Confirmation of the role of N-acetyltransferase 2 in teratogen-induced cleft palate using transgenics and knockouts

Previous work on Dilantin- and hydrocortisone-induced cleft palate and cleft lip with or without cleft palate using congenics for the N-acetyltransferase loci (Nat1 and Nat2 are closely linked) and recombinant inbred lines implicated the Nat1,2 region in susceptibility to teratogen-induced orofacial clefting. Since Nat1 does not differ between the two strains, Nat2 appeared to be responsible. We have now tested this conclusion using transgenics and knockouts. Transgenics for human NAT1 (equivalent to mouse Nat2) and knockouts for Nat2 were tested for susceptibility to Dilantin, hydrocortisone, and 6-aminonicotinamide-induced orofacial clefting. We found that Nat2 greatly influences teratogen-induced orofacial clefting on the A/J background but not on the C57BL/6J background. The magnitude and direction of the effects depended on which teratogen was used. The Nat2 knockout did not make C57BL/6J susceptible or A/J (already with very low activity) more susceptible but significantly decreased sporadic clefting in the A/J strain. We conclude that only the A/J strain, with several loci affecting orofacial clefting, is influenced by Nat2.     

Correlation of susceptibility to 6-aminonicotinamide and hydrocortisone-induced cleft palate

Our previous genome-wide Quantitative Trait Locus (QTL) mapping study using mouse A/J by C57BL/6J recombinant inbred (RI) lines suggested several chromosomal regions contain genes influencing susceptibility to phenytoin (PT)-induced cleft lip with or without cleft palate [CL(P)] and 6-aminonicotinamide (6-AN)-induced isolated cleft palate (CP). Importantly, the same chromosomal regions but different RI parental strain alleles were sometimes implicated in susceptibility to these different kinds of orofacial clefting. Here we report the susceptibility to hydrocortisone (HC)-induced CP in these RI lines. We treated pregnant females with HC and studied the incidence of CP in day 17 fetuses. RI lines showed highly correlated responses to HC and 6-AN. The A/J parental line and five RI lines showed very high levels of clefting in response to both of these teratogens. The C57BL/6J parental line and five other RI lines exhibited low incidence of CP for these teratogens. In contrast, there was no significant correlation between incidence of PT-induced CL(P) and HC-induced CP.     

Valproate, thalidomide and ethyl alcohol alter the migration of HTR-8/SVneo cells

Background  

Valproate, thalidomide and alcohol (ethanol) exposure during the first trimester of pregnancy is known to cause several developmental disorders. All these teratogens are known to pass the placental barrier and interfere directly with the normal development of the fetus. However, these teratogens also alter the formation and function of the placenta itself which may in turn affect the proper nourishment and development of the fetus. Optimum development of the placenta requires adequate invasion of trophoblast into the maternal uterine tissues. Changes in the migratory behavior of trophoblast by maternal exposure to these teratogens during placentogenesis may therefore alter the structure and function of the placenta.     

6-Aminonicotinamide-induced cleft palate in the mouse: the nature of the difference between the A/J and C57Bl/6J strains in frequency of response and its genetic basis.

Cleft palate induction by 6-aminonicotinamide (6-AN) was examined in the A/J and C57BL/6J strains of mice to determine the nature of the strain difference in frequency of cleft-palate response. Probit analysis of the cleft-palate response to dose of different genotypes revealed a family of linear and parallel dose-response curves. The genotypes differ only in dosage tolerance (log ED50) to 6-AN that is required for the cleft-palate response. No evidence for a maternal cytoplasmic effect on 6-AN-induced cleft palate was found under the conditions of the present study. When the difference is dosage tolerance to 6-AN between A/J and C57BL/6J was examined with a single dose and measured by differences in frequency of induced cleft palate on a probit scale, there was some departure from genetic additivity. There was an indication off dominance deviation of the F1 embryos in the direction of C57BL/6J.A3-locus, epistatic model is proposed to account for the difference in embryonic tolerance ot 6-AN-induced cleft palate. There was a suggestion of association with the brown (b) locus.     

