Measurement of Oxidatively Induced DNA Damage in <i>Caenorhabditis elegans</i> with High-Salt DNA Extraction and Isotope-Dilution Mass Spectrometry

<i>Caenorhabditis elegans</i> 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 <i>C. elegans</i> 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 <i>C. elegans</i> 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 250000 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 <i>C. elegans</i>. 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.