Biotransformation of nitrosobenzene, phenylhydroxylamine, and aniline in the isolated perfused rat liver

Abstract

1. Haemoglobin-free single-pass perfusion of isolated rat liver with [¹⁴C]aniline, [¹⁴C]phenylhydroxylamine, and [¹⁴C]nitrosobenzene was carried out.

2. Perfusion with aniline revealed apparent enzyme kinetics for 4-aminophenol formation with K_m = 144μM, V_max = 51 nmol/min per g liver wet; for 2-aminophenol K_m = 144μM, V_max = 16nmol/min per g; for acetanilide K_m = 33μM, V_max = 25 nmol/min per g. Formation of phenylhydroxylamine and nitrosobenzene was observed at a rate of 1.5 nmol/min per g provided that these metabolites had been trapped within red cells.

3. Perfusion with phenylhydroxylamine displayed a metabolic pattern similar to aniline with apparent phenylhydroxylamine reduction kinetics of K_m = 260μM and V_max = 600 nmol/min per g. In addition an acid-labile phenylhydroxylamine glucuronide was formed.

4. Perfusion with nitrosobenzene showed very rapid reduction to phenylhydroxylamine and to the metabolites observed with phenylhydroxylamine. In postmicrosomal supernatant, enzymic reduction of nitrobenzene by NADH and NADPH showed K_m = 12μM nitrosobenzene and V_max = 5000nmol/min per g.

5. Three per cent of nitrosobenzene was irreversibly bound to liver proteins. After 20min perfusion with nitrosobenzene, 0.95 μmol of liver glutathione was lost per 10μmol nitrosobenzene infused; 0.16μmol of glutathione was released with effusate and bile, 0.46μmol of glutathionesulphinanilide was produced, the rest, 0.33μmol, may have formed mixed disulphides.