Background matters: the effects of estrogen receptor alpha gene disruption on male sexual behavior are modified by background strain

One approach to study interactions between behavior and genetics is to use inbred mice with different genetic backgrounds. To examine the effect of background on a specific gene, we conducted a series of experiments with a well-characterized knockout (KO) mouse, the estrogen receptor alpha KO (ERalphaKO). The ERalphaKO mouse has so far been examined in one inbred line, C57BL/6J. Here, we examined the behavior of ERalphaKO mice within three different backgrounds mixed with C57BL/6J; DBA/2J, BALB/c, and A/J. First, we assessed masculine sexual behavior in both intact male and testosterone-treated female offspring. More ERalphaKO males in the DBA/2J (5/12) and BALB/c (5/13) backcrosses displayed intromissions and many ejaculated as compared with males in a C57BL/6J and A/J mixed background. Many fewer ERalphaKO females than males displayed masculine sexual behavior in any of the three hybrid crosses. We assessed fertility in males from the C57BL/6J by DBA/2J cross and found that one of 12 ERalphaKO males sired a litter. Several other characteristics of sexual behavior and physiology were unaffected by genetic background in ERalphaKO mice. Our data suggest that genetic background has dramatic effects on male sexual behavior and its dependence on the ERalpha gene.     

The effect of teratogens on maternal corticosterone levels and cleft incidence in A/J mice.

It is unknown whether orofacial clefting, one consequence of teratogenic exposure, results from a direct interaction between the teratogen and the embryonic palate, or indirectly from maternal alterations caused by the teratogen. In the current study pregnant A/J mice were exposed to one of three cleft-inducing agents in order to examine the relationship between drug-induced clefting and the response of maternal plasma corticosterone to drug administration. The agents used, haloperidol (HAL), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), or phenytoin (PHT), were administered in teratogenic doses between 0800 and 0930 on gestational day 10 (GD 10). For corticosterone determinations, mice were dosed on GD 10, and blood was collected at 1, 4, 24, or 48 hr after dosing. For fetal evaluation of cleft lip and/or cleft palate, mice were dosed on GD 10 and killed on GD 18. Phenytoin was the most potent inducer of cleft lip and palate and induced a sustained elevation of plasma corticosterone in maternal animals. The other treatments, in order of decreasing potency to induce clefting and/or cause an elevation of corticosterone in plasma were 2,4,5-T > HAL > controls. Correlations between maternal corticosterone levels and clefting incidence were very high at all time points examined; total exposure (area under the curve) was also highly correlated. A linear relationship between drug-induced increases in maternal corticosterone levels and the incidence of clefting in A/J mice was evident. Based on these findings, we believe that increased maternal corticosterone levels may play a role in orofacial clefting in A/J mice.     

Calcium transport genes are differently regulated in maternal and fetal placenta in the knockout mice of calbindin-D(9k) and -D(28k)

Calbindin-D(9k) (CaBP-9k) and -D(28k) (CaBP-28k) are cytosolic proteins with EF-hand motifs that have a high affinity for calcium ions. Many types of calcium channels and intracellular calcium binding proteins, such as sodium/calcium exchangers (NCXs) and transient receptor potential cation channels (TRPVs), have been detected in the placenta. In this study, the expression of calcium channels involved in maternal-fetal calcium transport were investigated in wild-type mice versus CaBP-9k, CaBP-28k, and CaBP-9k/28k double knockout (KO) mouse models. The expression of calcium transport genes in three dissected sections of the placenta (maternal, central, and fetal) was examined on gestational day 19 (GD 19). The expression of CaBP-9k, TRPV6, TRPV5, and NCX1 mRNA was high in fetal compared to maternal placenta, while CaBP-28k was abundant in the maternal placenta. CaBP-9k was enhanced in all sections of placenta in CaBP-28k KO mice, whereas CaBP-28k was reduced in CaBP-9k KO mice. The expression of TRPV6, TRPV5, and NCX1 were induced in both maternal and fetal placentas in CaBP-9k KO mice, but were upregulated in maternal and central placentas of CaBP-28k KO mice. The levels of these proteins showed similar patterns with those of their mRNA. Placental CaBP-9k, TRPV6, TRPV5, and NCX1 proteins were abundantly expressed in the intraplacental yolk sac located in the fetal placenta. CaBP-28k did not colocalize with other calcium transport genes, although it was enriched in the placental trophoblasts of the decidual zone in the maternal placenta. These results indicate that placental TRPV6, TRPV5, and NCX1 compensate for CaBPs in CaBP-9k and/or CaBP-28k KO mice, and may take over the roles of CaBP-9k and CaBP-28k to transfer calcium ions in the placenta. Taken together, these results indicate that TRPV6, NCX1, and CaBP-9k in the fetal placenta and CaBP-28k in the maternal placenta may play key roles in controlling calcium transport across the placenta during pregnancy.     

