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• W2034018732 abstract "We have studied the effect of various detergents on keratinocyte gene expression in vitro, using an anionic detergent (sodium dodecyl sulfate), a cationic detergent cetyltrimethylammoniumbromide (CTAB), and two nonionic detergents, Nonidet P-40 and Tween-20. We measured the effect of these detergents on direct cellular toxicity (lactate dehydrogenase release), on the expression of markers for normal differentiation (cytokeratin 1 and involucrin expression), and on disturbed keratinocyte differentiation (SKALP) by nothern blot analysis. As reported in other studies, large differences were noted in direct cellular toxicity. In a culture model that mimics normal epidermal differentiation we found that low concentrations of sodium dodecyl sulfate could induce the expression of SKALP, a proteinase inhibitor that is not normally expressed in human epidermis but is found in hyperproliferative skin. Sodium dodecyl sulfate caused upregulation of involucrin and downregulation of cytokeratin 1 expression, which is associated with the hyperproliferative/inflammatory epidermal phenotype found in psoriasis, wound healing, and skin irritation. These changes were not induced after treatment of cultures with CTAB, Triton X-100, and Nonidet-P40. This effect appeared to be specific for the class of anionic detergents because sodium dodecyl benzene sulfonate and sodium laurate also induced SKALP expression. These in vitro findings showed only a partial correlation with the potential of different detergents to induce clinical, biophysical, and cell biologic changes in vivo in human skin. Both sodium dodecyl sulfate and CTAB were found to cause induction and upregulation of SKALP and involucrin at low doses following a 24 h patch test, whereas high concentrations of Triton X-100 did not. Sodium dodecyl sulfate induced higher rates of transepidermal water loss, whereas CTAB treated skin showed more signs of cellular toxicity. We conclude that the action of anionic detergents on epidermal keratinocytes is qualitatively different from the other detergents tested, which might have implications for in vitro toxicology studies that use cell biologic parameters as a read-out. We would hypothesize that detergents cause skin injury by several mechanisms that include direct cellular toxicity, disruption of barrier function, and detergent specific effects on cellular differentiation, as demonstrated here for sodium dodecyl sulfate, sodium dodecyl benzene sulfonate, and sodium laurate. We have studied the effect of various detergents on keratinocyte gene expression in vitro, using an anionic detergent (sodium dodecyl sulfate), a cationic detergent cetyltrimethylammoniumbromide (CTAB), and two nonionic detergents, Nonidet P-40 and Tween-20. We measured the effect of these detergents on direct cellular toxicity (lactate dehydrogenase release), on the expression of markers for normal differentiation (cytokeratin 1 and involucrin expression), and on disturbed keratinocyte differentiation (SKALP) by nothern blot analysis. As reported in other studies, large differences were noted in direct cellular toxicity. In a culture model that mimics normal epidermal differentiation we found that low concentrations of sodium dodecyl sulfate could induce the expression of SKALP, a proteinase inhibitor that is not normally expressed in human epidermis but is found in hyperproliferative skin. Sodium dodecyl sulfate caused upregulation of involucrin and downregulation of cytokeratin 1 expression, which is associated with the hyperproliferative/inflammatory epidermal phenotype found in psoriasis, wound healing, and skin irritation. These changes were not induced after treatment of cultures with CTAB, Triton X-100, and Nonidet-P40. This effect appeared to be specific for the class of anionic detergents because sodium dodecyl benzene sulfonate and sodium laurate also induced SKALP expression. These in vitro findings showed only a partial correlation with the potential of different detergents to induce clinical, biophysical, and cell biologic changes in vivo in human skin. Both sodium dodecyl sulfate and CTAB were found to cause induction and upregulation of SKALP and involucrin at low doses following a 24 h patch test, whereas high concentrations of Triton X-100 did