Mitochondrial catalase overexpressed transgenic mice are protected against lung fibrosis in part via preventing alveolar epithelial cell mitochondrial DNA damage

Publication date: Available online 11 November 2016
Source:Free Radical Biology and Medicine
Author(s): Seok-Jo Kim, Paul Cheresh, Renea P Jablonski, Luisa Morales-Nebreda, Yuan Cheng, Erin Hogan, Anjana Yeldandi, Monica Chi, Raul Piseaux, Karen Ridge, C. Michael Hart, Navdeep Chandel, GR Scott Budinger, David W Kamp
RationaleAlveolar epithelial cell (AEC) injury and mitochondrial dysfunction are important in the development of lung fibrosis. Our group has shown that in the asbestos exposed lung, the generation of mitochondrial reactive oxygen species (ROS) in AEC mediate mitochondrial DNA (mtDNA) damage and apoptosis which are necessary for lung fibrosis. These data suggest that mitochondrial-targeted antioxidants should ameliorate asbestos-induced lung.ObjectiveTo determine whether transgenic mice that express mitochondrial-targeted catalase (MCAT) have reduced lung fibrosis following exposure to asbestos or bleomycin and, if so, whether this occurs in association with reduced AEC mtDNA damage and apoptosis.MethodsCrocidolite asbestos (100µg/50µl), TiO2 (negative control), bleomycin (0.025 units/50µl), or PBS was instilled intratracheally in 8–10 week-old wild-type (WT - C57Bl/6J) or MCAT mice. The lungs were harvested at 21d. Lung fibrosis was quantified by collagen levels (Sircol) and lung fibrosis scores. AEC apoptosis was assessed by cleaved caspase-3 (CC-3) / Surfactant protein C (SFTPC) immunohistochemistry (IHC) and semi-quantitative analysis. AEC (primary AT2 cells from WT and MCAT mice and MLE-12 cells) mtDNA damage was assessed by a quantitative PCR-based assay, apoptosis was assessed by DNA fragmentation, and ROS production was assessed by a Mito-Sox assay.ResultsCompared to WT, crocidolite-exposed MCAT mice exhibit reduced pulmonary fibrosis as measured by lung collagen levels and lung fibrosis score. The protective effects in MCAT mice were accompanied by reduced AEC mtDNA damage and apoptosis. Similar findings were noted following bleomycin exposure. Euk-134, a mitochondrial SOD/catalase mimetic, attenuated MLE-12 cell DNA damage and apoptosis. Finally, compared to WT, asbestos-induced MCAT AT2 cell ROS production was reduced.ConclusionsOur finding that MCAT mice have reduced pulmonary fibrosis, AEC mtDNA damage and apoptosis following exposure to asbestos or bleomycin suggests an important role for AEC mitochondrial H2O2-induced mtDNA damage in promoting lung fibrosis. We reason that strategies aimed at limiting AEC mtDNA damage arising from excess mitochondrial H2O2 production may be a novel therapeutic target for mitigating pulmonary fibrosis.



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Gut microbiota modulate host immune cells in cancer development and growth

Publication date: Available online 10 November 2016
Source:Free Radical Biology and Medicine
Author(s): Susan E Erdman, Theofilos Poutahidis
Emerging evidence shows that microbe interactions with the host immune system impact diverse aspects of cancer development and treatment. As a result, exciting new opportunities exist for engineering diets and microbe cocktails to lower cancer risks with fewer adverse clinical effects than traditional strategies. Microbe-based therapies may ultimately be used to reinforce host immune balance and extinguish cancer for generations to come.



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In situ visualization of carbonylation and its co-localization with proteins, lipids, DNA and RNA in Caenorhabditis elegans

Publication date: Available online 11 November 2016
Source:Free Radical Biology and Medicine
Author(s): Mira Kuzmic, Hélène Javot, Jean-Marc Bonzom, Catherine Lecomte-Pradines, Miroslav Radman, Jacqueline Garnier-Laplace, Sandrine Frelon
All key biological macromolecules are susceptible to carbonylation – an irreparable oxidative damage with deleterious biological consequences. Carbonyls in proteins, lipids and DNA from cell extracts have been used as a biomarker of oxidative stress and aging, but formation of insoluble aggregates by carbonylated proteins precludes quantification. Since carbonylated proteins correlate with and become a suspected cause of morbidity and mortality in some organisms, there is a need for their accurate quantification and localization. Using appropriate fluorescent probes, we have developed an in situ detection of total proteins, DNA, RNA, lipids and carbonyl groups at the level of the whole organism. In C. elegans, we found that after UV irradiation carbonylation co-localizes mainly with proteins and, to a lesser degree, with DNA, RNA and lipids. The method efficiency was illustrated by carbonylation induction assessment over 5 different UV doses. The procedure enables the monitoring of carbonylation in the nematode C. elegans during stress, aging and disease along its life cycle including the egg stage.

