Fasciola hepatica: Histological changes in the somatic and reproductive tissues of liver fluke following closantel treatment of experimentally-infected sheep.

Lambs infected with the Cullompton isolate of Fasciola hepatica were treated orally or subcutaneously with 10mg/kg of closantel at 16 weeks post-infection. Adult flukes were recovered from the liver of individual animals at 12h, 24h, or 36h post-treatment. The flukes were processed for histological analysis. In general, degenerative changes in the reproductive and somatic tissues were progressive, and were most marked in flukes exposed to closantel in vivo for 36h. However, flukes from a 12h subcutaneously-treated lamb showed marked deterioration of the testis, possibly because a portion of the dose has been delivered intravenously. Fewer intact eggs were seen in the uterus of flukes exposed to closantel for longer times (whether administered subcutaneously or orally to the host). The most conspicuous closantel-induced effect in flukes from treated hosts was progressive damage to the tegumental syncytium. While the flukes from 24h-treated hosts showed relatively minor damage to limited areas of the syncytium, towards the posterior end, the flukes from 36h-treated hosts (and flukes from the lamb that putatively received intravenous dosage) had lost large areas of the surface syncytium from the posterior end and dorsal surface, although the syncytium over the anterior end and the anterior ventral surface was largely spared. In areas where the syncytium had sloughed, the underlying structures such as the vitelline follicles, gut profiles and testis profiles, showed marked degeneration and breakdown. Other changes included cell depletion and early stage apoptosis in the testis, ovary and vitelline follicles. This study establishes a model for histological changes in closantel-sensitive F. hepatica exposed to closantel in vivo. Histopathological studies could be complementary to the efficacy controlled test for for closantel resistance in fluke populations.

A single amino acid substitution in isozyme GST mu in Triclabendazole resistant Fasciola hepatica (Sligo strain) can substantially influence the manifestation of anthelmintic resistance.

The helminth parasite Fasciola hepatica causes fascioliasis in human and domestic ruminants. Economic losses due to this infection are estimated in U$S 2000-3000 million yearly. The most common method of control is the use of anthelmintic drugs. However, there is an increased concern about the growing appearance of F. hepatica resistance to Triclabendazole (TCBZ), an anthelmintic with activity over adult and young flukes. F. hepatica has eight Glutathione S-Transferase (GST) isozymes, which are enzymes involved in the detoxification of a wide range of substrates through chemical conjugation with glutathione. In the present work we identified and characterized the GST mu gene isolated from the TCBZ-susceptible and TCBZ-resistant F. hepatica strains. Total RNA was transcribed into cDNA by reverse transcription and a 657 bp amplicon corresponding to the GST mu gene was obtained. The comparative genetic analysis of the GST mu gene of the TCBZ susceptible strain (Cullompton) and TCBZ resistant strain (Sligo) showed three nucleotide changes and one amino acid change at position 143 in the GST mu isozyme of the TCBZ-resistant strain. These results have potential relevance as they contribute better understand the mechanisms that generate resistance to anthelmintics.

Approach to molecular characterization of different strains of Fasciola hepatica using random amplified polymorphic DNA polymerase chain reaction.

The aim of the present study was to genetically characterize Fasciola hepatica strains from diverse ecogeographical regions (America and Europe), susceptible and resistant to Triclabendazole, using the random amplified polymorphic DNA fragments (RAPDs-PCR) technique to elucidate genetic variability between the different isolates. Ten different oligonucleotide primers of 10 bases with GC content varying from 50-70% were used. A polymerase chain reaction (PCR) was carried out in 25 µl of total volume. Duplicate PCR reactions on each individual template DNA were performed to test the reproducibility of the individual DNA bands. The size of the RAPD-PCR fragments was determined by the reciprocal plot between the delay factors (Rf) versus the logarithm of molecular weight ladder. The phenogram obtained showed three main clusters, the major of which contained European Strains (Cullompton and Sligo) showing a genetic distance of 27.2 between them. The American strains (Cedive and Cajamarca) on the other hand formed each their distinctive group but clearly maintaining a closer genetic relationship among them than that to their European counterparts, with which showed a distance of 33.8 and 37.8, respectively. This polymorphism would give this species enhanced adaptability against the host, as well as the environment. The existence of genetically different populations of F. hepatica could allow, against any selection pressure, natural or artificial (for use fasciolicides products and/or control measures), one or more populations of F. hepatica to be able to survive and create resistance or adaptability to such selective pressure.

Conversion of high and low pollen protein diets into protein in worker honey bees (Hymenoptera: Apidae).

