Toll-like receptor profiling of seven trophoblast cell lines warrants caution for translation to primary trophoblasts.

INTRODUCTION: Excessive placental inflammation is associated with pregnancy complications. Toll-like receptors (TLRs) are sensors for danger signals from infections and damaged tissue and initiate inflammation. Trophoblasts in the placenta broadly express TLRs. Trophoblast cell lines are used as surrogates for primary trophoblasts for in vitro studies, but the inflammatory translatability of trophoblast cell lines warrants examination. We aimed to assess TLR1-10 gene expression and activation in seven trophoblast cell lines and compare this to primary trophoblasts. METHODS: The five choriocarcinoma trophoblast cell lines BeWo, JAR, JEG-3, AC1M-32 and ACH-3P, and the two SV40 transfected trophoblast cell lines HTR-8/SVneo and SGHPL-5 were included and compared to primary first trimester trophoblasts (n = 6). TLR1-10 gene expression was analyzed by RT-qPCR. Cells were stimulated by specific TLR1-9 ligands for 24 h and cytokine release was measured by a 10-plex immunoassay. RESULTS: All choriocarcinoma cell lines demonstrated broad TLR gene expression, but lacked functional cytokine response to TLR ligand activation. In contrast, SV40 transfected cell lines showed restricted TLR gene expression, but SGHPL-5 cells displayed significantly increased levels of interleukin (IL)-6, IL-8, IL-12 and vascular endothelial growth factor A after TLR3 and/or TLR4 activation (P < 0.01), while TLR2 activation increased IL-6 and IL-8 levels (P < 0.05). HTR8/SVneo cells responded to TLR3 activation by increased IL-6 and interferon (IFN)-γ (P < 0.05). The SGHPL-5 TLR profile most closely resembled primary trophoblast. DISCUSSION: The characterized trophoblast cell line TLR profiles serve as a reference and warrant caution when selecting trophoblast cell lines as in vitro models for immune responses in primary trophoblasts.

Matrix metalloproteinase 1 in pre-eclampsia and fetal growth restriction: reduced gene expression in decidual tissue and protein expression in extravillous trophoblasts.

Superficial invasion of extravillous trophoblasts (EVTs) and impaired spiral artery remodelling are characterizing phenomena in pregnancies complicated by pre-eclampsia (PE) and fetal growth restriction (FGR). However, the underlying causes remain unclear. In this study, gene expression in decidua basalis tissue from pregnancies complicated with PE and/or FGR (n = 18) and normal pregnancies (n = 17) was assessed by Affymetrix HG Focus microarray to obtain hints of mechanisms involved in the pathogenesis. A total of 200 differentially expressed transcripts were detected at a false discovery rate (FDR)

Chronic in utero morphine exposure alters mu-agonist-stimulated [35S]-GTPgammaS binding in neonatal and juvenile guinea pig brainstem regions associated with breathing control.

Narcotic analgesics act acutely upon opioid receptors in the brainstem to depress respiration in neonates, whereas withdrawal from chronic in utero exposure to morphine is associated with hyperventilation in the newborn. Because of the association between breathing and exposure to exogenous opioids, opioid-stimulated [35S]-guanylyl-5'-O-(gamma-thio)-triphosphate ([35S]-GTPgammaS) binding was examined in respiratory control areas of the brainstem in 3-, 7-, and 14-day-old guinea pigs who were exposed during the last 2 weeks of gestation by maternal subcutaneous injections of 7.5 mg/kg of morphine or an equal volume of saline twice daily. Ten micromolar D-ala(2), MePhe(4), Gly(ol)(5)-enkephalin (DAMGO), a mu-specific opioid receptor agonist, stimulated [35S]-GTPgammaS binding in the caudal and rostral ventral respiratory groups (cVRG and rVRG), nucleus tractus solitarius (NTS), and the parabrachial nucleus (PB). There were no statistically significant age-related changes in DAMGO-stimulated binding for any of the respiratory areas. However, morphine-exposed animals had decreased DAMGO-stimulated [35S]-GTPgammaS binding in these brainstem areas compared to saline-exposed animals. We hypothesize that this morphine-induced decrease in DAMGO activation of the mu-opioid receptor may be a partial explanation for the hyperventilation observed during neonatal morphine withdrawal.

