Comparative study on some oxidative stress parameters in blood of vitiligo patients before and after combined therapy.

Currently accepted that oxidative stress is a triggering event in the melanocytic destruction and is probably involved in the etiopathogenesis of vitiligo disease. Despite numerous investigations, contradictory results were reported about the levels of oxidative stress biomarkers measured in the skin and blood of vitiligo patients. By Electron Paramagnetic Resonance spectroscopy (EPR) and spectrophotometry, we have investigated and compared some oxidative stress biomarkers in the blood of vitiligo patients' before and after UVB Narrow Band 311 nm phototherapy combined with the antioxidant nutritional supplement containing - Vitamin C, Vitamin B1, L -Cysteine, Lipoic Acid, and polyunsaturated fatty acids. Before therapy was found significantly higher levels of CAT activity and MDA compared to the patients after therapy and control group (p < 0.05). Moreover, levels of Asc* radicals in patients before therapy were significantly lower than those measured in controls and patients undergoing therapy (p < 0.05). Our finding, the combined therapy applied to vitiligo patients provoked an increase in the Asc* levels and a decrease in MDA products and also initial repigmentation in the vitiligo spots, made us believe that a combined antioxidant therapy, enriched with vitamin C could lead to improvement of the oxidant-antioxidant balance in vitiligo patients treated with UVB 311 phototherapy.

Protective effect of spin-labeled 1-ethyl-1-nitrosourea against oxidative stress in liver induced by antitumor drugs and radiation.

This study was carried out to investigate possible protection effect of 1-ethyl-3-[4-(2,2,6,6-tetramethylpiperidine-1-oxyl)]-1-nitrosourea (SLENU), synthesized in our laboratory, against oxidative liver injuries induced in mice treated by antitumor drugs: doxorubicin (DOX), bleomycin (BLM), or gamma irradiation (R). Specifically, alterations in some biomarkers of oxidative stress, such as lipid peroxidation products measured as malondialdehyde (MDA) levels and activities of the antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT), were studied in liver homogenates isolated from tumor bearing C57 black mice after i.p. treatment with solutions of DOX (60 mg/kg), BLM (60 mg/kg), or after total body gamma-irradiation with a single dose of 5 Gy. The same biomarkers were also measured after i.p. pretreatment of mice with SLENU (100 mg/kg). Statistical significant increased MDA levels and SOD and CAT enzymes activities were found in the liver homogenates of tumor bearing mice after alone treatment with DOX or gamma-irradiation compared to the control mice, while these parameters were insignificantly increased after BLM administration compared to the same controls.

Anion exchanger and the resistance against thermal haemolysis.

4,4'-Diiso-thiocyanato stilbene-2,2'-disulphonic acid (DIDS) is a membrane-impermeable, highly specific covalent inhibitor and powerful thermal stabiliser of the anion exchanger (AE1), the major integral protein of erythrocyte membrane (EM). Suspensions of control and DIDS-treated (15 µM, pH 8.2) human erythrocytes were heated from 20° to 70°C using various but constant heating rates (1-8°C/min). The cellular electrolyte leakage exhibited a sigmoidal response to temperature as detected by conductometry. The critical midpoint temperature of leakage, T(mo), extrapolated to low heating rate (0.5°C/min) was used as a measure for EM thermostability. T(mo) was greater for DIDS-treated erythrocytes, 63.2° ± 0.3°C, than for intact erythrocytes, 60.7° ± 0.2°C. The time, t(1/2), for 50% haemolysis of erythrocytes, exposed to 53°C was used as a measure for the resistance of erythrocytes against thermal haemolysis. The t(1/2) was also greater for DIDS-treated erythrocytes, 63 ± 3 min, than for intact erythrocytes, 38 ± 2 min. The fluorescent label N-(3-pyrenyl)maleimide and EPR spin label 3-maleimido-proxyl, covalently bound to sulphydryl groups of major EM proteins, were used to monitor the changes in molecular motions during transient heating. Both labels reported an intensification of the motional dynamics at the denaturation temperatures of spectrin (50°C) and AE1 (67°C), and, surprisingly, immobilisation of a major EM protein, presumably the AE1, at T(mo). The above results are interpreted in favour of the possible involvement of a predenaturational rearrangement of AE1 copies in the EM thermostability and the resistance against thermal haemolysis.

Antioxidative and radioprotective activities of semiquinone glucoside derivative (SQGD) isolated from Bacillus sp. INM-1.

