Environmentally Relevant Concentrations of Triphenyl Phosphate (TPhP) Impact Development in Zebrafish

Abstract

A common flame-retardant and plasticizer, triphenyl phosphate (TPhP) is an aryl phosphate ester found in many aquatic environments at nM concentrations. Yet, most studies interrogating its toxicity have used µM concentrations. In this study, we used the model organism zebrafish (Danio rerio) to uncover the developmental impact of nM exposures to TPhP at the phenotypic and molecular levels. At concentrations of 1.5-15 nM (0.5 µg/L-5 µg/L), chronically dosed 5dpf larvae were shorter in length and had pericardial edema phenotypes that had been previously reported for exposures in the µM range. Cardiotoxicity was observed but did not present as cardiac looping defects as previously reported for µM concentrations. The RXR pathway does not seem to be involved at nM concentrations, but the tbx5a transcription factor cascade including natriuretic peptides (nppa and nppb) and bone morphogenetic protein 4 (bmp4) were dysregulated and could be contributing to the cardiac phenotypes. We also demonstrate that TPhP is a weak pro-oxidant, as it increases the oxidative stress response within hours of exposure. Overall, our data indicate that TPhP can affect animal development at environmentally relevant concentrations and its mode of action involves multiple pathways.

Keywords: cardiotoxicity; natriuretic peptides; tbx5; triphenyl phosphate; zebrafish.

Figure 1

Experimental design and timing of measurements relative to TPhP dosing and development. The zebrafish images were created by BioRender.com.

Figure 2

(A) Proportion of scored heart development phenotypes in fish treated with DMSO vehicle control or 0.5, 1, or 5 µg/L TPhP. Larvae were initially scored 5-1; scores of 1 and 2 are grouped and represented as “severe abnormality” on this chart. n = 52, 38, 49, 52 per group. (B) Heart rates of treated larvae. n = 15 per group.

Figure 3

Pericardial areas of fish treated with DMSO vehicle control or 0.5, 1, or 5 µg/L TPhP. Data points are shown: black data points represent fish with an edema score of 5 (normal), and red points represent fish scored below 5 (i.e., displaying pericardial edema). Border: representative images of fish from all dosage groups with a range of heart development scores. n = 52, 28, 48, 52 per group, respectively.

Figure 4

TPhP exposure significantly affected body length at 5 dpf relative to the DMSO vehicle control in both the whole population (A) and individuals without pericardial edema (B). Individual data points are shown; lines connect mean values for each treatment and bars show standard deviation. In (A) n = 52, 39, 48, and 52 per group; in (B) n = 49, 35, 40, and 40 per group. p-values: ** p < 0.01, *** p < 0.001.

Figure 5

Expression of genes related to heart development and the RXR pathway in 5 dpf animals. Data are presented as the mean relative expression normalized to the housekeeping gene, b2m. Analysis of expression was performed in Prism using a Student’s t-test, adjusted for multiple testing, to compare the expression of a gene between the control and TPhP-treated samples, where an asterisk indicates a statistically significant change (p < 0.05) due to exposure.

Figure 6

Expression of gstp1 in 5 dpf animals dosed with TPhP for 1, 4, or 24 h prior to analysis of gene expression. Data are presented as the mean relative expression normalized to the housekeeping gene, b2m. Analysis of expression was performed in Prism using a two-way ANOVA with a Tukey’s HSD to compare the expression of the gene between the control and TPhP-treated samples, where an asterisk indicates a statistically significant change (p < 0.05) due to exposure and time.

Figure 7

Ratio of reduced (GSH) to oxidized (GSSG) glutathione in 5 dpf embryos. Ratios are presented as an average with standard deviation, where an asterisk indicates a statistically significant change (p < 0.05) compared to all other values, as calculated by a two-way ANOVA with a Tukey’s HSD test.