Neural HO-1/sterol interactions in vivo: implications for Alzheimer's disease.

BACKGROUND: Up-regulation of heme oxygenase-1 (HO-1) and altered cholesterol (CH) metabolism are characteristic of Alzheimer-diseased (AD) neural tissues. We previously provided evidence of significant HO-1/sterol interactions in vitro (cultured rat astroglia) and in post-mortem human AD brain (Religious Orders Study). METHODS: The current experiments were designed to further delineate these interactions in vivo by comparing the behavior of HO-1/sterol interactions in two mouse models; (1) a novel HO-1 transgenic mouse (GFAP.HMOX1) engineered to selectively express human HO-1 in the astrocytic compartment and (2) the previously described triple transgenic AD mouse (3xTg-AD). In samples of frontal cortex, total CH, CH precursors and relevant oxysterols were quantified by gas chromatography-mass spectrometry (GC-MS) and HO-1 protein expression was assessed by ELISA. The relationships of HO-1 expression to total CH, CH precursors and total oxysterols were determined for both mouse models using linear regression analysis. RESULTS: HO-1 expression is increased in GFAP.HMOX1 mice relative to wild type and in 11-12-month-old 3xTg-AD mice (with AD-like phenotype) relative to control mice and 5-6-month-old 3xTg-AD mice (no AD-like phenotype). Total oxysterols significantly decreased as HO-1 expression increased in GFAP.HMOX1 mice expressing high levels of HO-1, whereas total oxysterols increased as HO-1 expression increased in aged 3xTg-AD mice. Total CH and total CH precursors increased as HO-1 protein expression increased in 11-12-month-old 3xTg-AD mice relative to 5-6-month old 3xTg-AD mice. CONCLUSIONS: Our findings indicate a differential impact of HO-1 on patterns of brain sterol and redox homeostasis that is contingent on the presence or absence of AD-like neuropathology. These data provide fresh insight concerning the regulation of sterol homeostasis within the aging and degenerating CNS which may inform the development of novel therapeutic and preventive strategies for the management of AD and related conditions.

Reduced glutathione increases quercetin stimulatory effects on HDL- or apoA1-mediated cholesterol efflux from J774A.1 macrophages.

In our in vitro study, we analyzed the effects of incubation of J774A.1 macrophages with reduced glutathione (GSH) and quercetin on the extent of cellular cholesterol efflux by high-density lipoprotein (HDL) or apolipoprotein A1 (apoA1). This combination was the most potent one among other exogenous and endogenous antioxidant combinations, since it significantly increased the extent of HDL-mediated cholesterol efflux from macrophages by 47% versus control cells, whereas quercetin (20 µM) or GSH (200 µM) alone increased it by only 37% or 17%, respectively. Similarly, apoA1-mediated cholesterol efflux was increased by 11% or 22% in quercetin or quercetin + GSH-treated cells, respectively, versus control cells. These stimulatory effects were noted only after 20 h of cell incubation. The combination of quercetin + GSH demonstrated high scavenging capacity of free radicals versus quercetin or GSH alone. In addition, quercetin + GSH significantly decreased macrophage oxidative stress as measured by the scavenging capacity of free radicals in the cells, the formation of reactive oxygen species, and the levels of cellular glutathione and lipid peroxides. There was no significant effect of quercetin + GSH on cellular HDL binding, on ATP-binding cassette A1 (ABCA1) activity, or on ABCG1 messenger RNA (mRNA) levels. In contrast, mRNA levels for ABCA1 and peroxisome proliferator-activated receptor alpha (PPARα) were both significantly increased by 89% and 93%, respectively, in quercetin + GSH-treated cells versus control cells. Quercetin alone increased the mRNA levels for ABCA1 or PPARα by 42% or 77%, respectively, whereas GSH alone increased it by 22% or 28%, respectively. Mass spectra analysis revealed that oxidized quercetin reacts with GSH to form a new adduct product. We thus conclude that the stimulatory effects of quercetin + GSH on apoA1- or HDL-mediated macrophage cholesterol efflux are related to the ability of GSH to preserve quercetin in its reduced form.

