The role of tyrosinase in the inactivation of house fly microsomal mixed-function oxidases

The low mixed-function oxidase activity of house fly microsomes has been associated with low cytochrome P-450 content and NADPH-cytochrome c reductase activity. The microsomal cytochrome P-450 content and NADPH-cytochrome c reductase activity could be decreased by the addition of catechol and increased by the addition of cyanide to the homogenates. Similar results were obtained with rat liver microsomes treated with tyrosinase and catechol. During the inactivation of rat liver microsomal enzymes by tyrosinase and catechol, crosslinking of microsomal proteins occurred. These results suggest that the instability of house fly microsomal mixed-function oxidase may be due in part to the action of contaminating tyrosinase on endogenous substrates.     

Isolation of insect microsomal oxidases by rapid centrifugation

Only about 60% of the total relative gravitational force conventionally used to sediment microsomes is needed to prepare highly active microsomes from the midgut tissues of an insect larva. A rapid preliminary centrifugation for 2 min at 39,000g_max effectively removed contaminating microorganisms, tissue debris, nuclei, and mitochondria. The supernatant was recentrifuged for 20 min to 210,000g to sediment the microsomes. There were no losses of microsomal oxidase activities or degradation of cytochrome P-450 to the inactive form (P-420) resulting from the application of the higher gravitational force. Incorporation of 1 mM EDTA in the buffer and washing the microsomes resulted in an improved yield of the cytochrome compared to that in microsomes prepared in sucrose. Yields of microsomal protein, cytochrome P-450, and NADPH-cytochrome c reductase in the rapidly isolated microsomes were as good as those in conventionally prepared microsomes. The apparent kinetic characteristics of several microsomal oxidation activities and optical difference spectra of Types 1 and 2 ligands were identical in the rapidly and conventionally prepared microsomes. The morphological appearance of the microsomes was examined by electron microscopy. Microsomal pellets prepared by either method were indistinguishable. The rapid procedure saves significant time in microsome preparation and yields microsomal oxidase activities as good or slightly better than those prepared by usual centrifuged procedures.     

Prochloraz,a potent inducer of the microsomal cytochrome P-450 system

Prochloraz (N-propyl-N-[2-(2,4,6-trichlorophenoxy)ethyl]-imidazole-1-carboxamide), a recently developed agricultural fungicide, is a potent inducer of microsomal enzymes. Rats fed 7 days with a prochloraz-contaminated diet (2500 ppm) showed an increase in hepatic cytochrome P-450, cytochrome b₅, and microsomal protein level; aniline hydroxylase, 7-ethoxycoumarin dealkylase, 7-ethoxyresorufin dealkylase, NADPH-cytochrome c reductase, and epoxide hydrolase were significantly induced. At a lower dose (100 ppm), only an increase in cytochrome P-450 and 7-ethoxyresorufin dealkylase was noticed. As shown with aniline hydroxylase and 7-ethoxycoumarin dealkylase, prochloraz is also a potent inhibitor of drug-metabolizing enzymes. The interaction of prochloraz with hepatic microsomal fraction from rat liver was also studied. Prochloraz binds to oxidized cytochrome P-450 to produce a type II spectral change; the compound also binds to reduced cytochrome P-450. The binding of some ligands (7-ethoxycoumarin, n-octylamine, aniline, and imidazole) to oxidized cytochrome P-450 was determined after induction by prochloraz. Japanese quails (Coturnix coturnix) fed 7 days with a prochloraz-contaminated diet (2000 ppm) showed a dramatic increase in liver weight (2.5-fold) and both hepatic and duodenal cytochrome P-450 (9- and 12-fold, respectively).     

