Study on the mechanism of action of artemether against schistosomes: the identification of cysteine adducts of both carbon-centred free radicals derived from artemether

Reaction of the antimalarial and anti-schistosome drug artemether (1) and catalytic amount of ferrous ion in the presence of excess cysteine gave two adducts of cysteine and previous postulated primary and secondary carbon-centred free radicals besides their other rearrangement products. This piece of further evidence for the presence of carbon-centred radicals, especially the secondary carbon-centred free radical for the first time by the isolation of its coupling adduct, will be help to understand the mechanism of action of artemether and other qinghaosu derivatives against parasites.     

Study of the gametocytocidal/sporontocidal action of qinghaosu (artemisinin) by electron microscopy

The gametocytocidal efficacy of artemisinin (qinghaosu) was evaluated and scanning electron microscopical evidence is given for gametocytocidal action of a single 5-10-mg/kg dose of artemisinin against simian malarial parasite (Plasmodium cynomolgi B). No sporontocidal effect of artemisinin was observed.     

Antioxidant Activities of Some Extracts of Thymus zygis

The antioxidant activities of methanol and ethyl ether extracts obtained from Thymus zygis, collected during the flowering or non-flowering period, were evaluated and compared. To investigate this potential, extracts were tested on their capacity to react with diphenyl-picrylhydrazyl (DPPH) in a homogeneous medium, and to inhibit Fe²⁺/ascorbate-induced membrane lipid peroxidation, as estimated by the formation of thiobar-bituric acid-reactive substances (TBARS). Although methanol extracts reduce DPPH radicals more efficiently than ethyl ether extracts, suggesting a potent radical scavenger activity, the ethyl ether extracts were found to be most active in inhibiting lipid peroxidation in sarcoplasmic reticulum (SR) membranes. In addition, both extracts present peroxyl and superoxide radical scavenging activities. Peroxyl radicals were generated by the water soluble 2, 2A-azobis(2-amidinopropane) dihydrochloride (AAPH) azoinitiator, and the scavenging activities of the extracts were measured by the inhibition of cis-parinaric acid (PnA) fluorescence decay in SR. Superoxide radicals were generated either by an enzymatic or a non-enzymatic system, and the scavenger ability was evaluated by the inhibition of nitrob-lue tetrazolium reduction. Methanolic extracts are more potent as scavengers of peroxyl and super oxide radicals than the ethyl ether extracts. Apparently, there is a relationship between antioxidant potency and the total phenolic groups content in each extract.     

Antioxidant Activities of Some Extracts of Thymus zygis

The antioxidant activities of methanol and ethyl ether extracts obtained from Thymus zygis, collected during the flowering or non-flowering period, were evaluated and compared. To investigate this potential, extracts were tested on their capacity to react with diphenyl-picrylhydrazyl (DPPH) in a homogeneous medium, and to inhibit Fe²⁺/ascorbate-induced membrane lipid peroxidation, as estimated by the formation of thiobar-bituric acid-reactive substances (TBARS). Although methanol extracts reduce DPPH radicals more efficiently than ethyl ether extracts, suggesting a potent radical scavenger activity, the ethyl ether extracts were found to be most active in inhibiting lipid peroxidation in sarcoplasmic reticulum (SR) membranes. In addition, both extracts present peroxyl and superoxide radical scavenging activities. Peroxyl radicals were generated by the water soluble 2, 2A-azobis(2-amidinopropane) dihydrochloride (AAPH) azoinitiator, and the scavenging activities of the extracts were measured by the inhibition of cis-parinaric acid (PnA) fluorescence decay in SR. Superoxide radicals were generated either by an enzymatic or a non-enzymatic system, and the scavenger ability was evaluated by the inhibition of nitrob-lue tetrazolium reduction. Methanolic extracts are more potent as scavengers of peroxyl and super oxide radicals than the ethyl ether extracts. Apparently, there is a relationship between antioxidant potency and the total phenolic groups content in each extract.     

Molecular modeling studies of the artemisinin (qinghaosu)-hemin interaction: Docking between the antimalarial agent and its putative receptor

Artemisinin (qinghaosu, QHS) is a promising new antimalarial agent that is effective against drug-resistant strains of malaria. The antimalarial activity of this drug appears to be mediated by an interaction of the drug's endoperoxide bridge with intraparasitic hemin. We have carried out a computer-assisted docking of QHS with hemin from various starting configurations and found that, in the most stable docked configuration, the endoperoxide bridge is in close proximity to the hemin iron. In contrast, an inactive analog, deoxyartemisinin (DQHS), docks in a different manner. Further computer analysis of the drug-hemin interaction might aid in the design of new QHS congeners.     

