Enhanced repellency of binary mixtures of Zanthoxylum armatum seed oil, vanillin, and their aerosols to mosquitoes under laboratory and field conditions

The repellency of Zanthoxylum armatum seed oil (ZA-SO), alone or in combination with vanillin (VA), its six major constituents, and another four major previously known Zanthoxylum piperitum fruit oil constituents, as well as aerosol products containing 5 or 10% ZA-SO and 5% VA, was evaluated against female Aedes aegypti in laboratory and field studies. Results were then compared with those of N,N-diethyl-3-methylbenzamide (DEET) as a standard. Hand in cage laboratory tests showed that 0.2, 0.1, and 0.05 mg/cm2 ZA-SO resulted in > 92% protection through 30-min postexposure and was not significantly different than 0.05 mg/cm2 DEET. Skin treated with linalool and limonene (from Z. armatum) provided > 80% repellency to female Ae. aegypti at 10-min exposure, whereas cuminaldehyde, citronellal, geranyl acetate, and cuminyl alcohol (from Zanthoxylum piperitum) provided > 90% protection during this same time period. Only cuminaldehyde and citronellal provided complete protection comparable to DEET at 10-min postexposure. After that time, repellency of all plant constituents to mosquitoes was considerably decreased (< approximately 65%). An increase in repellency and duration of effectiveness was produced by a binary 1:4 mixture of ZA-SO and VA (0.05:0.2 mg/cm2) that was significantly more effective than 0.05 mg/cm2 DEET through 90 min. In field tests, an aerosol formulation containing 5 or 10% ZA-SO plus 5% VA gave 100% repellency at 60-min postexposure. Although these formulations were equal to the level of protection afforded by 10% DEET, repellency to the binary ZA-SO aerosol formulations at 90 min was significantly less effective than DEET. However, mixtures formulated from ZA-SO and VA merit further study as potential repellents for protection of humans and domestic animals from biting and nuisance caused by mosquitoes.     

In vitro effects of Artemisia dracunculus essential oil on ruminal and abomasal smooth muscle in sheep

Recently, essential oils have been substituted for synthetic antibiotics in improving rumen fermentation and animal performance. When ingested, along with their positive properties, some adverse effects may also be observed in animals. This study was primarily aimed at investigating the effects of the essential oils extracted from Artemisia dracunculus (EOAD) on ruminal and abomasal smooth muscle. EOAD was extracted using hydrodistillation. Sixteen compounds, representing 94.06% of the oil, were identified using gas chromatography/mass spectrometry analyses, Hinokitiol (17.47%), estragole (17.28%), pulegone (10.23%), limonene (7.57%), methyl eugenol (7.46%), and bornyl acetate (7.12%) were the major compound in the oil. In the in vitro study, except for isolated ruminal strips, abomasal preparations from 24 healthy sheep, EOAD (0.1–100?μg/mL) evoked a weak spasmogenic effect followed by relaxation, but complete abolishment of spontaneous contraction occurred at the highest dose (1,000?μg/mL) (P?<?0.05). EOAD significantly (P?<?0.05) inhibited the Ach-induced contraction when tissues were pre-incubated with the highest doses. When animal rations are supplemented with essential oils, a reduced basal tone and an impaired response of the rumen and abomasal muscles to acetylcholine reflect hypotony and subsequently predispose the animals to abomasal displacement, abomasal rotation, and or indigestion.     

Factors involved in the down-regulation of cytochrome P450 during Listeria monocytogenes infection

