AFB₁‐induced mutagenesis of the gpt gene in AS52 cells

Abstract

Aflatoxin B1 (AFB1) is a potent mutagen and an important risk factor for hepatocellular carcinoma (HCC) in humans. Transgenic mouse strains and cells in culture have been used to detect different types of mutations caused by AFB1 and investigate the molecular determinants of their location and frequency. The AFB1 mutational spectrum in the gpt gene was markedly different in AS52 cells compared to the liver in gpt delta B6C3F1 transgenic mice. The results demonstrate the importance of metabolism, chromosomal location, transcription and selection conditions on mutational spectra.

An engineered cell line lacking OGG1 and MUTYH glycosylases implicates the accumulation of genomic 8-oxoguanine as the basis for paraquat mutagenicity

“…Using this model, we have established that PQ is mutagenic and cytotoxic, its properties stemming from its ability to generate ROS and induce genomic accumulation of oxidative DNA lesions such as 8OG. The system described is well poised to enable deeper analysis of PQ mutagenesis in terms of the specific types of point mutations induced, and mutational spectra, both in the AS52 cells, and in an animal model (e.g., B6C3F1 gpt delta mouse [34, 49]).…”

“…To remove the spurious or pre-existing 6-thioguanine resistant (6-TG r ) mutants, cells were cultured in the presence of mycophenolic acid (MPA) (Sigma-Aldrich, USA) for 7 days. The MPA-containing medium was the complete medium (above) supplemented with 10 μg/ml MPA, 250 μg/ml xanthine (Sigma-Aldrich), 22 μg/ml adenine (Sigma-Aldrich), 11 μg/ml thymidine (Sigma-Aldrich), and 1.2 μg/ml aminopterin (Sigma-Aldrich) [33, 34]. Thereafter, cells were maintained in complete medium plus 11.5 μg/ml xanthine, 3 μg/ml adenine and 1.2 μg/ml thymidine for 3 additional days.…”

“…Using a forward mutagenesis assay, gpt mutants in AS52 cells can be selected by their ability to grow in the presence of 6-thioguanine (6-TG) [32–34]. However, for a weak mutagen such as PQ, the AS52 cell model is sensitivity-challenged, owing to the presence of only a single chromosomally integrated copy of gpt gene.…”

AFB1 hepatocarcinogenesis is via lipid peroxidation that inhibits DNA repair, sensitizes mutation susceptibility and induces aldehyde-DNA adducts at p53 mutational hotspot codon 249

“…Furthermore, we found that the mutational spectrum and types of mutations induced by these two aldehydes were distinctly different (Figure 5C & 5D ). Most notably Acet induced mainly G to T transversion mutations (69%), which are the same as AFB1-induced G to T mutations in the p53 and gpt genes in human cells and yeast systems [ 40 – 42 ]. Cro, on the other hand, induced both G to T (37.5%) and G to C (40.6%) mutations at similar levels [ 43 ].…”

“…Underlines represent tandem mutations. Note: The percentages of G to T and A mutations induced by Acet are similar to that of induced by AFB1 [ 40 – 42 ].…”