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Fish embryos exposed to complex mixtures of polycyclic aromatic hydrocarbons (PAHs) from petrogenic sources show a characteristic suite of abnormalities, including cardiac dysfunction, edema, spinal curvature, and reduction in the size of the jaw and other craniofacial structures. To elucidate the toxic mechanisms underlying these different defects, we exposed zebrafish (Danio rerio) embryos to seven non-alkylated PAHs, including five two- to four-ring compounds that are abundant in crude oil and two compounds less abundant in oil but informative for structure-activity relationships. We also analyzed two PAH mixtures that approximate the composition of crude oil at different stages of weathering. Exposure to the three-ring PAHs dibenzothiophene and phenanthrene alone was sufficient to induce the characteristic suite of defects, as was genetic ablation of cardiac function using a cardiac troponin T antisense morpholino oligonucleotide. The primary etiology of defects induced by dibenzothiophene or phenanthrene appears to be direct effects on cardiac conduction, which have secondary consequences for late stages of cardiac morphogenesis, kidney development, neural tube structure, and formation of the craniofacial skeleton. The relative toxicity of the different mixtures was directly proportional to the amount of phenanthrene, or the dibenzothiophene-phenanthrene total in the mixture. Pyrene, a four-ring PAH, induced a different syndrome of anemia, peripheral vascular defects, and neuronal cell death, similar to the effects previously described for potent aryl hydrocarbon receptor ligands. Therefore, different PAH compounds have distinct and specific effects on fish at early life history stages.

In utero exposure of male C57BL/6 mice to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) prevents prostatic epithelial buds from forming in the ventral region of the urogenital sinus (UGS) and reduces the number of buds that form in the dorsolateral region. This inhibition of budding is aryl hydrocarbon receptor (AHR) dependent and appears to be the primary cause of lobe-specific prostate abnormalities in TCDD-exposed mice. TCDD can inhibit prostatic epithelial bud formation by acting directly on the UGS in vitro, but whether it does so via AHR in UGS mesenchyme, epithelium, or both was unknown. To address this issue, UGS mesenchyme and epithelium from gestation day (GD) 15 wild-type C57BL/6J male mice were isolated, recombined, and cultured in vitro for 5 days with 10(-8) M 5alpha-dihydrotestosterone (DHT) and either 10(-9) M TCDD or vehicle. Prostatic epithelial buds were viewed by light microscopy after removal of mesenchyme. Effects depended greatly on which portions of the mesenchyme were used: TCDD had little if any effect when whole UGS epithelium (UGE) was recombined with ventral plus dorsolateral mesenchyme, tended to reduce bud numbers in recombinants made with UGE and dorsolateral mesenchyme, and severely reduced bud numbers in recombinants made with UGE and ventral mesenchyme (VM). [VM + UGE] recombinants prepared from wild-type and AHR knockout (Ahr(-/-)) mice were then cultured with DHT to determine the site of action of TCDD. AHR null mutation alone had no effect on budding. TCDD severely inhibited prostatic epithelial bud formation in recombinants that contained mesenchymal AHR, whereas bud formation was not inhibited by TCDD in recombinants lacking mesenchymal AHR, regardless of epithelial AHR status. These results demonstrate that UGS mesenchyme and not UGS epithelium is the site of action of TCDD. Therefore, the initial events responsible for abnormal UGS (and ultimately prostate) development occur within the UGS mesenchyme, and changes in gene expression in the UGS epithelium responsible for inhibited prostatic budding are secondary to the direct effects of TCDD on UGS mesenchyme.

Reactive oxygen species (ROS) can transduce intracellular signals or damage macromolecules, including mutational activation of the K-ras oncogene, particularly in A/J mice, which are reported to be highly sensitive to the ROS-initiating teratogen phenytoin. Here, we determined in embryo culture whether the Ras pathway mediated phenytoin embryopathy at the protein or gene level. Embryos from pregnant inbred A/J dams and outbred CD-1 dams were cultured with a therapeutic concentration of phenytoin, with or without alpha-hydroxyfarnesylphosphonic acid, an inhibitor of the enzyme farnesyl-protein transferase, which is required for posttranslational Ras activation. A/J and CD-1 embryos were similarly susceptible to phenytoin embryopathies, with reductions in anterior neuropore closure, turning, yolk sac diameter, and somite development (p < 0.05). The farnesyl-protein transferase inhibitor blocked phenytoin embryotoxicity in A/J embryos for all parameters except yolk sac diameter (p < 0.05) and completely blocked embryotoxicity in CD-1 embryos (p < 0.05). Embryonic DNA did not show phenytoin-initiated mutations in codon 12 of the K-ras gene in either A/J or CD-1 embryos, but phenytoin substantially increased the levels of GTP-bound Ras in both CD-1 and A/J embryos. These results provide the first direct evidence that Ras proteins may be involved in the teratogenicity of phenytoin, likely via a mechanism other than mutational activation.

Introduction: The persistent morbidity and mortality of congenital diaphragmatic hernia are largely due to associated pulmonary hypoplasia. We have shown previously that three antioxidants (vitamin C, glutathione, and vitamin E) could accelerate the growth of fetal hypoplastic lungs grown in culture. We hypothesize that this occurs via a reductant mechanism., Methods: Timed-pregnant rats were gavage-fed nitrofen (100 mg) on day 9.5 of gestation (term = day 22). Fetal lungs were harvested on day 13.5 and placed in organ culture containing serum-free BGJb medium with antibiotics. After randomization, the lung organ cultures were divided into a control group (n = 31) and an experimental group that received the antioxidant N-acetylcysteine (NAC, 100 microM, n = 31). The fetal lung organ cultures were grown for 4 days at 37 degrees C with 5% CO(2). Computer-assisted digital tracings of the airways were performed daily on live, unstained specimens, and lung bud count, perimeter, and area were measured. After 4 days, lungs were pooled, homogenized, and assayed for reduced and oxidized glutathione, normalized to protein, as an estimate of the tissue redox potential. Data were expressed as means +/- SEM, and statistical comparisons were performed using Student's unpaired t test, with P < 0.05 considered significant., Results: Area, perimeter, lung bud count, and complexity (as measured by the perimeter/square root of area) were all significantly increased with NAC treatment from day 2 onward. Reduced glutathione levels were significantly increased following NAC administration (67.1 +/- 5.8 versus 37.5 +/- 4.2 micromol/mg, P = 0.0004). The ratio of reduced to oxidized glutathione was 2.23., Conclusions: N-Acetylcysteine stimulates nitrofen-induced hypoplastic fetal lung growth in organ culture and increases the ratio of reduced to oxidized glutathione. These data support the concept that oxidation-reduction (redox) may be an important control mechanism for fetal lung growth.

The genetic mouse model BTBR-Pah(enu2) was used to more thoroughly investigate the pathogenesis of maternal phenylketonuria (MPKU). More specifically, it was used to examine the effect of maternal blood phenylalanine (PHE) level on the pregnancy outcome of MPKU offspring as determined by certain key measures of development at birth (i.e., head circumference, weight, and crown-rump length of offspring). In this study, we clearly observed that elevated maternal blood PHE levels, whether they were caused by the maternal diet or the maternal genotype, were responsible for fetal abnormalities. As in human MPKU, significant reductions (P < 0.0001) in birth weight, crown-rump length, and head circumference were seen in offspring gestated under the condition of high maternal blood PHE levels. These findings strongly suggest that there are sufficient similarities between human MPKU and MPKU in this mouse model to establish it as a very promising model for future studies designed to characterize human MPKU more thoroughly., (Copyright 2001 Elsevier Science.)

In this study, the mouse was used to evaluate paternal germline exposure to the organophosphate methamidophos for its potential to produce adverse effects on spermatozoa and in the offspring. There have been reports that organophosphate exposure can increase abnormal sperm morphology in mice. However, effects transmitted to the offspring following paternal exposure have not been reported previously. The maximum tolerated dose (MTD) was 7.5 mg kg(-1) body weight and this dose resulted in no deaths, although blood plasma cholinesterase activity was still decreased. Males were euthanized 4 weeks after an acute intraperitoneal injection of methamidophos (0.5, 3.75, 5.0, and 7.5 mg kg(-1) body wt) and the number of spermatids per gram testes and sperm morphology were analyzed. In this study, abnormal sperm morphology on a per group basis exhibited a dose-response significantly related to increased methamidophos exposure as indicated by regression analysis and a nested ANOVA (p < 0.0001). Preimplantation embryos that were conceived 6 weeks after paternal methamidophos exposure (5 mg kg(-1) body wt) exhibited a significant increase in cleavage arrest. Fertility of males was also affected as shown by a decrease in the number of two- to four-cell embryos per male (postexposure week 6) and an increase in the number of degenerated embryos (postexposure weeks 4-6). We conclude that methamidophos may have the potential to produce transmissible adverse embryonic effects following an acute paternal germline exposure., (Copyright 2000 Academic Press.)

New studies suggest that the teratogenicity of phenytoin (PHT) is linked to its membrane-stabilizing pharmacological action via the rapid component of the delayed rectified potassium channel (lkr), resulting in embryonic cardiac dysrhythmia during a restricted sensitive period. In order to further elucidate this theory, PHT was administered to Sprague-Dawley rats on gestation day (GD) 11 with either a single dose of 150 or 100 mg/kg ip or 150 mg/kg po and developmental toxicity at term (GD 21) was studied. In satellite animals blood samples were withdrawn (0.5-24 h after dose) and total and free maternal plasma concentrations of PHT were measured. Pharmacokinetic data correlated well with pregnancy outcome data. At 150 mg/kg ip high concentrations of long duration (C(max) 240 microM and AUC 5300 microMhl(-1) - total) and marked developmental toxicity (embryonic death, decreased fetal weights, and orofacial clefts) were observed. After 100 mg/kg ip (C(max) 150 microM, AUC 2600 microMhl(-1) - total) only slight developmental toxicity (decreased fetal weights) was recorded and after 150 mg/kg po the plasma concentrations were even lower (C(max) 63 microM and AUC 1100 microMhl(-1) - total) and no adverse effects at all were observed. In separate experiments the effect of different concentrations of PHT on the embryonic heart was studied by adding PHT to GD 11 rat embryos cultured in vitro or by culturing GD 11 embryos from exposed dams. The decrease in heart rates was 3, 16, and 32% after culture with 50, 100, and 200 microM of PHT, respectively. After maternal administration of 150 mg/kg ip or po, the embryonic heart rate in vitro decreased by 25 and 7%, respectively, compared to controls. Altogether the results suggest that the development toxicity of PHT is caused by concentration-dependent induction of embryonic dysrhythmia and hypoxia related damage., (Copyright 2000 Academic Press.)

