ATLA::Alternatives to Laboratory Animals

Volume 28, Number 3

Iron-induced lipid peroxidation in human skin-derived cell lines: protection by a red orange extract.

ATLA 28, 427-433, May/June 2000

Flora Morini,¹ Fabiola Dusatti,¹ Francesco P. Bonina,² Antonella Saija³ and Margherita Ferro¹

¹Department of Experimental Medicine, General Pathology Division, University of Genoa, Via L.B. Alberti 2, 16132 Genoa, Italy; ² Department of Pharmaceutical Sciences, University of Catania, Viale A. Doria 25, 95125 Catania, Italy; ³Department of Pharmacology of Natural Substances and General Physiology, University of Roma "La Sapienza", Piazzale A. Moro 6, 00185 Rome, Italy

SUMMARY

Although photodamage and photoprotection have already been extensively studied in cultured cells, few data have been reported in the literature regarding the in vitro behaviour of skin cells toward a chemical stress, such as iron-induced peroxidation. We investigated the susceptibilities of two human skin-derived cell lines (NCTC 2544 keratinocytes and HFFF2 fibroblasts) to lipid peroxidation induced by FeSO₄/histidine, FeSO₄/ascorbate and Fe₂(SO₄)₃/ADP. NCTC 2544 cells were more susceptible than HFFF2 cells to lipid peroxidation (assessed by measuring the content of malondialdehyde [MDA]) with iron/ascorbate and iron/ADP as pro-oxidants whereas, with iron/histidine, the same level of MDA production was achieved (about 10 nmol/mg protein) in the two cell populations. On the basis of these results, one experimental model (iron/histidine) was selected to assess the protective effect of a mixture of two classical antioxidants, Trolox C (50 mM) and Vitamin C (1 mM), added to the cell cultures according to various protocols. The maximal decrease of MDA production in both cell lines was obtained when the antioxidant mixture and the pro-oxidant were added simultaneously to the cultures. By using the same experimental design, NCTC 2544 and HFFF2 cells were exposed to a standardised extract of red oranges (ROE; 0.025-0.5 mg/ml), the main active principles of which (anthocyanins 3.1%, hydroxycinnamic acid 2.07%, and flavanone glycosides 8.1%) possess antioxidant activity. Cells treated with ROE, that were still over 90% viable, as evaluated by means of neutral red uptake and tetrazolium salt reduction tests, showed a significant and dose-dependent inhibition of MDA production. This study provides new information about the behaviour of cultured skin cells exposed to chemically induced oxidative stress, and provides further support to the possibility of using skin-derived human cell lines in the evaluation of the effectiveness of antioxidant ingredients for new drugs and/or cosmetics.

Keywords: keratinocytes, fibroblasts, NCTC 2544 cell line, HFFF2 cell line, iron-induced lipid peroxidation, ascorbic acid, Trolox C™, anthocyanins