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Polycyclic aromatic hydrocarbons (PAHs) are a ubiquitous class of environmental contaminants. The compound phenanthrene is a model PAH. A novel fluorometric method for measuring phenanthrene metabolism in vitro was developed and verified with direct measurement of [14C]phenanthrene using dog liver microsomes. The fluorometric assay and direct measurement of [14C]phenanthrene metabolism were used to show that CYP6D1, a house fly cytochrome P450, is the major house fly P450 involved in phenanthrene metabolism. Phenanthrene was metabolized by microsomes from the LPR strain of house fly that overexpresses CYP6D1, but metabolism was not observed in the CS strain that has a lower level of CYP6D1. Furthermore, the majority of phenanthrene metabolism was inhibited by a CYP6D1-specific antibody. This study increases the number of known substrates of CYP6D1 and identifies polyaromatic hydrocarbons as potential substrates of CYP6D1. The utility of CYP6D1 as an agent in bioremediation and the utility of the new fluorometric assay for understanding PAH metabolism in insects and mammals are discussed.

CYP6D1 is a housefly cytochrome P450 known to metabolize neurotoxic pyrethroid insecticides. To determine if the nervous system was capable of metabolizing pyrethroids, we examined CYP6D1-mediated in vitro metabolism in thoracic ganglia from pyrethroid-resistant (LPR) and -susceptible (CS) strains of housefly. SDS-PAGE/immunoblotting revealed that CYP6D1 was expressed in all tagmata and in thoracic ganglia of both strains, but in all cases the levels of CYP6D1 were higher in the LPR strain. Using a CYP6D1-specific antiserum, we found CYP6D1 to be the major, and possibly the only, P450 isozyme involved in cypermethrin metabolism in thoracic ganglia homogenates. Additionally, thoracic ganglia homogenates from LPR houseflies metabolize more cypermethrin than preparations from susceptible flies. This metabolism was inhibited by piperonyl butoxide and a CYP6D1-specific antibody. Our results indicate that thoracic ganglia of LPR houseflies are protected from the neurotoxin cypermethrin by virtue of the higher levels of CYP6D1 compared to the susceptible houseflies. This P450-mediated detoxification of an insecticide at the level of the target tissue helps to explain the high levels of resistance to pyrethroids in the LPR strain.

Cytochrome P450lpr is a xenobiotic metabolizing P450 that is found in house flies (Musca domestica). To better understand the regulation of cytochrome P450lpr, the effects of 21 potential monooxygenase inducers were examined for their ability to induce total cytochromes P450 and cytochrome P450lpr levels in adult flies. Six compounds caused induction of total cytochromes P450 per mg protein in adult susceptible (CS) house flies: ethanol (1.6-fold), phenobarbital in food (1.5-fold) or water (1.5-fold), naphthalene (1.3-fold), DDT (1.3-fold), xanthotoxin (1.4-fold), and alpha-pinene (1.2-fold). Six compounds were found to be inducers of cytochrome P450lpr: piperonyl butoxide in food (1.9-fold), phenobarbital in food (1.4-fold) and water (3.4-fold), clofibrate (1.3-fold), xanthotoxin (1.3-fold), methohexital (1.3-fold), and isosafrole (1.3-fold). Comparison of our results with house fly P450 6A1 indicates that there are specific inducers for each of these individual P450s as well as compounds that induce both P450s. Total P450s were inducible by PB in CS house fly larvae, but not in LPR larvae. Immunoblotting revealed no detectable P450lpr in control or PB-treated larvae in either strain. Thus, although total P450s are inducible in the susceptible strain larvae, P450lpr does not appear to be normally present or inducible with PB in larvae of either strain. Northern blots of phenobarbital (in water) treated CS flies indicated that there was a 4.2-fold increase in the P450lpr (i.e., CYP6D1) mRNA levels over the untreated flies. In the multiresistant LPR strain there was no apparent induction of CYP6D1 mRNA by phenobarbital. Following phenobarbital induction, the level of CYP6D1 mRNA in the CS strain was about half of the level in the LPR strain.