Furanocoumarin metabolism in Papilio polyxenes: biochemistry,genetic variability,and ecological significance

The ubiquitous occurrence of series of biosynthetically related plant secondary compounds within individual species has given rise to the suggestion that such multiplicity is adaptive; one possible mechanism that would serve to maintain such within-plant diversity is analog synergism. In a series of experiments, we provide evidence that synergism may account for the presence of multiple structurally related furanocoumarins in apiaceous plants. The black swallowtail, Papilio polyxenes, feeds exclusively on plant species containing furanocoumarins. Growth of larvae fed parsley leaves treated with both xanthotoxin and angelicin, two furanocoumarins that co-occur widely in swallowtail hostplants, was significantly slower than that of larvae fed leaves with an equimolar concentration of either xanthotoxin or angelicin. A multivariate combination of growth, food consumption and frass excretion differed significantly between larvae fed leaves treated with both xanthotoxin and angelicin and larvae fed leaves treated with angelicin alone. In addition, we measured rates of in vitro cytochrome P450-mediated metabolism of three furanocoumarins — bergapten, xanthotoxin, and angelicin. While bergapten and xanthotoxin, both linear furanocoumarins, were metabolized at similar rates (8.07 and 9.86 nmoles/min/g fw caterpillar, respectively), angelicin, an angular furanocoumarin, was metabolized more slowly (2.76 nmoles/min/g fw caterpillar). When all three furanocoumarins were assayed together, overall rates of metabolism were significantly reduced, suggesting substrate inhibition. Thus, the pattern of growth of larvae is consistent with the pattern of in vitro metabolism and is evidence in support of analog synergism. In a separate experiment, metabolism of xanthotoxin and angelicin individually and together were compared in six maternal families. Again, angelicin was metabolized more slowly than xanthotoxin and each furanocoumarin inhibited metabolism of the other. That significant family effects were found for rates of metabolism and for the ratio of moles of angelicin metabolized for each mole of xanthotoxin metabolized raises the possibility that genetic variation exists for the rate and specificity of metabolism and suggests that insect herbivores may be able to adapt to analog synergism.     

Selective breeding and characterization of a black mealworm strain of Tenebrio molitor Linnaeus (Coleoptera: Tenebrionidae)

Larvae of Tenebrio molitor Linnaeus are edible insects and are approved as a food ingredient in Korea. They are typically yellow; however, rare black larvae have been found in breeding boxes at insect farms. It is not clear whether black larvae represent a different species that invaded and hybridized with the yellow larvae of T. molitor or whether T. molitor shows intraspecific color variation. In this study, we characterized and identified black larvae for applications in industrial fields as well as accurate breeding and management. First, in a comparative analysis, we did not detect differences in the morphological characteristics of yellow and black larvae and adults, with the exception of larval body color. For accurate species identification, molecular analyses (p-distances and neighbor-joining) were performed based on partial COI sequences of 33 yellow and seven black larvae. Genetic divergence between yellow and black larvae ranged from 0.0% to 2.1%, revealing intraspecific variation. A neighbor-joining analysis strongly supported the classification of the two morphs as a single species. Black larvae were separated from yellow larvae and maintained by selective breeding. As a result, black larvae were completely fixed in the F2 generation (F1 = 96% and F2 = 100%). Yellow and black larvae showed no significant differences in developmental characteristics and fecundity. These findings improve our understanding of diversity within an important edible insect species and contribute to quality assurance in the food industry based on clear species identification.     

Antioxidant enzyme activities in subcellular fractions of larvae of the black swallowtail butterfly,Papilio polyxenes

The black swallowtail butterfly, Papilio polyxenes, larvae are specialized feeders of pro-oxidant rich plants of Apiaceae and Rutaceae. An important defense against toxic forms of oxygen species generated by ingestion of the pro-oxidants, are the antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), GSH-dependent glutathione peroxidases (selenium-dependent glutathione peroxidase [GPOX] and peroxidase activity of selenium-independent glutathione-S-transferase [GT_px]), and glutathione reductase (GR). The subcellular distribution of these enzymes in black swallowtail larvae was investigated and was found to resemble the patterns described for larvae of two other lepidopteran species: the southern armyworm, Spodoptera eridania, and the cabbage looper, Trichoplusia ni. The confinement of SOD in the cytosol and mitochondria was typically eukaryotic, but the relative proportion (1:1) was markedly different from the mammalian pattern (4:1; cytosol:mitochondria). The most obvious difference between the black swallowtail and other lepidoptera as a group, and mammalian species, is in very wide intracellular distributions of CAT, GTpx, and GR in insect species. Insects possess very low levels of a GPOX-like activity which reduces both H₂O₂ and organic peroxides. Consequently, insects have elaborate activities with a wide subcellular distribution of both CAT which decomposes H₂O₂, and GTpx which decomposes organic peroxides. The reduction of peroxides is dependent on GSH, which in this process is oxidized to GSSG. GR which reduces GSSG to GSH is also of wide subcellular distribution, analogous to the distribution pattern of GTpx.     

