Aflatoxin-Guanine DNA Adducts and Oxidatively Induced DNA Damage in Aflatoxin-Treated Mice <i>in Vivo</i> as Measured by Liquid Chromatography-Tandem Mass Spectrometry with Isotope Dilution

Abstract

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.