Biological effects of anionic meso-tetrakis (para-sulfonatophenyl) porphyrins modulated by the metal center. Studies in rat liver mitochondria

In this paper, we present a study about the influence of the porphyrin metal center and mesa ligands on the biological effects of meso-tetrakis porphyrins. Different from the cationic meso-tetrakis 4-N-methyl pyridinium (Mn(III)TMPyP), the anionic Mn(III) meso-tetrakis (para-sulfonatophenyl) porphyrin (Mn(III)TPPS4) exhibited no protector effect against Fe(citrate)-induced lipid oxidation. Mn(III)TPPS4 did not protect mitochondria against endogenous hydrogen peroxide and only delayed the swelling caused by tert-BuOOH and Ca(2+). Fe(III)TPPS4 exacerbated the effect of the tert-BuOOH, and both porphyrins did not significantly affect Fe(II)citrate-induced swelling. Consistently, Fe(III)TPPS4 predominantly promotes the homolytic cleavage of peroxides and exhibits catalytic efficiency ten-fold higher than Mn(III)TPPS4. For Mn(III)TPPS4, the microenvironment of rat liver mitochondria favors the heterolytic cleavage of peroxides and increases the catalytic efficiency of the manganese porphyrin due to the availability of axial ligands for the metal center and reducing agents such as glutathione (GSH) and proteins necessary for Compound II (oxomanganese IV) recycling to the initial Mn(III) form. The use of thiol reducing agents for the recycling of Mn(III)TPPS4 leads to GSH depletion and protein oxidation and consequent damages in the organelle. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Lipid hydroperoxide-induced and hemoglobin-enhanced oxidative damage to colon cancer cells

Epidemiological studies have indicated that Western diets are related to an increase in a series of malignancies. Among the compounds that are credited for this toxic effect are heme and lipid peroxides. We evaluated the effects of hemoglobin (Hb) and linoleic acid hydroperoxides (LAOOH) on a series of toxicological endpoints, such as cytotoxicity, redox status, lipid peroxidation, and DNA damage. We demonstrated that the preincubation of SW480 cells with Hb and its subsequent exposure to LAOOH (Hb + LAOOH) led to an increase in cell death, DCFH oxidation, malonaldehyde formation, and DNA fragmentation and that these effects were related to the peroxide group and the heme present in Hb. Furthermore, Hb and LAOOH alone exerted a toxic effect on the endpoints assayed only at concentrations higher than 100 mu M. We were also able to show that SW480 cells presented a higher level of the modified DNA bases 8-oxo-7,8-dihydro-2`-deoxyguanosine and 1,N(2)-etheno-2`-deoxyguanosine compared to the control. Furthermore, incubations with Hb led to an increase in intracellular iron levels, and this high level of iron correlated with DNA oxidation, as measured as EndoIII- and Fpg-sensitive sites. Thus, Hb from either red meat or bowel bleeding could act as an enhancer of fatty acid hydroperoxide genotoxicity, which contributes to the accumulation of DNA lesions in colon cancer cells. (C) 2011 Elsevier Inc. All rights reserved.

Redox regulation of the mitochondrial K(ATP) channel in cardioprotection

The mitochondrial ATP-sensitive potassium channel (mK(ATP)) is important in the protective mechanism of ischemic preconditioning (IPC). The channel is reportedly sensitive to reactive oxygen and nitrogen species, and the aim of this study was to compare such species in parallel, to build a more comprehensive picture of mK(ATP) regulation. mK(ATP) activity was measured by both osmotic swelling and Tl(+) flux assays, in isolated rat heart mitochondria. An isolated adult rat cardiomyocyte model of ischemia-reperfusion (IR) injury was also used to determine the role of mK(ATP) in cardioprotection by nitroxyl. Key findings were as follows: (i) mK(ATP) was activated by O(2)(center dot-) and H(2)O(2) but not other peroxides. (ii) mK(ATP) was inhibited by NADPH. (iii) mK(ATP) was activated by S-nitrosothiols, nitroxyl, and nitrolinoleate. The latter two species also inhibited mitochondrial complex II. (iv) Nitroxyl protected cardiomyocytes against IR injury in an mK(ATP)-dependent manner. Overall, these results suggest that the mK(ATP) channel is activated by specific reactive oxygen and nitrogen species, and inhibited by NADPH. The redox modulation of mK(ATP) may be an underlying mechanism for its regulation in the context of IPC. This article is part of a Special Issue entitled: Mitochondria and Cardioprotection. (C) 2010 Elsevier B.V. All rights reserved.

Mitochondrial ATP-sensitive K(+) channels as redox signals to liver mitochondria in response to hypertriglyceridemia

We have recently demonstrated that hypertriglyceridemic (HTG) mice present both elevated body metabolic rates and mild mitochondrial uncoupling in the liver owing to stimulated activity of the ATP-sensitive potassium channel (mitoK(ATP)). Because lipid excess normally leads to cell redox imbalance, we examined the hepatic oxidative status in this model. Cell redox imbalance was evidenced by increased total levels of carbonylated proteins, malondialdehydes, and GSSG/GSH ratios in HTG livers compared to wild type. In addition, the activities of the extramitochondrial enzymes NADPH oxidase and xanthine oxidase were elevated in HTG livers. In contrast, Mn-superoxide dismutase activity and content, a mitochondrial matrix marker, were significantly decreased in HTG livers. isolated HTG liver mitochondria presented lower rates of H(2)O(2) production, which were reversed by mitoK(ATP) antagonists. In vivo antioxidant treatment with N-acetylcysteine decreased both mitoKATP activity and metabolic rates in HTG mice. These data indicate that high levels of triglycerides increase reactive oxygen generation by extramitochondrial enzymes that promote MitoK(ATP) activation. The mild uncoupling mediated by mitoK(ATP) increases metabolic rates and protects mitochondria against oxidative damage. Therefore, a biological role for mitoK(ATP) is a redox sensor is shown here for the first time in an in vivo model of systemic and cellular lipid excess, (C) 2009 Elsevier Inc. All rights reserved.

