Effects of alginate oligomer on the expression of cell cycle- and stress-related genes in Chlamydomonas reinhardtii.

Enzymatically prepared alginate oligomer (AO) promoted the growth of Chlamydomonas reinhardtii in a concentration-dependent manner. AO at 2.5 mg/mL induced increase in expression levels of cyclin A, cyclin B, and cyclin D in C. reinhardtii. CuSO4 at 100 µM suppressed the growth of C. reinhardtiin, and AO at 2.5 mg/mL significantly alleviated the toxicity of CuSO4. Increased intracellular reactive oxygen species level in C. reinhardtii induced by CuSO4 was reduced by AO. After cultivation with CuSO4 at 100 µM, expression levels of ascorbate peroxidase and superoxide dismutase in C. reinhardtii were increased, and AO reduced the increased levels of these enzymes. These results suggest that AO exhibits beneficial effects on C. reinhardtii through influencing the expression of various genes not only at normal growth condition but also under CuSO4 stress.

Atorvastatin active metabolite inhibits oxidative modification of small dense low-density lipoprotein.

We tested the hypothesis that atorvastatin active metabolite (ATM), on the basis of its distinct structural features and potent antioxidant activity, preferentially inhibits lipid oxidation in human small dense low-density lipoprotein (sdLDL) and other small lipid vesicles. LDL, sdLDL, and various subfractions were isolated from human plasma by sequential ultracentrifugation, treated with ATM, atorvastatin, pravastatin, rosuvastatin, or simvastatin and were subjected to copper-induced oxidation. Lipid oxidation was measured spectrophotometrically as a function of thiobarbituric acid reactive substances formation. Similar analyses were performed in reconstituted lipid vesicles enriched in polyunsaturated fatty acids and prepared at various sizes. ATM was found to inhibit sdLDL oxidation in a dose-dependent manner. The antioxidant effects of ATM in sdLDL were 1.5 and 4.7 times greater (P < 0.001) than those observed in large buoyant LDL and very low-density lipoprotein subfractions, respectively. ATM had similar dose- and size-dependent effects in reconstituted lipid vesicles. None of these effects were reproduced by atorvastatin (parent) or any of the other statins examined in this study. These data suggest that ATM interacts with sdLDL in a specific manner that also confers preferential resistance to oxidative stress. Such interactions may reduce sdLDL atherogenicity and improve clinical outcomes in patients with cardiovascular disease.

Solid gastric emptying mediated by the serotonin (5-HT)3 receptor in mice is a simple marker to predict emesis.

Nausea and emesis are often observed as side effects with many medicines and may lead to poor treatment compliance. In the present study, we aimed to establish simple methods for predicting nausea and/or emesis in mice, which do not vomit, using drugs and chemicals known to evoke nausea and/or emesis. The gastrointestinal transit test, the liquid gastric emptying by phenol red solution (Phenol red method) and the solid gastric emptying by resin beads (Beads method) were used and the effects of antispasmogenics (atropine, 0.1-3 mg/kg i.p.; salmon calcitonin, 1-30 units/kg i.m.), nauseants (copper sulfate, 1-30 mg/kg p.o.; apomorphine, 0.01-0.3 mg/kg s.c.) and chemotherapeutics (cisplatin, 0.3-10 mg/kg i.v.; doxorubicin, 0.3-10 mg/kg i.v.) were evaluated. In addition, the effects of ondansetron, a serotonin (5-HT)(3) receptor antagonist, on the inhibition of solid gastric emptying induced by salmon calcitonin, copper sulfate, cisplatin and doxorubicin were also assessed. Only the solid gastric emptying method could detect changes of gastric emptying by all drugs and chemicals. We also found that the inhibition of solid gastric emptying induced by cisplatin and doxorubicin was dose-dependently antagonized by ondansetron. However, ondansetron failed to antagonize the salmon calcitonin-induced delay, but exerted only very weak effects with copper sulfate. Solid gastric emptying may be more suitable than gastrointestinal intestinal transit or liquid gastric emptying in mice to predict nausea and/or emesis. Our results also suggest that chemotherapeutic-induced delay of solid gastric emptying mediated via 5-HT(3) receptors in mice could also be useful for prediction purposes.

Hibiscus anthocyanins-rich extract inhibited LDL oxidation and oxLDL-mediated macrophages apoptosis.