Graphical abstract

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Interfacial Assembly at Silver Nanoparticle Enhances the Antibacterial Efficacy of Nisin

Publication date: Available online 11 November 2016
Source:Free Radical Biology and Medicine
Author(s): Manoranjan Arakha, Sapna M Borah, Mohammed Saleem, Anupam N Jha, Suman Jha
Nisin is a well-recognised antimicrobial peptide (AMP) used in food industry. However, efficacy of the peptide has been compromised due to development of resistance in different bacterial strains. Here, efficacy of the peptide upon assembly at a silver nanoparticle (AgNP) interface has been characterized. To this end, experimental and simulation studies are done to characterize the interfacial assembly of nisin and underlie antibacterial mechanism. Being an AMP, efficacy of an intact nisin is explored against Gram-positive and Gram-negative bacteria, and compared with antibacterial propensity of the interfacially assembled nisin. Antibacterial propensity, upon the assembly, increases against both kinds of bacteria. Interestingly, the growth inhibition studies of the interfacially assembled nisin indicate that the originally nisin resistant Gram-negative bacteria become sensitive to the nanomolar nisin concentrations. Furthermore, reactive oxygen species (ROS) measurements together with confocal microscopy imaging indicate that the increase in interfacial and intracellular ROS production upon the treatment is underling mechanism of enhanced antibacterial propensity of the assembled nisin. Thus, the study observed that the interfacial assembly of nisin at AgNP interface enhances the efficacy of nisin against different spectrum of bacteria, where the intact nisin is largely ineffective for the studied concentrations.

Graphical abstract

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Nitroxide free radicals protect macular carotenoids against chemical destruction (bleaching) during lipid peroxidation

Publication date: Available online 10 November 2016
Source:Free Radical Biology and Medicine
Author(s): M. Zareba, J. Widomska, J.M. Burke, W.K. Subczynski
Macular xanthophylls (MXs) lutein and zeaxanthin are dietary carotenoids that are selectively concentrated in the human eye retina, where they are thought to protect against age-related macular degeneration (AMD) by multiple mechanisms, including filtration of phototoxic blue light and quenching of singlet oxygen and triplet states of photosensitizers. These physical protective mechanisms require that MXs be in their intact structure. Here, we investigated the protection of the intact structure of zeaxanthin incorporated into model membranes subjected to oxidative modification by water- and/or membrane-soluble small nitroxide free radicals. Model membranes were formed from saturated, monounsaturated, and polyunsaturated phosphatidylcholines (PCs). Oxidative modification involved autoxidation, iron-mediated, and singlet oxygen-mediated lipid peroxidation. The extent of chemical destruction (bleaching) of zeaxanthin was evaluated from its absorption spectra and compared with the extent of lipid peroxidation evaluated using the thiobarbituric acid assay. Nitroxide free radicals with different polarity (membrane/water partition coefficients) were used. The extent of zeaxanthin bleaching increased with membrane unsaturation and correlated with the rate of PC oxidation. Protection of the intact structure of zeaxanthin by membrane-soluble nitroxides was much stronger than that by water-soluble nitroxides. The combination of zeaxanthin and lipid-soluble nitroxides exerted strong synergistic protection against singlet oxygen-induced lipid peroxidation. The synergistic effect may be explained in terms of protection of the intact zeaxanthin structure by effective scavenging of free radicals by nitroxides, therefore allowing zeaxanthin to quench the primary oxidant, singlet oxygen, effectively by the physical protective mechanism. The redox state of nitroxides was monitored using electron paramagnetic resonance spectroscopy. Both nitroxide free radicals and their reduced form, hydroxylamines, were equally effective. Obtained data were compared with the protective effects of α-tocopherol, which is the natural antioxidant and protector of MXs within the retina. The new strategies employed here to maintain the intact structure of MXs may enhance their protective potential against AMD.