Analysis of volatile organic compounds in rats with dopaminergic lesion: Possible application for early detection of Parkinson's disease.

Parkinson's disease (PD) is characterized by dopaminergic (DA) neuron depletion. Early detection of PD may help in selecting the appropriate treatment. Biomarkers of PD have been suggested, however none of these is currently in clinical use. The aim of this study was to identify volatile organic compounds (VOCs) as early biomarkers of PD. Our hypothesis was that during PD progression, specific VOCs are generated that are linked to the biochemical pathways characterizing PD. These VOCs can be detected by GC-MS combined with solid-phase microextraction (SPME) technique. Three groups of rats were studied: DA-lesioned rats injected with 6-hydroxydopamine (HDA; 250µg/rat n=11); control rats injected with saline (n=9), and control rats injected with DSP-4 (n=8), a specific noradrenergic neuron toxin. Blood and striatal tissue homogenate were analyzed. In the blood, 1-octen-3-ol and 2-ethylhexanol were found at significantly higher concentrations in HDA versus sham rats. In the striatal homogenate 1-octen-3-ol and other four compounds were found at significantly lower concentrations in HDA versus sham rats. 1-Octen-3-ol is a cytotoxic compound. These results may lead to the development of an early diagnostic test for PD based on profiling of VOCs in body fluids.

Mutual interaction of special phytoestrogenic compounds, their synthetic carboxy-derivatives and the less-calcemic vitamin D analog activities in human derived female cultured osteoblasts.

Cultured female-derived human bone cells (hObs) responded by different parameters to different phytoestrogenic and vitamin D compounds. Pre- and post-menopausal hObs express ERα and ERß mRNA with higher abundance of ERα. Pre-treatment with the less-calcemic vitamin D analog JKF 1624F(2)-2 (JKF) upregulated responsiveness to estrogens via modulation of ERs expression. These estrogenic compounds induce the expression and activity of 25 hydroxy-vitamin D(3)-1α hydroxylase (1OHase). We now analyzed the effects of carboxy-genistein (cG), carboxy-biocainin A (cBA) and carboxy-daidzein (cD), of BA, D or G and of licorice derived compounds glabridin (Glb) and glabrene (Gla) and estradiol-17ß (E(2)) on DNA synthesis, creatine kinase specific activity (CK), intracellular and membranal E(2) binding and their modulations by JKF in hObs. We also analyzed modulation by phytoestrogenic compounds of 1OHase mRNA expression and activity. We showed that: (1) all compounds stimulated DNA synthesis and CK. (2) JKF and all estrogenic compounds modulated ERα and ERß mRNA expression. (3) Pre-treatment with JKF increased DNA synthesis and CK responses only to E(2), D, G and Gla. (4) JKF increased the intracellular competitive binding only of E(2), D and G. (5) JKF abolished the membranal binding of all protein-bound estrogens. (6) JKF and all estrogenic compounds except the protein-bound ones up-regulated 1OHase expression and activity. In conclusion phytoestrogens and their analogs increase DNA synthesis and CK, and lead to increased production of 1,25(OH)(2)D(3) in hObs, while pre-treatment with JKF modulates the effect of estrogenic compounds via regulation of ERs mRNA expression in a yet unclear mechanism.

Alterations in striatal oxidative stress level produced by pharmacological manipulation of dopamine as shown by a novel synthetic marker molecule.

Oxidative stress (OS) is thought to participate in neurodegenerative diseases such as Parkinson's disease, but the contribution of dopamine metabolism and auto-oxidation to OS in Parkinson's and other diseases is not clear. Oxidative stress in rat striatum was measured by microdialysis using a novel synthetic compound composed of tyrosine and linoleic acid (LT), and determination of the oxidation products LT-OOH and LT-epoxy by HPLC-MS. Since LT is non-diffusible through the microdialysis membrane, the oxidized products formed in microdialyzate reflect oxidation state in the extracellular compartment. The extracellular oxidative stress (OS(ec)) was compared with intracellular oxidative stress (OS(ic)) as measured by tissue levels of oxidized and reduced glutathione and 7-ketocholesterol. Reserpinization caused an increase in OS(ic) but a reduction in OS(ec). Inhibition of both subtypes of monoamine oxidase (MAO-A and MAO-B) with tranylcypromine caused a reduction in both OS(ic) and OS(ec) whereas selective inhibition of MAO-A with clorgyline caused a reduction in Os(ic) but no change in OS(ec). A high dose (10 mg/kg) of amphetamine caused an increase in OS(ec) whereas a smaller dose (4 mg/kg) caused a reduction in OS(ec). Both doses of amphetamine reduced OS(ic). The present findings are consistent with a role of monoamine oxidase as well as dopamine auto-oxidation in production of striatal OS.

Specific oxidative stress profile associated with partial striatal dopaminergic depletion by 6-hydroxydopamine as assessed by a novel multifunctional marker molecule.

Real time oxidative stress in the extracellular compartment of rat striatum was characterized by microdialysis with synthetic non-dialyzable marker molecules composed of linoleic acid, tyrosine and guanosine (N-linoleoyl tyrosine (LT) and N-linoleoyl tyrosine 2'-deoxyguanosyl ester (LTG)). Partial dopaminergic deafferentation was induced by injection of 6-hydroxydopamine (250 microg) to the left lateral ventricle, which depleted ipsilateral striatal dopamine by 46% and dopaminergic cells in left substantia nigra by 44%, 5 weeks after administration. Resting microdialysate dopamine levels in dopamine-depleted striatum were not different from sham-operated rats, although the ratio of oxidized metabolites of dopamine to free dopamine was significantly increased. Hydroperoxide and epoxy products of the linoleoyl portion of LT and LTG were detected in the striatal microdialysate by LC/MS/MS following initial separation by HPLC and were significantly increased in dopamine-depleted compared with control striatum without an increase in guanosine or tyrosine oxidation or nitration. Systemic administration of N-acetyl cysteine (350 mg/kg i.p.) decreased the increment in hydroperoxide and epoxy metabolites to levels not significantly different from control. Oxidation activity towards polyunsaturated fatty acids is present in the extracellular space of partially dopamine-denervated striatum, whereas oxidized glutathione and oxysterol levels in striatal tissue are decreased, possibly indicative of a compensatory response.

The response of creatine kinase specific activity in rat pituitary to estrogenic compounds and vitamin d less-calcemic analogs.

We examined the response of rat female pituitary at different metabolic stages to treatments with estrogenic compounds and vitamin D analogs. Immature or ovariectomized (Ovx) female rats responded by increased creatine kinase specific activity (CK) to estradiol-17beta (E(2)), genistein (G), daidzein (D), biochainin A (BA), quecertin (Qu), carboxy- G (cG), carboxy- BA (cBA), and raloxifene (Ral). The response was inhibited when Ral was injected together with the estrogens. CK was increased when hormones were injected daily into Ovx rats for 4 different time periods. Pretreatment with the less-calcemic vitamin D analogs JK 1624 F(2)-2 (JKF) or QW 1624 F(2)-2 (QW) followed by estrogenic injection resulted in increased response and sensitivity to E(2) and loss of inhibition of E(2) by Ral. CK was also increased by feeding with E(2) or licorice or its components dose- and time- dependent in immature or Ovxrats. Diabetic female rats did not respond to increased doses of E(2). In conclusion, rat female pituitary is estrogens-responsive organ, suggesting to considerits response for HRT in postmenopausal women for both beneficial and hazardous aspects.

Estrogen-like activity of glabrene and other constituents isolated from licorice root.

