Pathways of chemical degradation of polypeptide antibiotic bacitracin

We described the main pathways of bacitracin (Bc) decomposition, chromatographically set the position of its major degradation products and evaluated microbiological activity of isolated components of Bc and its degradation products. All processes of Bc decomposition under stress and accelerated test conditions were monitored with HPLC, performed mainly on a new type reversed-phase (RP-18e) monolithic silica column (Chromolith) enabling fast separation times and some of them also on conventional HPLC columns. Diode array detection, preparative HPLC and FAB mass spectrometry were used for identification of individual Bc components. We found that the major decomposition mechanism in water solutions of Bc is oxidation, and in alkaline solutions, deamidation. In oxidation process the components B1, B2 and B3 and A are oxidized into their corresponding oxidative products H1, H2, H3 and F respectively by the same mechanism. A detailed study of oxidative degradation products revealed that HPLC separation with an acid mobile phase caused splitting of peaks of components H2, H3 and F into two peaks but the peak of component H1 did not split due to its special structural properties. For the component A we confirmed gradual formation of desamido product through an intermediate. We found oxidative degradation products of Bc to be relatively stable, and desamido degradation products to be rather unstable. The estimation of kinetics of Bc decomposition was presented with a semi-quantitative model. Microbiological activity of individual isolated active components of Bc was established and the negligible antimicrobial activity of the degradation products was confirmed.     

Deferoxamine inhibits methyl mercury-induced increases in reactive oxygen species formation in rat brain.

It has been suggested that methyl mercury may express its neurotoxicity by way of iron-mediated oxidative damage. Therefore, the effect of deferoxamine, a potent iron-chelator, on methyl mercury-induced increases in reactive oxygen species formation was studied in rat brain. The generation rate of reactive oxygen species was estimated in crude synaptosomal fractions using the probes 2',7'-dichlorofluorescin diacetate and dihydrorhodamine 123. The formation rate of the fluorescent oxidation products was used as the measure of reactive oxygen species generation. Seven days after a single injection of methyl mercury (5 mg/kg, ip), the formation rate of reactive oxygen species was significantly increased in the cerebellum. Pretreatment with deferoxamine (500 mg/kg, ip) completely prevented the methyl mercury-induced increase in cerebellar reactive oxygen species generation rates. The oxidative consequences of in vitro exposure to methyl mercury (20 microM) were also inhibited by deferoxamine (100 microM). The formation of the iron-saturated complex ferrioxamine was not affected by a 10-fold excess of methylmercuric chloride or mercuric chloride, suggesting that a deferoxamine-mercurial complex does not form. The findings in this study: (1) provide evidence that iron-catalyzed oxygen radical-producing reactions play a role in methyl mercury neurotoxicity, (2) demonstrate the potential of fluorescent probes as a measure of reactive oxygen species formation, and (3) provide support for iron-chelator therapy in protection against xenobiotic-induced oxidative damage.     

Chemical oxidation and metabolism of N-methyl-N-formylhydrazine. Evidence for diazenium and radical intermediates

N-Methyl N-formlhydrazine (1), a component of the mushroom Gyromitra esculenta, is a carcinogen. Its mode of action, however, is poorly understood. To determine the intermediates that may form during the metabolism of 1, we examined its oxidative chemistry, identified the products and inferred the intermediates on the basis of these products. The incubation of 1 with rat liver microsomes was also studied and the metabolites determined and quantified. Both the chemical and the microsome-mediated oxidation of 1 yielded formaldehyde and acetaldehyde. The formation of acetaldehyde requires (i) the oxidation of 1 to a diazenium ion (I) or diazene (II) and (ii) fragmentation of I/II to formyl and methyl radicals. It is suggested that these radical intermediates may be important in understanding and elucidating carcinogenesis by 1.     