Placenta as a selective barrier to cellular traffic

Transplacental passage of cells from mother to fetus during murine pregnancy was examined by using glucose phosphate isomerase (GPI) or fluorescein tagging as markers. Female mice of strains (BALB/cCr X C3H/HeJ)/F1 or (A/J X C57BL/6J)F1 (both Gpi-1a/b) were mated to the corresponding Gpi-la males (BALB/cCr or A/J, respectively). Cells of the liver, blood, and/or spleen in the offspring were typed at days 15, 16, 17, or 18 of gestation, the day of delivery, or 1 day postpartum. Only two of 172 Gpi-1a/a mice obtained from these matings showed evidence of maternal cell trafficking. Sensitivity of the assay was 1% Gpi-1a/a population. Fluorescein-labeled BALB/cCr peripheral red blood cells (RBC) or white blood cells (WBC) were injected i.v. into syngeneically mated BALB/cCr mothers on day 18. After 24 hr, the blood or liver of the neonates was formalin-fixed and examined in the fluorescence-activated cell sorter (FACS). Some RBC crossed the placenta, but WBC were usually not detected in fetal liver in significant numbers. This technique was very sensitive, and we estimated that no more than 225 WBC could enter the fetus via this route. Thus, we conclude that passage of significant numbers of maternal WBC into the fetus is rare, and perhaps this passage is related to a placental abnormality.     

Transport of maternal cholesterol to the fetus is affected by maternal plasma cholesterol concentrations in the Golden Syrian hamster

The fetus has a high requirement for cholesterol and synthesizes cholesterol at elevated rates. Recent studies suggest that fetal cholesterol also can be obtained from exogenous sources. The purpose of the current study was to examine the transport of maternal cholesterol to the fetus and determine the mechanism responsible for any cholesterol-driven changes in transport. Studies were completed in pregnant hamsters with normal and elevated plasma cholesterol concentrations. Cholesterol feeding resulted in a 3.1-fold increase in the amount of LDL-cholesterol taken up by the fetus and a 2.4-fold increase in the amount of HDL-cholesterol taken up. LDL-cholesterol was transported to the fetus primarily by the placenta, and HDL-cholesterol was transported by the yolk sac and placenta. Several proteins associated with sterol transport and efflux, including those induced by activated liver X receptor, were expressed in hamster and human placentas: NPC1, NPC1L1, ABCA2, SCP-x, and ABCG1, but not ABCG8. NPC1L1 was the only protein increased in hypercholesterolemic placentas. Thus, increasing maternal lipoprotein-cholesterol concentrations can enhance transport of maternal cholesterol to the fetus, leading to 1) increased movement of cholesterol down a concentration gradient in the placenta, 2) increased lipoprotein secretion from the yolk sac (shown previously), and possibly 3) increased placental NPC1L1 expression.     

Antinociceptive effects of morphine-6-glucuronide in homozygous MDR1a P-glycoprotein knockout and in wildtype mice in the hotplate test

Morphine-6-glucuronide (M6G), a major metabolite of morphine with agonist opioid-receptor activity, was reported to be a substrate of P-glycoprotein (P-gp). Inhibition of P-gp may thus result in higher brain uptake of M6G. The goal of this observer-blinded, placebo controlled study, was to compare the antinociceptive effects of M6G in homozygous P-gp knockout (mdr1a(-/-)) and wildtype (mdr1a(+/+)) mice. M6G was injected intraperitoneally as a single dose of 0, 0.5, 1, 2.5, 5, and 10 mg/kg. Eight P-gp knockout and eight wildtype mice were studied per dose. A hot plate test was performed before and 5, 15, 30, 60, 90, 120, and 150 min after M6G administration. Plasma-concentrations of M6G, morphine, and morphine-3-glucuronide (M3G) were measured after intraperitoneal injection of 5 mg/kg M6G in another 14 P-gp knockout and 14 wildtype mice. No difference neither in the dose response relationship, nor in the time course of response latency times were observed between P-gp knockout and wildtype mice. However, latency times increased with higher doses of M6G, with antinociception significantly different from placebo at a M6G dose of 5 and 10 mg/kg. P-gp knockout mice tended to have higher plasma concentrations than the wildtype. However, plasma concentrations widely overlapped between groups and therefore no statistical significant group difference could be detected. We conclude that despite reported doubling of M6G brain uptake, absence of mdr1a coded P-gp does not enhance antinociceptive effects of M6G in the hotplate test after acute single-dose administration in mdr1a(-/-) knockout mice.     

