Mechanisms of drug transfer across the human placenta.

In this review we summarized literature data on the mechanisms of human placental drug transport studied in the isolated perfused placental cotyledon, placental membrane vesicles or trophoblastic cell cultures. Overall human placental drug transport rarely exceeds the transfer of flow-dependent and membrane-limited marker compounds. Interestingly, relatively often placental drug transfer appeared to be much smaller, indicating impaired trans-placental transport, depending on the physico-chemical characteristics of the drug or placental factors such as tissue binding or metabolism. Although in perfusion studies overall human placental drug transport occurs by simple diffusion, at the membrane level several drug transport systems have been found, mainly for drugs structurally related to endogenous compounds.

Uptake of cimetidine into syncytial microvillus membrane vesicles of human term placenta.

Uptake of the H2-receptor antagonist, cimetidine, into syncytial microvillus membrane vesicles of human term placenta was investigated to clarify whether an active transport mechanism can be responsible for the observed barrier of the human placenta for cimetidine. Imposition of an outwardly directed H(+)-gradient stimulated cimetidine uptake, resulting in a small transient overshoot. The H(+)-gradient-dependent peak uptake was decreased under voltage-clamped conditions by carbonyl cyanide p-trifluoromethoxy-phenylhydrazone, suggesting the presence of an organic cation-proton exchange mechanism. Uptake was partially, but significantly, inhibited by organic cation transport inhibitors, H2-receptor antagonists and several other cationic drugs, providing further evidence for mediated uptake. H(+)-gradient-dependent cimetidine uptake was saturable and characterized by a low-affinity (Km) of 6.3 mM and Vmax of 17.5 nmol/mg protein/10 sec. We conclude that the system cannot play an important role in the barrier function of the human placenta in the transport of cimetidine. Rather than active transport, other factors, as for instance the degree of ionization of cimetidine at physiological pH, seem to be a more likely explanation for the low clearance of cimetidine across the human placenta.

Inhibition of choline uptake in syncytial microvillus membrane vesicles of human term placenta. Specificity and nature of interaction.

The potency and nature of the inhibitory effect of various cationic drugs on the transport of choline across the placental syncytial microvillus membrane was investigated. Tetraethylammonium, a model substrate for organic cation transport, was a poor inhibitor. Enlarging the degree of alkylation of the quaternary ammonium increased the inhibitory effect, in proportion with increasing lipophilicity. Log concentration vs % control uptake curves showed marked differences in inhibitory potency for the different cationic drugs. Hemicholinium-3 inhibited mediated choline uptake in the micromolar range, whereas atropine and mepiperphenidol were less potent. The H2-receptor antagonists cimetidine, ranitidine, and famotidine inhibited choline uptake in the millimolar ranges. Dixon analysis revealed a competitive nature of inhibition for hemicholinium-3 and atropine (Ki = 40 microM and 1.2 mM, respectively). Cimetidine interacted noncompetitively (Ki = 3.4 mM). Since relatively high concentrations were needed to reach half maximal inhibition, impairment of fetal choline supply due to maternal drug use during pregnancy is not to be expected.

Isolation of syncytial microvillous membrane vesicles from human term placenta and their application in drug-nutrient interaction studies.

The initial step in placental uptake of nutrients occurs across the syncytial microvillous membrane of the trophoblast. This study was designed to isolate syncytial microvillous membrane vesicles (SMMV) of human term placenta, to validate their purity and viability, and to investigate the interaction of several commonly used drugs with the transport of two essential nutrients: alanine and choline. SMMV were isolated according to an established procedure, but instead of homogenization the initial preparation step was replaced by mincing of placental tissue followed by gently stirring to loosen the microvilli. These modifications doubled the protein recovery and increased the enrichment in alkaline phosphatase, whereas no substantial contamination with basal membranes nor interfering subcellular organelles was found. The functional viability of the vesicles was evaluated through the transport of alanine. In accordance with literature, uptake was sodium-dependent, inhibitable by structural analogues, and saturable. A number of cationic drugs were were able to able to inhibit choline uptake, whereas no effect on alanine transport was observed. Anionic drugs, drugs of abuse, and catecholamines did not interfere with alanine transport either. In conclusion, our isolated SMMV provide a suitable tool for screening drug-nutrient interactions at the level of membrane transport. In view of the very low susceptibility of the alanine transporter to drug inhibition and the relatively high drug concentrations necessary to inhibit choline transport, it seems unlikely that clinically important drug interactions may occur with these nutrients.

