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.
Subject(s)
Androgen Antagonists/pharmacology , Dibutyl Phthalate/pharmacology , Flutamide/pharmacology , Testis/drug effects , Animals , Clusterin , Female , Fetus/drug effects , Gene Expression Regulation, Developmental/drug effects , Glycoproteins/genetics , Glycoproteins/metabolism , Immunohistochemistry , Male , Maternal Exposure , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Oligonucleotide Array Sequence Analysis , Pregnancy , Prenatal Exposure Delayed Effects , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Radioimmunoassay , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction , Testis/metabolism , Uterus/drug effectsABSTRACT
TA1 is a rat liver oncofetal cDNA and a member of an emerging family of evolutionarily conserved molecules with homology to amino acid transporters and permeases. The aim of these studies was to characterize the regulation and role of TA1 in acute rat liver injury by examining its relation to regeneration and metabolic stress. Following a single dose of CCl4, TA1 message was expressed 3-48 h. The major 3.3-kb TA1 transcript correlated temporally with c-myc expression. A novel 2.9-kb TA1 transcript was expressed more variably 24-48 h. TA1 protein was restricted to hepatocytes in G0 and G1 phases of the cell cycle. Relative to CCl4, a much smaller increase in TA1 was noted after partial hepatectomy and TA1 preceded the peak of c-myc expression. In vitro TA1 was not induced in hepatocytes by EGF or the acute-phase cytokines IL-6 and TNF-alpha, but was found to be modulated in response to amino acid availability. TA1 expression increased in media without arginine and glutamine and was repressed by total amino acid levels 5-fold over basal MEM. Together, these results contrast with the constitutive expression observed in transformed cells and suggest an adaptive role for TA1 during liver injury.
Subject(s)
DNA, Complementary/metabolism , Genes, myc/genetics , Liver Diseases/metabolism , Liver/metabolism , Membrane Proteins/genetics , Membrane Transport Proteins/metabolism , Amino Acid Transport Systems , Amino Acids/pharmacology , Animals , Carbon Tetrachloride , Cell Survival , Cells, Cultured , Chemical and Drug Induced Liver Injury , Cytokines/pharmacology , Hepatectomy , Kinetics , Large Neutral Amino Acid-Transporter 1 , Liver Regeneration , Male , Membrane Transport Proteins/genetics , RNA, Messenger/metabolism , Rats , Rats, WistarABSTRACT
TA1, a novel rat oncofetal cDNA, is the predicted homolog of the human lymphocyte activation gene E16. The encoded peptides share high homology with transport-associated and uncharacterized sequences in distant species, suggesting an important and conserved function in cellular homeostasis. Moderate steady-state levels of TA1 RNA were induced following acute and chronic CCl4-mediated liver injury. TA1 expression was either greatly reduced or absent in livers of animals receiving injury-protective doses of vitamin E in conjunction with CCl4. In contrast to the in vivo data, acute in vitro exposure of hepatocytes to CCl4 did not induce TA1 RNA. Our results indicate that TA1 is spatially and temporally associated with liver injury in vivo and may play an adaptive role in the hepatic response to environmental toxicants.
