Effects of combinations of methanol and formic acid on rat embryos in culture.

Acute human exposure to methanol (MeOH) results in elevated serum concentrations of both MeOH and formic acid. In order to better assess the risk of adverse developmental effects of MeOH exposure in humans, the effects of the combination of formate and MeOH, in addition to the individual toxicity profiles for MeOH and formate, need to be established. Gestational day 9 rat embryos were exposed to various concentrations of MeOH and formate in whole embryo culture (WEC) for 48 hr and the degree of embryotoxicity was evaluated using developmental score (DEVSC) as the parameter of comparison across exposure combinations. After establishing embryo toxicity of the individual compounds in previous studies, concentrations of MeOH and formate were chosen which would produce similar DEVSCs, and isoboles were plotted joining the equivalently toxic doses. These mixtures would be expected to have similar toxicity to the MeOH or formate concentrations according to a dose-addition model. The responses of embryos to the selected concentrations along each isobole were measured and tested for linearity to determine the nature of any interaction between the two agents. The concentrations of MeOH and formate used separately and in combination ranged from 0 to 8.75 mg/ml MeOH and 0 to 1.51 mg/ml formate. Increasing concentrations of either MeOH or formate resulted in significant decreases in DEVSC. Exposure to combinations of MeOH and formate had less effect on DEVSC than would be expected based on simple toxicity additivity. This observation was also true for embryonic crown-rump length, head length, and somite number. These results suggest that the embryotoxicity observed following low level exposure to MeOH is mechanistically different from that observed following exposure to formate.

Stereoselective dysmorphogenicity of the enantiomers of the valproic acid analogue 2-N-propyl-4-pentynoic acid (4-yn-VPA): cross-species evaluation in whole embryo culture.

We previously reported the in vitro differential stereoselective dysmorphogenic potential of the R(+) and S(-) enantiomers of 2n-propyl-4-pentynoic acid (4-yn-VPA) in mice. To determine whether this stereoselectivity is species specific, we evaluated the dysmorphogenic potential of these isomers as well as valproic acid (VPA) to gestational day 9 rat embryos using whole embryo culture (WEC). Aqueous solutions of the sodium salts of R-4-yn-VPA, S-4-yn-VPA, 50%R/ 50%S-4-yn-VPA or VPA were added to the culture medium to give 0, 0.075, 0.15, 0.3, 0.6, or 1.2 mmol/L and embryos were evaluated 48 hr later. The S-4-yn-VPA enantiomer gave clear concentration-dependent dysmorphology as well as effects on developmental score, somite number, crown rump length, and head length. Effects on rotation and defects of the neural tube, somites and heart were observed. Embryolethality was observed only at 1.2 mmol/L concentration. The R-4-yn-VPA enantiomer was neither embryo toxic nor dysmorphogenic at any concentration. VPA significantly reduced all parameters and was dysmorphogenic at the highest concentration but was not embryo lethal. The 50/50 mixture of R- and S-isomers appeared to elicit a degree of embryolethality and dysmorphology similar to VPA. The potency order for the four chemicals was S(-) > S(-)/R(+) = VPA > > > R(+), comparable to that observed in mice by either in vivo or in vitro exposure. These data demonstrate that the stereoselective dysmorphology for these enantiomers can be observed across species and is not related to maternal metabolism.

Validation of an in vitro teratology system using chiral substances: stereoselective teratogenicity of 4-yn-valproic acid in cultured mouse embryos.