The effects on embryonic development of a series of eight saturated (acetonitrile, propionitrile, and n-butyronitrile) and unsaturated (acrylonitrile, methacrylonitrile, allylnitrile, cis-2-pentenenitrile, and 2-chloroacrylonitrile) nitriles were compared in vitro using the whole embryo culture system. Day 10 rat embryos were cultured for 46 h in rat serum in the presence of either of these chemicals. All the tested chemicals produced concentration-dependent decreases in growth and differentiation and increases in the incidences of morphologically abnormal embryos. A wide range of embryotoxic potency was observed, with 2-chloroacrylonitrile and acetonitrile at the extremes (lowest effect levels of 50 microM and 40 mM, respectively). No common pattern could be drawn for all the eight nitriles tested in vitro, although there were some similarities between the malformations elicited by propionitrile and n-butyronitrile or between those elicited by the five unsaturated nitriles. Presence of a rat hepatic microsomal fraction and NADPH in the culture medium enhanced the embryotoxic effects of the five unsaturated nitriles tested but had no effects on saturated nitriles embryotoxicity. In addition to these in vitro experiments, pregnant rats were given a single oral dose of each compound on Day 10 of gestation and the embryos were evaluated on Day 12 of gestation, i.e., at a time of development corresponding to the developmental stage at the end of the whole embryo culture. All the nitriles investigated produced the characteristic defects developed by embryos exposed to sodium cyanide in utero or in culture. Our results provide further evidence that maternal production of cyanide may contribute to the developmental toxicity of saturated and unsaturated nitriles and suggest that distinct metabolites derived from microsomal metabolism of unsaturated nitriles may also play a role., (Copyright 2000 Academic Press.)

Effects of stage of development and 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) exposure on aryl hydrocarbon receptor (AhR) and AhR nuclear translocator (ARNT) protein concentrations in reproductive organs of male rats were determined. AhR protein levels in developing rat ventral and dorsolateral prostate decreased with age, declining approximately 70% between Postnatal Days (PND) 1 and 21. ARNT protein levels also decreased with age in dorsolateral, but not ventral prostate. The developmental decreases in prostatic AhR and ARNT protein were associated with decreases in AhR and ARNT mRNA. AhR and ARNT protein concentrations in fetal urogenital sinus on Gestation Days (GD) 16, 18, and 20 were similar to levels in ventral prostate on PND 7. TCDD exposure of adult male rats (0.2, 1, 5, or 25 micrograms/kg po, 24 h) decreased AhR but not ARNT protein in ventral and dorsolateral prostate, vas deferens, and epididymis. In utero and lactational TCDD exposure (1.0 micrograms/kg dam po, GD 15) did not alter ARNT levels but reduced prostatic AhR protein levels on PND 7 and delayed the developmental decrease in AhR protein in ventral and dorsolateral prostate. Finally, pretreatment of rat pups for 24 h with TCDD (5 micrograms/kg ip) down-regulated prostatic AhR protein on PND 7, but not on PND 1. Thus, prostatic AhR and ARNT protein and mRNA levels are regulated with age, whereas only AhR protein concentration is altered by TCDD exposure. Because in utero and lactational TCDD exposure only decreased prostatic AhR on PND 7, it is unlikely that down-regulation of AhR is the mechanism by which perinatal TCDD exposure impairs prostate development., (Copyright 1999 Academic Press.)

Previous studies have shown that, in general, young, postnatal animals are more sensitive than adults to the toxic effects of anticholinesterase (antiChE) pesticides. Paradoxically, often fetal brain cholinesterase (ChE) is less inhibited than maternal brain after gestational exposure to an antiChE, presumably due to placental and fetal detoxification of the antiChE. The present investigation was designed to study selected toxicokinetic and toxicodynamic factors surrounding the toxicity of chlorpyrifos (CPF; [O,O'-diethyl O-3,5,6-trichloro-2-pyridyl] phosphorothionate) in pregnant rats dosed repeatedly or singly during late gestation. Dams were dosed daily (po) with CPF in corn oil (0 or 7 mg/kg) on gestational days (GD) 14 to 18. Animals were euthanized at 2 to 120 h after the last dose and tissues were collected for enzyme analysis. Using this dosing regimen, we found that (1) the time of maximal ChE inhibition was the same (i.e., 5-10 h after dosing) for both maternal and fetal brain, (2) the degree of fetal brain ChE inhibition was 4.7 times less than maternal brain inhibition, and (3) the detoxification potential (i.e., carboxylesterase and chlorpyrifos-oxonase) of the fetal tissues was very low compared to the maternal tissues. A separate group of experiments showed that if pregnant dams received only one oral dose of 7 or 10 mg/kg CPF on GD18, the degree of ChE inhibition in the fetal brain was comparable to the maternal brain ChE inhibition. Taking into consideration the net increase (more than fourfold) in fetal brain ChE activity from GD14 to 18 in control animals, and the fact that maternal brain ChE was inhibited more than fetal brain ChE only in a repeated-dosing regimen, we conclude that the fetus is not genuinely protected from the toxic effects of a given dose of CPF. We propose that fetal brain ChE is simply able to recover more fully between each dose as compared to maternal brain ChE, giving the illusion that the fetal compartment is less affected than the maternal compartment.

In the male Holtzman rat, in utero and lactational 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure decreases prostate weight without inhibiting testicular androgen production or decreasing circulating androgen concentrations. Therefore, the present study sought to characterize effects of TCDD exposure on prostate development, from very early outgrowth from the urogenital sinus (Gestation Day [GD] 20) until rapid growth and differentiation are essentially complete (Postnatal Day [PND] 32). Pregnant Holtzman rats were administered a single dose of TCDD (1.0 microgram/kg po) or vehicle on GD 15 and offspring were exposed via placental transfer (GD 20 euthanasia) or placental and subsequent lactational transfer until euthanasia (if before PND 21) or weaning. Results show that the prostatic epithelial budding process was impaired by in utero TCDD exposure, as evidence by significant decreases in the number of buds emerging from dorsal, lateral, and ventral aspects of the GD 20 urogenital sinus. Ventral prostate cell proliferation index was significantly decreased on PND 1 but was similar to or higher than control at later times, whereas apoptosis was an extremely rare event in ventral prostates from both control and TCDD-exposed animals. Delays were noted in the differentiation of pericordal smooth muscle cells and luminal epithelial cells. In addition, ventral prostates from approximately 40% of TCDD-exposed animals examined on PNDs 21 and 32 exhibited alterations in the histological arrangement of cell types that could not be explained by a developmental delay. Compared to controls, these ventral prostates exhibited a disorganized, hyperplastic epithelium containing fewer luminal epithelial cells and an increased density or continuous layer of basal epithelial cells, as well as thicker periductal smooth muscle sheaths. In addition, in ventral prostates from TCDD-exposed animals, the intensity of androgen receptor staining was relatively low in the central and distal epithelium, and the number of androgen receptor-positive cells was relatively high in the periductal stroma. These data suggest that in utero and lactational TCDD exposure interferes with prostate development by decreasing very early epithelial growth, delaying cytodifferentiation, and, in the most severely affected animals, producing alterations in epithelial and stromal cell histological arrangement and the spatial distribution of androgen receptor expression that may be of permanent consequence.

Programmed cell death or apoptosis is an essential process during the morphogenesis of a large number of structures. Evidence obtained over the past few years indicates that, in some cases, the generation of reactive oxygen species (ROS) is an important event during the course of apoptosis. Using an in vitro culture system in which digit individualization of developing limbs normally occurs, we assayed the effect of different antioxidants on the cell death that takes place at interdigits. The addition of phenol, dimethyl sulfoxide, or 2',7'-dichlorodihydrofluorescein diacetate (DCDHF-DA) to murine developing limbs in culture prevented digit individualization as well as the typical interdigital cell death. Two ROS-sensitive dyes, 3-(4,5-dimethylthiazol)-2,5-diphenyl tetrazolium bromide and DCDHF-DA, stained interdigits and the so-called "necrotic zones," implying that they contain cells under oxidative stress. Very few interdigital cells were doubly stained with the ROS probes and two cell death indicators (i.e., acridine orange and propidium iodide), suggesting that they detect a different stage during the course of apoptosis. Furthermore, we found cells stained for ROS that did not express a specific macrophage marker and in a few cases were seen surrounded by a macrophage. Surprisingly, many regions of the midgestation mouse embryo that are undergoing cell death correlated with those that have a markedly higher level of ROS. Our data suggest that the generation of oxidative stress is a common requirement for cell death that occurs during mouse embryonic development.

