Expression of cytochrome P-450lpr Is developmentally regulated and limited to house fly

Expression of house fly cytochrome P-450_lpr was examined using immunoblotting in male and female adult LPR house flies, mixed sex adult house flies at 12 different ages, larvae, and pupae. P-450_lpr was expressed in both male and female adult house flies. P-450_lpr was clearly present in all adult stages examined, was barely detectable in pupae, and could not be detected in larvae. Thus, cytochrome P-450_lpr is developmentally regulated and present in both sexes of house fly. Expression of cytochrome P-450, immunologically homologous to house fly cytochrome P-450_lpr was examined in other species using immunoblot analysis. Eleven animal species were tested in the orders Diptera, Hymenoptera, Lepidoptera, Orthoptera, Acari, and Rodentia, using microsomes in some species from both induced and noninduced animals or insecticide-resistant and susceptible strains. P-450_lpr appears to be restricted to house flies, as none of these species contained cytochrome P-450 that reacted with antiserum to cytochrome P-450_lpr.     

The solubilization and separation of two forms of microsomal cytochrome P-450 from the house fly, Musca domestica l.

Microsomes prepared from house fly abdomens were solubilized with sodium deoxycholate and resolved on a DEAE cellulose column into two fractions containing cytochrome P-450. On the basis of spectral characteristics the cytochrome in a fraction eluting with 0.3M KCl is different from that eluting with 0.5M KCl. The two forms of P-450 were found in both susceptible and resistant strains of house flies. The P-450 in the 0.5M KCl eluting fraction appears to be more unstable than that in the 0.3M fraction. These results are in disagreement with those of other workers who have concluded that only one of two possible forms of cytochrome P-450 is present in a given strain of the house fly.     

Anti-P450lpr antiserum inhibits specific monooxygenase activities in LPR house fly microsomes.

Monooxygenase activity in microsomes from the LPR strain of house fly (Musca domestica L.) was inhibited by anti-P450lpr, and antiserum specific for house fly cytochrome P450lpr. Anti-P450lpr did not inhibit house fly cytochrome P450 reductase or rat cytochrome P450 monooxygenase assays, consistent with specific inhibition of P450lpr. Anti-P450lpr inhibited the ability of cytochrome P450 reductase to reduce carbon monoxide treated LPR microsomal cytochrome P450, up to 49% of the total, showing that inhibition of cytochrome P450 reduction is the major mechanism of inhibition. Anti-P450lpr inhibited 98% of methoxyresorufin-O-demethylase activity and all the benzo(a)pyrene hydroxylase activity in LPR microsomes, but none of the pentoxyresorufin-O-dealkylase activity. The antiserum partially inhibited ethoxyresorufin-O-dealkylase and ethoxycoumarin-O-dealkylase activity. These results demonstrate that methoxyresourfin-O-demethylase activity and benzo(a)pyrene hydroxylase activity are characteristic substrates for P450lpr activity in the LPR strain of house fly.     

Differences in the cytochrome P-450S from resistant and susceptible house flies

A comparison of difference spectra formed with microsomal cytochrome P-450 from a susceptible and a resistant strain of house flies shows both quantitative and qualitative differences. The differences are similar to those observed for the same spectra between normal and phenobarbital- or 3-methylcholanthrene-treated mammals. The relationship of these findings to resistance, synergism, and cytochrome P-450 in the house fly is discussed.     

