||Development of an Intact Hepatocyte Activation System for Routine Use with the Mouse Lymphoma Assay.
Brock, K. H. ;
Moore, M. M. ;
Oglesby, L. A. ;
||Health Effects Research Lab., Research Triangle Park, NC. Genetic Toxicology Div. ;Environmental Health Research and Testing, Inc., Research Triangle Park, NC. ;Northrop Services, Inc., Research Triangle Park, NC.
In viro analysis ;
Cell cultures ;
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The authors have developed a method for cocultivating primary rat hepatocytes with L5178Y/TK+/- 3.7.2C mouse lymphoma cells. The system should provide a means to simulate more closely in vivo metabolism compared to metabolism by liver homogenates, while still being useful for routine screening. Hepatocytes were isolated from 200-250 g adult male Sprague-Dawley rats. Rapid attachment of the hepatocytes (2 h) was obtained by using fibronectin-coated 25-cm2 tissue culture flasks. Several factors proved important in obtaining optimal cocultivated mouse lymphoma cell growth. These included maintaining the cocultivated cultures on a rocker platform instead of stationary and either (1) reducing the cocultivation time from 16 to 4 h or (2) utilizing less than 1 x 10 to the 6th power hepatocytes per flask for 16-h exposures. CP, DMN, DMBA, and B(a)P were used to demonstrate the capability of the system to detect mutagens that require metabolic activation.