Carin Thomas

PHOTOACTIVATION OF HYPERICIN GENERATES SINGLET OXYGEN IN MITOCHONDRIA AND INHIBITS SUCCINOXIDASE

Abstract— Photosensitized inhibition of mitochondrial succinoxidase by hypericin was measured in vitro and found to be drug‐dose, light‐dose, and wavelength dependent. Singlet oxygen generation, monitored using the singlet oxygen trap tetramethylethylene, and oxygen consumption in isolated mitochondria sensitized by hypericin were also light‐dose and wavelength dependent. Unequivocal evidence for the generation of singlet oxygen was obtained using kinetic isotope ratios of products from the reaction between singlet oxygen and geminally deuterated tetramethylethylene. An action spectrum for the inhibition of succinoxidase was measured at wavelengths between 400 and 700 nm and found to parallel the recorded visible absorption spectrum of hypericin in isolated mitochondria. The greatest singlet oxygen generation, oxygen consumption, and succinoxidase inhibition occurred with white light or 600 nm irradiation. These data are consistent with a type II singlet‐oxygen‐mediated mechanism for hypericin induced photosensitized inhibition of mitochondrial succinoxidase.

Inhibition of glutathione reductase by flavonoids. A structure-activity study.

A structure-activity study of fourteen chemically related flavonoids was conducted to evaluate their abilities to inhibit glutathione reductase (GR). By comparing the I50 values of flavonoids from different classes possessing an identical hydroxyl configuration, we determined the following order of potency for inhibition of GR: anthocyanidin > dihydroflavonol = chalcone > flavonol > catechin. Enzyme inhibition by delphinidin chloride and myricetin was partially prevented in a N2 atmosphere which implicates a role for oxygen in the mechanism of inhibition. To determine the role of oxygen species in enzyme inhibition, GR was preincubated with either mannitol, diethylenetriaminepenta-acetic acid (DETAPAC), superoxide dismutase (SOD), catalase (CAT), or SOD and CAT prior to assays for enzyme inhibition by flavonoids. Enzyme inhibition by delphinidin chloride and myricetin was suppressed by the addition of SOD, suggesting that superoxide (O2-.) is involved. However, inhibition by quercetin and morin was not sensitive to antioxidants. To further investigate the role of O2-. in GR inhibition, a superoxide generating system was utilized in the presence and absence of flavonoid. The O2-. generating system failed to inhibit GR in the absence of flavonoid but enhanced the inhibition by myricetin, indicating that the O2-. did not directly inhibit GR but reacted directly with certain flavonoids to form a reactive intermediate which, in turn, inhibited GR. These findings suggest that the mechanism of inhibition of GR by flavonoids is complex and may have oxygen-dependent and oxygen-independent components.

OXYGEN DEPENDENCE OF HYPERICIN‐INDUCED PHOTOTOXICITY TO EMT6 MOUSE MAMMARY CARCINOMA CELLS

Abstract

Hydroxyl radical is produced via the Fenton reaction in submitochondrial particles under oxidative stress: implications for diseases associated with iron accumulation

Abstract Mitochondrial dysfunction and reactive oxygen species (ROS) are often implicated in diseases involving oxidative stress and elevated iron. As mitochondria produce ATP by oxidative phosphorylation, ROS by-products are generated from the electron transport chain. Although superoxide and hydrogen peroxide have been thoroughly investigated, little evidence documents hydroxyl radical (HO•) production in mitochondria. In order to determine whether HO• is generated under oxidative stress conditions by a Fenton-type mechanism, bovine heart submitochondrial particles were examined for HO• in the presence and absence of iron ligands, antioxidant enzymes and HO• scavengers. HO• was measured as 2,3- and 2,5-dihydroxybenzoic acid (DHBA), using HPLC with electrochemical detection. The iron ligand desferrioxamine significantly decreased DHBAs, indicating that HO• generation required iron redox-cycling. In addition, results from exogenous SOD and catalase, exogenous hydrogen peroxide, and HO•-scavenger studies support a Fenton-type reaction mechanism. The results indicate that increased HO• levels occur in mitochondria under oxidative stress and that the HO• levels can be modulated with antioxidant enzymes and iron ligands. Our findings together with reports on iron accumulation in degenerative diseases highlight the importance of developing mitochondrial-targeted antioxidants for the therapeutic intervention of diseases associated with mitochondrial dysfunction and oxidative stress.

Hypochlorous acid disrupts the adhesive properties of subendothelial matrix.

