Human placental transport of cimetidine.

This study addresses the mechanism of transport of the H2-receptor antagonist, cimetidine, by the human placenta. A 4-h recycling perfusion of a single placental cotyledon of normal, term, human placenta was used. At a maternal concentration of 1 microgram/ml, cimetidine clearance from the maternal circulation was 0.58 +/- 0.16 ml/min per g placenta, a rate about one third that of antipyrine. There was no evidence of cimetidine metabolism by the placenta. Transfer of cimetidine from maternal to fetal compartments showed no saturation kinetics and was not inhibited by putative carrier competitors. Cimetidine did not accumulate against a drug concentration gradient. Fetal clearance of cimetidine was similar to maternal clearance. Studies with placental apical vesicles confirmed lack of saturability of cimetidine transport and of its concentration within vesicles. Thus, (a) cimetidine is transported across the human placenta bidirectionally at a rate about one third that of antipyrine, (b) the drug is not metabolized by the placenta, and (c) the transport is a passive one.

Arrest of epidermal growth factor-dependent growth in fetal hepatocytes after ethanol exposure.

Exposure of the fetal rat hepatocyte to ethanol in vitro blocks epidermal growth factor (EGF)-dependent cell replication. To define possible mechanisms for this growth arrest, we determined the effects of ethanol on EGF binding and EGF receptor (EGF-R) levels. During a 24-h exposure to ethanol (1.7 mg/ml, 31 mM), cell replication was completely blocked while EGF binding per cell doubled. This effect was no specific for EGF, with variable degrees of increased binding noted for insulin, transferrin, and glucagon. Significantly increased EGF binding was seen after 6 h of ethanol exposure, and both growth arrest and enhanced EGF binding were reversed within 12 h of ethanol withdrawal. Increases in both "high" and "low" affinity sites were seen, with no changes in the apparent Kd's. Total RNA, beta-actin mRNA, and EGF-R mRNA were increased 50-70% in ethanol exposed cells. However, direct measurements of EGF-R synthesis rates by [35S]methionine incorporation revealed no differences between control and ethanol exposed cells. Internalization of EGF-R was significantly altered by ethanol exposure. A 2-h incubation resulted in the internalization of 57% of the ligand in control cells, while only 31% of bound EGF was internalized in the ethanol exposed cells. Thus, the enhanced EGF binding may be due to decreased efficiency of internalization.

Effects of chronic prenatal ethanol exposure on mitochondrial glutathione and 8-iso-prostaglandin F2alpha concentrations in the hippocampus of the perinatal guinea pig.

It is hypothesised that oxidative stress is a key mechanism of ethanol neurobehavioural teratogenicity, resulting in altered endogenous antioxidant status and increased membrane lipid peroxidation in the hippocampus of chronic prenatal ethanol exposure (CPEE) offspring. To test this hypothesis, timed pregnant guinea-pigs (term, approximately gestational day (GD) 68) received chronic daily oral administration of (i) 4 g ethanol kg(-1) maternal bodyweight, (ii) isocaloric sucrose with pair feeding, or (iii) water. At GD 65 (term fetus) and postnatal day (PD) 0 (neonate), individual offspring were killed, the brain was excised and the hippocampi were dissected. Glutathione (GSH) concentration was measured in the cytosolic and mitochondrial fractions of hippocampal homogenate. The occurrence of lipid peroxidation was determined by measuring the concentration of 8-iso-prostaglandin F2+/- (8-iso-PGF2+/-). There was CPEE-induced decreased brain weight and hippocampal weight at GD 65 and PD 0, decreased mitochondrial GSH concentration in the hippocampus at PD 0, with no change in mitochondrial GSH concentration at GD 65 or cytosolic GSH concentration at GD 65 or PD 0, and no change in mitochondrial or whole-homogenate 8-iso-PGF2+/- concentration in the hippocampus at GD 65 or PD 0. The data demonstrate that CPEE produces selective mitochondrial dysfunction in the hippocampus of the neonatal guinea-pig, involving GSH depletion.

Inhibition of cytochrome c oxidase activity by 4-hydroxynonenal (HNE). Role of HNE adduct formation with the enzyme subunits.

