Development of a DNA adductomics approach as marker for eco-genotoxicity

The assessment of the biological effects of contaminants in the Baltic Sea is conducted by the Swedish National Marine Monitoring Program (SNMMP) through the analysis of several indicators that complement each other. One of these indicators is the aberration of the embryo in the benthic amphipod, Monoporeia affinis, which is a key species in the soft sediments of the Baltic. These aberrations may indicate an underlying but unknown genotoxicity mechanism, although some non-contaminant stress factors have also been suggested to stop embryonic development. Therefore, we must evaluate the relationship between the frequency of embryo aberration and changes in the DNA structure, if we want to use this indicator for the assessment of the contaminant effect. We analyzed DNA adducts in amphipods collected from SNMMP and grouped in healthy (without aberrant embryos in the brood envelope) and unhealthy (with at least 20% of aberrant embryos); 20 women per group were analyzed. The embryos were dissected and analyzed using the same method, where by the brood envelope and the females were used for DNA extraction and analysis using an adductomics approach, which means capturing all the modifications (or adducts) on the biomolecule. After enzymatic DNA splitting, liquid chromatography (LC) coupled with high-resolution Orbitrap mass spectrometry (MS) was used to screen for DNA modifications to deoxyribonucleosides generated within a defined mass range. MS was used to detect all parent ions giving a neutral loss of 116.0474 amu, which corresponds to the deoxyribose moiety of DNA. Subsequently, an MS / MS scan on the parent ions detected was performed to identify and quantify specific types of modification. In amphipods, using peak areas from the LC-MS analysis, we measured twenty-three types of DNA modifications. The partial squared regression model (PLSR) was performed with health status (healthy versus unhealthy) as the dependent variable and the DNA adduct values measured as predictors. A key predictor has been identified as 5-methyl-2’-deoxycytidine, which indicates the probable role of epigenetic alterations in reproductive aberrations in amphipods. Overall, the relationship between the measured DNA adducts and the amphipod embryonic aberrations indicates genotoxicity in polluted environments