RESUMEN
Skin sensitization assessment has progressed from the use of animal models towards the application of New Approach Methodologies (NAMs). Several skin sensitization NAMs are accepted for regulatory use, but a majority relies on submerged in vitro cell cultures that limit their applicability domain, posing challenges for testing hydrophobic chemicals and mixtures. A newly developed three-dimensional (3D) Nrf2 reporter epidermis model for skin sensitization assessment is reported. This NAM may help to overcome these limitations. The NAM combines the in vivo-like biology and exposure conditions of 3D epidermis models with the reliability, convenience, and cost-effectiveness of secreted reporter gene technology. The Keap1-Nrf2-ARE pathway was chosen as the reporter gene read-out, as it is induced by most skin sensitizers and already adopted in OECD Test guideline 442D. Immortalized human primary keratinocytes (Ker-CT) were stably transfected with the pIGB-Nrf2-SEAP vector to construct a Nrf2 reporter cell line. Ker-CT Nrf2 reporter cells showed negligible basal expression of the Secreted Embryonic Alkaline Phosphatase (SEAP) reporter, which was induced 13.5-fold by exposure to the skin sensitizer cinnamic aldehyde (CA). Co-exposure to CA and the Nrf2 inhibitor glucocorticoid clobetasol propionate significantly suppressed the CA-induced SEAP expression, confirming dependance of the SEAP expression on Nrf2 activation. Using air-liquid interface and animal constituent free culture conditions, the Ker-CT Nrf2 reporter cells differentiated to stratified 3D epidermis models with an in vivo-like skin architecture and functional skin barrier. Evaluation of a Ker-CT Nrf2 reporter cell-based 2D assay by testing 10 conventional reference chemicals showed a predictive accuracy for skin sensitization potential of 80% and 70% compared to LLNA and human data in two independent laboratories and a high intra- and interlaboratory reproducibility. Moreover, the 3D epidermis models predicted 3 sensitizing and 2 non-sensitizing reference chemicals correctly in a first proof-of-concept study. Further investigations foresee the testing of additional chemicals, including hydrophobic compounds and mixtures to confirm the potential of the 3D epidermis models to broaden the applicability domain for NAM-based skin sensitization assessment.
Asunto(s)
Dermatitis Alérgica por Contacto , Factor 2 Relacionado con NF-E2 , Animales , Humanos , Factor 2 Relacionado con NF-E2/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Reproducibilidad de los Resultados , Epidermis/metabolismo , Queratinocitos/metabolismo , Piel/metabolismo , Alternativas a las Pruebas en Animales , Ensayo del Nódulo Linfático LocalRESUMEN
A suite of in vitro assays and in silico models were evaluated to identify which best detected the endocrine-disrupting (ED) potential of 10 test chemicals according to their estrogenic, androgenic and steroidogenic (EAS) potential compared to the outcomes from ToxCast. In vitro methods included receptor-binding, CALUX transactivation, H295R steroidogenesis, aromatase activity inhibition and the Yeast oestrogen (YES) and Yeast androgen screen (YAS) assays. The impact of metabolism was also evaluated. The YES/YAS assays exhibited a high sensitivity for ER effects and, despite some challenges in predicting AR effects, is a good initial screening assay. Results from receptor-binding and CALUX assays generally correlated and were in accordance with classifications based on ToxCast assays. ER agonism and AR antagonism of benzyl butyl phthalate were abolished when CALUX assays included liver S9. In silico final calls were mostly in agreement with the in vitro assays, and predicted ER and AR effects well. The efficiency of the in silico models (reflecting applicability domains or inconclusive results) was 43-100%. The percentage of correct calls for ER (50-100%), AR (57-100%) and aromatase (33-100%) effects when compared to the final ToxCast call covered a wide range from highly reliable to less reliable models. In conclusion, Danish (Q)SAR, Opera, ADMET Lab LBD and ProToxII models demonstrated the best overall performance for ER and AR effects. These can be combined with the YES/YAS assays in an initial screen of chemicals in the early tiers of an NGRA to inform on the MoA and the design of mechanistic in vitro assays used later in the assessment. Inhibition of aromatase was best predicted by the Vega, AdmetLab and ProToxII models. Other mechanisms and exposure should be considered when making a conclusion with respect to ED effects.
Asunto(s)
Andrógenos , Disruptores Endocrinos , Andrógenos/metabolismo , Andrógenos/farmacología , Estrógenos/farmacología , Aromatasa , Saccharomyces cerevisiae/metabolismo , Receptores Androgénicos/metabolismo , Estrona , Disruptores Endocrinos/químicaRESUMEN
Skin metabolism is important to consider when assessing local toxicity and/or penetration of chemicals and their metabolites. If human skin supply is limited, pig skin can be used as an alternative. To identify any species differences, we have investigated the metabolism of 10 chemicals in a pig and human skin explant model. Phase I metabolic pathways in skin from both species included those known to occur via cytochrome P450s, esterases, alcohol dehydrogenases and aldehyde dehydrogenases. Common Phase II pathways were glucuronidation and sulfation but other conjugation pathways were also identified. Chemicals not metabolized by pig skin (caffeine, IQ and 4-chloroaniline) were also not metabolized by human skin. Six chemicals metabolized by pig skin were metabolized to a similar extent (percentage parent remaining) by human skin. Human skin metabolites were also detected in pig skin incubations, except for one unidentified minor vanillin metabolite. Three cinnamyl alcohol metabolites were unique to pig skin but represented minor metabolites. There were notable species differences in the relative amounts of common metabolites. The difference in the abundance of the sulfate conjugates of resorcinol and 4-amino-3-nitrophenol was in accordance with the known lack of aryl sulfotransferase activity in pigs. In conclusion, while qualitative comparisons of metabolic profiles were consistent between pig and human skin, there were some quantitative differences in the percentage of metabolites formed. This preliminary assessment suggests that pig skin is metabolically competent and could be a useful tool for evaluating potential first-pass metabolism before testing in human-derived tissues.
Asunto(s)
Cosméticos/farmacocinética , Absorción Cutánea/efectos de los fármacos , Piel/metabolismo , Administración Cutánea , Animales , Cosméticos/farmacología , Sistema Enzimático del Citocromo P-450/metabolismo , Glucuronosiltransferasa/metabolismo , Humanos , Fase I de la Desintoxicación Metabólica , Fase II de la Desintoxicación Metabólica , Técnicas de Cultivo de Órganos , Piel/efectos de los fármacos , Piel/enzimología , Especificidad de la Especie , Especificidad por Sustrato , Sulfotransferasas/metabolismo , Porcinos , Distribución TisularRESUMEN
Partition (K) and diffusion (D) coefficients are important to measure for the modelling of skin penetration of chemicals through the stratum corneum (SC). We compared the feasibility of three protocols for the testing of 50 chemicals in our main studies, using three cosmetics-relevant model chemicals with a wide range of logP values. Protocol 1: SC concentration-depth profile using tape-stripping (measures KSC/v and DSC /HSC2 , where HSC is the SC thickness); Protocol 2A: incubation of isolated SC with chemical (direct measurement of KSC/v only) and Protocol 2B: diffusion through isolated SC mounted on a Franz cell (measures KSC/v and DSC /HSC2 , and is based on Fick's laws). KSC/v values for caffeine and resorcinol using Protocol 1 and 2B were within 30% of each other, values using Protocol 2A were ~two-fold higher, and all values were within 10-fold of each other. Only indirect determination of KSC/v by Protocol 2B was different from the direct measurement of KSC/v by Protocol 2A and Protocol 1 for 7-EC. The variability of KSC/v for all three chemicals using Protocol 2B was higher compared to Protocol 1 and 2A. DSC /HSC2 values for the three chemicals were of the same order of magnitude using all three protocols. Additionally, using Protocol 1, there was very little difference between parameters measured in pig and human SC. In conclusion, KSC/v, and DSC values were comparable using different methods. Pig skin might be a good surrogate for human skin for the three chemicals tested. Copyright © 2017 The Authors Journal of Applied Toxicology published by John Wiley & Sons Ltd.
