Gene expression analysis of the rat testis after treatment with di(2-ethylhexyl) phthalate using cDNA microarray and real-time RT-PCR.
To investigate the effects of di(2-ethylhexyl) phthalate (DEHP) on gene expression in rat testis, 6-week-old male Sprague-Dawley rats were given a single oral dose of 20 or 2000 mg/kg and euthanized 3, 6, 24, or 72 h thereafter. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells were significantly increased in the testis at 24 and 72 h after the exposure to 2000 mg/kg of DEHP. On cDNA microarray analysis, in addition to apoptosis-related genes, genes associated with atrophy, APEX nuclease, MutS homologue (E. coli), testosterone-repressed-prostatic-message-2 (TRPM-2), connective tissue growth factor, collagen alpha 2 type V, and cell adhesion kinase were differentially expressed. To investigate the relationship between histopathological alteration and gene expression, we selected genes associated with apoptosis and analyzed their expression by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). With 20 mg/kg of DEHP treatment, bcl-2, key gene related to apoptosis, was increased. Up-regulation of bcl-2, inhibitor of Apaf-1/caspase-9/caspase-2 cascade of apoptosis, may be related to the fact that no morphological apoptotic change was induced after dosing of 20 mg/kg DEHP. With 2000 mg/kg of DEHP treatment, the apoptotic activator cascade, Fas/FasL, FADD/caspase-8/caspase-3 cascade, and Apaf-1/caspase-9/caspase-2 cascade were increased and bcl-2 was decreased. Thus, these gene regulations might lead the cells into apoptosis in the case of high exposure to DEHP. In contrast, FADD/caspase-10/caspase-6 cascade and caspase-11/caspase-3 cascade were not increased. These results indicate that the cascades of FADD/caspase-10/caspase-6 and caspase-11/caspase-3 are not related to apoptosis with DEHP treatment.
Kijima
Kazuyasu
K
Safety Research Laboratories, Dainippon Pharmaceutical Co, Ltd, Suita, Osaka 564-0053, Japan.
Toyosawa
Kaoru
K
Yasuba
Masashi
M
Matsuoka
Nobuo
N
Adachi
Tetsuya
T
Komiyama
Masatoshi
M
Mori
Chisato
C
D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics; D020169:Caspases / Q000235:genetics / Q000378:metabolism; D004051:Diethylhexyl Phthalate / Q000633:toxicity*; D053222:Fas Ligand Protein; D005786:Gene Expression Regulation / Q000187:drug effects*; D006651:Histocytochemistry; D020287:In Situ Nick-End Labeling; D007527:Isoenzymes; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D010968:Plasticizers / Q000633:toxicity*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D011897:Random Allocation; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013737:Testis / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology / Q000502:physiology; D051028:bcl-2-Associated X Protein; D019014:fas Receptor / Q000235:genetics / Q000378:metabolism
D053222:Fas Ligand Protein; C505662:Faslg protein, rat; D007527:Isoenzymes; D008562:Membrane Glycoproteins; D010968:Plasticizers; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D051028:bcl-2-Associated X Protein; D019014:fas Receptor; D004051:Diethylhexyl Phthalate; D020169:Caspases