Phenotypically anchored transcriptome profiling of developmental exposure to the antimicrobial agent, triclosan, reveals hepatotoxicity in embryonic zebrafish

Abstract

Triclosan (TCS) is an antimicrobial agent commonly found in a variety of personal care products and cosmetics. TCS readily enters the environment through wastewater and is detected in human plasma, urine, and breast milk due to its widespread use. Studies have implicated TCS as a disruptor of thyroid and estrogen signaling; therefore, research examining the developmental effects of TCS is warranted. In this study, we used embryonic zebrafish to investigate the developmental toxicity and potential mechanism of action of TCS. Embryos were exposed to graded concentrations of TCS from 6 to 120hours post-fertilization (hpf) and the concentration where 80% of the animals had mortality or morbidity at 120hpf (EC80) was calculated. Transcriptomic profiling was conducted on embryos exposed to the EC80 (7.37μM). We identified a total of 922 significant differentially expressed transcripts (FDR adjusted P-value≤0.05; fold change ≥2). Pathway and gene ontology enrichment analyses identified biological networks and transcriptional hubs involving normal liver functioning, suggesting TCS may be hepatotoxic in zebrafish. Tissue-specific gene enrichment analysis further supported the role of the liver as a target organ for TCS toxicity. We also examined the in vitro bioactivity profile of TCS reported by the ToxCast screening program. TCS had a diverse bioactivity profile and was a hit in 217 of the 385 assay endpoints we identified. We observed similarities in gene expression and hepatic steatosis assays; however, hit data for TCS were more concordant with the hypothesized CAR/PXR activity of TCS from rodent and human in vitro studies.

Keywords: Hepatotoxicity; Phenotypic anchoring; ToxCast; Transcriptomics; Triclosan; Zebrafish.

Figure 1

Developmental toxicity (mortality and morbidity) profile and logistic regression analysis of TCS exposure. (A) Concentration response profile for 0, 1, 4, 6, 8, and 10 µM TCS exposure across 11 phenotypic endpoints. MO24 and DP24 correspond to 24 hpf endpoints, and the remaining correspond to 120 hpf endpoints. (B) Logistic regression analysis of TCS developmental effects for any adverse phenotype, with logistic curve shown in red. Dashed lines indicate the EC₈₀.

Figure 2

TCS exposure induces robust transcriptional changes in embryonic zebrafish. Heatmap visualization with bi-hierarchical clustering of significant differentially expressed transcripts due to developmental exposure to the EC₈₀ of TCS (FDR corrected p-value ≤ 0.05, fold change ≥ 2.0). The two clusters are annotated by significant functional clusters of enriched GO terms as determined by DAVID.

Figure 3

TCS-induced transcriptional changes are significantly enriched in the liver and brain. Tissue-specific gene enrichment analysis of the significantly differentially expressed orthologous human genes was performed and visualized using the TSEA tool (http://genetics.wustl.edu/jdlab/tsea/). Tissue enrichment are visualized as hexagonal nodes aligned along a dendrogram indicating the similarity of tissue-enriched transcript lists across the 25 tissues.

Figure 4

Developmental time course study of the TCS EC₈₀. (A) Repeated logistic regression analysis for any adverse effect from 0–10 µM of the new TCS stock. Dashed lines indicate the EC₈₀. (B) Percent prevalence for mortality or morbidity at 0 or 6.46 µM TCS at 24, 48, 72, 96, and 120 hpf. (C) Images of zebrafish exposed to 0 (top panel) or 6.46 µM (lower panel) TCS across early development.

Figure 5

Quantitative RT-PCR validation of fifteen significantly differentially expressed genes identified in the microarray analysis at the EC₂₀, EC₅₀, and EC₈₀ of TCS. log₂ values are shown for comparison between the microarray and qRT-PCR data. A one-way ANOVA with Tukey’s post-hoc test or a Kruskal-Wallis with Dunn’s post-hoc test were used to test the significance of expression values between qRT-PCR treatment and controls (n=4; * p≤0.05; ** p ≤ 0.01; *** p ≤ 0.001).

Figure 6

TCS bioactivity profile across three ToxCast assay annotation levels. Hit-call data across the “biological_process_target” (A), “intended_target_family” (B), and the nuclear receptor subset of “intended_target_family” (C) assay annotation levels were visualized using radial pie diagrams. For reference, the height of a slice indicates the hit percent and the size/angle of the slice indicates the total number of assays within that slice.