Use this URL to cite or link to this record in EThOS:
Title: Strategies for the maintenance of the cytochrome P450 content of adult rat hepatocytes in primary culture
Author: Lindsay, Carol K.
Awarding Body: University of Aberdeen
Current Institution: University of Aberdeen
Date of Award: 1991
Availability of Full Text:
Access from EThOS:
Full text unavailable from EThOS. Please try the link below.
Access from Institution:
Primary cultures of adult rat hepatocytes could be used for the study of hepatic drug metabolism/toxicity provided enzyme activities were maintained at the fresh cell values. Adult rat hepatocytes cultured on a thin layer of rat tail collagen, in the presence of modified Earle's medium supplemented with 5% (v/v) foetal calf serum, lost much of their cytochrome P450 content within the first 5 days of culture. This loss of cytochrome P450 was accompanied by increasing levels of cytochrome P420, suggesting the loss of enzyme was due to degradation to the inactive cytochrome P420. Activities associated with the enzyme also declined rapidly. Pentoxyresorufin-O-dealkylase (PROD) activity could not be detected by the seventh day of culture, whilst ethoxyresorufin-O-deethylase (EORD) had declined to 4% of the fresh cell value. Co-culture of adult rat hepatocytes with rat liver derived epithelial cell lines did significantly (P< 0.05) increase the cytochrome P450 content on days 5 and 7 of culture if enzyme content was expressed per 100mm culture plate. PROD activity was not detectable by the seventh day of culture and EROD activity fell to 20% of the fresh cell value. Despite the observation that pure epithelial cell cultures grew poorly in modified Earle's medium epithelial cell overgrowth was a major disadvantage in the co-culture system. Cytochrome P450 content declined rapidly when hepatocytes were cultured on a layer of `Matrigel' or EHS extract. The loss was due to increased conversion to cytochrome P420 in culture. PROD activity was not detectable after 4-5 days in culture but EROD activity fell transiently during the first 2 days of culture and then rose to approximately twice the fresh cell value. The response of cells cultured on `Matrigel' or EHS extract to 1,2-benzanthracene (BA) was approximately 6 fold greater compared with hepatocytes cultured on collagen. The cytochrome P450 content of hepatocytes cultured in the presence of 2% (v/v) dimethylsulphoxide (DMSO) was 70% of the fresh cell value after 7 days in culture. The cytochrome P420 content was not significantly lowered in the presence of DMSO suggesting the solvent caused synthesis of new enzyme, rather than maintaining existing enzyme. PROD activity declined rapidly in the presence of DMSO whilst EROD activity was significantly (P< 0.05) increased on days 3,5 and 7 of culture. The increases in both cytochrome P450 content and EROD activity could be prevented by cycloheximide suggesting de novo synthesis of specific isozymes was occurring. Immunoblotting studies indicated de novo synthesis of cytochromes P450 lA1 and lA2 in the presence of DMSO. Simultaneous exposure to DMSO and BA caused a 40 fold increase in EROD activity. Immunoblotting studies revealed induction of cytochromes P450 1A1 and 1A2 indicating that culture of hepatocytes in the presence of DMSO allows hepatocytes in culture to respond to BA with a response qualitatively and quantitatively resembling the response in vivo.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Biochemistry