Genetic Differences in the Duration of the Lymphocyte Heat Shock Response in Mice

Lymphocytes from adult mice bearing a known difference in genetic susceptibility to teratogen-induced exencephaly (SWV/SD, and DBA/2J) were evaluated for changes in protein synthesis following an in vivo heat treatment. Particular attention was paid to changes indicative of the heat shock response, a highly conserved response to environmental insult consisting of induction of a few, highly conserved proteins with simultaneous decreases in normal protein synthesis. The duration of heat shock protein induction in lymphocytes was found to be increased by 1 hr in the teratogen-sensitive SWV/SD strain as compared to the resistant DBA/2J strain. Densitometric analysis revealed a significant decrease in the relative synthesis of at least two non-heat shock proteins (36 kD and 45 kD) in the SWV/SD lymphocytes as compared to DBA/2J cells. The increased sensitivity of protein synthesis to hyperthermia in the SWV/SD lymphocytes were lost in the F1 progeny of reciprocal crosses between SWV/SD and DBA/2J mouse strains. Sensitivity to hyperthermia-induced exencephaly is recessive to resistance in these crosses. The relationship between altered protein synthesis and teratogen susceptibility is discussed.     

Effect of indomethacin on alcohol-induced morphological anomalies in mice

The purpose of the present study was 1) to examine the effect of indomethacin (INDO), a prostaglandin synthesis inhibitor, on alcohol-induced growth and morphological impairment in C57BL/6J mice (Study 1) and 2) to determine if INDO crosses the placenta (Study 2). On day 10 of gestation, mice were injected (s.c.) acutely with either 0, 5, 10, or 20 mg/kg INDO, followed one hour later by alcohol (5.8 g/kg orally) or isocaloric sucrose. Fetuses were removed on day 19 of pregnancy, weighed, and examined for anomalous development. As expected, Study 1 demonstrated that maternal alcohol treatment decreased fetal weight and increased the number of fetuses with birth defects. INDO alone decreased fetal weight but did not affect morphologic development. More importantly, INDo antagonized alcohol-induced birth defects, but only at the highest dose. The results of Study 2 suggest that the relative ineffectiveness of INDO may be related to its inability to readily cross the placenta. Since high doses of INDO also caused maternal toxicity, the usefulness of this compound in future studies of this type was questioned.     

Placental essential fatty acid transport and prostaglandin synthesis

The studies reported here demonstrate two important aspects of placenta EFA transport and metabolism. (1) A mechanism exists within the placenta for the selective incorporation of 20:4 omega 6 into phosphoglycerides and the export of those phosphoglycerides to the fetal circulation. This mechanism allows the selective sequestering of 20:4 omega 6 in the fetoplacental unit and may provide the fetus with important performed structural membrane components. (2) Placental PG synthesis is directed mostly to the maternal circulation and stimulated placental PG synthesis is directed totally to the maternal circulation. This mechanism may protect the fetus from fluctuations in maternal and placental PG synthesis and may direct stimulated placental PG synthesis to a target organ, the myometrium. The perfused human placental model provides a valuable method for the study of a variety of biochemical phenomena in a whole human organ and its use may further elucidate the role of this tissue in the maintenance of pregnancy, the transport of EFA to the developing fetus and the involvement of placental PG synthesis in fetal development and parturition.     

Placenta-derived soluble MHC class I chain-related molecules down-regulate NKG2D receptor on peripheral blood mononuclear cells during human pregnancy: a possible novel immune escape mechanism for fetal survival

Mammalian pregnancy is an intriguing immunological phenomenon where the semiallogeneic fetus is not rejected. Tolerance toward the fetus involves a number of mechanisms associated with modifications of the immune status of the mother. In this study, we strongly suggest a novel mechanism for fetal evasion of maternal immune attack, based on the engagement and down-regulation of the activating NK cell receptor NKG2D on PBMC by soluble MHC class I chain-related proteins A and B (collectively termed MIC). A similar immune escape pathway was previously described in tumors. We found that MIC mRNA was constitutively expressed by human placenta and could be up-regulated upon heat shock treatment. Our immunomorphologic studies showed that the MIC expression in placenta was restricted to the syncytiotrophoblast. Immunoelectron microscopy revealed a dual MIC expression in the syncytiotrophoblast: on the apical and basal cell membrane and in cytoplasmic vacuoles as MIC-loaded microvesicles/exosomes. Soluble MIC molecules were present at elevated levels in maternal blood throughout normal pregnancy and were released by placental explants in vitro. Simultaneously, the cell surface NKG2D expression on maternal PBMC was down-regulated compared with nonpregnant controls. The soluble MIC molecules in pregnancy serum were able to interact with NKG2D and down-regulate the receptor on PBMC from healthy donors, with the consequent inhibition of the NKG2D-dependent cytotoxic response. These findings suggest a new physiological mechanism of silencing the maternal immune system that promotes fetal allograft immune escape and supports the view of the placenta as an immunoregulatory organ.     

