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Microsomal oxidase activity of three blowfly species and its induction by phenobarbital and β-naphtoflavone
PESTICIDE
BIOCHEMISTRY
AND
PHYSIOLOGY
14, 275-281 (1980)
Microsomal Oxidase Activity of Three Blowfly Species Induction by Phenobarbital and p-Naphthoflavone H. A.
ROSE*
L. C.
AND
and Its
TERRIERE
Department of Entomology, Oregon State University, Corvallis, Oregon 97331; Pathology and Agricultural Entomology, University of Sydney, New South
und *Department of Plunr Wales 2006, Ausfrulia
Received June 20, 1980; accepted November 20, 1980 Induction of the microsomal oxidase system by dietary phenobarbital and p-naphthoflavone was examined in three blowflies, Phormia regina (Mg.), Lucilia illustris (Mg.), and Eacolliphora lilicu (Walk.). Responses were similar in adults and larvae of all species. Phenobarbital increased cytochrome P-450 levels up to 9-fold and aldrin epoxidase up to 138-fold. Increases in cytochrome P-450 and aldrin epoxidase caused by p-naphthoflavone were minor relative to those produced by phenobarbital. In toxicity experiments with carbaryl and propoxur tolerance was associated with the amount of microsomat oxidase activity. Using piperonyl butoxide to synergize carbaryl and propoxur there was no clear indication for the use of either the synergist ratio or synergist difference as an indicator of microsomal oxidase activity. INTRODUCTION
Two aspects of the microsomal oxidase system that make it a particularly interesting mechanism of detoxication are its lack of substrate specificity and the ease with which its reaction rate can be accelerated in response to chemical stress. This provides the organism with a versatile response to potential biocides such as insecticides, insect growth regulators, and natural toxicants in its environment. Considering the number of insect species known, the microsomal oxidase system has been studied in a very small number and in most of these cases the examination has been rather superficial. However, since this system has the attributes mentioned above and since it has already been shown to be involved in such fundamental processes as hormone regulation (1, 2), response to plant substances (3, 4), and survival in a toxic environment (5), more knowledge of its activity in different species is highly desirable. The microsomal oxidase system has been studied in a few Diptera, the house fly Musca domestica (6-9), the blowfly Phormia regina, the fleshfly Sarcophaga bullata (lo-13), and the midge Chironomus riparius (14). In comparisons made so far
the house fly appears to contain the most microsomal oxidase activity on a body weight or protein basis and this level of activity correlates fairly well with susceptibility to insecticides in comparisons of house flies, fleshflies, and blowflies (13). Very few comparisons of this sort have been made, however, to determine whether species differences in susceptibility to insecticides can be explained by their respective microsomal oxidase levels. Species differences in the response of the microsomal oxidase system to dietary stimulants (inducers) probably occur also but, as yet, there has been very little experimentation in this area. Differences in morphology and feeding habits are serious limitations to comparative studies of both the microsomal oxidase system and its induction. We have overcome some of these limitations in experiments with three closely related Diptera with similar feeding habits and longevities. The results of our experiments are presented here. METHODS
Insects. P. regina was used from the laboratory culture of several years. Lucilia illustris and Eucalliphora lilica were reared
275 004%3575/80/060275-07$02.00/O Copyright All rights
Q 1980 by Academic Press. Inc. of reproduction in any form reserved.
276
ROSE
AND
TERRIERE
in the laboratory for 6 months prior to the Mortality, the criterion being the inability beginning of these experiments. The origito stand and walk, was checked after 24 hr. was replicated at least nal stocks were obtained locally from eggs Each experiment deposited in raw meat. The three species twice and the average mortality for each 80 were reared under a 14:lO L/D cycle at a dosage was based upon approximately temperature of 25 + 2°C. Adults were fed a flies. diet consisting of sugar, powdered milk, RESULTS and dry egg yolk (12:12:1) and larvae were reared on raw pork liver. Enzyme characteristics. Microsomes from both adults and larvae in all three Treatment with inducers. With adults, phenobarbital and @-naphthoflavone were blowfly species had no detectable aldrin administered by mixing thoroughly with the epoxidase activity when NADPH was diet as described by Yu and Terriere (15). omitted from the incubation medium. CarIn the case of larvae, the inducing chemibon monoxide which was bubbled gently cals were dusted onto the liver which had through the incubation medium for 1 min been pierced thoroughly with a pair of prior to incubation inhibited the activity by sharply pointed forceps. 70- lOO%, the greatest inhibition being seen Enzyme assays. Microsomal oxidase ac- in adult preparations. tivity in adults was measured with aldrin as Preliminary observations showed that substrate, the product, dieldrin, being mea- preparations of blowfly larvae contain insured by glc as described by Yu and Ter- hibitors of microsomal oxidase activity. Inriere (16). With larvae, the procedure was hibition was partly overcome by the use of identical except that the homogenizing BSA and KCN in the homogenizing mebuffer contained 1.5% bovine serum albu- dium. Results using different batches of min (BSA) and 2 x lop3 M KCN (17). larvae were somewhat erratic in actively Phenobarbital had no effect on aldrin growing larvae. Reliable comparisons, epoxidase activity nor on the amount of however, of enzyme activity could be made cytochrome P-450 when added in the at around the clear gut stage of larvae, i.e., homogenizing medium of either control or after the larvae had completed feeding. The