In this investigation, we studied the effects of ethanol (EtOH) on the development of noradrenergic (NE) neurons of the locus coeruleus. Fetal brainstem tissue from embryonic days 15-17 was grafted into the anterior chamber of the eye of adult rats. Two different experimental groups were exposed to 16% EtOH in the drinking water during different developmental windows. The first group received EtOH 24 h after transplantation and during the whole experimental period of 7 weeks (continuous EtOH), and the second group only during the last 5 weeks of the experimental period (delayed EtOH). The control group received water ad libitum. After 7 weeks, all the animals were sacrificed and morphological evaluations were performed. Immunohistochemical analysis showed that axon bundle formation and NE fiber outgrowth into the host iris was significantly reduced in the continuous EtOH-treated group compared to controls. We also studied the morphology of TH-positive neurons and processes in the intraocular transplants. A significant decrease in TH-positive staining intensity was observed in the continuous EtOH-treated group compared to controls. Moreover, we found a significant decrease in cell size and neuronal survival in both EtOH-treated groups compared to controls. The present results suggest that chronic EtOH exposure during development leads to an altered axonal outgrowth and decreased cell sizes and number of NE neurons in intraocular brain stem grafts. Furthermore, we found that NE neurons are more sensitive to EtOH exposure during the last prenatal days and the first postnatal week of development, compared to a later developmental period.

Triglyceride-containing lipid emulsions have been designed as caloric sources that can be administered intravenously to patients that cannot meet their nutritional needs by conventional parenteral therapies. In their study, we evaluate the developmental toxicity of a 20% lipid emulsion that contains a 3:1 ratio of medium chain triglyceride (MCT) to one long chain containing lipid emulsion (LCT). This emulsion was administered by intravenous infusion to rats and rabbits at dosages of 1 and 4.28 g lipid/kg body weight (g lipid/kg) at dose volumes of 5 and 21.4 mL/kg, respectively, once daily during organogenesis to assess the potential developmental toxicity of the test article. The control group received 0.9% saline at a dose volume of 21.4 mL/kg. Animals were observed for clinical signs of toxicity and adverse effects on body weights and feed consumption. On Day 20 (rats) or Day 29 (rabbits), females were necropsied and examined for maternal and embryo/fetal toxicity. Fetuses were removed, weighed, and examined for external, soft tissue, and skeletal abnormalities. Dosages of 4.28 g lipid/kg resulted in lower feed consumption for rats and rabbits, an expected finding based on the high-caloric nature of the test article. Potentially test article-related gross necropsy findings, including enlarged lymph nodes and spleen, small thymus, and enlarged renal pelvis, for rats given 4.28 g lipid/kg were present at a low incidence. There were no adverse effects on fetal parameters for rats even in the presence of some maternal toxicity. However, embryo and fetal toxicity (i.e., resorptions) and skeletal abnormalities were present for rabbits given 4.28 g lipid/kg. Under the conditions of this study, the no-observable-effect level for developmental toxicity was greater than or equal to 4.28 g lipid/kg for rats and greater than or equal to 1 g lipid/kg but less than 4.28 g lipid/kg for rabbits.

Pregnant Wistar rats (40/group) were administered monoethanolamine (MEA) as an aqueous solution by gavage at dose levels of 0, 40, 120, and 450 mg/kg/day on days 6 through 15 of gestation. On day 20 of gestation, 25 dams/group were euthanized and the fetuses were delivered by cesarean section, weighted, sexed, and examined for external, visceral, and skeletal alterations. The remaining dams (15/group) were allowed to litter and rear their pups to day 21 postpartum. The dams and pups were then euthanized and examined for gross pathologic changes. Gavage administration of 450 mg MEA/kg/day to pregnant rats resulted in maternal toxicity as evidenced by statistically significant (alpha = 0.05) decreases in feed consumption on gestation days 6-8 and 17-20 and on postpartum days 0-4. Additionally, statistically significant decreases in mean maternal body weights were observed on gestation days 15, 17, and 20 and on lactation days 0, 4, 7, and 21. Body weight gains of the 450 mg/kg/day dams were also significantly decreased (13% relative to controls) on gestation days 15-20. There was no evidence of maternal toxicity at 40 or 120 mg/kg/day of MEA. Despite the maternal effects observed at 450 mg/kg/day, no significant fetal effects were observed at this or any dose level tested, nor were there any indications of a treatment-related effect on postnatal growth or on the viability of offspring. Thus, it was concluded that MEA was not developmentally toxic to Wistar rats following repeated oral administration, even at maternally toxic dose levels as high as 450 mg/kg/day.

This study evaluates the developmental toxicity and placental and milk transfer of N,N-dimethylformamide (DMF) in rats. Sprague-Dawley rats were given 0, 50, 100, 200, and 300 mg DMF/kg/day, by gavage, on Gestational Days (GD) 6 through 20. Maternal toxicity was indicated by depressions in weight gain and food consumption at doses >/=100 mg/kg. Fetal toxicity was indicated by decreased fetal body weight at doses >/=100 mg/kg, and by increased incidences of two skeletal variations (absent or poorly ossified supraoccipital and sternebrae) at 200 and 300 mg/kg. Thus, the maternal and developmental no-observed-adverse-effect level was 50 mg/kg/day. The time course disposition of [14C]DMF was examined over a 48-hr period in GD12- and GD18-pregnant rats after a single oral dose of 100 mg [14C]DMF/kg. Peak concentrations of radiocarbon occurred within 1 hr after dosing. Embryonic (GD12) and fetal (GD18) tissues accounted for 0.15 and 6% of the administered dose, respectively. Levels of radiocarbon in embryonic and fetal tissues were equal or slightly less than in maternal plasma up to 8 and 24 hr, respectively, and higher thereafter. HPLC analysis performed at intervals from 1 to 8 hr on GD12 and 1-24 hr on GD18 indicated that unchanged DMF and metabolites were readily transferred to the embryonic and fetal tissues, where their levels were generally equal to those in maternal plasma. The parent compound accounted for most of the radioactivity until 4-8 hr and then decreased. N-Hydroxymethyl-N-methylformamide (HMMF) and N-methylformamide (NMF) were the predominent metabolites and increased with time. Much lower concentrations were found for formamide and N-acetyl-S-(N-methylcarbamoyl)cysteine. Transfer of radioactivity into milk was studied in dams given a single oral administration of 100 mg [14C]DMF on Lactation Day 14. DMF, HMMF, and NMF were found in the milk at concentrations equal to those in plasma., (Copyright 1997 Society of Toxicology.)

Effects of serine on restorative growth were characterized by comparing embryo/fetal responses after maternal exposure to 2-methoxyethanol (ME) and ME + serine by gavage on gestation day (gd) 13, a day of heightened limb sensitivity. Paws (gd 20) and limb buds (gd 15) were examined after ME alone at 50, 100, and 250 mg/kg, and after ME (either 100 or 250 mg ME/kg) + serine (1734 mg serine/kg) administered within minutes (0 hr) to 24 hr after ME. Paw development was not altered after ME at 100 mg/kg, but was highly sensitive to 250 mg ME/kg with all fetuses and litters exhibiting defects (ectrodactyly, syndactyly, and short digit) in the preaxial region. In contrast, the limb bud displayed dose-related incidences of abnormalities after maternal treatment with the low and high levels of ME. The condensing (precartilaginous, pentadactyl pattern) and noncondensing (undifferentiated mesenchymal cells) regions exhibited changes in their size, number, and location. Serine administration after 250 mg ME/kg was effective in reducing the occurrence of paw dysmorphogenesis with its protection potency inversely related to its delay of administration (i.e., 0% paw defect incidence after 0-hr delay, 25% after 4-hr delay, 41-45% after 8- and 12-hr delays, and 76% after 24-hr delay). The occurrences of limb bud pattern disturbances produced by ME were also markedly decreased by serine cotreatment. Higher incidences of embryonic defects versus those of fetal defects demonstrate that restorative growth followed ME exposure. Serine attenuation of ME teratogenicity appears to emanate from enhanced restorative growth so that tissue damage, which otherwise would be expressed as a defect at parturition, is repaired and replaced to resume development of the limb toward its normal structure.

To evaluate effects of in utero and lactational 2,3,7,8-tetrachlorodibenzo-rho-dioxin (TCDD) exposure on male and female reproductive system development of the mouse, the offspring of pregnant ICR mice administered 0, 15, 30, or 60 microg TCDD/kg on Gestation Day (GD) 14 were examined at the postweanling, pubertal, young adult, and adult stages of development. Dam and offspring body weights and prenatal and postnatal mortality were unaffected by TCDD exposure. The most sensitive endpoints in male offspring were decreased ventral prostate, coagulating gland, and thymus weights, accelerated eye opening, and hydronephrosis. Decreases in pituitary gland weight and epididymal sperm numbers were also found in TCDD-exposed male offspring. Testis, epididymis, and dorsolateral prostate weights, anogenital distance, latencies to testis descent and to preputial separation, and serum testosterone concentrations were unaffected. At the highest maternal TCDD dose uterus weights were decreased in female offspring evaluated during estrus and diestrus. No morphologic changes in the external genitalia of female offspring were found, nor were there alterations in ovary or pituitary gland weights. Cross-species comparisons showed that the mouse was not as sensitive to TCDD-induced developmental reproductive toxicity as the rat and hamster. Many endpoints affected by TCDD in rat and hamster offspring were either not affected or were less sensitive in mouse offspring. Endpoints of androgenic status were not affected in the mouse, decreases in accessory sex organ weights were restricted to fewer organs in the mouse, and decreases in daily sperm production were not found in the mouse. The only developmental reproductive endpoint observed in all three species was a reduction in epididymal sperm numbers.