In vitro systems are important for toxicity testing as well as for investigating the mechanism of action of xenobiotics. The validation of such in vitro systems is often incomplete and extrapolation to the in vivo situation is equivocal. In the present study, we studied the effects of enantiomers of an analogue of the antiepileptic drug valproic acid (VPA): R(+)- and S(-)-4-yn-VPA (R- and S-2-n-propyl-4-pentynoic acid), which have previously been shown to induce selective teratogenicity in mice after in vivo administration, in mouse whole-embryo culture (WEC). Aqueous solutions of the sodium salts of the pure R- and S-enantiomers as well as R,S-4-yn-VPA (racemic mixture) or VPA itself were added to the culture medium at 0, 0.075, 0.15, 0.3, 0.6, or 1.2 mmol/liter and embryos were evaluated 24 hr later. The S-4-yn-VPA enantiomer induced clear concentration-dependent dysmorphogenesis that was evident even at the lowest concentration. The primary anomalies were neural tube defects, erratic neural seams, blisters, and rotational defects. Embryolethality was observed at 1.2 mmol/liter. The R-4-yn-VPA enantiomer was neither embryotoxic nor dysmorphogenic at any tested concentration. The lack of biological activity over 24 hr in WEC with the R-enantiomer suggests also that, as previously shown in vivo, there was no racemization of this isomer to the more active S-enantiomer. The racemic mixture of R and S isomers appeared to be slightly more embryolethal and dysmorphogenic than VPA. Overall, the potency of the S-enantiomer was approximately four times that of VPA. Therefore, the rank order of the four chemicals tested was S(-) >> S(-), R(+) > VPA >>> R(+), which is in agreement with the effects observed in in vivo exposed mice. These data demonstrate a direct stereoselective effect of these compounds on the embryo. This is the first illustration of the stereoselectivity of a xenobiotic in the WEC in vitro test system. Pure and stable enantiomers, which induce stereoselective toxicity in vivo, are demonstrated to be valuable for validation of this in vitro system.

Utility of fluorescence microscopy in embryonic/fetal topographical analysis.

For topographical analysis of developing embryos, investigators typically rely on scanning electron microscopy (SEM) to provide the surface detail not attainable with light microscopy. SEM is an expensive and time-consuming technique, however, and the preparation procedure may alter morphology and leave the specimen friable. We report that by using a high-resolution compound epifluorescence microscope with inexpensive low-power objectives and the fluorochrome acridine orange, we were able to obtain surface images of fixed or fresh whole rat embryos and fetal palates of considerably greater topographical detail than those obtained using routine light microscopy. Indeed the resulting high-resolution images afford not only superior qualitative documentation of morphological observations, but the capability for detailed morphometry via digitization and computer-assisted image analysis.

Developmental toxicity of formate and formic acid in whole embryo culture: a comparative study with mouse and rat embryos.

Acute methanol (MeOH) toxicity in primates is attributed to the conversion of MeOH to formate and the resulting acidosis. MeOH has been shown to be developmentally toxic in mice and rats both in vivo and in vitro, but rodents neither accumulate formate nor develop acidosis after MeOH exposure. To further assess the potential human developmental toxicity of MeOH exposure, we evaluated the developmental effects of sodium (Na) formate and formic acid in rodent whole embryo culture (WEC). Day 9 rat embryos were cultured for 24 or 48 hours and day 8 mouse embryos were cultured for 24 hours in the presence of Na-formate or formic acid. Rat and mouse embryos exposed to either agent for 24 hours exhibited a trend toward reduced growth and development and the number of abnormalities increased at the higher concentrations. Rat embryos exposed for 48 hours to either Na-formate or formic acid showed a trend toward reduced growth and development with increasing concentration. Embryo lethality and incidence of abnormal embryos were also increased at the higher concentrations. The anomalies observed in both species after exposure to either compound were primarily open anterior and posterior neuropore with less frequent incidence of rotational defects, tail anomalies, enlarged pericardium and delayed heart development. Exposure to Na-formate or formic acid for comparable periods of time results in comparable degrees of embryotoxicity at concentrations (mMolar) at least 4-fold lower than those previously reported for methanol exposure.

Cell death in rat and mouse embryos exposed to methanol in whole embryo culture.

Methanol induces developmental toxicity in rats and mice producing exencephaly, cleft palate, cervical skeletal defects, reduced body weight, and increased embryo/fetal death. Exposure to methanol in whole embryo culture also induces developmental retardation, dysmorphogenesis, and embryo lethality. In the present study, cultured rat and mouse embryos were exposed to methanol and subsequently observed for morphological effects and increased cell death using modified Feulgen staining which allowed nuclei throughout the embryo to be examined in situ. Growth and developmental scores were reduced by methanol in both rat and mouse embryos and the mouse embryos were affected at lower concentrations when compared to the rat. Methanol increased cell death in specific regions of both rat and mouse embryos, including the forebrain, the visceral arches, otic and optic placodes. These regions form derivatives which manifest morphological abnormalities following exposure in vivo. Methanol did not increase cell death in the neuroepithelium or neural folds and neural tube defects cannot be explained by excess cell death. The results of this study suggest that increased cell death in specific regions of the exposed embryos has a role in producing cranial malformations, abnormalities of the eye and ear, and cleft palate.

