Evidence for a sex pheromone metabolizing cytochrome P-450 mono-oxygenase in the housefly.код для вставкиСкачать
Archives of Insect Biochemistry and Physiology 6:121-140 (1987) Evidence for a Sex Pheromone Metabolizing Cytochrome P-450 Mono-Oxygenase in the Housefly Sami Ahmad, Kenneth E. Kirkland, and Gary J. Blomquist Department of Biochemistry, University of Nevada, Reno , Direct evidence is presented for the role of a cytochrome P-450 monooxygenase (called mixed-function oxidase, or polysu bstrate mono-oxygenase, PSMO) in the metabolism of the sex pheromone (Z)-9-tricosene t o its corresponding epoxide and ketone in the housefly. A secondary alcohol, most likely an intermediate i n the conversion of the alkene t o the ketone, was also tentatively identified. The results of in vivo and in vitro experiments showed that the PSMO inhibitors, piperonyl butoxide (PB) and carbon monoxide, markedly inhibited the formation of epoxide and ketone from (9,10-3H)(Z)-9-tricosene.An examination of the relative rates of (a-9-tricosene metabolism showed that males exhibited a higher rate of metabolism than females with the antennae of males showing the highest activity of any tissue/ organ examined. The major product from all tissueslorgans was the epoxide. Data from experiments with subcellular fractions showed that the microsomal fraction had the majority of enzyme activity, which was strongly inhibited by PB and CO and required NADPH and O2 for activity. A carbon monoxide difference spectrum with reduced cytochrome showed maximal absorbance at 450 n m and allowed quantification of the cytochrome P-450 in the microsomal fraction of 0.410-nmol cytochrome P-450 m g - I protein. Interaction of (Z)-9-tricosene with the cytochrome P-450 resulted in a type I spectrum, indicating that the pheromone binds t o a hydrophobic site adjacent to the heme moiety of the oxidized cytochrome P-450. Key words: epoxidation, hydroxylation, Musca domestica, polysubstrate mono-oxygenase, (2)-9-tricosene INTRODUCTION The sex pheromone of the housefly, Muscu domesticu, consists of (3-9tricosene (CZ3alkene) [l]and related oxygenated compounds (Z)-9,lO-epoxAcknowledgments: This work was supported in part by National Science Foundation grant DCB-8416558 and i s a contribution of the Nevada Agricultural Experimental Station. We thank the Biology Section, S.C. Johnson and Sons, Racine, WI, for supplying housefly pupae. Received January29,1987; accepted May 26,1987. Address reprint requests to S. Ahmad, Department of Biochemistry, University of Nevada, Reno, NV 89557-0014. 0 1987 Alan R. Liss, Inc. 122 Ahmad, Kirkland, and Blomquist ytricosane (C23 epoxide) and (Z)-14-tricosen-lO-one (C23 ketone) , along with a series of C28-30 methylalkanes . Recently, the precise role for each component of the sex pheromone was established . Accordingly, (4-9tricosene is responsible for initiating the courtship ritual and especially inducing ”striking activity” in males. The C23 epoxide and ketone are ”sex recognition” factors, whereas the methylalkanes act as ”arrestants,” and promote and extend sexual contact and mating. The precursors and enzymatic pathways in the biosynthesis of the hydrocarbon components [(.Z)-9-tricoseneand methylalkanes] of the pheromone have been elucidated . It was demonstrated that the oxygenated components, C23 epoxide and C23ketone, are derived from (Z)-9-tricosene , and also hypothesized that these two components were products of oxidative attack by a “mixed function oxidase type enzyme,” also called a polysubstrate mono-oxygenase. This suggestion prompted a series of experiments on the in vivo and in vitro metabolism of (4-9-tricosene involving cytochrome P-450 mono-oxygenase inhibitors, piperonyl butoxide and carbon monoxide. The results, reported here, show that both C23 epoxide and C23 ketone are products of a microsomal cytochrome P-450 attack on (2J-9-tricosene. Aside from the well-established role of cytochrome P-450 in metabolism/ deactivation of foreign substances in organisms, these enzymes are further adapted for participation in the biosynthesis and regulation of substances physiologically important to insects [7,8]. This is the first report providing direct evidence for a role of the mono-oxygenase in pheromone synthesis and deactivation. MATERIALS AND METHODS Insects The housefly, Fales 1958 strain T-11, was supplied by the Biology Section, S.C. Johnson and Sons (Racine, WI). Within 12 h of emergence from the pupae, males and females were separated and held at 25 5 1”C, ca. 60-80% relative humidity, and provided ad libitum sucrose and low-fat powdered milk (l:l,wlw) and water. Chemicals [9,10-3H](Z)-9-Tricosene (60 Cilmmol) was prepared and purified as described earlier  and dissolved in either hexane or ethanol. The radiochemical purity of (4-9-tricosene was found to be 99+% by radio-GLC* . The use of high specific activity [3H](Z)-9-tricosenehas the advantage of reflecting the low amounts presumably transferred to males from females in courtship and mating. In addition, since females produce (Z)-9-tricosene, the addition *Abbreviations: BSA = bovine serum albumin (fraction V); HC = hydrocarbon; PB = piperonyl butoxide; PMSO = polysubstrate monooxygenase; NHC = nonhydrocarbon; radio-CLC = radio-gas-liquid chromatography; TLC = thin-layer chromatography. Pheromone Metabolismby Cytochrome P-450 123 of tracer amounts of labeled material does not significantly affect endogenous amounts on the insect. Unlabeled Q-9-tricosene was purchased from Sigma Chemical Co., St. Louis, MO . The (Z)-9,10-epoxytricosane,tricosan-12-one, and tricosan-12-01 were prepared according to the