Placental transfer of (125)Iodinated humanized immunoglobulin G2Δa in the Sprague Dawley rat.

Antibody-like biopharmaceuticals cross the placenta by utilizing transport pathways available for transfer of maternal antibodies to the conceptus. To characterize the timing and magnitude of this transfer in the rat, embryo/fetal biodistribution of maternally administered radiolabeled humanized IgG2 was quantified over the course of gestation using gamma counting and whole body autoradiography. The result was humanized IgG2 found in rat embryo/fetal tissues as early as gestation day 11 with a >1000-fold increase in the amount of total IgG2 by day 21. The concentration of IgG2 in rat embryo/fetal tissues generally remained unchanged from gestation day 11 to 17 with a slight increase from day 17 to 21. In addition, fetal-maternal tissue concentration ratios remained stable during organogenesis with a slight increase from gestation day 17 to 21. Based on the empirical amount of antibody present in the embryo/fetus during specific developmental windows, direct antibody binding to biological targets could potentially result in adverse developmental outcomes.

Embryo-fetal distribution of a biopharmaceutical IgG2 during rat organogenesis.

Embryo-fetal biodistribution of a maternally administered humanized IgG2 in rats was evaluated by enzyme-linked immunosorbent assay in dose response and time course studies. Fetal and maternal plasma IgG2 levels increased with dose from 10 to 300mg/kg but fetal:maternal ratio decreased with increasing dose. Plasma IgG2 levels decreased in fetal rat with increasing time post-dose but more slowly than maternal levels. This difference in post-dose kinetics resulted in an increased fetal:maternal ratio with increasing days since last dose. Lastly, IgG2 in embryo-fetal tissue was detected at very low levels on gestation day (GD) 10-12 and levels increased over 100-fold by GD 17. The profile of increasing IgG2 levels as gestation progressed continued in extra-embryonic fluid (GD 12-19) until the end of gestation in fetal plasma (GD 19-21). Based on the current study, there is a potential for direct effects on rat embryo-fetal development following maternal administration of a biopharmaceutical IgG2.

Inter-laboratory assessment of a harmonized zebrafish developmental toxicology assay - progress report on phase I.

This report provides a progress update of a consortium effort to develop a harmonized zebrafish developmental toxicity assay. Twenty non-proprietary compounds (10 animal teratogens and 10 animal non-teratogens) were evaluated blinded in 4 laboratories. Zebrafish embryos from pond-derived and cultivated strain wild types were exposed to the test compounds for 5 days and subsequently evaluated for lethality and morphological changes. Each of the testing laboratories achieved similar overall concordance to the animal data (60-70%). Subsequent optimization procedures to improve the overall concordance focused on compound formulation and test concentration adjustments, chorion permeation and number of replicates. These optimized procedures were integrated into a revised protocol and all compounds were retested in one lab using embryos from pond-derived zebrafish and achieved 85% total concordance. To further assess assay performance, a study of additional compounds is currently in progress at two laboratories using embryos from pond-derived and cultivated-strain wild type zebrafish.

Pubertal development and reproductive functions of Crl:CD BR Sprague-Dawley rats exposed to bisphenol A during prenatal and postnatal development.

Bisphenol A (BPA) is used on a large scale in the manufacture of polycarbonate plastics. BPA has been shown to bind weakly to both estrogen receptor (ER)alpha and ERbeta, and to transactivate reporter genes in vitro. The purpose of the present study was to determine whether exposure of rats to BPA during pre- and postnatal development affects estrogen-mediated end points related to pubertal development and reproductive functions. BPA was administered to pregnant Crl:CD BR Sprague-Dawley rats by gavage at 0, 3.2, 32, or 320 mg/kg/day from gestation day (GD) 11 through postnatal day (PND) 20. Diethylstilbestrol (DES) at 15 microg/kg/day was used as a reference chemical with known estrogenic effects. Female pubertal development was not affected by indirect BPA exposure of the offspring at any of the dose levels. Treatment with this chemical also did not produce detectable effects on the volume of the sexually dimorphic nucleus of the preoptic area (SDN-POA), estrous cyclicity, sexual behavior, or male reproductive organ weights of F(1) offspring. However, DES at 15 microg/kg/day increased the volume of the SDN-POA of female offspring and affected their normal estrous cyclicity following puberty. In this study, pre- and postnatal exposure of rats to BPA at 3.2, 32, or 320 mg/kg/day from GD 11 through PND 20 did not have any apparent adverse effects on female rat pubertal development and reproductive functions.

Characterization of cell death induced by 2-methoxyethanol in CD-1 mouse embryos on gestation day 8.

Cell death was analyzed in neurulating mouse embryos after in vivo doses of 2-methoxyethanol (2-ME) that produce anterior neural tube defects. Characterization of 2-ME-induced cell death was performed by evaluating: (1) vital fluorochrome staining in whole embryos applying confocal laser scanning microscopy; (2) characteristics of cell debris in conventional histological sections revealed by light microscopy; and (3) Apoptag in situ immunohistochemical staining for apoptosis using light microscopy. Methods for quantification of cell death identified by these three techniques were explored using computerized image analysis. Physiological cell death in control embryos primarily occurred in the neural crest region during neural fold elevation. Embryos exposed to 2-ME had expanded areas of cell death in the neural crest and also new areas of cell death in medial regions of the anterior neural tube. Both physiological and 2-ME-induced embryonic cell death had morphological, immunohistochemical, and fluorochrome staining characteristics of apoptosis. When fluorescence data from confocal microscopic analysis of vital fluorochrome-stained embryos were analyzed, a dose-dependent increase was found in embryos exposed to 2-ME. Similar results were obtained when cell death was analyzed in either conventional histological sections or sections prepared for immunohistochemical detection of apoptosis. The cell death data obtained in this study correlate with previously observed near-term malformation rates, suggesting that a quantitative relationship exists between 2-ME-induced embryonic cell death and neural tube defects.

Effects of 2-methoxyethanol on mouse neurulation.

The potent developmental toxicant, 2-methoxyethanol (2-ME), elicits exencephaly in near-term mouse fetuses following a single maternal treatment early on gestation day (gd) 8. Deleterious morphological consequences to the neurulating embryo shortly after exposure have not been reported. The present study was designed to fill this gap and to investigate the impact of 2-ME treatment on cell death patterns in the embryonic neural folds. Dams were injected subcutaneously with saline, 250 or 325 mg 2-ME/kg 2 hr prior to the beginning of gd 8. The effect of 2-ME on gross and microscopic neural development was examined in conceptuses on gd 9, 6 hr (9:6), 10:6, and 18:0. Compared to saline, 2-ME treatment increased the percentage of embryos with open neural tubes (ONTs) at all gestation days. Although few statistically significant differences (P < 0.05) existed among the ONT rates on the 3 observation days, an interesting biological response occurred. Both high and low 2-ME doses appeared to elicit the greatest incidence of neural tube patency on gd 9:6 (affecting approximately 27% of embryos). During the subsequent 24 hr, recovery occurred and many neural folds apparently closed. Consequently, the ONT incidences on gd 10:6 (approximately 11%) were quite similar to the gd 18 exencephaly rates elicited by both chemical treatments (approximately 15%). A dose response was not seen due to a substantial increase in resorption rates following the 325 mg/kg dose. Compared to the other treatment groups, the low 2-ME dose significantly inhibited embryonic growth as indicated by reduced crown-rump and head lengths and increased incidence of developmentally delayed brain maturation. To evaluate chemically induced changes in cell death, neurulating embryos were collected on gd 8:6 and either immersed in the vital dye, Nile blue sulfate (NBS), or processed for histopathology. In 2-ME-exposed embryos, excessive NBS uptake occurred in neural fold neuroepithelium at sites of nonclosure. Using histopathology, the extent of cell death in the cephalic neural folds was dependent on the 2-ME dose, and the neuroepithelium was more severely affected than the mesenchyme. These observations suggest 1) a trend toward repair and catch-up growth later in gestation which may ameliorate the overt early effects of 2-ME, and 2) an association between enhanced cell death and regions of the neural tube particularly vulnerable to nonclosure.

Dose effects on the excretion of urinary metabolites of 2-[1,2,methoxy-13C]methoxyethanol in rats and mice.

