In the present study, time-dependency of the induction effect of a selective inducer on the activity, protein and mRNA levels of cytochromes P450 1A1/2 (CYP1A1/2), NAD(P)H:quinone oxidoreductase 1 (NQO1) and glutathione S-transferases (GSTA), in primary culture of rat hepatocytes was tested and evaluated. To show the differences in responses of tested enzymes, the common aryl hydrocarbon receptor (AhR) ligand agonist, beta-naphthoflavone (BNF), was used. Induction of CYP1A1/2 by BNF was detected at all time intervals and at all levels (i.e., mRNA, protein, enzyme activity). Different responses of NQO1 and GSTA upon BNF treatment were observed. Our results demonstrate that the responses of different xenobiotic-metabolizing enzymes to the inducer vary in time and depend on the measured parameter. For these reasons, an induction study featuring only one-time interval treatment and/ or one parameter testing could produce misleading information.
- MeSH
- beta-naftoflavon metabolismus MeSH
- cytochrom P-450 CYP1A1 metabolismus MeSH
- cytochrom P-450 CYP1A2 metabolismus MeSH
- glutathiontransferasa metabolismus MeSH
- hepatocyty metabolismus MeSH
- játra metabolismus MeSH
- krysa rodu rattus MeSH
- ligandy MeSH
- messenger RNA metabolismus MeSH
- NAD(P)H dehydrogenasa (chinon) metabolismus MeSH
- receptory aromatických uhlovodíků metabolismus MeSH
- xenobiotika metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Benzimidazoles anthelmintics, which enter into environment primarily through excretion in the feces or urine of treated animals, can affect various organisms and disrupt ecosystem balance. The present study was designed to test the phytotoxicity and biotransformation of the three benzimidazole anthelmintics albendazole (ABZ), fenbendazole (FBZ) and flubendazole (FLU) in the harebell (Campanula rotundifolia). This meadow plant commonly grows in pastures and comes into contact with anthelmintics through the excrements of treated animals. Suspensions of harebell cells in culture medium were used as an in vitro model system. ABZ, FLU and FBZ were not found to be toxic for harebell cells, which were able to metabolize ABZ, FLU and FBZ via the formation of a wide scale of metabolites. Ultrahigh-performance liquid chromatography coupled with high mass accuracy tandem mass spectrometry (UHPLC-MS/MS) led to the identification of 24, 18 and 29 metabolites of ABZ, FLU and FBZ, respectively. Several novel metabolites were identified for the first time. Based on the obtained results, the schemes of the metabolic pathways of these anthelmintics were proposed. Most of these metabolites can be considered deactivation products, but a substantial portion of them may readily be decomposed to biologically active substances which could negatively affect ecosystems.
- MeSH
- albendazol metabolismus MeSH
- anthelmintika chemie metabolismus MeSH
- benzimidazoly chemie metabolismus MeSH
- biotransformace MeSH
- Campanulaceae cytologie metabolismus MeSH
- ekosystém MeSH
- feces chemie MeSH
- fenbendazol metabolismus MeSH
- kultivované buňky MeSH
- mebendazol analogy a deriváty metabolismus MeSH
- metabolické sítě a dráhy * MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The present in vitro study was designed to test and compare anthelmintic activity, hepatotoxicity, and biotransformation of four selected aminoacetonitrile derivatives (AADs): monepantel (MOP, anthelmintic approved for the treatment), AAD-970, AAD-1154, and AAD-1336. Micro-agar larval development test, MTT test of cytotoxicity, and biotransformation study coupled with Ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) technique were used for this purpose. Larvae of two Haemonchus contortus strains (drug susceptible and multi-drug resistant) and primary cultures of rat and ovine hepatocytes served as model systems. All AADs (including MOP) exhibited significant larvicidal effect in H. contortus susceptible as well as multi-resistant strains, much higher than those of reference anthelmintics thiabendazole and flubendazole. AAD-1154 provides the best results for most tested parameters among all AADs in this study. The cytotoxicity test showed that all AADs can be considered as nontoxic for hepatocytes. In the biotransformation study, Phase I and Phase II metabolites of AADs were identified and schemes of possible metabolic pathways in ovine hepatocytes were proposed. Biotransformation of MOP was much more extensive than biotransformation of other AADs. Based on obtained results, AAD-1154 and AAD-1336 can be considered as promising candidates for further in vivo testing.
