Effect of some ergosterol biosynthesis inhibiting fungicides on sterols and cytochrome P-450 from the Japanese quail Coturnix coturnix

Nine fungicides that inhibit ergosterol biosynthesis (diclobutrazol, fenarimol, fenpropimorph, imazalil, nuarimol, prochloraz, propiconazole, triadimefon, triadimenol) and one plant growth regulator (ancymidol) were administered to Japanese quails (Coturnix coturnix). Most of these compounds had a moderate or no effect, but prochloraz, imazalil and, to a lesser extent, propiconazole were shown to produce a dramatic increase in liver weight and cytochrome P-450 level. These three compounds were also found to be potent in-vitro inhibitors of 7-ethoxycoumarin O-de-ethylase and aniline hydroxylase, thus resulting in a biphasic effect on drug-metabolising enzymes. With these three compounds, and some others, an accumulation of lanosterol in liver was also observed, suggesting an inhibition of sterol synthesis.     

Binding of azole fungicides related to diclobutrazol to cytochrome P-450

Fungicides containing the imidazole and triazole groups are known to block the 14α-demethylation reaction in ergosterol biosynthesis, which is a cytochrome P-450 enzyme system. Fungicides related to diclobutrazol [(2RS, 3RS)-1-(2,4-dichlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)pentan-3-ol] bind to cytochrome P-450 in rat liver microsomes, whole yeast cells, yeast microsomes and to a partially purified cytochrome P-450 from yeast, with Type II spectral changes. The most fungicidally active isomer (2R, 3R) shows greater binding than the less active (2S, 3S)-enantiomer to yeast microsomes; when the cytochrome P-450 was purified, a preparation was obtained to which binding more closely matched the fungicidal activity. Binding to rat liver microsomes does not reflect the fungicidal activity of the compounds.     

Development of a cell-free assay from Botrytis cinerea as a biochemical screen for sterol biosynthesis inhibitors

An assay for measuring ergosterol synthesis in cell-free extracts of the filamentous plant pathogen Botrytis cinerea is described. The extracts capable of synthesizing C4-desmethyl sterols from [2- 14 C]mevalonate were derived by mechanical disruption of young conidial germlings in a Bead-Beater apparatus. The C4-desmethyl sterol fraction consisted of three distinct compounds and totalled 39% of the non-saponifiable lipids formed. Ergosterol accounted for 63% of the C4-desmethyl sterols. Only small amounts of C4-monomethyl sterols were synthesized, while C4, 4-dimethyl sterols made up 29% of the non-saponifiable lipids. The latter fraction mainly consisted of lanosterol (54%) and eburicol (28%). The cell-free system had a narrow pH optimum for synthesis of C4-desmethyl sterols of pH 7.3–7.4. Cell-free synthesis of C4-desmethyl sterols was inhibited by the imidazole fungicide imazalil, concomitant with an accumulation of eburicol. The IC₅₀ value (concentration of fungicide which inhibited cell-free synthesis of C4-desmethyl sterols by 50%) was 9.1 × 10 ?9 M. These results are consistent with the hypothesis that imazalil is a potent inhibitor of the cytochrome P450-dependent sterol 14x-demethylase of B. cinerea. The method described may be used to screen compounds biochemically for inhibition of sterol synthesis in an agriculturally important plant pathogen.     

Effect of the fungicide imazalil on hepatic microsomal cytochrome P-450 and cytochrome P-450 dependent activities in the Bobwhite quail (Colinus virginianus)

Bobwhite quails were treated with imazalil for 8 weeks. The fungicide was given admixed in the food at 0, 100, 300, 500 and 1000 mg kg^?1. Even at the highest dose tested, imazalil did not affect the liver weight or the hepatic microsomal protein content. In treated quails, no significant induction of cytochrome P-450 or NADPH-cytochrome c-reductase activity was observed. Furthermore, imazalil did not induce or inhibit 7-ethoxyresorufin or 7-ethoxycoumarin O-deethylase in quail microsomes. Only a slight but significant increase by 35% and 49% in aniline hydroxylase activity was measured for the 500- and 1000-mg kg^?1 dose levels, respectively. After a drug-free period of one week, aniline hydroxylase activity returned to control values, indicating that the effect was fully reversible. It is concluded that imazalil does not induce or inhibit drug-metabolizing enzymes in the quail, even at doses which exceed by far the maximum levels currently used to dress seed under field conditions (100 mg kg^?1).     