The administration of 2-methoxyethanol (2-ME) to pregnant rats, mice, or primates results in developmental toxicity. To assess the role of metabolism in the adverse response of 2-ME, carbon-13 NMR spectroscopy was used to examine, directly in the urine, metabolites produced after administering high (250 mg/kg) and low (25 mg/kg) doses of 2-[1,2,methoxy-13C]ME to pregnant CD-1 mice and male Fischer 344 rats. The high dose elicits teratogenic effects in mice and testicular toxicity in rats. The urinary disposition was also examined after dosing pregnant CD-1 mice with a developmentally toxic level of 2-ME together with serine or acetate (known attenuators of 2-ME embryotoxicity). Seven novel metabolites were found in rat urine, consistent with those assigned in our previous studies with mice. Metabolite composition was compared for the different dosing regimens. A lower percentage of metabolites derived after conversion of 2-ME to 2-methoxyacetic acid (2-MAA) was found following concurrent administration of 2-ME with acetate, D-serine, or L-serine. These differences are mainly attributed to higher levels of ethylene glycol and/or glycolic acid that arise for the 2-ME administrations with any of the attenuators. Acetate together with 2-ME also reduced the percentage of metabolites incorporated into intermediary metabolism. These data indicate that attenuators of 2-ME teratogenic effects may alter metabolism and distribution by decreasing the conversion of 2-ME to 2-MAA, decreasing the conversion of 2-MAA to a coenzyme A thioester (2-methoxyacetyl approximately CoA), altering the utilization of the coenzyme A thioester, and/or increasing the conversion of 2-ME to ethylene glycol and its further metabolism. These changes in metabolism may contribute to the attenuating effects of these agents on 2-ME.

Developmental phase alters dosimetry-teratogenicity relationship for 2-methoxyethanol in CD-1 mice.

The industrial solvent 2-methoxyethanol (2-ME) elicits phase-specific terata in mice through its primary metabolite and proximate toxicant, 2-methoxyacetic acid (2-MAA). Recent pharmacokinetic studies indicate that the incidence and severity of digit malformations induced in CD-1 mice by 2-ME exposure on gestation day (gd) 11 (copulation plug = gd 0) correlate better with the total 2-MAA exposure over time (= area under the curve; AUC) than with its peak concentrations (Cmax) in maternal plasma, embryo and extraembryonic fluid. In this study, the phase specificity of exencephaly induction by 2-ME was investigated to ascertain whether the 2-ME/2-MAA dosimetry-teratogenicity relationship remains consistent throughout organogenesis. Following a single intravenous (iv) bolus dose of 250 mg 2-ME/kg given to pregnant mice, exposure on gd 8 was decidedly the gestation day that best balanced low embryo lethality and high malformation incidence as recorded in near-term fetuses. Concentrations of 2-MAA were measured during distribution and elimination in maternal plasma and conceptuses following iv bolus doses of 175, 250, and 325 mg 2-ME/kg, as well as during and after termination of subcutaneous (sc) constant-rate infusion (4, 6, and 8 hr; 8 microliters/hr) of 277, 392, and 606 mg 2-ME/kg total doses. For all administration regimens, exencephaly incidence rates were determined in fetuses on gd 18. Similar plasma 2-MAA Cmax values (approximately 5 mmol/l) and fetal malformation frequencies (approximately 12%) were induced by sc infusion of 392 mg 2-ME/kg or a bolus dose of 250 mg 2-ME/kg. However, the AUC produced by infusion was significantly larger than that following the iv bolus dose (38 vs. 26 mmol.hr/l, respectively). In both maternal plasma and conceptuses, the correlation coefficients between Cmax and exencephaly rates, as well as developmental toxicity, were higher than they were for AUC and those end points. This outcome suggests that dosimetry-teratogenicity determinants may be quite specific for a distinct developmental phase during which a particular organ differentiates and a specific chemical acts upon the embryo.

Characterization of urinary metabolites from [1,2,methoxy-13C]-2-methoxyethanol in mice using 13C nuclear magnetic resonance spectroscopy.

