Altered Gene Profiles in Fetal Rat Testes after in Utero Exposure to Di(n-butyl) Phthalate

Abstract

Di(n-butyl) phthalate (DBP) has antiandrogenic-like effects on the developing reproductive tract in the male rat and produces regions of interstitial cell hyperplasia and gonocyte degeneration in the developing fetal testes at maternal doses of 100-500 mg/kg/day. Neither DBP nor its primary metabolites interact with the androgen receptor in vitro. The present study was performed to examine gene expression in the fetal rat testes following in utero DBP exposure. Pregnant Sprague-Dawley rats received corn oil, DBP (500 mg/kg/day), or flutamide (reference antiandrogen, 50 mg/kg/day) by gavage daily from gestation day (GD) 12 to 21. Dose levels were selected to maximize fetal response with minimal maternal toxicity. Testes were isolated on GD 16, 19, and 21. Global changes in gene expression were determined by microarray analysis. Selected genes were further examined by quantitative RT-PCR. DBP, but not flutamide, reduced expression of the steroidogenic enzymes cytochrome P450 side chain cleavage, cytochrome P450c17, and steroidogenic acute regulatory protein. Testicular testosterone and androstenedione were decreased on GD 19 and 21, while progesterone was increased on GD 19 in DBP-exposed testes. Testosterone-repressed prostate message-2 (TRPM-2) was upregulated, while c-kit (stem cell factor receptor) mRNA was downregulated following DBP exposure. TRPM-2 and bcl-2 protein staining was elevated in GD 21 DBP-exposed Leydig and Sertoli cells. Results of this study have led to the identification of several possible mechanisms by which DBP can induce its antiandrogenic effects on the developing male reproductive tract without direct interaction with the androgen receptor. Our results suggest that the antiandrogenic effects of DBP are due to decreased testosterone synthesis. In addition, enhanced expression of cell survival proteins such as TRPM-2 and bcl-2 may be involved in DBP-induced Leydig cell hyperplasia, whereas, downregulation of c-kit may play a role in gonocyte degeneration. Future studies will explore the link between these identified gene expression alterations and ultimate adverse responses.

Retraction: <i>In utero</i>-exposed di(<i>n</i>-butyl) phthalate induce dose dependent, age-related changes of morphology and testosterone-biosynthesis enzymes/associated proteins of Leydig cell mitochondria in rats

“…These abnormalities are consistent with inhibiting fetal testosterone production (Scott et al, 2009). Moreover, decreases in testicular or serum testosterone levels in juvenile male rats (≤ postnatal days 35) (Shultz et al, 2001;Mylchreest et al, 2002;Fisher et al, 2003;Fisher, 2004) and in adult male rats Wakui et al, 2013Wakui et al, , 2014Giribabu et al, 2014) were described after in utero DBP exposure. Leydig cells (LC) are the cells that synthesize testosterone.…”

“…In addition, in the fetal testis after treatment with DBP, it has been reported that reduction in fetal testicular testosterone levels occurs via several steroidogenesis enzymes/associated genes and proteins: SR-B1, StAR, P450scc, 3β-hydroxysteroid dehydrogenase/Δ 5-4 isomerase (3β-HSD), steroid 17 alpha-hydroxylase/17,20 lyase (P450c17), and 17β-Hydroxysteroid dehydrogenases (17β-HSD) Shultz et al, 2001;Barlow and Foster, 2003;Thompson et al, 2004). In the present study, the testosterone levels observed in the 100 mg DBP dose group were significantly lower than all other dose groups at all time-points investigated.…”

Novel molecular targets associated with testicular dysgenesis induced by gestational exposure to diethylhexyl phthalate in the rat: a role for estradiol

“…Before proceeding to discriminant and pathway analyses on these newly identified proteins, we probed western blots of extracts from control and DEHP-exposed testes with P450scc (CYP11A1) to confirm the absence of altered expression at both 10 and 100 mg exposures. This protein was selected, as several studies have demonstrated decreased gene expression of P450scc and other steroidogenic molecules following in utero exposure to phthalates at doses as low as 100 mg (Shultz et al 2001, Lehmann et al 2004, Hannas et al 2011. We observed no decrease across the DEHP exposures; a slight decrease at 100 mg was suggested.…”

“…In general, the expression of P450scc and P450c17 is not significantly altered until relatively high doses of phthalates are administered (500 mg DBP/kg; Shultz et al (2001) and Lehmann et al (2004)) whereas StAR's gene expression decreased at 50 mg DBP/kg (Lehmann et al 2004) and 100 mg DEHP/kg (Borch et al 2006). The importance of the posttranscriptional products (i.e.…”

Self-self Hybridization As An Alternative Experiment Design to Dye Swap for Two-color Microarrays

“…NA MICROARRAYS have been widely used for the discovery of the altered profiles of gene expression associated with toxicity and disease (Lander et al, 2001;Shultz et al, 2001;Yeoh et al, 2002), including many cancers (Khan et al, 2001;Perou et al, 2000;Shi et al, 2005c;Sorlie et al, 2001;Tothill et al, 2005;van 't Veer et al, 2002;Yeoh et al, 2002). Microarrays are commonly employed for identifying genes with expression differences in transcript concentrations between the sample classes (e.g., from diseased versus healthy tissue).…”