Dataset Information

The acute-phase response of cultured rat hepatocytes. System characterization and the effect of human cytokines.

ABSTRACT: Hepatocytes were isolated from adult livers and cultured for periods of up to 5 days as monolayers at an initial density of 10(6) cells/10cm2 in Williams E medium containing insulin, dexamethasone and 5% foetal-calf serum. The daily production of 11 plasma proteins was measured by electroimmunoassay and compared with the concentrations of the same proteins in the plasma of normal rats and of those with experimental inflammation. Hepatocytes from normal rats synthesized proteins in relative amounts which were similar to the relative proportions of the same proteins in the plasma of turpentine-injected animals. The pattern changed only slowly during 5 days in culture, but it did so profoundly either when the medium was devoid of dexamethasone or when human cytokines (from endotoxin-stimulated monocytes or unstimulated human squamous-carcinoma cell line COLO-16) were added. The cytokines consistently increased the synthesis of alpha 2-macroglobulin and fibrinogen and depressed that of albumin; variable increases in the synthesis of alpha 1-acute-phase globulin, alpha 1-acid glycoprotein, haptoglobin and alpha 1-proteinase inhibitor, and variable decreases in transferrin synthesis, were seen, whereas the synthesis of antithrombin III, alpha 1-macroglobulin and prothrombin remained virtually unaffected. The cytokine effects on protein synthesis required the presence of dexamethasone. The hepatocyte-stimulating activity derived from monocytes chromatographed on Sephadex G-100 corresponding to 30 000 Da, as opposed to the lymphocyte-activating factor, which was eluted as a molecule of approx. 15 000 Da. This suggests that both activities probably reside with distinct molecular species in the preparations of human cytokines.

SUBMITTER: Koj A

PROVIDER: S-EPMC1144459 | biostudies-other | 1984 Dec

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:Sandwich-cultured rat hepatocytes are used in drug discovery for pharmacological and toxicological assessment of drug candidates, yet their utility as a functional model for drug transporters has not been fully characterized. To evaluate the system as an in vitro model for drug transport, expression changes of hepatic transporters relative to whole liver and freshly isolated hepatocytes (day 0) were examined by real-time quantitative reverse transcription-polymerase chain reaction for 4 consecutive days of culture. No significant differences in transporter expression levels were observed between freshly isolated hepatocytes and whole liver. Two distinct mRNA profiles were detected over time showing 1) a more than 5-fold decline in levels of uptake transporters such as Na(+)-taurocholate cotransporting polypeptide (Ntcp), organic anion transporter (Oat) 2, organic anion-transporting polypeptide (Oatp) 1a1, Oatp1a4, and Oatp1b2 and 2) a greater than 5-fold increase of efflux transporters P-glycoprotein (P-gp), breast cancer resistance protein (Bcrp), and multidrug resistance-related proteins (Mrp) 1, 2, 3, and 4. In addition, protein levels and functional activities for selected transporters were also determined. Protein levels for Mrp2, Bcrp, P-gp, Ntcp, and Oatp1a4 corresponded to changes in mRNA. Functional activities of Oatps and Oct1 exhibited a 3- and 4-fold decrease on day 2 and day 4, respectively, relative to that on day 0, whereas a more than 10-fold reduction in Oat2 activity was observed. These results indicate that the cell culture conditions used herein did not provide an optimal environment for expression of all hepatic transporters. Significant time-dependent alterations in basal gene expression patterns of transporters were detected compared with those in liver or freshly isolated hepatocytes. Further work and new strategies are required to improve the validity of this model as an in vitro tool for in vivo drug transport or biliary clearance prediction.

Project description:DNA microarray is a powerful tool in biomedical research. However, transcriptomic profiling using DNA microarray is subject to many variations including biological variability. To evaluate the different sources of variation in mRNA gene expression profiles, gene expression profiles were monitored using the Affymetrix RatTox U34 arrays in cultured primary hepatocytes derived from six rats over a 26 hour period at 6 time points (0 h, 2h, 5h, 8h, 14 h and 26 h) with two replicate arrays at each time point for each animal. In addition, the impact of sample size on the variability of differentially expressed gene lists and the consistency of biological responses were also investigated. Excellent intra-animal reproducibility was obtained at all time points with 0 out of 370 present probe sets across all time points showing significant difference between the 2 replicate arrays (3-way ANOVA, p <or= 0.0001). However, large inter-animal biological variation in mRNA expression profiles was observed with 337 out of 370 present probe sets showing significant differences among 6 animals (3-way ANOVA, p <or= 0.05). Principal Component Analysis (PCA) revealed that time effect (PC1) in this data set accounted for 47.4% of total variance indicating the dynamics of transcriptomics. The second and third largest effects came from animal difference, which accounted for 16.9% (PC2 and PC3) of the total variance. The reproducibility of gene lists and their functional classification was declined considerably when the sample size was decreased. Overall, our results strongly support that there is significant inter-animal variability in the time-course gene expression profiles, which is a confounding factor that must be carefully evaluated to correctly interpret microarray gene expression studies. The consistency of the gene lists and their biological functional classification are also sensitive to sample size with the reproducibility decreasing considerably under small sample size.

Dataset Information

The acute-phase response of cultured rat hepatocytes. System characterization and the effect of human cytokines.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets