The PBDE metabolite 6-OH-BDE 47 affects melanin pigmentation and THRβ MRNA expression in the eye of zebrafish embryos

Abstract

Polybrominated diphenyl ethers and their hydroxyl-metabolites (OH-BDEs) are commonly detected contaminants in human serum in the US population. They are also considered to be endocrine disruptors, and are specifically known to affect thyroid hormone regulation. In this study, we investigated and compared the effects of a PBDE and its OH-BDE metabolite on developmental pathways regulated by thyroid hormones using zebrafish as a model. Exposure to 6-OHBDE 47 (10-100 nM), but not BDE 47 (1-50 μM), led to decreased melanin pigmentation and increased apoptosis in the retina of zebrafish embryos in a concentration-dependent manner in short-term exposures (4 - 30 hours). Six-OH-BDE 47 exposure also significantly decreased thyroid hormone receptor β (THRβ) mRNA expression, which was confirmed using both RT-PCR and in situ hybridization (whole mount and paraffin- section). Interestingly, exposure to the native thyroid hormone, triiodothyronine (T3) also led to similar responses: decreased THRβ mRNA expression, decreased melanin pigmentation and increased apoptosis, suggesting that 6-OH-BDE 47 may be acting as a T3 mimic. To further investigate short-term effects that may be regulated by THRβ, experiments using a morpholino gene knock down and THRβ mRNA over expression were conducted. Knock down of THRβ led to decreases in melanin pigmentation and increases in apoptotic cells in the eye of zebrafish embryos, similar to exposure to T3 and 6-OH-BDE 47, but THRβ mRNA overexpression rescued these effects. Histological analysis of eyes at 22 hpf from each group revealed that exposure to T3 or to 6-OH-BDE 47 was associated with a decrease of melanin and diminished proliferation of cells in layers of retina near the choroid. This study suggests that 6-OH-BDE 47 disrupts the activity of THRβ in early life stages of zebrafish, and warrants further studies on effects in developing humans.

Keywords: PBDE; apoptosis; melanin pigmentation; thyroid hormone receptor; thyroid hormones; whole mount in situ hybridization; zebrafish.

Figure 1

Relative pigmentation intensity in eyes. Embryos, with and without injection of THRβ mRNA at 1–2 cell stage, exposed to an aqueous solution containing 6-OH-BDE 47 (1, 10 and 100 nM) or T3 (0.01–100 nM) from 4 – to 30- hpf are shown. Levels of eye pigmentation (black coloration intensity) were quantified using ImageJ as stated in the methods. (A–J) Various representative embryos from different exposure groups. (K) (6-OH-BDE-47) and (L) (T3) are histograms of pigment intensity (integrated density) established from quantification of 20 embryos per group. Different letters indicate signifi-cant differences (P < 0.05; SEM indicated by vertical bar for each graph).

Figure 2

Expression of THRβ mRNA in different developmental stages of zebrafish embryos and larvae as shown by blue coloration in in-situ hybridization with whole mount or section. All scale bars are 100 μm. A-F shows whole mount in situ hybridization, G-K shows section in situ hybridization. (A) 50% epiboly; (B) (lateral orientation) and (C) (view showing rostral embryo and yolk sac) at 18-somite stage; (D) (24 hpf), (E and F) (48 hpf). (G) (30 hpf) control sense probe, (H) (30 hpf), (I) (48 hpf), (J and K) at 10 day stage. A has no expression. Red arrows (B–F and G–K) indicate the location of expression. Figures indicate that expression of THRβ is seen beginning at the 18 somite stage in head region (B and C). With further development (24hpf) definitive mRNA expression is in retina and periventricular regions of mid- and forebrain (D and H–K). At 48 hpf, extensive growth of embryo is apparent and expression is localized to periventricular region of midbrain (H–J). (F) shows greater detail in eye, with central blue localization apparent. Yellow to dark brown coloration in (E and F) is pigment. (G) Sense (control) reveals pigmentation around eye likely representing pigmented epithelial layer of retina and adjacent choroid. (H) (Antisense) Weak reaction in brain at 30 hpf. (I) 48 hpf shows THRβ mRNA localized to brain. At 10 dpf, THRβ mRNA expression in brain is similar to the 48 hpf figure. (J and K) Expression in brain and at higher magnification, (K) THRβ mRNA expression is localized to 2 layers in retina.

