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The microsomal mixed-function oxidase system of Heliothis punctiger Wallengren and H. Armiger (Hübner) (Lepidoptera: Noctuidae)
Comp. Biochem. Physiol. Vol. 77B, No. 4, pp. 849-855, 1984
0305-0491/84 $3.00 + 0.00 © t984 Pergamon Press Ltd
Printed in Great Britain
THE MICROSOMAL MIXED-FUNCTION OXIDASE SYSTEM OF H E L I O T H I S P U N C T I G E R WALLENGREN AND H. A R M I G E R (HUBNER) (LEPIDOPTERA: NOCTUIDAE) P. J. COLLINS* and G. H. S. HOOVER Department of Entomology, University of Queensland, St. Lucia. Q. 4067, Australia
(Received 25 August 1983) Abstract--1. The levels of cytochrome P-450, NADPH cytochrome c reductase, cytochrome b5and aldrin
epoxidation revealed quantitative differences between H. armiger and H. punctiger and among the tissues (midgut, fatbody and integument) of each species. 2. Qualitative differences between the species and among the tissues was indicated by P-450 spectral binding studies. Type I (substrate) binding occurred, in both species, only with midgut microsomes. Type II (ligand) binding occurred with n-octylamine in all tissues and with pyridine in only midgut and fatbody microsomes. Piperonyl butoxide added to reduced midgut microsomes illicited a type III spectrum. There was no reaction with either fatbody or integument microsomes. 3. n-Octylamine difference spectra also revealed the presence of high-spin P-450 in the midgut microsomes of both species. Fatbody and integument P-450 were predominantly in the low-spin form.
INTRODUCTION
In insects the microsomal mixed-function oxidases (MFO) comprise the most important system in the primary degradation of foreign compounds (Dauterman and Hodgson, 1978). Consequently, these enzymes play a central role in the metabolism of insecticides (Nakatsugawa and Morelli, 1976). Despite their significance, the major components of the M F O system (cytochrome P-450, cytochrome b 5 and N A D P H cytochrome c reductase) have been studied in only a few insect species. Cytochrome b5 and N A D P H cytochrome c reductase have been described from microsomes of the southern armyworm, Spodoptera eridania Cramer (Lepidoptera:Noctuidae) (McFadden et al., 1979; Crankshaw et al., 1981a) while the majority of studies of P-450 have been conducted on the housefly, Musca domestica L. (Kulkarni and Hodgson, 1976, 1980; Yu and Terriere, 1979). In addition, although these enzymes are well known from midguts of lepidopteran larvae (Brattsten, 1979) and housefly abdomens there have been few comparative studies of this system in other tissues (Brattsten et al., 1980; Tate et al., 1982). As the contribution of each tissue varies considerably among species there is a need not only to extend our knowledge of M F O ' s to new species but also to compare the activities of these enzymes in various tissues within species. This especially applies to pest species in which knowledge of detoxification systems is critical to the management of insecticide resistance. The cotton bollworm, H. arrniger and the native budworm, H. punctiger are appropriate candidates for this type of study as they are widespread and significant pests of field crops in Australia, and insecticides are still the major control method used against these species. *Present address: Entomology Branch, Department of Primary Industries, Meiers Road, Indooroopilly, Q. 4068, Australia.
In this investigation we have determined the concentration of the components of the mixed-function oxidase system and the binding spectra of cytochrome P-450 in midgut, fatbody and integument microsomes from H. armiger and H. punctiger. Aldrin epoxidase was used as a measure of overall activity. MATERIALS AND M E T H O D S
Insects
The H. punctiger culture was established from a laboratory strain maintained by the Queensland Department of Primary Industries. This strain was initiated from moths caught in 1972 at Redland Bay, in south-east Queensland. H. armiger was established from moths caught in the Gatton district, Queensland, in 1981. Heliothis armiger and H. punctiger larvae were reared on an artificial diet based on navy beans and wheat germ plus added vitamins and preservatives (Shorey and Hale, 1965). Adults and larvae were kept at 28 4- I°C and 70~o r.h. in a 12 hr light: 12 hr dark regime.
Chemicals Cytochrome c, NADPH, NADP, glucose-6-phosphate (G-6-P), giucose-6-phosphate dehydrogenase and bovine serum albumin (BSA) were purchased from either Sigma Chemical Co., USA or Boehringer-Mannheim Australia Pty., Sydney, Australia. n-Octylamine (> 99~o) was bought from Fluka AG, Buchs, Switzerland and pyridine (99.09/o) from Ajax Chemicals, Sydney, Australia. HHDN (aldrin) and HEOD (dieldrin), both >99.0~ pure, were a gift from Shell Research Limited, Sittingbourne, Kent, U.K. Carbaryl was synthesised from l-naphthol (Krishna et al., 1962), and DDT and permethrin had a purity of 95 and > 99~o respectively. Tissue preparation Last instar larvae, 24-36 hr since moulting, were used as the source of midgut and fatbody tissues and newly moulted, 0-6 hr, last instar larvae were the source of integuments. The midgut was dissected from the larva and the gut contents removed. Midguts were then rinsed and
849
850
P.J. COLLINS and G. H. S. HOOPER
placed in appropriate volume of ice-cold phosphate (NaH2PO 4 . 2H20/N2HP04) buffer pH 7.6 plus 1.15~o KCI. Once the midgut had been removed the fatbody could then be scraped away from the integumental tissue. The fatbody was placed directly into ice-cold buffer, as was the integument after being rinsed in buffer to remove any attached fatbody. The tissues, in ice-cold phosphate buffer, were homogenised in a ground-glass tube with either a motor-driven glass (for integument) or Teflon (for all other tissues) pestle. To prepare microsomal fractions the crude homogenate was immediately filtered through glass wool and spun in a Beckman L5~40 ultracentrifuge (at 0'C) at 10,000g average for 15 rain. The resulting supernatant was then centrifuged at 100,000g for 60 min. The pellet from this centrifugation was then resuspended in an appropriate volume of the standard phosphate buffer.
