Studies on the importance of microsomal epoxide hydrolase in the detoxification of arene oxides using the heterologous expression of the enzyme in mammalian cells.
In order to investigate the role of the microsomal epoxide hydrolase (mEH) in the detoxification of arene oxides in the presence of a high endogenous glutathione S-transferase (GST) activity-a situation found in several organs--we expressed the rat mEH cDNA in BHK21 Syrian hamster cells. These cells have high GST activities but contain an extremely low endogenous mEH enzyme activity. We obtained several cell clones which expressed the mEH heterologously, as determined by immunoblotting. The cell clone BHK21-mEH/Mz1 had the highest level of mEH protein. Immunofluorescence showed that the level of expression was almost homogeneous throughout the cell population. Total protein isolated from the cell line BHK21-mEH/Mz1 had a specific mEH activity of 123 pmol/min/mg protein, as determined with benzo[a]pyrene 4,5-oxide (B[a]P 4,5-oxide), which was 60 times higher than the activity in the parental cell line and eight times lower than the activity found in rat hepatocytes. However, BHK21-mEH/Mz1 cell homogenates were found to catalyze the conjugation of B[a]P 4,5-oxide to glutathione extremely well. The ratio of the GST enzyme activity to the mEH enzyme activity towards this substrate was 23 in the BHK21-mEH/Mz1 cell line. For hepatocytes this ratio was only six. Despite their already high potential to inactivate B[a]P 4,5-oxide by conjugation to glutathione, BHK21-mEH/Mz1 cells were better protected against the toxic and mutagenic effects of B[a]P 4,5-oxide than the parental cell line due to the expression of the mEH. The mEH, however, failed to protect the cells from the toxic and mutagenic effects of the bay region epoxide anti-7-methylbenz[a]anthracene-3,4-diol 1,2-oxide.
Friedberg
T
T
Institute of Toxicology, University of Mainz, Germany.
Becker
R
R
Oesch
F
F
Glatt
H
H
D000818:Animals; D001580:Benzopyrenes / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D004851:Epoxide Hydrolases / Q000235:genetics / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D008658:Inactivation, Metabolic; D008856:Microscopy, Fluorescence; D008861:Microsomes / Q000201:enzymology*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism
D001580:Benzopyrenes; D011994:Recombinant Proteins; C006707:benzo(a)pyrene 4,5-epoxide; D005982:Glutathione Transferase; D004851:Epoxide Hydrolases