Profile of procarbazine-induced embryotoxicity in an embryo hepatocyte co-culture system and after in utero glutathione depletion.

Procarbazine (PCZ) is an antineoplastic agent useful in the treatment of Hodgkin's disease, brain tumors, and chronic leukemia. PCZ is dysmorphogenic to developing embryos exposed in vivo or cultured in the serum of PCZ-treated rats. However, embryos directly cultured with PCZ (up to 400 micrograms/ml) or PCZ plus S-9 liver fractions are unaffected. Since intact liver cells provide several advantages over hepatic subcellular fractions for in vitro bioactivation, we exposed rat embryos to PCZ in an embryo/hepatocyte co-culture system. Gestation day (GD) 9.5 rat embryos exposed to 0, 200, 300, or 400 micrograms PCZ/ml in the presence of untreated or phenobarbital induced male rat hepatocytes failed to display toxicity. However, in a companion study GD 9.5 rat embryos cultured in the serum from PCZ-treated rats exhibited developmental deficiencies. Studies have shown that the formation of toxic metabolites can result from glutathione (GSH) conjugation of toxicants in the liver. Therefore, in a second set of experiments, rat embryos were cultured in serum from rats pretreated with two GSH depleters (phorone and buthionine sulfoximine) and subsequently dosed with PCZ. Effects on development were enhanced when embryos were cultured in the serum from PCZ-treated/GSH depleted rats. These data indicate that PCZ requires in vivo activation to be dysmorphogenic and further suggest that the metabolite(s) responsible for procarbazine embryo-toxicity are formed readily under conditions of low GSH levels. This argues against a glutathione conjugate as the ultimate toxicant.

Gamma-glutamyl transferase (GGT) activity and biochemical characterization of rat visceral yolk-sac during gestation with or without trypan blue exposure.

Yolk-sacs from untreated Sprague-Dawley rat conceptuses were removed on gestational days (GD) 9 to 18 and examined for gamma-glutamyl transferase (GGT), alkaline phosphatase (AP), lactate dehydrogenase (LDH), and glutamic-oxaloacetic transaminase (GOT) activities. All enzyme activities were found to increase through gestation in total activity as well as in specific activity. Protein (PRO) and urea nitrogen (UN) were also found to increase through gestation whereas triglyceride (TRI) increased steadily from GD 9 to 13 and then appeared to plateau through GD 19. Additional rats were treated on GD 8 with 75 mg trypan blue (TB)/kg body weight and yolk-sacs taken on GD 9 to 18. Yolk-sac GGT and GOT activities from TB-treated rats were significantly higher than the respective controls during early gestation but recovered to or were lower than control levels during midgestation. GGT activity in treated rats was significantly higher than the controls on GD 15 and 16, and both GGT and GOT were significantly lower than controls on GD 17 and 18. AP activity in the TB-treated yolk-sacs was significantly lower than that in controls during early and midgestation but was not significantly different from the control values late in gestation. Triglyceride concentration was not affected early in gestation but significantly decreased on GD 16 and 18. Thus, the yolk-sac enzymes monitored, which are associated with nutrition and normal growth, increased in activity through GD 18. The yolk-sac toxicant, trypan blue, significantly affected enzymatic activity at various time periods during gestation and resulted in significant changes in yolk-sac protein and triglyceride content.

Evaluation of the dysmorphogenic effects of procarbazine, benzylazoxyprocarbazine, and methylazoxyprocarbazine in whole embryo culture.

Serum from procarbazine (PCZ)-treated rats is dysmorphogenic to rat embryos cultured in vitro, but PCZ is not effective when added directly to culture medium, even with an exogenous metabolizing system. Methylazoxyprocarbazine (MPCZ) is a metabolite which we have identified by HPLC in the dysmorphogenic serum of PCZ-treated rats. PCZ, MPCZ, and benzylazoxyprocarbazine (BPCZ, an isomer of MPCZ) were tested in rat whole embryo culture to determine their effects on embryo development. The parent compound, PCZ, produced no effect on embryo growth or development at concentrations up to 200 micrograms/ml. MPCZ proved to be the most potent of the agents tested. There was significant embryo lethality at concentrations of > or = 10 micrograms/ml while 25 micrograms/ml had significantly reduced embryonic developmental score (DEVS), and 35 micrograms/ml reduced DEVS, head length, and somite number. There was 89% embryo lethality at the 50 micrograms/ml exposure level. At concentrations > 5 micrograms/ml, there were significant increases in anomalies, primarily, failure of neural tube closure, erratic neural seam, and microcephaly. In contrast, BPCZ produced embryo lethality and reductions in DEVS only at 100 micrograms/ml. These data suggest that MPCZ, which has been identified in PCZ-treated rat serum, may be the proximate dysmorphogenic metabolite of PCZ.