Development of methionine synthase, cystathionine-beta-synthase and S-adenosyl-homocysteine hydrolase during gestation in rats.

The developmental onset of three homocysteine metabolizing enzymes in the rat conceptus was investigated. Cystathionine-beta-synthase and methionine synthase were assayed from day 10 to day 20 of gestation in decidual and placental tissue, from day 10 to day 12 of gestation in embryonic tissue, from day 14 to day 20 of gestation in fetal liver and from day 14 to day 20 of gestation in fetal tissue without liver. On each day, material was obtained from at least four conceptuses from two dams. S-adenosylhomocysteine hydrolase was assayed in neurulating conceptuses in decidual tissue, parietal yolksac plus ectoplacental cone, visceral yolksac plus amnion and embryo proper. Conceptuses were pooled from seven (day 9.5 of gestation) or three (days 10.5 and 11.5 of gestation) dams. In embryonic and fetal tissue cystathionine-beta-synthase first occurred in fetal liver. During the organogenic phase it was present only in decidual tissue. Methionine synthase was present in all tissues from the first gestational day investigated and S-adenosylhomocysteine hydrolase was present in all tissues throughout the neurulating period. Our results indicate that the homocysteine-methionine cycle, which is crucial to transmethylation reactions, is functional during the neurulating period in embryonic tissue. Owing to the absence of cystathionine-beta-synthase at this stage of development in embryonic tissue, the homocysteinyl moiety is conserved in the homocysteine-methionine cycle.

Production of cyclophosphamide metabolites by primary hepatocyte cultures from male and pregnant rats: Effect of Aroclor 1254 pretreatment.

In a previous paper (VanAerts et al., Toxicology in Vitro 1993, 7, 769-775) we have shown that the embryotoxicity of Cyclophosphamide was greatly enhanced when Cyclophosphamide had been added to a primary hepatocyte culture derived from Aroclor 1254-pretreated male rats (M(A)) and the medium from this culture was added to a post-implantation rat embryo culture. However, when medium from hepatocytes that had been derived from untreated male rats (M(C)), untreated pregnant rats (P(C)) or Aroclor 1254-pretreated pregnant rats (P(A)) was used embryotoxicity was low. We now present data on the concentrations of 4-ketocyclophosphamide, carboxyphosphamide and nornitrogen mustard in the primary hepatocyte culture media. 4-Ketocyclophosphamide was absent in media from P(C) or P(A) and present in low concentrations in media from M(C) and M(A). Carboxyphosphamide and nornitrogen mustard were present in all media, but their concentrations were several times higher in medium from M(A) than in media from other sources. This indicates that Aroclor 1254 pretreatment enhances Cyclophosphamide metabolism much more in male rats than in pregnant rats. It is suggested that both a greater inducibility of CYP2B1 and augmented stabilization of 4-hydroxycyclophosphamide by glutathione conjugation may be processes that contribute to the observed differences in metabolite concentrations.

Prevention of neural tube defects by and toxicity of L-homocysteine in cultured postimplantation rat embryos.

Mild hyperhomocysteinemia is frequently observed in mothers who gave birth to a child with a neural tube defect (NTD). In a previous study we showed L-homocysteine was embryotoxic to gestational day 10 (GD10) rat embryos in culture, however, no NTDs were observed. We therefore investigated the effect of L-homocysteine on the development of neural plate stage (GD9.5) rat embryos. Other objectives of this study were investigation into whether the embryotoxicity of L-homocysteine could be attenuated by compounds related to its metabolism and clarification of the mechanism of L-homocysteine embryotoxicity. In GD9.5 rat embryos L-homocysteine was not toxic at 1- and 2-mM concentrations. Rather at these concentrations it promoted development of the rat embryos in serum that without supplementation caused NTDs in the embryos. L-Methionine had the same preventive effect at even lower concentrations, but folinic acid (1 mM) did not improve embryonic development. N5-Methyltetrahydrofolate (5-CH3-THF) (100 microM), L-serine (6 mM), and L-methionine (6 and 12 mM) attenuated the embryotoxicity of L-homocysteine (6 mM) in GD10 rat embryos. Vitamin B12 (10 microM) completely abolished the embryotoxicity of L-homocysteine, which was shown to be mediated by catalysis of the spontaneous oxidation of L-homocysteine to the less toxic L-homocystine. In GD11 rat embryos, both L- and D-homocysteine were readily taken up when added to the culture (3 mM) and increased embryonic S-adenosylhomocysteine (SAH) levels 14- and 3-fold, respectively. This difference was shown to be caused by the stereospecific preference of SAH hydrolase. We propose the basis for L-homocysteine embryotoxicity is an inhibition of transmethylation reactions by increased embryonic SAH levels.

