Validation of BPDE-Induced DNA Damage

BPDE was solved and stored in anhydrous THF/5 % TEA and although cellular BPDE treatment was performed in incomplete medium, a small portion of this solvent was always present. To exclude an influence of solvent on the assays performed, and to define non-cytotoxic solvent concentrations, an alamarBlue assay with increasing volume percentages of THF/TEA was conducted as described for chapter 4.5.8. In Figure 4.31A it can be seen, that THF/TEA concentrations ≥1 % in the medium had a negative influence on the cellular viability. As a consequence, in the following experiments, whenever possible, a volume percentage of exactly 0.1 % was used, giving a safety factor of 10. This threshold was only exceeded once, for the PAR induction with 50 µM BPDE and subsequent tandem MS measurement (0.5 % THF/TEA for one hour; chapter 4.5.5).

The direct influence of BPDE on DNA strand break formation was investigated by extracellular treatment of circular plasmid DNA and subsequent analysis of its structural features (Figure 4.31B).

When running plasmid DNA on an agarose gel, it can be detected in three forms, closed and circular supercoiled DNA migrating fastest; partially relaxed, nicked plasmids migrating at the slowest speed and linearized plasmids showing medium mobility. 24 h after BPDE treatment the migration patterns Figure 4.31: Analysis of the influence of THF on cellular health and the BPDE-induced strand-break formation.

A. An alamarBlue assay was performed 24 h after treatment of HeLa Kyoto cells with different concentrations of THF. With concentrations lower than 1 % no influence on cellular health could be observed. B. Plasmid DNA was treated with increasing concentrations of BPDE. After 24 h, the formation of DNA strand breaks was analysed in an agarose gel. Only at the highest concentrations of BPDE an increase of DNA strand breaks could be detected.

SC: solvent control; UT: untreated. Contributions by [E].

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were compared to plasmids nicked (lane 8) and linearized (lane 9) by restriction enzymes. With increasing BPDE concentrations a general loss of migration speed could be observed (lane 6 and 7).

But only at the second highest BPDE/DNA ratio (lane 6) an increase in the amount of nicked plasmid was detected. Since such ratios were not reached in cellular studies, only a minor influence of BPDE in direct strand-break induction is supposed by these experiments.

Formation of Reactive Oxygen Species

It has been previously reported that the metabolism of B[a]P, but also BPDE, can induce the formation of reactive oxygen species ³²⁷. These species, when reacting with DNA can trigger strand-incision and PARP-1 activation. To discriminate to which extent this mechanism is involved in PARylation after BPDE treatment (Figure 4.36), ROS levels in HeLa Kyoto cells were determined after BPDE exposure.

Figure 4.32: ROS formation in HeLa Kyoto cells after BPDE treatment. DHE was used after BPDE treatment as an indicator of ROS formation. A. Cells were treated with BPDE (10 or 50 µM) and after the indicated time points (≤ 1 h) DHE was added and fluorescence was detected. Using a high dose of 50 µM BPDE, a weak, but significant increase in ROS formation could be observed within the first hour after BPDE treatment. B. Longer exposure (5 h) to BPDE did not result in increased levels of ROS formation. C. Control treatments for ROS formation. HeLa Kyoto cells were treated first with the antioxidant NAC and subsequently with the ROS inducer TBHP (neg.

control) or only with TBHP (pos. control). A dose-dependent increase in ROS levels could be observed with H2O2. Data represent means ± SEM of three independent experiments, each performed in technical triplicates, normalized to untreated control. Statistical evaluation was performed using Two-Way ANOVA analysis followed by Sidak’s multiple comparison test. ** p>0.01. Contributions by [C].

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Cells were treated for up to 5 hours with high concentrations of BPDE (dose relates to the high dose used in Figure 4.36B and E) after which DHE was added. In the presence of ROS the blue fluorescent dye DHE is oxidized, intercalates into the DNA and exhibits a bright red fluorescence, detectable with a fluorescence reader (Figure 4.32). Treatment with the solvent alone decreased the fluorescence intensity of DHE treated cells by 10-15 %. Incubation with 10 or 50 µM BPDE resulted in a weak but stable induction of ROS formation, shown by mild increase in fluorescent intensity (Figure 4.32A). A significant increase in fluorescence signal, compared to solvent control, could only be observed after exposure to 50 µM BPDE for 60 min. On the other hand, neither concentration had an influence on the ROS level 5 hours after BPDE treatment (Figure 4.32B).

To have the possibility to relate the increase of ROS seen in Figure 4.32A to PAR induction, a dose-response with increasing concentrations of H2O2 was performed (Figure 4.32C). Already the lowest applied concentration of H2O2 caused a fluorescence increase of 25 %, which further increased in a dose-dependent manner. These data suggest a rather limited degree of ROS formation upon BPDE treatment in the cellular system used in this study.

Induction of BPDE-DNA Lesions

After a feasible solvent concentration without adverse effects was identified and the extent of unspecific DNA damage formation was analysed, control experiments on BPDE-DNA damage induction and the capabilities of damage detection were carried out. Therefore, genomic DNA was extracted from HeLa Kyoto cells and treated with various concentrations of BPDE. Treated DNA was subject to a slot-blot assay and induced lesions were detected with a BPDE-DNA specific antibody. Successful adduct visualization was possible in the lower nanomolar ranges of BPDE treatment (data not shown).

Figure 4.33: Treatment of HeLa Kyoto cells with BPDE induces bulky DNA adducts. A. Representative immunofluorescence images of BPDE-DNA adducts. Cells were treated for 1 h with 1 or 20 µM BPDE and DNA lesions were detected with a BPDE-DNA specific antibody. While 1 µM caused no detectable level of BPDE-DNA adducts, treatment with 20 µM BPDE resulted in a clear signal increase. B. Quantification of A. Depicted are the means of signal intensities of >100 cells of one experiment, normalized to solvent control. C. Cells were treated for 1 h with 0.5, 1 or 10 µM BPDE. Cellular DNA was extracted, blotted on a nylon membrane and BPDE adducts were detected using specific antibodies. D. Quantification of the chemiluminescence signal of C (n=1). Contributions by [E].

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The next step was the treatment of viable cells with BPDE and subsequent damage detection (Figure 4.33). HeLa Kyoto cells were treated with 1 or 20 µM BPDE for 1h. Afterwards cells were fixed and a modified immunofluorescence protocol, originally designed to detect 8-oxo DNA lesions (Trevigen, USA), was performed. With this it was possible to detect BPDE-adducts, but only at comparable high doses of BPDE (Figure 4.33A & B). To increase the sensitivity, and perceive damages induced by lower BPDE concentrations, cells were treated with increasing concentrations of the toxicant, genomic DNA was extracted and slot-blotted on a nylon membrane, followed by an immunochemical staining. With this procedure it was possible to lower the detectable BPDE concentration applied to cells to 0.5 µM.