Systematic evidence mapping informs a class-based approach to assessing personal care products and pubertal timing.

BACKGROUND: Personal care products (PCPs) contain many different compounds and are a source of exposure to endocrine disrupting chemicals (EDCs), including phthalates and phenols. Early-life exposure to EDCs commonly found in PCPs has been linked to earlier onset of puberty. OBJECTIVE: To characterize the human and animal evidence on the association between puberty-related outcomes and exposure to PCPs and their chemical constituents and, if there is sufficient evidence, identify groups of chemicals and outcomes to support a systematic review for a class-based hazard or risk assessment. METHODS: We followed the OHAT systematic review framework to characterize the human and animal evidence on the association between puberty-related health outcomes and exposure to PCPs and their chemical constituents. RESULTS: Ninety-eight human and 299 animal studies that evaluated a total of 96 different chemicals were identified and mapped by key concepts including chemical class, data stream, and puberty-related health outcome. Among these studies, phthalates and phenols were the most well-studied chemical classes. Most of the phthalate and phenol studies examined secondary sex characteristics and changes in estradiol and testosterone levels. Studies evaluating PCP use and other chemical classes (e.g., parabens) had less data. CONCLUSIONS: This systematic evidence map identified and mapped the published research evaluating the association between exposure to PCPs and their chemical constituents and puberty-related health outcomes. The resulting interactive visualization allows researchers to make evidence-based decisions on the available research by enabling them to search, sort, and filter the literature base of puberty-related studies by key concepts. This map can be used by researchers and regulators to prioritize and target future research and funding to reduce uncertainties and address data gaps. It also provides information to inform a class-based hazard or risk assessment on the association between phthalate and phenol exposures and puberty-related health outcomes.

Evaluating endocrine disrupting chemicals: A perspective on the novel assessments in CLARITY-BPA.

BACKGROUND: The Consortium Linking Academic and Regulatory Insights on Bisphenol A Toxicity (CLARITY-BPA) was a collaborative research effort to better link academic research with governmental guideline studies. This review explores the secondary goal of CLARITY-BPA: to identify endpoints or technologies from CLARITY-BPA and prior/concurrent literature from these laboratories that may enhance the capacity of rodent toxicity studies to detect endocrine disrupting chemicals (EDCs). METHODS: A systematic literature search was conducted with search terms for BPA and the CLARITY-BPA participants. Relevant studies employed a laboratory rodent model and reported results on 1 of the 10 organs/organ systems evaluated in CLARITY-BPA (brain and behavior, cardiac, immune, mammary gland, ovary, penile function, prostate gland and urethra, testis and epididymis, thyroid hormone and metabolism, and uterus). Study design and findings were summarized, and a risk-of-bias assessment was conducted. RESULTS: Several endpoints and methods were identified as potentially helpful to detect effects of EDCs. For example, molecular and quantitative morphological approaches were sensitive in detecting alterations in early postnatal development of the brain, ovary, and mammary glands. Hormone challenge studies mimicking human aging reported increased susceptibility of the prostate to disease following developmental BPA exposure. Statistical analyses for nonmonotonic dose responses, and computational approaches assessing multiple treatment-related outcomes concurrently in linked hormone-sensitive organ systems, reported effects at low BPA doses. CONCLUSIONS: This review provided an opportunity to evaluate the unique insights provided by nontraditional assessments in CLARITY-BPA to identify technologies and endpoints to enhance detection of EDCs in future studies.

Society for birth defects research and prevention's multidisciplinary research needs workshop 2022: A call to action.