Syntower bottoms (STB) was evaluated for subchronic and developmental toxicity. In the subchronic study, undiluted STB was applied on the shaved backs of male and female rats at dose levels of 0, 8, 30, 125, and 500 mg/kg for 13 weeks, 5 days per week. Exposure sites were not covered. In the developmental toxicity study, STB was similarly applied, but to pregnant rats at dose levels of 0, 8, 30, and 125 mg/kg on Gestation Days 0-19. In addition, 4 mg/kg was dosed as 8 mg/kg every other day, starting on Gestation Day 0, and 500 mg/kg was dosed on Gestation Days 10-12. Evidence of toxicity observed in the subchronic study included death, decreased body weights, aberrant serum chemistry and hematology values, altered organ weights, and histopathologic changes in a variety of organs. A no observed adverse effect level for systemic toxicity could not be established. Evidence of maternal toxicity was observed at all exposure levels in the development study. Regardless of the length of the exposure period, STB was toxic to the developing conceptus. Evidence of developmental toxicity observed included increased resorptions with a concomitant decrease in litter size and reduced fetal body weights. Cleft palate was observed in fetuses exposed in utero to STB during Gestation Days 10-12 at 500 mg/kg. No evidence of teratogenicity was observed when the exposure period was throughout gestation. Ossification delays were observed in fetuses exposed in utero to STB at doses in excess of 4 mg/kg. A no observed adverse effect level for maternal and developmental toxicity could not be established.

3,3',4,4',5-Pentachlorobiphenyl (pentaCB) caused a dose-dependent induction of chicken embryolethality, malformations, edema, and liver lesions at doses ranging from 0.5 to 12.0 microg/kg. In contrast, no embryotoxicity was observed after treatment with 10, 25, or 50 mg/kg 2,2',4,4',5,5'-hexaCB. In eggs cotreated with 2.0 microg/kg, 3,3',4,4',5-pentaCB plus 10, 25, or 50 mg/kg 2,2',4,4',5,5'-hexaCB, there was significant protection from 3,3',4,4',5-pentaCB-induced embryo malformations, edema, and liver lesions, whereas no inhibition of embryolethality was observed. These results further extend the response-specific nonadditive interactions of binary mixtures of polychlorinated biphenyls (PCBs) and should be considered in the development of approaches for hazard assessment of PCB mixtures and related compounds.

The developmental toxicity of ethylene oxide (EtO) was examined in Sprague-Dawley rats following inhalation exposure during Days 6 to 15 of gestation. Two different exposure regimens were used: (1) exposure for 0.5 hr once a day to 0, 400, 800, or 1200 ppm EtO; or (2) exposure for 0.5 hr three times a day to 0, 200, or 400 ppm EtO or 0, 800, or 1200 ppm EtO. Repeated brief exposures (3 x 0.5 hr/day) to EtO caused fetal toxicity indicated by reduced fetal weight at 800 and 1200 ppm, and overt maternal toxicity manifested as reduced body weight gain at 1200 ppm. Neither embryolethality nor teratogenicity occurred following any exposure regimen.

Timed-pregnant Sprague-Dawley (CD) outbred rats and New Zealand White rabbits were dosed by gavage with methacrylonitrile (MACR) in distilled water during major organogenesis. Rats were dosed on Gestational Days (GD) 6 through 15 (0, 5, 25, or 50 mg MACR/kg/day) and rabbits on GD 6 through 19 (0, 1, 3, or 5 mg MACR/kg/day). Maternal clinical status was monitored daily during treatment. At termination (GD 20, rats; GD 30, rabbits), confirmed-pregnant females (25-26 per group, rats; 17-22 per group, rabbits) were evaluated for clinical status and gestational outcome; each live fetus was examined for external, visceral, and skeletal malformations. In rats, no treatment-related maternal clinical signs or mortality were observed, nor was there any adverse effect on maternal body weight or food or water consumption. At necropsy, absolute, relative, and adjusted maternal liver weight was increased at the mid- and high-dose groups, an effect that may be indicative of induction of hepatic enzymes rather than toxicity. In the absence of any indication of maternal toxicity, the no-observed-adverse-effect level (NOAEL) for maternal toxicity in this study was >/=50 mg MACR/kg/day. The NOAEL for developmental toxicity in rats was also >/=50 mg MACR/kg/day. There was no effect of treatment on postimplantation loss, mean fetal body weight per litter, or morphological development. In rabbits, maternal mortality and clinical signs were not dose related. Maternal food consumption, body weight, and liver weight were not adversely affected by treatment. Thus, the maternal NOAEL was >/=5 mg MACR/kg/day. Maternal toxicity, including death, was observed >/=7.5 mg/kg/day in preliminary studies. The developmental NOAEL was also >/=5 mg MACR/kg/day. There was no adverse effect of treatment on postimplantation loss or fetal body weight. A significant decrease in the percentage male fetuses per litter was observed, although there was no effect on total live litter size, suggesting that the reduction in the ratio of live male fetuses in the high-dose group was not biologically significant. MACR had no adverse effect on morphological development. In summary, oral administration of MACR to rats and rabbits during organogenesis, at doses that did not cause persistent maternal toxicity (50 mg MACR/kg/day, rats; 5 mg MACR/kg/day, rabbits), also did not cause any adverse developmental effects.

Boric acid (BA), an ingredient of many pharmaceutical, cosmetic, and pesticide products, was previously shown to induce reproductive and developmental toxicity in laboratory rodents. In this study, BA (0, 62.5, 125, or 250 mg/kg/day, po) was administered on Gestational Days (GD) 6-19 to New Zealand White rabbits (18-23 pregnant/group). Maternal body weight, food consumption, and clinical condition were monitored at regular intervals throughout gestation. At termination (GD 30), the numbers of uterine implantations, resorptions, dead fetuses, and live fetuses were determined. Fetuses were weighed, and live fetuses examined for external, visceral, and skeletal defects. Maternal food intake decreased during treatment at 250 mg/kg/day and increased at >/=125 mg/kg/day after treatment. Maternal body weight (GD 9-30), weight gain during treatment, gravid uterine weight, and number of ovarian corpora lutea decreased at 250 mg/kg/day. In contrast, maternal corrected gestational weight gain increased at >/=125 mg/kg/day. Maternal liver weight was not affected. Relative (but not absolute) maternal kidney weight increased at 250 mg/kg/day, and microscopic evaluation revealed no treatment-related renal pathology. At 250 mg/kg/day, prenatal mortality was increased (90% resorptions/litter vs 6% for controls), the proportion of pregnant females with no live fetuses was increased (73% vs 0%), and live litter size was reduced (2.3 fetuses/litter vs 8.8). As a result, there were only 14 live fetuses (6 live litters) available for evaluation in the high-dose group, compared to 153-175 live fetuses (18-23 live litters) in the other groups. The percentage malformed fetuses/litter was increased at 250 mg/kg/day, primarily due to cardiovascular defects in 72% of high-dose fetuses vs 3% of controls. The most prevalent cardiovascular malformation (interventricular septal defect) was observed in 57% of high-dose fetuses compared to 0.6% among controls. At 250 mg/kg/day, average fetal body weight/litter was 92% of the average control weight (not statistically significant). In summary, no definitive maternal or developmental toxicity was observed at 62.5 or 125 mg/kg/day BA. Mild maternal effects and severe developmental toxicity were observed at 250 mg/kg/day.

Although much is known about the metabolism of environmental toxicants in adult organisms, little information exists on the role of cytochrome P450 (CYP) enzymes during development. The developing organism is remarkably dynamic, presenting a constantly changing metabolic profile as various enzyme systems are activated or repressed. This may explain the markedly different sensitivities to various toxicants that are exhibited throughout the developmental period. The application of molecular biological methods has provided important information on the roles of these enzymes in modulating the response of the developing organism to toxicological exposures. The first talk will focus on the identification and role of CYPs during early organogenesis, particularly on how these enzymes influence the response of the conceptus and early embryo to toxic chemicals. The second presentation will discuss the identification of CYPs expressed during human development, as many of the enzymes present in adults are not expressed in the fetus. The third speaker will discuss the developmental consequences of loss of expression of particular metabolic enzymes, focusing on recent studies employing knockout mice to examine the role of drug metabolic enzymes during development. The last two talks will discuss some of the short- and long-term consequences of in utero exposures to toxic chemicals and the role of CYP in modulating the toxic response of the developing organism. The first of these will focus on the role of CYP2E1 in human fetuses during late gestation and the response of this enzyme to inducing agents such as alcohol. The last talk will discuss the role of CYP1A1 in the activation of the Ki-ras oncogene following in utero exposure to carcinogens as a mechanism for lung tumor formation in a pharmacogenetic mouse model.

Mangafodipir trisodium injection (MnDPDP) is an intravenously administered manganese chelate undergoing clinical evaluation for magnetic resonance imaging contrast enhancement of the hepatobiliary system. The anticipated single clinical dose for adults is 5 micromol/kg body wt. MnDPDP, as well as the inorganic salt, MnCl2, was previously shown to induce a specific syndrome of skeletal abnormalities in rats. The syndrome malformations included angulated or irregularly shaped clavicle, femur, fibula, humerus, ilium, radius, scapula, tibia, and/or ulna. The objective of the present study was to assess the developmental toxicity of MnDPDP in a second mammalian species, the New Zealand White rabbit. MnDPDP was intravenously administered daily to groups of rabbits (22 per group) on Days 6 through 18 of pregnancy at doses of 0 (saline), 5, 20, 40, and 60 micromol/kg MnDPDP. Fetuses were examined on Day 29 of pregnancy for external, visceral, and skeletal abnormalities. Treatment with MnDPDP did not result in overt symptoms of maternal toxicity, and there were no significant effects on maternal body weight gains or feed consumption. The maternal no-observed-adverse-effect level (NOAEL), therefore, was 60 micromol/kg MnDPDP. Treatment with MnDPDP resulted in a significant increase in postimplantation loss at 60 micromol/kg, but there was no significant increase in external, visceral, or skeletal abnormalities at any dose. The developmental NOAEL for MnDPDP, therefore, was 40 micromol/kg. These results indicate that the developmental toxicity profile of MnDPDP differs considerably in the rat and rabbit. In the rat, this compound induces specific skeletal abnormalities, whereas in the rabbit, embryo/fetal toxicity is the most sensitive developmental endpoint with no evidence for the induction of specific skeletal abnormalities.

