Ronald S. Pardini

Inhibition of mitochondrial respiration and production of toxic oxygen radicals by flavonoids: A structure-activity study

A series of fourteen flavonoids were employed in a systematic structure-activity study to assess their abilities to inhibit succinoxidase and generate toxic oxygen species in beef heart mitochondria. By comparing I50 values toward succinoxidase activity, flavonoids with a catechol moiety on the b ring exhibited the following general order of potency: chalcone greater than flavone greater than flavonol greater than dihydroflavonol greater than anthocyanidin. Catechins were inactive. In a series of 3,5,7-trihydroxyflavones containing various configurations of the b ring hydroxyl groups, it was found that the flavonoids possessing adjacent trihydroxy (pyrogallol) and b ring ortho-hydroxy(catechol) configurations were the most potent inhibitors of succinoxidase, followed by those with meta-hydroxyl, monohydroxyl and unhydroxylated configurations. Four of the fifteen flavonoids tested exhibited substrate-independent, KCN-insensitive respiration. Two flavonols with a pyrogallol configuration, myricetin and quercetagetin, produced the largest respiratory bursts and were found to auto-oxidize. Evidence is presented that the mitochondrial respiratory bursts induced by both flavonols and their auto-oxidation resulted in the generation of O-2 and H2O2.

Electrochemistry of flavonoids: Relationships between redox potentials, inhibition of mitochondrial respiration, and production of oxygen radicals by flavonoids

We have investigated the redox behavior of a series of structurally related flavonoids employing cyclic voltammetry under physiological conditions. The flavonoids that auto-oxidized and produced oxygen radicals had oxidation potentials (E 1/2) significantly lower [-30 to +60 mV vs (SCE)] than those that did not undergo auto-oxidation (+130 to +340 mV vs SCE). The range of E 1/2 values for the auto-oxidizable flavonoids was comparable to the E 1/2 range reported for the optimum quinone induced production of superoxide (O2 pi) in mitochondrial NADH-CoQ reductase (complex I). The most potent flavonoid inhibitors of mitochondrial succinate-CoQ reductase (complex II) possessed hydroxyl configurations capable of supporting redox reactions. For a series of 3,5,7-trihydroxyflavones that differed by b-ring hydroxylation it was found that decreasing E 1/2 of the flavonoids was associated with decreasing I50 values towards succinoxidase. These findings suggest that the electrochemical properties of the flavonoids may contribute to their biological activity.

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.

Mechanisms for regulating oxygen toxicity in phytophagous insects

The antioxidant enzymatic defense of insects for the regulation of oxygen toxicity was investigated. Insect species examined were lepidopterous larvae of the cabbage looper (Trichoplusia ni), southern armyworm (Spodoptera eridania), and black swallowtail (Papilio polyxenes). These phytophagous species are subject to both endogenous and exogenous sources of oxidative stress from toxic oxygen radicals, hydrogen peroxide (H2O2) and lipid peroxides (LOOH). In general, the constitutive levels of the enzymes superoxide dismutase (SOD), catalase (CAT), glutathione transferase (GT), and its peroxidase activity (GTpx), and glutathione reductase (GR), correlate well with natural feeding habits of these insects and their relative susceptibility to prooxidant plant allelochemicals, quercetin (a flavonoid), and xanthotoxin (a photoactive furanocoumarin). Induction of SOD activity which rapidly destroys superoxide radicals, appears to be the main response to dietary prooxidant exposure. A unique observation includes high constitutive activity of CAT and a broader subcellular distribution in all three insects than observed in most mammalian species. These attributes of CAT appear to be important in the prevention of excessive accumulation of cytotoxic H2O2. Unlike mammalian species, insects possess very low levels of a GPOX-like activity toward H2O2. Irrefutable proof that this activity is due to a selenium-dependent GPOX found in mammals, is lacking at this time. However, the activity of selenium-independent GTpx is unusually high in insects, suggesting that GTpx and not GPOX plays a prominent role in scavenging deleterious LOOHs. The GSSG generated from the GPOX and GTpx reactions may be reduced to GSH by GR activity. A key role of SOD in protecting insects from prooxidant toxicity was evident when its inhibition resulted in enhanced toxicity towards prooxidants. The role of antioxidant compounds in protecting these insects from toxic forms of oxygen has not been explored in depth. A major finding, however, is that these insects are lutein accumulators. Lutein is a dihydroxy (diol) derivative of beta-carotene, and it is a good quencher of activated forms of oxygen and free radicals. Levels of lutein are highest in P. polyxenes which specializes in feeding on prooxidant-containing plants.

Effects of dietary fish oil on human mammary carcinoma and on lipid-metabolizing enzymes

The growth rate of a human mammary carcinoma, MX-1, was significantly reduced in athymic “nude” mice fed fish oil. Tumors from the fish oil-fed animals also showed a greater sensitivity to two anti-neoplastic agents, mitomycin C and doxorubicin. Mitochondria were isolated from control livers, host livers and tumors from fish oil-and corn oil-fed animals, and increased levels of 20∶5n−3 and 22∶6n−3 were found in mitochondrial lipids in all three tissues from the fish oil-fed animals. To investigate the effect of dietary n−3 fatty acids on lipid metabolism, the activity of the acyl-CoA:carnitine acyltransferase and three acyl-CoA desaturases were measured. Carnitine acyltransferase activity toward all four acyl-CoA substrates tested was markedly increased in mitochondria from liver by feeding fish oil. In mitochondria from tumors, feeding fish oil resulted in an increased activity toward only 18∶3n−3. These data suggest that fish oil may induce an increase in the oxidation of fatty acids. The Δ9-desaturase activity was decreased in microsomes from liver and tumor from fish oil-fed animals. However, both the Δ6 and Δ5 desaturases were increased in tumor and in control liver as a result of feeding fish oil. The Δ5 desaturase was not altered in microsomes from the host animals. The effect of fish oil on the Δ5 and Δ6 desaturases may involve alterations to metabolism of specific polyunsaturated fatty acids especially in the tumor tissue.