not. Sodium dodecyl sulfate induced higher rates of transepidermal water loss, whereas CTAB treated skin showed more signs of cellular toxicity. We conclude that the action of anionic detergents on epidermal keratinocytes is qualitatively different from the other detergents tested, which might have implications for in vitro toxicology studies that use cell biologic parameters as a read-out. We would hypothesize that detergents cause skin injury by several mechanisms that include direct cellular toxicity, disruption of barrier function, and detergent specific effects on cellular differentiation, as demonstrated here for sodium dodecyl sulfate, sodium dodecyl benzene sulfonate, and sodium laurate. cationic detergent cetyltrimethylammoniumbromide Exposure of the skin to irritants can induce various physiologic and cell biologic changes (Willis et al., 1990Willis C.M. Stephens C.J. Wilkinson J.D. Differential effects of structurally unrelated chemical irritants on the density and morphology of epidermal CD1+ cells.J Invest Dermatol. 1990; 95: 711-716Abstract Full Text PDF PubMed Google Scholar, Willis et al., 1993Willis C.M. Stephens C.J. Wilkinson J.D. Differential patterns of epidermal leukocyte infiltration in patch test reactions to structurally unrelated chemical irritants.J Invest Dermatol. 1993; 101: 364-370Abstract Full Text PDF PubMed Google Scholar, Willis et al., 1991Willis C.M. Stephens C.J. Wilkinson J.D. Selective expression of immune-associated surface antigens by keratinocytes in irritant contact dermatitis.J Invest Dermatol. 1991; 96: 505-511Abstract Full Text PDF PubMed Google Scholar). Application of irritants disrupts the barrier function of the horny layer that leads to activation of the keratinocyte. High concentrations of irritants may lead to direct cytotoxicity or necrosis as assessed by morphologic and biochemical criteria for cell death. Furthermore, application of irritants may lead to an inflammatory process and a cellular infiltrate of polymorphonuclear leucocytes and mononuclear cells (Andersen et al., 1987Andersen K.E. Benezra C. Burrows D. et al.Contact dermatitis. A review.Contact Dermatitis. 1987; 16: 55-78Crossref PubMed Scopus (62) Google Scholar, Thestrup Pedersen et al., 1989Thestrup Pedersen K. Larsen C.G. Ronnevig J. The immunology of contact dermatitis. A review with special reference to the pathophysiology of eczema.Contact Dermatitis. 1989; 20: 81-92Crossref Scopus (26) Google Scholar), depending on the strength of the detergent stimulus (Willis et al., 1991Willis C.M. Stephens C.J. Wilkinson J.D. Selective expression of immune-associated surface antigens by keratinocytes in irritant contact dermatitis.J Invest Dermatol. 1991; 96: 505-511Abstract Full Text PDF PubMed Google Scholar). These actions cannot be strictly separated from a mechanistic point of view because they are interdependent. Disruption of the barrier function is associated with loss of cohesion of corneocytes and desquamation and with increasing transepidermal water loss (TEWL), and consequently dehydration of the skin. The skin reacts through changes that are aimed at restoration of the epidermal barrier function. These changes include upregulation of differentiation associated proteins (e.g., involucrin) (Le et al., 1996Le T.K. Schalkwijk J. van Siegenthaler G. de Kerkhof P.C.M. Veerkamp J.H. van der Valk P.G.M. Changes in keratinocyte differentiation following mild irritation by sodium dodecyl sulphate.Arch Dermatol Res. 1996; 288: 684-690Crossref PubMed Scopus (47) Google Scholar), increased keratinocyte proliferation in the basal layer (Proksch et al., 1993Proksch E. Holleran W.M. Menon G.K. Elias P.M. Feingold K.R. Barrier function regulates epidermal lipid and DNA synthesis.Br J Dermatol. 1993; 128: 473-482Crossref PubMed Scopus (193) Google Scholar), increased lipid synthesis (Proksch et al., 1993Proksch E. Holleran W.M. Menon G.K. Elias P.M. Feingold K.R. Barrier function regulates epidermal lipid and DNA synthesis.Br J Dermatol. 