Adult specific expression and induction of cytochrome P450tpr in house flies

Cytochrome P450_tpr is a xenobiotic metabolizing P450 that is found in house flies (Musca domestica). To better understand the regulation of cytochrome P450_tpr, the effects of 21 potential monooxygenase inducers were examined for their ability to induce total cytochromes P450 and cytochrome P450_tpr levels in adult flies. Six compounds caused induction of total cytochromes P450 per mg protein in adult susceptible (CS) house flies: ethanol (1.6-fold), phenobarbital in food (1.5-fold) or water (1.5-fold), naphthalene (1.3-fold), DDT (1.3-fold), xanthotoxin (1.4-fold), and α-pinene (1.2-fold). Six compounds were found to be inducers of cytochrome P450_tpr: piperonyl butoxide in food (1.9-fold), phenobarbital in food (1.4-fold) and water (3.4-fold), clofibrate (1.3-fold), xanthotoxin (1.3-fold), methohexital (1.3-fold), and isosafrole (1.3-fold). Comparison of our results with house fly P450 6A1 indicates that there are specific inducers for each of these individual P450s as well as compounds that induce both P450s. Total P450s were inducible by PB in CS house fly larvae, but not in LPR larvae. Immunoblotting revealed no detectable P450_tpr in control or PB-treated larvae in either strain. Thus, although total P450s are inducible in the susceptible strain larvae, P450_tpr does not appear to be normally present or inducible with PB in larvae of either strain. Northern blots of phenobarbital (in water) treated CS flies indicated that there was a 4.2-fold increase in the P450_tpr (i.e., CYP6D1) mRNA levels over the untreated flies. In the multiresistant LPR strain there was no apparent induction of CYP6D1 mRNA by phenobarbital. Following phenobarbital induction, the level of CYP6D1 mRNA in the CS strain was about half of the level in the LPR strain. © 1996 Wiley-Liss, Inc.     

Partial purification and characterization of phenobarbital-induced house fly cytochrome P-450

Two forms of phenobarbital-induced cytochrome P-450 were partially purified from the Rutgers diazinon-resistant strain of house fly using cholate solubilization, polyethylene glycol 6000 precipitation, and chromatography on DEAE cellulose. The preparation of highest purity had an absorbance maximum of 452 nm, a specific content of 10.0 nmol/mg protein, and an apparent molecular weight of 60,000 when examined by sodium dodecyl sulfate polyacrylamide electrophoresis. The yield of the highly purified cytochrome P-450 was 2–3%. This form contained proportionately less cytochrome P-420 than the original cholate solubilized microsomes, and is thus apparently more stable. A second form of cytochrome P-450 having a specific content of 0.50–0.89 nmol/mg protein was eluted from DEAE cellulose with a 0-0.25 M salt gradient. This is consistent with a previously reported elution pattern for Emulgen 913-solubilized house fly microsomes. Several methods of solubilizing house fly microsomes were examined. High salt, 2M KCI, in the absence of detergents effectively solubilized cytochrome P-450 (50–70% recovery) with little or no conversion to cytochrome P-(420).     

Comparison of cytochrome P-450-dependent metabolism in different developmental stages of Drosophila melanogaster

The activities of several drug metabolizing enzymes were compared in microsomes from larvae and adult Drosophila. The cytochrome P-450 content and the benzo[a]pyrene (BP) hydroxylation, p-nitroanisole demethylation and 3- and 4-hydroxylation of biphenyl were 4-20-fold higher in microsomes from adult flies, while 7-ethoxycoumarin deethylase activity and cytochrome c reductase activity were about the same in the two stages. 2-OH-biphenyl was formed in trace amounts by microsomes from adult flies but not to any detectable amount by microsomes from larvae. Pretreatment with phenobarbital (PB), Aroclor 1254 (PCB) or beta-naphthoflavone (BNF) increased the cytochrome P-450 content and the various cytochrome P-450-mediated reactions up to 7-fold in larvae. The effects of the pretreatments were weaker in adult flies, where the increase never was more than 3-fold, and many reactions were unaffected by the pretreatments. BNF was thus inefficient in enhancing all reactions, except a slight (1.3-fold) increase in the formation of 4-OH-biphenyl. Microsomes from both stages exhibited increases in specific protein bands with apparent molecular weights of 51 000-58 000 in the sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis following treatment with PB, PCB and BNF. Differences were observed between larvae and adults with respect both to the number of and the molecular weights of the increased protein bands.     

