Detection of free radicals as a consequence of rat intestinal cellular drug metabolism

Because the intestine is the first pass organ for orally administered drugs and because some of these drugs are known to undergo oxidative metabolism leading to the formation of free radicals, we investigated the potential for this to occur in cell suspensions of rat enterocytes. As part of our study, the effect of intracellularly produced superoxide on cellular metabolism was investigated. The drugs chosen were the quinone, menadione and the aromatic nitro-containing compound, nitrazepam. On incubation of both drugs with isolated enterocytes and the spin trap, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), rapid appearance of an electron paramagnetic resonance (EPR) spectrum was recorded which was characteristic of hydroxyl radicals being spin trapped by DMPO giving 2,2-dimethyl-5-hydroxy-1-pyrrolidenyloxyl (DMPO-OH). Experiments were conducted which determined that the EPR spectrum of DMPO-OH resulted from the initial spin trapping of superoxide by DMPO to yield the corresponding nitroxide, 2,2-dimethyl-5-hydroxyl-1-pyrrolidenyloxyl (DMPO-OOH). Bioreduction of DMPO-OOH by glutathione peroxidase led to the rapid formation of DMPO-OH. We believe this enzymic pathway accounted for the EPR spectrum noted in incubations with either drug in the presence of the spin trap, DMPO. The incubation of enterocytes with both drugs did not mediate release of 51Cr nor lactate dehydrogenase. However, production of 14CO2 from [14C]glucose was severely inhibited (4-5-fold) in the presence of both drugs, while the incorporation of [14C]leucine into trichloroacetic acid precipitable protein was antagonized by menadione only. We conclude that superoxide can be demonstrated to arise as the result of enterocyte metabolism of menadione or nitrazepam. The consequence of oxidative metabolism of these drugs results in cellular dysfunction.