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Biomarkers of internal exposure/dose: Methods to quantify adducts to protein and DNA by LC/MS studied with benzo[a]pyrene and isocyanates
Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis focuses on methods for quantification by liquid chromatography/mass spectrometry (LC/MS) of specific biomarkers for internal dose of chemicals which induce toxicity through their electrophilic reactivity. In vivo such compounds are short-lived, and could feasibly be measured as their reaction products (adducts) with biomacromolecules. Analysis by MS methods of stable adducts offers the specificity and accuracy required to generate data on internal dose useful in risk estimation.

The primary aim was to develop a method for quantification by LC/MS of bulky adducts to serum albumin (SA) from polycyclic aromatic hydrocarbons, using the genotoxic diolepoxide (DE) of benzo[a]pyrene (BP) as a model. A method for analysis of the BPDE adducts to His146 in SA was developed which is robust, easy-to-use, has good reproducibility and which reached a high sensitivity. A method for quantification of BPDE adducts to N2-deoxyguanosine (dG) in DNA by LC/MS was also established.

In mice exposed to BP, adducts to SA and DNA from stereoisomers of BPDE were identified and quantified. The adduct level was shown to be >400 times higher in DNA than in SA, which from an in vitro study could be concluded to mainly depend on a large difference in the rates of adduct formation to His in SA and to dG in DNA. BPDE adduct levels to SA and DNA, and a biomarker of genotoxic effect (frequency of micronuclei), were compared in BP-exposed mice. The results were used to evaluate how these methods could be used in procedures for cancer risk estimation.

An LC/MS method for analysis of valine hydantoins (VH) formed as adducts from isocyanates to N-termini in haemoglobin was established. VH, formed from urea/isocyanic acid, was investigated in mice as a potential biomarker of renal failure and for dose adjustment during treatment with a radioactive cytostatic drug. The kidney dysfunction was not severe enough to give a significant increase of VH in the experiment. 

Place, publisher, year, edition, pages
Stockholm: Department of Materials and Environmental Chemistry, Stockholm University , 2015. , 66 p.
Keyword [en]
biomarker, PAH, serum albumin, adduct, DNA, isocyanate
National Category
Chemical Sciences
Research subject
Environmental Chemistry
Identifiers
URN: urn:nbn:se:su:diva-110490ISBN: 978-91-7649-074-7 (print)OAI: oai:DiVA.org:su-110490DiVA: diva2:772102
Public defence
2015-01-29, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.

Available from: 2015-01-07 Created: 2014-12-15 Last updated: 2015-08-17Bibliographically approved
List of papers
1. Conditions for sample preparation and quantitative HPLC/MS-MS analysis of bulky adducts to serum albumin with diolepoxides of polycyclic aromatic hydrocarbons as models
Open this publication in new window or tab >>Conditions for sample preparation and quantitative HPLC/MS-MS analysis of bulky adducts to serum albumin with diolepoxides of polycyclic aromatic hydrocarbons as models
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2014 (English)In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 406, no 5, 1519-1530 p.Article in journal (Refereed) Published
Abstract [en]

Stable adducts to serum albumin (SA) from electrophilic and genotoxic compounds/metabolites can be used as biomarkers for quantification of the corresponding in vivo dose. In the present study, conditions for specific analysis of stable adducts to SA formed from carcinogenic polycyclic aromatic hydrocarbons (PAH) were evaluated in order to achieve a sensitive and reproducible quantitative method. Bulky adducts from diolepoxides (DE) of PAH, primarily DE of benzo[a]pyrene (BPDE) and also DE of dibenzo[a,l]pyrene (DBPDE) and dibenzo[a,h]anthracene (DBADE), were used as model compounds. The alkylated peptides obtained after enzymatic hydrolysis of human SA modified with the different PAHDE were principally PAHDE-His-Pro, PAHDE-His-Pro-Tyr and PAHDE-Lys. Alkaline hydrolysis under optimised conditions gave the BPDE-His as the single analyte of alkylated His, but also indicated degradation of this adduct. It was not possible to obtain the BPDE-His as one analyte from BPDE-alkylated SA through modifications of the enzymatic hydrolysis. The BPDE-His adduct was shown to be stable during the weak acidic conditions used in the isolation of SA. Enrichment by HPLC or SPE, but not butanol extraction, gave good recovery, using Protein LoBind tubes. A simple internal standard (IS) approach using SA modified with other PAHDE as IS was shown to be applicable. A robust analytical procedure based on digestion with pronase, enrichment by HPLC or SPE, and analysis with HPLC/MS-MS electrospray ionisation was achieved. A good reproducibility (coefficient of variation (CV) 11 %) was obtained, and the achieved limit of detection for the studied PAHDE, using standard instrumentation, was approximately 1 fmol adduct/mg SA analysing extract from 5 mg SA.