p-aminohippurate uptake by syncytial microvillous membrane vesicles of human term placenta.

The mechanism of uptake of p-aminohippurate (PAH) by syncytial microvillous membrane vesicles of human term placenta was investigated. Initial PAH uptake and efflux were increased in the presence of a pH-gradient and a Cl(-)-gradient, respectively. Forced negative and positive membrane potentials did not influence the uptake, which indicated that the transport is not electrogenic. The pH-dependent increase is probably the result of a higher rate of diffusion due to a lower degree of dissociation of PAH. Because several organic anions failed to transstimulate PAH uptake and FCCP did not decrease the uptake in the presence of an inwardly directed H(+)-gradient, ruling out a PAH/OH- antiport, an anion exchange system does not appear to be present in these membranes. Since electrogenicity and anion exchange seem not to be involved in the Cl(-)-dependent increase, an allosteric effect of Cl- on the transporter might be possible. Various organic anions were able to inhibit pH-stimulated PAH uptake significantly. Kinetic analysis of the probenecid sensitive part of uptake provided further evidence for mediated transport of PAH (Km = 7.4 +/- 2.6 mM and Vmax = 2.0 +/- 0.4 nmol/mg/15 s). Non-inhibitable diffusion accounted for the main part of total transport. Concentration dependent inhibition of PAH transport by probenecid showed a Ki of 2.5 +/- 0.9 mM. It is concluded that human placental syncytial microvillous membrane vesicles possess a low affinity transport mechanism for PAH with low specificity. The importance of this system, for placental excretion of anionic drugs, will depend on the intrasyncytial concentration of these drugs, caused by the transport across the basal membrane.

Uptake of choline into syncytial microvillus membrane vesicles of human term placenta.

The uptake of the quaternary ammonium compound choline was studied in syncytial microvillus membrane vesicles of human term placenta. Uptake was stimulated by an inside negative membrane potential and by loading the vesicles with unlabeled choline. Imposition of an inwardly directed Na+ or outwardly directed H+ gradient did not stimulate choline uptake. Several organic cations were able to inhibit choline transport in the following order: hemicholinium-3 > or = choline > or = mepiperphenidol > cimetidine > or = famotidine. The kinetics of uptake involved a saturable process for choline with high affinity (Km = 550 microM). Our results confirm the presence of a carrier mediated transport system in human placental syncytial microvillus membranes. The system appears to be electrogenic, and able to transport choline efficiently from the maternal circulation into the placenta.

Study on the presence of homocysteine in ovarian follicular fluid.

OBJECTIVE: To study the presence of homocysteine, methionine and the vitamins folate, B12, and B6 in human ovarian follicular fluid (FF). DESIGN: Measurement of homocysteine, methionine, folate, and vitamins B12 and B6 in ovarian FF and blood. SETTING: Academic Department of Obstetrics and Gynecology at St. Radboud Hospital, Nijmegen, The Netherlands. PARTICIPANTS: Fourteen healthy women undergoing an IVF program. RESULTS: Detectable amounts of homocysteine and methionine were found in FF. Homocysteine concentrations were similar to those in serum. Methionine concentrations proved to be slightly but significantly lower than in corresponding serum samples. Concentrations of vitamins B12 and B6 were significantly lower in FF than in serum, whereas folate concentrations were not significantly different. A statistically significant correlation between corresponding serum and FF concentrations of homocysteine, folate, and vitamin B12 could be established. CONCLUSIONS: These data support the hypothesis that the ovum might be exposed to high homocysteine or low methionine concentrations, or both, and a lack of vitamins, which might be important in fertilization and early embryogenesis.

