• Development and application of biomarker methods in molecular environmental epidemiology for the detection of exposure to polycyclic aromatic hydrocarbons.

      Kovács, Katalin (University of Pécs, 2012, 2012)
      My research aimed at the further development of the 32P-postlabelling method by which, whilst maintaining the adduct-labelling efficiency, the amount of the radioisotope per sample can be reduced, and thereby the sample processing capacity of the laboratory is increased. The regular and the modified 32P-postlabelling methods were used for comparative determinations of the levels of DNA adducts from various types of DNA samples, i.e. BPDE-DNA adduct standard, DNA samples from MCF-7 cell line treated with B[a]P, DNA samples from human non-tumorous peripheral lung tissue, human peripheral blood lymphocytes and human buffy coat samples. In the modified 32P-postlabelling method, the final reaction volume of the radio-labelling was reduced with an evaporation-to-dryness step to one-third of the volume that was used in the regular method. This facilitated the reduction of the amount of [γ-32P]ATP substrate from 50 μCi per sample – depending on the DNS isolation method – by 50% for the Qiagen-isolated samples (i.e., 25 μCi per sample) and by 80% for the DNA samples isolated with the classic phenol extraction procedure (i.e.,10 μCi) for both experimental and human samples. The newly-developed BPDE-DNA direct sandwich chemiluminescence immunoassay (BPDE-DNA SCIA) for the determination of PAH-DNA adducts has been published in 2012. Whereas in the earlier competitive immunoassays the signal to be measured is coupled to the DNA adducts, in the SCIA the chemiluminescent signal is coupled to the non-adducted nucleotides. In SCIA, the strong end-point signal derives from the many orders of magnitude difference between the number of the non-adducted and the adducted mononucleotides. The limit of detection of the method is ~ 1.5 adducts/109 nucleotides from 5 μg DNA sample. For the validation of BPDE-DNA SCIA, I performed comparative measurements between the immunoassay and the 32P-postlabelling method. The DNA samples were obtained from MCF 7 cells treated with B[a]P, from the liver of mice, which were treated in vivo with several doses of B[a]P, B[b]F, and DB[a,h]A, respectively, and from human maternal peripheral blood and newborn cord blood samples. For the B[a]P-DNA adduct levels measured by SCIA and 32P-postlabelling from the MCF 7 cells, the ratio between the adduct values was about 0.5. For the animal samples, the adduct levels were several times lower by the immunoassay than by the 32P-postlabelling method (the ratios were ≈1:5 for B[a]P, ≈ 1:30 for B[b]F and ≈ 1:5 for DB[a,h]A). All the same, there was a very strong, highly significant positive correlation between the DNA adduct measurements of the dose-response curves by SCIA and 32P-postlabelling for each PAH compound (r = 0.87-0.99). For the human samples, the ratio between the SCIA and 32P-postlabelling values was approximately 1:10, but there was not correlation between the data-pairs measured by the two methods. For the human samples, the lack of correlation between the two methods may be explained by the different efficiency of detection of different structural types of DNA adducts that are derived from complex human environmental exposure.