Doctoral Dissertations

Date of Award

8-1988

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biomedical Sciences

Major Professor

Larry C. Waters

Committee Members

Stephen Kennel, Julian Preston, Bruce Jacobson, Carroll Nix

Abstract

The cytochrome P-450-dependent mixed function oxidase (MFC) system in insects appears to be particularly important in insecticide resistance. Because of the multiplicity of P-450 isozymes and their overlapping substrate specificities, qualitative and quantitative variations in the different forms of P-450 can influence both insecticide metabolism and normal metabolic processes such as hormone and pheromone biosynthesis and feeding habits. Resistance to various insecticides in Drosophila has been directly associated with increased or unique MFO activities.

The Drosophila strains Hikone-R(BG) and 91-R were originally selected for resistance to the organochlorine insecticide DDT and were shown in these experiments to also be cross-resistant to malathion, an organophosphate. The Hikone-R(BG), 91-R, and MH-II strains are all cross-resistant to various classes of insecticides, and all possess a unique microsomal protein band with a molecular mass of approximately 56 kDa, designated P-450-B, that is not discernible in the susceptible Drosophila strains Oregon-R, 91-C and Canton-S. All Drosophila strains examined contain an ubiquitous subset of P-450 with a molecular mass of 59 kDa identified as P-450-A. The Hikone-R(BG) and 91-R strains had higher total P-450 contents, and much greater (30 to 60-fold) N-nitrosodimethylamine (NDMA) demethylase activities than their susceptible counterparts Oregon-R and 91-C.

P-450-A and P-450-B were purified from Oregon-R and Hikone-R(BG) by sequential chromatography on octylamino-agarose, DEAE-cellulose, and hydroxyapatite. Purified Drosophila P-450-A and P-450-B had specific activities from 6-9 nmol P-450/mg protein and exhibited molecular weights exactly corresponding to P-450-A and P-450-B in microsomes.

Drosophila NADPH-cytochrome P-450 reductase was purified from Oregon-R by affinity chromatography on NADP-agarose and had a molecular weight of 65 kDa. Purified NADPH-cytochrome P-450 reductase was capable of reducing purified P-450 in vitro.

Analysis of purified P-450-A and P-450-B by isoelectric focusing and 2-D electrophoresis showed that P-450-A is composed of at least five forms with different isoelectric points while P-450-B contains a minimum of two different forms.

Purified preparations containing both P-450-A and P-450-B exhibited catalytic activity in a reconstituted system with phospholipid, NADPH-cytochrome P-450 reductase, and 7-ethoxycoumarin as substrate. Purified P-450-A, partially purified P-450 preparations, or solubilized microsomes did not metabolize 7-ethoxycoumarin or benzo(a)pyrene in the reconstituted system.

P-450-specific MoAbs were produced that recognized solubilized P-450 epitopes in solid-phase RIA and ELISA techniques and bound to denatured P-450 epitopes in immunoblots. One of these MoAbs, 433-8-1d was specific for P-450-B, while another, 433-13-2e, recognized P-450-A-specific epitopes.

Neither MoAb 433-8-1d or 433-13-2e could inhibit P-450-dependent NDMA-demethylase activity, but immune serum from the mouse utilized to produce these hybridomas suppressed over 50% of this activity compared to control levels. Purified MoAb 433-8-1d conjugated to Sepharose beads could not selectively remove NDMA-demethylase activity from microsomes.

P-450-A and P-450-B specific MoAbs were used to examine the expression of immunoreactive P-450 in insecticide resistant and sensitive Drosophila strains. Large amounts of P-450-B were detected in immunoblotting experiments using microsomes from resistant Hikone-R(BG), 91-R, and MH-II strains. Trace amounts of P-450-B were found in the susceptible Oregon-R, 91-C, and Canton-S strains. P-450-A constituted the bulk of the immunoreactive P-450 in susceptible strains, but P-450-B was the major P-450 form in the resistant strains. Immunoreactive P-450-B levels were 10 to 20-fold greater in resistant than susceptible strains. The expression of significant amounts of immunoreactive P-450-B required a positive second chromosome derived from a resistant strain and this P-450-B level was increased in strains containing both positive chromosomes II and III.

MoAbs to Drosophila P-450 cross-reacted with microsomes and partially purified preparations from the resistant Rutgers, and susceptible CSMA housefly strains in immunoblots, and the P-450-B-specific MoAb 433-8-1d bound microsomes from Southern armyworms in RIA and ELISA experiments. MoAb 433-8-1d also reacted with microsomal proteins prepared from both normal rat liver and animals induced with ethanol or phenobarbital while MoAb 13-2e did not recognize any rat liver proteins.

Cytochrome P-450-B expression related to insecticide resistance in Drosophila has been extensively characterized by biochemical, genetic, and immunochemical methods. The reagents necessary to expand this analysis to include the molecular biology of P-450 expression and P-450-related resistance are now available and the system is ready to be exploited to its fullest potential.

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