Hepato(Geno)Toxicity Assessment of Nanoparticles in a HepG2 Liver Spheroid Model

Abstract

1) In compliance with the 3Rs policy to reduce, refine and replace animal experiments, the development of advanced in vitro models is needed for nanotoxicity assessment. Cells cultivated in 3D resemble organ structures better than 2D cultures. This study aims to compare cytotoxic and genotoxic responses induced by titanium dioxide (TiO 2 ), silver (Ag) and zinc oxide (ZnO) nanoparticles (NPs) in 2D monolayer and 3D spheroid cultures of HepG2 human liver cells. (2) NPs were characterized by electron microscopy, dynamic light scattering, laser Doppler anemometry, UV-vis spectroscopy and mass spectrometry. Cytotoxicity was investigated by the alamarBlue assay and confocal microscopy in HepG2 monolayer and spheroid cultures after 24 h of NP exposure. DNA damage (strand breaks and oxidized base lesions) was measured by the comet assay. (3) Ag-NPs were aggregated at 24 h, and a substantial part of the ZnO-NPs was dissolved in culture medium. Ag-NPs induced stronger cytotoxicity in 2D cultures (EC 50 3.8 µg/cm 2 ) than in 3D cultures (EC 50 > 30 µg/cm 2 ), and ZnO-NPs induced cytotoxicity to a similar extent in both models (EC 50 10.1-16.2 µg/cm 2 ). Agand ZnO-NPs showed a concentration-dependent genotoxic effect, but the effect was not statistically significant. TiO 2 -NPs showed no toxicity (EC 50 > 75 µg/cm 2 ). (4) This study shows that the HepG2 spheroid model is a promising advanced in vitro model for toxicity assessment of NPs.Nanomaterials 2020, 10, 545 2 of 21 and silver (Ag) is used as a disinfection agent in medical equipment and consumer products, on account of its antimicrobial activity [5]. Thus, humans are likely to be exposed to NPs, either intentionally or accidentally, during production and usage [6]. Transport of NPs across biological barriers has been observed by elemental analysis in both rodents and humans [7][8][9][10][11]. As an example, gold NPs have been reported to reach the systemic circulation in humans, after inhalation, and translocate to other organs [8,9].Several in vivo studies show that NPs accumulate in the liver, which is an important target organ for NPs and other xenobiotics due to its metabolic activity [12][13][14][15][16][17][18]. Induction of hepatotoxicity is one of the most common reasons for a medicine to be rejected or removed from the market [19,20]. Therefore, there is a need for sensitive hepatotoxicity screening methods for drug development and hazard assessment of chemicals or new materials, such as NPs. When considering the 3Rs-replacement, reduction and refinement-to minimize the use of animal experiments, hepatotoxicity should be assessed by reliable in vitro models. A great advantage of in vitro hepatocellular models for studying hepatotoxicity is the possibility of using human cells, either as primary cells or cell lines. The use of human hepatocyte cell lines, such as HepG2, C3A, Huh7 and HepaRG, has many advantages compared to primary cells. They are relatively easy to culture and have an unlimited life span, a relatively stable phenotype, high availability and ...

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

“…The literature search resulted in 59 papers for “genotoxicity & 3D in vitro model”, 73 papers for “genotoxicity & advanced in vitro model”, 134 papers for “genotoxicity & high throughput”, eight papers for “genotoxicity & high throughput & nanomaterials” [ 15 , 16 , 17 , 18 , 19 , 20 , 21 ] and six papers for “genotoxicity & high throughput & nanoparticles” (4 are the same as for “genotoxicity & high throughput & nanomaterials”) [ 16 , 17 , 18 , 19 , 21 , 22 ], seven papers for “genotoxicity & organ on chip“ [ 23 , 24 , 25 , 26 , 27 , 28 , 29 ], seven papers for “genotoxicity & 3D models & nanoparticles” [ 30 , 31 , 32 , 33 , 34 , 35 , 36 ], 11 papers for “genotoxicity & 3D models & nanomaterials” [ 30 , 31 , 33 , 36 , 37 , 38 , 39 , 40 , 41 ] for the period of 20 years (2000–2020), whereby the year 2020 was considered only from January to August. ( Table 1 ).…”

“…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 ].…”

“…Wills et al [ 32 ] performed the RSMN with silica nanoparticles and the EpiDerm™ model and came to the conclusion that robust exposure characterization and uptake assessment methods are crucial to interpret nano(geno)toxicity studies successfully. However, 3D tissue-based assays definitely provide a more realistic test system to study the genotoxic potential of NMs, compared to 2D test systems [ 10 , 32 , 34 ].…”

From Basic Research to New Tools and Challenges for the Genotoxicity Testing of Nanomaterials

“…This issue contains mostly data from in vitro studies. In addition to classical cell lines such as hamster and human fibroblasts [ 1 , 2 ] or human lung cells [ 3 , 4 ], new models have been also used for investigating nanomaterials genotoxicity: ToxTracker reporter cell lines [ 5 ], human amniotic cells [ 6 ] and 3D HepG2 spheroids [ 7 ]. In this issue, three papers also deal with in vivo studies: on the plant Allium cepa [ 8 ], on the tadpoles of Xenopus laevis [ 9 ] and on rats [ 10 ].…”

“…Among the various assays available for investigating genotoxicity and mutagenicity, most of the papers used the comet assay to visualize the DNA fragmentation both in in vitro [ 3 , 4 , 6 , 7 ] and in vivo [ 10 ] studies. The micronucleus assay to detect chromosome and genome mutations was also largely performed in cell lines [ 2 , 4 ] as well as in whole organisms [ 8 , 9 , 10 ].…”