The developing nervous system is preferentially vulnerable to lead exposure with alterations in neuronal and glial cells of the brain. The present study examined early lead-induced alterations in the developing astrocyte population by examination of the developmentally regulated astrocyte specific protein, glial fibrillary acidic protein (GFAP). A developmental profile (Postnatal Day (PND) 6, 9, 12, 15, 20, and 25) for GFAP mRNA was generated for the cortex and hippocampus of developing Long-Evans hooded male rats under various lead exposure conditions: (1) prenatal (Gestational Day 13 to birth), (2) postnatal (Postnatal Day 1 to Postnatal Day 20), or (3) perinatal (Gestational Day 13 to Postnatal Day 20) exposure to lead acetate (0.2% in the drinking water of the dam). Control GFAP mRNA levels displayed a developmentally regulated profile of expression. In the cortex this was characterized by a transient elevation in peak level between PND 9 and PND 15 followed by a decline to within adult levels by PND 25. Under all lead acetate exposure conditions, the cortex showed an increase in the peak level of expression and extended the time of elevation of GFAP mRNA until PND 20. Levels of GFAP were elevated at PND 60 but not as early as PND 28. In the control hippocampus, levels of GFAP mRNA gradually increased until PND 20 followed by a sharp decline at PND 25. Postnatal and perinatal lead exposure followed a similar pattern; however, levels declined earlier at PND 20. Following prenatal lead exposure, levels of GFAP mRNA showed an earlier peak at PND 12 and a decrease as early as PND 15. By PND 60 protein level for GFAP was elevated in the postnatal lead exposure group only. As demonstrated by GFAP immunoreactivity, these lead-induced elevations were not associated with astrocyte hypertrophy. Following a physical injury in the cortex, astrocyte reactivity was similar between lead-exposed and control rats. These data suggest an alteration in the timing of astrocyte differentiation and maturation in the brain following developmental lead exposure.

Arsenic is a known teratogen and developmental toxicant in many animal models. The aim of the present study was to determine the influence of arsenic oxidation state, concentration, duration of exposure, and embryonic gestational age on arsenic-induced developmental toxicity. For these studies whole embryo culture was used since this experimental model allows an assessment of the direct effect of the toxicant on the embryo and precise control of the variables of interest. ICR and CD1 mouse embryos were prepared for whole embryo culture and exposed to concentrations of trivalent (1, 2, 5, 7.5, 10, 20, and 30 microM of sodium arsenite) and pentavalent arsenic (5, 10, 20, 50, and 100 microM of sodium arsenate) at different developmental stages (3, 4-6, 8-10, or 20-23 pairs of somites) and for different exposure periods (1, 4, 6, or 24 hr). Embryonic growth, development, malformation rates, and viability were evaluated. A comparison of the ED50s of the two oxidation states showed that arsenite was about three times more potent than arsenate with respect to both malformations and lethality. The pattern of malformations was similar for both arsenite and arsenate and involved nonclosure of the cranial neural tube, prosencephalic hypoplasia, dysmorphogenesis of the optic and otic anlagen, and pharyngeal arch defects. ICR conceptuses were more sensitive than CD1s with regard to perturbation in embryonic growth by both forms of arsenic. ICRs were also more sensitive to otic, pharyngeal arch, and somite dysmorphogenesis induced by arsenite. With increasing gestational age there was an increasing resistance to arsenic-induced effects. In comparison to the 4-6 somite stage, the ED50 for induction of dysmorphogenesis was increased about twice at the 8-10 somite and over three times in 20-23 somite stage embryos. Exposure to arsenite/arsenate for a 1-hr period was sufficient to induce maldevelopment. A 6-hr exposure induced prosencephalic, otic, and optic abnormal development at a rate similar to that produced by a 24-hr exposure. The malformation pattern produced by exposure to arsenite/arsenate in vitro closely corresponds to that produced by maternal administration at the same gestational stage. This indicates that the arsenic embryopathy may be the result of a direct impact of the agent on the conceptus.

Pregnant Sprague-Dawley rats and New Zealand White rabbits were exposed dermally to 0, 10, 25, and 75 mg/kg/day of monoethanolamine (MEA) for approximately 6 hr/day on Days 6 through 15 (rats) or 6 through 18 (rabbits) of gestation. A fifth dose group of 225 mg MEA/kg/day was evaluated in rats only. Dermal exposure of pregnant rats to 225 mg/kg/day and rabbits to 75 mg/kg/day resulted in significant increases in the incidence of skin irritation/lesions and maternal body weight effects. In general, the dermal irritation observed at the high dose was progressive, beginning with erythema and leading to necrosis, scabs, and scar formation. Doses of 25 mg/kg/day to rabbits produced only minor irritation. Despite maternal effects observed in rats and rabbits, no evidence of developmental or fetal toxicity was observed at any dose level tested. Thus, it was concluded that MEA was not developmentally toxic following dermal application at exposure levels up to and including 225 mg/kg/day for rats and 75 mg/kg/day for rabbits.

Distillate aromatic extract (DAE) was evaluated for subchronic and developmental toxicity. In the subchronic study, undiluted DAE was applied on the shaved backs of male and female rats at dose levels of 0, 30, 125, 500, and 1250 mg/kg for 13 weeks, 5 days per week. Exposure sites were not covered. In the developmental toxicity study, DAE was similarly applied, but to pregnant rats at dose levels of 0, 8, 30, and 125 mg/kg on Gestation Days 0-19, 500 mg/kg on Gestation Days 0-16, and 1000 mg/kg on Gestation Days 10-12. Evidence of toxicity observed in the subchronic study included death, decreased body weights, aberrant serum chemistry and hematology values, altered organ weights, and histopathologic changes in a variety of organs. Regardless of the length of the exposure period, DAE was toxic to the developing conceptus. Evidence of developmental toxicity observed included increased resorptions and reduced fetal body weights. Cleft palate and ossification delays were observed in fetuses exposed in utero to DAE on Gestation Days 10-12, but not when exposure spanned all (Gestation Days 0-19) or most (Gestation Days 0-16) of gestation.

Sodium fluoride (NaF; Cas No. 7681-49-4) is used in fluoridating municipal water supplies, resulting in chronic exposure of millions of people worldwide. Because of a lack of pertinent developmental toxicity studies in the literature, sodium fluoride was administered ad libitum in deionized/filtered drinking water (to mimic human exposure) to Sprague-Dawley-derived rats (26/group) on Gestation Days (GD) 6 through 15 at levels of 0, 50, 150, or 300 ppm and New Zealand White rabbits (26/group) on GD 6 through 19 at levels of 0, 100, 200, or 400 ppm. Higher concentrations via drinking water were not practicable due to the poor palatability of sodium fluoride. Drinking water (vehicle) contained less than 0.6 ppm sodium fluoride (limit of detection) and sodium fluoride content of the feed was 12.4 ppm fluoride (rats) and 15.6 ppm fluoride (rabbits). Maternal food, water, body weights, and clinical signs were recorded at regular intervals throughout these studies. Animals were killed on GD 20 (rats) or 30 (rabbits) and examined for implant status, fetal weight, sex, and morphological development. In the high-dose group of both studies there was an initial decreased maternal body weight gain which recovered over time and a decreased water consumption--attributed to decreased palatability. No clear clinical signs of toxicity were observed. Maternal exposure to sodium fluoride during organogenesis did not significantly affect the frequency of postimplantation loss, mean fetal body weight/litter, or external, visceral or skeletal malformations in either the rat or the rabbit. The NOAEL for maternal toxicity was 150 ppm sodium fluoride in drinking water (approximately 18 mg/kg/day) for rats, and 200 ppm (approximately 18/mg/kg/day rabbits. The NOAEL for developmental toxicity was > or = 300 ppm sodium fluoride (approximately 27 mg/kg/day) for rats and > or = 400 ppm (approximately 29 mg/kg/day) for rabbits administered during organogenesis in drinking water. The total exposure to fluoride (mg F/kg body weight/day from food and drinking water combined) in the mid- and high-dose groups for both species was > 100-fold higher than the range at 0.014-0.08 mg F/kg/day estimated for a 70-kg person from food and fluoridated (1 ppm) drinking water.

Rat fetuses proximal to birth process alcohol-derived cues when the drug is directly delivered into the amniotic fluid. Prior evidence indicates that chemosensory sensation is detected during gestational Day 17 (GD17). In the present study Wistar-derived pregnant females received 0, 1, or 2 g/kg/day of alcohol (intragastric intubation) during GDs 17-20. Prenatal treatment failed to affect different maternal-fetal and perinatal physical parameters, e.g., placenta weight, umbilical cord length, offspring's body weights, weights and/or size of the olfactory bulbs, cerebral hemispheres, and cerebellum. Alcohol chemosensory responsiveness assessed in a perinatal motor activity test, indicated that pups pretreated with the 1 and 2 g/kg alcohol dose exhibit significant decrements in their activity rate when alcohol odor is presented in the test chamber. Alcohol concentrations in maternal and fetal blood and in the amniotic fluid were also recorded through head-space chromatography 1 h after females received the last intubation procedure (GD20) with the 1 or 2 g/kg alcohol doses. Dose-dependent alcohol concentrations across the different sites of assessment were recorded. As indicated by previous studies, even the alcohol level in the amniotic fluid attained with the 1 g/kg alcohol dose is above threshold values in terms of allowing fetal chemosensory stimulation with alcohol-derived cues. The results suggest that maternal ethanol intoxication during the last days of pregnancy leads to fetal exposure to alcohol's sensory attributes and that this experience subsequently modifies responsiveness to these cues.

