Developmental toxicity assay using high content screening of zebrafish embryos

Abstract

Typically, time-consuming standard toxicological assays using the zebrafish (Danio rerio) embryo model evaluate mortality and teratogenicity after exposure during the first 2 days post-fertilization. Here we describe an automated image-based high content screening (HCS) assay to identify the teratogenic/embryotoxic potential of compounds in zebrafish embryos in vivo. Automated image acquisition was performed using a high content microscope system. Further automated analysis of embryo length, as a statistically quantifiable endpoint of toxicity, was performed on images post-acquisition. The biological effects of ethanol, nicotine, ketamine, caffeine, dimethyl sulfoxide and temperature on zebrafish embryos were assessed. This automated developmental toxicity assay, based on a growth-retardation endpoint should be suitable for evaluating the effects of potential teratogens and developmental toxicants in a high throughput manner. This approach can significantly expedite the screening of potential teratogens and developmental toxicants, thereby improving the current risk assessment process by decreasing analysis time and required resources.

Keywords: automated imaging; ethanol; high content screening; integrated morphometric analysis; ketamine; nicotine; zebrafish embryo.

Figure 1

Flow chart summarizing the steps used for the high content screening (HCS) assay of zebrafish embryos that include compound exposure, image acquisition from multi-well plates, endpoint selection and data analysis.

Figure 2

Schematic diagram explaining the difference between the two morphological endpoints the Integrated Morphometry Analysis (IMA) module quantifies, fiber length (A) and body length (B), obtained from the high content screening (HCS) assay of zebrafish embryos. Fiber length is $\frac{1}{4}\left(p+\sqrt{p^2-16a}\right)$, where ‘p’ is the perimeter and ‘a’ is the area of the image.

Figure 3

A low resolution montage of a region of a 384-well plate showing zebrafish embryos arrayed in the wells. Post-exposure to drugs and chemicals, embryos are manually placed into the wells of the plate using a transfer pipet. A multi-channel pipet is used to aspirate excess water from the wells and 50 μl fish water containing tricaine (0.016%) is added to each well for anesthesia.

Figure 4

Representative images from a 384-well plate used for high content automatic image capture and analysis of embryos (n = 30/each): treatment schedule shown in (A). A panel of control (untreated) and dimethyl sulfoxide (DMSO)-treated embryos [48 h post-fertilization (hpf)] is presented (B). Representative images of embryos captured using a FITC filter (green), threshold images (orange) and integrated morphometric analysis Integrated Morphometry Analysis (IMA) overlay images (orange plus green) are shown, respectively, for control (upper panel), and 0.1% DMSO (lower panel). Fiber lengths (C) and lengths (D) of the embryos in the control and 0.1% DMSO-treated groups are presented as means ± SDs.

Figure 5

Images (as described for Fig. 5) of control and ethanol (EtOH)-treated embryos from a 384-well plate captured by the high content automatic imager. Treatment schedule is shown in A. Representative images for control and EtOH-treated embryos are shown (B). Data for control and ethanol-treated embryos (n = 37/each) from a 384-well plate captured by the high content automatic imager. Fiber lengths and lengths were calculated from the archived images and representative images from the control, 1%, 1.5%, 2% and 2.5% EtOH exposure groups: values are presented as means ± SDs for fiber length (C) and length (D). * = P <0.05; ** = P <0.01.

Figure 6

Nicotine exposure schedule is shown in (A). Representative images (as described for Fig. 5) of control and nicotine-treated (30 and 50 μM) embryos (n = 51/group) from a 384-well plate captured by the high content automatic imager are presented in panel (B). Fiber lengths (C) and lengths (D) were calculated from the archived images and are presented as means ± SDs (B). * = P <0.05.

Figure 7

Treatment schedule for ketamine (A) and representative images (as described for Fig. 5) of control and 2 mM ketamine-treated embryos (n = 17/group) from a 384-well plate captured by the high content automatic imager (B). Fiber lengths (C) and lengths (D) were calculated from the archived images for control and ketamine-treated embryos and are presented as means ± SDs. * = P <0.05.

Figure 8

Schedule for caffeine treatment (A) and representative images of control and caffeine-treated embryos from a 384-well plate captured by the high content automatic imager. Images and representative images for control, 0.05 mM, 0.1 mM caffeine (B), control, 0.5 mM, 1.0 mM, 1.5 mM and 2 mM caffeine (C), and control and 5 mM caffeine (D), are shown. Values for fiber lengths (E) and lengths (F) of control and caffeine-treated zebrafish embryos (n = 39/group) are presented as means ± SDs obtained from multiple experiments. *P = <0.05; ** = P <0.01.

Figure 9

Representative images (as described for Fig. 5) of zebrafish embryos (n = 66/group) grown at two different temperatures (22 °C, top of panel and 28.5 °C, bottom of panel). A panel of embryos grown for 24 h starting at 24 h post-fertilization (hpf) at 22 °C and 28.5 °C were analyzed for fiber lengths and lengths. Images were captured at 48 hpf. Data were calculated from the archived images and are presented as means ± SDs for fiber length (B) and length (C). *P = <0.05.