Keyword
Bulky serum albumin adducts, Polycyclic aromatic hydrocarbons, Extraction (SPE vertical bar HPLC vertical bar butanol), Diol epoxides, Mass Spectrometry, Hydrolysis (pronase enzymatic vertical bar alkaline)
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-102079 (URN)10.1007/s00216-013-7540-7 (DOI)000331004700023 ()
Funder
Formas
Note

AuthorCount:7;

Funding agencies:

European Commission, QLK4-CT-2002-02402; Swedish Research Council FORMAS;    Swedish Cancer and Allergy Foundation 

Available from: 2014-03-27 Created: 2014-03-26 Last updated: 2017-12-05Bibliographically approved
2. Adduct levels from benzo[a]pyrenediol epoxide: Relative formation to histidine in serum albumin and to deoxyguanosine in DNA in vitro and in vivo in mice by LC/MS-MS methods
Open this publication in new window or tab >>Adduct levels from benzo[a]pyrenediol epoxide: Relative formation to histidine in serum albumin and to deoxyguanosine in DNA in vitro and in vivo in mice by LC/MS-MS methods
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2015 (English)In: Toxicology Letters, ISSN 0378-4274, E-ISSN 1879-3169, Vol. 232, no 1, 28-36 p.Article in journal (Refereed) Published
Abstract [en]

Stable and specific biomacromolecular adducts can be used to measure in vivo doses of reactive compounds. An LC/MS-MS method to measure adducts from the benzo[a]pyrene (BP) metabolite (±)-anti-BP-7,8-diol-9,10-epoxide ((±)-anti-BPDE) to His146 in serum albumin (SA), earlier evaluated on in vitro alkylated human SA, was tested for its applicability to mouse. It was shown that (+)-anti-BPDE form BPDE-His adducts to mouse SA. The method was applied to samples from BP-exposed mice (100 mg/kg of body weight for 1, 3, 7 and 28 days). BPDE-His in SA was close to the limit of quantification and showed the highest level (13 fmol/mg) 3 days after exposure. The level was 400 times lower (calculated per g macromolecule) than earlier measured level of BPDE-adduct to deoxyguanosine (dG) in DNA in the livers. The relative rate of formation of adducts from BPDE with His in SA and with dG in DNA was investigated. Quantification by LC/MS-MS of the adducts in human blood alkylated in vitro with (±)-anti-BPDE showed a 1850 times higher level of BPDE-dG compared to BPDE-His. The specific and stable BPDE-adducts to His in SA are potential biomarkers of in vivo dose of BPDE, though this requires a considerable improved analytical sensitivity of the LC/MS-MS method.

Keyword
DNA adduct, Serum albumin
National Category
Analytical Chemistry
Research subject
Environmental Chemistry
Identifiers
urn:nbn:se:su:diva-110326 (URN)10.1016/j.toxlet.2014.09.019 (DOI)000346175100004 ()
Funder
EU, European Research Council, QLK4-CT-2002-02402Swedish Research Council FormasSwedish Environmental Protection Agency
Available from: 2014-12-11 Created: 2014-12-11 Last updated: 2017-12-05Bibliographically approved
3. Comparison of specific biomarkers of internal dose and genotoxic effect after exposure to BP with the aim to use in risk assessment
Open this publication in new window or tab >>Comparison of specific biomarkers of internal dose and genotoxic effect after exposure to BP with the aim to use in risk assessment
Show others...
(English)Manuscript (preprint) (Other academic)
National Category
Chemical Sciences
Identifiers
urn:nbn:se:su:diva-110482 (URN)
Available from: 2014-12-16 Created: 2014-12-15 Last updated: 2016-01-29Bibliographically approved
4. A New General Pathway for Synthesis of Reference Compounds of N-Terminal Valine-Isocyanate Adducts
Open this publication in new window or tab >>A New General Pathway for Synthesis of Reference Compounds of N-Terminal Valine-Isocyanate Adducts
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2010 (English)In: Chemical Research in Toxicology, ISSN 0893-228X, E-ISSN 1520-5010, Vol. 23, no 3, 540-546 p.Article in journal (Refereed) Published
Abstract [en]