Commercial and naturally occurring fly parasitoids (Hymenoptera: Pteromalidae) as biological control agents of stable flies and house flies (Diptera: Muscidae) on California dairies

Filth fly parasites reared by commercial insectaries were released on two dairies (MO, DG) in southern California to determine their effect on populations of house flies, Musca domestica L., and stable flies, Stomoxys calcitrans (L.). Spalangia endius Walker, Muscidifurax raptorellus Kogan and Legner, and Muscidifurax zaraptor Kogan and Legner were released on the MO dairy from 1985 to 1987 in varying quantities. Parasitism by Muscidifurax zaraptor on the MO dairy was significantly higher (P less than 0.05) from the field-collected stable fly (4.4%) and house fly (12.5%) pupae, compared with a control dairy (0.1%, stable fly; 1.3%, house fly). Muscidifurax zaraptor, released from April through October during 1987 on the DG dairy (350,000 per month), was not recovered in a significantly higher proportion from either fly species relative to the corresponding control dairy. No specimens of Muscidifurax raptorellus were recovered from the MO dairy. Parasite treatments had no apparent effect on adult populations of either fly species or on overall parasitism rate of field-collected stable fly (16.8%, MO; 17.2%, DG) and house fly (23.3%, MO; 20.9%, DG) pupae. Spalangia spp. were the predominant parasites recovered from field-collected stable fly and house fly pupae on all four dairies. Sentinel house fly pupae placed in fly-breeding sites on both release dairies were parasitized at a significantly higher rate, as compared with sentinel pupae on control dairies. The generic composition of parasites emerging from sentinel house fly pupae was 20.6% Spalangia spp. and 73.2% Muscidifurax spp., whereas in field-collected house fly pupae, Spalangia spp. and Muscidifurax spp. constituted 74.3 and 19.6% of the parasites, respectively.     

Avian kidney mitochondrial hemeprotein P-4501 alpha: isolation, characterization and NADPH-ferredoxin reductase-dependent activity

We describe the isolation of cytochrome P-4501 alpha from chick-kidney mitochondria. Although, gel permeation HPLC yielded 41% of the total amount of P-450 present in cholate-solubilized hemeproteins, it produced a highly purified mixture from which the P-4501 alpha could be purified to homogeneity in a final detergent-free state by a single-step application of hydrophobic interaction HPLC using hydroxypropyl silica. The purified P-4501 alpha traveled as a single band in SDS gel electrophoresis with an apparent Mr = 57,000. The absolute spectrum of the P-4501 alpha (Fe3+) form gave a lambda max at 403 nm. This characteristic lends support to the anomalous high-spin heme electron paramagnetic resonance spectrum and the heme structure of P-4501 alpha which we have previously reported (Ghazarian et al. (1980) J. Biol. Chem. 255, 8275-8281; Pedersen et al. (1976) J. Biol. Chem. 251, 3933-3941). In reconstitution experiments with ferredoxin-dependent NADPH-cytochrome c (P-450) reductase complexes, P-4501 alpha catalyzed the hydroxylation of 25-hydroxy-9,10-secocholesta-5,7,10(19)-trien-3 beta-ol at the C-1 position exclusively with a turnover number of 0.03 min-1. This number is identical to that obtained from measurements of the catalytic activity in intact mitochondria, indicating that only one major species of cytochrome P-450 occurs in chick-kidney mitochondria. The complete responsiveness of cytochrome P-450 concentrations in intact mitochondria to the vitamin D status of chicks provided additional evidence that the major cytochrome P-450 species present in renal mitochondria is uniquely associated with vitamin D metabolism.     

Neonatal imprinting and hepatic cytochrome P-450 II. Partial purification of a sex-dependent and neonatally imprinted form(s) of cytochrome P-450