- MeSH
- aminoacetonitrily analogy a deriváty analýza farmakokinetika toxicita MeSH
- anthelmintika analýza farmakokinetika toxicita MeSH
- biotransformace MeSH
- Haemonchus účinky léků MeSH
- hepatocyty metabolismus MeSH
- krysa rodu rattus MeSH
- kultivované buňky MeSH
- larva MeSH
- mebendazol analogy a deriváty analýza farmakokinetika MeSH
- ovce MeSH
- potkani Wistar MeSH
- tandemová hmotnostní spektrometrie MeSH
- thiabendazol analýza farmakokinetika MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
The sheep tapeworm Moniezia expansa is very common parasite, which affects ruminants such as sheep, goats as well as other species. The benzimidazole anthelmintics albendazole (ABZ), flubendazole (FLU) and mebendazole (MBZ) are often used to treat the infection. The drug-metabolizing enzymes of helminths may alter the potency of anthelmintic treatment. The aim of our study was to assess the activity of the main drug-metabolizing enzymes and evaluate the metabolism of selected anthelmintics (ABZ, MBZ and FLU) in M. expansa. Activities of biotransformation enzymes were determined in subcellular fractions. Metabolites of the anthelmintics were detected and identified using high performance liquid chromatography/ultra-violet/VIS/fluorescence or ultra-high performance liquid chromatography/mass spectrometry. Reduction of MBZ, FLU and oxidation of ABZ were proved as well as activities of various metabolizing enzymes. Despite the fact that the conjugation enzymes glutathione S-transferase, UDP-glucuronosyl transferase and UDP-glucosyl transferase were active in vitro, no conjugated metabolites of anthelmintics were identified either ex vivo or in vitro. The obtained results indicate that sheep tapeworm is able to deactivate the administered anthelmintics, and thus protects itself against their action.
- MeSH
- albendazol farmakokinetika farmakologie MeSH
- alkoholoxidoreduktasy metabolismus MeSH
- anthelmintika farmakokinetika farmakologie MeSH
- biotransformace MeSH
- Cestoda účinky léků enzymologie ultrastruktura MeSH
- glukuronosyltransferasa metabolismus MeSH
- glutathiontransferasa metabolismus MeSH
- izoenzymy metabolismus MeSH
- katalasa metabolismus MeSH
- mebendazol analogy a deriváty farmakokinetika farmakologie MeSH
- monieziáza parazitologie MeSH
- multienzymové komplexy metabolismus MeSH
- NADH, NADPH oxidoreduktasy metabolismus MeSH
- nemoci ovcí parazitologie MeSH
- ovce MeSH
- oxidace-redukce MeSH
- oxygenasy se smíšenou funkcí metabolismus MeSH
- peroxidasa metabolismus MeSH
- superoxiddismutasa metabolismus MeSH
- systém (enzymů) cytochromů P-450 metabolismus MeSH
- tenké střevo parazitologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Monepantel (MOP), a new amino-acetonitrile anthelmintic for the treatment and control of gastrointestinal nematode infections and associated diseases in sheep, is approved and marketed as oral solution under the trade name Zolvix® (Novartis Animal Health Inc., Switzerland). The effect of MOP on hepatic cytochromes P450 (CYP) has been investigated in sheep. In an in vivo experiment, castrated rams (9-months old) were treated with the recommended therapeutic dose of MOP. Non-treated animals represented the controls. After 24 h, the animals were stunned and exsanguinated. Microsomal fractions and total RNA were prepared from liver homogenates. The activities towards alkyloxyresorufins, 7-methoxy-4-trifluoromethylcoumarin and midazolam were assayed and mRNAs of individual CYP isoforms were quantified. In an in vitro procedure, primary cultures of ovine hepatocytes were incubated with or without MOP (10 μM) for 24 h and then expression levels of individual CYP isoforms were analyzed. Results showed that MOP significantly increased all CYP-related activities and CYP3A24 mRNA in sheep. The induction effect of MOP on CYP3A was similar or even higher than those of dexamethasone and rifampicin, well-known CYP3A inducers. As CYP3A enzymes belongs to the most important biotransformation enzymes, their induction may have serious pharmacological and/or toxicological consequences. These facts should be taken into account when other drugs together with or after MOP (Zolvix®) are administered to sheep.