A[drin epoxidase Aldrin epoxidase activity was determined by gas-liquid chromatography using the epoxidation of aldrin to dieldrin (Krieger and Wilkinson, 1969). Incubations were carried out aerobically in 25 ml Erlenmeyer flasks and shaken in a water bath at 30C. Reaction time was 10 rain. The standard 5 ml incubation medium contained G-6-P (2.4mM). NADP (51 #m), phosphate (NaH2PO 4. 2HzO/NazHPO4) buffer pH 7.6, PTU (0.4mM) and KC1 (1.15,°,o). The reaction was initiated by the addition of 8 ~g aldrin in 20/~1 ethanol. The reaction was terminated with 1 ml of 1 M HC1 and aldrin and dieldrin were extracted with toluene after the method of Konrad et al. (1969). The amount of aldrin and dieldrin was quantified using a Packard Model 427 GLC fitted with an electron capture detector with a Ni63 radioactive source of 10 mCi, Pure N: was used as the carrier gas through a 1 m glass column (i.d. 2 mm, o.d. 6 mm) packed with 5~o silicone SE30 on Chromsorb WHP 100/12-mesh. A Shimadzu CRIA chromatopac recording data processor was used to quantify samples.
lished before the addition of any substrate or ligand to the microsomes. Solid samples (such as insecticides) were added directly to the sample cuvette which was inverted approx. 20 times and then allowed to equilibrate for a few minutes before scanning. Liquid samples were added directly to the sample cuvette in 10 ~tl of standard phosphate buffer and the sample cuvette was treated as for solids. An equivalent volume of buffer was added to the reference cuvette. These procedures were repeated until no further increase in spectral size occurred. Type I (substrate) and type It (ligand) binding spectra were classified according to Schenkman et al. (1967) and type III after Philpot and Hodgson (1971). n-Octylamine type II difference spectra were used to calculate o~, high- and low-spin P-450 using the method of Jefcoate et al. (1969). Type I binding was determined using the methylenedioxyphenyl synergist piperonyl butoxide and the insecticides DDT, carbaryl and permethrin. Type II binding was determined with pyridine and n-octylamine. Type IIl spectra were determined using piperonyl butoxide with reduced microsomes.
Protein determinations Crude homogenate protein was assayed using the biuret method (Layne, 1957) and diluted according to the activity of the particular tissue. The Bradford method (Bradford, 1976) was used to measure microsomal protein concentration. Bovine serum albumin was used as the standard for both assays,
Statistical analysis Aldrin epoxidase assays were replicated 4 times and data are reported as the mean + standard deviation. Spectral assays were replicated 4-6 times with fresh microsomes and data are reported as the mean + standard deviation. Spectral binding studies were each replicated 3 times. Students t-test was used to analyse the data. The enzyme source for each replicate was derived from a sample of 15-20 insects.
NADPH-cytochrome c reductase NADPH-cytochrome c reductase activity was measured at room temperature directly as the increase in the ~-band of reduced cytochrome c with time (Masters et al., 1965). The incubation mixture consisted of 0.8ml phosphate buffer (pH 7.6), 0.2 ml cytoehrome c solution (4.4 mg/ml in phosphate buffer) and 30-100 #g of microsomal protein was added in 50 #1 of phosphate buffer. Reactions were initiated by the addition of 20 #1 of NADPH (0.2 raM). Reductase activity was calculated from the initial change in absorbance at 550 nm using the millimolar extinction co-efficient of 21.1 cm i (Massey, 1959).
RESULTS
Aldrin epoxidase activity In b o t h species midgut specific activity was highest, followed by the fatbody and integument (Table 1). With all three tissues enzyme activity was apparently higher in H. armiger than in the respective tissues of H. punctiger, although only in fatbody and integument assays were the values for H. armiger significantly (P < 0.05) higher than for H. punctiger.
Cytoehrome P-450 Cytochrome b~ Cytochrome b 5 was measured by difference spectroscopy using the method of Omura and Sato (1964). Solid sodium dithionite was used to reduce the cytochrome b5 in the sample cuvette and the reduced minus oxidized difference spectrum was obtained. An extinction co-efficient of 1851/cm/mM was used for the change in absorbance between 426 and 409 nm. Microsomal protein concentration was 1.5 + 1 mg/ml.
Spectral maxima were at 450 nm for all tissues of both species. The level o f cytochrome P-450 from integument microsomes was about equal in the two species. F a t b o d y cytochrome P-450 from H. armiger was about 25% higher than that obtained from H. punctiger and with midgut microsomes H. armiger contained a b o u t 56% more P-450 than H. punctiger (Table 1).
Qvtochrome P-450
Cytochrome b5
Cytochrome P-450 was determined from the dithionitereduced CO difference spectrum according to the method of Omura and Sato (1964) using a molar extinction co-efficient of 91 1/cm/mM. Microsomal protein concentration was 1.5 _+ 1 mg/ml standard phosphate buffer.
The level o f cvtochrome b, (Table 1~ was much greater in midgut microsomes than that from either fatbody or integument. This was the case for both H. armiger and H. punctiger. In contrast to the relative amounts of cytochrome P-450 present in each tissue, there was more b 5 in integument than in fatbody microsomes. Integument microsomes from H. armiger contained about three-times the concentration o f cyto-
Spectral binding studies Oxidized cytochrome P-450 difference spectra were determined between 490 and 350 nm using a Varian Series 634 double beam spectrophotometer. A zero-baseline was estab-
MFO in Heliothis larvae
851
Table 1. Properties of the mixed-functionoxidase system of tissues from last instar larvae of Heliothis punctiger and H. armiger Species
MIDGUT
FATBODY
INTEGUMENT
Aldrin epoxidase specific activity , . . . . T nmol d l e l d n n produced/mg proteln/mxn
H. armiger
0.446 ~ 0.059
0.162 L 0.007
0.075 L 0.001
H. punctiger
0.424 ~ 0.057
0.108 ~ 0.015"
0.065 ~ 0.011"
nmol cytochrome c reduced/rain/mg protein H. armiger
51.33 ~ 2.55
H. punctiger
40.16 ~ 1.32"
17.26 ~ 7.5
5.84 ~ 0.30*
23.6
t
~ 3.7
15.98 ~ 1.4
Cytochrome b 5 content nmol/mg p r o t e i n
H. armiger
87.05 ~ 3.9
5.43 ~ 1.2
H. punctiger
62.90 L 5.3*
4.84 ~ 0 . i 0
21.0
~ 2.4
7.53 L 0.85*
Cytochrome P-450 conte~t nmol P-450/mg protein ~
H. armiger
0.403 + 0.012
0.150 + 0.038
0.058 + 0.011
H. punctiger
0.258 + 0.006*
0.120 + 0.013
0.057 + 0.011
*Significantly different (P < 0.05) from value for H. armiger. %Values are mean_4-SD, n = 4. :~Values are mean+ SD, n = 3. §Values are mean___SD, n = 4 or more separate experiments.
chrome bs as microsomes from H. punctiger and there was about 27~o more bdmg protein in H. armiger midgut microsomes than those from H. punctiger. There was no difference in b5 content of fatbody microsomes between the two species. The absorption maximum for all three tissues in both species was at 426 nm when reduced with sodium dithionite.
NADPH-cytochrome c (P-450) reductase Levels of N A D P H cytochrome c reductase activity followed a similar pattern to that of cytochrome bs. With both species midguts had the highest activity followed by integuments and then fatbody microsomes (Table 1). For midgut and fatbody microsomes H. armiger had significantly (P < 0.05) more activity than the respective tissue from H. punctiger. There was no significant difference between the integument values of the two species.
