Flubendazole carbonyl reduction in drug-susceptible and drug-resistant strains of the parasitic nematode Haemonchus contortus: changes during the life cycle and possible inhibition.

Carbonyl-reducing enzymes (CREs) catalyse the reduction of carbonyl groups in many eobiotic and xenobiotic compounds in all organisms, including helminths. Previous studies have shown the important roles of CREs in the deactivation of several anthelmintic drugs (e.g., flubendazole and mebendazole) in adults infected with the parasitic nematode Haemonchus contortus, in which the activity of a CRE is increased in drug-resistant strains. The aim of the present study was to compare the abilities of nematodes of both a drug-susceptible strain (ISE) and a drug-resistant strain (IRE) to reduce the carbonyl group of flubendazole (FLU) in different developmental stages (eggs, L1/2 larvae, L3 larvae, and adults). In addition, the effects of selected CRE inhibitors (e.g., glycyrrhetinic acid, naringenin, silybin, luteolin, glyceraldehyde, and menadione) on the reduction of FLU were evaluated in vitro and ex vivo in H. contortus adults. The results showed that FLU was reduced by H. contortus in all developmental stages, with adult IRE females being the most metabolically active. Larvae (L1/2 and L3) and adult females of the IRE strain reduced FLU more effectively than those of the ISE strain. Data from the in vitro inhibition study (performed with cytosolic-like fractions of H. contortus adult homogenate) revealed that glycyrrhetinic acid, naringenin, mebendazole and menadione are effective inhibitors of FLU reduction. Ex vivo study data showed that menadione inhibited FLU reduction and also decreased the viability of H. contortus adults to a similar extent. Naringenin and mebendazole were not toxic at the concentrations tested, but they did not inhibit the reduction of FLU in adult worms ex vivo.

Comparative Analysis of miRNA and EMT Markers in Metastatic Colorectal Cancer.

Colorectal cancer (CRC) is the fourth most commonly diagnosed malignant condition in the world. Micro RNAs (miRNAs) as well as epithelial to mesenchymal transition (EMT) play an important role in the pathogenesis of CRC. We performed a comparative analysis of the expression of selected miRNA genes and EMT markers in bioptic samples from patients (n = 45) with primary CRC or metastatic (m)CRC to the regional lymph node using reverse transcription-quantitative PCR and IHC staining. Results: Out of all miRNA analyzed, the miR-17 expression was most significantly different and associated with lower risk of CRC spread to the lymph node. In addition, significant relationships were found between the tumor side localization and several miRNAs expressions (miR-9, miR-29b, miR-19a, miR-19b, miR-21, miR-106a, miR-20a and miR-17). In addition, of the examined EMT markers, only VEGFA expression correlated with tumor progression (tumor grade G2). In the examined set of patient samples and their matched healthy tissue, several specific molecular markers (miRNAs associated with EMT and tumor progression) were identified with a promising prognostic potential. Their further examination in larger patient cohorts is planned to validate the present data.

Anthelmintics in the environment: Their occurrence, fate, and toxicity to non-target organisms.

Anthelmintics are drugs used for the treatment and prevention of diseases caused by parasitic worms (helminths). While the importance of anthelmintics in human as well as in veterinary medicine is evident, they represent emerging contaminants of the environment. Human anthelmintics are mainly used in tropical and sub-tropical regions, while veterinary anthelmintics have become frequently-occurring environmental pollutants worldwide due to intensive agri- and aquaculture production. In the environment, anthelmintics are distributed in water and soil in relation to their structure and physicochemical properties. Consequently, they enter various organisms directly (e.g. plants, soil invertebrates, water animals) or indirectly through food-chain. Several anthelmintics elicit toxic effects in non-target species. Although new information has been made available, anthelmintics in ecosystems should be more thoroughly investigated to obtain complex knowledge on their impact in various environments. This review summarizes available information about the occurrence, behavior, and toxic effect of anthelmintics in environment. Several reasons why anthelmintics are dangerous contaminants are highlighted along with options to reduce contamination. Negative effects are also outlined.

