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Ascorbic acid maintenance in HaCaT cells prevents radical formation and apoptosis by UV-B
I Savini1, I D'Angelo, M Ranalli
1Department of Experimental Medicine and Biochemical Sciences, University Tor Vergata, Rome, Italy.
Insights
HaCaT cells efficiently accumulate vitamin C (ascorbic acid) through uptake and regeneration, enhancing their antioxidant capacity and resistance to UV-B damage.
Area of Science:
- Cell Biology
- Biochemistry
- Dermatology
Background:
- Vitamin C (ascorbic acid) is a crucial antioxidant for skin health.
- Understanding its cellular mechanisms in epithelial cells is vital for dermatological applications.
Purpose of the Study:
- To investigate the enzymatic reduction and accumulation of vitamin C in HaCaT epithelial cells.
- To assess the impact of ascorbic acid 2-phosphate on cell proliferation and vitamin C levels.
- To evaluate the role of vitamin C in cellular protection against UV-B irradiation.
Main Methods:
- Enzymatic assays for ascorbate reductases.
- Subcellular fractionation to determine enzyme localization.
- Cell proliferation assays.
- Measurement of intracellular vitamin C concentrations.
- Assessment of antioxidant capacity using 2',7'-dihydrodichlorofluorescein staining.
- UV-B irradiation to induce oxidative stress.
Main Results:
- High ascorbyl free radical reductase activity was found in mitochondrial, microsomal, and plasma membrane fractions.
- Dehydroascorbate reductase activity was primarily localized in the post-microsomal supernatant.
- Ascorbic acid 2-phosphate derivative was taken up and accumulated as ascorbic acid without inhibiting cell growth.
- HaCaT cells demonstrated efficient mechanisms for maintaining high intracellular and extracellular vitamin C levels.
- Increased intracellular vitamin C enhanced antioxidant capacity and conferred resistance to UV-B-induced cell death.
Conclusions:
- HaCaT cells possess robust systems for vitamin C uptake and regeneration, maintaining high intracellular and extracellular concentrations.
- The regeneration and uptake of vitamin C significantly contribute to the intracellular antioxidant defense of HaCaT cells.
- Enhanced vitamin C levels in HaCaT cells provide protection against UV-B-induced oxidative stress and cell death, highlighting its potential in skin protection.
Abstract:
We have investigated the enzymatic reduction and accumulation of vitamin C in HaCaT epithelial cells. The subcellular localization and the activities of ascorbyl free radical reductase and dehydroascorbate reductase showed that mitochondrial, microsomal and plasma membranes fractions express high levels of ascorbyl free radical reductase activity, whereas dehydroascorbate reductase activity was found at low levels only in the post microsomal supernatant. We have also investigated cell proliferation and vitamin C accumulation induced by ascorbic acid 2-phosphate. This derivative caused no inhibition of cell growth, was uptaken from the extracellular medium and accumulated as ascorbic acid in mM concentrations. These results show that HaCaT cells possess very efficient systems to maintain high levels of both intracellular and extracellular ascorbic acid. The regeneration and uptake of ascorbic acid from extracellular medium contributes to the intracellular antioxidant capacity, as evaluated by 2',7'-dihydrodichlorofluorescein staining. Consequently, cells became more resistant to free radical generation and cell death induced by UV-B irradiation.