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Biochemical changes in the cytochrome P450 monooxygenases of seven insecticide-resistant house fly (Musca domestica L.) strains
PESTICIDE
BIOCHEMISTRY
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PHYSIOLOGY
36, 127-134 (19%)
Biochemical Changes in the Cytochrome P450 Monooxygenases of Seven Insecticide-Resistant House Fly (Musca domestica L.) Strains JEFFREY G. SCOTT,’ SUSANNA S.T.
LEE, AND TOSHIO SHONO~
Department of Entomology and institute for Comparative and Environmental Toxicology, Comstock Hall, Cornell University, Ithaca, New York 14853-0999 Received August 28, 1989; accepted October 30, 1989 The levels of cytochrome P45Os, cytochrome b,, NADPH-cytochrome c (P450) reductase, and three indices of monooxygenase activities (methoxyresorufin 0-demethylation (MROD), ethoxycoumarin 0-deethylation (ECOD), and aryl hydrocarbon hydroxylation (AHH)) were compared between one insecticide-susceptible and seven MFO-mediated insecticide-resistant house fly strains. Cytochrome b, and P450 reductase were elevated by 1.2- to 2.2-fold and 1.5 to 2.3-fold, respectively, in the resistant strains compared with the susceptible. The level of cytochrome P45Os was significantly increased (up to 2.1-fold) in all except two resistant strains compared with the susceptible strain. These results support the idea that all three monooxygenase components may play a role in insecticide resistance. Levels of MROD, ECOD, and AHH were significantly elevated (up to W-fold) in nearly all cases. Pyrethroid resistance was generally correlated with MROD and AHH activities in all except one strain. o 1990Academic press,I~C.
INTRODUCTION
Insecticide resistance is one of the major factors that limits our ability to control many key pests. Although the cytochrome P450 monooxygenases (MFOS)~ are known to be a very important mechanism for causing insecticide resistance (l), the biochemical basis underlying this type of resistance is not fully understood (2). It is generally believed that monooxygenase-mediated resistance is often associated with an increase in a cytochrome P450 isozyme(s) that can carry out a specific type of metabolism of the pesticide. However, it has been pointed out that MFO-mediated resistance is not always associated with increased levels of cytochrome P45Os (2). One possible expla’ To whom correspondence should be addressed. * Present address: National Institute of Health, lo35, 2-chome, Kamiosaki, Shinagawa-ku, Tokyo, JaPan. 3 Abbreviations used: P450 reductase, NADPHcytochrome c reductase; MROD, methoxyresorutin 0-demethylation; ECOD, ethoxycoumarin Odeethylation; AHH, aryl hydrocarbon hydroxylation; MFO, cytochrome P450 microsomal monooxygenases; PBO, piperonyl butoxide; LPR, Learn pyrethroidresistant house fly strain.
nation for this is that other components of the MFO system (i.e., NADPH-cytochrome c reductase (P450 reductase) and/or cytochrome b,) may also be involved in MFOmediated resistance (3, 6). There are relatively few cases where the levels of P450 reductase and/or cytochrome b5 have been examined in resistant species. DeVries and Georghiou (4) found 2.4-, 1.9-, and 2.2-fold increases in cytochrome P45Os, cytochrome bS, and P450 reductase, respectively, in a house fly strain having partially piperonyl butoxide (PBO)suppressible permethrin resistance. Vincent et al. (5) found 2.4- and 1.6-fold increases in cytochrome P45Os and P450 reductase, respectively, in the multiresistant Rutgers house fly strain. Scott and Georghiou (6) noted 3.8, 1.5-, and 2.8fold increases in cytochrome P45Os, cytochrome b,, and P450 reductase in the Learn-PyR (LPR) house fly strain having MFO-mediated pyrethroid resistance. These elevated levels were confirmed by Lee and Scott (7), although the relative amounts were slightly different. Subsequent studies with the LPR strain (8) showed a correlation between PBO127 0048-3575&O $3.00 Copyright All rights
8 1990 by Academic Press, Inc. of reproduction in any form reserved.
128
SCOTT,
LEE,
suppressible permethrin resistance and the presence of increased amounts of one or more of the MFO components on autosomes 1, 2, 3, and 5. In adult Tribolium castaneum, a DDT, organophosphate, and carbamate multiresistant strain (9), was found to have 3.0- and 2. l-fold more cytochrome P450 and cytochrome bs, respectively, compared with a susceptible strain (10). P450 reductase was not measured in these beetles. One difficulty encountered when studying the MFO system is the fact that there are multiple forms of cytochrome P450, and each may have its own unique substrate specificity. It is currently unknown if resistance is conferred by the action of several, a few, or perhaps only one P450. To investigate this question, we looked at three different MFO assays: methoxyresofurin Odemethylation (MROD), ethoxycoumarin 0-deethylation (ECOD), and aryl hydrocarbon hydroxylation (AHH). Because these were different types of MFO-mediated reactions, we reasoned that they might detect different populations of P45Os. Furthermore, from a previous study we found that pyrethroid resistance in a highly resistant strain of house fly was associated with a large (63.5-fold) increase in MROD activity (7), and we wished to further clarify this observation. We reasoned that we might be able to associate resistance to a specific insecticide class with a specific MFO assay, if the number of isozymes responsible for resistance to each class was relatively limited and showed a differential ability to oxidize methoxyresorufin, ethoxycoumarin, or benzo[a]pyrene. If successful, these studies could lead to the development of a diagnostic test for MFO-mediated insecticide resistance. The purpose of this study was to compare the levels of cytochrome P45Os, cytochrome b,, P450 reductase, and MFO activity between insecticide-resistant and susceptible house fly strains; to better understand the role of the MFO components in resistance and to determine
AND
SHONO
whether or not three common MFO assays could be correlated to resistance to one or more classes of insecticides. MATERIALS
AND
METHODS