1993; 128: 473-482Crossref PubMed Scopus (193) Google Scholar), and a strong upregulation of epidermal fatty acid binding protein (Le et al., 1996Le T.K. Schalkwijk J. van Siegenthaler G. de Kerkhof P.C.M. Veerkamp J.H. van der Valk P.G.M. Changes in keratinocyte differentiation following mild irritation by sodium dodecyl sulphate.Arch Dermatol Res. 1996; 288: 684-690Crossref PubMed Scopus (47) Google Scholar). This sequence of events finally leads to a new steady state of the cutaneous barrier. Several mediator systems have been implicated in the process of epidermal changes and inflammation. The release of cytokines by activated keratinocytes could be considered as a potential marker for irritation. Upon irritation and/or barrier disruption [e.g., by sodium dodecyl sulfate (SDS), acetone, or tape stripping], but also under pathologic conditions (e.g., psoriasis), enhanced production of pro-inflammatory cytokines on both protein and mRNA levels has been reported (Enk and Katz, 1992bEnk A.H. Katz S.I. Early molecular events in the induction phase of contact sensitivity.Proc Natl Acad Sci USA. 1992; 89: 1398-1402Crossref PubMed Scopus (582) Google Scholar; Nickoloff and Naidu, 1994Nickoloff B.J. Naidu Y. Perturbation of epidermal barrier function correlates with initiation of cytokine cascade in human skin.J Am Acad Dermatol. 1994; 30: 535-546Abstract Full Text PDF PubMed Scopus (408) Google Scholar; Wood et al., 1996Wood L.C. Elias P.M. Calhoun C. Tsai J.C. Grunfeld C. Feingold K.R. Barrier disruption stimulates interleukin-1 alpha expression and release from a pre-formed pool in murine epidermis.J Invest Dermatol. 1996; 106: 397-403Crossref PubMed Scopus (231) Google Scholar). Other investigations have focused on the expression of integrins and intercellular adhesion molecule-1 (ICAM-1). Under inflammatory conditions with involvement of the epidermis, and in wound healing models, upregulation and suprabasal expression of α2β1, α3β1, α6β4, αvβ5, and induction of α5β1 has been demonstrated (Adams and Watt, 1991Adams J.C. Watt F.M. Expression of β1 β3 β4, and β5 integrins by human epidermal keratinocytes and non-differentiating keratinocytes.J Cell Biol. 1991; 115: 829-841Crossref PubMed Scopus (159) Google Scholar). In line with these findings ICAM-1, a specific ligand for β2 integrins, is expressed by keratinocytes in vivo in irritant contact dermatitis (Griffiths and Nickoloff, 1989Griffiths C.E. Nickoloff B.J. Keratinocyte intercellular adhesion molecule-1 (ICAM-1) expression precedes dermal T lymphocytic infiltration in allergic contact dermatitis (Rhus dermatitis).Am J Pathol. 1989; 135: 1045-1053PubMed Google Scholar; Griffiths et al., 1990Griffiths C.E. Esmann J. Fisher G.J. Voorhees J.J. Nickoloff B.J. Differential modulation of keratinocyte intercellular adhesion molecule-I expression by gamma interferon and phorbol ester: evidence for involvement of protein kinase C signal transduction.Br J Dermatol. 1990; 122: 333-342Crossref PubMed Scopus (62) Google Scholar; Luger and Schwarz, 1990Luger T.A. Schwarz T. Evidence for an epidermal cytokine network.J Invest Dermatol. 1990; 95: 100S-104SAbstract Full Text PDF PubMed Google Scholar; Willis et al., 1991Willis C.M. Stephens C.J. Wilkinson J.D. Selective expression of immune-associated surface antigens by keratinocytes in irritant contact dermatitis.J Invest Dermatol. 1991; 96: 505-511Abstract Full Text PDF PubMed Google Scholar). This effect may be secondary, induced by the release of TNF-α (Willis et al., 1991Willis C.M. Stephens C.J. Wilkinson J.D. Selective expression of immune-associated surface antigens by keratinocytes in irritant contact dermatitis.J Invest Dermatol. 1991; 96: 505-511Abstract Full Text PDF PubMed Google Scholar). It has also been demonstrated that the plasminogen activator system is locally activated in vivo at the site of SDS induced skin irritation. The plasminogen activator system (active plasmin) is thought to exert pro-inflammatory capacity via activation of further enzyme systems, and/or via generation of chemotactic peptides and nonspecific tissue destruction (Reinartz et al., 1991Reinartz J. Boukamp P. Schickel E. Fusenig N.E. Kramer M.D. Activation of the plasminogen activator system in a keratinocyte cell line (HaCaT) by alkyl sulfates.Biomed Biochim Acta. 1991; 50: 743-748Google Scholar). In vitro SDS/alkyl sulfates are capable of strongly activating keratinocyte derived plasminogen. Irritancy tests in general are based on the concept of keratinocyte activation, i.e., that keratinocytes provide a signaling interface that transforms nonspecific harmful stimuli from the environment into endogenous signals, thereby activating local and systemic repair and defence mechanisms. These mechanisms include both hyperproliferative and inflammatory events (Kupper, 1989Kupper T.S. Mechanisms of cutaneous inflammation. Interactions between epidermal cytokines, adhesion molecules, and leukocytes [published erratum appears in Arch Dermatol 1989 Dec; 125 (12):1643].Arch Dermatol. 