Identification of differentially expressed genes in the testis of Sprague-Dawley rats treated with di( n -butyl) phthalate

• Published in: Toxicology (Amsterdam) 2007-05, Vol.234 (1), p.103-112
• Main Authors: Ryu, Ju Young, Lee, Byung Mu, Kacew, Sam, Kim, Hyung Sik
• Format: Article
• Language: English
• Subjects: Administration, Oral; Age Factors; Animals; Biological and medical sciences; Blotting, Western; Di( n-butyl) phthalate; Dibutyl Phthalate - administration & dosage; Dibutyl Phthalate - toxicity; Differentially expressed genes; Dose-Response Relationship, Drug; Emergency; Estrogen Receptor beta - genetics; Estrogen Receptor beta - metabolism; Gene Expression Profiling - methods; Gene Expression Regulation - drug effects; Genes, erbA - drug effects; Genes, erbA - genetics; Male; Medical sciences; Organ Size - drug effects; Plasticizers - administration & dosage; Plasticizers - toxicity; PPAR alpha - genetics; PPAR alpha - metabolism; Rats; Rats, Sprague-Dawley; Receptors, Androgen - genetics; Receptors, Androgen - metabolism; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger - genetics; RNA, Messenger - metabolism; Spermatogenesis; Spermatogenesis - drug effects; Spermatogenesis - genetics; Steroidogenesis; Testis; Testis - drug effects; Testis - metabolism; Testis - pathology; Toxicology
• ISSN: 0300-483X; 1879-3185
• DOI: 10.1016/j.tox.2007.02.003

Abstract The aim of this study was to identify the di( n -butyl) phthalate (DBP)-induced differentially expressed genes (DEGs) using a novel annealing control primer system in the testes of Sprague-Dawley male rats. Animals (4 weeks of age) were administered orally either corn oil only (vehicle control) or DBP (250, 500, or 750 mg/kg/day) for 30 days. Total RNA was isolated from the rat testes and GeneFishing PCR was used to determine the differential gene expression levels. Using this technique, a total of 59 DEG mRNA fragments were observed in the testes treated with DBP 750 mg/kg/day compared to vehicle control. Of these 59 genes, 31 genes were significantly altered after exposing rats to high dose DBP (750 mg/kg/day), and their sequences cloned. Based on the Basic Local Alignment Search Tool (BLAST), 4 expressed sequence tags (EST), 27 cloned genes ( Insl3, pgrp, H1SHR , etc.) and 3 genes (LDHA, lactate dehydrogenase A; Spag4, sperm associated antigen 4 and PBR, peripheral-type benzodiazepine receptor) were found to be involved in spermatogenesis and steroidogenesis. In addition, the expression patterns of the steroidogenesis-related genes such as scavenger receptor class B-1 (SR-B1), steroidogenic acute regulated protein (StAR), P450 side chain cleavage (P450scc), CYP17, and CYP19 were further analyzed by RT-PCR. Significant increases in the mRNA levels of steroidogenesis-related genes (PBR, SR-B1, StAR, P450scc, and CYP17) were observed in the high dose DBP-treated rats. However, DBP significantly decreased the CYP19 mRNA levels compared with controls. DBP (750 mg/kg/day) significantly increased the TR-α1 and PPAR γ expression in testes, whereas the AR and ERβ protein levels were significantly reduced in the same group. These data indicate that the steroidogenesis- or spermatogenesis-related genes identified in this study may provide insights into the molecular mechanisms underlying environmental pollutants-mediated male infertility.