Toxicological Research

Korean Society of Toxicology & Korea Environmental Mutagen Society (한국독성학회)
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Comparison of Sensitivity Between Balb/c 3T3 Cell and HaCaT Cell by NRU Assay to Predict Skin Phototoxicity Potential

  • Lee, Jong-Kwon (Department of Toxicology, National Institute of Toxicolosy Research, Korea Food and Drug Administration) ;
  • Lee, Eun-Hee (Department of Toxicology, National Institute of Toxicolosy Research, Korea Food and Drug Administration) ;
  • Lee, Sun-Hee (Department of Toxicology, National Institute of Toxicolosy Research, Korea Food and Drug Administration)
  • Published : 2002.09.01
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Abstract

In order to find out the appropriate in vitro method for high correlation with in vivo, we com-pared the sensitivities of phototoxicity (PT) in vitro method between in human keratinocytes, HaCaT cells and in 3T3 fibroblast cells derived from Balb/c mice. Both cells were exposed to six known phototoxic chemicals : promethazine, neutral red, chlortetracycline, amiodarone, bithionol, 8-methoxypsoralen, or non-phototoxic chemical, ALS (ammonium laureth sulfate) and then irradiated with 5 J/$cm^2$ of UVA. Cell viability ($IC_{50}$ ) was measured by neutral red uptake (NRU) assay. The ratio of $IC_{50}$ value of chemicals in the presence and absence of UVA was determined by the cut-off value. The phototoxic potential of test chemicals in NRU assay was determined by measuring the photoirriation factor (PIF) with a cut-off value of 5. In both 3T3 and HaCaT cells, all known phototoxic chemicals were positive (over 5 of PIF value), except that bithionol was found to be non-phototoxic to HaCaT cells, and ALS, non-phototoxic chemical was negative. These results suggest that Balb/c 3T3 cell was more sensitive than HaCaT cell to predict phototoxicity potential.

