Martina Štampar (Author), Sonja Žabkar (Author), Metka Filipič (Author), Bojana Žegura (Author)

Abstract

3D spheroids developed from HepG2 cells were used as a biosensor-like system for the detection of (geno)toxic effects induced by chemicals. Benzo(a)pyrene (B(a)P) and amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) with well-known mechanisms of action were used for system validation. HepG2 spheroids grown for 3 days were exposed to BaP and PhIP for 24 and 72 h. The growth and viability of spheroids were monitored by planimetry and Live/Dead staining of cells. Multi-parametric flow cytometric analysis was applied for simultaneous detection of specific end-effects including cell cycle analysis (Hoechst staining), cell proliferation (KI67 marker), and DNA double-strand breaks (ℽH2AX) induced by genotoxic compounds. Depending on the exposure concentration/time, BaP reduced spheroid growth, affected cell proliferation by arresting cells in S and G2 phase and induced DNA double-strand breaks (DSB). Simultaneous staining of ℽH2AX formation and cell cycle analysis revealed that after BaP (10 μM; 24 h) exposure 60% of cells in G0/G1 phase had DNA DSB, while after 72 h only 20% of cells contained DSB indicating efficient repair of DNA lesions. PhIP did not influence the spheroid size whereas accumulation of cells in the G2 phase occurred after both treatment times. The evaluation of DNA damage revealed that at 200 μM PhIP 50% of cells in G0/G1 phase had DNA DSB, which after 72-h exposure dropped to 40%, showing lower repair capacity of PhIP-induced DSB compared to BaP-induced. The developed approach using simultaneous detection of several parameters provides mechanistic data and thus contributes to more reliable genotoxicity assessment of chemicals as a high-content screening tool.

Keywords

biokemija;celični model in vitro 3D;HepG2;celični cikel;in vitro 3D cell model;flow cytometry;cell cycle;proliferation;DNA strand;breaks;

Data

Language: English
Year of publishing:
Typology: 1.01 - Original Scientific Article
Organization: NIB - National Institute of Biology
UDC: 577
COBISS: 84412419 Link will open in a new window
ISSN: 0045-6535
Views: 26
Downloads: 20
Average score: 0 (0 votes)
Metadata: JSON JSON-RDF JSON-LD TURTLE N-TRIPLES XML RDFA MICRODATA DC-XML DC-RDF RDF

Other data

Secondary language: Slovenian
Secondary keywords: biokemija;celični model in vitro 3D;HepG2;celični cikel;
Source comment: Št. članka: 132805;
Pages: str. 1-11
Volume: ǂVol. ǂ291
Issue: ǂpt. ǂ1
Chronology: 2022
DOI: 10.1016/j.chemosphere.2021.132805
ID: 24536859