Lidia Mazur

Radioprotective effects of the thiols GSH and WR-2721 against X-ray-induction of micronuclei in erythroblasts

The frequency of micronucleated polychromatic erythrocytes (MNPCEs) was assessed in the bone marrow and peripheral blood of adult male Swiss mice treated with reduced glutathione (GSH) and S-2-/3-aminopropylamino/ethyl phosphorothioic acid (WR-2721), at a dose of 400 mg/kg body weight, and exposed to 6 Gy X-rays. GSH or WR-2721 was applied alone, or 60 and 30 min, respectively, prior to X-ray-exposure. The number of MNPCEs was determined at 24 h after the thiol treatment and X-irradiation. The radioprotection and toxicity caused in the mouse erythroblasts by GSH and WR-2721, as indicated by the number of MNPCEs were dependent on the thiol applied. The stronger radioprotective effect is obtained following WR-2721 administration than after GSH application. WR-2721 showed greater toxicity than GSH. The combination of GSH and WR-2721 given before X-ray-exposure resulted in the most radioprotective effect as compared to the respective single-drug treatment of mice. Application of the both thiols, without subsequent X-irradiation appeared to be the most toxic, compared with administration of WR-2721 or GSH alone. The effective radioprotection by the combined action of GSH and WR-2721 against genomic instability induced in the mouse erythroblasts by X-rays was shown.

Flow cytometric estimation of the plasma membrane diversity of bone marrow cells in mice treated with WR-2721 and cyclophosphamide

The effects of S-2-/3-aminopropylamino/ethyl phosphorothioic acid (WR-2721, Amifostine) and cyclophosphamide (CP) on the cell surface exposure of phosphatidylserine (PS) and the plasma membrane impairment of bone marrow cells were assessed by flow cytometry assay with fluoresceinated annexin V (annexin V - FITC) and propidium iodide (PI). During the 96 h-period after treatment of adult male Swiss mice with WR-2721 (400 mg/kg b.wt.) and CP (200 mg/kg b.wt.), bone marrow cells expressing PS on the outer leaflet of the plasma membrane, which bound annexin V, and cells with a compromised cell membrane, which allowed PI to bind to the cellular DNA, were analysed. Temporary changes in the frequency of early apoptotic cells (annexin V - FITC positive/PI negative), late apoptotic and necrotic cells (annexin V - FITC positive/PI positive), and the number of live cells (annexin V - FITC negative/PI negative), were dependent on the drug(s) given. Application of CP distinctly triggered apoptotic and necrotic cell death, and WR-2721 pre-treatment of mice affected cell death induced by CP, causing reduction of the number of apoptotic and necrotic cells. The chemoprotective action of WR-2721 against PS externalisation and the plasma membrane impairment of normal bone marrow cells was shown.

Glufosfamide as a new oxazaphosphorine anticancer agent

Glufosfamide (&bgr;-D-glucose-isophosphoramide mustard, D-19575) belongs to the oxazaphosphorine class. Glufosfamide is a novel glucose conjugate of ifosfamide in which isophosphoramide mustard, the alkylating metabolite of ifosfamide, is glycosidically linked to the &bgr;-D-glucose molecule. Glufosfamide represents an attractive new agent for cancer therapy. Its mode of action on normal and pathological cells is still under experimental and clinical investigations. An assessment of the anticancer potential of glufosfamide is of key importance in therapy. The researchers reviewed the current knowledge available on glufosfamide tested in the preclinical studies/clinical trials, based on a collection of the original papers and conference abstracts published and relevant articles searched in the SCOPUS and MEDLINE database and websites.