Insects. Eight strains of house fly were used. (i) “S + ,” an insecticide-susceptible strain, was originally obtained from Dr. F. W. Plapp, Jr. of Texas A&M University. (ii) “Dairy,” a multiresistant strain (ll), was collected from the Learn dairy in Schuyler Co., New York, in 1986 and reared without insecticide selection. (iii) “LPR” is a multiresistant strain (12) with high levels of resistance to pyrethroids (e.g., > lO,OOO-fold to deltamethrin) (6). This strain was originally collected from the Learn dairy in 1980 and selected in the laboratory with permethrin for 40 generations (12). The mechanisms of pyrethroid resistance in this strain are increased MFOmediated detoxication, insensitivity of the nervous system (kdr), and decreased cuticular penetration (6). Each of these mechanisms has been mapped to specific autosomes (8). (iv) The “ASPR” strain was originally collected from Gunma Prefecture, Japan, and exposed to laboratory selection with permethrin for 20 generations. This strain exhibits higher levels of pyrethroid resistance in females compared with males (e.g., 3200- and 22-fold resistance to permethrin in females and males, respectively), and permethrin resistance in females is partially suppressible with PBO (13). (v) “Kashiwagura” is a pyrethroidresistant strain collected from a cattle farm in the Kashiwagura district of Miyagi village, Gunma Prefecture, Japan, in 1984 and selected with permethrin in the laboratory for 28 generations, becoming 3000-fold resistant to permethrin (T. Shono, unpublished). (vi) “RG” has the same origin as Kashiwagura; however, it was selected with permethrin for 24 generations and subsequently selected by resmethrin for four generations. (vii) “3rd-Y” is an organophosphate-resistant strain collected from the 3rd Yumenoshima island in 1979 and
MICROSOMAL
MONOOXYGENASES
AND
selected by malathion for 20 generations. 3rd-Y is lOOO- and 1600-fold resistant to fenitrothion and malathion, respectively, but is susceptible to pyrethroids (14). (viii) “EPR” is an organophosphate-resistant strain selected from the 3rd-Y strain using ethyl-fenitrothion for 20 generations and subsequently with parathion for four generations. EPR shows >4500-fold resistance to parathion, but is susceptible to pyrethroids (T. Shono, unpublished). Chemicals. Methoxyresorufin and benzo[a]pyrene were kindly provided by Dr. C. F. Wilkinson, Department of Entomology, Cornell University. Resorufin was from Eastman Kodak (Rochester, NY), while 7-hydroxycoumarin and 7-ethoxycoumarin were obtained from Aldrich Chemical Co. (Milwaukee, WI). All other chemicals used were of the highest purity grade and commercially available.
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Preparation of microsomes. Female house fly abdomens (80&2000) were used for preparing microsomes as described previously (15). Microsomal pellets were diluted to a final protein concentration of 1 mg/ml and stored in 2-ml aliquots in small glass vials at - 80°C. Microsomes were prepared from all strains on the same day for each experiment. The entire experiment was replicated two times. Protein was determined by the method of Bradford (16) using bovine serum albumin as the standard . Enzyme assays. Cytochrome P450 and b, were quantitatively analyzed at a final protein concentration of 1 mg/ml in triplicate by the method of Omura and Sato (17) using a Beckman DU-50 spectrophotometer. NADPH-cytochrome c (P450) reductase was measured in triplicate based on the method of Schonbrod and Teniere (18), as
TABLE
1
Levels of MFO Components and MFO Activity in a Susceptible (S +) Strain and Seven Insecticide-Resistant Strains of House Fly Measurement” Cytochrome x max f
P450
MlolP mg protein
Cytochrome 6, (nmol/ mg protein)
P450 reductaseb (nmoUmin/ mg protein)
MROD’ (nmoYmin/ mg protein)
ECODd (nmol/min/ mg protein)
AHH’ (nmol/min/ mg protein)
s+
552.5
0.53 (0.03)
0.38 (0.07)
74.3 (5.0)
0.05 (0.01)
0.04 (0.01)
3rd-Y
452.5
0.52 (0.02)
0.55 (0.01)
108.5
0.07 (0.01)
0.10 (0.01)
1.64 mw 1.56 (0.27)
451.5
(E,
0.50 (0.05)
126.3
0.92 (0.01)
0.30 ww
6.54 (0.24)
Dairy
450.5
0.88 (0.01)
0.56 (0.01)
6.18 (0.19)
453
0.69 (0.05)
0.53 (0.07)
0.38 (0.02)
1.77 (0.14)
RG
453
0.63 (0.03)
0.47 (0.05)
1.46 (0.W 0.69 wJf3 1.07 (0.03)
0.20 (0.01)
Kashiwagura
131.1 (3.01 113.9 (11.6)
0.35 (0.02)
4.64 (0.13)
ASPR
453
0.65 (0.07)
0.56 (0.W 0.82 (0.01)
(iii, 0.18 (0.01)
7.16 (0.W 20.1 (4.3)
EPR
LPR’
450
1.11 (0.W
a Values in parentheses are the SE of the mean (n = 6). b nmoles cytochrome c reduced. c nmoles resorufm. d nmoles 7-hydroxycoumarin. E mnoles benze[alpyrene. *All values are kO.5 nm or less. *n = 5. h Data for LPR from Lee and Scott (7).
(2.7) (3.8)
116.5 (2.1) 137.7 (10.6) 167.3
(9.6)
0.45 (0.02) 8.89 (0.33)
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SCOTT,
LEE,
previously described (15). P450 reductase was measured as nanomoles of cytochrome c reduced mini mg protein-’ and converted to nmoles reductase mg protein- ’ assuming a MW of 74,000 and 36 pmol cytochrome c reduced min- ’ mg reductase- ’ (29). The three indices of microsomal monooxygenase activities used were methoxyresorufin O-demethylation (19), ethoxycoumarin O-deethylation (20), and aryl hydrocarbon hydroxylation (21). All three reactions were measured in triplicate and carried out under atmospheric conditions in a lo-mm pathlength cuvette in an Aminco SPF-500 spectrofluorometer at 32°C as described previously (15). RESULTS
AND
DISCUSSION
The levels of the three MFO components and MFO activities present in the eight house fly strains are given in Table 1. The cytochrome P450 A,,, varied between strains, ranging from 453 nm for the ASPR, RG, and Kashiwagura strains to 450 nm for the LPR strain. This suggests that, although resistant strains may sometimes have a low Amax compared to a susceptible (22), no clear connection can be drawn. A likely explanation for this is that the P450 assay measures a summation of all the P450 isozymes. As many isozymes have a different A,,, the relative abundance of each P450 determines the overall A, observed, and thus it may be difficult to correlate resistance to the A,, in unrelated strains. Except for 3rd-Y and EPR, the level of cytochrome P45Os was higher in all the resistant strains (Fig. l), ranging from l.Zfold (RG) to 2.1-fold (LPR). The lack of correlation between cytochrome P450 levels and MFO-mediated resistance has been noted before (2). It is not clear from such results if MFO activity might increase solely by increasing the levels of P450 reductase and/or cytochrome 6,, or whether there actually is an increase in one of the cytochrome P45Os that is not detected when the sum of all P45Os is measured (3). For example, if re-
AND
SHONO Strain LPR ASPR
Kashiwagura Dairy EPR Brd-Y 0 Percent
50
100
150
of level in susceptible
200
250 strain
FIG. 1. Levels of cytochrome P45Os (nmollmg protein), cytochrome b, (nmollmg protein), and P450 reductase (nmol cytochrome c reduced minlmg protein) in seven insecticide-resistant housefly strains. Values are expressed as percentage of the level found in the susceptible strain.