Anthelmintic activity of European fern extracts against Haemonchus contortus.

Most drugs used in the treatment of helminthiasis in humans and animals have lost their efficacy due to the development of drug-resistance in helminths. Moreover, since anthelmintics, like many pharmaceuticals, are now recognized as hazardous contaminants of the environment, returning to medicinal plants and their products represents an environmentally friendly way to treat helminthiasis. The goal of the present study was to test the anthelminthic activity of methanol extracts of eight selected European ferns from the genera Dryopteris, Athyrium and Blechnum against the nematode Haemonchus contortus, a widespread parasite of small ruminants. Eggs and adults of H. contortus drug-susceptible strain ISE and drug-resistant strain WR were isolated from experimentally infected sheep. The efficacy of fern extracts was assayed using egg hatch test and adults viability test based on ATP-level measurement. Among the ferns tested, only Dryopteris aemula extract (0.2 mg/mL) inhibited eggs hatching by 25% in comparison to control. Athyrium distentifolium, Dryopteris aemula and Dryopteris cambrensis were effective against H. contortus adults. In concentration 0.1 mg/mL, A. distentifolium, D. aemula, D. cambrensis significantly decreased the viability of females from ISE and WR strains to 36.2%, 51.9%, 32.9% and to 35.3%, 27.0%, 23.3%, respectively in comparison to untreated controls. None of the extracts exhibited toxicity in precise cut slices from ovine liver. Polyphenol's analysis identified quercetin, kaempferol, luteolin, 3-hydroxybenzoic acid, caffeic acid, coumaric acid and protocatechuic acid as the major components of these anthelmintically active ferns.

Short-chain dehydrogenases in Haemonchus contortus: changes during life cycle and in relation to drug-resistance.

Short-chain dehydrogenases/reductases (SDRs) regulate the activities of many hormones and other signaling molecules and participate in the deactivation of various carbonyl-bearing xenobiotics. Nevertheless, knowledge about these important enzymes in helminths remains limited. The aim of our study was to characterize the SDR superfamily in the parasitic nematode Haemonchus contortus. Genome localization of SDRs was explored, and phylogenetic analysis in comparison with SDRs from free-living nematode Caenorhabditis elegans and the domestic sheep (Ovis aries, a typical host of H. contortus) was constructed. The expression profile of selected SDRs during the life cycle along with differences between the drug-susceptible and drug-resistant strains, were also studied. Genome sequencing enabled the identification of 46 members of the SDR family in H. contortus. A number of genes have no orthologue in the sheep genome. In all developmental stages of H. contortus, SDR1, SDR3, SDR5, SDR6, SDR14, and SDR18 genes were the most expressed, although in individual stages, huge differences in expression levels were observed. A comparison of SDRs expression between the drug-susceptible and drug-resistant strains of H. contortus revealed several SDRs with changed expression in the resistant strain. Specifically, SDR1, SDR12, SDR13, SDR16 are SDR candidates related to drug-resistance, as the expression of these SDRs is consistently increased in most stages of the drug-resistant H. contortus. These findings revealing several SDR enzymes of H. contortus warrant further investigation.

Albendazole from ovine excrements in soil and plants under real agricultural conditions: Distribution, persistence, and effects.