induced larvae. It was also shown that there results presented here were obtained with was no aldrin epoxidase activity or cyto- such larvae. chrome P-450 on liver on which the larvae In larvae microsomal aldrin epoxidase were reared from control or induced treatactivities, measured as picomoles of dielments assayed at the end of the experidrin produced per individual per minute, mental period. Cytochrome P-450 was were 7.7, 10.7, and 10.6 for P. regina, L. measured with an Aminco DW-2A dual illustris, and E. Mica, respectively. The beam spectrophotometer according to the corresponding figures for 8- to 9-day-old method of Omura and Sato (18). Protein headless adult females were 8.6, 7.6, and was determined by the Bradford method 11.8. Larval weights were 51.5, 40.6, and 45.2 mg and the headless adult females (19). Bioassays. Dosage-mortality relationweighed 41.5, 41.4, and 41.5 mg, respecships were obtained by topical application tively. of known amounts of carbaryl and proInduction. Preliminary experiments using poxur dissolved in acetone to 8- to 9-day-old P. regina larvae were undertaken to ascerfemale flies. Parallel bioassays with the tain the best way of applying the inducing synergist, piperonyl butoxide (5 parts chemicals to liver slabs. Chopped liver was synergist:l part insecticide) were also per- not used because the larvae did not develop formed. Following treatment the flies were normally. When solvents such as dimethyl provided with water and held at 25 -C 2°C. sulfoxide and xylene were injected into the
PHENOBARBITAL
ANDP-NAPHTHOFLAVONE
lNDucTIoN
liver a 2- to 4-fold increase compared with controls (untreated liver or liver injected with water) in aldrin epoxidase activity was obtained. Using phenobarbital, there was no difference in aldrin epoxidase activity with an aqueous solution being injected into the liver or crystals being dusted onto the liver which had been pierced thoroughly. The dusting method was adopted because it obviated the need for solvents and could be used for any inducing chemical. Table 1 shows that stimulation by 0.2% phenobarbital of cytochrome P-450 was 5.7-, 3.9-, and 3.5-fold for E. Mica, L. illustris, and P. regina, respectively. The data suggest that in the case of cytochrome P-450 the highest dose (0.2% phenobarbital) is at or near the upper threshold level. In the case of aldrin epoxidase, the dose giving maximum effect is probably above 0.2%. With higher doses than this, however, some mortality of larvae was noted. With p-naphthoflavone used at 0.1% the inductive effect was minor compared to that obtained with phenobarbital. There was no significant change in the level of cytochrome P-450 in P. regina or L. illustris but a 1.7-fold increase in E. lilica. The greatest increases in aldrin epoxidase activity were 3.6- and 3.0-fold obtained with E. lilica and P. regina, respectively.
IN THREE
277
BLOWFLIES
The results reported in Table 2 were obtained from abdomens only; abdomens had slightly less than 50% aldrin epoxidase activity of headless flies. Measurements of cytochrome P-450 of headless flies could not be made, however, because CO difference spectra revealed a deep trough at 446 nm and no peak around 450 nm indicative of the presence of cytochrome oxidase in the preparations (20). As in the case of larvae, phenobarbital is a strong inducer of cytochrome P-450 and of aldrin epoxidase in adults of the three species (Table 2). The threshold dose appears not to have been reached because in all cases the values for the 2% levels were the highest but with P. regina the increase from the 1% to the 2% level was small. The maximum increases noted in the case of the hemoprotein were 9.0-fold for P. regina, 3.5-fold for E. lilica, and 3.1-fold for L. illustris. The amount of the hemoprotein in the three species at the 2% level was quite similar ranging from 374 to 463 pmol/mg protein. Aldrin epoxidase activity ranged from 3.0- to 12.6-fold higher in P. regina than in the other two species at the 2% level. At l%, fi-naphthoflavone increased aldrin epoxidase activity 2.3- and 2.1-fold in the case of P. regina and E. lilica, respec-
TABLE
1
Microsomul Cytochrome P-450 and Aldrin Epoxidase in Blonfly Lurlsae ufter Induction by Phenobarbital und PNaphthojluvone Cytochrome Dose” (%I
P. regitu
P-450 (pmol/mg
Dieldrin
produced
(pmoYmin/mg
protein)’
L. illustris
E. lilica
P. reginu
L. illustri.7
E. lilicu
24.8 t 0.4
35.6 t 3.6
23.4 + 1.0
24.8 L 0.7
36.9 + 0.7
Control
21.8
2 3.4
Phenobarbital 0.02 0.05 0.10 0.20
41.1 59.5 68.7 75.7
t 2 -e k
7.6 0.5 5.9 4.3
41.0 51.1 102.0 96.1
/SNaphthoflavone 0.10
23.0 f 1.3
24.4
u Percentage in diet (pork liver) ’ Mean r SE of two experiments. c Mean f SE of two experiments,
protein)”
2 + L +
5.1 8.0 6.5 1.3
+ 0.4
for 1% days duplicate
prior
84.6 111.2 164.6 203.0
5 + t k
3.2 5.3 0.1 4.1
60.6 f 0.8 to assay.
incubations.
419.4 638.5 829.6 943.1
4 _t -+ -t
34.3 31.9 9.4 14.6
69.5 + 0.3
90.9 136.5 335.6 649.6
? 2 -t 5
2.4 11.2 44.2 15.6
34.2 z? 2.6
189.8 382.8 639.5 839.7
k t t +
5.7 8.5 7.3 65.3
133.9 k 0.8
278
ROSE AND TERRIERE TABLE Microsomal after
Dose” (%) Control Phenobarbital 0.2 0.5 1.0 2.0 P-Naphthoflavone 1.0
Cytochrome Induction
2
P-450 and Aldrin epoxidase in blo@y by Phenoburbital and /SNaphthoJavone
Adults
Cytochrome P-450 (pmol/mg protein)*
Dieldrin produced (pmoUmin/mg protein)’
P. regina
P. reginu
L. illustris
12.7 2 0.6
31.9 r 3.2
L. illustris
47.8 i 2.6 180.5 354.0 399.6 431.9
E. lilicu
149.2 k 19.4 107.4 r 8.7
k 12.7 rc_36.9 re_7.9 i 14.0
172.5 189.4 270.5 462.6
79.0 _f 7.5
L k k -t
26.0 1.9 38.2 8.5
131.4 t 22.4
169.7 270.5 323.2 373.8
+ 2 t 2
44.2 329.2 rt_ 37.5 46.7 1195.3 2 26.6 10.2 1475.0 s 32.9 45.1 1755.9 5 68.3
136.5 -t 24.5
28.9 + 1.2
61.3 102.6 211.5 554.9
-t + rt +
E. lilica
7.4 5.1 30.3 14.3
35.0 2 4.8
21.0 c 4.1 36.7 95.1 337.2 583.9
t 3.0 Y?6.1 t 17.8 t 34.8
44.8 s 0.9
” Females, 2-3 days old, were fed with the inducer in the diet for 3 days prior to enzyme assays. ’ Mean ? SE of two experiments. ’ Mean 2 SE of two experiments, duplicate incubations.