Asunto(s)
Cosméticos/química , Cosméticos/metabolismo , Epidermis/metabolismo , Absorción Cutánea/efectos de los fármacos , Adulto , Animales , Cafeína/metabolismo , Cumarinas/metabolismo , Difusión/efectos de los fármacos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Modelos Animales , Modelos Biológicos , Permeabilidad/efectos de los fármacos , Resorcinoles/metabolismo , PorcinosRESUMEN
The Cosmetics Europe Skin Bioavailability and Metabolism Task Force aims to improve the measurement and prediction of the bioavailability of topically-exposed compounds for risk assessment. Key parameters of the experimental design of the skin penetration studies were compared. Penetration studies with frozen human and pig skin were conducted in two laboratories, according to the SCCS and OECD 428 guidelines. The disposition in skin was measured 24h after finite topical doses of caffeine, resorcinol and 7-ethoxycoumarin. The bioavailability distribution in skin layers of cold and radiolabelled chemicals were comparable. Furthermore, the distribution of each chemical was comparable in human and pig skin. The protocol was reproducible across the two laboratories. There were small differences in the amount of chemical detected in the skin layers, which were attributed to differences in washing procedures and anatomical sites of the skin used. In conclusion, these studies support the use of pig skin as an alternative source of skin should the availability of human skin become a limiting factor. If radiolabelled chemicals are not available, cold chemicals can be used, provided that the influence of chemical stability, reactivity or metabolism on the experimental design and the relevance of the data obtained is considered.
Asunto(s)
Cafeína/farmacocinética , Cosméticos/farmacocinética , Cumarinas/farmacocinética , Técnicas In Vitro/métodos , Resorcinoles/farmacocinética , Piel/metabolismo , Administración Tópica , Adulto , Animales , Disponibilidad Biológica , Femenino , Humanos , Masculino , Persona de Mediana Edad , Medición de Riesgo , Absorción Cutánea , Porcinos , Adulto JovenRESUMEN
The sensitizing potential of chemicals is usually identified and characterized using one of the available animal test methods, such as the mouse local lymph node assay. Due to the increasing public and political concerns regarding the use of animals for the screening of new chemicals, the Colipa Skin Tolerance Task Force collaborates with and/or funds research groups to increase and apply our understanding of the events occurring during the acquisition of skin sensitization. Knowledge gained from this research is used to support the development and evaluation of novel alternative approaches for the identification and characterization of skin sensitizing chemicals. At present one in chemico (direct peptide reactivity assay (DPRA)) and two in vitro test methods (cell based assays (MUSST and h-CLAT)) have been evaluated within Colipa inter-laboratory ring trials and accepted by the European Centre for the Validation of Alternative Methods (ECVAM) for pre-validation. Data from all three test methods will be used to support the development of testing strategy approaches for skin sensitizer potency prediction. The replacement of the need for animal testing for skin sensitization risk assessment is viewed as ultimately achievable and the next couple of years should set the timeline for this milestone.
Asunto(s)
Alérgenos/toxicidad , Alternativas a las Pruebas en Animales , Haptenos/efectos de los fármacos , Pruebas de Irritación de la Piel/métodos , Piel/efectos de los fármacos , Alérgenos/clasificación , Alérgenos/farmacocinética , Animales , Línea Celular Tumoral , Cromatografía Líquida de Alta Presión , Biología Computacional , Haptenos/análisis , Humanos , Medición de Riesgo , Piel/metabolismo , Células U937RESUMEN
Desquamation in human skin is a well-balanced process of de novo production of corneocytes and their shedding from the skin surface. The proteolysis of corneodesmosomes is an important step in the final desquamation process. In the degradation of these adhesion molecules, the stratum corneum tryptic enzyme (SCTE) plays a key role. In initial studies with extracts of porcine epidermis, SCTE was shown to be inactivated by low concentrations of sodium lauryl ether sulphate (SLES). These in vitro findings were supported by in situ results obtained by measuring the release of fluorescent dyes coupled to trypsin-specific substrates incubated on human skin cross-sections. Moreover, in further studies, it could be demonstrated that the SCTE activity in the human horny layer decreases after in vivo application of cleansing products containing SLES. After repeated washing of human volunteers with tap water, a standard market cleansing product (SLES/betaine system) or a new improved cleansing product (SLES/betaine/disodium cocoyl glutamate system), the specific SCTE activity was determined in extracts from the uppermost layers of the stratum corneum. It could be shown that after application of the new formula the remaining SCTE activity was significantly higher than after use of the standard market formula. This ex vivo approach has proven to be very helpful for measuring surfactant effects on human skin enzymes. Using this assay, we developed an improved shower gel formula, which leads to a significantly higher skin enzyme activity after application, compared to a standard market formula.