The activation of host defense mechanisms has been shown to cause a depression in hepatic cytochrome P450-mediated metabolism in rodents and humans. In a previous study, it was demonstrated that the Gram-positive bacteria Listeria monocytogenes causes a down-regulation of hepatic cytochrome P450 and related substrate metabolism as a result of a pretranslational depression of apoprotein synthesis. The objectives of this study were to determine whether the effect of listeria on hepatocyte cytochrome P450 involves hepatic nonparenchymal cells and whether the hemolysin, secreted only by hemolytic forms of the bacteria, plays any part in mediating this effect. Total cytochrome P450 levels as well as ethoxyresorufin-O-dealkylase (EROD) and benzyloxyresorufin-O-dealkylase (BROD) activities were significantly reduced in hepatic microsomes isolated from mice infected in vivo for 48 h with 15U listeria, whereas the same dose of the avirulent non-hemolytic M3D strain had no effect. Listeria (15U) significantly depressed hepatocyte EROD and BROD activities after 24 h incubations with liver cell cultures containing hepatocytes and nonparenchymal cells, as the result of both a direct effect on the hepatocyte and an interaction of listeria with hepatic nonparenchymal cells. The M3D strain of listeria had no effect on cytochrome P-450-mediated metabolism in isolated cells, confirming that hemolysin is an essential component of the mechanism responsible for the down-regulation of cytochrome P450 during listeria infections.     

1.8%AVM乳油对小鼠肝微粒体CYP450酶系与GST影响的初探

目的观察1.8%阿维菌素（AVM）乳油对小鼠肝微粒体细胞色素P450（CYP450）酶系与谷胱甘肽S-转移酶（GST）的影响,初步探讨1.8%AVM在肝脏的可能的代谢过程和毒性机理。方法 40只清洁级昆明种小鼠（雌雄各半）灌胃给予1.8%AVM乳油（70、35、17.5mg/kg）,连续7d,以0.9%氯化钠溶液作对照。末次给药后处死小鼠,采用差速离心法制备大鼠肝微粒体,Brandford法测定微粒体蛋白浓度,CO还原差示光谱法检测肝微粒体CYP450含量,差示光谱法则定肝微粒体b5（Cyt-b5）含量,紫外分光光度法则定肝微粒红霉素N-脱甲基酶活性（ERD）和氨基比林-N-脱甲基酶（ADM）和GST的活性。结果各1.8%AVM乳油染毒剂量组ERD、ADM活性均低于对照组,差异有统计学意义（P〈0.05）;各剂量组小鼠肝脏指数、CYP450与b5的含量及GST的活性与对照组比较差异均无统计学意义（P〉0.05）,且不同剂量组间差异亦无统计学意义（P〉0.05）。结论 1.8%AVM乳油可抑制小鼠肝微粒体ERD（主要反映CYP3A活性）和ADM（主要反映CYP2E1）的活力,而对CYP450和b5含量及GST活性影响小,未观察到1.8%AVM乳油对小鼠肝微粒体重要的Ⅰ相酶CYP450和Ⅱ相酶GST的诱导或抑制作用。     

Noncompetitive mixed-type inhibition of rainbow trout CYP1A catalytic activity by clotrimazole

Over the past two decades a number of antifungal imidazole derivatives have been approved for use in agricultural. The purpose of this study was to characterize the interaction of a model antifungal imidazole compound with a cytochrome P450 isozyme in a species of fish. Clotrimazole inhibited rainbow trout (Oncorhyncus mykiss) hepatic CYP1A-catalyzed ethoxyresorufin O-deethylase (EROD) activity in vivo and in vitro. Although clotrimazole inhibited EROD activity in vivo, it did not effect CYP1A mRNA levels. Addition of clotrimazole to microsomes produced a type II binding spectrum and clotrimazole was determined to be a noncompetitive mixed-type inhibitor of EROD activity with an IC50 of 190 nM. Since antifungal imidazole compounds may be co-applied with other pesticides, inhibition of cytochrome P450 activity by antifungal imidazole compounds may lead to unexpected toxicological interactions.     