Study of the Mode of Action of Some Nitrodiphenyl Ethers

Nitrosoderivatives of the nitrodiphenyl ether herbicides (nitrofen, bifenox) have been studied. UV irradiation in different organic solvents gives degradation products. In buffered aqueous media, in the presence of chloroplasts and spin traps such as DMPO, hydroxy and peroxy radicals have been characterized.

In organic media and in the presence of spin traps such as DMPO, PBN, 4-POBN, solvent radicals (CHCIl₂, CCI₃, CH₂O) have been formed.

Nitro-derivatives have been studied under UV irradiation and in the presence of tetramethylethylene (TME), alkenylhydroxylamines are formed which autoxidize in nitroxide radicals. The formation of the stable nitroxide radical occurs in the dark process after continuous irradiation. The intensity of the signal decreases strongly when a new irradiation is applied. Radical species, with analogous ESR spectral characteristics are formed on reaction with nitrodiphenyl ethers and fatty acids.

The reactivity of these herbicides in micellar media (SDS, Brij 35, and CTAB) has been investigated. The kinetics of formation of the ESR signal corresponding to the photoreduction of the nitrodiphenyl ether in the presence of TME behave differently in a micellar environment as compared to solution. The intensity of the formation of the nitroxide increases under irradiation and decreases in the dark; the rotational correlation time t_c has been determined for each type of micelle.

Synthetic nitrosodiphenyl ether made by the reduction of nitrodiphenyl ether using hydrogen gas and PtO₂ as a catalyst gives the corresponding amine, which is oxidized with rneta-chloroperbenzoic acid (m.CPBA). The nitrosodiphenyl ether in the presence of soja azolectin liposorne containing a fluorescent probe has been analysed. When this synthetic nitrosodiphenyl ether is added to a medium containing soja azolectin liposomes and a carboxyfluorescein, fluorescent probe placed inside the liposornes, a rapid increase in the fluorescence of the medium is observed. The nitrosodiphenyl ether induce a break in the liposorne membrane.     

Plasmodium vinckei petteri: identification of the stages sensitive to arteether.

Antimalarial activity of arteether, a derivative of artemisinin (qinghaosu) against blood-induced infections of the highly synchronous Plasmodium vinckei petteri rodent species of malaria was evaluated in Swiss mice. A single subcurative dose of arteether of 2.2 mg/kg body weight was injected subcutaneously to mice, either during the prepatent period or during the patent infection, when different stages of the parasitic cycle were present in the blood. It was shown that rings and young trophozoites were the most susceptible stages to arteether. The drug had no effect on merozoites and little effect on mid-term trophozoites which is the stage most sensitive to chloroquine. The alcoholic solution (10% alcohol in sterile water) had an immediate effect while the oily solution (miglyol 840) was active between 3 and 21 hr after injection.     

Singlet Oxygen-Trapping Reaction as a Method of 1O2 Detection: Role of Some Reducing Agents

The production of singlet oxygen by H₂O₂ disproportionation and via the oxidation of H₂O₂ by NaOCl in a neutral medium was monitored by spin trapping with 2,2,6,6 tetramethyl-4-piperidone (TMPone). The singlet oxygen formed in both reactions oxidized 2,2,6,6 tetramethyl-4-piperidone to give nitroxide radicals. However the production of nitroxide radicals was relatively small considering the concentrations of H₂O₂ and NaOCl used in the reaction systems. Addition of electron donating agents: ascorbate, Fe²⁺ and desferrioxamine leads to an increase in the production of nitroxide radicals. We assumed that a very slow step of the reaction sequence, the homolytic breaking of the O-O bond of N-hydroperoxide (formed as an intermediate product during the reaction of ¹O₂ with TMPone) could be responsible for the relatively small production of nitroxide radicals. Electron donating agents added to the reaction system probably raise the rate of the hydroperoxide decomposition by allowing a more rapid heterolytic cleavage of the O-O bond leading to a greater production of nitroxide radicals. The largest effect was observed in the presence of desferrioxamine. Its participation in this process is proved by the concomitant appearance of desferrioxamine nitroxide radicals. The results obtained demonstrate that the method proposed by several authors and tested in this study to detect singlet oxygen is not convenient for precise quantitative studies. The reactivity of TMPone towards O₂^-7HO₂' and 'OH has been also investigated. It has been found that both O₂^-7HO₂' and 'OH radicals formed in a phosphate buffer solution (pH 7.4, 37°C), respectively by a xanthine-oxidase/hypoxanthine system and via H₂O₂ UV irradiation, do not oxidize 2,2,6,6 tetramethyl-4-piperidone to nitroxide radicals.     