sistance is due to elevated levels of a major isozyme, it may be detected as an increase in total P45Os, while resistance due to elevated levels of a minor isozyme may not measurably increase the level of total P45Os (3). P450 reductase and cytochrome b, were elevated in all of the resistant strains compared with the susceptible (Table 1, Fig. 1). Increases ranged from 1.5-fold (3rd-Y) to 2.3-fold (LPR) for P450 reductase and 1.2fold (RG) to 2.2-fold (LPR) for cytochrome b,. This suggests that elevated P450 reductase and/or cytochrome bS levels may be involved in the increased MFO activity (i.e., MFO-mediated insecticide resistance) found in these strains. Recent reports of NADH stimulation of MFO activity in house flies (23) and diamondback moth (24) support the idea that cytochrome b5 can be important in determining monooxygenase activity in insects. In order to investigate if any relationship existed between the monooxygenase components , we attempted to correlate levels of
MICROSOMAL
MONOOXYGENASES
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INSECTICIDE
RESISTANCE
0.9
0.7
0.5
P-450
0.07
y = 0.0084 + 0.,0707x
_
P=O.O03
l
0.3
I
I
1.0
1.2
(nmoles / mg protein)
r
o&6
0.02
I 0.8
I 0.6
0.3 ’ 0.4
R = 0.89
/
I
1
1
0.5
0.7
0.9
b 5 (nmoles / mg protein) y = 0.019 + 0.0398x
0.06 -
R = 0.80
PzO.018
0.04 q 0.02
0.4
L
1
0.6
0.8
P-450
I
1.0
I
1.2
(nmoles / mg protein)
Plots of the levels of cytochrome b, vs P45Os (A), P450 reductase vs cytochrome b, (B), and P450 reductase vs cytochrome P45Os (C)from eight strains of house fry. The regression equation for each line, its correlation coeficient, and the P value are given for each plot. FIG. 2.
131
132
SCOTT,
LEE,
individual components (Table 1) across the eight strains studied. Levels of cytochrome b, were somewhat correlated with levels of cytochrome P45Os (Fig. 2A), as were levels of P450 reductase vs cytochrome b, (Fig. 2B) and P450 reductase vs cytochrome P45Os (Fig. 2C). There was good correlation among the levels of P450, b,, and P450 reductase, suggestive of some type of coordinated control of the levels of these three components. If this is true, it suggests a sophisticated control mechanism because P450 reductase, cytochrome b,, and certain P450 isozymes appear to be coded for by genes on different autosomes (8, 31). The P450:cytochrome bS:P450 reductase ratio suggested by Fig. 2 (approx. 12: 10: 1) is different from the 12:4: 1 ratio proposed previously (25). While our results are consistent with the ideas that there are l&14 P45Os per reductase (25, 26) our results suggest an approximate 1: 1 ratio between cytochrome P45Os and cytochrome b,. The levels of the three monooxygenase activities (MROD, ECOD, AHH) were elevated in all the resistant strains compared with the susceptible, except for AHH activity in the 3rd-Y strain, whether comparisons were based on specific activity (Fig. 3A) or on turnover numbers (Fig. 3B). MROD and AHH activities were highest in the LPR strain, which has the highest levels of pyrethroid resistance, and these activities were lowest in the pyrethroid-susceptible (3rd-Y) strain. Additionally, MROD and AHH activities -vere increased in the LPR strain compared with the Dairy strain, which was collected from the same site but had a lower level of pyrethroid resistance. A similar correlation between levels of monooxygenase-mediated pyrethroid resistance and MROD activity has recently been reported in larvae of the diamondback moth Plutella xylostella (27). Furthermore, monooxygenase-mediated DDT, carbaryl, and parathion resistance in the Hikone-R strain of Drosophila melanogaster, which is pyrethroid susceptible, was not correlated with large (Gthree-fold) increases in
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ECOD or AHH activities (28). Our correlation between MROD or AHH activities and pyrethroid resistance was not perfect, however, as intermediate levels were noted in one pyrethroid-susceptible strain (EPR). ECOD activities were similar in most of the resistant strains, but were again lowest in 3rd-Y. We have recently purified the major cytochrome P450 from the LPR strain (30). Production of antisera and reconstitution experiments will help to clarify the substrate (i.e., methoxyresorufin, benzo[a]pyrene, etc.) specificity of this putative pyrethroid-metabolizing cytochrome P450. In conclusion, we found that MFO&&I LPR ASPR RG Kashiwagura Dairy EPR 3rd-Y 100 Fold increase
in monooxygenase
activity
Strain LPR ASPR RG Kashiwagura Dairy EPR 3rd-Y 100 Fold
increase
in
monooxygenase
TON
Levels of the three monooxygenase activities in seven insecticide-resistant housefly strains, expressed as a fold increase in specific activity (A) or as a fold increase in TON (turnover number, i.e., activity per nanomole P450) (E), compared with the susceptible strain. MROD, ECOD, and AHH refer to methoxyresoru$n 0-demethylation, ethoxycoumarin Odeethylation, and aryl hydrocarbon hydroxylation, respectively. FIG.
3.
