Short Communication Natural Allelic Variants of Bovine ATP-Binding Cassette Transporter ABCG2: Increased Activity of the Ser581 Variant and Development of Tools for the Discovery of New ABCG2 Inhibitors

ATP-binding cassette transporter ABCG2 [breast cancer resistance protein (BCRP)] is a member of the ABC transporter superfamily that actively extrudes xenotoxins from cells and is a major determinant of the bioavailability of many compounds. ABCG2 expression is strongly induced during lactation in the mammary gland and is related to the active secretion of drugs into the milk. The presence of drug residues and environmental pollutants in milk is an outstanding problem for human milk consumption and milk industrial processes, involving important risks to public health and the dairy industry. In cows, a single nucleotide polymorphism (SNP) in this protein has been described previously (Tyr581) and is associated with higher fat and protein percentages and lower milk yield. However, whether this amino acid substitution affects ABCG2-mediated drug transport in cows, including milk secretion, required further exploration. We cloned the two variants of bovine ABCG2 and evaluated the effect of this SNP on mitoxantrone accumulation assays performed in ovine primary fibroblasts transiently expressing either of the variants. It is interesting to note that statistically significant differences in activity between both variants were observed, and the Ser581 variant was related with an increased efflux activity. In addition, we demonstrated that genistein is a very good inhibitor of bovine ABCG2 and identified new inhibitors of the transporter, such as the macrocyclic lactones, ivermectin, and selamectin. Moreover, the inhibitory effect of these compounds on human and murine ABCG2 homologs was confirmed using transduced Marbin-Dabin canine kidney II cells. These findings may have important implications regarding the presence of drug residues in milk and drug interactions affecting the pharmacological behavior of ABCG2 substrates. Efflux transporters of the ATP-binding cassette (ABC) family play major physiological and pharmacological roles in mammals. These transmembrane proteins actively extrude a variety of drugs, carcinogens, and other xenotoxins across the cellular plasma membrane. This way, ABC transporters restrict the systemic exposure of many compounds by reducing their net intestinal uptake and by mediating their active excretion (van Herwaarden and Schinkel, 2006). One member of this family, ABCG2/breast cancer resistance protein (BCRP), has been detected in alveolar epithelial cells in the mammary gland (Jonker et al., 2005; Wu et al., 2008), where it is strongly expressed during lactation. It has been demonstrated that ABCG2 is the major factor involved in the active secretion of xenotoxins into milk (Jonker et al., 2005), although some physiological nutrients (such as riboflavin and vitamin K3) have also been identified as ABCG2 substrates (van Herwaarden et al., 2007; Shukla et al., 2007). However, in apparent contradiction with the protective role of this transporter in reducing drug bioavailability, the contamination of milk exposes suckling individuals as well as dairy consumers to xenotoxins. The presence of drug residues and environmental pollutants in milk is an outstanding problem for human milk consumption and milk industrial processes, involving important risks to public health and the dairy industry. In addition, the health of the suckling calves could be impaired. Because ABCG2 transporter activity is one of the main determinants of xenotoxin presence in milk, polymorphisms in this protein become of great relevance and may contribute to differences in drug bioavailability and presence of drug residues in milk. Human single nucleotide polymorphisms (SNPs) of ABCG2 have been extensively studied because of their relevance in drug pharmacokinetics. For instance, the significance of the Q141K SNP has been shown in vitro (Mizuarai et al., 2004) and in clinical studies (Sparreboom et al., 2004; Yamasaki et al., 2008). With regard to domestic animals, a single nucleotide change (A/C) in exon 14 of the bovine ABCG2 gene has been reported, encoding a substitution of tyrosine581 to serine (Cohen-Zinder et al., 2005). This polymorphism has been proposed as a quantitative trait nucleotide on bovine chromosome 6 (Cohen-Zinder et al., 2005; Olsen et al., 2007), with the Tyr581 variant being associated with higher fat and protein percentages and lower milk yield. However, whether this SNP influences ABCG2-mediated drug transport in cattle and the presence of drug residues in milk remained to be addressed. To further explore this matter, we cloned the two variants of the This work was supported by Ministerio de Ciencia y Tecnología (Spain) [Grant BIO2006-00430]; a Ramón y Cajal grant from the Ministerio de Ciencia y Tecnología (Spain) [Grant RyC-2006-001896]; and University of Leon (Spain) [Grant