We have investigated whether the cell adhesion-promoting properties of the subendothelial matrix are affected by exposure to neutrophil-derived oxidants. Native subendothelial matrix was exposed to increasing doses of H2O2 in the presence of myeloperoxidase and Cl- or to reagent hypochlorous acid (HOCl). Increasing doses of either oxidant system resulted in progressive loss in the adhesive properties of the matrix, and phase contrast microscopy showed that the cells failed to attach to and spread on the oxidant-treated surface. When cells were replated on the treated matrix in the presence of 20% serum, they did attach, but showed abnormal spreading and morphology in longer-term culture. In a modified ELISA system, binding of antibodies specific to fibronectin, thrombospondin and laminin was also disrupted by prior exposure of the matrix to HOCl. Of these components, the cell-binding region of fibronectin was most affected by HOCl, thrombospondin and laminin were less sensitive, and the collagen-binding region of fibronectin was the most resistant. SDS-PAGE of 35S-labelled subendothelial matrix proteins indicated that there was no major irreversible crosslink formation or fragmentation after exposure to HOCl or the myeloperoxidase system, although formation of disulfides is quite likely.

FREE RADICAL INACTIVATION OF RABBIT MUSCLE CREATINE KINASE: CATALYSIS BY PHYSIOLOGICAL AND HYDROLYZED ICRF-187 (ICRF-198) IRON CHELATES

Creatine kinase is a sulfhydryl containing enzyme that is particularly susceptible to oxidative inactivation. This enzyme is potentially vulnerable to inactivation under conditions when it would be used as a diagnostic marker of tissue damage such as during cardiac ischemia/reperfusion or other oxidative tissue injury. Oxidative stress in tissues can induce the release of iron from its storage proteins, making it an available catalyst for free radical reactions. Although creatinine kinase inactivation in a heart reperfusion model has been documented, the mechanism has not been fully described, particularly with regard to the role of iron. We have investigated the inactivation of rabbit muscle creatine kinase by hydrogen peroxide and by xanthine oxidase generated superoxide or Adriamycin radicals in the presence of iron catalysts. As shown previously, creatine kinase was inactivated by hydrogen peroxide. Ferrous iron enhanced the inactivation. In addition, micromolar levels of iron and iron chelates that were reduced and recycled by superoxide or Adriamycin radicals were effective catalysts of creatinine kinase inactivation. Of the physiological iron chelates studied, Fe(ATP) was an especially effective catalyst of inactivation by what appeared to be a site-localized reaction. Fe(ICRF-198), a non-physiological chelate of interest because of its putative role in alleviating Adriamycin-induced cardiotoxicity, also catalyzed the inactivation. Scavenger studies implicated hydroxyl radical as the oxidant involved in iron-dependent creatine kinase inactivation. Loss of protein thiols accompanied loss of creatine kinase activity. Reduced glutathione (GSH) provided marked protection from oxidative inactivation, suggesting that enzyme inactivation under physiological conditions would occur only after GSH depletion.

Ultrafine Particulate Ferrous Iron and Anthracene Associations with Mitochondrial Dysfunction

The ultrafine size fraction of ambient particles (ultrafine particles [UFP], diameter < 100 nm) has been identified as being particularly potent in their adverse health effects, yet, the detailed mechanisms for why UFP display such distinctive toxicity are not well understood. In the present study, mitochondria were exposed to ambient UFP while monitoring mitochondrial electron transport chain (ETC) activity as a model system for biochemical toxicity. UFP samples were collected in rural and urban environments, and chemically characterized for trace metals, ferrous (Fe(II)) and easily reducible ferric (Fe(III)) iron, polycyclic aromatic hydrocarbons (PAHs), and surface constituents with X-ray photoelectron spectroscopy (XPS). Fixed doses of UFP (8 μg mL−1) inhibited mitochondrial ETC function compared to controls in 94% of the samples after the 20 min of exposure. Significant moderate to weak correlations exist between initial %ETC inhibition (0 – 10 min) and Fe(II) (R = 0.55, P = 0.03, N = 15), anthracene (R = 0.74, P < 0.01, N = 13), and %C–O surface bonds (R = 0.56, P = 0.03, N = 15), whereby anthracene and %C–O correlate with each other (R = 0.58, P = 0.03, N = 14). Multivariate linear regression showed that when combined, Fe(II) and anthracene best describe the initial %ETC inhibition (R = 0.91, P = 0.00, N = 14). No significant associations were identified with total Fe and other trace metals. Results from this study indicate that Fe(II) and anthracene-related, C–O containing, surface structures may contribute to the initial detrimental behavior of UFP, also supporting the idea that the Fe(II)/Fe(III) and certain efficient hydroquinone/quinone redox pairs play important roles due to their potential to produce reactive oxygen species (ROS).