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.     

Characterization of the hepatic mixed function oxidase system in endrin-resistant and -susceptible pine voles

Studies were conducted to assess the contribution of the hepatic microsomal mixed function oxidase system to a 7.2-fold difference in susceptibility to the lethal effects of endrin between endrin-resistant and -susceptible pine voles, Microtus pinetorum. Evaluations of microsomal enzyme systems were conducted for basal and endrin-treated pine voles of both strains. The microsomal activity of ICR white mice was investigated to provide a species comparison. Maximal microsomal mixed function oxidase activities were determined in in vitro incubations for the model substrates ethylmorphine, aniline, and benzo(a)pyrene. V_max values were estimated for the rate of disappearance of benzo(a)pyrene in in vitro incubations. No significant strain differences in basal microsomal enzyme activity were found for the model substrates investigated, although activity was invariably higher in the resistant strain. The concentration of cytochrome P-450 was significantly higher in the resistant vole though actually less than 20% different. The occurrence of significant strain differences in the levels of microsomal enzyme activity induced by endrin were rare. Significant endrin treatment effects on the levels of microsomal enzyme activity for the pine vole were observed but the degree and direction of change depended on the substrate used. A marked species difference in microsomal mixed function oxidase activity was noted between pine voles and white mice. This was particularly evident for endrin-treated animals. The microsomal activity of endrin-treated white mice was greatly induced relative to basal levels. The degree of induction depended on the substrate used. The small strain differences in microsomal enzyme activity for the systems investigated were judged to be insufficient to explain the strain difference in susceptibility to endrin.     

Enzyme activities and cytochrome P-450 levels of some Japanese quail tissues with respect to their metabolism of several pesticides

In the Japanese quail, cytochrome P-450, A- and B-esterase, amidase, and glutathione S-aryl transferase were assayed in postmitochondrial centrifugal fractions, in microsomes, and supernatant fractions of liver, lungs, kidneys, and testes. Liver microsomes contained the highest A-esterase activity and P-450 levels. B-esterase was more generally distributed and higher in the microsomal tissue fractions. Microsomal amidase activity was highest in quail lung and kidney, and lowest in the liver (per mg protein). Very little difference in glutathione S-aryl transferase activity was noted among the tissues assayed. In vitro metabolism of carbaryl, phosphamidon, and chlorotoluron by the various centrifugal fractions revealed that the production of 1-naphthyl-N-hydroxymethylcarbamate and 1-naphthol, the major metabolites, was greatest in the postmitochondrial fraction of the liver. The major carbaryl metabolite in all other quail tissue fractions was 1-naphthol. Phosphamidon metabolism in postmitochondrial preparations of quail liver was higher than in the supernatant and microsomes. Chlorotoluron metabolism occurred only in the postmitochondrial fractions of quail liver. The major products were the oxidative metabolites, N-(3-chloro-4-methylphenyl)-N′-methylurea and N-(3-chloro-4-hydroxymethylphenyl)-N′-methylurea.     

Methoxyresorufin as a substrate for the fluorometric assay of insect microsomal O-dealkylases

Methoxy-, ethoxy-, propoxy-, and butoxyresorufin were prepared and tested as substrates for the fluorometric assay of O-dealkylation by the mixed-function oxidase system in house flies, Musca domestica L. Methoxyresorufin proved to be the most suitable substrate because of the favorable reaction rates. This sensitive assay can be performed with a minimum of microsomal protein (<0.3 mg/ml) in 5 min or less. The apparent K_m and maximum velocity were calculated as 2.88 μM and 0.27 nmol of resorufin produced/min/mg of microsomal protein, respectively. The O-dealkylation reaction required O₂ and NADPH and was inhibited by CO.     

