Development and validation of a direct sandwich chemiluminescence immunoassay for measuring DNA adducts of benzo[a]pyrene and other polycyclic aromatic hydrocarbons.

2.50
Hdl Handle:
http://hdl.handle.net/10146/232052
Title:
Development and validation of a direct sandwich chemiluminescence immunoassay for measuring DNA adducts of benzo[a]pyrene and other polycyclic aromatic hydrocarbons.
Authors:
Georgiadis, Panagiotis; Kovacs, Katalin; Kaila, Stella; Makedonopoulou, Paraskevi; Anna, Livia; Poirier, Miriam C.; Knudsen, Lisbeth E.; Schoket, Bernadette; Kyrtopoulos, Soterios A.
Abstract:
We have developed and validated a sandwich chemiluminescence immunoassay (SCIA) which measures polycyclic aromatic hydrocarbon (PAH)-DNA adducts combining high throughput and adequate sensitivity, appropriate for evaluation of adduct levels in human population studies. Fragmented DNA is incubated with rabbit antiserum elicited against DNA modified with r7,t8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) and subsequently trapped by goat anti-rabbit IgG bound to a solid surface. Anti-single-stranded (ss) DNA antibodies binds in a quantity proportional to the adduct levels and is detected by chemiluminescence. The BPDE-DNA SCIA has a limit of detection of 3 adducts per 10(9) nucleotides with 5 μg DNA per well. We have validated the BPDE-DNA SCIA using DNA modified in vitro, DNA from benzo[a]pyrene (BP)-exposed cultured cells and mice. The levels of adduct measured by SCIA were lower (30-60%) than levels of bulky DNA adducts measured in the same samples by (32)P-postlabelling. The BPDE-DNA SCIA also detected adducts produced in vivo by PAHs other than BP. When blood DNA samples from maternal/infant pairs were assayed by BPDE-DNA SCIA, the adduct levels obtained were significantly correlated. However, there was no correlation between (32)P-postlabelling and SCIA values for the same samples. The SCIA can be extended to any DNA adduct and is expected to provide, when fully automated, a valuable high-throughput approach in large-scale population studies.
Citation:
Mutagenesis 2012, 27 (5):589-597
Journal:
Mutagenesis
Issue Date:
18-May-2012
URI:
http://hdl.handle.net/10146/232052
DOI:
10.1093/mutage/ges024
PubMed ID:
22610669
Additional Links:
http://mutage.oxfordjournals.org/content/early/2012/05/17/mutage.ges024.long
Type:
Article
Language:
en
ISSN:
1464-3804
Sponsors:
This work was supported by contracts from the European Union (to S.A.K): Network of Excellence ECNIS—‘Environmental cancer risk, nutrition and individual susceptibility’ and Integrated Project NewGeneris—‘Newborns and genotoxic exposure risks’ (contract numbers FOOD-CT-2005-513943, FOOD-CT-2005-016320). The work was also supported in part by the intramural program of the Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA (to M.C.P.).
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Full metadata record

DC FieldValue Language
dc.contributor.authorGeorgiadis, Panagiotisen_GB
dc.contributor.authorKovacs, Katalinen_GB
dc.contributor.authorKaila, Stellaen_GB
dc.contributor.authorMakedonopoulou, Paraskevien_GB
dc.contributor.authorAnna, Liviaen_GB
dc.contributor.authorPoirier, Miriam C.en_GB
dc.contributor.authorKnudsen, Lisbeth E.en_GB
dc.contributor.authorSchoket, Bernadetteen_GB
dc.contributor.authorKyrtopoulos, Soterios A.en_GB
dc.date.accessioned2012-07-04T09:14:07Z-
dc.date.available2012-07-04T09:14:07Z-
dc.date.issued2012-05-18-
dc.identifier.citationMutagenesis 2012, 27 (5):589-597en_GB
dc.identifier.issn1464-3804-
dc.identifier.pmid22610669-
dc.identifier.doi10.1093/mutage/ges024-
dc.identifier.urihttp://hdl.handle.net/10146/232052-
dc.description.abstractWe have developed and validated a sandwich chemiluminescence immunoassay (SCIA) which measures polycyclic aromatic hydrocarbon (PAH)-DNA adducts combining high throughput and adequate sensitivity, appropriate for evaluation of adduct levels in human population studies. Fragmented DNA is incubated with rabbit antiserum elicited against DNA modified with r7,t8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) and subsequently trapped by goat anti-rabbit IgG bound to a solid surface. Anti-single-stranded (ss) DNA antibodies binds in a quantity proportional to the adduct levels and is detected by chemiluminescence. The BPDE-DNA SCIA has a limit of detection of 3 adducts per 10(9) nucleotides with 5 μg DNA per well. We have validated the BPDE-DNA SCIA using DNA modified in vitro, DNA from benzo[a]pyrene (BP)-exposed cultured cells and mice. The levels of adduct measured by SCIA were lower (30-60%) than levels of bulky DNA adducts measured in the same samples by (32)P-postlabelling. The BPDE-DNA SCIA also detected adducts produced in vivo by PAHs other than BP. When blood DNA samples from maternal/infant pairs were assayed by BPDE-DNA SCIA, the adduct levels obtained were significantly correlated. However, there was no correlation between (32)P-postlabelling and SCIA values for the same samples. The SCIA can be extended to any DNA adduct and is expected to provide, when fully automated, a valuable high-throughput approach in large-scale population studies.en_GB
dc.description.sponsorshipThis work was supported by contracts from the European Union (to S.A.K): Network of Excellence ECNIS—‘Environmental cancer risk, nutrition and individual susceptibility’ and Integrated Project NewGeneris—‘Newborns and genotoxic exposure risks’ (contract numbers FOOD-CT-2005-513943, FOOD-CT-2005-016320). The work was also supported in part by the intramural program of the Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA (to M.C.P.).en_GB
dc.languageENG-
dc.language.isoenen
dc.relation.urlhttp://mutage.oxfordjournals.org/content/early/2012/05/17/mutage.ges024.longen_GB
dc.rightsArchived with thanks to Mutagenesisen_GB
dc.subjectPolycyclic Aromatic Hydrocarbons (PAHs)en_GB
dc.subjectBenzo[a]pyreneen_GB
dc.subjectDNA adductsen_GB
dc.subjectHumanen_GB
dc.subjectChemiluminescenceen_GB
dc.subjectImmunoassaysen_GB
dc.subject(32)P-postlabellingen_GB
dc.subject.mesh7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide-
dc.subject.meshAdult-
dc.subject.meshAnimals-
dc.subject.meshDNA Adducts-
dc.subject.meshEnzyme-Linked Immunosorbent Assay-
dc.subject.meshFemale-
dc.subject.meshHep G2 Cells-
dc.subject.meshHumans-
dc.subject.meshInfant, Newborn-
dc.subject.meshLeukocytes, Mononuclear-
dc.subject.meshLuminescent Measurements-
dc.subject.meshMCF-7 Cells-
dc.subject.meshMale-
dc.subject.meshMice-
dc.subject.meshPolycyclic Hydrocarbons, Aromatic-
dc.subject.meshReference Standards-
dc.subject.meshReproducibility of Results-
dc.subject.meshSensitivity and Specificity-
dc.subject.meshStaining and Labeling-
dc.titleDevelopment and validation of a direct sandwich chemiluminescence immunoassay for measuring DNA adducts of benzo[a]pyrene and other polycyclic aromatic hydrocarbons.en
dc.typeArticleen
dc.identifier.journalMutagenesisen_GB

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