Experimental Evidence of the Occurrence of Intramolecular Electron Transfer in Catalyzed 1,2-Dioxetane Decomposition

The activation parameters for the thermal decomposition of 13 acridinium-substituted 1,2-dioxetanes, bearing an aromatic moiety, were determined and their chemiluminescence emission quantum yields estimated, utilizing in situ photosensitized 1,2-dioxetane generation and observation of its thermal decomposition kinetics, without isolation of these highly unstable cyclic peroxides. Decomposition rate constants show linear free-energy correlation for electron-withdrawing substituents, with a Hammett reaction constant of rho = 1.3 +/- 0.1, indicating the occurrence of an intramolecular electron transfer from the acridinium moiety to the 1,2-dioxetane ring, as postulated by the intramolecular chemically initiated electron exchange luminescence (CIEEL) mechanism. Emission quantum yield behavior can also be rationalized on the basis of the intramolecular CIEEL mechanism, additionally evidencing its occurrence in this transformation. Both relations constitute the first experimental evidence for the occurrence of the postulated intramolecular electron transfer in the catalyzed and induced decomposition of properly substituted 1,2-dioxetanes.

Direct Kinetic Observation of the Chemiexcitation Step in Peroxyoxalate Chemiluminescence

A high-energy intermediate in the peroxyoxalate reaction can be accumulated at room temperature under specific reaction conditions and in the absence of any reducing agent in up to micromolar concentrations. Bimolecular interaction of this intermediate, accumulated in the reaction of oxalyl chloride with hydrogen peroxide, with an activator (highly fluorescent aromatic hydrocarbons with low oxidation potential) added in delay shows unequivocally that this intermediate is responsible for chemiexcitation of the activator. Activation parameters for the unimolccular decomposition of this intermediate (Delta H(double dagger) = 11.2 kcal mol(-1); Delta S(double dagger) = -23.2 cal mol(-1) K(-1)) and for its bimolecular reaction with 9,10-diphenylanthracene (Delta H(double dagger) = 4.2 kcal mol(-1); Delta S(double dagger) = -26.9 cal mol(-1) K(-1)) show that this intermediate is much less stable than typical 1,2-dioxetanes and 1,2-dioxetanones and demonstrate its highly favored interaction with the activator. Therefore, it can be inferred that structural characterization of the high-energy intermediate in the presence of an activator must be highly improbable. The observed linear free-energy correlation between the catalytic rate constants and the oxidation potentials of several activators definitely confirms the occurrence of the chemically initiated electron-exchange luminescence (CIEEL) mechanism in the chemiexcitation step of the peroxyoxalate system.

Fenchyl substituted 1,2-dioxetanes as an alternative to adamantyl derivatives for bioanalytical applications

The synthesis and study of the chemiluminescence parameters and thermal stability of 1,2-dioxetanes containing a spirofenchyl substituent are reported. Three fenchyl-substituted 1,2-dioxetanes were synthesized by photooxygenation of the corresponding alkenes, obtained by Barton-Kellogg olefination of the readily available (-)-fenchone. The fenchyl-substituted 1,2-dioxetanes showed thermal stabilities similar to those of the corresponding spiroadamantyl-substituted derivatives, although being slightly more labile with respect to unimolecular decomposition than the latter derivatives, which are widely utilized as labels in a great variety of chemiluminescent immunoassays. Fluoride induced decomposition of one triggerable fenchyl 1,2-dioxetane derivative showed kinetic parameters similar to those of the corresponding adamantyl-substituted derivative. The chemiluminescence quantum yields in the one percent range are also similar to that of other widely utilized chemiluminescence systems as the luminol reaction. These results indicate that fenchyl-substituted 1,2-dioxetanes can potentially be utilized as a cheaper alternative to substitute the corresponding spiroadamantyl derivatives in bioanalytical applications. (C) 2010 Elsevier B.V. All rights reserved.

Antiparasitic, antineuroinflammatory, and cytotoxic polyketides from the marine sponge Plakortis angulospiculatus collected in Brazil

Investigation of the bioactive crude extract from the sponge Plakortis angulospiculatus from Brazil led to the isolation of plakortenone (1) as a new polyketide, along with five known polyketides (2-6) previously isolated from other Plakortis sponges. The known polyketides were tested in antileishmanial, antitrypanosomal, antineuroinflammatory, and cytotoxicity assays. The results show that plakortide P (3) is a potent antiparasitic compound, against both Leishmania chagasi and Trypanosona cruzi, and exhibited antineuroinflammatory activity. The known polyketides 2-6 were tested for cytotoxicity against four human cancer cell lines, but displayed only moderate cytotoxic activity.