Inhibition by chloramphenicol of the microsomal monooxygenase complex of rat liver

Abstract

Received 17 October 1978 1. Introduction In several species of animal the concomitant administration of chloramphenicol with a number of other therapeutic agents has been shown to prolong the actions of these agents [I--4]. Consistent with these observations was the finding that chli~r- amphenicol could inhibit the drug metabolizing activity of a 9000 X g supernatant of an homogenate of liver tissue [5], and the antibiotic was further shown to be a competitive inhibitor of liver microsomal mono- oxygenase activity [6]. More recently however there have been reports [7-9] which have indicated that incubation of chloramphenicol with an oxygenated preparation of liver microsomes containing a system for generating reduced NADP results in the produc- tion of a metabolite of the antibiotic that binds covalenfly to microsomal protein. These f'mdings would be consistent with the antibiotic being able to bring about irreversible inhibition ofmicrosomal monooxygenase activity and they suggested to us the need to assess the claim, made in [1] where the experimental result~ were not given, that the antibiotic could cause irreversible inhibition in vitro of the monooxygenase activity of a 9000 X g supematant fraction of an homogenate of liver tissue. However, since such a microsomal preparation contains unknown, and possibly variable, concentrations of endogenous substrates of monooxygenase activity, together with a variety of systems for generating reduced pyridine nucleotides, we selected as our enzyme preparation for the present studies resus- pended washed liver microsomes. In initial rate studies using this preparation we showed that chlorampheni¢ol does both competitively and irreversibly inhibit monooxygenase activity with a half-life for inactivation of 11.5 min. We further showed that o-nitroanisole, a substrate for mono- oxygenase activity, profoundly inhibits the specific binding of radiolabeled material to microsomes incubated with 14C.labeled chloramphenicol, and we consider that these collected results are sufficient evidence to warrant an investigation into the possible involvement of an active site-directed mechanism [10] in the inhibitory action of the antibiotic on microsomal monooxygenase activity. 2. Methods and results Batches of microsomes were prepared at ,-~17 mz protein/ml by the procedure in [11] from liver tissue taken, on each occasion, from 2-4 sexually mature male Wistar rats, protein being determined on each batch by the Folin-phenol method [ 12]. The animals were all obtained from the University Central Animal Breeding Facility, and fed a stock laboratory diet until the day prior to sacrifice by decapitation. The mono- oxygenase activities of these microsomal preparations were measured at 37°C by amodification of a spectro- photometric method [11 ] in which O-demethylation of o-nitroanisole was measured. This method was chosen because: 0) In principle it allows initial rates of enzymic activity to be measured directly, this being due to the development in theassay system of an absorption band associated with the production of o-nitrophenol;