Induction of micronucleated erythrocytes by MEA, AET, WR-2721 and X-rays

The induction of micronucleated polychromatic erythrocytes (MNPCEs) was assessed in the bone marrow of adult male Swiss mice treated with MEA (cysteamine HCl), AET (2-aminoethylisothiouronium Br.HBr), or WR-2721 (S-2-(3-aminopropylamino)ethyl phosphorothioic acid), at a dose of 200 mg/kg body weight, and/or exposed to 6 Gy X-rays. MEA, AET, or WR-2721 was given alone or 15 min prior to X-ray exposure, and the frequency of MNPCEs was determined 24 h after the aminothiol treatment and X-irradiation of mice. A genotoxic effect was shown for MEA, AET, WR-2721, and X-rays, as well as a protective effect of the aminothiols against X-ray-induced genotoxicity in the mouse erythropoietic system. The aminothiol drugs given alone, without subsequent X-irradiation, elevated the frequency of MNPCEs, and WR-2721 appeared to be less toxic than AET and MEA. After exposure of mice to X-rays, the number of MNPCEs was distinctly increased. MEA, AET, or WR-2721 administration prior to X-irradiation resulted in a reduction of the X-ray-induced elevation of the frequency of micronuclei, but a stronger radioprotective effect was obtained following WR-2721 and AET treatment than after MEA application. So, the genotoxic and radioprotective effect of the aminothiols was dependent on the compound applied.

Influence of different levels of traffic pollution on haemoglobin content in the earthworm Lumbricus terrestris

Abstract Our aim was to determine haemoglobin content in earthworm populations from sites with different amounts of soil pollution. The study was done on Lumbricus terrestris specimens from four sites: the median strip of a busy street in Cracow; a city park; forest about 30 km from Cracow (100 m from a low-traffic local road); and a forest on calcareous soil about 30 km from Cracow (distant from traffic). There were statistically significant differences in haemoglobin content between study sites. The lowest amount of haemoglobin was found in earthworms from the median strip (2.19 g 100 ml−1), the highest in those from the ‘cleanest’ forest on calcareous soil (3.18 g 100 ml−1). The highest lead concentration was found in specimens from the median strip (12.3 μg g−1) and only trace amounts were found in those from the forest on calcareous soil. There was a negative correlation between haemoglobin and lead concentrations in earthworms. Species composition, density and biomass of earthworm populations at the four sampling sites as well as the body mass of adult individuals of L. terrestris were estimated. No correlation was found between any of these variables and the extent of exposure to traffic pollution.

Flow cytometric detection of apoptotic bone marrow cells with fractional DNA content after application of WR-2721, cyclophosphamide, cisplatin, and exposure of mice to gamma rays.

Little is known about the mechanisms of apoptosis triggered in normal cells of the haemopoietic system by the aminothiol WR-2721 (Amifostine), chemotherapeutic drugs, and ionizing radiation; thus, the present study was undertaken to evaluate the effects of WR-2721, cyclophosphamide (CP), cisplatin (CDDP), and 60Co gamma rays on induction of apoptotic DNA degradation in bone marrow cells. Adult male Swiss mice were treated with WR-2721 (400 mg/kg b.wt.), CP (200 mg/kg b.wt.), and CDDP (10 mg/ kg b.wt.), and exposed to 6 Gy 60Co gamma rays. Alterations in the number of apoptotic cells with fractional DNA content and also the cell cycle position of the non-apoptotic cells were determined in the bone marrow at 7 and 24 hours after treatment of mice with these agents, using flow cytometric assay of the controlled extraction of low-MW DNA from apoptotic cells. The chemotherapeutic drugs CP and CDDP and 60Co gamma rays triggered apoptosis and affected the cell cycle position of the non-apoptotic cells in the mouse bone marrow. The pretreatment of mice with WR-2721 resulted in the modulatory action of the aminothiol on induction of apoptotic cell death and changes in the cell cycle distribution of the non-apoptotic cells caused by the DNA-damaging agents. The patterns of changes in the frequency of apoptotic cells and the cell cycle position of the non-apoptotic cells, observed in the bone marrow, were dependent on the agent(s) applied and the time interval after application of the drug(s) and exposure of mice to gamma rays. Understanding of the mechanisms responsible for triggering of apoptotic cell death and disturbing of the cell cycle by the DNA-damaging agents, and modulation of the apoptotic and cell cycle pathways by the aminothiol WR-2721, can lead to more effective therapy and chemo-and radio-protection of normal cells.

