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8‐oxo‐7,8‐dihydroguanine, commonly referred to as 8‐oxoG, is considered one of the most predominant oxidative lesions formed in DNA. Due to its ability to pair with adenines in its syn configuration, this lesion has a strong mutagenic potential in both eukaryotes and prokaryotes. Escherichia coli cells are endowed with the GO system, which protects them from the mutagenic properties of this lesion when formed both in cellular DNA and the nucleotide pool. MutY and MutM (Fpg) DNA glycosylases are crucial components of the GO system. A strong mutator phenotype of the Escherichia coli mutM mutY double mutant underscores the importance of 8‐oxoG repair for genomic stability. Here, we report that in Caulobacter crescentus, a widely studied alpha‐proteobacterium with a GC‐rich genome, the combined lack of MutM and MutY glycosylases produces a more modest mutator phenotype when compared to E. coli. Genetic analysis indicates that other glycosylases and other repair pathways do not act synergistically with the GO system for spontaneous mutation prevention. We also show that there is not a statistically significant difference in the spontaneous levels 8‐oxodGuo in E. coli and C. crescentus, suggesting that other yet to be identified differences in repair or replication probably account for the differential importance of the GO system between these two species. Environ. Mol. Mutagen. 61:246–255, 2020. © 2019 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Caloric restriction is the most effective non-genetic intervention to enhance lifespan known to date. A major research interest has been the development of therapeutic strategies capable of promoting the beneficial results of this dietary regimen. In this sense, we propose that compounds that decrease the efficiency of energy conversion, such as mitochondrial uncouplers, can be caloric restriction mimetics. Treatment of mice with low doses of the protonophore 2,4-dinitrophenol promotes enhanced tissue respiratory rates, improved serological glucose, triglyceride and insulin levels, decrease of reactive oxygen species levels and tissue DNA and protein oxidation, as well as reduced body weight. Importantly, 2,4-dinitrophenol-treated animals also presented enhanced longevity. Our results demonstrate that mild mitochondrial uncoupling is a highly effective in vivo antioxidant strategy, and describe the first therapeutic intervention capable of effectively reproducing the physiological, metabolic and lifespan effects of caloric restriction in healthy mammals. [ABSTRACT FROM AUTHOR]

The decomposition of lipid hydroperoxides (LOOH) into peroxyl radicals is a potential source of singlet molecular oxygen (1O2) in biological systems. Recently, we have clearly demonstrated the generation of 1O2 in the reaction of lipid hydroperoxides with biologically important oxidants such as metal ions, peroxynitrite and hypochlorous acid. The approach used to unequivocally demonstrate the generation of 1O2 in these reactions was the use of an isotopic labeled hydroperoxide, the 18O-labeled linoleic acid hydroperoxide, the detection of labeled compounds by HPLC coupled to tandem mass spectrometry (HPLC-MS/MS) and the direct spectroscopic detection and characterization of 1O2 light emission. Using this approach we have observed the formation of 18O-labeled 1O2 by chemical trapping of 1O2 with anthracene derivatives and detection of the corresponding labeled endoperoxide by HPLC-MS/MS. The generation of 1O2 was also demonstrated by direct spectral characterization of 1O2 monomol light emission in the near-infrared region (λ = 1270 nm). In summary, our studies demonstrated that LOOH can originate 1O2. The experimental evidences indicate that 1O2 is generated at a yield close to 10% by the Russell mechanism, where a linear tetraoxide intermediate is formed in the combination of two peroxyl radicals. In addition to LOOH, other biological hydroperoxides, including hydroperoxides formed in proteins and nucleic acids, may also participate in reactions leading to the generation 1O2. This hypothesis is currently being investigated in our laboratory. [ABSTRACT FROM AUTHOR]

Porphyrias are defined as either inborn or acquired diseases related to enzymatic deficiencies in the heme biosynthetic pathway. Lead poisoning, hereditary tyrosinemia, and acute intermittent porphyria (AIP) are characterized by the absence of photosensitivity and the accumulation of 5-aminolevulinic acid (ALA) together with its increased urinary excretion. The main clinical manifestations of AIP are intermittent attacks of abdominal pain, neuromuscular weaknesses and neuropsychiatry alterations, and also an association with primary liver cancer, in which may be involved the oxidative potential of ALA which is able to cause DNA damage. The use of antioxidants in the treatment of ALA-induced oxidative stress is not well established. In the current work, we show the antioxidant efficacy of several compounds including melatonin, quercetin, resveratrol andN1-acetyl-N2-formyl-5-methoxykynuramine (AFMK), a melatonin oxidation product, in terms of their ability to limit DNA damage induced by ALA/Fe2+ in an in vitro system. Damage was measured by plasmid DNA strand breaks and detection of 8-oxo, 7-8-dihydro,2′-deoxyguanosine (8-oxodGuo) by high-performance liquid chromatography coupled with electrochemical detection. All compounds tested showed a dose-dependent protective action against free radical damage. These results could be the first step toward studies of the possible use of these antioxidants in oxidative stress promoted by ALA or other pro-oxidants. [ABSTRACT FROM AUTHOR]

