Biological effects of 6-formylindolo[3,2-b]carbazole (FICZ) in vivo are enhanced by loss of CYP1A function in an Ahr2-dependent manner.
6-Formylindolo[3,2-b]carbazole (FICZ) is a potent aryl hydrocarbon receptor (AHR) agonist that is efficiently metabolized by AHR-regulated cytochrome P4501 enzymes. FICZ is a proposed physiological AHR ligand that induces its own degradation as part of a regulatory negative feedback loop. In vitro studies in cells show that CYP1 inhibition in the presence of FICZ results in enhanced AHR activation, suggesting that FICZ accumulates in the cell when its metabolism is blocked. We used zebrafish (Danio rerio) embryos to investigate the in vivo effects of FICZ when CYP1A is knocked down or inhibited. Embryos were injected with morpholino antisense oligonucleotides targeting CYP1A (CYP1A-MO), Ahr2, or a combination of both. FICZ exposure of non-injected embryos or embryos injected with control morpholino had little effect. In CYP1A-MO-injected embryos, however, FICZ dramatically increased mortality, incidence and severity of pericardial edema and circulation failure, reduced hatching frequency, blocked swim bladder inflation, and strongly potentiated expression of Ahr2-regulated genes. These effects were substantially reduced in embryos with a combined knockdown of Ahr2 and CYP1A, indicating that the toxicity was mediated at least partly by Ahr2. Co-exposure to the CYP1 inhibitor alpha-naphthoflavone (αNF) and FICZ had similar effects as the combination of CYP1A-MO and FICZ. HPLC analysis of FICZ-exposed embryos showed increased levels of FICZ after concomitant CYP1A-MO injection or αNF co-exposure. Together, these results show that a functioning CYP1/AHR feedback loop is crucial for regulation of AHR signaling by a potential physiological ligand in vivo and further highlights the role of CYP1 enzymes in regulating biological effects of FICZ.
Institute of Environmental Medicine, Karolinska Institutet, 17177 Stockholm, Sweden; Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543-1050, USA. Electronic address: emma.wincent@swetox.se.
Emma
E
Wincent
Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543-1050, USA. Electronic address: akubota@obihiro.ac.jp.
Akira
A
Kubota
Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543-1050, USA. Electronic address: aliciat@schoolph.umass.edu.
Alicia
A
Timme-Laragy
Department of Environmental Toxicology, Uppsala University, 75236 Uppsala, Sweden. Electronic address: maria.jonsson@ebc.uu.se.
Maria E
ME
Jönsson
Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543-1050, USA. Electronic address: mhahn@whoi.edu.
Mark E
ME
Hahn
Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543-1050, USA. Electronic address: jstegeman@whoi.edu.
John J
JJ
Stegeman
D000818:Animals; D001571:Benzoflavones / Q000494:pharmacology; D002227:Carbazoles / Q000494:pharmacology*; D019363:Cytochrome P-450 CYP1A1 / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000235:genetics*; D004487:Edema / Q000139:chemically induced / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D004625:Embryo, Nonmammalian; D025461:Feedback, Physiological; D018507:Gene Expression Regulation, Developmental; D007267:Injections; D060172:Morpholinos / Q000235:genetics / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000235:genetics / Q000378:metabolism; D010496:Pericardium / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D018336:Receptors, Aryl Hydrocarbon / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D015027:Zebrafish; D029961:Zebrafish Proteins / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism
C111855:6-formylindolo(3,2-b)carbazole; C474372:AHR2 protein, zebrafish; D001571:Benzoflavones; D002227:Carbazoles; D060172:Morpholinos; D016376:Oligonucleotides, Antisense; D018336:Receptors, Aryl Hydrocarbon; D029961:Zebrafish Proteins; C011512:alpha-naphthoflavone; D019363:Cytochrome P-450 CYP1A1
6-Formylindolo[3,2-b]carbazole; Aryl hydrocarbon receptor; Cytochrome P4501; Enzyme inhibition; Synergistic receptor activation; Zebrafish embryo toxicity