Effect of liver enzyme induction on paraoxon metabolism in the rat

Orally administered [1-¹⁴C]ethyl paraoxon, O,O-diethyl-O-p-nitrophenyl phosphate, is readily absorbed from the gastrointestinal tract of male albino rats. Radioactivity is essentially eliminated in 72 hr by excretion into urine and feces and by expiration as ¹⁴CO₂. Compounds with radioactivity in the urine are tentatively identified as diethyl phosphoric acid, desethyl paraoxon, ethanol, metabolites conjugated with amino acids, and paraoxon; the first compound is the predominant radioactive metabolite. Intraperitoneally injected phenobarbital, DDT, dieldrin, and endrin are inducers of microsomal enzymes that degrade paraoxon. The aryl phosphate-cleaving activity in vitro is not dependent on the addition of NADPH. O-Dealkylation of paraoxon is catalyzed by microsomal enzymes that require NADPH and oxygen and are inhibited by carbon monoxide. Microsomal enzymes from rats pretreated with enzyme inducers give an increased rate of O-dealkylation of paraoxon. Reduced glutathione has little or no effect on paraoxon degradation by either microsomal or soluble enzymes. Actinomycin D inhibits O-dealkylation of paraoxon in vivo, as indicated by reduction of ¹⁴CO₂ formation, and in vitro, as indicated by decreased activity of microsomal O-dealkylase. The role of microsomal mixed-function oxidases and NADPH-dependent O-dealkylase in the metabolism of organophosphorus insecticides is discussed.     

Metabolism of metalaxyl in cell suspension cultures of Lactuca sativa L. and Vitis vinifera L

The metabolic fate of metalaxyl in suspension cultures of Lactuca sativa has been investigated as a function of explant source, culture age, and nutrient medium quality. In all cases, the fungicide was the subject of diverse metabolic attack yielding products which arose from O- and N-dealkylation, alkyl and aryl hydroxylation, and ester hydrolysis. Substantial amounts of metabolites were found in the culture filtrates although products arising from aryl hydroxylation and combined O-/N-dealkylation accumulated exclusively as cellular conjugates. Cultures newly isolated from the cotyledon, hypocotyl, and root of seedlings displayed very little variation in the rate and qualitative nature of breakdown and in the relative distribution of metabolites among the polar conjugate fraction, unconjugated cellular fraction, and culture filtrate. Similarly, metabolism proceeded independently of changes in nutrient medium composition. A suspension culture initiated from 4-year-old hypocotyl callus produced less identifiable metabolites and had an impaired ability to perform alkyl hydroxylation, O-dealkylation, and ester hydrolysis. A greater proportion of metabolites occurred as polar conjugates in the 4-year-old hypocotyl cells than that in the newly isolated hypocotyl line. The metabolic profile seen in Vitis vinifera was similar to that in L. sativa and a time-course study indicated some interconversion of metabolites and a change in the ratio of two atropisomers formed by alkyl hydroxylation.     

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.     

Biphenyl hydroxylation and its induction differ between montane voles and Swiss Webster mice

Hepatic microsomes from female montane voles, Microtus montanus, metabolized biphenyl to 4-hydroxybiphenyl (359 ng/mg microsomal protein/min), 3-hydroxybiphenyl (49 ng/mg/min), and 2-hydroxybiphenyl (29 ng/mg/min). Phenobarbital treatment of the voles (ip) induced in vitro biphenyl hydroxylations by about 324, 865, and 445% for 4-, 3- and 2-hydroxylation, respectively. Comparable studies were made of female, white Swiss Webster mice. The major microsomal metabolite was 4-hydroxybiphenyl (1010 ng/mg/min) but more 2-hydroxybiphenyl (118 ng/mg/min) was formed than 3-hydroxybiphenyl (92 ng/mg/min). Phenobarbital treatment of the mice barely changed biphenyl 2-hydroxylation (?1.3%) but did raise 3- and 4-hydroxylation (55 and 211%, respectively). Treatment of voles with 3-methylcholanthrene raised biphenyl 2-, 3-, and 4-hydroxylation by about 81, 50, and 47%, respectively, whereas in mice the increases were 176, 41, and 31%, respectively. β-Naphthoflavone treatment had similar effects. The vole liver microsomes were dependent upon NADPH for biphenyl hydroxylation. The carbon monoxide difference spectra of reduced cytochrome in the microsomes show a peak at 450.4 nm. This was unchanged by phenobarbital and only slightly shifted (?0.8 nm) by 3-methylcholanthrene or β-naphthoflavone treatment. Voles and mice represent different families of the order Rodentia. The results obtained with voles more closely resemble those reported for hamsters which are in the same family but a different subfamily than voles. Comparative consideration of the taxonomic relations of the rodents, therefore, may be useful in interpreting their comparative toxicology.     