tively, with the corresponding increases in cytochrome P-450 being only 1.7 and 1.3. The effect of the inducer on aldrin epoxidase activity and cytochrome P-450 of L. illustris was negligible. The Ama, of the CO difference spectra in control and induced larvae of the three species are shown in Table 3. Absorption maxima of larvae were lower than those of adults. Also, the A,,, for phenobarbital was lower than the corresponding control by about 1 nm. The A,,, for /?-naphthoflavone was higher than that of the corresponding phenobarbital figure, being the same or slightly lower than the control figure. Readings of P-450 were taken l-2 min after the addition of Na,$O, to the cuvette
as it was observed that there was a shift to a higher value for A,,, of about 1.5 nm in 7- 10 min. The amount of P-450 was variable. In general it was higher in larvae than adults and the amount increased with the higher doses of phenobarbital. Insecticide toxicity experiments. Results of the bioassay tests are summarized in Table 4. Experiments with the synergist were included in order to test the usefulness of this microsomal oxidase inhibitor as an indicator of the role of these enzymes in metabolizing the two insecticides. The synergist data are expressed as synergist ratios as suggested by Brattsten and Metcalf (21) and as synergist differences as suggested by Brindley (22).
TABLE Absorption Maxima Induced Adults
3
of CO Difference Spectra in Control and Larvae of Three f3lowjly Species
and
Absorption maxima (nm) P. regina
L. illustris
E. lilica
Adults
Larvae
Adults
Larvae
Adults
Larvae
Control
452.0
451.5
452.0
449.0
453.0
451.0
Induced Phenobarbital P-Naphthoflavone
450.5 452.0
450.0 451.0
451.0 452.0
448.5 449.0
452.0 453.0
449.5 450.0
PHENoBARBITALAND/~-NAPHTHOFLAVONE
INDUCTION
IN THREE
BLOWFLIES
279
TABLE4 Toxicity
Data
for
Carbaryl
Dose effect parameter LD,,, carbaryl” LD,, carbaryl + piperonyl butoxide Synergist ratio” Synergist difference’ LD,o propoxur LD,” propoxur + piperonyl butoxide Synergist ratio Synergist difference
and Propoxur
.
with
and without
Piperonyl
Butoxide
in Blowjly
Adults
P. regina
L. illustris
E. lilica
2014 23.8
1005 10.5
2804 18.0
85 1990 7.7 4.2
96 995 3.6 2.1
1.56 2786 14.0 6.8
1.8 3.4
1.7 1.5
2.1 7.2
cfLD,, given as mg/kg. b LD,, carbaryVLD,, carbaryl + piperonyl butoxide. ’ LD,,, carbaryl -(LD,,, carbaryl + piperonyl butoxide).
Arranging the LD,, values of carbaryl and propoxur with and without synergist the approximate relationship is 1:2:3 for L. illustris, P. regina, and E. lilica. The microsomal aldrin epoxidase activities expressed in picomoles of dieldrin produced per milligram of individual in 8 to 9-day-old headless female adults were 184, 207, and 283, respectively, the ratios being 1: 1.1: 1.5. Thus the relative position of the three species with respect to microsomal oxidase activity is maintained but the differences are much less than would be indicated by the LD,, values of the two insecticides. DISCUSSION
These results show that dietary phenobarbital is capable of greatly stimulating aldrin epoxidase in blowfly adults and larvae. The reason for such large increases is not clear although the fleshfly (S. buflata) is also highly inducible (IO) and so are adults of susceptible house fly strains (15). Dietary phenobarbital also increased the cytochrome P-450 content in adults and larvae but induction was much less when compared with aldrin epoxidase. The reason for this is not clear. It is known that cytochrome P-450 exists in multiple forms in insects (7-9, 23) and mammals, and there is a degree of substrate specificity
with each of the different forms (24). It is possible, therefore, that one or more forms of cytochrome P-450 with a good specific epoxidase activity was induced by phenobarbital. Another possibility is that induction may have also changed the activity of some or all of the cytochrome, especially that accepting aldrin as a substrate. This is based on evidence that active forms of cytochrome P-450 can occur in house . flies (8). Only small variations in the response of both larvae and adults to phenobarbital in L. illustris and E. lilica are evident. Thus, the increases in P-450 content and aldrin epoxidase activity are fairly similar. The aldrin epoxidase of P. regina, however, appears to be more inducible than the aldrin epoxidase of the other two species with adult and larval activity being 138- and 40-fold higher than the control values at the highest dose levels. P-Naphthoflavone was added in the diet because it behaves similarly to 3-methylcholanthrene in vertebrates (25). Based on substrate specificities, these inducers act very differently from phenobarbital. Moreover, unlike phenobarbital, p-naphthoflavone and 3-methylcholanthrene typically induce a form of P-450 which is designated P-448 due to the shift in the CO difference spectrum of the hemoprotein. In