- MeSH
- aminoacetonitrily analogy a deriváty farmakologie MeSH
- cytochrom P-450 CYP3A genetika metabolismus MeSH
- hepatocyty cytologie účinky léků enzymologie MeSH
- játra účinky léků enzymologie MeSH
- kultivované buňky MeSH
- messenger RNA metabolismus MeSH
- ovce MeSH
- protein - isoformy genetika metabolismus MeSH
- systém (enzymů) cytochromů P-450 genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Monepantel (MOP) is a new anthelmintic drug intended for the treatment and control of gastrointestinal roundworms (nematodes) infection and associated disease in sheep. The aim of our study was to find out metabolic pathways of MOP in sheep in vivo and in its parasite Haemonchus contortus ex vivo. MOP biotransformation in two H. contortus strains with different sensitivity to anthelmintics was also compared. Ultra high-performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) technique is used for the identification of MOP metabolites in ovine urine, faeces, and nematodes. MOP biotransformation study in sheep in vivo led to the identification of 13 MOP metabolites; 7 of them have not been described previously in in vitro study. The study of MOP biotransformation in H. contorus ex vivo reveals four MOP metabolites. The nitrile hydrolysis as a new biotransformation pathway in helminths ex vivo is reported here for the first time. Unlike sheep, H. contorus nematodes are not able to metabolize MOP via phase II biotransformation. Nematodes of resistant White river (WR) strain form more types of MOP metabolites than nematodes of sensitive inbred susceptible Edinburgh (ISE) strain. Based on obtained results, schemes of metabolic pathways of MOP in sheep and nematodes are proposed.
- MeSH
- aminoacetonitrily analogy a deriváty analýza metabolismus MeSH
- anthelmintika analýza metabolismus MeSH
- Haemonchus účinky léků metabolismus MeSH
- ovce MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Monepantel (MOP) belongs to a new class of anthelmintic drugs known as aminoacetonitrile derivatives. It was approved for use in veterinary practice in Czech Republic in 2011. So far, biotransformation and transport of MOP in target animals have been studied insufficiently, although the study of metabolic pathways of anthelmintics is very important for the efficacy of safety of therapy and evaluation of the risk of drug-drug interactions. The aim of this study was to identify MOP metabolites and to suggest the metabolic pathways of MOP in sheep. For this purpose, primary culture of ovine hepatocytes was used as a model in vitro system. After incubation, medium samples and homogenates of hepatocytes were extracted separately using solid-phase extraction. Analysis was performed using a hybrid quadrupole-time-of-flight analyzer with respect to high mass accuracy measurements in full scan and tandem mass spectra for the confirmation of an elemental composition. The obtained results revealed S-oxidation to sulfoxide and sulfone and arene hydroxylation as MOP phase I biotransformations. From phase II metabolites, MOP glucuronides, sulfates, and acetylcysteine conjugates were found. Based on the obtained results, a scheme of the metabolic pathway of MOP in sheep has been proposed.
- MeSH
- aminoacetonitrily analogy a deriváty metabolismus MeSH
- anthelmintika metabolismus MeSH
- extrakce na pevné fázi MeSH
- hepatocyty metabolismus MeSH
- kultivované buňky MeSH
- metabolické sítě a dráhy MeSH
- ovce metabolismus MeSH
- tandemová hmotnostní spektrometrie MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Ivermectin (IVE), one of the most important anthelmintics, is often used in the treatment of haemonchosis in ruminants. The objective of our work was (1) to find and identify phase I and II metabolites of IVE formed by the Barber's pole worm (Haemonchus contortus), and (2) to compare IVE metabolites in helminths with IVE biotransformation in sheep (Ovis aries) as host species. Ultrahigh-performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS) was used for this purpose. During in vitro incubations, microsomes (from adult worms or from ovine liver) and a primary culture of ovine hepatocytes were incubated with IVE. In the ex vivo study, living H. contortus adults were incubated in the presence of 1 μM IVE for 24 h. The results showed that the H. contortus enzymatic system is not able to metabolize IVE. On the other hand, 7 different phase I as well as 9 phase II IVE metabolites were detected in ovine samples using UHPLC/MS/MS analyses. Most of these metabolites have not been described before. Haemonchus contortus is not able to deactivate IVE through biotransformation; therefore, biotransformation does not contribute to the development of IVE-resistance in the Barber's pole worm.
