Evaluation of the Aryl Hydrocarbon Receptor Response in LMH 3D Spheroids

Abstract

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

Concentration‐ and time‐dependent induction of Cyp1a and DNA damage response by benzo(a)pyrene in LMH three‐dimensional spheroids

“…Cyp1a activity and upregulation of Cyp1a4 or Cyp1a5 mRNA expression were the only significant effects detected at 8 hr in both 2D and 3D cultures. In a previous study, LMH spheroids had higher Cyp1a activity, and Cyp1a4 and Cyp1a5 expression than 2D monolayer cells following exposure to PCB‐126 (Sharin et al ., 2020). CYP1A1 expression was 14‐fold higher in HepG2 spheroids compared to 2D monolayer cells after 24 hr BaP exposure (Stampar et al ., 2019).…”

“…The LMH cell line (CRL‐2117) was acquired from Cedarlane (Burlington, ON) and cultured as described elsewhere (Sharin et al ., 2020). For 3D spheroids, 10,000 cells in 250 μL of media were added to each well of ultra‐low attachment (ULA) round bottom 96 well microplates (Corning, Corning, NY).…”

“…Cyp1a activity was determined using the EROD assay as described previously (Sharin et al ., 2020). The presence of the EROD product, resorufin, was measured at an excitation wavelength of 530 nm and an emission wavelength of 590 nm.…”

“…3D cell culturing techniques, such as growing cells as spheroids, enhances intercellular interactions and better represents in vivo conditions (Edmondson et al ., 2014). Even immortalized hepatocytes, which typically have poor metabolic activity, have improved metabolic competence when cultured as spheroids, including increased CYP450 and other metabolizing enzyme activity (Gunness et al ., 2013; Ramaiahgari et al ., 2017; Lammel et al ., 2019; Sharin et al ., 2020).…”

“…The immortalized chicken hepatic cell line, LMH, is an alternative, animal‐free in vitro model for screening chemicals for avian toxicity testing. In a previous study, LMH cells cultured as 3D spheroids had improved Cyp1a activity and corresponding Cyp1a4 and Cyp1a5 mRNA expression along with the altered expression of additional aryl hydrocarbon receptor (AhR)‐regulated genes (Sharin et al ., 2020). The objectives of the present study were to: (a) determine if Cyp1a metabolism‐dependent genotoxic endpoints could be detected in LMH cells cultured as 2D monolayers or 3D spheroids exposed to BaP and; (b) evaluate whether LMH 3D spheroids could be a suitable alternative to CEH for avian genotoxicity testing.…”