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.

Tacrine First-Phase Biotransformation and Associated Hepatotoxicity: A Possible Way to Avoid Quinone Methide Formation.

Tacrine was withdrawn from clinical use as a drug against Alzheimer's disease in 2013, mainly due to drug-induced liver injury. The culprit of tacrine-associated hepatotoxicity is believed to be the 7-OH-tacrine metabolite, a possible precursor of quinone methide (Qmeth), which binds to intracellular -SH proteins. In our study, several different animal and human models (liver microsomes, primary hepatocytes, and liver slices) were used to investigate the biotransformation and hepatotoxicity of tacrine and its 7-substituted analogues (7-methoxy-, 7-phenoxy-, and 7-OH-tacrine). Our goal was to find the most appropriate in vitro model for studying tacrine hepatotoxicity and, through rational structure modifications, to develop derivatives of tacrine that are less prone to Qmeth formation. Our results show that none of animal models tested accurately mimic human tacrine biotransformation; however, the murine model seems to be more suitable than the rat model. Tacrine metabolism was overall most accurately mimicked in three-dimensional (3D) spheroid cultures of primary human hepatocytes (PHHs). In this system, tacrine and 7-methoxytacrine were hydroxylated to 7-OH-tacrine, whereas 7-phenoxytacrine formed, as expected, only trace amounts. Surprisingly, however, our study showed that 7-OH-tacrine was the least hepatotoxic (7-OH-tacrine < tacrine < 7-methoxytacrine < 7-phenoxytacrine) even after doses had been adjusted to achieve the same intracellular concentrations. The formation of Qmeth-cysteine and Qmeth-glutathione adducts after human liver microsome incubation was confirmed by all of the studied tacrine derivatives, but these findings were not confirmed after incubation with 3D PHH spheroids. Therefore, the presented data call into question the suggested previously hypothesized mechanism of toxicity, and the results open new avenues for chemical modifications to improve the safety of novel tacrine derivatives.

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.

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.

Soybean (Glycine max) Is Able to Absorb, Metabolize and Accumulate Fenbendazole in All Organs Including Beans.

Although manure is an important source of minerals and organic compounds it represents a certain risk of spreading the veterinary drugs in the farmland and their permeation to human food. We tested the uptake of the anthelmintic drug fenbendazole (FBZ) by soybean, a common crop plant, from the soil and its biotransformation and accumulation in different soybean organs, including beans. Soybeans were cultivated in vitro or grown in a greenhouse in pots. FBZ was extensively metabolized in roots of in vitro seedlings, where sixteen metabolites were identified, and less in leaves, where only two metabolites were found. The soybeans in greenhouse absorbed FBZ by roots and translocated it to the leaves, pods, and beans. In roots, leaves, and pods two metabolites were identified. In beans, FBZ and one metabolite was found. FBZ exposure did not affect the plant fitness or yield, but reduced activities of some antioxidant enzymes and isoflavonoids content in the beans. In conclusion, manure or biosolids containing FBZ and its metabolites represent a significant risk of these pharmaceuticals entering food consumed by humans or animal feed. In addition, the presence of these drugs in plants can affect plant metabolism, including the production of isoflavonoids.

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.

The Uptake of Ivermectin and Its Effects in Roots, Leaves and Seeds of Soybean (Glycine max).

In recent years interest has grown in the occurrence and the effects of pharmaceuticals in the environment. The aim of this work is to evaluate the risk of fertilizing crops with manure from livestock treated with anthelmintics. The present study was designed to follow the fate of the commonly used anthelmintic drug, ivermectin (IVM) and its metabolites in soybeans (Glycine max (L.) Merr.), a plant that is grown and consumed world-wide for its high content of nutritional and health-beneficial substances. In vitro plantlets and soybean plants, cultivated in a greenhouse, were used for this purpose. Our results showed the uptake of IVM and its translocation to the leaves, but not in the pods and the beans. Four IVM metabolites were detected in the roots, and one in the leaves. IVM exposure decreased slightly the number and weight of the beans and induced changes in the activities of antioxidant enzymes. In addition, the presence of IVM affected the proportion of individual isoflavones and reduced the content of isoflavones aglycones, which might decrease the therapeutic value of soybeans. Fertilization of soybean fields with manure from IVM-treated animals appears to be safe for humans, due to the absence of IVM in beans, the food part of plants. On the other hand, it could negatively affect soybean plants and herbivorous invertebrates.

The Identification of Metabolites and Effects of Albendazole in Alfalfa (Medicago sativa).

Albendazole (ABZ), a widely used anthelmintic drug, enters the environment mainly via livestock excrements. To evaluate the environmental impact of ABZ, the knowledge of its uptake, effects and metabolism in all non-target organisms, including plants, is essential. The present study was designed to identify the metabolic pathway of ABZ and to test potential ABZ phytotoxicity in fodder plant alfalfa, with seeds and in vitro regenerants used for these purposes. Alfalfa was chosen, as it may meet manure from ABZ-treated animals in pastures and fields. Alfalfa is often used as a feed of livestock, which might already be infected with helminths. The obtained results showed that ABZ did not inhibit alfalfa seed germination and germ growth, but evoked stress and a toxic effect in alfalfa regenerants. Alfalfa regenerants were able to uptake ABZ and transform it into 21 metabolites. UHPLC-MS/MS analysis revealed three new ABZ metabolites that have not been described yet. The discovery of the parent compound ABZ together with the anthelmintically active and instable metabolites in alfalfa leaves shows that the contact of fodder plants with ABZ-containing manure might represent not only a danger for herbivorous invertebrates, but also may cause the development of ABZ resistance in helminths.