RESUMEN
OBJECTIVE: A model approach is presented to the in vivo inflammation cascade, in which the activities of key enzymes (phospholipase A2 [3.1.1.4], prostaglandin synthase [EC 1.14.99.1], and lipoxygenases [EC 1.13.11.12]) are determined simultaneously in a single in vitro assay. METHODS AND RESULTS: Detection of phospholipids (up to 50 pM) and arachidonic acid (up to 33 pM) is attained with high sensitivity and without radiolabelling using a SEDERE light scattering detector. CONCLUSIONS: Thus, in combination with a diode array UV-detector, lipids, prostaglandins, HETEs and other metabolites of the inflammation cascade can be determined with high efficiency using a reversed phase-high performance liquid chromatograph equipped with two highly sensitive detectors in series.
Asunto(s)
Cromatografía Líquida de Alta Presión , Dermatitis/metabolismo , Lipooxigenasa/metabolismo , Fosfolipasas A/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/análisis , Animales , Araquidonato 12-Lipooxigenasa/metabolismo , Ácido Araquidónico/análisis , Dermatitis/enzimología , Masculino , Fosfolipasas A2 , Fosfolípidos/análisis , Prostaglandina H2 , Prostaglandinas H/análisis , Ovinos , PorcinosRESUMEN
A new method for the selective and quantitative determination of phosphotyrosine residues is presented using a differential iodination technique. Characterization of tyrosine-phosphorylated proteins was performed in a biological system using human U937 myeloid leukemia cells. The method is based on the saturation of free iodine binding sites using non-radioactive iodine. Samples are then treated with alkaline phosphatase. New iodine binding sites in dephosphorylated tyrosines are subsequently radio-iodinated, resulting in specific labeling of tyrosine phosphates. Separation is performed by RP-HPLC or sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radiolabeled proteins are then identified using a radioactivity detector or autoradiography.
Asunto(s)
Yodo/metabolismo , Oligopéptidos/química , Fosfotirosina/química , Fosfatasa Alcalina/química , Fosfatasa Alcalina/metabolismo , Animales , Anticuerpos Monoclonales , Autorradiografía , Sitios de Unión , Western Blotting , Bovinos , Cromatografía Líquida de Alta Presión , Citosol/química , Electroforesis en Gel de Poliacrilamida , Humanos , Inmunoglobulina G/química , Inmunoglobulina G/inmunología , Intestinos/enzimología , Yodo/química , Radioisótopos de Yodo/química , Ratones , Fosfotirosina/inmunología , Fosfotirosina/metabolismo , Conejos , Radiactividad , Espectrofotometría Ultravioleta , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
The purpose of this study was to assess the possibility of isolating biologically active peptides from human blood using large volumes of blood filtrate, which are available from patients undergoing extracorporeal ultrafiltration because of renal insufficiency. This filtrate was submitted to six chromatographic separation steps, yielding one purified peptide which was completely analysed in its primary structure. It was found to be strikingly similar to proteins, described initially as rabbit uteroglobin (or blastokinin) and, more recently, from human bronchial lavage as the '10 kDa Clare cell protein', as well as from human urine as 'protein-1'. The natural molecule contains two chains of identical amino acid sequences of 70 residues which are arranged as an antiparallel dimer due to the disulphide bonds between two cysteines at positions 3 and 69. Mass analysis of the molecular forms yielded molecular weights from 15827 Da (non-oxidized form) to 15859 Da (bi-oxidized form). We conclude that this peptide isolated from the filtrate represents the human uteroglobin, and we demonstrate for the first time that this peptide may be involved as a humoral factor in reproductive or other physiological functions.
Asunto(s)
Uteroglobina/sangre , Secuencia de Aminoácidos , Animales , División Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Dimerización , Hemofiltración , Humanos , Fallo Renal Crónico/sangre , Espectrometría de Masas , Datos de Secuencia Molecular , Peso Molecular , Mapeo Peptídico , Conformación Proteica , Conejos , Ratas , Alineación de Secuencia , Células Tumorales CultivadasRESUMEN
Several degradation products of fibrinogen have been shown to possess regulatory functions. Using peptide extracts from human blood filtrate, a large number of fibrinogen A alpha fragments was identified. These fragments are generated at known plasmin attack sites and at several novel cleavage sites especially at hydrophobic and basic amino acid residues. One fragment containing the cell attachment site (RGD sequence) of fibrinogen A alpha efficiently inhibits fibrinogen binding and platelet aggregation (IC50:20-50 microM) in vitro. We conclude that in vivo degradation of fibrinogen A alpha results in generation of endogenous antithrombotic peptides with local importance in fibrinolysis and platelet aggregation.
