Daniel M. Sheehan

QSAR Models Using a Large Diverse Set of Estrogens

Endocrine disruptors (EDs) have a variety of adverse effects in humans and animals. About 58,000 chemicals, most having little safety data, must be tested in a group of tiered assays. As assays will take years, it is important to develop rapid methods to help in priority setting. For application to large data sets, we have developed an integrated system that contains sequential four phases to predict the ability of chemicals to bind to the estrogen receptor (ER), a prevalent mechanism for estrogenic EDs. Here we report the results of evaluating two types of QSAR models for inclusion in phase III to quantitatively predict chemical binding to the ER. Our data set for the relative binding affinities (RBAs) to the ER consists of 130 chemicals covering a wide range of structural diversity and a 6 orders of magnitude spread of RBAs. CoMFA and HQSAR models were constructed and compared for performance. The CoMFA model had a r2 = 0.91 and a q2LOO = 0.66. HQSAR showed reduced performance compared to CoMFA with r2 = 0.76 and q2LOO = 0.59. A number of parameters were examined to improve the CoMFA model. Of these, a phenol indicator increased the q2LOO to 0.71. When up to 50% of the chemicals were left out in the leave-N-out cross-validation, the q2 remained significant. Finally, the models were tested by using two test sets; the q2pred for these were 0.71 and 0.62, a significant result which demonstrates the utility of the CoMFA model for predicting the RBAs of chemicals not included in the training set. If used in conjunction with phases I and II, which reduced the size of the data set dramatically by eliminating most inactive chemicals, the current CoMFA model (phase III) can be used to predict the RBA of chemicals with sufficient accuracy and to provide quantitative information for priority setting.

Evaluation of Quantitative Structure−Activity Relationship Methods for Large-Scale Prediction of Chemicals Binding to the Estrogen Receptor†

Three different QSAR methods, Comparative Molecular Field Analysis (CoMFA), classical QSAR (utilizing the CODESSA program), and Hologram QSAR (HQSAR), are compared in terms of their potential for screening large data sets of chemicals as endocrine disrupting compounds (EDCs). While CoMFA and CODESSA (Comprehensive Descriptors for Structural and Statistical Analysis) have been commercially available for some time, HQSAR is a novel QSAR technique. HQSAR attempts to correlate molecular structure with biological activity for a series of compounds using molecular holograms constructed from counts of sub-structural molecular fragments. In addition to using r2 and q2 (cross-validated r2) in assessing the statistical quality of QSAR models, another statistical parameter was defined to be the ratio of the standard error to the activity range. The statistical quality of the QSAR models constructed using CoMFA and HQSAR techniques were comparable and were generally better than those produced with CODESSA. It is notable that only 2D-connectivity, bond and elemental atom-type information were considered in building HQSAR models. Since HQSAR requires no conformational analysis or structural alignment, it is straightforward to use and lends itself readily to the rapid screening of large numbers of compounds. Among the QSAR methods considered, HQSAR appears to offer many attractive features, such as speed, reproducibility and ease of use, which portend its utility for prioritizing large numbers of potential EDCs for subsequent toxicological testing and risk assessment.

QSAR Models for Binding of Estrogenic Compounds to Estrogen Receptor α and β Subtypes

We have developed Quantitative Structure-Activity Relationship (QSAR) models based on Comparative Molecular Field Analysis (CoMFA) for 31 estrogenic chemicals whose relative binding affinity (RBA) is available for both ER-α and ER-β. The models demonstrated a significant correlation (r2 > 0.95) between the CoMFA-calculated steric/electrostatic fields and corresponding RBA data and a good predictive capability (q2 > 0.6) based on cross-validation. The CoMFA models and contour plots obtained for ER-α and ER-β suggest a close similarity between the receptors in terms of mode of binding and provide a rational basis for ligand selectivity.