2-Methoxyethanol (2-ME) is an industrial solvent that induces developmental and testicular toxicity in laboratory animals. Oxidation of 2-ME to 2-methoxyacetic acid (2-MAA) is required for the generation of these adverse effects. The urinary metabolites of 2-ME were investigated to characterize the fate of 2-ME and 2-MAA. 13C NMR spectroscopy was used to detect and assign metabolites in the urine of pregnant CD-1 mice following administration of 250 mg/kg of [1,2,methoxy-13C]-2-ME. Two-dimensional NMR methods were used to correlate signals from the labeled carbons in each 2-ME metabolite and to determine the number of hydrogens attached to each carbon. Structures were assigned from the NMR data together with calculated values of shift for biochemically feasible metabolites and by comparison to standards. Pathways involved in forming metabolites assigned in this study include transformation of 2-ME via ethylene glycol, conjugation with glucuronide or sulfate, and oxidation to 2-MAA. Additional metabolites were assigned that can be formed from further conversion of 2-MAA to glycine and glucuronide conjugates, as well as metabolites derived from the incorporation of 2-methoxyacetyl CoA derivatives into intermediary metabolism. Elucidation of the further metabolism of 2-MAA may be important for understanding the mechanisms by which 2-ME induces adverse effects.

2-Methoxyethanol metabolism in pregnant CD-1 mice and embryos.

Upon oxidation to 2-methoxyacetic acid (2-MAA), 2-methoxyethanol (2-ME) causes malformations in all animal species that have been examined. Commonly, 2-MAA is thought to be the proximate toxicant. However, our previous studies with [1,2-14C]2-ME and the present data obtained with [1-14C]2-MAA, [2-14C]2-ME and [methoxy-14C]2-ME revealed that metabolism beyond 2-MAA occurs. Regardless of the 14C position, dams exhaled approximately 5% of the radioactivity administered as a single teratogenic oral dose (3.3 mmol/kg on Gestation Day [gd] 11) as 14CO2. With all isotopic variants urine contained 70-80% of the dose within 24 hr after administration and 13-18% in the next 24 hr. Three labeled products were resolved using HPLC: an unidentified Peak A (12-18% of dose), 2-MAA (approximately 50%), and the glycine conjugate of 2-MAA (approximately 25%). Short-term (4 hr) whole embryo culture on gd 11 with 3 mM 2-MAA and a tracer dose of [1-14C]2-MAA, [2-14C]2-MAA, or [methoxy-14C]2-MAA showed that 14CO2 evolved from the former two substrates, while there was none detectable from the latter. The data indicate that dams metabolized [methoxy-14]2-MAA to 14CO2, while embryos apparently did not. The production of labeled CO2 from [2-14C]2-ME suggests that 2-methoxyacetyl approximately CoA (the precursor for amino acid conjugation with glycine) entered into the tricarboxylic acid (TCA) cycle. This interpretation is supported by the inhibition of 14CO2 evolution elicited by fluoroacetate (0.1 or 1.0 mM) and sodium acetate (5 mM). It is not yet clear whether entry of 2-methoxyacetyl approximately CoA as a "false substrate" in the TCA cycle is of significance for the embryotoxic effects of 2-ME/2MAA.

Inhibition of DNA synthesis in mouse whole embryo culture by 2-methoxyacetic acid and attenuation of the effects by simple physiological compounds.

Gestation day 11 CD-1 mouse embryos were cultured in serum containing or serum-free media for 6 h in the presence of 2-methoxyacetic acid (2-MAA), the proximate teratogen arising from 2-methoxyethanol. The rate of DNA synthesis was determined following exposure of embryos to [3H]thymidine during the final hour of culture. 2-MAA (25 mM) produced a 50% reduction of [3H]thymidine incorporation into embryos cultured in serum-containing medium, whereas only 5 mM 2-MAA were required for a similar inhibitory effect in embryos cultured in serum-free medium. When embryos were concomitantly exposed to 2-MAA (5 mM) and to formate (1 mM), acetate (5 mM), or sarcosine (2 mM), a significant attenuation of the 2-MAA-induced inhibition of [3H]thymidine incorporation occurred. The findings suggest that these agents, which also attenuated developmental toxicity in vivo, may compete with 2-MAA actions which inhibit DNA synthesis in the embryo.

Effect of growth state, tumor promoters, and transformation upon intercellular communication between C3H/10T1/2 murine fibroblasts.

