Richard T. Mayer

Ethoxy-, pentoxy- and benzyloxyphenoxazones and homologues: a series of substrates to distinguish between different induced cytochromes P-450

The individual members of a homologous series of phenoxazone ethers related to ethoxyresorufin were O-dealkylated, and the parent compound phenoxazone was ring-hydroxylated, each at different rates with hepatic microsomes of untreated rats. A structure-activity relationship (SAR) was plotted, relating the rate of O-dealkylation to the length and type of the ether side-chain. Phenobarbitone (PB), 3-methylcholanthrene (MC), Aroclor 1254 (ARO), isosafrole (ISO) and SKF-525A each induced preferentially the O-dealkylation of different members of the homologous series, resulting in the appearance of 5 different SAR plots, which characterized and differentiated between the 5 different inducers. beta-Napthoflavone (BNF) had a similar effect to MC, whereas pregnenolone 16 alpha-carbonitrile treatment caused no large change in the metabolism of any of the substrates tested. For characterizing the effects of the different inducers it was largely sufficient to compare the O-dealkylations of just 4 of the ethers: methoxy-, ethoxy-, pentoxy- and benzyloxyphenoxazone. Very high degrees of induction were seen. MC and ARO each induced preferentially the O-dealkylation of ethoxyphenoxazone (51- and 61-fold respectively). PB and SKF-525A each induced preferentially the O-dealkylation of pentoxyphenoxazone (283- and 324-fold respectively). ISO induced preferentially the O-dealkylation of benzyloxyphenoxazone (43-fold). For any particular induced type of microsomes the substrate with the fastest metabolism was not necessarily the substrate whose metabolism was induced the most, so that in order to characterize each of the 5 different inducers (PB, MC/BNF, ARO, ISO, SKF) it was necessary to compare both the degrees of induction and the specific activities of the reactions. Experiments with purified cyt. P-450 isozymes showed that ethoxyphenoxazone and pentoxyphenoxazone were highly selective substrates for the major isozymes induced by MC and PB respectively, whilst benzyloxyphenoxazone was a good substrate for both isozymes. Experiments using the organic inhibitors metyrapone and alpha-naphthoflavone and inhibitory antibodies against individual cyt. P-450 isozymes indicated that similar substrate selectivities occurred with the monooxygenase system in the microsomal membrane. It is suggested that the use of some or all of these homologous phenoxazone ethers will provide both a simple routine test for the characterization of several types of inducing agents and a powerful tool for investigating the biochemical basis for cyt. P-450 isozyme substrate selectivity.

Dealkylation of pentoxyresorufin: A rapid and sensitive assay for measuring induction of cytochrome(s) P-450 by phenobarbital and other xenobiotics in the rat

The O-dealkylation of pentoxyresorufin (7-pentoxyphenoxazone) by rat liver microsomes was examined. The reaction appeared highly specific for certain phenobarbital inducible forms of cytochrome P-450 and was increased 95- to 140-fold by animal pretreatment with phenobarbital (75 mg/kg/day, four ip injections) and approximately 50-fold by Aroclor 1254 (500 mg/kg, one ip injection) while animal pretreatment with 3-methylcholanthrene (50 mg/kg/day, three ip injections) resulted in less than a 2-fold increase over the rate detected in control microsomes. It was observed that this activity, in microsomes for Aroclor-pretreated rats, was dependent on O2 and was inhibited by metyrapone and SKF 525-A, indicative of cytochrome(s) P-450 mediation in the reaction. When antibodies directed against purified cytochrome(s) P-450s were employed to inhibit the pentoxyresorufin O-dealkylation reaction, antibodies to P-450PB-B greatly inhibited the reaction (greater than 90%), while antibodies to P-450PB-C or P-450PB/PCN-E had minimal effects. Assay of hepatic microsomes from rats which were pretreated with varying doses of phenobarbital (0.9-75 mg/kg/day, four ip injections) indicated that while aminopyrine-N-demethylase activity was induced only 2-fold at the maximum dose (75 mg/kg/day), pentoxyresorufin O-dealkylase activity was induced approximately 140-fold at this dose and approximately 4-fold by a dose of phenobarbital as low as 0.9 mg/kg.

The Role of Plant Rapidly Induced Responses in Asymmetric Interspecific Interactions Among Insect Herbivores

The role of induced responses of tomato, Lycopersicon esculentum, in interspecific interactions between two polyphagous herbivores, the silverleaf whitefly, Bemisia argentifolii (WF), and the vegetable leafminer, Liriomyza trifolii (LM), was characterized in laboratory and field experiments. Feeding by LMs and WFs induced local and systemic production of putative defensive proteins, i.e., chitinases, peroxidases, β-1,3-glucanases, and lysozymes. The magnitude of the induction for each defensive protein varied between species. Unlike WFs, LMs caused a 33% local reduction in total foliar protein content. In a whole-plant choice experiment, adult LM feeding, oviposition, and larval survival were reduced by 47.7%, 30.7%, and 26.5%, respectively, for the WF-infested host compared with the controls. Early WF infestations also had negative systemic (plant-mediated) effects on LMs. Adult LMs preferred leaves from control plants to leaves of plants that had been previously infested with WFs; no reciprocal effect of LMs on WFs were found. Feeding by Helicoverpa zea larvae, which has been shown previously to affect LM performance, had no effect on WF survival and development. LM natural population dynamics were monitored on WF-preinfested and control plants in a field experiment. WF-infested plants were less suitable for LM development with an overall 41% reduction in LM population density. These results demonstrate asymmetric direct and plant-mediated interspecific interactions between generalist herbivores feeding simultaneously on the same host. Possible mechanisms by which WFs overcome plant defenses are suggested. This ability may also contribute to WF success that makes them a major pest worldwide. The study supports the idea that over an evolutionary time scale, herbivores sharing the same host plant will automatically compete.