DNA存在下Cu(Ⅱ)—去甲斑螫酸络合物催化抗坏血酸氧化的动力学

在pH 7.40,20.00±0.05℃,磷酸盐缓冲液中,研究了Cu(Ⅱ)及Cu(Ⅱ)络合物的可见光谱,以及DNA存在下,Cu(Ⅱ)的去甲斑蝥酸络合物Cu(Ⅱ)/H₂DCA催化抗坏血酸(H₂A)有氧氧化动力学,检测了Cu(Ⅱ)/H₂DCA催化H₂A有氧氧化过程中·OH生成的速度。实验结果表明,在Cu(Ⅱ)/H₂DCA存在下,抗坏血酸断裂DNA链的反应体系中,存在Cu(Ⅱ)与H₂DCA和DNA的三元络合。据此,推测H₂A的催化有氧氧化对DNA链的断裂作用按特定部位的Fenton反应机理进行。该机理能够解释前文的DNA链断裂实验的所有结果。     

A prediction system of oxidation reaction as a solid-state stress condition: Applied to a pyrrole-containing pharmaceutical compound

Stress conditions for predicting oxidative degradation products in solid-state pharmaceutical compounds were investigated. 4-Methyl-2-(3,4-dimethylphenyl)-1-(4-sulfamoylphenyl)pyrrole, Compound A, was used as the model compound for this study and its four main degradation products were due to oxidation, as identified by LC–MS and LC-¹H NMR. In order to develop a prediction system for the oxidation reaction, solid-state Compound A was stored under moisture-saturated conditions. Hydrogen peroxide was added to the solution used to saturate the headspace with moisture and oxygen was substituted for the headspace air, in order to stimulate the oxidation reaction. After optimizing the conditions, a similar degradation product profile to that actually observed in the stability studies was obtained in only 3 days under conditions using 3% hydrogen peroxide at 40 °C. The prediction of the oxidative degradation products in a solid-state pharmaceutical compound was successfully achieved in a short term utilizing this newly developed prediction system.     

Chemical Pathways of Peptide Degradation. X: Effect of Metal-Catalyzed Oxidation on the Solution Structure of a Histidine-Containing Peptide Fragment of Human Relaxin

Purpose. To elucidate the major degradation products of the metal-catalyzedoxidation of (cyclo S-S) AcCys-Ala-X-Val-Gly-CysNH₂(X = His, cyclic-His peptide), which is a fragment of the proteinrelaxin, and the effect of this oxidation on its solution structure. Methods. The cyclic-His peptide and its potential oxidative degradationproducts, cyclic-Asp peptide (X = Asp) and cyclic-Asn peptide(X = Asn), were prepared by using solid phase peptide synthesisand purified by preparative HPLC. The degradation of the cyclic-Hispeptide was investigated at pH 5.3 and 7.4 in an ascorbate/cupricchloride/oxygen [ascorbate/Cu(II)/O₂] system in the absence or presenceof catalase (CAT), superoxide dismutase (SOD), isopropanol, andthiourea. The oxidation of the cyclic-His peptide was also studied in thepresence of hydrogen peroxide (H₂O₂). All reactions were monitored byreversed-phase HPLC. The main degradation product of the cyclic-Hispeptide formed at pH 7.4 in the presence of ascorbate/Cu(II)/O₂was isolated by preparative HPLC and identified by ¹H NMR andelectrospray mass spectrometry. The complexation of Cu(II) with thecyclic-His peptide was determined with ¹H NMR. The solution structureof the cyclic-His peptide in the presence and absence of Cu(II) at pH5.3 and 7.4 and the solution structure of the main degradation productwere determined using circular dichroism (CD). Results. CAT and thiourea were effective in stabilizing the cyclic-Hispeptide to oxidation by ascorbate/Cu(II)/O₂, while SOD and isopropanolwere ineffective. Cyclic-Asp and cyclic-Asn peptides were notobserved as degradation products of the cyclic-His peptide oxidized atpH 5.3 and 7.4 in an ascorbate/Cu(II)/O₂ system. The main degradationproduct formed at pH 7.4 was the cyclic 2-oxo-His peptide (X = 2-oxo-His).At pH 5.3, numerous degradation products were formed inlow yields, including the cyclic 2-oxo-His peptide. The cyclic 2-oxo-Hispeptide appeared to have a different secondary structure than didthe cyclic-His peptide as determined by CD. ¹H NMR results indicatecomplexation between the cyclic-His peptide and Cu(II). CD resultsindicated that the solution structure of the cyclic-His peptide in thepresence of Cu(II) at pH 5.3 was different than the solution structureobserved at pH 7.4. Conclusions. H₂O₂ and superoxide anion radical ( ) were deducedto be the intermediates involved in the ascorbate/Cu(II)/O₂-inducedoxidation of cyclic-His peptide. H₂O₂ degradation by a Fenton-typereaction appears to form secondary reactive-oxygen species (i.e.,hydroxyl radical generated within complex forms or metal-bound formsof hydroxyl radical) that react with the peptide before they diffuse intothe bulk solution. CD results indicate that different complexes areformed between the cyclic-His peptide and Cu(II) at pH 5.3 and pH7.4. These different complexes may favor the formation of differentdegradation products. The apparent structural differences between thecyclic-His peptide and the cyclic 2-oxo-His peptide indicate that conformationof the cyclic-His peptide was impacted by metal-catalyzedoxidation.     