Interactions of allelochemicals with detoxication enzymes of insecticide-susceptible and resistant fall armyworms

The induction of glutathione S-transferases and microsomal oxidases by host plants and allelochemicals was examined in sixth-instar larvae of insecticide-susceptible and resistant strains of the fall armyworm, Spodoptera frugiperda (J. E. Smith). Among 11 host plants studied, parsnip and parsley were the best inducers of glutathione S-transferase, resulting in increases of 39- and 19-fold, respectively, compared with the artificial diet. The inducer in parsnip leaves was identified by mass spectrometry, high-pressure liquid chromatography, gas chromatography, and thin-layer chromatography as xanthotoxin, a furanocoumarin. Xanthotoxin also showed a bimodal effect on the microsomal oxidase systems, increasing cytochrome P-450 content and heptachlor epoxidase activity but inhibiting aldrin epoxidase, biphenyl 4-hydroxylase, and p-chloro-N-methylaniline N-demethylase. Using indole 3-acetonitrile, indole 3-carbinol, and flavone as inducers, the inducing pattern of glutathione S-transferases was the same toward 3,4-dichloronitrobenzene, 1-chloro-2,4-dinitrobenzene, and methyl iodide. Microsomal oxidase and glutathione S-transferase were also inducible by host plants and allelochemicals in larvae of a carbaryl-resistant strain.     

In vitro metabolism and interaction of profenofos by human, mouse and rat liver preparations

Biotransformations of profenofos were studied in vitro. Two metabolites, desthiopropylprofenofos and hydroxyprofenofos, were detected by LC-MS after incubation of profenofos with human liver homogenates and different mammalian liver microsomes. The rank order of desthiopropylprofenofos formation in liver microsomes based on intrinsic clearance (V_max/K_m) was mouse > human > rat, while for profenofos hydroxylation it was mouse > rat > human. In view of the ratio between desthiopropylation and hydroxylation intrinsic clearance rates, human liver microsomes were most active in profenofos bioactivation. The interspecies differences and interindividual variation were within range of the default uncertainty/safety factors for chemical risk assessment. CYP3A4, CYP2B6 and CYP2C19 were identified as profenofos-oxidizing enzymes in human liver on the basis of recombinant expressed enzymes and correlation with CYP model activities. The rank order of CYPs in profenofos activation was CYP3A4 > CYP2B6 > CYP2C19, whereas it was the contrary for profenofos hydroxylation. Profenofos inhibited relatively potently several human liver microsomal activities: the lowest IC₅₀ values were about 3 μM for CYP1A1/2 and CYP2B-associated activities. Profenofos is extensively metabolized by liver microsomal CYP enzymes and its interaction potential with several CYP activities is considerable.     

Insect juvenile hormones: Chemistry and action: Edited by Julius J. Menn and Morton Beroza. Academic Press,New York, 1972, $11.00

Heptachlor metabolism was determined in young male rats fed for 10 days diets containing 18% protein supplied either by gluten or casein. Rats on the casein diet were either pair-fed on an individual basis to the gluten-fed animals or they were fed ad lib. Saline, sodium phenobarbital (80 mg/kg) or diethylaminoethyl 2,2-diphenylvalerate HCl (SKF 525-A, 50 mg/kg) was administered ip to the animals before sacrifice. Weight gain, liver microsomal protein, and heptachlor metabolism (nmole of product/g liver/10 min), assayed in 9000g rat liver supernatants, were found to be significantly reduced in the gluten-fed animals. Animals pair-fed the casein diet had higher heptachlor epoxidase activity than those fed ad lib. Phenobarbital pretreatment significantly enhanced the metabolism of heptachlor in animals in all groups to varying magnitudes. Heptachlor epoxidase activity (units/g liver) was increased 11-fold in the gluten-fed animals, 6-fold in the pair-fed animals on casein diet, and 7-fold in the animals fed casein ad lib. Heptachlor metabolism was less inhibited by SKF 525-A in the rats fed gluten compared to the rats on casein diet, either pair-fed or ad lib. The degree of induction or inhibition of enzyme activity was not influenced by restriction of food intake (pair-fed vs ad lib). The results of these studies suggest an interaction of protein inadequacy with drug metabolism and its induction or inhibition.     

