The worldwide use of chemicals, drugs and other pharmaceuticals is significant. However, they represent toxic pollutants and their presence in the environment seriously endangers human health. Pharmaceutical residues can interact with biological targets and thus exert their different toxic effects at very low concentrations. These often-irreversible interactions triggered serious damages in lipids, proteins, and DNA molecules (Farrugia and Balzan 2012). Our ability to predict toxicological outcomes of exposure to drugs as well as understanding the mechanisms underlying toxicity in biological systems are of significant importance for human health and safety. Saccharomyces cerevisiae is one of the best-explored models of eukaryotic cells for studying cellular mechanisms that occur under stress conditions. The main advantage of using yeasts in drug testing is the possibility to identify sensitive biomarkers and mechanisms of drug-mediated cell toxicity in the cases when the toxic compounds are still unknown. In addition, S. cerevisiae is currently the only system that allows evaluation of all targets in the cell, simultaneously and in vivo (Smith et al. 2010). However, most of the yeast-based toxicological studies involve the use of proliferating Saccharomyces cerevisiae cells (Gasch et al. 2000; Ericson et al. 2008; Dos Santos et al. 2012; Braconi et al. 2016). Since quiescence is the most common cellular state found on Earth, using Saccharomyces cerevisiae in quiescent state will be the more valuable tool for prediction of cellular response to exposure of environmental toxic compounds. Correspondingly, this study compares the toxic response of proliferating and stationary phase (Q and NQ) S. cerevisiae BY4741 cells to Zeocin. With its radiomimetic properties, this compound is known to induce single (Miné-Hattab and Rothstein 2013) and double-strand breaks (Chankova et al. 2007; Kopaskova et al. 2012; Todorova et al. 2015a) as well as basic substitutions in the DNA molecule (Guénolé et al. 2013). Moreover, it has been shown that Zeocin has pro-oxidative, mutagenic, and carcinogenic effects in S. cerevisiae (Todorova et al. 2015b). Taking this into consideration, we investigated the effect of Zeocin (IC₅₀) on the level of intracellular reactive oxygen species (ROS), total intracellular glutathione, oxidized lipids, and proteins as sensor molecules for measuring induced oxidative stress during drug toxicity. The DNA damaging potential of high concentrations Zeocin on proliferating, Q and NQ cells was also estimated.

Used data represent the mean values with Standard error of the mean (±SEM) of three independent experiments. The statistical analysis was performed using MICROSOFT OFFICE 365 EXCEL 2020 software. Differences in means were analysed using Student’s t test with independent measures. Differences were considered statistically significant at the p < 0.05 level.

Currently, the yeast Saccharomyces cerevisiae remains one of the highly important experimental models in the field of toxicogenomics. Studying the biology of yeast cells, especially those in quiescence, could reveal the potential of this microorganism for investigating the cellular response to a particular environment. The evaluation of toxicological response and stress mechanisms in this microorganism could further be helpful in the clarification of equivalent mechanisms in higher eukaryotes. The environmental conditions can change dramatically, which includes progressive depletion of nutrients, rising ambient temperatures, or sudden contamination with xenobiotics. Regardless of their nature, such changes in the environment invariably cause stress to the organisms, to which they must effectively adapt in order to survive. This stress is often associated with ROS accumulation (Avery 2011). Results obtained through this study confirmed that the exposure to Zeocin led to disturbance of the cellular redox balance and induced an increase in the levels of toxic oxygen radicals independently of the growth phase. The fraction of NQ cells probably rapidly lost the ability to divide and accumulated almost 4 times higher levels of ROS (Fig. 1). Meanwhile, the stationary cells which have entered in G₀ cell cycle, showed significantly higher resilience to the effect of Zeocin. The treatment with this toxic chemical led to only a 2-fold increase in the levels of ROS, which was probably due to their specific morphological and physiological properties. It was well-known that S. cerevisiae quiescent cells are characterised by thicken cell walls, condensed chromosomes and increased termo- and osmotolerance (Gray et al. 2004). On the other hand, the NQ cells differ significantly, possessing genomic instability, being easily lysed, and providing nutrients for those cells entering G₀ (Aragon et al. 2008). As regards the logarithmic cells, they are characterised by higher metabolic activity, elevated ROS levels and respectively increased susceptibility to toxic compounds, in that case Zeocin (Cabral et al. 2003).

