br Methods br Results br Discussion Chronic stress

Methods
Results
Discussion Chronic stress renders individuals vulnerable to diseases, and this effect might be, at least partially, mediated by stress hormone-induced immunosuppression. Therefore, investigating the effects of catecholamines on immune Sotalol is of high relevance. We hypothesized that stress hormones might affect PARP-1 leading to accumulation of DNA strand breaks after chronic treatment with isoproterenol. In order to confirm our hypothesis, we first established an experimental setup mimicking chronic release of epinephrine in response to stress (Fig. 1). However, our experimental set up has some limitations. Since foetal calf serum (FCS) is an ill-defined mixture of components, with albumin being one of the main components, and since the latter has a strong binding effect on different kinds of compounds, all our experiments were performed using serum-free medium. Although a defined medium was used, serum might have some physiological effects on epinephrine-mediated DNA damage response in vivo. Furthermore, isoproterenol bioavailability might be higher without serum. Therefore, whether the isoproterenol concentration in medium mimics the blood levels accurately, remains unclear. Our results showed decreased cell viability due to ex vivo incubation (Fig. 1c) and the percentage of dead cells was increased after 48 and 72 hour incubation (data not shown). Therefore, later adaptive responses to the treatment cannot be addressed. Since changing medium is always accompanied by cell loss, especially the loss of dead cells and therefore cell selection, cells were kept in the same medium, which led to an accumulation of isoproterenol in medium. Regarding the stimulation of β-AR, we are not aware of any other similar ex vivo study. The repeated isoproterenol treatment in 30 or 60-minute time intervals did not lead to an additional increase in cAMP formation with increasing times of stimulation (Fig. 2). This could be attributed to the receptor internalisation and desensitisation but also to ATP depletion or both.
Conclusion Chronic release of epinephrine might lead to a long-lasting DNA damage response (DDR), including PARP-1 activation, which compromises cellular function due to induction of genomic instability and/or energy depletion. As a consequence, cells can undergo programmed cell death, cellular senescence or malignant transformation. Evidence for the implication of chronic stress on the development of senescence and tumour progression has been summarized recently (Moreno-Villanueva and Bürkle, 2016). On the other hand, epinephrine seems to protect tumour cells from apoptosis (Sastry et al., 2007). Therefore, epinephrine antagonists may possibly be considered as adjuvant cancer therapy. Indeed propranolol, an epinephrine antagonist, potentiates the anti-angiogenic effects and anti-tumour efficacy of chemotherapeutic agents (Pasquier et al., 2011). However, further studies are necessary in order to address this question. Our experiments show that chronic stimulation of peripheral blood mononuclear cells with isoproterenol leads to β-AR-dependent and β-AR-independent induction of DNA strand breaks, which are only partially repaired within 24 h. Furthermore, our findings indicate that increasing cycles of isoproterenol decreases PARP-1 activity and PARP-1 protein level. And finally, the number of apoptotic cells increased with isoproterenol treatment. Interestingly, our experiments revealed that some of the demonstrated isoproterenol-induced effects, especially on PARP-1 protein, might be subject-dependent, even among healthy subjects. However, this study was not designed to detect individual variability. Additional, more extensive, experiments addressing open questions such as receptor desensitisation, receptor density, and individual PARP-1 levels need to be performed taking into consideration the subject susceptibility to isoproterenol. Nevertheless, our findings provide a starting point for the study of individual vulnerability to chronic stress-associated diseases and may have important implications for future personalized medicine approaches. For instance, NAD+ supplementation under chronic stress could enhance PARP-1 activity supporting DNA repair and preserving ATP pools. The role of nicotinamide in the protection from carcinogenesis, DNA repair, and maintenance of genomic stability has been summarized by Surjana et al. (2010). Alternatively, supplementation of nicotinic acid, another NAD+ precursor, which in contrast to nicotinamide lacks the ability to inhibit PARP or sirtuins activity, has recently been shown to boost NAD+ levels and strand break-induced PARylation in normal human PBMCs and to protect the cells from cytotoxicity and genomic instability (Weidele et al., 2017). Furthermore, β-AR antagonists could prevent the epinephrine-mediated decrease in PARP-1 activity. Finally, in case of stress-induced PARP-1 hyperactivation PARP inhibitors could be considered. We conclude that an adequate clinical intervention for counteracting the detrimental effects of epinephrine on DNA damage and repair might depend on the individual PARP response and/or NAD+ availability (Fig. 9).