Biochemical aspects of the fungitoxic and growth regulatory action of fenarimol and other pyrimidin-5-ylmethanols

The C-14 demethylation of the sterols, dependent on cytochrome P-450, and the C-22(23) desaturation of sterols are reactions in the ergosterol biosynthesis pathway that are regarded as primary target sites in the toxicity of fungicides of the pyrimidin-5-ylmethanol type. Currently, there is no evidence for target sites in other pathways of comparable sensitivity, although the failure of added ergosterol to reverse the fungitoxicity suggests the existence of such sites. The mitochondrial respiratory systems in Ustilago maydis and Aspergillus nidulans are insensitive to this type of fungicide and are not regarded as primary targets of fungitoxicity in these organisms. Appreciable evidence indicates that the primary targets in higher plant growth regulation are reactions, dependent on cytochrome P-450, that assist in the conversion of kaurene to kaurenoic acid in the gibberellin (GA) biosynthesis pathway. Growth retardation by high concentrations of pyrimidin-5-ylmethanols, which is not reversable by GA, apparently involves action at sites outside the GA biosynthesis pathway. The data derived from various studies of the mechanisms of fungitoxicity and growth regulation suggest that any undetected primary targets of the pyrimidin-5-ylmethanols are likely to be haem enzymes, similar to the cytochrome P-450 involved in sterol C-14 demethylation.     

Cytochrome P-450 Inducers and Inhibitors Interfering with Ecdysone 20-Monooxygenases and Their Activities during Postembryonic Development of Neobellieria bullata Parker

The cytochrome P-450-dependent microsomal and mitochondrial ecdysone 20-monooxygenase systems convert ecdysone into 20-hydroxyecdysone. The microsomal fraction of fat bodies of zero h wandering stage fleshfly larvae (Neobellieria bullata; Diptera: Sarcophagidae) has a high ecdysone 20- monooxygenase activity. The effects of cytochrome P-450 inhibitors were investigated in vitro on microsomal ecdysone 20-monooxygenase. Metyrapone, fenarimol and certain imidazole derivatives (KK-42, KK-110, KK-135 and PIM) are strong inhibitors. The IC₅₀ value of KK-110, which is the strongest inhibitor, is 2 × 10^?7 M. A triazolyl and two cyclopropylamine derivatives have low activity. The activities of different NADPH-cytochrome c (P-450) reductase inhibitors were also assessed; diquat dibromide is a moderate inhibitor of microsomal ecdysone 20-monooxygenase, while paraquat dichloride has no activity. In-vivo experiments with cytochrome P-450 inducers and inhibitors gave the following results: (a) fenarimol, FI-121, precocene-2 caused “permanent” first-instar larvae; (b) barbital, phenobarbital and their sodium salts caused significant delay in larval development; (c) PIM, PTM, metyrapone, KK-42, KK-135, J-2710, RH 5849 and colchicine caused moulting disturbances; (d) J-2710, PIM, PTM, KK-42, KK-135, RH 5849 and colchicine caused lethal spiracle and mandible malformation; (e) KK-110, fenarimol, barbital and phenobarbital caused precocious pupariation.     

