Quantification of epoxide metabolite specific n-terminal globin adducts: a biomarker of internal dosimetry of 1,3-butadiene exposure and metabolism

Butadiene (BD) carcinogenicity in rodents shows gender, species and concentration dependency, making the extrapolation of animal results to humans complex. BD is a multispecies multisite carcinogen, with mice being a much more sensitive species than rats. This is considered to be related to the metabolism of BD to its epoxy metabolites, 1,2-epoxy-3-butene (EB), 1,2;3,4-diepoxybutane (DEB) and 1,2-epoxy-butanediol (EB-diol). The mutagenic potency of individual epoxides varies up to 200-fold, with DEB being the most mutagenic metabolite. For accurate risk assessment it is important to elucidate species differences in the internal formation of the individual epoxides in order to assign the relative risks associated with their different mutagenic potencies. N-terminal globin adducts have been widely used for measurements of the formation of BD derived epoxides. In this study, the formation of each epoxide was evaluated in globin samples from both genders of mice and rats exposed to BD by inhalation for 10 days. The numbers of globin adducts were then converted into EB dose equivalents. These were calculated on the basis of the combined internal dose (globin adducts) and the relative genotoxic potency of the respective epoxides inferred from the efficiency of inducing mutations at the Hprt locus. Then, the multiplicative cancer risk model was applied to quantitatively estimate tumor incidence by using the EB dose equivalent and long-term cancer bioassay data. Based on the EB equivalent, higher exposures formed lower amounts per ppm BD. This indicates that metabolism of BD to epoxides is most effective at low exposures. The EB equivalent for mice was about 40-fold higher than that of rats at similar exposures. No gender differences were noted in globin adducts of mice or rats at all exposures. As such, EB dose equivalents provide quantitative data on biomarkers of exposure that can be extended to a scientific basis for BD risk assessment

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

Studies on the repair and conformation of DNA containing O6-alkylguanine and O4-alkylthymine

The carcinogenic N-nitroso compounds alkylate DNA. Among the different products of alkylation, O6-alkylguanine and O4-alkylthymine have attracted most of the attention since they are highly mutagenic and a correlation exists between their formation and persistence, and oncogenesis in animal model systems. Both DNA adducts in E. coli and at least O6-alkylguanine in mammalian cells are repaired by enzymes, the so-called O6-alkylguanine-DNA-alkyltransferases. Based on the HPLC separation of short, self-complementary oligonucleotides containing O6-methylguanine, O6-ethylguanine, or O4-methylthymine from the respective parent non-alkylated oligomers, the rate constants for their repair by the E. coli ada and ogt and the human alkyltransferases were determined. Although all alkyltransferases were able to repair O6-methylguanine, O6-ethylguanine and O4-methylthymine, the relative efficiencies were found to differ significantly. Using an immunoprecipitation assay, the rates of repair of an O6-methylguanine residue in various positions in 15 base-pair DNA duplexes were measured. The sequence of the oligomers was that of the rat H-ras sequence around codon 12 and the rates of repair were found to vary up to 25-fold depending on the sequence flanking the methylguanine. An O6-methylguanine in the second position of the GGA codon 12 was the least well repaired. The combination of this slow repair and sequence selectivity in alkylation appears to be the explanation of the selective mutation of this position observed in rat mammary tumours. The avidity constants between antibody and O6-methylguanine were also dependent on the sequence flanking the adduct, with the most rapidly repaired being those most easily bound to the antibody. It is suggested that the rate of repair is a reflection of the conformation of the oligomers containing O6-methylguanine. An unusual feature of DNA which is often associated with protein-DNA interactions is DNA curvature. A characteristic of curved DNA is that it has less electrophoretic mobility than normal DNA. In order to assess if alkylated adducts in DNA induce DNA curvature or flexibility, DNA duplexes containing O4-alkylthymine or O6-methylguanine were synthesized and self-ligated to form multimers with the alkylated bases out of phase (16 base-pairs apart) or in phase (21 base-pairs apart) with the helical repeat of DNA. All the sequences containing O4-alklylthymine migrated more slowly than expected in a non-denaturing polyacrylamide gel. In general the effect was seen when the alkylated base was out of phase or in phase with the helical repeat suggesting that the altered base-pair confers flexibility which is largely isotropic, i.e has no preferred direction, rather than anisotropic flexibility or bending. The effect of O4-methylthymine in the mobility of the oligonucleotides was much greater than that of O6-methylguanine and the effect of O4-ethylthymine slightly greater than that of O4-methylthymine. DNA duplexes containing O4-alkylT:A base-pairs were more retarded, and had lower thermal point (Tm) than DNA duplexes containing O4-alkylT:G base-pairs