Albendazole (ABZ), a broad-spectrum anthelmintic drug frequently used in livestock against parasitic worms (helminths), enters the environment mainly via faeces of treated animals left in the pastures or used as dung for field fertilization. To obtain information about the subsequent fate of ABZ, the distribution of ABZ and its metabolites in the soil around faeces along with uptake and effects in plants were monitored under real agricultural conditions. Sheep were treated with a recommended dose of ABZ; faeces were collected and used to fertilize fields with fodder plants. Soil samples (in two depths) and samples of two plants, clover (Trifolium pratense) and alfalfa (Medicago sativa), were collected at distances 0-75 cm from the faeces for 3 months after fertilization. The environmental samples were extracted using QuEChERS and LLE sample preparation procedures. The targeted analysis of ABZ and its metabolites was conducted by using the validated UHPLC-MS method. Two main ABZ metabolites, ABZ-sulfoxide (anthelmintically active) and ABZ-sulfone (inactive), persisted in soil (up to 25 cm from faeces) and in plants for three months when the experiment ended. In plants, ABZ metabolites were detected even 60 cm from the faeces and abiotic stress was observed in the central plants. The considerable distribution and persistence of ABZ metabolites in soil and plants amplify the negative environmental impact of ABZ documented in other studies.

Role of N-Cadherin in Epithelial-to-Mesenchymal Transition and Chemosensitivity of Colon Carcinoma Cells.

(1) Background: N-cadherin expression, epithelial-to-mesenchymal transition (EMT) and aggressive biological phenotype of tumor cells are linked although the underlying mechanisms are not entirely clear. (2) Methods: In this study, we used two different in vitro cell models with varying N-cadherin expression (stabilized lines and primocultures) and investigated their select biological features including the degree of their chemoresistance both in vitro as well as in vivo. (3) Results: We report that although enforced N-cadherin expression changes select morphological and behavioral characteristics of exposed cells, it fails to successfully reprogram cells to the aggressive, chemoresistant phenotype both in vitro as well as in vivo as verified by implantation of those cells into athymic mice. Conversely, primocultures of patient-colonic cells with naturally high levels of N-cadherin expression show fully aggressive and chemoresistant phenotype pertinent to EMT (in vitro and in vivo), with a potential to develop new mutations and in the presence of dysregulated regulatory pathways as represented by investigated miRNA profiles. (4) Conclusions: The presented results bring new facts concerning the functional axis of N-cadherin expression and related biological features of colon cancer cells and highlight colon cancer primocultures as a useful model for such studies.

Image recognition based on deep learning in Haemonchus contortus motility assays.

Poor efficacy of some anthelmintics and rising concerns about the widespread drug resistance have highlighted the need for new drug discovery. The parasitic nematode Haemonchus contortus is an important model organism widely used for studies of drug resistance and drug screening with the current gold standard being the motility assay. We applied a deep learning approach Mask R-CNN for analysing motility videos containing varying rates of motile worms and compared it to other commonly used algorithms with different levels of complexity, namely the Wiggle Index and the Wide Field-of-View Nematode Tracking Platform. Mask R-CNN consistently outperformed the other algorithms in terms of the detection of worms as well as the precision of motility forecasts, having a mean absolute percentage error of 7.6% and a mean absolute error of 5.6% for the detection and motility forecasts, respectively. Using Mask R-CNN for motility assays confirmed the common problem with algorithms that use non-maximum suppression in detecting overlapping objects, which negatively impacts the overall precision. The use of intersect over union as a measure of the classification of motile / non-motile instances had an overall accuracy of 89%, indicating that it is a viable alternative to previously used methods based on movement characteristics, such as body bends. In comparison to the existing methods evaluated here, Mask R-CNN performed better and we anticipate that this method will broaden the number of possible approaches to video analysis of worm motility.

The induction and inhibition of UDP-glycosyltransferases in Haemonchus contortus and their role in the metabolism of albendazole.

