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Cryopreservation and long-term storage of primary rat hepatocytes: Effects on substrate-specific cytochrome P450-dependent activities and unscheduled DNA synthesis

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Abstract

The effects of cryopreservation and long-term storage on substrate-specific cytochrome P45O-dependent activities and unscheduled DNA synthesis were studied in freshly isolated and cryopreserved hepatocytes derived from adult male Fischer 344 and Sprague-Dawley rats. Primary rat hepatocytes were isolated via an in situ collagenase perfusion technique, cryopreserved at −196°C, and thawed at 5 weeks and 104 and 156 weeks post-freezing. In Fischer 344 and Sprague-Dawley rats, cryopreserved hepatocytes were equivalent or similar to freshly isolated hepatocytes in substrate-specific activities for 7-ethoxyresorufin-0-deethylase and dimethylnitrosamine-N-demethylase and unscheduled DNA synthesis responses. No significant differences in activities toward 7-ethoxyresorufin-0-deethylase and dimethylnitrosamine-N-demethylase, the substrate-specific activities for cytochromes P4501A1 and P4501A2 and cytochrome P4502E1, respectively, were observed between freshly isolated and cryopreserved hepatocytes. Similar unscheduled DNA synthesis responses, a measure of DNA damage and repair, were observed after exposure to the genotoxic carcinogens 2-acetylaminofluorene, 7,12-dimethyEbenz[a]anthracene, and dimethylnitrosamine; although some decreases were also observed in Fischer 344 hepatocytes after 104 weeks and Sprague-Dawley hepatocytes after 156 weeks in the highest concentrations tested. These results suggest that cryopreserved hepatocytes, stored for extended periods of time in liquid nitrogen, are metabolically equivalent to freshly isolated hepatocytes in their ability to activate precarcinogens.

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Abbreviations

2-AAF:

2-acetylaminofluorene

DDH2O:

distilled deionized water

DMBA:

7,12-dimethyIbenz[a]anthracene

DMN:

dimethylnitrosamine

DMNA:

dimethylnitrosamine-N-demethylase

DMSO:

dimethyl sulfoxide

EROD:

7-ethoxyresorufin-O-deethylase

F344:

Fischer 344

FBS:

fetal bovine serum

%IR:

percentage of cells in repair

LN2 :

liquid nitrogen

LSD:

least significant difference

CG:

cytoplasmic grains

NNG:

net nuclear grains

SD:

Sprague-Dawley

UDS:

unscheduled DNA synthesis

WE:

Williams' Medium E

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Authors and Affiliations

  1. Division of Genetic Toxicology, National Center for Toxicological Research, NCTR Drive, 72079, Jefferson, Arkansas, USA

    J. G. Shaddock & D. A. Casciano

  2. Division of Biochemical Toxicology, National Center for Toxicological Research, NCTR Drive, 72079, Jefferson, Arkansas, USA

    J. E. Snawder

  3. Departments of Biochemistry and Molecular Biology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA

    D. A. Casciano

Authors
  1. J. G. Shaddock
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  2. J. E. Snawder
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  3. D. A. Casciano
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Shaddock, J.G., Snawder, J.E. & Casciano, D.A. Cryopreservation and long-term storage of primary rat hepatocytes: Effects on substrate-specific cytochrome P450-dependent activities and unscheduled DNA synthesis. Cell Biol Toxicol 9, 345–357 (1993). https://doi.org/10.1007/BF00754463

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