The effects of cocaine and nicotine on amino acid transport across the human placental cotyledon perfused in vitro.

The inhibitory effects of cocaine and nicotine on placental amino acid transport, as a mechanism contributing to intrauterine growth restriction, were investigated in the in vitro placental perfusion model. Amino acids that represent substrates for known placental transporters were selected: alanine (system A), glutamine (system N), phenylalanine and valine (system l), and arginine (system y(+)). Amino acid accumulation on the fetal side was measured in the absence of cocaine or nicotine (n = 7) and in the presence of 1.2 microg/ml cocaine (n = 6), 120 ng/ml nicotine (n = 6), or both (n = 6). Neither cocaine nor nicotine alone significantly inhibited alanine transport, whereas their combination did (P =.02). Significant inhibition of arginine transport was detected with nicotine (P =.007), cocaine (P =.01), and their combination (P =.01), whereas phenylalanine (P =.03, P =.04) and valine (P =.03, P =.04) transport was affected by cocaine and the combination of cocaine and nicotine, respectively. For glutamine, neither cocaine, nicotine, nor their combination had a statistically significant inhibitory effect. In conclusion, both cocaine and nicotine may contribute to fetal growth restriction by interfering with the activity of amino acids transporters that are necessary to maintain the nutrient gradients associated with normal fetal growth.

Maternal cocaine use and cigarette smoking in pregnancy in relation to amino acid transport and fetal growth.

This review covers the weight of evidence that shows the association of cocaine and cigarette smoking in pregnancy with the impaired transplacental amino acid transport which might give rise to fetal growth restriction (IUGR). Vasoconstrictive effects of both cocaine and nicotine on the placental vasculature are clearly not the only cause for inhibition of placental amino acid uptake and transfer. In vitro studies strongly suggest that cocaine decreases the activity of placental amino acid transport system A and system N, and possibly system l and system y(+), while nicotine decreases the activity of system A. These findings are supported by cordocentesis studies in human IUGR pregnancies not resulting from drug abuse. More work is needed to be done in order to understand the potential additive or synergistic effect of cocaine and cigarette smoking on fetal growth and to determine the underlying cellular mechanisms of interaction with placental amino acid transporters.

Transplacental transfer and biotransformation studies of nicotine in the human placental cotyledon perfused in vitro.

Our objective was to study the characteristics of transfer and biotransformation of nicotine in the human term placenta. Nicotine transfer was studied by dually perfusing an isolated cotyledon of the human placenta in vitro. Nicotine metabolism to cotinine was investigated in intact tissue during perfusion and in placental microsomal fractions. Following the addition of nicotine (40 ng/ml) to the maternal side of the placenta, distribution into placental tissue (0.43 +/- 0.13 ng/ml/min) was three times higher than transfer to the fetal side of the placenta (0.15 +/- 0.01 ng/ml/min). The steady-state maternal-to-fetal transfer of nicotine was approximately 90% that of antipyrine (a marker of flow-dependent transfer). There was no evidence of nicotine metabolism to cotinine by intact placental tissue or in microsomal fractions. The observation that nicotine readily crosses the human placenta with no evidence of metabolism suggests that nicotine has the potential to cause adverse affects on the developing fetus.

The transfer, of cocaethylene across the human term placental cotyledon perfused in vitro.

Cocaethylene is produced by transesterification of cocaine in the presence of ethanol, and there is evidence that it is more neurotoxic than cocaine. Because many women of reproductive age use cocaine and because many cocaine users also consume alcohol, the fetal toxicology of cocaethylene is of great concern. At the present time the placental transfer of cocaethylene has not been fully characterized. The objective of this article was to measure the transfer of cocaethylene across the human term placenta. The transfer of cocaethylene was measured using the in vitro dual perfusion of the human term placental cotyledon. Using a "closed-circuit" design, the extraction fraction of cocaethylene was measured to be 0.009 microgram/mL.min-1 and the transfer fraction was measured to be 0.013 microgram/mL.min-1, suggesting that the placental tissue retained some of the administered dose. Using an "open circuit" design, the clearance of the compound by the placenta was found to be 78 +/- 14% that of antipyrine clearance. The metabolism of cocaethylene by the perfused placental cotyledon was also measured using an "open circuit" design and was found to be negligible. These results indicate that the placenta does not serve as a significant physical or metabolic barrier to cocaethylene transfer from mother to child. As compared to previously reported results, the transfer of this compound across the human placenta is similar to that of cocaine. Variability in placental handling of cocaethylene may therefore determine fetal exposure to this compound.

