Characterization of heme oxygenase and biliverdin reductase gene expression in zebrafish (Danio rerio): Basal expression and response to pro-oxidant exposures

Abstract

While heme is an important cofactor for numerous proteins, it is highly toxic in its unbound form and can perpetuate the formation of reactive oxygen species. Heme oxygenase enzymes (HMOX1 and HMOX2) degrade heme into biliverdin and carbon monoxide, with biliverdin subsequently being converted to bilirubin by biliverdin reductase (BVRa or BVRb). As a result of the teleost-specific genome duplication event, zebrafish have paralogs of hmox1 (hmox1a and hmox1b) and hmox2 (hmox2a and hmox2b). Expression of all four hmox paralogs and two bvr isoforms were measured in adult tissues (gill, brain and liver) and sexually dimorphic differences were observed, most notably in the basal expression of hmox1a, hmox2a, hmox2b and bvrb in liver samples. hmox1a, hmox2a and hmox2b were significantly induced in male liver tissues in response to 96 hour cadmium exposure (20 µM). hmox2a and hmox2b were significantly induced in male brain samples, but only hmox2a was significantly reduced in male gill samples in response to the 96 hour cadmium exposure. hmox paralogs displayed significantly different levels of basal expression in most adult tissues, as well as during zebrafish development (24 to 120 hpf). Furthermore, hmox1a, hmox1b and bvrb were significantly induced in zebrafish eleutheroembryos in response to multiple pro-oxidants (cadmium, hemin and tert-butylhydroquinone). Knockdown of Nrf2a, a transcriptional regulator of hmox1a, was demonstrated to inhibit the Cd-mediated induction of hmox1b and bvrb. These results demonstrate distinct mechanisms of hmox and bvr transcriptional regulation in zebrafish, providing initial evidence of the partitioning of function of the hmox paralogs.

Spatiotemporal expression and transcriptional regulation of heme oxygenase and biliverdin reductase genes in zebrafish (Danio rerio) suggest novel roles during early developmental periods of heightened oxidative stress

“…Furthermore, since we have previously demonstrated a role for Nrf2a in regulating the induction of bvrb in older zebrafish eleutheroembryos in response to Cd exposure (Holowiecki et al, 2016), we also investigated any changes in spatial expression of bvra and bvrb in Nrf2a morphants after Cd-treatment. Finally, although spatial expression of hmox1a has been well characterized in older zebrafish eleutheroembryos in response to pro-oxidant exposures, we included an assessment of hmox1a expression for greater context and to demonstrate successful Nrf2a knockdown.…”

“…We have previously characterized the sex-specific differences in basal expression of all four hmox zebrafish paralogs and both bvr genes, as well as differences in induction by cadmium (Cd) exposure in adult zebrafish tissues (Holowiecki et al, 2016). Furthermore, we have also characterized the basal expression of all six hmox and bvr genes during zebrafish development and in response to multiple pro-oxidant exposures (Cd, tert-butylhydroquinone, and hemin) (Holowiecki et al, 2016).…”

“…Furthermore, we have also characterized the basal expression of all six hmox and bvr genes during zebrafish development and in response to multiple pro-oxidant exposures (Cd, tert-butylhydroquinone, and hemin) (Holowiecki et al, 2016). To expand this characterization, we sought to investigate any changes in the spatial and temporal expression of bvra and bvrb during early zebrafish development under both basal conditions and in response to pro-oxidant exposure.…”

“…To expand this characterization, we sought to investigate any changes in the spatial and temporal expression of bvra and bvrb during early zebrafish development under both basal conditions and in response to pro-oxidant exposure. Cd was chosen as the pro-oxidant since we have previously demonstrated that the Cd-inducibility of hmox1a and bvrb during zebrafish development is regulated in part by Nrf2a (Holowiecki et al, 2016). hmox1a expression was also characterized to add greater context to the observed changes in bvra and bvrb expression.…”

“…Similarly, bvrb has been shown to be expressed within the intermediate cell mass (ICM), the site of embryonic erythrocyte formation during early zebrafish development, and this expression appears to be dependent on the erythroid specific transcription factor GATA-binding factor 1 (Gata-1) (Galloway et al, 2005), which is also known as nuclear factor erythroid 1 (Nf-e1). Furthermore, it is well established that HMOX1 is regulated by nuclear factor erythroid 2-related factor 2 (NRF2), the master regulator of the oxidative stress response (Alam et al, 1999), and previous studies have provided evidence of a role for NRF2 in regulating BVR expression (Moon et al, 2012; Wu et al, 2011; Holowiecki et al, 2016). Interestingly, recent studies have suggested a novel developmental role for NRF2 as a regulator of hematopoietic stem cell (HSC) homeostasis (Merchant et al, 2011; Tsai et al, 2013).…”