Licorice root extract and its major isoflavan, glabridin, exhibited varying degrees of estrogen receptor (ER) agonism in different tissues in vitro and in vivo. Animals fed with licorice extract, compared with estradiol and glabridin, showed an increase in creatine kinase (CK) activity, a known marker for estrogen responsive genes, which was higher than expected from the levels of glabridin in the extract. This led us to test for other components that may contribute to this strong estrogen agonist activity. Results indicated that glabrene and isoliquiritigenin, (2',4',4-three hydroxy chalcone) (ILC) in the licorice extract can bind to the human ER with higher affinity (IC50, 1 and 0.5 microM) than glabridin (IC50, 5 microM). The stimulatory effects of glabrene in vivo were tissue specific and similar to those of estradiol. The effect of increasing concentrations of glabrene and ILC on the growth of breast tumor cell were biphasic. Both showed an ER-dependent growth-promoting effect at low concentrations (10 nM-10 microM), and ER-independent antiproliferative activity at concentrations >15 microM. This is the first study to indicate that glabrene, an isoflavene exerted varying degrees of ER agonism in different tissues.

Pomegranate juice supplementation to atherosclerotic mice reduces macrophage lipid peroxidation, cellular cholesterol accumulation and development of atherosclerosis.

Inhibition of lipid peroxidation contributes to the attenuation of macrophage cholesterol accumulation, foam-cell formation and atherosclerosis. Evidence suggests that nutritional antioxidants such as pomegranate juice (PJ) can contribute to the reduction of oxidative stress and atherogenesis. The goals of the present study were to determine whether such beneficial effects of PJ exist when supplemented to apolipoprotein E-deficient (E(0)) mice with advanced atherosclerosis and to analyze the antiatherosclerotic activity of a tannin-fraction isolated from PJ. Mice (4-mo-old) were supplemented with PJ in their drinking water for 2 mo and compared with age-matched placebo-treated mice, as well as to young (4-mo-old) control mice, for their mouse peritoneal macrophage (MPM) oxidative state, cholesterol flux and mice atherosclerotic lesion size. PJ supplementation reduced each of the proatherogenic variables determined in the present study compared with age-matched placebo-treated mice. It significantly induced serum paraoxonase activity and reduced MPM lipid peroxide content compared with placebo-treated mice and control mice. PJ administration to E(0) mice significantly reduced the oxidized (Ox)-LDL MPM uptake by 31% and MPM cholesterol esterification and increased macrophage cholesterol efflux by 39% compared with age-matched, placebo-treated mice. PJ consumption reduced macrophage Ox-LDL uptake and cholesterol esterification to levels lower than those in 4-mo-old, unsupplemented controls. PJ supplementation to E(0) mice with advanced atherosclerosis reduced the lesion size by 17% compared with placebo-treated mice. In a separate study, supplementation of young (2-mo-old) E(0) mice for 2 mo with a tannin fraction isolated from PJ reduced their atherosclerotic lesion size, paralleled by reduced plasma lipid peroxidation and decreased Ox-LDL MPM uptake. PJ supplementation to mice with advanced atherosclerosis reduced their macrophage oxidative stress, their macrophage cholesterol flux and even attenuated the development of atherosclerosis. Moreover, a tannin-fraction isolated from PJ had a significant antiatherosclerotic activity.

Paradoxical interactions among estrogen receptors, estrogens and SERMS: mutual annihilation and synergy.