Mu opioid receptor efficacy and potency of morphine-6-glucuronide in neonatal guinea pig brainstem membranes: comparison with transfected CHO cells.

The major side effect of morphine and its active metabolite, morphine-6-glucuronide (M6G), is respiratory depression, which is mediated by mu opioid receptors in the medulla and pons. Although the effect of morphine on coupling between mu opioid receptors and G proteins has been studied, the effect of M6G on this coupling has not. Therefore, stimulation of guanylyl-5'-O-([gamma(35)S]-thio)-triphosphate ([(35)S]-GTPgammaS) binding by these two narcotic analgesic drugs was compared to the mu-specific synthetic opioid peptide [D-Ala(2), N-MePhe(4), Gly-ol(5)]enkephalin in Chinese hamster ovarian cells stably transfected with the murine mu opioid receptor and in brainstem membranes prepared from 3-, 7-, and 14-day-old guinea pigs. All three agonists stimulated [(35)S]-GTPgammaS binding in transfected cells and neural tissue, and the stimulation was antagonized by naloxone. In brainstem membranes, but not transfected cells, M6G was less efficacious but more potent than morphine, which may be due to differences between murine and guinea pig mu opioid receptors or in the G proteins in these two tissues. Efficacy of the agonists did not change during development, but overall potency decreased between 3 and 14 days after birth. In vivo potency differences for respiratory depression between morphine and M6G are qualitatively similar to in vitro potency differences of these drugs to stimulate [(35)S]-GTPgammaS binding in neonatal guinea pig brainstem membranes. Tolerance to opioid effects on [(35)S]-GTPgammaS binding developed in transfected cells incubated with morphine with the maximum decrease in potency occurring 18 h later than the maximum decline in efficacy.

Morphine metabolism in the pregnant guinea pig and her pups.

The study of chronic in utero exposure to heroin and morphine in the human is limited by polysubstance abuse. The guinea pig was used as a model for the human to determine the in vivo and in vitro effect of chronic morphine exposure on morphine metabolism in the pregnant dam and her offspring. In vivo pharmacokinetics of morphine were examined in pregnant guinea pigs following pretreatment with either saline or morphine. In vitro hepatic enzyme kinetics were also examined in a similar group of pregnant dams and their fetuses. Additional pregnant dams were allowed to give birth and their pups' enzyme kinetics were studied at 1, 3, and 7 days. Apparent V(MAX) for the formation of both morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) formation was significantly increased in the morphine-treated pregnant guinea pig. However, no effect of morphine treatment was detectable on the in vivo pharmacokinetics of morphine in the pregnant dam. The apparent morphine K(M) for the formation of M3G was significantly different than the apparent K(M) for the formation of M6G. Significant age effects on the enzyme kinetics were found. The apparent V(MAX) for the formation of both glucuronides increased through the neonatal period. Through literature comparisons, the guinea pig was shown to have in vivo pharmacokinetics similar to the pregnant human, and the guinea pig pups were found to have enzyme development consistent with in vivo pharmacokinetic development seen in human neonates, infants and children.

Morphine regulation of a novel uridine diphosphate glucuronosyl-transferase in guinea pig pups following in utero exposure.

