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ToxSci Advance Access published online on March 16, 2005
Toxicological Sciences, doi:10.1093/toxsci/kfi145
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Published by Oxford University Press 2005.
Received November 29, 2004
Accepted March 14, 2005

In Vitro Toxicology

Characterization of Chemically Induced Hepatotoxicity in Collagen Sandwiches of Rat Hepatocytes

Dora Farkas 1 and Steven R. Tannenbaum 2*

1 Biological Engineering Division, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge 02139, Massachusetts
2 Biological Engineering Division, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge 02139, Massachusetts; Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge 02139, Massachusetts

* To whom correspondence should be addressed.
Steven R. Tannenbaum, E-mail: srt{at}mit.edu


  Abstract

It has been shown that hepatocytes cultured in a collagen sandwich configuration maintain cell viability, morphology and drug metabolizing activities for several weeks. The purpose of this study was to characterize chemically induced general toxicity in this system by exposing hepatocytes to eight different hepatotoxic compounds. Cell function and viability was measured by analyzing the secretions of urea and albumin and the release of lactate dehydrogenase. Significant decreases in urea and albumin secretions were detected after treatments with 32 nM aflatoxin B1 and 1mM doses of cadmium and the alkylating agents N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and methyl methane sulfonate (MMS). However, no significant toxicity could be measured following exposures to 5mM carbon tetrachloride, 1 mM N, N-dimethylformamide (DMF), 1 mM vinyl acetate and 1mM acetaminophen. Western Blots of cell lysates showed that hepatocytes maintained CYP1A, 2B, 3A2 but gradually lost CYP2E1, which is the main metabolic enzyme for acetaminophen, carbon tetrachloride, and DMF. The metabolites of acetaminophen were identified using liquid chromatography and electrospray mass spectrometry. It was determined that the hepatocytes converted most of the acetaminophen to the glucuronide and sulfate metabolites and only formed a small amount of the glutathione adduct. This research shows that the collagen sandwich culture system can only be used selectively for detecting hepatotoxicity and for identifying major metabolites of xenobiotic compounds.

Keywords: toxicity; collagen; sandwich; hepatocytes; aflatoxin; acetaminophen.
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