The phenomenon of mutual annihilation of action between 17beta estradiol (E(2)) and a selective estrogen receptor modulator (SERM), previously described in prepubertal rat diaphysis, epiphysis and uterus, has been investigated in ROS 17/2.8 rat osteoblastic cells and in transiently co-transfected cells in culture. In ROS 17/2.8 cells, the estrogen-induced marker enzyme creatine kinase B (CKB) was stimulated by raloxifene, tamoxifen and tamoxifen methiodide to a specific activity equal to or greater than that induced by 10 nM E(2). However, when a fully inhibitory dose of any of these SERMS was given simultaneously with E(2), no stimulation of CK activity resulted. Therefore, SERMS can be full agonists when acting alone, but complete antagonists to a super-physiological dose of estrogen. It is expected that excess tamoxifen would prevent the action of a SERM, but that the agonist activity of a SERM is abolished by 1000-fold less estrogen is a phenomenon without obvious explanation by classical pharmacology of competitive inhibition. To probe the mechanism of this interaction further, a ckb-CAT reporter plasmid, plus the human receptor expression plasmid, HEO, was transfected transiently into several cell types. In MCF-7 cells, a 1:10 ratio of E(2) to tamoxifen produced mutual annihilation, but the same ratio in ROS 17/2.8 or HeLa cells led to synergistic stimulation. In HeLa cells, co-transfected with the more efficient wild-type estrogen receptor plasmid, HEGO, synergy was demonstrated only at sub-saturation levels of HEGO. We speculate that, in the presence of estradiol and a SERM, not only active homodimers would be formed, but also hetero-dimers of estrogen-liganded and tamoxifen-liganded receptor monomers, depending on the molar ratio of their ligands and their relative affinities. The resulting hetero-dimer conformation would change the specific receptor surface for interactions with the growing number of co-activators and co-repressors, structural changes which could help to explain the mutual annihilation and synergy phenomena and their cell selectivity.

Selective distribution of oxysterols in atherosclerotic lesions and human plasma lipoproteins.

The presence of oxidized sterols (oxysterols) in human serum and lesions has been linked to the initiation and progression of atherosclerosis. Data concerning the origin, identity and quantity of oxysterols in biological samples are controversial and inconsistent. This inconsistency may arise from different analytical methods or handling conditions used by different investigators. In the present study, oxysterol levels and distribution were analyzed by an optimized GC-MS method, in human atherosclerotic coronary and carotid lesions, in atherosclerotic apolipoprotein E deficient mice (E degrees mice) and in native and in vitro oxidized human low and high density lipoproteins. Oxysterol levels were analyzed with a limit of detection of 0.06 - 0.24 ng, with 25-hydroxycholesterol (25-OH) being the least sensitive. In human coronary and carotid lesions, obtained from endatherectomic samples, 27-hydroxycholesterol (27-OH) was the major oxysterol, with about 85% as sterols esterified to fatty acids. While total cholesterol and oxysterols levels were similar in both kinds of human lesions, oxysterol distribution was significantly different. In coronary lesions the mean levels of 27-OH and 7beta-hydroxycholesterol (7beta-OH) were 38% and 20% of total oxysterols, whereas in carotid lesions their mean levels were 66% and 5%, respectively. Unlike in human aortic lesions, 27-OH was entirely absent in E degrees mice, whereas the level of 7alpha-hydroxycholesterol (7alpha-OH) was 28% of the total oxysterols, vs. 5% in human coronary lesions. As 27-OH is an enzymatic product of cholesterol oxidation, this finding may indicate that such an enzymatic process does not take place in E degrees mice.

Estrogenic and antiproliferative properties of glabridin from licorice in human breast cancer cells.

There is an increasing demand for natural compounds that improve women's health by mimicking the critical benefits of estrogen to the bones and the cardiovascular system but avoiding its deleterious effects on the breast and uterus. The estrogenic properties of glabridin, the major isoflavan in licorice root, were tested in view of the resemblance of its structure and lipophilicity to those of estradiol. The results indicate that glabridin is a phytoestrogen, binding to the human estrogen receptor and stimulating creatine kinase activity in rat uterus, epiphyseal cartilage, diaphyseal bone, aorta, and left ventricle of the heart. The stimulatory effects of 2.5-25 microg/animal glabridin were similar to those of 5 microg/animal estradiol. Chemical modification of glabridin showed that the position of the hydroxyl groups has a significant role in binding to the human estrogen receptor and in proliferation-inducing activity. Glabridin was found to be three to four times more active than 2'-O-methylglabridin and 4'-O-methylglabridin, and both derivatives were more active than 2',4'-O-methylglabridin. The effect of increasing concentrations of glabridin on the growth of breast tumor cells was biphasic. Glabridin showed an estrogen receptor-dependent, growth-promoting effect at low concentrations (10 nM-10 microM) and estrogen receptor-independent antiproliferative activity at concentrations of > 15 microM. This is the first study to indicate that isoflavans have estrogen-like activities. Glabridin and its derivatives exhibited varying degrees of estrogen receptor agonism in different tests and demonstrated growth-inhibitory actions on breast cancer cells.