Adequate protein levels are necessary to maintain strong honey bee [Apis mellifera (L.)] colonies. The aim of this study was to quantify how pollens with different crude protein contents influence protein stores within individual honey bees. Caged bees were fed one of three diets, consisting of high-protein-content pollen, low-protein-content pollen, or protein-free diet as control; measurements were made based on protein content in hemolymph and fat body, fat body weight, and body weight. Vitellogenin in hemolymph was also measured. Bees fed with high crude protein diet had significantly higher levels of protein in hemolymph and fat bodies. Caged bees did not increase pollen consumption to compensate for the lower protein in the diet, and ingesting approximately 4 mg of protein per bee could achieve levels of 20 microg/microl protein in hemolymph. Worker bees fed with low crude protein diet took more time in reaching similar protein content of the bees that were fed with high crude protein diet. The data showed that fat bodies and body weight were not efficient methods of measuring the protein status of bees. The determination of total protein or vitellogenin concentration in the hemolymph from 13-d-old bees and protein concentration of fat bodies from 9-d-old bees could be good indicators of nutritional status of honey bees.

Increase of gluthatione S-transferase, carboxyl esterase and carbonyl reductase in Fasciola hepatica recovered from triclabendazole treated sheep.

Fasciolasis is a zoonotic parasitic disease caused by Fasciola hepatica and its control is mainly based on the use of triclabendazole (TCBZ). Parasite resistance to different anthelmintics is growing worldwide, including the resistance of F. hepatica to TCBZ. In the present work we evaluate "in vivo" the activity of xenobiotic metabolizing enzymes of phase I (carboxyl esterases) and phase II (glutathione S-transferases and carbonyl reductases) recovered of flukes from sheep treated with TCBZ. All three enzymes showed increased activity in TCBZ flukes returning 60h post-treatment at similar to baseline unexposed flukes. TCBZ action may induce secondary oxidative stress, which may explain the observed increment in activities of the analyzed enzymes as a defensive mechanism. The enzymes analyzed are candidates to participate actively in the development of resistance at TCBZ in F. hepatica.

Resistance of Fasciola hepatica against Triclabendazole in cattle in Cajamarca (Peru): a clinical trial and an in vivo efficacy test in sheep.

Fasciolosis caused by Fasciola hepatica, is the most prevalent parasitic disease in dairy cattle from the northern region of Cajamarca, Peru. The control of this parasite is based on the use of Triclabendazole (TCBZ), a drug that has been used for more than fifteen years in this area. Recent studies, however, have reported a lack of clinical efficacy after treating dairy cattle. This research was aimed to determine the efficacy of TCBZ in a clinical trial. Eleven dairy cows all positive to F. hepatica identified by presence of eggs in feces, were treated with TCBZ (Fasinex(®) 10%) at 12 mg/kg body weight. Fourteen and thirty days after treatment, the animals were analyzed for F. hepatica eggs in their feces by the fecal egg count reduction test. The results found show an overall efficacy of 31.05% and 13. 63% (14 and 30 days post treatment, respectively). Furthermore, an in vivo efficacy test was conducted in sheep with metacercariae obtained from eggs isolated from a cow clinically resistant to TCBZ. Eleven sheep divided in two groups, a control group with no treatment (n=5) and a treated group (n=6) were all infected with two hundred metacercariae. One hundred and six days after infection all the animals demonstrated F. hepatica eggs in their feces, confirming the presence of adult parasites in their livers. The animals were then treated with TCBZ (Fasinex(®) 10%) at 10mg/kg body weight. Fifteen days later, the animals were sacrificed and the number of F. hepatica in their livers counted. The results of this experiment showed an efficacy of the flukicide of 25.2% confirming the resistance to TCBZ of the F. hepatica isolated from dairy cattle in Cajamarca, Peru.

Increase of carboxylesterase activity in Fasciola hepatica recovered from triclabendazole treated sheep.

In the present work, we evaluate in vivo the activity of carboxylesterase of Fasciola hepatica exposed to triclabendazole. We observed a statistically significant increase in enzyme activity at 24 and 48 h post treatment (P<0.01 and P<0.001, respectively). The zymogram of cytosolic fractions identified a protein of 170 kDa containing the carboxylesterase activity. The densitograms of the zymograms confirmed the phenomenon of enzyme induction under the experimental conditions of the assay. These results provide not only the understanding of the importance of this metabolic pathway in flukes but carboxylesterase would also be an enzyme that could participate more actively in the development of anthelmintic resistance at TCBZ.

Early onset of changes to the reproductive system of Fasciola hepatica following in vivo treatment with triclabendazole.