procedures given by Blomquist and Jackson  and Blomquist et al . The ketone and secondary alcohol were used for TLC standards, where they comigrated with (Z)-14-tricosen-lO-one and (414-tricosen-10-01. Glucose-6-phosphate, glucose-6-phosphate dehydrogenase (450 units mg-' protein), Tris buffer, sodium dithionite, and BSA were obtained from Sigma Chemical Co. Piperonyl butoxide was purchased from Chem Service, Inc., West Chester, PA. Coomassie brilliant blue G-250 was obtained from Bio-Rad Laboratories, Richmond, CA. All other chemicals used were of highest purity available (98+%), and all reagent grade solvents were redistilled prior to use. In Vivo Assays Three-day-old unmated male and female flies were seperated into two batches; one batch of each sex was pretreated topically with 5 p g of PB dissolved in 1pl acetone to the fly dorsum, the other with 1p1 acetone. Equal batches of PB-treated and solvent control flies were held in glass beakers covered with glass petri dishes for 30 min. Flies from both batches were then treated with 1p1 of [9,10-3H]((Z)-9-tricosenesolution in hexane. Another batch of flies treated with labeled(Z)-9-tricosenewere held under a gentle stream of CO:N2:02 (60:20:20) 20 cclmin. Each fly received ca. 100,000 dpm of [3H](Z)-9-tricosene.The flies were transferred into 10-ml glass vials chilled over dry ice 30 and 60 min after the treatment with the labeled material. Two ml of hexane was immediately added (2 ml) and the glass containers were capped and stored at -20°C for later extraction. Each replicate consisted of ten flies, and there were three to four replicates to each treatment group. Organ Tissue Assays Male and female flies were dissected in isotonic 1.15%KC1 solution chilled to ca. 4°C. The following organsltissues from eight flies were collected: abdominal and thoracic epithelia including the cuticle, antennae, fat bodies, and legs. Tissues from eight flies comprised a replicate; the experiment was duplicated. Each organltissue replicate was placed in 0.5 ml of the isotonic KCl solution. The abdominal and thoracic epithelia were placed with the cell layer facing down and in contact with the KC1 solution; the cuticle was dorsally exposed. Approximately 1,000,000 dpm of [3H](Z)-9-tricosenein 4 pl ethanolic solution was carefully spread over the upper surface of the pooled tissues. The tissues were then equilibrated at 30°C in a shaker bath with minimal agitation. After 5 min, 1.5 ml of isotonic KC1 and Tris buffer, 50 mM, pH 7.5, was added. After 15 min, the reaction was terminated by adding 6 ml of a methanol-chloroform (2:l) mixture and the samples were capped and stored at -20°C. In some tissuelorgan incubations, the incubates were treated with 5 pgl replicate PB (in 1 p1 of ethanol) 10 min prior to starting the assay by the 124 Ahmad, Kirkland, and Blomquist addition of the labeled substrate. Controls were pretreated with 1 pl of ethanol. Subcellular Assays Subcellular fractions containing mitochondria and microsomes were prepared by the following procedure. From 3-day-old unmated male and female houseflies, 200 intact abdomens, and, in a separate experiment, 200 abdomens with guts removed, were ground in a glass homogenizer with a Teflon pestle. The homogenization medium was 20 ml of cold (2-4°C) 50 mM Tris buffer (pH 7.5) containing 1.15" (wh) KC1. The homogenate was immediately fractionated into subcellular components by differential centrifugation. The 750 g (for 15 min) precipitate was discarded. The 750 g supernatant was centrifuged at 10,800 g for 20 min at 2°C to pellet the mitochondrial fraction. The supernatant was centrifuged at 100,900 g for 1 h at 1°C to obtain the microsomal pellet. The mitochondrial and microsomal fractions were washed by resuspension in the homogenization buffer and recentrifuged to minimize cross contamination. Protein concentration was determined by the Bio-Rad Coomassie blue method using BSA as standard. Both mitochondrial and microsomal fractions were adjusted to 2 mg protein ml-I with the Tris buffer-KC1 solution. An incubation volume of 2 ml contained 100 pmol Tris Buffer, pH 7.5, 24.4 pmol KCI, 4.8 pmol NADP, 4.8 pmol glucose-6-phosphate, 0.48 units of glucose-6-phosphate dehydrogenase, and 2 mg of mitochondrial or microsoma1 protein. The reaction was started by adding ca. 1,000,000 dpm of [3H(Z)9-tricosene in 4 pl of ethanol. Incubations were carried out aerobically for 20 min with agitation in a shaker water bath at 30°C. The reaction was terminated by adding 6 ml of methanol-chloroform (2:1), and immediately stored at -20°C. In some samples, 5 pg of piperonyl butoxide in 1pl of ethanol was added 10 min prior to the addition of the labeled substrate, whereas 1 pl methanol was added to controls. Each experiment was duplicated. In another experiment using microsomal preparations from whole abdomens, instead of the NADPH-generating system, varying amounts of NADPH were directly added and incubations were carried out aerobically under continuous gassing with water-saturated N2:02 (80:20). To demonstrate oxygen dependence, additional incubations were performed under water-saturated 100% N2. The inhibition by CO was demonstrated by continuously gassing the incubation mixtures with water-saturated C0:02 (80:20), whereas controls were gassed with N2:02(80:20). Extraction, Partitioning, and Clean-Up of Metabolites Insects from in vivo experiments were extracted by immersion in 2 ml of hexane for 10 min, and then rinsed twice with 2 ml of hexane for 1min. The rinses were combined, the volume reduced under N2, and one-tenth aliquots were used for assaying radioactivity. Rinsed insects from the