Expression and activity of a house-fly cytochrome P450, CYP6D1, in Drosophila melanogaster.

The cytochrome P450 system of animals comprises many individual cytochromes P450 in addition to a single cytochrome P450 reductase and cytochrome b5. Although individual genes of the cytochrome P450 superfamily are highly diverged, the P450 reductase and cytochrome b(5) remain more conserved across taxa. Here, we describe the transformation of Drosophila melanogaster with a house-fly-specific cytochrome P450, CYP6D1. Functional activity of ectopically expressed cytochromes P450 requires successful interaction between the transgenic P450 and the requisite coenzymes of the host organism. Transformed Drosophila, but not controls, contained CYP6D1 protein as identified by protein immunoblotting, elevated total P450 and elevated CYP6D1 enzymatic activity. These data demonstrate that house-fly CYP6D1 can interact with low to moderate efficiency with Drosophila P450 reductase and cytochrome b(5).

Metabolism of phenanthrene by house fly CYP6D1 and dog liver cytochrome P450.

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.

Induction of hepatic cytochromes P450 in dogs exposed to a chronic low dose of polychlorinated biphenyls.

Induction of cytochrome P450 isoforms, specifically CYP1A1, and their catalytic activities are potential biomarkers of environmental contamination by polychlorinated biphenyls (PCBs). In this study, dogs were exposed to 25 ppm or 5 ppm Aroclor 1248 (PCB mixture) daily in their diet for 10 or 20 weeks, respectively. Relative to controls, hepatic microsomes from dogs dosed with PCBs had higher levels of CYP1A1 detected in immunoblots and higher levels of EROD activity, but low levels of induction for CYP2B and PROD activity. Concentrations of 96 PCB congeners in serum and liver were evaluated using capillary chromatography. Results showed that all dogs exposed to PCB mixtures had higher levels of PCB in serum and liver. Dogs preferentially sequestered highly chlorinated PCB congeners in liver relative to serum. With these experiments, we demonstrated that EROD activity was a potentially sensitive marker of PCB exposure at 5 and 25 ppm. Furthermore, CYP1A1 and EROD activity were maximally induced in dogs consuming dietary concentrations only 2.5 times the maximal permissible level for human food (FDA). The value of CYP1A1 induction as a biomarker of PCB exposure was tenuous because neither CYP1A1 levels nor EROD activity correlated with total PCB body burden. However, a small subset of congeners were identified in liver that may strongly influence EROD and PROD induction. Finally, two dogs in the 25 ppm dose group were fasted for 48 h. After 24 h of fasting, several new congeners appeared in the serum and remained in the serum for the remainder of the fast. The fast caused a 293% increase in PCB concentration in serum. This increase has strong implications regarding mobilization of toxic PCBs in wildlife during fasting (e.g., migration, hibernation).

Nitric oxide and prostaglandin E2 formation parallels blood-brain barrier disruption in an experimental rat model of bacterial meningitis.

During meningitis, the host produces a plethora of signaling agents as part of a coordinated defense mechanism against invading pathogens. Nitric oxide (NO) and prostaglandin E2 (PGE2) are two such inflammatory mediators produced in response to bacterial endotoxins. Disruption of the blood-brain barrier (BBB) is one of many pathophysiological consequences of meningitis. The present objective was to examine the time-course of NO and PGE2 production in relationship to BBB permeability alterations during experimentally-induced meningitis. Meningeal inflammation was elicited by intracisternal administration of the bacterial endotoxin, lipopolysaccharides (LPS; 200 microg), and NO, PGE2, and BBB integrity were monitored over the next 24 h. Meningeal NO production was assessed by headspace chemiluminescence; cerebrospinal fluid PGE2 was determined by enzyme-linked immunosorbent assay (ELISA) immunoassay; and BBB integrity was determined by the brain accumulation of 14C-sucrose. Similar time-course profiles for NO and PGE2 were observed, with a peak effect for both inflammatory mediators observed within 6-8 h after intracisternal LPS dosing. Statistically significant (p < 0.05) disruption of the BBB was observed in various brain regions. Strikingly similar temporal relationships were observed for NO and PGE2 production and BBB disruption. These results suggest the hypothesis that NO and PGE2 may act in conjunction to disrupt the BBB during experimental meningitis.

CYP6D1 protects thoracic ganglia of houseflies from the neurotoxic insecticide cypermethrin.

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.

Pharmacological characterization of nitric oxide production in a rat model of meningitis.

Depending on its concentration and target site, nitric oxide (NO) is an intracellular messenger or inflammatory mediator. Recent research supports an expanded role for NO in the pathophysiology of neuroinflammatory diseases. Using analytical and pharmacological techniques, the present study identifies NO as a potential inflammatory mediator in experimental meningitis in the rat. Intracisternal administration of lipopolysaccharide (LPS) induced NO synthesis from the lateral and third ventricle choroid plexi and surface meninges but not from systemic white blood cells, suggesting that meningeal inflammation was restricted to the central nervous system. The time course of NO production revealed at 3 hr lag after intracisternal LPS, followed by a peak of 8 hr and subsequent decrease to baseline 24 hr after LPS dosing. The pharmacological rank order of NO synthase inhibitors in meningeal preparations (NG-aminoarginine > NG-methylarginine approximately aminoguanidine) was slightly different than the rank order for the LPS-stimulated monocyte-macrophage cell line, J774A.1 (NG-aminoarginine approximately NG-methylarginine > aminoguanidine). A prolonged inhibition of NO production was observed in cultured meningeal preparations or J774A.1 cells briefly exposed to and washed free from NO synthase inhibitors. These findings implicate NO as an inflammatory mediator during experimental meningitis, and suggest that NO synthase inhibitors might be potentially useful agents for meningeal inflammation.