Comparative toxicity,absorption, and metabolism of chlorpyrifos and its dimethyl homologue in methyl parathion-resistant and -susceptible tobacco budworms

Chlorpyrifos (Dowco 179) and its dimethyl homologue, chlorpyrifosmethyl (Dowco 214), were used to study the influence of the O,O-dialkyl group of organophosphorus insecticides on toxicity, absorption, and metabolism among larvae of the tobacco budworm [Heliothis virescens (F.)] from strains that were resistant (R) and susceptible (S) to methyl parathion. In toxicity tests, chlorpyrifos and chlorpyrifosmethyl were more toxic than methyl parathion to 3rd-stage R larvae but less toxic to S larvae. Chlorpyrifosmethyl was more toxic (3–4 ×) than chlorpyrifos to both strains of larvae, and the results of absorption studies indicated that the toxicity differential of the homologues may be explained in part by the more rapid absorption of the dimethyl form. Studies of the in vivo metabolism of both Dowco compounds indicated that each was degraded mainly by the cleavage of the pyridylphosphate linkage. In vitro tests demonstrated that the NADPH-dependent microsomal oxidases were of primary importance in detoxification, while glutathione (GSH)-dependent mechanisms (aryl- and alkyltransferases) present in the soluble cell fractions were of lesser importance. O-dealkylation occurred only with chlorpyrifosmethyl. The R larvae demonstrated greater capability in detoxifying both compounds in the comparative in vivo and in vitro studies of metabolism, but the differences were more apparent during the 5th instar than during the 3rd instar.     

The effect of hydrogen peroxide on the viability of tomato cells and of the fungal pathogen Cladosporium fulvum

An oxidative burst was previously demonstrated to be induced in tomato plants by race specific elicitors of the fungal pathogen Cladosporium fulvum . The in planta levels of H₂O₂estimated to occur during elicitor treatment, were compared with the levels required to show toxicity to host cells and to the fungal pathogen. Injection of Cf-9 tomato leaves with 100 m m H₂O₂caused an insignificant degree of necrosis and 1m H₂O₂was required to cause complete leaf necrosis comparable to that induced by the AVR9 elicitor. Assays with Cf-5 tomato cell suspensions confirmed the low toxicity of H₂O₂to tomato cells but, as expected, the addition of Fe²⁺with H₂O₂(or with intercellular fluids containing AVR5 elicitor) enhanced cell death as determined by the Evans Blue assay. Germination and germ tube growth of conidia of C. fulvum were significantly retarded by 4–5 m m H₂O₂, and at higher concentrations, death of germ tubes was observed (ED₅₀=22 m m), as determined by the fluorescein diacetate assay. The addition of Fe²⁺with H₂O₂had little effect on fungal growth or viability in vitro . These results suggest that the amount of H₂O₂accumulating during an elicitor-induced response in leaves may be sufficient to affect fungal colonization but not to affect viability of host cells unless the Fe²⁺status in the apoplast is in some way altered by the elicitor to facilitate OH^.production via the Fenton reaction.     