280
ROSE
AND
a study using adult house flies, 3-methylcholanthrene had no effect on microsomal oxidase activity (15). Our results show a minor effect with P-naphthoflavone compared to that of phenobarbital at the same dose. Increases in the amount of cytochrome P-450 and aldrin epoxidase were only slight. In the case of P. regina and E. lilica, however, increases in aldrin epoxidase activity were greater than those of cytochrome P-450 suggesting that P-naphthoflavone had produced a change in the P-450 of those species but not with L. illustris (Tables 1 and 2). There was no evidence that /3-naphthoflavone induced a P-448 type of cytochrome P-450 as the CO difference spectra from individuals treated with this compound were always higher than the phenobarbital-treated individuals. In vertebrates, the reverse would be true. Another difference between blowflies and vertebrates was evident. Induction by phenobarbital in blowflies yielded a difference spectrum with a lower A,,, compared to controls, a result also obtained with house flies (26). Our carbaryl LD,, value for P. regina was 22 times higher than that reported by Brattsten and Metcalf (11) although our figure for synergized carbaryl was only 2.2 times higher. Perhaps their holding temperatures of 28°C could partially explain their much lower figure for carbaryl LD,,. This study showed that the greatest tolerance to carbaryl and propoxur was associated with the highest amount of microsomal oxidase activity. The correlation, however, was not 1: 1. No clear picture of a preference for the use of either the synergist ratio (21) or synergist difference (22) as an indicator of microsomal oxidase activity emerged from the results. There is strong synergist activity with carbaryl, however, indicating that all three species must depend to a large extent on microsomal oxidases for carbaryl detoxication. The microsomal oxidases of two more blowfly species have been investigated with the completion of this study. It is evident
TERRIERE
that in common with other Diptera studied, the enzyme system requires NADPH and is inhibited by CO. The results in this paper are characterized more by similarities rather than differences between the three species. The species belong to three different tribes (Lucilia:Luciliini, Eucuiliphoru: Calliphorini, and Phormiu:Phormiini), the latter belonging to subfamily Chrysomyinae while the other two belong to Calliphorinae. The biology of most blowflies, however, is fairly similar (27) and in this investigation the same culture conditions were used for all species. It is not surprising therefore that no marked differences were observed in any of the results from one species to another. ACKNOWLEDGMENTS
This research was supported by USPHS Grant ES00362-21. The authors are grateful for the advice of Dr. S. J. Yu, for the identification of L. illusrris and E. Mica by Dr. B.F. Eldridge. and for the technical assistance of Mr. Dan Farnsworth. REFERENCES
1. R. Feyereisen and F. Durst, Ecdysterone biosynthesis: A microsomal cytochrome P450-linked ecdysone 20-monooxygenase from tissues of the African migratory locust, ENT. J. Biochem.
88, 37 (1978).
2. S. J. Yu and L.C. Terriere, Metabolism of juvenile hormone I by microsomal oxidase, esterase, and epoxide hydrase of Musca domestica and some comparisons with Phormia regina and Sarcophaga bullatn, Pestic. Biochem. Physiol. 9, 237 (1978). 3. R. 1. Krieger, P. P. Feeny, and C. F. Wilkinson, Detoxication enzymes in the guts of caterpillars: An evolutionary answer to plant defenses? Science
172, 579 (1971).
4. L. B. Brattsten, C. F. Wilkinson, and T. Eisner, Herbivore-plant interactions: Mixed-function oxidases and secondary plant substances, Science 196, 1349 (1977). 5. T. Nakatsugawa and M. A. Morelli, Microsomal oxidation and insecticide metabolism, in “lnsecticide Biochemistry and Physiology (C. F. Wilkinson, Ed.). p. 61, Plenum, New York, 1976. 6. E. Hodgson, L. G. Tate, A. P. Kulkami, and F. W. Plapp, Jr., Microsomal cytochrome P-450: Characterization and possible role in insecticide resistance in Muscu domestica. J. Agr. Food Chem. 22, 360 (1974).
PHENoBARBIT,~LAND/~-NA~HTH~FLAV~NE
7. R. D. Schonbrod and L. C. Terriere, The solubilization and separation of two forms of microsomal cytochrome P-450 from the house fly, Musca domestica, L., Biochem. Biophys. Res. Commun. 64, 829 (1975). 8. S. J. Yu and L. C. Terriere, Cytochrome P-450 in insects: 1. Differences in the forms present in insecticide resistant and susceptible house flies, Pestic.
Biochem.
Physiol.
INDUCTION
19.
20.
12, 239 (1979).