The Society for Birth Defects Research and Prevention (BDRP) strives to understand and protect against potential hazards to developing embryos, fetuses, children, and adults by bringing together scientific knowledge from diverse fields. The theme of 62nd Annual Meeting of BDRP, "From Bench to Bedside and Back Again", represented the cutting-edge research areas of high relevance to public health and significance in the fields of birth defects research and surveillance. The multidisciplinary Research Needs Workshop (RNW) convened at the Annual Meeting continues to identify pressing knowledge gaps and encourage interdisciplinary research initiatives. The multidisciplinary RNW was first introduced at the 2018 annual meeting to provide an opportunity for annual meeting attendees to participate in breakout discussions on emerging topics in birth defects research and to foster collaboration between basic researchers, clinicians, epidemiologists, drug developers, industry partners, funding agencies, and regulators to discuss state-of-the-art methods and innovative projects. Initially, a list of workshop topics was compiled by the RNW planning committee and circulated among the members of BDRP to obtain the most popular topics for the Workshop discussions. Based on the pre-meeting survey results, the top three discussion topics selected were, A) Inclusion of pregnant and lactating women in clinical trials. When, why, and how? B) Building multidisciplinary teams across disciplines: What cross-training is needed? And C) Challenges in applications of Artificial Intelligence (AI) and machine learning for risk factor analysis in birth defects research. This report summarizes the key highlights of the RNW workshop and specific topic discussions.

Developing a database of systematic reviews of animal studies.

Systematic reviews (SRs) are common practice in clinical and public health research, but less common in non-human animal research. Systematic reviews of animal studies can be valuable to inform clinical research, to evaluate the need for further animal experiments on a given topic, and to assess the hazard of an environmental exposure in the evaluation of toxicological studies. In the last 10 years, there has been an increase in the number of SRs of animal research, as well as several publications with detailed guidance on how to perform high-quality systematic reviews of experimental animal studies. In order to evaluate current analytical approaches used in SRs of animal studies, easily identify all systematic reviews on a specific topic, and subsequently the original animal studies and their results and promote awareness and understanding of these emerging approaches, we compiled a database of SRs of animal studies. The database was developed using a rigorous, systematic approach and covers a broad range of research fields: preclinical research, toxicology, environmental health, and veterinary medicine. The database currently includes 3113 SRs of animal studies (search date June 2019). In addition to bibliographical information, data on whether or not a risk of bias assessment and meta-analysis were conducted were extracted. For future users, the search features of the database provide users with a platform to identify and select SRs with a particular characteristic for export to Microsoft Word or Microsoft Excel. From there, users may perform additional data extraction to meet their research needs. The database is freely available at www.Mendeley.com (link). The database provides methodologists a comprehensive source that can be used to explore and advance the current methodology applied to SRs of animal studies, and can help researchers to easily identify all systematic reviews on a specific topic, and subsequently the original animal studies and their results and avoid duplication and unnecessary animal research.

Cumulative effects of antiandrogenic chemical mixtures and their relevance to human health risk assessment.

Toxicological studies of defined chemical mixtures assist human health risk assessment by establishing how chemicals interact with one another to induce an effect. This paper reviews how antiandrogenic chemical mixtures can alter reproductive tract development in rats with a focus on the reproductive toxicant phthalates. The reviewed studies compare observed mixture data to mathematical mixture model predictions based on dose addition or response addition to determine how the individual chemicals in a mixture interact (e.g., additive, greater, or less than additive). Phthalate mixtures were observed to act in a dose additive manner based on the relative potency of the individual phthalates to suppress fetal testosterone production. Similar dose additive effects have been reported for mixtures of phthalates with antiandrogenic pesticides of differing mechanisms of action. Overall, data from these phthalate experiments in rats can be used in conjunction with human biomonitoring data to determine individual hazard indices, and recent cumulative risk assessments in humans indicate an excess risk to antiandrogenic chemical mixtures that include phthalates only or phthalates in combination with other antiandrogenic chemicals.

Dose Addition Models Based on Biologically Relevant Reductions in Fetal Testosterone Accurately Predict Postnatal Reproductive Tract Alterations by a Phthalate Mixture in Rats.