Components of the electron transport system in the microsomal mixed-function oxidase system in wild birds

Notable differences were found among six species of wild-caught birds in the levels of cytochrome P-450, cytochrome b₅, NADPH-cytochrome c reductase, and NADH-cytochrome c reductase. Ethyl isocyanide difference spectra showed significant variations among the species in peak height and in the ratios of the $\text{430}\text{455}\text{-}\text{nm}$ peaks. Substantial aldrin epoxidase activity was found in all species, and the amounts of dieldrin produced compared favorably with pigeon and rat liver microsomes. Higher content of cytochrome P-450 was not always accompanied by a similar rise in specific catalytic activity. Thus, no correlation could be established between these two parameters. Aldrin epoxidase activity with NADH as the sole electron donor was 25–49% as effective as with the NADPH-generating system. Addition of both NADH and NADPH-generating systems to the incubation mixture produced a synergistic effect with liver microsomes of two species but not with two other species. DDE and polychlorinated biphenyls residues were found in the heart tissue of all species examined, and this might indicate a possible inductive effect on the microsomal mixed-function oxidase system by environmental contaminants.     

The effects of dicofol on induction of hepatic microsomal metabolism in rats

In a comparative study, the induction effects of dicofol, technical Kelthane, and DDT on hepatic microsomal and cytosolic enzyme activities in rats were compared with those effects produced by phenobarbital (PhB) and β-naphthoflavone (BNF). Male rats (ca. 250 g) were injected (ip) for 4 consecutive days with 1.0 ml of vehicle containing either dicofol (1.5, 15.0, 29.5, or 59.0 mM, Kelthane (dicofol content equal to 29.5 or 59.0 mM), DDT (59.0 mM), or BNF (36.7 mM). Liver weights, microsomal protein, and cytochrome P-450 concentrations and microsomal and cytosolic enzyme specific activities were measured. Dicofol produced dose-related increases in all of the parameters measured except liver weight and cytosolic epoxide hydrolase activity. At a concentration of 59.0 mM, dicofol increased the concentrations of microsomal protein (1.7-fold) and cytochrome P-450 (2.9-fold), and the specific activities of cytochrome c reductase (1.6-fold), ethoxycoumarin O-deethylase (2.3-fold), aminopyrine N-demethylase (3.0-fold), microsomal epoxide hydrolase (2.6-fold), and glutathione S-transferase (2.9-fold). The induction potency of dicofol was equivalent to Kelthane, DDT, and PhB at equimolar (59.0 mM) concentrations of chemical.     

Blood meal and cytochrome P-450 monooxygenases in the northern house mosquito,Culex pipiens

Conditions for the measurement of aldrin epoxidation by microsomes prepared from abdominal tissues (fat body + integument) of adult female Culex pipiens were characterized. The enzyme activity had a pH optimum of 7.2 and an apparent K_m of 3.4 μM. Aldrin epoxidation and NADPH-cytochrome c reductase had similar patterns of inhibition by a rabbit antiserum to house fly NADPH-cytochrome P-450 reductase, thus implicating cytochrome P-450 monooxygenase(s) in the epoxidation of aldrin. Low (71 pmol/mg protein) levels of cytochrome P-450 were detected in abdominal tissue microsomes. In non-blood-fed insects, aldrin epoxidation and NADPH-cytochrome c reductase activities did not change between Day 1 and Day 12 after adult emergence, except for a small peak on Day 2. In insects fed a blood meal on Day 6 after emergence both activities increased (two- to threefold) to a plateau maintained between 2 and 4 days after the blood meal. Aldrin epoxidation and NADPH-cytochrome c reductase activities decreased to normal values between 4 and 6 days after the blood meal.     

