Simultaneous RNA and DNA Adductomics Using Single Data-Independent Acquisition Mass Spectrometry Analysis.

Adductomics studies are used for the detection and characterization of various chemical modifications (adducts) of nucleic acids and proteins. The advancements in liquid chromatography coupled with high-resolution tandem mass spectrometry (HRMS/MS) have resulted in efficient methods for qualitative and quantitative adductomics. We developed an HRMS-based method for the simultaneous analysis of RNA and DNA adducts in a single run and demonstrated its application using Baltic amphipods, useful sentinels of environmental disturbances, as test organisms. The novelty of this method is screening for RNA and DNA adducts by a single injection on an Orbitrap HRMS instrument using full scan and data-independent acquisition. The MS raw files were processed with an open-source program, nLossFinder, to identify and distinguish RNA and DNA adducts based on the characteristic neutral loss of ribonucleosides and 2'-deoxyribonucleosides, respectively. In the amphipods, in addition to the nearly 150 putative DNA adducts characterized earlier, we detected 60 putative RNA adducts. For the structural identification of the detected RNA adducts, the MODOMICS database was used. The identified RNA adducts included simple mono- and dimethylation and other larger functional groups on different ribonucleosides and deaminated product inosine. However, 54 of these RNA adducts are not yet structurally identified, and further work on their characterization may uncover new layers of information related to the transcriptome and help understand their biological significance. Considering the susceptibility of nucleic acids to environmental factors, including pollutants, the developed multi-adductomics methodology with further advancement has the potential to provide biomarkers for diagnostics of pollution effects in biota.

DNA Adductomics for the Biological Effect Assessment of Contaminant Exposure in Marine Sediments.

Exposure to chemical pollution can induce genetic and epigenetic alterations, developmental changes, and reproductive disorders, leading to population declines in polluted environments. These effects are triggered by chemical modifications of DNA nucleobases (DNA adducts) and epigenetic dysregulation. However, linking DNA adducts to the pollution load in situ remains challenging, and the lack of evidence-based DNA adductome response to pollution hampers the development and application of DNA adducts as biomarkers for environmental health assessment. Here, we provide the first evidence for pollution effects on the DNA modifications in wild populations of Baltic sentinel species, the amphipod Monoporeia affinis. A workflow based on high-resolution mass spectrometry to screen and characterize genomic DNA modifications was developed, and its applicability was demonstrated by profiling DNA modifications in the amphipods collected in areas with varying pollution loads. Then, the correlations between adducts and the contaminants level (polycyclic aromatic hydrocarbons (PAHs), trace metals, and pollution indices) in the sediments at the collection sites were evaluated. A total of 119 putative adducts were detected, and some (5-me-dC, N6-me-dA, 8-oxo-dG, and dI) were structurally characterized. The DNA adductome profiles, including epigenetic modifications, differed between the animals collected in areas with high and low contaminant levels. Furthermore, the correlations between the adducts and PAHs were similar across the congeners, indicating possible additive effects. Also, high-mass adducts had significantly more positive correlations with PAHs than low-mass adducts. By contrast, correlations between the DNA adducts and trace metals were stronger and more variable than for PAHs, indicating metal-specific effects. These associations between DNA adducts and environmental contaminants provide a new venue for characterizing genome-wide exposure effects in wild populations and apply DNA modifications in the effect-based assessment of chemical pollution.

nLossFinder-A Graphical User Interface Program for the Nontargeted Detection of DNA Adducts.

DNA adductomics is a relatively new omics approach aiming to measure known and unknown DNA modifications, called DNA adducts. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become the most common method for analyzing DNA adducts. Recent advances in the field of mass spectrometry have allowed the possibility to perform a comprehensive analysis of adducts, for instance, by using a nontargeted data-independent acquisition method, with multiple precursor m/z windows as an inclusion list. However, the generated data are large and complex, and there is a need to develop algorithms to simplify and automate the time-consuming manual analysis that has hitherto been used. Here, a graphical user interface (GUI) program was developed, with the purpose of tracking a characteristic neutral loss reaction from tandem mass spectrometry of the nucleoside adducts. This program, called nLossFinder, was developed in the MATLAB platform, available as open-source code. Calf thymus DNA was used as a model for method optimization, and the overall adductomics approach was applied to DNA from amphipods (Monoporeia affinis) collected within the Swedish National Marine Monitoring Program. In the amphipod DNA, over 150 putative adducts were found in comparison to 18 using a manual approach in a previous study. The developed program can improve the processing time for large MS data, as it processes each sample in a few seconds, and hence can be applicable for high-throughput screening of adducts.

DNA epigenetic marks are linked to embryo aberrations in amphipods.

Linking exposure to environmental stress factors with diseases is crucial for proposing preventive and regulatory actions. Upon exposure to anthropogenic chemicals, covalent modifications on the genome can drive developmental and reproductive disorders in wild populations, with subsequent effects on the population persistence. Hence, screening of chemical modifications on DNA can be used to provide information on the probability of such disorders in populations of concern. Using a high-resolution mass spectrometry methodology, we identified DNA nucleoside adducts in gravid females of the Baltic amphipods Monoporeia affinis, and linked the adduct profiles to the frequency of embryo malformations in the broods. Twenty-three putative nucleoside adducts were detected in the females and their embryos, and eight modifications were structurally identified using high-resolution accurate mass data. To identify which adducts were significantly associated with embryo malformations, partial least squares regression (PLSR) modelling was applied. The PLSR model yielded three adducts as the key predictors: methylation at two different positions of the DNA (5-methyl-2'-deoxycytidine and N6-methyl-2'-deoxyadenosine) representing epigenetic marks, and a structurally unidentified nucleoside adduct. These adducts predicted the elevated frequency of the malformations with a high classification accuracy (84%). To the best of our knowledge, this is the first application of DNA adductomics for identification of contaminant-induced malformations in field-collected animals. The method can be adapted for a broad range of species and evolve as a new omics tool in environmental health assessment.