A new form of cytochrome P-450 was partially purified from hepatic microsomes of neonatally imprinted rats (adult male and adult male castrated at four weeks of age). This new form of cytochrome P-450 appears to have an apparent molecular weight of approximately 50,000 daltons as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It appears that this form of cytochrome P-450 is either absent or present in low concentrations in cytochrome P-450 preparations isolated from neonatally nonimprinted rats (adult female and adult male castrated at birth). Reconstitution of testosterone hydroxylase and benzphetamine N-demethylase activities of this partially purified cytochrome P-450 revealed that the presence of testosterone 16α-hydroxylase activity, an imprintable microsomal enzyme, was in parallel with the imprinting status of the animals; a significantly higher activity was detected in the neonatally imprinted than that of the nonimprinted animals. This was in contrast to the nonimprintable benzphetamine N-demethylase, testosterone 7α-and 6β-hydroxylase activities which exhibited no correlation with the imprinting status of the animals. We have prepared antisera from rabbits using the partially purified cytochrome P-450 preparations from adult male rats as antigens. These antisera inhibited microsomal testosterone 16α- and 7α-hydroxylase activities in a concentration-dependent manner, without impairing 6β-hydroxylase activity. These data suggest that the partially purified cytochrome P-450 from adult male rats consists of both imprintable (16α-) and nonimprintable (7α-) testosterone hydroxylase activities. The antisera formed immunoprecipitant lines in the Ouchterlony double diffusion plates with partially purified cytochrome P-450 from both neonatally imprinted and nonimprinted adult rats. The immunoprecipitant lines, as stained by coomassie blue, suggest the homology of the cytochrome P-450 preparations from neonatally imprinted and nonimprinted rats. Immunoabsorption of the antisera against neonatally nonimprinted, partially purified cytochrome P-450 completely removed the immunoprecipitant lines without appreciably impairing the inhibitory effects of antisera on the microsomal testosterone 16α-and 7α-hydroxylase activities. In contrast, immunoabsorption of the antisera against partially purified cytochrome P-450 from adult male rats (imprinted) abolished completely both the immunoprecipitant lines and the inhibition on microsomal testosterone hydroxylation reaction (16α and 7α). The inhibitory actin of antisera on testosterone hydroxyulation was also abolished upon boiling the antisera at 100°C for 5 minutes. The biochemical and immunochemical data in this study suggest that the neonatally imprintable form or forms of hepatic microsomal cytochrome P-450 accounts for a small fraction of the bulk of total cytochrome P-450. However, the existence of this form of cytochrome P-450 is regulated by gonadal hormones during the neonatal period and accounts for the major imprintable sex difference in drug and steroid metabolism in adulthood.     

Preparation of homogenous NADPH cytochrome c (P-450) reductase from house flies using affinity chromatography techniques.

NADPH-cytochrome c (P-450) reductase (EC 1.6.2.4) was purified to apparent homogeneity from microsomes of house flies, Musca domestica L. The purification procedure involves column chromatography on three different resins. The key step in the purification scheme is the chromatography of the enzyme mixture on an affinity column of agarose-hexane-nicotinamide adenine dinucleotide phosphate. The enzyme has an estimated molecular weight of 83,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contains 1 mol each of FAD and FMN per mol of enzyme. The enzyme exhibited a Bi Bi ping-pong kinetic mechanism with NADPH and cytochrome c. The Vmax and Km for cytochrome c were 42.3 mumol min-1 mg-1 and 12.7 muM, respectively. Turnover numbers based on micromoles of enzyme were 2,600 min-1. NADP+ and 2'-AMP both inhibited the reductases with apparent Ki values of 6.9 and 187 muM, respectively. These preparations of NADPH-cytochrome c reductase were found to reduce purified house fly cytochrome P-450 in the presence of NADPH.     

Effects of host age, host density and parent age on reproduction of the filth fly parasite Urolepis rufipes (Hymenoptera: Pteromalidae)

Urolepis rufipes Ashmead, a pteromalid wasp, was recently discovered parasitizing house fly and stable fly pupae in eastern Nebraska dairies. Studies have been conducted on the biology of this parasite to evaluate its potential as a biological control agent of stable flies (Stomoxys calcitrans (L.] and house flies (Musca domestica L.). House fly pupae were suitable as hosts for U.rufipes at all ages; however, significantly higher parasitism occurred on host pupae aged 96-120 h. Parasite-induced mortality (host mortality without progeny production) was higher than for other pteromalid parasites of filth flies under similar conditions. Parasitism increased with parasite--host ratio at 20 degrees C; however, the opposite was noted at 30 degrees C for parasite--host ratios ranging from 5:50 to 50:50. Fly eclosion decreased as parasite--host ratio increased at 20 degrees C, and no host eclosion occurred at the highest parasite--host ratios (20:50 and 50:50) at 30 degrees C. Females produced an average of 18.6 female and 7.6 male progeny. 88% of the progeny were produced during the first 6 days post parental eclosion. The short life span, low progeny emergence rate and high per cent host eclosion, in comparison with other parasite species, suggests that the Nebraska strain of U.rufipes may not an effective biological control agent of house flies.     