1989; 125: 1406-1412Crossref PubMed Scopus (134) Google Scholar; Sauder, 1989Sauder D.N. Interleukin 1.Arch Dermatol. 1989; 125: 679-682Crossref PubMed Scopus (28) Google Scholar; Luger and Schwarz, 1990Luger T.A. Schwarz T. Evidence for an epidermal cytokine network.J Invest Dermatol. 1990; 95: 100S-104SAbstract Full Text PDF PubMed Google Scholar). Keratinocyte activation is thought to initiate a humoral cascade originating from immediate release of preformed key mediators such as arachidonic acid and its metabolites and IL-1α. These mediators in turn trigger the biosynthesis and release of other cytokines and proinflammatory mediators (Kupper, 1989Kupper T.S. Mechanisms of cutaneous inflammation. Interactions between epidermal cytokines, adhesion molecules, and leukocytes [published erratum appears in Arch Dermatol 1989 Dec; 125 (12):1643].Arch Dermatol. 1989; 125: 1406-1412Crossref PubMed Scopus (134) Google Scholar; Griffiths et al., 1990Griffiths C.E. Esmann J. Fisher G.J. Voorhees J.J. Nickoloff B.J. Differential modulation of keratinocyte intercellular adhesion molecule-I expression by gamma interferon and phorbol ester: evidence for involvement of protein kinase C signal transduction.Br J Dermatol. 1990; 122: 333-342Crossref PubMed Scopus (62) Google Scholar; Barker et al., 1991Barker J.N. Mitra R.S. Griffiths C.E. Dixit V.M. Nickoloff B.J. Keratinocytes as initiators of inflammation [see comments].Lancet. 1991; 337: 211-214Abstract PubMed Scopus (609) Google Scholar; Hunziker et al., 1992Hunziker T. Brand C.U. Kapp A. Waelti E.R. Braathen L.R. Increased levels of inflammatory cytokines in human skin lymph derived from sodium lauryl sulphate-induced contact dermatitis.Br J Dermatol. 1992; 127: 254-257Crossref PubMed Scopus (65) Google Scholar). The different effects of irritants on keratinocytes are difficult to disentangle in vivo, and we have therefore applied an in vitro culture model to study the direct effects of irritants on keratinocytes without the involvement of inflammatory cells, and without the effects secondary to barrier disruption. Furthermore, in vitro irritants do not have to pass the skin barrier and bioavailability is not variable. An in vitro model allows discrimination between keratinocyte gene expression and cytotoxicity. Finally, environmental conditions can be controlled, a high degree of reproducibility can be obtained, and a wide range of concentrations of various test agents can be applied. In this study we compared the potential of different detergents to induce the hyperproliferative/inflammatory differentiation phenotype (as described by van Ruissen et al., 1996van Ruissen F. de Jongh G.J. Zeeuwen P.L. van Erp P.E. Madsen P. Schalkwijk J. Induction of normal and psoriatic phenotypes in submerged keratinocyte cultures.J Cell Physiol. 1996; 168: 442-452Crossref PubMed Scopus (59) Google Scholar). Using keratinocyte cultures three different classes of detergents were evaluated: e.g., anionic detergents [SDS, sodium dodecyl benzene sulfonate (SDBS), and sodium laurate (SL)]; cationic detergents [cetyltrimethyl-ammoniumbromide (CTAB)]; and nonionic detergents [sorbitan mono-oleate (Tween-20), nonylphenyl-polyethylene glycol (Nonidet-P40), and polyethylene glycol tert-octylphenyl ether (Triton X-100)]. The in vitro effects of these detergents were measured on direct cellular toxicity [lactate dehydrogenase (LDH) release] and on the expression of several markers for normal differentiation [cytokeratin 1 (CK1) and involucrin]. As an inducible marker for disturbed differentiation we used the expression of the protease inhibitor SKALP/elafin, a member of the recently described Trappin gene family (Zeeuwen et al., 1997Zeeuwen Pljm Hendriks W. de Jong W.W. Schalkwijk J. Identification and sequence analysis of two new members of the SKALP/elafin and SPAI-2 gene family.J Biol Chem. 1997; 272: 20471Crossref PubMed Scopus (51) Google Scholar). Furthermore, induction of inflammation in vivo was evaluated by the assessment of erythema and TEWL. In addition, expression of involucrin and SKALP was evaluated immunohistochemically. Our results reveal differential effects of detergents on cell biologic markers, which stresses the complexity of irritant reactions in vivo. Keratinocytes were seeded in 6 well culture dishes and cultured until confluence in keratinocyte growth medium (KGM) as described before (van Ruissen et al., 1996van Ruissen F. de Jongh G.J. Zeeuwen P.L. van Erp P.E. Madsen P. Schalkwijk J. Induction of normal and psoriatic phenotypes in submerged keratinocyte cultures.J Cell Physiol. 