MICROSOMAL
MONOOXYGENASES
mediated insecticide resistance in the house fly was associated with increases in cytochrome P45Os, P450 reductase, and cytochrome b5. Additionally, our results suggest that MROD activity may be a useful assay to monitor for MFO-mediated pyrethroid resistance, although further work will be needed to clarify this idea. ACKNOWLEDGMENT This work was supported in part by a gift from Toagosei Chemical Industry Co. and Hatch Grant 139414. REFERENCES 1. L. B. Brattsten, C. W. Holyoke, Jr., J. R. Leeper, and K. F. Raffa, Insecticide resistance: Challenge to pest management and basic research, Science 231, 1255 (1986). 2. E. Hodgson, The significance of cytochrome P450 in insects, Insect Biochem. 13, 237 (1983). 3. J. G. Scott, Insecticide resistance in insects, in “Handbook of Pest Management” (D. Pimentel, Ed.), CRC Press, Boca Raton, FL, In press. 4. D. H. DeVries and G. P. Georghiou, Absence of enhanced detoxication of permethrin in pyrethroid-resistant house flies, Pestic. Biochem. Physiol. 15, 242 (1981). 5. D. R. Vincent, A. F. Moldenke, D. E. Famsworth, and L. C. Terriere, Cytochrome P-450 in insects. 6. Age dependency and phenobarbital induction of cytochrome P-450, P-450 reductase, and monooxygenase activities in susceptible and resistant strains of Musca domestica, Pestic. Biochem. Physiol. 23, 171 (1985). 6. J. G. Scott and G. P. Georghiou, Mechanisms responsible for high levels of permethrin resistance in the house fly, Pestic. Sci. 17, 195 (1986). 7. S. S. T. Lee and J. G. Scott, Microsomal cytochrome P450 monooxygenases in the house fly (Musca domestica L.): Biochemical changes associated with pyrethroid resistance and phenobarbital induction, Pestic. Biochem. Physiol. 35, 1 (1989). 8. J. G. Scott and G. P. Georghiou, The biochemical genetics of permethrin resistance in the LeamPyR strain of house fly, Biochem. Genet. 24,25 (1986). 9. C. E. Dyte, Resistance to synthetic juvenile hormone in a strain of flour beetle, Tribolium castaneum, Nature (London) 238, 48 (1972). 10. E. Cohen, Studies on several microsomal enzymes in two strains of Tribolium castaneum (Tenebrionidae: Coleoptera), Comp. Biochem. Physiol. C 71, 123 (1982).
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11. J. G. Scott, R. T. Roush, and D. A. Rutz, Insecticide resistance of house flies from New York dairies (Diptera: Muscidae), J. Agric. Entomoi. 6, 53 (1989). 12. J. G. Scott and G. P. Georghiou, Rapid development of high-level permethrin resistance in a field-collected strain of the house fly (Diptera: Muscidae) under laboratory selection, .I. Econ. Entomol. 78, 316 (1985). 13. T. Shono and J. Scott, Autosomal sex-associated pyrethroid resistance in a strain of house fly (Diptera: Muscidae), possessing a maledetermining factor on chromosome three, .I. Econ. Entomol., in press. 14. H. Yoshikawa, T. Shono, and M. Eto, Synthesis and insecticidal activity of methylphosphonothiolates, J. Pestic. Sci. 11, 15 (1986). 15. S. S. T. Lee and J. G. Scott, An improved method for preparation, stabilization and storage of house fly (Diptera: Muscidae) microsomes, J. Econ. Entomol. 82, 1559 (1989). 16. M. M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding, Anal. Biochem. 72, 248 (1976). 17. T. Omura and R. Sato, The carbon monoxidebinding pigment of liver microsomes. I. Evidence for its hemoprotein nature, J. Biol. Chem. 239, 2370 (1964). 18. R. D. Schonbrod and L. C. Terriere, Inhibition of housefly microsomal epoxidase by the eye pigment, xanthommatin, Pestic. Biochem. Physiol. 1, 409 (1972). 19. R. T. Mayer, J. W. Jermyn, M. D. Burke, and R. A. Prough, Methoxyresorufin as a substrate for the fluorometric assay of insect microsomal 0-dealkylases, Pestic. Biochem. Physiol. 7,349 (1977). 20. V. Ullrich and P. Weber, The 0-dealkylation of 7-ethoxycoumarin by liver microsomes: A direct fluorometric test, Hoppe-Seyler’s Z. Physiol. Chem. 353, 1171 (1972). 21. M. S. Denison, M. Murray, and C. F. Wilkinson, Microsomal aryl hydrocarbon hydroxylase: Comparison of the direct, indirect and radiometric assays, Anal. Lett. 16, 381 (1983). 22. A. S. Perry, W. E. Dale, and A. J. Buckner, Induction and repression of microsomal mixedfunction oxidases and cytochrome P-450 in resistant and susceptible houseflies, Pestic. Biothem. Physiol. 1, 131 (1971). 23. M. J. J. Ronis, E. Hodgson, and W. C. Dauterman, Characterization of multiple forms of cytochrome P-450 from an insecticide resistant strain of house fly (Musca domestica), Pestic. Biochem. Physiol. 32, 74 (1988). 24. M.-C. Yao, C.-F. Hung, and C.-N. Sun, Fenvalerate resistance and aldrin epoxidation in larvae
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of the diamondback moth, Pesric. Biochem. Physiol. 30, 272 (1988). 25. M. Agosin, Role of microsomal oxidations in insecticide degradation, in “Comprehensive Insect Physiology, Biochemistry and Pharmacology” (G. A. Kerkut and L. I. Gilbert, Eds.), Vol. 12, p. 647, Pergamon Press, Oxford, 1985. 26. J. A. Peterson, R. E. Ebel, D. H. O’Keeffe, T. Matsubara, and R. W. E&brook, Temperature dependence of cytochrome P-450 reduction. A model for NADPH-cytochrome P-450 reductase: Cytochrome P-450 interaction, J. Biol. Chem. 251, 4010 (1976). 27. C.-F. Hung and C.-N. Sun, Microsomal monooxygenases in diamondback moth larvae resistant to fenvalerate and piperonyl butoxide, Pestic. Biochem. Physiol. 33, 168 (1989).
28. L. C. Waters and C. E. Nix, Regulation of insecticide resistance-related cytochrome P-450 expression in Drosophila melanogaster, Pestic. Biochem. Physiol. 30, 214 (1988). 29. D. R. Vincent, A. F. Moldenke, and L. C. Terriere, NADPH-cytochrome P-450 reductase from the house fly, Musca domestica, Insect Biothem. 13, 559 (1983). 30. G. D. Wheelock and J. G. Scott, Simultaneous purification of a cytochrome P-450 and cytochrome b, from the house fly, Musca domestica L., Insect Biochem. 19,481 (1989). 31. R. Feyereisen, J. F. Keener, F. Carino, and A. S. Daggett, Cytochrome P450 in insects, Amer. Chem. Sot. Div. Agrochem. Sept. 1989. [Abstract 421
BIOCHEMISTRY
AND
PHYSIOLOGY
36, 127-134 (19%)
Biochemical Changes in the Cytochrome P450 Monooxygenases of Seven Insecticide-Resistant House Fly (Musca domestica L.) Strains JEFFREY G. SCOTT,’ SUSANNA S.T.
