Dose-Dependent Alterations in Gene Expression and Testosterone Synthesis in the Fetal Testes of Male Rats Exposed to Di (n-butyl) phthalate

CIIT Centers for Health Research, Research Triangle Park, NC 27709, USA.
Toxicological Sciences (Impact Factor: 3.85). 10/2004; 81(1):60-8. DOI: 10.1093/toxsci/kfh169
Source: PubMed


Exposure to di (n-butyl) phthalate (DBP) in utero impairs the development of the male rat reproductive tract. The adverse effects are due in part to a coordinated decrease in expression of genes involved in cholesterol transport and steroidogenesis with a resultant reduction in testosterone production in the fetal testis. To determine the dose-response relationship for the effect of DBP on steroidogenesis in fetal rat testes, pregnant Sprague-Dawley rats received corn oil (vehicle control) or DBP (0.1, 1.0, 10, 50, 100, or 500 mg/kg/day) by gavage daily from gestation day (GD) 12 to 19. Testes were isolated on GD 19, and changes in gene and protein expression were quantified by RT-PCR and Western analysis. Fetal testicular testosterone concentration was determined by radioimmunoassay. DBP exposure resulted in significant dose-dependent reductions in mRNA and protein concentration of scavenger receptor, steroidogenic acute regulatory protein (StAR), cytochrome P450 side-chain cleavage, 3beta-hydroxysteroid dehydrogenase, and cytochrome P450c17. Testicular testosterone was reduced at doses of 50 mg/kg/day and above. Whole-testis expression of peripheral benzodiazepine receptor (PBR) mRNA, which functions with StAR to transport cholesterol across the mitochondrial membrane, was upregulated following exposure to DBP at 500 mg/kg/day. By immunocytochemistry, however, PBR protein was reduced in interstitial cells and also expressed but not reduced in gonocytes. Our results demonstrate a coordinate, dose-dependent reduction in the expression of key genes and proteins involved in cholesterol transport and steroidogenesis and a corresponding reduction in testosterone in fetal testes following maternal exposure to DBP, at dose levels below which adverse effects are detected in the developing male reproductive tract. Alterations in gene and protein expression and testosterone synthesis may serve as sensitive indicators of testicular response to DBP.