Decomposition of lipid hydroperoxides (LOOH) is known to generate toxic products capable to induce tissue injury. We have recently confirmed that decomposition of LOOH into peroxyl radicals is a potential source of singlet oxygen (^1O_2) in biological system. Using ^{18}O-labeled linoleic acid hydroperoxide (LA^{18}O^{18}OH) in the presence of Ce^{4+} or Fe^{2+}, we observed the formation of ^{18}O-labeled ^1O_2 (^{18}[^1O_2]) by chemical trapping of ^1O_2 with 9,10-diphenylanthracene (DPA) and detecting the corresponding ^{18}O-labeled DPA endoperoxide (DPA^{18}O^{18}O) by HPLC coupled to tandem mass spectrometry (HPLC-MS/MS). ^{18}O-Labeled alcohol and ketone were also detected providing further evidence for the generation of ^1O_2by the Russell mechanism. Similarly the reaction of LA^{18}O^{18}OH with peroxynitrite also generated^{18}[^1O_2].In conclusion, these results indicates that the use of ^{18}O-labeled LOOH associated with HPLC-MS/MS can be an useful tool to clarify mechanistic features involved in the reaction of LOOH in biological media. [ABSTRACT FROM AUTHOR]

Melatonin ( N-acetyl-5-methoxytryptamine) is implicated in physiologic changes related to light–dark cycles and has been recently found to display antioxidant properties. It is known that the reaction of melatonin with certain reactive oxygen and nitrogen species, such as hydrogen peroxide and singlet oxygen, produces N1-acetyl- N2-formyl-5-methoxykynuramine (AFMK). We report herein on the development of a new liquid chromatography/tandem mass spectrometry (LC/ESI/MS-MS) assay to quantitatively determine melatonin and AFMK. The stable isotopic internal standard of melatonin-D3 was synthesized by the reaction of 5-methoxytryptamine with deuterated acetyl chloride (CD3COCl). Labeled AFMK (AFMK-D3) was obtained after photooxidation of melatonin-D3. The predominant ion [M + H]+ in the full scan mass spectra of melatonin, melatonin-D3, AFMK and AFMK-D3 were located, respectively, at m/ z = 233, 236, 265 and 268. The collision-induced dissociation of the molecules revealed a predominant fragment at m/ z = 174 for melatonin and melatonin-D3 (loss of the N-acetyl group), and at m/ z = 178 for AFMK and AFMK-D3 (loss of both the N-acetyl and the N-formyl groups). The m/ z transitions from 233 to 174 (melatonin), from 236 to 174 (melatonin-D3), from 265 to 178 (AFMK), and from 268 to 178 (AFMK-D3) were therefore chosen for the multiple reaction monitoring detection experiments, ensuring a high specificity and an accurate quantification of melatonin and AFMK in human plasma. [ABSTRACT FROM AUTHOR]

It has been shown that melatonin exhibits antioxidant properties. Chemical structures of some of the products formed by the interaction of melatonin with reactive oxygen and nitrogen species have been elucidated. Despite some evidence that the reaction of melatonin with singlet molecular oxygen (O2 (1 Δg )) produces N 1 -acetyl-N 2 -formyl-5-methoxykynurenine (AFMK), it has not been fully documented. In this investigation, melatonin was oxidized by photosensitization with methylene blue or by a clean chemical source of O2 (1 Δg ), the thermodecomposition of N ,N ′-di(2,3-dihydroxypropyl)-1,4-naphtalenedipropanamide (DHPNO2 ). The resulting product was characterized by high performance liquid chromatography, coupled to electrospray ionization mass spectrometry and also by 1 H, 13 C and dept135 nuclear magnetic resonance spectroscopy. An isotopically labeled DHPN18 O2 was also prepared and used as a chemical source of labeled 18 [O2 (1 Δg )] to unequivocally characterize the end product. The results uncovered by this work confirm the hypothesis that oxidation of melatonin by O2 (1 Δg ) produces AFMK. [ABSTRACT FROM AUTHOR]