Human serum paraoxonases (PON1) Q and R selectively decrease lipid peroxides in human coronary and carotid atherosclerotic lesions: PON1 esterase and peroxidase-like activities.

BACKGROUND: Human serum paraoxonase (PON1) exists in two polymorphic forms: one that differs in the amino acid at position 192 (glutamine and arginine, Q and R, respectively) and the second one that differs in the amino acid at position 55 (methionine and leucine, M and L, respectively). PON1 protects LDL from oxidation, and during LDL oxidation, PON1 is inactivated. METHODS AND RESULTS: The present study compared PON1 isoforms Q and R for their effect on lipid peroxide content in human coronary and carotid lesions. After 24 hours of incubation with PON1Q or PON1R (10 arylesterase units/mL), lipid peroxides content in both coronary and carotid lesion homogenates (0.1 g/mL) was reduced up to 27% and 16%, respectively. The above incubation was associated with inactivation of PON1Q and PON1R by 15% and 45%, respectively. Lesion cholesteryl linoleate hydroperoxides and cholesteryl linoleate hydroxides were hydrolyzed by PON1 to yield linoleic acid hydroperoxides and linoleic acid hydroxides. Furthermore, lesion and pure linoleic acid hydroperoxides were reduced to yield linoleic acid hydroxides. These results thus indicate that PON1 demonstrates esterase-like and peroxidase-like activities. Recombinant PON1 mutants in which the PON1-free sulfhydryl group at cysteine-284 was replaced with either alanine or serine were no longer able to reduce lipid peroxide content in carotid lesions. CONCLUSIONS: We conclude that PON1 may be antiatherogenic because it hydrolyzes lipid peroxides in human atherosclerotic lesions.

Oxidized monocyte-derived macrophages in aortic atherosclerotic lesion from apolipoprotein E-deficient mice and from human carotid artery contain lipid peroxides and oxysterols.

Oxidative stress is thought to play an important role in atherogenesis. The present study demonstrated, for the first time, that macrophages (originally derived from blood monocytes) isolated from aortas of the atherosclerotic apolipoprotein E deficient (E degrees ) mice or from human carotid artery, are oxidized as they contain lipid peroxides and oxysterols. The major oxysterol in arterial macrophages was found to be 7-ketocholesterol (51% of total oxysterols). To find out whether lipid peroxidation of monocytes occurs in vivo already in the blood, we analyzed the oxidative state of monocytes derived from E degrees mice in comparison to monocytes from control mice. Cellular lipid peroxides and total oxysterols were four and sevenfold higher respectively, in monocytes derived from E degrees mice in comparison to monocytes from control mice. The results of the present study thus demonstrated the presence of lipid-peroxidized monocytes already in the blood, which are further oxidized in the arterial wall after their conversion into macrophages. The arterial oxidized macrophages could be considered key contributors to foam cell formation, the hallmark of early atherosclerosis.

Macrophage enrichment with the isoflavan glabridin inhibits NADPH oxidase-induced cell-mediated oxidation of low density lipoprotein. A possible role for protein kinase C.

