No abstract

Both exogenous and endogenous agents are a threat to DNA integrity. Exogenous environmental agents such as the ultraviolet (UV), ionizing radiation, genotoxic chemicals and endogenous byproducts of metabolism including reactive oxygen species can cause alterations in DNA structure (DNA damage). Unrepaired DNA damage has been linked to a variety of human disorders including cancer and neurodegenerative disease. Thus, efficient mechanisms to detect DNA lesions, signal their presence and promote their repair have been evolved in cells. If DNA is effectively repaired, DNA damage response is inactivated and normal cell functioning resumes. In contrast, when DNA lesions cannot be removed, chronic DNA damage triggers specific cell responses such as cell death and senescence. Recently, DNA damage has been shown to induce autophagy, a cellular catabolic process that maintains a balance between synthesis, degradation, and recycling of cellular components. But the exact mechanisms by which DNA damage triggers autophagy are unclear. More importantly, the role of autophagy in the DNA damage response and cellular fate is unknown. In this review we analyze evidence that supports a role for autophagy as an integral part of the DNA damage response.

Adenoviruses with deletion of E1b have been used in clinical trials to treat cancers that are resistant to conventional therapies. The efficacy of viral replication within cancer cells determines the results of oncolytic therapy, which remains poorly understood and requires further improvement. In this report, we show that adenoviruses induce autophagy by increasing the conversion of LC3-I to LC3-II and the formation of the Atg12-Atg5 complex. Inhibition of autophagy with 3-methyladenine (3MA) resulted in a decreased synthesis of adenovirus structural proteins, and thereby a poor viral replication; promotion of autophagy with rapamycin increased adenovirus yield. This study indicates that adenovirus-induced autophagy correlates positively with virus replication and oncolytic cell death, and that autophagy may generate nutrients that can be used for building viral progeny particles. These results further suggest that chemotherapeutic agents that increase cancer cell autophagy may improve the efficacy of oncolytic virotherapy.

Parkinson’s disease (PD) is a multifactorial disorder with a complex etiology including genetic risk factors, environmental exposures, and aging. While energy failure and oxidative stress have largely been associated with the loss of dopaminergic cells in PD and the toxicity induced by mitochondrial/environmental toxins, very little is known regarding the alterations in energy metabolism associated with mitochondrial dysfunction and their causative role in cell death progression. In this study, we investigated the alterations in the energy/redox-metabolome in dopaminergic cells exposed to environmental/mitochondrial toxins (paraquat, rotenone, 1-methyl-4-phenylpyridinium [MPP+], and 6-hydroxydopamine [6-OHDA]) in order to identify common and/or different mechanisms of toxicity. A combined metabolomics approach using nuclear magnetic resonance (NMR) and direct-infusion electrospray ionization mass spectrometry (DI-ESI-MS) was used to identify unique metabolic profile changes in response to these neurotoxins. Paraquat exposure induced the most profound alterations in the pentose phosphate pathway (PPP) metabolome. 13C-glucose flux analysis corroborated that PPP metabolites such as glucose-6-phosphate, fructose-6-phosphate, glucono-1,5-lactone, and erythrose-4-phosphate were increased by paraquat treatment, which was paralleled by inhibition of glycolysis and the TCA cycle. Proteomic analysis also found an increase in the expression of glucose-6-phosphate dehydrogenase (G6PD), which supplies reducing equivalents by regenerating nicotinamide adenine dinucleotide phosphate (NADPH) levels. Overexpression of G6PD selectively increased paraquat toxicity, while its inhibition with 6-aminonicotinamide inhibited paraquat-induced oxidative stress and cell death. These results suggest that paraquat “hijacks” the PPP to increase NADPH reducing equivalents and stimulate paraquat redox cycling, oxidative stress, and cell death. Our study clearly demonstrates that alterations in energy metabolism, which are specific for distinct mitochondiral/environmental toxins, are not bystanders to energy failure but also contribute significant to cell death progression.

Metabolomics datasets are commonly acquired by either mass spectrometry (MS) or nuclear magnetic resonance spectroscopy (NMR), despite their fundamental complementarity. In fact, combining MS and NMR datasets greatly improves the coverage of the metabolome and enhances the accuracy of metabolite identification, providing a detailed and high-throughput analysis of metabolic changes due to disease, drug treatment, or a variety of other environmental stimuli. Ideally, a single metabolomics sample would be simultaneously used for both MS and NMR analyses, minimizing the potential for variability between the two datasets. This necessitates the optimization of sample preparation, data collection and data handling protocols to effectively integrate direct-infusion MS data with one-dimensional (1D) 1H NMR spectra. To achieve this goal, we report for the first time the optimization of (i) metabolomics sample preparation for dual analysis by NMR and MS, (ii) high throughput, positive-ion direct infusion electrospray ionization mass spectrometry (DI-ESI-MS) for the analysis of complex metabolite mixtures, and (iii) data handling protocols to simultaneously analyze DI-ESI-MS and 1D 1H NMR spectral data using multiblock bilinear factorizations, namely multiblock principal component analysis (MB-PCA) and multiblock partial least squares (MB-PLS). Finally, we demonstrate the combined use of backscaled loadings, accurate mass measurements and tandem MS experiments to identify metabolites significantly contributing to class separation in MB-PLS-DA scores. We show that integration of NMR and DI-ESI-MS datasets yields a substantial improvement in the analysis of neurotoxin involvement in dopaminergic cell death.