Diminished intercellular communication has been associated with heightened sensitivity of cultured cells to morphological transformation and enhancement of transformation by tumor promoters. Microinjection of Lucifer yellow dye was employed to evaluate intercellular communication between transformable C3H/10T1/2 murine fibroblasts under a variety of culture conditions. Intercellular communication assayed by dye transfer from injected cells to surrounding cells in contact occurred in logarithmically growing cultures, declined to very low levels as confluence was attained, and then resumed upon the formation of mature confluent monolayers. Dye-transfer networks of 50 or more cells resulted from injection of single monolayer cells. Freeze-fracture electron microscopy confirmed the presence of gap junction structures in confluent cultures. Treatment with the initiating agent N-methyl-N'-nitro-N-nitrosoguanidine and/or the tumor promoter 2,3,7,8-tetrachlorodibenzo-p-dioxin did not inhibit intercellular communication between C3H/10T1/2 cells during 6-week transformation experiments. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate produced a transient inhibition of dye-coupling upon first introduction to cultures and prolonged the period of diminished dye-coupling at the attainment of confluence, but did not inhibit subsequent interactions between monolayer cells. A simple relationship thus could not be established between levels of dye-coupling within monolayers and focus formation events. Curiously, although the cells of foci in early phases of development did not exhibit dye-transfer capacity, dye-coupling was observed in mass cultures of most transformed cell lines cloned from foci. Co-cultivation of communication-competent transformed cells with nontransformed cells to produce reconstructed foci generally resulted in a cessation of dye-transfer by transformed cells. An often reversible loss of communication competence thus accompanies the growth of transformed C3H/10T1/2 cells as foci and may constitute an adaptive response which facilitates focus growth in the presence of intercellular communication between monolayer cells.

Alterations of intercellular communication associated with the transformation of C3H/10T1/2 cells.

Cultures of C3H/10T1/2 mouse embryo cells were treated in accordance with several treatment regimens that induced the focal growth of morphologically transformed cells. Intercellular communication between focus cells, and between focus and monolayer cells, was examined in late stages of transformation experiments by microinjection of Lucifer yellow dye into cells and observation of dye transfer to surrounding cells. Transformed foci produced by treatment with 3-methylcholanthrene, by initiation with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA), or by initiation with MNNG and promotion with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were studied. These included focus types thought to possess tumorigenic potential (Types II and III) and those believed to lack such potential (Type I). Cells within all focus types exhibited only limited communication with each other or with surrounding monolayer cells. In contrast, microinjection of monolayer cells typically resulted in dye transfer to an average of approximately 50 other monolayer cells. The presence of the tumor promoters TPA or TCDD did not alter intercellular communication between monolayer cells. These studies demonstrate that alterations in intercellular communication are evident during the growth of transformed foci. These changes are relatively independent of both the treatment regimen used to produce foci and the presumed oncogenic potential of different focus types.

Karyotype, growth, and cell cycle analysis of human embryonic palatal mesenchymal cells: relevance to the use of these cells in an in vitro teratogenicity screening assay.

Human embryonic palatal mesenchyme (HEPM) is an established cell line that is presently under investigation as an in vitro prescreening assay used to determine the teratogenic potential of chemicals. We describe here general growth characteristics, karyotype, and cell cycle analysis of these cells. HEPM cells had plating efficiencies of less than 95% and displayed notable contact growth inhibition following an exponential growth phase that lasted for approximately 6 days. These cells had the diploid karyotype of a female human embryo. The chromosomal complement showed no dramatic change between passage 5 and 14. Flow cytofluorometry analysis using bromodeoxyuridine (BrdU) pulse labeling and a direct immunofluorescence anti-BrdU FITC probe revealed that the total cell cycle transit time was approximately 22 hr: the duration of G1 was 12.2 hr, S was 6.1 hr, and G2-M lasted for 3.7 hr. The results indicate that HEPM cells met the criteria regarding karyotype stability that were assumed by the National Toxicology Program of the USA.

Effects of 6-thioguanine on communication competence and factors affecting reversibility of phorbol ester inhibition in V79 cells measured by the metabolic cooperation assay and by dye coupling.