Challenges in cumulative risk assessment of anti-androgenic phthalate mixtures include a lack of data on all the individual phthalates and difficulty determining the biological relevance of reduction in fetal testosterone (T) on postnatal development. The objectives of the current study were 2-fold: (1) to test whether a mixture model of dose addition based on the fetal T production data of individual phthalates would predict the effects of a 5 phthalate mixture on androgen-sensitive postnatal male reproductive tract development, and (2) to determine the biological relevance of the reductions in fetal T to induce abnormal postnatal reproductive tract development using data from the mixture study. We administered a dose range of the mixture (60, 40, 20, 10, and 5% of the top dose used in the previous fetal T production study consisting of 300 mg/kg per chemical of benzyl butyl (BBP), di(n)butyl (DBP), diethyl hexyl phthalate (DEHP), di-isobutyl phthalate (DiBP), and 100 mg dipentyl (DPP) phthalate/kg; the individual phthalates were present in equipotent doses based on their ability to reduce fetal T production) via gavage to Sprague Dawley rat dams on GD8-postnatal day 3. We compared observed mixture responses to predictions of dose addition based on the previously published potencies of the individual phthalates to reduce fetal T production relative to a reference chemical and published postnatal data for the reference chemical (called DAref). In addition, we predicted DA (called DAall) and response addition (RA) based on logistic regression analysis of all 5 individual phthalates when complete data were available. DA ref and DA all accurately predicted the observed mixture effect for 11 of 14 endpoints. Furthermore, reproductive tract malformations were seen in 17-100% of F1 males when fetal T production was reduced by about 25-72%, respectively.

In utero phthalate effects in the female rat: a model for MRKH syndrome.

Mayer-Rokitansky-Kuster-Hauser (MRKH) syndrome is characterized by uterine and vaginal canal aplasia in normal karyotype human females and is a syndrome with poorly defined etiology. Reproductive toxicity of phthalate esters (PEs) occurs in rat offspring exposed in utero, a phenomenon that is better studied in male offspring than females. The current study reports female reproductive tract malformations in the Sprague-Dawley rat similar to those characteristic of MRKH syndrome, following in utero exposure to a mixture of 5 PEs. We determined that females are ∼2-fold less sensitive to the effects of the 5-PE mixture than males for reproductive tract malformations. We were not fully successful in defining the critical exposure period for females; however, incidence of malformations was 88% following dosing from GD8 to 19 versus 22% and 0% for GD8-13 and GD14-19, respectively. Overall, this study provides valuable information regarding female vulnerability to in utero phthalate exposure and further characterizes a potential model for the human MRKH syndrome.

Dose-response assessment of fetal testosterone production and gene expression levels in rat testes following in utero exposure to diethylhexyl phthalate, diisobutyl phthalate, diisoheptyl phthalate, and diisononyl phthalate.

Several phthalate esters have been linked to the Phthalate Syndrome, affecting male reproductive development when administered to pregnant rats during in utero sexual differentiation. The goal of the current study was to enhance understanding of this class of compounds in the Sprague Dawley (SD) fetal rat following exposure on gestational days (GDs) 14-18 by determining the relative potency factors for several phthalates on fetal testes endpoints, the effects of a nine phthalate mixture on fetal testosterone (T) production, and differences in SD and Wistar (W) strain responses of fetal T production and testicular gene expression to di(2-ethylhexyl) phthalate (DEHP). We determined that diisobutyl phthalate (DIBP) and diisoheptyl phthalate (DIHP) reduced fetal testicular T production with similar potency to DEHP, whereas diisononyl phthalate (DINP) was 2.3-fold less potent. DINP was also less potent at reducing StAR and Cyp11a gene expression levels, whereas DIBP was slightly more potent than DEHP. We observed that administration of dilutions of a mixture of nine phthalates (DEHP, DIHP, DIBP, dibutyl-, benzyl butyl-, dicyclohexyl-, diheptyl-, dihexyl-, and dipentyl phthalate) reduced fetal T production in a dose-dependent manner best predicted by dose addition. Finally, we found that the differential effects of in utero DEHP treatment on epididymal and gubernacular differentiation in male SD and W rats (0, 100, 300, 500, 625, 750, or 875 mg DEHP/kg/day) are likely due to tissue-specific strain differences in the androgen and insl3 signaling pathways rather than differential effects of DEHP on fetal testis T and insl3 production.