Albendazole (ABZ) is an anthelmintic frequently used to treat haemonchosis, a common parasitosis of ruminants caused by the gastrointestinal nematode Haemonchus contortus. This parasite is able to protect itself against ABZ via the formation of inactive ABZ-glycosides. The present study was designed to deepen the knowledge about the role of UDP-glycosyltransferases (UGTs) in ABZ glycosylation in H. contortus. The induction effect of phenobarbital, a classical inducer of UGTs, as well as ABZ and ABZ-sulphoxide (ABZSO, the main active metabolite of ABZ) on UGTs expression and UGT activity toward ABZ was studied ex vivo in isolated adult nematodes. The effect of three potential UGT inhibitors (5-nitrouracil, 4,6-dihydroxy-5-nitropyrimidine and sulfinpyrazone) on ABZ glycosylation was tested. Pre-incubation of nematodes with ABZ and ABZSO led to increased expression of several UGTs as well as ABZ-glycosides formation in subsequent treatment. Phenobarbital also induced UGTs expression, but did not affect ABZ biotransformation. In the nematode's subcellular fraction, sulfinpyrazone inhibited UGT activity toward ABZ, although no effect of other inhibitors was observed. The inhibitory potential of sulfinpyrazone on the formation of ABZ-glycosides was also proved ex vivo in living nematodes. The obtained results confirmed the role of UGTs in ABZ biotransformation in H. contortus adults and revealed sulfinpyrazone as a potent inhibitor of ABZ glycosylation in this parasite. The possible use of sulfinpyrazone with ABZ in combination therapy merits further research.

The role of UDP-glycosyltransferases in xenobioticresistance.

Uridine diphosphate sugar-utilizing glycosyltransferases (UGTs) are an enzyme superfamily that catalyzes glycosyl residues transfer from activated nucleotide sugars to acceptor molecules. In addition to various endogenous compounds, numerous xenobiotics are substrates of UGTs. As the glycosides formed are generally less active/toxic and more hydrophilic than aglycones, UGTs effectively protect organisms from potentially harmful xenobiotics. Therefore, increased UGT expression and/or activity improve the protection of the organism and may contribute to the development of individuals that become more resistant to certain xenobiotics. While the function of UGTs in the resistance of human cancer cells to chemotherapy is now well known, other organisms and other xenobiotics have attracted much less attention. This review was designed to fill this knowledge gap by presenting complex information about the role of UGTs in xenobiotic-resistance in various organisms. This summarization and evaluation of the available information reveals that UGTs play an important role in defense against xenobiotics not only in humans, but in countless other organisms such as parasites, insects, and plants. Moreover, many recent studies clearly show the participation of UGTs in the resistance of nematodes to anthelmintics, insects to insecticides, weeds to herbicides as well as humans to various drugs (not only those used in cancer therapy but also in the treatment of epilepsy, psychiatric disorders, hypertension, hypercholesterolemia, and HIV infection). Nevertheless, although the contribution of UGTs to xenobiotic resistance in diverse organisms has become obvious, many pieces of information remain missing, for example with regard to the mechanisms of UGT regulation.

Environmental circulation of the anthelmintic drug albendazole affects expression and activity of resistance-related genes in the parasitic nematode Haemonchus contortus.

Veterinary anthelmintics excreted from treated animals pass to soil, subsequently to plants and then to their consumers. This circulation might have various consequences, including drug-resistance promotion in helminths. The present study was designed to follow the effect of the environmental circulation of the common anthelmintic drug albendazole (ABZ) in real farm conditions on the parasitic nematode Haemonchus contortus in vivo. Two fields with fodder plants (clover and alfalfa) were fertilized, the first with dung from ABZ-treated sheep (at the recommended dosage), the second with dung from non-treated sheep (controls). After a 10-week growth period, the fresh fodder from both fields was used to feed two groups of sheep, which were infected with H. contortus. Eggs and adult nematodes from the animals of both groups were isolated, and various parameters were compared. No significant changes in the eggs' sensitivity to ABZ and thiabendazole were observed. However, significantly increased expression of several cytochromes P450 and UDP-glycosyl transferases as well as increased oxidation and glycosylation of ABZ and ABZ-sulfoxide (ABZ-SO) was found in the exposed nematodes. These results show that ABZ environmental circulation improves the ability of the helminths to deactivate ABZ.

Sertraline as a new potential anthelmintic against Haemonchus contortus: toxicity, efficacy, and biotransformation.