9. J. Capdevila and M. Agosin, Multiple forms of housefly cytochrome P-450, in “Microsomes and Drug Oxidations” (V. Ullrich, Ed.), p. 144, 21. Pergamon, London, 1977. 10. L. C. Terriere and S. J. Yu, Microsomal oxidases in the flesh fly (Sarcophaga bullata, Parker) and black blow fly (Phormia regina (Meigen)), Pestic. Biochem. Physioi. 6, 223 (1976). 11. L. B. Brattsten and R. L. Metcalf, Age-dependent 22. variations in the response of several species of Diptera to insecticidal chemicals, Pestic. Biochem. Physiol. 3, 189 (1973). 12. P. Y. Lu, S. Y. Yang, and R. L. Metcalf, Influ23. ence of aldrin, methoxychlor, and parathion on longevity of Musca domestica and Phormia regina, J. Econ. Entomol. 71, 407 (1978). 13. L. C. Terriere and S. J. Yu, Cytochrome P-450 in insects. 2. Multiple forms in the flesh fly (Sarcophaga bullatu. Parker). and the blow fly 24. (Phormia regina (Meigen)), Pestic. Biochem. Physiol. 12, 249 (1979). 14. J. F. Estenik and W. J. Collins, In vivo and in vitro studies of mixed-function oxidase in an aquatic insect, Chironomus riparius, in “Pesticide and Xenobiotic Metabolism in Aquatic Organisms” (M.A.Q. Khan. J.J. Lech. and J.J. Menn, Eds.), ACS Symposium Ser. No. 99, p. 349, 25. Amer. Chem. Sot., Washington, D.C., 1979. 15. S. J. Yu and L. C. Terriere, Phenobarbital induction of detoxifying enzymes in resistant and susceptible houseflies, Pestic. Biochem. Physiol. 3, 141 (1973). 16. S. J. Yu and L. C. Terriere, Enzyme induction in the housefly: The specificity of the cyclodiene 26. insecticides, Pestic. Biochem. Physiol. 2, 184 (1972). 17. L. C. Terriere, S. J. Yu, D. E. Farnsworth, and H. A. Rose, Tyrosinase as an inhibitor of aldrin epoxidase in house fly microsomes, Pestic. Biochem. Physiol. 13, 274 (1980). 27. 18. T. Omura and R. Sato, Carbon monoxide-binding
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pigment of liver microsomes, J. Biol. Chem. 239, 2370 (1964). 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). R. W. Estabrook, J. Peterson, J. Baron, and A. Hildebrandt, The spectrophotometric measurement of turbid suspensions of cytochrome associated with drug metabolism, in “Methods in Pharmacology” (C.F. Chignell, Ed.), Vol. 2, p. 303. Meredith Corp., New York, 1972. L. B. Braltsten and R. L. Metcalf, The synergistic ratio of carbaryl with piperonyl butoxide as an indicator of the distribution of multifunction oxidases in the Insecta, J. Econ. Entomol. 63, 101 (1970). W. A. Brindley, Synergist differences as an afternate interpretation of carbaryl-piperonyl butoxide toxicity data, Environ. Entomol. 6, 885 (1977). J. Capdevila. N. Ahmad, and M. Agosin, Soluble cytochrome P-450 from house fly microsomes. Partial purification and characterization of two hemoprotein forms. J. Biol. Chem. 250, 1048 (1975). A. Y. H. Lu, W. Levin, S. B. West, M. Jacobson, D. Ryan, R. Kuntzman, and A. H. Conney, Reconstituted liver microsomal enzyme system that hydroxylates drugs, other foreign compounds, and endogenous substrates. VI. Different substrate specificities of the cytochrome P-450 fractions from control and phenobarbital-treated rats, J. Biol. Chem. 248, 456 (1973). A. R. Boobis, D. W. Nebert, and J. S. Felton, Comparison of @-naphthoflavone and 3methylcholanthrene as inducers of hepatic cytochrome P-448 and aryl hydrocarbon (benzo[a]pyrene) hydroxylase activity, Mol. Pharmacol. 13. 259 (1977). J. Capdevila, A. Morello, A. S. Perry, and M. Agosin, Effect of phenobarbital and naphthalene on some of the components of the electron transport system and the hydroxylating activity of housefly microsomes. Biochemistry 12, 1445 (1973). M. T. James, The flies that cause myiasis in man. U.S. Dep. Agr. Misc. Pub/. No. 631 (1947).
BIOCHEMISTRY
AND
PHYSIOLOGY
14, 275-281 (1980)
Microsomal Oxidase Activity of Three Blowfly Species Induction by Phenobarbital and p-Naphthoflavone H. A.
ROSE*
L. C.
AND
and Its
TERRIERE
Department of Entomology, Oregon State University, Corvallis, Oregon 97331; Pathology and Agricultural Entomology, University of Sydney, New South
und *Department of Plunr Wales 2006, Ausfrulia
Received June 20, 1980; accepted November 20, 1980 Induction of the microsomal oxidase system by dietary phenobarbital and p-naphthoflavone was examined in three blowflies, Phormia regina (Mg.), Lucilia illustris (Mg.), and Eacolliphora lilicu (Walk.). Responses were similar in adults and larvae of all species. Phenobarbital increased cytochrome P-450 levels up to 9-fold and aldrin epoxidase up to 138-fold. Increases in cytochrome P-450 and aldrin epoxidase caused by p-naphthoflavone were minor relative to those produced by phenobarbital. In toxicity experiments with carbaryl and propoxur tolerance was associated with the amount of microsomat oxidase activity. Using piperonyl butoxide to synergize carbaryl and propoxur there was no clear indication for the use of either the synergist ratio or synergist difference as an indicator of microsomal oxidase activity. INTRODUCTION
Two aspects of the microsomal oxidase system that make it a particularly interesting mechanism of detoxication are its lack of substrate specificity and the ease with which its reaction rate can be accelerated in response to chemical stress. This provides the organism with a versatile response to potential biocides such as insecticides, insect growth regulators, and natural toxicants in its environment. Considering the number of insect species known, the microsomal oxidase system has been studied in a very small number and in most of these cases the examination has been rather superficial. However, since this system has the attributes mentioned above and since it has already been shown to be involved in such fundamental processes as hormone regulation (1, 2), response to plant substances (3, 4), and survival in a toxic environment (5), more knowledge of its activity in different species is highly desirable. The microsomal oxidase system has been studied in a few Diptera, the house fly Musca domestica (6-9), the blowfly Phormia regina, the fleshfly Sarcophaga bullata (lo-13), and the midge Chironomus riparius (14). In comparisons made so far
the house fly appears to contain the most microsomal oxidase activity on a body weight or protein basis and this level of activity correlates fairly well with susceptibility to insecticides in comparisons of house flies, fleshflies, and blowflies (13). Very few comparisons of this sort have been made, however, to determine whether species differences in susceptibility to insecticides can be explained by their respective microsomal oxidase levels. Species differences in the response of the microsomal oxidase system to dietary stimulants (inducers) probably occur also but, as yet, there has been very little experimentation in this area. Differences in morphology and feeding habits are serious limitations to comparative studies of both the microsomal oxidase system and its induction. We have overcome some of these limitations in experiments with three closely related Diptera with similar feeding habits and longevities. The results of our experiments are presented here. METHODS
Insects. P. regina was used from the laboratory culture of several years. Lucilia illustris and Eucalliphora lilica were reared
275 004%3575/80/060275-07$02.00/O Copyright All rights
Q 1980 by Academic Press. Inc. of reproduction in any form reserved.