in vivo experiment, incubated organsltissues, and subcellular assays were extracted by the method of Bligh and Dyer [lo]. Phase separation was facilitated by the addition of ca. 50 mg anhydrous Pheromone Metabolism by Cytochrome P-450 125 Na2S04 and by refrigeration overnight at 2°C. The aqueous and the organic phases were each reduced in volume under a stream of N2, the organic phase to 1ml and the aqueous phase to 5 ml, and one-tenth aliquots of each were used for determination of radioactivity. The organic phase was separated into HC and NHC fractions on 7 cm by 0.5-cm columns packed with activated Biosil A. The HCs were eluted with 7 ml of hexane. The NHCs were eluted with 8 ml diethyl ether-hexane: 3:2. Each fraction was reduced in volume under N2 and reconstituted to 1ml; one-tenth aliquots of each sample were used to assay radioactivity. Metabolite Separation and Identification Radio-GLC was performed as described by Blomquist et al. . The nonhydrocarbons (diethyl ether-hexane fractions) were separated by TLC developed in hexane-diethyl ether (85:15). With the use of standards, the zones corresponding to the epoxide, ketone, and secondary alcohol metabolites were ascertained, and, moreover, additional metabolites were detected by sequential cm-by-cm scan of the TLC plates for radioactivity. The Rfs of the identified metabolites and of unknowns were: (Z)-14-tricosen-lO-one (0.89Ifr 0.01), (Z)-9,10-epoxytricosane (0.85~frO.Ol), unknown 3 (0.61f0.032), (Z)-14-tricosen-l0-o1(0.461t0.03), unknown 2 (0.35+0.02), and unknown 1 (0.10 Ifr 0.01) 1 Assay for Radioactivity Radioactivity was determined by assaying sample aliquots by liquid scintillation spectrometry at 45-50% efficiency. All data were corrected for background. Aliquots of organic phases were assayed by placing in 10 ml of a toluene-based (PPO, 0.4%, wlv) scintillation cocktail and aqueous samples were assayed in 10 ml Formula 963 Scintillation cocktail (NEN Research Products, Boston, MA). Cytochrome P-450 Spectral Analysis The CO-difference (of the reduced cytochrome P-450-carbon monoxide complex) spectrum was obtained essentially as described by Omura and Sat0 [ll]. The dithionite used in reducing the cytochrome P-450 often converts it to substantial amounts of the inactive form, cytochrome P-420, also accompanied by a reduction in maximum absorbance of cytochrome P-450 at 450 nm from an erroneously apparent wavelength shift, thus resulting in incorrect estimation of the cytochrome content . Microsomes were therefore reconstituted in 50 mM Tris buffer, pH 7.75, containing 1mM EDTA and 20% (vh) glycerol. This procedure minimized cytochrome P-450 to cytochrome P420 conversion. The amount of cytochrome P-450 was determined using the extinction coefficient, 91 mM-' cm-', for the absorbance between 450 and 490 nm, and the cytochrome P-420 was also measured using the extinction coefficient, 110 mM-l cm-' at 420 nm [ll]. An Aminco DW-2 spectrophotometer in the split beam mode was used to measure the absorbances of the reduced CO-cytochromes P-450 and P-420 difference spectra. 126 Ahmad, Kirkland, and Blomquist A difference spectrum of (Z)-9-tricosene binding to the oxidized cytochrome P-450 was obtained. The ligand was added gradually to the oxidized cytochrome preparation as a 25 nm solution in acetone. An equal volume of acetone was added to the reference cuvette. A difference spectrum appeared when the ligand concentration reached 25 pM. Spectra were recorded for microsomes from both male and female flies. RESULTS In Vivo Metabolism of (3-9-Tricosene Most of the recovered radioactivity from topically administered [3H](Z)-9tricosene in both male and female houseflies was present in the hexane extract, representing material on the surface of the insect (Table 1).In males, between 83% and 94% of the recovered radioactivity was in external washes, and in females, between 94% and 98%. The metabolites of [3H](Z)-9-tricosenepresent on the surface of the insect increased with time, from 27.4% to 36.6% in males and 5.5% to 10.8% in females at 30 and 60 min, respectively (Table 1).Metabolism of the alkene in males proceeded more rapidly during the first 30 min, amounting to 75% of the metabolism observed at 60 min. In females, however, the total amount of (3-9-tricosene metabolized by 60 min was doubled of that at 30 min, suggesting that in females metabolism proceeds at a constant rate up to 1h. In addition to this difference, metabolism of (3-9-tricosene was significantly greater in males than in females (ANOVA, P > F = 0.01; metabolism ratio ca. 37:11 at 60 min). However, direct comparison of rates of conversion in males and females is not possible. Males do not produce the pheromone, but the females past day 2 are producing (Z)-9-tricosene1 which dilutes the specific activity of topically administered [3H](Z)-9-tricosene. Effects of Cytochrome P-450 Inhibitors on the Metabolism of [3H](2n-9-Tricosene Pretreatment with PB (5 pglinsect) markedly inhibited [3H](Z)-9-tr* icosene metabolism. There was a decrease in the amount of metabolites in both males (66% and 72% at 30 and 60 min, respectively) and females (55% and 61% at 30 and 60 min, respectively) (Table 1).Carbon monoxide also markedly inhibited the alkene metabolism. there was a decrease in the amount of metabolites in both males (86%) and females (55%) in 30 rnin (Table 1). Formation of Metabolites Radio-GLC analyses of the HC fractions from both the external and internal extracts (and those derived from control, PB, or CO-treated insects) all indicated that (G-9-tricosene was the sole constituent. TLC was used to separate each component of the NHC