Exposure to BPDE Triggers PAR Formation

Although the mechanism of PARP-1 activation in NER is still under debate, a rising number of publications have shown an induction of PAR formation and its essential role for an efficient repair of NER lesions. But so far these reports dealt only with UV-induced DNA damages, and direct evidence of PARylation could often only be made under special conditions 483,485,488. These conditions enhanced Figure 4.34: PARP inhibition with ABT888 modulates PARP-1 protein levels. H1299 cells were incubated with or without ABT888 and either treated with BPDE or with THF as a solvent control. After the indicated time points cells were harvested and subjected to a SDS-PAGE and immunoblotting. Independent of BPDE treatment, the inhibition of PARP activity strongly increased the levels of detectable PARP-1 protein. Shown is one representative blot of three independent experiments.

- 112 -

PARylation or caused its continuous accumulation (e.g. PARG knockdown), resulting in stronger signals. Anyhow, NER substrates are a wide class of lesions which have only in common their DNA helix distortion and kinking. Although all these lesions are targeted by this repair pathway, the response of the NER might not always be the same. It has been reported that not all lesions are recognized by all factors in same degree. Different NER repair complexes preferentially deal with different DNA damages and some lesions need longer to be repaired than others. To clarify, if PARylation is evident in the context of BPDE exposure, induction of PARP-1 activity was analysed.

To be able to compare the dynamics of BPDE-induced PARylation to that of an established PAR-inducer, an immunofluorescence analysis of PAR formation was performed in a time series after H2O2

treatment (Figure 4.35). H2O2 induced a rapid PARylation response and already after the first few minutes PAR levels ascended fast. Its maximum was reached after about 6.5 min and continuously decreased thereafter. After 15 min the amount of PAR was again close to basal levels.

H2O2 treatment results in an immediate and strong PARP-1 response, which is essential for the repair of oxidative lesions and DNA strand breaks. In the overall slower process of NER, PARP activity seems to be more of a modulatory role and PARylation in UV-treated cells was comparably weaker ⁴⁸³. As it was expected, that PARylation after BPDE exposure would behave similar to PARylation after UV-irradiation, methodology was switched from immunofluorescence based microscopy to the more sensitive method of PAR detection via tandem MS (Figure 4.36).

Figure 4.35: Time series of PAR formation and degradation after H2O2 damage induction. A. Representative immunofluorescence images to analyse the time course of PAR levels after treating HeLa Kyoto cells with 0.5 mM H2O2. Scale bar represents 30 µm. B. Quantification of A. Immediately after damage induction, PAR levels started to increase, reaching a level of ~6-fold after 6.5 min. After 15 min PAR signals were again comparable to signals in untreated cells. Data represent means ± SEM (n=3) normalized to untreated control. Statistical evaluation was performed using One-Way ANOVA analysis followed by Sidak’s multiple comparison test.

** p<0.01, *** p<0.001. Contributions by [C].

- 113 -

First, a dose-response experiment was performed. Cells were treated with increasing concentrations of BPDE for 1 h, samples were prepared and PAR levels determined via LC-MS/MS (Figure 4.36A).

Exposure to the lower BPDE concentrations did not trigger PARylation, however, with 10 and 50 µM BPDE a significant 2-3 fold increase in PAR formation could be observed. Therefore, these two concentrations were used for the following PAR time series. The higher dose of 50 µM BPDE was used in a short-term incubation for up to 90 min. Already after 10 min PAR levels started to rise and reached Figure 4.36: PAR formation in HeLa Kyoto cells after BPDE treatment detected with LC-MS/MS. A. Dose-response curve of BPDE-PAR formation. Cells were treated for 1 hour with increasing concentrations of BPDE and PAR levels were detected by tandem MS. 10 and 50 µM BPDE induced a significant increase in PAR formation. B. Short time series of PAR formation after BPDE treatment. Cells were incubated in 50 µM BPDE in incomplete medium until PAR detection. 30 and 60 min after damage induction PAR formation was triggered.

Cells preincubated in 10 µM ABT888 showed no response to BPDE in respect of PAR detection. C. Representative chromatogram of B of ABT888-untreated cells. D. Representative chromatogram of B of ABT888-treated cells.

E. Long time series of PAR formation after BPDE treatment. Cells were treated with 10 µM BPDE and after the indicated time points, PAR levels were analysed using tandem MS. PAR levels continuously increased for the first 5 h after damage induction, followed by a slow but steady decrease. F. Representative chromatogram of E. A-B

& E. Data represent means ± SEM (n=3) normalized to solvent control. Statistical evaluation was performed using One-Way ANOVA (A & E) or Two-Way ANOVA (B) analysis followed by Dunnett’s or Sidak’s multiple comparison test, respectively. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Contributions by [C].

- 114 -

a significant 3-fold induction after 30 min (Figure 4.36B). ABT888 was used as a specificity control of the method, and completely blocked any formation of PAR above basal levels. Appropriate, representative chromatograms for samples with and without inhibitor treatment are depicted in Figure 4.36C & D. The red line marks the retention time and amount of the internal standard, the black line the detected PAR level of the sample (Figure 4.36D, no PAR detected). In Figure 4.36E-F the results of the long-term time series up to 8 hours are presented. Here, cells were treated with 10 µM BPDE and every hour samples were prepared for LC-MS/MS measurement. Within the first 5 hours a steady increase of the PAR signal could be observed and also thereafter the PAR signal was persistent and only diminished slowly within the time frame analysed.

The overall dynamics in PAR formation unfolded to be completely different for the two genotoxins, H2O2 and BPDE. While the oxidant caused a strong but rather short peak of PARylation (<15 minutes), BPDE triggered a rather weak but sustained PARylation event (> 8 hours), probably reflecting the velocity of the responding repair pathways.