Some histochemical aspects of keratinization of oral, esophageal and gastric membrane of the albino rat.

Using histochemical methods a comparative study was carried out, to identify important substances at the keratinization metabolism. With the results obtained, we may conclude that: a) in the four layers of all regions studied, it was possible to detect the presence of the characteristic active radicals of the amino acids cysteine, cystine and arginine, mainly in the corneus layer; b) the presence of -SH radicals of cysteine in the keratohyalin granules was evident in all regions studied.     

A Comparison of the Free Radical Properties of Several Anthracycline Anti-Tumour Drugs and Some of Their Analogues

NADPH consumption and esr spectroscopy have been used to study the rate of formation and signal intensity of free radicals produced by various anthracycline anti-tumour drugs in rat liver microsomal extract. The drugs investigated were Adriamycin, 4'Deoxyadriamycin, Daunorubicin, 4 Demethoxydaun-orubicin and Carminomycin. Pulse radiolysis was also used to determine the ease of reduction of each of the analogues to its semiquinone radical and some kinetic properties of the radicals produced. It is believed that the Occurrence of reactions other than dismutation could be responsible for the shortened lifetimes of the semiquinone radicals observed in biological systems.     

A Comparison of the Free Radical Properties of Several Anthracycline Anti-Tumour Drugs and Some of Their Analogues

NADPH consumption and esr spectroscopy have been used to study the rate of formation and signal intensity of free radicals produced by various anthracycline anti-tumour drugs in rat liver microsomal extract. The drugs investigated were Adriamycin, 4′Deoxyadriamycin, Daunorubicin, 4 Demethoxydaun-orubicin and Carminomycin. Pulse radiolysis was also used to determine the ease of reduction of each of the analogues to its semiquinone radical and some kinetic properties of the radicals produced. It is believed that the Occurrence of reactions other than dismutation could be responsible for the shortened lifetimes of the semiquinone radicals observed in biological systems.     

The potentiating action of tetrandrine in combination with chloroquine or qinghaosu against chloroquine-sensitive and resistant falciparum malaria

Using chloroquine-sensitive (CS) and chloroquine-resistant (CR) strains of Plasmodium falciparum in vitro, interactions between tetrandrine (TT) and either chloroquine (CQ) or qinghaosu (QHS, artemisinin) were assessed using isobolograms. Sums of the fractional inhibitory concentration for the combination of the two drugs are less than one and therefore, we can conclude that in vitro TT and CQ or QHS act synergistically against CS and CR falciparum malaria. Remarkably, using CR malaria, TT can lower the IC50 dose of CQ as much as 40 fold. These drug combinations may impair the advantage that the development of CQ resistance conveys on the parasite.     

Some roles of free radicals in malaria

Malaria parasites are very vulnerable to oxidant stress during the part of their life cycle when they inhabit erythrocytes. As the infection progresses they also activate macrophages, one consequence of which is extracellular release of reactive oxygen species. For these reasons free radicals are frequently discussed in the literature on antimalarial drugs, malarial immunity, and disease pathogenesis. They are also central to arguments explaining how the genetic mutations that lead to sickle cell disease, thalassemia and glucose-6-phosphate dehydrogenase have become so common in tropical regions. This review summarizes how these links between free radicals and this disease came to be understood, and the present state of the field.     