Effects of the synergist piperonyl butoxide on metabolism of pesticides in green sunfish

The effects of piperonyl butoxide on metabolism of ¹⁴C-labeled methoxychlor, aldrin, and trifluralin were investigated in green sunfish, Lepomis cyanellus. Piperonyl butoxide inhibited epoxidation of aldrin to dieldrin, O-dealkylation of methoxychlor, and N-dealkylation of trifluralin, resulting in higher levels of total radioactivity in animals exposed to the combination compared to those exposed to pesticide alone. Where piperonyl butoxide was present a greater proportion of the total radioactivity in the fish extract occurred as parent compound compared to metabolites than in fish exposed to pesticide alone. After 16 days of exposure piperonyl butoxide increased the proportion of parent compound eight times for methoxychlor, 17 times for aldrin, and 15 times for trifluralin.     

Rosewood polyphenols alter phenoloxidase activity from the mantle of the marine bivalve mollusc, Modiolus demissus dillwyn

Phenoloxidase from the mantle of the marine bivalve Modiolus demissus Dillwyn slowly catalyzes the oxidation of the rosewood ingredient obtusastyrene (K_m 0.13 mM, V_max 0.30 mM/min/mg). However, in the presence of another rosewood ingredient, obtusaquinone, the oxidation rate is increased to a limiting maximal velocity of 11 mM obtusastyrene/min/mg, without a concommitant change in the K_m. The oxidation products of either the slow reaction or the obtusaquinone-enhanced reaction inhibit the catechol dehydrogenase function of phenoloxidase. The phenoloxidase-modifying properties of obtusaquinone and obtusastyrene may be related to the inhibition of shell formation in wood-boring bivalve larvae settling on rosewood.     

Metabolic O-dealkylation of 1-(4′-ethylphenoxy)-3,7-dimethyl-7-methoxy or ethoxy-trans-2-octene,potent juvenoids

1-(4′-Ethylphenoxy)-3,7-dimethyl-7-methoxy or ethoxy-trans-2-octene (the ethyl-methoxide and ethyl-ethoxide) are more stable in some biological systems and less stable in others than 1-(4′-ethylphenoxy)-3,7-dimethyl-6,7-epoxy-trans-2-octene (the ethyl-epoxide). In housefly adults and mealworm pupae the persistence increases in the order of the ethyl-epoxide, -methoxide and -ethoxide but with adult stable flies the epoxide is of intermediate stability. The alkoxides are metabolized in living insects and microsomal oxidase systems of houseflies and mouse liver mainly by O-dealkylation, at a higher rate for the methoxide than the ethoxide, but benzylic oxidation of the ethyl group also occurs and is more important in the degradation of the ethyl-epoxide than the ethyl-alkoxides. The photostability on silica gel is slightly better for the ethyl-ethoxide than the -methoxide or -epoxide.     

Biotransformation of s-Triazine Herbicides and Related Degradation Products in Liquid Cultures by the White Rot Fungus Phanerochaete chrysosporium

The ability of the white rot basidiomycete Phanerochaete chrysosporium to transform s-triazine herbicides has been investigated in laboratory experiments. The chlorinated metabolites formed during atrazine N-dealkylations were not further transformed by the fungus, whereas hydroxy-atrazine was converted to an unknown product. P. chrysosporium was also able to carry out the N-dealkylation of the herbicides simazine, propazine and terbuthylazine. Herbicide metabolism was not supported by purified peroxidases. The highest rates of herbicide N-dealkylation were obtained in liquid cultures maintained under moderate temperature allowing a long mycelium growing phase. Atrazine transformation was found to be supported by the mycelium, which contained significant amounts of microsomal cytochrome P450. Herbicide N-dealkylation was decreased in the presence of 1-aminobenzotriazole, in agreement with the involvement of P450 monooxygenases in atrazine metabolism. © 1997 SCI.     