Delta-aminolevulinic acid synthetase in the house fly and its possible role in oxidative resistance to insecticides

δ-Aminolevulinic acid synthetase (ALA synthetase EC 2.3.1.37) is the initial and rate-limiting enzyme in the biosynthetic pathway leading to heme and cytochrome formation in animals. The occurrence of ALA synthetase in house fly mitochondria was established and its possible relationship to oxidative resistance to insecticides was investigated.Levels of ALA synthetase in five house fly strains were measured and compared with levels of microsomal oxidases and cytochrome P-450 in the same strains. ALA synthetase was elevated in those strains with elevated levels of microsomal oxidases and cytochrome P-450 and was highest in the strain with the highest levels of microsomal oxidases and P-450. A possible regulatory role for ALA synthetase in relation to oxidative resistance to insecticides in the house fly is discussed.     

Dehydrogenation: A previously unreported pathway of lindane metabolism in mammals

This study presents evidence for the dehydrogenation of lindane by a hepatic microsomal mixed-function oxidase system. Preliminary investigation established that the incubation of lindane with rat liver homogenates produces a chlorinated, nonpolar compound identified as hexachlorocyclohexene. Differential centrifugation resulted in the sedimentation of most of the dehydrogenase activity in the microsomal fraction. Optimum in vitro assay conditions were established and it was found that the dehydrogenase system required molecular oxygen and reduced pyridine nucleotide coenzyme for maximum activity. Inhibition by SKF 525-A and CO suggested that the enzyme was cytochrome P-450 dependent. Lack of inhibition by cyanide indicated that the cytochrome b₅ desaturase system was probably not involved. Pretreatment of rats with DDT, which stimulates lindane metabolism, also induced significantly higher dehydrogenase activity. Both the in vivo and in vitro metabolism of hexachlorocyclohexene produced previously identified lindane metabolites. The existence of a cytochrome P-450 dependent mixed-function oxidase which catalyzes the dehydrogenation of lindane has not previously been reported and may be of importance in the metabolism of other xenobiotics.     

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.     

吡唑解草酯对小麦细胞色素P450的诱导作用及其光谱特征

用吡唑解草酯浇灌小麦,试验结果表明,50μmol/L吡唑解草酯处理抗6号小麦,可使其细胞色素P450含量达到最大值108.18 pmol/mg蛋白质,为对照组的1.67倍;100μmol/L吡唑解草酯处理敏18号小麦,可使其细胞色素P450含量达到最大值80.97 pmol/mg蛋白质,为对照组的1.86倍。吡唑解草酯对两个小麦品种的细胞色素P450均有诱导作用,抗6号小麦更容易被诱导,这与两小麦品种的耐药性一致。室温(20±1)℃下扫描不同时间的细胞色素P450-CO结合光谱,结果表明,微粒体粗提液室温(20±1)℃保存200 min后,细胞色素P450完全转变为细胞色素P420。     

Effects of sterol biosynthesis-inhibiting (SBI) fungicides on cytochrome P-450 oxygenations in fungi

The fungicides miconazole, fenarimol, and etaconazole block ergosterol biosynthesis in fungi by inhibiting sterol 14α-demethylation, which is mediated by a cytochrome P-450 enzyme. The sensitivity of cytochrome P-450-dependent hydroxylation or demethylation of several substrates to these fungicides and similar compounds was compared to that of fungal growth and sterol 14α-demethylation. Demethylation of p-chloro-N-methylaniline (PCMA) by sporidia of Ustilago maydis and 11α-hydroxylation of progesterone by Aspergillus nidulans were relatively insensitive to these compounds and to metyrapone. The ability of a sterol 14α-demethylation-deficient mutant to demethylate PCMA indicates that this substrate is not demethylated by the sterol 14α-demethylation system of U. maydis. The 14α-hydroxylation of progesterone by cells of Curvularia lunata was quite sensitive to the three fungicides, and also to metyrapone and isopropylphenylimidazole. This system was less sensitive to the three fungicides than sterol 14α-demethylation, but was appreciably more sensitive than PCMA demethylation. A study of progesterone 14α-hydroxylation in cell-free preparations of C. lunata showed the reaction to be inhibited by CO, and to be competitively inhibited by low concentrations of miconazole. These data suggest that the primary action of sterol biosynthesis-inhibiting (SBI) fungicides is competitive inhibition of sterol/steroid-type cytochrome P-450 enzymes rather than interference with the function of sterol carrier proteins or enzyme-modulating phospholipids.     