A direct indicator of the onset of the redox balance disturbance is the appearance of carbonyl groups in the proteins. Oxidative damage to proteins affects the processes maintaining the cellular homeostasis, which compromises their viability (Farrugia and Balzan 2012). In this respect as the Q-cells were characterised by very low metabolic levels and lower ability to sequestered damaged molecules, the observed elevated oxidative modifications in their proteins was not surprising. This might be also due to the coordinated toxic effects of Zeocin, and stress caused by the lack of nutrients in the environment. In NQ cells, the amount of carbonylated proteins after treatment with Zeocin was comparable to that in control cells - 6.56 and 6.06 µM/mg, respectively (Fig. 2). This indicated that here the appearance of oxidized proteins is rather a consequence of the physiological state of the cell population and is not directly resulting from the Zeocin-induced oxidative stress.

The measured higher intracellular concentration of malonaldehyde in Zeocin exposed cells further confirmed that one of the cytotoxic effects of this antibiotic was related to the induction of oxidative stress in the cell. It resulted in impairment of membrane functionality and permeability, probably causing the release of the intracellular content (Hodges et al. 1999). Obviously, when the metabolic activity of the cell is higher and the transport across the membrane - dynamic, the yeast cells are more vulnerable to the action of xenobiotics, including the Zeocin. This eventually explains the measured excess levels of oxidized lipids in the logarithmic cells comparing to the Q and NQ ones (Fig. 3).

To prevent the unbalanced accumulation of ROS and consecutive cellular damages, yeast cells react with specific induction of both non-enzymatic and enzymatic antioxidant defence mechanisms. Key molecule acting as first line of defence against oxidative injuries is the glutathione. That is why the total intracellular amount of this tripeptide is an important parameter for measuring the oxidative stress levels. In this study we confirmed that after exposure to IC₅₀ Zeocin the three types of yeast populations showed increased intracellular levels of glutathione ranging from 1.5-fold (for Q cells) to 2.5-fold (for logarithmic cells) (Fig. 4). Having in mind that in actively proliferating cells the rate of transcription and translation are the highest, it is not surprising that in these yeast populations the biosynthesis of glutathione was augmented with more than 100%. By contrast, in quiescent yeast cells, characterised by very low metabolic profile, the measured increase in glutathione is only about 30%. These findings also correlated with the observed tendency in the intracellular ROS levels of the studied yeasts. Non-quiescent cells had the highest measured concentrations of glutathione possibly related with the excess levels of intracellular ROS in general (Aragon at al. 2008).

Furthermore, this study aimed to assess the potential of Zeocin to induce DNA double-strand breaks (DSB) in dependence to the growth phase. The highest levels of spontaneous DSBs were observed in non-quiescent cell. Such results are not surprising, considering the fact that they represent the fraction of stationary cells having very low ability to reproduce and tendency to enter apoptosis. Such differences in the spontaneous DSB levels being dependent on the growth phase were also observed previously in other strains of S. cerevisiae (Todorova et al. 2015b, 2019). The statistically significant induction of DSB in logarithmic cells after the treatment with concentrations equal or higher than 200 μg/ml Zeocin is in a good correspondence with the one obtained on strain D7ts1 (Todorova et al. 2019). In late stationary cells significantly higher levels of DSBs were detected in comparison with those in exponential and early stationary cells. However, the entrance into G₀ cell cycle considerably affects the levels of DSB, rendering quiescent cells with a low susceptibility to DNA damages. When Zeocin is used as an inducing agent for DSB formation the tendency has been preserved. Most robust to its action were the cells already entered the G₀ state. A possible explanation for their resistance to this radiomimetic chemical could be again assigned to their specific cellular features. As mentioned above they are characterised by thickened cell walls, highly condensed chromosomes and, as a result, restricted ability of the Zeocin to penetrate the cell and reach its target molecule (Gray et al. 2004; Allen et al. 2006).

The comparative analysis of different yeast cell populations – logarithmic, quiescent, and non-quiescent – revealed that the cellular physiological state is a critical factor determining the resistance to xenobiotics. More robust to Zeocin exposure were the quiescent cells showing lower levels of ROS and DSBs. By contrast, the logarithmically grown yeasts are more susceptible to the action of this compound with observed higher damages in DNA, proteins and lipids.

The data underpinning the analysis reported in this paper are deposited at Figshare at https://figshare.com/s/e89e88bfc6f1247a4755.