Unlike HOCl, HOSCN can be detoxified by thioredoxin reductase, and reacts selectively with thiols to result in reversible modifications, which could potentially reduce the extent of MPO-induced damage during chronic inflammation. reversible modifications, which could potentially reduce the extent of MPO-induced damage during chronic inflammation. In this study, we show that exposure of macrophages, a key inflammatory cell type, to HOSCN results in the reversible modification of multiple mitochondrial proteins, leading to increased mitochondrial membrane permeability, decreased oxidative phosphorylation and reduced formation of ATP. The increased permeability and reduction in ATP could be reversed by pre-treatment of the macrophages with cyclosporine A, implicating a Protopanaxatriol role for the mitochondrial permeability transition pore. HOSCN also drives cells to utilise fatty acids as an energetic substrate after the inhibition of oxidative phosphorylation. Raman imaging studies highlighted the ability of HOSCN to perturb the electron transport chain of mitochondria Protopanaxatriol and redistribute these organelles within the cell. Taken together, these data provide new insight into the pathways by which HOSCN can induce cytotoxicity and cellular damage, which may have relevance for the development of inflammatory disease, and therapeutic strategies to reduce HOCl-induced damage by supplementation with SCN-. and and and and and and molecules compared to the cytochromes molecules (I747/I1124 and I747/I1124 with SDT; Fig. S4). Similarly, there was also no significant difference between the relative amount of lipids compared to proteins in the cells after HOSCN treatment (I1445/I1660, Fig. S4). Open in a separate windows Fig. 7 HOSCN treatment of J774A.1?cells alters the Raman intensity of mitochondrial from your inner mitochondrial space into the cytosol. The loss of the mitochondrial membrane potential is usually significant at 50?M HOSCN treatment concentration, where a significant loss in basal respiration, ATPClinked respiration, maximum respiration, coupling efficiency and cellular ATP levels is also apparent. The loss in ATP production could be largely mitigated by pre-treatment of the cells with CsA, which suggests that HOSCN induces MPTP formation, as CsA blocks the peptidylprolyl isomerase activity of cyclophilin D, which is required for the formation of a complex with the adenine nucleotide translocator (ANT) during MPTP formation (examined [50]). The mechanism by which HOSCN induces MPTP formation is not known. Given that there is no significant switch in intracellular Ca2+ concentrations in the J774A.1?cells exposed to HOSCN under conditions where changes in mitochondrial function are apparent, it is perhaps more likely that the effects observed are related to oxidation of key proteins involved in the assembly of the MPTP [51]. For example, oxidation of Cys residues on ANT and the formation Protopanaxatriol of a cross-link between Cys160 and Cys257 around the matrix facing loops prevents ADP-mediated inhibition of the MPTP opening, and results in sensitisation of the MPTP to Ca2+ [50,52,53]. It is also possible that HOSCN could induce a conformation switch in the MPTP to increase the Ca2+ binding sites, as reported previously in studies with isolated mitochondria exposed to and contribute to the Raman spectrum of cells with 532?nm laser excitation, as the Raman scattering of oxidized cytochromes is negligible [[34], [35], [36], [37], [38], [39]], and cytochrome in cytochrome oxidase (complex IV) does not absorb light with this energy [34,38,39]. The Raman data allow us to estimate the relative amount of reduced and in the intermembrane space of mitochondria following addition of HOSCN to the cells. The cytochrome present in Complex II is not involved in electron transport, and exists in its oxidized form and therefore does not contribute to the Raman spectra of cells Protopanaxatriol [58]. Our data are consistent with a decrease in the relative amount of reduced and [60]. In summary, we show that HOSCN induces mitochondrial dysfunction by inducing a decrease in mitochondrial membrane potential, respiration and ATP production, which is usually associated with formation of the MPTP and alterations to the mitochondrial redox state. These changes are believed Rabbit Polyclonal to PLD2 (phospho-Tyr169) to reflect the ability of HOSCN Protopanaxatriol to target multiple mitochondrial proteins.