Analysis of the effect of the bovine adenosine triphosphate-binding cassette transporter G2 single nucleotide polymorphism Y581S on transcellular transport of veterinary drugs using new cell culture models1

Abstract

In commercial dairy production, the risk of drug residues and environmental pollutants in milk from ruminants has become an outstanding problem. One of the main determinants of active drug secretion into milk is the ATP-binding cassette transporter G2/breast cancer resistance protein (ABCG2/BCRP). It is located in several organs associated with drug absorption, metabolism, and excretion, and its expression is highly induced during lactation in the mammary gland of ruminants, mice, and humans. As a consequence, potential contamination of milk could expose suckling infants to xenotoxins. In cows, a SNP for this protein affecting quality and quantity of milk production has been described previously (Y581S). In this study, our main purpose was to determine whether this polymorphism has an effect on transcellular transport of veterinary drugs because this could alter substrate pharmacokinetics and milk residues. We stably expressed the wild-type bovine ABCG2 and the Y581S variant in Madin-Darby canine kidney epithelial cells (MDCKII) and MEF3.8 cell lines generating cell models in which the functionality of the bovine transporter could be addressed. Functional studies confirmed the greater functional activity in mitoxantrone accumulation assays for the Y581S variant with a greater relative V(MAX) value (P = 0.040) and showed for the first time that the Y581S variant presents greater transcellular transport of the model ABCG2 substrate nitrofurantoin (P = 0.024) and of 3 veterinary antibiotics, the fluoroquinolone agents enrofloxacin (P = 0.035), danofloxacin (P = 0.001), and difloxacin (P = 0.008), identified as new substrates of the bovine ABCG2. In addition, the inhibitory effect of the macrocyclic lactone ivermectin on the activity of wild-type bovine ABCG2 and the Y581S variant was also confirmed, showing a greater inhibitory potency on the wild-type protein at all the concentrations tested (5 μM, P = 0.017; 10 μM, P = 0.001; 25 μM, P = 0.008; and 50 μM, P = 0.003). Differential transport activity depending on the genotype together with the differential inhibition pattern might have clinical consequences, including changes in substrate pharmacokinetics (and subsequently pharmacodynamics) and more specifically, changes in secretion of ABCG2 substrates into milk, potentially implying important consequences to veterinary therapeutics.

Role of ABCG2 in Transport of the Mammalian Lignan Enterolactone and its Secretion into Milk in Abcg2 Knockout Mice

“…In addition, mean plasma concentrations of endogenous enterolactone in wild-type mice (57 6 13 nM) (Table 1) were in agreement with the values reported in humans (Kuijsten et al, 2005;Adolphe et al, 2010). Recently, the secretion of compounds into ruminant milk has been reported to be affected by ABCG2 polymorphisms and ABCG2 inhibition by drugs and flavonoids (Real et al, 2011;Otero et al, 2013). Thus, these ABCG2-related factors presumably could affect enterolactone concentrations in milk and thus the possible health benefits of lignans from the consumption of milk.…”

“…In recent years, its role as a transporter of phytoestrogens and their conjugated metabolites has gained special relevance (Zhu et al, 2010;Alvarez et al, 2011;Tan et al, 2013).…”

Effect of bovine ABCG2 polymorphism Y581S SNP on secretion into milk of enterolactone, riboflavin and uric acid

“…MDCK-II subclones stably transduced with the bovine ABCG2 (WT-bABCG2) and its Y581S variant (Y581S-bABCG2) were generated by our group (Real et al, 2011). These transduced cell lines express bABCG2 protein at similar levels.…”

“…Recently, ovine and bovine variants of ABCG2 have been cloned to facilitate the understanding of the interspecies variations of the transporter function (Real et al, 2011;Gonzalez-Lobato et al, 2014). In these terms, Mealey (2012) reviewed the relevance of the interaction between drugs and ABC transporters in the veterinary field with a special mention to the reported polymorphisms.…”

“…Y581S SNP frequency can reach 20% in some Holstein populations (Ron et al, 2006). Moreover, this SNP has been described in vitro as a gain-of-function polymorphism revealing a higher capacity of the Y581S variant (Real et al, 2011) that produces a higher in vivo transport of antimicrobials such as danofloxacin into milk 2015). The involvement of ABCG2 in milk secretion and plasma levels of endogenous compounds has been extensively studied in humans.…”