Parasites that attack stable fly and house fly (Diptera: Muscidae) puparia during the winter on dairies in northwestern Florida

Throughout the winter and early spring months, stable fly, Stomoxys calcitrans (L.), and house fly, Musca domestica L., puparia were collected from silage, hay, and manure from six dairies in northwestern Florida and evaluated for parasitism. Of the puparia producing flies or parasites, 23% of the stable flies and 46% of the house flies were parasitized. The predominant parasite observed attacking muscoid flies (76% for stable flies and 58% for house flies) was Spalangia cameroni Perkins. Muscidifurax sp. was recovered from 11 and 36% of the stable fly and house fly pupae, respectively. Other parasite species encountered were Spalangia endius Walker and S. nigroaenea Curtis. Significantly more parasitized fly pupae were collected from silage than from hay residues or manure. Winter and early spring parasite populations in northwestern Florida appear to be present as long as viable fly pupae are available to support the developing parasites.     

NADPH: cytochrome P-450 reductase in olfactory epithelium. Relevance to cytochrome P-450-dependent reactions.

The presence of a very active cytochrome P-450-dependent drug-metabolizing system in the olfactory epithelium has been confirmed by using 7-ethoxycoumarin, 7-ethoxyresorufin, hexobarbitone and aniline as substrates, and the reasons for the marked activity of the cytochrome P-450 in this tissue have been investigated. The spectral interaction of hexobarbitone and aniline with hepatic and olfactory microsomes has been examined. By this criterion there was no evidence for marked differences in the spin state of the cytochromes of the two tissues, or for the olfactory epithelium containing a greater amount of cytochrome capable of binding hexobarbitone, a very actively metabolized substrate. Rates of NADPH and NADH: cytochrome c reductase activity were found to be higher in the olfactory epithelium than in the liver, and direct evidence was obtained for a greater amount of the NADPH-dependent flavoprotein in the olfactory microsomes. Investigation of male rats and male and female mice, as well as male hamsters, demonstrated that, in all cases, the cytochrome P-450 levels of the olfactory epithelium were lower than those of the liver, while the 7-ethoxycoumarin de-ethylase and NADPH:cytochrome c reductase activities were higher. A correlation was found between 7-ethoxycoumarin de-ethylase and NADPH:cytochrome c reductase activities for both tissues in all species examined. The ratio of reductase to cytochrome P-450 was found to be considerably higher in the olfactory epithelium (1:2-1:3) than in the liver (1:11-1:15), regardless of the species examined, suggesting that facilitated electron flow may contribute significantly to the cytochrome P-450 catalytic turnover in the olfactory tissue.     

Larval Competition, Adult Fitness, and Reproductive Strategies in the Acoustically Orienting Ormiine Homotrixa alleni (Diptera: Tachinidae)

Homotrixa alleni is a gregarious endoparasitoid fly that attacks adult male Sciarasaga quadrata (Orthoptera: Tettigoniidae) in southwestern Western Australia. Gravid female flies acoustically orient to their host's call and deposit live first-instar larvae upon or near their calling host. Up to 16 larvae may be found developing in the one host, and since only calling adult male S. quadrata are parasitized, host size and hence larval resources are essentially fixed at parasitism. This study examines parasitism by H. alleni in relation to intraspecific larval competition and adult fitness. The mean number of larvae emerging per host failed to increase significantly beyond a clutch size of four. Mean pupal weight and survival to the adult stage decreased linearly with increasing clutch size across the entire range of clutch sizes examined. Within a clutch, heavier pupae successfully completed pupal development significantly more often than lighter pupae. Pupal weight was directly related to adult size, with adult males being significantly larger than adult females at any given pupal weight. Female body size was positively correlated with fecundity. The size distribution of emerging females was normally distributed, while the distribution of searching gravid females collected at acoustic traps in the field was significantly skewed toward larger flies, suggesting yet another fitness benefit associated with large size. Using fecundity and survival to adulthood as our measure of fitness we calculated the optimal clutch size maximizing fitness per host to be seven, which exceeds the majority of observed clutch sizes in the field. Uncertainties associated with larvae successfully entering the host following larviposition are likely to reduce clutch sizes of H. alleni below this optimum in the field.     

