment with isoprene monoxide or butadiene monoxide
Open this publication in new window or tab >>Hemoglobin adducts and micronuclei in rodents after treatment with isoprene monoxide or butadiene monoxide
2005 (English)In: Mutation Research, ISSN 1383-5742, E-ISSN 1388-2139, Vol. 585, no 1-2, 21-32 p.Article in journal (Refereed) Published
Abstract [en]

1,3-Butadiene and isoprene (2-methyl-1,3-butadiene) are chemically related substances that are carcinogenic to rodents. The overall aim of this work is to elucidate the role of the genotoxic action of diepoxide metabolites in the carcinogenesis of the dialkenes. In vivo doses of the diepoxide metabolites were measured through reaction products with hemoglobin (Hb adducts) in studies of induced micronuclei (MN) in rodents. In the reaction with N-terminal valine in Hb, diepoxybutane and isoprenediepoxide form ring-closed adducts, pyrrolidines [N,N-(2,3-dihydroxy-1,4-butadiyl)valine and N,N-(2,3-dihydroxy-2-methyl-1,4-butadiyl)valine, respectively]. The method applied for Hb-adduct measurement is based on tryptic degradation of the protein and liquid chromatography electrospray ionisation tandem mass spectrometry (LC–ESI-MS/MS) analysis. Mice were given single i.p. injections of the monoepoxides of butadiene and isoprene, 1,2-epoxy-3-butene or 1,2-epoxy-2-methyl-3-butene, respectively. Rats were treated in the same way with 1,2-epoxy-3-butene. In mice pyrrolidine adduct levels increased with increasing administered doses of the monoepoxides. The in vivo dose of diepoxybutane was on average twice as high (0.29 ± 0.059 mMh) as the in vivo dose of isoprenediepoxide (0.15 ± 0.053 mMh) per administered dose (mmol/kg body weight) of the monoepoxides. In mice the genotoxic effects of the two monoepoxides, measured as the increase in the frequencies of micronuclei (MN), were approximately linearly correlated to the in vivo doses of the diepoxides (except at the highest dose of diepoxybutane). In rats the pyrrolidine-adduct levels from diepoxybutane were below the limit of quantification at all administered doses of 1,2-epoxy-3-butene and no significant increase was observed in the frequency of MN. Measurement of the ring-closed adducts to N-termini in Hb by the applied method permits analysis of in vivo doses of diepoxybutane and isoprenediepoxide, which may be further used for the elucidation of the mechanisms of carcinogenesis of butadiene and isoprene.

Place, publisher, year, edition, pages
Elsevier B.V., 2005
Keyword
Isoprene; Butadiene; Metabolites; LC–MS; Micronucleus; Hemoglobin adduct
National Category
Environmental Sciences
Identifiers
urn:nbn:se:su:diva-25173 (URN)10.1016/j.mrgentox.2005.03.009 (DOI)
Note
Part of urn:nbn:se:su:diva-78Available from: 2004-03-25 Created: 2004-03-25 Last updated: 2017-12-13Bibliographically approved
5. Characterization of alkyl-cobalamins formed in trapping of epoxide metabolites of 1,3-butadiene
Open this publication in new window or tab >>Characterization of alkyl-cobalamins formed in trapping of epoxide metabolites of 1,3-butadiene
Show others...
2004 (English)In: Journal of Separation Science, ISSN 1615-9306, E-ISSN 1615-9314, Vol. 27, no 7-8, 607-612 p.Article in journal (Refereed) Published
Abstract [en]

Analytical methods facilitating studies of electrophilically reactive and genotoxic compounds in vitro and in vivo are needed. The strong nucleophile, cob(I)alamin, formed by reduction of Vitamin B12 [cob(III)alamin], may be used for trapping and analysis of 1,2-epoxides and other electrophiles. In the present study, cob(I)alamin is evaluated as an analytical tool for 1,2-epoxide metabolites (oxiranes) of 1,3-butadiene. Products of reaction of cob(I)alamin with 1,2-epoxy-3-butene (EB), 1,2:3,4-diepoxybutane (DEB), and 1,2-epoxy-3,4-butanediol (EBdiol) have been analyzed by reversed phase high performance liquid chromatography (HPLC) coupled on-line to electrospray ionization mass spectrometry (ESI-MS) and ultraviolet diode array detection (UV-DAD). It was shown that a specific alkyl-Cbl complex is formed for each metabolite and that it was possible to discriminate between the products by HPLC-UV and by LC-MS. Quantification of DEB with the method by use of another 1,2-epoxide as an internal standard was successfully performed. The possibility of using cob(I)alamin for trapping and analysis of the three oxirane metabolites of 1,3-butadiene will facilitate quantitative comparisons of species in vitro with regard to metabolism of 1,3-butadiene.

Keyword
1, 3-Butadiene • Diepoxybutane • Cobalamin • Reactive metabolites • LC-MS
National Category
Environmental Sciences
Identifiers
urn:nbn:se:su:diva-25174 (URN)10.1002/jssc.200301689 (DOI)
Note
Part of urn:nbn:se:su:diva-78Available from: 2004-03-25 Created: 2004-03-25 Last updated: 2017-12-13Bibliographically approved

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