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Updated: Jun 25, 2025
Using Alizarin Red Staining to Detect Chemically Induced Bone Loss in Zebrafish Larvae
Published on: December 28, 2021
Biological variability hampers the use of skeletal staining methods in zebrafish embryo developmental toxicity assays
Jente Hoyberghs1, Jonathan Ball2, Maciej Trznadel2
1University of Antwerp, Wilrijk, Belgium.
Abstract:
Zebrafish embryo assays are used by pharmaceutical and chemical companies as new approach methodologies (NAMs) in developmental toxicity screening. Despite an overall high concordance of zebrafish embryo assays with in vivo mammalian studies, false negative and false positive results have been reported. False negative results in risk assessment models are of particular concern for human safety, as developmental anomalies may be missed. Interestingly, for several chemicals and drugs that were reported to be false negative in zebrafish, skeletal findings were noted in the in vivo studies. As the number of skeletal endpoints assessed in zebrafish is very limited compared to the in vivo mammalian studies, the aim of this study was to investigate whether the sensitivity could be increased by including a skeletal staining method. Three staining methods were tested on zebrafish embryos that were exposed to four teratogens that caused skeletal anomalies in rats and/or rabbits and were false negative in zebrafish embryo assays. These methods included a fixed alizarin red-alcian blue staining, a calcein staining, and a live alizarin red staining. The results showed a high variability in staining intensity of larvae exposed to mammalian skeletal teratogens, as well as variability between control larvae originating from the same clutch of zebrafish. Hence, biological variability in (onset of) bone development in zebrafish hampers the detection of (subtle) treatment-related bone effects that are not picked-up by gross morphology. In conclusion, the used skeletal staining methods did not increase the sensitivity of zebrafish embryo developmental toxicity assays.
Insights
Skeletal staining methods did not improve the accuracy of zebrafish embryo assays for detecting developmental toxicity. Biological variability in zebrafish bone development hinders the identification of subtle drug effects, impacting human safety risk assessment.
Area of Science:
- Toxicology
- Developmental Biology
- Drug Safety
Background:
- Zebrafish embryo assays are New Approach Methodologies (NAMs) for developmental toxicity screening in pharmaceutical and chemical industries.
- While generally concordant with mammalian studies, false negatives in zebrafish assays pose risks to human safety by potentially missing developmental anomalies.
- Skeletal findings in mammalian studies of false-negative zebrafish assays suggest a need to assess skeletal endpoints more thoroughly.
Purpose of the Study:
- To determine if incorporating skeletal staining methods can enhance the sensitivity of zebrafish embryo assays in detecting developmental toxicity.
- To investigate the utility of alizarin red-alcian blue, calcein, and live alizarin red staining in identifying skeletal effects of known teratogens.
Main Methods:
- Zebrafish embryos were exposed to four teratogens known to cause skeletal anomalies in mammals but were false negatives in prior zebrafish assays.
- Three skeletal staining techniques were applied: fixed alizarin red-alcian blue, calcein staining, and live alizarin red staining.
- Staining intensity and developmental effects were evaluated in treated and control zebrafish larvae.
Main Results:
- High variability in staining intensity was observed in larvae exposed to teratogens and even in control larvae from the same clutch.
- This biological variability in zebrafish bone development onset and expression interfered with detecting subtle, treatment-related skeletal effects.
- The tested skeletal staining methods did not improve the detection sensitivity compared to gross morphological assessment.
Conclusions:
- Skeletal staining methods, as applied, did not increase the sensitivity of zebrafish embryo assays for developmental toxicity.
- Biological variability in zebrafish skeletal development is a significant challenge for detecting subtle teratogenic effects.
- Current skeletal staining approaches do not fully resolve the issue of false negatives in zebrafish developmental toxicity screening.