Binding spectra of cytochrome P-450 Both species exhibited a similar pattern of response to the addition of various compounds to the microsomal suspension. Type I spectra were elicited by the three insecticides and the synergist piperonyl butoxide with midgut microsomes. These spectra had the characteristic (type I) peak at 390 nm and a trough between 415 and 420 nm. Type I spectra could not be detected with fatbody or integument microsomes.
The extent of type I binding (Table 2) was greatest with piperonyl butoxide in both species but particularly with H. punctiger when compared with the compounds tested. The two species had about the same affinity for carbaryl and D D T but there was more than 50~o greater binding with permethrin using H. armiger microsomes than those from H. punctiger. Pyridine gave rise to a type 1I spectrum with midgut and fatbody microsomes in both species but there was no reaction with integument microsomes. With H. punctiger midgut and fatbody microsomes pyridine addition produced characteristic type II spectra with a trough at 395nm and a peak at 425 nm. The fatbody A absorbance was about half that of the midgut. On the other hand, the spectra produced by tissues from H. armiger, although still typical II spectra, differed from the pattern shown by H. punctiger with a larger A absorbance and a peak at 427 nm and a trough at 398 nm. Typical type II spectra were also formed when n-octylamine was added to midgut, fatbody and integument microsomes from both species. H. armiger and H. punctiger fatbody and integument spectra had peaks at 430-432 nm and a single, though broad, trough at 410-416 nm. In contrast, midgut n-octylamine spectra from both species revealed a trough with two distinct minima at 410 and 392 nm. With all three tissues spectra formed with microsomes from H.
852
P, J. COLLINSand G. H. S. HOOPER Table 2. Type I (substrate) binding of microsomal cytochrome P-450 from midguts of H. armiger and H. punctiger
Compound
H. armiger
H. punctiger
carbaryl
0.010 + 0.002
0.012 + 0.002
DDT
0.016 + 0.002
0.013 + 0.002
permethrin
0.037 + 0.007
0.015 + 0.003
piperonyl butoxide
0.045 + 0.004
0.065 + 0.003
Values are the mean +_ SD. n = 3 or more separate experiments. Type I binding = A absorbance/nmol P-450. A Absorbance was measured between peak (ca 390 nm) and trough (ca 415 nm).
armiger were larger than the corresponding spectra from H. punctiger. There were large differences among the various tissues of the two Heliothis species in the extent of binding to the type II compounds (Table 3). Midgut microsomes from H. punctiger and H. armiger demonstrated about the same binding capacity for pyridine but their affinity for n-octylamine differed, i.e. H. armiger type II binding was about 30% greater than that for H. punctiger. In contrast, H. armiger fatbody had a greater affinity for pyridine than that from H. punctiger, although the reverse was the case with n-octylamine. Integument microsomes from both species bound comparatively extensively (especially H. armiger) with n-octylamine. The percentage of high-spin P-450 in midgut microsomes of the two species was about the same. There was little high-spin P-450 in the other tissues assayed (Table 4). When piperonyl butoxide was added to reduced midgut microsomes a spectrum with two peaks (type III) occurred. There was no reaction with fatbody or integument reduced microsomes. With midgut spectra in both species the larger peak had a maximum at 427 nm, H. punctiger having the higher A absorbance (Table 5). The second peak had a maximum at 455 nm with H. armiger and 458 nm with H. punc-
tiger. The 455/427 nm peak height ratio was higher in H. armiger than H. punctiger. Ratios of 0.698 for the 427 nm peak and 1.143 for the 455 nm (approx.) peak are obtained when the magnitude of the peaks of the two species are compared (H. armiger/H, punctiger). Also, when the 445 nm peak of each spectrum was compared to the respective A absorbance for the CO-reduced P-450 spectrum, it was apparent that the magnitude of this peak was lower in H. armiger than H. punctiger (Table 5). DISCUSSION
The results reveal that the midgut, fatbody and integument tissues of Heliothis punctiger and H. armiger contain active microsomal mixed-function oxidase systems. Further, these systems can be characterised both quantitatively and qualitatively. The midgut aldrin epoxidase specific activity of both species, although relatively high are within the range reported for other noctuid larvae (Krieger and Wilkinson, 1969; Williamson and Schechter, 1970; Chandran and Khan, 1972; Gould and Hodgson, 1980; Farnsworth et al., 1981). in contrast, fatbody epoxidase activity was high compared to the limited published data (Brattsten et al., 1980; Tate et al.,
Table 3. Type II (ligand) binding of microsomal cytochrome P-450 from various tissues of H. armiger and H. punctiger
H. armiger midgut
fatbody
integument
pyridine
0.037 + 0.002
0.060 + 0.003
NR +
n-octylamine
0.046 + 0.003
0.046 + 0.010
0.149 + 0.013
H. punctiger midgut
fatbody
integument
pyridine
0.038 ~ 0.004
0.035 ~ 0.006
NR +
n-octylamine
0.032 + 0.011
0.078 + 0.012
0.070 + 0.005
Values are mean _+ SD, n = 3 or more separate experiments. Type II binding = A absorbance/nmol P-450. A Absorbance was measured between peak (ca 430 nm) and trough (410-415 nm). ?No reaction.
853
MFO in Heliothis larvae Table 4. n-Octylamine difference spectra of oxidized microsomes from tissues of last instar H. armiger and H. punctiger
Tissue
Amax.
Amin.
~A(410-500nm) AA(392-500nm)
% high-spin P-450 #
H. armiger
midgut fatbody integument
430 430 432
392/410 412 416
1.0 ~ 0.05 t 5.0 + 1.00 6.0 ~ 0.50
16
H. punctiger
midgut fatbody integument
432 430 432
392 410 415
0.94 + 0.06 3.00 ~ 0.50 3.25 + 1.06
17
tCalcalated from the standard curve of Jefcoate et al. (1970). +Values are mean_ SD, n = 3 or more separate experiments. 1982). Reports of integument epoxidase activity are not available for comparison. Cytochrome b5 concentration of midgut microsomes from both Heliothis species are close to the level reported by Tate et al. (1982) for M . sexta. These authors, however, found about three-times more b5 in fatbody microsomes than occurred in H. punctiger and H. armiger. Levels of midgut N A D P H cytochrome c reductase are within the range reported for other noctuid species (Crankshaw et al., 1981b). However, Tate et al. (1982) report about a 100-fold lower level of reductase activity in fatbody microsomes from M . sexta than we found with H. armiger and H. punetiger. It is interesting that unlike the levels of aldrin epoxidase and cytochrome P-450, in both Heliothis species, the amount of cytochrome b5 and cytochrome c reductase was higher in integument microsomes than fatbody microsomes (Table 1). This may be a reflection of different functions of the microsomal enzymes in these tissues.