Glutathione S-transferase activities in phytophagous insects: Induction and inhibition by plant phototoxins and phenols

The effects of two plant phototoxins (xanthotoxin and harmine) and three plant phenols (quercetin, ellagic acid, and juglone) on detoxification enzymes were studied in the polyphagous cabbage looper, Trichoplusia ni, and the oligophagous black swallowtail, Papilio polyxenes. In P. polyxenes, glutathione S-transferase (GST) activities toward 1-chloro-2,4-dinitrobenzene (CDNB) were 1840 and 1750 nmol CDNB conjugate/mg protein/min in the cytosolic fraction of midgut and fat body, respectively. Dietary xanthotoxin (0.1% fw) increased the activity 2.5 and 2.9-fold in the midgut and fat body, respectively. Xanthotoxin-conjugating GST activity was absent in both tissues. In T. ni, GST activity, 513 nmol CDNB conjugate/mg protein/min in the cytosolic fraction of midgut, was increased almost twofold by dietary xanthotoxin and harmine. Plant phenols effectively inhibited in vitro GST and Se-independent glutathione peroxidase (GPOX) activities in a dose-dependent manner in the two species. Both GST and GPOX of P. polyxenes were 2-fold less sensitive to phenol inhibitors than T. ni. GST inhibition differed according to the nature of the inhibitor in P. polyxenes. Quercetin is competitive with CDNB and is non-competitive with respect to GSH. In contrast, inhibition by ellagic acid is non-competitive with CDNB and competitive with GSH. Juglone showed competitive inhibition with both GSH and CDNB.     

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.     

Molecular analysis of CYP321A1, a novel cytochrome P450 involved in metabolism of plant allelochemicals (furanocoumarins) and insecticides (cypermethrin) in Helicoverpa zea

Cytochrome P450 monooxygenases play a significant role in the detoxification of hostplant allelochemicals and synthetic insecticides in Lepidoptera. In the corn earworm Helicoverpa zea, a noctuid of considerable economic importance, metabolisms of xanthotoxin, a toxic furanocoumarin, and alpha-cypermethrin, an insecticide, are mediated by at least one P450 with a catalytic site capable of accepting both substrates. To further the characterization of P450s in this species, we have cloned three full-length cDNAs encoding two CYP4M subfamily members and a novel CYP321A subfamily member. RNA analyses have demonstrated that the CYP321A1 gene is highly induced (51-fold) in larval midguts in response to xanthotoxin but not cypermethrin. Both CYP4M genes are expressed at negligible levels that are not increased by xanthotoxin or cypermethrin. Baculovirus-mediated expression of the full-length CYP321A1 cDNA has demonstrated that the CYP321A1 protein metabolizes xanthotoxin and angelicin, like the CYP6B1 protein in the furanocoumarin specialist Papilio polyxenes, and alpha-cypermethrin, like the CYP6B8 protein previously characterized in H. zea. In contrast, the CYP4M7 protein does not metabolize xanthotoxin at any detectable level. We conclude that at least two xanthotoxin-inducible P450s from highly divergent subfamilies (CYP6B and CYP321A) contribute to the resistance of H. zea larvae to toxic furanocoumarins and insecticides. Genomic PCR analysis indicates that the CYP321A1 gene has evolved independently from the CYP6B genes known to be present in this insect.     