Toxicokinetics and structure-activity relationships of nine para-substituted phenols in rat embryos in vitro.

The purpose of this study was to examine the toxicokinetics of embryo uptake following exposure to a variety of chemically related phenols in rat embryo culture. The uptake of nine radiolabeled para-substituted phenols by day 10 (9-13 somite stage) rat embryos in vitro was determined from 1 to 42 hrs after being placed in culture media containing various phenols. Uptake was rapid, having a half-life of 3 hr or less, with 7 of the nine compounds having uptake half-times of less than one hour. The equilibrium concentration in the embryo ranged from 53 to 136% of the media concentration, indicating only a factor of 2 in maximum discrimination against the compound for any of the phenols studied. The fraction of radioactivity remaining unbound in the media decreased with increasing log P (octanol/water partition coefficient). The binding was calculated to be 50% for log P = 1.77 from the fitted regression equation and decreased by a factor of 5.9 for every decade increase in P. When hepatocytes were also present in the media the equilibrium concentration in the embryos was less than when hepatocytes were absent. With the limited data, four of the phenols appeared to have no (i.e., zero) equilibrium level when hepatocytes were present. Thus the metabolites produced by the hepatocytes appeared to have less affinity for the embryo than the parent phenol. Toxicodynamic information as given by the effective concentration of the phenol in the embryo to cause somite or tail teratological effects was best predicted by the measured unbound fraction.

Effects of developmental stage and tissue type on embryo/fetal DNA distributions and 5-fluorouracil-induced cell-cycle perturbations.

Cell-cycle analysis of nuclei obtained from the circulating erythroblasts (gestational day [GD] 11-16), livers (GD 14-19), and whole embryos (GD 10-13) or remaining (extrahepatic) tissues (GD 14-16) of rat embryos/fetuses revealed age- and tissue-dependent variations in the relative percentages of cells in the G0/G1, S, and G2/M phases of the cell cycle. With development, the rate of cell proliferation declined resulting in decreases in the relative percentage of S-phase cells and increases in the G0/G1 percentage, while the percentage of G2/M-phase cells remained relatively constant. Comparing tissue cell-cycle profiles during development, erythroblasts exhibited the most rapid age-dependent decline in S-phase percentage (from 75% at GD 11 to 8% by GD 14), embryos/extrahepatic tissues exhibited a more gradual reduction (from 55% at GD 10 to 14% by GD 15), while the hepatic isolates exhibited a relatively constant S-phase percentage of approximately 40% from GD 14 to GD 18 before decreasing to 23% at GD 19. These age-dependent variations suggest that cell-cycle distribution may be useful in staging embryogenesis and in detecting abnormal development. To determine how these developmental and organ-specific cell-cycle variations affect toxic response, we sampled GD 11-13 embryos 6 hr after maternal administration of a teratogenic dose of 5-fluorouracil (5-FU), a thymidylate synthetase inhibitor that induces S-phase accumulation. The results indicate that, on a relative basis, the amount of induced S-phase accumulation in erythroblasts and whole embryos 6 hr postdosing increased with development.(ABSTRACT TRUNCATED AT 250 WORDS)

Developmental toxicity of methanol in whole embryo culture: a comparative study with mouse and rat embryos.

