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Flow cytometric determination of HPRT-variantsin human peripheral blood lymphocytes
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology. (genetisk toxikologi)
2002 (English)In: Mutation research, ISSN 0027-5107, E-ISSN 1873-135X, Vol. 499, no 1, 63-71 p.Article in journal (Refereed) Published
Abstract [en]

Hypoxanthine guanine phosphoribosyl transferase (HPRT) deficient human peripheral blood lymphocytes are usually enumerated either by the cloning assay or by the autoradiographic short-term assay. The short-term approach presented here is based on flow cytometric (FCM) scoring of 6-thioguanine (6-TG) resistant lymphocytes. HPRT-variants are enumerated on the basis of both DNA synthesis (by use of immunofluorescent detection of incorporated 5-bromo-2-deoxyuridine, BrdU) and total DNA content (by propidium iodide (PI) incorporation) of proliferating cells, i.e. the cells must both be labelled with BrdU and reside in late-S or G(2) phase in order to be scored as a HPRT-variant. This approach is combined with a stringent discrimination of false-positive events, minimising occurrence of phenocopies or other non-specifically labelled cells that might falsely be scored as true HPRT-variants. The HPRT-variant frequency (V-f) found by the presented method varied between 0.8 x 10(-5) and 5.8 x 10(-5) for healthy male and female donors aged between 20 and 74 years. There was no significant gender difference in V-f. A strong linear correlation was found between HPRT-variant frequency and age, showing an increase of 0.56 x 10(-6) per year of age (r(2) = 0.62, P < 0.001). The frequencies of false-positive events found showed a mean of 0.22 x 10(-5) in comparison with a pooled mean V-f of 2.87 x 10(-5). There was no significant age effect on the frequency of false events (r(2) = = 0.15, P < 0.095). The method presented here may provide a rapid and sensitive alternative to the autoradiographic technique for the short-term enumeration of HPRT-variants.

Place, publisher, year, edition, pages
2002. Vol. 499, no 1, 63-71 p.
Keyword [en]
HPRT; human lymphocyte; 5-bromo-2-deoxyuridine; flow cytometry; biomonitoring
National Category
URN: urn:nbn:se:su:diva-45905OAI: diva2:370298
Available from: 2010-11-16 Created: 2010-11-16 Last updated: 2013-06-19Bibliographically approved
In thesis
1. Biomarkers for DNA damage in human biomonitoring
Open this publication in new window or tab >>Biomarkers for DNA damage in human biomonitoring
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Genomic DNA in humans is constantly exposed to different kinds of damage. Therefore, it is desirable to implement methods for detecting and measuring of inflicted body burden. Human biomonitoring (HBM) can here be a useful tool as a link between environmental exposure and disease outcome. The present thesis aims to monitor DNA damage in humans by studies on: 1) urinary thymidine dimer (T=T) as a novel biomarker (BM) of human exposure to UV-light; 2) enumeration of variants in HPRT gene in human peripheral blood lymphocytes by developing a sensitive flow cytometric (FCM) analysis; 3) the impact of dietary habits on genomic stability in vegetarians and omnivores in terms of micronuclei (MN) induction detected by FCM. Urinary T=T was quantified by a 32P-postlabeling technique, the kinetics of T=T excretion was studied and the method was validated by delivering controlled UV-doses. The major conclusion was that the amount of urinary T=T was determined by the UV dose, and hence T=T can be used as a BM of UV exposure. Moreover, a new approach for rapid and sensitive enumeration of HPRT-variants by FCM was developed. The obtained HPRT-frequencies were comparable to those previously published by others. Finally, the FCM assay for MN enumeration was applied to study effects of dietary habits in vegetarians and omnivores. The main finding was that vegetarians had significantly lower MN frequencies compared to omnivores. In summary, the applied BMs and respective methods have high sensitivity and/or throughput possibility which are important factors considered in HBM.


Place, publisher, year, edition, pages
Stockholm: Department of Genetics, Microbiology and Toxicology, Stockholm University, 2010. 55 p.
biomonitoring, biomarkers, DNA damage, cancer, diet, UV-light
Research subject
Molecular Genetics
urn:nbn:se:su:diva-45910 (URN)978-91-7447-183-0 (ISBN)
Public defence
2010-12-17, sal E306, Arrheniuslaboratorierna, Svante Arrhenius väg 20C, Stockholm, 14:00 (English)
At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manscript.Available from: 2010-11-25 Created: 2010-11-16 Last updated: 2010-11-16Bibliographically approved

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