Molecular docking of substrates and inhibitors in the catalytic site of CYP6B1, an insect cytochrome p450 monooxygenase

Furanocoumarins represent plant toxins that are used in the treatment of a variety of skin diseases and are metabolized by cytochrome p450 monooxygenases (p450s) existing in insects such as Papilio polyxenes (the black swallowtail). To elucidate the active site in the CYP6B1 protein that is the principal p450 existing in this species, we have constructed a homology model of it based on sequence and structure alignments with the bacterial CYP102 protein whose crystal structure has been defined and with the insect CYP6B4 protein that also metabolizes furanocoumarins. In the derived CYP6B1 model, Phe116 and His117 in SRS1, Phe371 in SRS5 and Phe484 in SRS6 contribute to the formation of a resonant network that stabilizes the p450's catalytic site and allows for interactions with its furanocoumarin substrates. The first two of these residues are absolutely conserved in all members of the insect CYP6B subfamily and the last two are variable in different members of the CYP6B subfamily. A combination of theoretical and experimental docking analyses of two substrates (xanthotoxin and bergapten) and two inhibitors (coumarin and pilocarpine) of this p450 provide significant information on the positioning of furanocoumarins within this catalytic pocket. Molecular replacement models based on the results of variations at two of these critical amino acids provide support for our furanocoumarin-docked model and begin to rationalize the altered substrate reactivities observed in experimental analyses.     

Cytochrome P-450 from mitochondria of bovine adrenal cortex Comparison of cholesterol side-chain cleavage P-450 with steroid 11β-hydroxylation P-450 and immunochemical cross-reactivity between adrenal mitochondrial and liver microsomal cytochromes P-450

Adrenocortical mitochondrial cytochrome P?450 specific to the cholesterol side-chain cleavage (desmolase) reaction differs from that for the 11β-hydroxylation reaction of deoxycorticosterone. The former cytochrome appears to be more loosely bound to the inner membrane than the latter. Upon ageing at 0°C or by aerobic treatment with ferrous ions, the desmolase P-450 was more stable than the 11β-hydroxylase P-450. By utilizing artificial hydroxylating agents such as cumene hydroperoxide, H₂O₂, and sodium periodate, the hydroxylation reaction of deoxycorticosterone to corticosterone in the absence of NADPH was observed to a comparable extent with the reaction in the presence of adrenodoxin reductase, adrenodoxin and NADPH. However, the hydroxylation reaction of cholesterol to pregnenolone was not supported by these artificial agents.Immunochemical cross-reactivity of bovine adrenal desmolase P-450 with rabbit liver microsomal P-450_LM4 was also investigated. We found a weak but significant cross-reactivity between the adrenal mitochondrial P-450 and liver microsomal P-450_LM4, indicating to some extent a homology between adrenal and liver cytochromes P-450.     

Cross-resistance to alpha-cypermethrin after xanthotoxin ingestion in Helicoverpa zea (Lepidoptera: Noctuidae)

Cytochrome P450 monooxygenases (P450) are membrane-bound hemoproteins that play important roles in conferring protection against both naturally occurring phytochemicals and synthetic organic insecticides. Despite the potential for common modes of detoxification, cross-resistance between phytochemicals and synthetic organic insecticides has rarely been documented. In this study, we examined the responses of a susceptible strain of corn earworm, Helicoverpa zea (Boddie), a polyphagous noctuid, to exposure by an allelochemical infrequently encountered in its host plants and by an insecticide widely used for control purposes. Within a single generation, survivors of xanthotoxin exposure displayed higher levels of tolerance to alpha-cypermethrin than did unexposed control larvae. The F1 offspring of xanthotoxin-exposed survivors also displayed higher alpha-cypermethrin tolerance than did offspring of unexposed control larvae, suggesting that increased alpha-cypermethrin tolerance after xanthotoxin exposure represents, at least in part, heritable resistance. Administration of piperonyl butoxide, a P450 synergist, demonstrated that resistance to both xanthotoxin and alpha-cypermethrin is P450-mediated. Alpha-cypermethrin-exposed survivors, however, failed to show superior growth on xanthotoxin diets. Assays with control larvae, larvae induced by both xanthotoxin and alpha-cypermethrin, and survivors of LD50 doses of both compounds indicated that H. zea midgut P450s are capable of metabolizing both xanthotoxin and alpha-cypermethrin. Metabolism of each compound is significantly inhibited by the presence of the other compound, suggesting that at least one form of P450 in H. zea midguts degrades both compounds and may constitute the biochemical basis for possible cross-resistance. Compared with control larvae, xanthotoxin- and alpha-cypermethrin-induced larvae displayed 2- to 4-fold higher P450-mediated metabolism of both compounds. However, xanthotoxin- and alpha-cypermethrin-exposed survivors exhibited much higher (2.5- to 11-fold) metabolism of both compounds than did the induced larvae. The metabolism results, like the bioassay results, are consistent with the interpretation that increased alpha-cypermethrin tolerance after xanthotoxin exposure is attributable mainly to heritable resistance.     

