Abstract
Research has been carried out into the mechanisms of activation of chemical carcinogens, using the Ames mutagenicity test as an indication of this parameter. As the mixed-function oxidase system has been the major enzyme system investigated, initial studies were carried out to evaluate the specificity of assays for the two major forms of the terminal oxygenase, cytochromes P-450 and P-448. A combined use of inducers, inhibitors and purified enzymes confirmed the specificity of the ethoxyresorufin O-deethylation assay for cytochrome P-448, and further detailed studies emphasised the advantages of this activity over biphenyl 2-hydroxylase and aryl hydrocarbon hydroxylase. In addition, 9-hydroxyellipticine was found to be a specific inhibitor and inducer of cytochrome P-448, and provided a useful tool for both in vivo and in vitro investigations. Furthermore, a detailed study of the spectral interactions of substrates with cytochromes P-450 and P-448 indicated that these two cytochromes have distinctly different substrate binding sites, which could be related to their disparate metabolic specificities and contrasting roles in activation and detoxification of xenobiotics. The role of cytochromes P-450 and P-448, as well as other oxidative enzymes (i. e. the FAD-monooxygenase or Ziegler enzyme, and monoamine oxidase) in the activation of three major groups of carcinogens (nitrosamines, aromatic amines and polycyclic aromatic hydrocarbons) has been studied. An excellent positive correlation was observed between the activation of benzo(a)pyrene to mutagens and the O-deethylation of ethoxyresorufin, indicating that these are catalysed by the same form of cytochrome P-448. No similar clear-cut correlations could be obtained for dimethylnitrosamine and 2-acetylaminofluorene, and indeed, activation of dimethylnitrosamine could also not be related to its demethylation. In addition, a comprehensive study of species and sex differences in the activation of these three groups of carcinogens revealed that the rat (both male and female) was generally poor at activation, while in contrast, the hamster consistently provided a good activating system, especially in the case of the nitrosamines. Finally, preliminary studies have been carried out on the metabolic capacity of cytochrome P-450 in yeast and plant cells, and on the ability of iron-based chemical systems and haemoglobin to mimic the activity of cytochrome P-450. The cytochrome P-450 models were able to hydroxylate aniline and demethylate dimethylnitrosamine, while the yeast and plant preparations were most efficient at hydroxylating benzo(a)pyrene.