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.     

Fungitoxicity and growth regulation involving aspects of lipid biosynthesis

Triarimol and triforine inhibit ergosterol biosynthesis in fungi and cause accumulation of free fatty acids, 24-methylenedihydrolanosterol, obtusifoliol and 14α-methyl-δ^8,24(28)-ergostadienol. Triparanol also inhibits ergosterol synthesis and causes accumulation of free fatty acids, but not of the latter 3 sterols. Triparanol appears to inhibit prior to lanosterol in the sterol biosynthetic pathway of Ustilago maydis and at unidentified sites subsequent to lanosterol which lead to the accumulation of a sterol which migrates with desmethylsterols on TLC plates. Quantitative abnormalities in sterols and free fatty acids in U. maydis are not produced by the fungicides carbendazim, chloroneb, carboxin and cycloheximide. A deficiency in nitrogen leads to a marked increase in triglycerides, but a normal distribution pattern for other lipids.Inhibition of oxidative demethylation of the sterol 14α-methyl group is probably the prime mechanism of inhibition of ergosterol biosynthesis by triarimol. Rates of formation of obtusifoliol and 14α-methyl-δ^8,24(28)-ergostadienol in triarimol-treated U. maydis cells suggest that C-4 demethylation occurs along an abnormal pathway which operates effectively only at high substrate concentrations. The growth retardant action of triarimol and ancymidol in higher plants most likely results from inhibition of a reaction in the gibberellin biosynthetic pathway analogous to sterol C-14 demethylation.Free fatty acid accumulation in U. maydis cells treated with inhibitors of sterol synthesis are derived mainly from polar lipid degradation and from de novo synthesis as a consequence of the disproportionality between fatty acid synthesis and utilization. The free fatty acids may play a significant role in the lethality of these inhibitors in this organism.     

Prochloraz,a potent inducer of the microsomal cytochrome P-450 system

Prochloraz (N-propyl-N-[2-(2,4,6-trichlorophenoxy)ethyl]-imidazole-1-carboxamide), a recently developed agricultural fungicide, is a potent inducer of microsomal enzymes. Rats fed 7 days with a prochloraz-contaminated diet (2500 ppm) showed an increase in hepatic cytochrome P-450, cytochrome b₅, and microsomal protein level; aniline hydroxylase, 7-ethoxycoumarin dealkylase, 7-ethoxyresorufin dealkylase, NADPH-cytochrome c reductase, and epoxide hydrolase were significantly induced. At a lower dose (100 ppm), only an increase in cytochrome P-450 and 7-ethoxyresorufin dealkylase was noticed. As shown with aniline hydroxylase and 7-ethoxycoumarin dealkylase, prochloraz is also a potent inhibitor of drug-metabolizing enzymes. The interaction of prochloraz with hepatic microsomal fraction from rat liver was also studied. Prochloraz binds to oxidized cytochrome P-450 to produce a type II spectral change; the compound also binds to reduced cytochrome P-450. The binding of some ligands (7-ethoxycoumarin, n-octylamine, aniline, and imidazole) to oxidized cytochrome P-450 was determined after induction by prochloraz. Japanese quails (Coturnix coturnix) fed 7 days with a prochloraz-contaminated diet (2000 ppm) showed a dramatic increase in liver weight (2.5-fold) and both hepatic and duodenal cytochrome P-450 (9- and 12-fold, respectively).     