LEE, AND TOSHIO SHONO~
Department of Entomology and institute for Comparative and Environmental Toxicology, Comstock Hall, Cornell University, Ithaca, New York 14853-0999 Received August 28, 1989; accepted October 30, 1989 The levels of cytochrome P45Os, cytochrome b,, NADPH-cytochrome c (P450) reductase, and three indices of monooxygenase activities (methoxyresorufin 0-demethylation (MROD), ethoxycoumarin 0-deethylation (ECOD), and aryl hydrocarbon hydroxylation (AHH)) were compared between one insecticide-susceptible and seven MFO-mediated insecticide-resistant house fly strains. Cytochrome b, and P450 reductase were elevated by 1.2- to 2.2-fold and 1.5 to 2.3-fold, respectively, in the resistant strains compared with the susceptible. The level of cytochrome P45Os was significantly increased (up to 2.1-fold) in all except two resistant strains compared with the susceptible strain. These results support the idea that all three monooxygenase components may play a role in insecticide resistance. Levels of MROD, ECOD, and AHH were significantly elevated (up to W-fold) in nearly all cases. Pyrethroid resistance was generally correlated with MROD and AHH activities in all except one strain. o 1990Academic press,I~C.
INTRODUCTION
Insecticide resistance is one of the major factors that limits our ability to control many key pests. Although the cytochrome P450 monooxygenases (MFOS)~ are known to be a very important mechanism for causing insecticide resistance (l), the biochemical basis underlying this type of resistance is not fully understood (2). It is generally believed that monooxygenase-mediated resistance is often associated with an increase in a cytochrome P450 isozyme(s) that can carry out a specific type of metabolism of the pesticide. However, it has been pointed out that MFO-mediated resistance is not always associated with increased levels of cytochrome P45Os (2). One possible expla’ To whom correspondence should be addressed. * Present address: National Institute of Health, lo35, 2-chome, Kamiosaki, Shinagawa-ku, Tokyo, JaPan. 3 Abbreviations used: P450 reductase, NADPHcytochrome c reductase; MROD, methoxyresorutin 0-demethylation; ECOD, ethoxycoumarin Odeethylation; AHH, aryl hydrocarbon hydroxylation; MFO, cytochrome P450 microsomal monooxygenases; PBO, piperonyl butoxide; LPR, Learn pyrethroidresistant house fly strain.
nation for this is that other components of the MFO system (i.e., NADPH-cytochrome c reductase (P450 reductase) and/or cytochrome b,) may also be involved in MFOmediated resistance (3, 6). There are relatively few cases where the levels of P450 reductase and/or cytochrome b5 have been examined in resistant species. DeVries and Georghiou (4) found 2.4-, 1.9-, and 2.2-fold increases in cytochrome P45Os, cytochrome bS, and P450 reductase, respectively, in a house fly strain having partially piperonyl butoxide (PBO)suppressible permethrin resistance. Vincent et al. (5) found 2.4- and 1.6-fold increases in cytochrome P45Os and P450 reductase, respectively, in the multiresistant Rutgers house fly strain. Scott and Georghiou (6) noted 3.8, 1.5-, and 2.8fold increases in cytochrome P45Os, cytochrome b,, and P450 reductase in the Learn-PyR (LPR) house fly strain having MFO-mediated pyrethroid resistance. These elevated levels were confirmed by Lee and Scott (7), although the relative amounts were slightly different. Subsequent studies with the LPR strain (8) showed a correlation between PBO127 0048-3575&O $3.00 Copyright All rights
8 1990 by Academic Press, Inc. of reproduction in any form reserved.
128
SCOTT,
LEE,
suppressible permethrin resistance and the presence of increased amounts of one or more of the MFO components on autosomes 1, 2, 3, and 5. In adult Tribolium castaneum, a DDT, organophosphate, and carbamate multiresistant strain (9), was found to have 3.0- and 2. l-fold more cytochrome P450 and cytochrome bs, respectively, compared with a susceptible strain (10). P450 reductase was not measured in these beetles. One difficulty encountered when studying the MFO system is the fact that there are multiple forms of cytochrome P450, and each may have its own unique substrate specificity. It is currently unknown if resistance is conferred by the action of several, a few, or perhaps only one P450. To investigate this question, we looked at three different MFO assays: methoxyresofurin Odemethylation (MROD), ethoxycoumarin 0-deethylation (ECOD), and aryl hydrocarbon hydroxylation (AHH). Because these were different types of MFO-mediated reactions, we reasoned that they might detect different populations of P45Os. Furthermore, from a previous study we found that pyrethroid resistance in a highly resistant strain of house fly was associated with a large (63.5-fold) increase in MROD activity (7), and we wished to further clarify this observation. We reasoned that we might be able to associate resistance to a specific insecticide class with a specific MFO assay, if the number of isozymes responsible for resistance to each class was relatively limited and showed a differential ability to oxidize methoxyresorufin, ethoxycoumarin, or benzo[a]pyrene. If successful, these studies could lead to the development of a diagnostic test for MFO-mediated insecticide resistance. The purpose of this study was to compare the levels of cytochrome P45Os, cytochrome b,, P450 reductase, and MFO activity between insecticide-resistant and susceptible house fly strains; to better understand the role of the MFO components in resistance and to determine
AND
SHONO
whether or not three common MFO assays could be correlated to resistance to one or more classes of insecticides. MATERIALS
AND
METHODS