fraction. In addition to the original position of the spot (TLC origin), six metabolites were clearly resolved. Of the six metabolites, three cochromatographed with authentic standards of the C23 epoxide, ketone, and secondary alcohol. The remaining were designated as unknowns (U) 1,2 and 3, and, together with the origin, are included 60 60 30 30 30 30 30 30 60 60 Incubation time (min) 94.0 94.9 92.5 94.9 97.6 f 1.0 f 1.0 f 0.2 f 0.6 f 0.3 87.8 f 0.8 93.8 f 0.4 90.4 rt 0.5 83.4 f 0.5 91.5 f 1.6 5.3 f 0.4 2.4 f 0.2 2.4 f 0.1 10.8 f 0.8 4.2 f 0.1 27.4 f 1.6 9.3 f 2.1 6.6 rt 0.5 36.6 f 1.6 10.4 f1.4 metabolized % Alkene rt 2.0 f 7.6 f 2.8 f 6.7 f 6.1 53.8 f 4.4 63.2 f 5.1 63.6 f 5.4 61.4 f 5.0 54.0 f 2.8 39.8 41.7 37.3 42.6 45.1 Epoxide 21.8 f 3.9 19.1 f 5.0 16.9 ir: 0.9 24.5 f 1.4 29.9 f 2.2 14.8 f 2.6 16.6 f 1.3 17.3 f 0.8 13.3 ir: 3.4 14.8 f 4.8 4.2 4.3 4.0 2.0 5.8 4.5 4.4 3.7 2.9 3.2 ir: 2.3 f 0.5 f 0.7 f 0.4 f 0.4 f 1.6 f 0.9 f 0.3 f 1.2 f 1.8 20.2 f 4.1 13.2 ir: 0.2 15.5 f 5.7 12.1 f 3.2 10.3 f 2.0 40.9 f 6.1 37.3 f 8.3 41.7 f 2.0 41.2 f 9.4 36.9 f 5.7 % Composition of external metabolites Secondary Ketone alcohol Unknowns *Data are means f SD derived from four replicates, except for the CO experiments, where means are derived from three replicates. A batch of ten insects comprised a replicate, and each insect was topically treated with ca. 100,000 dpm [3H] (Z)-9-tricosene.Total average recovery of the label including Bligh and Dyer extracts, was 98%, the range being 96-102%. PB, piperonyl butoxide. + Females +PB +CO PB +PB +co + Males PB Inhibitor (PB or CO) % Recovered in hexane extracts TABLE 1. The Effect of the Cytochrome P-450 Inhibitors, PB and CO, on the In Vivo Metabolism of [3H](Z)-9-Tricosene in 3-Day-Old Male and Female Houseflies* 128 Ahrnad, Kirkland, and Blornquist as "unknowns" in Table 1. Radio-GLC  was used to confirm that the radioactivity in the epoxide and ketone fractions was entirely in the C23 components. A C23 secondary alcohol was predicted to be the precursor of the C23 ketone  and its resolution and quantification here supports that hypothesis. The relative proportions of C23 epoxide, ketone, secondary alcohol, and unknown metabolites are given in Table 1.The external metabolite composition in both males and females was C23epoxide > ketone > secondary alcohol, but the internal composition was C23 epoxide > secondary alcohol > ketone. These data indicate that the less polar epoxide and ketone are more efficiently extruded to the cuticular surface than the more polar secondary alcohol. The internal pool of secondary alcohol is apparently designated for oxidation to the ketone, much of which is then transported to the cuticle. Cytochrome P-450 epoxidation and hydroxylation (in the formation of alcohols) reactions are strongly inhibited by PB and CO 17,121, a finding consistent with a profound decrease of (9-9-tricosene metabolism (Table 1). The decrease in ketone formation is undoubtedly due to the inhibition of the enzyme that forms its precursor, the secondary alcohol. The radioactivity in the aqueous phase showed that the total water-soluble radioactivity was much higher in males compared with females. For example, 42.0 k 2.3% of the internal radioactivity from 30-min incubations in males was in the aqueous phase, compared with 15.2 If: 4.2% for females. This is consistent with the greater amounts of [3H](Z)-9-tricosenemetabolized by males to NHC, which are then presumably converted to water-soluble products by phase I1 enzymes . Comparative Profile of (23-9-TricoseneMetabolism by Body Parts (Organs/ Tissues) of Male and Female Houseflies (3-9-Tricosene metabolism by isolated organs/tissues was greater in males than in females (Table 2). Male: female ratios, depicting metabolic rate differences were: antennae, 4.0:l.O; legs, 1.6:l.O; fat bodies, 1.5:l.O; and abdominal epithelial tissue, 1.8:l.O. The ratio for thoracic epithelial tissue was 0.7:1.0, indicating that in this tissue, in females, the metabolic capacity surpasses that of males. Also, within each sex, the rate of metabolism greatly varied: the order in males being: antennae > > legs 2 fat bodies 2 abdominal epithelial tissue > > thoracic epithelial tissue. In females, however, the rate of metabolism was thoracic epithelial tissue = legs > fat bodies 2 abdominal epithelial tissue > antennae. Data in Table 2 further show that in males, more water-soluble than NHC metabolites were formed by antennae and legs: 67% and 154% more, respectively. In the remaining body parts, the NHC components were in excess of water-soluble metabolites. In females, nearly equal amounts of radioactivity were in the NHC fraction and the aqueous phase from antennae, and were greater in the NHC fraction than other fractions in all other tissues. These data suggest that the male antennae and legs have the highest activity of phase-I1 enzymes . Piperonyl butoxide markedly inhibited the metabolism of (Z)-9-tricosene by all organsltissues, presumably by blocking the initial enzymatic attack on 3.9 (3.7-4.1) 6.1 (6.1-6.1) 5.2 (4.7-5.6) 4.7 (4.5-4.9) 6.1 (6.0-6.2) 15.5 (15.1-15.8) 9.9 (8.4-11.4) 9.0 (8.4-9.5) 8.5 (8.3-8.6) 4.2 (3.9-4.4) (%I 1.9 (1.7-2.0) 3.4 (3.43.4) 4.1 (3.7-4.4) 3.8 (3.6-4.0) 4.2 (4.1-4.3) 5.8 (5.7-5.8) 2.8 (2.5-3.1) 5.0 (4.7-5.2) 4.5 (4.44.5) 2.9 (2.6-3.1) 2.1 (2.0-2.1) 2.7 (2.7-2.7) 1.1 (1.0-1.2) 0.9 (0.9-0.9) 1.9 (1.9-1.9) 9.7 (9.4-10.0) 7.1 (5.9-8.3) 4.0 (3.7-4.3) 4.0 (3.9-4.1) 1.3 (1.3-1.3) Distribution (%) of metabolitesa NHC Aqueous 49.8 (43.7-55.9) 65.3 (65.0-65.0) 57.4 (56.6-58.1) 47.8 (43.0-52.6) 56.8 (56.6-56.9) 75.2 (74.1-76.3) 72.0 (71.4-72.6) 60.5 (60.4-60.6) 56.2 (54.6-57.8) 