Changes of drug metabolizing enzymes in the liver of male sheep exposed to either cypermethrin or dimethoate

Xenobiotics such as insecticides are metabolized to more or less toxic metabolites by drug-metabolizing enzymes including cytochrome P450 (Cyp P450), cytochrome b5 (Cyp b5), NADPH-cytochrome c reductase (Cyt.c R), N-nitrosdimethylamine-N-demethylase I (NDMA-dI), glutathione (GSH), glutathione s-transferase (GST), and glutathione reductase (GR). Therefore, the present study showed the influence of oral administration of cypermethrin (6 and 12 mg/kg/day) and dimethoate (1.6 and 3.2 mg/kg/day) for 63 consecutive days on the activities of the above mentioned enzymes in the livers of male sheep. Low and high-treatments of sheep with cypermethrin significantly increased the levels of Cyp P450 by 56% and 98%, Cyp b5 by 65% and 80%, GSH by 68% and 74%, and Cyt.c R by 67% and 98%, respectively in a dose-dependent manner. However, low dose of cypermethrin increased the activities of GST and GR by 56% and 91% respectively. In addition, low and high dose-treatments with dimethoate increased the hepatic contents of Cyp P450 by 27% and 40%, GSH by 259% and 132%, whereas NDMA-dI decreased by 27 and 55% respectively, and no change in the content of Cyp b5 and the activity of Cyt.c-R at any given dose of this compound. It is concluded that exposure to cypermethrin and dimethoate significantly changed the hepatic activity of phases I & II drugmetabolizing enzymes in sheep, and these changes are mainly dependent on the administred dose, and also on the type of the tested insecticides. Also, such changes should be considered when therapeutic drugs administered to people exposed to such insecticides.     

Protection from N-nitrosodimethylamine-mediated liver damage by indole-3-carbinol

Indole-3-carbinol (I-3-C) was examined for its ability to protect mice against 24-hr N-nitrosodimethylamine (NDMA)-mediated hepatotoxicity. NDMA (20 mg/kg body weight) alone produced extensive hemorrhagic and centrolobular necrotic lesions, with a necrotic severity index of 3.0 +/- 0.4 (scale of 0-5). Treatment with 50 mg/kg body weight of I-3-C by gavage, 1 hr prior to NDMA, substantially protected against hemorrhagic lesions. Furthermore, I-3-C lowered the NDMA-mediated tissue necrotic index to 1.5 +/- 0.3, by reducing the extent of tissue necrosis rather than the severity in the necrotic region. Release of liver enzymes into the blood correlated with the histopathology; I-3-C reduced NDMA-mediated elevated activities of plasma alanine transaminase and ornithine transcarbamylase by 84 and 51.3%, respectively. Although no changes in nonprotein sulfhydryls were evident at 24-hr after NDMA, ascorbate levels were reduced to 40% of control values. However, treatment with I-3-C prior to NDMA prevented the decline in tissue ascorbate concentrations. In vitro, I-3-C was found to be a type II ligand for cytochrome P-450, with a Ks value of 237 microM. However, if such binding occurs in vivo, it does not protect against the approximately 60% decrease in hepatic cytochrome P-450 or the 80% decrease in NDMA demethylase I activity produced by NDMA. Since I-3-C slightly enhances cytochrome P-450 content and NDMA demethylase activity, the histopathologic protection by I-3-C must be due to factors other than inhibiting metabolic activation of NDMA.     

Selective enhancement of cytochrome p-450 activity in rat hepatocytes by in vitro heat shock

We investigated the effect of heat shock on cytochrome P-450 activity in rat hepatocytes and report a significant, selective, and time-dependent enhancement of cytochrome P-450 activity in heatshocked hepatocytes. Stable long-term cultures of rat hepatocytes were heat shocked (42.5 degrees C) for 1 to 3 h and allowed to recover at 37 degrees C. Cytochrome P-450-dependent ethoxyresorufin O-dealkylase (EROD) and benzyloxyresorufin O-dealkylase (BROD) activities were measured up to 48 h after heat shock treatment. In general, the optimal heat shock exposure time was between 2 and 3 h. BROD activity (induced by sodium phenobarbital) increased approximately 6-fold in hepatocytes heat shocked for 3 h in comparison with hepatocytes maintained at 37 degrees C. EROD activity (induced by 3-methylcholanthrene) increased 2-fold on exposure to heat shock for 2 h. The expression of inducible heat shock proteins Hsp70 and Hsp32 was verified by Western immunoblot analyses. In the absence of the appropriate inducer, heat shock treatment did not enhance cytochrome P-450 activity. Furthermore, enhanced P-450 enzyme activity was delayed for heat-shocked hepatocytes. It is hypothesized that heat shock treatment attenuates the negative effects triggered by the addition of the toxic inducers and possibly stabilizes the levels of cytochrome P-450 proteins. These results suggest that heat shock treatment may be used to enhance the functionality of hepatocytes, specifically, in bioartificial liver assist devices.     