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.     

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.     

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.     

Transformation of fenuron induced by photochemical excitation of humic acids

When neutral solutions containing the herbicide 3-phenyl-1,1-dimethylurea (fenuron) and a humic acid are irradiated at 365 nm, 3-(4-hydroxyphenyl)-1,1-dimethylurea and three biphenyl products are formed as main products. The apparent quantum yield of fenuron disappearance is evaluated as 6·2 × 10⁻⁵ mole E⁻¹. Upon irradiation of the same mixture at 253·7 nm, both direct and induced phototransformations of fenuron occur. Direct photooxidation yields 2- and 4-amino-N,N-dimethylbenzamide. The induced phototransformation leads to 2- and 4-hydroxylation of the aromatic ring in accordance with the fact that hydroxyl radicals are involved in the oxidation.     

Alterations in microsomal cytochrome P-450-catalyzed reactions as a function of chlordecone (Kepone) induction

The effects of chlordecone treatment on the hepatic microsomal monooxygenase system of male rats were investigated. Chlordecone increased the microsomal content of cytochrome P-450, NADPH-cytochrome P-450 (c) reductase and, to a lesser extent, cytochrome b₅ in a time- and dose-dependent manner. The content of NADH-cytochrome b₅ (c) reductase was reduced. The turnover of seven substrates was studied in detail and, with the exception of aniline, was found to be increased between 1.3- and 2.2-fold. The apparent K_m's for these substrates were increased 2.1- to 16.7-fold. In addition, zoxazolamine paralysis time was reduced as a result of chlordecone treatment. These kinetic changes are explained on the basis of alterations in the cytochrome P-450 pool together with residual chlordecone acting as an inhibitor of substrate turnover. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein pattern of microsomes isolated from chlordecone-treated rats more closely resembled that of microsomes isolated from untreated rats than that of microsomes isolated following phenobarbital or 3-methylcholanthrene treatment.     

Human metabolism of atrazine

Atrazine (ATZ) metabolism by human liver microsomes (HLM), cytochrome P450 (CYP) isoforms, and human liver (HL) S9 fractions, was investigated using HPLC/PDA and LC/MS/MS. CYP-dependent metabolites from pooled HLM are desethylatrazine (DEA), desisopropylatrazine (DIA), 1-hydroxyisopropylatrazine (HIATZ), and 2-hydroxyethyl atrazine (HEATZ). DEA and DIA were major metabolites in pooled HLM. CYP1A2 and 2C19, respectively, were major isoforms for DEA and DIA production. CYP3A4, while less active, is generally at high concentrations, produces both DEA and DIA and is significant. The percent total normalized rates (%TNR) for CYP1A2 and 3A4 in pooled HLM were 63% and 24% for DEA, and 35% and 56% for DIA production. Single donor HLM samples, showed correlations for CYP1A2 (r = 0.92) and 3A4 (r = 0.81) for DEA and DIA production, while variations in production of DEA and DIA were 8.5- and 6.0-fold, respectively. Pooled S9 fractions also mediate glutathione conjugation of atrazine, DEA and DIA.     

Induction of the mixed-function oxidase system in the liver of the barn owl Tyto alba by PCBs

Injection of 30 mg/kg body wt of polychlorinated biphenyl (Aroclor 1254) into liver parenchymal tissue of nestling and adult barn owls Tyto alba resulted in increases in the level of cytochrome P-450. Concomitantly, there were increases in catalytic activity of the microsomal enzyme system as measured by aldrin epoxidation and aminopyrine N-demethylation. However, the ratio $\text{455}\text{430}\text{nm}$ in the ethylisocyanide-difference spectrum remained unchanged. Of particular interest is the sudden drop in the level and catalytic activity of cytochrome P-450 in nestling owls at age 40 days. Treatment with Aroclor 1254 produced small hemorrhages in the liver of nestling owls and the liver appeared much enlarged (hepatomegaly), indicating a toxic effect and resulting in little induction of microsomal enzymes. In adult owls the inductive effect was much greater. Aroclor 1254 produced a spectral shift in the cytochrome P-450-difference spectrum from 450 to 448 nm and in the ethylisocyanide-difference spectrum from 455 to 453 nm and from 430 to 427 nm.     