Adducts to Hb could be used as biomarkers to monitor exposure to isocyanates. Particularly useful is the measurement of carbamoylation of N-terminal valines in Hb, after detachment as hydantoins. The synthesis of references from the reactive isocyanates, especially diisocyanates, has been problematic due to side reactions and polymerization of the isocyanate starting material. A simpler, safer, and more general method for the synthesis of valine adducts of isocyanates has been developed using N-[(4-nitrophenyl)-carbamate]valine methylamide (NPCVMA) as the key precursor to adducts of various mono- and diisocyanates of interest. By reacting NPCVMA with a range of isocyanate-related amines, carbamoylated valines are formed without the use of the reactive isocyanates. The carbamoylated products synthesized here were cyclized with good yields of the formed hydantoins. The carbamoylated derivative from phenyl isocyanate also showed quantitative yield in a test with cyclization tinder the conditions used in blood. This new pathway for the preparation of N-carbamoylated model compounds overcomes the above-mentioned problems in the synthesis and is a general and simplified approach, which could make such reference compounds of adducts to N-terminal valine from isocyanates accessible for biomonitoring purposes. The synthesized hydantoins corresponding to adducts from isocyanic acid, methyl isocyanate, phenyl isocyanate, and 2,6-toluene diisocyanate were characterized by LC-MS analysis. The background level of the hydantoin from isocyanic acid in human blood was analyzed with the LC-MS conditions developed.

National Category
Chemical Sciences
Identifiers
urn:nbn:se:su:diva-52218 (URN)10.1021/tx900278p (DOI)000275411700015 ()
Note

authorCount :6

Available from: 2011-01-13 Created: 2011-01-13 Last updated: 2017-12-11Bibliographically approved
5. Evaluation of retinol binding protein 4 and carbamoylated haemoglobin as potential renal toxicity biomarkers in adult mice treated with Lu-177-octreotate
Open this publication in new window or tab >>Evaluation of retinol binding protein 4 and carbamoylated haemoglobin as potential renal toxicity biomarkers in adult mice treated with Lu-177-octreotate
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2014 (English)In: EJNMMI Research, ISSN 2191-219X, E-ISSN 2191-219X, Vol. 4, no 59Article in journal (Refereed) Published
Abstract [en]

Background: The kidneys are regarded as one of the main dose-limiting organs in the treatment of neuroendocrine tumours with Lu-177-[DOTA(0), Tyr(3)]-octreotate (Lu-177-octreotate), despite the successful use of kidney uptake blocking agents such as lysine and arginine. To avoid renal toxicity but still give each patient as high amount of Lu-177-octreotate as possible, there is a need for methods/biomarkers that indicate renal injury in an early stage of the treatment. The aim of this study was to investigate the potential of using urinary retinol binding protein 4 (RBP4) and carbamoylated haemoglobin (Hb) in blood as biomarkers of nephrotoxic effects on adult mice after Lu-177-octreotate treatment. Methods: Adult BALB/c nude mice were injected with 60 MBq or 120 MBq of Lu-177-octreotate or with saline (control). Urine was collected before injection and concentrations of urinary RBP4 and creatinine were determined 14 to 90 days after injection Blood samples were collected after 90 days, and carbamoylated N-terminal valine in Hb, formed from urea, was measured as valine hydantoin (VH) after detachment from Hb. Results: The RBP4 values increased with administered activity and time. For the 60 and 120 MBq groups, statistically significantly higher RBP4 levels (p <0.05) were found at day 60 and 90 compared to baseline, also at day 30 for 120 MBq group. For VH, the mean values were similar for the 60 MBq and control groups, while a small increase was observed for the 120 MBq group; but there were no statistically significant differences between any of the groups (p >0.05). No morphological changes in the kidney tissue were found. Conclusions: Urinary RBP4 is a promising new biomarker for radiation-induced renal toxicity. For the conditions used in this experiment, carbamoylated Hb (from urea) measured as VH may not be a sufficiently sensitive biomarker to be used for renal toxicity.

Keyword
Peptide receptor radionuclide therapy, Nephrotoxicity, RBP, Creatinine, Urea, Carbamoylated haemoglobin, Valine hydantoin
National Category
Chemical Sciences
Identifiers
urn:nbn:se:su:diva-110342 (URN)10.1186/s13550-014-0059-x (DOI)000358122900001 ()
Available from: 2014-12-11 Created: 2014-12-11 Last updated: 2017-12-05Bibliographically approved

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