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Cob(I)alamin as a Quantitative Tool for Analysis, Metabolism and Toxicological Studies of Electrophilic Compounds: Butadiene Epoxides, Glycidamide and Sucralose
Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
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: urn:nbn:se:su:diva-55481ISBN: 978-91-7447-242-4 (print)OAI: oai:DiVA.org:su-55481DiVA: diva2:404290
Public defence
2011-04-14, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
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
List of papers
1. Cob(I)alamin for trapping butadiene epoxides in metabolism with rat S9 and for determining associated kinetic parameters
Open this publication in new window or tab >>Cob(I)alamin for trapping butadiene epoxides in metabolism with rat S9 and for determining associated kinetic parameters
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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.

National Category
Natural Sciences
Identifiers
urn:nbn:se:su:diva-30098 (URN)10.1021/tx900088w (DOI)000269892600005 ()19764821 (PubMedID)
Available from: 2009-10-12 Created: 2009-10-02 Last updated: 2017-12-12Bibliographically approved
2. Cob(I)alamin for measuring interspecies variation in butadiene epoxide metabolism in S9 liver fractions and evaluation of in vivo doses estimated from enzyme kinetics and from hemoglobin adducts
Open this publication in new window or tab >>Cob(I)alamin for measuring interspecies variation in butadiene epoxide metabolism in S9 liver fractions and evaluation of in vivo doses estimated from enzyme kinetics and from hemoglobin adducts
(English)In: Toxicology and Applied Pharmacology, ISSN 0041-008X, E-ISSN 1096-0333Article in journal (Other academic) Submitted
National Category
Natural Sciences
Identifiers
urn:nbn:se:su:diva-55414 (URN)
Available from: 2011-03-14 Created: 2011-03-14 Last updated: 2017-12-11Bibliographically approved
3. Quantitative analysis by liquid chromatography-tandem mass spectrometry of glycidamide using cob(I)alamin trapping method: Validation and application to in vitro metabolism of acrylamide
Open this publication in new window or tab >>Quantitative analysis by liquid chromatography-tandem mass spectrometry of glycidamide using cob(I)alamin trapping method: Validation and application to in vitro metabolism of acrylamide
(English)In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778Article in journal (Other academic) Submitted
National Category
Natural Sciences
Identifiers
urn:nbn:se:su:diva-55416 (URN)
Available from: 2011-03-14 Created: 2011-03-14 Last updated: 2017-12-11Bibliographically approved
4. Cob(I)alamin reacts with sucralose to afford an alkylcobalamin: Relevance to in vivo cobalamin and sucralose interaction
Open this publication in new window or tab >>Cob(I)alamin reacts with sucralose to afford an alkylcobalamin: Relevance to in vivo cobalamin and sucralose interaction
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2011 (English)In: Food and Chemical Toxicology, ISSN 0278-6915, E-ISSN 1873-6351, Vol. 49, no 4, 750-757 p.Article in journal (Refereed) Published
Abstract [en]

Vitamin B(12), viz., cyano- or hydroxo-cobalamin, can be chemically or enzymatically converted into the derivatives methyl- and adenosyl-cobalamin, which are complex organometallic cofactors associated with several cobalamin-dependent enzymes. The reduced form of vitamin B(12), cob(I)alamin {Cbl(I)}, obtained by reduction of hydroxocobalamin (OH-Cbl) with e.g. sodium borohydride, is one of the most powerful nucleophiles known. Cbl(I) was shown to react readily with the synthetic sweetener sucralose (1,6-dichloro-1,6-dideoxy-β-d-fructofuranosyl-4-chloro-4-deoxy-α-d-galactopyranoside) in an aqueous system to form an alkylcobalamin (Suc-Cbl). This occurred by replacement of one of the three chlorine atoms of sucralose with a cobalamin moiety. The efficiency of trapping sucralose in presence of excess Cbl(I) was estimated to be >90%. Furthermore, in an in vitro study using human liver S9 with NADPH regeneration, in presence of OH-Cbl and sucralose, Suc-Cbl was shown to be formed. The Suc-Cbl was characterized primarily by LC-ESI(+)-MS/MS. Given the human consumption of sucralose from food and beverages, such a reaction between the sweetener and reduced vitamin B(12) could occur in vivo.

Keyword
Vitamin B12; Sucralose; NADPH; Alkylcobalamin; LC-MS/MS
National Category
Chemical Sciences
Identifiers
urn:nbn:se:su:diva-53222 (URN)10.1016/j.fct.2010.11.037 (DOI)000289136100006 ()21130828 (PubMedID)
Available from: 2011-01-21 Created: 2011-01-21 Last updated: 2017-12-11Bibliographically approved
5. Formation of an alkylcobyrinate from sucralose and mechanistic aspects of its Co-C bond cleavages
Open this publication in new window or tab >>Formation of an alkylcobyrinate from sucralose and mechanistic aspects of its Co-C bond cleavages
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(English)In: European Journal of Inorganic Chemistry, ISSN 1434-1948, E-ISSN 1099-1948Article in journal (Other academic) Submitted
National Category
Natural Sciences
Identifiers
urn:nbn:se:su:diva-55417 (URN)
Available from: 2011-03-14 Created: 2011-03-14 Last updated: 2017-12-11Bibliographically approved

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