Šuteková Eva and Jakub Hofman RECETOX, Masaryk University, Faculty of Science, Kamenice 126/3, Brno, CZ-62500, Czech Republic 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 ► 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 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 INCHEMBIOL). Acknowledgements Römbke, J., & Knacker, Th. (1989). Aquatic toxicity test for enchytraeids. Hydrobiologia, 180, ISO/DIS (2002). Soil quality – Effects of pollutants on Enchytraeidae (Enchytraeus sp.) – Determination of effects on reproduction and survival. Selected results Materials and Methods 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 References 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. * 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? *