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A market for bisphenol A (BPA) replacement compounds has emerged as a result of restrictions on the use of BPA. Some of these compounds have been detected in the environment; however, little is known about their toxicological properties. In the present study, an avian in vitro toxicogenomic approach was used to compare the effects of 5 BPA alternatives. Cell viability and mRNA expression were compared in primary embryonic hepatocytes of chicken (CEH) and double‐crested cormorant (DCEH) exposed to 4,4′‐(propane‐2,2‐diyl) diphenol (BPA), bis (4‐hydroxyphenyl) methane (BPF), bis (3‐allyl‐4‐hydroxyphenyl) sulfone (TGSH), 7‐bis (4‐hydroxyphenylthio)‐3,5‐dioxaheptane (DD‐70), 2,2‐bis (4‐hydroxyphenyl) hexafluoropropane (BPAF), and 4‐hydroxyphenyl 4‐isoprooxyphenylsulfone (BPSIP). Changes in gene expression were determined using 2 polymerase chain reaction (PCR) arrays: 1) species‐specific ToxChips that contain genes representing toxicologically relevant pathways, and 2) chicken‐specific AestroChip that measures estrogen responsive genes. In CEH and DCEH, BPA alternatives TGSH, DD‐70, and BPAF were most cytotoxic. Some of the replacement compounds changed the expression of genes related to xenobiotic metabolism, bile acid, and cholesterol regulation. The rank order based on the number of genes altered on the chicken ToxChip array was TGSH > DD‐70 > BPAF = BPF > 17β estradiol (E2) > BPSIP > BPA. On the cormorant ToxChip array, BPSIP altered the greatest number of genes. Based on the chicken AestroChip data, BPSIP and BPF were slightly estrogenic. These results suggest that the replacement compounds have comparable or even greater toxicity than BPA and act via different mechanisms. Environ Toxicol Chem 2021;40:1368–1378. © 2021 Her Majesty the Queen in Right of Canada. Reproduced with the permission of the Minister of Environment and Climate Change Canada.

In the present study, we investigated whether the immortalized chicken hepatocellular carcinoma cell line, leghorn male hepatoma (LMH), had a comparable aryl hydrocarbon receptor (AhR) response to primary chicken embryonic hepatocytes (CEHs) when used in a well‐established assay for chemical screening and prioritization. The LMH cells were grown as 2‐dimensional (2D) confluent cells and 3D spheroids to determine the optimal cell culture states for chemical screening. Cytochrome P450 1A4 and 1A5 (CYP1A) activity and gene expression were compared between CEHs and LMH cells grown in 2 culture states following exposure to the dioxin‐like compound 3,3′,4,4′,5‐pentachlorobiphenyl (PCB‐126). The CYP1A activity was measured using the ethoxyresorufin‐O‐deethylase (EROD) assay, and changes in mRNA expression associated with the AhR pathway were determined using a custom‐designed polymerase chain reaction array. Among LMH cell culture states (i.e., 2D vs 3D), EROD induction was observed only in 3D LMH spheroids. Similarly, 3D spheroids had the greatest number of changes in AhR‐related genes compared with confluent cells. Overall, these results suggest that LMH cells grown as 3D spheroids have a metabolic and gene expression profile that is comparable to that of CEH, and may represent a suitable animal‐free alternative for in vitro screening of chemicals. Environ Toxicol Chem 2020;39:1693–1701. © 2020 SETAC

An avian in vitro screening approach was used to determine the effects of 21 bisphenol A (BPA) alternatives. Cytotoxicity and dysregulation of genes associated with estrogen response and other toxicologically relevant pathways evoked by these alternatives were compared with BPA. Most of the BPA alternatives (15/21) were equally or more cytotoxic than BPA in chicken embryonic hepatocytes; variability in cell viability was associated with chemical structure and the log octanol-water partition coefficient (logP) values. A negative linear relationship (r 2 = 0.745; p = 0.49 -07 ; n = 18) was observed between logP and the log median lethal concentration (logLC50) values. The least cytotoxic BPA alternatives elicited the greatest gene dysregulation and, overall, most of the alternatives altered more genes than BPA (measured with a custom polymerase chain reaction array). This overall approach shows promise for use as a screen for hazard-based prioritization of BPA replacement alternatives and to ideally identify those that may be less harmful and/or require additional toxicity testing.

In vitro liver toxicity tests performed using cell lines cultured as two‐dimensional (2D) monolayer have limited CYP450 activity and may be inadequate for screening chemicals that require activation to exert toxicity. Metabolic competence is greatly improved using three‐dimensional (3D) cell culture. In this study, Cyp1a induction, and subsequent DNA damage response induced by benzo(a)pyrene (BaP) were compared in 2D monolayer cells and 3D spheroids of the chicken hepatic cell line, LMH. Cells were exposed to BaP (0.1–100 μM) for different durations: 8, 24, 35, or 48 hr. Cyp1a activity, mRNA expression of Cyp1a and DNA damage response (DDR) genes, and phosphorylation of H2AX (γH2AX) were determined using the EROD assay, a customized PCR array, and flow cytometry, respectively. EROD activity was induced at 8 hr and achieved maximal induction at 24 hr in spheroids; earlier time points than for monolayer cells. In spheroids, BaP exposure resulted in a concentration‐dependent increase in Cyp1a4 mRNA expression at 8 hr followed by upregulation of DDR genes at 24 hr, whereas Cyp1a4 mRNA induction was only observed at 48 hr in monolayer cells. Cyp1a5 mRNA was induced at 8 hr in monolayer cells but maximum induction was greater in spheroids. An increase in γH2AX was observed at 24 hr in spheroids; this endpoint was not evaluated in monolayer cells. These results suggest that BaP metabolism precedes the DNA damage response and occurs earlier in 3D spheroids. This study demonstrates that LMH 3D spheroids could be a suitable metabolically‐competent in vitro model to measure genotoxicity of chemicals that require metabolic activation by Cyp1a.

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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.

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