Cytochrome P-450 concentration in midgut microsomes from H, punctiger is comparable to levels of 0.032-0.29 nmol/mg protein reported for other lepidopteran species (Yu et al., 1979; Brattsten et al., 1980; Farnsworth et al., 1982), while that for H. armiger is relatively high. Fatbody P-450 concentrations of H. armiger and H. punctiger were higher than those reported for S. eridania (Brattsten et al., 1980) and M. sexta (Tate et al., 1982). There have been no studies published of larval integument P-450 from the Lepidoptera or any other insects for comparison. The cytochrome P-450 spectral binding patterns reveal qualitative differences between the species and among the tissues of each species, The lack of type I binding in either fatbody or integument microsomes is a fundamental difference between these tissues and the midgut. Fatbody and integument hemoprotein were also distinguished by the latter's apparent lack of type I! binding with pyridine. Although these
Table 5. Type III binding of cytochrome P-450 from midgut microsomes of H. armiger and H. punctiger with piperonyl butoxide determined at pH 7.6 Peak
Species
H. armiger
H. punct~ger
max.
Aabsorbance t
455
0.004 ~ 0.001
427
0.037 + 0.007
458
0.00S ~ 0.001
427
0.053 + 0.004
Peak height r a t i o s 455#
455§
427
CO P-450
0.108
0.125
0.066
0.130
tReference wavelength: 490 nm. Values are mean _+SD, n - 3 separate experiments. :~Approx. §Magnitude of type III piperonyl butoxide 455 nm peak relative to CO-reduced P-450 A absorbance value.
854
P.J. COLt,lYS and G. H. S. Hooper
results seem to indicate important differences between the hemoprotein species in each tissue, it is also possible that type I and type lI binding did not occur in some cases because the added compounds may not be able to compete with endogenous substrates present in the microsomal suspension. It is also possible that spectra formed but were too small to detect. Midgut microsomes contained a significant amount of high-spin P-450 while microsomes from the other tissues were composed predominantly of low-spin hemoprotein. In houseflies the presence of high-spin state hemoprotein has been associated with an increase in epoxidase activity (Yu and Terriere, 1979), In this investigation presence of high-spin P-450 was associated with not only higher aldrin epoxidase activity but also the presence of type I spectral binding and higher P-450 concentration. Where binding occurred there were also distinctive responses to various compounds in the two Heliothis species. The difference in type I binding to permethrin and D D T in midgut microsomes (Table 2) suggests that H. armiger might more readily metabolise insecticides than H. punctiger and may therefore have a greater potential for developing resistance to insecticides. Higher aldrin epoxidase and cytochrome P-450 specific activities of H. armiger midgut microsomes than those from H. punctiger support this proposition. The piperonyl butoxide type III spectrum formed with reduced P-450 was also elicited only with midgut microsomes. The 455/427 peak height ratio and the 455/CO P-450 ratio have been used to qualify cytochromes P-450 from various strains of housefly. Capdevila et al. (1974) and Tate et al. (1973) demonstrated that at pH 7.5 the 455 nm peak was reduced in insecticide resistant strains while in susceptible strains it was about equal in magnitude to the CO reduced P-450 peak. Also the 455/430 ratio of type Ill piperonyl butoxide spectrum was lower in R- than S-strain microsomes (Philpot and Hodgson, 1971/2). The piperonyl butoxide type III spectra for H. punctiger and H. armiger (Table 5) midgut microsomes varied particularly in the 455/427 peak height ratio. There was little difference between the 455/CO P-450 ratios of the two species, however. The former may be indicative of qualitative differences in the nature of the P-450 cytochromes in the two Heliothis species. It has been shown in mammals with ethylisocyanide that the ratio of the 455/430 nm peaks increases with increased hydrophobicity of the cytochrome P-450 (Schwen and Mannering, 1982). This is further evidence that the hemoprotein from H. armiger is more capable of xenobiotic metabolism than that from H. punctiger. REFERENCES
Bradtord M. M. (1976) A rapid and sensitive method [or the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem. 72, 248-254. Brattsten L. (1979) Ecological significance of mixedfunction oxidations. Drug metab. Rev. 10, 35 58. Brattsten L. B., Price S. L. and Gunderson C. A. (1980) Microsomal oxidases in midgut and fatbody tissues of a broadly herbivorous insect larva, Spodoptera eridania
Cramer (Noctuidae). Comp. Biochem. Physiol. 66C, 231 237. Capdevila J., Perry A. S. and Agosin M. (1974) Spectral and catalytic properties ofcytochrome P-450 from a diazinoneresistant housefly strain. Chem. biol. Interact. 9, 105 116. Chandran S. R. and Khan M. A. Q. (1972) Microsomes: mixed function oxidase in the midgut of the corn earworm. J. econ. Ent. 65, 1510-1512. Crankshaw D. L., Hetnarski H. K. and Wilkinson C. F. (1981a) The functional role of NADPH-cytochrome c reductase in southern armyworm Spodoptera eridania midgut microsomes. Insect. Biochem. !1, 515-522. Crankshaw D. L., Hetnarski H. K. and Wilkinson C. F. (1981 b) Interspecies cross-reactivity of an antibody to the southern armyworm Spodoptera eridania midgut NADPH-cytochrome c reductase, lnsect Bioehem. II, 593-598, Dauterman W. C. and Hodgson E. (1978) Detoxicalion mechanisms in insects. In Biochemist O' qf lnsects (Edited by Rockstein M.), pp. 541 577. Academic Press. New York. Farnsworth D. E., Berry R. E., Yu S. J~ and Terriere L. C. (1981) Aldrin epoxidase activity and cytochrome P-450 content ofmicrosomes prepared from alfalfa and cabbage looper larvae fed various plant diets. Pestic. Biochem. Physiol. 15, 158-165. Gould F. and Hodgson E. (1980) Mixed function oxidase and glutathione transferase activity in last instar Heliothis virescens larvae. Pestic. Bioehem. Physiol. 13, 34--40. Jefcoate C. R. E., Gaylor J. L. and Calabrese R. L. (1969) Ligand interactions with cytochrome P-450. I. Binding of primary amines. Biochemistr) 8, 3455-3463. Konrad J. G., Pionke H. B. and Chester G. (1969) An improved method for extraction of organochlorine and organophosphate insecticides from lake waters. Analyst 94, 490-492. Krieger R. I. and Wilkinson C. F. 11969) Microsomal mixed function oxidises in insects--1. Localization and properties of an enzyme system effecting aldrin epoxidation in larvae of the southern armyworm (Prodenia eridania). Bioehem. Pharmae. 18, 1403-1415. Krishna J. G., Dorough H. W. and Casida J. E. (1962) Synthesis of N-methylcarbamates via methylisocyanate ~4Cand chromatographic purifications. J. Agric../d Chem. 10, 462 466. Kulkarni A. P. and Hodgson E. (1976) Microsomal electron transport systems in the housefly, Musca domestiea. A model for the study of detoxication systems in insects. Israel J. Ent. XI, 93 108. Kulkarni A. P. and Hosgson E. (1980) Multiplicity of cytochrome P-450 in rnicrosomal membranes from the housefly, Musca domestiea. Biochem. biophys. Aeta 632, 573 588. Layne E. (1957) Spectrophotometric and turbidimetric methods for measuring proteins. In Met6ocA' #l Enz),molog)' II1 (Edited by Colowick S. P. and Kaplan N. O.), p. 447. Academic Press, New York. McFadden K. A., Crankshaw D. L,. Hetnarski H, K. and Wilkinson C. F. (1979) Purification and properties of cytochrome b 5 from the larval midgut of the southern armyworm (Spodoptera eridania). Insect Biochem. 9, 301-308. Massey V. (1959) The microestimation of succinate and the extinction coefficient of cytochrome c. Bioehim. Biophys. Acta 34, 255 256. Masters B. S. W., Kamin H., Gibson Q. H. and Williams C. H. (1965) Studies on the mechanism of microsomal triphosphopyridine nucleotide-cytochrome c reductase. J. biol Chem. 240, 921 931. Nakatsugawa T. and Morelli M. A. (1976) Microsomal oxidation and insecticide metabolism. In Insecticide Biochemistry and Physiology (Edited by Wilkinson C. F.), p. 61. Plenum Press, New York.
