Zebrafish: A Predictive Model for Assessing Cancer Drug-Induced Organ Toxicity - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 133

Stemi 2000C stereomicroscope (Zeiss, Thornwood, NY) equipped with a SPOT
Insight digital camera (MVI, Avon, MA). Three percent methylcellulose (Sigma, St.
Louis, MO) was used to immobilize zebrafish to facilitate imaging.
11.2.6 End Points for Organ Morphology/Function
Toxicity
Heart: Abnormal heart rate: tachycardia (increased heart rate), bradycardia (de-
creased heart rate), arrhythmia; abnormal circulation, pericardial edema, abnormal
heart chamber morphology, and stagnant blood flow, which can lead to thrombosis.
CNS: Misshapen brain, brain hemorrhage, or degenerated brain tissue, which is
opaque, brown/gray in color.
Liver: Liver size and color. Degenerated liver tissue is opaque, brown/gray. Normal
liver is transparent and clear/light brown, and blood flow can be visualized.
Kidney: Fluid accumulation around the kidney, cyst formation, or trunk edema.
11.2.7 Assessment of Cell DeathAfter 24hTreatment
The same compound treatment strategy described in Section 11.2.3 was used for
the cell death assay. Cell death in target organs was assessed using fluorescent
acridine orange (AO) dye that penetrates dead cells and labels nucleotides.
Zebrafish samples were stained with AO (1mg/mL) for 1 h, and then washed
to remove excess dye. After washing, stained zebrafish were anesthetized with
MESAB and dead cells in heart, CNS, liver, and kidney were visually assessed
using a fluorescence microscope (Zeiss M2Bio fluorescence microscope)
equipped with a rhodamine (red) cube, a FITC (green) filter, and a chilled CCD
camera (AxioCam MRm, Zeiss, NY).
11.2.8 Assessment of Reversibility of Morphology/
Function Organ Toxicity
In order to determine if compound-induced acute toxicity was reversible, after
treatment for 24 h, compounds were removed by washing and incubating zebrafish
in fresh fish water for an additional 48 h, and organ toxicity was reassessed.
11.2.9 NOEC Determination
To determine the concentration that did not induce observable toxicity, for each
condition, zebrafish exhibiting toxicity in any of the four organs were counted and
expressed as percent incidence. Percent incidence and concentration were then
correlated and NOEC was estimated for each drug.