How caterpillars avoid overheating: behavioral and phenotypic plasticity of pipevine swallowtail larvae

We tested the hypothesis that larvae of the pipevine swallowtail butterfly, Battus philenor, employ behavioral and phenotypic plasticity as thermoregulatory strategies. These larvae are phenotypically varied across their range with predominantly black larvae (southeastern USA and California) and red larvae (western Texas, Arizona) occurring in different regions. Two years of field observations in south Texas indicate that the proportion of red larvae increases with increasing daily temperatures as the growing season progresses. Larvae were also observed to shift their microhabitats by climbing on non-host vegetation and avoided excessive heat in their feeding microhabitat. Larvae of ten half-sib families from populations in south Texas and California, reared under different temperature regimes in common garden experiments, exhibited plasticity in larval phenotype, with larvae from both populations producing the red phenotype at temperatures greater than 30°C and maintaining the black phenotype at cooler temperatures. However, larvae from Texas were more tolerant of higher temperatures, showing no decrease in growth rate in the highest temperature (maximum seasonal temperature) treatment, compared to the California population. In a field experiment, black larvae were found to have higher body temperatures when exposed to sunlight compared to red larvae. These results suggest that microhabitat shifts and the color polyphenism observed in pipevine swallowtail larvae may be the adaptive strategies that enable larvae to avoid critical thermal maximum temperatures.     

6种植物次生物质对斜纹夜蛾解毒酶活性的影响

草食性昆虫取食植物时遇到宿主植物中大量次生物质的化学防御,研究昆虫适应植物毒素的反防御策略具有重要的科学意义。分别添加0.01%肉桂酸、0.01%水杨酸、0.01%花椒毒素、0.02%槲皮素、0.05%黄酮和0.1%香豆素等6种植物次生物质的人工饲料饲养斜纹夜蛾(Spodoptera litura)五龄幼虫48 h后,测定斜纹夜蛾幼虫中肠和脂肪体中谷胱甘肽S-转移酶(GSTs)、羧酸酯酶(CarE)、P450的酶含量及头部乙酰胆碱酯酶(AChE)的活性,利用半定量RT-PCR检测中肠和脂肪体中CYP4M14和CYP4S9的基因表达水平。结果表明:取食肉桂酸和香豆素后,斜纹夜蛾中肠中CarE的酶活性分别提高了1.67和1.37倍,取食6种次生物质均能显著提高斜纹夜蛾脂肪体中GSTs酶活性。取食肉桂酸和香豆素48 h后,脂肪体中P450酶含量比对照增加2.93和14.50倍。取食肉桂酸、花椒毒素、槲皮素和香豆素后,斜纹夜蛾头部AchE酶活性与对照相比提高了1.53、1.80、2.36和1.56倍。6种次生物质均可诱导脂肪体中CYP4M14基因表达,槲皮素、肉桂酸和香豆素强烈诱导CYP4S9在脂肪体中表达。表明,斜纹夜蛾具有利用植物次生物质诱导其解毒酶的能力,进而提高其对毒素的抗性。     

The metabolism of drugs and carcinogens in isolated subcellular fractions of Drosophila melanogaster. II. Enzyme induction and metabolism of benzo[a]pyrene

The effect of various pretreatments on the activities of several drug metabolizing enzymes was investigated in microsomes and postmicrosomal supernatant fractions isolated from whole body homogenates of Drosophila melanogaster larvae of different strains. Pretreatments of larvae with either phenobarbital (PB), β-naphthoflavone (BNF) or a mixture of polychlorinated biphenyls (Aroclor 1254, PCB) for 24 h increased microsomal benzo[a]pyrene (BP) monooxygenase activity 2- to 6-fold in all strains as compared to untreated larvae. A simultaneous increase in the contents of cytochrome P-450 occurred after pretreatment with PB and PCB. Comparison of the turnover rates of BP per molecule of cytochrome P-450 indicated that BP was a poor substrate for control cytochrome P-450 whereas BNF induced a most active hemoprotein for this metabolism. Marked differences in the qualitative pattern of BP metabolites were obtained between microsomes isolated from BNF-treated larvae or rat liver microsomes. 3-Hydroxy-BP (3-OH-BP) was the dominating metabolite with both preparations, while the BP dihydrodiols were formed in minor quantities in Drosophila as compared to rat liver. Metyrapone and SKF 525-A inhibited BP metabolism in microsomes isolated from untreated and BNF treated larvae of all strains. In contrast, α-naphthoflavone (ANF) stimulated the BP monooxygenase activity of microsomes isolated from untreated larvae approx. 3-fold but only slightly influenced the activity of microsomes from BNF treated larvae indicating that the latter species of cytochrome P-450 was less sensitive to ANF.In all strains, PCB and PB treatments approximately doubled microsomal epoxide hydrolase activity and increased cytosolic glutathione-S-transferase activity 25–60%, significant only in strain Berlin K after PB treatment. The activities of epoxide hydrolase and glutathione-S-transferase in control larvae were comparable in the different strains, whereas the content of cytochrome P-450 and BP monooxygenase activity was higher in the Hikone R strain. Variability in the induction response to the various pretreatment was observed among the three strains.     