Macrophage-mediated oxidation of low density lipoprotein (LDL) is considered to be of major importance in early atherogenesis; therefore, intervention means to inhibit this process are being extensively studied. In the present study, we questioned the ability of the isoflavan glabridin (from licorice) to accumulate in macrophages and to affect cell-mediated oxidation of LDL. We first performed in vitro studies, using mouse peritoneal macrophages (MPMs) and the J-774 A.1 macrophage-like cell line. Both cells accumulated up to 1.5 micrograms of glabridin/mg of cell protein after 2 h of incubation, and this process was time- and glabridin dose-dependent. In parallel, in glabridin-enriched cells, macrophage-mediated oxidation of LDL was inhibited by up to 80% in comparison with control cells. Glabridin inhibited superoxide release from MPMs in response to phorbol 12-myristate 13-acetate, or to LDL when added together with copper ions, by up to 60%. Translocation of P-47, a cytosolic component of NADPH oxidase to the plasma membrane was substantially inhibited. In glabridin-enriched macrophages, protein kinase C activity reduced by approximately 70%. All of the above effects of glabridin required the presence of the two hydroxyl groups on the flavonoid's B phenol ring. In order to assess the physiological significance of these results, we next performed in vivo studies, using the atherosclerotic apolipoprotein E-deficient (E0) mice. MPMs harvested from glabridin-treated E0 mice (20 micrograms/mouse/day for a period of 6 weeks) demonstrated reduced capability to oxidize LDL by 80% in comparison with placebo-treated mice. This latter phenomenon was associated with a reduction in the lesion oxysterols and a 50% reduction in the aortic lesion size. We thus conclude that glabridin accumulation in macrophages is associated with reduced cell-mediated oxidation of LDL and decreased activation of the NADPH oxidase system. These phenomena could be responsible for the attenuation of atherosclerosis in E0 mice, induced by glabridin.

Bioflavonoid effects on the mitochondrial respiratory electron transport chain and cytochrome c redox state.

The polyphenolic structure common to flavonoids enables them to donate electrons and exert antioxidant activity. Since the mitochondrial electron transport chain consists of a series of redox intermediates, the effect of flavonoids in a complex mixture of polyphenols, as well as related pure flavonoids, was evaluated on the rat liver mitochondrial electron transport chain. A French maritime pine bark extract (PBE), a complex mixture of polyphenols and related pure flavonoids, was able to reduce cytochrome c reversibly, possibly by donation of electrons to the iron of the heme group; the donated electrons can be utilized by cytochrome c oxidase. Among single flavonoids tested, (-)-epicatechin gallate had the greatest ability to reduce cytochrome c. In addition, PBE competitively inhibited electron chain activity in both whole mitochondria and submitochondrial particles. A 3.5-fold increase in the apparent Km value for succinate was calculated from reciprocal plots. Among the flavonoids tested, taxifolin and (-)-epicatechin gallate showed minor inhibitory effects, while (+/-)-catechin and (+)-epicatechin were ineffective. Activities of NADH-ubiquinone, succinate-ubiquinone, and ubiquinol-cytochrome c reductases were inhibited by low concentrations of PBE to a similar extent. However, inhibition of cytochrome c oxidase activity required 4-fold higher PBE concentrations. These results suggest that flavonoids reduce cytochrome c and that PBE inhibits electron transport chain activity mainly through NADH-ubiquinone, succinate-ubiquinone, and ubiquinol-cytochrome c reductases.

Structural aspects of the inhibitory effect of glabridin on LDL oxidation.

