Prenatal exposure of mice to the human liver carcinogen aflatoxin B₁ reveals a critical window of susceptibility to genetic change

Abstract

It has become axiomatic that critical windows of susceptibility to genotoxins exist and that genetic damage in utero may be a trigger for later life cancers. Data supporting this critical window hypothesis are remarkably few. This study provides a quantitative bridge between DNA damage by the liver carcinogen aflatoxin B1 (AFB1) during prenatal development and the risk of later life genetic disease. AFB1 was given to pregnant C57BL/6J mice, carrying F1 gestation day 14 (GD14) embryos of the B6C3F1 genotype. Ultra-high performance liquid chromatography and mass spectrometry (UPLC-MS) using aflatoxin-15N5-guanine adduct standards afforded measurement of the AFB1-N7-Gua and AFB1-FAPY adducts six hours post dosing in liver DNA of mothers and embryos. A parallel cohort gave birth and the livers of the F1 were analyzed for mutations in the gpt gene at three and ten weeks of age. The data revealed mutational spectra dominated by G:C to T:A mutations in both the mother and offspring that are characteristic of AFB1 and distinct from background. It was shown that adducts in GD14 embryos were 20-fold more potent inducers of mutagenesis than adducts in parallel-dosed adults. This sensitivity enhancement correlated with Ki67 staining of the liver, reflecting the proliferative potential of the tissue. Taken together, these data provide insight into the relative genetic risks of prenatal and adult exposures to AFB1. Early life exposure, especially during the embryonic period, is strikingly more mutagenic than treatment later in life. Moreover the data provide a baseline against which risk prevention strategies can be evaluated.

Mutational spectra of aflatoxin B 1 in vivo establish biomarkers of exposure for human hepatocellular carcinoma

“…In the traditional application of the assay, mutations in the gpt gene are phenotypically enumerated by 6-thioguanine resistance and characterized by conventional DNA sequencing. However, the traditional gpt assay results are limited in that mutations are detected only if they disrupt the functionality of the GPT protein; thus, only a biased set of mutations (i.e., a selected spectrum) in relatively few sequence contexts can be identified (15,16). By contrast, the DS strategy (Fig.…”

“…The G:C→T:A dominance in the spectra was expected due to the mutational properties of individual AFB 1 -DNA adducts (all of which occur at guanines) (12,13,29,30), the mutational spectra in vitro and in vivo of AFB 1 in cells and tissues (12)(13)(14)(15)(16)(17)(18), and observed patterns of mutations in human tumors (27,28). Unexpected, however, was the high G→T mutation yield at selected guanyl residues in the 16 possible three-base contexts (Fig.…”

“…In the infant mouse, AFB 1 forms strongly mutagenic adducts during an early-life period of rapid replicative growth (15,16), resulting in the efficient conversion of adducts into mutations; these early mutations likely constitute the bulk of the genetic events depicted in A-10. By contrast to the single-dose mouse model, the liver cells of chronically exposed humans suffer repeated, but probably sublethal, insults from dietary AFB 1 .…”

“…The currently leading model is that estradiol production in postpubescent females provides an antiinflammatory environment that lowers ultimate cancer risk (44). From previous work, we know that the MF attributable to AFB 1 at 10 wk of life is identical in males and females, leading to the conclusion that later life events, most likely inflammation or epigenetic modifications in response to the presence of a developing tumor, accelerate male hepatocarcinogenesis (15,16). The composite spectrum of AFB 1 -induced tumors (A-72T) is likely embedded with subspectra characteristic of the mutational processes associated with the hypothetical inflammatory and other events that accelerate male carcinogenesis.…”

“…Owing to its persistence and high inherent mutability, the AFB 1 -FAPY adduct likely accounts for the majority of the mutations induced by aflatoxin (12); when traversed by replicative or translesion polymerases, the AFB 1 -FAPY adduct causes primarily G→T mutations. This defining mutation of AFB 1 has been documented following replication in vitro (12,13), in cell culture (14), in animal models (15,16), and even in human specimens (17,18).…”