Šuteková Eva and Jakub Hofman

1. Šuteková Eva and Jakub Hofman RECETOX, Masaryk University, Faculty of Science, Kamenice 126/3, Brno, CZ-62500, Czech Republic eva.sutek@quick.cz,hofman@recetox.muni.cz OXIDATIVE STRESS IN ENCHYTRAEIDAE (E. albidus, E. crypticus) – ESTABLISHMENT AND OPTIMALIZATION OF METHODS • Oxidative stress is one of a main toxicity mechanisms of toxic compounds. • In cells, redox imbalance may occur due to enhanced production of reactive oxygen species (ROS) and/or antioxidants inhibition. • Consenquences of oxidative stress are damage of important macromolecules (DNA, proteins, lipids) and finally disturbance of cell´s or whole organism´s fyziology. • Oxidative stress has not been studied in soil enchytraeids so far, although it might be expected for some types of organic pollutants (e.g. PAHs and their derivates) • Synchronization of cultures results in: • decrease in natural variability - obtained adults are more uniform and bigger with comparison to non-synchronized ones (aprox. 2fold increase in proteins for E. crypticus) X Values are means ± SD (n=3); S – synchronized, N – non-synchronized • increase of statistical significance X Values shown with asterisk were significantly different (p<0,05); S – synchronized, N – non-synchronized • breeding substrate modulates sensitivity S – synchronized, N – non-synchronized • allows to observe developmental profiles • X • Values are means ± SD (n=3); with asterisk were significantly different (p<0,05) from embryonal stage (w.3,4) Introduction Selected results • Objectives of the study: • to establish methods for biomarkers of oxidative stress (GST, GR, GPX, GSH, LP) for soil enchytraeids E. albidus and E. crypticus • to assess influence of breeding conditions on observed parametres • to characterize developmental profile of detoxification and antioxidant aparatus • to use these tests for assessment the effects of model substance (DMNQ) and selected PAHs * • Main steps of the study: • optimalization of methods • - including modifications in animal number, reagent and sample concentrations, • pH of testing medium • synchronization of cultures • - preparation of new breeds to obtain well-developed adults with minimal • variability in age (±3days) and size • dose-response tests with DMNQ and selected pollutants • - redox cyclator DMNQ (2,3-dimethoxy-1,4-naphtochinone) • - PAHs: antracene and phenantrene • - aza-PAHs: acridin (N-derivate of antracene) • and 1,10-phenantroline (2N-derivate of phenantrene) What has been done? * • Enchytraeids: - adults were used with well-developed clitellum - Enchytraeus albidus(both synchronized and non-synchronized culture were kept in mixture of field and commercial garden soils) - Enchytraeus crypticus(synchronized culture was kept on agar plates and non-synchronized stock culture was kept in artificial soil) • Aquatic toxicity test: - closed jars, exposure for 96h, 20°C, darkness - using the fact that Enchytraeids survive in water, without reproduction - artificial, unreal, but there is no problem of bioavailability - recommended as fast screening or preliminary test - 10 E. albidus or 20 E. crypticus per sample were used • Biomarker assessment: - all assays are spectrophotometrical methods modified for microtitre plates - formation of color product or dissapearance of reagent during biochemical reaction were measured - observed biomarkers are glutathion-S-transferase (GST), glutathion reductase (GR), glutathion peroxidase (GPX), glutathion reduced (GSH) and lipid peroxidation (LP) - all results are expressed on protein content basis • Statistical analysis: • - One-Way ANOVA (Dunnett test), T-test, correlation by Spearman test Materials and Methods • Conclusions • In the present study, procedures were optimalized for determination of biomarkers of oxidative stress in two enchytraeid species – E. albidus and E. crypticus. • Optimal numbers of individuals for determination of biomarkers were found to be 10 for E. albidus and 20 for E. crypticus. • Synchronization of enchytraeids improved statistical significance. • Significant differences between cultures breeded on various substrate (agar, soil mixture) were observed. • These parametres were used to characterization of developmental profile of detoxication and antioxidant aparatus. GST showed no significant trend, but GSH and GR increased in time. Römbke, J., & Knacker, Th. (1989). Aquatic toxicity test for enchytraeids. Hydrobiologia, 180, 235-242 ISO/DIS 16387 (2002). Soil quality – Effects of pollutants on Enchytraeidae (Enchytraeus sp.) – Determination of effects on reproduction and survival. References Research on this topic was enabled by financial support of Grant Agency of Czech Republic (GACR 525/04/P159) and by Ministry of Education of the Czech Republic (project no. MSM 0021622412 INCHEMBIOL). Acknowledgements