MFO in Heliothis larvae Omura T. and Sato R. (1964) The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. J. biol. Chem. 239, 2370-2378. Philpot R. M. and Hodgson E. (1971) A cytochrome P-450-piperonyl butoxide spectrum similar to that produced by ethyl isocyanide. Life Sci. 10 (Part II), 503-512. Philpot R. M. and Hodgson E. (1971/2) Differences in the cytochrome P-450s from resistant and susceptible houseflies. Chem. biol. Interact. 4, 399-408. Schwen R. J. and Mannering G. J. (1982) Hepatic cytochrome P-450-dependent monooxygenase systems on the trout, frog and snake---I. Components. Comp. Biochem. Physiol. 71B, 431-436. Shenkman J. B., Remmer H, and Estabrook R. W. (1967) Spectral studies of drug interaction with hepatic microsomal cytochrome. Molec. Pharmac. 3, 113-123. Shorey H. H. and Hale R. L. (1965) Mass rearing of the larvae of nine noctuid species on a single artificial medium. J. econ. Ent. 58, 522-524.
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Tate L. G., Plapp F. W. and Hodgson E. (1973) Cytochrome P-450 difference spectra of microsomes from several insecticide-resistant and susceptible strains of the housefly. Musca domestica. Chem. biol. Interact. 6, 237-47. Tate L. G., Nakat S. S. and Hodgson E. (1982) Comparison of detoxication activity in midgut and fatbody during fifth instar development of the tobacco hornworm, Manduca sexta. Comp. Biochem. Physiol. 72C, 75-81. Williamson R. L. and Schechter M, S. (1970) Microsomal epoxidation of aldrin in lepidopterous larvae. Biochem. Pharmac. 19, 1719-1727. Yu S. J. and Terriere L. C. (1979) Cytochrome P-450 in insects. 1. Differences in the forms present in insecticide resistant and susceptible houseflies. Pestic. Biochem. Physiol. 9, 237-246. Yu S. J., Berry R. E. and Terriere L. C. (1979) Host plant stimulation of detoxifying enzymes in a phytophagous insect. Pestic. Biochem. Physiol. 12, 280-284.
0305-0491/84 $3.00 + 0.00 © t984 Pergamon Press Ltd
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THE MICROSOMAL MIXED-FUNCTION OXIDASE SYSTEM OF H E L I O T H I S P U N C T I G E R WALLENGREN AND H. A R M I G E R (HUBNER) (LEPIDOPTERA: NOCTUIDAE) P. J. COLLINS* and G. H. S. HOOVER Department of Entomology, University of Queensland, St. Lucia. Q. 4067, Australia
(Received 25 August 1983) Abstract--1. The levels of cytochrome P-450, NADPH cytochrome c reductase, cytochrome b5and aldrin
epoxidation revealed quantitative differences between H. armiger and H. punctiger and among the tissues (midgut, fatbody and integument) of each species. 2. Qualitative differences between the species and among the tissues was indicated by P-450 spectral binding studies. Type I (substrate) binding occurred, in both species, only with midgut microsomes. Type II (ligand) binding occurred with n-octylamine in all tissues and with pyridine in only midgut and fatbody microsomes. Piperonyl butoxide added to reduced midgut microsomes illicited a type III spectrum. There was no reaction with either fatbody or integument microsomes. 3. n-Octylamine difference spectra also revealed the presence of high-spin P-450 in the midgut microsomes of both species. Fatbody and integument P-450 were predominantly in the low-spin form.
INTRODUCTION
In insects the microsomal mixed-function oxidases (MFO) comprise the most important system in the primary degradation of foreign compounds (Dauterman and Hodgson, 1978). Consequently, these enzymes play a central role in the metabolism of insecticides (Nakatsugawa and Morelli, 1976). Despite their significance, the major components of the M F O system (cytochrome P-450, cytochrome b 5 and N A D P H cytochrome c reductase) have been studied in only a few insect species. Cytochrome b5 and N A D P H cytochrome c reductase have been described from microsomes of the southern armyworm, Spodoptera eridania Cramer (Lepidoptera:Noctuidae) (McFadden et al., 1979; Crankshaw et al., 1981a) while the majority of studies of P-450 have been conducted on the housefly, Musca domestica L. (Kulkarni and Hodgson, 1976, 1980; Yu and Terriere, 1979). In addition, although these enzymes are well known from midguts of lepidopteran larvae (Brattsten, 1979) and housefly abdomens there have been few comparative studies of this system in other tissues (Brattsten et al., 1980; Tate et al., 1982). As the contribution of each tissue varies considerably among species there is a need not only to extend our knowledge of M F O ' s to new species but also to compare the activities of these enzymes in various tissues within species. This especially applies to pest species in which knowledge of detoxification systems is critical to the management of insecticide resistance. The cotton bollworm, H. arrniger and the native budworm, H. punctiger are appropriate candidates for this type of study as they are widespread and significant pests of field crops in Australia, and insecticides are still the major control method used against these species. *Present address: Entomology Branch, Department of Primary Industries, Meiers Road, Indooroopilly, Q. 4068, Australia.
In this investigation we have determined the concentration of the components of the mixed-function oxidase system and the binding spectra of cytochrome P-450 in midgut, fatbody and integument microsomes from H. armiger and H. punctiger. Aldrin epoxidase was used as a measure of overall activity. MATERIALS AND M E T H O D S
Insects
The H. punctiger culture was established from a laboratory strain maintained by the Queensland Department of Primary Industries. This strain was initiated from moths caught in 1972 at Redland Bay, in south-east Queensland. H. armiger was established from moths caught in the Gatton district, Queensland, in 1981. Heliothis armiger and H. punctiger larvae were reared on an artificial diet based on navy beans and wheat germ plus added vitamins and preservatives (Shorey and Hale, 1965). Adults and larvae were kept at 28 4- I°C and 70~o r.h. in a 12 hr light: 12 hr dark regime.
