Project title: Comparative Analysis of Short-term vs. Long-term Culture of Primary Mouse Hepatocytes for Modeling in vivo Responses to Toxicants
Completed in: 2010
Towards the goal of establishing useful in vitro models of in vivo responses to toxicants, this work characterizes several advantages and disadvantages of primary mouse hepatocytes (PMHs) cultured for a short term vs those maintained for three weeks with 2.25% DMSO. PMHs examined 1 day (short term, ST) after isolation demonstrated a dramatic loss in ATP (~27 fold lower compared to in vivo) while those cultured for 3 weeks (3 wk) over-produced ATP. The important liver proteins albumin, Cyp1a1 and Gsta were found to be higher in the 3 wk cultures. Gene expression analysis revealed the ST cultures have unregulated numerous pathways involved in compensatory hyperplasia and oxidative injury.
Aflatoxin and acetaminphen (APAP) were used to investigate the metabolic capability of each system. Metabolism of aflatoxin B1 by S9 fractions harvested from ST and 3 wk cultures induced for 24 hours with 3-methylcholanthrene, pregnenolone-16a-carbonitrile, and dexamethasone indicated the ST cultures had higher basal Cyp1a1/2 and Cyp3a11 activity, but did not respond to inducing agents at the concentrations used in this study. In contrast, the 3 wk cultures demonstrated an increase in Cyp1a1/2 and Cyp3a11 activity with induction to levels equivalent to or higher than ST cultures. Cyp3a11 activity was most pronounced in the 3 wk cultures with removal of DMSO prior to induction. In contrast, Cyp1a1 activity was greater in the 3 wk cultures with DMSO present at the time of induction.
The ST cultures produced expected metabolites of acetaminophen, whereas the 3 wk system showed a marked depletion of the parent compound, with little to no appearance of metabolites. Given that the transcript levels of several multidrug resistance proteins.
Exposure to Tri-n-octylphosphine oxide, Poly(maleic anhydride-alt-1-tetradecene)- (PMAT) coated CdSe Qdots did not produce overt toxicity in either culture system, as measured by WST-1 assay, cellular glutathione, or changes in heme oxygenase. Qdot treatment did not appear to cause autophagy. Results from gene expression profiling suggest Qdots may subtly affect cell adhesion and cell division in the PMH cultures.