Measurement of Oxidatively-Induced DNA Damage in Caenorhabditis elegans with High-Salt DNA Extraction and Isotope-Dilution Mass Spectrometry.

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ID: 21637
2019
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Abstract
Caenorhabditis elegans is used extensively as a medical and toxicological model organism. However, little is known about background levels of oxidatively-induced DNA damage in the nematode or how culturing methods affect DNA damage levels. The tough C. elegans cuticle makes it challenging to extract genomic DNA without harsh procedures that can artifactually increase DNA damage. Therefore, a mild extraction protocol based on enzymatic digestion of the C. elegans cuticle with high-salt phase-separation of DNA has been developed and optimized. This method allows for efficient extraction of > 50 µg DNA using a minimum of 250,000 nematodes grown in liquid culture. The extracted DNA exhibited acceptable RNA levels (< 10 % contamination), functionality in polymerase chain reaction assays, and reproducible DNA fragmentation. Gas chromatography/tandem mass spectrometry (GC-MS/MS) with isotope-dilution measured lower lesion levels in high-salt extracts than in phenol extracts. Phenolic extraction produced a statistically significant increase in 8-hydroxyguanine, a known artifact, and additional artifactual increases in 2,6-diamino-4-hydroxy-5-formamidopyrimidine, 4,6-diamino-5-formamidopyrimidine and 8-hydroxyadenine. The high-salt DNA extraction procedure utilizes green solvents and reagents, and minimizes artifactual DNA damage, making it more suitable for molecular and toxicological studies in C. elegans. This is, to our knowledge, the first use of GC-MS/MS to measure multiple 8,5´-cyclopurine-2´-deoxynucleosides in a toxicologically important terrestrial organism.
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Authors Scanlan, Leona D;Coskun, Sanem Hosbas;Jaruga, Pawel;Hanna, Shannon K;Sims, Christopher M;Almeida, Jamie L;Catoe, David;Coskun, Erdem;Golan, Rachel;Dizdaroglu, Miral;Nelson, Bryant C;
Journal Analytical chemistry
Year 2019
DOI
10.1021/acs.analchem.9b01503
URL
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