O3对日光温室黄瓜病虫害的防治效果

通过室内与大田试验，明确O3对日光温室黄瓜病虫害的防治效果。研究结果表明，在病害发生初期，用O3发生器隔天施放O3，每天施放3次，每次40min，间隔20min，O3浓度维持约1mg／kg，施放后7天，对黄瓜霜霉病、白粉病、细菌性角斑病及灰霉病防治效果分别为63．4％、53．6％、68．7％和65．0％。室内施放O3 60s，间隔20min，持续12h，对温室白粉虱、南美斑潜蝇成虫杀伤力为100％。O3对黄瓜植株安全，但随着黄瓜生育期的推进，对O3的忍受能力依次减弱。苗期忍耐O3最大浓度为1～2mg／kg，结果期0．4～1mg/kg，后期0．2～0．4mg／kg。O3防治黄瓜病虫害新技术具有安全、有效、方便、无二次污染等特点，尤其在黄瓜定植前对棚室进行大剂量消毒，可不留死角，且有效地解决了硫磺等药物消毒所造成棚内一些设施的破坏及对生态环境污染的难题，具有广阔的推广应用前景。     

Triazine absorption by excised corn root tissue and isolated corn root protoplasts

Absorption of four triazine herbicide analogs [ametryn (2-(ethylamino)-4-(isopropylamino)-6-(methylthio)-s-triazine), atrazine (2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine), atratone (2-methoxy-4-(ethylamino)-6-(isopropylamino)-s-triazine), and hydroxyatrazine (2-hydroxy-4-(ethylamino)-6-(isopropylamino)-s-triazine)] was compared using excised corn (Zea mays L.) root segments and isolated corn root protoplasts. The tissue absorbed ametryn, atrazine, and atratone for only 20 min. Ametryn and atrazine permeated tissue to passive equilibrium with the ambient solution in 10 min. Atratone permeated to 65 and 82% of passive equilibrium in 10 and 30 min, respectively. In contrast, hydroxyatrazine concentration in tissue was only 15 and 70% of the ambient concentration at 30 min and 24 hr, respectively. However, hydroxyatrazine permeated frozen/thawed tissue to 90% of passive equilibrium in 10 min. Protoplast absorption of ametryn and atratone was complete in 10 sec; hydroxyatrazine absorption by protoplasts did not reach a plateau until 5 min. Protoplasts absorbed the triazines to greater than passive equilibrium. Three kinetically homogeneous pools were detected for ametryn, atrazine, and atratone, whereas elution of hydroxyatrazine produced four pools. The three pools for atrazine were confounded by metabolism of atrazine to hydroxyatrazine. Pools for the triazines could not be identified as the free space, cytoplasm, and vacuole as proposed previously for mineral ions. Although the plasma membrane impeded diffusion of hydroxyatrazine, all analogs penetrated into the symplast.     

糠菌唑在大鼠、小鼠、兔、狗和人肝微粒体中的立体选择性代谢

采用高效液相色谱-串联质谱 (HPLC-MS/MS) 手性色谱柱法定量分析肝微粒体中的糠菌唑,通过密度泛函理论计算光谱与振动圆二色光谱 (VCD) 和红外光谱 (IR) 比对,确定了糠菌唑4种对映体的绝对构型;以大鼠、小鼠、兔、狗和人肝微粒体为模型,研究了糠菌唑的立体选择性降解行为。结果表明:在供试肝微粒体中,4种异构体的降解均遵循一级反应动力学方程,且在人和小鼠肝微粒体中的代谢速率相对较慢。(2S, 4R)-和 (2R, 4S)-糠菌唑在5种供试肝微粒体中的立体选择性趋势一致,而 (2R, 4R)- 和 (2S, 4S)-糠菌唑只在兔肝微粒体中的降解有显著的立体选择性差异,在小鼠肝微粒体中几乎没有立体选择性。酶促反应动力学结果也证实了糠菌唑代谢的立体选择性,并显示 (2R, 4R)- 和 (2S, 4S)-糠菌唑在供试肝微粒体中的酶促反应趋势存在种属差异。     

