Zeocin is a member of the bleomycin family of antibiotics, compounds prized in molecular biology for their ability to cut DNA but feared for their toxicity. The drug mimics the effects of ionizing radiation, inducing both single- and double-strand breaks in the genetic material as well as base substitutions in the DNA molecule. Earlier work has also linked Zeocin to pro-oxidative, mutagenic and carcinogenic effects in yeast, making it an ideal probe for studying how cells cope with one of the most dangerous classes of chemical insults. Understanding precisely how such compounds injure cells — and which cellular states confer protection — matters far beyond the laboratory, because pharmaceutical residues released into the environment can interact with biological targets at very low concentrations and inflict lasting damage on lipids, proteins and DNA.

The research team, working with the widely used laboratory strain S. cerevisiae BY4741, took the unusual step of comparing three distinct cell populations side by side. The first consisted of logarithmically growing cells, the fast-dividing workhorses of most yeast experiments. The second and third came from stationary-phase cultures that had been starved into dormancy for 168 hours: using a Percoll density gradient, the scientists separated these cultures into true quiescent cells, which retain the capacity to re-enter the cell cycle, and non-quiescent cells, a more damaged fraction that has largely lost the ability to reproduce. Because quiescence is thought to be the most common cellular state found on Earth, the authors argue that testing drugs on resting cells may yield far more realistic predictions of how environmental toxicants affect natural microbial communities than the standard practice of testing only on proliferating cultures.

The experiments focused on a battery of molecular sensors of oxidative stress. Cells were exposed to 50 micrograms per millilitre of Zeocin — a concentration corresponding to the IC50, the dose that halves survival — for one hour, after which the researchers measured intracellular reactive oxygen species, carbonylated proteins, oxidized lipids and total glutathione. Reactive oxygen species, the toxic radicals generated when cellular redox balance collapses, rose in all three populations, but not equally. Proliferating cells and non-quiescent cells each accumulated roughly four times more ROS than their untreated counterparts, while quiescent cells showed only a modest 1.5-fold increase, the smallest response of any group.

Damage to macromolecules told a complementary story. Carbonyl groups, the chemical fingerprints of oxidized proteins, increased about threefold in quiescent cells after Zeocin treatment, whereas logarithmic and non-quiescent cells showed only slight elevations. The authors interpret this paradoxical result as a consequence of the quiescent cells’ very low metabolic rate: with reduced capacity to sequester and degrade damaged molecules, oxidized proteins simply accumulate. Lipid peroxidation, measured as malonaldehyde equivalents, followed a different pattern again, with the highest levels of oxidized lipids appearing in Zeocin-treated proliferating and quiescent cells at 1.88 and 1.4 nanomoles per mole respectively, while non-quiescent cells showed negligible lipid damage whether treated or not. Because lipid peroxidation compromises membrane functionality and permeability, the findings suggest that metabolically active cells, with dynamic transport across their membranes, are simply more vulnerable to xenobiotic penetration.

Glutathione, the tripeptide that serves as the cell’s first line of non-enzymatic antioxidant defence, rose in every treated population, confirming that Zeocin provokes a coordinated stress response. The magnitude of the response tracked metabolic activity: proliferating cells boosted glutathione biosynthesis roughly 2.5-fold, consistent with their high rates of transcription and translation, while quiescent cells managed only about a 30 percent increase. Non-quiescent cells carried the highest absolute concentrations of the tripeptide, likely reflecting their chronically elevated baseline ROS levels rather than a specific response to the drug.

The most striking results concerned DNA. Using constant field gel electrophoresis, which quantifies double-strand breaks as the fraction of DNA released from agarose plugs, the team found that spontaneous break levels depended strongly on growth phase, with non-quiescent cells carrying about 1.5 times more spontaneous breaks than either logarithmic or quiescent cells — an expected consequence of their genomic instability and tendency toward apoptosis. When the researchers then challenged cells with much higher Zeocin concentrations, from 100 to 300 micrograms per millilitre, logarithmic and non-quiescent cells responded with statistically significant induction of double-strand breaks, while quiescent cells showed no significant break induction at any concentration tested. Their DNA remained essentially as intact as that of untreated controls.

Repair capacity added a final layer to the picture. When given 30 and 60 minutes to recover after treatment, logarithmic cells demonstrated the best repair performance, with calculated repair capacity rising to 3.21 at the highest Zeocin dose after an hour of recovery. Non-quiescent cells, by contrast, proved unable to repair the induced breaks at all, with repair capacity values hovering near or below 1 regardless of dose or recovery time. For quiescent cells, repair capacity could not even be calculated, simply because the treatment had failed to produce a statistically significant increase in breaks in the first place. The authors attribute the extraordinary resilience of quiescent cells to their distinctive physiology: thickened cell walls, highly condensed chromosomes and increased thermo- and osmotolerance, features that likely restrict Zeocin’s ability to penetrate the cell and reach its DNA target.

Taken together, the study delivers a clear message for toxicology: the physiological state of a cell is a critical determinant of its resistance to environmental stress. Actively proliferating yeast, with their high metabolic activity, elevated baseline ROS and dynamic membranes, proved the most susceptible to Zeocin’s combined assault on DNA, proteins and lipids, while cells that had entered the G0 quiescent state emerged as the most robust. Because S. cerevisiae shares a high degree of genetic conservation with higher eukaryotes and is currently the only experimental system that permits simultaneous in vivo evaluation of all cellular targets, the findings carry implications well beyond microbiology. They suggest that toxicity screening programmes relying exclusively on dividing cells may systematically overestimate — or in some cases mischaracterize — the hazards that drugs and environmental pollutants pose to the dormant microbial populations that dominate natural ecosystems. Incorporating quiescent cells into standard yeast-based test panels, the authors argue, would make such assays smarter, more sensitive and far more representative of the biological reality that toxic compounds encounter in the wild.

Subject of Research: Assessing Zeocin-induced oxidative stress and DNA damage in proliferating, quiescent and non-quiescent Saccharomyces cerevisiae cells

Article Title: Saccharomyces cerevisiae yeast cells as a test system for assessing Zeocin toxicity

Article References: Marinovska, P., Todorova, T., Tomova, A., Pisareva, E., Boyadzhiev, K., Dimitrov, M., Parvanova, P., Dimitrova, M., Chankova, S., & Petrova, V. (2022). Saccharomyces cerevisiae yeast cells as a test system for assessing Zeocin toxicity. BioRisk, 17, 105-116. https://doi.org/10.3897/biorisk.17.77227

Image Credits: AI Generated

DOI: 10.3897/biorisk.17.77227

Keywords: Saccharomyces cerevisiae, Zeocin, toxicity testing, quiescent cells, oxidative stress, reactive oxygen species, glutathione, DNA double-strand breaks, lipid peroxidation, radiomimetic antibiotic, yeast model, cellular stress response