Prenylation enhances cytotoxicity of apigenin and liquiritigenin in rat H4IIE hepatoma and C6 glioma cells.

Antioxidative as well as cytotoxic effects of the prenylated flavonoids licoflavone C (8-prenylapigenin) and isobavachin (8-prenylliquiritigenin) were investigated in comparison to the corresponding non-prenylated flavonoids (apigenin, liquiritigenin) and vitexin (apigenin-C8-glucoside) using metabolically active H4IIE hepatoma and metabolically poorly active C6 glioma cells. None of the substances showed radical scavenging activities in the 2,2-diphenyl-1-picrylhydrazyl (DPPH)-assay nor were they effective in protection against H2O2-induced intracellular 2',7'-dichlorodihydrofluorescein (H2DCF) oxidation (fluorescent probe for oxidative stress) in H4IIE and C6 cells. When the intrinsic effects of the substances were investigated, licoflavone C and isobavachin exerted a pronounced toxicity in both H4IIE (IC50 values of 42+/-5 and 96+/-19 micromol/L) and C6 cells (IC50 values of 37+/-6 and 69+/-3 micromol/L) while the non-prenylated analogues as well as the glycosylated derivate vitexin showed almost no cytotoxic effect up to 250 micromol/L. In H4IIE cells the induction of apoptotic cell death by licoflavone C and icobavachin was detected as an activation of caspase 3/7 (6- and 3.3-fold, respectively). Based on these experiments we suggest that C8-prenylation of a flavonoid enhances the cytotoxicity inducing an apoptotic cell death in H4IIE cells without affecting antioxidative properties.     

维生素E抑制冠心病患者低密度脂蛋白氧化修饰作用的临床研究

目的探讨维生素E(VitE)对冠心病低密度脂蛋白氧化修饰作用。方法健康对照组32例,将45例冠心病患者分两个剂量组 ,VitE0.1g/d及0.3g/d。采用给药前后自身对照的方法测定血清氧化低密度脂蛋白(oxLDL)、血清脂质过氧化物(LPO)、全血谷胱甘肽过氧化酶(GSH_Px)、全血超氧化物歧化酶(SOD)。结果冠心病组oxLDL、LPO显著升高(P<0.01,P<0.05),GSH_Px显著降低(P<0.01)。VitE0.3g/d治疗后 ,oxLDL和LPO显著降低(P<0.01) ,GSH_Px升高(P<0.01)。结论VitE是一种良好的抗氧化剂 ,其抑制低密度脂蛋白的氧化修饰作用是防治冠心病的重要机制之一     

Activation of the carcinogen N-hydroxy-N-(2-fluorenyl)benzamide via chemical and enzymatic oxidations. Comparison to oxidations of the structural analogue N-hydroxy-N-(2-fluorenyl)acetamide.