Sex difference of cytochrome P-450 in the rat: Purification,characterization, and quantitation of constitutive forms of cytochrome P-450 from liver microsomes of male and female rats

One of each constitutive form of cytochrome P-450 from liver microsomes of adult male and female rats was purified essentially following the same method to an apparent homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weights estimated by the electrophoresis were 52,000 and 50,000 for forms of cytochrome P-450, P-450-male, and P-450-female, purified from male and female rats, respectively. In addition, the purified preparations of P-450-male and P-450-female showed properties different from each other with respect to spectral characteristics and catalytic activities. In Ouchterlony double diffusion plates, partially purified rabbit immunoglobulin G (IgG) raised against P-450-male and P-450-female showed very weak or no cross-reactivity with P-450-female and P-450-male, respectively. From these results, P-450-male was confirmed to be a form distinct from P-450-female. The anti-P-450-male and anti-P-450-female antibodies, which had been further purified by immunoadsorption, did not form any apparent precipitation bands with liver microsomes from untreated female and male rats, respectively. Supporting this, radial immunodiffusion analysis for P-450-male and P-450-female with an agarose gel impregnated with the rabbit antibodies showed that P-450-male and P-450-female appear in liver microsomes rather specifically depending on the sex hormones. Based on these results, sex differences in drug metabolism in the rat were confirmed as explicable, at least in part, by the presence of distinct forms of cytochrome P-450 in microsomes of male and female rats.     

Immunological evidence for phenobarbital-stimulated synthesis of cytochrome P-450 in primary cultures of chick embryo hepatocytes

The induction of cytochrome P-450 by phenobarbital was studied in primary cultures of chick embryo hepatocytes. The rate of the de novo synthesis of the induced form of cytochrome P-450 was measured directly and specificially, using form-specific anti-cytochrome antibodies that quantitatively immunoprecipitated this form from the radiolabeled hepatocytes. Additionally, the steady-state levels of the cytochrome were estimated spectrophotometrically and electrophoretically. In the presence of phenobarbital the synthesis of cytochrome P-450_PB by cultured hepatocytes was markedly accelerated. Furthermore, the same cytochrome P-450_PB form was induced by phenobarbital in vivo in chicken liver and in the cultured chick embryo hepatocytes. Their identity was judged from immunological and electrophoretic properties of these induced cytochromes. Immunological cross-reactivity was also detected between the cytochrome P-450_PB forms from chick embryo hepatocytes and from adult rat liver. The immunological cross-reactivity observed between the phenobarbital-induced cytochrome P-450 forms from different species was not observed between the different cytochrome forms with the same liver (Thomas, P.E., Reik, L.M., Ryan, D.E. and Levin, W. (1981) J. Biol. Chem. 256, 1044–1052). Implications as to the evolutionary origin of the different cytochrome forms are discussed.     

Cytochromes P450 from Papilio polyxenes: Adaptations to host plant allelochemicals

1. Papilio polyxenes, a caterpillar which feeds on xanthotoxin-containing plants, has cytochromes P450 that are six- to 100-fold less sensitive to the suicide substrate inhibitor, xanthotoxin, than cytochromes P450 from Manduca sexta, which does not survive on xanthotoxin-containing plants.2. Xanthotoxin is a suicide substrate inhibitor of O-demethylation of p-nitroanisole by M. sexta microsomes but a reversible inhibitor of O-demethylation by P. polyxenes microsomes.3. Aldrin epoxidation is irreversibly inhibited by xanthotoxin in both species.4. Patterns of cross inhibition demonstrate that O-demethylase and aldrin epoxidase from both species and the P. polyxenes xanthotoxin-metabolizing cytochrome P450 are distinct enzymes.