Biochemical characteristics of insecticide resistance in the fall armyworm, Spodoptera frugiperda (J.E. Smith)

A strain of the fall armyworm, Spodoptera frugiperda (J.E. Smith), collected from corn in Citra, Florida, showed high resistance to carbaryl (562-fold) and methyl parathion (354-fold). Biochemical studies revealed that various detoxification enzyme activities were higher in the field strain than in the susceptible strain. In larval midguts, activities of microsomal oxidases (epoxidases, hydroxylase, sulfoxidase, N-demethylase, and O-demethylase) and hydrolases (general esterase, carboxylesterase, β-glucosidase) were 1.2- to 1.9-fold higher in the field strain than in the susceptible strain. In larval fat bodies, various activities of microsomal oxidases (epoxidases, hydroxylase, N-demethylase, O-demethylases, and S-demethylase), glutathione S-transferases (CDNB, DCNB, and p-nitrophenyl acetate conjugation), hydrolases (general esterase, carboxylesterase, β-glucosidase, and carboxylamidase) and reductases (juglone reductase and cytochrome c reductase) were 1.3- to 7.7-fold higher in the field strain than in the susceptible strain. Cytochrome P450 level was 2.5-fold higher in the field strain than in the susceptible strain. In adult abdomens, their detoxification enzyme activities were generally lower than those in larval midguts or fat bodies; this is especially true when microsomal oxidases are considered. However, activities of microsomal oxidases (S-demethylase), hydrolases (general esterase and permethrin esterase) and reductases (juglone reductase and cytochrome c reductase) were 1.5- to 3.0-fold higher in the field strain than in the susceptible strain. Levels of cytochrome P450 and cytochrome b₅ were 2.1 and 1.9-fold higher, respectively, in the field strain than in the susceptible strain. In addition, acetylcholinesterase from the field strain was 2- to 85-fold less sensitive than that from the susceptible strain to inhibition by carbamates (carbaryl, propoxur, carbofuran, bendiocarb, thiodicarb) and organophosphates (methyl paraoxon, paraoxon, dichlorvos), insensitivity being highest toward carbaryl. Kinetics studies showed that the apparent K_m value for acetylcholinesterase from the field strain was 56% of that from the susceptible strain. The results indicated that the insecticide resistance observed in the field strain was due to multiple resistance mechanisms, including increased detoxification of these insecticides by microsomal oxidases, glutathione S-transferases, hydrolases and reductases, and target site insensitivity such as insensitive acetylcholinesterase. Resistance appeared to be correlated better with detoxification enzyme activities in larval fat bodies than in larval midguts, suggesting that the larval fat body is an ideal tissue source for comparing detoxification capability between insecticide-susceptible and -resistant insects.     

Cytochrome P-450 in insects. 7. Age dependency and phenobarbital induction of cytochrome P-450, P-450 reductase,and monooxygenase activities in the black blow fly (Phormia regina,Meigen)

Development and phenobarbital (PB) induction of microsomal cytochrome P-450, NADPH-cytochrome c (P-450) reductase, two epoxidation, and two O-demethylation activities were examined in chronologically timed populations of female black blow flies (Phormia regina, Meigen). Measurements of these enzymes started with the pharate adult stage and ended 5 days following eclosion. Induction occurred in all enzymes, even at 0.005% PB, and was maximum at 0.15%. Dramatic induction of the O-demethylation of 7-methoxy-4-methylcoumarin was observed in flies dosed with the maximum concentration of the drug. This monooxygenase activity increased to nearly 1400 times the level in control flies, whereas the other O-demethylation (methoxyresorufin) and the two epoxidation reactions exhibited considerably less change. Induction of the structural enzymes of this enzyme system were 10-fold for cytochrome P-450 and 5-fold for NADPH-cytochrome c (P-450) reductase. These data suggest that PB induces several P-450's in the blow fly, particularly one bearing a high degree of specificity for 7-methoxy-4-methycoumarin.     