Insects. Eight strains of house fly were used. (i) “S + ,” an insecticide-susceptible strain, was originally obtained from Dr. F. W. Plapp, Jr. of Texas A&M University. (ii) “Dairy,” a multiresistant strain (ll), was collected from the Learn dairy in Schuyler Co., New York, in 1986 and reared without insecticide selection. (iii) “LPR” is a multiresistant strain (12) with high levels of resistance to pyrethroids (e.g., > lO,OOO-fold to deltamethrin) (6). This strain was originally collected from the Learn dairy in 1980 and selected in the laboratory with permethrin for 40 generations (12). The mechanisms of pyrethroid resistance in this strain are increased MFOmediated detoxication, insensitivity of the nervous system (kdr), and decreased cuticular penetration (6). Each of these mechanisms has been mapped to specific autosomes (8). (iv) The “ASPR” strain was originally collected from Gunma Prefecture, Japan, and exposed to laboratory selection with permethrin for 20 generations. This strain exhibits higher levels of pyrethroid resistance in females compared with males (e.g., 3200- and 22-fold resistance to permethrin in females and males, respectively), and permethrin resistance in females is partially suppressible with PBO (13). (v) “Kashiwagura” is a pyrethroidresistant strain collected from a cattle farm in the Kashiwagura district of Miyagi village, Gunma Prefecture, Japan, in 1984 and selected with permethrin in the laboratory for 28 generations, becoming 3000-fold resistant to permethrin (T. Shono, unpublished). (vi) “RG” has the same origin as Kashiwagura; however, it was selected with permethrin for 24 generations and subsequently selected by resmethrin for four generations. (vii) “3rd-Y” is an organophosphate-resistant strain collected from the 3rd Yumenoshima island in 1979 and
MICROSOMAL
MONOOXYGENASES
AND
selected by malathion for 20 generations. 3rd-Y is lOOO- and 1600-fold resistant to fenitrothion and malathion, respectively, but is susceptible to pyrethroids (14). (viii) “EPR” is an organophosphate-resistant strain selected from the 3rd-Y strain using ethyl-fenitrothion for 20 generations and subsequently with parathion for four generations. EPR shows >4500-fold resistance to parathion, but is susceptible to pyrethroids (T. Shono, unpublished). Chemicals. Methoxyresorufin and benzo[a]pyrene were kindly provided by Dr. C. F. Wilkinson, Department of Entomology, Cornell University. Resorufin was from Eastman Kodak (Rochester, NY), while 7-hydroxycoumarin and 7-ethoxycoumarin were obtained from Aldrich Chemical Co. (Milwaukee, WI). All other chemicals used were of the highest purity grade and commercially available.
INSECTICIDE
RESISTANCE
129
Preparation of microsomes. Female house fly abdomens (80&2000) were used for preparing microsomes as described previously (15). Microsomal pellets were diluted to a final protein concentration of 1 mg/ml and stored in 2-ml aliquots in small glass vials at - 80°C. Microsomes were prepared from all strains on the same day for each experiment. The entire experiment was replicated two times. Protein was determined by the method of Bradford (16) using bovine serum albumin as the standard . Enzyme assays. Cytochrome P450 and b, were quantitatively analyzed at a final protein concentration of 1 mg/ml in triplicate by the method of Omura and Sato (17) using a Beckman DU-50 spectrophotometer. NADPH-cytochrome c (P450) reductase was measured in triplicate based on the method of Schonbrod and Teniere (18), as
TABLE
1
Levels of MFO Components and MFO Activity in a Susceptible (S +) Strain and Seven Insecticide-Resistant Strains of House Fly Measurement” Cytochrome x max f
P450
MlolP mg protein
Cytochrome 6, (nmol/ mg protein)
P450 reductaseb (nmoUmin/ mg protein)
MROD’ (nmoYmin/ mg protein)
ECODd (nmol/min/ mg protein)
AHH’ (nmol/min/ mg protein)
s+
552.5
0.53 (0.03)
0.38 (0.07)
74.3 (5.0)
0.05 (0.01)
0.04 (0.01)
3rd-Y
452.5
0.52 (0.02)
0.55 (0.01)
108.5
0.07 (0.01)
0.10 (0.01)
1.64 mw 1.56 (0.27)
451.5
(E,
0.50 (0.05)
126.3
0.92 (0.01)
0.30 ww
6.54 (0.24)
Dairy
450.5
0.88 (0.01)
0.56 (0.01)
6.18 (0.19)
453
0.69 (0.05)
0.53 (0.07)
0.38 (0.02)
1.77 (0.14)
RG
453
0.63 (0.03)
0.47 (0.05)
1.46 (0.W 0.69 wJf3 1.07 (0.03)
0.20 (0.01)
Kashiwagura
131.1 (3.01 113.9 (11.6)
0.35 (0.02)
4.64 (0.13)
ASPR
453
0.65 (0.07)
0.56 (0.W 0.82 (0.01)
(iii, 0.18 (0.01)
7.16 (0.W 20.1 (4.3)
EPR
LPR’
450
1.11 (0.W
a Values in parentheses are the SE of the mean (n = 6). b nmoles cytochrome c reduced. c nmoles resorufm. d nmoles 7-hydroxycoumarin. E mnoles benze[alpyrene. *All values are kO.5 nm or less. *n = 5. h Data for LPR from Lee and Scott (7).
(2.7) (3.8)
116.5 (2.1) 137.7 (10.6) 167.3
(9.6)
0.45 (0.02) 8.89 (0.33)
130
SCOTT,
LEE,
previously described (15). P450 reductase was measured as nanomoles of cytochrome c reduced mini mg protein-’ and converted to nmoles reductase mg protein- ’ assuming a MW of 74,000 and 36 pmol cytochrome c reduced min- ’ mg reductase- ’ (29). The three indices of microsomal monooxygenase activities used were methoxyresorufin O-demethylation (19), ethoxycoumarin O-deethylation (20), and aryl hydrocarbon hydroxylation (21). All three reactions were measured in triplicate and carried out under atmospheric conditions in a lo-mm pathlength cuvette in an Aminco SPF-500 spectrofluorometer at 32°C as described previously (15). RESULTS
AND
DISCUSSION
The levels of the three MFO components and MFO activities present in the eight house fly strains are given in Table 1. The cytochrome P450 A,,, varied between strains, ranging from 453 nm for the ASPR, RG, and Kashiwagura strains to 450 nm for the LPR strain. This suggests that, although resistant strains may sometimes have a low Amax compared to a susceptible (22), no clear connection can be drawn. A likely explanation for this is that the P450 assay measures a summation of all the P450 isozymes. As many isozymes have a different A,,, the relative abundance of each P450 determines the overall A, observed, and thus it may be difficult to correlate resistance to the A,, in unrelated strains. Except for 3rd-Y and EPR, the level of cytochrome P45Os was higher in all the resistant strains (Fig. l), ranging from l.Zfold (RG) to 2.1-fold (LPR). The lack of correlation between cytochrome P450 levels and MFO-mediated resistance has been noted before (2). It is not clear from such results if MFO activity might increase solely by increasing the levels of P450 reductase and/or cytochrome 6,, or whether there actually is an increase in one of the cytochrome P45Os that is not detected when the sum of all P45Os is measured (3). For example, if re-
AND
SHONO Strain LPR ASPR
Kashiwagura Dairy EPR Brd-Y 0 Percent
50
100
150
of level in susceptible
200
250 strain
FIG. 1. Levels of cytochrome P45Os (nmollmg protein), cytochrome b, (nmollmg protein), and P450 reductase (nmol cytochrome c reduced minlmg protein) in seven insecticide-resistant housefly strains. Values are expressed as percentage of the level found in the susceptible strain.