Transgenerational effects of Di (2-ethylhexyl) phthalate in the male CRL:CD(SD) rat: added value of assessing multiple offspring per litter.

In the rat, some phthalates alter sexual differentiation at relatively low dosage levels by altering fetal Leydig cell development and hormone synthesis, thereby inducing abnormalities of the testis, gubernacular ligaments, epididymis, and other androgen-dependent tissues. In order to define the dose-response relationship between di(2-ethylhexyl) phthalate (DEHP) and the Phthalate Syndrome of reproductive alterations in F1 male rats, Sprague-Dawley (SD) rat dams were dosed by gavage from gestational day 8 to day 17 of lactation with 0, 11, 33, 100, or 300 mg/kg/day DEHP (71-93 males per dose from 12 to 14 litters per dose). Some of the male offspring continued to be exposed to DEHP via gavage from 18 days of age to necropsy at 63-65 days of age (PUB cohort; 16-20/dose). Remaining males were not exposed after postnatal day 17 (in utero-lactational [IUL] cohort) and were necropsied after reaching full maturity. Anogenital distance, sperm counts and reproductive organ weights were reduced in F1 males in the 300 mg/kg/day group and they displayed retained nipples. In the IUL cohort, seminal vesicle weight also was reduced at 100 mg/kg/day. In contrast, serum testosterone and estradiol levels were unaffected in either the PUB or IUL cohorts at necropsy. A significant percentage of F1 males displayed one or more Phthalate Syndrome lesions at 11 mg/kg/day DEHP and above. We were able to detect effects in the lower dose groups only because we examined all the males in each litter rather than only one male per litter. Power calculations demonstrate how using multiple males versus one male/litter enhances the detection of the effects of DEHP. The results at 11 mg/kg/day confirm those reported from a National Toxicology Program multigenerational study which reported no observed adverse effect levels-lowest observed adverse effect levels of 5 and 10 mg/kg/day DEHP, respectively, via the diet.

Pubertal administration of DEHP delays puberty, suppresses testosterone production, and inhibits reproductive tract development in male Sprague-Dawley and Long-Evans rats.

Although is clear that exposure to high dosage levels of some phthalates delays the onset of puberty in the male rat, it has been hypothesized that low levels of di(2-ethylhexyl) phthalate (DEHP) accelerate puberty by enhancing testicular androgen synthesis. The current study was designed to determine if the dose response to DEHP was nonmonotonic, as hypothesized. Pubertal administration of DEHP delayed the onset of puberty and reduced androgen-dependent tissue weights in both Long-Evans (LE) and Sprague-Dawley (SD) male rats 300 and 900 mg DEHP/kg/day. These effects were generally of greater magnitude in LE than SD rats. By contrast, alterations in testis histopathology (300 and 900 mg/kg/day) were more severe in SD than in LE rats. Taken together, these results suggest that DEHP may be acting on the pubertal male rat testis via two modes of action; one via the Leydig cells and the other via the Sertoli cells. Treatment with DEHP generally reduced serum testosterone and increased serum luteinizing hormone (LH) levels, demonstrating that the reduction in testosterone was due to the effect of DEHP on the testis and not via an inhibition of LH from hypothalamic-pituitary axis. Testosterone production ex vivo (with and without human chorionic gonadotropin stimulation) was consistently reduced in males at the time of puberty and shortly thereafter. DEHP treatment did not accelerate the age at puberty or enhance testosterone levels at 10 or 100 mg/kg/day in either LE or SD rats, as some have hypothesized. Taken together, these results do not provide any evidence of a nonmonotonic dose response to DEHP during puberty.

The herbicide linuron reduces testosterone production from the fetal rat testis during both in utero and in vitro exposures.

