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Reproductive Toxicology Division

Reproductive Toxicology Division

Drinking Water Team (DW)

Overview

The Safe Drinking Water Act of 1974 mandates the EPA to review regulated contaminants, consider unregulated contaminants, and protect source water. RTD investigators provide information to the Office of Water (OW) on the identification and classification of reproductive and developmental risks associated with disinfection byproducts (DBPs). Research in this project is focused on evaluating the potential toxicity of regulated DBPs, including trihalomethanes and haloacetic acids. It has evolved into an evaluation of (1) the effects of a complex, undefined mixture of DBPs in rodents and (2) associations between consumption of disinfected water and altered semen quality in humans. RTD investigators conduct animal studies on the toxicity of DBPs including effects on male and female fertility, pregnancy maintenance, pregnancy outcome, and developmental defects. In addition to DBP studies, RTD is also evaluating reproductive and developmental effects of algal toxin contaminants and is developing in vitro tools to screen novel DBPs and other contaminants in drinking water for developmental toxicity.

The team's research is focused on these the following Long-Term Goals (LTG) in EPA's Multi-Year Plans (MYP):

DW Team Projects

Studies of the Developmental Effects of DBPs and Haloacetic Acids

Contact: Sid Hunter

The haloacetic acids (HAAs) are a family of chemical disinfectant byproducts (DBPs). In animals, all of the HAAs that have been evaluated are developmental toxicants and teratogens. However, not all of the HAAs have been fully tested in vivo. The three objectives of this project are to evaluate the (1) effects of DBPs using in vivo models; (2) effects of HAAs on rodent development in vitro; and (3) potential mechanisms responsible for HAA-induced dysmorphogenesis. Standard in vivo toxicity studies are performed on a range of DBPs in an effort to identify DBPs that pose a developmental hazard and establish dose response relationships. In vitro studies of the dysmorphic effects of novel DBPs are designed to help prioritize compounds for in vivo testing, aid in the identification of the proximate toxicant, and provide comparisons within and between classes of DBPs. The mechanism(s) by which the HAAs produce toxicity are unknown. The goal of mechanistic studies is to use a systems approach to evaluate protein, transcription factor, and gene expression changes in the embryonic response to HAAs. This research continues to provide important information and fill data gaps for the Office of Water.

Recent Publications. Current RTD scientists are in bold.

Johnson C, Sulik KK, Zucker RM, Hunter ES. 2007. Perturbation of retinoic acid (RA)-mediated limb development suggests a role for diminished RA signaling in the teratogenesis of ethanol. Birth Defects Res A Clin Mol Teratol. 79:631-641. Abstract

Hunter ES, Blanton MR, Rogers EH, Mole M, Andrews JE, Chernoff N. 2006. Short-term exposures to dihaloacetic acids produce dysmorphogenesis in mouse conceptuses in vitro. Reprod Toxicol. 22:443-448. Abstract

Hunter ES, Rogers EH, Blanton MR, Richard AM, Chernoff N. 2006. Bromochloro-haloacetic acids: effects on mouse embryos in vitro and QSAR considerations. Reprod Toxicol. 21:260-266. Abstract

Karoly ED, Schmid JE, Hunter ES. 2005. Ontogeny of transcription profiles during mouse early craniofacial development. Reprod Toxicol. 19:265-280. Abstract

Johnson C, Blanton MR, Hunter ES. 2004. Effects of ethanol and hydrogen peroxide on mouse limb bud mesenchyme differentiation and cell death. In Vitro Cell Dev Biol Anim. 40:108-112. Abstract

Trihalomethanes, Hormones, and Pregnancy Loss: Evaluation in Rodent and In Vitro Models

Contact: Michael Narotsky

Some epidemiologic data suggest that exposure to chlorination disinfection by-products (DBPs) via drinking water increases the risk of pregnancy loss (i.e. spontaneous abortion and stillbirth). This project addresses whether experimental models can be developed to understand modes of action of this effect and investigate whether pregnancy loss in a rodent model is analogous to spontaneous abortion in humans. Work has shown that F344 rats are sensitive to toxicant-induced pregnancy loss from chemicals such as bromoform, bromodichloromethane (BDCM), and carbon tetrachloride. Further work has demonstrated that BDCM-induced pregnancy loss in rats is mediated by disruption of pituitary luteinizing hormone (LH) and that LH-mediated pregnancy loss in rats may be analogous to spontaneous abortion. Results from studies using trophoblast cultures and low concentrations of BDCM are consistent with the rat model of BDCM-induced pregnancy loss and provide insight into the epidemiological association of BDCM with spontaneous abortion and stillbirth.