276
ROSE
AND
TERRIERE
in the laboratory for 6 months prior to the Mortality, the criterion being the inability beginning of these experiments. The origito stand and walk, was checked after 24 hr. was replicated at least nal stocks were obtained locally from eggs Each experiment deposited in raw meat. The three species twice and the average mortality for each 80 were reared under a 14:lO L/D cycle at a dosage was based upon approximately temperature of 25 + 2°C. Adults were fed a flies. diet consisting of sugar, powdered milk, RESULTS and dry egg yolk (12:12:1) and larvae were reared on raw pork liver. Enzyme characteristics. Microsomes from both adults and larvae in all three Treatment with inducers. With adults, phenobarbital and @-naphthoflavone were blowfly species had no detectable aldrin administered by mixing thoroughly with the epoxidase activity when NADPH was diet as described by Yu and Terriere (15). omitted from the incubation medium. CarIn the case of larvae, the inducing chemibon monoxide which was bubbled gently cals were dusted onto the liver which had through the incubation medium for 1 min been pierced thoroughly with a pair of prior to incubation inhibited the activity by sharply pointed forceps. 70- lOO%, the greatest inhibition being seen Enzyme assays. Microsomal oxidase ac- in adult preparations. tivity in adults was measured with aldrin as Preliminary observations showed that substrate, the product, dieldrin, being mea- preparations of blowfly larvae contain insured by glc as described by Yu and Ter- hibitors of microsomal oxidase activity. Inriere (16). With larvae, the procedure was hibition was partly overcome by the use of identical except that the homogenizing BSA and KCN in the homogenizing mebuffer contained 1.5% bovine serum albu- dium. Results using different batches of min (BSA) and 2 x lop3 M KCN (17). larvae were somewhat erratic in actively Phenobarbital had no effect on aldrin growing larvae. Reliable comparisons, epoxidase activity nor on the amount of however, of enzyme activity could be made cytochrome P-450 when added in the at around the clear gut stage of larvae, i.e., homogenizing medium of either control or after the larvae had completed feeding. The induced larvae. It was also shown that there results presented here were obtained with was no aldrin epoxidase activity or cyto- such larvae. chrome P-450 on liver on which the larvae In larvae microsomal aldrin epoxidase were reared from control or induced treatactivities, measured as picomoles of dielments assayed at the end of the experidrin produced per individual per minute, mental period. Cytochrome P-450 was were 7.7, 10.7, and 10.6 for P. regina, L. measured with an Aminco DW-2A dual illustris, and E. Mica, respectively. The beam spectrophotometer according to the corresponding figures for 8- to 9-day-old method of Omura and Sato (18). Protein headless adult females were 8.6, 7.6, and was determined by the Bradford method 11.8. Larval weights were 51.5, 40.6, and 45.2 mg and the headless adult females (19). Bioassays. Dosage-mortality relationweighed 41.5, 41.4, and 41.5 mg, respecships were obtained by topical application tively. of known amounts of carbaryl and proInduction. Preliminary experiments using poxur dissolved in acetone to 8- to 9-day-old P. regina larvae were undertaken to ascerfemale flies. Parallel bioassays with the tain the best way of applying the inducing synergist, piperonyl butoxide (5 parts chemicals to liver slabs. Chopped liver was synergist:l part insecticide) were also per- not used because the larvae did not develop formed. Following treatment the flies were normally. When solvents such as dimethyl provided with water and held at 25 -C 2°C. sulfoxide and xylene were injected into the
PHENOBARBITAL
ANDP-NAPHTHOFLAVONE
lNDucTIoN
liver a 2- to 4-fold increase compared with controls (untreated liver or liver injected with water) in aldrin epoxidase activity was obtained. Using phenobarbital, there was no difference in aldrin epoxidase activity with an aqueous solution being injected into the liver or crystals being dusted onto the liver which had been pierced thoroughly. The dusting method was adopted because it obviated the need for solvents and could be used for any inducing chemical. Table 1 shows that stimulation by 0.2% phenobarbital of cytochrome P-450 was 5.7-, 3.9-, and 3.5-fold for E. Mica, L. illustris, and P. regina, respectively. The data suggest that in the case of cytochrome P-450 the highest dose (0.2% phenobarbital) is at or near the upper threshold level. In the case of aldrin epoxidase, the dose giving maximum effect is probably above 0.2%. With higher doses than this, however, some mortality of larvae was noted. With p-naphthoflavone used at 0.1% the inductive effect was minor compared to that obtained with phenobarbital. There was no significant change in the level of cytochrome P-450 in P. regina or L. illustris but a 1.7-fold increase in E. lilica. The greatest increases in aldrin epoxidase activity were 3.6- and 3.0-fold obtained with E. lilica and P. regina, respectively.