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Sestrin2 inhibits hypoxia-inducible factor-1α accumulation via AMPK-mediated prolyl hydroxylase regulation

Publication date: Available online 11 November 2016
Source:Free Radical Biology and Medicine
Author(s): Kyuhwa Seo, Suho Seo, Sung Hwan Ki, Sang Mi Shin
Sestrin2 (SESN2) is an antioxidant protein that modulates cellular redox homeostasis through regeneration of peroxiredoxins. It has beneficial effects in oxidative or metabolic stress conditions as an upstream regulator of AMP-activated protein kinase (AMPK). Since hypoxia causes oxidative and metabolic stress, this study investigated the effect of SESN2 on signaling pathways altered by hypoxia in colon cancer cells. SESN2 overexpression in HEK293 cells inhibited hypoxia-inducible factor-1α (HIF-1α), which plays a crucial role in tumor growth and development in hypoxia. Moreover, infection with adenovirus-SESN2 (Ad-SESN2) decreased hypoxia or CoCl2-induced HIF-1α accumulation in colorectal cancer cells. Ad-SESN2 also reduced CoCl2-induced hypoxia response element (HRE)-luciferase activity and mRNA level of HIF-1α-driven genes. Furthermore, Ad-SESN2 infected cells showed anti-metastatic effects in serum-induced cell migration and invasion in vitro. Ad-SESN2 facilitated the ubiquitination of HIF-1α protein and increased hydroxyl-HIF-1α (OH-HIF-1α) level. In contrast, treatment with dimethyloxalylglycine (DMOG), an inhibitor of prolyl hydroxylase (PHD), reversed Ad-SESN2-induced OH-HIF-1α and subsequently suppressed HIF-1α level. The inhibitory effects of SESN2 on the serum-induced in vitro cell migration and invasion were also abrogated by DMOG treatment. Furthermore, knockdown of AMPKα reversed Ad-SESN2-mediated increase of OH-HIF-1α and inhibition of HIF-1α. Dominant-negative form of AMPK also restored the Ad-SESN2 mediated decrease in HIF-1α accumulation. Lastly, Ad-SESN2 suppressed tumor growth in a mouse xenograft model. Taken together, these results suggest that SESN2 increases degradation of HIF-1α via AMPK-PHD regulation that contributes to inhibition of in vitro and in vivo tumorigenesis.



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Connexin43 regulates high glucose-induced expression of fibronectin, ICAM-1 and TGF-β1 via Nrf2/ARE pathway in glomerular mesangial cells

Publication date: Available online 11 November 2016
Source:Free Radical Biology and Medicine
Author(s): Zhiquan Chen, Xi Xie, Junying Huang, Wenyan Gong, Xiaoyu Zhu, Qiuhong Chen, Jiani Huang, Heqing Huang
Nrf2/ARE signaling pathway is a crucial cellular defense system to cope with oxidative stress, which is adaptively activated, in diabetic condition that is not efficient enough to resist the oxidative stress provoked by hyperglycemia. We have previously demonstrated that Connexin43 (Cx43) attenuates renal fibrosis through c-Src. However, the underlying mechanisms need to be further clarified. It has been reported that Cx43 possesses the ability of anti-oxidative. The current study aimed to determine if Cx43 exerts protective effects on renal fibrosis in diabetes via activation of Nrf2/ARE pathway and explore the underlying molecular mechanisms. The following findings were observed: (1) Cx43 expression decreased and c-Src activity increased in kidneys of diabetic animals; (2) Over-expressed Cx43 in high glucose treated GMCs inhibited protein levels of FN, ICAM-1 and TGF-β1; (3) Nrf2/ARE signaling adaptively responded to high glucose treatment in GMCs; (4) Cx43 reduced ROS generation by boost Nrf2/ARE signaling under high glucose condition; (5) Inhibition of c-Src activity promoted nucleus accumulation of Nrf2; (6) Over-expressed Cx43 inhibited c-Src activity and the interaction between c-Src and Nrf2 in GMCs cultured in high glucose. Thus we propose that Cx43 might enhance the activation of Nrf2/ARE pathway by means of inhibiting c-Src activity to hinder the nuclear export of Nrf2, and then reduce expression of FN, ICAM-1 and TGF-β1, ultimately attenuating renal fibrosis in diabetes.

Graphical abstract

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