Cytochrome P-450 in insects. 6. Age dependency and phenobarbital induction of cytochrome P-450, P-450 reductase,and monooxygenase activities in susceptible and resistant strains of Musca domestica

Development and phenobarbital (PB) induction of microsomal cytochrome P-450, cytochrome P-450 reductase, two epoxidation, and two O-demethylation activities were examined in chronologically timed populations of insecticide-susceptible (NAIDM) and -resistant (Rutgers) house flies. Measurements of these enzymes started with the pharate adult stage and ended 5 days following eclosion. Untreated insects of both strains had comparable reductase levels, whereas cytochrome P-450 and associated monooxygenase activities were 1.5-fold or more higher in Rutgers. Maximum induction, as well as toxicity, occurred at a lower PB concentration in NAIDM than Rutgers. The drug caused consistently higher increases in enzymes and activities within 12 hr of starting treatment in both strains. When PB was withdrawn from treated flies (both strains) 48 hr after treatment began, specific activities (product min^?1 mg protein^?1) in all enzymes returned to control values in 24 hr while metabolic capacity (product min^?1 insect^?1) achieved control values within 48 hr. The changes in turnover numbers (pmol product min^?1 pmol P-450^?1), in conjunction with the differences in the monooxygenation of the four substrates, suggest that PB treatment induced both a quantitative and qualitative change in NAIDM monooxygenation but only a quantitative change in Rutgers monooxygenation.     

Hepatic microsomal oxidative metabolism of pesticides and other xenobiotics in pregnant CD1 mice

Pregnancy-related changes in oxidative metabolism of several xenobiotics including pesticides were examined in the hepatic microsomes of CD₁ mice. The effect of pregnancy on hepatic microsomal cytochrome P-450-catalyzed substrate oxidation was found to be dependent upon the type of reaction examined. Not all substrates undergoing the same reaction showed identical changes during pregnancy. Those enzyme activities which exhibited a decline in specific activity during pregnancy generally exhibited no change in total hepatic capacity. Enzymes posting no change in specific activity throughout gestation generally showed large increases in total hepatic activity. Phorate S-oxidation was catalyzed by both microsomal flavin-containing monooxygenase (MFMO) and cytochrome P-450. Moreover, there was no pregnancy-related change in either MFMO or total enzymatic (MFMO plus cytochrome P-450) phorate S-oxidation.     

In vitro effects of chlordecone (Kepone) on hepatic microsomal cytochrome P-450

The interaction of chlordecone (decachlorooctahydro-1,3,4-metheno-2H-cyclobuta[cd]-pentalene-2-one, Kepone) with hepatic microsomal cytochrome P-450 was studied. Chlordecone binds to cytochrome P-450 to produce a Type I spectral change the magnitude of which is dependent upon the chemical pretreatment of the animal. In kinetic studies of chlordecone was found to be a competitive inhibitor of aminopyrine N-demethylase and p-nitroanisole O-demethylase and a noncompetitive inhibitor of aniline p-hydroxylase.     

Polysubstrate monooxygenases (cytochrome P-450) in larvae of susceptible and resistant strains of house flies

The polysubstrate monooxygenases (PSMO or cytochrome P-450) of house fly larvae were studied at the mature larval or “clear gut” stage. Fat body and gut tissues were most efficient in the conversion of aldrin to dieldrin. Microsomal fractions of larval homogenates had the highest PSMO activities, with lower PSMO activities also found associated with mitochondrial fractions. Microsomes from Rutgers (resistant) larvae had higher levels of NADPH:cytochrome c reductase (2×), cytochrome P-450 (2×), aldrin (4×), and heptachlor (9×) epoxidases than microsomes from CSMA (susceptible) larvae. Cytochrome P-450 of Rutgers larvae had an absorption maximum at 449 nm, 2 nm lower than the cytochrome P-450 of CSMA larvae. n-Octylamine spectra showed that the level of high-spin cytochrome P-450 was higher in Rutgers larvae. NADPH:cytochrome c reductase, cytochrome P-450, and aldrin epoxidase were induced by phenobarbital, and Rutgers larvae were shown to be more sensitive to this inducer than CSMA larvae. Induction of larval PSMO by phenobarbital did not affect the expression or the inducibility of PSMO in adults.     