IN THREE
277
BLOWFLIES
The results reported in Table 2 were obtained from abdomens only; abdomens had slightly less than 50% aldrin epoxidase activity of headless flies. Measurements of cytochrome P-450 of headless flies could not be made, however, because CO difference spectra revealed a deep trough at 446 nm and no peak around 450 nm indicative of the presence of cytochrome oxidase in the preparations (20). As in the case of larvae, phenobarbital is a strong inducer of cytochrome P-450 and of aldrin epoxidase in adults of the three species (Table 2). The threshold dose appears not to have been reached because in all cases the values for the 2% levels were the highest but with P. regina the increase from the 1% to the 2% level was small. The maximum increases noted in the case of the hemoprotein were 9.0-fold for P. regina, 3.5-fold for E. lilica, and 3.1-fold for L. illustris. The amount of the hemoprotein in the three species at the 2% level was quite similar ranging from 374 to 463 pmol/mg protein. Aldrin epoxidase activity ranged from 3.0- to 12.6-fold higher in P. regina than in the other two species at the 2% level. At l%, fi-naphthoflavone increased aldrin epoxidase activity 2.3- and 2.1-fold in the case of P. regina and E. lilica, respec-
TABLE
1
Microsomul Cytochrome P-450 and Aldrin Epoxidase in Blonfly Lurlsae ufter Induction by Phenobarbital und PNaphthojluvone Cytochrome Dose” (%I
P. regitu
P-450 (pmol/mg
Dieldrin
produced
(pmoYmin/mg
protein)’
L. illustris
E. lilica
P. reginu
L. illustri.7
E. lilicu
24.8 t 0.4
35.6 t 3.6
23.4 + 1.0
24.8 L 0.7
36.9 + 0.7
Control
21.8
2 3.4
Phenobarbital 0.02 0.05 0.10 0.20
41.1 59.5 68.7 75.7
t 2 -e k
7.6 0.5 5.9 4.3
41.0 51.1 102.0 96.1
/SNaphthoflavone 0.10
23.0 f 1.3
24.4
u Percentage in diet (pork liver) ’ Mean r SE of two experiments. c Mean f SE of two experiments,
protein)”
2 + L +
5.1 8.0 6.5 1.3
+ 0.4
for 1% days duplicate
prior
84.6 111.2 164.6 203.0
5 + t k
3.2 5.3 0.1 4.1
60.6 f 0.8 to assay.
incubations.
419.4 638.5 829.6 943.1
4 _t -+ -t
34.3 31.9 9.4 14.6
69.5 + 0.3
90.9 136.5 335.6 649.6
? 2 -t 5
2.4 11.2 44.2 15.6
34.2 z? 2.6
189.8 382.8 639.5 839.7
k t t +
5.7 8.5 7.3 65.3
133.9 k 0.8
278
ROSE AND TERRIERE TABLE Microsomal after
Dose” (%) Control Phenobarbital 0.2 0.5 1.0 2.0 P-Naphthoflavone 1.0
Cytochrome Induction
2
P-450 and Aldrin epoxidase in blo@y by Phenoburbital and /SNaphthoJavone
Adults
Cytochrome P-450 (pmol/mg protein)*
Dieldrin produced (pmoUmin/mg protein)’
P. regina
P. reginu
L. illustris
12.7 2 0.6
31.9 r 3.2
L. illustris
47.8 i 2.6 180.5 354.0 399.6 431.9
E. lilicu
149.2 k 19.4 107.4 r 8.7
k 12.7 rc_36.9 re_7.9 i 14.0
172.5 189.4 270.5 462.6
79.0 _f 7.5
L k k -t
26.0 1.9 38.2 8.5
131.4 t 22.4
169.7 270.5 323.2 373.8
+ 2 t 2
44.2 329.2 rt_ 37.5 46.7 1195.3 2 26.6 10.2 1475.0 s 32.9 45.1 1755.9 5 68.3
136.5 -t 24.5
28.9 + 1.2
61.3 102.6 211.5 554.9
-t + rt +
E. lilica
7.4 5.1 30.3 14.3
35.0 2 4.8
21.0 c 4.1 36.7 95.1 337.2 583.9
t 3.0 Y?6.1 t 17.8 t 34.8
44.8 s 0.9
” Females, 2-3 days old, were fed with the inducer in the diet for 3 days prior to enzyme assays. ’ Mean ? SE of two experiments. ’ Mean 2 SE of two experiments, duplicate incubations.
tively, with the corresponding increases in cytochrome P-450 being only 1.7 and 1.3. The effect of the inducer on aldrin epoxidase activity and cytochrome P-450 of L. illustris was negligible. The Ama, of the CO difference spectra in control and induced larvae of the three species are shown in Table 3. Absorption maxima of larvae were lower than those of adults. Also, the A,,, for phenobarbital was lower than the corresponding control by about 1 nm. The A,,, for /?-naphthoflavone was higher than that of the corresponding phenobarbital figure, being the same or slightly lower than the control figure. Readings of P-450 were taken l-2 min after the addition of Na,$O, to the cuvette
as it was observed that there was a shift to a higher value for A,,, of about 1.5 nm in 7- 10 min. The amount of P-450 was variable. In general it was higher in larvae than adults and the amount increased with the higher doses of phenobarbital. Insecticide toxicity experiments. Results of the bioassay tests are summarized in Table 4. Experiments with the synergist were included in order to test the usefulness of this microsomal oxidase inhibitor as an indicator of the role of these enzymes in metabolizing the two insecticides. The synergist data are expressed as synergist ratios as suggested by Brattsten and Metcalf (21) and as synergist differences as suggested by Brindley (22).