Dietary fat impacts fetal growth and metabolism: uptake of chylomicron remnant core lipids by the placenta

The fetus requires significant energy for growth and development. Although glucose is a major source of energy for the fetus, other maternal nutrients also appear to promote growth. Thus, the goal of these studies was to determine whether triglyceride-rich remnants are taken up by the placenta and whether maternal dietary lipids, independently of adiposity, can impact fetal growth. To accomplish our first goal, chylomicron particles were duallly labeled with cholesteryl ester and triglycerides. The placenta took up remnant particles/core lipids at rates greater than adipose tissue and skeletal muscle but less than the liver. Although the placenta expresses apoE receptors, uptake of chylomicron remnants and/or core lipids can occur independently of apoE. To determine the impact of dietary lipid on fetal growth, independent of maternal adiposity, females were fed high-fat diets (HFD) for 1 mo; there was no change in adiposity or leptin levels prior to or during pregnancy of dams fed HFD. Fetal masses were greater in dams fed HFD, and mRNA levels of proteins involved in fatty acid oxidation (CPT I, PPARα), but not glucose oxidation (pyruvate kinase) or other regulatory processes (HNF-4α, LXR), were increased with maternal dietary fat. There was also no change in mRNA levels of proteins involved in placental glucose and fatty acid transport, and GLUT1 protein levels in microvillous membranes were similar in placentas of dams fed either diet. Thus, the ability of the placenta to take up chylomicron remnant core lipids likely contributes to accelerated fetal growth in females fed high fat diets.     

Embryotoxicity of phenytoin in adrenalectomized CD-1 mice.

It has been proposed that the anticonvulsant drug phenytoin (PHT) and glucocorticoids induce orofacial clefting by the same mechanism. Previous work had demonstrated that PHT treatment significantly increased endogenous maternal corticosterone concentrations for approximately 48 hr after dosing in A/J mice. The purpose of the present investigation was to determine whether PHT is embryotoxic in the absence of endogenous maternal glucocorticoids. Maternal adrenal glands were removed on Day 7 of gestation, and the incidence of clefting after PHT treatment was determined. There was a high level of maternal toxicity following adrenalectomy (ADX) and PHT treatment at either 60 or 75 mg/kg. This increased toxicity did not appear to be due to altered maternal drug levels in ADX mice. There was a significant increase in the clefting incidence among offspring of ADX dams treated with PHT at 60 mg/kg. This dose of PHT did not elevate maternal corticosterone levels in ADX dams. These data suggest that PHT is capable of producing clefts in the absence of endogenous maternal corticosterone.     

3'-azido-3'-deoxythymidine (AZT) transplacental perfusion kinetics and DNA incorporation in normal human placentas perfused with AZT.

Vertical transmission of the human immunodeficiency virus 1 (HIV-1) is reduced from approximately 25% to approximately 7% as a result of 3'-azido-3'-deoxythymidine (AZT) therapy given during pregnancy; however, the consequences of transplacental AZT exposure to the fetus remain unknown. To address the extent and kinetics of AZT transfer across the human placenta, perfusion studies have been performed with fresh uninfected human placentas perfused with 0.5, 1. 0 and 5.0 mg AZT/ml for 2 h using a dual recirculating single cotyledon perfusion apparatus [T.I. Ala-Kokko, P. Pienimaki, R. Herva, A.I. Hollmen, O. Pelkonen, K. V?h?kangas, Transfer of lidocaine and bupivacaine across the isolated perfused human placenta, Pharmacol. Toxicol. 77 (1995) 142-148]. For two placentas, samples of perfusion effluent were taken every 15 min from the maternal and fetal sides of the apparatus and AZT levels were determined by AZT radioimmunoassay (RIA). At the end of the perfusion, AZT-DNA incorporation into placental DNA was determined by AZT-RIA. The concentration of AZT in the fetal perfusate increased with time, along with a concomitant slow decrease in the concentration of AZT in the maternal perfusates. For three different placentas, at 2 h after the start of perfusion, AZT-DNA incorporation values (molecules of AZT/10(6) nucleotides) were 11.8 for the 0.5 mg AZT/ml perfusate, 13.7 for the 1.0 mg AZT/ml perfusion, and 42.0 for the 5 mg AZT/ml perfusion. An additional placenta perfused with 1 mg AZT/ml did not have detectable values of AZT incorporated into DNA (data not shown). The data show that AZT crosses the human placenta and becomes rapidly incorporated into DNA of placental tissue in a dose-dependent fashion, suggesting that even short exposures to this drug might induce fetal genotoxicity and might also inhibit maternal-fetal viral transmission.