Comparison of Primary Human Fibroblasts and Keratinocytes with Immortalized Cell Lines Regarding their Sensitivity to Sodium Dodecyl Sulfate in a Neutral Red Uptake Cytotoxicity Assay

Abstract

Cell lines present a valuable tool for in vitro assessment of skin damage caused by application of cosmeticals or pharmaceuticals. They form a reproducible test system under controllable test conditions and, in many cases, can be used as alternatives to animal testing in order to assess the compatibility of drugs or cosmetics and human skin. Yet, it can not necessarily be assumed that the behavior of cultured cells, when treated with different substances, is exactly consistent with the behavior of cells being part of a live organism. Becoming immortal, cells exhibit changes in genotype and/or phe-notype, possibly resulting in modified reactions to external influences. Therefore, to obtain results close to in vivo studies, it seems apparent to use primary cells for testing that have not yet undergone any modifications. To compare the properties of primary fibroblasts (Normal Human Dermal Fibroblasts, NHDF) and primary keratinocytes (Normal Human Epidermal

Keratinocytes, NHEK) with those of immortal cell lines (3T3 (ACC 173) Swiss albino mouse fibroblasts and HaCaT (human, adult, low calcium, high temperature, human adult skin keratinocytes) cells), their sensitivities in cytotoxicity assays have been assessed. While both fibroblast cell cultures showed similar sensitivities towards sodium dodecyl sulfate (SDS), primary keratinocytes died at SDS concentrations about three times lower than the immortal HaCaT cells.

Key words

Cytotoxicity assays - Immortalized cell lines - Primary human fibroblasts - Primary human keratinocytes - Sodium dodecyl sulfate

• References

• 1 ECVAM DB-ALM INVITTOX protocol. BALB/C 3T3 Cytotoxicity Test. INVITTOX n° 46. Eye Irritation, Basal Cytotoxicity. http:llecvam-dbalm.jrc.ec.europa.eu
• 2 Lee JK, Kim DB, Kim JI, Kim PY. In vitro cytotoxicity tests on cultured human skin fibroblasts to predict skin irritation potential of surfactants. Toxicol In Vitro. 2000; 14 (4) 345-349
• 3 Osborne R, Perkins MA. An approach for development of alternative test methods based on mechanisms of skin irritation. Food Chem Toxicol. 1994; 32 (2) 133-142
• 4 Benassi L, Bartazzoni G, Seidenari S. In vitro testing of ten-sides employing monolayer cultures: a comparison with results of patch tests on human volunteers. Contact Dermatitis. 1999; 40 (l) 38-44
• 5 Martinez V, Corsini E, Mitjans M, Pinazo A, Vinardell MP. Evaluation of eye and skin irritation of arginine-derivative surfactants using different in vitro endpoints as alternatives to the in vivo assays. Toxicol Lett. 2006; 164 (3) 259-267
• 6 Wilhelm KP, Samblebe M, Siegers CP. Quantitative in vitro assessment of N-alkyl sulphate-induced cytotoxicity in human keratinocytes (HaCaT). Comparison with in vivo human irritation tests. Br J Dermatol. 1994; 130 (l) 18-23
• 7 Krebs FC, Miller SR, Catalone BJ, Fichorova R, Anderson D, Malamud D, Howett MK, Wigdahl B. Comparative in vitro sensitivities of human immune cell lines, vaginal and cervical epithelial cell lines, and primary cells to candidate mi-crobicides Nonoxynol 9, C31G, and sodium dodecyl sulfate. Antimicrob Agents Chemother. 2002; 46 (7) 2292-2298
• 8 Bae D-S, Gennings C, Carter Jr WH, Yang RSH, Campain JA. Toxicological interactions among arsenic, cadmium, chromium, and lead in human keratinocytes. Tox Sci. 2001; 63: 132-142
• 9 Boukamp P, Petrussevska RT, Breitkreutz D, Hornung J, Markham A, Fusenig NE. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. J Cell Biol0. 1988; 106: 761-771
• 10 Borenfreund E, Puerner JA. Toxicity determination in vitro by morphological alterations and Neutral Red absorption. Toxicol Lett. 1985; 24: 119-124
• 11 Korting HC, Herzinger T, Hartinger A, Kerscher M, Anger-pointer T, Maibach HI. Discrimination of the irritancy potential of surfactants in vitro by two cytotoxicity assays using normal human keratinocytes, HaCaT cells and 3T3 mouse fibroblasts: correlation with in vivo data from a soap chamber assay. J Derm Sci. 1994; 7: 119-129