Recent Publications. Current RTD scientists are in bold.

Bielmeier SR, Murr AE, Best DS, Harrison RA, Pegram RA, Goldman JM, Narotsky MG. 2007. Effects of bromodichloromethane on ex vivo and in vitro luteal function and bromodichloromethane tissue dosimetry in the pregnant F344 rat. Toxicol In Vitro. 21:919-928. Abstract

Bielmeier SR, Best DS, Narotsky MG. 2004. Serum hormone characterization and exogeneous hormone rescue of bromodichloromethane-induced pregnancy loss in the F344 rat. Toxicol Sci. 77:101-108. Abstract

Bielmeier SR, Best DS, Narotsky MG. 2004. Serum hormone characterization and exogeneous hormone rescue of bromodichloromethane-induced pregnancy loss in the F344 rat. Toxicol Sci. 77:101-8. Abstract

Chen J, Thirkill TL, Lohstroh PN, Bielmeier SR, Narotsky MG, Best DS, Harrison RA, Natarajan K, Pegram RA, Overstreet JW, Lasley BL, Douglas GC. 2004. Bromodichloromethane inhibits human placental trophoblast differentiation. Toxicol Sci. 78:166-74. Abstract

The Male Reproductive Effects Associated with Haloacid By-Products of Drinking Water Disinfection: Impact on Decision Making and Validation of a Sperm Biomarker

Contact: Gary Klinefelter

Work has shown that the haloacetic acid (HAA) by-products have consistently altered both spermatogenesis and puberty in the male, and the changes in spermatogenesis include decreases in both sperm quality and sperm quantity. Further work demonstrated that both fertilizing ability and levels of the sperm membrane biomarker SP22 are reduced significantly by treatment with either dibromoacetic acid (DBA) or bromochloroacetic acid (BCA). In a binary mixture study, these HAA-induced changes were found to be both dose and effect additive. SP22 is now considered for incorporation with other parameters of semen quality in emerging epidemiology studies. In other work, DBA and BCA were each shown to delay puberty in males and females, and studies on mixtures showed similar effects. Investigations into the mode of action by which the DBPs alter sperm quality and delay puberty are important aspects of this project.

Recent Publications. Current RTD scientists are in bold.

Veeramachaneni D, Palmer JS, Klinefelter GR. 2007. Chronic exposure to low levels of dibromoacetic acid, a water disinfection by-product, adversely affects reproductive function in male rabbits. J Androl. 28:565-577. Abstract

Kaydos E, Suarez JD, Roberts NL, Bobseine KL, Zucker RM, Laskey JW, Klinefelter GR. 2004. Haloacid induced alterations in fertility and the sperm biomarker SP22 in the rat are additive: Validation of an ELISA. Toxicol Sci. 81:430-42. Abstract

Klinefelter GR, Strader LF, Suarez JD, Roberts NL, Goldman JM, Murr AS. 2004. Continuous exposure to dibromoacetic acid delays pubertal development and compromises sperm quality in the rat. Toxicological Sciences 81:419-29. Abstract

Bodensteiner KJ, Sawyer HR, Moeller CL, Kane CM, Pau K, Klinefelter GR, Veeramachaneni D. 2004. Chronic exposure to dibromoacetic acid, a water disinfection by-product, diminishes primordial follicle populations in the rabbit. Toxicol Sci. 80:83-91. Abstract

Klinefelter GR, Strader LF, Suarez JD, Roberts NL. 2002. Bromochloroacetic acid exerts qualitative effects on rat sperm: Implications for a novel biomarker. Toxicol Sci. 68:164-73. Abstract