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Available from: Kim Priscilla Lehmann, Jan 15, 2014
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    • "In utero exposure of rats to high doses of certain phthalate esters, such as diethylhexyl phthalate (DEHP) or di(n-butyl) phthalate (DBP), impairs steroido genesis by the fetal testis, resulting in post natal disorders such as hypospadias, cryptorchidism, and impaired spermato genesis (Drake et al. 2009; Johnson et al. 2012; Lehmann et al. 2004; Thompson et al. 2004). In humans, these disorders are thought to comprise a " testicular dysgenesis syndrome " (TDS) (Skakkebaek et al. 2001), for which the DBP-exposed Wistar rat may be a useful model to dissect the underlying mechanisms (Fisher et al. 2003; Sharpe and Skakkebaek 2008). "
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    ABSTRACT: Phthalate exposure induces germ cell effects in the rat fetal testis. Whilst experimental models have shown that the human fetal testis is insensitive to the steroidogenic effects of phthalates, the effects on germ cells are less explored. To identify the effects of phthalate exposure on human fetal germ cells in a dynamic model and to establish if the rat is an appropriate model for investigating such effects. Immunohistochemistry, immunofluorescence and quantitative real-time PCR for Sertoli and germ cell markers on vehicle/di(n-butyl) phthalate (DBP)-exposed rat testes and human fetal testis xenografts. This included analysis of germ cell differentiation markers, proliferation markers and cell adhesion proteins. In both rat and human fetal testes, DBP exposure induced similar germ cell effects, namely germ cell loss (predominantly undifferentiated), induction of multinucleated gonocytes (MNGs) and aggregation of differentiated germ cells, although the latter occurred rarely in the human. The mechanism for germ cell aggregation/MNG induction appears to be loss of Sertoli-germ cell membrane adhesion, probably due to Sertoli cell microfilament redistribution. Our findings provide the first comparison of DBP effects in vivo on germ cell number, differentiation and aggregation in human and rat. They show comparable effects on germ cells in both species, but the effects in the human were muted compared with the rat. Nevertheless, phthalate effects on germ cells have potential implications for the next generation, which merits further study. Our results show that the rat is a human-relevant model in which to explore the mechanisms for germ cell effects.
    Environmental Health Perspectives 12/2014; 123(3). DOI:10.1289/ehp.1408248 · 7.98 Impact Factor
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    • "ect of design of protein expression experiments . Indeed , protein expression data are typically not collected for the entire proteome level at this time . In the five publications analyzed here , these problems were overcome by examining the expression of relevant proteins based on existing knowledge of testicular toxicity ( Chen et al . , 2008 ; Lehmann et al . , 2004 ) or following global gene expression analysis experi - ments that identified relevant pathways affected by a toxicant ( Ludwig et al . , 2011 ) . Illustrated here is the value of BMD analysis in considering temporal and dose – response concordance for protein analysis for MOAs / AOPs for which weight of evidence is sufficient , which l"
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    ABSTRACT: Reliable quantification of gene and protein expression has potential to contribute significantly to the characterization of hypothesized modes of action (MOA) or adverse outcome pathways for critical effects of toxicants. Quantitative analysis of gene expression by benchmark dose (BMD) modeling has been facilitated by the development of effective software tools. In contrast, protein expression is still generally quantified by a less robust effect level (no or lowest [adverse] effect levels) approach, which minimizes its potential utility in the consideration of dose-response and temporal concordance for key events in hypothesized MOAs. BMD modeling is applied here to toxicological data on testicular toxicity to investigate its potential utility in analyzing protein expression relevant to the proposed MOA to inform human health risk assessment. The results illustrate how the BMD analysis of protein expression in animal tissues in response to toxicant exposure: (1) complements other toxicity data, and (2) contributes to consideration of the empirical concordance of dose-response relationships, as part of the weight of evidence for hypothesized MOAs to facilitate consideration and application in regulatory risk assessment. Lack of BMD analysis in proteomics has likely limited its use for these purposes. This paper illustrates the added value of BMD modeling to support and strengthen hypothetical MOAs as a basis to facilitate the translation and uptake of the results of proteomic research into risk assessment. Copyright © 2014 Her Majesty the Queen in Right of Canada. Journal of Applied Toxicology © 2014 John Wiley & Sons, Ltd.
    Journal of Applied Toxicology 11/2014; 34(11). DOI:10.1002/jat.3071 · 2.98 Impact Factor
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    • "Although the proximate molecular target for phthalate action in the developing male fetus has not been identified, the mode of action involves alterations in fetal Leydig cell differentiation, causing a reduction in the expression of genes associated with steroid synthesis/transport, and consequently, lowered fetal androgen levels during the masculinization programming window. These alterations can be used as quantitative predictors of postnatal phenotypes (Barlow and Foster 2003; Foster et al. 1983; Howdeshell et al. 2008; Lehmann et al. 2004; McKinnell et al. 2005; Thompson et al. 2004). "
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    ABSTRACT: Sex differentiation of the male reproductive tract in mammals is driven, in part, by fetal androgen production. In utero, some phthalate esters (PEs) alter fetal Leydig cell differentiation, reducing the expression of several genes associated with steroid synthesis/transport, and consequently, lowering fetal androgen and Insl3 hormone levels. Simvastatin (SMV) is a cholesterol-lowering drug that directly inhibits HMG-CoA reductase. SMV may also disrupt steroid biosynthesis, but through a different mode of action (MOA) than the PEs. As cholesterol is a precursor of steroid hormone biosynthesis, we hypothesized that in utero exposure to SMV during the critical period of sex differentiation would lower fetal testicular testosterone (T) production without affecting genes involved in cholesterol and androgen synthesis and transport. Secondly, we hypothesized that a mixture of SMV and a PE, which may have different MOAs, would reduce testosterone levels in an additive manner. Pregnant Sprague Dawley rats were dosed orally with SMV, dipentyl phthalate (DPeP), or SMV plus DPeP from gestational days 14-18, and fetuses were evaluated on GD18. On GD18, SMV lowered fetal T production and serum triglycerides, LDL, HDL, and total cholesterol levels, and downregulated two genes in the fetal testis that were different from those altered by PEs. When SMV and DPeP were administered as a mixture, fetal T production was significantly reduced in an additive manner, thus demonstrating that a mixture of chemicals can induce additive effects on fetal T production even though they display different MOAs.
    Toxicological Sciences 07/2014; 141(2). DOI:10.1093/toxsci/kfu149 · 3.85 Impact Factor
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