Marian T. Ebron-McCoy

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.

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.

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)

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.

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.

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 Waymouths embryo-hepatocyte co-cultivation medium (WEHC), 70% McCoys 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.