Biotransformation of cyclophosphamide in post-implantation rat embryo culture using maternal hepatocytes in co-culture.

The post-implantation rat embryo culture technique is employed to study embryotoxic effects of xenobiotic compounds in the absence of the maternal compartment. For compounds biotransformed in vivo the embryo culture technique must be adapted in order to mimick the in vivo effects. In the present study the possibility of co-culturing metabolically active maternal hepatocytes suspended in the standard culture system with rat serum as a medium was investigated. Cyclophosphamide (CP) was used as a model compound as it needs bioactivation to display embryotoxicity. Morphologic and histologic effects were studied. Neither hepatocytes nor CP alone affected embryo development, whereas in the presence of hepatocytes embryotoxicity was observed at 30 micrograms/ml CP. Embryotoxicity was decreased in the additional presence of metyrapone, a monoxygenase inhibitor. Hepatocyte suspensions prepared via slicing or perfusion of livers were equally effective. In conclusion, co-culture of embryos and suspended hepatocytes can be performed under optimal conditions for embryo development and in the presence of biotransforming activity.

An ultrastructural study of the response of normal skin to epicutaneous application of leukotriene B4.

The ultrastructural appearance of normal skin following epicutaneous application of leukotriene B4 (LTB4) was studied using transmission electronmicroscopy. The acute phase (first 24 h) after LTB4 challenge is characterized by extravasation of polymorphonuclear leukocytes (PMN) and endothelial changes such as focal necrosis, formation of fenestrations, gaps, and a multilayered basal lamina. In the 'late phase' (24-48 h), the endothelium still possesses many fenestrations and gaps. The density of endothelial cell nuclei is increased, and the relatively large nucleoli and high density of mitochondria are indicative of a hyperactive endothelium. The PMN in this phase show pycnotic nuclei and a vacuolated cytoplasm. Macrophages are observed phagocytosing these degenerating PMN. Monocytes and lymphocytes extravasate without any signs of endothelial or epidermal damage in their vicinity. The mast cells show no degranulation throughout the observation period.

Growth and liver morphology after long-term ethanol consumption of rats.

Ethanol was administered to female and male Wistar rats by mixing it with their drinking water. Ethanol concentrations were gradually increased up to either 8% or 15%. Female rats receiving 8% ethanol in their drinking water consumed 5-13 g, males 4-10 g daily. The ethanol/total food caloric intake percentages were 13 to 20% and 9 to 15% for female and male rats, respectively. There was no difference in body weight and relative liver weight between treated rats and their controls. Female and male rats receiving 15% of ethanol in their drinking water consumed 8-14 g ethanol per kg body weight per day. The percentages of ethanol/total food caloric intake were stabilized at about 25% for both sexes. Growth of the rats differed only slightly from controls; a tendency for a higher increase of body weight of the control rats was found. No difference in relative liver weight between ethanol-treated and control rats was observed. Microscopic examinations revealed that the ethanol treatment resulted in fat accumulation in the liver cells. A proliferation of the Smooth Endoplasmic Reticulum (SER) was more marked in the 15% dosed rats than in the 8% dosed rats and more distinct in female rats than in male rats in both dosage groups.

An ultrastructural study of transcutaneous migration of polymorphonuclear leukocytes following application of leukotriene B4.

In the present study the ultrastructural aspects of the migration of polymorphonuclear leukocytes (PMN) in apparently normal skin following the epicutaneous application of leukotriene B4 (LTB4) were studied and it was investigated whether this model is representative of the migration of PMN in psoriatic skin. At the ultrastructural level the following features were observed: degranulation of PMN, formation of fenestrations and gaps of the endothelium, a multilayered basal lamina and hyperactive endothelial cells with protrusions and focal necrosis of these cells. Epicutaneous application of LTB4 is a practical approach to study the dynamics of the interaction between PMN and the endothelium in vivo.

The intake of cadmium in the Kempen, an area in the south of The Netherlands.

