Biological effects of 6-formylindolo[3,2-b]carbazole (FICZ) in vivo are enhanced by loss of CYP1A function in an Ahr2-dependent manner

Abstract

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-naphtoflavone 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.

Transcriptional profiling of cytochrome P450 genes in the liver of adult zebrafish, <i>Danio rerio</i>

“…The overall enzymatic activities of CYP1D1 seem to be lower than those of other CYP1s (Scornaienchi et al, 2010b;Stegeman et al, 2015). Biological effects of the potent Ahr agonist 6-formylindolo[3,2-b]carbazole (FICZ) in vivo in zebrafish embryos were found to be enhanced by loss of CYP1A function in an Ahr2-dependent manner, indicating a crucial role of a functioning CYP1A/Ahr2 feedback loop in the regulation of Ahr signaling by a potential physiological ligand in vivo (Wincent et al, 2016). Thus, higher transcript levels of CYP1A expression in the adult liver might indicate its physiological roles in addition to roles in xenobiotic metabolism.…”

The Aryl Hydrocarbon Receptor senses the Henna pigment Lawsone and mediates Yin-Yang effects on skin homeostasis

“…Supplement 1H). Inhibition of CYP1A1 can lead to indirect AhR activation in a mechanism involving Trp [19,27]. Using the EROD assay [28], CYP1A1 enzymatic activity was increased by Lawsone in HEK cells (Fig.…”

The tryptophan derivative 6-formylindolo[3,2-b]carbazole, FICZ, a dynamic mediator of endogenous aryl hydrocarbon receptor signaling, balances cell growth and differentiation

“…Wincent et al (2016) demonstrated that FICZ dramatically increased mortality and the incidence and severity of pericardial edema and circulatory failure, while reducing hatching and blocking inflation of the swim bladder in zebrafish embryos. Importantly, these toxic manifestations were only seen when CYP1A was inhibited by morpholino antisense oligonucleotides or a-naphthoflavone (aNF), this latter compound augmenting the mortality caused by FICZ even at concentrations as low as 5 pM (Wincent et al 2016). Proper development of zebrafish embryos appears to require crosstalk between the AHR and the Wnt/b-catenin pathway (Yin et al 2011;Yoshioka et al 2011) and this latter pathway was a key target for FICZ during early zebrafish development (Wincent et al 2015).…”

“…FICZ is a strong agonist of fish AHR2, inducing CYP1A and CYP1B1 in zebrafish embryos approximately fivefold more potently than the polybrominated biphenyl PCB126 (Jonsson et al 2009). Wincent et al (2016) demonstrated that FICZ dramatically increased mortality and the incidence and severity of pericardial edema and circulatory failure, while reducing hatching and blocking inflation of the swim bladder in zebrafish embryos. Importantly, these toxic manifestations were only seen when CYP1A was inhibited by morpholino antisense oligonucleotides or a-naphthoflavone (aNF), this latter compound augmenting the mortality caused by FICZ even at concentrations as low as 5 pM (Wincent et al 2016).…”