The role of 4-hydroxynonenal (HNE), a major lipid peroxidation product, in oxidative damage to mitochondrial cytochrome c oxidase (COX) was examined. Oxidative stress was induced in mitochondria isolated from livers of male Sprague-Dawley rats by tert-butylhydroperoxide (t-BHP). COX activity was inhibited, with a concomitant increase in endogenous HNE level in mitochondria. COX activity was also inhibited following incubation of mitochondria with 50-450 microM HNE. Blocking HNE degradation intensified COX inhibition by HNE and by t-BHP-induced oxidative stress, the latter accompanied by a simultaneous increase in endogenous HNE production. On the other hand, COX inhibition by HNE was markedly reduced by potentiating HNE degradation via enhancing conjugation of HNE with reduced glutathione (GSH). Incubation of purified COX with 10-400 microM HNE resulted in HNE adduct formation with specific subunits of COX, correlated with inhibition of the enzyme activity. These data suggest that HNE may inhibit mitochondrial COX by forming adducts with the enzyme, and that this could be one mechanism underlying mitochondrial damage caused by oxidative stress. The findings also illustrate a role for GSH in protecting mitochondria from the deleterious effects of HNE.

The transfer of cocaine and its metabolites across the term human placenta.

This study defines human placental transport of cocaine and its two minor, but pharmacologically active, metabolites--norcocaine and cocaethylene. The experimental system was the single, isolated perfused cotyledon of a normal term human placenta, and antipyrine served as a freely diffusible marker. Cocaine was transferred rapidly by the placenta at a rate about 80% that of antipyrine. The transfer had characteristics of passive transport consistent with the high lipid solubility of the drug. We found no evidence of significant placental metabolism of cocaine during its rapid placental transfer. Ethanol did not alter the cocaine transfer rate. Norcocaine and cocaethylene were equally as rapidly transferred. Thus the placenta is no barrier to the transfer of cocaine and its derivatives to the fetus.

Ethanol, oxidative stress, reactive aldehydes, and the fetus.

The fetotoxic effects of maternal ethanol (E) consumption have been documented for over two decades, yet the mechanisms underlying this devastating phenomenon remain uncertain. The wide variety of cellular/biochemical effects of E on fetal tissues is itself a puzzle and strongly suggests that fetotoxic responses to E reflect a multifactorial setting. Many of these responses can be conceptually connected to effects on membrane structure and function. Representative of this, are studies in our laboratory documenting E effects on fetal cell replication, membrane transport systems, membrane fluidity, Na+-K+ pump expression, and EGF receptor expression. Recent studies have provided evidence that oxidative stress may be one mechanism by which E produces these membrane-related events. We initially observed E-induced oxidative stress in cultured fetal rat hepatocytes, the latter exhibiting morphological and biochemical signs of mitochondrial damage. E increased H2O2, O2-, lipid peroxidation products, along with signs of membrane damage. Supplementation with antioxidants or agents that enhance glutathione stores reversed these effects. E was found to inhibit activities of mitochondrial respiratory chain components (a potential source of the enhanced levels of H2O2, and O2-) and this could be reversed by antioxidant treatment. Subsequent studies have documented oxidative stress and membrane lipid peroxidation in fetal brain and liver (gestation day 19) following a two day maternal E consumption and in gestation day 14 and 17 "embryos" immediately following a single dose of E to the pregnant dam. The means by which E can induce oxidative stress in fetal cells is under investigation. We have examined effects of E on activities of key antioxidant enzymes and found no depressant responses. However, the low levels of antioxidants in fetal tissues and an exaggerated response of fetal mitochondria to prooxidant stimulation in vitro, suggest that fetal cells are strongly predisposed to oxidative stress. Additionally, recent studies have suggested that fetal tissues are likewise prone to the formation and subsequent accumulation of at least one toxic lipid peroxidation product, 4-hydroxynonenal. We conclude that maternal E consumption induces oxidative stress in fetal tissues and that this is responsible for some toxic responses to E. Additionally, the low antioxidant defenses in fetal tissues and accumulation of toxic aldehyde products of lipid peroxidation predispose the fetus to oxidative damage.

Ethanol effects on active Na+ and K+ transport in cultured fetal rat hepatocytes.