Evaluation of novel insecticides for control of dengue vector Aedes aegypti (Diptera: Culicidae)

Insecticides are one of the major tools for controlling vector populations and for reducing the transmission of human pathogens. However, there are few new insecticides being developed and marketed for vector control. Herein, we report on the toxicity of six novel insecticides to both adult and larval Aedes aegypti (L). and the toxicity of three novel insect growth regulators (IGRs) to larvae. Four insecticides were highly or moderately toxic to larvae with LC50 values of 16 (chlorfenapyr), 70 (hydramethylnon), 79 (indoxacarb), and 84 ng/ml (imidacloprid). Diafenthiuron and chlorfenapyr were moderately toxic to adult mosquitoes with LC50 values of 13 and 92 ng/cm2, respectively. Imidacloprid was strongly synergized by piperonyl butoxide (PBO) in Ae. aegypti adults, suggesting that neonicotinoids are intrinsically very toxic to adult mosquitoes (in the absence of detoxification). The effect of PBO on the toxicity in adults and larvae was considerably different, both in terms of the insecticides that were synergized (or antagonized for chlorfenapyr versus adults) and in terms of the degree of synergism. This result implies that the cytochrome P450s involved in metabolism of these insecticides are different between adults and larvae. Pyriproxyfen was confirmed as a potent IGR (EC50 of 0.0017 ng/ml) for mosquitoes, although tebufenozide lacked activity. The potential for use of these materials in mosquito control is discussed.     

Laboratory evaluation of pyriproxyfen and spinosad, alone and in combination, against Aedes aegypti larvae

In this study, the efficacy of pyriproxyfen and spinosad, alone and in combination, was evaluated against the dengue vector Aedes aegypti (L.). Larval bioassays were carried out on susceptible mosquito larvae to determine the concentration-mortality responses of mosquitoes exposed to each insecticide alone and in mixture. Synergism between pyriproxyfen and spinosad was determined by the calculation of a combination index (CI) by using the isobologram method. For pyriproxyfen, LC50 and LC95 were 1.1 x 10(-4) (1.0 x 10(-4)-1.1 x 10(-4)) and 3.2 x 10(-4) (2.9 x 10(-4)-3.6 x 10(-4)) mg/liter, respectively. Pyriproxyfen acted at very low concentrations by inhibiting the adult emergence of Ae. aegypti (97% inhibition rates at 3.3 x 10(-4) mg/liter). Spinosad activity was -500 times lower than that of pyriproxyfen against the Bora strain, with LC50 and LC95 values estimated at 0.055 (0.047-0.064) and 0.20 (0.15-0.27) mg/liter, respectively. A binary mixture of pyriproxyfen and spinosad was realized at the ratio 1:500 by considering the values of the LC50 obtained for each product. The LC50 and LC95 of the mixture were 0.019 (0.016 - 0.022) and 0.050 (0.040 - 0.065) mg/liter, respectively. The mixture combined both the larvicidal activity of spinosad and the juvenoid action of pyriproxyfen. From the LC70 to LC99 a significant synergism effect was observed between the two insecticides (CI ranged from 0.74 to 0.31). This strong synergism observed at high concentrations allows a reduction by five and nine-fold of pyriproxyfen and spinosad amounts to kill almost 100% mosquitoes. Combination of pyriproxyfen and spinosad may then represent a promising strategy to improve mosquito control in situations with insecticide-resistant Aedes dengue vectors.     