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.

Pharmaceuticals in environment: the effect of ivermectin on ribwort plantain (Plantago lanceolata L.).

The anthelmintic drug ivermectin (IVM), used frequently especially in veterinary medicine, enters the environment mainly via excrements in pastures and could negatively affect non-target organisms including plants. The present study was designed to follow up on our previous investigations into IVM metabolism and its effects in the common meadow plant ribwort plantain (Plantago lanceolata L.) during long-term exposure of both cell suspensions and whole plant regenerants. IVM uptake, distribution, and biotransformation pathways were studied using UHPLC-MS analysis. In addition, the IVM effect on antioxidant enzymes activities, proline concentration, the content of all polyphenols, and the level of the main bioactive secondary metabolites was also tested with the goal of learning more about IVM-induced stress in the plant organism. Our results showed that the ribwort plantain was able to uptake IVM and transform it via demethylation and hydroxylation. Seven and six metabolites respectively were detected in cell suspensions and in the roots of regenerants. However, only the parent drug IVM was detected in the leaves of the regenerants. IVM accumulated in the roots and leaves of plants might negatively affect ecosystems due to its toxicity to herbivorous invertebrates. As IVM exposition increased the activity of catalase, the concentration of proline and polyphenols, as well as decreased the activity of ascorbate peroxidase and the concentration of the bioactive compounds acteoside and aucubin, long-term exposition of the ribwort plantain to IVM caused abiotic stress and might decrease the medicinal value of this herb.

Anthelmintics in the future: current trends in the discovery and development of new drugs against gastrointestinal nematodes.

The control of gastrointestinal nematodes (GINs), the most abundant and serious parasites of livestock, has become difficult because of the limited number of available drugs and fast development of drug resistance. Thus, considerable efforts have been devoted to developing new anthelmintics that are efficient against nematodes, especially resistant species. Here, we summarize the most recent results using various approaches: target-based or high-throughput screening (HTS) of compound libraries; the synthesis of new derivatives or new combinations of current anthelmintics; the repurposing of drugs currently approved for other indications; and lastly, the identification of active plant products. We also evaluate the advantages and disadvantages of each of these approaches.

UDP-glycosyltransferase family in Haemonchus contortus: Phylogenetic analysis, constitutive expression, sex-differences and resistance-related differences.

UDP-glycosyltransferases (UGT), catalysing conjugation of UDP-activated sugar donors to small lipophilic chemicals, are widespread in living organisms from bacteria to fungi, plant, or animals. The progress of genome sequencing has enabled an assessment of the UGT multigene family in Haemonchus contortus (family Trichostrongylidae, Nematoda), a hematophagous gastrointestinal parasite of small ruminants. Here we report 32 putative UGT genes divided into 15 UGT families. Phylogenetic analysis in comparison with UGTs from Caenorhabditis elegans, a free-living model nematode, revealed several single member homologues, a lack of the dramatic gene expansion seen in C. elegans, but also several families (UGT365, UGT366, UGT368) expanded in H. contortus only. The assessment of constitutive UGT mRNA expression in H. contortus adults identified significant differences between females and males. In addition, we compared the expression of selected UGTs in the drug-sensitive ISE strain to two benzimidazole-resistant strains, IRE and WR, with different genetic backgrounds. Constitutive expression of UGT368B2 was significantly higher in both resistant strains than in the sensitive strain. As resistant strains were able to deactivate benzimidazole anthelmintics via glycosylation more effectively then the sensitive strain, UGT368B2 enhanced constitutive expression might contribute to drug resistance in H. contortus.

The metabolism of flubendazole in human liver and cancer cell lines.

Flubendazole (FLU), a benzimidazole anthelmintic drug widely used in veterinary medicine, has been approved for the treatment of gut-residing nematodes in humans. In addition, FLU is now considered a promising anti-cancer agent. Despite this, information about biotransformation of this compound in human is lacking. Moreover, there is no information regarding whether cancer cells are able to metabolize FLU in order to deactivate it. For these reasons, the present study was designed to identify all metabolites of Phase I and Phase II of FLU in human liver and in various cancer cells using ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis. Precision-cut human liver slices and 9 cell lines of different origin (breast, colon, oral cavity) were used as in vitro model systems. Our study showed that FLU with a reduced carbonyl group (FLUR) is the only FLU metabolite formed in the human liver. All human cancer cell lines were able to form FLUR. In addition, methylated FLUR was detected in breast cells MCF7 and intestinal SW480 cells. The accumulation of FLU and its reduction to FLUR markedly differed among cells. The extent of FLU reduction was in a good correlation with the detected expression level of carbonyl reductase 1. In most cases, FLU entered in a higher amount and was reduced to a lesser extent in proliferating (metastatic) cells than in differentiated (non-cancerous, non-metastatic) ones. These results support the promising potential of FLU in anti-cancer therapy.