sistance is due to elevated levels of a major isozyme, it may be detected as an increase in total P45Os, while resistance due to elevated levels of a minor isozyme may not measurably increase the level of total P45Os (3). P450 reductase and cytochrome b, were elevated in all of the resistant strains compared with the susceptible (Table 1, Fig. 1). Increases ranged from 1.5-fold (3rd-Y) to 2.3-fold (LPR) for P450 reductase and 1.2fold (RG) to 2.2-fold (LPR) for cytochrome b,. This suggests that elevated P450 reductase and/or cytochrome bS levels may be involved in the increased MFO activity (i.e., MFO-mediated insecticide resistance) found in these strains. Recent reports of NADH stimulation of MFO activity in house flies (23) and diamondback moth (24) support the idea that cytochrome b5 can be important in determining monooxygenase activity in insects. In order to investigate if any relationship existed between the monooxygenase components , we attempted to correlate levels of
MICROSOMAL
MONOOXYGENASES
AND
INSECTICIDE
RESISTANCE
0.9
0.7
0.5
P-450
0.07
y = 0.0084 + 0.,0707x
_
P=O.O03
l
0.3
I
I
1.0
1.2
(nmoles / mg protein)
r
o&6
0.02
I 0.8
I 0.6
0.3 ’ 0.4
R = 0.89
/
I
1
1
0.5
0.7
0.9
b 5 (nmoles / mg protein) y = 0.019 + 0.0398x
0.06 -
R = 0.80
PzO.018
0.04 q 0.02
0.4
L
1
0.6
0.8
P-450
I
1.0
I
1.2
(nmoles / mg protein)
Plots of the levels of cytochrome b, vs P45Os (A), P450 reductase vs cytochrome b, (B), and P450 reductase vs cytochrome P45Os (C)from eight strains of house fry. The regression equation for each line, its correlation coeficient, and the P value are given for each plot. FIG. 2.
131
132
SCOTT,
LEE,
individual components (Table 1) across the eight strains studied. Levels of cytochrome b, were somewhat correlated with levels of cytochrome P45Os (Fig. 2A), as were levels of P450 reductase vs cytochrome b, (Fig. 2B) and P450 reductase vs cytochrome P45Os (Fig. 2C). There was good correlation among the levels of P450, b,, and P450 reductase, suggestive of some type of coordinated control of the levels of these three components. If this is true, it suggests a sophisticated control mechanism because P450 reductase, cytochrome b,, and certain P450 isozymes appear to be coded for by genes on different autosomes (8, 31). The P450:cytochrome bS:P450 reductase ratio suggested by Fig. 2 (approx. 12: 10: 1) is different from the 12:4: 1 ratio proposed previously (25). While our results are consistent with the ideas that there are l&14 P45Os per reductase (25, 26) our results suggest an approximate 1: 1 ratio between cytochrome P45Os and cytochrome b,. The levels of the three monooxygenase activities (MROD, ECOD, AHH) were elevated in all the resistant strains compared with the susceptible, except for AHH activity in the 3rd-Y strain, whether comparisons were based on specific activity (Fig. 3A) or on turnover numbers (Fig. 3B). MROD and AHH activities were highest in the LPR strain, which has the highest levels of pyrethroid resistance, and these activities were lowest in the pyrethroid-susceptible (3rd-Y) strain. Additionally, MROD and AHH activities -vere increased in the LPR strain compared with the Dairy strain, which was collected from the same site but had a lower level of pyrethroid resistance. A similar correlation between levels of monooxygenase-mediated pyrethroid resistance and MROD activity has recently been reported in larvae of the diamondback moth Plutella xylostella (27). Furthermore, monooxygenase-mediated DDT, carbaryl, and parathion resistance in the Hikone-R strain of Drosophila melanogaster, which is pyrethroid susceptible, was not correlated with large (Gthree-fold) increases in
AND
SHONO
ECOD or AHH activities (28). Our correlation between MROD or AHH activities and pyrethroid resistance was not perfect, however, as intermediate levels were noted in one pyrethroid-susceptible strain (EPR). ECOD activities were similar in most of the resistant strains, but were again lowest in 3rd-Y. We have recently purified the major cytochrome P450 from the LPR strain (30). Production of antisera and reconstitution experiments will help to clarify the substrate (i.e., methoxyresorufin, benzo[a]pyrene, etc.) specificity of this putative pyrethroid-metabolizing cytochrome P450. In conclusion, we found that MFO&&I LPR ASPR RG Kashiwagura Dairy EPR 3rd-Y 100 Fold increase
in monooxygenase
activity
Strain LPR ASPR RG Kashiwagura Dairy EPR 3rd-Y 100 Fold
increase
in
monooxygenase
TON
Levels of the three monooxygenase activities in seven insecticide-resistant housefly strains, expressed as a fold increase in specific activity (A) or as a fold increase in TON (turnover number, i.e., activity per nanomole P450) (E), compared with the susceptible strain. MROD, ECOD, and AHH refer to methoxyresoru$n 0-demethylation, ethoxycoumarin Odeethylation, and aryl hydrocarbon hydroxylation, respectively. FIG.
3.