Effects of inhibition and induction of cytochrome P-450 isozymes on hyperoxic lung injury in rats.

Pulmonary oxygen toxicity most likely results from excessive production of reactive oxygen species. The role of the cytochromes P-450 in this process is controversial because these enzymes have been reported both to enhance hyperoxic lung injury and to protect from the damaging effects of 100% oxygen. We sought to further determine the role of the cytochromes P-450 in hyperoxic lung injury by inhibiting and inducing pulmonary cytochrome P-450 isozymes in rats. Treatment with the cytochrome P-450 inhibitor cimetidine or 8-methoxypsoralen did not improve survival or reduce lung edema in rats exposed to 100% oxygen. The activity of cytochrome P-450IIB1, the major pulmonary cytochrome P-450 isozyme in rats, was clearly inhibited by 8-methoxypsoralen. beta-Naphthoflavone (beta NF), a selective inducer of cytochrome P-450IA1, was administered in two-dose and five-dose regimens. The two-dose regimen produced significant and sustained induction of cytochrome P-450IA1 activity, but survival in these rats was not improved when exposed to 100% oxygen. In rats treated with five doses of beta NF, a small increase in survival time was found from 71.1 +/- 8.7 to 88.0 +/- 20.2 h; however, there was no difference in the induction of cytochrome P-450IA1 activity between this five-dose regimen and the two-dose regimen. The small improvement in survival after five doses of beta NF is thus unrelated to cytochrome P-450IA1 induction. We conclude that neither inhibition of cytochrome P-450IIB1 activity nor induction of cytochrome P-450IA1 activity protects adult rats against hyperoxic lung injury.     

Combined effect of heat, essential oils and salt on fungicidal activity against Trichophyton mentagrophytes in a foot bath.

This work was originally undertaken to determine the effective conditions of essential oils against Trichophyton mentagrophytes in vitro for the treatment of tinea pedis in a foot bath. Agar blocks implanted with T. mentagrophytes were immersed in 0.1% aqueous agar containing two-fold dilutions of essential oils with or without sodium chloride at 27 degrees C, 37 degrees C and 42 degrees C for 10 and 20 min. The number of surviving mycelia on the agar blocks was determined from the standard curves of the colony diameter and original inocula of the conidia. At the same time, the thermal effect on the cellular morphology was examined using SEM. Most fungal mycelia (99.7%) were killed after treatment at 42 degrees C for 20 min without essential oil. The fungicidal activity of essential oils was markedly enhanced by treating at 42 degrees C for 20 min as compared with that at 27 degrees C, showing 1/4 - 1/32-fold reduction of minimum fungicidal concentration (MFC to kill 99.99%). The order of the fungicidal activity of 11 essential oils was oregano, thyme thymol, cinnamon bark > lemongrass > clove, palmarose, peppermint, lavender > geranium Bourbon, tea tree > thyme geraniol oils. MFCs were further reduced to 1/2 - 1/8 by the addition of 10% sodium chloride. The salt effect was explained, at least partly, by an increase in mycelial adsorption of antifungal constituents in the presence of sodium chloride. Considerable hyphal damage was done at 27 degrees C by the essential oils, but no further alteration in morphology of the hyphae treated at 42 degrees C with or without oil was observed by SEM. The inhibitory effect of heat and oils was also observed against mycelia of T. rubrum and conidia of T. mentagrophytes. Thermotherapy combined with essential oils and salt would be promising to treat tinea pedis in a foot bath.     

