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.

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.

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.

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.

Increase of cadmium-thiolate clusters as a measure of morphological non-toxic cadmium accumulation in the rat liver.

When cadmium is chronically administered to rats, an increase by more than 10% of protein bound disulphides and cadmium-thiolate clusters appears to be an indicator for non-toxic accumulation of cadmium in liver and kidney and probably in other organs as well. Using enzyme histochemistry, no damage could be observed in these livers, on the contrary, even signs of increased cellular activity could be demonstrated with specific staining for single stranded RNA. It is clearly demonstrated that in the case of 2 livers with the same quantity of accumulated cadmium morphological damage is completely dependent on dose and schedule of administration. However, despite the fact that cadmium is retained very well in rat livers showing an increase in protein-bound disulphides and cadmium-thiolate clusters, there are still small morphological changes, especially in cells and tissues that appear to have a relatively small potency for producing cadmium-binding proteins.

Light microscopic aspects of the rat placenta after chronic cadmium administration.

Female Wistar rats were injected daily with different doses of CdCl2 during the first 19 days of pregnancy. The placentas of highly exposed animals showed reduced weights as well as some light microscopic changes with respect to the amount of collagen in the basal membranes around fetal blood vessels and to the relative volume densities of the fetal blood vessels. These results are in accordance with changes, found in the placentas of smoking mothers, probably due to cadmium in cigarette smoke.

Large increase in disulphide bonds containing cytosol proteins after chronic cadmium administration, estimated in isolated rat liver cells.

Using histochemical staining, followed by cytophotometric quantitation of disulphide bonds and total protein in isolated liver cells of rats treated for a long time with low doses of CdCl2, a large increase in disulphide bonds containing proteins could be demonstrated in cells of one ploidy class. This increase seems to be due to an increase in high molecular weight (HMW) cytosol proteins as estimated biochemically. They probably represent polymers of metallothionein.

Histochemical changes in protein disulphide bonds in rat liver and kidney after chronic cadmium administration, and the possible relation to metallothionein.

After chronic exposure to low doses of CdCl2 an increase in disulphide bonds has been established in rat liver using a specific staining method for disulphide bonds and cytophotometric quantitation. This increase is dependent on doses and length of exposure time. Evidence is presented that this increase might be related to the accumulation of metallothionein or some other cadmium binding protein. Using the same staining method after long exposure to low doses of CdCl2 a large number of large dark blue stained granules were observed in the proximal tubule cells, with blue stained deposits in the lumen of the proximal and some renal medulla tubules of the kidney. Evidence is presented that this staining pattern corresponds to the destruction of the proximal tubule cell by the cadmium thionein complex.

Structural changes in the placenta of smoking mothers: a quantitative study.

Placentae of matched pairs of smoking and non-smoking mothers have been investigated by quantitative light microscopic techniques. A smaller diameter of the villous capillaries, a decrease in vasculosyncytial membranes and basement membrane thickening were observed in the placenta of smoking mothers. Quantitative morphometric methods showed a significant decrease in volume density of the fetal vessels in the terminal villi of the smoker's placenta. The exchange area of the smoker's placenta was shown to be decreased. The pathophysiological mechanisms leading to these changes and the possible role of toxic agents (i.e., cadmium) are discussed.