One assay used to study the role of intracellular communication in tumor promotion examines chemically-induced inhibition of metabolic cooperation between 6-thioguanine (6-TG) sensitive (HGPRT+) V79 Chinese hamster cells in co-culture with 6-TG resistant (HGPRT-) cells. In the present study metabolic cooperation and Lucifer Yellow dye coupling were applied to detect intercellular communication. HGPRT+ cells were still communication competent when pre-incubated with 6-TG for up to 48 h before HGPRT- cells were plated. In both assays the phorbol esters 12-O-tetradecanoylphorbol-13-acetate (TPA) and phorbol-12,13-dibutyrate (PDBu) inhibited intercellular communication completely at 1 ng/ml. Using 6-TG metabolic cooperation, extensive washing of cells failed to reverse the inhibition caused by 100 ng TPA/ml after exposure for as little as 1 h. However, the effect of PDBu was completely eliminated if the drug was removed within 24 h. Complete blockade of dye coupling by 1, 10 or 100 ng TPA/ml was reversible after washing only at 1 ng/ml. Cells cultured in the presence of these TPA concentrations displayed significant reversal of dye coupling inhibition after 40 h. The results indicate that the onset of communication competence impairment between V79 cells by 6-TG is much slower than hitherto implied. Failure of intercellular communication to recover even from short exposure to 100 ng TPA/ml appears to be attributable to its lipophilic nature. Not enough TPA can be removed by washing to overcome its potent inhibitory action. These observations indicate that the physicochemical properties of a chemical must be taken into consideration before making judgements about the duration of inhibition.

Effects of a teratogen on [3H]uridine nucleotide transfer between human embryonal cells and on gap junctions.

Intercellular communication (IC) mediated by gap junctions (gj) occurs during embryonal development and appears to be important for normal differentiation through the exchange of morphogenetic signalling substances. Disruption of IC by chemicals may induce abnormal development resulting from failed cell-cell interactions. It was established in the present study that genotypically normal human embryonal palate mesenchyme (HEPM) cells displayed IC in cell culture and that the transfer of [3H]uridine nucleotides was inhibited by the potent embryotoxin and teratogen 12-O-tetradecanoyl-phorbol-13-acetate (TPA). IC was mediated by gj which were revealed by freeze-fracture and electron microscopy. Quantitative morphometric analysis showed that inhibition of IC by TPA coincided with a significant reduction in the number of gj. The observations suggest that inhibition of IC by the teratogen TPA may be one among the many mechanisms believed to be responsible for the induction of abnormal development by chemical teratogens.

Inhibition of intercellular communication between normal human embryonal palatal mesenchyme cells by teratogenic glycol ethers.

Cell-cell communication, possibly through gap junctions, is a fundamental event for the differentiation of embryonal tissues. Chemical substances which can interfere with this process may be able to disrupt embryogenesis. We have examined the response of a normal diploid human embryonal palatal mesenchymal (HEPM) cell line to glycol ethers. These cells have gap junctions whose function in cell-cell communication was inhibited by a model teratogen. Potential HEPM donor cells (2000 to 4000) were pulse labeled with 3H-uridine (10 microCi/mL; 3 hr) and then cocultured for 3 hr with 200,000 to 400,000 potential recipient cells in the absence or presence of 2-methoxyethanol (ME; 0.13-0.30 M) or 2-isopropoxyethanol (IPE; 0.05-0.1 M). Computer-assisted quantitative autoradiography was applied to assess the effects on metabolic cooperation. Although this phenomenon was inhibited by ME, the effect was probably not attributable to interference with gap junction-mediated transfer of labeled nucleotides but rather to the lack of formation of gap junctions resulting from cytotoxicity and poor physical contact between cells. The inhibition obtained with IPE was apparently not due to adverse effects on HEPM cells as judged by light microscopy and cell counts of recipients surrounding a given donor. The results suggest that HEPM cells are suitable to study disruption of cell-cell communication as a mechanism responsible for teratogenesis and offer the unique possibility to apply human embryonal cells. However, care has to be taken to assess potential cytotoxicity of xenobiotics, and further refinement of the criteria to detect adverse effects is required.

