Effect of dioxins on regulation of tyrosine hydroxylase gene expression by aryl hydrocarbon receptor: a neurotoxicology study.
BACKGROUND
Dioxins and related compounds are suspected of causing neurological disruption. Epidemiological studies indicated that exposure to these compounds caused neurodevelopmental disturbances such as learning disability and attention deficit hyperactivity disorder, which are thought to be closely related to dopaminergic dysfunction. Although the molecular mechanism of their actions has not been fully investigated, a major participant in the process is aryl hydrocarbon receptor (AhR). This study focused on the effect of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) exposure on the regulation of TH, a rate-limiting enzyme of dopamine synthesis, gene expression by AhR.


METHODS
N2a-Rbeta cells were established by transfecting murine neuroblastoma Neuro2a with the rat AhR cDNA. TH expression induced by TCDD was assessed by RT-PCR and Western blotting. Participation of AhR in TCDD-induced TH gene expression was confirmed by suppressing AhR expression using the siRNA method. Catecholamines including dopamine were measured by high-performance liquid chromatography. A reporter gene assay was used to identify regulatory motifs in the promoter region of TH gene. Binding of AhR with the regulatory motif was confirmed by an electrophoretic mobility shift assay (EMSA).


RESULTS
Induction of TH by TCDD through AhR activation was detected at mRNA and protein levels. Induced TH protein was functional and its expression increased dopamine synthesis. The reporter gene assay and EMSA indicated that AhR directly regulated TH gene expression. Regulatory sequence called aryl hydrocarbon receptor responsive element III (AHRE-III) was identified upstream of the TH gene from -285 bp to -167 bp. Under TCDD exposure, an AhR complex was bound to AHRE-III as well as the xenobiotic response element (XRE), though AHRE-III was not identical to XRE, the conventional AhR-binding motif.


CONCLUSIONS
Our results suggest TCDD directly regulate the dopamine system by TH gene transactivation via an AhR-AHRE-III-mediated pathway. The AhR- mediated pathway could have a particular AhR-mediated genomic control pathway transmitting the effects of TCDD action to target cells in the development of dopaminergic disabilities.
Akahoshi
Eiichi
E
Functional Material Laboratory, Corporate Research & Development Center, Toshiba Corporation, 1 Komukai-Toshiba cho, Saiwai-ku, Kawasaki 212-8582, Japan. eiichi.akahoshi@toshiba.co.jp
Yoshimura
Seiko
S
Uruno
Saeko
S
Ishihara-Sugano
Mitsuko
M
D000818:Animals; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D004298:Dopamine / Q000096:biosynthesis; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D008636:Mesencephalon / Q000737:chemistry; D051379:Mice; D009447:Neuroblastoma; D009498:Neurotoxins / Q000633:toxicity*; D000072317:Polychlorinated Dibenzodioxins / Q000633:toxicity*; D051381:Rats; D018336:Receptors, Aryl Hydrocarbon / Q000032:analysis / Q000378:metabolism*; D015533:Transcriptional Activation / Q000187:drug effects; D014162:Transfection; D014446:Tyrosine 3-Monooxygenase / Q000032:analysis / Q000235:genetics* / Q000378:metabolism
D009498:Neurotoxins; D000072317:Polychlorinated Dibenzodioxins; D018336:Receptors, Aryl Hydrocarbon; D014446:Tyrosine 3-Monooxygenase; D004298:Dopamine