Haemonchus contortus is a parasitic nematode of ruminants which causes significant losses to many farmers worldwide. Since the drugs currently in use for the treatment of haemonchosis are losing their effectiveness due to the drug-resistance of this nematode, a new or repurposed drug is highly needed. As the antipsychotic drug sertraline (SRT) has been shown to be effective against the parasitic nematodes Trichuris muris, Ancylostoma caninum and Schistosoma mansoni, the aim of the present study was to evaluate the possible effect of SRT on H. contortus. The potential hepatotoxicity of SRT was tested in sheep, a common H. contortus host. In addition, the main metabolic pathways of SRT in H. contortus and the ovine liver were identified. While no effect of SRT on H. contortus egg hatching was observed, SRT was found to significantly decrease the viability of H. contortus adults in drug-sensitive and resistant strains, with its effect comparable to the commonly used anthelmintics levamisole and monepantel. Moreover, SRT in anthelmintically active concentrations showed no toxicity to the ovine liver. Biotransformation of SRT in H. contortus was weak, with most of the drug remaining unmetabolized. Production of the main metabolite hydroxy-SRT did not differ significantly between strains. Other minor metabolites such as SRT-O-glucoside, dihydroxy-SRT, and SRT-ketone were also identified in H. contorts adults. Compared to H. contortus, the ovine liver metabolized SRT more extensively, mainly via desmethylation and glucuronidation. In conclusion, the potency of SRT against H. contortus was proven, and it should be tested further toward possible repurposing.

The ATP bioluminescence assay: a new application and optimization for viability testing in the parasitic nematode Haemonchus contortus.

The parasitic gastrointestinal nematode Haemonchus contortus causes serious economic losses to agriculture due to infection and disease in small ruminant livestock. The development of new therapies requires appropriate viability testing, with methods nowadays relying on larval motility or development using procedures that involve microscopy. None of the existing biochemical methods, however, are performed in adults, the target stage of the anthelmintic compounds. Here we present a new test for the viability of H. contortus adults and exsheathed third-stage larvae which is based on a bioluminescent assay of ATP content normalized to total protein concentration measured using bicinchoninic acid. All the procedure steps were optimized to achieve maximal sensitivity and robustness. This novel method can be used as a complementary assay for the phenotypic screening of new compounds with potential antinematode activity in exsheathed third-stage larvae and in adult males. Additionally, it might be used for the detection of drug-resistant isolates.

Proof of the environmental circulation of veterinary drug albendazole in real farm conditions.

Anthelmintics, drugs against parasitic worms, are frequently used in livestock and might act as danger environmental microcontaminants. The present study was designed to monitor the possible circulation of common anthelmintic drug albendazole (ABZ) and its metabolites in the real agriculture conditions. The sheep were treated with the recommended dose of ABZ. Collected faeces were used for the fertilization of a field with fodder plants (alfalfa and clover) which served as feed for sheep from a different farm. The selective ultrasensitive mass spectrometry revealed surprisingly high concentrations of active ABZ metabolite (ABZ-sulphoxide) in all samples (dung, plants, ovine plasma, rumen content and faeces). Our results prove for the first time an undesirable permeation of ABZ metabolites from sheep excrement into plants (used as fodder) and subsequently to other sheep in real agricultural conditions. This circulation causes the permanent exposition of the ecosystems and food-chain to the drug and can promote the development of drug resistance in helminths.

MicroRNAs mediated regulation of glutathione peroxidase 7 expression and its changes during adipogenesis.