Effect of subchronic administration of methyl parathion on in vivo protein synthesis in pregnant rats and their conceptuses.

Pregnant rats received daily po doses of the organophosphate methyl parathion (MPTH) from Day 6 through Day 15 or 19 of gestation at doses causing no (1.0 mg/kg) or minimal (1.5 mg/kg) signs of maternal toxicity. Following the dose of MPTH on Day 15 or 19, in vivo protein synthesis was measured 0.5, 1.0, and 2.0 hr after sc injection of L-[1-14C]valine at a dose of 5 microCi/mmol/100 g body wt. The specific activity of [14C]valine in the free amino acid pool and protein bound pool was significantly reduced in various regions of maternal brain and in maternal viscera, placenta, and whole embryos (Day 15), and in fetal brain and viscera (Day 19). The inhibitory effect of MPTH on net protein synthesis was dose dependent, greater on Day 19 than 15 of gestation and more pronounced in fetal than in maternal tissues.

Inhibition of metabolic cooperation between Chinese hamster V79 cells by structurally diverse teratogens.

Cell-cell communication through chemical messengers is a fundamental event required for the differentiation of embryonal cells. Interference with this process by xenobiotics may disrupt embryogenesis. Chinese hamster cells (V79) which display a specific form of cell-cell communication called metabolic cooperation were cultured in the presence of structurally diverse chemical teratogens. Among them were 12-O-tetradecanoyl-phorbol-13-acetate (TPA), diphenylhydantoin (DPH), warfarin, and a series of monoalkyl ethers of ethylene glycol with alcohol chain lengths from methyl to butyl. Sodium saccharin and ascorbic acid were examined to represent two chemicals which have been thoroughly tested for teratogenic effects in laboratory animals and cause no birth defects. Recovery of 100 6-thioguanine-resistant V79 (6-TGr) cells in coculture with 400,000 6-thio-guanine-sensitive V79 (6-TGs) cells in the presence of 6-thioguanine (6-TG) and the chemical agent was measured. In amounts that neither interfered with colony forming ability nor caused cytostasis when 100 6-TGr cells were plated alone, all of the substances except for saccharin and vitamin C increased the number of surviving 6-TGr cells in a concentration-related manner. The recovery was increased by the presence of TPA (to 100% by 4 ng/ml), DPH (from 26% at 91 microM to 43% at 274 microM), warfarin (from 15.5% at 162 microM to 44.5% at 487 microM) and to variable extents by all five glycol ethers. The most efficacious in the latter group of compounds was the isopropyl ether which raised 6-TGr recovery from 8% at 0.017 M to 66% at 0.087 M. Based on the evidence accumulated by previous studies involving TPA, we postulate that the teratogens employed inhibited metabolic cooperation. These observations suggest that V79 cells may be suitable to study inhibition of cell-cell communication as a mechanism of teratogenesis.

Release of acetylcholine from fragments of human term placenta and effects of the choline acetyltransferase inhibitor (2-benzoylethyl)trimethylammonium iodide.

Intact tissue from human term placenta contained 102 +/- 17 (n = 8) nmoles of acetylcholine (ACh)/g fresh tissue when analyzed by pyrolysis gas chromatography (GC). Free-hand dissection followed by extensive washing led to reductions of tissue ACh content (57 +/- 11 nmoles/g; n = 5) presumably as a result of release into the bathing medium. Presence or absence of physostigmine as an anticholinesterase agent in all solutions used had no significant effect on the ACh content of intact fragments immediately after dissection. Upon incubation at 37 degrees C a new steady state ACh concentration was established within 40-50 min (177 +/- 14 nmoles/g; n = 11) which reflected the content typical for purified villus. ACh was released continuously in amounts linearly related to incubation time up to 80 min, the longest time point examined. When the incubation volume was doubled there was an apparently immediate significant effect on release of ACh. Thereafter liberation of ACh continued at the rate observed prior to the volume perturbation. The stable choline acetyltransferase inhibitor (2-benzoylethyl)trimethylammonium (BETA) inhibited the enzyme rapidly and almost completely in tissue fragments. The drug affected the rate of ACh release significantly 40 min after addition to the bathing solution. At the same time it led to a depletion of the total ACh content that could be extracted from human placenta fragments.