EDTA affects cytochrome P450-dependent biotransformation reactions during incubations for the liver microsomal assay.
In order to optimize the condition of the liver microsomal assay (LMA), studies were carried out to determine the effects of EDTA on mixed-function oxidase activity and its stability under the exact incubation conditions for the LMA. Aminopyrine N-demethylase (APD) and p-nitroanisole O-demethylase (p-NAD) activities as well as lipid peroxidation development (LP) in S9 liver fractions from beta-naphthoflavone and sodium phenobarbital (beta-NF + PB)- or Aroclor 1254 (AC)-treated mice were examined during a period of preincubation with EDTA ranging from 1 to 40 mM. At 5 mM EDTA, we obtained a strong inhibition of the microsomal LP as well as the greatest value of the mean specific activity (Asp) for both APD and pNAD activities. In agreement with the biochemical data, the presence of 5 mM EDTA in the incubation mixtures for the LMA significantly increased the mitotic gene conversion, mitotic crossing-over and point-reverse mutation of the well-known premutagen cyclophosphamide (30 mM) on the diploid D7 strain of Saccharomyces cerevisiae as the outcome of a greater metabolic activity. We concluded that the systematic use of 5 mM EDTA in LMA mixtures could improve the reliability and sensitivity of such a test.
D000633:Aminopyrine N-Demethylase / Q000378:metabolism; D000818:Animals; D001711:Biotransformation / Q000187:drug effects*; D003434:Crossing Over, Genetic / Q000187:drug effects; D003520:Cyclophosphamide / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004492:Edetic Acid / Q000494:pharmacology*; D005785:Gene Conversion / Q000187:drug effects; D066298:In Vitro Techniques; D008054:Lipid Peroxides / Q000378:metabolism; D008862:Microsomes, Liver / Q000378:metabolism*; D009152:Mutagenicity Tests / Q000379:methods*; D010090:Oxidoreductases, O-Demethylating / Q000378:metabolism; D011355:Prodrugs / Q000494:pharmacology; D051381:Rats
D008054:Lipid Peroxides; D011355:Prodrugs; D003520:Cyclophosphamide; D003577:Cytochrome P-450 Enzyme System; D004492:Edetic Acid; C026048:4-nitroanisole O-demethylase; D010090:Oxidoreductases, O-Demethylating; D000633:Aminopyrine N-Demethylase