Glutathione peroxidase 7 (GPx7) acts as an intracellular stress sensor/transmitter and plays an important role in adipocyte differentiation and the prevention of obesity related pathologies. For this reason, finding the regulatory mechanisms that control GPx7 expression is of great importance. As microRNAs (miRNAs) could participate in the regulation of GPx7 expression, we studied the inhibition of GPx7 expression by four selected miRNAs with relation to obesity and adipogenesis. The effect of the transfection of selected miRNAs mimics on GPx7 expression was tested in three cell models (HEK293, SW480, AT-MSC). The interaction of selected miRNAs with the 3'UTR of GPx7 was followed up on using a luciferase gene reporter assay. In addition, the levels of GPx7 and selected miRNAs in adipose tissue mesenchymal stem cells (AT-MSC) and mature adipocytes from four human donors were compared, with the changes in these levels during adipogenesis analyzed. Our results show for the first time that miR-137 and miR-29b bind to the 3'UTR region of GPx7 and inhibit the expression of this enzyme at the mRNA and protein level in all the human cells tested. However, no negative correlation between miR-137 nor miR-29b level and GPx7 was observed during adipogenesis. Despite the confirmed inhibition of GPx7 expression by miR-137 and miR-29b, the action of these two molecules in adipogenesis and mature adipocytes must be accompanied by other regulators.

Stress Effect of Food Matrices on Viability of Probiotic Cells during Model Digestion.

The aim of this study was to evaluate the influence of model (alcohol, sugar, salt, protein and acid) and real foods and beverages on the viability of probiotics during incubation and artificial digestion. Viability of monocultures Lactobacillus acidophilus CCM4833 and Bifidobacterium breve CCM7825T, and a commercial mixture of 9 probiotic bacterial strains, was tested by cultivation assay and flow cytometry. In model foods, the best viability was determined in the presence of 0.2 g/L glucose, 10% albumin and 10% ethanol. As the most suitable real food for probiotic survival, complex protein and carbohydrate substrates were found, such as beef broth, potato salad with pork, chicken with rice, chocolate spread, porridge and yoghurt. The best liquid was milk and meat broth, followed by Coca-Cola, beer and coffee. Viability of probiotics was higher when consumed with meals than with beverages only. Addition of prebiotics increased the viability of probiotics, especially in presence of instant and fast foods. Generally, the highest viability of probiotics during artificial digestion was observed in mixed culture in the presence of protein, sugar and fat, or their combination. The increase of cell viability observed in such foods during model digestion may further contribute to the positive effect of probiotics on human health.

UDP-Glycosyltransferases and Albendazole Metabolism in the Juvenile Stages of Haemonchus contortus.

The nematode Haemonchus contortus, a gastrointestinal parasite of ruminants, can severely burden livestock production. Although anthelmintics are the mainstay in the treatment of haemonchosis, their efficacy diminishes due to drug-resistance development in H. contortus. An increased anthelmintics inactivation via biotransformation belongs to a significant drug-resistance mechanism in H. contortus. UDP-glycosyltransferases (UGTs) participate in the metabolic inactivation of anthelmintics and other xenobiotic substrates through their conjugation with activated sugar, which drives the elimination of the xenobiotics due to enhanced solubility. The UGTs family, in terms of the biotransformation of commonly used anthelmintics, has been well described in adults as a target stage. In contrast, the free-living juvenile stages of H. contortus have attracted less attention. The expression of UGTs considerably varies throughout the life cycle of the juvenile nematodes, suggesting their different roles. Furthermore, the constitutive expression in a susceptible strain with two resistant strains shows several resistance-related changes in UGTs expression, and the exposure of juvenile stages of H. contortus to albendazole (ABZ) and ABZ-sulfoxide (ABZSO; in sublethal concentrations) leads to the increased expression of several UGTs. The anthelmintic drug ABZ and its primary metabolite ABZSO biotransformation, tested in the juvenile stages, shows significant differences between susceptible and resistant strain. Moreover, higher amounts of glycosidated metabolites of ABZ are formed in the resistant strain. Our results show similarly, as in adults, the UGTs and glycosidations significant for resistance-related differences in ABZ biotransformation and warrant further investigation in their individual functions.

Atmospheric Pressure Plasma Polymerized 2-Ethyl-2-oxazoline Based Thin Films for Biomedical Purposes.