Reproductive Effects in Female Rats in Response to Haloacetic Acid Drinking Water Disinfection By-Products

Contact: Jerome Goldman

The major research objective of this project was to evaluate potential adverse effects of haloacetic acids on reproductive activity in non-pregnant female rats. Studied endpoints include ovarian cyclicity, serum hormone concentrations, hormone secretion from endocrine tissues in vitro, and oocyte release. A number of studies have reported that trihalomethanes and haloacetic acids (HAAs) can act as mammalian reproductive toxicants. Initial experiments with dibromoacetic acid (DBA) on non-pregnant female rats showed a disruption of estrous cyclicity, and work on bromochloroacetic acid (BCA) showed similar effects. Parallel studies characterized endocrine alterations underlying this effect. Additional studies on BCA using a multigenerational design showed delays in male and female pubertal development at the highest dose. Most recently, the research focus has shifted from potential health effects of individual disinfection byproducts (DBPs) to an evaluation of DBP mixtures that encompass a range of heretofore unstudied impurities in finished drinking water.

Recent Publications. Current RTD scientists are in bold.

Murr AS, Goldman JM. 2005. Twenty week exposures to the drinking water disinfection by-product dibromoacetic acid: Reproductive cyclicity and steroid concentrations in the female Sprague-Dawley rat. Reprod Toxicol. 20:73-80. Abstract

Goldman JM, Murr AS. 2003. Dibromoacetic acid-induced elevations in circulating estradiol: Effects in both cycling and ovariectomized/steroid-primed female rats. Reprod Toxicol. 17:585-592. Abstract

Goldman JM, Murr AS. 2002. Alterations in ovarian follicular progesterone secretion by elevated exposures to the drinking water disinfection by-product dibromoacetic acid: examination of the potential site(s) of impact along the steroidogenic pathway. Toxicology. 171:83-93. Abstract

The Healthy Men Study: Is There an Association Between DBP Exposure and Semen Quality?

Contact: Sally Perreault Darney

The question posed by this project is whether disinfection byproducts (DBPs) pose a risk to reproductive health in healthy men as reflected in changes in semen quality. Objectives include: (1) the evaluation of semen quality in 3 cohorts of men exposed to different levels of DBPs in drinking water; (2) the analysis of semen indicators of genetic integrity of the sperm (SCSA and CMA3) and a sperm fertility biomarker (SP22) in addition to indicators of sperm production and function; (3) the development and evaluation of improved epidemiological methods for sampling remote populations such as home semen collection and shipment; and (4) the determination of whether certain polymorphisms might affect the susceptibility of men to adverse reproductive effects of DBPs. Recent analyses of results do not support any significant associations between exposure and adverse changes in semen quality or quantity.

Recent Publications. Current RTD scientists are in bold.

Luben TJ, Olshan AF, Herring AH, Jeffay SC, Strader LF, Buus RM, Chan RL, Savitz DA, Singer PC, Weinberg HS, Perreault SD. 2007. The healthy men study: An evaluation of exposure to disinfection by-products in tap water and sperm quality. Environ Health Perspect. 115:1169-76. Abstract

Olshan AF, Perreault SD, Bradley L, Buus RM, Strader LF, Jeffay SC, Lansdell L, Savitz DA, Herring A. 2007. The healthy men study: design and recruitment considerations for environmental epidemiologic studies in male reproductive health. Fertil Steril 87(3):554-564. Abstract

Reproductive Toxicity Assessment of Defined and Complex Mixtures of Disinfection By-Products

Contact: Michael Narotsky

Disinfected water has been found to contain more than 500 compounds, and many disinfection byproducts (DBPs) remain unidentified. This project uses a "whole mixture" approach to evaluate the toxicity of complex mixtures produced by chlorination. Chlorinated drinking water is concentrated to form a complex DBP mixture and provided to rats in a multigenerational study design. Reproductive and developmental endpoints examined include pregnancy maintenance, parturition, prenatal development, postnatal development, onset of puberty, estrous cyclicity, and fertility. Defined mixtures of regulated DBPs are evaluated as well. This effort addresses both a regulatory need to develop new approaches to the study of complex mixtures, such as those found in drinking water, and a research need to characterize the risk from exposure to DBP mixtures. Results from this research will assist EPA risk assessors in identifying risk associated with regulated DBPs both in combination and in the "whole mixture" containing known and unidentified DBPs.