Studies on a fluorescent insect growth regulator metabolite complex with house-fly microsomal cytochrome P-450

The fluorescent insect growth regulator 5[[[5-(dimethylamino)-1-naphthalenyl]amino]-1,3-benzodioxole (DNSAB) forms a metabolite complex with house-fly microsomal cytochrome P-450. Formation of the metabolite complex is dependent on the presence of NADPH and O₂; NADH supports the reaction at a reduced rate. The presence of antibodies to house-fly cytochrome c (P-450) reductase in reaction mixtures inhibits the complex formation, indicating that the reductase is necessary for transfer of electrons from NADPH to cytochrome P-450 to complete the reaction. In the oxidized form, the metabolite complex has a single absorbance maximum at 431 nm, whereas the reduced form has two absorbance maxima at 426 (major) and 455 nm (minor). The pH of the media affects the extinction of the 426- and 455-nm Soret bands; increased pH decreases the extinction of the 426-nm band and increases the extinction of 455-nm band. Formation of the DNSAB metabolite-cytochrome P-450 complex decreases the amount of CO-reactive cytochrome P-450 by 24%. The metabolite complex is not dissociable by treatment with ferricyanide or by using centrifugation techniques. Dissociation is accomplished by addition of DNSAB to the oxidized metabolite complex. Kinetic analysis of the complex formation gives apparent K_m and V_max values at 2.55 ± 1.0 μM and 1.1 ± 0.4 × 10^?2 ΔA min^?1 nmol^?1 cytochrome P-450, respectively. Addition of juvenile hormone [(E,E)-cis-methyl-10,11-epoxy-7-ethyl-3,11-dimethyl-2,6-tridecadienoate; JH] to the reaction medium competitively inhibits the formation of the metabolite complex giving an inhibition constant of 16 μM. DNSAB synergized the lethal effects of JH against Aedes aegypti larvae threefold; however, JH did not synergize DNSAB. These data suggest that DNSAB may acquire its hormonal qualities by complexing a species of cytochrome P-450 that metabolizes JH, thereby prolonging the in vivo lifetime of this hormone.     

Cytochrome P-450 in insects: 4. Reconstitution of cytochrome P-450-dependent monooxygenase activity in the house fly

Two cytochrome P-450-containing fractions were isolated from detergent-solubilized house fly microsomes by hydrophobic chromatography on a tryptamine-Sepharose gel. These fractions (designated P-450-1 and P-450-2) were distinctive in their spectral characteristics and in their profiles following electrophoresis in the presence of sodium dodecyl sulfate. Both fractions exhibited NADPH-dependent epoxidase activity when reconstituted with purified house fly cytochrome P-450 reductase and phospholipid. The aldrin epoxidase activity of fraction P-450-1 was twice that of P-450-2 even though heptachlor epoxidase activity of the fractions was equivalent. O-Demethylase activity with 7-methoxy-4-methylcoumarin was detectable only in the P-450-2 fraction.     

Temperature and diet modulate cytochrome P-450 activities in southern armyworm,Spodoptera eridania (Cramer), caterpillars

Larvae of the southern armyworm, Spodoptera eridania (Cramer), grew well in the 15–30°C temperature range. Pupae survived poorly at 15°C but moths emerged from 85% of the pupae at 30°C. The time for development was prolonged at 15°C and larvae grew significantly bigger than at 30°C. Cytochrome P-450 content, cytochrome P-450 reductase, p-chloro N-methylaniline N-demethylation, methoxyresorufin 0-demethylation, and aldrin epoxidation activities were higher at 15°C than at 30°C. All cytochrome P-450 activities were more inducible by dietary pentamethylbenzene at 30°C than at 15°C. High cytochrome P-450-catalyzed activities were associated with increases in microsomal protein rather than with changes in membrane lipid or phospholipid content. Phosphatidylcholine was the major midgut membrane phospholipid. There was only a tendency towards increased unsaturation of the phospholipid fatty acyl moieties and lowered membrane phase transition temperature in cold-adapted larvae. Acute oral carbaryl toxicity was generally inversely correlated with cytochrome P-450 catalyzed activities. Carbaryl toxicity was decreased about 10-fold by pentamethylbenzene induction and about 3-fold by the lower acclimatization temperature.     