Chemicals Cytochrome c, NADPH, NADP, glucose-6-phosphate (G-6-P), giucose-6-phosphate dehydrogenase and bovine serum albumin (BSA) were purchased from either Sigma Chemical Co., USA or Boehringer-Mannheim Australia Pty., Sydney, Australia. n-Octylamine (> 99~o) was bought from Fluka AG, Buchs, Switzerland and pyridine (99.09/o) from Ajax Chemicals, Sydney, Australia. HHDN (aldrin) and HEOD (dieldrin), both >99.0~ pure, were a gift from Shell Research Limited, Sittingbourne, Kent, U.K. Carbaryl was synthesised from l-naphthol (Krishna et al., 1962), and DDT and permethrin had a purity of 95 and > 99~o respectively. Tissue preparation Last instar larvae, 24-36 hr since moulting, were used as the source of midgut and fatbody tissues and newly moulted, 0-6 hr, last instar larvae were the source of integuments. The midgut was dissected from the larva and the gut contents removed. Midguts were then rinsed and
849
850
P.J. COLLINS and G. H. S. HOOPER
placed in appropriate volume of ice-cold phosphate (NaH2PO 4 . 2H20/N2HP04) buffer pH 7.6 plus 1.15~o KCI. Once the midgut had been removed the fatbody could then be scraped away from the integumental tissue. The fatbody was placed directly into ice-cold buffer, as was the integument after being rinsed in buffer to remove any attached fatbody. The tissues, in ice-cold phosphate buffer, were homogenised in a ground-glass tube with either a motor-driven glass (for integument) or Teflon (for all other tissues) pestle. To prepare microsomal fractions the crude homogenate was immediately filtered through glass wool and spun in a Beckman L5~40 ultracentrifuge (at 0'C) at 10,000g average for 15 rain. The resulting supernatant was then centrifuged at 100,000g for 60 min. The pellet from this centrifugation was then resuspended in an appropriate volume of the standard phosphate buffer.
A[drin epoxidase Aldrin epoxidase activity was determined by gas-liquid chromatography using the epoxidation of aldrin to dieldrin (Krieger and Wilkinson, 1969). Incubations were carried out aerobically in 25 ml Erlenmeyer flasks and shaken in a water bath at 30C. Reaction time was 10 rain. The standard 5 ml incubation medium contained G-6-P (2.4mM). NADP (51 #m), phosphate (NaH2PO 4. 2HzO/NazHPO4) buffer pH 7.6, PTU (0.4mM) and KC1 (1.15,°,o). The reaction was initiated by the addition of 8 ~g aldrin in 20/~1 ethanol. The reaction was terminated with 1 ml of 1 M HC1 and aldrin and dieldrin were extracted with toluene after the method of Konrad et al. (1969). The amount of aldrin and dieldrin was quantified using a Packard Model 427 GLC fitted with an electron capture detector with a Ni63 radioactive source of 10 mCi, Pure N: was used as the carrier gas through a 1 m glass column (i.d. 2 mm, o.d. 6 mm) packed with 5~o silicone SE30 on Chromsorb WHP 100/12-mesh. A Shimadzu CRIA chromatopac recording data processor was used to quantify samples.
lished before the addition of any substrate or ligand to the microsomes. Solid samples (such as insecticides) were added directly to the sample cuvette which was inverted approx. 20 times and then allowed to equilibrate for a few minutes before scanning. Liquid samples were added directly to the sample cuvette in 10 ~tl of standard phosphate buffer and the sample cuvette was treated as for solids. An equivalent volume of buffer was added to the reference cuvette. These procedures were repeated until no further increase in spectral size occurred. Type I (substrate) and type It (ligand) binding spectra were classified according to Schenkman et al. (1967) and type III after Philpot and Hodgson (1971). n-Octylamine type II difference spectra were used to calculate o~, high- and low-spin P-450 using the method of Jefcoate et al. (1969). Type I binding was determined using the methylenedioxyphenyl synergist piperonyl butoxide and the insecticides DDT, carbaryl and permethrin. Type II binding was determined with pyridine and n-octylamine. Type IIl spectra were determined using piperonyl butoxide with reduced microsomes.
Protein determinations Crude homogenate protein was assayed using the biuret method (Layne, 1957) and diluted according to the activity of the particular tissue. The Bradford method (Bradford, 1976) was used to measure microsomal protein concentration. Bovine serum albumin was used as the standard for both assays,
Statistical analysis Aldrin epoxidase assays were replicated 4 times and data are reported as the mean + standard deviation. Spectral assays were replicated 4-6 times with fresh microsomes and data are reported as the mean + standard deviation. Spectral binding studies were each replicated 3 times. Students t-test was used to analyse the data. The enzyme source for each replicate was derived from a sample of 15-20 insects.
NADPH-cytochrome c reductase NADPH-cytochrome c reductase activity was measured at room temperature directly as the increase in the ~-band of reduced cytochrome c with time (Masters et al., 1965). The incubation mixture consisted of 0.8ml phosphate buffer (pH 7.6), 0.2 ml cytoehrome c solution (4.4 mg/ml in phosphate buffer) and 30-100 #g of microsomal protein was added in 50 #1 of phosphate buffer. Reactions were initiated by the addition of 20 #1 of NADPH (0.2 raM). Reductase activity was calculated from the initial change in absorbance at 550 nm using the millimolar extinction co-efficient of 21.1 cm i (Massey, 1959).
RESULTS
Aldrin epoxidase activity In b o t h species midgut specific activity was highest, followed by the fatbody and integument (Table 1). With all three tissues enzyme activity was apparently higher in H. armiger than in the respective tissues of H. punctiger, although only in fatbody and integument assays were the values for H. armiger significantly (P < 0.05) higher than for H. punctiger.
Cytoehrome P-450 Cytochrome b~ Cytochrome b 5 was measured by difference spectroscopy using the method of Omura and Sato (1964). Solid sodium dithionite was used to reduce the cytochrome b5 in the sample cuvette and the reduced minus oxidized difference spectrum was obtained. An extinction co-efficient of 1851/cm/mM was used for the change in absorbance between 426 and 409 nm. Microsomal protein concentration was 1.5 + 1 mg/ml.
Spectral maxima were at 450 nm for all tissues of both species. The level o f cytochrome P-450 from integument microsomes was about equal in the two species. F a t b o d y cytochrome P-450 from H. armiger was about 25% higher than that obtained from H. punctiger and with midgut microsomes H. armiger contained a b o u t 56% more P-450 than H. punctiger (Table 1).