MICROSOMAL
MONOOXYGENASES
mediated insecticide resistance in the house fly was associated with increases in cytochrome P45Os, P450 reductase, and cytochrome b5. Additionally, our results suggest that MROD activity may be a useful assay to monitor for MFO-mediated pyrethroid resistance, although further work will be needed to clarify this idea. ACKNOWLEDGMENT This work was supported in part by a gift from Toagosei Chemical Industry Co. and Hatch Grant 139414. REFERENCES 1. L. B. Brattsten, C. W. Holyoke, Jr., J. R. Leeper, and K. F. Raffa, Insecticide resistance: Challenge to pest management and basic research, Science 231, 1255 (1986). 2. E. Hodgson, The significance of cytochrome P450 in insects, Insect Biochem. 13, 237 (1983). 3. J. G. Scott, Insecticide resistance in insects, in “Handbook of Pest Management” (D. Pimentel, Ed.), CRC Press, Boca Raton, FL, In press. 4. D. H. DeVries and G. P. Georghiou, Absence of enhanced detoxication of permethrin in pyrethroid-resistant house flies, Pestic. Biochem. Physiol. 15, 242 (1981). 5. D. R. Vincent, A. F. Moldenke, D. E. Famsworth, and L. C. Terriere, Cytochrome P-450 in insects. 6. Age dependency and phenobarbital induction of cytochrome P-450, P-450 reductase, and monooxygenase activities in susceptible and resistant strains of Musca domestica, Pestic. Biochem. Physiol. 23, 171 (1985). 6. J. G. Scott and G. P. Georghiou, Mechanisms responsible for high levels of permethrin resistance in the house fly, Pestic. Sci. 17, 195 (1986). 7. S. S. T. Lee and J. G. Scott, Microsomal cytochrome P450 monooxygenases in the house fly (Musca domestica L.): Biochemical changes associated with pyrethroid resistance and phenobarbital induction, Pestic. Biochem. Physiol. 35, 1 (1989). 8. J. G. Scott and G. P. Georghiou, The biochemical genetics of permethrin resistance in the LeamPyR strain of house fly, Biochem. Genet. 24,25 (1986). 9. C. E. Dyte, Resistance to synthetic juvenile hormone in a strain of flour beetle, Tribolium castaneum, Nature (London) 238, 48 (1972). 10. E. Cohen, Studies on several microsomal enzymes in two strains of Tribolium castaneum (Tenebrionidae: Coleoptera), Comp. Biochem. Physiol. C 71, 123 (1982).
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11. J. G. Scott, R. T. Roush, and D. A. Rutz, Insecticide resistance of house flies from New York dairies (Diptera: Muscidae), J. Agric. Entomoi. 6, 53 (1989). 12. J. G. Scott and G. P. Georghiou, Rapid development of high-level permethrin resistance in a field-collected strain of the house fly (Diptera: Muscidae) under laboratory selection, .I. Econ. Entomol. 78, 316 (1985). 13. T. Shono and J. Scott, Autosomal sex-associated pyrethroid resistance in a strain of house fly (Diptera: Muscidae), possessing a maledetermining factor on chromosome three, .I. Econ. Entomol., in press. 14. H. Yoshikawa, T. Shono, and M. Eto, Synthesis and insecticidal activity of methylphosphonothiolates, J. Pestic. Sci. 11, 15 (1986). 15. S. S. T. Lee and J. G. Scott, An improved method for preparation, stabilization and storage of house fly (Diptera: Muscidae) microsomes, J. Econ. Entomol. 82, 1559 (1989). 16. M. M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding, Anal. Biochem. 72, 248 (1976). 17. T. Omura and R. Sato, The carbon monoxidebinding pigment of liver microsomes. I. Evidence for its hemoprotein nature, J. Biol. Chem. 239, 2370 (1964). 18. R. D. Schonbrod and L. C. Terriere, Inhibition of housefly microsomal epoxidase by the eye pigment, xanthommatin, Pestic. Biochem. Physiol. 1, 409 (1972). 19. R. T. Mayer, J. W. Jermyn, M. D. Burke, and R. A. Prough, Methoxyresorufin as a substrate for the fluorometric assay of insect microsomal 0-dealkylases, Pestic. Biochem. Physiol. 7,349 (1977). 20. V. Ullrich and P. Weber, The 0-dealkylation of 7-ethoxycoumarin by liver microsomes: A direct fluorometric test, Hoppe-Seyler’s Z. Physiol. Chem. 353, 1171 (1972). 21. M. S. Denison, M. Murray, and C. F. Wilkinson, Microsomal aryl hydrocarbon hydroxylase: Comparison of the direct, indirect and radiometric assays, Anal. Lett. 16, 381 (1983). 22. A. S. Perry, W. E. Dale, and A. J. Buckner, Induction and repression of microsomal mixedfunction oxidases and cytochrome P-450 in resistant and susceptible houseflies, Pestic. Biothem. Physiol. 1, 131 (1971). 23. M. J. J. Ronis, E. Hodgson, and W. C. Dauterman, Characterization of multiple forms of cytochrome P-450 from an insecticide resistant strain of house fly (Musca domestica), Pestic. Biochem. Physiol. 32, 74 (1988). 24. M.-C. Yao, C.-F. Hung, and C.-N. Sun, Fenvalerate resistance and aldrin epoxidation in larvae
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of the diamondback moth, Pesric. Biochem. Physiol. 30, 272 (1988). 25. M. Agosin, Role of microsomal oxidations in insecticide degradation, in “Comprehensive Insect Physiology, Biochemistry and Pharmacology” (G. A. Kerkut and L. I. Gilbert, Eds.), Vol. 12, p. 647, Pergamon Press, Oxford, 1985. 26. J. A. Peterson, R. E. Ebel, D. H. O’Keeffe, T. Matsubara, and R. W. E&brook, Temperature dependence of cytochrome P-450 reduction. A model for NADPH-cytochrome P-450 reductase: Cytochrome P-450 interaction, J. Biol. Chem. 251, 4010 (1976). 27. C.-F. Hung and C.-N. Sun, Microsomal monooxygenases in diamondback moth larvae resistant to fenvalerate and piperonyl butoxide, Pestic. Biochem. Physiol. 33, 168 (1989).
28. L. C. Waters and C. E. Nix, Regulation of insecticide resistance-related cytochrome P-450 expression in Drosophila melanogaster, Pestic. Biochem. Physiol. 30, 214 (1988). 29. D. R. Vincent, A. F. Moldenke, and L. C. Terriere, NADPH-cytochrome P-450 reductase from the house fly, Musca domestica, Insect Biothem. 13, 559 (1983). 30. G. D. Wheelock and J. G. Scott, Simultaneous purification of a cytochrome P-450 and cytochrome b, from the house fly, Musca domestica L., Insect Biochem. 19,481 (1989). 31. R. Feyereisen, J. F. Keener, F. Carino, and A. S. Daggett, Cytochrome P450 in insects, Amer. Chem. Sot. Div. Agrochem. Sept. 1989. [Abstract 421