Barlow Norman J, Phillips Suzanne L, Wallace Duncan G, Sar Madhabananda, Gaido Kevin W, Foster Paul M D
CIIT Centers for Health Research, Research Triangle Park, North Carolina 27709, USA.
Toxicol Sci. 2003 Jun;73(2):431-41. doi: 10.1093/toxsci/kfg087. Epub 2003 Apr 15.
Di(n-butyl) phthalate (DBP) alters male reproductive development by decreasing testicular testosterone (T) production when fetuses are exposed on gestation days (GD) 12-21. Previous studies have shown altered gene expression for enzymes in the T biosynthetic pathway following exposure to DBP. The objectives of this study were to develop a more detailed understanding of the effect of DBP on steroidogenesis, using a robust study design with increased numbers of dams and fetuses, compared with previous studies, and to explore messenger RNA (mRNA) expression for other critical genes involved in androgen biosynthesis and signaling. Additionally, immunohistochemical localization of protein expression for several key genes was performed to further confirm mRNA changes. Fetal Leydig cell lipid levels were also examined histochemically, using oil red O. Six to seven pregnant Crl:CD(SD)BR rats per group were gavaged with corn oil or DBP at 500 mg/kg/day on GD 12-19. Testicular RNA isolated from three randomly selected GD 19 fetuses per litter was used for real-time RT-PCR for the following genes: scavenger receptor class B-1 (SRB1), steroidogenic acute regulatory protein (StAR), P450 side-chain cleavage enzyme (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), P450c17, 17beta-hydroxysteroid dehydrogenase (17beta-HSD), androgen receptor (AR), luteinizing hormone receptor (LHR), follicle-stimulating hormone receptor (FSHR), stem cell factor tyrosine kinase receptor (c-kit), stem cell factor (SCF), proliferating cell nuclear antigen (PCNA), and testosterone-repressed prostate message-2 (TRPM-2). mRNA expression was downregulated for SRB1, StAR, P450scc, 3beta-HSD, P450c17, and c-kit following DBP exposure, and TRPM-2 was upregulated. 17beta-HSD, AR, LHR, FSHR, and PCNA were not significantly changed. Immunohistochemical staining for c-kit was seen in fetal Leydig cells, which has not been previously reported. Downregulation of most of the genes in the T biosynthetic pathway confirms and extends previous findings. Diminished Leydig cell lipid content and alteration of cholesterol transport genes also support altered cholesterol metabolism and transport as a potential mechanism for decreased T synthesis following exposure to DBP.
邻苯二甲酸二丁酯（DBP）在妊娠第12至21天使胎儿暴露时，通过降低睾丸睾酮（T）的产生来改变雄性生殖发育。先前的研究表明，暴露于DBP后，T生物合成途径中酶的基因表达发生了改变。本研究的目的是，与先前的研究相比，采用更强大的研究设计，增加母鼠和胎儿数量，以更详细地了解DBP对类固醇生成的影响，并探索参与雄激素生物合成和信号传导的其他关键基因的信使核糖核酸（mRNA）表达。此外，对几个关键基因的蛋白质表达进行免疫组织化学定位，以进一步确认mRNA的变化。还使用油红O对胎儿睾丸间质细胞的脂质水平进行了组织化学检查。每组6至7只怀孕的Crl:CD(SD)BR大鼠在妊娠第12至19天，每天按500 mg/kg的剂量灌胃玉米油或DBP。从每窝中随机选择3只妊娠第19天的胎儿分离睾丸RNA，用于以下基因的实时逆转录聚合酶链反应（RT-PCR）：清道夫受体B1类（SRB1）、类固醇生成急性调节蛋白（StAR）、细胞色素P450侧链裂解酶（P450scc）、3β-羟基类固醇脱氢酶（3β-HSD）、细胞色素P450c17、17β-羟基类固醇脱氢酶（17β-HSD）、雄激素受体（AR）、黄体生成素受体（LHR）、促卵泡激素受体（FSHR）、干细胞因子酪氨酸激酶受体（c-kit）、干细胞因子（SCF）、增殖细胞核抗原（PCNA）和睾酮抑制的前列腺信息-2（TRPM-2）。DBP暴露后，SRB1、StAR、P450scc、3β-HSD、P450c17和c-kit的mRNA表达下调，TRPM-2上调。17β-HSD、AR、LHR、FSHR和PCNA无显著变化。在胎儿睾丸间质细胞中观察到c-kit的免疫组织化学染色，这在以前尚未见报道。T生物合成途径中大多数基因的下调证实并扩展了先前的发现。睾丸间质细胞脂质含量减少和胆固醇转运基因改变也支持胆固醇代谢和转运改变是暴露于DBP后T合成减少的潜在机制。