Investigation of azole resistance in the Ustilago maydis sterol demethylase mutant erg 40

Resistance to azole fungicides in Ustilago maydis (DC) Corda has been examined using the mutant erg 40, a newly isolated mutant Tri^R-1 and erg 40 revertants. Azole-induced growth arrest of the wild type did not support an obvious role for 3,6-diol in the mode of action has is clear for Saccharomyces cerevisiae Meyer ex Hansen. The level of microsomal P450 of erg 40 was identical to that of the parent, and reversion analysis showed no evidence of mutation in the sterol Δ⁵⁽⁶⁾ desaturase, as would be expected for a S. cerevisiae mutant accumulating 14α-methylfecosterol. Resistance appeared to be due to a single mutation in P450 14αdm. It is proposed that the orthologous forms of fungal sterol Δ⁵⁽⁶⁾ desaturases have varied responses when attempting to utilise 14α-methylated substrates.     

Conversion of α-pinene to α-pinene oxide by rat liver and the bark beetle Dendroctonus terebrans microsomal fractions

The metabolism of α-pinene, a major monoterpene in Pinus spp. in the United States, has been examined utilizing microsomal fractions from larval and adult Dendroctonus terebrans and rat liver. Under hydroxylating conditions, both insect and rat liver microsomes convert α-pinene into α-pinene oxide and several other undentified products. α-Pinene oxide was identified by mass spectrometry. α-Pinene is an inducer of cytochrome P-450 in rat liver microsomes and its effect on the pattern of α-pinene metabolism is very similar to β-naphthoflavone. No increase in cytochrome P-450 was observed when insects were treated with α-pinene; however, the quantity of α-pinene metabolic products was increased by α-pinene pretreatment. The role of cytochrome P-450 linked reactions in the production of insect pheromones via the α-pinene epoxide intermediate is discussed.     

Biochemical genetics of oxidative resistance to diazinon in the house fly

The genetics and biochemistry of oxidative resistance to diazinon were investigated in a diazinon-resistant strain of the house fly, Musca domestica L. The resistant strain was crossed with a multimarker susceptible strain and substrains containing portions of the resistant strain genome were prepared. Resistance, microsomal oxidase, and cytochrome P-450 spectral characteristics were then compared in the different strains. The major gene for resistance to diazinon is semidominant and is located on chromosome II, 13 crossing over units from the recessive mutant stubby wing. Additional resistance genes occur on chromosome II and on other chromosomes as well. Resistance to diazinon was introduced into a susceptible mutant-marked strain via genetic crossing over. Increases in parathion oxidase, total and P-450-specific N- and O-demethylase activity, and resistant strain type I binding spectrum were introduced along with resistance, indicating genes controlling these parameters and resistance are either identical or closely linked. No increase in activity of cytochrome P-450 itself was introduced into the mutant strain. Additional genes controlling the amount of cytochrome P-450 and several spectral changes characteristic of the resistant strains are apparently controlled by genes located at different loci on chromosome II. Resistance factors on other chromosomes are also present, but were not characterized.     

Resistance in mutagen-induced mutants of Ustilago maydis to fungicides which inhibit ergosterol biosynthesis

Resistance to a number of inhibitors of sterol C-14 demethylation, (clotrimazole, imazalil, miconazole, fenarimol, nuarimol and triadimefon), as well as resistance to inhibitors of sterol C-14(15) double bond reduction, (tridemorph and fenpropi-morph), was readily induced in Ustilago maydis. Resistant mutants were obtained after mutagenic treatment by ultraviolet irradiation, or by treatment with 1-methyl-3-nitro-1-nitrosoguanidine, of sporidia of the wild-type strain, followed by selection in the presence of the toxicant. The level of resistance of these mutants varied appreciably. Although not always reciprocal, cross-resistance to fungicides which inhibit ergosterol biosynthesis (EBIs) appeared to be present in most cases. Several of the U. maydis mutants which were resistant to inhibitors of sterol C-14 demethylation lacked cross-resistance to tridemorph and fenpropimorph, or displayed increased sensitivity to fenpropimorph (negatively correlated cross-resistance). Cross-resistance between EBIs and the antimicrobial agents climbazole and lombazole was also established. It is suggested that fungal mutants that possess a resistance mechanism based on a deficiency in sterol C-14 demethylation or sterol C-14(15) double bond reduction, have a greatly reduced chance of survival.     

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.