Figure 3

Six-OH-BDE 47 decreased THRβ mRNA expression in the periventricular zone of zebra-fish embryonic brain. Embryos were exposed from 4- to 22- hpf to 6-OH-BDE 47 (1, 10 and 100 nM). Note the prominent blue coloration in (A) (control) when compared to 6-OH-BDE exposed embryos (B–D). Expression was diminished at 1, 10 and 100 nM respectively. Red arrows indicate localization of expression. The average color intensity recorded in brain at 22 somite stage is shown in the histograms (E). (F) Histogram of the quantitative PCR (qRT-PCR) for THRβ at 22 somite stage. The 1, 10 and 100 nM 6-OH-BDE 47 was significantly different from the control (*, P <0.05, 3 replicates of 20 embryos each were used).

Figure 4

Effect of T3 on THRβ mRNA expression in the periventricular zone of zebrafish embryonic brain. Embryos were exposed from 4- to 22- hpf to T3. Note the prominent blue coloration in control, (A) localized to fore- and midbrain regions (red arrow). By comparison, panels B,C and E show less blue coloration (red arrows) upon exposure to a range of T3 from 0.1 nM to 100 nM. The average intensity of THRβ expression recorded in brain at 22 somite stage showed dose dependent reduction (histogram, E). (F) Bar chart of THRβ expression based on quantitative PCR (qRT-PCR, total RNA from whole embryos) at 22 somite stage. The 10 and 100 nM T3 was significantly different from the control (*, P < 0.05, 3 replicates of 20 embryos eyes each were used). No significant differences were seen at the various concentrations.

Figure 5

Panel 1. Exposure of a THRβ morpholino to zebrafish embryos decreased eye pigmentation and THRβ mRNA rescued the decreased pigmentation in eye. After injection of morpholino and/or THRβ mRNA into the 1–2 cell stage embryos (n = 20 control and exposed) at 30 hpf and eye pigment was quantified using ImageJ. The effect of the morpholino was rescued by coexposure to THRβ mRNA. Letters in the bar graph indicate significant differences (P < 0.05). (Panel 2) Exposure of a THRβ morpholino to zebrafish increased discrete TUNEL positive reactions (apoptosis) in the retina of zebrafish embryos. THR Morpholino and/or THRβ mRNA was injected into the 1–2 cell stage embryos (n &Equals; 20 control and exposed embryos), at 22 hpf discrete apoptotic bodies were quantified using light microscopy manually. Different letters in the bar graph indicate significant differences (P < 0 ). Clearly, THRβ mRNA rescued the phenotype.

Figure 6

Panel 1. (A) TUNEL staining in eyes of paraffin-embedded and sectioned embryos at 22 hpf. Embryos, with and without injection of THRβ mRNA at 1–2 cell stage, were exposed to 6-OH-BDE 47 (10 and 100 nM) or T3 (10 and 100 nM) from 4 – to 22- hpf. The location of apoptosis (brown color) was observed by light microscopy on sections. TUNEL-positive, brown colored cells (i.e., apoptotic cells) were exclusively found in the retina (6A– J). (A–J) Apoptotic cells (TUNEL analysis) in eye (red arrows). The effect of 6-OH-BDE 47 and T3 was rescued by overexpression of THRβ mRNA (G–J). In rescued animals, the number of apoptotic cells decreased to a level comparable with the control (A). Pigment (melanin) was seen as thin black lines of varying length and intensity. Panel 2. THRβ mRNA rescued the increased discrete apoptosis in the retina of zebrafish embryos caused by exposure to 6-OH-BDE 47. THRβ mRNA was injected into the 1–2 cell stage embryos (n &Equals; 20 control and exposed). When embryos were exposed to aqueous 6-OH-BDE 47 (1, 10 and 100 nM) from 4- to 22- hpf discrete apoptosis in eye was seen. (A–F) show apoptotic cells in eyes. By comparison of (B, D, and E), a concentration effect was seen, and THRβ mRNA rescued 6-OH-BDE 47 induced apoptosis in eye. (G) Histogram of the number of individual apoptotic cells in eye. Numbers of discrete TUNEL positive reactions per eye of each of 10 individual embryos are shown. Injected THRβ mRNA and/or exposure to 10 and or 100 nM concentrations resulted in quantitative differences (increase) in apoptosis that were different from values for the low concentration, control, and THRβ mRNA treated group and different from each other (different letters over histograms; P < 0.05). Panel 3. THRβ mRNA rescued T3 induced apoptosis in the retina of zebrafish embryos. THRβ mRNA was injected into the 1–2 cell stage embryos (n &Equals; 20 control and exposed embryos), secondly, embryos were exposed by aqueous route to T3 (1, 10 and 100 nM) from 4- to 22- hpf. (A–F) TUNEL positive reactions, some likely involving more than one cell (apoptosis) in the eye. Comparing Figures 6B, D, and E to 6A, C, or F, a general increase is suggested. (C and F) Show THRβ mRNA rescue of T3- induced apoptosis. (G) A histogram based on the number of TUNEL positive reactions in retina (n &Equals; 10 individual embryos per concentration) showed that only the 100nM concentration produced apoptotic cell numbers different from those of controls, lower concentrations and THRβ treated groups. (p <0 .05).