On the intracellular localization of the ecdysone 20-monooxygenase in Musca domestica. L

The cytochrome P-450-dependent 20-monooxygenation of ecdysone is catalyzed both by mitochondria and microsomes isolated from Musca domestica (L.) larvae; however, about 50% of the activity is associated with mitochondria, and 37% is associated with microsomes. Pretreatment of larvae with ecdysone results in an increase in Vmax and a decrease in Km values in mitochondria but not in microsomes. Phenobarbital, a known cytochrome P-450 inducer, increases the cytochrome P-450 levels in microsomes without affecting the 20-monooxygenase activity, but both the cytochrome P-450 levels and monooxygenase activity are depressed in mitochondria from phenobarbital-pretreated larvae. The ecdysone 20-monooxygenase activity is equally distributed between mitochondria and microsomes in adult insects. Pretreatment of the insects with ecdysone does not significantly modify the 20-monooxygenase activity of either mitochondrial or microsomal fractions, but the cytochrome P-450 levels are reduced in mitochondria. Phenobarbital also depresses the mitochondrial cytochrome P-450 levels while markedly increasing the microsomal cytochrome P-450 levels. However, no significant changes in ecdysone 20-monooxygenase activity are produced by phenobarbital pretreatment. The effects of ecdysone on adult cytochrome P-450 are mostly evidenced in mitochondria isolated from females, whereas in males the changes are not statistically significant. It is concluded that the mitochondrial ecdysone 20-monooxygenase is under regulatory control by ecdysone in the larval stage, which suggests that only the mitochondrial activity has a physiological role during insect development in M. domestica. In adults, both the mitochondrial and microsomal ecdysone 20-monooxygenase activities are not responsive to ecdysone, which, coupled to their high Km values, indicates that the reaction may not be of physiological importance in adult insects and that the mitochondrial cytochrome P-450 species being depressed by ecdysone in females are possibly not involved in ecdysone metabolism.     

Regulation of cytochrome P-450j, a high-affinity N-nitrosodimethylamine demethylase, in rat hepatic microsomes

Polyclonal antibodies were produced in rabbits against purified cytochrome P-450j isolated from isoniazid-treated adult male rats. The monospecificity of immunoadsorbed antibody to cytochrome P-450j was demonstrated by Ouchterlony double diffusion analyses, enzyme-linked immunosorbent assays, and immunoblots. Immunoquantitation results indicated that rat liver microsomal cytochrome P-450j content decreases between 3 and 6 weeks of age in both the male and female animal. Several xenobiotics, such as Aroclor 1254, mirex, and 3-methylcholanthrene, repressed cytochrome P-450j levels when administered to male rats. Isoniazid, dimethyl sulfoxide, pyrazole, 4-methylpyrazole, and ethanol were inducers of cytochrome P-450j in rat liver although these compounds showed different inducing potencies. Microsomes from adult male rats with chemically induced diabetes also contained elevated levels of cytochrome P-450j compared to untreated animals. Cytochrome P-450j levels were measurable in kidney, whereas this isozyme was barely detectable in lung, ovaries, and testes; however, extrahepatic cytochrome P-450j was inducible by isoniazid. Approximately 80-90% of microsomal N-nitrosodimethylamine demethylation was inhibited by antibody to cytochrome P-450j whether the microsomes were isolated from untreated rats or animals administered inducers or repressors of cytochrome P-450j. The residual catalytic activity resistant to antibody inhibition may be a reflection of the inaccessibility of a certain amount of cytochrome P-450j due to interference by NADPH-cytochrome P-450 reductase based on results obtained with the reconstituted system. There was a good correlation (r2 = 0.87) between cytochrome P-450j content and N-nitrosodimethylamine demethylase activity in microsomes from rats of different ages and treated with various xenobiotics. The evidence presented indicates that cytochrome P-450j is the primary, and perhaps sole, microsomal catalyst of N-nitrosodimethylamine demethylation at substrate concentrations relevant to hepatocarcinogenesis induced by N-nitrosodimethylamine.     

Role of microsomal cytochrome P-450 in the formation of ecdysterone in larval house fly.