The uridine diphosphate glucuronosyltransferases (UGTs) catalyze conjugation reactions between various substrates and glucuronic acid, UDPGA (uridine diphosphate glucuronic acid), within the endoplasmic reticulum. Conjugation with UDPGA (glucuronidation) is an important pathway in the elimination, detoxification, and activation of compounds including steroid hormones, xenobiotics, and quaternary ammonium substrates. The guinea pig, which has a placental structure and a glucuronidation profile for morphine that are similar to the human, serves as a good small animal model to study the ontogeny of UGTs and the effect of in utero exposure to morphine on UGTs. We examined type 2 UGTs expressed in the guinea pig using amplification and cloning of partial cDNAs from liver RNA. Sequence analysis revealed a novel UGT2 (subsequently named UGT2A3),(2) that has a 64% amino acid sequence similarity to a known UGT2.(3) Full-length cDNAs were isolated from a guinea pig liver cDNA library. Tissue distribution of UGT2A3 using Northern blot analysis showed expression of three distinct size UGT2A3 mRNAs with unique expression in liver and small intestine. UGT2A3 mRNA is expressed at high levels in liver and lower levels in kidney and small intestine. In utero exposure to chronic intermittent morphine resulted in the up regulation of mRNA in 7-day-old female pups' liver and kidney as determined by quantitative RT-PCR analysis. The conjugation profile for UGT2A3 using stable expression in CHO cells and thin-layer chromatography demonstrated active conjugation of phenolic substrates. Regulation of UGTs by in utero morphine exposure may play an important role in fetal development.

Chronic intermittent in utero exposure to morphine: effects on respiratory control in the neonatal guinea pig.

This study was done to determine if chronic intermittent in utero exposure to morphine (MOR) during the last half of gestation results in hypoventilation and decreased ventilatory sensitivity to CO2 in the neonate. Pregnant guinea pigs were randomly assigned to once-daily treatment with saline and 1.5, 5.0, or 15.0 mg/kg MOR. Neonates were studied for 3 weeks. Prenatal exposure to 5.0 and 15.0 mg/kg MOR significantly increased neonatal minute ventilation and central inspiratory drive on day 7 while breathing room air or 5% CO2. The increase in minute ventilation was part of a withdrawal syndrome that included increased locomotor activity, but was not due to an increase in metabolic rate or sensitivity to CO2. We conclude that neonatal guinea pigs exposed once daily to MOR during the last half of gestation hyperventilate during the 1st week after birth. These changes are neither permanent nor followed by hypoventilation or depressed sensitivity to CO2.

In vivo pharmacokinetics and in vitro production of cocaethylene in pregnant guinea pigs.

Simultaneous exposure to cocaine and ethanol results in the formation of cocaethylene, an active metabolite of cocaine. The concurrent abuse of both cocaine and ethanol is common during human pregnancy, but the kinetics of elimination and formation of this ethyl ester of cocaine have not been studied during pregnancy in any species. In the late gestation guinea pig (61 to 63 days), cocaethylene, at doses of 2 to 4 mg.kg-1, is rapidly eliminated with a half-life of 29 min and a total body clearance of 77 ml.min-1.kg-1. It is formed enzymatically by hepatic microsomal preparations from fetal, neonatal and maternal guinea pigs. The maximum rate of cocaethylene production (apparent Vmax) when either ethanol or cocaine are varied while the other substrate is held constant, increases with age, from the late fetal period (65 days gestation, term 70 days) to adulthood. However, the Michaelis-Menten constant (apparent KM) does not change with age. The rapid elimination of cocaethylene, coupled with the slow rate of formation (apparent Vmax of 140 pmol.min-1.mg microsomal protein-1) and the small amount of plasma analyzed most likely explains the inability to detect coacethylene in vivo after concomitant cocaine and ethanol administration.

Developmental change of mu opioid receptors in neonatal guinea pig brain stem.

Opioid receptor binding of morphine-6-beta-D-glucuronide (M6G), morphine and [3H][D-Ala2,N-methylPhe4-Glyol5]enkephalin (DAMGO) were determined in neonatal guinea pigs. Pontomedullary membranes specifically bound [3H]DAMGO, which was displaced by M6G and morphine. The KI for M6G and morphine were 15.1 nM and 5.0 nM, respectively, and did not change between day 3 and day 7 after birth. KD for [3H]DAMGO binding was constant (1.1nM), however Bmax increased from 62.2 to 88.3 fmol/mg protein between days 3 and 7 (P < 0.01). This 42% increase in mu receptors may play a role in the increased potency of M6G respiratory effects for guinea pigs during the first week after birth.