In utero exposure to linuron, an urea-based herbicide, results in a pattern of malformations of androgen-dependent tissues in adult male rat offspring resembling that produced by some phthalate esters which are known to decrease fetal testosterone production. This study investigated the impact of in utero linuron treatment on fetal testis gene expression and testosterone production. Timed-pregnant Sprague Dawley rats were administered corn oil vehicle, 12.5, 25, 50 or 75mg linuron/day/kg orally from GD13 to 18. Ex vivo testosterone (T) production was significantly decreased at 50 and 75mg/kg when analyzed on a per litter basis. Unlike the phthalate esters, linuron treatment did not affect insl3, cyp17a, cyp11a or StAR mRNA expression. Control GD18 fetal testes were then incubated with increasing concentrations of linuron (1-300microM) to evaluate if linuron inhibited T production in vitro. T production was significantly reduced at 30microM and above. Progesterone production was not affected in any of the studies indicating that linuron directly inhibited testosterone production in the absence of cytotoxicity. These results indicate the malformations induced by linuron and phthalate esters in male offspring are similar because both reduce fetal T levels during the critical period of sex differentiation but suggest that the mechanisms differ.

Cumulative effects of in utero administration of mixtures of "antiandrogens" on male rat reproductive development.

Although risk assessments are typically conducted on a chemical-by-chemical basis, the 1996 Food Quality Protection Act (FQPA) required the Environmental Protection Agency (EPA) to consider cumulative risk of chemicals that act via a common mechanism of toxicity. To this end, we are conducting studies with mixtures to provide a framework for assessing the cumulative effects of "antiandrogenic" chemicals. Rats were dosed during pregnancy with antiandrogens singly or in pairs at dosage levels equivalent to about one half of the ED50 for hypospadias or epididymal agenesis. The pairs include: AR antagonists (vinclozolin plus procymidone), phthalate esters (DBP plus BBP and DEHP plus DBP), a phthalate ester plus an AR antagonist (DBP plus procymidone), and linuron plus BBP. We predicted that each chemical by itself would induce few malformations; however, by mixing any two chemicals together, about 50% of the males would be malformed. All binary combinations produced cumulative, dose-additive effects on the androgen-dependent tissues. We also conducted a mixture study combining seven "antiandrogens" together. These chemicals elicit antiandrogenic effects at two different sites in the androgen signaling pathway (i.e., AR antagonist or inhibition of androgen synthesis). In this study, the complex mixture behaved in a dose-additive manner. Our results indicate that compounds that act by disparate mechanisms of toxicity display cumulative, dose-additive effects when present in combination.

Mechanisms of action of phthalate esters, individually and in combination, to induce abnormal reproductive development in male laboratory rats.

Phthalate esters are high production volume chemicals used to impart flexibility to polyvinyl chloride products as well as other applications. In the male laboratory rat, the period of sexual differentiation in utero is particularly sensitive to certain phthalate esters, which induce a suite of reproductive malformations, including epididymal and gubernacular agenesis. The fetal rat testes are a main target for phthalate esters as evidenced by a reduction in testosterone production and insulin-like hormone 3 (insl3) expression, a peptide hormone critical for testis descent. Histopathology of fetal and postnatal testes reveals that in utero exposure to phthalate esters disrupts Leydig and Sertoli cell maturation leading to a reduction in germ cells in the malformed seminiferous tubules in adulthood as well as an increased incidence of multinucleated germ cells. There are some strain-specific differences in the target organs in the male reproductive tract in rats affected by phthalate esters. Mixtures of phthalate esters with one another and with other anti-androgenic compounds exhibit cumulative, largely dose-additive effects on male reproductive tract development when administered during sexual differentiation in utero. Since phthalate ester metabolites are detected in maternal and fetal body fluids, and androgen-signaling and insl3 are highly conserved among mammals, phthalates may potentially affect human reproductive development.

A mixture of five phthalate esters inhibits fetal testicular testosterone production in the sprague-dawley rat in a cumulative, dose-additive manner.