Investigation of activity of monoterpenes and phenylpropanoids against immature stages of Amblyomma cajennense and Rhipicephalus sanguineus (Acari: Ixodidae)

The objective of this study was to assess the acaricidal activity of carvacrol, thymol, eugenol, and (E)-cinnamaldehyde on unengorged larvae and nymphs of Amblyomma cajennense and Rhipicephalus sanguineus, using the modified larval packet test. Carvacrol, eugenol, and (E)-cinnamaldehyde were tested at concentrations of 2.5, 5.0, 10.0, 15.0, and 20.0 μl/ml, while thymol was tested at concentrations of 2.5, 5.0, 10.0, 15.0, and 20.0 mg/ml, in all cases with 10 repetitions per treatment. For the A. cajennense larvae, mortality rates caused by carvacrol, thymol, eugenol, and (E)-cinnamaldehyde at the lowest concentration were 45.0, 62.7, 10.2, and 81.6 %, respectively, reached 100 % at the concentration of 5.0 μl/ml for carvacrol and (E)-cinnamaldehyde and 5.0 mg/ml for thymol, while this mortality was observed at 15.0 μl/ml for eugenol. For the nymphs of this species, carvacrol and thymol caused 100 % mortality starting at a concentration of 5.0 μl/ml and 10.0 mg/ml, respectively, while eugenol caused 100 % mortality at 20.0 μl/ml and the mortality caused by (E)-cinnamaldehyde did not exceed 64 %. In the tests with R. sanguineus larvae, the lowest concentration of carvacrol and (E)-cinnamaldehyde resulted in 100 % mortality, while this percentage was observed starting at 10.0 μl/ml for eugenol. For nymphs, carvacrol and thymol at the smallest concentration caused 100 % lethality, unlike the results for eugenol and (E)-cinnamaldehyde, where 100 % mortality was only observed starting at the concentration of 10.0 μl/ml. The results obtained indicate that the tested substances have acaricidal activity on unengorged larvae and nymphs of A. cajennense and R. sanguineus.     

Enzymes-based resistant mechanism in pyrethroid resistant and susceptible Aedes aegypti strains from northern Thailand

Previous studies have shown that permethrin resistance in our selected PMD-R strain of Aedes aegypti from Chiang Mai, Thailand, was associated with a homozygous mutation in the knockdown resistance (kdr) gene and other mechanisms. In this study, we investigated the metabolic mechanism of resistance of this strain compared to the PMD strain which is susceptible to permethrin. The permethrin susceptibility of larvae was determined by a dose–response bioassay. Two synergists, namely piperonyl butoxide (PBO) and bis(4-nitrophenyl)-phosphate (BNPP), were also added to determine if the resistance is conferred by oxidase or esterase enzymes, respectively. The LC₅₀ value for PMD-R (25.42 ppb) was ∼25-fold higher than for PMD (1.02 ppb). The LC₅₀ was reduced 3.03-fold in PMD-R and 2.27-fold in PMD when the oxidase inhibitor (PBO) was added, but little or no reduction was observed in the presence of BNPP, indicating that oxidative enzymes play an important role in resistance. However, the LC₅₀ previously observed in the heterozygous mutation form was reduced ∼eightfold, indicating that metabolic resistance is inferior to kdr. The levels of cytochrome P450 (P450) extracted from fourth instar larvae were similar in both strains and were about 2.3-fold greater in microsomal fractions than in crude supernatant and cytosol fractions. Microsome oxidase activities were determined by incubation with each of three substrates, i.e., permethrin, phenoxybenzyl alcohol (PBOH), and phenoxybenzaldehyde (PBCHO), in the presence or absence of nicotinamide adenine dinucleotide phosphate (NADPH), nicotinamide adenine dinucleotide (NAD⁺), PBO, and BNPP. It is known that hydrolysis of permethrin produces PBOH which is further oxidized to PBCHO by alcohol dehydrogenase (ADH) and then to phenoxybenzoic acid (PBCOOH) by aldehyde dehydrogenase (ALDH). When incubated with permethrin, a small amount of PBCOOH was detected in both strains (about 1.1–1.2 nmol/min/mg protein), regardless of the addition of NADPH. The addition of PBO resulted in about 70% and 50% reduction of PBCOOH in PMD and PMD-R, respectively. The addition of BNPP reduced PBCOOH about 50% and 35% in PMD and PMD-R, respectively. Using PBOH as substrate increased PBCOOH ∼16-fold and ∼40-fold in PMD and PMD-R, respectively. Using PBCHO as substrate increased PBCOOH ∼26-fold and ∼50-fold in PMD and PMD-R, respectively. The addition of NADPH, and particularly NAD⁺, increased the level of PBCOOH. Together, the results have indicated the presence of a metabolic metabolism involving P450, ADHs, and ALDHs in both PMD and PMD-R strains, with greater enzyme activity in the latter.     

