RESUMEN

This study describes the characterization of pharmacokinetic (PK) properties of acetaminophen (APAP) in the Two-Organ-Chip platform (2-OC), a two-chamber device able to cultivate 3D tissues under flow. The APAP intestinal absorption and hepatic metabolism were emulated by human intestine and liver equivalents respectively. The intestinal barrier was produced using Caco-2 and HT-29 cells. The liver spheroids were produced with HepaRG and HHSTeC cells. Cell viability and toxicity were assessed by MTT assay, histology, confocal immunohistochemistry, and multiparametric high content analysis. Gene expression of intestine and liver equivalents were assessed by real-time PCR. Three assemblies of Microphysiological System (MPS) were applied: Intestine 2-OC, Liver 2-OC, and Intestine/Liver 2-OC. The oral administration was emulated by APAP placement over the apical side of the intestinal barrier and the intravenous routes were mimic by the application in the medium. Samples were analyzed by HPLC/UV. APAP 12 µM or 2 µM treatment did not induce cytotoxicity for the intestinal barrier (24 h time-point) or for the liver spheroids 12 h time-point), respectively. All preparations showed slower APAP absorption than reported for humans: Peak time (Tmax) = 12 h for Intestine 2-OC and 6 h for Intestine/Liver 2-OC in both static and dynamic conditions, against reported Tmax of 0,33 to 1,4 h after oral administration to humans. APAP metabolism was also slower than reported for humans. The APAP half-life (T1/2) was 12 h in the dynamic Liver 2-OC, against T1/2 = 2 ±â€¯0,4 h reported for humans. Samples taken from the Liver 2-OC static preparation did not show APAP concentration decrease. These findings show the MPS capability and potential to emulate human PK properties and highlight the critical role of mechanical stimulus over cell functionality, especially by demonstrating the clear positive influence of the microfluidic flow over the liver equivalents metabolic performance.

Asunto(s)

Acetaminofén/farmacología , Técnicas de Cultivo de Célula/métodos , Absorción Intestinal/efectos de los fármacos , Hígado/efectos de los fármacos , Acetaminofén/análisis , Acetaminofén/farmacocinética , Células CACO-2 , Técnicas de Cultivo de Célula/instrumentación , Supervivencia Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Expresión Génica/efectos de los fármacos , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Células HT29 , Semivida , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Hígado/metabolismo , Microfluídica , Microscopía Confocal , Transportador 1 de Sodio-Glucosa/genética , Transportador 1 de Sodio-Glucosa/metabolismo , Espectrofotometría Ultravioleta

RESUMEN

The hepatitis B virus (HBV) protein HBx has been implicated to induce liver cancer in transgenic mice and transactivates a variety of viral and cellular promoters. The 17 kDa protein HBx consists of 154 amino acids, contains 10 cysteine residues and is translated during the viral infection. It has been shown previously that the HBx protein is able to bind to singlestranded DNA and RNA. This nucleic acid binding activity might be relevant for HBx oncogenic character. Furthermore, HBx has been reported to interact with a series of cellular proteins, especially with transcription factors, including the tumor suppressor protein p53. To evaluate the importance of the cysteine residues in HBx for its interaction with RNA and p53 we expressed full-length HBx-wt as well as several truncated mini-HBx(18-142) proteins with multiple cysteine to serine point mutations as 6xHis fusion proteins in Escherichia coli. Using UV cross-linking assays we demonstrate that all truncated mini-HBx proteins with cysteine/serine point mutations maintained the ability to bind to an AU-38 RNA oligonucleotide. Furthermore, we performed in vitro binding assays of selected HBx mutants with GST-p53, circular dichroism spectroscopic analysis of the mutant HBx protein secondary structure and a p53 based transcription activation assay in yeast cells. In summary, our data suggest that the cysteine residues in the HBx protein are of minor importance for its interaction with both RNA and the p53 protein.

Asunto(s)

Cisteína/fisiología , Virus de la Hepatitis B/fisiología , ARN/metabolismo , Transactivadores/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Sustitución de Aminoácidos , Dicroismo Circular , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Mutación Missense , Unión Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Transactivadores/química , Activación Transcripcional/fisiología , Proteínas Reguladoras y Accesorias Virales