Phthalate diesters are chemicals to which humans are ubiquitously exposed. Exposure to certain phthalates during sexual differentiation causes reproductive tract malformations in male rats. In the fetal rat, exposure to the phthalates benzylbutyl phthalate (BBP), di(n)butyl phthalate (DBP), and diethylhexyl phthalate (DEHP) decreases testicular testosterone production and insulin-like 3 hormone mRNA levels. We characterized the dose-response effects of six individual phthalates (BBP, DBP, DEHP, diethyl phthalate [DEP], diisobutyl phthalate [DiBP], and dipentyl phthalate [DPP]) on gestation day (GD) 18 testicular testosterone production following exposure of Sprague-Dawley rats on GD 8-18. BBP, DBP, DEHP, and DiBP were equipotent (ED50 of 440 +/- 16 mg/kg/day), DPP was about threefold more potent (ED50 = 130 mg/kg/day) and DEP had no effect on fetal testosterone production. We hypothesized that coadministration of these five antiandrogenic phthalates would reduce testosterone production in a dose-additive fashion because they act via a common mode of toxicity. In a second study, dams were dosed at 100, 80, 60, 40, 20, 10, 5, or 0% of the mixture. The top dose contained 1300 mg of total phthalates/kg/day including BBP, DBP, DEHP, DiBP (300 mg/kg/day per chemical), and DPP (100 mg DPP/kg/day). This mixture ratio was selected such that each phthalate would contribute equally to the reduction in testosterone. As hypothesized, testosterone production was reduced in a dose-additive manner. Several of the individual phthalates and the mixture also induced fetal mortality, due to pregnancy loss. These data demonstrate that individual phthalates with a similar mechanism of action can elicit cumulative, dose additive effects on fetal testosterone production and pregnancy when administered as a mixture.

Low phytoestrogen levels in feed increase fetal serum estradiol resulting in the "fetal estrogenization syndrome" and obesity in CD-1 mice.

BACKGROUND: Although estrogenic chemicals can disrupt development of the reproductive system, there is debate about whether phytoestrogens in soy are beneficial, benign, or harmful. OBJECTIVES: We compared reproductive and metabolic characteristics in male and female mice reared and maintained on non-soy low-phytoestrogen feed or soy-based high-phytoestrogen feed. METHODS: The low-phytoestrogen diet was non-soy PMI 5K96 (verified casein diet), and the high-phytoestrogen diet consisted of soy-based PMI 5008 during pregnancy and lactation and soy-based PMI 5001 maintenance feed after weaning. RESULTS: In fetuses whose mothers consumed the low-phytoestrogen PMI 5K96 feed, we found a paradoxical significant elevation in endogenous serum estradiol, which was associated postnatally with adverse reproductive outcomes referred to as the "fetal estrogenization syndrome (FES)". In females, this syndrome included early puberty and increased uterine responsiveness to estrogen, and in males, it included reduced testis, epididymis, and seminal vesicle size, but an enlarged prostate. The low-phytoestrogen-fed males and females were lighter at birth, but, between weaning and adulthood, they became obese and developed abnormally high serum leptin levels; these males, but not females, showed impaired glucose regulation. CONCLUSIONS: Removing phytoestrogens from mouse feed produces an obese phenotype consistent with metabolic syndrome, and the associated reproductive system abnormalities are consistent with FES due to elevated endogenous fetal estradiol. Laboratory rodents may have become adapted to high-phytoestrogen intake over many generations of being fed soy-based commercial feed; removing all phytoestrogens from feed leads to alterations that could disrupt many types of biomedical research.

Gestational and lactational exposure to ethinyl estradiol, but not bisphenol A, decreases androgen-dependent reproductive organ weights and epididymal sperm abundance in the male long evans hooded rat.

