Barbara A. Elswick

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.

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.

Comments to the editor concerning the paper entitled "Reproductive malformation of the male offspring following maternal exposure to estrogenic chemicals" by C. Gupta.

P otential effects on the endocrine system elicited by the weak estrogen bisphenol A (BPA) in laboratory rats and mice are highly controversial. The increased prostate gland weights in adult male offspring whose mothers were exposed to very low doses of BPA during pregnancy (I) were not found by other investigators (2, 3). An association of the low-dose effects on mouse prostate glands of BPA based on the effects of other much more potent estrogenic compounds with human prostatic disease has been postulated (4). Humans are exposed to exceedingly low levels of BPA from ingestion of canned foods and beverages where container linings contain BPA, raising the possibility that human health ma~ be at ris~. These discrepancies in animal study data require resolution based on the weight of scientific evidence, and, if deemed necessary, action needs to be taken to protect human health. To determine whether exposure to BPA and other weak estrogenic chemicals elicits detectable adverse biological effects in rodents, stringent study design and careful data analysis are nccessary. Examination of the data presented in a recently published paper by C. Gupta (5) raises concern regarding statistical analysis of the observations and the resulting conclusions. Several questions should be addressed so that the

Respiration in Sprague-Dawley Rats During Pregnancy

Minute ventilation and tidal volume increase in humans during pregnancy. Little data exists, however, on the respiration in pregnant rats, despite their widespread use as an animal model. Since respiration will affect the pharmacokinetics of volatile compounds and ultimately the dose to the fetus, we conducted a study to evaluate respiration in rats during pregnancy. Whole-body plethysmography was used to measure the breathing frequency and tidal volume approximately every other day from gestation day (GD) 1 to 21 in 16 timed pregnant and 16 nonpregnant, female, Sprague-Dawley rats. Minute ventilation was calculated as a product of the breathing frequency and tidal volume, and the body weight of each rat was used to determine the scaled ventilation. Multivariate analysis of variance methods for a repeated-measures design were used to analyze the respiratory data. Breathing frequency was not affected by pregnancy; however, tidal volume was somewhat greater in pregnant versus nonpregnant rats. The increase in tidal volume resulted in significantly increased minute ventilation in pregnant rats compared to nonpregnant rats during the latter period of gestation. Due to the increased body weight of the pregnant rats, the scaled ventilation at the end of gestation was significantly lower in pregnant rats compared to nonpregnant rats. This study provides important reference values that can be used in pharmacokinetic models during pregnancy.