1. Six cytochrome P-450 species have been purified to varying extents from microsomes obtained from ecdysone-induced house fly larvae by the use of octylamino Sepharose-4B, Synchropak AX-300, Synchropak CM-300 and TSK-DEAE-5 PW column chromatography. 2. One of the fractions apparently corresponded to a mixture of low- and high-spin cytochrome P-450 as judged by spectral characteristics. 3. Molecular weights of the cytochrome P-450 species ranged from 50,000 to 57,000. 4. In a reconstituted system, all the microsomal species hydroxylated ecdysone at rates within the range of microsomal suspensions, as it occurs with mitochondrial fractions 1, 2, 3, 5, and 6 (Srivatsan et al., 1990, Biochem, biophys. Res. Commun. 166, 1372-1377); whereas, mitochondrial fraction 4 hydroxylates ecdysone at significantly higher rates. 5. It is postulated that the 20-monooxygenation of ecdysone is a mitochondrial event which requires the induction of a low-Km cytochrome P-450 species by ecdysone. 6. Microsomal hydroxylation of ecdysone may not be of physiological significance, as Km values for the reaction are above the normal concentrations of the hormone and the activity is not inducible by ecdysone (Agosin et al., 1988, Arch. Insect Biochem. Physiol. 9, 107-117).     

High activity of cytochrome P-450-linked aminopyrine N-demethylase in mouse brain microsomes, and associated sex-related difference.

The presence of cytochrome P-450 and associated mono-oxygenase activities was examined in brain microsomes from male and female mice. Although the cytochrome P-450 level in male mouse brain was very low as compared with mouse liver, the aminopyrine N-demethylase and morphine N-demethylase specific activities in male mouse brain were much higher than those observed in mouse liver. Ethoxycoumarin O-de-ethylase and aniline hydroxylase activities were, however, not detected in mouse brain. Sex-related differences were observed in both the cytochrome P-450 levels and aminopyrine N-demethylase activity in mouse brain, the levels of both being higher in male mouse brain as compared with female mouse brain. Aminopyrine N-demethylase activity in mouse brain microsomes was dependent on the presence of oxygen and NADPH and could be inhibited by piperonyl butoxide, N-octyl imidazole and carbon monoxide. Antiserum raised to the phenobarbital-inducible form of rat liver cytochrome P-450 [P-450(b+e)] inhibited mouse brain aminopyrine N-demethylase activity by around 80+ mouse brain microsomal protein exhibited cross-reactivity against this antiserum when examined by Ouchterlony double diffusion and immunoblotting. The present results indicate the presence of a phenobarbital-inducible form of cytochrome P-450 (or a form of cytochrome P-450 that is similar immunologically) in mouse brain microsomes, which is associated with a sex-related difference.     

Susceptibility of black soldier fly (Diptera: Stratiomyidae) larvae and adults to four insecticides

Dosage-mortality regressions were determined for black soldier fly, Hermetia illucens (L.), larvae fed cyromazine or pyriproxifen treated media. Cyromazine LC50 for larvae dying before becoming prepupae ranged from 0.25 to 0.28 ppm with dosage-mortality regression slopes between 5.79 and 12.04. Cyromazine LC50s for larvae dying before emergence ranged from 0.13 to 0.19 ppm with dosage-mortality regression slopes between 3.94 and 7.69. Pyriproxifen dosage-mortality regressions were not generated for larvae failing to become prepupae since <32% mortality was recorded at the highest concentration of 1,857 ppm. LC50s for larvae failing to become adults ranged from 0.10 to 0.12 ppm with dosage mortality-regression slopes between 1.67 and 2.32. Lambda-cyhalothrin and permethrin dosage-mortality regressions were determined for wild adult black soldier flies and house flies, Musca domestica L., and for susceptible house flies. Our results indicate that the wild house fly, unlike the black soldier fly, population was highly resistant to each of these pyrethroids. Regression slopes for black soldier flies exposed to lambda-cyhalothrin were twice as steep as those determined for the wild house fly strain. Accordingly, LC50s for the black soldier fly and susceptible house fly were 10- to 30-fold lower than those determined for wild house flies. The differential sensitivity between wild black soldier flies and house flies might be due to behavioral differences. Adult house flies usually remain in animal facilities with the possibility of every adult receiving pesticide exposure, while black soldier fly adults are typically present only during emergence and oviposition thereby limiting their exposure.