62.3 (62.3-63.9) 54.2 (51.4-56.1) 69.8 (68.7-70.1) 37.5 (33.7-41.3) 54.6 (48.6-60.6) 75.2 (74.6-75.8) 83.3 (82.6-84.0) 79.6 (78.4-80.7) 68.3 (67.5-69.1) 65.3 (63.4-67.2) 78.7 (77.7-79.6) Inhibition (%) by PBa NHC Aqueous *Metabolism of [3H] (Z)-9-tricosene directly applied to separate pools of antennae, legs, fat bodies, abdominal epithelium (abd. ep.), and thoracic epithelium from eight insects, and incubated in isotonic KC1-Tris, pH 7.5, solution for 20 min at 30°C. The total recovery of 3H was in the 96-99% range. Data in the last column are transformed to show percentage inhibition of 3H metabolites partitioning either in NHC or aqueous fractions. PB, piperonyl butoxide; NHC, nonhydrocarbon. aData are averages of duplicate determinations, with the range given in parentheses. Thoriac ep. Abd. ep. Fat Bodies Legs Females Antennae Thoriac ep. Abd. ep. Fat Bodies Legs Males Antennae OrgandTissues Total (Z)-9-tricosene metabolizeda TABLE 2. Profile of [3H] (Z)-g-Tricosene Metabolism by Isolated OrganslTissues of 3-Day-Old Male and Female Houseflies* Ahmad, Kirkland, and Blornquist 130 60y i "1-7 40: Male 0 - ~~~ 2 2 c 20 3 m 01 - 40- z * u) I Female ANTENNAE ANTENNAE 0 a 00 - - ~ - a a -1 - 1 LEGS LEGS + + - _. __c_ & FAT BODIES - + ~ - ~ 1 - ~ a - FQl===I --c FAT BODIES - Fig. 1. Relative percent distribution of the metabolites from selected organsitissuesof 3-dayold male and female houseflies incubated with [3H](Z)-9-tricosenefor 20 min at 30° C. Metabolites shown are: epoxide (E), ketone (K), secondary alcohol (A), unknowns (UI-U3), and radioactivity at the original thin-layer chromatography spot (0).Epithelium is abbreviated as Ep. In each data bar, the upper and lower segments represent the relative amount of metabolite formed in the control and piperonyl butoxide (PBI-treated tissues, respectively. Each determination contained a pool of organs/tissues from eight insects, and the data are averages of duplicate determinations with the error bars representing the range. Pheromone Metabolism by Cytochrome P-450 131 indicate relative proportions of each of the metabolites. In all organsltissues of the males, epoxide was by far the most abundant metabolite, followed by secondary alcohol and ketone. In females, the relative amounts of epoxide, ketone, and secondary alcohol were more variable. The relative metabolite composition from antennae, legs, and thoracic epithelial tissue is essentially the same in males (Fig. 1).A different pattern was observed for fat bodies and abdominal epithelial tissue. These two tissues form significantly greater (P > F = 0.05) amounts of C23 ketone, compared with antennae and legs. In females, the antennae and legs produce high levels of C23 ketone, whereas the fat bodies and the abdominal and thoracic epithelial tissues produce high amounts of the epoxide, followed by the ketone and secondary alcohol. Subcellular Assays Metabolism of (Z)-9-tricosene was much more active in microsomal than in mitochondria1 preparations from 3-day-old male and female houseflies. Data presented in Table 3 show that the microsomal preparation from whole abdomens converted 22.1% and 17.2% of the alkene to oxygenated products in 20 min by males and females, respectively. The difference in the metabolic rate between the two sexes was significant (P > F = 0.05). The metabolic rate by microsomes from abdomens without the gut tissues was 10.7% (males) and 12.9% (females), the decrease in the rate indicating that the gut component of the enzyme in the intact abdominal microsomes contributed to its high activity. (Z)-9-Tricosene metabolism by the microsomal enzymes was markedly inhibited by PB (Table 3), with at least a 50% inhibition of the formation of NHC metabolites and between 27.6% and 82.6% inhibition of formation of aqueous metabolites. Carbon monoxide inhibition of the microsoma1 metabolism of the alkene was even more pronounced (Table 4), with 87.6% to 89.6% of inhibition of the formation of NHCs and between 86.4% and 91.1% inhibition of formation of water-soluble metabolites. The relative profiles of metabolites formed, largely in the NHC fraction, are shown in Figure 2. Common to both sexes is the following pattern of metabolite formation by the microsomal enzymes: 1. The C23 epoxide comprised an average of 85% of all NHC components, whereas the secondary alcohol averaged 7.5%. This indicates that the epoxidation, rather than hydroxylation, is a more favored reaction. 2. All other metabolites were formed in very small quantities, including the C23 ketone. Apparently the ketone is a further oxidation product of the secondary alcohol, by another enzyme. The inhibition of the ketone formation by PB parallels the inhibition of its precursor. Both PB and CO inhibited the formation of all metabolites of the NHC fraction, indicating that they all arise from mono-oxygenation reaction directly, or, like the ketone, via secondary metabolism of primary products from a mono-oxygenase-catalyzed reaction. Furthermore, the microsomal preparation containing only the peripheral tissues’ enzyme, compared to the other preparation containing the gut tissues, was less sensitive to the inhibition of the epoxidation reaction by PB but more sensitive to the inhibition of hydroxylation. This pattern suggests the possibility of a qualitative difference 17.2 (16.7-17.7) 12.9 (12.5-13.3) 22.1 (20.8-23.4) 10.7 (10.4-11.1) Total (Z)-9-tricosene metabolized (%) 14.3 (13.8-14.7) 10.2 (10.0-10.4) 17.6 (16.7-18.4) 8.8 (8.5-9.2) 2.9 (2.9-2.9) 2.7 (2.5-2.9) 4.5 (4.1-5.0) 1.9 (1.9-1.9) Distribution (%) of metabolites NHC Aqueous 60.6 (59.6-61.6) 57.0 (49.3-52.6) 53.1 (51.5-54.6) 51.2 (51.1-51.3) NHC 49.5 (43.0-55.9) 82.6 (79.3-85.9) 27.6 (28.0-37.2) 68.7 (64.6-72.8) Aqueous Inhibition by PB (%)b *Labeled substrate was incubated with 2 mg microsomal protein for 20 min at 30°C, in total volume of 2.0 ml and buffered with KC1-Tris, pH 7.5. aAbd.