THE INFLUENCE OF TITANIUM DIOXIDE AND SILVER NANOPARTICLES ON GENOTOXICITY IN DIFFERENT CELL TYPES OF WISTAR RATS M. Dobrzyńska1, J. Radzikowska,1 A. Gajowik1, J. Gromadzka-Ostrowska2, M. Oczkowski2, A. Krawczyńska2, K. Dziendzikowska2, G. Brunborg3, M. Dusinska4, A. Lankoff5, M. Wojewódzka5, M. Kruszewski5 1National Institute of Public Health – National Institute of Hygiene, Poland, 2Warsaw University of Life Sciences, Poland, 3Norwegian Institute of Public Health, Norway, 4Norwegian Institute of Air Research, Norway, 5Institute of Nuclear Chemistry and Technology, Poland R E S U L T S INTRODUCTION In recent years the number of applications of nanomaterials in many fields of human life growing fast. Nanomaterials possess new properties and their use constitutes new opportunities. Nanoparticles are used in drug delivery systems, medical devices, food products, cosmetics, electroanalysis. Human are at increasing of exposure to nanoparticles, which may enter the body through the skin, lungs or intestinal tract during manufacture, use and disposal of nanoproducts. From the site of deposition, the nanoparticles are translocated to different parts of the body through the circulatory or lymphatic system. The potential health effects from exposure to nanoparticles are increasing, but so far not exactly known, because for some nanoparticles the toxicological data are limited. Silver nanoparticles (Ag NPs) are widely used in medicine, physics, material sciences and chemistry. Many medical products are coated or embedded with nanosilvers, for example contraceptive devices, surgical instruments, bone prostheses, dental alloys. Silver naoparticles are the component of room spays, detergents, wall paints, textiles, clothing, socks, cosmetics. AgNPs are used in lining of washing machines, dishwashers, refrigerators and toilet seats. Titanium dioxide (TiO2 NPs) nanoparticles are manufactured in large quantities for use in a wide range of application. They are used as pigments for paints, varnishes, enamels, lacquers and paper coatings to impact whiteness, opacity and brightness. Exposure can also occur from their use in cosmetics such as in sunscreams, dusting powder, ointments and from radioactive decontamination of the skin. The study was designed to evaluate the effects of titanium dioxide and silver nanoparticles on Wistar male rats. Fig. 1: Induction of micronuclei in the bone marrow reticulocytes (MN/1000 cells) Fig 2: Comet tail moment in lung cells of male rats MATERIALS AND METHODS Silver nanoparticles (20 nm and 200 nm) were purchased from PlasmaChem (Germany). Titanium dioxide nanoparticles P-25 were prepared for Project Nanoform „NAPIRA” (European Commission - Joint Research Centre). Nanoparticles were dispersed in deionized H2O containing DMSO (50µl DMSO/1 ml H2O). The solution was sonicated for 5 min (output 20) (Branson Sonifier, USA) in plastic test tube surrounded by ice in the same manner before each experiment. Adult Wistar male rats were housed in plastic cages After one week acclimatisation they were assigned randomly to either control or exposed groups. Animals were injected intravenously with 5 mg/kg of size 20 nm (AgI) or 10 mg/kg Ag NPs of size 20 nm (AgII) or with 5 mg/kg of size 200 nm (AgIII) as well as with 5 mg/kg TiO2 NPs. Control groups were untreated or received NaCl solution. Groups of animals were sacrificed at 24 h, 1 week and 4 weeks after exposure. Cell samples were analysed using the micronucleus test (bone marrow reticulocytes), sperm count, and the Comet assay (germ cells and lung cells). For induction of micronuclei estimation, the bone marrow cells were flushed out from femora with fetal bovine serum. Homogenous cell suspension was obtained by pipetting. 5 l of the cell suspension were placed on the slide previously coated with acridine orange solution and covered with cover slip. All slides were coded and examined using fluorescent microscope. For estimation of the frequency of micronuclei, 1000 reticulocytes were counted and simultaneously the number of micronuclei was registered For sperm count, one epididymidis was macerated in 0.8 ml of 1 % solution of trisodium citrate for 7-8 min and minced. Then the solution was made up to 8 ml and mixed for about 1 min. The sperm suspension was diluted 1:1 in 10 % buffered formalin. The spermatozoa were counted using improved Neubauer haemocytometer. For Comet assay, the part of one testis and the part of one lung from each animal was placed in RMPI 1640 medium and minced with scissors. Finally, single cells remained in the suspension. 3-5 µl of cell suspension were mixed in an Ependorff tube with 75 µl of low melting point agarose (LMPA) for embedding on the slides previously covered with normal melting point agrarose (NMPA). After solidify the agarose at 4°C another layer of LMPA was added and allow to solidify at 4°C again. The slides were immersed in lysing solution at 4°C for at least 24 h. Then slides were removed from lysing solution, drained and placed in a gel electrophoresis tank, and incubated in the electrophoresis solution for 20 min to allow the unwinding of DNA. The alkaline electrophoresis was conducted for 20 min at 4°C using 24 V and 300 mA. After neutralisation, slides were stained with ethidum bromide soltion and examined using fluorescence microscope. Images of 100 randomly selected cells from each animal were recorded and analyzed using CASP image-analysis software. The DNA tail moment was chosen as parameter for further analysis. Fig 3: Sperm count (x106) in control and exposed males Fig 4: Comet tail moment in germ cells of male rats SUMMARY AND CONCLUSIONS Titanium dioxide slightly enhanced the frequency of micronuclei at 24 h after exposure and 1 week later, but there were no significant differences. Silver nanoparticles at different doses and particle sizes induced slight, not significant increase in the levels of micronuclei at 24 h and 4 weeks later (Fig. 1). A dose of 5 mg/kg of size 200 nm Ag NPs significantly (p<0.05, Student t-test) increased comet tail moment in lung cells at 24 h and caused cytotoxicity 4 weeks later. A dose of 10 mg/kg of size 20 nm Ag NPs slightly enhanced comet tail moment one week following exposure (Fig. 2). Sperm counts were slightly reduced at 24 h after exposure to TiO2 NPs and to 5 mg/kg of Ag NPs of size 20 nm, and after 4 weeks in the group exposed to TiO2 NPs only. There were no significant differences compared to control (Fig 3). Silver nanoparticles in all doses and sizes significantly (p<.05, Student t-test) increased the comet tail moments in germ cells at 24 h following exposure. TiO2 NPs markedly increased the level of comet tail moments 1 and 4 weeks after exposure, whereas 5 mg/kg of size 20 nm of AgNPs gave increased tail moments at one week following the injection. The results show that both silver and titanium dioxide nanoparticles may induce DNA damage in lung and male germ cells of Wistar rats. ACKNOWLEDGMENT This work was funded by Polish Norwegian Research Foundation (2008-2010), Project no PNRF-122-AI-1/07.