Sensing chemical-induced genotoxicity and oxidative stress via yeast-based reporter assays using NanoLuc luciferase

Abstract

Mutagens and oxidative agents damage biomolecules, such as DNA; therefore, detecting genotoxic and oxidative chemicals is crucial for maintaining human health. To address this, we have developed several types of yeast-based reporter assays designed to detect DNA damage and oxidative stress. This study aimed to develop a novel yeast-based assay using a codon-optimized stable or unstable NanoLuc luciferase (yNluc and yNluCP) gene linked to a DNA damage- or oxidative stress-responsive promoter, enabling convenient sensing genotoxicity or oxidative stress, respectively. End-point luciferase assays using yeasts with a chromosomally integrated RNR3 promoter (PRNR3)-driven yNluc gene exhibited high levels of chemiluminescence via NanoLuc luciferase and higher fold induction by hydroxyurea than a multi-copy plasmid-based assay. Additionally, the integrated reporter system detected genotoxicity caused by four different types of chemicals. Oxidants (hydrogen peroxide, tert-butyl hydroperoxide, and menadione) were successfully detected through transient expressions of luciferase activity in real-time luciferase assay using yeasts with a chromosomally integrated TRX2 promoter (PTRX2)-linked yNlucCP gene. However, the luciferase activity was gradually induced in yeasts with a multi-copy reporter plasmid, and their expression profiles were notably distinct from those observed in chromosomally integrated yeasts. The responses of yNlucCP gene against three oxidative chemicals, but not diamide and zinc oxide suspension, were observed using chromosomally integrated reporter yeasts. Given that yeast cells with chromosomally integrated PRNR3-linked yNluc and PTRX2-linked yNlucCP genes express strong chemiluminescence signals and are easily maintained and handled without restrictive nutrient medium, these yeast strains with NanoLuc reporters may prove useful for screening potential genotoxic and oxidative chemicals.

Fig 1. Response of ^PRNR3-linked yNluc gene in two reporter strains after hydroxyurea (HU) treatment.

The luciferase-derived luminescence in yeast cells carrying a multi-copy reporter plasmid (A) and a chromosomally integrated reporter gene (B) were measured after exposure to various concentrations of HU for 24 h. The luminescence intensities were normalized using the A₆₀₀ value and plotted against the HU concentrations with the standard deviations. The fold inductions in two yeast strains are shown in (C). * Statistically significant (p < 0.01, Student’s t-test). The raw dataset for Fig 1 and statistical analysis are shown in S3 and S4 Tables, respectively.

Fig 2. Nluc activity and fold induction in genotoxicant-treated yeast cells with a chromosomally integrated ^PRNR3-yNluc gene.

The luminescence intensity was normalized by the cell density and fold induction in the integrated reporter yeasts after exposure to the indicated concentrations of methyl methanesulfonate (MMS) (A, E), phleomycin (Phl) (B, F), mitomycin C (MMC) (C, G), and camptothecin (CPT) (D, H). The raw dataset for Fig 2 is shown in S5 Table.

Fig 3. Response of ^PTRX2-yNlucCP gene in yeast after exposure to hydrogen peroxide (H₂O₂).

The luminescence intensities in yeast cells carrying a multi-copy reporter plasmid (A) and a chromosomally integrated reporter gene (B) were measured in the presence of H₂O_2; the normalized intensity is shown with the standard deviations. (C) Fold inductions calculated from the activity of a panel (B) were plotted against the culture period. (D) The relative maximal luciferase activity was calculated as the percentage of each peak activity divided by the highest peak activity and plotted against the concentrations as shown in panel b with the standard deviations. The raw dataset for Fig 3 is shown in S6 Table.

Fig 4. Response of a chromosomally integrated ^PTRX2-yNlucCP gene in yeast after exposure to tert-butyl hydroperoxide (t-BHP).

The luminescence intensities in yeast cells carrying a multi-copy reporter plasmid (A) and a chromosomally integrated reporter gene (B) were measured in the presence of the indicated concentrations of t-BHP. (C) Fold inductions calculated from the activity of a panel (B) are plotted against the culture period. (D) The relative maximal activities in yeasts treated with the indicated concentrations of t-BHP were calculated from the activity of a panel (B) and plotted against the concentrations with the standard deviations. The raw dataset for Fig 4 is shown in S7 Table.

Fig 5. Response of a chromosomally integrated ^PTRX2-yNlucCP gene in yeast exposed to menadione.

The luminescence intensities in yeast cells carrying a multi-copy reporter plasmid (A) and a chromosomally integrated reporter gene (B) were measured in the presence of the indicated concentrations of menadione. (C) The fold inductions were calculated by the activity of a panel (B) during the culture period. (D) The relative maximal activities in yeasts treated with the indicated concentration of menadione were determined by the activity of a panel (B) and plotted against the concentrations with the standard deviations. (E) and (F) Fold induction and relative maximal activity plots, respectively, obtained from another experiment. The raw dataset for Fig 5 is shown in S8 Table.

Fig 6. Response of a chromosomally integrated ^PTRX2-yNlucCP gene in yeast cells treated with three chemicals.

Normalized luciferase activities (A, D, G), fold inductions (B, E, H), and relative maximal activities (C, F, I) in yeasts after exposure to t-BHP (A–C), diamide (D–F), and zinc oxide suspension (G–I). The raw dataset for Fig 6 is shown in S9 Table.