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Cob(I)alamin for trapping butadiene epoxides in metabolism with rat S9 and for determining associated kinetic parameters
Stockholm University, Faculty of Science, Department of Environmental Chemistry.
Stockholm University, Faculty of Science, Department of Environmental Chemistry.
Stockholm University, Faculty of Science, Department of Environmental Chemistry.
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2009 (English)In: Chemical Research in Toxicology, ISSN 0893-228X, E-ISSN 1520-5010, Vol. 22, no 9, 1509-1516 p.Article in journal (Refereed) Published
Abstract [en]

The reduced state of vitamin B(12), cob(I)alamin, acts as a supernucleophile that reacts ca. 10(5) times faster than standard nucleophiles, for example, thiols. Methods have been developed for trapping electrophilically reactive compounds by exploiting this property of cob(I)alamin. 1,3-Butadiene (BD) has recently been classified as a group 1 human carcinogen by the International Agency for Research on Cancer (IARC). The carcinogenicity of BD is considered to be dependent on the activation or deactivation of the reactive metabolites of BD, that is, the epoxides (oxiranes) 1,2-epoxy-3-butene (EB), 1,2:3,4-diepoxybutane (DEB), and 1,2-epoxy-3,4-butanediol (EBdiol). Cytochrome P450 (P450) isozymes are involved in oxidation of BD to EB and further activation to DEB. EB and DEB are hydrolyzed by epoxide hydrolases (EH) to 3,4-dihydroxy-1-butene (BDdiol) and EBdiol, respectively. EBdiol can also be formed by oxidation of BDdiol. In the present study, cob(I)alamin was used for instant trapping of the BD epoxide metabolites generated in in vitro metabolism to study enzyme kinetics. The substrates EB, DEB, and BDdiol were incubated with rat S9 liver fraction, and apparent K(m) and apparent V(max), were determined. The ratio of conversion of EB to DEB (by P450) to the rate of deactivation of DEB by EH was 1.09. Formation of EBdiol from hydrolysis of DEB was ca. 10 times faster than that from oxidation of BDdiol. It was also found that the oxidation of EB to DEB was much faster than that of BDdiol to EBdiol. The study offers comparative enzyme kinetic data of different BD metabolic steps, which is useful for quantitative interspecies comparison. Furthermore, a new application of cob(I)alamin was demonstrated for the measurement of enzyme kinetics of compounds that form electophilically reactive metabolites.

Place, publisher, year, edition, pages
2009. Vol. 22, no 9, 1509-1516 p.
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:su:diva-30098DOI: 10.1021/tx900088wISI: 000269892600005PubMedID: 19764821OAI: oai:DiVA.org:su-30098DiVA: diva2:271615
Available from: 2009-10-12 Created: 2009-10-02 Last updated: 2011-03-17Bibliographically approved
In thesis
1. Cob(I)alamin as a Quantitative Tool for Analysis, Metabolism and Toxicological Studies of Electrophilic Compounds: Butadiene Epoxides, Glycidamide and Sucralose
Open this publication in new window or tab >>Cob(I)alamin as a Quantitative Tool for Analysis, Metabolism and Toxicological Studies of Electrophilic Compounds: Butadiene Epoxides, Glycidamide and Sucralose
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Vitamin B12 can be reduced to cob(I)alamin [Cbl(I)], which is one of the most powerful nucleophiles known and referred to as a “supernucleophile”. Cbl(I) was applied as a tool in toxicological studies of the air pollutant 1,3-butadiene (BD), the toxicant acrylamide (AA) present in many foods, and the artificial sweetener sucralose.

BD, a human carcinogen, is metabolised to genotoxic epoxides, two monoepoxides and the most potent diepoxybutane (DEB). AA, classified as a probable human carcinogen, is metabolised to the genotoxic epoxide glycidamide (GA). Due to their reactivity, quantitative analysis of the epoxides presents an analytical challenge. By using Cbl(I) for trapping, a sensitive and accurate method to quantify the epoxides as alkylcobalamins by LC-MS/MS in metabolism studies was developed and validated.

Using the Cbl(I) method, enzyme kinetic parameters, Vmax and Km, were determined for the metabolic steps associated with the BD epoxides and with the formation of GA from AA, in liver S9 fractions of human, mouse and rat.

An approach to estimate dose in vivo (i.e. area under concentration time curve, AUC) of BD epoxides by scaling the enzyme kinetic data was designed. The AUCs obtained from in vitro were evaluated by comparing with AUCs in vivo that were calculated from published haemoglobin adduct data. The AUCs from in vitro and in vivo showed to be in agreement with each other for mouse and rat, and this evaluation allowed prediction of the unknown AUC of DEB in human from BD exposure. This approach has a potential to reduce animal experiments in the future.

Sucralose is of concern due to its chlorinated structure and persistence in the aquatic environment. It was demonstrated that Cbl(I) reacts with sucralose, also under in vitro physiological conditions, which might have toxicological significance. The demonstrated reaction also suggested a potential role for Cbl(I) in dehalogenation/degradation of sucralose. This was evaluated and shown possible using heptamethyl cobyrinate, a model compound for cobalamin.

Place, publisher, year, edition, pages
Stockholm: Department of Materials and Environmental Chemistry, Stockholm University, 2011. 64 p.
National Category
Environmental Sciences
Research subject
Environmental Chemistry
Identifiers
urn:nbn:se:su:diva-55481 (URN)978-91-7447-242-4 (ISBN)
Public defence
2011-04-14, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
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Note
At the time of the doctoral defense, the following papers were unpublished  and had a status as follows: Paper 2: Submitted. Paper 3: Submitted. Paper 5: Submitted.Available from: 2011-03-23 Created: 2011-03-16 Last updated: 2011-03-17Bibliographically approved

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