Low ozone concentrations stimulate cytoskeletal organization, mitochondrial activity and nuclear transcription

Background: why the ozone dose matters at the level of the cell

Medical ozone is used at carefully controlled, low concentrations, and a long-standing idea in the field is that its benefits follow a hormetic pattern - a mild, precisely dosed stress that prompts cells to adapt and strengthen, whereas too much becomes damaging. To test that idea directly, researchers needed to look inside living cells and see how different ozone concentrations affect the structures that underpin cell function: the cytoskeleton (the internal scaffolding that governs shape, adhesion and movement), the mitochondria (the organelles that generate energy), and the nucleus (where genes are transcribed into RNA). This laboratory study set out to map those responses across a range of doses.

Study design: HeLa cells exposed to graded ozone concentrations

This was an in vitro experiment using cultured human HeLa cells. The cells were exposed to a gaseous oxygen-ozone mixture at three concentrations - 1, 10 and 20 micrograms of ozone per millilitre of oxygen - with air and pure oxygen used as controls. The researchers combined several techniques to build a detailed picture of the cellular response. Bright-field, fluorescence and transmission electron microscopy assessed cell shape, adhesion and the fine structure of mitochondria. Fluorescent phalloidin labelling was used to visualise the actin cytoskeleton. Flow cytometry with JC-1 staining measured mitochondrial membrane potential, and a cytochemical stain detected reactive oxygen species. Nuclear transcription was tracked by measuring incorporation of a labelled RNA precursor, and immunolabelling identified stress-response and mitochondrial proteins. Quantitative morphometry was applied throughout to compare responses between doses and over time.

Findings: 10 micrograms per millilitre produced the strongest, most durable response

The cellular effects varied clearly with ozone concentration. The lowest dose, 1 microgram per millilitre, produced only transient, short-lived changes. The intermediate dose of 10 micrograms per millilitre produced positive and long-lasting responses: improved cell adhesion and flattening, better-organised actin filaments, more developed mitochondrial cristae with raised membrane potential, and increased nuclear RNA transcription. At the highest dose, 20 micrograms per millilitre, the pattern reversed - cell adhesion was reduced and cytoskeletal reorganisation was impaired. The nucleus and the cytoplasmic organelles did not respond identically, indicating that different cell compartments can be affected differently by the same exposure.

What the authors concluded

The authors concluded that the effects of ozone administration depend on the gas concentration, and that the nucleus and cytoplasmic organelles may be affected differently from one another. In this model, an intermediate concentration of around 10 micrograms per millilitre gave the most favourable and sustained activation of cytoskeletal organisation, mitochondrial activity and nuclear transcription. They suggested that, with further work, ozone administration protocols might be refined to reliably and durably activate cell metabolism, while taking account of the specific features of different tissues.

Limitations of this study

The authors were explicit that these data were obtained in an in vitro cell model and cannot be directly translated to therapeutic use in the body. The work used a single immortalised human cell line under controlled laboratory conditions, so the responses seen may differ from those of other cell and tissue types, and from the more complex environment of a living organism. The study is mechanistic and preclinical: it helps explain how low ozone concentrations might act at the cellular level, but it does not measure clinical outcomes and should not be read as evidence of benefit in patients.