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References

  1. Toxicology Letters v.24 Toxicity determined in vitro by morphological alterations and neutral red absorption Borenfreund,E.;Puerner,J.A. https://doi.org/10.1016/0378-4274(85)90046-3
  2. J. Cell Biol. v.106 Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line Boukamp,P.;Petrussevska,R.T.;Britkreutz,B.;Hornung,J.;Markham, A.;Fusenig,N.E. https://doi.org/10.1083/jcb.106.3.761
  3. Alternatives to laboratory animals v.27 The use of human keratinocytes in the EU/COLIPA interantional in vitro phototoxicity test validation study and the ECVAM/COLIPA study on UV filter chemicals Clothier,R.;Willshaw,A.;Cox,H.;Garle,M.;Bowler,H.;Combes,R.
  4. Toxicology in vitro v.8 A screen for photoirritants in cultured cells Dixit,M.B.;West,M.;Earl,L.K.;Lovell,W.W. https://doi.org/10.1016/0887-2333(94)90061-2
  5. Toxicology in vitro v.14 In vitro cytotoxicity tests on cultured human skin fibroblasts to predict skin irritation potential of surfactants Lee,J.K.;Kim,J.I.;Kim,P.Y. https://doi.org/10.1016/S0887-2333(00)00028-X
  6. Journal of Toxicology and Public Health v.15 In vitro skin test of humectants with human skin fibroblasts Lee,J.K.;Kim,D.B.;Lee,E.H.;Ryu,S.R.;Kim,Y.j.;An,S.S.;Lee,B.M.;Lee,Y.S.;Kim,P.Y.
  7. Journal of Toxicology and Public Health v.16 In vitro alternative study of phototoxicity by neutral red uptake assay using human fibroblast Lee,J.K.;Lee,E.H.;Kim,H.S.;Hong,J.T.;Ryu,S.R.;Park,K.S.;Kim,D.B.;Kim,P.Y.;Cho,D.H.;Lee,H.S.
  8. Alternative Methods in Toxicology. In Vitro Skin Toxicology-Irritation, v.10 First results of the EC/COLIPA validation project in vitro phototoxicity testing Liebsch,M.;Spielmann,H.;Balls,M.;Brand,M.;Doering,B.;Dupuis,J.;Hoszhutter,H.G.;Klecak,G.;Moldenhauer,F.;Morre,L.;Pape,W.J.W.;Pfannenbecker,U.;Potthast,J.;De Silva,O.;Steiling,W.;Willshaw,A.;A.Raugler;A.M.Goldberg;H.I.Maibach(eds.)
  9. Alternative Methods in Toxicology. In Vitro Skin Toxicology-Irritation, Phototoxicity, Sensitization v.10 The use of keratinocyte and fibroblast cultures to predict phototoxicity of chemicals Maier,K.;A.Raugier;A.M.Goldberg;H.I.Maibach(eds.)
  10. Alternatives to laboratory animals v.27 Vitro alternatives and phototoxicity testing Ⅱ. Effects of reactive oxygen species in in vitro phototoxicity assays Okamoto,Y.;Ryu,A.;Ohkoshi,K.
  11. Toxicology in vitro v.15 Hydrogen peroxide induced stress in human keratinocytes and its effect on bithionol toxicity Reid,L.;Clothier,R.H.;Khammo,N. https://doi.org/10.1016/S0887-2333(01)00049-2
  12. Toxicology in vitro v.15 Lessons learned from validation of in vitro test: from failure to acceptance into regulatory practice Spielmann,H.;Liebsch,M. https://doi.org/10.1016/S0887-2333(01)00070-4
  13. Toxicology in vitro v.8 EEC/COLIPA project on in vitro phototoxicity testing: First results obtained with a Balb/c 3T3 cell phototoxicity assay Spielmann,H.;Balls,M.;Brand,M.;Doring,B.;Holzhutter,H.G.;Kalweit,S.;Klecak,G.;Eplattenier,H.L.;Liebsch,M.;Lovell,W.W.;Maurer, T.;Moldenhauer,F.;Moore,L.;Pape,W.J.W.;Pfannenbecker,U.;Potthast,J.;de Silva,O.;Steiling,W.;Willshaw,A. https://doi.org/10.1016/0887-2333(94)90069-8
  14. Toxicology in vitro v.12 The international EU/COLIPA in vitro phototoxicity validation study: Results of phase Ⅱ(Blind trial). Part Ⅰ: The 3T3 NRU phototoxicity test Spielmann,H.;Balls,M.;Dupuis,J.;Pape,W.J.;Pechovitch,G.;de Silva,O.;Holzhutter,H.G.;Clothier,R.;Desolle,P.;Gerberick,F.;Liebsch,M.;Lovell,W.W.;Maurer,T.;Pfannenbdcker,U.;Potthast,J.M.;Csato,M.;Sladowski,D.;Stelling,W.;Brantom,P. https://doi.org/10.1016/S0887-2333(98)00006-X
  15. Alternatives to laboratory animals v.26 A Study on UV filter chemicals from Annex Ⅶof European Union Directive 76/768/EEC, in the in vitro 3T3 NRU phototoxicity test Spielmann,H.;Balls,M.;Dupuis,J.;Pape,W.W.J.;Pechovitch,G.;de Silva,O.;Holzhutter,H.G.;Gerberick,F.;Liebsch,M.;Lovell, W.W.;Pfannenbdcker,U.
  16. Alternatives to laboratory animals v.22 In vitro phototoxicity testing. The report and recommendations of ECVAM workshop 2 Spielmann,H.;Lovell,W.W.;Holze,E.;Johnson,B.E.;Maurer,T.;Miranda,M.A.;Pape,W.W.J.;Sapora,O.;Sladowski,D.
  17. Toxicology in vitro v.15 Hydrogen peroxide induced stress in human keratinocytes and its effect on bithionol toxicity Reid,L.;Clothier,R.H.;Khammo,N. https://doi.org/10.1016/S0887-2333(01)00049-2
  18. Alternative Methods in Toxicology. In Vitro Skin Toxicology-Irritation, v.10 First results of the EC/COLIPA validation project in vitro phototoxicity testing Liebsch,M.;Spielmann,H.;Balls,M.;Brand,M.;Doering,B.;Dupuis,J.;Hoszhutter,H.G.;Klecak,G.;Moldenhauer,F.;Morre,L.;Pape,W.J.W.;Pfannenbecker,U.;Potthast,J.;De Silva,O.;Steiling,W.;Willshaw,A.;A.Raugier;A.M.Goldberg;H.I.Maibach(eds.)