A kinetic model of the cell culture cytotoxicity of metabolically activated agents: N-methyl-N-(acetoxymethyl)nitrosamine, methylnitrosourethane, and (methylazoxy)methanol acetate.
The activity of three metabolically activated methylating agents, N-methyl-N-(acetoxymethyl)nitrosamine (DMN-OAc), methylnitrosourethane (MNUT), and (methylazoxy)methanol acetate (MAM-Ac), were determined in cell culture by using a P388 cell growth rate inhibition assay. Experiments were conducted with normal P388 cells in Fischer's medium and under conditions in which the esterase-mediated activation was modified by pretreating cells with the irreversible esterase inhibitor paraoxon and by adding acetylcholinesterase to the medium. Inhibition of intracellular esterase had a much greater effect on activity than did addition of enzyme to medium. These experiments provided data that were used to assess the utility of kinetic models as a means to gain a more detailed understanding of the cytotoxicity process in cell culture. Growth rate inhibition was related to the amount of intracellular alkylation resulting from formation of metabolic intermediates and their subsequent chemical reaction to form methyldiazonium ion and methylation products by using kinetic rate laws and measured rate constants. The model is applicable to systems that form unstable metabolites that can, in part, partition between the cell volume and incubation medium. When growth rate inhibition effects were related to cumulative intracellular alkylation [P], the ED50 values were the same for all three agents and for three previously reported chemically activated methylating agents, N-methyl-N-nitrosourea, streptozotocin, and N-methyl-N'-nitro-N-nitrosoguanidine, which are also thought to act through the methyldiazonium ion. This observation is consistent with a growth rate inhibition effect of the methyldiazonium ion that reflects the intrinsic activity of this species that is independent of the precursor molecule.(ABSTRACT TRUNCATED AT 250 WORDS)
Weinkam
R J
RJ
Drug Metabolism Department, Allergan, Inc., Irvine, California 92715.
Plakunov
I
I
D000110:Acetylcholinesterase / Q000378:metabolism; D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D002800:Cholinesterase Inhibitors; D004128:Dimethylnitrosamine / Q000031:analogs & derivatives* / Q000493:pharmacokinetics / Q000633:toxicity; D006868:Hydrolysis; D007941:Leukemia P388 / Q000378:metabolism / Q000473:pathology; D008746:Methylazoxymethanol Acetate / Q000493:pharmacokinetics* / Q000633:toxicity; D051379:Mice; D008954:Models, Biological; D009605:Nitrosomethylurethane / Q000493:pharmacokinetics* / Q000633:toxicity; D010261:Paraoxon / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology
D002800:Cholinesterase Inhibitors; C014659:methyl(acetoxymethyl)nitrosamine; D008746:Methylazoxymethanol Acetate; D009605:Nitrosomethylurethane; D000110:Acetylcholinesterase; D004128:Dimethylnitrosamine; D010261:Paraoxon