Methanol (MeOH), a widely used industrial solvent, has been proposed as an alternative motor vehicle fuel. Inhaled MeOH is developmentally toxic in both rats and mice but the mouse is more sensitive than is the rat. The contribution of the embryo to this differential sensitivity was studied in whole embryo culture (WEC) using equivalent stage rat (day 9) and mouse (day 8) embryos (plug day = day 0). Rat embryos were explanted and cultured in 0, 2, 4, 8, 12 or 16 mg MeOH/ml rat serum for 24 h and then transferred to rat serum alone for 24 h. Embryonic development of the 2 and 4 mg MeOH/ml groups was not significantly different from the controls whereas the higher concentrations resulted in a concentration related decrease in somite number, head length and developmental score. The 12 mg/ml dose resulted in some embryolethality as well as dysmorphogenesis, while the highest dose was embryolethal. MeOH was dysmorphogenic in vitro in rat embryos at a MeOH concentration comparable to that reported in maternal serum following teratogenic in vivo exposures. Day 8 mouse embryos were explanted and cultured in 0, 2, 4, 6 or 8 mg MeOH/ml culture medium (75% rat serum, 25% Tyrode's salt solution) for 24 h. Embryonic development in the 2 mg/ml MeOH group was not significantly different from the controls but all higher concentration groups had a significant decrease in developmental score and crown-rump length. The high concentration group also suffered 80% embryolethality. Thus, mouse embryos were affected at MeOH concentrations which were not dysmorphogenic or embryotoxic in the rat, suggesting that the higher sensitivity of the mouse to the developmental toxicity of inhaled methanol is due, at least in part, to greater intrinsic embryonal sensitivity of this species to methanol.

Developmental toxicity of bromohydroquinone (BHQ) and BHQ-glutathione conjugates in vivo and in whole embryo culture.

Glutathione conjugates of 2-bromohydroquinone (GSyl-BHQ) cause renal proximal tubular necrosis that is dependent upon the activity of gamma-glutamyl transferase (GGT). GGT is present in embryonic yolk sac and its activity increases with gestational age, suggesting that the developing embryo might be at risk from maternal exposure to glutathione conjugates or compounds which are shown to form glutathione conjugates. Studies in pregnant rats exposed on Day 9 of gestation to 400 or 800 mumol/kg BHQ or 20 mumol/kg 2-Br-(di-GSyl)HQ and examined on Day 11 of gestation suggested that the parent compound (BHQ) or a metabolite was nephrotoxic in the adult and dysmorphogenic in the embryo and that 2-Br-(di-GSyl)HQ was nephrotoxic but not dysmorphogenic at the dose tested (20 mumol/kg). We therefore exposed Day 9 rat embryos to BHQ, 2-Br-6-(GSyl)HQ, or 2-Br-(di-GSyl)HQ in vitro for 48 hr to determine the relative dysmorphogenic activity of the parent compounds and the two conjugates. In vitro exposure to BHQ (0-40 microM) resulted in dose-related decreases in somite number (SN), total protein, and developmental score (DEVSC), with no effect on yolk sac diameter (YSD), crown rump length (CR), head length (HL), or percentage abnormal embryos (%AE); 60 microM BHQ was embryolethal. Embryos exposed to 2-Br-6-(GSyl)HQ (0-120 microM) were not affected at concentrations below 120 microM, at which dose there were significant effects on protein, YSD, CR, HL, DEVSC, SN, and %AE. Embryos exposed to 2-Br-(di-GSyl)HQ had a significantly lower DEVSC at the 80 microM concentration and significantly lower YSD, protein, and DEVSC and significantly higher %AE at the 10, 25, and 120 microM concentrations. CR, HL, and SN were not affected at any exposure level with this compound. In conclusion, BHQ was found to be developmentally toxic in vitro and in vivo at doses which also produced severe maternal renal necrosis. The doses of 2-Br-(di-GSyl)HQ in vivo which caused only mild maternal renal necrosis did not produce developmental toxicity. Conjugation of BHQ with either one or two molecules of GSH decreased the embryolethality of BHQ. The conjugates appeared to be of comparable toxicity as measured by the incidence of abnormal embryos in vitro. The role of maternal toxicity and GSH conjugation in 2-BHQ-mediated developmental toxicity remains to be determined.

Lowering pH increases embryonic sensitivity to formate in whole embryo culture.