A semiquinone glucoside derivative (SQGD) was isolated from a radioresistant bacterium Bacillus sp. INM-1 and its antioxidant and radioprotective activities were evaluated using in vitro assays. Natural stable free radical properties of SQGD in solid as well as in solution form were estimated using Electron Paramagnetic Resonance (EPR) spectrometry. Results of the study were demonstrated high reducing power (1.267 ± 0.03356 U(abs)) and nitric oxide radicals scavenging activity (34.684 ± 2.132%) of SQGD. Maximum lipid peroxidation inhibitory activity of SQGD was found to be 74.09 ± 0.08% at 500 µg/ml concentration. Similarly, significant (39.54%; P < 0.05) protection to the liposomal artificial membrane against gamma radiation was observed by SQGD in terms of neutralization of gamma radiation-induced TBARS radicals in vitro. OH(-) radicals scavenging efficacy of SQGD was estimated in terms of % inhibition in deoxy D: -ribose degradation by non-site-specific and site-specific assay. The maximum (54.01 ± 1.01%) inhibition of deoxy D: -ribose degradation was observed in non-site-specific manner, whereas, site-specific inhibition was observed to be 46.36 ± 0.5% at the same concentration (250 µg/ml) of SQGD. EPR spectroscopic analysis of the SQGD indicated ~80% reduction of DPPH radicals at 6.4% concentration. EPR spectral analysis of SQGD was revealed an appearance of very strong EPR signal of 2.00485 (crystalline form) and 2.00520 (solution form) g(y) tensor value, which were an established characteristic of o-semiquinone radicals. Therefore, it can be concluded that SQGD is a natural stable o-semiquinone-type radical, possessing strong antioxidant activities and can effectively neutralize radiation induced free radicals in biological system.

Two glycine containing 2-chloroethylnitrosoureas--a comparative study on some physicochemical properties, in vivo antimelanomic effects and immunomodulatory properties.

Physicochemical properties such as alkylating and carbamoylating activity and in vivo antimelanomic effects against B16 melanoma of the spin labeled (nitroxyl free radical containing) glycine nitrosourea (SLCNUgly) and its nonlabeled analogue (ChCNUgly), synthesized in our laboratory are studied and compared to those of antitumour drug 3-cyclohexyl-1-(2-chloroethyl)-1-nitrosourea (CCNU). We have demonstrated that introducing of glycine moiety in the nitrosourea structure in practice does not affect either alkylating or carbamoylating activity. On the other hand replacement of cyclohexyl moiety in ChCNUgly structure with nitroxyl free radical leads to a decrease in carbamoylating activity and an increase in alkylating activity. Compound ChCNUgly showed in vivo a higher antimelanomic activity against B16 melanoma in comparison with CCNU and SLCNUgly. It completely inhibited B16 melanoma growth (TGI=100%) at a dose 64.0 mg/kg. Moreover, we established that joint i.p. application in normal mice of SLCNUgly plus a new immunostimulator (C3bgp) formerly isolated in our laboratory led to a 75% restoration in immune function with respect to antibody production measured by Jerne hemolytic plaque assay. In contrast, no immunostimulation was found after joint application of C3bgp plus ChCNUgly or CCNU at the same experimental conditions. Based on these preliminary results, a possibility for developing of new combination immunochemotherapy schemes for treatment of human cancers is discussed.

Spin labelled nitrosoureas and triazenes and their non-labelled clinically used analogues--a comparative study on their physicochemical properties and antimelanomic effects.

Physicochemical properties, such as half life time (tau0.5), alkylating and carbamoylating activity and in vivo antimelanomic effects against B16 melanoma of spin labeled (containing nitroxyl free radical moiety) amino acid nitrosoureas, synthesized in our laboratory, have been studied and compared to those of the antitumor drug N'-cyclohexyl-N-(2-chloroethyl)-N-nitrosourea (lomustine, CCNU). We have shown that the introduction of amino acid moieties and the replacement of cyclohexylamine with nitroxyl moiety leads to a faster decomposition, higher alkylating, lower carbamoylating activity, better antimelanomic activity and lower general toxicity, when compared to those of CCNU. It was also established that spin labeled triazenes, previously synthesized by us, were more stable in phosphate saline than their nonlabeled analogue, 5-(3,3-dimethyltriazene-1-yl)-imidazole-4-carboxamide (dacarbazine, DTIC). A higher cytotoxicity to B16 melanoma cells than to YAC-1 and lymphocytes was demonstrated for all spin labeled triazenes, in comparison with DTIC. An assumption has been made to explain the lower general toxicity of the spin labeled nitrosoureas compared to that of CCNU. Based on the results presented, we accept that a new trend for synthesis of more selective and less toxic nitrosourea and triazene derivatives as potential antimelanomic drugs might be developed.