1996; 168: 442-452Crossref PubMed Scopus (59) Google Scholar). KGM was composed of keratinocyte basal medium (Biowhittaker, Walkersville, MD; 0.15 mM calcium), supplemented with ethanolamine (0.1 mM) (Sigma, St. Louis, MO), phosphoethanolamine (0.1 mM) (Sigma), bovine pituitary extract (0.4% vol/vol) (Biowhittaker, Walhusville, MD), insulin (5 μg per ml) (Sigma), hydrocortisone (Collaborative Research, Lexington, MA), mouse epidermal growth factor (10 ng per ml) (Sigma), penicillin (100 U per ml) (Gibco, Breda, the Netherlands), and streptomycin (100 μg per ml) (Gibco). At confluence, keratinocyte differentiation was established by switching the cells to KGM supplemented with 5% vol/vol fetal calf serum (Seralab, Nistelrode, the Netherlands) (KGM/FCS) or to KGM depleted of growth factors (bovine pituitary extract, insulin, and epidermal growth factor) and hydrocortisone (KGM/–GF) for 48 h. Addition of KGM/FCS induced a hyperproliferative/inflammatory differentation marked by strong expresssion of SKALP, involucrin, and transglutaminase and absence of CK1 expression. Cultures grown in KGM/–GF exhibited a normal differentiation characterized by expression of CK1, involucrin, and transglutaminase and the absence of SKALP expression (van Ruissen et al., 1996van Ruissen F. de Jongh G.J. Zeeuwen P.L. van Erp P.E. Madsen P. Schalkwijk J. Induction of normal and psoriatic phenotypes in submerged keratinocyte cultures.J Cell Physiol. 1996; 168: 442-452Crossref PubMed Scopus (59) Google Scholar). Detergents were added at different concentrations to KGM/–GF. After 48 h of incubation, media were collected, cultures were washed twice with phosphate-buffered saline, and total RNA was isolated. The following detergents were applied: SDS, Triton X-100, Tween-20 (Biorad Laboratories, Richmond, CA), Nonidet P40 (BDH Chemicals, Poole, U.K.), CTAB, SDBS, and LS (Sigma). The LDH release was measured in media of cultured human keratinocytes treated with detergents. Aliqouts of 100 μl media were transferred to a 96 microtiter plate and mixed with 100 μl reaction mixture (Boehringer, Mannheim, Germany). After 30 min incubation, performed in the dark at room temperature, the absorbance of the formazan salt (red) was measured at 495 nm. As a reference wavelength we used 655 nm. LDH release was measured at different time points during treatment (0, 3, 6, and 24 h after treatment). To exclude errors due to the substances, we performed a control for both LDH activity contained within the test substance and whether the substance itself interfered with the LDH activity. Cytotoxicity measurements were perfomed in triplicate, the values were calculated as suggested by the manufacturer, including a background control for LDH activity in the media, a control for untreated cells (0% lysis of the cells), and a control for maximum LDH release (100% lysis of the cells). Cells were lysed in 1 ml RNase-All [2.1 M Guanidine-thiocyanate (Research Organics, Cleveland, OH), 8.5 mM N-lauroylsarcosine (Sigma), 12.5 mM NaAc pH 5.2, 0.35% vol/vol β-mercaptoethanol (Merck, Darmstadt, Germany), and 50% vol/vol Tris-saturated biophenol pH 8.0 (Biosolve, Amsterdam, the Netherlands)]. After lysis, 100 μl chloroform was added. The samples were centrifuged for 15 min (13000 rpm, 4°C) and the aqueous phase was precipitated with 500 μl isopropanol on ice for 45 min, and subsequently centrifuged at 4°C for 15 min. The pellet was washed with 70% ethanol and dried at room temperature for 5 min. This RNA pellet was resuspended in 150 μl NSE (50 mM NaAc, 0.2% SDS, and 2 mM ethylenediamine tetraacetic acid) and 562.5 μl 100% ethanol was added. For quantitation 62.5 μl of this RNA suspension was pelleted, resuspended in 1 ml sterile H2O, and spectrophotometrically analyzed at 260/280 