Effect of Neem Seed Extract on the Brown Citrus Aphid (Homoptera: Aphididae) and its Parasitoid Lysiphlebus testaceipes (Hymenoptera: Aphidiidae)

Abstract The biological effects of a commercially available neem seed extract (Neemix. 4.5, 4.5% azadirachtin, AZ) were assessed on the brown citrus aphid, Toxoptera citricida (Kirklady), a recently introduced insect pest of citrus in the United States and its parasitoid, Lysiphlebus testaceipes. When small citrus seedlings were dipped with the neem extract at 11–180 ppm AZ, 0–8% of nymphs and 0–17.5% of adults survived 7 d after the treatment while 95% of nymphs and 42.5% of adults in the control survived for the same period. The extract drastically reduced longevity of both adults and nymphs, adult fecundity, and molting of nymphs at all tested concentrations. Spraying neem extract (11–180 ppm AZ) onto potted citrus plants in the greenhouse also significantly reduced aphids by 20–100%, while control aphid populations increased by 950% 7 d after treatment. Application of the extract had little impact on the survival of adult parasitoids and developing parasitoids within aphids because parasite emergences were similar between treated and untreated parasitized aphids. These results indicate that neem extract may be compatible with integrated pest management programs in citrus and should be evaluated for field efficacy.

Induction of systemic acquired resistance in cotton by BTH has a negligible effect on phytophagous insects

Whether or not chemical changes in plants in response to pests (insects and pathogens) are general or specific remains unclear. Some evidence indicates that an induced response (IR) to arthropods via the octadecanoid pathway represents a distinct mechanism from the salicylic acid‐based pathway of systemic acquired resistance (SAR) to pathogens. To further test this hypothesis, young cotton seedlings were activated with benzo (1,2,3) thiadiazole‐7‐carbothioic acid (S) methyl ester (BTH), an elicitor of SAR. The enzymatic activities of a number of pathogenesis‐related (PR) proteins in young and old leaves of control and BTH treated plants were measured. BTH applications elicited marked increases in the activity levels of chitinase, peroxidase, and β‐1,3‐glucanase both locally and systemically. The highest levels of induction were detected systemically in young leaves. Except for some local effects on whitefly oviposition, the induction of SAR by BTH had no effect on either host preference of whiteflies Bemisia tabaci (Gennadius) or on feeding efficiency of cotton bollworms Helicoverpa armigera (Hübner). We conclude that SAR induction via the salicylic acid pathway in ‘Acala’ cotton has negligible effect on the tested insect herbivores.

Immersion of cucumber fruit in heated water alters chilling-induced physiological changes

Abstract Cucumber (Cucumis sativus cv. Dasher 3) fruit were immersed in water at 25, 38, or 42 °C for 30 min prior to storage at 12 (nonchilling) or 2.5 °C (chilling) for two weeks to determine the effects of these treatments on chilling-induced changes in electrolyte leakage, and CO2, and ethylene evolution. Storage at 2.5 °C resulted in a significant increase in electrolyte leakage, which decreased significantly as immersion temperature increased. Chilled fruit had higher rates of CO2 production than did nonchilled fruit following transfer to 21 °C, but there was no difference due to immersion temperature. Following transfer to 21 °C, nonchilled fruit produced no detectable ethylene whereas chilled fruit produced significant amounts of ethylene. The amount of chilling-induced ethylene production decreased with increased temperature of immersion, and this difference persisted for at least 72 h after transfer to 21 °C. The amount of 1-aminocyclopropane-1-carboxylic acid (ACC) was significantly higher in chilled than in nonchilled fruit although the amount of ACC in chilled fruit decreased significantly as immersion temperature increased. Nonchilled fruit had significantly higher ACC oxidase activity than did chilled fruit at the time of transfer to 21 °C, and ACC oxidase activity decreased as immersion temperature increased.