Polyoxazoline thin coatings were deposited on glass substrates using atmospheric pressure plasma polymerization from 2-ethyl-2-oxazoline vapours. The plasma polymerization was performed in dielectric barrier discharge burning in nitrogen at atmospheric pressure. The thin films stable in aqueous environments were obtained at the deposition with increased substrate temperature, which was changed from 20 ∘C to 150 ∘C. The thin film deposited samples were highly active against both S. aureus and E. coli strains in general. The chemical composition of polyoxazoline films was studied by FTIR and XPS, the mechanical properties of films were studied by depth sensing indentation technique and by scratch tests. The film surface properties were studied by AFM and by surface energy measurement. After tuning the deposition parameters (i.e., monomer flow rate and substrate temperature), stable films, which resist bacterial biofilm formation and have cell-repellent properties, were achieved. Such antibiofouling polyoxazoline thin films can have many potential biomedical applications.

The Modulation of Phase II Drug-Metabolizing Enzymes in Proliferating and Differentiated CaCo-2 Cells by Hop-Derived Prenylflavonoids.

Prenylflavonoids in the human organism exhibit various health-beneficial activities, although they may interfere with drugs via the modulation of the expression and/or activity of drug-metabolizing enzymes. As intestinal cells are exposed to the highest concentrations of prenylflavonoids, we decided to study the cytotoxicity and modulatory effects of the four main hop-derived prenylflavonoids on the activities and mRNA expression of the main drug-conjugating enzymes in human CaCo-2 cells. Proliferating CaCo-2 cells were used for these purposes as a model of colorectal cancer cells, and differentiated CaCo-2 cells were used as an enterocyte-like model. All the tested prenylflavonoids inhibited the CaCo-2 cells proliferation, with xanthohumol proving the most effective (IC50 8.5 µM). The prenylflavonoids modulated the activities and expressions of the studied enzymes to a greater extent in the differentiated, as opposed to the proliferating, CaCo-2 cells. In the differentiated cells, all the prenylflavonoids caused a marked increase in glutathione S-transferase and catechol-O-methyltransferase activities, while the activity of sulfotransferase was significantly inhibited. Moreover, the prenylflavonoids upregulated the mRNA expression of uridine diphosphate (UDP)-glucuronosyl transferase 1A6 and downregulated that of glutathione S-transferase 1A1/2.

Sub-lethal doses of albendazole induce drug metabolizing enzymes and increase albendazole deactivation in Haemonchus contortus adults.

The efficacy of anthelmintic therapy of farm animals rapidly decreases due to drug resistance development in helminths. In resistant isolates, the increased expression and activity of drug-metabolizing enzymes (DMEs), e.g. cytochromes P450 (CYPs), UDP-glycosyltransferases (UGTs) and P-glycoprotein transporters (P-gps), in comparison to sensitive isolates have been described. However, the mechanisms and circumstances of DMEs induction are not well known. Therefore, the present study was designed to find the changes in expression of CYPs, UGTs and P-gps in adult parasitic nematodes Haemonchus contortus exposed to sub-lethal doses of the benzimidazole anthelmintic drug albendazole (ABZ) and its active metabolite ABZ-sulfoxide (ABZSO). In addition, the effect of ABZ at sub-lethal doses on the ability to deactivate ABZ during consequent treatment was studied. The results showed that contact of H. contortus adults with sub-lethal doses of ABZ and ABZSO led to a significant induction of several DMEs, particularly cyp-2, cyp-3, cyp-6, cyp-7, cyp-8, UGT10B1, UGT24C1, UGT26A2, UGT365A1, UGT366C1, UGT368B2, UGT367A1, UGT371A1, UGT372A1 and pgp-3, pgp-9.1, pgp-9.2, pgp-10. This induction led to increased formation of ABZ metabolites (especially glycosides) and their increased export from the helminths' body into the medium. The present study demonstrates for the first time that contact of H. contortus with sub-lethal doses of ABZ (e.g. during underdose treatment) improves the ability of H. contortus adults to deactivate ABZ in consequent therapy.