Furosemide pharmacokinetics following intratracheal instillation in the guinea pig.

Inhaled furosemide has been shown to attenuate bronchospasm in asthmatics and to increase lung compliance in infants with bronchopulmonary dysplasia (BPD). The reports involving BPD used a dose of 1 mg/kg and some have failed to show an effect with that dose. We determined the pharmacokinetics of furosemide administered directly to the airway in 7 young adult male guinea pigs who received intravenous and intratracheal doses of furosemide. Each animal received a 3 mg/kg i.v. bolus, 1, 3 and 6 mg/kg i.t. in 2 ml/kg normal saline and 3 mg/kg i.t. in 2 ml/kg bovine extract surfactant. Blood was sampled multiple times after each dose. The mean fraction of the intratracheal dose absorbed was 0.50-0.60 for all doses. Surfactant delayed the absorption of furosemide but did not alter the fraction absorbed.

Neonatal ventilatory changes following in utero cocaine exposure: a minireview of the guinea pig and rabbit models.

Only three animal studies have been published on the consequences of prenatal cocaine exposure for neonatal ventilation. The one in guinea pigs examines the ventilatory response to carbon dioxide inhalation and two in rabbits consider the response to hypoxia. Cocaine exposure during intrauterine development causes modest and reversible increases in room air breathing and the ventilatory response to carbon dioxide in the neonatal guinea pig. These changes were not dose-dependent over the range studied. Increase of ventilation while breathing room air may be due to drug-related increases in oxygen consumption or neural output. Increased sensitivity to carbon dioxide inhalation, however, is most likely due to an increase in central respiratory drive. Changes in ventilation may be related to persistence of active metabolites or to cocaine withdrawal. In the first published cocaine study in newborn rabbits, ventilation while breathing room air was not altered, whereas the ventilatory response to hypoxia was absent compared to controls. The second study by the same authors, which differed from the first in several respects, again suggested that cocaine did not alter room air breathing in the neonates. A ventilatory response to hypoxia was observed in the cocaine exposed neonates and was even greater than that in control animals. However, the response to severe, prolonged hypoxia in the cocaine-exposed pups was not as effective in maintaining oxygen saturation of blood and heart rate compared to control animals. This finding suggests that total body oxygen consumption and/or cardiovascular function is affected by cocaine in addition to the alteration in ventilation.(ABSTRACT TRUNCATED AT 250 WORDS)

Potential mechanisms of cocaine-induced developmental neurotoxicity: a minireview.

There is great concern about the health effects of cocaine use during pregnancy. Cocaine alters maternal physiology and crosses the placenta to interact with fetal tissues including the brain. Neurotoxicity, defined as structural and/or functional changes which result in a neurobehavioral deficit, is not unequivocally documented in the human. It is difficult to ascertain duration, intensity and frequency of cocaine exposure in humans as well as the effects of multiple drug use, poor nutrition, lack of prenatal care, lead exposure and infectious diseases. In addition, plasticity of the central nervous system makes the postnatal environment as important as the intrauterine milieu for the developing organism. Animal studies, which allow quantitation of drug exposure and reduction of confounding variables, suggest several possible mechanisms for neurotoxicity induced by cocaine or its active metabolites. The possible mechanisms include: alteration of sodium channel and monoamine transporter development, release of epinephrine from the adrenal medulla with subsequent hyperglycemia, vasoconstriction with subsequent hypoxia and decrease of nutrient supply, calcium ion chelation, superoxide formation or infarction following repeated ischemia and reperfusion, enzyme inhibition, reduced neurotrophic activity, altered gene expression and plasma membrane changes. Alterations in the above parameters may cause acute and reversible effects as well as chronic and permanent effects. Not all alterations in structure and function are deficits, and no single mechanism may explain a given alteration.