Urban Tree Canopy and Asthma, Wheeze, Rhinitis, and Allergic Sensitization to Tree Pollen in a New York City Birth Cohort

Background: Urban landscape elements, particularly trees, have the potential to affect airflow, air quality, and production of aeroallergens. Several large-scale urban tree planting projects have sought to promote respiratory health, yet evidence linking tree cover to human health is limited. Objectives: We sought to investigate the association of tree canopy cover with subsequent development of childhood asthma, wheeze, rhinitis, and allergic sensitization. Methods: Birth cohort study data were linked to detailed geographic information systems data characterizing 2001 tree canopy coverage based on LiDAR (light detection and ranging) and multispectral imagery within 0.25 km of the prenatal address. A total of 549 Dominican or African-American children born in 1998–2006 had outcome data assessed by validated questionnaire or based on IgE antibody response to specific allergens, including a tree pollen mix. Results: Tree canopy coverage did not significantly predict outcomes at 5 years of age, but was positively associated with asthma and allergic sensitization at 7 years. Adjusted risk ratios (RRs) per standard deviation of tree canopy coverage were 1.17 for asthma (95% CI: 1.02, 1.33), 1.20 for any specific allergic sensitization (95% CI: 1.05, 1.37), and 1.43 for tree pollen allergic sensitization (95% CI: 1.19, 1.72). Conclusions: Results did not support the hypothesized protective association of urban tree canopy coverage with asthma or allergy-related outcomes. Tree canopy cover near the prenatal address was associated with higher prevalence of allergic sensitization to tree pollen. Information was not available on sensitization to specific tree species or individual pollen exposures, and results may not be generalizable to other populations or geographic areas.

Prenatal dexamethasone exposure in rats : effects of dose, age at exposure, and drug-induced hypophagia on malformations and fetal organ weights

Glucocorticoids cause stunting and cleft palate in rodents. The aim of this study is to identify fetal organs and developmental periods sensitive to stunting induced by maternal exposure to dexamethasone (DEX). DEX (0.2 or 0.4 mg/kg) or saline was given sc to pregnant CD albino rats on Gestation Days (GD) 9-14 or 14-19. On GD 20 dams were euthanized. Fetuses were weighed and examined for cleft palate. Eight fetuses/litter were randomly selected, and weights were obtained. Fetal skeletons were examined for abnormalities, and long bone measurements were taken. A dose-related decrease in maternal and fetal body weights occurred at both exposure periods. Developmental stage-specific malformations were noted in the high-dose group on GD 9-14 (cleft palate) and on GD 14-19 (wavy ribs). A dose-response in stunting occurred in all organs except cerebellum in at least one exposure period. Across both exposure periods the brain, heart, testes, and long bones were relatively resistant to DEX. Sensitive organs included thymus, spleen, adrenals, lungs, liver, and kidneys. DEX substantially reduced maternal food intake and increased water intake in some dams. Pair-feeding experiments suggested that the hypophagic effect of DEX was not responsible for the noted malformations and had little impact on growth stunting. The present findings have identified fetal organs, skeletal regions, and developmental periods sensitive to DEX exposure.

Teratology Society Public Affairs Committee position paper: developmental toxicity of endocrine disruptors to humans.

SUSAN BARLOW,1 ROBERT J. KAVLOCK,2 JOHN A. MOORE,3 SUSAN L. SCHANTZ,4 DANIEL M. SHEEHAN,5 DANA L. SHUEY,6 AND JOSEPH M. LARY7* 1Consultant Toxicologist, Brighton, East Sussex, BN1 6RE United Kingdom 2Reproductive Toxicology Division, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711 3Institute for Evaluating Health Risks, Washington, DC 20006 4Department of Veterinary Biosciences, College of Veterinary Medicine, University of Illinois, Urbana, Illinois 61802 5Genetic and Reproductive Toxicology Division, National Center for Toxicological Research, Jefferson, Arkansas 72079 6DuPont Pharmaceuticals Company, Stine-Haskell Research Center, Newark, Delaware 19714 7Division of Birth Defects and Pediatric Genetics, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, Georgia 30341

Methylation Profile and Amplification of Proto-Oncogenes in Rat Pancreas Induced with Phytoestrogens