Cytochrome P-450 content and aldrin epoxidation to dieldrin in isolated rat hepatocytes

A rat hepatocyte suspension effectively epoxidized aldrin to dieldrin with a V_max of 7.19 mol/mol P-450/min and a K_m of 9.27 μM. Viability and metabolic activity were stable for 6 hr after isolation when cells were maintained at room temperature (20°C) with the gentle introduction of $\text{O}{}_2\text{CO}{}_2$ onto the surface of the suspension. The cytochrome P-450 content of the suspension was 303 pmol/10⁶ cells. Primary maintenance culture of the cells also epoxidized aldrin. During culture for 3 days, metabolic activity decreased slowly day by day. Metabolic activity of microsomal fraction from rat liver was also examined. Microsomes epoxidized aldrin with a V_max of 5.11 mol/mol P-450/min and a K_m of 1.64 μM. Significant loss of some subspecies of cytochrome P-450 during fractionation of liver homogenate was indicated.     

Structure-activity relationships of 1,2,3-benzothiadiazole insecticide synergists as inhibitors of microsomal oxidation

The activities of 47 substituted 1,2,3-benzothiadiazoles as inhibitors of microsomal epoxidation and/or hydroxylation in enzyme preparations from rat liver or armyworm (Spodoptera eridania) gut have been evaluated. Many were found to be effective inhibitors of microsomal oxidation, the most active being the 6-butyl and 6-propoxy derivatives with I₅₀ values of 4.9 × 10^?7 and 7.0 × 10^?7M, respectively, for the epoxidation reaction. Regression analyses have established that activity of the 5-, 6-, and 5,6-substituted compounds can be satisfactorily described in equations in terms of π², π, and σ whereas that of the 4-substituted derivatives depends on π and the steric parameter E₈.     

Properties of Tribolium gut chitin synthetase

An insect chitin synthetase (CS) is readily assayed using the microsomal fraction (～0.5 mg protein) from an homogenate of Tribolium castaneum larvae. This enzyme preparation is incubated at 22°C with uridine 5′-diphospho-N-acetyl[³H]glucosamine in 355 μl of 25 mM Tris-HCl buffer containing 10 mM MgCl₂, 17 mM N-acetylglucosamine, and 1 mM dithiothreitol. Other divalent cations and amino sugars are less effective activators or are inhibitory. T. castaneum CS is strongly inhibited by polyoxin D and uridine 5′-diphosphate. These activation and inhibition properties of Tribolium castaneum gut CS are similar to those of fungal CS. The polymerization product formed by the Tribolium enzyme is stable in alkali but hydrolyzed by chitinase. Enzymes of Tribolium confusum, Tribolium brevicornis, Tenebrio molitor, and Galleria mellonella are also active under the same conditions. These enzymes are from the gut and probably from the peritrophic membrane. Integumental CS activity is not detected under the indicated assay conditions.     

Metabolism of O,O,S-trimethyl phosphorothioate in rats after oral administration of a toxic dose,and the effect of coadministration of its antagonistic thionate isomer

The in vivo metabolism of [¹⁴CH₃S]- and [¹⁴CH₃O]O,O,S-trimethyl phosphorothioate (OOS) was followed in rats after oral administration of threshold or LD₅₀ toxic doses of 20 or 60 mg/kg. Similar metabolic studies were conducted with coadministration of 1% O,O,O-trimethyl phosphorothionate (OOO), which prevented all signs of delayed toxicity, including weight loss. When administered alone, OOS was metabolized mainly (50–60%) via removal of the CH₃S moiety, which was largely converted to expired CO₂. Approximately 20% of the compound was O-demethylated, presumably by conjugation with glutathione, and then further metabolized to CO₂. Major urinary products were identified as O,O-dimethyl phosphoric acid (50–60%) and O,S-dimethyl phosphorothioic acid (～20%). Coadministration of OOO caused a slight decrease (～5%) in the cleavage of the CH₃S moiety, indicated by a reduction in ¹⁴CO₂ from [¹⁴CH₃S]OOS and a quantitatively similar increase in the formation of O,S-dimethyl phosphoric acid. Limited pharmacokinetic studies indicated that OOS was rapidly absorbed and distributed throughout the body. Coadministration of 1% OOO caused a slight increase in the blood half-life of parent OOS when administered at 60 mg/kg. It was concluded that a small proportion of the cleavage of the CH₃S moiety from OOS is involved in the intoxication process, and that this intoxication reaction is specifically inhibited by OOO.     