Pharmacokinetics and metabolism of cocaine in maternal and fetal guinea pigs.

The pharmacokinetics and metabolism of cocaine (COC) were determined in late gestation maternal and fetal guinea pigs. After a single i.v. dose of 2-12 mg/kg, the average +/- SD total body clearance of COC was 59 +/- 16 ml/min/kg and was not dose dependent. However, volume of distribution was 2.1 and 3.9 l/kg, mean resident time (MRT) was 42 and 57 min, and elimination half-life was 34 and 49 min at the 2 and 4 mg/kg dose of COC, respectively. With the exception of an increased MRT, the pharmacokinetics were similar after s.c. COC administration. Benzoylecgonine (BE) and benzoylnorecgonine (BN) were major and persistent metabolites. Norcocaine (NOR) concentrations were low and transient. After chronic maternal administration of 6 mg/kg COC s.c., there was no difference between maternal and fetal plasma COC concentrations one hour after the last injection, but COC and BN accumulated in amniotic fluid. Examination of in vitro metabolism of COC in fetal and maternal guinea pig hepatic microsomes demonstrated minimal fetal N-demethylation and induction of maternal N-demethylation by chronic COC exposure. The minimal fetal N-demethylation suggest BN seen previously in vivo after chronic maternal COC administration resulted from maternal formation of NOR and subsequent maternal and/or fetal hydrolysis to BN.

Placental permeability for drugs of abuse and their metabolites.

Placental passage of drugs, including peptides, is determined by the physical and chemical properties of the drug and the physiology of the placenta. Fetal blood concentrations of drugs administered to the mother depend upon time after maternal drug administration, dose, placental blood flow and permeability, plasma protein binding, blood pH, placental biotransformation, and fetal elimination.

Effects of morphine and cocaine on breathing control in neonatal animals: a minireview.

From observational studies in humans and experimental studies in animal models, there is a strong indication that prenatal exposure to morphine or cocaine causes at least transient abnormalities of breathing control in the neonate. If these abnormalities persist, sudden death may occur in the infant. Mechanisms responsible for alterations in breathing control by drugs of abuse and the relationship of these alterations to SIDS are not proven at this time. The complex nature of breathing control and neurodevelopment, as well as the confounding variables in human drug abuse, have so far prevented a definitive understanding of the link between drug abuse during pregnancy and SIDS. Further investigations are required to explore the biology of this important public health problem.

Diffusion of morphine-6-beta-D-glucuronide into the neonatal guinea pig brain during drug-induced respiratory depression.

Morphine-6-beta-D-glucuronide (M6G) is an active metabolite of morphine. In a previous study, M6G depressed respiration in the neonatal guinea pig, becoming more potent with aging, a finding that is confirmed in the current study. After s.c. injection, M6G is absorbed into plasma, crosses the blood-brain barrier and is present in the central nervous system at the time of maximal M6G-induced ventilatory depression. No hydrolysis of M6G to morphine is detected in either plasma or brain tissue by high-performance liquid chromatography. About 30% more M6G is in plasma in 3-day-old than in 7-day-old pups after drug administration (P < .05). Mean brain concentrations of M6G are 12% higher on day 3 than day 7, but the difference is not statistically significant. Brain-to-plasma ratios of M6G do not differ after 5 or 15 mg/kg of M6G or with age (mean ratio = 0.037). Brain drug concentration is a linear function of plasma drug levels (r2 = 0.84), suggesting M6G crosses the blood brain barrier by diffusion. Differential systemic absorption or central nervous system distribution of M6G cannot explain enhanced respiratory depression with aging. Morphine-3-beta-D-glucuronide (M3G) also crossed the blood-brain barrier, but is less permeable than M6G (mean brain-to-plasma ratio = 0.022). Contrary to reports in the literature, M3G at a dose of 75 mg/kg, does not stimulate respiration in this study. Morphine administration to neonatal guinea pigs produces measurable plasma and brain levels of M6G and M3G.(ABSTRACT TRUNCATED AT 250 WORDS)

Cocaine metabolites in the neonate: potential for toxicity.

