Low-concentration methylene blue delayed senescence in cultured human fibroblasts
Mark Kemp
In cultured human fibroblasts, nanomolar methylene blue extended replicative lifespan by over 20 population doublings and raised complex IV activity 30%. A plain summary of Atamna et al., FASEB J 2008.
In cultured human IMR90 lung fibroblasts, methylthioninium chloride (methylene blue) at nanomolar concentration extended replicative lifespan by more than 20 population doublings and delayed senescence (Atamna et al., FASEB J 2008). Mitochondrial complex IV (cytochrome c oxidase) activity rose by 30%, cellular oxygen consumption by 37–70%, and haem synthesis increased; the authors proposed that redox cycling between methylene blue and leucomethylene blue within mitochondria accounts for the effect.
Citation: Atamna H, Nguyen A, Schultz C, et al. FASEB J 2008;22(3):703–12. PMID 17928358. doi:10.1096/fj.07-9610com.
What the study did
The authors used IMR90 cells — human diploid lung fibroblasts, a standard model for replicative senescence. Cells of this kind divide a limited number of times in culture and then stop. The number of divisions before they stop is the measurable outcome.
They grew the cells with methylene blue at nanomolar concentrations and counted population doublings. Alongside that, they measured mitochondrial complex IV activity, whole-cell oxygen consumption and haem synthesis, and tested whether methylene blue could reverse premature senescence induced by hydrogen peroxide or cadmium. Other diaminophenothiazines were tested in parallel. A separate experiment in HepG2 liver cells examined phase-2 antioxidant enzyme induction, and rat liver mitochondrial lysates were used to assess the methylene blue-to-cytochrome c ratio.
What it found
Methylene blue-treated IMR90 cells divided more than 20 additional times before senescence, compared with untreated cells.
Complex IV activity increased by 30%. Cellular oxygen consumption increased by 37–70%. Haem synthesis increased.
Premature senescence induced by hydrogen peroxide or cadmium was reversed.
In HepG2 cells, methylene blue induced phase-2 antioxidant enzymes. In rat liver mitochondrial lysates, the ratio of methylene blue to cytochrome c was found to matter for the protective effect.
The authors proposed that the delay in senescence is caused by methylene blue cycling between its oxidised form and its reduced form, leucomethylene blue, inside mitochondria — flavin-dependent enzymes reduce it using NAD(P)H, and cytochrome c reoxidises it — and that this cycling may reduce oxidant production by the mitochondrion.
| Model | IMR90 human lung fibroblasts, in vitro; HepG2 cells; rat liver mitochondrial lysates |
| Compound | Methylthioninium chloride; other diaminophenothiazines for comparison |
| Concentration | Nanomolar |
| Measured | Population doublings to senescence; complex IV activity; oxygen consumption; haem synthesis; recovery from H₂O₂- and cadmium-induced senescence; phase-2 enzyme induction |
| Result | > 20 additional population doublings; complex IV +30%; oxygen consumption +37–70%; haem synthesis up; premature senescence reversed |
| Not tested | Animals; humans; oral administration; any dose |
What it means, and what it doesn't
The measurable finding is that cells in a dish divided more times before stopping, and that several mitochondrial activities were higher in treated cells. That is what the study shows.
It does not show anything about ageing in an animal or a person. “Cellular senescence” is a property of cells in culture; it is related to ageing biology, but the study did not test ageing. The authors’ suggestion that methylene blue “may be useful” against age-related mitochondrial decline is their hypothesis, stated as such in the paper, and not a result.
The concentrations are nanomolar bath concentrations in culture medium. The paper does not attempt to translate them to any amount a person might take, and this page does not either.
Read the paper
Atamna H, Nguyen A, Schultz C, Boyle K, Newberry J, Kato H, Ames BN. Methylene blue delays cellular senescence and enhances key mitochondrial biochemical pathways. FASEB Journal 2008;22(3):703–712.
This summarises a laboratory study on cultured cells. It is not evidence of an effect in humans and is not a claim about any product.