Effect of different sampling designs on outcome of endocrine disruptor studies.

In this article, we demonstrate how sampling strategy can influence the outcome of endocrine disruptor studies. In a study of the weak xenoestrogen bisphenol A (BPA), possible treatment-related effects on ventral prostate (VP) fresh weight were found in rat offspring at 6 months of age when only one or two male pups were randomly selected from each litter. In subsequent BPA and di-n-butyl phthalate studies, large intralitter variability of this specific end point was apparent when the VP weights from entire litter complements were examined. We modeled the effects of sampling 1, 2, or 3 pups from each litter using the full-litter complement data. When one pup was randomly selected, a substantial percentage of incorrect conclusions about the presence or absence of treatment effects occurred. These statistical modeling analyses raise significant concern about the selection of one pup per litter for highly variable end points.

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

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

Development of a physiologically based pharmacokinetic model of 2-methoxyethanol and 2-methoxyacetic acid disposition in pregnant rats.

An accurate description of developing embryos' exposure to a xenobiotic is a desirable component of mechanism-based risk assessments for humans exposed to potential developmental toxicants during pregnancy. 2-Methoxyethanol (2-ME), a solvent used in the manufacture of semiconductors, is embryotoxic and teratogenic in all species tested including nonhuman primates. 2-Methoxyacetic acid (2-MAA) is the primary metabolite of 2-ME and the proximate embryotoxic agent. The objective of the work described here was to adapt an existing physiologically based pharmacokinetic (PBPK) model for 2-ME and 2-MAA kinetics during midorganogenesis in mice to rats on gestation days (GD) 13 and 15. Blood and tissue data were analyzed using the extrapolated PBPK model that was modified to simulate 2-ME and 2-MAA kinetics in maternal plasma and total embryo tissues in pregnant rats. The original mouse model was simplified by combining the embryos and placenta with the richly perfused tissue compartment. The model includes a description of the growth of the developing embryo and changes in the physiology of the dam during pregnancy. Biotransformation pathways of 2-ME to either ethylene glycol (EG) or to 2-MAA were described as first-order processes based on the data collected from rats by Green et al., (Occup. Hyg. 2, 67-75, 1996). Tissue partition coefficients (PCs) for 2-ME and 2-MAA were determined for a variety of maternal tissues and the embryos. Model simulations closely reflected the biological measurement of 2-ME and 2-MAA concentrations in blood and embryo tissue following gavage or iv administration of 2-ME or 2-MAA. The PBPK model for rats as described here is well suited for extrapolation to pregnant women and for assessment of 2-MAA dosimetry under various conditions of possible human exposure to 2-ME.

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

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

Development of a physiologically based pharmacokinetic model describing 2-methoxyacetic acid disposition in the pregnant mouse.

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.

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

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

Pharmacokinetics of 2-methoxyethanol and 2-methoxyacetic acid in the pregnant mouse: a physiologically based mathematical model.

A physiologically based pharmacokinetic (PBPK) model was created to describe the disposition of 2-methoxyethanol (2-ME) and its teratogenic metabolite, 2-methoxyacetic acid (2-MAA), in the pregnant CD-1 mouse. The model's foundation is a mathematical description of the physiological changes that occur during gestation (O'Flaherty et al., Toxicol. Appl. Pharmacol. 112, 245-256, 1992). The PBPK model was developed and validated with data collected on Gestation Day (GD) 11. Absorption, distribution, and oxidation of 2-ME to 2-MAA and ethylene glycol (EG) were simulated. Flow-limited disposition of 2-ME in maternal tissues was described using in vitro-determined tissue partition coefficients (PCs). The maximum velocity (Vmax) of 2-ME oxidation to 2-MAA was calculated from literature-based in vitro data. Vmax for EG formation, and Michaelis constants for 2-MAA and EG pathways, were estimated from optimized simulations of plasma 2-ME and metabolite levels obtained after intravenous injection of 5-600 mg 2-ME.kg-1.2-MAA disposition and elimination in the dam were described by a nonphysiological one-compartment model, which was linked to the 2-ME model, based on the volume of distribution (0.510 liters.kg-1) and overall elimination rate constant (0.124 hr-1) calculated from iv 2-MAA plasma concentration-time courses. Transfer of 2-MAA between the placenta and conceptus was described as a diffusion-limited process to more accurately simulate the higher concentrations of 2-MAA determined in embryonic compartments compared with maternal plasma levels. Subsequent 2-MAA disposition within the embryo proper and surrounding fluid of the GD 11 conceptus was adequately described using embryo/blood (0.94) and extraembryonic fluid/blood (1.33) PCs. Extension of the PBPK model to oral and subcutaneous 2-ME administrations required optimization of first-order absorption rates; model simulations agreed closely with measured 2-ME/2-MAA levels. With refinements and further validation, the PBPK model of 2-ME/2-MAA disposition should prove helpful for extrapolation throughout gestation and between species.