Advancing Discovery of Polar Polycyclic Aromatic Hydrocarbons via UHPLC-HRMS

Industrielle utslipp fra offshore petroleumsvirksomhet introduserer polysykliske aromatiske hydrokarboner (PAH-er) i marine miljøer. Miljøovervåkning av petroleum-relaterte PAH-er i fisk innebærer måling av et sett av deres metabolitter, som er produkter fra avgiftningsprosesser. Til tross for at PAH-er er velkjente skadelige forurensninger, er det fulle omfanget av PAH-metabolitter og deres assosierte mutagene og kreftfremkallende versjoner bundet til DNA (DNA addukter) uklart. Utviklingen innen væskekromatografi (LC) og elektrosprayionisering kombinert med tandem massespektrometri (MS/MS), og den mer avanserte ion mobility quadrupole time-of-flight MS (IMS-QTOF MS), gir nå nye muligheter for separasjon, deteksjon og identifikasjon av et bredere spekter av polare forbindelser i biologiske prøver. Denne doktorgradsforskningen siktet på å bruke ultra-high performance LC (UHPLC), koplet til IMS-QTOF MS for kvalitativ screening for å (i) forbedre forståelsen av PAH-avgiftning i fisk og (ii) identifisere PAH-DNA addukter. I tillegg forsøkte forskningen å undersøke kvantitativ analyse av PAH-metabolitter ved bruk av LC-MS/MS. Det presenterte arbeidet er basert på galle- og DNA ekstrakter fra kontrollerte forsøk med eksponering for enkeltkomponenter på atlantisk hyse (Melanogrammus aeglefinus). Den omfattende screeningsmetoden integrerte funksjonsprioritering basert på fragmenteringsmønstre, mistenktlister og in silico verktøy i egendefinerte arbeidsflyter som muliggjorde påvisningen av et bredt spekter av PAH-metabolitter. Dette resulterte i oppdagelsen av både kjente og ukjente metabolske veier, samt utviklingen av et omfattende internt bibliotek av massespektra, med hydroksy-, dihydrodiol-, karboksylsyre-, glukuronid-, sulfat-, glutation- og merkaptursyre-avledede metabolitter. Foreløpige LC-MS/MS resultater antyder betydelig potensial for metabolittkvantifisering i overvåkning av PAH-er i fisk. DNA addukter ble ikke identifisert til tross for omfattende innsats, men optimismen for adductomics vedvarer. Dette doktorgradsarbeidet presenterer den mest detaljerte oversikten over PAH-metabolitter i fisk som er tilgjengelig. Implementeringen av nye metodikker i dette arbeidet signaliserer et behov for overgang fra gammel til ny overvåkingspraksis, da disse metodikkene i betydelig grad øker forståelsen av PAH-avgiftning i fisk.Industrial offshore petroleum discharges introduce polycyclic aromatic hydrocarbons (PAHs) into marine environments. Environmental monitoring of petroleum-related PAHs in fish involves measuring a set of their metabolites, which are products formed by detoxification processes. Although PAHs are well-known harmful contaminants, the full scope of PAH metabolites and their associated mutagenic and carcinogenic species coupled to DNA (DNA adducts) remains unclear. Developments in liquid chromatography (LC) and electrospray ionization coupled with tandem mass spectrometry (MS/MS), and the more advanced ion mobility quadrupole time-of-flight MS (IMS-QTOF MS), now offer new opportunities for the separation, detection and identification of a wider array of polar compounds in biological samples. This PhD research aimed to use ultra-high performance LC (UHPLC) coupled with IMS-QTOF MS for qualitative screening to (i) enhance the understanding of PAH detoxification in fish and (ii) identify PAH-DNA adducts. Additionally, the research sought to investigate the quantitative analysis of PAH metabolites using LC-MS/MS. The presented work is based on bile- and DNA extracts from controlled single-component exposure experiments on Atlantic haddock (Melanogrammus aeglefinus). The comprehensive screening approach integrated feature prioritization based on fragmentation patterns, suspect lists, and in silico tools in custom workflows, enabling the detection of a diverse range of PAH metabolites. This resulted in the discovery of both known and unknown metabolic pathways and the development of an extensive in-house mass spectral library, featuring hydroxy-, dihydrodiol-, carboxylic acid-, glucuronide-, sulfate-, glutathione-, and mercapturic acid-derived metabolites. Preliminary LC-MS/MS results suggest significant potential for metabolite quantification in the monitoring of PAHs in fish. DNA adducts were not identified despite extensive efforts, but optimism for adductomics persists. This PhD presents the most detailed overview of PAH metabolites in fish available. The implementation of new methodologies in this work signals a need to transition from old to new monitoring practices, as these methodologies significantly enhance the understanding of PAH detoxification in fish.Doktorgradsavhandlin