Some aspects of the role of cytochrome P-450 isozymes in the n-oxidative transformation of secondary and tertiary amine compounds

Indirect evidence of the participation of cytochrome P-450 (P-450) in the microsomal N-oxygenation of secondary and tertiary nitrogen functions is presented by studies employing diagnostic modifiers of the hemoprotein system as well as antibodies directed toward the diverse P-450 isoforms and NADPH-cytochrome P-450 reductase. Experiments with recombinant hemoproteins or P-450 isozymes directly purified from the tissues of various animal species support the results obtained by the inhibitor assays. Although the intermediacy of aminium radicals is thought to be restrictive to P-450-catalyzed N-oxygenation of secondary and tertiary amine groups bearing accessible hydrogens on the α-carbon, numerous exceptions to this rule are documented. It is proposed that aminium radicals partition between oxygen rebound and α-hydrogen abstraction to yield a finite level of N-oxygenated product in all P-450-mediated amine oxidations, the partition ratio depending on the amine structure and particular P-450 isozyme operative. In some instances, N-oxygenation appears to proceed by peroxidatic mechanisms. The relative contribution of P-450 to the N-oxygenation of secondary and tertiary amines in crude preparations or live animals, where competition with the flavin-containing monooxygenase (FMO) occurs, seems to be a function of the relative amounts and catalytic capacities of the two enzyme systems. Both parameters are species and tissue dependent. Accordingly, the extent to which P-450 contributes to total N-oxidative turnover of the amine substrates varies from minor to major. © 1996 John Wiley & Sons, Inc.     

Electrochemical Oxidations of Some Purine Nucleosides. Formation of Some Novel Purine Oligonucleosides

Abstract

The electrochemical oxidation of the purine nucleosides xanthosine and guanosine at carbon electrodes has been studied. The initial electrochemical event is a 1e-, 1H+ oxidation to give free radicals which undergo a series of follow-up chemical and electrochemical reactions which lead to formation of a new class of purine oligonucleosides.     

Some features of nucleo-cytoplasmic RNA transport from isolated nuclei

Messenger RNA is released preferentially from isolated rat liver nuclei in the presence of the ATP-generating system and cytosol. The release is suppressed by spermidine, while cytoplasmic RNase inhibitor was ineffective and PCMB like some other thiol-blocking agents inhibitory. Cytoplasmic SOD added to the system strongly suppressed RNA release. A similar effect could be obtained by anaerobiosis due to addition of SMP. In both cases the inhibition is reversed by cyanide.In contrast to normal liver where the generation of superoxide radicals takes place almost exclusively in microsomes and is coupled with the oxidation of NADPH, in mouse ascites hepatoma 22a the generation of superoxide radicals occurs mainly in the nuclear envelope and is coupled with the oxidation of both NADPH and NADH and inhibited by cyanide.Abbreviations PCMB p-Chloromercuri benzoate - SMP Submitochondrial particles - SOD Superoxide dismutase     

Myocardial Tissue Preparation for ESR Spectroscopy: Some Methods May Cause Artifactual Generation of Signals

It has been suggested that some techniques of tissue preparation for esr spectroscopy may artifactually generate radicals. We have investigated this, together with the possibility that the susceptibility of the tissue to preparation artifacts may be altered by ischaemia and reperfusion. Three different methods of tissue processing have been assessed: (i) freeze-clamping (- 196 °C), using grooved, aluminium tongs which produce frozen cylinders of tissue (3 mm diameter) which fit directly into esr tubes; (ii) grinding of freeze-clamped tissue with a porcelain pestle and mortar; (iii) lyophilisation of ground, freeze-clamped, tissue. Isolated rat hearts (n = 7 or n = 5/group) were subjected to aerobic perfusion (10 min, 37 °C), total, global ischaemia (15 min) and reperfusion (30 sec). Hearts were freeze-clamped at the end of each period. Tissue was prepared by each of the three methods and esr spectra recorded at - 100 °C. In spectra from tissue which had been freeze-clamped only, broad high- and low-spin iron III signals (g = 1.9, g = 2.2-2.9 and g = 4.6) were seen together with a narrow, well-defined signal (g = 2.005), possibly from a semiquinone radical. In spectra from ground samples, an anisotropic signal (g_∥ = 2.040 and g_⊥ = 2.008), probably from a peroxyl radical, was observed in addition to the iron III signals. The intensity of the anisotropic signal varied with perfusion conditions; in ischaemic tissue it was decreased to 33 ± 10% of the control value and in reperfused tissue it was decreased to 76 ± 26%. In spectra from lyophilised samples, a narrow signal (g = 2.009), probably from a protein radical, was observed in addition to the iron III signals. The intensity of the signal at g = 2.009 was increased in ischaemic tissue to 170 ± 57% of the control value and in reperfused tissue to 241 ± 85%. In conclusion, artifactual generation of radicals can occur upon grinding (peroxyl radical) and lyophilisation (protein radical). Ischaemia and reperfusion may alter not only radical content per se but may also modify the susceptibility of the tissue to the artifactual production of radicals.     