Cocaine inhibits hCG secretion by the human term placental cotyledon perfused in vitro.

Cocaine has been shown to adversely affect pregnancy outcome in humans, but the mechanism(s) are not well understood. Using the technique of perfusing the human term placental cotyledon in vitro, we measured the rate of hCG appearance in the maternal circulation in the presence and absence of cocaine in the maternal circulation. At a dose of 0.80 microgram/mL, hCG secretion was reduced by 46%. This reduction in hCG concentration in the maternal circulation may effect normal steroidogenesis required to maintain pregnancy and may contribute to our understanding of the reproductive toxicology of cocaine.

Drug transfer across the placenta. Considerations in treatment and research.

Using the pregnant woman as a means to medicate her fetus represents a new and exciting therapeutic approach in clinical pharmacology. In years to come, we will see an increase in the use of the currently accepted fetal therapies and an increase in the number of therapies available to the caregiver or clinician aimed at assisting the unborn child. The pharmacologist, through in vitro research that aids our understanding of the role of the placenta in fetal drug therapy, can contribute to the growth of this new field within the realm of obstetric medicine.

Biotransformation of carcinogenic arylamines and arylamides by human placenta.

Placental biotransformation reactions may modulate the effect a xenobiotic has on the developing fetus. However, in spite of the critical role the placenta plays in supporting fetal life, little is known about the pharmacology and toxicology of the human placenta. Our laboratory has previously characterized the N-acetylation activity of the human term placenta. This activity is predominantly attributable to the NAT1 form of arylamine N-acetyltransferase (NAT). Although acetylation is generally thought to be a detoxifying reaction, both N-acetylation and deacetylation reactions play an important role in the activation of carcinogenic arylamines to their reactive and toxic forms. In the current study we characterized the activity of human placental NAT and deacetylase toward the carcinogenic arylamine, 2-aminofluorene (AF) and its acetylated metabolite, 2-acetylaminofluorene (AAF). 2-Aminofluorene is a synthetic, prototype carcinogenic arylamine compound, and its metabolism has been extensively studied in the laboratory. Our data show that the affinity (Km = 24.2 +/- 1.66 mumol/L; mean +/- SEM, n = 6) and maximal velocity (Vmax = 4.29 +/- 0.33 nmol/min/mg; mean +/- SEM, n = 6) of AF N-acetylation by human placenta are similar to those in human liver. The deacetylation of AAF to AF by placental microsomes may be catalyzed by a carboxylesterase. However, our studies with inhibitors reveal that the characteristics of human placental deacetylation activity differ from that of human liver.(ABSTRACT TRUNCATED AT 250 WORDS)

Arylamine N-acetyltransferase activity of the human placenta.

The fetus is exposed to almost all of the substances found in the maternal circulation whether nutrients or foreign chemicals ("xenobiotics"). The main route of exposure is the placenta. The placenta is metabolically active toward xenobiotics and the nature of the compounds reaching the fetal circulation will, in part, depend on placental biotransformation reactions. Arylamine N-acetyltransferase (NAT) catalyzes the acetyl CoA-dependent N-acetylation of primary arylamine and hydrazine substrates such as sulfamethazine, isoniazid, p-aminobenzoic acid as well as arylamine carcinogens such as 2-aminofluorene and benzidine. NAT activity is multigenically determined and can be attributed to two independently expressed proteins: NAT1 and NAT2. The acetylation capacity of the human placenta has not been investigated extensively. In the current study we identified and characterized the NAT activity of human term placenta. The kinetic data show that the activity of NAT in human placenta predominantly reflects the NAT1 enzyme. The apparent affinity (19.1 +/- 0.97 microM; mean +/- S.E.M., n = 22) and maximal velocity (3.84 +/- 0.32 nmol/min/mg; mean +/- S.E.M., n = 22) of p-aminobenzoic acid N-acetylation are similar to those measured in other tissues such as liver, blood lymphocytes, neutrophils and monocytes. In addition, there is evidence of NAT2 activity in some of the placental samples assayed, although the contribution of a small amount of NAT2 activity to the overall acetylation capacity of the placenta is likely to be small.