In The Netherlands many areas with soil pollution have been detected. The largest polluted area is a zone of 350 km2 in the Kempen, in the south of The Netherlands. This Kempen zone is polluted with heavy metals, especially cadmium (Cd) and zinc (Zn), emitted from metal factories in the Netherlands and in Belgium. Because of the high Cd in soil, vegetables grown in that area contain relatively high Cd concentrations. The Cd uptake by inhabitants of these areas--especially individuals consuming vegetables from their own gardens--therefore is considerably increased. This Cd intake is shown to be higher than the provisionally tolerated weekly uptake of Cd set by the WHO. The role of smoking in Cd intake is discussed.

Glycogen content of placenta and of fetal and maternal liver of cadmium-exposed rats. II: A quantitative histochemical study.

Quantitative data are presented of the glycogen contents in the placental labyrinth, fetal liver and maternal liver of 14-, 16-, 18-, 19- and 20-day pregnant rats exposed to cadmium during pregnancy. The values are obtained from periodic acid-Schiff-stained sections by microdensitometry. No changes due to cadmium exposure were observed in the glycogen content of maternal and fetal livers. However, at 18, 19 and 20 days of pregnancy, significantly higher amounts of glycogen were observed in the trophoblastic labyrinth of cadmium-exposed rats compared with control animals.

Light and electron microscopic investigation of the rat placenta after cadmium administration during pregnancy.

The morphology of the rat placenta was studied after exposure to cadmium chloride during pregnancy, using optimal fixation conditions. In contrast to previous observations, no differences were observed after cadmium administration in relative volume densities of trophoblastic tissue, maternal lacunae, fetal capillaries and connective tissue, nor in trophoblastic thickness or other morphometric features. At the ultrastructural level, the amount of glycogen in trophoblast layer II was elevated in cadmium exposed rats, but other electron microscopic features (amount and localization of lipid, degenerative vesicles, thickness and general appearance of the trophoblastic and endothelial layers and thickening or multiplication of the basal lamina) were not changed. Results obtained from the present experiments do not support the suggestion that cadmium is responsible for structural changes in the placentae of human smokers.

The effects of long-term exposure to cadmium on the small blood vessels in the rat uterus: a light microscopic study.

A group of female Wistar rats was exposed to 0.5 mg/kg cadmium three times a week for a period of 29 weeks. The cadmium was administered as the chloride in saline by subcutaneous injection. A second group of female Wistars was divided into a control group and and two experimental groups. The animals in the last two groups were exposed to 0.23 and 0.046 mg/kg cadmium three times a week for a period of 82 weeks, likewise administered by subcutaneous injection, to study the long-term effects of cadmium on the microvasculature of the uterus. The small blood vessels in the myometric layer of the uteri were studied. The thickness of the media was analyzed and an inventory was made on the morphology of the media, of the endothelial layer, and of the perivascular connective tissue. A dose- and time-related increase of the thickness of the media could be demonstrated. In the highest dose group, signs of perivascular inflammatory reaction could be observed.

Effects of chronic cadmium administration on placental and fetal development.

Cadmium was administered subcutaneously to pregnant Wistar rats: 0.49 mg/kg as CdCl2 in saline daily, starting at the day of conception. Placentas and fetal livers were collected on day 14, 16, 18, 19 and 20 of gestation. Livers and thymuses from the newborns were collected 5 hours after delivery (day 22) and 1, 2 and 5 weeks after delivery. In these tissues concentrations of cadmium and zinc were determined by solid sampling ETA-AAS. Furthermore, the effect of cadmium administration on the glycogen content of the trophoblastic labyrinth and the fetal liver was studied. The concentration of cadmium in the placenta increased with time of exposure, indicating accumulation of cadmium in this organ. In the fetal liver, cadmium was present in a very low concentration, which slightly increased with longer exposure. The concentration of zinc in the placenta tends to decrease between day 14 and day 20. This decrease was observed both in control and in cadmium-exposed animals. Zinc levels increased in fetal livers from control dams, whereas this rise was markedly reduced in fetuses from cadmium-exposed animals. Placentas from cadmium-exposed animals had a changed glycogen pattern as compared to the controls, namely higher glycogen contents of the labyrinth at the end of pregnancy. However, notwithstanding lower zinc levels in the fetus and changed glycogen deposition in the placenta, it is not quite clear whether cadmium affects fetal development. No changes were observed in fetal weights or birthweights, nor in glycogen deposition of the fetal liver. Indications were obtained for reduced neonatal thymic weights.