Stereoselective and regioselective hydroxylation of warfarin and selective O-dealkylation of phenoxazone ethers in human placenta

The oxidative metabolism of warfarin and a series of phenoxazone ethers was studied in two groups of human placentas which exhibited high or low levels of aryl hydrocarbon hydroxylase (AHH). Warfarin metabolism was stereoselective (mean R/S = 2.48) for the R-enantiomer and regioselective for the 6- and 8- positions in the high AHH group whereas warfarin metabolism in the low AHH group displayed no significant overall stereoselectivity (mean R/S = 1.24) and was regioselective for the 7- position. The high AHH group metabolized the methyl, ethyl, propyl and butyl ethers of phenoxazone rapidly, while the low AHH group catalyzed their biotransformation at very low or negligible rates. Neither group detectably metabolized phenoxazone or pentyloxyphenoxazone whereas both groups metabolized benzyloxyphenoxazone at low but similar rates. Rates of warfarin R-6 and R-8 hydroxylation were highly correlated with metabolism of benzo(alpha)pyrene (r = 0.99) and the C1-C4 phenoxazone ethers (r greater than 0.87), but poorly correlated with metabolism of benzyloxyphenoxazone (r less than 0.50). These data support the use of warfarin and the phenoxazone ethers as sensitive biochemical probes for P-450 isozymes in human extrahepatic tissues. They indicate the presence of a multiplicity of xenobiotic metabolizing P-450s in placental tissue which has not been exposed to inducing agents that elevate AHH.

Constituents from the periderm and outer cortex of Ipomoea batatas with antifungal activity against Rhizopus stolonifer

Abstract Rhizopus stolonifer invades sweetpotato roots through injuries and infected roots are rapidly consumed by a soft rot. However, not all injuries are equally susceptible to infection; shallow injuries (1–2 mm deep) are less prone to infection than deeper injuries (>5 mm deep). The presence of antifungal compounds in external tissues may partially explain the resistance of shallow injuries to infection. To test this hypothesis, we developed a quantitative bioassay for measuring the growth of R. stolonifer utilizing the vital stain 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). Acetone extracts of the fresh interior flesh of four cultivars had no antifungal activity, but extracts of the exterior 2 mm of the root were inhibitory. We used the R. stolonifer bioassay to guide the purification of the active components. Two active fractions were isolated. One active fraction contained predominately caffeic acid, but this compound was determined not to be the most biologically active component. The second active fraction contained 3,5-dicaffeoylquinic acid (3,5-DCQA), and this compound was found to be active, with an EC50 of 2.2 g l−1. The presence of antifungal compounds in the external tissues helps explain why shallow injuries are resistant to infection. Additionally, we demonstrated that interior flesh tissues accumulate antifungal compounds when elicited and incubated under curing conditions (30°C and 90–95% RH) for 24 h.

Effects of Nutrient Supply on Citrus Resistance to Root Herbivory by Diaprepes abbreviatus L. (Coleoptera: Curculionidae)

Abstract We treated two citrus cultivars with a complete fertilizer diluted to 25, 100, 200, or 400 ppm N to test whether increasing fertilizer concentration alters root and leaf chemistry and decreases resistance of citrus to root-feeding larvae of Diaprepes abbreviatus L. Roots and leaves of better-nourished ‘sour orange’ (Citrus aurantium L.) had larger amounts of total proteins and increased activities of enzymes associated with resistance than did plants given 25 ppm N. The fertilizer effect was less consistent for ‘Swingle citrumelo’ (C. paradise Macf. × Poncirus trifoliate L.), which has greater resistance to D. abbreviatus. Herbivory increased root protein content and peroxidase but decreased activities of chitinase and β-1,3-glucanase, which are enzymes associated with resistance to microbial pathogens. When significant, the effect of root herbivory on enzyme activities in leaves was opposite the effects on roots. Fertilizer and herbivory rarely interacted, indicating enzyme induction was not a function of nutrient supply. Fertilizer did not affect total phenolics in roots of either citrus, but root herbivory increased levels in ‘sour orange’. Despite elevated levels of putative defense proteins, ‘sour orange’ given ≥100 ppm N produced 50% greater total larval mass per pot than did plants given 25 ppm N. Fertilizer concentration did not affect mass of larvae on ‘Swingle citrumelo’ roots and did not affect larval mortality for either citrus cultivar. Our results concerning a root herbivore are consistent with the body of studies of folivores that have demonstrated that increased fertilizer has no effect or increases herbivore performance.

EFFECT OF DIETARY COPPER ON LARVAL DEVELOPMENT OF DIAPREPES ABBREVIATUS (COLEOPTERA: CURCULIONIDAE)

Abstract Larvae of the Diaprepes root weevil, Diaprepes abbreviatus (L.), were reared from hatching on an artificial diet containing four concentrations of two copper compounds, cupric sulfate (CuSO4) or cupric hydroxide [Cu(OH)2]. Negative effects of copper on insect development were observed only for early instars. Survival of larvae from hatching to 4 weeks of age was significantly affected by the copper compounds compared with the artificial diet alone, and greater mortality was associated with CuSO4 compared with Cu(OH)2. The two compounds had equivalent effects on larval weight gain of early instars. Weight gain was negatively correlated with increasing copper concentration. No effect of copper was observed on late instars maintained on these diets beyond the initial 4 weeks. Larval and pupal period, weight gain, and survival of late instars were statistically similar. No effect on larval survival or weight gain was observed when copper solutions were applied at nonphytotoxic levels to two varieties of citrus rootstock. The potential for manipulating citrus tree copper content to control this pest is discussed.