Sensitivity of alpha-amanitin to oxidation by a lactoperoxidase-hydrogen peroxide system.

Oxidation of alpha-amanitin - a potent hepatotoxin found in the mushroom Amanita phalloides - by a lactoperoxidase-hydrogen peroxide system was investigated by different techniques. (i). UV spectroscopy of the mixture after 24 h incubation reveals a significant decrease in the absorbance range characteristic of the putative reactive moiety of the toxin, the tryptathionine group. (ii). Formation of a new product was detected by Thin Layer Chromatography. (iii). In vivo experiments with non-inbred male albino mice showed a lowered toxicity of the modified toxin in comparison with that of the native one. Taking into account the latter results concerning the sensitivity of the toxin towards an oxidising system, the formation and reactivity of an alpha-amanitin derivative is discussed in the course of A. phalloides poisoning (inhibition of RNA polymerase type II and development of damaging radical species).

Modulating effect of new potential antimelanomic agents, spin-labeled triazenes and nitrosoureas on the DOPA-oxidase activity of tyrosinase.

The modulating effect of newly synthesized alkylating spin labeled triazene and spin labeled nitrosourea derivatives on the DOPA-oxidase activity of mushroom tyrosinase has been investigated by Bumett's spectrophotometric method (Burnett et al., 1967). All spin labeled triazenes have exhibited activating effect on DOPA-oxidase activity of tyrosinase, whereas clinically used triazene (DTIC), which does not contain nitroxide moiety, have showed inhibiting effect. At the same experimental conditions the spin labeled aminoacid nitrosoureas have showed dual effect - activating, in the beginning of the enzyme reaction and inhibiting later on. It is deduced that the activating effect of the spin labeled compounds is due to the nitroxide moiety and the inhibiting effect of all compounds depends on their half-life time. This study might contribute to make more clear the mechanism of action of the new compounds and on the other hand would come in quite useful as a preliminary prognosis for their antimelanomic activity.

Spin labeled amino acid nitrosourea derivatives--synthesis and antitumour activity.

The synthesis of three spin labeled derivatives of N-[N'-(chloroethyl)-N'-nitrosocarbamoyl] amino acids is reported. The new nitrosoureas are obtained by condensation of the corresponding N-[N'-(2-chloroethyl)-N'-nitrosocarbamoyl] amino acid with 2,2,6,6-tetramethyl-1-oxyl-4-aminopiperidine using dicyclohexylcarbodiimide. Their chemical structures are confirmed by elemental analysis, IR, MS, and EPR spectroscopy. All newly synthesized compounds showed high antitumour activity against the lymphoid leukemia L1210 in BDF1 mice.

Cytotoxicity on L1210 leukemic cells of beta-amanitin-concanavalin A and phallacidin-concanavalin A conjugates.

The conjugates beta-amanitin-concanavalin A and phallacidin concanavalin A were tested for direct cytotoxicity on L1210 lymphocytic leukemia cells by a combined in vitro-in vivo bioassay. Both conjugates exerted strong direct cytotoxicity on the tumour cells.

Activation of the anticancer drug CCNU into a free radical intermediate via UV irradiation--an ESR study.

We studied the formation of a free radical induced by UV irradiation of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) in benzene. It was determined a stable nitroxide radical by ESR spectroscopy. We confirmed that sterically hindered cyclic amine 2,2,6,6-tetramethyl-4-piperidone (TMPone) was transformed into a corresponding stable free nitroxyl radical during UV irradiation. When CCNU was present, the rate of free radical formation from TMPone increased considerably.

Preparation of a beta-amanitin-concanavalin A conjugate of low toxicity.

Pure beta-amanitin was isolated by combined adsorption chromatography of a crude methanol extract from Amanita phalloides on Sephasorb HP Ultrafine and Sephadex LH-20. The beta-amanitin was coupled to concanavalin A by the carbodiimide method. The conjugate was purified by fractionation on a column of Sephadex G-75. The molar ratio beta-amanitin to concanavalin A in this conjugate was 4.14. The purified conjugate was tested by thin-layer chromatography and showed characteristic (for amatoxins) bright purple staining but different mobility. The ultraviolet spectrum of the conjugate was different from the spectra of the native beta-amanitin and the native concanavalin A. The toxicity of the conjugate was determined by in vivo toxicological studies and was four-fold lower than that of the native beta-amanitin. These results suggest that this conjugate may be used for immunization procedures and for the production of amatoxin-specific antibodies.