INCUMPLIMIENTO A LAS OBLIGACIONES PACTADAS EN EL ANEXO "SSPA"

An imbalance of reactive oxygen species (ROS) production and antioxidant status leads to oxidative stress and has been associated with tissue injury and disease processes, including the pathogenesis of AP (Dziurkowska-Marek et al. 2004; Leung et al. 2009; Booth et al. 2011; Hackert et al. 2011). The generation of ROS in pancreatic acinar cells has been shown to inhibit the ATPase pump PMCA, suggesting a role for oxidative stress in modulating Ca2+ overload, a major driver of necrosis (Bruce et al. 2007; Baggaley et al. 2008). Many preclinical and clinical investigations have demonstrated increased free radical activities, levels of superoxide anions, hydrogen peroxide, and hydroxyl free radicals in affected tissues (Guyan et al. 1990; Szuster-Ciesielska et al. 2001). Elevated levels of lipid peroxides in blood, plasma and tissue have been shown alongside diminished antioxidant

120 | P a g e defences. Depletion of antioxidants such as glutathione (Rahman et al. 2004), Vitamins A and E and Carotenoid (Curran et al. 2000) have been associated with AP (De Waele et al. 1992; Scott et al. 1993). Yet restoring antioxidant levels in the clinical setting has provided conflicting results (Virlos et al. 2003; Siriwardena et al. 2007; Armstrong et al. 2013).

Many clinical attempts to regulate oxidative stress have encountered a reduction in biomarkers of oxidative stress with little or no therapeutic benefit. A meta-analysis indicated that antioxidant therapies such as β-carotene, vitamin A and vitamin E do not improve the outcome of a number of diseases and may actually be detrimental, causing an increase in mortality (Bjelakovic et al. 2007). Although there has been a lack of success of antioxidants in the clinic, there is still evidence for a pivotal role of ROS in the development of AP, amongst many other oxidative stress-related diseases such as hypertension, atherosclerosis, diabetes and kidney disease. Therefore, targeted antioxidants were developed, including MitoQ, MitoE and MitoSOD (Smith et al. 2008).

Most small molecule antioxidants are only up-taken into the mitochondria in small amounts and are distributed around the body. MitoQ is targeted to the mitochondria by conjugation to a TPP moiety and can be actively recycled by ETC complex II to the active antioxidant form within the mitochondria (James et al. 2005; Smith et al. 2008). MitoQ is an effective antioxidant against lipid peroxidation, peroxynitrite and superoxide. In pancreatic acinar cells, MitoQ can effectively inhibit ROS increases and Ca2+ oscillations induced by CCK (10pM) in pancreatic acinar cells

121 | P a g e (Camello-Almaraz et al. 2006). On the other hand, MitoQ and other ubiquinol based antioxidants have demonstrated the ability to act also as a prooxidant, enhancing superoxide production via redox cycling and therefore H2O2 through dismutation

(Boveris et al. 1976; James et al. 2004; Doughan et al. 2007; Plecitá-Hlavatá et al. 2009). MitoQ can also inhibit the ETC in a manner dependent on the targeting component TPP+ and acyl chain (Trnka et al. 2015). MitoQ (1-10µM) demonstrated no detectable prooxidant potential in CM-H2DCFDA loaded pancreatic acinar cells.

CM-H2DCFDA is a general oxidative stress indicator predominantly effective at

detecting hydrogen peroxide, hydroxyl radicals, nitrogen dioxide and carbonate radicals (Wojtala et al. 2014). Levels of oxidant production induced by MitoQ in pancreatic acinar cells may be too diminutive to detect using this technique. It is possible that applying a more specific probe (optimised with spectral analysis) such as DHE (Dihydroethidium) or MitoSOX (DHE plus TPP+) or electron spin resonance (ESR) spectroscopy in future work may have the potential to highlight more specific increases in superoxide production seen in other cell types (Bindokas et al. 1996; Li et al. 2003; Rivera et al. 2005). To establish if any superoxide production by MitoQ was adequate to cause mitochondrial damage, the activity of TCA dehydrogenases such as aconitase could be assessed.

MitoQ has demonstrated the ability to block numerous redox signalling pathways induced by extracellular H2O2, but the mechanisms are not defined

(Echtay et al. 2002; Saretzki et al. 2003; Schäfer et al. 2003; Chen et al. 2004; Dhanasekaran et al. 2004; Ross et al. 2005). MitoQ does not react with alkyl peroxides or H2O2 directly, although successfully inhibits lipid peroxidation (Kelso et

122 | P a g e al. 2001; Asin-Cayuela et al. 2004; James et al. 2005). This would indicate that the effectiveness of MitoQ is predominately downstream of H2O2, acting on

OH•- formed from iron catalysed Fenton reaction and to inhibit the peroxidation chain reaction rather than directly upon H2O2 (Sutton et al. 1984; Braughler et al.

1986; Winterbourn 1987).

The capabilities of MitoQ to inhibit high levels of extracellular H2O2- (1mM)

induced ROS increases were assessed, administering MitoQ as a pre-treatment. There are a variety of parameters, such as the energised state of the mitochondria (Kelso et al. 2001), which affect the rate of MitoQ accumulation into the mitochondria. Pre-treatment with MitoQ may improve the effectiveness and comparing pre-treatment to treatment provided a key observational tool and comparison to clinical failures encountered with non-targeted antioxidants (Demols et al. 2000). MitoQ (1µM) proved to be an effective antioxidant in pancreatic acinar cells, providing protection against 1mM H2O2-induced ROS increases compared to

the non-antioxidant moiety dTPP control. Reports have shown that excessive accumulation of lipophilic cations in the mitochondria are toxic due to disruption to ATP synthesis (Smith et al. 2003) membrane integrity and respiration (Murphy 2008). To assess the cellular effects of excessive accumulation in pancreatic acinar cells on ROS levels, a ten-fold concentration of each compound (10µM) was applied to isolated pancreatic acinar cells prior to H2O2 treatment (1mM). MitoQ and dTPP

(10µM) both reduced 1mM H2O2-induced ROS increases, indicating that this

reduction is not dependent on the antioxidant moiety but the TPP+ component and/or acyl chain.

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