Spatial Mapping of the DNA Adducts in Cancer

DNA adducts and strand breaks are induced by various exogenous and endogenous agents. Accumulation of DNA damage is implicated in many disease processes, including cancer, aging, and neurodegeneration. The continuous acquisition of DNA damage from exogenous and endogenous stressors coupled with defects in DNA repair pathways contribute to the accumulation of DNA damage within the genome and genomic instability. While mutational burden offers some insight into the level of DNA damage a cell may have experienced and subsequently repaired, it does not quantify DNA adducts and strand breaks. Mutational burden also infers the identity of the DNA damage. With advances in DNA adduct detection and quantification methods, there is an opportunity to identify DNA adducts driving mutagenesis and correlate with a known exposome. However, most DNA adduct detection methods require isolation or separation of the DNA and its adducts from the context of the nuclei. Mass spectrometry, comet assays, and other techniques precisely quantify lesion types but lose the nuclear context and even tissue context of the DNA damage. The growth in spatial analysis technologies offers a novel opportunity to leverage DNA damage detection with nuclear and tissue context. However, we lack a wealth of techniques capable of detecting DNA damage in situ. Here, we review the limited existing in situ DNA damage detection methods and examine their potential to offer spatial analysis of DNA adducts in tumors or other tissues. We also offer a perspective on the need for spatial analysis of DNA damage in situ and highlight Repair Assisted Damage Detection (RADD) as an in situ DNA adduct technique with the potential to integrate with spatial analysis and the challenges to be addressed

Defining the Scope of Exposome Studies and Research Needs from a Multidisciplinary Perspective

The concept of the exposome was introduced over 15 years ago to reflect the important role that the environment exerts on health and disease. While originally viewed as a call-to-arms to develop more comprehensive exposure assessment methods applicable at the individual level and throughout the life course, the scope of the exposome has now expanded to include the associated biological response. In order to explore these concepts, a workshop was hosted by the Gunma University Initiative for Advanced Research (GIAR, Japan) to discuss the scope of exposomics from an international and multidisciplinary perspective. This Global Perspective is a summary of the discussions with emphasis on (1) top-down, bottom-up, and functional approaches to exposomics, (2) the need for integration and standardization of LC- and GC-based high-resolution mass spectrometry methods for untargeted exposome analyses, (3) the design of an exposomics study, (4) the requirement for open science workflows including mass spectral libraries and public databases, (5) the necessity for large investments in mass spectrometry infrastructure in order to sequence the exposome, and (6) the role of the exposome in precision medicine and nutrition to create personalized environmental exposure profiles. Recommendations are made on key issues to encourage continued advancement and cooperation in exposomics

Applications of Fourier Transform Ion Cyclotron Resonance (FT-ICR) and Orbitrap Based High Resolution Mass Spectrometry in Metabolomics and Lipidomics

This review explores the latest developments in Fourier transform mass spectrometry and Orbitrap based metabolomics technology, its advantages and drawbacks for using in metabolomics and lipidomics studies, and development of novel approaches for processing high resolution mass spectrometry data

Bacterial Response to Nanoparticles at the Molecular Level

University of Minnesota Ph.D. dissertation. May 2018. Major: Chemistry. Advisor: Christy Haynes. 1 computer file (PDF); xiv, 222 pages.Nanotechnology has been an emerging field due to the promising properties of engineered nanomaterials, materials with at least one dimension less than 100 nanometers. With increasing application of NPs, the risk of these novel materials to environment requires thorough investigation to prevent negative impacts. NPs have enormous variety due to combinations of chemical compositions, sizes, shapes, structures and surface modifications. Building predictive models that link NP properties to biological outcomes is the key to proactive safer NP design. High-throughput toxicity screening and investigating toxicity mechanisms are the common two strategies building towards predictive models of nanotoxicity. These two strategies work together: high-throughput assays facilitate preliminary screening of potentially toxic materials for further mechanistic studies to discover biomarkers and molecular pathways of interest, which will in turn be validated on multiple materials and organisms with high-throughput screening. My thesis work combines both strategies to develop high-throughput screening assays and mechanistic understanding at different molecular levels of how an environmental bacterium, Shewanella oneidensis MR-1, responds to various NP exposures. In this work, Chapter 1 reviews recent advances in analytical nanotoxicology and identifies four key areas that would further bring the field to its maturity. Chapter 2 represents a comprehensive mechanistic study on bacteria responding to TiO2 NPs with UVA illumination. Chapter 3 uses gene expression to explore molecular response among two organisms at different trophic levels to positively and negatively charged gold NPs. Chapter 4 identifies that purification method can be one neglected source of apparent NP toxicity. A high-throughput bacterial viability assay that is free of NP interference is presented in Chapter 5. Finally, in Chapter 6, DNA damage is revealed as a toxicity mechanism for nanoscale complex metal oxide nanomaterials to bacteria