Phenobarbital induction of detoxifying enzymes in resistant and susceptible houseflies

Houseflies, Musca domestica, L., were treated with the drugs phenobarbital and 3-methylcholanthrene to study the effects of these compounds as inducing agents of the microsomal oxidases, heptachlor epoxidase, and p-nitroanisole O-demethylase, and of DDT-dehydrochlorinase. Phenobarbital was active when applied by injection or as part of the diet but inactive when topically applied. The resulting increases in heptachlor epoxidase activity were as much as 25-fold that of the untreated controls. The net increase in enzyme activity after phenobarbital treatment was greater in an insecticide-susceptible strain, WHO-SRS strain, than in a carbamate-resistant strain. However, the phenobarbital induced increases in DDT-dehydrochlorinase were greater, about 2-fold, in the resistant strains than in the susceptible strain.The optimum dose for phenobarbital was 1% in the diet for a period of 3 days. None of the treatments with 3-MC, feeding, injection, exposure to residues, or topical, were effective in induction.     

高效液相色谱法测定元参、黄芩和杭白菊中的 多菌灵残留

建立了一种测定元参、黄芩和杭白菊3种中药材中多菌灵残留的反相高效液相色谱分析方法(R-HPLC)。样品用丙酮提取,柱层析(弗罗里硅土-中性氧化铝=7∶ 4,质量比)和液液分配净化,以甲醇-水(体积比60∶ 40)为流动相,在波长281 nm下对待测组份进行检测,外标法定量。结果表明,多菌灵在3种中药材中的添加回收率分别为元参:69.08% ~88.31%,相对标准偏差(RSD)为3.35% ~4.67%;黄芩:71.39% ~88.40%,RSD为3.73% ~8.62%;杭白菊:73.33% ~97.31%,RSD为2.83% ~9.44%;均符合农药残留分析的要求。该杀菌剂在0.05~10 mg/L之间具有良好的线性关系,r=0.999 7,仪器的最小检出量(按S/N=3计)分别为0.3、0.5和0.3 ng,方法的最低检测浓度分别为0.01、0.05和0.01 mg/kg。该方法简单易行,净化效果较好,适合各种中药材中多菌灵残留的检测。     

Comparative enzyme activities and cytochrome P-450 levels of some rat tissues with respect to their metabolism of several pesticides

Cytochrome P-450, A- and B-esterase, amidase, and glutathione S-aryl transferase were assayed in the postmitochondrial centrifugal fraction, microsomes, and supernatant of rat liver, lungs, kidneys, and testes. Liver microsomes contained the highest P-450 levels and A-esterase activity. B-esterase activity was more generally distributed and higher in the microsomal tissue fractions. Microsomal amidase activity was highest in rat lung and lowest in the liver (per mg protein). Glutathione S-aryl transferase activity was highest in the liver. The in vitro metabolism of carbaryl, phosphamidon, and chlorotoluron by the various centrifugal fractions revealed many differences. Carbaryl metabolism was greater in the liver microsomal fractions than in any other preparation. 1-Naphthol was the major metabolite in all tissue fractions. Although very little metabolism of phosphamidon occurred in the rat, metabolism in the rat liver postmitochondrial fraction was slightly higher with respect to the production of metabolites than in the supernatant and microsomes combined. Chlorotoluron was not metabolized by any of the tissue fractions of the rat. At least a low level of activity toward some compounds was observed in all tissues, but this study confirmed that the liver was the most active metabolizing tissue as well as having the highest levels of enzymatic activity usually associated with pesticide metabolism.