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Abstract |
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). 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.
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Materials and Methods |
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TopAbstractMaterials and MethodsResults and DiscussionReferences |
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). Dulbecco's
modified Eagle's medium with Ham's F12 medium was obtained from Invitrogen
(Carlsbad, CA); hydrocortisone and insulin were from Sigma-Aldrich (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 µg/ml), penicillin (100U/ml), and streptomycin (100 µg/ml).
The cells were seeded on 3407 Snapwell polycarbonate 0.4 µm, 1
cm2 filter inserts (Corning Inc., Acton, MA), then differentiated
in a secretion medium, in which epidermal growth factor was replaced with
prolactin (3 µg/ml) and hydrocortisone (3 µg/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 14C-nitrofurantoin
concentrative transport permeability were determined by placing
14C-nitrofurantoin on either the basolateral
(B1) or the apical (A)
side and using cold nitrofurantoin on none, one, or both sides of the
diffusion chambers. Cold nitrofurantoin (where present) was dissolved in the
buffer to give a final concentration of 250 µM, about 7.5 times the
Km for nitrofurantoin transport
(Gerk et al., 2002). Humidified
95% oxygen 5% carbon dioxide was bubbled through both sides of each diffusion
chamber at 20 to 25 ml/min. As described previously
(Gerk et al., 2002), the
diffusion chambers were assembled and filled with 5 ml of the buffer with or
without cold nitrofurantoin. Samples (100 µl) were taken from the
basolateral and apical sides at 1, 20, 40, 60, 80, 100, and 120 min and
analyzed by liquid scintillation counting. Basolateral to apical (B-A) or
apical to basolateral (A-B) effective permeabilities (Pe) were determined, by
rearranging the equation J = A*Pe*Cd
to solve for Pe = J/(A*Cd), where J is
flux, A is snapwell area (1 cm2), and Cd is the
14C-nitrofurantoin concentration on the donor side. Mass balance
was complete, and less than 5% of the radiolabel was transferred to the
opposite side in 120 min.
At the end of the experiments, the diffusion chambers were disassembled,
the snapwells were rinsed quickly three times in ice-cold buffer, excess fluid
was drained, and the snapwells were placed in 50-ml centrifuge tubes, then
lysed overnight with 2 ml of 10% trichloroacetic acid. A 1-ml aliquot of the
lysate was taken for liquid scintillation counting. Permeability and
association data were analyzed by one-way ANOVA with Bonferroni's multiple
comparisons post hoc with 
= 0.05, where the same prospectively
determined comparisons are indicated in
Fig. 1, B and C.
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Results and Discussion |
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TopAbstractMaterials and MethodsResults and DiscussionReferences |
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).
The effects of a saturating concentration of nitrofurantoin on the
directional permeability of nitrofurantoin shown in
Fig. 1B indicate a basolateral
uptake mechanism as an important step in nitrofurantoin active transport
across this model. First, the direction of active transport is basolateral to
apical, as expected for active transport into milk and as demonstrated
previously (Toddywalla et al.,
1997; Gerk et al.,
2002). The difference in directional transport was eliminated when
cold nitrofurantoin was present on both sides, consistent with a saturable
mechanism. Basolateral to apical permeability was inhibited as effectively by
placing cold nitrofurantoin only on the basolateral (cis) side as it was by
placing it on both sides. B-A permeability was less effectively inhibited when
cold nitrofurantoin was placed only on the apical (trans) side. This suggests
a basolateral active uptake mechanism. A-B permeability increased when cold
nitrofurantoin was present on either or both sides, consistent with inhibiting
a basolateral active uptake mechanism trying to maintain a concentration
gradient. Since nitrofurantoin has a high diffusional permeability (half of
total B-A permeability), diffusion may result in concentrations near the
transporter high enough to inhibit it.