Lambs infected with the Cullompton triclabendazole (TCBZ)-susceptible isolate of Fasciola hepatica were treated with TCBZ at a dosage of 10mg/kg at 16 weeks post-infection. Adult flukes were recovered from the liver at 3h, 24h, 48 h and 60 h post-treatment (pt). They were processed for histological analysis of the uterus, Mehlis' gland, vitellaria, ovary and testis. At 3h pt, the flukes were essentially similar to the controls and were producing normal eggs. Egg production had ceased by 24h pt. At this time period, the cells of the Mehlis' gland showed some evidence of vacuolation, but otherwise were relatively normal. A shift in the population of vitelline cells towards mature cells was observed at 24h pt, and this trend continued at later time-periods. It was accompanied by a breakdown of the cells and the presence of apoptotic bodies. Marked changes to the ovary were first noted at 48 h pt, as evidenced by vacuolation and the presence of apoptotic bodies. Some disruption to the testis was seen at 24h pt, with a reduction in the population of spermatogenic cells, the appearance of apoptotic bodies and some peripheral vacuolation of the tubules. These abnormalities increased in severity with longer time periods pt. The results bring forward the time-line of cessation of egg production by 24h, demonstrating that this process is affected very rapidly pt.

Expression differential of microsomal and cytosolic glutathione-S-transferases in Fasciola hepatica resistant at triclabendazole.

In the present work, we evaluate in vitro the cytosolic and microsomal activity of glutathione-S-transferases in adults of Fasciola hepatica susceptible (Cullompton Strain) and resistant (Sligo Strain) to triclabendazole. The triclabendazole resistant (Sligo) fluke expressed significant major metabolic activity of glutathione-S-transferases compared to that measured in the cytosolic and microsomal fractions obtained from susceptible flukes (Cullompton Strain). The results associated with previous data where the Sligo Strain overexpress Flavin Monooxigenase confirm a multienzymatic response involving more than one metabolic pathway.

Reproductive disruption in Fasciola hepatica associated with incomplete efficacy of a new experimental formulation of triclabendazole.

The objective of the present study was to analyse the reproductive viability (using histopathologic studies) of Fasciola hepatica from cattle artificially infected and treated subcutaneously with a new experimental formulation of triclabendazole (8mg/kg b.w.). The results of the efficacy controlled test, which only takes into account the presence of live adult flukes, indicated that, whilst in the control group (n=7) 533 live specimens were recovered, in the test groups (doses of 8 and 12mg/kg b.w.) only 195 and 47 adults were recovered, respectively. These numbers indicate efficacies of 69% and 95.6%, respectively. It was observed in that dose of 8mg/kg b.w. some specimens remained viable, but they were infertile, which severely compromises the biological cycle of the trematode. In the testis tubules of flukes treated with the low dose of TCBZ (8mg/kg), very few cells were present and the vitelline follicles were markedly reduced in size and each follicle contained very few cells. This would have direct implications for the pathogenesis of the parasitosis since the remaining parasites would produce little clinical-productive manifestations, would stimulate the immune response and would find it difficult to establish future re-infestations/re-infections. Consequently, these observations will also prompt a review of certain methodological and interpretative aspects related to efficacy tests, where the only discriminative factor is the reduction of the adult parasite load. On one hand, histopathological studies could be complementary to the efficacy controlled test for TCBZ or other BZD formulations.

Albendazole enantiomeric metabolism and binding to cytosolic proteins in the liver fluke Fasciola hepatica.

Fascioliasis causes important economic losses in ruminant species all over the world. Its control is largely based on the use of the flukicidal compound triclabendazole (TCBZ). However, its chemically related benzimidazole anthelmintic albendazole (ABZ) is being successfully used to control TCBZ-resistance flukes. This research gains some insights into the comparative molecular behaviour of both anthelmintics within the target fluke. The goals of the current work were: (i) to assess the competitive binding of ABZ and TCBZ to cytosolic proteins of F. hepatica, and (ii) to evaluate the enantioselective biotransformation of ABZ in microsomal fractions obtained from TCBZ-susceptible and TCBZ-resistant strains of the liver fluke. Cytosolic proteins from fluke specimens bound TCBZ with greater affinity (83%) than ABZ (44%) and the fraction of TCBZ bound to cytosolic proteins was not displaced by ABZ. The microsomes from both -susceptible and resistant flukes sulphoxidized ABZ into ABZ sulphoxide (ABZSO). However, this oxidative activity was 49% higher in microsomes from TCBZ-resistant flukes (P < 0.001) with a predominant production of the (+) ABZSO enantiomer. As earlier shown for TCBZ, the results reported here confirm an enhanced ability for ABZ oxidation in TCBZ-resistant flukes. While this enhanced oxidative metabolism of ABZ may cooperate to the resistance phenomenon, other pharmacodynamic-based mechanisms may be involved, which would explain why, although being chemically-related, ABZ remains efficacious against TCBZ resistant flukes under field conditions.

Understanding triclabendazole resistance.

