title: In vivo and in vitro Ah-receptor activation by commercial and fractionated pentabromodiphenylether using zebrafish (Danio rerio) and the DR-CALUX assay.
abstract: The present study addresses the toxicity of a commercial pentabrominated diphenylether (PeBDE) flame retardant mixture, DE-71, in a model aquatic vertebrate. Four weeks' exposure of juvenile zebrafish (Danio rerio) to water-borne DE-71 resulted in dose-dependent induction of CYP1A immunoreactivity, predominantly in the endocardium and the endothelium of larger blood vessels, such as ventral aorta and branchial arteries, as well as the larger hepatic and pancreatic blood vessels. To investigate the impact of possible contaminating PBDD/Fs in the DE-71 product, the study was repeated after DE-71 had been fractionated into a non-planar (cleaned PBDEs) and a planar fraction (PBDD/Fs). Zebrafish were exposed under similar conditions to the planar and cleaned DE-71 fractions, and to uncleaned DE-71. In addition, the above fractions were chemically analyzed and tested in a reporter gene assay (DR-CALUX) for their aromatic hydrocarbon-receptor (AhR) stimulating potencies. A relatively strong CALUX response was detected from the planar DE-71 fraction (19.7ng TCDD equivalent (TEQ)/g DE-71), coinciding with a strong induction of CYP1A immunoreactivity in zebrafish. CYP1A immunoreactivity in zebrafish exposed to uncleaned DE-71 was intense, although the CALUX response was 10-fold less compared to the planar fraction. Only weak CYP1A immunoreactivity was found in fish exposed to cleaned DE-71, and none in control animals; no CALUX response was detected in cleaned DE-71. The present findings indicate that chemical impurities of the commercial PeBDE product account for AhR-mediated effects. Analytical isolation of a planar fraction from the commercial product increased the in vitro (DR-CALUX) signal 10 times. Immunohistochemistry showed a strong tissue specific reaction to DE-71 in vivo at these relatively low TEQ levels regardless of chemical pretreatment of the mix, reflecting the sensitivity of CYP1A induction in juvenile zebrafish to AhR agonists.
authors.affiliation: Department of Pathobiology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, P.O. Box 80158, 3508 TD Utrecht NL, The Netherlands. R.V.Kuiper@vet.uu.nl
authors.forename: R V
authors.initials: RV
authors.lastname: Kuiper
authors.forename: A J
authors.initials: AJ
authors.lastname: Murk
authors.forename: P E G
authors.initials: PE
authors.lastname: Leonards
authors.forename: G C M
authors.initials: GC
authors.lastname: Grinwis
authors.forename: M
authors.initials: M
authors.lastname: van den Berg
authors.forename: J G
authors.initials: JG
authors.lastname: Vos
mesh_terms: D000818:Animals; D000906:Antibodies / Q000032:analysis / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000187:drug effects* / Q000276:immunology; D004305:Dose-Response Relationship, Drug; D004699:Endocardium / Q000187:drug effects; D004727:Endothelium / Q000187:drug effects; D005411:Flame Retardants / Q000633:toxicity*; D017930:Genes, Reporter / Q000187:drug effects; D005880:Gills / Q000187:drug effects; D055768:Halogenated Diphenyl Ethers; D008099:Liver / Q000187:drug effects; D010647:Phenyl Ethers / Q000737:chemistry / Q000633:toxicity*; D011075:Polybrominated Biphenyls / Q000737:chemistry / Q000633:toxicity*; D011897:Random Allocation; D018336:Receptors, Aryl Hydrocarbon / Q000187:drug effects* / Q000378:metabolism; D015027:Zebrafish / Q000502:physiology*
chemical_list: D000906:Antibodies; D005411:Flame Retardants; D055768:Halogenated Diphenyl Ethers; D010647:Phenyl Ethers; D011075:Polybrominated Biphenyls; D018336:Receptors, Aryl Hydrocarbon; C086401:pentabromodiphenyl ether; D003577:Cytochrome P-450 Enzyme System