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.

Elucidation of chromatographic peak shifts in complex samples using a chemometrical approach.

Chromatographic retention time peak shifts between consecutive analyses is a well-known fact yet not fully understood. Algorithms have been developed to align peaks between runs, but with no specific studies considering the causes of peak shifts. Here, designed experiments reveal chromatographic shift patterns for a complex peptide mixture that are attributable to the temperature and pH of the mobile phase. These results demonstrate that peak shifts are highly structured and are to a high degree explained by underlying differences in physico-chemical parameters of the chromatographic system and also provide experimental support for the alignment algorithm called the generalized fuzzy Hough transform which exploits this fact. It can be expected that the development of alignment algorithms enters a new phase resulting in increasingly accurate alignment by considering the latent structure of the peak shifts.

Adductomic Screening of Hemoglobin Adducts and Monitoring of Micronuclei in School-Age Children.

Electrophilic compounds/metabolites present in humans, originating from endogenous processes or exogenous exposure, pose a risk to health effects through their reactions with nucleophilic sites in proteins and DNA, forming adducts. Adductomic approaches are developed to screen for adducts to biomacromolecules in vivo by mass spectrometry (MS), with the aim to detect adducts corresponding to unknown exposures from electrophiles. In the present study, adductomic screening was performed using blood samples from healthy children about 12 years old (n = 51). The frequencies of micronuclei (MN) in erythrocytes in peripheral blood were monitored as a measure of genotoxic effect/genotoxic exposure. The applied adductomic approach has been reported earlier by us and is based on analysis of N-terminal valine adducts in hemoglobin (Hb) by liquid chromatography tandem mass spectrometry (LC-MS/MS). High resolution MS was introduced for refined screening of previously unknown N-terminal Hb adducts. Measured adduct levels were compared with MN frequencies using multivariate data analysis. In the 51 individuals, a total of 24 adducts (whereof 12 were previously identified) were observed and their levels quantified. Relatively large interindividual variations in adduct levels were observed. The data analysis (with partial least-squares regression) showed that as much as 60% of the MN variation could be explained by the adduct levels. This study, for the first time, applies the combination of these sensitive methods to measure the internal dose of potentially genotoxic chemicals and genotoxic effects, respectively. The results indicate that this is a valuable approach for the characterization of exposure to chemical risk factors for the genotoxic effects present in individuals of the general population.

Selective iteratively reweighted quantile regression for baseline correction.

Extraction of qualitative and quantitative information from large numbers of analytical signals is difficult with drifted baselines, particularly in multivariate analysis. Baseline drift obscures and "fuzzies" signals, and even deteriorates analytical results. In order to obtain accurate and clear results, some effective methods should be proposed and implemented to perform baseline correction before conducting further data analysis. However, most of the classic methods require user intervention or are prone to variability, especially with low signal-to-noise signals. In this study, a novel baseline correction algorithm based on quantile regression and iteratively reweighting strategy is proposed. This does not require user intervention and prior information, such as peak detection. The iteratively reweighting strategy iteratively changes weights of residuals between fitted baseline and original signals. After a series of tests and comparisons with several other popular methods, using various kinds of analytical signals, the proposed method is found to be fast, flexible, robust, and easy to use both in simulated and real datasets.

Ovarian intratumoral 21-hydroxylase deficiency in a postmenopausal hirsute woman / Deficiência de 21-hidroxilase ovariana intratumoral na virilização da mulher pós-menopausa

Virilising ovarian tumours are a rare cause of hyperandrogenism in women, accounting for less than 5% of all ovarian neoplasms. It occurs most often in - and postmenopausal women. We report a case of a 64 year-old woman with signs of virilisation that had started 3 years before. Blood hormone analysis revealed increased levels of testosterone, and 17-hydroxyprogesterone. The tetracosactin test revealed 21-hydroxylase deficiency. Radiological imaging demonstrated a nodule in her left ovary. The patient was submitted to bilateral laparoscopic oophorectomy, and histopathological examination revealed a luteoma of the left ovary. Postoperative serum testosterone level and 17-hydroxyprogesterone returned to normal levels in one month. Virilism regressed within six months. Our patient also showed an elevation in 17-OHP serum levels. Normalization of 17-OHP after oophorectomy suggests a case of intratumoral 21-hydroxylase deficiency. To our knowledge, this is the first description of ovarian intratumoral 21-hydroxylase deficiency in a postmenopausal woman. Arq Bras Endocrinol Metab. 2012;56(9):672-6.

Tumores ovarianos virilizantes são uma causa rara de hiperandrogenismo em mulheres, contabilizando menos de 5% de todos as neoplasias ovarianas. Esses tumores ocorrem mais comumente em mulheres em peri ou pós-menopausa. Relatamos aqui o caso de uma mulher de 64 anos de idade com sintomas de virilização que começaram 3 anos antes. O perfil hormonal revelou níveis aumentados de testosterona e de 17-hidroxiprogesterona (17-OHP). O teste de tetracosactin demonstrou deficiência de 21-hidroxilase. Exames radiológicos mostraram um nódulo no ovário esquerdo. A paciente foi submetida à ooforectomia laparoscópica bilateral e o exame histopatológico revelou um luteoma no ovário esquerdo. A concentração sérica de testosterona e de 17-hidroxiprogesterona após a cirurgia retornou aos níveis normais em um mês. A virilização regrediu em 6 meses. Nossa paciente também revelou uma elevação dos níveis séricos de 17-OHP. A normalização da 17-OHP após a ooforectomia sugere um caso de deficiência de 21-hidroxilase intratumoral. Esta é a primeira descrição de deficiência de 21-hidroxilase intratumoral em uma mulher na pós-menopausa. Arq Bras Endocrinol Metab. 2012;56(9):672-6.

Ovarian intratumoral 21-hydroxylase deficiency in a postmenopausal hirsute woman.

Virilising ovarian tumours are a rare cause of hyperandrogenism in women, accounting for less than 5% of all ovarian neoplasms. It occurs most often in - and postmenopausal women. We report a case of a 64 year-old woman with signs of virilisation that had started 3 years before. Blood hormone analysis revealed increased levels of testosterone, and 17-hydroxyprogesterone. The tetracosactin test revealed 21-hydroxylase deficiency. Radiological imaging demonstrated a nodule in her left ovary. The patient was submitted to bilateral laparoscopic oophorectomy, and histopathological examination revealed a luteoma of the left ovary. Postoperative serum testosterone level and 17-hydroxyprogesterone returned to normal levels in one month. Virilism regressed within six months. Our patient also showed an elevation in 17-OHP serum levels. Normalization of 17-OHP after oophorectomy suggests a case of intratumoral 21-hydroxylase deficiency. To our knowledge, this is the first description of ovarian intratumoral 21-hydroxylase deficiency in a postmenopausal woman.