To define further the influence of ethanol on membranes, its effects on Na+ pump function were studied in monolayer cultures of fetal rat hepatocytes. The effects of ethanol (2 and 4 mg/ml) on total K+ influx, ouabain-sensitive K+ influx, Na+ pump density (from specific [3H]ouabain binding), pump turnover rates and intracellular Na+ were measured following exposure of the cells to ethanol for 1-24 hr. In parallel studies, the effects of ethanol (2 mg/ml) on cell water content and membrane fluidity were measured. Ethanol had no immediate effect on K+ influx, but after 1 hr ethanol in concentrations of 2 and 4 mg/ml decreased the total K+ influx (mumol/10(11) cells/sec) from a control of 8.5 +/- 0.64 to 4.46 +/- 0.50 and 4.09 +/- 0.26 respectively (N = 6 for each experiment; P less than 0.001). This represented the maximum effect of ethanol since after 6 and 24 hr of ethanol treatment the K+ influx had increased towards control levels but remained significantly (P less than 0.01 for 2 mg/ml and P less than 0.001 for 4 mg/ml) below that in control cells even at 24 hr. The decrease in K+ influx reflected a decrease in mean ouabain-sensitive K+ influx from a control of 5.87 to 3.24 and 2.70 (mumol/10(11) cells/sec) after a 1-hr treatment with 2 and 4 mg ethanol/ml medium respectively. Ethanol (2 mg/ml) treatment for 1-hr decreased Na+ pump density (x 10(5) molecules ouabain per cell) from a control of 2.80 +/- 0.30 to 1.70 +/- 0.11 (P less than 0.001). At 6 and 24 hr [3H]ouabain binding showed a pattern similar to that seen with the K+ influx, tending to return to pretreatment levels. There was no change in individual pump turnover rates in the presence of ethanol. Following exposure to ethanol, cellular Na+ content steadily increased over the first 6 hr and then returned to control levels. When corrected for parallel changes in cell volume, however, intracellular Na+ concentration increased by 17% (P less than 0.01) after 1 hr and thereafter remained at this higher level throughout the 24-hr period. Measurements of membrane fluidity showed that it was increased markedly by ethanol at a concentration of 2 mg/ml and that the effect bore a close temporal relationship to the changes in active K+ influx and Na+ pump density. We conclude that ethanol has a depressant effect on hepatic Na+ pump function, resulting in an increase in intracellular Na+ and an eventual gain in cell water.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of aging on hepatic elimination of cimetidine and subsequent interaction of aging and cimetidine on aminopyrine metabolism.

Aging and cimetidine may each impair hepatic microsomal drug metabolism. To test if and by what mechanisms advanced age may increase sensitivity to the inhibitory effects of cimetidine, the interaction of these two factors with aminopyrine metabolism in the rat was studied using a correlative approach. Initial studies using the aminopyrine breath test indicated that a 40 mg/kg dose of cimetidine, i.p., impaired the 14CO2 exhaled by up to 76% more in aged (26-month) than in young (3- to 4-month-old) rats. Using an isolated liver perfusion to dissect out hepatic components of this phenomenon, it was found that various doses of cimetidine impaired aminopyrine clearance to a greater degree (P less than 0.05) in aged than in young livers. However, cimetidine metabolism in this system ranged from 36 to 78% less in aged versus young livers (P less than 0.05). Subsequent in vitro studies indicated that microsomes isolated from aged livers also averaged a 76% lower rate of cimetidine metabolism (P less than 0.05). A fixed cimetidine concentration, however, inhibited aminopyrine demethylation to the same degree in aged versus young rats (P less than 0.05). In vivo pharmacokinetics showed an age-related decrease in both aminopyrine and cimetidine systemic clearance. In the young rat the liver contributed about 30% to total systemic clearance of cimetidine. In the aged rat, all clearance was renal. Despite a decrease in glomerular filtration rate, net tubular cimetidine secretion was well-maintained. Despite this, absence of the hepatic component resulted in decreased overall systemic clearance of the drug in aged rats. It is concluded that (1) the aged rat liver exhibits impaired cimetidine metabolism, resulting in decreased overall systemic clearance of the drug despite normal net renal tubular secretion, (2) there is no age-related enhanced sensitivity to cimetidine of the hepatic microsomal oxidizing system using aminopyrine as the probe drug, and (3) the larger inhibition of aminopyrine metabolism in aged rats following various doses of cimetidine is due to decreased overall cimetidine clearance, resulting in higher concentrations of the inhibitor in the liver of aged rats.

Placental amino acid uptake in normal and complicated pregnancies.

Amino acids are the essential substrates for fetal growth and catabolism. The fetus is dependent on the placenta for the provision of amino acids, the first step being concentration of amino acids within the syncytiotrophoblast for subsequent transfer to the fetus. A reliable technique for the isolation of human syncytiotrophoblast plasma membrane has been described, and the suitability of this preparation for the study of amino acid uptake and membrane transport has been well documented. Using this technique, the microvillous vesicle uptake of alpha-aminoisobutyric acid (AIB), a nonmetabolizable amino acid, has been determined over multiple time points for normal (NL) pregnancies and those complicated by pregnancy-induced hypertension (PIH), non-insulin-dependent diabetes mellitus (NIDDM) and those delivering small-for-gestational-age (SGA) neonates. There was no significant difference in AIB uptake between NL pregnancies and those complicated by PIH or NIDDM. Compared to each of the above, AIB uptake by the SGA group was significantly less at each time point. These results suggest that normal placental amino acid transport mechanisms may be altered in SGA pregnancies. If so, such alterations may interfere with the normal provision of nutrients to the fetus and ultimately contribute to impaired growth in utero.

A comparative evaluation of the transport of H2-receptor antagonists by the human and baboon placenta.

Using a single cotyledon perfusion model, the placental transport of four H2-receptor antagonists, cimetidine, famotidine, nizatidine, and ranitidine, was determined and compared using normal term human and normal preterm baboon placentas. In both the human and baboon placentas, the transport of each agent was similar whether administered singly or in combination with the other drugs. Drug transport was the same in both directions, maternal-to-fetal and vice versa, indicating a lack of preferential transfer. The H2-receptor antagonists were transported at about 40% the rate of the freely diffusable reference compound, antipyrine. There were no significant differences between the human and baboon in any of the parameters of placental function evaluated. Placental glucose and oxygen consumptions, and lactate production were comparable in the human and baboon preparations. The transport and clearance of each of the H2-antagonists were similar in each species.

Azidothymidine (zidovudine) transport by the human placenta.

The diagnosis of Acquired Immunodeficiency Syndrome (AIDS) is increasingly made in pregnant women, and the disease may be transmitted to the fetus. Azidothymidine (AZT, Zidovudine) is the one therapeutic agent of some promise in this condition. As there is no information on the transport of this drug by the human placenta, such studies were carried out using the single cotyledon placental perfusion system and human placental vesicles. AZT crossed the placenta readily and bidirectionally. The transfer rate was about 70% that of a freely diffusible reference marker, antipyrine, and was comparable in both directions. There was no evidence of active or carrier-mediated transport and no glucuronidated metabolites of the drug were identified in either maternal or fetal compartments. The authors believe that the drug crosses the placenta by diffusion, consistent with its lipophilicity and transport into various blood cells.

Ganciclovir transfer by human placenta and its effects on rat fetal cells.

Cytomegalovirus is a common cause of intrauterine infection. Ganciclovir is an accepted therapeutic agent for this infection, but is proscribed in pregnancy, except when there is a life-threatening maternal infection, because of its known teratogenic and embryotoxic effects in experimental animals. There are no such data in humans and the human transplacental transfer of this drug has not been studied. This study defines the rate and mechanism of human-placental ganciclovir transport using maternal-facing syncytiotrophoblast vesicles and the perfused, isolated single-cotyledon system and determines further the effects of ganciclovir on fetal tissue, using cultured rat fetal hepatocytes. Ganciclovir was taken up by the maternal-facing placental membrane by a carrier-dependent, Na-independent system inhibited by adenine, guanine, and acyclovir, but not by cytosine and thymine or thymidine and uridine. By contrast, the overall transfer of the drug by the placenta was passive and without drug metabolism. Therefore, the drug is concentrated initially at the maternal placental surface and then crosses passively into the fetal compartment, with the latter process being rate-limiting. There was little or no toxic effect of high concentrations of ganciclovir on cultured fetal-rat hepatocytes.

Ethanol-induced oxidative stress and enzymatic defenses in cultured fetal rat hepatocytes.