Mode of action of naphthalic anhydride as a safener for the herbicide AC 263222 in maize

In hydroponic experiments, seed-dressing with the herbicide safener 1,8-naphthalic anhydride (NA), significantly enhanced the tolerance of maize, (Zea mays L., cv. Monarque) to the imidazolinone herbicide, AC 263222, (2-[4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl]-5-methylnicotinic acid). Uptake, distribution and metabolism studies where [¹⁴C]AC 263222 was applied through the roots of hydroponically grown maize plants showed that NA treatment reduced the translocation of radiolabel from root to shoot tissue and accelerated the degradation of this herbicide to a hydroxylated metabolite. Reductions in the lipophilicity and, therefore, mobility of this compound following hydroxylation may account for NA-induced retention of radiolabel in the root system. Hydroxylation of AC 263222 suggested that NA may stimulate the activity of enzymes involved in oxidative herbicide metabolism, such as the cytochrome P₄₅₀ mono-oxygenases. In agreement with this theory, the cytochrome P₄₅₀ inhibitor, 1-aminobenzotriazole (ABT), synergized AC 263222 activity and inhibited its hyroxylation in vivo. NA seed-dressing enhanced the total cytochrome P₄₅₀ and b₅ content of microsomes prepared from etiolated maize shoots. Isolated microsomes catalyzed AC 263222 hydroxylation in vitro. This activity possessed the characteristics of a cytochrome P₄₅₀ mono-oxygenase, being NADPH-dependent and susceptible to inhibition by ABT. Activity was stimulated four-fold following NA seed treatment. Differential NA enhancement of AC 263222 hydroxylase and the cytochrome P₄₅₀-dependent cinnamic acid-4-hydroxylase (CA4H) activity, suggested that separate P₄₅₀ isozymes were responsible for each activity. These results indicate that the protective effects of NA result from enhancement of AC 263222 hydroxylation and concomitant reduction in herbicide translocation. This may be attributed to the stimulation of a microsomal cytochrome P₄₅₀ system. © 1998 SCI.     

Absence of enhanced detoxication of permethrin in pyrethroid-resistant house flies

The mechanisms of resistance to pyrethroids were studied in a permethrin-selected (147-R) strain of the house fly, Musca domestica L. Approximately 12-fold synergism was obtained with a mixture of (1R)-trans-permethrin:piperonyl butoxide (1:5) so that the resistance decreased from 97-fold to 22-fold. Tests with the esterase inhibitor S,S,S-tributyl phosphorotrithioate produced very little synergism in either the resistant (R) strain (1.6-fold) or the susceptible (S) strain (1.9-fold). An investigation of the microsomal components revealed that compared to the S strain, the R strain demonstrated twice as much cytochrome P-450 and cytochrome b₅ and double the rate of NADPH-cytochrome c reductase activity. In addition, the rate of p-nitroanisole O-demethylation was found to be six times greater in the R strain. An in vivo accumulation study showed that the R strain displayed a decreased rate of penetration of trans-[¹⁴C]permethrin. When treated at equitoxic doses the R strain was found to tolerate 50-fold more internal permethrin than the S strain. An in vitro metabolism study indicated that there was no difference between strains in the overall rate of metabolism of trans-[¹⁴C]permethrin. The evidence obtained supports the conclusion that several resistance factors are involved but that decreased sensitivity of the nervous system to the action of pyrethroids is the principal mechanism of resistance in the 147-R strain.     

