Assessing the role of the ABCG2 transporter in plasma levels and secretion into milk of aflatoxins B2 and G1

Aflatoxins are highly toxic food contaminants frequently detected in cereals and animal-derived products, with milk representing a relevant route of exposure for both animals and humans. Although milk safety assessments have predominantly focused on aflatoxin M₁ (AFM₁), aflatoxins B₂ (AFB₂) and G₁ (AFG₁) have been detected in dairy products and human breast milk. However, the physiological mechanisms governing their biodistribution and secretion into milk remain poorly characterized. Given the central role of ABCG2 in xenobiotic disposition, and the previous evidence of ABCG2-mediated transport of aflatoxin B₁ (AFB₁) and AFM₁, we investigated the contribution of this transporter to the in vitro transport, plasma levels and milk secretion of AFB₂ and AFG₁. Transepithelial transport assays were conducted using polarized MDCK-II cells overexpressing species-specific ABCG2 variants, followed by in vivo lactation studies in wild-type and Abcg2^-/- mice. In vitro, both AFB₂ and AFG₁ exhibited pronounced basolateral-to-apical transport, with up to 40-fold higher relative efflux ratios in ABCG2-expressing subclones, confirming them as ABCG2 substrates across murine, ovine, bovine, and human variants. In vivo, Abcg2 deficiency resulted in significantly reduced milk concentrations and milk-to-plasma ratios of both aflatoxins, accompanied by increased plasma levels. For AFB₂, milk concentrations were approximately two-fold higher and milk-to-plasma ratios three-fold higher in wild-type compared with Abcg2^-/- mice, with comparable trends observed for AFG₁. Together, these results identify ABCG2 as a key determinant of AFB₂ and AFG₁ plasma levels and transfer into milk, providing novel mechanistic insight into the active transport of aflatoxins across biological barriers, particularly the mammary gland.