nm. Equal quantities of this total RNA (10 μg) were loaded on a 1% agarose gel and electrophoretically separated in 10 mM sodium phosphate buffer (Sambrook et al., 1989Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning. A Laboratory Manual. Cold Sprin Harbor Laboratory Press, New York1989Google Scholar). The pH of the electrophoresis buffer was kept within acceptable limits by constant circulation of the buffer. The gels were blotted on positively charged nylon membranes (Boehringer) using 10 × sodium citrate/chloride buffer (1.5 M NaCl, 0.15 M NaCitrate). After RNA transfer, the membranes were washed in 2 × sodium citrate/chloride buffer, and the RNA was cross-linked to the membrane using ultraviolet irradiation (312 nm, 0.2 J per cm2). Subsequently, blots were stained with ethidium bromide dissolved in sterile water to visualize the RNA. The blots were (pre)-hybridized overnight at 65°C as described by Church and Gilbert, 1984Church G.M. Gilbert W. Genomic sequencing.Proc Natl Acad Sci USA. 1984; 81: 1991-1994Crossref PubMed Scopus (7202) Google Scholar using random labeled probes for CK1, SKALP, involucrin, and human acidic ribosomal phosphoprotein PO (hARP), which functions as a control to quantitate the amount of total RNA loaded in each lane (Laborda, 1991Laborda J. 36B4 cDNA used as an estradiol-independent mRNA control is the cDNA for human acidic ribosomal phosphoprotein PO.Nucl Acids Res. 1991; 19 (3998): 3998Crossref PubMed Scopus (427) Google Scholar). Autoradiography was performed using Kodak X-Omat X-ray films and Kodak Biomax MS films (Eastman Kodak, Rochester, NY). A group of six healthy volunteers with no past or present history of skin disease participated in the study. Their ages ranged from 21 to 27 y, and all subjects gave written informed consent. Detergents were applied (day 0) for 24 h using a patch test. Aliquots of 200 μl detergent solutions were pipetted on to several patches as previously described (Le et al., 1996Le T.K. Schalkwijk J. van Siegenthaler G. de Kerkhof P.C.M. Veerkamp J.H. van der Valk P.G.M. Changes in keratinocyte differentiation following mild irritation by sodium dodecyl sulphate.Arch Dermatol Res. 1996; 288: 684-690Crossref PubMed Scopus (47) Google Scholar). Patches were applied to the skin of the upper back parallel to the vertebral column. The detergents were applied at the following concentrations: SDS, 1%; CTAB, 2%; and Triton-X100, 10%. Visual examination and TEWL measurements were performed at 1 h (day 1) and 24 h (day 2) after removal of the patches. The erythema was graded using the following visual scoring; 1, mild patchy erythema; 2, diffuse mild erythema; 3, moderate erythema; 4, intense erythema; and 5, intense erythema with edema. TEWL measurements were performed using a Tewameter TM 210 (Courage & Khazaka, Germany), according to the standard guidelines (Pinnagoda et al., 1990Pinnagoda J. Tupker R.A. Agner T. Serup J. Guidelines for transepidermal water loss (TEWL) measurement. A report from the Standardization Group of the European Society of Contact Dermatitis.Contact Dermatitis. 1990; 22: 164-178Crossref PubMed Scopus (1009) Google Scholar). Before the actual assessments, a delay period of 15 min was introduced to reduce the effects of sweating (Baker and Kligman, 1967Baker H. Kligman A.M. Measurement of transepidermal water loss by electrical hygrometry. Instrumentation and responses to physical and chemical insults.Arch Dermatol. 1967; 96: 441-452Crossref PubMed Scopus (87) Google Scholar). During the measurements the room temperature was kept at a constant temperature of 20°C. Relative humidity varied from 35% to 49% (mean 39.3%). Punch biopsies (3 mm in diameter) were taken from each of the patch tests. A maximum of two biopsies per volunteer were taken and a total of 12 biopsies were obtained per time point. After 4 h of fixation in formalin the samples were embedded in paraffin, sectioned at 6 μm, and prepared for immunohistochemistry. Sections were deparaffinized in xylene for 20 min, followed by rehydration through an ethanol series ranging from 100% to 50%, and a final wash in phosphate-buffered saline during 10 min. Sections stained for anti-involucrin received a pretreatment with citrate buffer and needed antigen retrieval in the microwave oven. Additionally, sections were preincubated for 15 min with 