The oxidative modification of low-density lipoprotein (LDL) plays a key role in the pathogenesis of atherosclerosis. Anti-oxidative reagents, which can effectively inhibit LDL oxidation, may prevent atherosclerosis via reducing early atherogenesis, and slowing down the progression to advance stages. As shown in previous studies Hibiscus sabdariffa L. is a natural plant containing a lot of pigments that was found to possess anti-oxidative of activity. Therefore, in this study, we evaluated the anti-oxidative activity of Hibiscus anthocyanins (HAs) by measuring their effects on LDL oxidation (in cell-free system) and anti-apoptotic abilities (in RAW264.7 cells). HAs have been tested in vitro examining their relative electrophoretic mobility (REM), Apo B fragmentation, thiobarbituric acid relative substances (TBARS) and radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activity assay. The anti-oxidative activity of HAs was defined by relative electrophoretic mobility of oxLDL (decrease of 50% at 2 mg/ml), fragmentation of Apo B (inhibition of 61% at 1mg/ml), and TBARS assay (IC(50): 0.46 mg/ml) in the Cu(2+)-mediated oxidize LDL. Furthermore, the addition of >0.1 mg/ml of HAs could scavenge over 95% of free DPPH radicals, HAs showed strong potential in inhibiting LDL oxidation induced by copper. In addition, to determine whether oxLDL-induced apoptosis in macrophages is inhibited by HAs, we studied the viability, morphology and caspase-3 expression of RAW 264.7 cells. MTT assay, Leukostate staining analysis and Western blotting reveals that HAs could inhibit oxLDL-induced apoptosis. According to these findings, we suggest that HAs may be used to inhibit LDL oxidation and oxLDL-mediated macrophage apoptosis, serving as a chemopreventive agent. However, further investigations into the specificity and mechanism(s) of HAs are needed.

The receptor binding domain of apolipoprotein E is responsible for its antioxidant activity.

Apolipoprotein E (apoE) is a 34-kDa lipid-associated protein present in plasma and in the central nervous system. Previous studies have demonstrated that apoE has multiple functions, including the ability to transport lipids, regulate cell homeostasis, and inhibit lipid oxidation. The lipid binding domain of apoE has been localized to the carboxyl-terminal domain, whereas a cluster of basic amino acid residues within the N-terminal domain is responsible for its receptor binding activity. This study was undertaken to identify the domain in apoE responsible for its antioxidant activity. Results showed that apoE inhibits Cu(2+)-induced LDL oxidation by delaying conjugated diene formation in a concentration-dependent manner. Reductive methylation of lysine residues or cyclohexanedione modification of arginine residues in apoE abolished its ability to inhibit LDL oxidation. Additional studies showed that a 22-kDa peptide containing the N-terminal domain of apoE3 was more effective than a similar peptide with the apoE4 sequence in inhibiting Cu(2+)-induced LDL oxidation. In contrast, the 10-kDa peptide that contains the C-terminal domain of apoE was ineffective. Inhibition of Cu(2+)-induced LDL oxidation can also be accomplished with a peptide containing either a single sequence or a tandem repeat sequence of the receptor binding domain (residues 141-155) of apoE. Taken together, these results localized the antioxidant domain of apoE to its receptor binding domain and the basic amino acids in this domain are important for its antioxidant activity.

Pyridoxal isonicotinoyl hydrazone (PIH) prevents copper-mediated in vitro free radical formation.

Pyridoxal isonicotinoyl hydrazone (PIH) is an iron chelator with antioxidant activity, low toxicity and is useful in the experimental treatment of iron-overload diseases. Previous studies on x-ray diffraction have revealed that PIH also forms a complex with Cu(II). Since the main drug of choice for the treatment of Wilson's disease, d-penicillamine, causes a series of side effects, there is an urgent need for the development of alternative copper chelating agents for clinical use. These chelators must also have antioxidant activity because oxidative stress is associated with brain and liver copper-overload. In this work we tested the ability of PIH to prevent in vitro free radical formation mediated by Cu(II), ascorbate and dissolved O2. Degradation of 2-deoxyribose mediated by 10 microM Cu(II) and 3 mM ascorbate was fully inhibited by 10 microM PIH (I50 = 6 microM) or 20 microM d-penicillamine (I50 = 10 microM). The antioxidant efficiency of PIH remained unchanged with increasing concentrations (from 1 to 15 mM) of the hydroxyl radical detector molecule, 2-deoxyribose, indicating that PIH does not act as a hydroxyl scavenger. On the other hand, the efficiency of PIH (against copper-mediated 2-deoxyribose degradation and ascorbate oxidation) was inversely proportional to the Cu(II) concentration, suggesting a competition between PIH and ascorbate for complexation with Cu(lI). An almost full inhibitory effect by PIH was observed when the ratio PIH:copper was 1:1. A similar result was obtained with the measurement of copper plus ascorbate-mediated O2 uptake. Moreover, spectral studies of the copper and PIH interaction showed a peak at 455 nm and also indicated the formation of a stable Cu(II) complex with PIH with a 1:1 ratio. These data demonstrated that PIH prevents hydroxyl radical formation and oxidative damage to 2-deoxyribose by forming a complex with Cu(II) that is not reactive with ascorbate (first step of the reactions leading to hydroxyl radical formation from Cu(II), ascorbate and O2) and does not participate in Haber-Weiss reactions.

Inhibitory effect of quercetin metabolites and their related derivatives on copper ion-induced lipid peroxidation in human low-density lipoprotein.