TABLE Absorption Maxima Induced Adults
3
of CO Difference Spectra in Control and Larvae of Three f3lowjly Species
and
Absorption maxima (nm) P. regina
L. illustris
E. lilica
Adults
Larvae
Adults
Larvae
Adults
Larvae
Control
452.0
451.5
452.0
449.0
453.0
451.0
Induced Phenobarbital P-Naphthoflavone
450.5 452.0
450.0 451.0
451.0 452.0
448.5 449.0
452.0 453.0
449.5 450.0
PHENoBARBITALAND/~-NAPHTHOFLAVONE
INDUCTION
IN THREE
BLOWFLIES
279
TABLE4 Toxicity
Data
for
Carbaryl
Dose effect parameter LD,,, carbaryl” LD,, carbaryl + piperonyl butoxide Synergist ratio” Synergist difference’ LD,o propoxur LD,” propoxur + piperonyl butoxide Synergist ratio Synergist difference
and Propoxur
.
with
and without
Piperonyl
Butoxide
in Blowjly
Adults
P. regina
L. illustris
E. lilica
2014 23.8
1005 10.5
2804 18.0
85 1990 7.7 4.2
96 995 3.6 2.1
1.56 2786 14.0 6.8
1.8 3.4
1.7 1.5
2.1 7.2
cfLD,, given as mg/kg. b LD,, carbaryVLD,, carbaryl + piperonyl butoxide. ’ LD,,, carbaryl -(LD,,, carbaryl + piperonyl butoxide).
Arranging the LD,, values of carbaryl and propoxur with and without synergist the approximate relationship is 1:2:3 for L. illustris, P. regina, and E. lilica. The microsomal aldrin epoxidase activities expressed in picomoles of dieldrin produced per milligram of individual in 8 to 9-day-old headless female adults were 184, 207, and 283, respectively, the ratios being 1: 1.1: 1.5. Thus the relative position of the three species with respect to microsomal oxidase activity is maintained but the differences are much less than would be indicated by the LD,, values of the two insecticides. DISCUSSION
These results show that dietary phenobarbital is capable of greatly stimulating aldrin epoxidase in blowfly adults and larvae. The reason for such large increases is not clear although the fleshfly (S. buflata) is also highly inducible (IO) and so are adults of susceptible house fly strains (15). Dietary phenobarbital also increased the cytochrome P-450 content in adults and larvae but induction was much less when compared with aldrin epoxidase. The reason for this is not clear. It is known that cytochrome P-450 exists in multiple forms in insects (7-9, 23) and mammals, and there is a degree of substrate specificity
with each of the different forms (24). It is possible, therefore, that one or more forms of cytochrome P-450 with a good specific epoxidase activity was induced by phenobarbital. Another possibility is that induction may have also changed the activity of some or all of the cytochrome, especially that accepting aldrin as a substrate. This is based on evidence that active forms of cytochrome P-450 can occur in house . flies (8). Only small variations in the response of both larvae and adults to phenobarbital in L. illustris and E. lilica are evident. Thus, the increases in P-450 content and aldrin epoxidase activity are fairly similar. The aldrin epoxidase of P. regina, however, appears to be more inducible than the aldrin epoxidase of the other two species with adult and larval activity being 138- and 40-fold higher than the control values at the highest dose levels. P-Naphthoflavone was added in the diet because it behaves similarly to 3-methylcholanthrene in vertebrates (25). Based on substrate specificities, these inducers act very differently from phenobarbital. Moreover, unlike phenobarbital, p-naphthoflavone and 3-methylcholanthrene typically induce a form of P-450 which is designated P-448 due to the shift in the CO difference spectrum of the hemoprotein. In
280
ROSE
AND
a study using adult house flies, 3-methylcholanthrene had no effect on microsomal oxidase activity (15). Our results show a minor effect with P-naphthoflavone compared to that of phenobarbital at the same dose. Increases in the amount of cytochrome P-450 and aldrin epoxidase were only slight. In the case of P. regina and E. lilica, however, increases in aldrin epoxidase activity were greater than those of cytochrome P-450 suggesting that P-naphthoflavone had produced a change in the P-450 of those species but not with L. illustris (Tables 1 and 2). There was no evidence that /3-naphthoflavone induced a P-448 type of cytochrome P-450 as the CO difference spectra from individuals treated with this compound were always higher than the phenobarbital-treated individuals. In vertebrates, the reverse would be true. Another difference between blowflies and vertebrates was evident. Induction by phenobarbital in blowflies yielded a difference spectrum with a lower A,,, compared to controls, a result also obtained with house flies (26). Our carbaryl LD,, value for P. regina was 22 times higher than that reported by Brattsten and Metcalf (11) although our figure for synergized carbaryl was only 2.2 times higher. Perhaps their holding temperatures of 28°C could partially explain their much lower figure for carbaryl LD,,. This study showed that the greatest tolerance to carbaryl and propoxur was associated with the highest amount of microsomal oxidase activity. The correlation, however, was not 1: 1. No clear picture of a preference for the use of either the synergist ratio (21) or synergist difference (22) as an indicator of microsomal oxidase activity emerged from the results. There is strong synergist activity with carbaryl, however, indicating that all three species must depend to a large extent on microsomal oxidases for carbaryl detoxication. The microsomal oxidases of two more blowfly species have been investigated with the completion of this study. It is evident
TERRIERE
that in common with other Diptera studied, the enzyme system requires NADPH and is inhibited by CO. The results in this paper are characterized more by similarities rather than differences between the three species. The species belong to three different tribes (Lucilia:Luciliini, Eucuiliphoru: Calliphorini, and Phormiu:Phormiini), the latter belonging to subfamily Chrysomyinae while the other two belong to Calliphorinae. The biology of most blowflies, however, is fairly similar (27) and in this investigation the same culture conditions were used for all species. It is not surprising therefore that no marked differences were observed in any of the results from one species to another. ACKNOWLEDGMENTS
This research was supported by USPHS Grant ES00362-21. The authors are grateful for the advice of Dr. S. J. Yu, for the identification of L. illusrris and E. Mica by Dr. B.F. Eldridge. and for the technical assistance of Mr. Dan Farnsworth. REFERENCES
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PHENoBARBIT,~LAND/~-NA~HTH~FLAV~NE
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