Human placental transfer and metabolism of p-aminobenzoic acid.

Studies in our laboratory have shown that the N-acetylation activity of the human term placenta is a predominantly attributable to the NAT1 form of arylamine N-acetyltransferase (NAT). To further assess the acetylation capacity of the placenta, the N-acetylation of the prototype NAT1-selective substrate, p-aminobenzoic acid (PABA), was studied using the in vitro human placental perfusion model. This study compared the net N-acetylation of PABA in intact placental tissue with the PABA acetylation activity observed in a subcellular fraction (cytosol). Such studies with intact tissue can permit assessment of the exposure of the fetus in vivo to drugs and their metabolites. Acetylated metabolite (N-acetyl-p-aminobenzoic acid) was detectable in fetal and maternal venous samples taken less than 5 min from the start of perfusion with PABA. In a closed recirculating system, the rate of placental PABA transfer decreased as PABA concentrations equilibrated across the placenta. In contrast, the rate of N-acetyl-p-aminobenzoic acid formation continued to increase throughout the entire time of perfusion. Kinetic parameters of PABA N-acetylation measured in cytosol prepared from perfused placental tissue show that the placenta retains its ability to N-acetylate PABA at fresh tissue levels even after 6 hr of in vitro perfusion (Vmax = 5.75 +/- 0.42 nmol/min/mg (fresh) vs. Vmax = 7.24 +/- 0.31 nmol/min/mg (perfused); mean +/- S.E.M., n = 6). These studies indicate that the human placenta has a significant capacity to N-acetylate NAT1-selective substrates of NAT and that it maintains its ability to metabolize xenobiotics during in vitro perfusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Transfer of cocaine and benzoylecgonine across the perfused human placental cotyledon.

OBJECTIVE: Our aim was to measure the transfer of cocaine and its major metabolite benzoylecgonine across the human term placenta. STUDY DESIGN: By means of in vitro perfusion of the human term placental cotyledon the transfer of these compounds was measured. RESULTS: The steady-state maternal-to-fetal transfer of cocaine (0.18 +/- 0.05 microgram/ml/min) was significantly greater than benzoylecgonine transfer (0.02 +/- 0.01 microgram/ml/min) (p < 0.05). When the perfused tissue was analyzed 32% +/- 7% of the maternal cocaine dose was retained by the placental tissue, whereas only 12% +/- 12% of the maternal benzoylecgonine dose was retained by the placental compartment. CONCLUSIONS: These results suggest (1) the placenta may serve as a depot for large amounts of cocaine, thus offering some degree of fetal protection after bolus administration; (2) fetal exposure may be prolonged by placental retention and subsequent release of cocaine and benzoylecgonine; and (3) benzoylecgonine does not cross the placenta as readily as does cocaine. Variability in placental handling of cocaine and benzoylecgonine may therefore determine fetal exposure to these agents.

Acetylcholinesterase and butyrylcholinesterase activity in the human term placenta: implications for fetal cocaine exposure.

The characterization of the enzymes responsible for drug metabolism in the human placenta is of great importance in determining the possible role the placenta plays in protecting the fetus from potentially fetotoxic drugs. We speculate that the placenta metabolizes cocaine, serving to protect the fetus from the drug's ill effects. Cholinesterase, the principle enzyme that metabolizes cocaine, has been hypothesized to be present yet is not well characterized in the human placenta. The purpose of this study was to quantify human placental acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activity. Human placentas were obtained from elective cesarean sections, and several lobules were thoroughly perfused with cold buffer to ensure minimal contamination from erythrocyte AChE. Subcellular fractions were then prepared from these lobules by using standard differential centrifugation techniques. Microsomes and cytosol were assayed for AChE and BChE activity by using a spectrophotometric assay. BChE activity was found in the cytosolic fraction of the placental villous tissue, whereas AChE activity was measured in the microsomal fraction. By demonstrating that BChE activity is present in human term placenta we have shown that this organ has the capacity to metabolize cocaine and may therefore serve as a metabolic barrier to fetal exposure to cocaine.