Previously, we have documented an ethanol (E)-induced oxidative stress (OS) in cultured fetal rat hepatocytes (FRH). The cause of this is uncertain, but an inhibition of key antioxidant enzymes could be a/the factor. OS was also observed in fetal liver (FL) during in utero E exposure, but not in maternal liver, a difference that might be related to selectively lower enzymatic defenses in the fetus. Here, we record effects of E on activities of catalase (Cat), superoxide dismutase (Cu, Zn SOD and Mn SOD), glutathione peroxidase (GPX), and glutathione-S-transferase (GST) in FRH isolated from 20-day-old fetuses and exposed to E (2 mg/ml) for up to 24 h and we compare these to adult rat liver data. E treatment decreased fetal liver reduced glutathione (GSH) pools by 23% (p < 0.05) and increased malondialdehyde (MDA) by 14% (p < 0.05) within 24 h of E exposure. E caused an increase in fetal liver Cat by 18%, 32%, and 47% by 3, 6, and 24 h of E, respectively (p < 0.05). A 24-h E exposure increased Cu, Zn SOD by 22% (p < 0.05) and Mn SOD by 21% (p < 0.05). A 24 h E treatment increased GPX by 18% (p < 0.05) and GST by 17% (p < 0.05). Cat in whole FL was 26% of adult (p < 0.05) whereas Cu, Zn SOD and Mn SOD in whole FL were 12% and 11% of adult levels (p < 0.05). GPX and GST in FL were 11% and 28% of adult values (p < 0.05). It is concluded that in FRH, E-induced OS is not caused by impaired activities of these enzymes, but their low basal activities (vs. adult) may predispose the fetus to OS.

Effects of nicotine and nicotine/ethanol on human placental amino acids transfer.

Ethanol abuse and smoking during pregnancy both result in decreased offspring weight. One explanation for this may be impaired placental nutrient transport. This study assessed this possibility utilizing the 4-hr perfused human placental system and human placental vesicles exposed to "physiological," 0.2 microM and large (about 20 microM) nicotine concentrations alone, as well as nicotine combined with ethanol, 200 or 400 mg/ml, for up to 48 hr. Two nonmetabolizable amino acids, alpha-aminoisobutyric acid (AIB) and cycloleucine (CLEU) were used as probes. Nicotine was measured by gas chromatography in the placental perfusion system and vesicles and verified as to concentration. There was no statistically significant evidence of decreased transport of these amino acids with exposure to nicotine alone or nicotine and ethanol together in either test system. Thus, brief exposure to nicotine and ethanol does not impair amino acid transport by the human placenta.

Effect of ethanol on thromboxane and prostacyclin production in the human placenta.

Fetal alcohol syndrome (FAS) is frequently associated with intrauterine growth retardation (IUGR). One cause of ethanol-induced IUGR is thought to be related to increased pressor activity in the human placenta, resulting in decreased oxygenation and nutrient transport to the fetus. Thus, we have investigated the effect of ethanol on paracrine substances, such as thromboxane and prostacyclin, that act as vasoregulators within the intrauterine tissues. In these studies we have utilized the perfused single human cotyledon system to study the effect of ethanol on placental prostanoid production. We assessed the effect of longer (240 min) and more acute (60 min) exposure to ethanol on release of thromboxane B(2) (TxB(2)) and 6-keto-prostaglandin F(1 alpha) (6-keto-PGF(1 alpha)) at the maternal and fetal sides of the placenta. Thromboxane was increased by both longer and shorter ethanol exposure, especially on the fetal side of the placenta. Prostacyclin was essentially unchanged with exposure to ethanol. The thromboxane:prostacyclin ratio also tended to increase with both 60- and 240-min ethanol exposure, but a statistically significant increase was seen only at a few time points. In the 60-min ethanol exposure, an increase in thromboxane was observed both during and following exposure to ethanol. The increase in the thromboxane milieu observed with ethanol exposure may lead, at least in part, to the IUGR which is frequently associated with FAS. Prevention of this effect of ethanol on thromboxane production might be a beneficial intervention for FAS.

Histochemical method for localization of hydrogen peroxide and oxygen radicals in the intact neonatal brain.

The generation of reactive oxygen species has been implicated in the pathogenesis of a wide variety of diseases of the central nervous system. Often these pathological conditions involve damage to specific cell types within selected areas of the brain. Thus, there is a marked need for a method which allows microscopic visualization/detection of these oxygen radicals in discrete brain areas. We are reporting a method to histochemically localize, with single cell resolution, hydrogen peroxide (H2O2) and oxygen radicals in the neonatal brain in vivo. This method expands on the technique developed to visualize H2O2 and the superoxide anion radical (O2-) in isolated perfused organs (e.g., lung, heart) (Bobbs, 1994). With our technique, the intact brain is perfused intracardially with warm oxygenated saline to remove blood, followed by perfusion with buffers containing either iron and diethylenetriaminepentaacetate for the detection of H2O2 or manganese for the detection of oxygen radicals. The free radical oxidizes its respective metal, which in turn oxidizes diaminobenzidine (DAB) to form a brown reaction product which can be visualized using light microscopy.