To determine the antioxidant activity of dietary quercetin (3,3',4', 5,7-pentahydroxyflavone) in the blood circulation, we measured the inhibitory effect of quercetin metabolites and their related derivatives on copper ion-induced lipid peroxidation of human low-density lipoprotein (LDL). Conjugated quercetin metabolites were prepared from the plasma of rat 1 h after oral administration of quercetin aglycone (40 micromol/rat). The rate of cholesteryl ester hydroperoxide (CE-OOH) accumulation and the rate of alpha-tocopherol consumption in mixtures of LDL solution (0.4 mg/ml) with equal volumes of this preparation were slower than the rates in mixtures of LDL with preparations from control rats. The concentrations of CE-OOH after 2 h oxidation in the mixtures of LDL with preparations of conjugated quercetin metabolites were significantly lower than those in the control preparation. It is therefore confirmed that conjugated quercetin metabolites have an inhibitory effect on copper ion-induced lipid peroxidation in human LDL. Quercetin 7-O-beta-glucopyranoside (Q7G) and rhamnetin (3,3',4', 5-tetrahydroxy-7-methoxyflavone) exerted strong inhibition and their effect continued even after complete consumption, similarly to quercetin aglycone. The effect of quercetin 3-O-beta-glucopyranoside (Q3G) did not continue after its complete consumption, indicating that the antioxidant mechanism of quercetin conjugates lacking a free hydroxyl group at the 3-position is different from that of the other quercetin conjugates. The result that 4'-O-beta-glucopyranoside (Q4'G) and isorhamnetin (3,4',5, 7-tetrahydroxy-3'-methoxyflavone) showed little inhibition implies that introduction of a conjugate group to the position of the dihydroxyl group in the B ring markedly decreases the inhibitory effect. The results of azo radical-induced lipid peroxidation of LDL and the measurement of free radical scavenging capacity using stable free radical, 1,1,-diphenyl-2-picrylhydrazyl, demonstrated that the o-dihydroxyl structure in the B ring is required to exert maximum free radical scavenging activity. It is therefore likely that conjugation occurs at least partly in positions other than the B ring during the process of metabolic conversion so that the inhibitory effect of dietary quercetin is retained in blood plasma after absorption.

Oxidative modification of apolipoprotein E in human very-low-density lipoprotein and its inhibition by glycosaminoglycans.

The mechanism of metal ion-catalyzed oxidative modification of apolipoprotein E (apoE) in human very-low-density lipoprotein (VLDL) and its inhibition by glycosaminoglycan (GAG) was investigated in vitro. The VLDL oxidation catalyzed by Cu2+ led to the lipid peroxidation, the formation of aggregates, and covalent modification of apoE. The modified apoE lost heparin-binding activity. These results suggest that the lipid peroxidation of VLDL and modification of apoE cause impairment of lipid uptake by cells and deposit the oxidized lipids in the tissues. The lipid peroxidation and oxidative modification of apoE in VLDL mediated by Cu2+ and an aqueous radical generator were suppressed by GAG, heparan sulfate, heparin, and chondroitin sulfate A, even though GAGs demonstrated no ability to scavenge alpha,alpha-diphenyl-beta-picrylhydrazyl radical. There were no relationships between inhibitory activity of GAGs in the VLDL oxidation and their number of sulfate groups which possess chelating activity of metal ion. Therefore, it can be considered that the inhibition of VLDL oxidation by GAGs is possibly due to the interaction between GAG and VLDL which bring about the steric hindrance, interference with the reaction between VLDL particle and the reactive oxygen species. These studies suggest that GAGs preserve the biological functions of apoE from oxidative stress.

Potential protective effect of calcium carbonate as liming agent against copper toxicity in the African tilapia Oreochromis mossambicus.

The lipid peroxidative effects of copper sulfate singly (4 mg/l CuSO4.5H2O) and in combination with calcium carbonate (4 mg/l CuSO4.5H2O + 50 mg/l CaCO3) were determined in the liver of the African tilapia Oreochromis mossambicus following exposures of the fish to the chemicals for 24, 48, 72, 96 and 168 h. Lipid peroxidative effects of the treatment with calcium carbonate (50 mg/l CaCO3) and with a known hepatotoxicant, carbon tetrachloride (0.25 ml/l CCl4) were also determined. Fish not exposed to any chemical served as negative controls. The extent of lipid peroxidation was based on hepatic malondialdehyde (MDA) levels as assayed using the thiobarbituric acid reaction test. Results suggested the lipid peroxidative property of the copper salt which was associated with the toxic nature of the heavy metal, although, this effect was not as potent as that of CCl4. Findings also indicated a measure of protection against copper hepatotoxicity provided by the addition of calcium carbonate as a liming agent in the water.

Aged garlic extract and its constituents inhibit Cu(2+)-induced oxidative modification of low density lipoprotein.

Oxygen radical injury and lipid peroxidation have been suggested as major causes of atherosclerosis, cancer, liver disease, and the aging process. More specifically, oxidative modification of low density lipoprotein (LDL) has been recognized as an important process of atherosclerosis. In this study, we determined the effects of aged garlic extract (AGE), four of its constituents, and a metabolite on Cu(2+)-induced oxidative modification of LDL using an in vitro system. All these compounds were shown to inhibit oxidative modification of LDL.