Chemical or enzymatic oxidations of the carcinogen N-hydroxy-N-(2- fluorenyl)benzamide (N-OH-2-FBA) were investigated under the conditions facilitating one-electron oxidation or oxidative cleavage of N-hydroxy-N-(2-fluorenyl)acetamide (N-OH-2-FAA). HPLC methods were developed for separation and quantitation of the above hydroxamic acids and their respective oxidation products. To identify the products of oxidation of N-OH-2-FBA, N-(benzoyloxy)-2-FBA (N-BzO-2-FBA) was synthesized and shown to undergo ortho rearrangement to 1- and 3-BzO-2-FBA. Oxidation of N-OH-2-FBA (4.88 mM) with alkaline K3Fe(CN)6 in benzene was complete and yielded equimolar amounts of 2-nitrosofluorene (2-NOF) and the ester (chiefly N-BzO-2-FBA), indicative of one-electron oxidation to nitroxyl free radical which undergoes bimolecular dismutation. However, one-electron oxidation of N-OH-2-FBA (30 or 10 microM) by horseradish peroxidase/H2O2 at pH 7 or myeloperoxidase/H2O2 at pH 6.5 yielded only approximately 10% as much product as N-OH-2-FAA (30 microM). The addition of 0.1 mM Br- +/- 0.1 M Cl- at pH 4 to 6.5 increased 2-NOF formation in MPO/H2O2-catalyzed oxidations. Simulations of these oxidations with HOCl/Cl- or HOBr/Br- showed that the latter was more efficient, converting N-OH-2-FAA almost completely and less than or equal to 62% of N-OH-2-FBA to 2-NOF. The amounts of the ester (N- and o-BzO-2-FBA), which by itself did not contribute to 2-NOF formation or significant substrate regeneration, indicated that approximately 10% of 2-NOF originated from one-electron oxidation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antioxidant activity of maslinic acid,a triterpene derivative obtained from Olea europaea

We investigated the effect of maslinic acid (a triterpene derivative obtained from olive pomace), on the susceptibility of plasma or hepatocyte membranes to lipid peroxidation (LPO), induced respectively by the hydroxyl radical (OH*) generated by Fe2+/H2O2 ex vivo and by the system Fe3+/ascorbate in vitro; moreover, three groups of animals used in the plasma study were pretreated with CCl4 (to generate CCl3-*). Endogenous plasma lipoperoxide levels and susceptibility to LPO were decreased in rats treated with maslinic acid, after exposure to OH* by Fe2+/H2O2 (Fenton reaction). Co-incubation with maslinic acid prevented hepatocyte membrane LPO as shown by the reduction of TBARS. In conclusion, maslinic acid may offer some advantages in the resistance of oxidative stress in the animals.     

Menadione (2-methyl-1,4-naphthoquinone)-induced Ca2+ release from rat-liver mitochondria is caused by NAD(P)H oxidation

Incubation of rat-liver mitochondria with menadione in the presence of succinate and rotenone resulted in rapid glutathione and NAD(P)H oxidation followed by Ca2+ release and mitochondrial swelling. Ca2+ release, NAD(P)H oxidation and mitochondrial swelling, were also observed in mitochondria from selenium-deficient rats. Glutathione was only slowly oxidized, suggesting that glutathione oxidation, and subsequent NAD(P)H oxidation via the glutathione peroxidase-glutathione reductase system were not required for Ca2+ release by menadione. Isocitrate prevented and reversed Ca2+ release dose-dependently but dicoumarol had no effect indicating that NADH-ubiquinone oxidoreductase and not DT-diaphorase was responsible for NAD(P)H oxidation. Superoxide anion radical was formed by cyanide-resistant respiration, suggesting that menadione undergoes a one-electron reduction to an autoxidizable semiquinone radical by NADH-ubiquinone oxidoreductase. The inability of menadione to oxidize glutathione in selenium-deficient mitochondria indicates that the metabolism of the superoxide dismutation product, H2O2, by glutathione peroxidase was probably responsible for the glutathione oxidation in selenium-replete mitochondria.     

Induction of oxidative stress in paraquat formulating workers

Paraquat as a bipyridyl compound is widely used as an effective herbicide worldwide. In this study, oxidative stress was investigated in blood samples of workers in a pesticide factory, formulating paraquat products for use in agriculture. Controls were age-matched workers with no history of pesticide exposure. They were measured for lipid peroxidation (LPO), antioxidant power and total thiol (SH) groups in blood. The results expressed as mean+/-SD show induction of oxidative stress in workers as revealed by increased plasma LPO (11.46+/-0.99 vs 10.11+/-0.69, P<0.001), decreased plasma antioxidant capacity (1.35+/-0.03 vs 1.54+/-0.05, P<0.001) and plasma SH groups (0.16+/-0.01 vs 0.21+/-0.01, P<0.001) in comparison to those of controls. It is concluded that paraquat-formulating factory workers have elevated LPO and decreased antioxidant power, which may put them in further consequences of oxidative stress.     

