ISSN 1819-7124, Neurochemical Journal, 2009, Vol. 3, No. 2, pp. 145–148. © Pleiades Publishing, Ltd., 2009. Original Russian Text © A.A. Galoyan, K.S. Margaryan, G.G. Hovhannisyan, G.H. Gasparyan, D.N. Aroutiounian, R.M. Aroutiounian, 2009, published in Neirokhimiya, 2009, Vol. 26, No. 2, pp. 156–160. 145 INTRODUCTION We previously isolated and studied a proline-rich polypeptide (PRP-1) containing 15 amino acids that is secreted by the cells of the N. Paraventricularis and N. Supraopticus of the hypothalamus [1]. We observed the antibacterial effects of this polypeptide against several types of infection [2], including anthrax (Bacillus anthracis) under in vivo conditions. This effect was possibly due to the activation of the release of oxygen free radicals from neutrophils and macrophages [3]. The polypeptide is also involved in the regulation of myelo- and lymphopoiesis [3–8], and increases the production of antibodies in B-lympho- cytes [8], stimulating the stem cells [9–10]. Taking into account the inhibitory effects of PRP-1 on myeloid and leukemic cells, we proposed to use this peptide as a pharmaceutical. It is known that the development of new drugs must include an assessment of the risks of their appli- cation, which includes estimation of their negative effects on cells, tissues, and the body. An important part of the risk assessment program is studying the chronic consequences of drug application, specifically its genotoxicity. During recent years, the comet assay was widely used in genetic toxicology as a high-resolution method [11, 12]. This method was firstly proposed by Ostling and Johnson [13] and was based on single-cell gel electrophoresis. However, a modified method, which is based on the use of alkaline gel electrophore- sis [14], is more often used because of its higher sen- sitivity and standardization capabilities. The comet assay reveals DNA breaks in single and double-strand alkaline-labile sites DNA–DNA and DNA–protein crosslinks, and DNA reparation in eukaryotic cell populations. Here, we investigated the in vitro dependence of PRP-1’s genotoxic effects on its dose and incubation time. MATERIALS AND METHODS Cell culture. Suspended cells of the human mye- loid leukemia cell line KCL-22 were used. Cells were cultured at 37°ë in a RPMI-1640 medium containing 10% fetal calf serum, and gentamicin (50 mg/ml). PRP-1, at concentrations of 0.5–4 μg/ml, was added to the cell culture (10 6 per ml) and incubated for 2 or 24 hours. Cell viability was estimated using the trypan blue exclusion test. Trypan blue was supplied by Sigma (United States). Comet assay. We used this method with the modi- fications described in [14]. Two agarose layers were applied onto slides. Firstly, the slides were covered with 1% normal melting agarose (RM–273, HiMedia, India) and dried in a thermostat at 37°C for 12– 24 hours. Then, these slides were covered with a sec- ond layer, which consisted of a mixture of 15 μl of cell suspension, preliminarily incubated with PRP-1 at Study of the Genotoxic Effects of a Proline-Rich Polypeptide Using the Comet Assay A. A. Galoyan a , K. S. Margaryan a, b , G. G. Hovhannisyan b , G. H. Gasparyan b , D. N. Aroutiounian b , and R. M. Aroutiounian b, 1 a Buniatian Institute of Biochemistry, National Academy of Sciences of Republic of Armenia, Yerevan, Armenia b Yerevan State University, Yerevan, Armenia Received December 16, 2008 Abstract—The genotoxicity of a proline-rich polypeptide was investigated in vitro in the human myeloid leu- kemia KCL-22 cell line. A comet assay, based on single-cell gel electrophoresis, was used for the analysis of the DNA damage and reparation rate. After electrophoresis, images resembling comets were formed; the inten- sity of the “tail” relative to the “head” reflected the number of DNA breaks. We demonstrated that the level of DNA damage depended on the polypeptide concentration and incubation time. Estimation of DNA damage lev- els by the comet assay is recommended for the evaluation of the genotoxicity of new pharmaceuticals. Key words: proline-rich polypeptide, genotoxicity, single cell gel-electrophoresis, comet assay DOI: 10.1134/S1819712409020111 METHODICAL ARTICLES 1 Corresponding author; ul. Manukyana, 1, Yerevan, 0025 Arme- nia, e-mail:genetik@ysu.am