Defining the Scope of Exposome Studies and Research Needs from a Multidisciplinary Perspective

The concept of the exposome was introduced over 15 years ago to reflect the important role that the environment exerts on health and disease. While originally viewed as a call-to-arms to develop more comprehensive exposure assessment methods applicable at the individual level and throughout the life course, the scope of the exposome has now expanded to include the associated biological response. In order to explore these concepts, a workshop was hosted by the Gunma University Initiative for Advanced Research (GIAR, Japan) to discuss the scope of exposomics from an international and multidisciplinary perspective. This Global Perspective is a summary of the discussions with emphasis on (1) top-down, bottom-up, and functional approaches to exposomics, (2) the need for integration and standardization of LC- and GC-based high-resolution mass spectrometry methods for untargeted exposome analyses, (3) the design of an exposomics study, (4) the requirement for open science workflows including mass spectral libraries and public databases, (5) the necessity for large investments in mass spectrometry infrastructure in order to sequence the exposome, and (6) the role of the exposome in precision medicine and nutrition to create personalized environmental exposure profiles. Recommendations are made on key issues to encourage continued advancement and cooperation in exposomics

The effect of charge location in ion mobility mass spectrometry for small molecule analytes

In travelling wave IM-MS an electric field is applied across the IMS cell and analyte collisions with the inert gas give rise to a relationship between the drift time and the molecular shape and overall charge. Recent findings show near baseline IMS separation (R>1) where shape and overall charge appear not to explain the IMS separation. Multiple ion mobility peaks are observed for a single m/z including the fluoroquinolone antibiotics norfloxacin and lomefloxacin, the pesticide indoxacarb and a number of steroids

High throughput exposomic studies for new insights into smoke exposures in occupational and population health

Exposomics aims to characterize the totality of exposures over the lifespan, and their impact on human health. Currently, chronic exposure to harmful chemicals from air pollution and/or tobacco smoke, along with a suboptimal diet, remain leading causes for preventable mortality and morbidity worldwide. As a result, new analytical methods are needed to measure robust biomarkers of smoke exposure and food intake for improved risk assessment of clinical events. This thesis aims to develop high throughput methods to rapidly quantify urinary biomarkers of environmental smoke in high-risk occupations, and diverse global populations using multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS) technology. Chapter II outlines an inter-laboratory method comparison for the targeted analysis of urinary 1-hydroxypyrene (HP) when using gas chromatography-high resolution mass spectrometry (GC-HRMS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) on urine samples collected from firefighters. This work revealed the critical role of incomplete enzymatic deconjugation on method bias and underreporting of true smoke exposures. Chapter III introduces a high throughput MSI-CE-MS/MS method (< 3 min/sample) to directly analyze the intact glucuronide conjugate of HP (HP-G) in urine without complex pre-column enzyme deconjugation and derivatization procedures. Importantly, firefighters deployed under emergency conditions at the 2016 Fort McMurray wildfire had creatinine normalized HP-G concentrations below the biological exposure index, likely caused by delays in urine collection under emergency conditions, at early stages of firefighting. Chapter IV extends from targeted biomonitoring of occupational smoke exposure, towards elucidating the relative risk of tobacco smoking in an international cohort of participants (n=1000) from the Prospective Urban and Rural Epidemiological (PURE) study. Comprehensive analysis of nicotine metabolites in urine by MSI-CE-MS allowed for reliable determination of the total nicotine equivalent and nicotine metabolic ratio as robust indicators of recent tobacco smoke exposure and nicotine dependence, respectively. This method also offers a more accurate approach for biochemical verification of smoking status in large-scale epidemiological studies that are prone to social desirability and gender bias when relying on standardized questionnaires. Lastly, Chapter V employs a nontargeted metabolomics workflow using MSI-CE-MS to identify urinary metabolites that may serve as objective dietary biomarkers of food intake in participants across 14 countries from the PURE cohort. A panel of robust and generalizable metabolites were validated for biomonitoring of complex dietary exposures, that may further exacerbate the hazards of tobacco smoking. In summary, this thesis contributes high throughput analytical tools for characterizing the human urine exposome to better decipher the roles of smoke exposure, and suboptimal diet on chronic disease burden among diverse populations and regions worldwide.ThesisDoctor of Philosophy (PhD