+G, intact abdomen; abd. -G, abdomen without guts. bData are averages of duplicate determinations, with the range given in parentheses. NHC, nonhydrocarbons; PB, piperonyl butoxide. Abd.-G Females Abd.+G Abd.-G Males Abd. +G Source of microsomesa TABLE 3. Profile of [3H](Z)-9-TricoseneMetabolism by Microsomal Preparations From Intact Abdomens and Abdomens Without Guts of 3-Day-Old Male and Female Houseflies* Pheromone Metabolism by Cytochrome P-450 133 TABLE 4. Effect of CO on Microsomal Metabolism of [3H](Z)-9-Tricosene by Microsomal Preparations From Whole Abdomens of 3-Day-Old Male and Female Houseflies* %(Z)-9Tricosene metabolized Incubation atmosphere Males N2:02 (80:20) C0:02 (80120) Females N2:02 (80:20) C0:Oz (80:20) Relative (%) partitioning of metabolites NHC Aqueous YO Inhibition by CO NHC Aqueous 20.9 f 0.2 2.2 < 0.1 * 16.4 f 0.2 1.7 f 0.1 4.5 f 0.1 0.4 f < 0.1 89.6 91.1 18.3 f 0.3 2.3 f 0.1 16.1 f 0.4 2.0 f 0.1 2.2 f 0.1 0.3 I < 0.1 87.6 86.4 *All values are means f SD derived from three replicates. A batch of ten insects comprised a replicate, and each insect was topically treated with [3H](Z)-9-tricosene.Values below 0.05 are shown as < 0.1. Incubation compositions and conditions were the same as given in the footnote of Table 3. NHC, nonhydrocarbons . Male microsornes U U 6 A iti h E K A U l U 2 U 3 0 Female microsornes U c> A 0 E K A U l U 2 U 3 0 E R RR K A UlU2U30 0 E K A UlU2U3 0 Fig. 2. Relative percent distribution of the metabolites from incubations of microsomal preparations from whole abdomens (A) and abdomens without the gut tissues (B) of 3-dayold-houseflies, with [3H](Z)-9-tricosene.Labeled substrate was incubated with 2 mg microsoma1 protein for 20 min at 30OC. Metabolites shown are; epoxide (E), ketone (K), secondary alcohol (A), unknowns (UI-U3), and the radioactivity at the origin thin-layer chromatography spot (0).In each data bar, the upper and lower segments represent the relative amount of metabolite formed in the control and piperonyl butoxide (PB)-treated microsomal preparatons, respectively. Data are averages of duplicate determinations with the error bars representing the range. 134 Ahrnad, Kirkland, and Blornquist between the peripheral cytochrome P-450 monooxygenase vs. the gut enzyme in (2)-9-tricosene metabolism in both sexes. The monooxygenations catalyzed by microsomes prepared from both male and female houseflies was highly dependent on NADPH (optimal amount was 2.0 pmol) and molecular oxygen, as shown in Figure 3. In the absence of any exogenous NADPH, the alkene metabolism was only 0.9% (males) and 0.6% (females); these values were subtracted from each data point in depicting the NADPH-dependency curves (Fig. 3). It is also evident from Figure 1that there is an absolute requirement of 0 2 in the mono-oxygenation reactions, because under N2 the metabolism of (9-q-tricosene was drastically reduced. Minimal metabolism observed under gassing with N2 could be from incomplete elimination of 0 2 from the incubation mixture. The mitochondrial fraction produced much lower amounts of oxygenated products of (3H](z)-9-tricosene (between 2.6% and 4.3% of recovered radioactivity) than did the microsomal fraction. Similar products were produced by both fractions, but the mitochondrial activity was not inhibited by piperonyl butoxide (data not shown). Because of the low activity in the mitochondrial fraction, it was not further characterized. 22 20 24 zf !2 $ N2:02* 80:20 0 la- i- Male Female 16- I w 6 3 I- 6 0 6 a. 141210- a6 - 4- 0 I I I I 1 2 3 4 prnol NADPH Fig. 3. NADPH and O2 dependency computer-generated plots for the metabolism of 13H](D9-tricosene by microsomes prepared from whole abdomens of 3-day old male and female houseflies. The incubations were conducted in total volume of 2.0 ml buffered by KCI-Tris, pH 7.5, and contained 2 mg microsomal protein labeled substrate, and varying amounts of NADPH. Incubations were carried out at 3OoC and terminated at 20 min. NADPH dependency of alkene metabolism was established under aerobic conditions by continuous gassing withsaturated N2:02 (80:20). Oxygen dependency was demonstrated by gassing incubation mixtures with 100% N2. The data points are means derived from three replicates. The SDs of the means were all less than 10% and the majority were under 2% of the means. Pheromone Metabolism by Cytochrome P-450 135 Difference Spectra: Evidence of Cytochrorne P-450 and Its Binding to (Z)-f)-Tricosene As shown in Figure 4, microsomal preparations from 3-day-old male and female houseflies produced a CO-difference spectrum with reduced cytochrome with maximal absorbance at 450 nm. This feature is typical of cytochrome P-450 and forms the basis of its quantification. The absorbance mg-' of the Fales 1958 strain I1 is 0.0375, amounting to 0.41 nmol cytochrome P450 m8-l protein; it compares well with absorbances of 0.0335 and 0.0310 established for the FC and CSMA strains of the housefly, respectively [q. There was some conversion of cytochrome P-450 to the inactive form, cytochrome P-420, which also complexes with CO. The amount was calculated to be 0.06 nmol cytochrome P-420 mg-' protein. The conversion is affected by sulfuric acid formed by the oxidation of the cytochrome reductant, sodium dithionite Figure 4 also shows the (.Z)-9-tricosene-oxidized cytochrome P-450 difference spectrum, which resembles a type I