Tyrosyl radical production by myeloperoxidase: a phagocyte pathway for lipid peroxidation and dityrosine cross-linking of proteins

To kill invading bacteria, viruses, and fungi, phagocytes secrete hydrogen peroxide (H(2)O(2)) and the heme enzyme myeloperoxidase. We have explored the possibility that myeloperoxidase might use H(2)O(2) to convert L-tyrosine to tyrosyl radical. Activated human neutrophils and monocytes used the system to oxidize free L-tyrosine to o,o'-dityrosine, a stable product of tyrosyl radical. Protein-bound tyrosyl residues exposed to myeloperoxidase, H(2)O(2), and L-tyrosine were also oxidized to o,o'-dityrosine. The cross-linking reaction required free L-tyrosine, suggesting that myeloperoxidase converts the amino acid to a diffusible radical catalyst that promotes protein oxidation. We used electron paramagnetic resonance to provide direct evidence that the oxidizing intermediate is free tyrosyl radical. Myeloperoxidase-generated tyrosyl radical also initiates lipid peroxidation, suggesting that activated phagocytes might also be able to oxidize lipids in host tissues. Moreover, myeloperoxidase is present and active in human atherosclerotic tissue, and levels of protein-bound dityrosine are elevated in such lesions. Our recent studies indicate that activated neutrophils use oxidants generated by the phagocyte NADPH oxidase to produce protein-bound dityrosine during acute inflammation. Collectively, these findings suggest that generation of tyrosyl radical by myeloperoxidase allows activated phagocytes to damage both proteins and lipids. Elevated levels of o,o'-dityrosine have been detected in inflammatory lung disease, neurodegenerative disorders, and aging. Thus, oxidation of tyrosine to tyrosyl radical might play a role in the pathogenesis of many diseases.     

Involvement of oxidative stress in bee venom-induced inhibition of Na+/glucose cotransporter in renal proximal tubule cells

1. The present study was conducted to examine the involvement of oxidative stress in bee venom-induced inhibition of the Na+/glucose cotransporter (alpha-methyl-d-glucopyranoside (alpha-MG) uptake), a typical functional marker of proximal tubules, in primary cultured rabbit renal proximal tubule cells (PTC). 2. Bee venom (> or = 1 microg/mL) increased lipid peroxide (LPO) formation over 30 min. The increase in [(3)H]-arachidonic acid (AA) release and LPO formation and the inhibition of alpha-MG uptake induced by bee venom (1 microg/mL) and melittin (a major component of bee venom; 0.5 microg/mL) were blocked by N-acetyl-l-cysteine, vitamin C and vitamin E, anti-oxidants. 3. Bee venom- and melittin-induced increases in LPO formation and inhibition of alpha-MG uptake were significantly prevented by mepacrine and AACOCF(3), phospholipase A(2) inhibitors. In addition, nordihydroguaiareic acid (a lipoxygenase inhibitor) and econazole (a cytochrome P-450 epoxygenase inhibitor), but not indomethacin (a cyclo-oxygenase inhibitor), prevented bee venom- and melittin-induced increases in LPO formation and inhibition of alpha-MG uptake. 4. Nordihydroguaiareic acid prevented bee venom- and melittin-induced increases in Ca(2+) uptake. Moreover, anti- oxidants significantly prevented bee venom- and melittin-induced increases in Ca(2+) uptake. 5. In conclusion, bee venom inhibits alpha-MG uptake via the phospholipase A(2)-oxidative stress-Ca(2+) signalling cascade in primary cultured rabbit renal proximal tubule cells.     

Stress degradation studies on lornoxicam using LC, LC-MS/TOF and LC-MSn

Lornoxicam was subjected to forced degradation studies under hydrolytic (acidic, basic and neutral), oxidative, photolytic and thermal stress conditions, as defined under ICH guideline Q1A (R2). The drug degraded significantly in hydrolytic, oxidative and photoneutral conditions, leading to the formation of eight degradation products in total. It was stable on exposure to light and dry heat in the solid state. The stressed samples in which degradation was observed were mixed together and used to develop a stability-indicating HPLC method wherein degradation products were separated from the drug and also from each other. To characterize the degradation products, a complete mass fragmentation pathway of the drug was first established with the help of MS/TOF, MSⁿ and H/D exchange mass studies. The same was followed by LC-MS/TOF and on-line H/D exchange experiments on the degradation products. The degradation pathway of the drug was outlined, justified by the mechanisms of formation of the degradation products.     