Immunological and mass spectrometry-based approaches to determine thresholds of the mutagenic DNA adduct O 6 -methylguanine in vivo

© 2018, Springer-Verlag GmbH Germany, part of Springer Nature. N-nitroso compounds are alkylating agents, which are widespread in our diet and the environment. They induce DNA alkylation adducts such as O 6 -methylguanine (O 6 -MeG), which is repaired by O 6 -methylguanine-DNA methyltransferase (MGMT). Persistent O 6 -MeG lesions have detrimental biological consequences like mutagenicity and cytotoxicity. Due to its pivotal role in the etiology of cancer and in cytotoxic cancer therapy, it is important to detect and quantify O 6 -MeG in biological specimens in a sensitive and accurate manner. Here, we used immunological approaches and established an ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) to monitor O 6 -MeG adducts. First, colorectal cancer (CRC) cells were treated with the methylating anticancer drug temozolomide (TMZ). Immunofluorescence microscopy and an immuno-slot blot assay, both based on an adduct-specific antibody, allowed for the semi-quantitative, dose-dependent assessment of O 6 -MeG in CRC cells. Using the highly sensitive and specific UPLC–MS/MS, TMZ-induced O 6 -MeG adducts were quantified in CRC cells and even in peripheral blood mononuclear cells exposed to clinically relevant TMZ doses. Furthermore, all methodologies were used to detect O 6 -MeG in wildtype (WT) and MGMT-deficient mice challenged with the carcinogen azoxymethane. UPLC–MS/MS measurements and dose–response modeling revealed a non-linear formation of hepatic and colonic O 6 -MeG adducts in WT, whereas linear O 6 -MeG formation without a threshold was observed in MGMT-deficient mice. Collectively, the UPLC–MS/MS analysis is highly sensitive and specific for O 6 -MeG, thereby allowing for the first time for the determination of a genotoxic threshold upon exposure to O 6 -methylating agents. We envision that this method will be instrumental to monitor the efficacy of methylating chemotherapy and to assess dietary exposures

Adduct-specific monoclonal antibodies for the measurement of cisplatin-induced DNA lesions in individual cell nuclei

The anticancer drug cisplatin executes its cytotoxic activity via formation of intra- and interstrand crosslinks in DNA. The relative contribution of structurally defined cisplatin adducts to induce apoptosis and the cellular processing of these lesions is still poorly understood mostly due to the lack of sensitive analytical tools for in vivo studies. Here we describe a new method to establish and characterize monoclonal antibodies (Mab) for structurally defined DNA adducts. The two major reaction products of cisplatin, the guanine–guanine (Pt-[GG]) and adenine–guanine (Pt-[AG]) intrastrand crosslinks are recognized by Mab R-C18 and R-B3, respectively. Both antibodies were employed in an immuno-cytological assay allowing the quantification of drug-induced lesions in individual cell nuclei at clinically relevant doses. Analyzing various tissues of cisplatin-treated C57Bl/6 mice the accumulation of Pt-(GG) was highest in kidney tubular cells compared with 30, 50 and 90% lower levels in kidney stroma, liver and peripheral blood cells, respectively. Adduct kinetics revealed that wild type mouse cells remove up to 80% of the crosslinks in contrast to their complete persistence in nucleotide excision repair-deficient (XPC(−/−)) mice. The aptitude of the immunoassay for human molecular dosimetry studies was demonstrated by measuring adduct levels in tumor biopsies from patients treated with cisplatin