Dataset Information

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

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 ?g 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 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.

SUBMITTER: Scanlan LD

PROVIDER: S-EPMC6996937 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Publications

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

Scanlan Leona D LD Coskun Sanem Hosbas SH Jaruga Pawel P Hanna Shannon K SK Sims Christopher M CM Almeida Jamie L JL Catoe David D Coskun Erdem E Golan Rachel R Dizdaroglu Miral M Nelson Bryant C BC

Analytical chemistry 20190910 19

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 ...[more]

PMID: 31454479

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Project description:Dietary exposure to aflatoxin B1 (AFB1) is a significant contributor to the incidence of hepatocellular carcinomas globally. AFB1 exposure leads to the formation of AFB1-N7-guanine (AFB1-N7-Gua) and two diastereomers of the imidazole ring-opened 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxyaflatoxin B1 (AFB1-FapyGua) in DNA. These adducts lead to G ? T transversion mutations with the ring-opened adduct being more mutagenic than the cationic species. Accurate measurement of these three adducts as biomarkers in DNA and urine will help identify dietary exposure to AFB1 as a risk factor in the development of hepatocellular carcinoma worldwide. Herein, we report an improved methodology for the measurement of AFB1-N7-Gua and the two diastereomers of AFB1-FapyGua using liquid chromatography-tandem mass spectrometry with isotope dilution. We measured the levels of these compounds in liver DNA of six control mice and six AFB1-treated mice. Levels varying from 1.5 to 45 lesions/106 DNA bases in AFB1-treated mice were detected depending on the compound and animal. No background levels of these adducts were detected in control mice. We also tested whether the AFB1 treatment caused oxidatively induced DNA base damage using gas chromatography-tandem mass spectrometry with isotope dilution. Although background levels of several pyrimidine- and purine-derived lesions were detected, no increases in these levels were found upon AFB1 treatment of mice. On the other hand, significantly increased levels of (5' R)- and (5' S)-8,5'-cyclo-2'-deoxyadenosines were observed in liver DNA of AFB1-treated mice. The impact of this work is expected to achieve the accurate measurement of three AFB1-DNA adducts and oxidatively induced DNA lesions as biomarkers of AFB1 exposure as germane to investigations designed for the prevention of aflatoxin-related hepatocellular carcinomas and for determining the effects of genetic deficiencies in human populations.

Dataset Information

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

Publications

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

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