Inhibition of mitochondrial respiration and cyanide-stimulated generation of reactive oxygen species by selected flavonoids

A continuation of our structure-activity study on flavonoids possessing varied hydroxyl ring configurations was conducted. We tested six additional flavonoids for their ability to inhibit beef heart mitochondrial succinoxidase and NADH-oxidase activities. In every case, the IC50 observed for the NADH-oxidase enzyme system was lower than for succinoxidase activity, demonstrating a primary site of inhibition in the complex I (NADH-coenzyme Q reductase) portion of the respiratory chain. The order of potency for inhibition of NADH-oxidase activity was robinetin, rhamnetin, eupatorin, baicalein, 7,8-dihydroxyflavone, and norwogonin with IC50 values of 19, 42, 43, 77, 277 and 340 nmol/mg protein, respectively. Flavonoids with adjacent tri-hydroxyl or para-dihydroxyl groups exhibited a substantial rate of auto-oxidation which was accelerated by the addition of cyanide (CN-). Flavonoids possessing a catechol configuration exhibited a slow rate of auto-oxidation in buffer that was stimulated by the addition of CN-. The addition of superoxide dismutase (SOD) and catalase in the auto-oxidation experiments each decreased the rate of oxygen consumption, indicating that O2- and H2O2 are generated during auto-oxidation. In the CN(-)-stimulated oxidation experiments, the addition of SOD also slowed the rate of oxygen consumption. These findings demonstrate that the CN-/flavonoid interaction generated O2- non-enzymatically, which could have biological implications.

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

Abstract

Cabbage looper antioxidant enzymes: Tissue specificity

Abstract A unique pattern of antioxidant enzymes exists in phytophagous insects for defense against endogenous and exogenous sources of toxic forms of oxygen, and data presented herein describe a profile of these enzymes in many tissues of larvae of the cabbage looper moth, Trichplusia ni . The specific activities of the antioxidant enzymes were high in tissues of high metabolic activities, i.e. Malpighian tubules, hindgut, muscles and gonads. A unique finding was the high constitutive activity of a superoxide dismutase (SOD) in hemocytes, probably consisting predominantly of the CuZn-SOD, which is analogous to the exclusive presence of this form of SOD in vertebrate erythrocytes and leukocytes. In all other tissues, the activity of Mn-SOD was higher than that of the CuZn-SOD which is converse to the pattern in vertebrate tissues. The glutathione peroxidase (GPOX) activity, present in all tissues and with highest levels in the gonads, did not seem to be the selenoprotein typical of the mammalian GPOX. Glutathione -S- transferase (GST) activity paralleled that of its glutathione peroxidase activity (GSTPX). The high GSTPX activity suggests that GSTPX and not GPOX, forms a sequential team with glutathione reductase (GR) to reduce deleterious lipid hydroperoxides and to reduce the oxidized glutathione, GSSG, back to GSH. Catalase (CAT) which decomposes H 2 O 2 has very high activity apparently correlated with the low GPOX activity. Finally, the integumental epithelium, and the gut (combined sections) possessed higher amounts of antioxidant enzymes than other tissues. Thus, a physiological relationship may occur between the antioxidant enzyme levels in tissues of T. ni with particularly high metabolic activity and associated endogenous oxidative stress. In addition, another physiological role of these enzymes may be to protect from exogenous oxidative stress exerted by dietary redox-active pro-oxidants in the gut, and to the potential of photodynamically mediated oxygen toxicity in peripheral organs such as the integument.

Docosahexaenoic acid alters epidermal growth factor receptor-related signaling by disrupting its lipid raft association

Docosahexaenoic acid (DHA), a 22:6 n-3 polyunsaturated fatty acid, is the longest and most highly unsaturated fatty acid found in most membranes and has been shown to inhibit cancer cell growth in part by modifying cell signaling. In the current study, alterations to epidermal growth factor receptor (EGFR) signaling upon DHA supplementation are examined in A549 lung adenocarcinoma, WiDr colon carcinoma and MDA-MB-231 breast carcinoma cell lines. Interestingly, EGFR phosphorylation, most notably at the tyrosine 1068 residue, is dramatically upregulated, and EGFR association with the Sos1 guanine nucleotide exchange factor is concomitantly increased upon DHA supplementation. However, guanosine triphosphate-bound Ras and phosphorylated extracellular signal-regulated kinase (Erk)1/2 are paradoxically downregulated in the same treatments. Previous reports have noted changes in membrane microdomains upon DHA supplementation, and our findings confirmed that EGFR, but not Ras, is excluded from caveolin-rich lipid raft fractions in DHA-treated cells, resulting in a decreased association of Ras with Sos1 and the subsequent downregulation of Erk signaling. Xenografts of the A549 cell line implanted in athymic mice fed a control high-fat diet or a diet high in DHA confirmed our in vitro data. These results demonstrate for the first time a functional consequence of decreased EGFR protein in lipid raft microdomains as a result of DHA treatment in three different cancer models. In addition, we report the ability of DHA to enhance the efficacy of EGFR inhibitors on anchorage-independent cell growth (soft agar), providing evidence for the potential development of enhanced combination therapies.