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Embryonic Vascular Disruption Adverse Outcomes: Linking High Throughput Signaling Signatures with Functional Consequences

Ellis-Hutchings RG, Settivari RS, McCoy AT, Kleinstreuer N, Franzosa J, Knudsen TB, Carney EW.
Reproductive Toxicology (2017) DOI: https://doi.org/10.1016/j.reprotox.2017.05.005 PMID: 28527947


Publication


Abstract

Embryonic vascular disruption is an important adverse outcome pathway (AOP) as chemical disruption of cardiovascular development induces broad prenatal defects. High throughput screening (HTS) assays aid AOP development although linking in vitro data to in vivo apical endpoints remains challenging. This study evaluated two anti-angiogenic agents, 5HPP-33 and TNP-470, across the ToxCastDB HTS assay platform and anchored the results to complex in vitro functional assays: the rat aortic explant assay (AEA), rat whole embryo culture (WEC), and the zebrafish embryotoxicity (ZET) assay. Both were identified as putative vascular disruptive compounds (pVDCs) in ToxCastDB and disrupted angiogenesis and embryogenesis in the functional assays. Differences were observed in potency and adverse effects: 5HPP-33 was embryolethal (WEC and ZET); TNP-470 produced caudal defects at lower concentrations. This study demonstrates how a tiered approach using HTS signatures and complex functional in vitro assays might be used to prioritize further in vivo developmental toxicity testing.

Figures


Figure 1. Box and whisker plot.

Box and whisker plot of (A) ToxCast BioSeek AC50 Values, (B) ToxCast lowest 25th Percentile AC50 Values. Line in the middle of the box is plotted at the median and the box extends from the 25th to 75th percentiles. The whiskers go down to the smallest value and up to the largest. * Statistically lower than the 5HP3 group via t-test at P < 0.05.

Figure 2. Microvessel Outgrowth Inhibition Dose-response.

Rat aortic explants were incubated in the absence or presence of increasing concentrations of (A) 5HPP-33 or (B) TNP-470 for four days followed by image acquisition and outgrowth quantification. Integrated density values of the microvessel outgrowth were normalized to the values at the beginning of treatment. Data are expressed as the mean relative (normalized) integrated density ± SD. Samples sizes are shown in the respective figures. Nonlinear regression analysis was used to estimate the concentration that inhibited 50% of the microvessel outgrowth relative to the untreated explants (EC50) using nonlinear curve fitting software. * Statistically different from the untreated group via 1-way ANOVA with Dunnett’s Multiple Comparison Test at P < 0.05.

Figure 3. Photomicrographs of Microvessel Outgrowth Inhibition.

Images of representative rat aortic explants following four days of treatment (test day 8). Untreated control (A, B), 5HPP-33 (46 μM; C, D), and TNP-470 (0.0025 μM; E, F). Magnification = 20 x (A, C, E) or 100 x (B, D, F). The explant is visible as the dark circle while microvessel outgrowth can be seen extending out from the explant.

Figure 4. Reductions in Embryo Viability following 5HPP-33 Exposure.

(A) Rat Whole Embryo Culture. (B) Zebrafish Embryotoxicity Test. Gestation day (GD) 10 rat embryos and 1–4 h post fertilization (hpf) zebrafish embryos were cultured under static conditions in the absence or presence of increasing concentrations of 5HPP-33 for two or five days, respectively, followed by evaluation of viability and morphology. 5HPP-33 exposure primarily resulted in reductions in mean% embryo viability. Samples sizes are shown in the respective figures. Nonlinear regression analysis was used to estimate the concentration lethal to 50% of the embryo (LC50) using nonlinear curve fitting software. * Statistically different from the untreated group via 1-way ANOVA with Dunnett’s Multiple Comparison Test at P < 0.05.

Figure 5. Dose-dependent Embryo Dysmorphology following TNP-470 Exposure.

(A) Rat Whole Embryo Culture. (B) Zebrafish Embryotoxicity Test. Gestation day (GD) 10 rat embryos and 1–4 h post fertilization (hpf) zebrafish embryos were cultured under static conditions in the absence or presence of increasing concentrations of TNP-470 for two or five days, respectively, followed by evaluation of viability and morphology. A total of 18 or 12 separate structural features were evaluated in WEC and ZET, respectively (see methods for more details). TNP-470 exposure resulted in developmental abnormalities in the absences of lethality. The mean% of abnormal embryos is shown along with the samples sizes in the respective figures. Nonlinear regression analysis was used to estimate the concentration that caused a 50% incidence of abnormal embryos (EC50) using nonlinear curve fitting software. * Statistically different from the untreated group via 1-way ANOVA with Dunnett’s Multiple Comparison Test at P < 0.05.

Figure 6. TNP-470-induced Embryo Dysmorphology in Rat WEC (Representative photomicrographs).

Photomicrographs of representative rat embryos following two days of culture in the absence or presence of increasing concentrations of TNP-470. Untreated control (A, D), TNP-470 (2.5 μM; B, C, E). Magnification = 20x (A,B, and C) or 50x (D, E). TNP-470 was teratogenic causing primarily caudal extension defects and abnormal somite patterning. The asterisks in 6 B refer to reduced caudal length and kinking, blisters in the caudal and fronto-nasal process, and distended hindbrain. The asterisks in 6C refers to the somite region most affected in TNP-470-exposed embryo. Figs. 6D and E show higher magnification of this region where reductions in somite size, definition and altered shape are apparent with TNP-470 exposure (6E) compared to untreated embryos (6D).

Figure 7. TNP-470-induced Zebrafish Embryo Dysmorphology (Representative photomicrographs).

Fig. 7. TNP-470-induced Zebrafish Embryo Dysmorphology (Representative photomicrographs). Photomicrographs of representative zebrafish embryos following five days of culture in the absence or presence of increasing concentrations of TNP-470. Magnification = 100x. Untreated control (A), TNP-470 (7B, 0.1 μM; 7C, 10 μM; 7D, 1 μM). TNP-470 was teratogenic causing primarily caudal defects and abnormal somite patterning. The asterisks in 7 B shows tail fin abnormalities; in 7C shows tail fin abnormalities, disorganized somites, and forebrain blisters; in 7D shows blisters.

Tables


Table 1. Functional and ToxCast HTS Assay Characteristics.

Table 2. ToxCast in vitro HTS Assay Findings.

Table 3. ToxCast in vitro HTS Assay Data: 25th Percentile AC50 Values.

Table 4. Comparison of Intermediate Tier Functional and Selected ToxCast HTS Assay Findings.

Supplemental Materials


Supplementary data