Cytochrome P-450 in insects: 2. Multiple forms in the flesh fly (Sarcophaga bullata,Parker), and the blow fly (Phormia regina (Meigen))

Microsomes prepared from the abdomens of the flesh fly (Sarcophaga bullata, Parker) and the blow fly (Phormia regina (Meigen)) contain approximately one-fifth and one-eighth as much cytochrome P-450, respectively, as those prepared from house fly (Musca domestica, L.) abdomens. These values correlate well with the microsomal aldrin epoxidase activity of the three species and with their respective susceptibilities to the insecticide, propoxur. When the microsomes of the flesh fly and the blow fly are solubilized by treatment with deoxycholate and resolved by ion-exchange chromatography on DEAE-cellulose and hydroxylapatite, four chromatographically distinct fractions containing cytochrome P-450 are obtained. Spectrophotometric assays of the cytochrome P-450 in these fractions indicate purifications of two-to sixfold for the flesh fly hemoprotein and two-to eightfold for that of the blow fly. SDS-Polyacrylamide gel electrophoresis of the four column fractions from the flesh fly microsomes indicates that six hemoproteins in the 40,000–60,000 molecular weight range are present. In similar experiments with blow fly fractions containing approximately the same amount of cytochrome P-450 no high molecular weight hemoproteins could be detected. This result is interpreted, with other evidence, as an indication of the greater instability of the blow fly hemoprotein. The results indicate that multiple forms of cytochrome P-450 are present in both species but there is insufficient data on which to estimate the number of such forms.     

Microsomal oxidase activity of three blowfly species and its induction by phenobarbital and β-naphtoflavone

Induction of the microsomal oxidase system by dietary phenobarbital and β-naphthoflavone was examined in three blowflies, Phormia regina (Mg.), Lucilia illustris (Mg.), and Eucalliphora lilica (Walk.). Responses were similar in adults and larvae of all species. Phenobarbital increased cytochrome P-450 levels up to 9-fold and aldrin epoxidase up to 138-fold. Increases in cytochrome P-450 and aldrin epoxidase caused by β-naphthoflavone were minor relative to those produced by phenobarbital. In toxicity experiments with carbaryl and propoxur tolerance was associated with the amount of microsomal oxidase activity. Using piperonyl butoxide to synergize carbaryl and propoxur there was no clear indication for the use of either the synergist ratio or synergist difference as an indicator of microsomal oxidase activity.     

Host plant induction of microsomal monooxygenase activity in relation to diazinon metabolism and toxicity in larvae of the tobacco budworm Heliothis virescens (F.)

The effect of the wild tomato, Lycopersican hirsutam F. glabratam (accession PI 134417), on susceptibility to and metabolism of diazinon, 0,0-diethyl-0-(2-isopropyl-4-methyl-6-pyrimidinyl) phosphorothioate, in larvae of the tobacco budworm, Heliothis virescans F., was studied. The larvae were over 4-fold more tolerant to diazinon toxicity when fed on leaves of wild tomato than when fed on an artificial diet. Diazinon injected into fifth instar larvae is converted into two chloroform-soluble and five water-soluble metabolites. Degradation of diazinon was faster in larvae fed tomato leaves (88.1%) than in larvae fed on the artificial diet (68.4%). Some oxon (13.0%) was detected in the latter case but none in larvae fed tomato leaves. The major metabolite was hydroxypyrimidine and its formation was higher (73.2%) in larvae fed tomato leaves than in larvae fed the artificial diet (49.2%). In vitro studies revealed that both diazinon and its oxon were metabolized primarily by the microsomal cytochrome P-450-dependent monooxygenase system which was induced 2.5- to 3.7-fold by feeding on tomato leaves. It was concluded that diazinon was degraded in H. virescens larvae through desulfuration, hydroxylation of the ring-alkyl side chain, and oxidative dearylation reactions, all of which were increased by varying amounts after feeding on tomato leaves. Treatment of the larvae with 2-tridecanone, a naturally occurring toxin in tomato leaves, resulted in increased tolerance to diazinon and increased in vitro degradation of diazinon and its oxon, the induction being dependent on the magnitude of 2-tridecanone treatment. The microsomes of tomato fed larvae had a 1.5- to 2.1-fold higher concentration of cytochrome P-450, accompanied by a 1–2 nm shift in the λmax of the cytochrome P-450 carbon monoxide complex.