Figure 1.
Phenotype analysis of BPA early-life exposure toxicity in wild-type zebrafish larvae.
(A) Percentage of mortality and (B) percentage of larvae with edema when developing zebrafish were exposed to BPA at 50, 100, 500, 1500 and 4500 µg/L BPA or vehicle control for 7 days from 3 hour post-fertilization (hpf) onwards. Error bars represent mean ± SD. Asterisk indicate significant (P<0.05) differences when compared to control group (n = 4 replicate batches of embryos; each treatment group consisted of 50 embryos). Inset in (A) represents another set of acute toxicity experiment performed under similar conditions indicating percentage of mortality for developing zebrafish exposed to BPA at 500, 5000,10000, 15000 and 20000 µg/L. Representative samples of zebrafish larvae exposed to: (C) vehicle (control), (D) 50 µg/L, (E) 100, (F) 500, (G) 1500, and (H) 4500 µg/L, of BPA for 7 days from 3 hpf. Adverse toxic effects was observed at higher frequency in 1500 and 4500 µg/L of BPA which include cardiac edema (ce), cranio-facial abnormality (cf) and swimbladder (sb) development/inflation problem. Scale bar = 500 µm.
Figure 2.
Phenotype analysis of BPA early-life exposure toxicity in GFP transgenic fish larvae.
(A) Tg(Nkx2.2a-mEGFP) zebrafish larvae exposed to vehicle (control), 500 µg/L BPA and 5000 µg/L BPA. Panel on the right represent the inset region marked by white-outlined box in the corresponding left panel. White arrows indicate axon branching which are normal in control fish but marginally and severely affected in fish exposed to 500 and 5000 µg/L BPA, respectively. (B) ET(krt8-EGFP)sqet20 zebrafish larvae exposed to vehicle (control), 500 µg/L BPA and 5000 µg/L BPA. Panel on the right represent the inset region marked by white-outlined box in the corresponding left panel. White arrows indicate neuromast cells which are rosette circular-like shape in control fish but severely deformed in fish exposed to 500 and 5000 µg/L BPA. (C) Tg(Flia-EGFP)y1 zebrafish larvae exposed to vehicle (control), 500 µg/L and 5000 µg/L BPA. White arrow indicate vascularization in abdominal region which is normal in control fish but suppressed in fish exposed to 500 and (R) 5000 µg/L BPA.
Figure 3.
Transcriptomic changes induced by BPA early-life exposure toxicity.
(A) Venn-diagrams showing number (percentage) of genes that were considered significantly deregulated among the three treatment groups (BPA_500 µg/L, BPA_1500 µg/L and BPA_4500 µg/L). B) Expression profiles of gene probes categorized based on their significant deregulation in one, two and three treatment groups. The colored cells reflect the mean expression level of four biological replicates in each concentration group when compared to the control (vehicle) group (n = 4). Shades of red cells reflect up-regulated, green cells reflect down-regulated and black cells reflect unchanged, expression levels when compared to control (vehicle) group.
Figure 4.
Transcriptomic analysis and inference of BPA early-life exposure toxicity.
Selected top functional sub-categories within (A) molecular and cellular function, (B) physiological system development and function, (C) disease and disorder, and (D) canonical pathway, which were all significantly enriched (except for those labeled with asterisk ‘*’where Fisher's Exact Test P>0.05) with human homologs of zebrafish genes deregulated in BPA_500 µg/L, BPA_1500 µg/L and BPA_4500 µg/L groups. Refer to Additional Files 2–5 for more information. The figure legend and Y-axis title for the histogram in (A) are applicable to all other histograms.
Figure 5.
Connectivity network for common-targeted genes with enriched functional subcategories.
The network shows the connectivity between significantly (Fisher's Exact Test P<0.05) enriched functional subcategories with the endocrine-regulated human homologs of zebrafish genes that were commonly deregulated in all the three treatment groups (BPA_500 µg/L, BPA_1500 µg/L and BPA_4500 µg/L). Refer to Additional Files 6 and 7 for more information.
Figure 6.
Real-time PCR validation of selected up-regulated zebrafish genes.
Using samples from a separate validation experiment (n = 5–6 biological replicates; each replicate consist of five pooled larvae), selected up-regulated genes were validated by real-time PCR and compared with corresponding microarray data. Axis titles on the upper left histogram and figure legends are applicable to all histograms.
Table 1.
Human homolog of zebrafish genes (38) that were significantly deregulated in all three BPA exposed groups (500 µg/L, 1500 µg/L and 4500 µg/L).
Figure 7.
Real-time PCR validation of selected down-regulated zebrafish genes.
Using samples from a separate validation experiment (n = 5–6 biological replicates; each replicate consist of five pooled larvae), selected down-regulated genes were validated by real-time PCR and compared with corresponding microarray data. Axis titles on the upper left histogram and figure legends are applicable to all histograms.