spectrum. The absorbance peaks and troughs vary for type I spectra, but the peak for both male and female enzymes is at ca. 385 nm, which is usual for type I spectra  and is accompanied by a broad trough (400-450 nm) that varies for the two sexes. A type I spectrum indicates that (2)-9-tricosene resembles the majority of lipophilic substrates in binding to a hydrophobic site adjacent to the heme moeity of the oxidized (Fe3') cytochrome P-450. In a complete cytochrome P-450 system substrate complexed with oxidized cytochrome P-450 under- [n. 0.1 - 0.1 A B w 0 z a E 51 m a 0.0 i 400 i 350 I I I I 450 500 400 450 I I i 425 500 350 WAVELENGTH (nm) i _ _ _ . . . A 500 _ _ 425 - i 500 WAVELENGTH (nm) Fig. 4. Difference spectra of the microsomal cytochrome P-450 of 3-day-old houseflies. A and B depict spectra of male and female reduced (Fe+') cytochrome P-450-carbon monoxide complex. C (males) and D (females) show type I binding spectra of the oxidized cytochrome P-450 (Fe+3)with the ligand, (Z)-9-tricosene. Baseline for the spectra are also given. 136 Ahmad, Kirkland, and Blomquist goes reduction. The reduced complex then binds with molecular oxygen, and following oxygen activation the substrate is mono-oxygenated 1121. Evidence that (3-9-tricosene complex with the cytochrome follows this sequence is apparent from the data from the in vivo as well as in vitro studies. DISCUSSION The pathways for the biosynthesis of the housefly pheromone components, the alkene (Z)-9-tricosene, and C28-3 methyl-branched alkanes, have been previously established [13,14]. Accordingly, the alkene is synthesized by the elongation of oleic acid followed by conversion to the alkene, and the methyl-branched alkanes originate by the elongation of methyl-branched precursors. Except for the suggestion that a mixed-function oxidase may be involved , until now there has been no direct evidence for the enzymatic pathway responsible for the formation of the remaining two oxygenated hydrocarbon pheromone components, the C23 epoxide and the C23 ketone. The data reported here strongly implicate a cytochrome P-450-dependent microsomal PSMO (E.C. 18.104.22.168) in the synthesis of these two pheromone components. Thus we now have a more complete profile on the precursors and pathways for the biosynthesis of all pheromone components of the housefly. A number of criteria necessary to ascertain the involvement of cytochrome P-450 biotransformation [12,15] in (3-9-tricosene metabolism in the housefly have been met. The in vitro and in vivo products (the epoxide and secondary alcohol) are typical of a PSMO-catalyzed reaction. The inhibition by the PSMO inhibitors, i.e., PB and CO, both in vivo and in vitro, are consistent with a cytochrome P-450 involvement. Further support for the involvement of cytochrome P-450 is provided by NADPH and 0 2 dependence for the enzymatic activity. Likewise, the occurrence of a type I binding spectrum in both male and female microsomes with the addition of (3-9-tricosene is consistent with a cytochrome P-450 dependent PSMO, since most lipophilic substrates and some competitive inhibitors (that are slowly metabolized) bind at a hydrophobic site in the cytochrome P-450 protein in close enough proximity to the heme iron to allow both perturbations of the absorbance spectrum (type I) and interaction with the activated oxygen . Most of the enzyme activity was microsomal rather than mitochondrial. Cytochrome P-450 PSMO apparently metabolizes the C23 olefin to the corresponding epoxide and ketone, the latter through an intermediate PSMOcatalyzed hydroxylated product, the secondary alcohol, (Z)-9-tricosene-lO-o1. Reuttinger and Fulco 1161 concluded that in a bacterium, Bucillus meguterium, a single PSMO was capable of both epoxidizing double bonds and hydroxylating saturated bonds. This also appears to be the case with the housefly in which both PSMO reactions occur; the epoxidation of the olefin (at carbons 9 and 10) is predominant over hydroxylation (at carbon 10 from the other end of the molecule). Well-known and commonly used inhibitors of the microsoma1 PSMO, a methylene dioxyphenyl compound, PB, and CO inhibit the in vivo and in vitro formation of C23 epoxide, secondary alcohol, and the ketone; formation of the latter is suppressed, presumbaly due to the inhibi- Pheromone Metabolismby Cytochrome P-450 137 tion of its alcohol precursor. In addition, both PB and CO inhibited the formation of the organic soluble unknowns and water-soluble metabolites, thus indicating that these products were also either direct products or due to further metabolism of a PSMO reaction on (2)-9-tricosene. Recent studies have established that an epoxide hydrolase (E.C.22.214.171.124.) and a glutathione-S-transferase (E.C.126.96.36.199) readily convert epoxides to corresponding diols and glutathione conjugates (while opening the epoxide ring)/ respectively 112,201. Thus the epoxide formation is a convenient way to deactivate the main sex-attractant and to mobilize it in a highly polar form for excretion. In this context, we have tentatively identified a polar metabolite, unknown 1, as a diol. The glutathione conjugate could well be a component of the uncharacterized metabolites in the aqueous phase. Both enzymes occur ubiquitously in insects. Because the secondary alcohol is quite polar, it may be excreted as such, or after conjugation (12). As to how the ketone is deactivated, one possibility is a cytosolic ketone reductase-mediated conversion to an alcohol . The alcohol could then be disposed of in a manner described above. The distinct differences between the PSMO activity ratios of microsomes prepared