Asunto(s)
Fibrinógeno/metabolismo , Fibrinopéptido A/química , Fibrinopéptido A/farmacología , Inhibidores de Agregación Plaquetaria/química , Agregación Plaquetaria/efectos de los fármacos , Adenosina Difosfato/farmacología , Secuencia de Aminoácidos , Sitios de Unión , Plaquetas/efectos de los fármacos , Plaquetas/fisiología , Fibrinolisina/metabolismo , Fibrinopéptido A/aislamiento & purificación , Humanos , Datos de Secuencia Molecular , Oligopéptidos , Inhibidores de Agregación Plaquetaria/aislamiento & purificación , Inhibidores de Agregación Plaquetaria/farmacologíaRESUMEN
Specific labeling of tyrosine sulfate-containing peptides was achieved using a differential iodination approach. In a complex peptide mixture from human hemofiltrate, cold iodination to saturate free iodine binding sites was followed by mild acidic desulfation of tyrosine sulfate and subsequent radioiodination using iodine-125. Reaction steps were controlled by amino acid analysis using o-phthaldialdehyde precolumn derivatization and by spiking with a sulfated cholecystokinin fragment (CCK4-S). Separation of the peptide mixture with RP-HPLC on a C18 column coupled to a radioactivity monitor led to the sensitive (< or = 5 pM) and specific determination of tyrosine sulfate-containing peptides.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Radioisótopos de Yodo , Péptidos/sangre , Sulfatos/sangre , Aminoácidos/sangre , Cromatografía Líquida de Alta Presión/estadística & datos numéricos , Hemofiltración , Humanos , Concentración de Iones de Hidrógeno , Marcaje Isotópico , Sincalida/sangre , Tirosina/análogos & derivados , Tirosina/sangre , Tirosina/químicaRESUMEN
A sequential approach was developed to label tyrosine sulfate and peptides containing tyrosine sulfate selectively. Amino acids and peptides containing tyrosine and tyrosine sulfate were first iodinated using chloramine-T-method. Reaction products were determined by RP-HPLC. Mono- and biiodination of tyrosine and several model peptides was achieved within 120 s incubation time. Iodination of free tyrosine sulfate and sulfated cholecystokinin26-33 was less than 5%. After desulfation of the reaction products with 1 N HCl successful radioiodination of desulfated tyrosine was carried out whereas tyrosine did incorporate radioactive iodine only 10%. As shown by RP-HPLC specific labeling of tyrosine sulfate containing peptides with 125iodine was achieved.
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Yodo/química , Péptidos/análisis , Sulfatos/análisis , Secuencia de Aminoácidos , Cloraminas , Cromatografía Líquida de Alta Presión , Indicadores y Reactivos , Radioisótopos de Yodo , Cinética , Datos de Secuencia Molecular , Sincalida/análogos & derivados , Sincalida/análisis , Radioisótopos de Azufre , Compuestos de Tosilo , Tirosina/químicaRESUMEN
Human hemofiltrate (HF) was evaluated regarding its content of free amino acids, proteins, and regulatory peptides. Human HF was obtained from patients with end stage renal disease (ESRD). In contrast to plasma it mainly contains low and middle weight molecules < or = 45 kDa. The content of free amino acids, peptides, and proteins in pooled filtrate was determined by amino acid analysis using ortho-phthaldialdehyde/fluorenyl methyl chloroformate (OPA/FMOC) precolumn derivatization. The total amount of peptides and proteins in human HF is 49.4 mg/L (n = 8). The levels of all free amino acids (230 mg/L) and the concentration of some regulatory peptides like insulin, endothelin, gastrin, vasopressin and angiotensin II were similar compared with blood plasma. The amount of peptides and proteins detected in the filtrate was around 0.07% of total plasma proteins, and consisted mainly of smaller proteins and peptides as shown by size exclusion chromatography (SEC). The presence of large proteins in plasma is reduced by a factor of 1500 after filtration. We conclude that human hemofiltrate is a valuable source for the large-scale extraction of regulatory peptides.