Abnormalities of Sexual Development in Male Rats with in Utero and Lactational Exposure to the Antiandrogenic Plasticizer Di(2-ethylhexyl) Phthalate (DEHP)

Environmental Health Perspectives v.109, n.3, Mar01

Robert W. Moore,1,2 Thomas A. Rudy,1 Tien-Min Lin,1 Kinarm Ko,3 and Richard E. Peterson1,2,3

1School of Pharmacy, 2Environmental Toxicology Center, and 3Endocrinology-Reproductive Physiology Program, University of Wisconsin, Madison, Wisconsin, USA

Abstract
Several members of the phthalate ester family have antiandrogenic properties, yet little is known about how exposure to these ubiquitous environmental contaminants early in development may affect sexual development. We conducted experiments to determine effects of in utero and lactational exposure to the most prevalent phthalate ester, di(2-ethylhexyl) phthalate (DEHP), on male reproductive system development and sexual behavior. Sprague-Dawley rats were dosed with corn oil or DEHP (0, 375, 750, or 1,500 mg/kg/day, per os) from gestation day 3 through postnatal day (PND) 21. Dose-related effects on male offspring included reduced anogenital distance, areola and nipple retention, undescended testes, and permanently incomplete preputial separation. Testis, epididymis, glans penis, ventral prostate, dorsolateral prostate, anterior prostate, and seminal vesicle weights were reduced at PND 21, 63, and/or 105-112. Additional dose-related effects included a high incidence of anterior prostate agenesis, a lower incidence of partial or complete ventral prostate agenesis, occasional dorsolateral prostate and seminal vesicle agenesis, reduced sperm counts, and testicular, epididymal, and penile malformations. Many DEHP-exposed males were sexually inactive in the presence of receptive control females, but sexual inactivity did not correlate with abnormal male reproductive organs. These results suggest that in utero and lactational DEHP exposure also inhibited sexually dimorphic central nervous system development. No major abnormalities were found in any of eight control litters, but DEHP caused severe male reproductive system toxicity in five of eight litters at 375 mg/kg/day, seven of eight litters at 750 mg/kg/day, and five of five litters at 1,500 mg/kg/day. These results demonstrate that the male reproductive system is far more sensitive to DEHP early in development than when animals are exposed as juveniles or adults. The effects of DEHP on male reproductive organs and sexual behaviors and the lack of significant effects on time to vaginal opening and first estrus in their littermates demonstrate that DEHP (and/or its metabolites) affects development of the male reproductive system primarily by acting as an antiandrogen. The pattern of effects of in utero and lactational DEHP exposure differed from patterns caused by other phthalate esters, and the preponderance of anterior prostate agenesis appears to be unique among all chemicals. These results suggest that DEHP acts partly by mechanisms distinct from those of other antiandrogens. Key words: antiandrogens, di(2-ethylhexyl) phthalate, in utero exposure, lactational exposure, male reproductive system development, masculine sexual behaviors, reproductive organ agenesis. Environ Health Perspect 109:229-237 (2001). [Online 28 February 2001]

http://ehpnet1.niehs.nih.gov/docs/2001/109p229-237moore/abstract.html

 

Address correspondence to R.W. Moore, School of Pharmacy, University of Wisconsin, 425 N. Charter Street, Madison, WI 53706 USA. Telephone: (608) 265-2531. Fax: (608) 265-3316. E-mail: rwmoore@pharmacy.wisc.edu
We thank O. Li for sperm analyses.
This research was supported by NIH grants ES 06806 and ES 01332 and by the University of Wisconsin EHS Center for Developmental and Molecular Toxicology.
This article is contribution 331 from the Environmental Toxicology Center, University of Wisconsin, Madison.
Received 15 August 2000; accepted 13 October 2000.

Introduction

The development of the male reproductive system is androgen-dependent and is therefore vulnerable to antiandrogens. Among the chemicals humans are routinely exposed to are phthalate esters, several of which have antiandrogenic properties. Phthalate esters are used most commonly as plasticizers. They constitute 10-60% by weight of many plastics because they impart flexibility, transparency, and other desirable physical properties. Because phthalate esters are not covalently bound to the polymers with which they are mixed, they can leach into the foods, beverages, or other materials contained by these plastics. Consequently, because plastics are so commonplace, phthalate esters are ubiquitous in foods and the environment (1,2).

Many phthalate esters have long been known to be reproductive toxicants when animals are dosed as juveniles or adults, and their teratogenicity is well established (1,3), yet little has been published on the effects of in utero and lactational (or continuous multigenerational) exposure to any phthalate ester on postnatal development of the male or female reproductive systems or sexual differentiation of the central nervous system (CNS).

Of the approximately 20 phthalate esters in common use, di(2-ethylhexyl) phthalate (DEHP) constitutes approximately half the total; 1-4 million tons are produced per year (1,2). DEHP is used in numerous consumer products, especially those made of flexible polyvinyl chloride. The use of DEHP in teething rings, pacifiers, and toys for young children has largely been discontinued, but DEHP continues to be used in clothing, toys, food containers, and a variety of building, household, and automotive products (3,4). Typical human exposure is estimated to be 4-30 µg DEHP/kg/day, but some individuals have substantially greater exposure resulting from DEHP-plasticized medical devices such as blood bags, hemodialysis tubing and membranes, autophoresis equipment, and nasogastric feeding tubes (5). The average long-term dialysis patient is reported to receive approximately 12 g of DEHP over the course of a year (6).

The impetus for our investigations of effects of DEHP was provided by a report that in utero and lactational di(n-butyl) phthalate exposure disrupts male reproductive system development by what appears to be an antiandrogenic mechanism (7). Because DEHP was already known to have antiandrogenic properties, we hypothesized that in utero and lactational DEHP exposure would cause similar responses. Effects of DEHP on juvenile or adult rodents include reductions in testis (8-11) and epididymis (12) weights, severe reductions in sperm production (12,13), and various pathological effects on the testis (8,10,11,14-17). Accessory sex organs are highly androgen-dependent, and reductions in seminal vesicle (10,11,18) and ventral prostate (10,11,18) weights have commonly been observed in DEHP-treated rats. Reductions in serum testosterone concentrations have also been seen (9,10). In all these studies, males were not exposed until after weaning, when the male reproductive system is far less vulnerable to many toxicants than before weaning, and doses were typically 1,000-2,000 mg DEHP/kg/day. In other studies, in utero DEHP exposure (alone) and lactational DEHP exposure (alone) each reduced testis weight and epididymal sperm counts in rats (19-21).

The primary information gap when we began this research was that there was not a single publication on the effects of in utero and lactational or continuous multigenerational DEHP exposure on any aspect of postnatal development of the male (or female) reproductive system or sexual differentiation of the CNS in any species, except for a report that fertility was not impaired in male or female rats (22). We therefore conducted a dose-response, time course experiment to test the hypothesis that male reproductive system development and sexually dimorphic CNS development in rats are vulnerable to in utero and lactational DEHP exposure. While this work was in progress, Arcadi et al. (23) reported that low-level in utero and lactational DEHP exposure reduces testis weight and alters testicular morphology in rats. And after completing the in-life portion of this research, Gray et al. (24) published results of a study in which effects of a single daily maternal dose of DEHP on male reproductive system development in rats were determined.

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