Figure 6

Panel 1. (A) TUNEL staining in eyes of paraffin-embedded and sectioned embryos at 22 hpf. Embryos, with and without injection of THRβ mRNA at 1–2 cell stage, were exposed to 6-OH-BDE 47 (10 and 100 nM) or T3 (10 and 100 nM) from 4 – to 22- hpf. The location of apoptosis (brown color) was observed by light microscopy on sections. TUNEL-positive, brown colored cells (i.e., apoptotic cells) were exclusively found in the retina (6A– J). (A–J) Apoptotic cells (TUNEL analysis) in eye (red arrows). The effect of 6-OH-BDE 47 and T3 was rescued by overexpression of THRβ mRNA (G–J). In rescued animals, the number of apoptotic cells decreased to a level comparable with the control (A). Pigment (melanin) was seen as thin black lines of varying length and intensity. Panel 2. THRβ mRNA rescued the increased discrete apoptosis in the retina of zebrafish embryos caused by exposure to 6-OH-BDE 47. THRβ mRNA was injected into the 1–2 cell stage embryos (n &Equals; 20 control and exposed). When embryos were exposed to aqueous 6-OH-BDE 47 (1, 10 and 100 nM) from 4- to 22- hpf discrete apoptosis in eye was seen. (A–F) show apoptotic cells in eyes. By comparison of (B, D, and E), a concentration effect was seen, and THRβ mRNA rescued 6-OH-BDE 47 induced apoptosis in eye. (G) Histogram of the number of individual apoptotic cells in eye. Numbers of discrete TUNEL positive reactions per eye of each of 10 individual embryos are shown. Injected THRβ mRNA and/or exposure to 10 and or 100 nM concentrations resulted in quantitative differences (increase) in apoptosis that were different from values for the low concentration, control, and THRβ mRNA treated group and different from each other (different letters over histograms; P < 0.05). Panel 3. THRβ mRNA rescued T3 induced apoptosis in the retina of zebrafish embryos. THRβ mRNA was injected into the 1–2 cell stage embryos (n &Equals; 20 control and exposed embryos), secondly, embryos were exposed by aqueous route to T3 (1, 10 and 100 nM) from 4- to 22- hpf. (A–F) TUNEL positive reactions, some likely involving more than one cell (apoptosis) in the eye. Comparing Figures 6B, D, and E to 6A, C, or F, a general increase is suggested. (C and F) Show THRβ mRNA rescue of T3- induced apoptosis. (G) A histogram based on the number of TUNEL positive reactions in retina (n &Equals; 10 individual embryos per concentration) showed that only the 100nM concentration produced apoptotic cell numbers different from those of controls, lower concentrations and THRβ treated groups. (p <0 .05).

Figure 7

H&E stains of paraffin sectioned eyes at 22 hpf. Controls are shown in (A and D). Eyes from T3- exposed individuals are shown in (B and E) and the effects of 6-OH-BDE 47 are shown in (C and F). Note enhanced cellularity of retina in controls vs. T3 and/or 6-OH-BDE 47 exposed individuals. The effect of exposure on pigment at bases of future retinal pigmented epithelial cells is shown. Both T3 and 6-OH-BDE 47 exposures reduced the thickness of the pigmented layer and higher magnification revealed gaps in this structure. Double arrows indicate pigmented layer. Single arrows point to pyknotic cells.

Figure 8

Flow diagram of hypothesized mechanism for 6-OH-BDE 47 and T3 induction of apoptosis and decreased eye pigmentation of zebra-fish embryos. Time frame is shown in left panel. Responses are in center panel. Right panel shows exposure initiation and duration. Six-OH-BDE 47 or T3 combined with THRβ and decreased its expression. This may promote formation of NO [data not from this paper but from Hu et al. (2009) and Santos et al. (2006). Increased NO may induce active oxygen stress which in turn leads to apoptosis. Subsequently, pigmentation was decreased at 30 hpf in eye of zebrafish embryos.