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Effects of reduced mitochondrial DNA content on secondary mitochondrial toxicant exposure in Caenorhabditis elegans.

ABSTRACT: The mitochondrial genome (mtDNA) is intimately linked to cellular and organismal health, as demonstrated by the fact that mutations in and depletion of mtDNA result in severe mitochondrial disease in humans. However, cells contain hundreds to thousands of copies of mtDNA, which provides genetic redundancy, and creates a threshold effect in which a large percentage of mtDNA must be lost prior to clinical pathogenesis. As certain pharmaceuticals and genetic mutations can result in depletion of mtDNA, and as many environmental toxicants target mitochondria, it is important to understand whether reduced mtDNA will sensitize an individual to toxicant exposure. Here, using ethidium bromide (EtBr), which preferentially inhibits mtDNA replication, we reduced mtDNA 35-55% in the in vivo model organism Caenorhabditis elegans. Chronic, lifelong, low-dose EtBr exposure did not disrupt nematode development or lifespan, and induced only mild alterations in mitochondrial respiration, while having no effect on steady-state ATP levels. Next, we exposed nematodes with reduced mtDNA to the known and suspected mitochondrial toxicants aflatoxin B1, arsenite, paraquat, rotenone or ultraviolet C radiation (UVC). EtBr pre-exposure resulted in mild sensitization of nematodes to UVC and arsenite, had no effect on AfB1 and paraquat, and provided some protection from rotenone toxicity. These mixed results provide a first line of evidence suggesting that reduced mtDNA content may sensitize an individual to certain environmental exposures.

SUBMITTER: Luz AL

PROVIDER: S-EPMC5023498 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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Effects of reduced mitochondrial DNA content on secondary mitochondrial toxicant exposure in Caenorhabditis elegans.

Luz Anthony L AL Meyer Joel N JN

Mitochondrion 20160823

The mitochondrial genome (mtDNA) is intimately linked to cellular and organismal health, as demonstrated by the fact that mutations in and depletion of mtDNA result in severe mitochondrial disease in humans. However, cells contain hundreds to thousands of copies of mtDNA, which provides genetic redundancy, and creates a threshold effect in which a large percentage of mtDNA must be lost prior to clinical pathogenesis. As certain pharmaceuticals and genetic mutations can result in depletion of mtD ...[more]

PMID: 27566481

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Project description:Mitochondrial respiratory chain disease is caused by a wide range of individually rare genetic disorders that impair cellular energy metabolism. While fluorescence microscopy analysis of nematodes fed MitoTracker Green (MTG) and tetramethylrhodamine ethyl ester (TMRE) can reliably quantify relative mitochondrial density and membrane potential, respectively, in C. elegans models of mitochondrial dysfunction, it is a tedious process with limitations in the number and age of animals that can be studied. A novel, large particle, flow cytometry-based method reported here accelerates and automates the relative quantitation of mitochondrial physiology in nematode populations. Relative fluorescence profiles of nematode populations co-labeled with MTG and TMRE were obtained and analyzed by BioSorter (Union Biometrica). Variables tested included genetic mutation (wild-type N2 Bristol versus nuclear-encoded respiratory chain complex I mutant gas-1(fc21) worms), animal age (day 1 versus day 4 adults), classical respiratory chain inhibitor and uncoupler effects (oligomycin, FCCP), and pharmacologic therapy duration (24h versus 96h treatments with glucose or nicotinic acid). A custom MATLAB script, which can be run on any computer with MATLAB runtime, was written to automatically quantify and analyze results in large animal populations. BioSorter analysis independently validated relative MTG and TMRE changes that we had previously performed by fluorescence microscopy in a variety of experimental conditions, with notably greater animal population sizes and substantially reduced experimental time. Older, fragile animal populations that are difficult to study by microscopy approaches were readily amenable to analysis with the BioSorter method. Overall, this high-throughput method enables efficient relative quantitation of in vivo mitochondrial physiology over time in a living animal in response to gene mutations and candidate therapies, which can be used to accelerate the translation of basic research into optimization of clinical therapies for mitochondrial disease.

Project description:BackgroundThe present study was designed to investigate the protective effects and mechanisms of carnosine on lipopolysaccharide (LPS)-induced injury in Caenorhabditis elegans.MethodsC. elegans individuals were stimulated for 24 h with LPS (100 μg/mL), with or without carnosine (0.1, 1, 10 mM). The survival rates and behaviors were determined. The activities of superoxide dismutase (SOD), glutathione reductase (GR), and catalase (CAT) and levels of malondialdehyde (MDA) and glutathione (GSH) were determined using the respective kits. Reverse transcription polymerase chain reaction (RT-PCR) was performed to validate the differential expression of sod-1, sod-2, sod-3, daf-16, ced-3, ced-9, sek-1, and pmk-1. Western blotting was used to determine the levels of SEK1, p38 mitogen-activated protein kinase (MAPK), cleaved caspase3, and Bcl-2. C. elegans sek-1 (km2) mutants and pmk-1 (km25) mutants were used to elucidate the role of the p38 MAPK signaling pathway.ResultsCarnosine improved the survival of LPS-treated C. elegans and rescued behavioral phenotypes. It also restrained oxidative stress by decreasing MDA levels and increasing SOD, GR, CAT, and GSH levels. RT-PCR results showed that carnosine treatment of wild-type C. elegans up-regulated the mRNA expression of the antioxidant-related genes sod-1, sod-2, sod-3, and daf-16. The expression of the anti-apoptosis-related gene ced-9 and apoptosis-related gene ced-3 was reversed by carnosine. In addition, carnosine treatment significantly decreased cleaved caspase3 levels and increased Bcl-2 levels in LPS-treated C. elegans. Apoptosis in the loss-of-function strains of the p38 MAPK signaling pathway was suppressed under LPS stress; however, the apoptotic effects of LPS were blocked in the sek-1 and pmk-1 mutants. The expression levels of sek-1 and pmk-1 mRNAs were up-regulated by LPS and reversed by carnosine. Finally, the expression of p-p38MAPK and SEK1 was significantly increased by LPS, which was reversed by carnosine.ConclusionCarnosine treatment protected against LPS injury by decreasing oxidative stress and inhibiting apoptosis through the p38 MAPK pathway.

Dataset Information

Effects of reduced mitochondrial DNA content on secondary mitochondrial toxicant exposure in Caenorhabditis elegans.

Publications

Effects of reduced mitochondrial DNA content on secondary mitochondrial toxicant exposure in Caenorhabditis elegans.

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