The roles of thiol-derived radicals in the use of 2',7'-dichlorodihydrofluorescein as a probe for oxidative stress

2',7'-Dichlorodihydrofluorescein (DCFH2) is one of the most widely used probes for detecting intracellular oxidative stress, but requires a catalyst to be oxidized by hydrogen peroxide or superoxide and reacts nonspecifically with oxidizing radicals. Thiyl radicals are produced when many radicals are "repaired" by thiols, but are oxidizing agents and thus potentially capable of oxidizing DCFH2. The aim of this study was to investigate the reactivity of thiol-derived radicals toward DCFH2 and its oxidized, fluorescent form 2',7'-dichlorofluorescein (DCF). Thiyl radicals derived from oxidation of glutathione (GSH) or cysteine (CysSH) oxidized DCFH2 with rate constants at pH 7.4 of approximately 4 or approximately 2x10(7) M(-1) s(-1), respectively. Both the rates of oxidation and the yields of DCF were pH-dependent. Glutathione-derived radicals interacted with DCF, resulting in the formation of DCFH* absorbing at 390 nm and loss of fluorescence; in contrast, cysteine-derived radicals did not cause any depletion of DCF fluorescence. We postulate that the observed apparent difference in reactivity between GS* and CysS* toward DCF is related to the formation of carbon-centered, reducing radicals from base-catalyzed isomerization of GS*. DCF formation from interaction of DCFH2 with GS* was inhibited by oxygen in a concentration-dependent manner over the physiological range. These data indicate that in applying DCFH2 to measure oxidizing radicals in biological systems, we have to consider not only the initial competition between thiols and DCFH2 for the oxidizing radicals, but also subsequent reactions of thiol-derived radicals, together with variables--including pH and oxygen concentration--which control thiyl radical chemistry.     

Reactive oxygen species are physiological mediators of the noradrenergic signaling pathway in the mouse supraoptic nucleus

Free radicals are essential for the vasopressin (AVP) response to plasmatic hyperosmolarity. Noradrenergic afferents are the major projections on the supraoptic nucleus (SON) of the hypothalamus and stimulate the expression of AVP via a nitric oxide (NO) pathway. In this study, we investigated the mechanisms linking free radicals and noradrenaline (NA)-induced regulation of AVP. Analysis of Tg8 transgenic mice, invalidated for the monoamine oxidase-A gene and with consequently high levels of brain monoamines and AVP in the SON, showed that free radicals are more abundant in their SON than in that of wild-type mice (WT). Antioxidant superoxide dismutase 1 and 2 and catalase enzyme activities were also higher in these mice than in WT. This may explain the observed absence of cytotoxicity that would otherwise be associated with such high level of free radicals. Treatment of Tg8 mice with α-MPT, a blocking agent for NA synthesis, decreased both the production of free radicals and the AVP levels in the SON. Furthermore, incubation of ex vivo slices including the SON with NA increased the production of free radicals and AVP levels in wild-type mice. When NA was associated with α-lipoic acid, an antioxidant blocking the production of free radicals, AVP remained at its control level, indicating that free radicals are required for the effect of NA on the expression of AVP. In slices incubated with SNP, a producer of NO, free radicals and AVP levels increased. When NA was associated with L-NAME (a NO synthase blocker), the levels of free radicals and AVP were the same as in controls. Thus, the noradrenaline–NO pathway, which stimulates the expression of vasopressin, involves free radicals. This study provides further evidence of the physiological importance of free radicals, which should no longer be considered solely as cytotoxic factors.     

Generation of alloxan free radicals in chemical and biological systems: implication in the diabetogenic action of alloxan

Electron spin resonance (ESR) studies that on reaction with NADPH, alloxan was reduced forming labile anion radicals giving a 7-line signal with g = 2.005. These radicals were also produced on incubation of alloxan with rat liver subcellular fractions and their production was greatly enhanced by NADPH. Alloxan effectively scavenged superoxide anion generated by a xanthine-xanthine oxidase (XOD) system in association with its reduction to these anion radicals. These radicals were also formed during incubation of alloxan with rat pancreatic beta-cells. These results suggest that the cytotoxicity of alloxan is related to the formation of alloxan anion radicals.