Formation and reduction of glutathione-protein mixed disulfides during oxidative stress. A study with isolated hepatocytes and menadione (2-methyl-1,4-naphthoquinone)

Incubation of isolated rat hepatocytes with menadione (2-methyl-1,4-naphthoquinone) resulted in a dose-dependent depletion of intracellular reduced glutathione (GSH), most of which was oxidized to glutathione disulfide (GSSG). Menadione metabolism was also associated with a dose- and time-dependent inhibition of glutathione reductase, impairing the regeneration of GSH from GSSG produced during menadione-induced oxidative stress. Inhibition of glutathione reductase by pretreatment of hepatocytes with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) greatly potentiated both GSH depletion and GSSG formation during the metabolism of low concentrations of menadione. Concomitant with GSH oxidation, mixed disulfides between glutathione and protein thiols were formed. The amount of mixed disulfides produced and the kinetics of their formation were dependent on both the intracellular GSH/GSSG ratio and the activity of glutathione reductase. The mixed disulfides were mainly recovered in the cytosolic fraction and, to a lesser extent, in the microsomal and mitochondrial fractions. The removal of glutathione from protein mixed disulfides formed in hepatocytes exposed to oxidative stress was dependent on GSH and/or cysteine and appeared to occur predominantly via a thiol-disulfide exchange mechanism. However, incubation of the microsomal fraction from menadione-treated hepatocytes with purified glutathione reductase in the presence of NADPH also resulted in the reduction of a significant portion of the glutathione-protein mixed disulfides present in this fraction. Our results suggest that the formation of glutathione-protein mixed disulfides occurs as a result of increased GSSG formation and inhibition of glutathione reductase activity during menadione metabolism in hepatocytes.     

Stability of aqueous solutions of mibolerone.

The kinetics and mechanism of degradation of mibolerone were studied in aqueous buffered solutions in the pH range of 1-8 at 67.5 degrees. Mibolerone showed maximum stability between pH 5.5 and 6.4. At pH 1-2, the major degradative pathway was dehydration followed by migration of the 18-methyl group to form 7alpha,17,17-trimethylgona-4,13-dien-3-one. While there was only one degradation product at pH 1-2, the degradation at pH 7-8 was complex. As many as 12 degradation products were detected by GLC. Mass spectral data indicated that the majority of these products were either oxidation products or isomers. At pH 7.6, the apparent first-order rate constants exhibited marked dependency on buffer concentration. Incorporation of a sequestering agent into the solutions eliminated this dependency, suggesting that trace metal impurities from the buffer reagents were catalyzing the degradation. This was confirmed by degradation studies of solutions in water for injection containing 5 ppm of trace metal ions. Sn+2, Cu"2, and Fe+2 accelerated the degradation, with Fe+2 having the most catalytic effect. The temperature dependence of the rate of degradation was studied in 0.05 M phosphate buffer at pH 6.4. The activation energy was 19.6 +/- 1.63 kcal/mole.     

Cu2+、Zn2+胁迫对蛹虫草(Cordyceps militaris) 抗氧化酶活性及脂氧化水平的影响

研究了Cu^2＋、Zn^2＋对蛹虫草菌丝体抗氧化酶活性及脂氧化水平的影响。结果表明：高浓度的Cu^2＋抑制菌丝体的生长和细胞内抗氧化酶的活性；高浓度的Zn^2＋对菌丝体的生长也有抑制作用但抗氧化酶的活性变化不大。脂氧化水平随细胞内过氧化状态的变化而变化。蛹虫草菌丝体对Cu^2＋、Zn^2＋有较高的耐受度。     

Lipid peroxidation and cataracts: N-acetylcarnosine as a therapeutic tool to manage age-related cataracts in human and in canine eyes