The reproductive toxicity and growth effects of developmental lead exposure were assessed using a rat model in which 0.6% (w/v) lead acetate was administered in the drinking water ad libitum. Three series of experiments were conducted in which lead exposure was initiated beginning in utero, prepubertally, or postpubertally. Lead effects were measured on reproductive physiology and endocrinology, sexually dimorphic hepatic testosterone hydroxylation, and growth rates in both male and female animals. In male animals secondary sex organ weights were significantly decreased only in animals exposed prepubertally. In addition, serum testosterone levels were significantly suppressed, most severely in animals exposed from in utero (in the in utero group). Little effect was observed in adult female rats. However, in female animals exposed prepubertally, delayed vaginal opening and disrupted estrus cycling was observed. More severe reproductive disruption was accompanied by suppression of circulating estradiol in the in utero group. Effects on circulating sex steroids were accompanied by variable effects on circulating luteinizing hormone (LH) levels, pituitary LH, and pituitary LH beta mRNA, suggesting a dual site of lead action: (a) at the level of the hypothalamic pituitary unit, and (b) directly at the level of gonadal steroid biosynthesis. Prepubertal growth in both sexes was suppressed 25% in the in utero group. However, pubertal growth rates were significantly suppressed only in male animals and postpubertal growth was not significantly different from controls in any of the experiments, despite continued exposure to high lead levels in the drinking water. In addition, at age 85 days, male-specific hepatic hydroxylation of testosterone at positions 2 alpha and 16 alpha, which is catalyzed by a cytochrome P450 isozyme CYP 2C11, itself regulated by sexually dimorphic growth hormone secretion, was unaffected. This suggests that the growth effects of lead are possibly due to a delay in the development of sex-specific pituitary growth hormone secretion patterns rather than a persistent developmental defect. Thus, the reproductive and growth effects of lead are complex and sex-dependent, and appear to involve multiple sites on the hypothalamic-pituitary-gonadal axis.

Chlorpyrifos (O,O-diethyl-O-(3,5,6-trichloro-2-pyridyl)-phosphorothioate), an organophosphate insecticide, was evaluated for its potential to produce developmental and reproductive toxicity in rats following oral exposure. Pregnant Fischer 344 rats were given doses of 0 (corn oil vehicle), 0.1, 3.0, or 15 mg chlorpyrifos/kg/day, by gavage, on Gestation Days 6 through 15. Maternal effects noted at the two higher dose levels included decreased cholinesterase levels at 3.0 mg/kg/day and cholinergic signs (excessive salivation and tremors), decreased cholinesterase levels, and decreased body weight gain at 15 mg/kg/day. No maternal effects were apparent at 0.1 mg/kg/day. Although maternal toxicity was observed at these two higher exposure levels, no developmental effects were noted at any dose. In a two-generation reproduction study, Sprague-Dawley rats were maintained on diets supplying 0, 0.1, 1.0, or 5.0 mg chlorpyrifos/kg/day. Parental effects included decreased plasma and erythrocyte cholinesterase at 1.0 mg/kg/day, and decreased plasma, erythrocyte, and brain cholinesterase and histopathologic alterations of the adrenal zona fasciculata at 5.0 mg/kg/day. The histopathologic alterations of the adrenal were characterized as very slight to slight vacuolation (consistent with fatty change) in males, and very slight vacuolation and/or altered tinctorial properties in females. No effects on the reproductive or fertility indices or on the histopathology of reproductive tissues were observed at any dose level, and no neonatal effects were observed at 0.1 or 1.0 mg/kg/day in the F1 or F2 litters. Parental toxicity at the high dose was accompanied by decreased pup body weight and increased pup mortality in the F1 litters only. These data show that oral administration of chlorpyrifos to rats at parentally toxic dose levels was not embryolethal, embryo/fetotoxic, or teratogenic and did not adversely affect fertility or the function or structure of the reproductive organs. Although effects on neonatal growth and survival were observed at a maternally toxic dose level in one generation, this effect was not observed in the subsequent generation and, therefore, may not have been related to treatment.

Methoxyethanol (ME) produces embryotoxic effects in rodents, rabbits, and nonhuman primates. Mechanistic evaluations of ME dysmorphogenesis have focused mainly on developmental insults and chemical disposition in the mouse. These assessments in mice were based on developmental phase specificity (DPS) and dose-response relationship (DRR) of ME. DPS and DRR indicated treatments for selectively inducing defects to study ME disposition and expressed dysmorphogenesis. This study was conducted to establish DPS and DRR of ME in the rat. DPS was determined by injecting 500 mg ME/kg (6.6 mmol/kg) into the tail vein on Gestational Day (gd; sperm-positive day = gd 0) 10, 11, 12, 13, 14, or 15 (n = 6 dams/gd; saline controls on gd 12). On gd 20, embryolethality incidence was 100% after gd 10 dosing; at gd 11 through 15, it was 50, 32, 15, 2, and 5% respectively (control, 2%). Incidences of external defects in live fetuses exposed on gd 11-15 were 97, 98, 100, 44, and 0% and those of viscera were 100, 62, 44, 10, and 0%, respectively. The predominant anomalies observed were ectrodacyly and renal agenesis. DRR was determined on gd 13, when live embryos/litter and external malformations (ectro- and syndactyly, micromelia) were maximal. Dams (n = 8/dose group) were injected intravenously with 0, 100, 250, 350, or 500 mg ME/kg. On gd 20, fetal defect rates were 0, 0, 82.5, 83.0, and 100% at these concentrations, respectively. Based on these studies, appropriate ME doses, times of maternal exposure, and critical phases of development in the rat model are available for reproducing selective defects to investigate biochemical and pharmacokinetic determinants underlying their expression.

The developmental toxicity of N-methylformamide (MMF), an industrial chemical intermediate used in the production of agrichemicals, was examined in pregnant rats. MMF was administered by nose-only inhalation, 6 hr daily on Days 7-16 of gestation (the day copulation was confirmed was termed Day 1 of gestation, Day 1G) at exposure concentrations of 0, 15, 50, or 150 ppm. Dams were regularly monitored throughout gestation for body weight gain, feed consumption, and clinical signs. Cesarean sections were performed on Day 22G and the offspring were examined. Maternal toxicity was evident in dams exposed to 50 or 150 ppm; one dam exposed to 150 ppm died on Day 14G (considered to be treatment-related) and dams in the 50 and 150 ppm groups exhibited concentration-related clinical findings. Clinical signs of wheezing and rattling were observed both during and after the exposure period. The 150 ppm group also showed significant decreases in weight gain and feed consumption. A significant increase in the mean number of resorptions per litter at the 150 ppm level indicated an embryolethal effect. Developmental toxicity was apparent by a significant decrease in mean fetal body weight and increases in fetal malformations (subcutaneous cysts on the head, microphthalmia, anophthalmia, fused ribs and/or vertebra, and distended brain ventricles) and variations (misaligned and fused sternebrae) due to retarded development at 150 ppm. Significant fetal body weight decreases were also present at 50 ppm. Thus, in this study, the no-observable-adverse-effect level for both dam and fetus was 15 ppm MMF, indicating that for the parameters included in this study, the conceptus is not uniquely sensitive to MMF.

Cocaine use has been associated with adverse developmental effects in humans. However, clinical reports both confirm and deny an association between cocaine use and malformations. Similarly, differences in species and strain, as well as route and timing of cocaine administration, have added to the difficulties in determining the teratogenicity of cocaine in animal models. This study was undertaken to compare the effects of dose, route, and timing of cocaine administration in ICR mice during early organogenesis. A single intraperitoneal (ip) administration of cocaine ( > or = 60 mg/kg) on Day 9 of gestation (plug day = 1) produced maternal lethality. The predominant developmental effect of cocaine administration was an increase in the percentage of litters exhibiting an enlarged renal pelvis. Despite a high incidence of affected pups at these doses, the enlargement was not severe. These results, in agreement with previous reports, provide further evidence that the developing urogenital system is sensitive to cocaine administration. When cocaine was administered using a subcutaneous route, pup weights were greater and the incidence of enlarged renal pelvis was lower than when an ip route was used. To better mimic human binge cocaine abuse, the toxicity of a "split dose" was determined. A 60 mg/kg dose was administered using one administration of 60 mg/kg, two treatments of 30 mg/kg, or three administrations of 20 mg/kg with 1 hr separating the treatments. The incidence of enlarged renal pelvis was similar when cocaine was administered as one or two but was decreased when cocaine was administered as three treatments. Both the route and split-dose studies suggest that high-peak serum concentrations are required to perturb development. There were no differences in the incidence or severity of enlarged renal pelvis when cocaine was administered on Day 8, 9, or 10 or on all 3 days of gestation. This suggested that the increase in enlarged renal pelvis may not be a specific teratogenic effect of cocaine administration but may be a delay of normal development induced by cocaine exposure during this early period of organogenesis. To address this hypothesis, cocaine was administered on Day 9 using an ip route and the pups were allowed to be naturally born. In pups whose mothers received cocaine there was an increase in postnatal deaths and a trend toward a reduction in pup body weight/litter at Postnatal Day 21. However, when renal morphology was assessed on Postnatal Day 21 no abnormal kidneys were seen. This supports the hypothesis that enlarged renal pelvis produced by cocaine administration during early organogenesis represents a developmental delay and not a persistent teratogenic defect. These studies suggest that high peak cocaine concentrations are required to delay normal kidney morphogenesis in mice.