The effects of formate exposure on mammalian embryo development were investigated using the rat whole embryo culture system as a model. Day 9.5 (presomite) rat embryos were explanted and cultured for 48 hr in rotating bottles containing rat serum with 0, 0.2, 0.4, 0.8, 1.2 or 1.6 mg sodium formate/ml culture medium at pH 8.13, 7.75, 7.00, 6.50 or 6.00 to determine whether the pH of the culture medium affects the in vitro developmental toxicity of formate. Several parameters of embryonic development decreased in the presence of decreasing pH, suggesting that altered pH alone could have a negative impact on embryo development. Exposure to 1.6 mg formate/ml affected protein concentration, somite number (SN), head length (HL), developmental score (DS), crown-rump length (CRL) and yolk-sac diameter of embryos at all pH levels. Formate became more toxic with decreasing pH of the culture media. There was an apparent pH-dependent increase in embryolethality at 1.6 mg formate/ml and 100% lethality at pH 6.00. The 1.2-mg/ml formate concentration affected DS, CR, HL and protein content at the pH 7.75 level whereas 0.8 mg formate/ml resulted in reduced DS, HL, CR, SN and protein content at pH levels of 7.00 and lower. At pH 6.5, embryos that were not exposed to formate were not significantly different from the other control groups except in reduced CR but at this pH, all exposure levels of formate resulted in microcephaly and reduction in embryonic protein as well as reduced CR. These data demonstrate that sensitivity to formate-induced embryo toxicity and dysmorphogenesis in whole embryo culture is increased in the presence of lower pH.

Developmental effects of methyl benzimidazolecarbamate following exposure during early pregnancy.

Methyl 2-benzimidazolecarbamate (MBC) and its parent compound benomyl are used as agricultural fungicides. Both chemicals are embryotoxic if administered during organogenesis, and benomyl is teratogenic. Based on a previous study indicating a lack of maternal effects of MBC following exposure during early pregnancy, the current experiments were designed to evaluate the effect of exposure to MBC during early pregnancy on developmental parameters of offspring. Rats were administered MBC at 0, 100, 200, 400, or 600 mg/kg/day during Days 1-8 of pregnancy and killed on Day 11 or Day 20 of gestation. On Day 11, embryos were assessed for survival rate, growth parameters, and anomalies. On Day 20, standard developmental toxicity evaluations were performed. Doses of 200 to 600 mg/kg/day MBC reduced embryonic survival by Day 11; exposure to MBC at 100 to 600 mg/kg/day reduced the number of fetuses surviving on Day 20. Evidence of developmental delay was apparent on Day 11 at all doses, and fetal weight was reduced by Day 20. MBC produced a dose-dependent increase in developmental defects seen on Day 11 and in several malformations observed on Day 20. MBC exposure during the first week of pregnancy was shown to be embryotoxic, resulting in embryonic death, growth retardation, and developmental abnormalities when evaluated on Days 11 or 20 of gestation.

In vitro embryotoxicity of a series of para-substituted phenols: structure, activity, and correlation with in vivo data.

The embryotoxicity of phenol and twelve para-substituted congeners on mid-gestation rat embryos was evaluated in vitro. Through application of correlative procedures and stepwise regression, equations describing the relationship between physical-chemical properties and various measures of activity were developed. Embryotoxicity was quantified by the log of the reciprocal of the potency estimates for reduction in selected growth parameters and induction of four morphological defects. In general, co-cultured hepatocytes ameliorated embryotoxicity; only phenol-induced embryotoxicity was enhanced by the presence of hepatocytes. In the absence of hepatocytes, measures of growth retardation were positively correlated with molar refractivity of the phenols. With hepatocytes, lipophilicity became important for describing the potential to induce growth deficits. The structural defects had varying correlation patterns in both culture systems. Potencies of these congeners in vitro were also compared to maternal and developmental potencies observed in vivo (Kavlock, Teratology, 41:43-59, '90). Two of the congeners were very toxic in both systems. For the remaining congeners, one maternal toxicity measure was found to be positively correlated with embryotoxicity for growth and development in vitro without hepatocytes. With hepatocytes, a broad spectrum of correlations, both positive and negative, were observed between in vivo developmental toxicity endpoints and in vitro embryotoxicity. Data from preliminary dosimetry studies suggest that phenol congeners may accumulate in embryos exposed in vitro more readily than with in vivo exposure. Potency calculations based on dosimetry information may demonstrate better correlations between data and allow additional relationships between chemical structure and activity to be developed.

In vitro/in vivo comparison of yolk-sac function and embryo development.

