Hypothalamus region-specific global gene expression profiling in early stages of central endocrine disruption in rat neonates injected with estradiol benzoate or flutamide.
To identify genes linked to early stages of disruption of brain sexual differentiation, hypothalamic region-specific microarray analyses were performed using a microdissection technique with neonatal rats exposed to endocrine-acting drugs. To validate the methodology, the expression fidelity of microarrays was first examined with two-round amplified antisense RNAs (aRNAs) from methacarn-fixed paraffin-embedded tissue (PET) in comparison with expression in unfixed frozen tissue (UFT). Decline of expression fidelity when compared with the 1x-amplified aRNAs from UFTs was found as a result of the preferential amplification of the 3' side of mRNAs in the second round in vitro transcription. However, expression patterns for the 2x-amplified aRNAs were mostly identical between methacarn-fixed PET and UFT, suggesting no obvious influence of methacarn fixation and subsequent paraffin embedding on expression levels. Next, in the main experiment, neonatal rats at birth were treated subcutaneously either with estradiol benzoate (EB; 10 microg/pup) or flutamide (FA; 250 microg/pup), and medial preoptic area (MPOA)-specific microarray analysis was performed 24 h later using 2x-amplified aRNAs from methacarn-fixed PET. Numbers of genes showing constitutively high expression in the MPOA predominated in males, implying a link with male-type growth supported by perinatal testosterone. Around 60% of genes showing sex differences in expression demonstrated altered levels after EB treatment in females, suggesting an involvement of genes necessary for brain sexual differentiation. When compared with EB, FA affected a rather small number of genes, but fluctuation was mostly observed in females, as with EB. Moreover, many selected genes common to EB and FA showed down-regulation in females with both drugs, suggesting a common mechanism for endocrine center disruption in females, at least at early stages of post-natal development.
Shibutani
Makoto
M
Division of Pathology, National Institute of Health Sciences, Setagaya-ku, Tokyo 158-8501, Japan. shibutan@nihs.go.jp
Lee
Kyoung-Youl
KY
Igarashi
Katsuhide
K
Woo
Gye-Hyeong
GH
Inoue
Kaoru
K
Nishimura
Tetsuji
T
Hirose
Masao
M
D019342:Acetic Acid / Q000494:pharmacology; D000726:Androgen Antagonists / Q000494:pharmacology*; D000818:Animals; D002725:Chloroform / Q000494:pharmacology; D003270:Contraceptive Agents / Q000494:pharmacology*; D004958:Estradiol / Q000031:analogs & derivatives* / Q000494:pharmacology; D005260:Female; D005404:Fixatives; D005485:Flutamide / Q000494:pharmacology*; D005629:Frozen Sections; D015870:Gene Expression / Q000187:drug effects*; D020869:Gene Expression Profiling / Q000379:methods; D018507:Gene Expression Regulation, Developmental; D007031:Hypothalamus / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D000432:Methanol / Q000494:pharmacology; D046228:Microarray Analysis / Q000379:methods; D042282:Microdissection; D051381:Rats; D012733:Sex Differentiation / Q000187:drug effects*
D000726:Androgen Antagonists; D003270:Contraceptive Agents; D005404:Fixatives; C046544:methacarn; C074283:estradiol 3-benzoate; D004958:Estradiol; D005485:Flutamide; D002725:Chloroform; D019342:Acetic Acid; D000432:Methanol