The inhibitory effects of glabridin, an isoflavan isolated from licorice (Glycyrrhiza glabra) root, and its derivatives on the oxidation of LDL induced by copper ions or mediated by macrophages were studied, in order to evaluate the contribution of the different parts of the isoflavan molecule to its antioxidant activity. The peak potential (E1/2) of the isoflavan derivatives, their radical scavenging capacity toward 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radical and their ability to chelate heavy metals were also analyzed and compared to their inhibitory activity on LDL oxidation. In copper ion-induced LDL oxidation, glabridin (1), 4'-O-methylglabridin (2), hispaglabridin A (3), and hispaglabridin B (4), which have two hydroxyl groups at positions 2' and 4' or one hydroxyl at position 2' on ring B, successfully inhibited the formation of conjugated dienes, thiobarbituric acid reactive substances (TBARS) and lipid peroxides, and inhibited the electrophoretic mobility of LDL under oxidation. Compounds 1-3 exhibited similar activities, whereas compound 4 was less active. In macrophage-mediated LDL oxidation, the TBARS formation was also inhibited by these isoflavans (1-4) at a similar order of activity to that obtained in copper ion-induced LDL oxidation. On the other hand, 2'-O-methylglabridin (5), a synthesized compound, whose hydroxyl at 2'-position is protected and the hydroxyl at 4'-position is free, showed only minor inhibitory activity in both LDL oxidation systems. 2',4'-O-Dimethylglabridin (6), whose hydroxyls at 2'- and 4'-positions are both protected, was inactive. Resorcinol (7), which is identical to the phenolic B ring in glabridin, presented low activity in these oxidation systems. The isoflavene glabrene (8), which contains an additional double bond in the heterocyclic C ring, was the most active compound of the flavonoid derivatives tested in both oxidation systems. The peak potential of compounds 1-5 (300 microM), tested at pH 7.4, was similar (425-530 mV), and that for compound 6 and 8 was 1078 and 80 mV, respectively. Within 30 min of incubation, compounds 1, 2, 3, 4, 8 scavenged 31%, 16%, 74%, 51%, 86%, respectively, of DPPH radical, whereas compounds 5 and 6, which almost did not inhibit LDL oxidation, also failed to scavenge DPPH. None of the isoflavan derivatives nor the isoflavene compound were able to chelate iron, or copper ions. These results suggest that the antioxidant effect of glabridin on LDL oxidation appears to reside mainly in the 2' hydroxyl, and that the hydrophobic moiety of the isoflavan is essential to obtain this effect. It was also shown that the position of the hydroxyl group at B ring significantly affected the inhibitory efficiency of the isoflavan derivatives on LDL oxidation, but did not influence their ability to donate an electron to DPPH or their peak potential values.

The antioxidative effects of the isoflavan glabridin on endogenous constituents of LDL during its oxidation.

The effect of the consumption of glabridin, an isoflavan isolated from Glycyrrhiza glabra (licorice) root, on the susceptibility of low density lipoprotein (LDL) to oxidation was studied in atherosclerotic apolipoprotein E deficient (E[o] mice) and was compared with that of the known flavonoids, quercetin and catechin. Glabridin inhibitory activity on in vitro oxidation of human LDL was also investigated by determining the formation of lipid peroxides and oxysterols and the consumption of LDL-associated lipophilic antioxidants. Determination of the extent of LDL oxidation by measuring the formation of thiobabituric acid reactive substances (TBARS) after 2 h of LDL incubation with CuSO4 (10 microM) or 2,2'-azobis (2-amidino-propane) dihydrochloride (AAPH) (5 mM), revealed that glabridin or quercetin consumption resulted in a 53 and 54% reduction in copper ion induced oxidation, respectively, and a 95 and 83% reduction in AAPH induced LDL oxidation, respectively. No inhibition was obtained with consumption of catechin. About 80% of glabridin was found to bind to the LDL human particle. In the in vitro oxidation of LDL induced by AAPH (5 mM), glabridin inhibited the formation of TBARS, lipid peroxides and cholesteryl linoleate hydroperoxide (CLOOH) at all the concentrations tested (5-60 microM), while in oxidation induced by copper ions (10 microM), glabridin exhibited a pro-oxidant activity at concentrations lower than 20 microM, and a clear antioxidant activity at concentrations greater than 20 microM. Glabridin (30 microM) inhibited the formation of cholest-5-ene-3,7-diol (7-hydroxycholesterol), cholest-5-ene-3-ol-7-one (7-ketocholesterol) and cholestan-5,6-epoxy-3-ol (5,6-epoxycholesterol) after 6 h of AAPH induced LDL oxidation, by 55, 80 and 40%, respectively, and after 6 h of copper ion induced LDL oxidation, by 73, 94 and 52%, respectively. Glabridin also inhibited the consumption of beta-carotene and lycopene by 38 and 52%, respectively, after 0.5 h of LDL oxidation with AAPH, but failed to protect vitamin E. The in vivo and in vitro reduction of the susceptibility of LDL to oxidation obtained with glabridin, may be related to the absorption or binding of glabridin to the LDL particle and subsequent protection of LDL from oxidation by inhibiting the formation of lipid peroxides and oxysterols, and by protecting LDL associated carotenoids.