The yolk-sac function and development of rat embryos grown in vitro for 24 hr, starting on day 10.5, were compared with those of embryos grown in utero. The embryos grown in vitro had significantly fewer somites, shorter crown-rump length and smaller yolk-sac diameter when compared with the embryos grown in vivo but all values were within the normal range for this stage of gestation. Head length was not significantly different between the two groups. The cellular and nuclear volumes (Coulter counter) of nucleated yolk-sac red blood cells did not differ significantly between the two groups. RBC cell-cycle analyses by flow cytometry did not reveal any difference between in vitro and in vivo embryos. The clinical chemistries of embryo-yolk-sac homogenates were compared. Protein, triglyceride, lactate dehydrogenase, cholesterol, urea nitrogen and glutamic-oxalacetic transaminase concentrations did not differ significantly between the two groups. The in vitro embryos had significantly lower gamma-glutamyl transferase (GGT) and sorbitol dehydrogenase activities. GGT activity is almost entirely in the yolk sac in the day 10.5 conceptus. alpha-Foetoprotein is synthesized by the yolk sac at this stage of development and was significantly lower in the in vitro embryos. Transferrin is transported across the yolk sac to the embryo and was significantly higher in the in vitro embryos. These data indicate that impaired yolk-sac function could, in part, be responsible for the developmental delays and the short survival times of cultured embryos.

In vivo and in vitro structure-dosimetry-activity relationships of substituted phenols in developmental toxicity assays.

Structure-dosimetry-activity relationships (SDARs) of a series of substituted phenols were evaluated following exposure of gestation day 11 rats in vivo and in comparable stage embryos in vitro. In the in vivo study, 27 congeners were assayed and log P (a term used synomously with lipophilicity in this paper) and Hammett sigma values (a measure of the electronic withdrawing ability of the substituent) were shown to correlate with maternal toxicity; however, no relationships between these parameters and developmental effects were observed. In the in vitro system, 13 congeners were evaluated and molar refractivity and/or lipophilicity were shown to correlate with the ability of the phenols to induce embryonic growth retardation and structural defects in the absence of the hepatocytes. In contrast, when a metabolic activating system (primary hepatocytes) was present in the in vitro system, the potential to induce growth retardation was inversely related to lipophilicity, although the relationships were weaker than the positive relationship seen without the hepatocytes. The binding of the phenols to macromolecules in the culture medium was highly correlated with log P. Correcting the in vitro potency data for the variable amount of binding improved the predictiveness of the quantitative structure-activity relationships (QSARs). The potential to induce embryotoxicity in vitro was not well correlated with the potential to induce developmental toxicity in vivo: whereas the in vitro data demonstrates that the phenols are intrinsically embryotoxic, few of them actually produced significant developmental toxicity in the in vivo system, and there were few positive correlations between effects observed in the two systems.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro culture of postimplantation hamster embryos.

In vitro culture of intact rat and mouse embryos has been described extensively, but information on the culture of other species is sparse. The present study examined some culture requirements of early somite stage hamster embryos and assessed the embryotoxic effects of sodium salicylate (SS), a direct acting chemical and cyclophosphamide (CP), a proteratogen, on these embryos. Hamster embryos explanted on gestation days (GD) 8 and 9 were cultured in Waymouth's embryo-hepatocyte co-cultivation medium (WEHC), 70% McCoy's 5A medium-30% male rat serum (MMRS) or 100% male rat serum (MRS) for 24 hours under various oxygen concentrations. Embryos cultured GD 8 to 9 in the various media grew and differentiated much as they did in vivo, while embryos cultured GD 9 to 10 grew best in MMRS as compared to embryos at the same stage in vivo. Embryos exposed to SS in MMRS at concentrations of 250, 300, or 400 micrograms/ml showed dose related embryotoxicity which included CNS defects, absence of hind limb bud formation, and lack of axial rotation. Hamster embryos co-cultivated with pregnant hamster hepatocytes and treated with 2.5, 6.25 and 12.5 micrograms/ml of CP, showed dose-dependent toxicity when compared to co-cultivated controls. Hamster embryos develop extensively in culture over a 24 hour period. This system may therefore provide a valuable tool for evaluating the species differences of a variety of potential teratogens and embryotoxins and allow the comparison of these embryotoxic effects between rat, mouse and hamster during similar stages of organogenesis.