WR-2721: inhibitor of cisplatin-induced micronuclei.

The modulatory effect of S-2-/3-aminopropylamino/ethyl-phosphorothioic acid, (WR-2721, Amifostine) on induction of micronuclei by cis-diamminedichloroplatinum[II] (CDDP) was studied. The adult, male Swiss mice were treated with WR-2721, at a dose of 200 mg/kg or 400 mg/kg body weight, and/or with CDDP, at a dose 5 mg/kg or 10 mg/kg body weight. WR-2721 was given alone or 30 min before CDDP administration. The frequency of micronucleated polychromatic erythrocytes (MNPCEs) and also the number of polychromatic erythrocytes (PCEs) in the bone marrow and peripheral blood, at 24 h after the drug application, were determined. After administration of CDDP, the frequency of MNPCEs distinctly increased, and the number of PCEs decreased. As compared with the animals injected with CDDP only, in mice treated with WR-2721 before CDDP application, the number of MNPCEs was reduced and the frequency of PCEs was increased. However, WR-2721 given alone, without subsequent administration of cis-diamminedichloroplatinum[II], caused an increase in the number of micronucleated polychromatic erythrocytes and a decrease in the number of PCEs. The geno- and cyto-toxicity and chemoprotection were dependent on the doses of the agents WR-2721 and CDDP applied. In mice injected with CDDP and/or WR-2721, the patterns of changes in the frequency of MNPCEs and PCEs were similar in the bone marrow and peripheral blood, respectively. The protective effect of the aminothiol compound WR-2721 against induction of micronuclei and apoptotic cell death in the erythropoietic system by CDDP was shown.

Inhibition of the clastogenic effect of cyclophosphamide by WR-2721 in the bone marrow of mice

The frequency of micronucleated polychromatic erythrocytes in the bone marrow of Swiss mice treated with WR-2721, at a dose of 200 mg/kg or 400 mg/kg body weight, 15 or 30 min prior to cyclophosphamide (CP) administration, at a dose of 200 mg/kg body weight, was determined 24 h after CP treatment. In mice injected with CP, the number of micronuclei in polychromatic erythrocytes was significantly increased in comparison with the controls, and in mice treated with WR-2721 and CP, the frequency of micronucleated polychromatic erythrocytes was distinctly decreased in comparison to those given CP alone. The protective effect of WR-2721 against cyclophosphamide-induced clastogenicity was shown. The effect was dependent on the dose of the thiol agent given, and it was more expressed when WR-2721 was applied at the higher dose, 400 mg/kg body weight. However, the protection by the aminothiol appeared not to depend on the time intervals between WR-2721 and CP administration to the mouse organism.

Combination of ABT-737 and resveratrol enhances DNA damage and apoptosis in human T-cell acute lymphoblastic leukemia MOLT-4 cells

ABT-737 belongs to a new class of anticancer agents named BH3 mimetics. ABT-737 competitively binds to surface hydrophobic grooves of anti-apoptotic proteins of Bcl-2 family, counteracting their protective effect. Resveratrol is a natural polyphenol that has been shown to inhibit the proliferation and/or induce apoptosis in a number of different types of cancer cells. The present study was designed to analyze the combined effects of ABT-737 and resveratrol on human acute lymphoblastic leukemia cells. The in vitro cytotoxic activity of these agents against MOLT-4 leukemia cells was determined using the Coulter electrical impedance method, comet assay, and flow cytometry, light microscopy and western blot techniques. The results are the first data showing that ABT-737 combined with resveratrol markedly decreased the cell viability, increased DNA damage, caused the cell cycle perturbation, and synergistically enhanced apoptosis in MOLT-4 cells, when compared to the data obtained after application of the single agent. Moreover, the simultaneous treatment of leukemia cells with ABT-737 and resveratrol resulted in a reduction in mitochondrial membrane potential, an increase of p53 protein level and up-regulation of the Bax/Bcl-2 ratio. The obtained data indicate that the combination of ABT-737 and resveratrol is a promising approach for acute lymphoblastic leukemia treatment that should be further explored.