alterations under controlled conditions. Sensitivity and specificity of the assay, in revealing the exposure to the main classes of genotoxic pollutants, have to be determined. Sources of variability that may influence the expression of the cytogenetic damage such as climate change, nutritional status, and reproductive status have to be evaluated in field experiment. doi:10.1016/j.cbpa.2010.06.031 3. Genotoxicity and genetic changes in RTG-2 fish cell line upon exposure to benzo[a]pyrene and ethyl methanesulfonate detected by the Comet assay and AFLP marker analysis M. Šrut, A. Štambuk, G.I.V. Klobučar (Department of Zoology, Faculty of Science, University of Zagreb, Croatia) Genotoxicity is often one of the earliest signs of toxicant impact and is frequently assessed using in vitro models in biomonitoring programmes. In this study Comet assay and amplified fragment length polymorphism (AFLP) were applied to assess DNA damage in RTG-2 fish cell line after 3 days of exposure to a concentration range of model genotoxic agents (benzo[a]pyrene (B[a]P) and ethyl methanesulfonate (EMS)). Quantitative modifications arising in AFLP profiles as a measure of DNA effects were analyzed in order to establish permanent genetic changes arising from mutation events (e.g. rearrangements, point mutations, small inserts or deletions of DNA) induced by toxicant exposure. Significant induction of DNA damage measured by the Comet assay was noticed in RTG-2 cells after 3 days of B[a]P treatment at all concentrations used (0.1 μM, 1 μM, 5 μM, 10 μM). After 3 days of recovery all the values returned to the control level. In contrast, 3 days of exposure to EMS (10 μM, 50 μM,100 μM, 500 μM,1000 μM) induced significant DNA damage only at the highest concentration used and 3 days of recovery resulted in more pronounced genotoxic effect. The changes occurring in AFLP profiles of RTG-2 cells following both toxicant treatments included loss of normal bands and appearance of new bands at higher toxicant concentrations in comparison to the band profile of the control. Our results indicate that the AFLP method could be a useful biomarker for detection of permanent genotoxic influence of toxicant exposure and encourage the use of Comet assay on fish cell lines as a versatile tool for genotoxicity assessment. doi:10.1016/j.cbpa.2010.06.032 4. Use of the Comet and micronucleus assays for in vivo genotoxicity assessment in the coelomocytes of the earthworm Eisenia andrei S. Sforzini, I. Saggese, L. Oliveri, A. Viarengo (University of Piemonte Orientale, DiSAV, Italy); C. Bolognesi (Environmental Carcinogenesis Unit IST, Genova, Italy) The assessment of genotoxic effects caused by environmental stressors represents an essential part of ecotoxicology because of its relevance in carcinogenesis. Many POPs are genotoxicant, i.e. they can alter both the structure and integrity of DNA, either directly or indirectly. The Comet assay is a method now widely used on different vertebrates and invertebrates to detect DNA damage (e.g. single- and double-strand breaks, alkali-labile sites, oxidative DNA base damage, etc.) in individual cells. Notwithstanding, the micronucleus test has emerged as one of the more powerful methods for assessing chromosome damage (both chromosome loss and chromosome breakage) accumulated during life- span of the cell. In this study, earthworms of the species Eisenia andrei were exposed to artificial standard soils contaminated with environmen- tally relevant concentrations of 2,3,7,8 tetrachlorodibenzodioxin (1 × 10 -4 , 1 × 10 -5 , 2×10 -3 mg/kg) and benzo[a]pyrene (0.1, 10, 50mg/kg) for 10 and 28 days. The DNA damage, using both the neutral and alkaline versions of the Comet assay, was assessed in earthworm coelomocytes in the different experimental conditions. Moreover, on these same cells, the presence of cytoplasmic micronuclei (MNi) was also studied by cytochemical staining of DNA by the fluorescent dye DAPI. The results obtained by the Comet assay showed, only in alkaline condition, significant effects at the different concentrations of the chemicals utilised and the times of exposure. Induction of MNi in coelomocytes, identified according to standardized criteria, was demonstrated, indicating a significant increase in MNi frequency particularly relevant in animals exposed to the higher concentrations of both chemicals. This study represents the first step of validation work of this test on earthworm cells. doi:10.1016/j.cbpa.2010.06.033 5. Potential genotoxic effects induced by different superficial morphologies and different dimensions of amorphous silica powders evaluated in murine alveolar macrophages (Raw 264.7) cell lines P. Guidi, V. Scarcelli, M. Bernardeschi, P. Lucchesi, M. Nigro, G. Frenzilli (University of Pisa, Italy) No sufficient evidence exists about the health hazards caused by nanosized amorphous silica, nor as regard its superficial conformity. The aim of the study was to investigate the potential genotoxic effects of silica particles with different superficial morphologies (mesoporous and dense spheres), and dimensions (250 and 500 nm) by in vitro experimental models. Pure quartz, vitreous silica and imogolite (1 nm diameter) were also investigated. Murine alveolar machrophages (Raw 264.7) cell lines have been used as representative of occupational and environmental exposures. Genotoxicity was evaluated by Comet Assay (4 and 24 h exposure) and Micronucleus Test. Cytotoxicity was tested using Trypan Blue method. Cell lines have been treated with 5–10–20–40–80 μg/cm 2 of different silica-based compounds. The same doses of exposure were tested by Micronucleus test. MMS was used as positive control in Comet assay, Mytomicin C for micronucleated cells. Comet assay results showed that the particle superficial morphology had an effect on DNA integrity, as mesoporous ones induced higher damage level with respect to the dense nanoparticles. On the contrary, particle dimension did not seem to have any effect. Clear dose–effect relationships were not detected, but the highest dose of exposure induced significant DNA fragmentation in powders tested. An increase in micronucleated cells was observed at intermediate doses of imogolite. Cell suspensions exposed to the lowest dimension of MCM-41 show a particular distribution of these particles inside the cell, with respect to the one across the slide. A deeper analysis of this phenomenon is in progress (TEM observation). doi:10.1016/j.cbpa.2010.06.034 POSTER PRESENTATIONS 6. Genotoxicity of marine sediment samples assessed by the Comet assay on PLHC-1 fish hepatoma cell line M. Šrut, A. Štambuk, S. Kralj, G.I.V. Klobučar (Department of Zoology, Faculty of Science, University of Zagreb, Croatia); L. Traven, V. Mićović S13 Abstracts / Comparative Biochemistry and Physiology, Part A 157 (2010) S12–S14