The transport of digoxin across the perfused human placental lobule.

The cardiac glycoside, digoxin, is clinically used to treat fetal tachyarrhythmias and congestive heart failure. The time course of digoxin transfer across the human placenta was studied by dually perfusing an isolated lobule of the human placenta in vitro. Viability of the placental preparation was validated by measuring the rates of glucose and oxygen consumption, lactate production and synthesis of the protein hormone, chorionic gonadotropin. Following administration of 5 ng/ml digoxin to the maternal circulation, digoxin appeared in the fetal circulation within 5 min. The disappearance of digoxin from the maternal circulation was biexponential and best fit a two-compartment pharmacokinetic model. Mean calculated volume of the central compartment (257 +/- 6.3 ml) was consistent with the actual volume of the in vitro maternal circulation (246 +/- 7.4 ml). The half-life of the distribution phase was 9.7 +/- 3.3 min, and half-life of the terminal elimination phase was 362 +/- 83 min. After 30 min of perfusion, the amount of digoxin leaving the maternal circulation and appearing in the fetal circulation was constant at a fetomaternal mass ratio of 0.36 +/- 0.04. This ratio was maintained through to the end of the 3-hr experiment. All of the digoxin leaving the maternal circulation could be accounted for either in the fetal circulation or bound to placental tissue. The time to achieve equal concentrations on both sides of the placenta was estimated to be 268 +/- 34 min. These data are consistent with in vivo data obtained in humans, and support the relevance of using the in vitro placental perfusion model to obtain information regarding placental drug transfer in humans.

Ionized calcium measurement in vitro: is it necessary?

Experimental data obtained from a study on the inhibition of placental Ca-ATPase by ethacrynic acid were graphically analyzed using either total calcium or ionized calcium as the independent variable. Correct interpretation of the results required actual measurement of ionized calcium in the incubation medium. The assumption that ionized calcium is equal to the total calcium resulted in artifacts and led to erroneous conclusions. These observations emphasize the necessity for accurate measurement of ionized calcium in test systems where calcium effects are being examined.

Transplacental 45Ca and 32P flux in the guinea pig: effect of maternal hypercalcaemia and hypocalcaemia.

Transplacental 45Ca and 32P flux was measured across the in situ perfused guinea-pig placenta under conditions of acute maternal hypocalcaemia and hypercalcaemia. Maternal hypercalcaemia induced acutely by calcium gluconate infusion caused an increase in maternal-to-fetal 45Ca flux which was proportional to the increase in maternal plasma ionized calcium concentration. Acute maternal hypocalcaemia was induced by EGTA infusion and resulted in a decrease in maternal plasma ionized calcium concentration proportional to a corresponding decrease in transplacental 45Ca transfer. A bolus of calcium gluconate caused a transient decrease in 32P flux, whereas EGTA administration was without significant effect on transplacental 32P transfer. Calcium transport across the placenta is not saturated under conditions of maternal normocalcaemia and may be altered according to acute changes in maternal plasma calcium concentration. Thus, control of maternal-to-fetal calcium transfer does not appear to be at the placental level. This suggests that fetal calcium homeostasis may be regulated by the fetus itself.

Hepoxilin A, hydroxyepoxide metabolite of arachidonic acid, stimulates transport of 45Ca across the guinea pig visceral yolk sac.

The effect of hepoxilin A, a newly isolated hydroxyepoxide metabolite of arachidonic acid, on calcium transport across the visceral yolk sac membrane of the guinea pig was investigated in vitro in Ussing chambers. While 1-14C-labelled hepoxilin A itself was not transported across the membrane, it increased the rate of transport of calcium toward the side to which hepoxilin A was added. The degree of increase in calcium transport was similar whether hepoxilin A was added to the maternal side or to the fetal side of the membrane. The observed effect was dependent on the concentration of hepoxilin A over a narrow range (0.5-1.0 X 10(-6) M). It was also dependent on the time of incubation reaching maximal effect by 25 min. We have recently observed that hepoxilin A is formed from platelet-derived 12-hydroperoxyeicosatetraenoic acid (12-HPETE) through hemin and hemoglobin catalysis as well as during perifusion of 12-HPETE through isolated pancreatic islets. The present study suggests that hepoxilin A, if formed in vivo, could play a role in the mobilization of calcium.

