Dihydroxyacetone suppresses mTOR nutrient signaling and induces mitochondrial stress in liver cells

Dihydroxyacetone (DHA) is the active ingredient in sunless tanning products and a combustion product from e-juices in electronic cigarettes (e-cigarettes). DHA is rapidly absorbed in cells and tissues and incorporated into several metabolic pathways through its conversion to dihydroxyacetone phosphate (DHAP). Previous studies have shown DHA induces cell cycle arrest, reactive oxygen species, and mitochondrial dysfunction, though the extent of these effects is highly cell-type specific. Here, we investigate DHA exposure effects in the metabolically active, HepG3 (C3A) cell line. Metabolic and mitochondrial changes were evaluated by characterizing the effects of DHA in metabolic pathways and nutrient-sensing mechanisms through mTOR-specific signaling. We also examined cytotoxicity and investigated the cell death mechanism induced by DHA exposure in HepG3 cells. Millimolar doses of DHA were cytotoxic and suppressed glycolysis and oxidative phosphorylation pathways. Nutrient sensing through mTOR was altered at both short and long time points. Increased mitochondrial reactive oxygen species (ROS) and mitochondrial-specific injury induced cell cycle arrest and cell death through a non-classical apoptotic mechanism. Despite its carbohydrate nature, millimolar doses of DHA are toxic to liver cells and may pose a significant health risk when higher concentrations are absorbed through e-cigarettes or spray tanning

Genotoxic effects induced by DHA.

(A) HepG3 cells were dosed with 7mM DHA for the corresponding time points, and strand break analysis was performed with γH2AX staining. (B) A significant increase in intensity was observed starting at 24 h and continued until 96h. The statistical significance level was marked as ****p (TIF)</p

Mitochondrial ROS is increased when cells are exposed to DHA.

(A) HepG3 cells were dosed with 7 mM DHA for 24 and 48 h and imaged to evaluate mitochondrial ROS; scale bars 25 μm. (B) A significant increase in fluorescent intensity can be observed at 24 and 48h DHA exposure cells compared to the control. The level of statistical significance was marked as follows: *p < 0.05; **p < 0.01.</p

Cytochrome c localization and intensity were evaluated through immunofluorescence.

(A) An increase was found in cells dosed with DHA starting at 48 h until 96 h. Cytochrome c is found in the mitochondrial compartment only. (B) Cytochrome c intensity was quantified, where a 2-fold increase was calculated in cells dosed with 96 h DHA. Significance is indicated as **p (TIF)</p

LC3BII intensity was calculated in addition to the ratio calculated in Fig 4.

A significant increase was found in cells dosed with 96 h DHA. Three biological replicates were quantified, and the significance level is marked as follows: ***p (TIF)</p

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Growth inhibition in cells treated with chloroquine and DHA.

(A) Cells were exposed to DHA, chloroquine, or a combination of chloroquine and DHA and counted after 5 days. The graph displays the percentage survival of cells relative to control. (B) Immunoblotting autophagic cell death markers using a select dose of chloroquine alone and in combination with DHA. LC3BII was found to reduce after DHA and chloroquine treatment compared to DHA and return to control level. (C) Immunoblotting of apoptotic using a select dose of chloroquine alone and in combination with DHA. No change in apoptotic markers caspase 3 and PARP1 were observed in combination treatment. Three biological replicates were quantified, and the significance level is marked as follows compared to control: *p p p ##p  (TIF)</p

Cell growth and viability decrease confirmed by growth inhibition in HepG3.

Cells were exposed to a range of DHA doses and counted after 5 days. The graph displays the percentage of cells relative to control. An IC50 of 1.5 mM ± 0.98 and an IC90 of 4.6 mM ± 0.72 were calculated. (TIF)</p

Glycolysis is modulated after DHA exposure.

(A) Extracellular acidification rate (ECAR) displayed a shift in cells dosed with 7 mM DHA for 24 h. (B) All measured parameters showed a decrease with a significant difference in the glycolysis and glycolytic capacity. The statistical significance level was marked as **p < 0.01.</p