Environment and Climate Change Canada, National Wildlife Research Centre, Ottawa, Ontario, Canada.
Department of Biology, University of Ottawa, Ottawa, Ontario, Canada.
Environ Mol Mutagen. 2021 Jun;62(5):319-327. doi: 10.1002/em.22433. Epub 2021 Jun 1.
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.
体外肝毒性试验使用二维（2D）单层培养的细胞系进行，CYP450 活性有限，可能不足以筛选需要激活才能发挥毒性的化学物质。使用三维（3D）细胞培养可大大提高代谢能力。在这项研究中，比较了二维单层细胞和鸡肝细胞系 LMH 的 3D 球体中苯并（a）芘（BaP）诱导的 Cyp1a 诱导和随后的 DNA 损伤反应。细胞暴露于 BaP（0.1-100 μM）不同时间：8、24、35 或 48 小时。使用 EROD 测定法、定制的 PCR 阵列和流式细胞术分别测定 Cyp1a 活性、Cyp1a 和 DNA 损伤反应（DDR）基因的 mRNA 表达以及 H2AX 的磷酸化（γH2AX）。在球体中，8 小时即可诱导 EROD 活性，24 小时达到最大诱导；比单层细胞更早。在球体中，BaP 暴露导致 Cyp1a4 mRNA 表达在 8 小时呈浓度依赖性增加，随后在 24 小时上调 DDR 基因，而在单层细胞中仅在 48 小时观察到 Cyp1a4 mRNA 诱导。单层细胞中 Cyp1a5 mRNA 在 8 小时诱导，但球体中最大诱导更大。在球体中 24 小时观察到 γH2AX 增加；在单层细胞中未评估该终点。这些结果表明，BaP 代谢先于 DNA 损伤反应，并在 3D 球体中更早发生。这项研究表明，LMH 3D 球体可能是一种合适的代谢能力体外模型，可用于测量需要 Cyp1a 代谢激活的化学物质的遗传毒性。