Effects of pharmacological and physiological modulators on Ca-ATPase and alkaline phosphatase activities from guinea-pig placenta in vitro.

In a search for modulators of Ca-ATPase and AP activities, we examined three pharmacological agents and the cations Ca2+ and Zn2+. Placental Ca-ATPase specific activity was uncompetitively inhibited in vitro by millimolar concentrations of the diuretics ethacrynic acid and furosemide. Cysteine, a sulphydryl donor, partially reversed the ethacrynic acid inhibition but enhanced the furosemide inhibition, indicating that sulphydryl-binding may be part of the mechanism of the inhibition of Ca-ATPase by ethacrynic acid but not by furosemide. In contrast to Ca-ATPase, AP activity was enhanced by both ethacrynic acid and furosemide. Zinc inhibited Ca-ATPase activity at all concentrations tested, but enhanced and, at higher concentrations, inhibited AP activity. The inhibition of AP activity by D-penicillamine was reversed by Zn, supporting the view that this drug acts by chelating Zn which is essential for AP activity. D-penicillamine had no significant effect on Ca-ATPase activity. Calcium activated both enzyme activities but inhibited only AP activity at higher concentrations. These results indicate that placental Ca-ATPase and AP activities may be distinct and dissociable based on responses to various pharmacological and physiological modulators.

Placental calcium and phosphorus transfer in the guinea pig: lack of effect of modulators of Ca-ATPase and alkaline phosphatase activity.

The effects of modulators of Ca-ATPase and alkaline phosphatase (AP) activity on placental calcium and phosphorus transfer were studied using the in situ perfused guinea pig placenta. The diuretics ethacrynic acid and furosemide had no significant effect on placental calcium and phosphorus transfer when injected into the mother (1.0 or 10.0 mg X kg-1) or added to the solution perfusing the fetal side of the placenta (0.25 or 2.0 mM). These two drugs have previously been shown to inhibit placental Ca-ATPase and enhance AP activity in vitro. D-Penicillamine, which inhibits placental AP but not Ca-ATPase activity in vitro, also had no significant effect on net calcium and phosphorus transfer from mother to fetus either when given to the mother (50 mg X kg-1) or added to the placental perfusion solution (0.25 or 2.0 mM). These results suggest that placental transfer of calcium and phosphorus in the guinea pig may not be directly related to placental Ca-ATPase and AP activities.

Placental transfer of calcium (Ca) and phosphorus (Pi) in the guinea pig: lack of correlation with placental Ca-ATPase and alkaline phosphatase (AP) activities.

In summary, it has been observed that in vitro inhibitors of placental Ca-ATPase and AP activities (EA, F, D-pen) and activators of placental AP (EA,F) are not associated with changes in Ca and Pi transfer across the in situ perfused guinea pig placenta. Assuming that the two enzyme activities were altered in vivo by these drugs, it may be that they are not directly related to active transport of Ca and Pi across the placenta.

Calcium and phosphate fluxes across the fetal membranes of the guinea pig: in vitro measurement.

Bidirectional calcium (45Ca) and phosphate (32P) fluxes across the amnion and visceral yolk sac of the guinea pig were measured in vitro in modified Ussing chambers. The net flux of these ions across both fetal membranes was in the maternal-to-fetal direction. The net flux of 45Ca and 32P across the yolk sac was significantly greater than that across the amnion. This difference was due to a greater maternal-to-fetal flux across the yolk sac. In addition, net 32P flux was greater than net 45Ca flux across the yolk sac, while in the amnion, there was no significant difference in the net flux of the two ions. It is suggested that the fetal membranes, especially the visceral yolk sac, contribute significantly to fetal acquisition of calcium and phosphate in mammals possessing a functional yolk sac placenta.