Delayed acute toxicity of O,S,S-trimethyl phosphorodithioate and O,O,S-trimethyl phosphorothioate to the rat

The toxicity and LD₅₀ of O,S,S-trimethyl phosphorodithioate were reexamined in the rat. Animals treated orally (single dose) with this compound exhibited early cholinergic signs followed at approximately 5 hr by delayed toxic signs, with an LD₅₀ of 43 mg/kg. Contamination of O,S,S-trimethyl phosphorodithioate by as much as 5% (w/w) O,O,O-trimethyl phosphorothioate provided only limited antagonism to the dithioate's toxicity. In contrast, the addition of 5% O,O,S-trimethyl phosphorodithioate to O,O,S-trimethyl phosphorothioate gave protection against the toxic effects of the latter compound up to 80 mg/kg of toxicant. Pretreatment of rats with as little as 5% O,O,O-trimethyl phosphorothioate, 24 hr prior to treatment with 200 mg/kg O,O,S-trimethyl phosphorothioate, gave complete protection against the toxic effects of this compound. Conversely, administration of 10% (w/w) O,O,O-trimethyl phosphorothioate 4 or 24 hr after treatment with 60 or 80 mg/kg of O,O,S-trimethyl phosphorothioate provided only partial protection at 4 hr and no protection from the effects of the toxicant at 24 hr. The ability of O,O,O-trimethyl phosphorothioate to antagonize the toxicity of this compound depended markedly on the route of administration (oral, intravenous, or intraperitoneal). At 4 hr past treatment with toxicant, only oral administration of the antagonist provided full protection. Intraperitoneal and intravenous administration of antagonist 4 hr after treatment with toxicant were partially effective and completely ineffective, respectively, in halting the toxic effects of this compound.     

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

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

The relationship between brain levels of cismethrin and bioresmethrin in female rats and neurotoxic effects

The oral toxicity of 5-benzyl-3-furylmethyl-(1R, cis)-chrysanthemate (cismethrin) to female rats decreased as their environmental temperature was raised. Acute oral LD₅₀ values increased from 157 mg/kg at 4°C to 197 mg/kg at 20°C and to > 1000 mg/kg at 30°C. Cismethrin was much more toxic given intravenously when the LD₅₀ was 4.5 mg/kg. This value did not change at different environmental temperatures. Irrespective of the environmental temperature, or route of adminstration, following the respective LD₅₀'s cismethrin caused tremors in rats when brain levels of 0.5–1.0 μg/g were reached and, at death, brain concentrations were 3.9–5.1 μg/g. These results suggested that the accumulation of cismethrin by the brain could be used as a model for the nervous system as a whole. The isomeric 5-benzyl-3-furylmethyl-(1R, trans)-chrysanthemate (bioresmethrin) was about 50 times less toxic to rats than cismethrin. After an intravenous LD₅₀, tremors started when brain concentrations were 4–5 μg/g. At death, brain levels were 25–35 μg/g. Plasma esterases were about equally active in hydrolysing cismethrin and bioresmethrin, whereas liver microsomal esterases hydrolyzed bioresmethrin over 10 times more rapidly than cismethrin. It is suggested that the lower toxicity of bioresmethrin is not only due to its faster metabolism but to an intrinsically lower toxicity at the critical site of action in the nervous system.     

