Quiescent Yeast Cells Prove Most Vulnerable to Oxidative Stress from Menadione

Quiescent cells — the dormant, non-dividing state that most cells in human tissues occupy for much of their lives — have long been difficult to study, yet a new study suggests they may be far more vulnerable to a common oxidative stressor than their actively dividing counterparts. Using baker’s yeast, Saccharomyces cerevisiae, researchers compared how three distinct cell populations respond to menadione, a synthetic form of vitamin K and a well-known inducer of oxidative stress. The findings, published in the journal BioRisk, reveal a striking asymmetry: quiescent cells suffered the greatest lethality and the only measurable DNA damage, while actively dividing cells mounted a stronger biochemical defense.

The research team, working with the standard laboratory haploid strain BY4741, separated yeast cultures into logarithmically growing (log) cells, quiescent (Q) cells, and non-quiescent (NQ) cells. Quiescent and non-quiescent fractions were isolated from stationary-phase populations grown for 168 hours by layering the biomass on a Percoll density gradient and centrifuging it. The denser lower fraction contained rounded G0 cells with thickened cell walls and no budding — hallmarks of true quiescence — while the upper fraction held a heterogeneous mix of budding, elliptical, and deformed non-quiescent cells. This separation allowed the investigators to interrogate each population independently, an approach that matters because quiescence is believed to function through mechanisms and gene sets shared with mammalian and human cells.

Menadione, chemically 2-methyl-1,4-naphthoquinone, was applied across a concentration range of 1 to 200 micromolar for 60 minutes at 30 degrees Celsius. The researchers then calculated two lethality benchmarks — the doses killing 20 percent (LD20) and 50 percent (LD50) of each population. The contrast was dramatic. Log cells tolerated menadione with an LD20 of 35 micromolar and an LD50 of 199 micromolar. Quiescent cells, by comparison, succumbed at an LD20 of just 0.65 micromolar and an LD50 of only 9 micromolar — roughly a 22-fold difference in the dose required to kill half the population. Survival curves showed a dose-dependent decline in both populations, but the effect was far more pronounced in the quiescent fraction.

To understand whether this heightened susceptibility translated into actual genetic damage, the team employed constant field gel electrophoresis (CFGE), a technique that detects DNA double-strand breaks by quantifying the fraction of DNA released from the wells of a gel. The results confirmed the survival data in a specific and telling way: dose-dependent double-strand break induction appeared only in quiescent cells. After treatment with 150 micromolar menadione, the level of DNA released was 1.5-fold higher than spontaneous levels. In contrast, neither log nor non-quiescent cells showed any measurable increase in double-strand breaks, even at the same concentrations. This selectivity is significant because double-strand breaks are among the most dangerous forms of DNA damage, capable of triggering mutations, chromosomal rearrangements, or cell death if left unrepaired.

The biochemical analysis painted a complementary picture of why quiescent cells fare so poorly. Reactive oxygen species (ROS) levels in quiescent cells rose approximately three-fold above untreated controls after menadione exposure, while the effect on log cells was statistically detectable but biologically negligible. Superoxide dismutase (SOD), a key enzymatic antioxidant that converts superoxide radicals into less harmful species, was also significantly induced in quiescent cells after treatment — another sign that these cells were experiencing genuine oxidative assault. Catalase levels, by contrast, remained unchanged across all populations, suggesting that the hydrogen peroxide-scavenging arm of the antioxidant system was not the primary responder in this scenario.

Interestingly, the actively dividing log cells showed a different pattern of stress markers. Rather than accumulating ROS, they exhibited higher induction of oxidized proteins — measured as carbonyl groups — with approximately a six-fold increase compared to only a two-fold rise in quiescent cells, which already carried around seven-fold higher constitutive carbonyl levels due to starvation. Lipid peroxidation, tracked through malondialdehyde (MDA), the toxic end product of membrane lipid oxidation, also rose about two-fold in log cells after treatment, while remaining similar between control and treated quiescent cells. The researchers suggest that the thicker cell wall of quiescent cells may partially explain this muted lipid peroxidation response.

Glutathione, the tripeptide that serves as a cellular redox buffer and protects against ROS through detoxification mechanisms, told yet another part of the story. Untreated quiescent cells carried three-fold higher glutathione concentrations than log cells, but menadione treatment produced only a modest two-fold rise in the quiescent fraction. In log cells, the same treatment triggered a seven-fold increase in glutathione levels. Critically, the ratio of oxidized to reduced glutathione (GSSG/GSH) remained relatively constant in quiescent cells before and after treatment. The authors interpret this stability as evidence that menadione’s toxicity in these cells operates through its redox-cycling mechanism — generating ROS — rather than through direct conjugation with reduced glutathione, which would have caused a sharp drop in intracellular glutathione as menadione-S-glutathione conjugates formed.

The non-quiescent population added further nuance. These cells carried significantly higher constitutive levels of ROS, oxidized proteins, and MDA than either log or quiescent cells, reflecting their heterogeneous composition and advanced stationary-phase state. Yet after menadione treatment, their ROS levels actually decreased below control values. The researchers attribute this counterintuitive result to a higher percentage of non-quiescent cells being in a terminal state and entering apoptosis, a process that would reduce measurable ROS as cells progressively lose metabolic function.

The mechanistic explanation for quiescent cells’ vulnerability likely lies in their metabolic profile. Quiescent cells are characterized by lower energy consumption and reduced ADP content, conditions that may lead to increased intracellular oxygen levels and a greater prevalence of single-electron oxygen reducers. Because quinones like menadione depend on oxygen presence to catalyze ROS generation through redox cycling, the oxygen-rich, metabolically quiescent environment may amplify the compound’s genotoxic effects. This finding carries broader implications, since most cells in human tissues are non-dividing, making quiescence a major form of cellular life whose stress responses remain understudied in multicellular organisms due to signal complexity.

Taken together, the study establishes that Saccharomyces cerevisiae quiescent cells are the most susceptible population to menadione’s action, and it suggests that the DNA-damaging and genotoxic effects observed in these cells are related to ROS production. Because yeast genes and protein functions are homologous to those of mammals, the results position quiescent yeast as a practical platform for screening the effects of toxic compounds on mammalian and human cells — particularly relevant for understanding how dormant cells in tissues respond to oxidative challenges that dividing cells can more readily withstand.

Subject of Research: Comparative susceptibility and oxidative stress response of logarithmic, quiescent, and non-quiescent Saccharomyces cerevisiae cell populations to menadione

Article Title: Cellular susceptibility and oxidative stress response to menadione of logarithmic, quiescent, and nonquiescent Saccharomyces cerevisiae cell populations

Article References: Marinovska, P. G., Todorova, T. I., Boyadzhiev, K. P., Pisareva, E. I., Tomova, A. A., Parvanova, P. N., Dimitrova, M., Chankova, S. G., & Petrova, V. Y. (2022). Cellular susceptibility and oxidative stress response to menadione of logarithmic, quiescent, and nonquiescent Saccharomyces cerevisiae cell populations. BioRisk, 17, 127-138. https://doi.org/10.3897/biorisk.17.77320

Image Credits: AI Generated

DOI: 10.3897/biorisk.17.77320

Keywords: Saccharomyces cerevisiae, quiescent cells, menadione, oxidative stress, reactive oxygen species, DNA double-strand breaks, glutathione, superoxide dismutase, lipid peroxidation, cell survival, yeast, genotoxicity