The effect of serum from liver cancer patients on the growth and function of primary and immortalised hepatocytes.

A limiting factor in the efficacy of bioartificial liver (BAL) for the treatment of liver failure is the toxicity of the patients' serum to the hepatocytes in the device. This study investigates the interaction of liver cancer patient serum with primary and immortalised rat hepatocytes. Liver cancer serum increased the growth rate of immortalised hepatocytes, without affecting reduced glutathione levels. The activities of DT-diaphorase and pi glutathione-S-transferase (GST), enzymes associated with de-differentiation, were also increased. Exposure of primary hepatocytes to liver cancer serum resulted in a decrease in cytochrome P450 (CYP) content, and in P450 dependent metabolism of testosterone. Formation of 2-alpha- and 6-beta- hydroxy testosterone was decreased. These reactions are predominantly associated with CYP 2C11 and 3A1 respectively in normal rat liver. The activity of total GST was also decreased, although that of the pi isoenzyme of GST was not affected. Our results suggest that exposure of hepatocytes in a bioreactor to liver cancer patient serum will result in overgrowth of cells, if proliferating cells are being used, and in de-differentiation. The serum may have to be pretreated with adsorbants to remove toxins prior to BAL treatment.     

Antimicrobial activity of five essential oils against origin strains of the Enterobacteriaceae family

An in vitro assay measuring the antimicrobial activity of essential oils of Coridothymus capitatus (Spanish origanum), Satureja montana, Thymus mastichina (Spanish Origanum majorana), Thymus zygis (Spanish variety of Thymus vulgaris) and Origanum vulgare has been carried out against poultry origin strains of Escherichia coli, Salmonella enteritidis and Salmonella essen, and pig origin strains of enterotoxigenic E. coli (ETEC), Salmonella choleraesuis and Salmonella typhimurium. Using the broth microdilution method, all the essential oils showed an MIC > or = 2% (v/v) for the two strains of E. coli. The essential oil that showed the highest antimicrobial activity against the four strains of Salmonella was Origanum vulgare (MIC < or = 1% v/v), followed by Thymus zygis (MIC < or =2% v/v). Thymus mastichina inhibited all the microorganisms at the highest concentration, 4% (v/v), while the rest of the essential oils showed highly variable results. By chemotyping, higher inhibitory capacity was observed in the oils with a higher percentage of phenolic components (carvacrol and thymol) in comparison with oils containing the monoterpenic alcohol linalool. The results of this work confirm the antimicrobial activity of some essential oils, as well as their potential application in the treatment and prevention of poultry and pig diseases caused by salmonella.     

Determination of cytochrome P450 1A activities in mammalian liver microsomes by high-performance liquid chromatography with fluorescence detection

A sensitive method for the determination of cytochrome P450 (P450 or CYP) 1A activities such as ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-demethylase (MROD) in liver microsomes from human, monkey, rat and mouse by high-performance liquid chromatography with fluorescence detection is reported. The newly developed method was found to be more sensitive than previous methods using a spectrofluorimeter and fluorescence plate reader. The detection limit for resorufin (signal-to-noise ratio of 3) was 0.80 pmol/assay. Intra-day and inter-day precisions (expressed as relative standard deviation) were less than 6% for both enzyme activities. With this improved sensitivity, the kinetics of EROD and MROD activities in mammalian liver microsomes could be determined more precisely. EROD activities in human and monkey liver microsomes, and MROD activities in liver microsomes from all animal species exhibited a monophasic kinetic pattern, whereas the pattern of EROD activities in rat and mouse liver microsomes was biphasic. In addition, the method could determine the non-inducible and 3-methylcholanthrene-inducible activities of EROD and MROD in rat and mouse liver microsomes under the same assay conditions. Therefore, this method is applicable to in vivo and in vitro studies on the interaction of xenobiotic chemicals with cytochrome CYP1A isoforms in mammals.     

