The mechanism of the suicidal, reductive inactivation of microsomal cytochrome P-450 by carbon tetrachloride

Abstract

1. Stoichiometric losses of microsomal haem and cytochrome P-450 were observed when carbon tetrachloride (CCl 4) was incubated anaerobically with rat liver microsomes using NADPH or sodium dithionite as a reducing agent. A rapid destruction of haem was also observed during the nonenzymatic reductive incubation of CCl 4 with soluble haem preparations (methaemalbumin) in presence of sodium dithionite. The results indicate that haem is both the site and the target of the suicidal activation of CCl 4 by cytochrome P-450. 2. When an additional, fluorimetric assay for haem determination was used, an equimolar loss of protoporphyrin IX fluorescence was also observed in both the enzymatic and non-enzymatic system, indicating that the haem moiety of cytochrome P-450 has undergone a structural change, involving either loss or labilization of the porphyrin tetrapyrrolic structure. In both systems the loss of porphyrin was prevented by carbon monoxide (CO). 3. A dichlorocarbene-cytochrome P-450 ligand complex is partially responsible for the difference spectrum obtained on addition of CCl 4 to anaerobically reduced rat liver microsomes. A molar extinction coefficient for this complex has been calculated. The carbene trapping agent 2,3-dimethyl-2-butene (DMB) strongly inhibited (>95%) the formation of this spectrum but did not modify the loss of haem in reduced CCl 4-supplemented microsomal incubations. The results suggest that dichlorocarbene (:CCl 2) is not significantly involved in CC 4-dependent haem destruction. 4. Pretreatment of rats with different microsomal enzyme inducers was responsible for similar but not identical patterns of :CCl 2 and CO formation and haem loss during incubation of CCl 4 with reduced microsomes. This indicates a critical role of CCl 4 metabolism in the suicidal destruction of cytochrome P-450 haem and suggests that the apoprotein of cytochrome P-450 is capable of modulating not only the metabolism of CCl 4 to :CCl 2 but also the hydrolysis of :CCl 2 to CO. 5. Inactivation of cytochrome P-450 by CCl 4 with reduced microsomes from Aroclor-pretreated rats was saturable and followed pseudo first-order kinetics. This provides further evidence to conclude that CCl 4 activation is a suicidal process where the reactive metabolite(s) formed bind to haem, we predict, in a one to one stoichiometry. 6. The partition ratio between loss of cytochrome P-450 haem and CCl 4 metabolism by liver microsomes from Aroclor pretreated rats has been investigated using limiting concentrations of CCl 4. It was calculated that approximately 26 molecules of CCl 4 had to be metabolised to achieve the loss of one molecule of haem.