Geoffrey D. Wheelock

The role of cytochrome P450lpr in deltamethrin metabolism by pyrethroid-resistant and susceptible strains of house flies

Abstract The LPR strain of house fly is highly resistant to pyrethroid insecticides and this resistance is associated with high levels of monooxygenase activity and total cytochromes P450. To evaluate the role of P450 lpr (the major P450 in LPR house flies) in pyrethroid resistance, an antiserum specific for house fly cytochrome P450 lpr was tested for its ability to inhibit cytochrome P450 monooxygenase-dependent metabolism of the pyrethroid insecticide deltamethrin. Treatment of microsomes from pyrethroid resistant LPR house flies with anti-P450 lpr antiserum resulted in 97% of the radiolabel being recovered as deltamethrin compared to 76% in the normal serum control. There was no significant difference in the amount of deltamethrin recovered from microsomes of susceptible (S+) flies treated with normal serum or anti-P450 lpr antiserum compared to the carbon monoxide-inhibited control. However, there was statistically significant inhibition of specific metabolite production in both strains. Using LPR microsomes, seven NADPH-dependent products of [ 14 C]deltamethrin metabolism could be identified by thin-layer chromatography, designated as B, C, D, E, F, G, and H (origin). Metabolites B, C, D, and H were the major metabolites. Formation of metabolites B, C, and H was inhibited by anti-P450 lpr while D was increased. In S+ microsomes, only metabolites C, D, and E were consistently detected and anti-P450 lpr inhibited formation of C and D while E was unchanged. The primary metabolites inhibited by the antiserum in LPR appear to be modifications of the acid portion of the deltamethrin molecule, and the major metabolites increased by antiserum treatment were oxidations on the alcohol portion of the molecule, suggesting that P450 lpr preferentially attacks the gem-dimethyl group of deltamethrin. Pyrethroid-resistant LPR flies, topically dosed with a sublethal dose of deltamethrin, rapidly metabolized and excreted polar metabolic products. Similarly, microsomes from LPR house flies exhibited rapid cytochrome P450-dependent metabolism of deltamethrin. This study indicates that P450 lpr contributes to monooxygenase-dependent deltamethrin resistance in LPR house flies.

Simultaneous purification of a cytochrome P-450 and cytochrome b5 from the house fly, Musca domestica L.

Abstract Cytochrome P-450, cytochrome b5 and cytochrome P-450 reductase were purified from house fly abdomens using high performance liquid chromatography (HPLC). Using a new technique, cytochrome P-450 was separated from the bulk of other proteins after polyethylene glycol fractionation and hydrophobic interaction chromatography (HIC) using a phenyl-5PW column. This technique resulted in 91% recovery of the cytochrome P-450s in a single concentrated fraction that also contained the remaining cytochrome b5 and cytochrome P-450 reductase activity. Further purification by anion exchange on a DEAE-5SW column resolved the cytochrome P-450s, cytochrome b5 and cytochrome P-450 reductase into individual fractions. The ion exchange step yielded one fraction that contained a high specific content of P-450 (14.4 nmol/mg protein). This cytochrome P-450 fraction ran as a single band at 54.3 kDa in sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gel electrophoresis and had a carboxy ferrocytochrome absorbance maximum at 447 nm. Further purification of the anion exchange cytochrome b5 fraction, by C8 reverse phase HPLC, resulted in a cytochrome b5 fraction with a specific content of 51.8 nmol/mg protein and an apparent molecular mass of 19.7 kDa by SDS-PAGE. The anion exchange HPLC fraction containing the cytochrome P-450 reductase activity was further purified by NADP-agarose affinity chromatography. This step yielded cytochrome P-450 reductase with an apparent molecular mass of 72 kDa.

