Abstract
Previously, we showed that the chicken LMH cell line cultured as 3D spheroids may be a suitable animal free alternative to primary chicken embryonic hepatocytes (CEH) for avian in vitro chemical screening. In this study, cytotoxicity and mRNA expression were determined in LMH 3D spheroids following exposure to bisphenol A (BPA), five BPA replacement compounds (BPF, TGSH, DD-70, BPAF, BPSIP), and 17β estradiol (E2). Results were compared to an earlier study that evaluated the same endpoints for these chemicals in CEH. BPA and the replacement compounds had LC50 values ranging from 16.6 to 81.8 μM; DD-70 and BPAF were the most cytotoxic replacements (LC50 = 17.23 ± 4.51 and 16.6 ± 4.78 μM). TGSH and DD-70 modulated the greatest number of genes, although fewer than observed in CEH. Based on the expression of apovitellenin and vitellogenin, BPAF was the most estrogenic compound followed by BPF, BPSIP, and BPA. More estrogen-responsive genes were modulated in LMH spheroids compared to CEH. Concentration-dependent gene expression revealed that DD-70 and BPAF altered genes related to lipid and bile acid regulation. Overall, cytotoxicity and clustering of replacements based on gene expression profiles were similar between LMH spheroids and CEH. In addition to generating novel gene expression data for five BPA replacement compounds in an in vitro avian model, this research demonstrates that LMH spheroids may represent a useful animal free alternative for avian toxicity testing.
Highlights
In recent years, toxicity testing has shifted focus towards mechanistic approaches to characterize the toxicity of the large number of chemicals in the environment (Krewski et al 2009)
Bisphenol A (BPA) can bind to endocrine receptors and interfere with other biological pathways, which led to a ban on use in applications including infant products, food packaging, and thermal paper (Siracusa et al 2018; ECHA 2020)
The production and use of BPA replacement compounds are increasing globally, and they have been detected in various environmental samples (Chen et al 2016), yet their toxic
Summary
Toxicity testing has shifted focus towards mechanistic approaches to characterize the toxicity of the large number of chemicals in the environment (Krewski et al 2009). Primary embryonic hepatocytes are frequently used for avian toxicity testing (Mundy et al 2019; Porter et al 2014). Bisphenol A (BPA) replacement compounds are a group of chemicals for which toxicity data are limited. BPA can bind to endocrine receptors and interfere with other biological pathways, which led to a ban on use in applications including infant products, food packaging, and thermal paper (Siracusa et al 2018; ECHA 2020). Such restrictions have led to the production of “BPA-free” products, many of which contain structural analogs of BPA. The production and use of BPA replacement compounds are increasing globally, and they have been detected in various environmental samples (Chen et al 2016), yet their toxic
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