RESUMEN
Acetaminophen, a safe analgesic when dosed properly but hepatotoxic at overdoses, has been reported to induce DNA strand breaks but it is unclear whether this event preceeds hepatocyte toxicity or is only obvious in case of overt cytotoxicity. Moreover, it is not known whether the formation of reactive oxygen species (ROS) is involved in the formation of the DNA strand breaks. In the present study, the dose-response curves for cytotoxicity and DNA strand breaks and the response to antioxidant protection have been compared. In primary hepatocytes from untreated male rats, cytotoxicity as measured by the MTT test and by Neutral Red accumulation was obvious at 10 mM acetaminophen but DNA strand breaks as measured by the comet assay were only found at 25-30 mM acetaminophen. Non-cytotoxic concentrations of three compounds with antioxidant activity, the glutathione precursor N-acetylcysteine (100 micro M), the plant polyphenol silibin (25 micro M) and the antioxidant vitamin alpha-tocopherol (50 micro M), were not able to inhibit acetaminophen toxicity at any acetaminophen concentration, while they completely prevented the formation of DNA strand breaks at 25-30 mM acetaminophen. The occurrence of oxidative stress in our experiments was indicated by a slight increase of malondialdehyde formation at 40 mM acetaminophen and by an adaptive increase in catalase mRNA concentration. We conclude that in acetaminophen-treated hepatocytes ROS-independent cell death and ROS-dependent DNA strand breaks occur which appear not to be causally related as judged from their dose dependency and their response to antioxidants.
Asunto(s)
Acetaminofén/toxicidad , Analgésicos no Narcóticos/toxicidad , Antioxidantes/farmacología , Enfermedad Hepática Inducida por Sustancias y Drogas , Daño del ADN , Acetilcisteína/farmacología , Animales , Northern Blotting , Muerte Celular/efectos de los fármacos , Ensayo Cometa , Relación Dosis-Respuesta a Droga , Formazáns/metabolismo , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Peróxidos Lipídicos/metabolismo , Hepatopatías/genética , Masculino , Rojo Neutro/metabolismo , ARN/química , ARN/genética , Ratas , Ratas Wistar , Silibina , Silimarina/farmacología , Sales de Tetrazolio/metabolismo , alfa-Tocoferol/farmacologíaRESUMEN
The human calcium-binding protein (hS100A15) was first identified in inflamed hyperplastic psoriatic skin, where the S100A15 gene is transcribed into two mRNA splice variants, hS100A15-S and hS100A15-L. To compare the contribution of the human S100A15 (hS100A15) isoforms in skin inflammation and differentiation, we determined the expression, distribution and regulation of hS100A15-S and hS100A15-L in psoriasis and chronic atopic eczema compared with normal skin. We found that both hS100A15 transcripts were mainly distributed in the epidermis of normal and inflamed skin with hS100A15-L being the predominantly expressed mRNA isoform in both psoriasis and atopic eczema. In cultured keratinocytes, IL-1beta and Th1 cytokines significantly induced hS100A15-L compared with hS100A15-S. In contrast, Th2-derived cytokines had no influence on the expression of either hS100A15 splice variant. Differentiation of human keratinocytes induced by 1.2 mm calcium resulted in the upregulation of both hS100A15 mRNA isoforms. Our data show that both hS100A15 splice variants are differentially regulated and expressed with epidermal differentiation and skin inflammation. Overexpression of hS100A15 in chronic inflammatory skin diseases and regulation by inflammatory cytokines and calcium suggest that hS100A15 is involved in Th1-associated epithelial responses and epidermal maturation in normal and diseased human skin.
Asunto(s)
Dermatitis Atópica/inmunología , Isoformas de Proteínas/inmunología , Psoriasis/inmunología , Proteínas S100/genética , Células TH1/inmunología , Calcio/metabolismo , Células Cultivadas , Dermatitis Atópica/metabolismo , Epidermis/inmunología , Epidermis/metabolismo , Regulación de la Expresión Génica/inmunología , Humanos , Interleucina-1beta/metabolismo , Queratinocitos/citología , Queratinocitos/inmunología , Isoformas de Proteínas/metabolismo , Psoriasis/metabolismo , ARN Mensajero/metabolismo , Proteína A7 de Unión a Calcio de la Familia S100 , Proteínas S100/metabolismoRESUMEN
E. coli is a gram-negative bacterium rarely found on human skin. We investigated whether direct interaction of E. coli with keratinocytes might induce an innate immune response through recognition by pattern recognition receptors. The capacity of E. coli to activate innate immune responses and IL-8 induction was investigated. We found that E. coli significantly induced human S100A7 and S100A15 transcript abundance and IL-8 release in cultured primary human keratinocytes. S100A15 is a member of the S100 protein family with previously unknown function. E. coli induced effects could be inhibited by neutralizing Toll-like receptor 4 (TLR4) antibodies, suggesting that E. coli-induced IL-8 and S100A15 expression in human keratinocytes are TLR4 dependent. TLR4-/- mice lacked elevated mS100A15 expression after infection with E. coli in contrast to wild-type mice. In vitro, human S100A15 displayed antimicrobial activity against E. coli. Our findings suggest that E. coli modulates S100A15 and IL-8 expression of keratinocytes by recognition through TLR4.