Effect of PSK, a protein-bound polysaccharide from Coriolus versicolor, on drug-metabolizing enzymes in sarcoma-180 bearing and normal mice

The effects of PSK and Propionibacterium acnes (anaerobic Corynebacterium) on hepatic drug-metabolizing enzymes were studied using sarcoma-180 bearing and non-tumor bearing mice. PSK had no influence on aminopyrine N-demethylase and aniline hydroxylase activities, cytochrome P-450 concentration in hepatic microsomes, and the reductase activity of cytochrome c in normal mice. The content of cytochrome P-450 was not significantly reduced in S-180 bearing mice. On the other hand, P. acnes administration significantly decreased the amount of cytochromes P-450 and b5 and aminopyrine N-demethylase activity. When FT-207 (Tegafur) was administered orally to S-180 bearing mice combined with the immunoadjuvants, only P. acnes significantly reduced the 5-FU levels in the serum and some organs.     

Some effects of the fungicide propiconazole on cytochrome P450 and glutathione S-transferase in brown trout (Salmo trutta).

The fungicide propiconazole (1-(2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-ylmethyl) -1H-1,2,4-triazole) induced the hepatic cytochrome P4501A (CYP1A) activity towards ethoxyresorufin-O-deethylase (EROD), the content of CYP1A protein as quantified by enzyme-linked immunosorbent assay (ELISA) and the glutathione S-transferase (GST) activity towards the three commonly used substrates CDNB(1-chloro-2,4-dinitrobenzene), cumene hydroperoxide (CU) and ethachrynic acid (EA) in brown trout (Salmo trutta) depending on dose and body weight. An exponential dose response relationship existed between propiconazole exposure and CYP1A activity. A 2. order polynomial regression of the propiconazole concentration (square root transformed) on the data for CDNB, EU and CU revealed a bell-shaped pattern of the GST induction. Reverse-phase HPLC of the GSH-affinity chromatography purified GST isozymes in trout exposed to respectively 8.3, 23, 93, 313 and 606 microg l(-1) propiconazole in the water indicated that the propiconazole treatment may lead to changes in the composition of the subunits compared to the controls. Thus, propiconazole exposure through the water changed the properties of the brown trout hepatic CYP1A and GST, and these changes may be used as a bioindicator on the molecular level of exposure and effect of propiconazole in controlled experiments. The use in monitoring of propiconazole exposure under natural field conditions is possible, however needs further investigation.     

The mosquitocidal activity of methanolic extracts of Lantana cramera root and Anacardium occidentale leaf: role of glutathione S-transferase in insecticide resistance

Larvicidal activity of methanolic plant extracts of Lantana cramera (P1) root and Anacardium occidentale (P2) leaf was investigated against the larvae of the three mosquito species (Culex quinquefasciatus, Anopheles stephensi, and Aedes aegypti reared in the laboratory), and the respective glutathione S-transferase (GST) activity was analyzed as an index of protection against the extracts. The LC50 (extract concentration that shows 50% mortality) values of P1 extract for An. stephensi, Ae. aegypti, and Cx. quinquefasciatus were 132.55, 27.82, and 11.68 ppm, respectively, whereas those of P2 extract were 56.81, 912, and 10.79 ppm, respectively. In general, in the untreated groups, the level of GST activity was significantly higher in Ae. aegypti in comparison with An. stephesi and Cx. quinquefasciatus. However, the enzyme activity failed to show any response when treated with either of the plant extracts in Ae. aegypti. However, an increase in the GST activity was recorded in extract-treated larvae of both An. stephensi and Cx. quinquefasciatus. The results of the current study suggest that both the plant extracts show species-specific mosquitocidal potential. Induction of GST activities in survived An. stephensi and Cx. quinquefasciatus larvae suggests the role of this enzyme in conferring resistance to the plant extracts.