The Cytoprotective Effect Of Amifostine Against Hydroquinone-induced Apoptosis In Bone Marrow

Background Benzene is an important solvent and used broadly in themanufacture of leather and print. Chronic benzene exposure may impair hematopoiesis, and give rise to aplastic anemia and leukemia. Therefore, it is very necessary to search for a safe and effective cytoprotective agent and protect workers of benzene exposure from benzene damage.1,4-hydroquinone and 1,4-benzoquinone are two main metabolites of benzene. Recently research found that these two elements play a critical role in hematopoietic damage. The mechanism is production of free radical of oxygen and impairing DNA, promoting apoptosis of hematopoietic stem cell. Amifostine is a broad-spectrum cytoportective agent, can selectively protect cells from chemotherapeutic and radiative damage, without decrease the effects of chemotherapy and radiotherapy. Amifostine also is a potential hematopoiesis-stimulating agent. It may protect multiple hematopoietic stem cells. It can stabilize DNA molecule, eliminate free radical of oxygen and delay apoptosis by cytokines.It is established that amifostine can protect cell from damage of chemotherapy and radiotherapy. Whether amifostine can protect normal cellfrom other toxic agents is unknown. Recent reports do not involve this aspect. Through in vitro culture, our goals were evaluation of the protective effect of amifostine against hydroquinone-induced apoptosis of bone marrow mononuclear cells. Expect to provide a theoretical basis for protective effect of amifostine against benzene poisoning.Material and method1. Collect of normal volunteer bone marrow in a sterilized tube containing 50U/mL heparin, after separation of mononuclear cells with Ficoll-Hypaque, stained by trypan blue. The rate of trypan blue stain-resistance is more than 95%.2. Use RPMI basic medium supplemented with heat-inactivated 10% FBS, culture under 37癈, saturated humidity and 5%CC>2.3. Hydroquinone AR from shanghai LingFeng chemical agent Ltd, prepare solution with distilled water. Amifostine (for injection) from Hunan sliver liver biochemical engineering Ltd, prepare solution with saline. Diluted needed concentration.4. Methods for examination of apoptosis: HT (apoptosis agent II) stain, observe cell morphology under fluorescent microscope (Olympus 70DX). Apoptotic cell present nucleus dark-colored, nucleus pycnosis, chromatin condensed round the nucleus. Normal cell present nucleus light-colored and uniform. 1.2% agarose gel electrophoresis under 30V voltage, after EB staining, the typical presentation is DNA ladder, due to DNA degradation. Add anti-Annexin V FITC and PI staining, examination apoptotic and necrotic rate by flow cytometer. Early apoptotic cells present Annexin+/PI-, late apoptotic and necrotic cells present Annexin+/PI+ and normal cells present7Annexin-/PI-, respectively.5. Add final concentration of 0, 25, 50, 75 and ISO^M hydroquinone, determine apoptosis after culture for 2, 6 and 12 hours. Acquired the optimal concentration of apoptosis-induced by hydroquinone. Based on this optimal concentration, decide next experimental concentration of hydroquinone.6. According to optimal concentration of hydroquinone, after culture for 0, 2, 4. 6, 8, 10, 12. 18 and 24 hours, determine cell apoptosis, found the peak time of apoptosis, as the optimal culture time.7. Add final concentration of 0, 2, 10, 50, 100 and 50Q\ig/m\ amifostine with or without hydroquinone, determine the peak apoptotic time. Compare apoptotic rate under different concentration of amifostine, found the optimal concentration of amifostine which is lowest toxic effect and most effective apoptosis-inhibited.8. Add amifostine before adding hydroquinone 30 minutes, after adding hydroquinone 30 minutes and at the same time of adding hydroquinone. Determine the apoptotic and necrotic rate at the peak apoptotic time.9. Divide into four groups: ゜lank control group: add distilled water 60ml and saline 60ml; ゛mifostine group: add d...