The surface of rat visceral yolk sacs (VYS) of intact, viable rat conceptuses were continuously monitored with a microfiberoptic sensor optimized for detection of the reduced pyridine nucleotides, NADH and NADPH. Model chemical toxins, cyanide and alloxan, were used and evaluated on the basis of their differential ability to modulate NAD(H)- and NADP(H)-dependent cellular pathways, respectively. Exposure with 2 mM sodium cyanide for 5 min caused a reversible fluorescence increase of 325 arbitrary fluorescence units (AFU) and 225 AFU on Gestational Days (GD) 10 and 11, respectively. Exposure with 40 mM alloxan for 5 min resulted in a fluorescence decrease of 170 and 120 AFU on GD 10 and 11, respectively. Glutathione (GSH) levels in the VYS, as determined by HPLC, showed a marked decrease from 27.3 +/- 2.1 to 2.9 +/- 0.4 pmol/mg protein, within the 5-min alloxan exposure period on GD 10. No decrease in GSH levels was noted for the same exposure duration on GD 11. A 2-hr pretreatment with 25 microM BCNU [(1,3 bis(2-chloroethyl)-1-nitrosourea], to inhibit glutathione disulfide reductase (GSSG-Rd), resulted in an elimination of the fluorescence decrease, but still led to a significant drop in GSH levels as seen on both days of gestation. These results are consistent with overall changes in intracellular pyridine nucleotide concentrations, where the relative amounts of NADPH increase significantly and disproportionately from GD 10 to 11. The net oxidation of NADPH, through GSSG-Rd activity, appears to be responsible for the alloxan-induced decrease in surface fluorescence. Conversely, the cyanide-induced fluorescence increases appear to be the result of NAD+ reduction, mediated through the inhibition of the terminal cytochrome oxidase in the electron transport chain.

Acute, subacute, and subchronic inhalation toxicity studies, developmental toxicity studies, a cardiac sensitization evaluation, and mutagenicity assays were conducted with pentafluoroethane (HFC-125). In the acute study, rats were exposed to a single concentration of 800,000 ppm for 4 hr. Ataxic gait and abnormal respiration were observed during exposure but not after exposure. There was no mortality or other signs of toxicity. Repeated exposures of rats to 50,000 ppm, 6 hr/day, 5 days/week for either 4 or 13 weeks elicited no effects on body weight, food consumption, clinical signs, hematology, biochemistry, urinalysis, organ weight, or tissue morphology. Positive evidence of cardiac sensitization in response to an intravenous epinephrine challenge in dogs was seen at 100,000 ppm and above, but not at 75,000 ppm. HFC-125 was not mutagenic in Salmonella typhimurium and Escherichia coli strains at concentrations of 20 to 100% (v/v) with and without activation. No evidence of clastogenic activity was observed in cultured Chinese hamster ovary (CHO) cells or human lymphocytes at < or = 70% HFC-125 when treatments were conducted for 3-4 hr with activation or for 24 and 48 hr (human lymphocytes only) without activation. However, a statistically significant increase in chromosomally aberrant cells was observed in CHO cells at 60% HFC-125 when treatment without activation was extended to 48 hr. The biological significance of this effect is questionable since signs of severe toxicity were also present. In vivo, no micronuclei were induced in mouse bone marrow at concentrations as high as 600,000 ppm HFC-125 for a 6-hr exposure. In addition, HFC-125 did not induce embryotoxic or teratogenic effects in either the rat or the rabbit at exposure concentrations as high as 50,000 ppm.

This study evaluates the developmental toxicity and placental transfer of 1,2-dichloroethane (DCE) in rats. Sprague-Dawley rats were given 0-2.4 mmol DCE kg-1 day-1 by gavage, or were exposed for 6 hr per day to 0-300 ppm DCE by inhalation, from Day 6 to 20 of gestation. Maternal toxicity was observed after inhalation exposure to 300 ppm DCE and oral administration of 2.0 or 2.4 mmol DCE kg-1. There was no evidence of altered growth nor teratogenic effects after either inhalation or oral administration of DCE at any concentration tested. The time course disposition of 14C was examined over a 48-hr period in 12- and 18-day pregnant rats after a single oral dose of 1.6 mmol [14C]DCE kg-1. Peak concentrations of radiocarbon occurred between 2 and 4 hr postdose. Conceptus (Day 12) and fetal (Day 18) tissues accounted for 0.06 and 0.4% of the administered dose, respectively. Up to 4 hr, levels of radiocarbon in placenta and fetus were slightly less than in maternal plasma of 18-day pregnant rats and were two to five times higher at later periods. At 2 hr, unchanged DCE accounted for most of radioactivity (78-86%) recovered in maternal plasma, placenta, and fetus. Acidic metabolites and radioactivity bound to macromolecules increased up to 24 hr (0.01 mumol-eq DCE g-1) in either placental or fetal tissues. Thereafter, their levels declined more slowly than those in the maternal plasma. Results from this developmental toxicity study in rats confirm embryonic exposure to radiocarbon associated with [14C]DCE and/or its metabolites and has demonstrated the lack of observable teratogenic effects.

Developmental hypothyroidism causes growth deficits, motor dysfunction, and hearing disorders in humans and animals. Therefore, environmental toxicants, such as polychlorinated biphenyls (PCBs), may secondarily affect these endpoints via thyrotoxicity. In this study, Long-Evans rats were given Aroclor 1254 (po), at 0, 1, 4, or 8 mg/kg from Gestation Day 6 through Postnatal Day (PND) 21. We evaluated the offspring at various age intervals for circulating thyroid hormone concentrations [thyroid-stimulating hormone, and free and total triiodothyronine (T3) and thyroxin (T4)], body weight, eye opening, survival, motor activity development, auditory startle response, and auditory thresholds. Circulating T4 concentrations were sharply reduced in a dose-dependent fashion in PCB-exposed groups at PND 1, 7, 14, 21, and 30 but recovered to control levels by PND 45. Moderate reductions in T3 concentrations were apparent in the 4 and 8 mg/kg groups on PND 21 and 30. Deficits in body weight gain and early eye opening were apparent in the treated pups; by weaning, pup mortality was 20% in the 4 mg/kg group and 50% at the highest dose. Motor activity was also transiently reduced in 15 day old offspring from the 8 mg/kg group. At this dose, animals showed reduced auditory startle amplitudes at PND 24, but not when tested as adults. Importantly, Aroclor 1254 caused permanent auditory deficits (20-30 dB threshold shift) at the lowest frequency tested (1 kHz) in both the 4 and 8 mg/kg groups, whereas auditory thresholds were not significantly affected at higher frequencies (4, 16, 32, or 40 kHz). These data indicate that while some effects of Aroclor 1254 exposure are dissimilar to drug-induced hypothyroidism (e.g., age of eye opening), effects on hormone levels and body weight are comparable. Detection of auditory deficits in PCB-treated animals is a novel finding and may reflect the effects of thyroid hormone disruption on the development of the cochlea.

Ethylene glycol (EG; CAS No. 107-21-1) is teratogenic to mice by whole-body (WB) exposure to aerosol (1000-2500 mg/m3). The WB results were confounded by possible exposure from ingestion after grooming and/or from percutaneous absorption. Therefore, CD-1 mice were exposed to EG aerosol (MMAD 2.6 +/- 1.7 microns) on Gestational Days (GD) 6 through 15, 6 hr/day, by nose-only (NO) (0, 500, 1000, or 2500 mg/m3) or WB exposures (0 or 2100 mg/m3, as positive control), 30/group. Five additional "satellite" females each at 2500 mg/m3 NO and 2100 mg/m3 WB were exposed on GD 6 for measurement of EG on fur. Control environments were water aerosol (4200 mg/m3 for NO; 2700 mg/m3 for WB). Females were weighed and evaluated for clinical signs and water consumption throughout gestation. On GD 18, maternal uterus, liver, and kidneys (2) were weighed, with kidneys examined microscopically. Corpora lutea and implantation sites were recorded. Live fetuses were weighed, sexed, and examined for structural alterations. For NO dams, kidney weights were increased at 1000 and 2500 mg/m3; no renal lesions and no other treatment-related maternal toxicity were observed. There were no effects on pre- or postimplantation loss; fetal body weights/litter were reduced at 2500 mg/m3. At 2500 mg/m3, incidences of fused ribs and skeletal variations were increased. The 2500 mg/m3 NO satellite animals had approximately 330 mg/kg extractable EG. The WB group exhibited maternal and developmental toxicity including increased fetal skeletal malformations and variations, confirming previous results, with 1390 mg/kg extractable EG on fur. Therefore, exposure of CD-1 mice to a respirable EG aerosol during organogenesis by NO inhalation resulted in minimal maternal toxicity at 1000 and 2500 mg/m3 and developmental toxicity at 2500 mg/m3. The NOAEL was 500 mg/m3 NO for maternal and 1000 mg/m3 NO for developmental toxicity. This study supports the interpretation of the initial EG WB results as due to systemic exposure from noninhalation routes since limiting noninhalation routes prevented almost all of the effects (including teratogenicity) observed in mice after WB exposure.

