Adaptation of the in vitro micronucleus assay for genotoxicity testing using 3D liver models supporting longer-term exposure durations

Abstract

Following advancements in the field of genotoxicology, it has become widely accepted that 3D models are not only more physiologically relevant but also have the capacity to elucidate more complex biological processes that standard 2D monocultures are unable to. Whilst 3D liver models have been developed to evaluate the short-term genotoxicity of chemicals, the aim of this study was to develop a 3D model that could be used with the regulatory accepted in vitro micronucleus (MN) following low-dose, longer-term (5 days) exposure to engineered nanomaterials (ENMs). A comparison study was carried out between advanced models generated from two commonly used liver cell lines, namely HepaRG and HepG2, in spheroid format. While both spheroid systems displayed good liver functionality and viability over 14 days, the HepaRG spheroids lacked the capacity to actively proliferate and, therefore, were considered unsuitable for use with the MN assay. This study further demonstrated the efficacy of the in vitro 3D HepG2 model to be used for short-term (24 h) exposures to genotoxic chemicals, aflatoxin B1 (AFB1) and methyl-methanesulfonate (MMS). The 3D HepG2 liver spheroids were shown to be more sensitive to DNA damage induced by AFB1 and MMS when compared to the HepG2 2D monoculture. This 3D model was further developed to allow for longer-term (5 day) ENM exposure. Four days after seeding, HepG2 spheroids were exposed to Zinc Oxide ENM (0–2 µg/ml) for 5 days and assessed using both the cytokinesis-block MN (CBMN) version of the MN assay and the mononuclear MN assay. Following a 5-day exposure, differences in MN frequency were observed between the CBMN and mononuclear MN assay, demonstrating that DNA damage induced within the first few cell cycles is distributed across the mononucleated cell population. Together, this study demonstrates the necessity to adapt the MN assay accordingly, to allow for the accurate assessment of genotoxicity following longer-term, low-dose ENM exposure.

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 [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).…”

Understanding the impact of more realistic low-dose, prolonged engineered nanomaterial exposure on genotoxicity using 3D models of the human liver

“…To assess this, a cell-line based 3D in vitro HepG2 spheroid model able to evaluate cytotoxicity, (pro-)inflammatory response and genotoxicity associated with both acute and longer-term (≤ 10 days) ENM exposure upon the liver was utilised. In this system, hepatocyte spheroids recapitulated basic in vivo hepatic functions and structure, whilst maintaining specific parameters required for multiple biochemical endpoint testing [ 28 , 29 ]. Applying this liver model, a range of five different ENMs were evaluated, across a low concentration range of 0.2–10.0 µg/mL, to determine whether the individual physico-chemical characteristics would elicit a different biological response following either an acute (24 h) or longer-term (120 h) exposure scenario in vitro.…”

“…This is further supported with evidence that the microtissues were shown to rotate within the wells, and thus ENM exposure is likely to be even across the surface of the spheroids [ 14 ]. Similarly, with the addition of an agarose coating at the base of the spheroids, the HepG2 spheroids are also free to move and rotate within the well enabling the ENMs to access the entire surface layer of actively proliferating HepG2 cells [ 28 ].…”

“…viability, hepatic functionality, metabolic activity and pro-inflammatory response, they do not actively proliferate and so cannot be used for genotoxicity testing which often requires proliferating cells [ 14 ]. To overcome this, a proliferating cell-line based 3D in vitro HepG2 spheroid model has been developed, which can be utilised to evaluate multiple toxicological endpoints including genotoxicity [ 28 , 29 ]. HepG2 cells have shown phenotypic secretion of hepatic plasma proteins (e.g.…”

In Vitro Three‐Dimensional Liver Models for Nanomaterial DNA Damage Assessment

“…highlighted this when assessing the suitability of HepaRG and HepG2 cell lines to support the CBMN assay, whereby HepaRG, regardless of cytochalasin B concentration and exposure time, only yields a binucleate frequency of <10% whilst HepG2 exhibited a binucleate frequency of >30%. [ 70,73,83 ] As a result, DNA damage and genotoxicity assessment are usually performed using alternative methods such as the comet assay or integrated GFP‐reporters. However, recently the CBMN assay has been successfully used to detect DNA damage and genotoxicity in 3D HepG2 hanging drop spheroids following acute exposure to aflatoxin B1 and benzopyrene.…”