Qvtochrome P-450
Cytochrome b5
Cytochrome P-450 was determined from the dithionitereduced CO difference spectrum according to the method of Omura and Sato (1964) using a molar extinction co-efficient of 91 1/cm/mM. Microsomal protein concentration was 1.5 _+ 1 mg/ml standard phosphate buffer.
The level o f cvtochrome b, (Table 1~ was much greater in midgut microsomes than that from either fatbody or integument. This was the case for both H. armiger and H. punctiger. In contrast to the relative amounts of cytochrome P-450 present in each tissue, there was more b 5 in integument than in fatbody microsomes. Integument microsomes from H. armiger contained about three-times the concentration o f cyto-
Spectral binding studies Oxidized cytochrome P-450 difference spectra were determined between 490 and 350 nm using a Varian Series 634 double beam spectrophotometer. A zero-baseline was estab-
MFO in Heliothis larvae
851
Table 1. Properties of the mixed-functionoxidase system of tissues from last instar larvae of Heliothis punctiger and H. armiger Species
MIDGUT
FATBODY
INTEGUMENT
Aldrin epoxidase specific activity , . . . . T nmol d l e l d n n produced/mg proteln/mxn
H. armiger
0.446 ~ 0.059
0.162 L 0.007
0.075 L 0.001
H. punctiger
0.424 ~ 0.057
0.108 ~ 0.015"
0.065 ~ 0.011"
nmol cytochrome c reduced/rain/mg protein H. armiger
51.33 ~ 2.55
H. punctiger
40.16 ~ 1.32"
17.26 ~ 7.5
5.84 ~ 0.30*
23.6
t
~ 3.7
15.98 ~ 1.4
Cytochrome b 5 content nmol/mg p r o t e i n
H. armiger
87.05 ~ 3.9
5.43 ~ 1.2
H. punctiger
62.90 L 5.3*
4.84 ~ 0 . i 0
21.0
~ 2.4
7.53 L 0.85*
Cytochrome P-450 conte~t nmol P-450/mg protein ~
H. armiger
0.403 + 0.012
0.150 + 0.038
0.058 + 0.011
H. punctiger
0.258 + 0.006*
0.120 + 0.013
0.057 + 0.011
*Significantly different (P < 0.05) from value for H. armiger. %Values are mean_4-SD, n = 4. :~Values are mean+ SD, n = 3. §Values are mean___SD, n = 4 or more separate experiments.
chrome bs as microsomes from H. punctiger and there was about 27~o more bdmg protein in H. armiger midgut microsomes than those from H. punctiger. There was no difference in b5 content of fatbody microsomes between the two species. The absorption maximum for all three tissues in both species was at 426 nm when reduced with sodium dithionite.
NADPH-cytochrome c (P-450) reductase Levels of N A D P H cytochrome c reductase activity followed a similar pattern to that of cytochrome bs. With both species midguts had the highest activity followed by integuments and then fatbody microsomes (Table 1). For midgut and fatbody microsomes H. armiger had significantly (P < 0.05) more activity than the respective tissue from H. punctiger. There was no significant difference between the integument values of the two species.
Binding spectra of cytochrome P-450 Both species exhibited a similar pattern of response to the addition of various compounds to the microsomal suspension. Type I spectra were elicited by the three insecticides and the synergist piperonyl butoxide with midgut microsomes. These spectra had the characteristic (type I) peak at 390 nm and a trough between 415 and 420 nm. Type I spectra could not be detected with fatbody or integument microsomes.
The extent of type I binding (Table 2) was greatest with piperonyl butoxide in both species but particularly with H. punctiger when compared with the compounds tested. The two species had about the same affinity for carbaryl and D D T but there was more than 50~o greater binding with permethrin using H. armiger microsomes than those from H. punctiger. Pyridine gave rise to a type 1I spectrum with midgut and fatbody microsomes in both species but there was no reaction with integument microsomes. With H. punctiger midgut and fatbody microsomes pyridine addition produced characteristic type II spectra with a trough at 395nm and a peak at 425 nm. The fatbody A absorbance was about half that of the midgut. On the other hand, the spectra produced by tissues from H. armiger, although still typical II spectra, differed from the pattern shown by H. punctiger with a larger A absorbance and a peak at 427 nm and a trough at 398 nm. Typical type II spectra were also formed when n-octylamine was added to midgut, fatbody and integument microsomes from both species. H. armiger and H. punctiger fatbody and integument spectra had peaks at 430-432 nm and a single, though broad, trough at 410-416 nm. In contrast, midgut n-octylamine spectra from both species revealed a trough with two distinct minima at 410 and 392 nm. With all three tissues spectra formed with microsomes from H.
852
P, J. COLLINSand G. H. S. HOOPER Table 2. Type I (substrate) binding of microsomal cytochrome P-450 from midguts of H. armiger and H. punctiger
Compound
H. armiger
H. punctiger
carbaryl
0.010 + 0.002
0.012 + 0.002
DDT
0.016 + 0.002
0.013 + 0.002
permethrin
0.037 + 0.007
0.015 + 0.003
piperonyl butoxide
0.045 + 0.004
0.065 + 0.003
Values are the mean +_ SD. n = 3 or more separate experiments. Type I binding = A absorbance/nmol P-450. A Absorbance was measured between peak (ca 390 nm) and trough (ca 415 nm).
armiger were larger than the corresponding spectra from H. punctiger. There were large differences among the various tissues of the two Heliothis species in the extent of binding to the type II compounds (Table 3). Midgut microsomes from H. punctiger and H. armiger demonstrated about the same binding capacity for pyridine but their affinity for n-octylamine differed, i.e. H. armiger type II binding was about 30% greater than that for H. punctiger. In contrast, H. armiger fatbody had a greater affinity for pyridine than that from H. punctiger, although the reverse was the case with n-octylamine. Integument microsomes from both species bound comparatively extensively (especially H. armiger) with n-octylamine. The percentage of high-spin P-450 in midgut microsomes of the two species was about the same. There was little high-spin P-450 in the other tissues assayed (Table 4). When piperonyl butoxide was added to reduced midgut microsomes a spectrum with two peaks (type III) occurred. There was no reaction with fatbody or integument reduced microsomes. With midgut spectra in both species the larger peak had a maximum at 427 nm, H. punctiger having the higher A absorbance (Table 5). The second peak had a maximum at 455 nm with H. armiger and 458 nm with H. punc-
tiger. The 455/427 nm peak height ratio was higher in H. armiger than H. punctiger. Ratios of 0.698 for the 427 nm peak and 1.143 for the 455 nm (approx.) peak are obtained when the magnitude of the peaks of the two species are compared (H. armiger/H, punctiger). Also, when the 445 nm peak of each spectrum was compared to the respective A absorbance for the CO-reduced P-450 spectrum, it was apparent that the magnitude of this peak was lower in H. armiger than H. punctiger (Table 5). DISCUSSION
The results reveal that the midgut, fatbody and integument tissues of Heliothis punctiger and H. armiger contain active microsomal mixed-function oxidase systems. Further, these systems can be characterised both quantitatively and qualitatively. The midgut aldrin epoxidase specific activity of both species, although relatively high are within the range reported for other noctuid larvae (Krieger and Wilkinson, 1969; Williamson and Schechter, 1970; Chandran and Khan, 1972; Gould and Hodgson, 1980; Farnsworth et al., 1981). in contrast, fatbody epoxidase activity was high compared to the limited published data (Brattsten et al., 1980; Tate et al.,
Table 3. Type II (ligand) binding of microsomal cytochrome P-450 from various tissues of H. armiger and H. punctiger
H. armiger midgut
fatbody
integument
pyridine
0.037 + 0.002
0.060 + 0.003
NR +
n-octylamine
0.046 + 0.003
0.046 + 0.010
0.149 + 0.013
H. punctiger midgut
fatbody
integument
pyridine
0.038 ~ 0.004
0.035 ~ 0.006
NR +
n-octylamine
0.032 + 0.011
0.078 + 0.012
0.070 + 0.005
Values are mean _+ SD, n = 3 or more separate experiments. Type II binding = A absorbance/nmol P-450. A Absorbance was measured between peak (ca 430 nm) and trough (410-415 nm). ?No reaction.
