Transcriptomic and physiological analysis of endocrine disrupting chemicals Impacts on 3D Zebrafish liver cell culture system

In recent decades, extensive efforts have focused on developing in vitro platforms mimicking fish livers to better understand the acute or chronic effects of toxicants on lower aquatic vertebrates. Fish liver cell lines have emerged as a promising culture system for these in vitro platforms because they complement the currently limited in vitro tools that mostly consist of mammalian cell lines and adhere to the 3Rs: replacement, reduction, and refinement of living animal tests. However, monolayer cell lines have lower transcriptional and physiological responses upon exposure to toxic chemicals than freshly isolated primary cells. To overcome this challenge, we utilized a three-dimensional (3D) spheroid-based in vitro platform, in which hepatocyte cells had self-organized into spheroid forms via E-cadherin bonds. This platform exhibited augmented transcriptomic and phenotypic regulation of liver cells in comparison to monolayer cells. We examined the organoid platform using the zebrafish liver (ZFL) cell line as a model system. ZFL cells spontaneously clustered into 3D spheroids with long-term viability by optimizing cell seeding density on a non-adherent substrate. Interestingly, 3D ZFL spheroids treated with estrogenic chemicals were activated to synthesize a higher level of vitellogenin (Vtg) than monolayer cells. Whole-transcriptome sequencing analysis confirmed that 3D ZFL spheroids had greater transcriptional regulation of genes related to reproductive toxicological response and liver functions, such as the urea cycle, estrogen receptors, and vitellogenin, compared to monolayer cells. These results may contribute to the engineering of novel 3D in vitro platforms for screening harmful chemicals and improving understanding of the underlying liver toxicity mechanisms at the molecular and cellular levels.

Automated summary

In recent years, there have been efforts to develop in vitro platforms that mimic fish livers in order to better understand the effects of toxicants on aquatic vertebrates. Fish liver cell lines have emerged as a promising culture system in these platforms, as they complement existing in vitro tools and adhere to the 3Rs principles of animal testing. However, monolayer cell lines have shown lower transcriptional and physiological responses to toxic chemicals compared to freshly isolated primary cells. To overcome this challenge, a three-dimensional (3D) spheroid-based in vitro platform was utilized, which showed enhanced regulation of liver cells compared to monolayer cells. This platform was examined using the zebrafish liver (ZFL) cell line as a model system, and the 3D ZFL spheroids demonstrated increased synthesis of vitellogenin (Vtg) when treated with estrogenic chemicals. Transcriptome sequencing analysis confirmed that the 3D ZFL spheroids had greater transcriptional regulation of genes related to reproductive toxicological response and liver functions compared to monolayer cells. These findings contribute to the development of novel 3D in vitro platforms for screening harmful chemicals and improving understanding of liver toxicity mechanisms.