Aflatoxin B1-DNA adduct formation and mutagenicity in livers of neonatal male and female B6C3F1 mice

Abstract

Exposure to genotoxic chemicals at a young age increases cancer incidence later in life. Aflatoxin B(1) (AFB(1)) is a potent genotoxin that induces hepatocellular carcinoma (HCC) in many animal species and in humans. Whereas adult mice are insensitive to aflatoxin-induced carcinogenesis, mice treated with AFB(1) shortly after birth develop a high incidence of HCC in adulthood. Furthermore, the incidence of HCC in adult male mice treated as infants is much greater than in females, reasons for which are unclear. In this study, treatment with AFB(1) produced similar levels of DNA damage and mutations in the liver of newborn male and female gpt delta B6C3F1 mice. Twenty-four hours after dosing with AFB(1) (6 mg/kg), the highly mutagenic AFB(1)-FAPY adduct was present at twice the level of AFB(1)-N(7)-guanine in liver DNA of males and females. A multiple dose regimen (3 × 2 mg/kg), while delivering the same total dose, resulted in lower AFB(1) adduct levels. Mutation frequencies in the gpt transgene in liver were increased by 20- to 30-fold. The most prominent mutations in AFB(1)-treated mice were G:C to T:A transversions and G:C to A:T transitions. At this 21-day time point, no significant differences were found in mutation frequency or types of mutations between males and females. These results show that infant male and female B6C3F1 mice experience similar amounts of DNA damage and mutation from AFB(1) that may initiate the neoplastic process. The gender difference in the subsequent development of HCC highlights the importance of elucidating additional factors that modulate HCC development.

FIG. 1.

Experimental scheme for treatment of newborn gpt delta B6C3F1 mice with AFB₁, analysis of AFB₁-DNA adducts and mutations in the gpt gene in liver.

FIG. 2.

Amounts of AFB₁-N⁷-guanine in liver DNA of 4-day old male (-▴-) and female (-▪-) gpt delta B6C3F1 mice after administration of 6 mg/kg AFB₁. Points = mean ± SD.

FIG. 3.

AFB₁ DNA adducts in liver of gpt delta B6C3F1 mice 24 h after a single 6 mg/kg dose (A) or 3 × 2 mg/kg administered every third day (B). Plotted data = mean ± SD.

FIG. 4.

MF in neonatal gpt delta B6C3F1 mice treated with AFB₁ or DMSO vehicle. Mice were administered a single 6 mg/kg dose of AFB₁ (single) or treated with 3 × 2 mg/kg doses of AFB₁ (multiple). At 25 (single) or 31 (multiple) days of age DNA was isolated from liver and the number of 6-TG-resistant colonies determined. Plotted data = mean ± SD.

FIG. 5.

Nucleotide sequence of the gpt gene indicating the position and type of base substitutions, deletions (□) and insertions (V) induced by a single 6 mg/kg AFB₁ dose administered to 4-day-old gpt delta B6C3F1 mice. Mutations were assayed 21 days after dosing.