杨梅叶抑菌活性成分初步分离与鉴定

用石油醚、氯仿、乙酸乙酯和正丁醇分别对杨梅叶甲醇提取物进行了萃取,得不同萃取相。测定了其对番茄灰霉病菌Botrytis cinerea、番茄早疫病菌Alternaria solani、水稻立枯丝核Rhizoctonia solani、小麦禾谷镰刀菌Fusarium graminearum、 黄瓜炭疽病菌Colletotrichum lagenarium、玉米大斑病菌Setosphaeria turcica等植物病原菌的抑菌活性。结果表明,乙酸乙酯相和正丁醇相的抑菌活性较好。对乙酸乙酯相进一步分离得到7个化合物,经电喷雾质谱(ESI-MS)、核磁共振氢谱(1H NMR)和碳谱(13C NMR)鉴定其分别为已知化合物山楂酸( 1 )、3-O-反-对-香豆酰基马斯里酸( 2 )、3-O-没食子酰二氢杨梅素( 3 )、斛皮素-7-O-鼠李糖甙( 4 )、杨梅素3-O-鼠李糖甙( 5 )、杨梅素-3-O-鼠李糖(3-O-没食子酰)甙( 6 )和杨梅素-3-O-鼠李糖(2-O-没食子酰)甙( 7 )。孢子萌发法测定结果表明,化合物 3~6 对番茄灰霉病菌和玉米大斑病菌的孢子萌发具有较好的抑制作用,EC50值分别在3.44~4.59 mg/L和8.85~11.6 mg/L之间。     

冠菌素诱导甘蓝幼苗抗黑腐病及其机理初步研究

以甘蓝 Brassica oleracea L.感病品种"庆丰"为材料,通过叶面喷施冠菌素(coronatine,COR)和人工接种黑腐病菌——野油菜黄单胞菌野油菜致病变种(Xanthomonas campestris pv.campestris)的方法,研究了COR诱导甘蓝幼苗抗黑腐病的效果及其生理生化机制。结果表明, COR在质量浓度为0.01、0.1和1.0 mg/L下均具有诱导甘蓝幼苗抗黑腐病的效果,其中1.0 mg/L 处理的病情指数为38.2,诱抗率为43.7%,诱导抗病效果最好。与对照相比,随着COR质量浓度的提高,叶片中过氧化氢酶(CAT)活性和超氧阴离子(O2-)产生的速率明显增加,而过氧化物酶(POD)的活性增加不明显,超氧化物歧化酶(SOD)活性和丙二醛(MDA)的含量明显下降。表明COR具有诱导甘蓝幼苗抗黑腐病的作用,而这种作用可能与COR能调控甘蓝幼苗中抗氧化酶活性、提高O2-产生速率以及降低MDA的含量相关。     

Metabolism of cisanilide (Cis-2,5-dimethyl-1-pyrrolidinecarboxanilide) by rat liver microsomes

Metabolism of [phenyl-¹⁴C] and [(2,5) pyrrolidine-¹⁴C] cisanilide was investigated in vitro with microsomal preparations from rat liver. Microsomal activity was associated with a mixed-function oxidase system that required O₂ and NADPH and was inhibited by CO. Two major ether-soluble metabolites were isolated. They were identified as primary oxidation products: 2-hydroxy-2,5-dimethyl-1-pyrrolidinecarboxanilide (A) and 4′-hydroxy-2,5-dimethyl-1-pyrrolidinecarboxanilide (B). Minor ether-soluble metabolites were also isolated. Precursor product studies and qualitative thin layer chromatography analysis of [pyrrolidine-¹⁴C] and methylated [phenyl-¹⁴C] hydrolysis products suggested that these metabolites were secondary oxidation products formed from metabolites A or B. One of these metabolites appeared to be the dihydroxy product 2,4′-dihydroxy-2,5-dimethyl-1-pyrrolidinecarboxanilide. Crude microsomal preparations (postmitochondrial supernatant fractions) also formed small quantities (<10%) of polar metabolites. Enzyme hydrolysis with β-glucuronidase (Escherichia coli) indicated that approximately 50% of these metabolites were glucuronides. Similarities and differences in cisanilide oxidation in vivo in plants and in vitro with rat liver microsomal preparations were discussed.