[In vitro inhibition of oxidative N-demethylation with carbon disulfide].
Earlier findings have shown that in experimental animals (rat) and in man inhaled carbon disulphide (CS2) reversibly inhibits the non-specific oxidative drug metabolism caused by hepatic microsomal enzymes. Very little is known concerning the underlying mechanism. The present investigations were undertaken to throw light on this question. After addition of an NADPH-regenerating system to liver microsomes isolated from adult female Wistar rats, the oxidative N-demethylation of aminopyrine was measured under simultaneous exposure to CS2 by quantitatively determining the formaldehyde obtained; the resulting data were evaluated using enzyme-kinetic parameters according to Lineweaver-Burk: 1. Following acute exposure to low and medium-grade CS2 concentrations (20-400 ppm/8 h), the pattern of inhibition in rat liver microsomes is identical to that obtained in normal liver microsomes to which CS2 had been added. This finding seems to suggest that the inhibitory process under in vivo and in vitro conditions is based on one and the same molecular mechanism. 2. Upon addition of CS2 the in vitro pattern of inhibition corresponds to a strong mixed-type inhibition. 3. It is concluded from the enzyme-kinetic behaviour that CS2 attacks at two different sites of the enzyme molecule: Binding to the first site is followed by inhibition, as evidenced by the rise of Km; after saturation of this site, a second site is occupied resulting in a reactivation and, beyond this, an activation of enzyme output, as shown by the decrease of Km. CS2 exhibits a high affinity for the first site, and a low affinity for the second site. Binding at the first site is reversible. The possibility that the active centre in the enzyme molecule is the site where binding of the inhibitor occurs is ruled out. 4. A continuous decrease of Vmax at increasing inhibitor concentration is causally related with the formation of an enzyme/substrate inhibitor complex. 5. The CS2 added to the microsomes can be eliminated by helium gas; this is followed by the return of the original enzyme activity. It is concluded from this behaviour that under in vitro conditions CS2 itself (rather than its metabolites) acts as the inhibitor. 6. Oxygen treatment of the microsome-containing reaction mixture enhances the inhibition. In substrate-free control mixtures, addition of CS2 was followed by the dose-dependent formation of formaldehyde; a causal explanation is not readily available at this time.
[In vitro inhibition of oxidative N-demethylation with carbon disulfide]. Earlier findings have shown that in experimental animals (rat) and in man inhaled carbon disulphide (CS2) reversibly inhibits the non-specific oxidative drug metabolism caused by hepatic microsomal enzymes. Very little is known concerning the underlying mechanism. The present investigations were undertaken to throw light on this question. After addition of an NADPH-regenerating system to liver microsomes isolated from adult female Wistar rats, the oxidative N-demethylation of aminopyrine was measured under simultaneous exposure to CS2 by quantitatively determining the formaldehyde obtained; the resulting data were evaluated using enzyme-kinetic parameters according to Lineweaver-Burk: 1. Following acute exposure to low and medium-grade CS2 concentrations (20-400 ppm/8 h), the pattern of inhibition in rat liver microsomes is identical to that obtained in normal liver microsomes to which CS2 had been added. This finding seems to suggest that the inhibitory process under in vivo and in vitro conditions is based on one and the same molecular mechanism. 2. Upon addition of CS2 the in vitro pattern of inhibition corresponds to a strong mixed-type inhibition. 3. It is concluded from the enzyme-kinetic behaviour that CS2 attacks at two different sites of the enzyme molecule: Binding to the first site is followed by inhibition, as evidenced by the rise of Km; after saturation of this site, a second site is occupied resulting in a reactivation and, beyond this, an activation of enzyme output, as shown by the decrease of Km. CS2 exhibits a high affinity for the first site, and a low affinity for the second site. Binding at the first site is reversible. The possibility that the active centre in the enzyme molecule is the site where binding of the inhibitor occurs is ruled out. 4. A continuous decrease of Vmax at increasing inhibitor concentration is causally related with the formation of an enzyme/substrate inhibitor complex. 5. The CS2 added to the microsomes can be eliminated by helium gas; this is followed by the return of the original enzyme activity. It is concluded from this behaviour that under in vitro conditions CS2 itself (rather than its metabolites) acts as the inhibitor. 6. Oxygen treatment of the microsome-containing reaction mixture enhances the inhibition. In substrate-free control mixtures, addition of CS2 was followed by the dose-dependent formation of formaldehyde; a causal explanation is not readily available at this time.