Recent Publications. Current RTD scientists are in bold.

Simmons JE, Teuschler LK, Gennings C, Speth TF, Richardson SD, Miltner RJ, Narotsky MG, Schenck KD, Hunter ES, Hertzberg RC, Rice G. 2004. Component-based and whole-mixture techniques for addressing the toxicity of drinking-water disinfection by-product mixtures. J Toxicol Environ Health A. 67:741-54. Abstract

Andrews JE, Nichols HP, Schmid JE, Mole LM, Hunter ES, Klinefelter GR. 2004. Developmental toxicity of mixtures: The water disinfection by-products dichloro-, dibromo- and bromochloro acetic acid in rat embryo culture. Reprod Toxicol. 19:111-6. Abstract

Potential Developmental Effects of Algal Toxins - Microcystin, Anatoxin, Cylindrospermopsin

Contact: Neil Chernoff

The cyanobacteria (blue green algae) constitute a division containing some 2000 species that are present globally in a wide range of aquatic and terrestrial habitats. Species of cyanobacteria produce a variety of potent toxins as secondary metabolites. Human exposures result primarily from drinking untreated or contaminated water. There are a number of water treatment techniques, including disinfection by chlorination or ozonation and adsorption with activated carbon, that remove or deactivate cyanotoxins, but they are variable in both efficiency and efficacy. Prior to work in this project, virtually nothing was known about potential developmental toxicity of these toxins. Work is concentrated on three toxins: microcystin-LR, anatoxin-a, and cylindrospermopsin. Objectives of this work include the elucidation of effects of the toxins on in vivo and/or in vitro development in mammalian (mice) and non-mammalian (a toad) species.

Recent Publications. Current RTD scientists are in bold.

Rogers EH, Zehr RD, Gage MI, Humpage AR, Falconer IR, Marr M, Chernoff N. 2007. The cyanobacterial toxin, cylindrospermopsin, induces fetal toxicity in the mouse after exposure late in gestation. Toxicon. 49:855-64. Abstract

Rogers EH, Hunter ES, Moser VC, Phillips PM, Herkovits J, Munoz L, Hall LL, Chernoff N. 2005. Potential developmental toxicity of anatoxin-a, a cyanobacterial toxin. J Appl Toxicol. 25:527-34. Abstract

Chernoff N, Hunter ES, Hall LL, Rosen MB, Brownie CF, Malarkey D, Marr M, Herkovits J. 2002. Lack of teratogenicity of microcystin-LR in the mouse and toad. J Appl Toxicol. 22:13-7. Abstract

Stem Cells as a Model System for Toxicant Evaluation

Contact: Sid Hunter

Many of the compounds identified in drinking water have not been tested for toxicity. Therefore, there is a need to create standard tools to prioritize and potentially screen xenobiotics for developmental effects. The goal of this project is to use embryonic stem cells to evaluate and compare xenobiotics for their potential as developmental toxicants. Although the Embryonic Stem cell Test (EST) protocol has been established, the test has not been validated as a predictive tool for developmental toxicity. An objective of this project is to modify the endpoints assessed by EST to provide a more complete assessment of differentiation outcome and, in so doing, establish a protocol that may more accurately reflect the potential for a chemical to disrupt morphogenesis. Specific aims for this objective are: (1) to establish a set of molecular markers for assessing the differentiation outcome of mouse embryonic stem cells (mESC); (2) evaluate xenobiotic-induced effects on differentiation outcome of mESC; and (3) establish a protocol for assessing the effects of xenobiotics on outcome of human embryonic stem cells.

Environmental Carcinogenesis Division | Experimental Toxicology Division | Human Studies Division 
 Neurotoxicology Division | Reproductive Toxicology Division

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