Development of in vitro 3D cell model from hepatocellular carcinoma (HepG2) cell line and its application for genotoxicity testing

Štampar, Martina; Tomc, Jana; Filipič, Metka; Žegura, Bojana

doi:10.1007/s00204-019-02576-6

Cited by 47 publications

(50 citation statements)

References 61 publications

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“…For genotoxicity assessment, robust protocols for 3D models have been established for skin, airways and liver tissue equivalents [ 10 , 197 ]. Many of the 3D cell culture systems applied in genotoxicity testing (of NMs) have been spheroids, such as liver spheroids constructed from primary hepatocytes, HepG2 hepatocellular carcinoma cells or the HepaRG cell line applied to the comet assay [ 14 , 31 , 34 , 198 , 199 ] and micronucleus assay [ 200 ]. Human 3D airway models usually consist of a functional and differentiated respiratory epithelium with cilia and mucus and are cultured on the physiological relevant air-liquid-interface (ALI) [ 19 ].…”

Section: New Advanced In Vitro Models (3d Organ-on-a-chip)mentioning

confidence: 99%

Genotoxicity of Nanomaterials: Advanced In Vitro Models and High Throughput Methods for Human Hazard Assessment—A Review

et al. 2020

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Changes in the genetic material can lead to serious human health defects, as mutations in somatic cells may cause cancer and can contribute to other chronic diseases. Genotoxic events can appear at both the DNA, chromosomal or (during mitosis) whole genome level. The study of mechanisms leading to genotoxicity is crucially important, as well as the detection of potentially genotoxic compounds. We consider the current state of the art and describe here the main endpoints applied in standard human in vitro models as well as new advanced 3D models that are closer to the in vivo situation. We performed a literature review of in vitro studies published from 2000–2020 (August) dedicated to the genotoxicity of nanomaterials (NMs) in new models. Methods suitable for detection of genotoxicity of NMs will be presented with a focus on advances in miniaturization, organ-on-a-chip and high throughput methods.

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Section: New Advanced In Vitro Models (3d Organ-on-a-chip)mentioning

confidence: 99%

Genotoxicity of Nanomaterials: Advanced In Vitro Models and High Throughput Methods for Human Hazard Assessment—A Review

et al. 2020

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“…Traditional two-dimensional (2D) cell culture models and in vivo animal models are commonly used to evaluate nanotoxicity; however, 2D models do not accurately predict in vivo toxicity and biological effects due to the simple element and lack of a microenvironment, 14,15 and animal models have a complex physiological structure that renders it difficult to elucidate molecular mechanisms. Therefore, it is necessary to develop a novel model for the rapid assessment of nanotoxicity and investigation into the toxic mechanisms of nanoparticles.…”

Section: Introductionmentioning

confidence: 99%

“…Three-dimensional (3D) culture models are a promising alternative, since they provide cell−cell and cell−matrix interactions that better mimic the physiological in vivo aspects. [15][16][17] Human osteoblast-like spheroids treated with nano-TiO 2 have been shown to augment collagen deposition and proinflammatory cytokine and growth factor secretion, although no significant differences in morphology, proliferation, or cell cycle have been observed. 16 Nevertheless, the molecular mechanisms underlying the osteoblast spheroid alterations caused by nano-TiO 2 are not well understood.…”

Section: Introductionmentioning

confidence: 99%

<p>Involvement of TGF-β and ROS in G1 Cell Cycle Arrest Induced by Titanium Dioxide Nanoparticles Under UVA Irradiation in a 3D Spheroid Model</p>

Ren

Geng

et al. 2020

IJN

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Background: As one of the most widely produced engineered nanomaterials, titanium dioxide nanoparticles (nano-TiO 2 ) are used in biomedicine and healthcare products, and as implant scaffolds; therefore, the toxic mechanism of nano-TiO 2 has been extensively investigated with a view to guiding application. Three-dimensional (3D) spheroid models can simplify the complex physiological environment and mimic the in vivo architecture of tissues, which is optimal for the assessment of nano-TiO 2 toxicity under ultraviolet A (UVA) irradiation. Methods and Results: In the present study, the toxicity of nano-TiO 2 under UVA irradiation was investigated in 3D H22 spheroids cultured in fibrin gels. A significant reduction of approximately 25% in spheroid diameter was observed following treatment with 100 μg/mL nano-TiO 2 under UVA irradiation after seven days of culture. Nano-TiO 2 under UVA irradiation triggered the initiation of the TGF-β/Smad signaling pathway, increasing the expression levels of TGF-β1, Smad3, Cdkn1a, and Cdkn2b at both the mRNA and protein level, which resulted in cell cycle arrest in the G1 phase. In addition, nano-TiO 2 under UVA irradiation also triggered the production of reactive oxygen species (ROS), which were shown to be involved in cell cycle regulation and the induction of TGF-β1 expression. Conclusion: Nano-TiO 2 under UVA irradiation induced cell cycle arrest in the G1 phase and the formation of smaller spheroids, which were associated with TGF-β/Smad signaling pathway activation and ROS generation. These results reveal the toxic mechanism of nano-TiO 2 under UVA irradiation, providing the possibility for 3D spheroid models to be used in nanotoxicology studies.

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“…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). In LMH monolayer cells and spheroids, upregulation of Cyp1a4 and Cyp1a5 occurred before the expression of cell cycle and DNA damage response genes (Tables 1 and 2), suggesting that BaP is metabolized prior to DNA damage.…”

Section: Discussionmentioning

confidence: 99%

“…Markers of DNA damage like γH2AX accumulate in S‐phase cells in response to genotoxicants (Friesner et al ., 2005). DSBs were observed after 24 hr exposure to BaP in HepG2 cells cultured as 3D spheroids and 2D monolayers; however, the concentration of BaP required to induce DSBs in spheroids was lower than monolayer cells (Stampar et al ., 2019). Similar to the current study, BaP treatment in HepaRG and HepG2 cells resulted in an increase in γH2AX at 24 hr, which decreased at 48 hr (van Delft et al ., 2010; Quesnot et al ., 2016).…”

Section: Discussionmentioning

confidence: 99%

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

Sharin

Gyasi

Jones

et al. 2021

Environ and Mol Mutagen

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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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Development of in vitro 3D cell model from hepatocellular carcinoma (HepG2) cell line and its application for genotoxicity testing

Cited by 47 publications

References 61 publications

Genotoxicity of Nanomaterials: Advanced In Vitro Models and High Throughput Methods for Human Hazard Assessment—A Review

Genotoxicity of Nanomaterials: Advanced In Vitro Models and High Throughput Methods for Human Hazard Assessment—A Review

<p>Involvement of TGF-β and ROS in G1 Cell Cycle Arrest Induced by Titanium Dioxide Nanoparticles Under UVA Irradiation in a 3D Spheroid Model</p>

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

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