Immunological detection of cytochrome P450 from insecticide resistant and susceptible house flies (Musca domestica)

Abstract An antiserum was raised in a rabbit against a cytochrome P450, termed P450 lpr , purified from the pyrethroid resistant LPR strain of house fly. This polyclonal antiserum was specific for P450 lpr as judged by both rocket immunoelectrophoresis and immunoblotting. The antiserum was used to detect immunoreactive cytochrome P450s in insecticide susceptible and resistant house flies. Single immunoreactive bands of a slightly variable position were obtained when crude microsomes from six insecticide resistant house fly strains (LPR, Dairy, Kashiwagura, 3rd-Y, EPR, ASPR) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting, while immunoreactive bands were barely detectable in insecticide susceptible ( aabys , S+) strains. The immunoreactive cytochrome P450s were isolated from each of eight house fly strains by high-performance liquid chromatography. The isolated immunoreactive cytochrome P450s from all strains were immunologically identical to P450 lpr by fused rocket immunoelectrophoresis, and only those from the Kashiwagura and ASPR strains differed in chromatographic and/or electrophoretic properties from P450 lpr . Amounts of immunoreactive P450 were determined in house fly microsomes. Immunoreactive P450 represented 68% (specific content: 0.48 nmol/mg protein) of the total (0.72 nmol/mg) cytochrome P450 in LPR microsomes and 6.6% (0.011 nmol/mg) of the total (0.15 nmol/mg) cytochrome P450 in S+. Thus, immunoreactive P450 was constituitively expressed at 44-fold higher levels in insecticide resistant LPR flies compared to the susceptible S+ strain.

Discordant expression of the cyclin-dependent kinases and cyclins in rat liver following acute administration of the hepatocarcinogen [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio] acetic acid (WY14,643)

Cellular proliferation is an essential aspect of chemical carcinogenesis. At the core of cell cycle regulation is a family of serine/threonine protein kinases termed cyclin-dependent kinases (cdk). Cdk activity, which directs progression through the cell cycle, is dependent upon cdk binding to the appropriate, phase-specific cyclin proteins. Alterations in hepatic cdk1, cdk2, cdk4, cdk5, and cyclin protein expression were determined in response to acute dosing of the prototypic peroxisome proliferator and hepatocarcinogen [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio] acetic acid (WY14,643). Intraperitoneal dosing of 45 mg WY14,643/kg daily for 4 days to young, male rats produced dramatic increases in hepatic protein expression of all cdk analyzed as well as cyclins B, D2, D3, and proliferating cell nuclear antigen (PCNA). The largest relative increases, 6.1-, 2.8-, 11-, 83-, and 7.9-fold, were seen with cdk1, cdk4, cyclin B, cyclin D3, and PCNA, respectively. Increases of only 1.8-, 2-, 1.6-, and 1.4-fold were noted, respectively, for cdk2, cdk5, cyclin D2, and cyclin E. Analysis of gel filtration fractions indicated that PCNA co-eluted with cdk1 from the WY14,643-treated rats as a 70-80 kDa molecular complex. In contrast, cdk4, cdk5 and D cyclins migrated as much larger complexes with an estimated MW of approximately 180-190 kDa.

Bioimmunoassay of Aryl Hydrocarbon (Ah) Receptor Transformation in Vitro by 2,3,7,8- Tetrachlorodibenzo-p-Dioxin(TCDD)

An assay is described that detects 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) indirectly by quantitating transformation of guinea pig aryl hydrocarbon (Ah) receptor in vitro. Transformed Ah receptor is isolated on an affinity column and measured by dot-blot immunoassay. Antisera were developed against the highly conserved amino-terminal region of mammalian Ah receptor and affinity-purified. Hepatic cytosol from guinea pigs was treated with TCDD. Treatment resulted in the transformation of the Ah receptor to the DNA binding form. Affinity chromatography of the treated cytosol using dioxin-responsive element (DRE) oligonucleotides isolated a single immunoreactive and TCDD treatment-dependent protein with a molecular mass of 105 kDa. The protein was identified as guinea pig Ah receptor based on TCDD treatment dependence, molecular weight, and immunoreactivity. A bioimmunoassay was designed to detect the transformation of guinea pig Ah receptor by TCDD. Transformation of guinea pig Ah receptor by TCDD was dos...

Rapid isolation of a neurohormone from mosquito heads by high-performance liquid chromatography.

