Standardization of Caiman latirostris lymphocyte culture for the application of Chromosomal aberration test.

LÓPEZ GONZÁLEZ, EVELYN C.; ANDRIOLI, NANCI; SIROSKI, PABLO A.; POLETTA, GISELA L.

Buenos Aires

Congreso; SETAC Latin America 11th Biennial Meeting - Buenos Aires, Argentina. 7?10 September.; 2015

SOCIETY OF ENVIRONMENTAL AND CHEMISTRY TOXICOLOGY (SETAC)

The aim of this study was to standardized the Chromosomal Aberration (CA) test on C. latirostris lymphocytes, as there were no previous reports of its application in any crocodilian species.In order to obtain suitable metaphases for CA analysis, different methodological adjustments were done to the conventional technique for karyotype analysis on C. latirostris. First, serial cultures were performed at differents times in order to establish the duration of lymphocyte cycle, data not known for this species. Then, different variations were tested in various parametres of culture and harvest processes, as well as fixed and drip conditions, includying: culture time and temperature, culture of whole blood or isolated lymphocytes, type of culture mediums, amount and action time of mitogen (Phytohemagglutinin -PHA), type and action time of hypotonic solution, type of fixed solution and drip conditions (temperature and slides pre-treatment).Once optimal culture conditions were adcquire, we conducted an assay for the standardization of the CA technique. We exposed whole blood in vitro to the known CA inductor agent Methyl methanesulfonate (MMS) at 10 and 20 µM, and a negative control (NC) without exposure, all groups with and without colchicine, to evaluate CA induction at metaphase, post-metaphase and existing basal cell damage in this population.Our results constitute the first report on the application of the CA test in C. latirostris, as well as in all crocodilian species, and allow us to propose this technique as a new biomarker of genotoxicity to be incorporated into the battery of tests (Micronucleus test, Nuclear Abnormalities test, Comet Assay, Repair assay) routinely applied by our group in the assessment of the genotoxic effect produced by different agents in this and other native reptile species.