Cataract formation represents a serious problem in the elderly, with approximately 25% of the population aged >65 years and about 50% aged >80 years experiencing a serious loss of vision as a result of this condition. Not only do cataracts diminish quality of life, they also impose a severe strain on global healthcare budgets. In the US, 43% of all visits to ophthalmologists by Medicare patients are associated with cataract. Surgery represents the standard treatment of this condition, and 1.35 million cataract operations are performed annually in the US, costing 3.5 billion US dollars (year of costing, 1998). Unfortunately, the costs of surgical treatment and the fact that the number of patients exceeds surgical capacities result in many patients being blinded by cataracts worldwide. This situation is particularly serious in developing countries; worldwide 17 million people are blind because of cataract formation, and the problem will grow in parallel with aging of the population. In any event, surgical removal of cataracts may not represent the optimal solution. Although generally recognised as being one of the safest operations, there is a significant complication rate associated with this surgical procedure. Opacification of the posterior lens capsule occurs in 30-50% of patients within 2 years of cataract removal and requires laser treatment, a further 0.8% experience retinal detachments, approximately 1% are rehospitalised for corneal problems, and about 0.1% develop endophthalmitis. Although the risks are small, the large number of procedures performed means that 26,000 individuals develop serious complications as a result of cataract surgery annually in the US alone. Thus, risk and cost factors drive the investigation of pharmaceutical approaches to the maintenance of lens transparency. The role of free radical-induced lipid oxidation in the development of cataracts has been identified. Initial stages of cataract are characterised by the accumulation of primary (diene conjugates, cetodienes) lipid peroxidation (LPO) products, while in later stages there is a prevalence of LPO fluorescent end-products. A reliable increase in oxiproducts of fatty acyl content of lenticular lipids was shown by a direct gas chromatography technique producing fatty acid fluorine-substituted derivatives. The lens opacity degree correlates with the level of the LPO fluorescent end-product accumulation in its tissue, accompanied by sulfhydryl group oxidation of lens proteins due to a decrease of reduced glutathione concentration in the lens. The injection of LPO products into the vitreous has been shown to induce cataract. It is concluded that peroxide damage of the lens fibre membranes may be the initial cause of cataract development. N-acetylcarnosine (as the ophthalmic drug Can-C), has been found to be suitable for the nonsurgical prevention and treatment of age-related cataracts. This molecule protects the crystalline lens from oxidative stress-induced damage, and in a recent clinical trial it was shown to produce an effective, safe and long-term improvement in sight. When administered topically to the eye in the form of Can-C, N-acetylcarnosine functions as a time-release prodrug form of L-carnosine resistant to hydrolysis with carnosinase. N-acetylcarnosine has potential as an in vivo universal antioxidant because of its ability to protect against oxidative stress in the lipid phase of biological cellular membranes and in the aqueous environment by a gradual intraocular turnover into L-carnosine. In our study the clinical effects of a topical solution of N-acetylcarnosine (Can-C) on lens opacities were examined in patients with cataracts and in canines with age-related cataracts. These data showed that N-acetylcarnosine is effective in the management of age-related cataract reversal and prevention both in human and in canine eyes.     

Quercetin protects the hydrogen peroxide-induced apoptosis via inhibition of mitochondrial dysfunction in H9c2 cardiomyoblast cells

Quercetin possesses a broad range of pharmacological properties, including protection of LDL from oxidation. However, little is known about the mechanism by which quercetin rescues cardiomyoblasts from oxidative damage. This study was designed to investigate the protective mechanism of quercetin on H(2)O(2)-induced toxicity of H9c2 cardiomyoblasts. Oxidative stress, such as H(2)O(2), ZnCl(2), and menadione, significantly decreased the viability of H9c2 cells, which was accompanied with apparent apoptotic features, including fragmentation of genomic DNA as well as activation of caspase protease. However, quercetin markedly inhibited the apoptotic characteristics via reduction of intracellular reactive oxygen species generation. Also, it prevented the H(2)O(2)-mediated mitochondrial dysfunction, including disruption of mitochondria membrane permeability transition as well as an increase in expression of apoptogenic Bcl-2 proteins, Bcl-2 and Bcl-X(L). Furthermore, pretreatment of quercetin inhibited the activation of caspase-3, thereby both cleavage of poly(ADP-ribose) polymerase and degradation of inhibitor of caspase-activated DNase/DNA fragmentation factor by H(2)O(2) were completely abolished. Taken together, these data suggest that protective effects of quercetin against oxidative injuries of H9c2 cardiomyoblasts may be achieved via modulation of mitochondrial dysfunction and inhibition of caspase activity.