Methods were developed for the isolation of the egg development neurosecretory hormone, EDNH, from heads of the mosquito Aedes aegypti. This hormone stimulates ecdysone production by ovaries. Methods used for the successful isolation of insulin-like peptides from vertebrate tissues were modified to develop a four-step procedure involving extraction in acidified ethanol, precipitation by neutralization, followed by sequential separation on size-exclusion, ion-exchange and reversed-phase high-performance liquid chromatography columns.

Rapid microsome preparation from limited numbers of house flies

Microsomal cytochrome P450 dependent monooxygenases are important in metabolism of insecticides, allelochemicals and hormones. Among insects, microsomal monooxygenases are commonly studied in the house fly, which is often utilized as a model for insect monooxygenases (Hodgson, 1985). However, study of monooxygenases is difficult in many (especially small) insects due to the relatively large amount of tissue needed to isolate microsomes. Standard methods for isolating microsomes by differential ultracentrifugation require large amounts of starting material due to the size limit of ultracentrifugation apparatus, typically having centrifuge tubes that require 10-50 ml (Lake, 1987), making them ill suited to process microsomes from small amounts of tissue. Additional drawbacks of standard methods include long ultracentrifugation times (typically one hour) and expensive equipment. Methods suitable for isolating small amounts of microsomes include gel filtration (Tangen et al., 1973), calcium aggregation (Schenkman & Cinti, 1972), and isoelectric precipitation (Fry & Bridges, 1975). Gel filtration is unsuitable for processing large amounts of samples (Lake, 1987), while calcium aggregation can result in alteration of microsome composition (Schenkman & Cinti, 1972) and isoelectric precipitation can result in loss of enzyme activity (Fry & Bridges, 1975). However, differential centrifugation has none of these drawbacks and is the most popular method for isolating microsomes (Lake, 1987). The desirability of a micro method to quickly process small amounts of microsomes from multiple insects utilizing differential centrifugation prompted this study.

Detection of cytochrome b5 from the house-fly, Musca domestica : comparison of immunological and spectrophotometric methods

Spectrophotometric assay of microsomal cytochrome b5 in house-flies produces different results depending on whether sodium dithionite or NADH is used as the reducing agent and whether or not detergent is present. Microsomes assayed for cytochrome b5 with dithionite in the presence of detergent gave the highest values, followed by dithionite alone, NADH plus detergent, and then NADH alone. Isopropanol treatment of microsomes extracted cytochrome b5 free of spectrophotometrically interfering cytochrome P-450. Studies using immunoblotting and rocket immunoelectrophoresis with polyclonal antisera raised against the purified cytochrome b5 showed that isopropanol treatment quantitatively extracted cytochrome b5.

Purification and characterization of cytochrome b5 from the housefly (Musca domestica)

Abstract 1. 1. Isopropanol treatment of Musca domestica (house fly) microsomes extracted cytochrome b5 and resulted in a minimum 5.2-fold purification. 2. 2. Cytochrome b5 was highly purified from the isopropanol extract using ion exchange and reverse phase high performance liquid chromatogrphy. 3. 3. A polyclonal antiserum was produced against the purified cytochrome b5. 4. 4. Purified house fly cytochrome b5 had a molecular mass of 19.7 kDa, slightly higher than those reported for rabbit and rat microsomal cytochrome b5 (16.6–16.7 kDa), but lower than cytochrome b5 from Mediterranean fruit fly (21 kDa). 5. 5. Reduced house fly cytochrome b5 absorbed maximally at 424 nm, similar to other cytochromes b5. 6. 6. Amino acid analysis of house fly cytochrome b5 produces at higher percentage of hydrophobic amino acid residues and less acidic residues than other cytochromes b5. 7. 7. In immuno-blot analysis, house fly cytochrome b5 shared antigenic epitope(s) with the dipteran species Musca autumnalis (face fly), Stomoxys calcitrans (stable fly) and to a lesser extent, Drosophila melanogaster (fruit fly). 8. 8. Anti-house fly cytochrome b5 did not recognize cytochrome b5 from Apis mellifera (honey bee), Tricoplusia ni (cabbage looper), Tetranychus urticae (two-spotted spider mite) or Rattus rattus (rat).