Exposure to pesticides or toxic substances that disrupt the endocrine system during sex differentiation can permanently alter reproductive function and produce morphological pseudohermaphrodism. While some developmental toxicants affect either the male or the female, in utero exposure to 0.5 micrograms TCDD/kg/day from Gestational Day (GD) 6 to GD 15 induces infertility in both sexes (K.S. Khera and J.A. Ruddick, Chlorodioxins--Origins and Fate, pp. 70-84, Am. Chem. Soc., Washington, DC, 1973). Although a number studies have focused on the effects of a single dose of TCDD on sex differentiation of the male rat and hamster, the reproductive alterations that account for female-mediated infertility after in utero exposure to TCDD have not been described. Hence, it was our objective to describe the anatomical and functional reproductive alterations in female progeny after gestational administration of TCDD. In the first experiment, LE Hooded rats were given a single dose of 1 microgram TCDD/kg by gavage on CD 8 (i.e., a period that includes major organogenesis) or GD 15 (i.e., a period prior to sex differentiation and a dosing regime that alters sex differentiation of the male LE rat). In a second experiment, Holtzman rats were dosed with TCDD at 1 microgram/kg on GD 15, to determine if the progeny of this strain displayed malformations of the external genitalia and vaginal orifice as did LE rats. TCDD-treated female LE offspring displayed a number of unusual reproductive alterations. In the GD 15 group, puberty was delayed, more than 65% of the female offspring displayed complete to partial clefting of the phallus, and 80% displayed a permanent "thread" of tissue across the opening of the vagina. In the GD 8 treatment group, 25% displayed partially cleft phallus and 14% had a vaginal thread. GD 15 TCDD administration also induced a high incidence of malformations in Holtzman female progeny (100% clefting and 83% with a vaginal thread). At necropsy (> 550 days old), ovarian weight was significantly reduced by 23% in both rat strains. In the LE rat, vaginal and behavioral estrous cyclicity, estrous cycle-mediated running wheel activity, and female sexual behaviors at proestrus (darting and lordosis to mount ratios) were not affected by gestational GD 15 TCDD treatment. However, untreated stud males had difficulty attaining intromission and took longer to ejaculate and vaginal bleeding was displayed during mating by GD 15 TCDD-exposed female offspring.(ABSTRACT TRUNCATED AT 400 WORDS)

Indium is increasingly used in a variety of industries, and while there are few studies of its developmental toxicity, ther are no reports of its potential reproductive toxicity. These studies were undertaken to investigate the possible reproductive toxicity of indium and to determine the relative vulnerability of males and females. We used, initially, a 21-day combined developmental/reproductive toxicity protocol. Oral exposures to InCl3 ( < or = 250 mg/kg) were without effect on the male reproductive system or liver. A kidney effect was demonstrated in males by a decrease in urinary N-acetyl glucosaminidase. The ability of females to become pregnant was unaffected. However, fetal development was adversely affected, manifested as increased intrauterine deaths in the presence of reduced maternal weight gain. A developmental toxicity study identified no increase in fetal malformations, but verified the increased fetal deaths, in the absence of effects on adjusted maternal body weight. In vitro toxicity studies showed that the embryolethality was at least in part a result of direct toxicity to the conceptus, with effective doses in the low micromolar range. A limited disposition study showed that fetuses contained low micromolar concentrations of indium, more indium than maternal liver, and comparable to levels that were toxic in vitro. Although studies of greater exposure duration are required for risk assessment, these data indicate that fetal development is likely to be more affected by indium than female or male reproduction, with adverse effects occurring at low micromolar levels in vivo and at exposures that may or may not affect body weight.

Recently, most attention on the application of benchmark dose (BMD) techniques to toxicology data has focused on quantal measures of response. Before the advantages of the BMD approach can be exploited in the risk assessment process, it is important that continuous measures of response also be modeled appropriately. In this study, we examined a variety of approaches to estimating BMDs for a change in fetal weight following chemical exposure from a total of 85 developmental toxicity experiments. We modeled the change in the mean fetal weight of a litter in response to treatment using a continuous power model, as well as reductions in the weight of individual fetuses within litters (defined as falling below a preset level) using a log-logistic model which incorporates litter size as a covariable and considers intralitter correlations. For the litter-based approach, several methods of defining a benchmark effect (BME) were considered, including a percentage change in mean litter weight, a change in mean litter weight relative to variability in the control group, and a reduction in the mean litter weight to some point on the control group distribution curve. For the fetus-based approach, we examined several BME options on the cumulative frequency distribution of the control fetuses for defining a low weight fetus and calculated several levels of additional risk. BMDs for four litter-based BMEs (a difference of 5% in mean fetal weight, a decrease to the 25th percentile mean weight of control litters, a decrease in the mean weight by 2 standard errors, and a decrease of 0.5 standard deviation units) and two fetus-based BMEs (a 5% added risk of weighing less than the 5th percentile of control weights and a 10% added risk of weighing less than the 10th percentile) showed strong similarities to each other and to statistically derived NOAELs. In addition to providing comparison with the NOAEL as a reference value, these analyses provided confirmation of the advantages of the BMD approach over the NOAEL in terms of the influence of dose spacing and dose selection. Combined with our previous analyses of quantal endpoints of fetal effects, this information provides a firm basis upon which to implement the benchmark dose concept in developmental toxicity risk assessments.

Timed-pregnant CD-1 outbred albino Swiss mice and CD Sprague-Dawley rats were administered hydrochlorothiazide (HCTZ, USP) in corn oil by gavage during major organogenesis, Gestational Days (GD) 6 through 15. The doses administered were 0, 300, 1000, or 3000 mg/kg/day for mice and 0, 100, 300, or 1,000 mg/kg/day for rats. Maternal clinical status was monitored daily during treatment. At termination (GD 17, mice; GD 20, rats), confirmed pregnant females (20-27 per group, mice; 36-39 per group, rats) were evaluated for clinical status and gestational outcome; each live fetus was examined for external, visceral, and skeletal malformations. In mice, no maternal mortality was observed. However, clinical signs including dehydration, piloerection, lethargy, and single-day weight loss appeared to be dose-related. HCTZ had no effect on maternal weight gain or water consumption, gravid uterine weight, relative maternal liver weight, or relative maternal kidney weight. There was no definitive evidence of embryotoxicity or fetal toxicity for mice on GD 17. Thus, the no observed adverse effect level (NOAEL) for both maternal and developmental toxicity was 3000 mg/kg/day. In rats, HCTZ had no effect on maternal survival, clinical signs, or water consumption. Clinical signs were not dose-related. Maternal weight gain during treatment was depressed at 1000 mg/kg/day. Gravid uterine weight and relative maternal liver weight were unaffected. Relative maternal kidney weight was slightly (7-8%) increased at all dose levels, but there was no evidence of a dose response. Thus, the maternal NOAEL for rats was 300 mg/kg/day, based on decreased maternal weight gain during treatment at 1000 mg/kg/day.(ABSTRACT TRUNCATED AT 250 WORDS)

The potential developmental toxicity of trifluralin was evaluated in rats and rabbits. Pregnant rats and rabbits were dosed once daily by gavage on Gestation Days 6-15 and 6-18, respectively. Doses for rats were 0, 100, 225, 475, or 1000 mg/kg; doses for rabbits were 0, 100, 225, or 500 mg/kg. Cesarean sections were performed on rats and rabbits on Gestation Days 20 and 28, respectively. In rats, maternal toxicity was indicated in the 475 and 1000 mg/kg treatment groups by depression of body weights and food consumption. Fetal viability and morphology were not adversely affected at any dose level. Developmental toxicity was indicated at the 1000-mg/kg dose level by depression of fetal weight. The NOAEL for maternal toxicity in the rat was 225 mg/kg; the NOAEL for developmental toxicity in the rat was 475 mg/kg. In rabbits, maternal toxicity was indicated at the 225 and 500 mg/kg dose levels by abortions and/or deaths in conjunction with anorexia and cachexia. Developmental toxicity was indicated at the 500 mg/kg dose level by depressed fetal viability and weight. Fetal morphology was not adversely affected at any dose level. The NOAELs for maternal and developmental toxicity in the rabbit were 100 and 225 mg/kg, respectively. Based on these data, trifluralin did not exhibit selective toxicity toward the developing conceptus.

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.

The relative developmental toxicity of four direct acting, alkylating agents was determined in primary cultures of differentiating rat embryo midbrain (CNS) and limb bud (LB) cells and compared with that observed in the rat whole embryo postimplantation culture system. The alkylating agents tested include methylnitrosourea (MNU), ethylnitrosourea (ENU), methyl methanesulfonate (MMS), and ethyl methanesulfonate (EMS). These alkylating agents have been shown to produce developmental toxicity following either in vitro or in vivo exposure. Viability for both CNS and LB was assessed by a neutral red dye assay. Differentiation of CNS cells was assessed by hematoxylin staining of neurons; differentiation of LB cells was assessed by Alcian blue staining of extracellular proteoglycans. Relative potencies of these compounds in the cell culture system were not the same as those observed in the embryo culture system. Whereas rank order of potency in the cell culture system, for viability and differentiation, was MMS > MNU > ENU > EMS, rank order in the embryo culture system, for embryo lethality and malformations, was MNU > ENU > MMS > EMS. Effective concentrations for cell culture viability and differentiation by MNU and ENU in cell culture were about three to nine times higher than comparable values previously reported for embryos, while effective concentrations for MMS and EMS were two to seven times lower than those observed in the embryos. Differences in potency between the two culture systems may be related to differences in formation and repair of DNA adducts, as well as differences in culture conditions.

Using the potent developmental toxicant 2-methoxyethanol (2-ME) as a prototypical compound, a physiologically based pharmacokinetic (PBPK) model was developed to describe the disposition of its primary metabolite and proximate toxicant 2-methoxyacetic acid (2-MAA) in the pregnant CD-1 mouse. Data were collected during early, mid, and late organogenesis, specifically Gestation Days (GD) 8, 11, and 13 (GD 0 = plug-positive date). Pharmacokinetics and tissue partition coefficients for 2-MAA were determined in maternal plasma and conceptus on GD 8 and in maternal plasma, embryo, and extraembryonic/amniotic fluid (EAF) on GD 11 and 13. For simulation of GD 8 data, the conceptus was described as a single compartment, combining the yolk sac placenta, embryo, EAF, and decidua. For GD 11 and 13, the placenta, embryo, and EAF were explicitly described. Several hypotheses were tested for their ability to predict 2-MAA dosimetry. These hypotheses were encoded as alternative models having (a) blood flow-limited delivery of 2-MAA to model compartments, (b) pH trapping of ionized 2-MAA within compartments, (c) active transport of 2-MAA into compartments, and (d) reversible binding of 2-MAA within compartments. While the flow-limited description adequately predicted GD 8 dosimetry, the best simulations of the pharmacokinetic data collected on GD 11 and 13 were obtained with the active transport models. Since the mechanism by which 2-MAA accumulates into the embryo and EAF has not yet been elucidated, these mathematical descriptions are empirical. Further development of this PBPK model for 2-MAA in pregnant mice, in particular its scale-up to humans, will facilitate more realistic human risk assessments for the developmental toxicity of 2-ME and related compounds.