Short Communication BASOLATERAL ACTIVE UPTAKE OF NITROFURANTOIN IN THE CIT3 CELL CULTURE MODEL OF LACTATION

Nitrofurantoin and other agents are actively transported into human and rodent milk. The purpose of this study was to determine whether nitrofurantoin active transport across mammary epithelia occurs basolaterally or apically, using the CIT3 cell culture model of lactation. The CIT3 model actively transports nitrofurantoin in the basolateral to apical direction. Basolateral to apical permeability [92.9 6.6 ( l/h)/cm] was differentially decreased by unlabeled nitrofurantoin (250 M) on the basolateral, apical, or both sides [49.5 1.8, 57.9 1.4, or 48.5 1.6 ( l/h)/cm, respectively]. Apical to basolateral permeability [27.6 1.8 ( l/h)/cm] was increased in the presence of unlabeled nitrofurantoin (250 M) on the basolateral, apical, or both sides [36.4 1.5, 39.9 0.7, 42.4 1.1 ( l/h)/cm, respectively]. These data indicate a basolateral active uptake mechanism for nitrofurantoin, which remains to be identified. This mechanism may influence the exposure of suckling infants to xenobiotics, as well as having potentially toxic effects on the lactating mammary epithelium and possibly altering the nutritional quality of the milk. Breastfeeding has numerous benefits but also some disadvantages, including potential xenobiotic exposure to the suckling infant (Gerk et al., 2001a). Although most drugs enter milk by passive diffusion (Fleishaker and McNamara, 1988), some drugs are actively transported into milk. Previously, we have demonstrated active transport of nitrofurantoin into human and rat milk, reaching milk-to-serum concentration ratios of 20 to 100 times those predicted by diffusion, respectively (Gerk et al., 2001a,b). Furthermore, active, saturable transport of nitrofurantoin across a murine cell culture model of lactation has been demonstrated (Toddywalla et al., 1997). Previously published data in lactating rats and/or the CIT3 model shows that nitrofurantoin transport is also sodium-dependent; inhibited by cimetidine, dipyridamole, and purine nucleosides; but insensitive to probenecid, pyrimidine nucleosides, and nucleobases (Gerk et al., 2001b, 2002). However, the mechanism has not been identified and further elucidation is needed to gain a better understanding of how certain drugs are concentrated into milk. For nitrofurantoin to be concentratively transported from the blood (or serum) into milk, it must cross the basolateral (blood-facing) and apical (milk-facing) cell membranes. As a result, the active transport step may occur as either basolateral uptake or apical efflux. A basolateral active uptake mechanism would result in high intracellular substrate concentrations, but an apical active efflux mechanism will minimize intracellular substrate concentrations. Although it is not known whether active nitrofurantoin transport occurs apically or basolaterally, the location of the active transport step has functional consequences and may yield insights into the nitrofurantoin transport mechanism. Therefore, the purpose of this study was to determine whether nitrofurantoin active transport across mammary epithelia occurs basolaterally or apically. Materials and Methods The C-nitrofurantoin (58 mCi/mmol) was obtained from Chemsyn Labs (Lexena, KS) and was purified as described previously (Gerk et al., 2002). Dulbecco’s modified Eagle’s medium with Ham’s F12 medium was obtained from Invitrogen (Carlsbad, CA); hydrocortisone and insulin were from SigmaAldrich (St. Louis, MO); epidermal growth factor was from BD Biosciences Discovery Labware (Bedford, MA); ovine prolactin was from the National Hormone and Pituitary Program (Dr. A. F. Parlow, Torrance, CA); and fetal bovine serum was from Gemini BioProducts (Calabasas, CA). All other chemicals were obtained from Sigma-Aldrich. CIT3 cells were cultured according to the previously published protocol (Toddywalla et al., 1997). Briefly, the cells (passages 15 to 21) were grown in a growth medium containing Dulbecco’s modified Eagle’s medium with Ham’s F12 medium supplemented with 2% fetal bovine serum, epidermal growth factor (5 ng/ml), insulin (10 g/ml), penicillin (100U/ml), and streptomycin (100 g/ml). The cells were seeded on 3407 Snapwell polycarbonate 0.4 m, 1 cm filter inserts (Corning Inc., Acton, MA), then differentiated in a secretion medium, in which epidermal growth factor was replaced with prolactin (3 g/ml) and hydrocortisone (3 g/ml). The experiments were performed at pH 7.4 in an isotonic saline buffer free of antibiotics, serum, hormones, or proteins, containing 111 mM NaCl, 22 mM NaHCO3, 4.2 mM KHCO3, 1.05 mM CaCl2, 0.41 mM MgSO4, 0.14 mM MgCl2, 20 mM glucose, and 10 mM HEPES. The calculated osmolarity was adjusted to 280 to 305 mOsm/liter with mannitol. The directionality and inhibition of C-nitrofurantoin concentrative transport permeability were determined by placing C-nitrofurantoin on either the basolateral (B1) or the apical (A) side and using cold nitrofurantoin on This work was supported by the University of Kentucky Women’s Health Initiative and National Institutes of Health Grant HD37463. This work was published as a part of P. M. G.’s doctoral dissertation at the University of Kentucky. These results were presented in part at the annual meeting of the American Association of Pharmaceutical Scientists in 2000. Address correspondence to: Dr. Patrick J. McNamara, Room 401A Pharmacy Building, University of Kentucky College of Pharmacy, Lexington, KY 40536-0082. E-mail: [email protected] 1 Abbreviations used are: B, basolateral; A, apical; Pe, permeabilities; J, flux; Cd, C-nitrofurantoin concentration on the donor side; ANOVA, analysis of variance. 0090-9556/03/3106-691–693$7.00 DRUG METABOLISM AND DISPOSITION Vol. 31, No. 6 Copyright © 2003 by The American Society for Pharmacology and Experimental Therapeutics 937/1064665 DMD 31:691–693, 2003 Printed in U.S.A. 691 at A PE T Jornals on A uust 7, 2017 dm d.aspurnals.org D ow nladed from