AN ANALYSIS OF THE CYTOTOXIC ACTIVITY OF METHYLATING AGENTS AND THE METABOLISM OF POLYCYCLIC AROMATIC HYDROCARBONS IN CELL CULTURE

A series of cytotoxic, carcinogenic methylating agents that include methylnitrosoureas, methyltriazenes, methylazoxymethanol acetate, N-methyl-N-nitrosourethane and N-methyl-N-acetoxymethylnitrosamine were tested for their effects on the growth rate of P388 mouse leukemia cells in culture. Although these methylating agents display different modes of activation, they are precursors to the same methylating intermediate, the methyldiazonium ion. Pharmacokinetic models were developed to reflect the amount of the active methylating species generated during the period of exposure of cells to the drug. These models permit cytotoxicity data for different compounds obtained through different cell treatment schedules and conditions to be compared directly. The dose-response models were tested for a range of initial drug concentrations, different exposure periods and in the presence of extracellular enzyme and/or an enzyme inhibitor. The results demonstrate that the activity of these agents depends only on the amount of active species generated within the cell and is independent of the structure of the parent compound. These results suggest that the activity of these agents is the same with respect to the number of methyldiazonium ion alkylation events occurring within the cell volume and is unrelated to the duration of exposure of the cells to that intermediate. A large proportion of the metabolites formed from polycyclic aromatic hydrocarbons in cell cultures are water-soluble metabolites including glutathione, sulfate and glucuronic acid conjugates. An ion-pair H.P.L.C. system was developed for the separation of the water-soluble metabolites formed from benzo(a)pyrene and 7,12-dimethylbenz(a)anthracene in cell cultures from rodents (Sencar mouse, Syrian hamster and Wistar rat embryos), fish (bluegill fry, brown bullhead) and humans (hepatoma cell line, HepG2). Ion-pair chromatography allowed rapid separation and analysis of the three classes of hydrocarbon conjugates. The major water-soluble metabolites formed from benzo(a)pyrene and 7,12-dimethylbenz(a)anthracene in rodent cultures were phenolic glucuronides. In HepG2 cells, 40% of the water-soluble metabolites were sulfate conjugates of benzo(a)pyrene phenols. The fish cells formed glucuronide conjugates of both 3-hydroxy-benzo(a)pyrene and benzo(a)pyrene-7,8-diol. Ion-pair chromatography permits the analysis of the conjugation pathways of benzo(a)pyrene and 7,12-dimethylbenz(a)anthracene and allows comparison of hydrocarbon metabolism in cell cultures derived from different species