20% normal rabbit serum for anti-involucrin (MON-150) or 20% normal swine serum for anti-SKALP staining. Sections were incubated for 60 min with anti-involucrin (MON-150; 1:15) and anti-SKALP (SSK-9201; 1:500) diluted in phosphate-buffered saline/azide containing 1% bovine serum albumin. Finally, sections were incubated for 30 min with either peroxidase-conjugated rabbit anti-mouse or peroxidase-conjugated swine anti-rabbit immunoglobulins diluted in phosphate-buffered saline containing 5% human serum. All sections were developed using 3-amino-9-ethylcarbazol as chromogenic substrate for 10 min at 37°C. Stained sections were washed twice with demineralised water and mounted in glycerol-gelatine. Keratinocyte gene expression is induced in vitro after application of detergentsDetergents were tested during a period of 48 h on confluent keratinocyte cultures in a protein free medium (KGM/–GF). Using northern blot analysis we evaluated the effect of detergents on keratinocyte gene expression as described previously (van Ruissen et al., 1996van Ruissen F. de Jongh G.J. Zeeuwen P.L. van Erp P.E. Madsen P. Schalkwijk J. Induction of normal and psoriatic phenotypes in submerged keratinocyte cultures.J Cell Physiol. 1996; 168: 442-452Crossref PubMed Scopus (59) Google Scholar). The concentrations of detergents ranged from 0 μg per ml to 50 μg per ml for SDS, from 0 μg per ml to 3 μg per ml for CTAB, Nonidet-P40, and Triton X-100, and from 0 mg per ml to 3 mg per ml for Tween-20. We used gene expression induced by KGM/FCS (hyperproliferative phenotype) and KGM/-GF (normal phenotype) as a positive and negative control, respectively. Northern blot analyses of keratinocyte cultures treated with five different detergents are summarized in Figure 1. Addition of SDS leads to a strong induction of SKALP expression, upregulation of involucrin expression, and downregulation of CK1 as illustrated in Figure 1d. The induction of SKALP expression was found in a narrow concentration range between 3.13 and 6.25 μg per ml. At concentrations of 12 μg per ml and higher, cytotoxicity was induced and RNA isolated from these cultures was totally degraded as shown by the absence of any positive signal of the control household gene (hARP). The strong induction of SKALP and the downregulation of CK1 appears to be coupled. This demonstrates the switch from a normal differentiation program where SKALP is absent and CK1 is highly expressed, to a hyperproliferative/inflammatory differentiation where CK1 expression is downregulated and SKALP expression is strongly upregulated. The strong induction of SKALP expression and the changes in involucrin and CK1 expression are similar to those found under conditions of regenerative maturation, as described by Mansbridge and Knapp, 1987Mansbridge J.N. Knapp A.M. Changes in keratinocyte maturation during wound healing.J Invest Dermatol. 1987; 89: 253-263Abstract Full Text PDF PubMed Google Scholar. Examination of the northern blots demonstrates that CTAB, Triton X-100, Nonidet-P40, and Tween-20 do not induce changes in SKALP, involucrin, or CK1 expression, compared with the control medium without detergent. To investigate if the induction of the hyperproliferative/inflammatory gene expression is due to the physicochemical properties of anionic detergents, we examined the induction of SKALP expression by other anionic detergents, e.g., SDBS and SL. Figure 2 illustrates the induction of SKALP expression by SDBS. SDBS and LS induce SKALP expression in a similar concentration range as found for SDS. SKALP expression is induced at concentrations ranging from 3.13 μg per ml to 6.25 μg per ml. In addition, involucrin expression showed the same expression pattern as described by SDS. Using SDBS we see involucrin expression at concentrations ranging from 1.56 μg per ml to 6.25 μg per ml. Also the toxicity profile was simi" @default.
• W2034018732 created "2016-06-24" @default.
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• W2034018732 date "1998-04-01" @default.
• W2034018732 modified "2023-09-27" @default.
• W2034018732 title "Differential Effects of Detergents on Keratinocyte Gene Expression" @default.
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• W2034018732 doi "https://doi.org/10.1046/j.1523-1747.1998.00155.x" @default.
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