Genotoxicity assessment by micronucleus assay in peripheral blood lymphocytes and buccal epithelial cells of children with chronic kidney disease

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Abstracts / Toxicology Letters 196S (2010) S37–S351

P202-038 Genotoxicity assessment by micronucleus assay in peripheral blood lymphocytes and buccal epithelial cells of children with chronic kidney disease G. Cakmak Demircigil 1 , B. Aykanat 1 , K. Fidan 2 , U.S. Bayrakci 3 , B. Buyukkaragoz 2 , H. Karakayali 4 , M. Haberal 4 , E. Baskin 3 , N. Buyan 2 , S. Burgaz 1 1

Gazi University, Faculty of Pharmacy, Department of Toxicology, Ankara, Turkey, 2 Gazi University, Faculty of Medicine, Department of Pediatric Nephrology, Ankara, Turkey, 3 Baskent University, Faculty of Medicine, Department of Pediatric Nephrology, Ankara, Turkey, 4 Baskent university, Faculty of Medicine, Department of General Surgery, Ankara, Turkey The investigation of the patients with chronic kidney disease (CKD) and their treatment approaches are crucial. One consequence of CKD, dialysis and post-renal transplantation immun supressor treatment is an elevated cancer risk. There is no genomic data in children with CKD and after transplantation although children deserve the highest attention as they have to live with the disease longer and vulnerable to the treatment strategies. Therefore, in this study the genotoxicity in peripheral blood lymphocytes (PBL) and buccal epithelial cells (BEC) of children in pre-dialysis stage (Pre-D) (n = 17), on regular hemodialysis (HD) (n = 15) and transplanted (Tx) (n = 17) has been compared to the healthy children (n = 20) by micronucleus (MN) assay. The cytokinesis-blocked MN (CBMN) assay in PBL has been used in this study which has recently been proved to be predictor of cancer risk. Secondly, MN in BEC has been evaluated as they can be obtained easily by non-invasive methods from children. Finally, we combined fluorescence in situ hybridization (FISH) technique to CBMN in order to discriminate chromosome breakage as centromere negative (C−) MN and chromosome loss as centromere positive (C+) MN. In our study, gender and age were consistent among the groups. The results revealed that, MN frequencies were statistically significantly higher in CKD patients in PBL and BEC (8.06 ± 4.58, 8.40 ± 7.82, respectively) vs. control group (1.60 ± 0.99, 0.90 ± 1.41, respectively) (p < 0.001). Similarly, FISH-CBMN assay showed that both C− and C+ MN frequencies were statistically significantly higher in CKD patients (3.09 ± 2.47, 4.39 ± 2.38, respectively) vs. control group (0.65 ± 0.67, 1.10 ± 0.97, respectively) (p < 0.001). Concurrently, each subgroup of children (Pre-D, HD and Tx) had the statistically significant MN increase against to the healthy children (p < 0.001). Pre-D children had the highest MN frequencies in PBL whereas TX children had the highest MN frequencies in BEC. As a conclusion, MN assay in two different tissues put the genotoxic outcome clearly for children either with CRD or under HD and Tx. doi:10.1016/j.toxlet.2010.03.574

P202-039 Biomonitoring in children exposure to heavy metals ˜ 1 , M. C. Herrera Maldonado 1 , M.E. Sánchez 1 , C. Paz Y. Mino Segovia 2 , C. Carrera 2 , M. Arévalo 2 , V.H. Espín 3 , M. Novillo 3 1

U.D.L.A. Instituto de Investigaciones Biomédicas, Spain, 2 Pontificia Universidad Católica del Ecuador, Ecuador, 3 Universidad Central del Ecuador, Ecuador The biomonitoring evaluate the health status of exposed populations to mutagens or carcinogens trough biomarkers of exposure, effect and susceptibility. The constant exposure to heavy metals

independently of their anthropological or environmental origin represent a potential risk for children, existing a larger tendency in order to develop genetic, dermises, neurological and digestive pathologies, during pregnancy or childhood. Diverse studies in vitro and in vivo demonstrate that heavy metals can generate permanent modifications in the genetic material, resultant of the oxidative damage at cellular level induced by reactive kinds of oxygen, which play an important role in the development of human cancers, this toxicity liberates noxious action mechanisms at cell level which produces cytogenetic alterations, fractures to the ADN, mutations, alterations in the reparation or replication processes, which can drive to genomic instability, changes on the progression of cell cycle or apoptosis. The cytogenetic markers in monitoring studies in human populations exposed to genotoxic substances: chromosome aberrations, of chromatid sisters interchanges, micronuclei and comet assay are utilize to identify the standing level of individuals and populations risk. In Ecuator the presence of arsenic and heavy metals of natural origin has been found in the provinces of Carchi, Imbabura, Pichincha, Cotopaxi and Tungurahua at dregs, geothermal, subterranean and superficial waters. The project outlines the monitory of exposed children to heavy metals, with the objective of determine the damage in the DNA, through the applications of exposure markers (mineralogram) and of effect (comet assay and chromosomic aberrations). This study will count with 20 exposed children located in Valle de Tumbaco and a control group of 20 children from Valle de los Chillos, who will be evaluated by medical and toxicological diagnosis. 30 metaphases and 100 nucleoids will be analyzed per individual. doi:10.1016/j.toxlet.2010.03.575

P202-040 Genotoxicity of combined exposure to benz(e)acephenanthrylene and UV-A by using human skin cell line D. Ali Indian Institute of Toxicology Research, Lucknow, India Benz(e)acephenanthrylene (BeA), a polycyclic aromatic hydrocarbon (PAH) and UV-A radiation are two harmful agents. When combined, their deleterious properties are synergistic. However, less information is available on its DNA damaging potential in skin cells using genotoxic biomarkers. The objective of the present study was to detect DNA damage, induced by UV-radiation and BeA in human skin cell line (A-375) using the comet assay. A reduction in cell viability as a function of both BeA + UV-A as well as exposure time were observed through 3-(4,5-dimethyl2-thiazolyl)-2,5-diphenyl-tetrazolium bromide (MTT) and neutral red uptake (NRU) assays. BeA + UV-A demonstrated DNA damaging potential as evident from an increased percentage tail DNA of 17.96 ± 0.46 (2.5 ␮g/ml) compared to control 4.29 ± 0.27 in the comet assay after an exposure. Thus our data demonstrates that even at low concentration of BeA with UV-A induce genotoxicity in human skin cells. This study also explored the utility of the comet assay for in vitro laboratory studies using cell lines for screening the genotoxic potential of various agents. Keywords: Polycyclic aromatic hydrocarbon; DNA damage; Cell line; Comet assay doi:10.1016/j.toxlet.2010.03.576