853
MFO in Heliothis larvae Table 4. n-Octylamine difference spectra of oxidized microsomes from tissues of last instar H. armiger and H. punctiger
Tissue
Amax.
Amin.
~A(410-500nm) AA(392-500nm)
% high-spin P-450 #
H. armiger
midgut fatbody integument
430 430 432
392/410 412 416
1.0 ~ 0.05 t 5.0 + 1.00 6.0 ~ 0.50
16
H. punctiger
midgut fatbody integument
432 430 432
392 410 415
0.94 + 0.06 3.00 ~ 0.50 3.25 + 1.06
17
tCalcalated from the standard curve of Jefcoate et al. (1970). +Values are mean_ SD, n = 3 or more separate experiments. 1982). Reports of integument epoxidase activity are not available for comparison. Cytochrome b5 concentration of midgut microsomes from both Heliothis species are close to the level reported by Tate et al. (1982) for M . sexta. These authors, however, found about three-times more b5 in fatbody microsomes than occurred in H. punctiger and H. armiger. Levels of midgut N A D P H cytochrome c reductase are within the range reported for other noctuid species (Crankshaw et al., 1981b). However, Tate et al. (1982) report about a 100-fold lower level of reductase activity in fatbody microsomes from M . sexta than we found with H. armiger and H. punetiger. It is interesting that unlike the levels of aldrin epoxidase and cytochrome P-450, in both Heliothis species, the amount of cytochrome b5 and cytochrome c reductase was higher in integument microsomes than fatbody microsomes (Table 1). This may be a reflection of different functions of the microsomal enzymes in these tissues.
Cytochrome P-450 concentration in midgut microsomes from H, punctiger is comparable to levels of 0.032-0.29 nmol/mg protein reported for other lepidopteran species (Yu et al., 1979; Brattsten et al., 1980; Farnsworth et al., 1982), while that for H. armiger is relatively high. Fatbody P-450 concentrations of H. armiger and H. punctiger were higher than those reported for S. eridania (Brattsten et al., 1980) and M. sexta (Tate et al., 1982). There have been no studies published of larval integument P-450 from the Lepidoptera or any other insects for comparison. The cytochrome P-450 spectral binding patterns reveal qualitative differences between the species and among the tissues of each species, The lack of type I binding in either fatbody or integument microsomes is a fundamental difference between these tissues and the midgut. Fatbody and integument hemoprotein were also distinguished by the latter's apparent lack of type I! binding with pyridine. Although these
Table 5. Type III binding of cytochrome P-450 from midgut microsomes of H. armiger and H. punctiger with piperonyl butoxide determined at pH 7.6 Peak
Species
H. armiger
H. punct~ger
max.
Aabsorbance t
455
0.004 ~ 0.001
427
0.037 + 0.007
458
0.00S ~ 0.001
427
0.053 + 0.004
Peak height r a t i o s 455#
455§
427
CO P-450
0.108
0.125
0.066
0.130
tReference wavelength: 490 nm. Values are mean _+SD, n - 3 separate experiments. :~Approx. §Magnitude of type III piperonyl butoxide 455 nm peak relative to CO-reduced P-450 A absorbance value.
854
P.J. COLt,lYS and G. H. S. Hooper
results seem to indicate important differences between the hemoprotein species in each tissue, it is also possible that type I and type lI binding did not occur in some cases because the added compounds may not be able to compete with endogenous substrates present in the microsomal suspension. It is also possible that spectra formed but were too small to detect. Midgut microsomes contained a significant amount of high-spin P-450 while microsomes from the other tissues were composed predominantly of low-spin hemoprotein. In houseflies the presence of high-spin state hemoprotein has been associated with an increase in epoxidase activity (Yu and Terriere, 1979), In this investigation presence of high-spin P-450 was associated with not only higher aldrin epoxidase activity but also the presence of type I spectral binding and higher P-450 concentration. Where binding occurred there were also distinctive responses to various compounds in the two Heliothis species. The difference in type I binding to permethrin and D D T in midgut microsomes (Table 2) suggests that H. armiger might more readily metabolise insecticides than H. punctiger and may therefore have a greater potential for developing resistance to insecticides. Higher aldrin epoxidase and cytochrome P-450 specific activities of H. armiger midgut microsomes than those from H. punctiger support this proposition. The piperonyl butoxide type III spectrum formed with reduced P-450 was also elicited only with midgut microsomes. The 455/427 peak height ratio and the 455/CO P-450 ratio have been used to qualify cytochromes P-450 from various strains of housefly. Capdevila et al. (1974) and Tate et al. (1973) demonstrated that at pH 7.5 the 455 nm peak was reduced in insecticide resistant strains while in susceptible strains it was about equal in magnitude to the CO reduced P-450 peak. Also the 455/430 ratio of type Ill piperonyl butoxide spectrum was lower in R- than S-strain microsomes (Philpot and Hodgson, 1971/2). The piperonyl butoxide type III spectra for H. punctiger and H. armiger (Table 5) midgut microsomes varied particularly in the 455/427 peak height ratio. There was little difference between the 455/CO P-450 ratios of the two species, however. The former may be indicative of qualitative differences in the nature of the P-450 cytochromes in the two Heliothis species. It has been shown in mammals with ethylisocyanide that the ratio of the 455/430 nm peaks increases with increased hydrophobicity of the cytochrome P-450 (Schwen and Mannering, 1982). This is further evidence that the hemoprotein from H. armiger is more capable of xenobiotic metabolism than that from H. punctiger. REFERENCES
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