The snapwell association data are consistent with a basolateral active uptake mechanism. Since only 1.3 to 15% of the radioactivity was bound to the blank filters, the majority of 14C-nitrofurantoin bound to the snapwells was associated with the cell layer (85 to 98.7%), so these data mainly represent accumulation and binding in the cell layer rather than the filter. 14C-Nitrofurantoin association with CIT3 snapwell filters was greater when 14C-nitrofurantoin was presented on the basolateral side than when it was presented on the apical side, or in the presence of cold nitrofurantoin on either or both sides. The other comparisons performed were not significant. The data are consistent with a basolateral uptake mechanism, which would be expected to result in less accumulation in the cell layer when the transporter is saturated or when nitrofurantoin was presented on the opposite side. By contrast, an apical efflux mechanism would function to minimize cellular entry from the apical compartment, and inhibiting it would increase the intracellular concentration. However, when nitrofurantoin was presented to the apical side, there was no increased association with the snapwells in the presence of cold nitrofurantoin on either or both sides, again supporting a basolateral active uptake mechanism.
An implication of a basolateral active uptake mechanism concentrating its
substrates into cells would be the potential for cellular toxicity, as seen
with organic anion transporter 1 concentrating ochratoxin A into renal
proximal tubule cells (Tsuda et al.,
1999). For nitrofurantoin, oxidative stress occurs through
reduction to its radical anion by NADPH-cytochrome P450 reductase, which
generates superoxide anion radicals, and causes formation of hydrogen
peroxide, leading to depletion of reduced glutathione and protein thiols, and
lipid peroxidation in rat lungs (Suntres
and Shek, 1992). Also, nitrofurantoin causes dose-dependent
depletion of reduced glutathione and protein thiols as well as increased bile
flow in the isolated perfused rat liver
(Hoener et al., 1989), and
time- and concentration-dependent enhancement of membrane damage due to
hydroperoxides or diamide in rat liver mitochondria
(Carbonera et al., 1988). As a
result of basolateral active uptake, high intracellular concentrations of
nitrofurantoin could cause oxidative stress, in the lactating mammary
epithelial cells, depending on the activity of NADPH-cytochrome P450 reductase
or other enzymes in the lactating mammary epithelium. Furthermore, if
nitrofurantoin displaces uptake of an endobiotic needed to support cell
metabolism and/or milk production, the health of the lactating mammary
epithelial cells and/or the nutritive qualities of the milk could also be
adversely affected. The uptake activity of this transporter could expose the
suckling infant to xenobiotics, and toxicity to the lactating mammary
epithelium could affect the mother as well as the nutritional quality of the
milk.
The present results, along with previously published data on nitrofurantoin
active transport in lactating rats and/or the CIT3 model, can help eliminate
several nitrofurantoin transporter candidates. Previous data in lactating rats
and/or the CIT3 model show that nitrofurantoin transport is saturable,
sodium-dependent, inhibited by cimetidine, dipyridamole, and purine
nucleosides, but relatively insensitive to probenecid, pyrimidine nucleosides,
and nucleobases (Gerk et al.,
2001b,
2002). These data indicate that
nitrofurantoin active transport is inconsistent with the ABC drug-resistance
transporters (Litman et al.,
2001), bile salt transporters
(Meier and Stieger, 2002),
organic anion transporters (Dresser et al.,
2001), and known nucleoside/nucleobase transporters. Future
studies could compare the expression patterns of other transporter candidates
with the basolateral location of the nitrofurantoin active transporter.
In conclusion, nitrofurantoin transport across the CIT3 model involves a basolateral active uptake mechanism as well as nonsaturable components. This mechanism may influence the exposure of suckling infants to xenobiotics as well as potentially having toxic effects on the lactating mammary epithelium. Further studies are needed to determine the identity of the nitrofurantoin active transporter.
Phillip M. Gerk
Jeffrey A. Moscow
Patrick J. McNamara
University of Kentucky College of Pharmacy, Division of Pharmaceutical Sciences (P.M.G., P.J.M.); and University of Kentucky College of Medicine, Department of Pediatrics (J.A.M.), Lexington, Kentucky
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Acknowledgments |
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Footnotes |
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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.
1 Abbreviations used are: B, basolateral; A, apical; Pe, permeabilities; J,
flux; Cd, 14C-nitrofurantoin concentration on the donor side;
ANOVA, analysis of variance. 
Address correspondence to: Dr. Patrick J. McNamara, Room 401A Pharmacy Building, University of Kentucky College of Pharmacy, Lexington, KY 40536-0082. E-mail: pmcnamar{at}email.uky.edu
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TopAbstractMaterials and MethodsResults and DiscussionReferences |
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0.05 and solid arrows where p < 0.05.