- MeSH
- anthelmintika chemie metabolismus terapeutické užití MeSH
- chromatografie kapalinová MeSH
- Haemonchus účinky léků růst a vývoj metabolismus MeSH
- hemonchóza farmakoterapie parazitologie veterinární MeSH
- hepatocyty metabolismus MeSH
- hmotnostní spektrometrie MeSH
- ivermektin chemie metabolismus terapeutické užití MeSH
- kultivované buňky MeSH
- mikrozomy metabolismus MeSH
- nemoci ovcí farmakoterapie parazitologie MeSH
- ovce domácí metabolismus MeSH
- ovce metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Benzimidazole anthelmintics, the drugs against parasitic worms, are widely used in human as well as veterinary medicine. Following excretion, these substances may persist in the environment and impact non-target organisms. In order to test phytoremediation as a possible tool for detoxification of anthelmintics in environment, the biotransformation pathways of albendazole (ABZ) and flubendazole (FLU) were studied in reed (Phragmites australis) in vitro. Reed cells were able to uptake and biotransform both anthelmintics. Ten ABZ metabolites and five FLU metabolites were found. Some atypical biotransformation reactions (formation of glucosylglucosides, acetylglucosides and xylosylglucosides), which have not been described previously, were identified. Based on the obtained results, the schemes of metabolic pathways of ABZ and FLU in reed were proposed. Most of ABZ and FLU metabolites can be considered as anthelmintically less active; therefore uptake and biotransformation of these anthelmintics by reed could be useful for decrease of their toxicity in environment.
- MeSH
- albendazol chemie metabolismus MeSH
- anthelmintika chemie metabolismus MeSH
- benzimidazoly chemie metabolismus MeSH
- biodegradace MeSH
- biotransformace MeSH
- lidé MeSH
- lipnicovité cytologie metabolismus MeSH
- mebendazol analogy a deriváty chemie metabolismus MeSH
- metabolická inaktivace * MeSH
- metabolické sítě a dráhy MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The increased activity of drug-metabolizing enzymes can protect helminths against the toxic effect of anthelmintics. The aim of this study was to compare the metabolism of the anthelmintic drug albendazole (ABZ) and the activities of selected biotransformation and antioxidant enzymes in three different strains of Haemonchus contortus: the ISE strain (susceptible to common anthelmintics), the BR strain (resistant to benzimidazole anthelmintics) and the WR strain (multi-resistant). H. contortus adults were collected from the abomasum of experimentally infected lambs. In vitro (subcellular fractions of H. contortus homogenate) as well as ex vivo (living nematodes cultivated in flasks with medium) experiments were performed. HPLC with spectrofluorimetric and mass-spectrometric detection was used in the analysis of ABZ metabolites. The in vitro activities of oxidation/antioxidation and conjugation enzymes toward model substrates were also assayed. The in vitro data showed significant differences between the susceptible (ISE) and resistant (BR, WR) strains regarding the activities of peroxidases, catalase and UDP-glucosyltransferases. S-oxidation of ABZ was significantly lower in BR than in the ISE strain. Ex vivo, four ABZ metabolites were identified: ABZ sulphoxide and three ABZ glucosides. In the resistant strains BR and WR, the ex vivo formation of all ABZ glucosides was significantly higher than in the susceptible ISE strain. The altered activities of certain detoxifying enzymes might partly protect the parasites against the toxic effect of the drugs as well as contribute to drug-resistance in these parasites.
- MeSH
- aktivace enzymů účinky léků MeSH
- albendazol chemie metabolismus farmakologie MeSH
- anthelmintika metabolismus farmakologie MeSH
- Haemonchus účinky léků enzymologie metabolismus MeSH
- katalasa metabolismus MeSH
- léková rezistence * MeSH
- molekulární struktura MeSH
- nemoci ovcí parazitologie MeSH
- ovce MeSH
- peroxidasa metabolismus MeSH
- superoxiddismutasa metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