from whole abdomens and abdomens without gut tissues suggests that the housefly gut PSMO is very active in oxidizing (Z)-Ptricosene. This is not surprising, for insect gut is known to contain high levels of cytochrome P-450 activity, and other tissues have lower enzyme activities, including the PSMO in corpora allata, which catalyzes the synthesis of the epoxide juvenile hormone 17,121. Furthermore, the gut PSMO of the southern armyworm, Spodoptera evidunia, is capable of epoxidating an olefin to an epoxide, disparlure, which is the sex pheromone of the gypsy moth, Lyrnantvia dispar [ l q . Nevertheless: 1)there was PSMO activity in housefly tissues peripheral to the gut; 2) the organltissue assays indicated substantial PSMO activity in the abdominal tissue; and 3) the cuticle-related tissues are recognized sites for pheromone synthesis in the housefly . We therefore suggest that a cuticular enzyme rather than the gut enzyme is responsible for the synthesis of the oxygenated pheromone components from (Z)-9-tricosene. Each of the housefly tissues investigated, i.e., abdominal epithelium, thoracic epithelium, fat bodyr legs, and antennae, metabolized (3-9-tricosene to essentially the same metabolites, although the capacity varied from one organltissue to another. The substantial microsomal PSMO activity in abdominal tissues peripheral to the gut tissues in all probability represents the sum of enzyme activities of the cuticle-related abdominal epithelium and fat body. The dectection of PSMO activity in an insect’s antennae and legs has important implications in catabolism of the pheromone. Consideration of the data showing differences among PSMO activities of organsltissues and between sexes, reveals possible interpretations in addition to the possibility that separate cytochrome P-450s are responsible. One alternative would include the same cytochrome P-450, but differing in specific activity and working in concert with other enzyme to yield different product distributions. This is probably the case and explains: 1)high malelfemale ratios in all organsltissues and especially the antennae; and 2) higher ketonelepoxide ratio in the female’s antennae and legs, reflecting a shunt through either a 138 Ahrnad, Kirkland, and Blomquist more abundant or higher specific activity enzyme that oxidizes the alcohol to the ketone. In addition, there is evidence for the existence of separate cytochrome P-450s in the abdomen’s peripheral vs. gut microsomal PSMO. Although much of the (3-9-tricosene synthesis takes place in abdominal cuticle tissue, it is likely that synthesis also occurs elsewhere. Also, grooming redistributes (Z)-Ptricosene to other body parts. The presence of large amounts of pheromone on female housefly legs has been indicated to occur in this manner . Thus, the metabolism of (Z)-9-tricosene could be useful for the females to maintain an effective ratio of the pheromone components for optimizing activity. We have shown that the oxidative pathway in the synthesis of the oxygenated hydrocarbon pheromone components is dominated by a PSMO, and that the enzyme is active in both males and females, despite the fact that females, not males, synthesize the pheromone. The production of the pheromone components in females occurs in synchrony with vitellogenesis and concommital increase in ecdysteriod titer . The ecdysteriod initiates (23-9tricosene synthesis probably by inducing the synthesis of an enzyme that transforms a 24-carbon fatty acid moiety to an alkene one carbon shorter. Thus the oxidative pathway in the synthesis of the epoxide and ketone pheromone components is not under direct humoral control. The very active PSMO in males probably serves to deactivate the olefinic pheromone to prevent attenuation of the pheromone receptors. We suggest that the same pathway the females use for the synthesis of the oxygenated pheromone components is fortuitously used by the males for deactivation. (Z)-9-Tricosene, being highly lipophilic and lacking functional groups, is ideally suited as a PSMO substrate. All of the NHC products may represent early steps in the deactivation processes involving other enzymes that act in concert with the PSMO and convert (23-9-tricoseneto water-soluble products. Males of several insect species readily metabolize the female ester sexpheromones by esterase(s)present in sensillae of antennae, legs, and located elsewhere, and several body parts including wings . Tibia of housefly legs are reported to possess receptors for the sex pheromone , and, based on the metabolic profile of the antennae, it also seems likely that such receptors are present on this sensilla-rich organ. Thus the deactivation most likely occurs in both body parts; this suggestion is consistent with the high metabolic capacity of these organs. The PSMO system of insects has been shown to play a central role in the primary degradation of xenobiotics, both man-made and naturally occurring phytochemicals [12,23]. Recently, the PSMO’s participation in the biogenesis of physiologically important substances to insects was reviewed [7,8]. Brattsten  previously speculated that in danaid butterflies, pyrrolizidine alkaloids are converted to male courtship pheromones by a PSMO-catalyzed reaction. A PSMO has been postulated to convert an olefin to an epoxide, disparlure, a sex pheromone of the female gypsy month. To date, however, neither one has been experimentally validated, except that the formation of disparlure was shown to occur with the gut PSMO of the southern armyworm larva, which does not normally produce this pheromone. 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