Kinetic studies of benzpyrene and hydroxybenzpyrene metabolism

The kinetics of benzypyrene (BP) metabolism were examined in liver microsomes, and in accordance with the results of Hansen and Fouts [9] exhibited curcilinear Lineweaver-Burk plots. The problem was exacerbated in microsomes of 3-methylcholanthrene (MC-ms) treated rats. The Km for BP, measuring hydroxybenzypyrene (OHBP) appearance was about 0.3 muM in MC-treated adult rats and about 1.0 muM in untreated rats. These values were obtained using a substrate range of 0.2-2.0 muM benzpyrene, 20 mug of microsomal protein/ml and a 3 min assay time. With longer assay times and with higher microsomal protein concentrations curvilinear reciprocal plots were obtained. This was found to be due to a combination of three factors, namely non-specific binding of BP to the microsomes, rapid depletion of substrate, and further metabolism of hydroxy products. At 100 mug microsomal protein/ml about 50% of added BP was non-specifically bound to the microsomes in the range of 0.2-2.0 muM BP. Addition of albumin to the medium (1 mg/ml) greatly enhanced the BP hydroxylase activity but only slightly increased the amount of BP remaining in the medium after sedimentation of the microsomes by centrifugation. 3-OHBP, one of the phenolic products of BP metabolism was found to be metabolized to a non-fluorescent products(s); the Km for this compound was similar to that for BP. Differences were seen in the Vmax rates of BP disappearance and OHBP appearance. Disappearance of BP is several fold faster than OHBP appearance and has a larger Km. The latter may be due to the need to use higher amounts of protein and to allow depletion of enough substrate to make measurements significantly reproducible or the higher Km may reflect a composite value for different routes of BP metabolism.