Oxidative and conjugative metabolism of p-nitroanisole and p-nitrophenol in isolated rat liver cells.

Isolated rat liver cells were used in the study of p–nitroanisole and p–nitrophenol metabolism. p–Nitroanisole was o–dealkylated to form p–nitrophenol which was subsequently conjugated to form predominantly sulphate esters and β–glucuronides. 1. At low concentrations of p–nitrophenol, sulphate conjugation was predominant but with increasing substrate concentration the glucuronidation activity was increased. At 250 μM p–nitrophenol the major conjugate formed was glucuronide while sulphate conjugation was inhibited. 2. With p–nitroanisole in low concentration, resulting in low oxidation rate, all the p–nitrophenol formed was further conjugated to sulphate esters. With higher phenol production, glucuronide conjugates were also formed. 3. Phenobarbital treatment of the rats increased the rate of p–nitroanisole oxidation by more than four times but had very little effect on the conjugation reactions. In these cells the major conjugate formed was glucuronide. 4. The rate of glucuronidation in the isolated liver cells was higher than in microsomes and responded differently to various inhibitors. Alprenolol, a substrate of the hepatic mono–oxygenase system, had no inhibitory effect on glucuronidation in microsomes in the absence of NADPH. However, low concentrations of alprenolol showed similar inhibitory effects in microsomes in the presence of NADPH as in isolated liver cells. 5. Nitrophenol glucuronide was strongly inhibitory to the glucuronidation in microsomes while almost no inhibition was observed in the isolated liver cells, probably due to poor penetration of the glucuronide through the cell membrane. 6. Rotenone, menadione and ethanol had little effect in microsomes but were potent inhibitors of glucuronidation in the isolated liver cells, most probably by interfering with the synthesis of UDPGlcA. The effect of ethanol was maximal at as low a concentration as 10 mM and was shown to be due to alcohol dehydrogenase–dependent ethanol oxidation. The inhibition was presumably caused by the the increased cytoplasmic NADH/NAD4 ratio resulting from the metabolism of ethanol.