Abstract
Mechanisms of chemical toxicity and carcinogenesis have been critically reviewed. Apart from activation by cytochrome P-450-dependent mixed-function oxidation, flavoprotein-mediated one-electron oxidation appears to be a general mechanism for the metabolism of chemicals and carcinogens. The consequent generation of free radical metabolites, and/or free radical forms of oxygen, may result in interaction with tissue membranes and macromolecules followed by toxicity, mutations and cancer. The possible involvement of free radical-mediated autoxidative damage in carbon disulphide toxicity was investigated. It was found that after intraperitoneal administration of carbon disulphide to rats cytochrome P-450 is destroyed by converting to cytochrome P-420, with loss of mixed-function oxidase activities. This is followed by the degradation of both cytochromes until about 12 hours after administration of the CS2 when synthesis of new cytochrome P-450 occurs. The spectral maximum of the carbon monoxide ligand complex of the new cytochrome is 488.8 nm, indicating either preferential synthesis of cytochrome P-448 or the preferred degradation of cytochrome P-450. Loss of cytochrome P-450 caused by CS2 in vitro is totally prevented by addition of EDTA. This provides indirect evidence for the involvement of lipid peroxidation (autoxidation) in cytochrome P-450 destruction. Increased formation of ethane by rat liver microsomal preparations incubated in the presence of both CS2 and NADPH, gives supportive evidence of a lipid peroxidative mechanisms of the microsomal membrane damage caused by CS2. A mechanism for the cytochrome P-450-mediated formation of free radical oxygen species by CS2 has been postulated. The present study also provides evidence that chemically-induced lipid peroxidation, as monitored by ethane formation, is associated with a loss of cytochrome P-450. Thus, the determination of cytochrome P-450 concentration, and the rate of its denaturation to cytochrome P-450 and subsequent destruction, could prove to be a valuable in vitro method for studying the potential of environmental chemicals for causing autoxidative damage. The use of human lymphocytes as an in vitro system for the detection of carcinogens and mutagens by the induction of aryl hydrocarbon hydroxylase, has proved to be unreliable. Studies have shown that lack of specificity of the aryl hydrocarbon hydroxylase assay and numerous variable factors involved in the culture of the lymphocytes contribute to poor reproducibility of this published method. In an attempt to find an alternative substrate for aryl hydrocarbon hydroxylase, biphenyl, ethoxyresorufin O-de-ethylase and benzo(a)pyrene hydroxylase assays have been examined with respect to sensitivity, which was found to increase in the order of biphenyl 4-hydroxylase < biphenyl 2-hydroxylase < ethoxyresorufin O-de-ethylase < benzo(a)pyrene hydroxylase. The very high sensitivity of ethoxyresorufin O-de-ethylase assay and its high specificity for cytochrome P-448 (which plays a key role in the activation of carcinogens and mutagens), indicates that it may provide a highly suitable method for the determination of cytochrome P-448-dependent activity, and for the detection of chemical carcinogens.