Pure MnTBAP selectively scavenges peroxynitrite over superoxide: comparison of pure and commercial MnTBAP samples to MnTE-2-PyP in two models of oxidative stress injury, an SOD-specific Escherichia coli model and carrageenan-induced pleurisy

Abstract

MnTBAP is often referred to as an SOD mimic in numerous models of oxidative stress. We have recently reported that pure MnTBAP does not dismute superoxide, but commercial or poorly purified samples are able to perform O2.- dismutation with low-to-moderate efficacy via non-innocent Mn-containing impurities. Herein, we show that neither commercial nor pure MnTBAP could substitute for SOD enzyme in a SOD-deficient Escherichia coli model, whereas MnTE-2-PyP-treated SOD-deficient E. coli grew as well as a wild-type strain. This SOD-specific system indicates that MnTBAP does not act as an SOD mimic in vivo. In another model, carrageenan-induced pleurisy in mice, inflammation was evidenced by increased pleural fluid exudate and neutrophil infiltration and activation: these events were blocked by 0.3 mg/kg MnTE-2-PyP and, to a slightly lesser extent, by 10 mg/kg of either MnTBAP. Also, 3-nitrotyrosine formation, an indication of peroxynitrite existence in vivo, was blocked by both compounds; again MnTE-2-PyP was 33-fold more effective. Pleurisy model data indicate that MnTBAP exerts some protective actions in common with MnTE-2-PyP, which are not O2.- related and can be fully rationalized if one considers that the common biological role shared by MnTBAP and MnTE-2-PyP is related to their reduction of peroxynitrite and carbonate radical, the latter arising from ONOOCO2 adduct. The log kcat (O2.-) value for MnTBAP is estimated to be about 3.16, which is approximately 5 and approximately 6 orders of magnitude smaller than the SOD activities of the potent SOD mimic MnTE-2-PyP and Cu,Zn-SOD, respectively. This very low value indicates that MnTBAP is too inefficient at dismuting superoxide to be of any biological impact, which was confirmed in the SOD-deficient E. coli model. The peroxynitrite scavenging ability of MnTBAP, however, is only approximately 2.5 orders of magnitude smaller than that of MnTE-2-PyP and is not significantly affected by the presence of the SOD-active impurities in the commercial MnTBAP sample (log k red (ONOO-) = 5.06 for pure and 4.97 for commercial sample). The reduction of carbonate radical is equally fast with MnTBAP and MnTE-2-PyP. The dose of MnTBAP required to yield oxidative stress protection and block nitrotyrosine formation in the pleurisy model is > 1.5 orders of magnitude higher than that of MnTE-2-PyP, which could be related to the lower ability of MnTBAP to scavenge peroxynitrite. The slightly better protection observed with the commercial MnTBAP sample (relative to the pure MnTBAP) could arise from its impurities, which, by scavenging O2.-, reduce consequently the overall peroxynitrite and secondary ROS/RNS levels. These observations have profound biological repercussions as they may suggest that the effect of MnTBAP observed in numerous studies may conceivably relate to peroxynitrite scavenging. Moreover, provided that pure MnTBAP is unable to dismute superoxide at any significant extent, but is able to partially scavenge peroxynitrite and carbonate radical, this compound may prove valuable in distinguishing ONOO-/CO3.- from O2.- pathways.

Figure 1

Structural diagram of MnTE-2-PyP and MnTBAP.

Figure 2

Linear relationship between the SOD-like and peroxynitrite scavenging activities of Mn porphyrins (y = −6.98 + 2.02 x; R² = 0.96). Data points from [27]. The MnTBAP point was not included in the linear regression as log k_cat (O₂^•−) was too low to be measured accurately (< 3.50); this value was estimated to be ~3.16 based on log k_red (ONOO⁻) of 5.02 determined for pure MnTBAP (see Table 1).

Figure 3

Aerobic growth of SOD-deficient (JI132) and wild type (SOD-proficient, AB1157) E. coli strains in the presence and absence of 20 or 200 μM of commercial MnTBAP, pure MnTBAP, and MnTE-2-PyP in (A) 5 amino acid-medium after 20 hours, and (B) in nutrient rich M9CA medium at 10 hours. Figures are representative of at least 3 experiments performed on different days.

Figure 4

Effects of commercial MnTBAP, pure MnTBAP, and MnTE-2-PyP on carrageenan-induced pleural exudate production, accumulation of polymorphonuclear cells in pleural cavity and myeloperoxidase activity (MPO). A significant production in pleural exudate (A), polymorphonuclear and (B) cells infiltration was observed in pleural cavity from vehicle-treated mice at 4h after carrageenan administration. Furthermore, MPO activity in lung tissues was significantly elevated at 4 h after carrageenan administration in vehicle-treated mice (C). The treatment with commercial or pure MnTBAP, and MnTE-2-PyP significantly reduced the presence of pleural exudate (A) and the number of inflammatory cells (B) as well as lung tissue levels of MPO (C). Data are means ± SEM of 10 mice for each group. *p < 0.01 vs. SHAM; °p < 0.01 vs. carrageenan group.

Figure 5

Effects of commercial MnTBAP, pure MnTBAP and MnTE-2-PyP on lung injury. Lung tissues collected at 4 h were stained with hematoxylin and eosin. No injury was observed in the lung tissues collected from sham (vehicle-treated) mice (a). Histological examination of lung sections collected at 4 h from all carrageenan-injected mice showed tissue injury as well as inflammatory cells infiltration (see arrows, b and b1). The treatment of mice with commercial MnTBAP (c), pure MnTBAP (d), and MnTE-2-PyP (e) significantly reduced the lung injury at 4 h after carrageenan injection. Figure is representative of at least 3 experiments performed on different days.

Figure 6

Effects of MnTE-2-PyP on nitrotyrosine formation in the lung. No positive staining for nitrotyrosine was observed in lung tissues obtained from sham mice (a). In contrast, tissue sections obtained from carrageenan-treated mice at 4h after carrageenan administration demonstrate positive staining for nitrotyrosine (see arrows b) and this was blocked by MnTE-2-PyP (c). Figure is representative of at least 3 experiments performed on different experimental days.

Figure 7

Effects of commercial MnTBAP on nitrotyrosine formation in the lung. No positive staining for nitrotyrosine was observed in lung tissues obtained from sham mice (a). On the contrary, tissue sections obtained 4h after the administration of carrageenan demonstrate positive staining for nitrotyrosine (see arrows, b) and this was blocked by MnTBAP (10 mg/kg, c). Figure is representative of at least 3 experiments performed on different experimental days.

Figure 8

Comparison of the reactivity of MnTBAP and MnTE-2-PyP. The numbers are the log k for the Mn(III) porphyrins and the reactants linked with each line (numbers in parenthesis correspond to the reactions with the corresponding Mn(II) species). Data are from [4,18,27,55] and this work.