Human placental transport of vinblastine, vincristine, digoxin and progesterone: contribution of P-glycoprotein
Introduction
Vinblastine and vincristine, antineoplastic vinca alkaloids, are commonly used for the treatment of breast cancer and Hodgkin's disease, malignancies that are often found in gravida. Their teratogenic potencies have been examined in a variety of animal models Joneja and Ungthavorn, 1969, Courtney and Valerio, 1968, Ferm, 1963, Tanaki et al., 1967. In hamsters, intravenous injection of vinblastine (0.25 mg/kg) or vincristine (0.1 mg/kg) was reported to induce malformations such as microphthalmia, anophthalmia, exencephaly and spina bifida (Ferm, 1963). Similar results were reported in mice (Joneja and Ungthavorn, 1969), rats (Tanaki et al., 1967) and rhesus monkeys (Courtney and Valerio, 1968). In humans, two cases of vinca alkaloid-induced teratogenicity were reported after maternal treatment with vinblastine or vincristine Thomas, 1976, Mannuti et al., 1975. Digoxin was also reported to be teratogenic (Johnny and Menachem, 1987). However, the permeability and transport mechanisms of these drugs across the placenta remain to be investigated.
Throughout gestation, the placenta plays important roles in regulating the exchange of various materials between the maternal and the fetal circulations (Stulc, 1997). Many investigators have examined the transport mechanisms of nutrients, such as amino acids, vitamins and glucose, across the blood–placental barrier, which consists of trophoblast cells (placental microvillous membrane epithelial cells) shown in Fig. 1 (Hay, 1994). Several transport systems for amino acids, including the Na+-dependent A or ASC system and the Na+-independent L system, were reported to be present in trophoblasts Furesz et al., 1993, Moe et al., 1994, Moe, 1995, Ramamoorthy et al., 1992. It was also reported that glucose transporter 1 (GLUT 1) was expressed both on the brush-border (maternal side) and the basal (fetal side) membranes, while glucose transporter 3 (GLUT 3) was expressed only on the brush-border membrane Bissonnette, 1981, Reid and Boyd, 1994, Hahn and Desoye, 1996. Thiamine (vitamin B1) is transported in exchange for H+ via a Na+- and membrane potential-dependent transport system (Grassl, 1998). Biotin, lipoate and pantothenate are cotransported with Na+ Hu et al., 1994, Schenker et al., 1992, Prasad et al., 1998. However, little is known about the transport of drugs or xenobiotics across the blood–placental barrier, though, several drugs are known to cross the placenta and reach the developing fetus when administered during pregnancy. The fetal blood concentration of drugs is not always equal to the maternal blood concentration Pacifici and Nottoli, 1995, Van der Aa et al., 1998, suggesting that there are active transport systems for drugs in the placenta. In several tissues such as kidney, adrenal gland, vessels at blood–brain barrier sites, liver, intestine and testis, P-glycoprotein was found to be expressed and to extrude a range of hydrophobic natural products and drugs against a concentration gradient Thiebaut et al., 1987, Tsuji et al., 1992, Cordon-Cardo et al., 1989, Sugawara et al., 1988, Sugawara et al., 1997. P-Glycoprotein is encoded by a multidrug resistance gene (MDR1), and can confer multidrug resistance by extruding a wide range of structurally unrelated, amphiphilic hydrophobic drugs from cells in an ATP-dependent manner Juliano and Ling, 1976, Kartner et al., 1983. In recent studies, P-glycoprotein was shown to be expressed in the placenta Sugawara et al., 1997, Nakamura et al., 1997. However, these reports were based on immunohistochemical techniques, and functional studies have not been carried out. Therefore, it is essential to investigate whether P-glycoprotein operates as a drug-efflux pump in the human placenta. Human placental choriocarcinoma epithelial cells (BeWo cells) are commonly used for studies of the blood–placental barrier, including transport mechanisms. BeWo cells have similar properties to normal trophoblasts in terms of morphology, biochemical markers and hormone secretion (Liu et al., 1997).
In the present study, we aimed to elucidate the role of P-glycoprotein in the placenta by means of immunoblotting studies and uptake and transcellular transport studies using BeWo cells. We employed vinblastine, vincristine, digoxin and progesterone as model drugs. Immunoblot analysis was carried out with cultured BeWo cells, isolated human placental trophoblast cells, human placental brush-border membrane vesicles and human placental basolateral membrane vesicles.
Section snippets
Materials and reagents
[3H]Vinblastine sulphate (15.5 Ci/mmol) and [3H]vincristine sulphate (5.70 Ci/mmol) were purchased from Amersham International (Buckinghamshire, UK). [3H]Digoxin (19.0 Ci/mmol) and [1, 2-3H]progesterone (52.0 Ci/mmol) were purchased from NEN Research Products (MA, USA). [1-14C]d-mannitol (53 Ci/mmol) was purchased from Moravek Biochemicals (CA, USA). Trypsin was purchased from GIBCO BRL Life Technologies (Rockville, MD, USA). Collagenase Type I was purchased from Worthington Biochemical
Purity of human placental brush-border membrane vesicles and human placental basolateral membrane vesicles
The purity of human placental brush-border membrane vesicles or human placental basolateral membrane vesicles was confirmed by examining the enzyme activities of alkaline phosphatase and γ-glutamyl transpeptidase or dihydroalprenolol binding activity, respectively. In the case of human placental brush-border membrane vesicles, the activities of alkaline phosphatase and γ-glutamyl transpeptidase for the vesicles and homogenate were 7.14±0.03 and 0.41±0.01 (pmol/mg protein/min) and 26.79±0.43 and
Discussion
P-Glycoprotein can confer multidrug resistance by extruding a wide range of structurally unrelated, amphiphilic hydrophobic drugs from cells in an ATP-dependent manner Juliano and Ling, 1976, Kartner et al., 1983. In recent studies, P-glycoprotein was reported to be expressed in trophoblast cells but not endothelial cells of the placenta Sugawara et al., 1997, Cordon-Cardo et al., 1989, Nakamura et al., 1997. Therefore, P-glycoprotein in the trophoblast cells is likely to be involved in the
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