Many chemicals released into the environment are capable of disrupting normal sex steroid balance, including the oral contraceptive ethinyl estradiol (EE) and the plastic monomer bisphenol A (BPA). EE and BPA are reported to impair reproductive organ development in laboratory animals; however, effects of lower doses of these chemicals have been debated. The goal of the current study was to determine whether relatively low oral doses of EE or BPA would alter male reproductive morphology and associated hormone levels of Long Evans hooded rat. Dams were gavaged with corn oil vehicle, EE (0.05-50 mug/kg/day) or BPA (2, 20, and 200 mug/kg/day) during pregnancy through lactation from gestational day 7 to postnatal day (PND) 18. Anogenital distance was measured at PND2 and nipple retention was measured at PND14 in male pups. Male offspring were euthanized beginning at PND150, and sera and organs were collected for analyses. Adult body weight was significantly decreased in males exposed to 50 mug EE/kg/day. Developmental EE exposure reduced androgen-dependent tissue weights in a dose-dependent fashion; for example, seminal vesicle and paired testes weights were reduced with >/= 5 mug EE/kg/day. Epididymal sperm counts were also significantly decreased with 50 mug EE/kg/day. In contrast, treatment with 2, 20, or 200 mug BPA/kg/day or EE at 0.05-1.5 mug/kg/day did not significantly affect any male endpoint in the current study. These results demonstrate that developmental exposure to oral micromolar doses of EE can permanently disrupt the reproductive tract of the male rat.

A role for basic transcription element-binding protein 1 (BTEB1) in the autoinduction of thyroid hormone receptor beta.

Thyroid hormone (T(3)) induces gene regulation programs necessary for tadpole metamorphosis. Among the earliest responses to T(3) are the up-regulation of T(3) receptor beta (TRbeta; autoinduction) and BTEB1 (basic transcription element-binding protein 1). BTEB1 is a member of the Krüppel family of transcription factors that bind to GC-rich regions in gene promoters. The proximal promoter of the Xenopus laevis TrbetaA gene has seven GC-rich sequences, which led us to hypothesize that BTEB1 binds to and regulates TrbetaA. In tadpoles and the frog fibroblast-derived cell line XTC-2, T(3) up-regulated Bteb1 mRNA with faster kinetics than TrbetaA, and Bteb1 mRNA correlated with increased BTEB1 protein expression. BTEB1 bound to GC-rich sequences in the proximal TrbetaA promoter in vitro. By using chromatin immunoprecipitation assay, we show that BTEB1 associates with the TrbetaA promoter in vivo in a T(3) and developmental stage-dependent manner. Induced expression of BTEB1 in XTC-2 cells caused accelerated and enhanced autoinduction of the TrbetaA gene. This enhancement was lost in N-terminal truncated mutants of BTEB1. However, point mutations in the zinc fingers of BTEB1 that destroyed DNA binding did not alter the activity of the protein on TrbetaA autoinduction, suggesting that BTEB1 can function in this regard through protein-protein interactions. Our findings support the hypothesis that BTEB1 associates with the TrbetaA promoter in vivo and enhances autoinduction, but this action does not depend on its DNA binding activity. Cooperation among the protein products of immediate early genes may be a common mechanism for driving developmental signaling pathways.

Differential expression of the phthalate syndrome in male Sprague-Dawley and Wistar rats after in utero DEHP exposure.

Exposure to phthalate esters during sexual differentiation disrupts testosterone and insulin-like three hormones resulting in malformations of androgen- and insulin-like three-dependent tissues. The current study was designed to test the hypothesis that gubernacular lesions would be more prevalent in the DEHP-treated (750 mg/kg/day, gestational days 14-18) Wistar male than in the SD rat offspring, whereas the SD rat would display a higher incidence of epididymal agenesis. As hypothesized, striking differences were seen in the incidences of epididymal (67% in SD versus 8% in Wistar) and gubernacular lesions (0% in SD versus 64% in Wistar) among the two strains. In addition, fetal androgen and insl3 mRNA levels differed among the strains. SD fetal males had higher insl3 mRNA and lower T levels than Wistar males. The ratio of insl3 mRNA to T differed among DEHP-treated SD and Wistar fetal males, indicating that the steroidogenic pathway was more affected in the SD strain than in the Wistar strain. Taken together, these results suggest that the different malformation profiles produced by in utero phthalate treatment arise, at least in part, from strain differences in fetal Leydig cell function and the manner in which these cells respond to DEHP treatment.