Recent reports indicate that cocaine metabolites have biologic activity and could be toxic. To explore this possibility, two studies were initiated. The first study aimed to define the distribution of cocaine species by quantifying levels of cocaine and its metabolites norcocaine, benzoylecgonine, and benzoylnorecgonine in newborn cord blood and meconium. The second study sought to determine whether they produced a clinical effect. Compared to cord blood, meconium had a greater number of metabolites and a higher concentration of cocaine metabolites, including the previously undetectable norcocaine and benzoylnorecgonine derivatives. Benzoylecgonine was the most common species found in both sources and was usually lower in concentration in blood. An inverse relation existed between meconium benzoylecgonine levels and the serum catabolic enzyme pseudocholinesterase, implying genetic variability in cocaine metabolism. To determine whether cocaine and/or its metabolites could be linked to a distinct clinical state, a second study focusing on newborn behavior was performed with an independent large cohort of cocaine-exposed infants. Neonates with increased signs of "neuroexcitation" had benzoylecgonine and no cocaine in urine, whereas lethargic neonates had detectable urinary cocaine. These findings support the hypothesis that cocaine metabolites, especially benzoylecgonine, may play a role in altering newborn behavior and produce a clinical syndrome distinct from that related to the parent compound.

Morphine-6-beta-D-glucuronide respiratory pharmacodynamics in the neonatal guinea pig.

Morphine-6-beta-D-glucuronide (M6G) is a metabolite of morphine with opioid activity in adults. No data are available, however, on the developmental pharmacology of M6G including investigation of the respiratory effects of M6G in the neonate. A randomized, placebo-controlled study comparing the time-action, dose-response and potency of the respiratory effects of M6G to morphine was done using a nonanesthetized neonatal guinea pig model and a noninvasive computerized plethysmograph technique. Respiration was measured while the neonate breathed room air followed by 5% CO2 in air. M6G (0.5-5.0 mg/kg) and morphine (1.5-15 mg/kg) administered subcutaneously decreased ventilation in 3-, 7- and 14-day-old neonatal guinea pigs given a 5% CO2 challenge. During CO2 inhalation, time-to-peak action for M6G occurred 21 min later than for morphine. At maximal ventilatory depression on day 3, a dose of 1.5 mg/kg morphine or M6G decreased minute ventilation while breathing 5% CO2 by 30% compared to placebo. Ventilation also decreased as a function of age in both placebo and drug-treated animals. The percent respiratory depression relative to placebo remained constant for a given dose of morphine as the neonate aged, but not for M6G, which increased in potency. M6G was equipotent to morphine on day 3 after birth, but was 8-fold more potent by day 7. This increase in potency persisted through day 14. The increased potency of M6G that accompanies aging may be caused by either a change in M6G disposition or a change in opioid receptors during development of the neonatal guinea pig.

Quantitation of benzoylnorecgonine and other cocaine metabolites in meconium by high-performance liquid chromatography.

A method for simultaneous extraction of cocaine and metabolites benzoylnorecgonine, benzoylecgonine and norcocaine from meconium was developed. The procedure uses solid-phase extraction columns with both cation-exchange and hydrophobic properties after vortex-mixing meconium with methanol. Chromatography utilizes reversed-phase high-performance liquid chromatography with a C18 column and phosphate buffer-acetonitrile as mobile phase. The method is specific and sensitive to 50 ng/g meconium for all compounds. Standard curves are linear from 0.05 to 5.0 micrograms/g (r2 > or = 0.989). Intra-assay coefficients of variation were < or = 6.9%. Meconium from infants exposed to cocaine in utero contained varying combinations of the four drugs.