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

Reversible Aggregation of DNA-Decorated Gold Nanoparticles Controlled by Molecular Recognition

The programmable assembly of functional nanomaterials has been extensively addressed; however, their selective reversible assembly in response to an external stimulus has been more difficult to realize. The specificity and programmable interactions of DNA have been exploited for the rational self-assembly of DNA-conjugated nanoparticles, and here we demonstrate the sequence-controlled disaggregation of DNA-modified gold nanoparticles simply by employing two complementary oligonucleotides. Target oligonucleotides with perfectly matching sequence enabled dissociation of aggregated nanoparticles, whereas oligonucleotides differing by one nucleotide did not cause disassembly of the aggregated nanoparticles. Physical aspects of this process were characterized by UV–vis absorption, light scattering, and transmission electron microscopy. This strategy for programmed disassembly of gold nanoparticles in response to biological stimuli demonstrates a fundamentally important concept anticipated to be useful for diverse applications involving molecular recognition

In-Gene Quantification of <i>O</i>⁶‑Methylguanine with Elongated Nucleoside Analogues on Gold Nanoprobes

Exposure of DNA to chemicals can result in the formation of DNA adducts, a molecular initiating event in genotoxin-induced carcinogenesis. <i>O</i>⁶-Methylguanine (<i>O</i>⁶-MeG) is a highly mutagenic DNA adduct that forms in human genomic DNA upon reaction with methylating agents of dietary, environmental, or endogenous origin. In this work, we report the design and synthesis of novel non-natural nucleoside analogues 1′-β-[1-naphtho­[2,3-<i>d</i>]­imidazol-2­(3<i>H</i>)-one)]-2′-deoxy-d-ribofuranose and 1′-β-[1-naphtho­[2,3-<i>d</i>]­imidazole]-2′-deoxy-d-ribofuranose and their use for quantifying <i>O</i>⁶-MeG within mutational hotspots of the human KRAS gene. The novel nucleoside analogues were incorporated into oligonucleotides conjugated to gold nanoparticles to comprise a DNA hybridization probe system for detecting <i>O</i>⁶-MeG in a sequence-specific manner on the basis of colorimetric readout of the nanoparticles. The concept described herein is unique in utilizing new nucleoside analogues with elongated hydrophobic surfaces to successfully measure in-gene abundance of <i>O</i>⁶-MeG in mixtures with competing unmodified DNA

Quantitative Bioluminometric Method for DNA-Based Species/Varietal Identification in Food Authenticity Assessment

A method is reported for species quantification by exploiting single-nucleotide polymorphisms (SNPs). These single-base changes in DNA are particularly useful because they enable discrimination of closely related species and/or varieties. As a model, quantitative authentication studies were performed on coffee. These involved the determination of the percentage of Arabica and Robusta species based on a SNP in the chloroplastic trnL­(UAA)-trnF­(GAA) intraspacer region. Following polymerase chain reaction (PCR), the Robusta-specific and Arabica-specific fragments were subjected to 15 min extension reactions by DNA polymerase using species-specific primers carrying oligo­(dA) tags. Biotin was incorporated into the extended strands. The products were captured in streptavidin-coated microtiter wells and quantified by using oligo­(dT)-conjugated photoprotein aequorin. Aequorin was measured within 3 s via its characteristic flash-type bioluminescent reaction that was triggered by the addition of Ca²⁺. Because of the close resemblance between the two DNA fragments, during PCR one species serves as an internal standard for the other. The percentage of the total luminescence signal obtained from a certain species was linearly related to the percent content of the sample with respect to this species. The method is accurate and reproducible. The microtiter well-based assay configuration allows high sample throughput and facilitates greatly the automation

Iron phosphate nanoparticles for food fortification: Biological effects in rats and human cell lines

Nanotechnology offers new opportunities for providing health benefits in foods. Food fortification with iron phosphate nanoparticles (FePO4 NPs) is a promising new approach to reducing iron deficiency because FePO4 NPs combine high bioavailability with superior sensory performance in difficult to fortify foods. However, their safety remains largely untested. We fed rats for 90 days diets containing FePO4 NPs at doses at which iron sulfate (FeSO4), a commonly used food fortificant, has been shown to induce adverse effects. Feeding did not result in signs of toxicity, including oxidative stress, organ damage, excess iron accumulation in organs or histological changes. These safety data were corroborated by evidence that NPs were taken up by human gastrointestinal cell lines without reducing cell viability or inducing oxidative stress. Our findings suggest FePO4 NPs appear to be as safe for ingestion as FeSO4