title: Keratinocyte ATP release assay for testing skin-irritating potentials of structurally diverse chemicals.
abstract: Irritant dermatitis represents innate inflammatory responses to toxic chemicals. We have reported recently that ATP released from chemically injured keratinocytes may serve as a causative mediator for irritant dermatitis. In this study, we examined whether ATP release from keratinocytes would serve as a reliable readout for predicting skin irritating potentials of structurally diverse compounds. A vast majority (19/20) of the tested compounds, i.e., strong and weak irritant chemicals selected from the literature, induced rapid (<10 min) and significant (P<0.05) ATP release from Pam 212 keratinocytes. Two compounds caused no detectable skin inflammation in our standard mouse model, documenting relatively high sensitivity (false negative rate of 0/18) and specificity (false positive rate of 1/20) of our ATP release assay. Selected compounds, primarily those containing phenol residues or hydrophobic hydrocarbon chains, triggered rapid (<10 min) and robust leakage of a fluorescence probe from liposomes, suggesting that lipid bilayers serve as one, but not the only, target moiety on keratinocytes. Not only do our data support the pathogenic role for keratinocyte-derived ATP in irritant dermatitis, they also form the basis for a formal validation study to evaluate the utility of the keratinocyte-based in vitro assay in screening environmental and industrial chemicals.
authors.affiliation: Department of Dermatology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
authors.forename: Norikatsu
authors.initials: N
authors.lastname: Mizumoto
authors.forename: Mark E
authors.initials: ME
authors.lastname: Mummert
authors.forename: David
authors.initials: D
authors.lastname: Shalhevet
authors.forename: Akira
authors.initials: A
authors.lastname: Takashima
mesh_terms: D000251:Adenosine Triphosphatases / Q000502:physiology; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D015703:Antigens, CD / Q000502:physiology; D001081:Apyrase; D002118:Calcium / Q000378:metabolism; D006241:Haptens / Q000494:pharmacology; D007509:Irritants / Q000494:pharmacology*; D015603:Keratinocytes / Q000187:drug effects* / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D012867:Skin / Q000187:drug effects*
chemical_list: D015703:Antigens, CD; D006241:Haptens; D007509:Irritants; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D001081:Apyrase; C067869:CD39 antigen; D002118:Calcium