Abstract Specific gene hypermethylation has been shown in DNA from neonatal rats exposed to the phytoestrogens, coumestrol, and equol. The pancreas is an organ in which estrogen receptors have been shown to be present. Studies have correlated the development of acute pancreatitis with rising levels of human estrogen binding proteins. Neonatal rats were dosed with 10 or 100 μg of coumestrol or equol on postnatal day (PND) 1-10. The animals were sacrificed at Day 15. The pancreas was excised and pancreatic acinar cells isolated for molecular analysis. DNA was isolated from the cells by lysis in TEN-9 buffer supplemented with proteinase K and 0.1% SDS. High molecular weight (HMW) DNA was digested with the methylated DNA specific restriction enzymes, Hpa II and Msp I, for determination of methylation profiles. Both coumestrol and equol at high doses caused hypermethylation of the c-H-ras protooncogene. No hypermethylation or hypomethylation was observed in the protooncogenes, c-myc or c-fos. Methylation is thought to be an epigenetic mechanism involved in the activation (hypomethylation) or inactivation (hypermethylation) of cellular genes which are known to play a role in carcinogenesis. Epidemiology studies have shown that equol may have anti-carcinogenic effects on some hormone-dependent cancers. Additional studies are needed to further understand the role of phytoestrogens and methylation in relation to pancreatic disorders.

Comparative molecular field analysis (CoMFA) model using a large diverse set of natural, synthetic and environmental chemicals for binding to the androgen receptor

A large number of natural, synthetic and environmental chemicals are capable of disrupting the endocrine systems of experimental animals, wildlife and humans. These so-called endocrine disrupting chemicals (EDCs), some mimic the functions of the endogenous androgens, have become a concern to the public health. Androgens play an important role in many physiological processes, including the development and maintenance of male sexual characteristics. A common mechanism for androgen to produce both normal and adverse effects is binding to the androgen receptor (AR). In this study, we used Comparative Molecular Field Analysis (CoMFA), a three-dimensional quantitative structure–activity relationship (3D-QSAR) technique, to examine AR-ligand binding affinities. A CoMFA model with and was developed using a large training data set containing 146 structurally diverse natural, synthetic, and environmental chemicals with a 106-fold range of relative binding affinity (RBA). By comparing the binding characteristics derived from the CoMFA contour map with these observed in a human AR crystal structure, we found that the steric and electrostatic properties encoded in this training data set are necessary and sufficient to describe the RBA of AR ligands. Finally, the CoMFA model was challenged with an external test data set; the predicted results were close to the actual values with average difference of 0.637 logRBA. This study demonstrates the utility of this CoMFA model for real-world use in predicting the AR binding affinities of structurally diverse chemicals over a wide RBA range.

Development and deployment of the Computer Assisted Neighborhood Visual Assessment System (CANVAS) to measure health-related neighborhood conditions.

Public health research has shown that neighborhood conditions are associated with health behaviors and outcomes. Systematic neighborhood audits have helped researchers measure neighborhood conditions that they deem theoretically relevant but not available in existing administrative data. Systematic audits, however, are expensive to conduct and rarely comparable across geographic regions. We describe the development of an online application, the Computer Assisted Neighborhood Visual Assessment System (CANVAS), that uses Google Street View to conduct virtual audits of neighborhood environments. We use this system to assess the inter-rater reliability of 187 items related to walkability and physical disorder on a national sample of 150 street segments in the United States. We find that many items are reliably measured across auditors using CANVAS and that agreement between auditors appears to be uncorrelated with neighborhood demographic characteristics. Based on our results we conclude that Google Street View and CANVAS offer opportunities to develop greater comparability across neighborhood audit studies.

Developing a laboratory animal model for perinatal endocrine disruption: the hamster chronicles.

At the biomedical, regulatory, and public level, considerable concern surrounds the concept that inappropriate exposure to endocrine-disrupting chemicals, especially during the prenatal and/or neonatal period, may disrupt normal reproductive tract development and adult function. The intent of this review was to 1. Describe some unique advantages of the hamster for perinatal endocrine disruptor (ED) studies, 2. Summarize the morphological and molecular consequences of exposure to the established perinatal ED, diethylstilbestrol, in the female and male hamster, 3. Present some new, histomorphological insight into the process of neonatal diethylstilbestrol-induced disruption in the hamster uterus, and 4. Introduce recent efforts and future plans to evaluate the potency and mechanism of action of other putative EDs in the hamster experimental system. Taken together, the findings indicate that the hamster represents a unique and sensitive in vivo system to probe the phenomenon of endocrine disruption. The spectrum of candidate endpoints includes developmental toxicity, neoplasia, and more subtle endpoints of reproductive dysfunction.