Triclabendazole (TCBZ) has been the drug of choice to treat liver fluke infections in livestock for >20 years, due to its high activity against both adult and juvenile flukes. More recently, it has been used successfully to treat human cases of fascioliasis. Resistance to TCBZ first appeared in the field in Australia in the mid-1990s. Since then, resistance has been reported from a number of countries throughout Europe: Ireland, Scotland, Wales, Spain and The Netherlands. The heavy reliance on a single drug puts treatment strategies for fascioliasis at risk. Should resistance develop further, the prospect is an alarming one. This review will present an overview of progress in understanding the mechanism of resistance to TCBZ, examining possible changes in the target molecule, in drug influx/efflux mechanisms and in the metabolism of TCBZ by the fluke. The review will also consider ways to deal with resistance, covering drug-oriented options such as: the use of alternative drugs, drug combinations and the search for new compounds.

Altered drug influx/efflux and enhanced metabolic activity in triclabendazole-resistant liver flukes.

Triclabendazole (TCBZ) is a halogenated benzimidazole compound that possesses high activity against immature and adult stages of the liver fluke, Fasciola hepatica. The intensive use of TCBZ in endemic areas of fascioliasis has resulted in the development of liver flukes resistant to this compound. TCBZ sulphoxide (TCBZSO) and TCBZ sulphone (TCBZSO2) are the main molecules recovered in the bloodstream of TCBZ-treated animals. In order to gain some insight into the possible mechanisms of resistance to TCBZ, the goals of the work described here were: to compare the ex vivo transtegumental diffusion of TCBZ parent drug and its sulpho-metabolites (TCBZSO and TCBZSO2) into TCBZ-susceptible and -resistant liver flukes; and to assess the comparative pattern of TCBZ biotransformation by TCBZ-susceptible and -resistant F. hepatica. For the tegumental diffusion studies, TCBZ-susceptible (Cullompton) and -resistant (Sligo) adult flukes collected from untreated infected sheep were incubated (15-180 min) in KRT buffer containing either TCBZ, TCBZSO or TCBZSO2 (5 nmol.ml-1). For the metabolism studies, microsomal fractions obtained from TCBZ-susceptible and -resistant flukes were incubated for 60 min with TCBZ (40 microM), and the amount of the formed metabolic product (TCBZSO) was measured. Drug/metabolite concentrations were quantified by HPLC. All the assayed TCBZ-related molecules penetrated through the tegument of both TCBZ-susceptible and -resistant flukes. However, significantly lower (approximately 50%) concentrations of TCBZ and TCBZSO were recovered within the TCBZ-resistant flukes compared to the TCBZ-susceptible ones over the 180 min incubation period. The rate of TCBZ sulphoxidative metabolism into TCBZSO was significantly higher (39%) in TCBZ-resistant flukes. The flavin-monooxigenase (FMO) enzyme system appears to be the main metabolic pathway involved in the formation of TCBZSO in both TCBZ-susceptible and -resistant flukes. The altered drug influx/efflux and enhanced metabolic capacity identified in TCBZ-resistant liver flukes may account for the development of resistance to TCBZ.

Triclabendazole biotransformation and comparative diffusion of the parent drug and its oxidized metabolites into Fasciola hepatica.

Triclabendazole (TCBZ) is an halogenated trematodicidal benzimidazole compound extensively used in veterinary medicine. It is active against immature and adult stages of the liver fluke Fasciola hepatica. Free and conjugated TCBZ metabolites have been identified in the bile of treated sheep. The experimental aims were to characterize the in vitro patterns of TCBZ biotransformation both in the animal host (sheep liver microsomes) and target parasite (F. hepatica microsomal preparation); and to compare the ex vivo diffusion of TCBZ parent drug and its oxidized metabolites (TCBZ sulphoxide [TCBZSO], TCBZ sulphone [TCBZSO2], and TCBZ-hydroxy derivatives) into F. hepatica. Additionally, the octanol-water partition coefficients for TCBZ and all its metabolites were estimated as an indicator of the relationship between drug lipophilicity and diffusion into the target parasite. Drug/metabolites concentrations were quantified by HPLC after sample clean up and a solvent-mediated chemical extraction. Sheep liver microsomes metabolized TCBZ into its sulphoxide and sulphone metabolites after 30 min of incubation. The rate of TCBZ sulphoxidation in the liver was significantly greater (p < 0.01) than that observed for the sulphonation of TCBZSO. The trematode parasite oxidized TCBZ into its sulphoxide metabolite after 60 min of incubation at a metabolic rate of 0.09 nmol min(-1) mg protein(-1). TCBZ and all its oxidized metabolic products were recovered from F. hepatica as early as 15 min after their ex vivo incubation in a Kreb's Ringer Tris buffer. However, the diffusion of the hydroxy-derivatives into the fluke was lower than that observed for TCBZ, TCBZSO and TCBZSO2. There was a high correlation (r=0.82) between drug lipophilicity (expressed as octanol-water partition coefficients) and drug availability measured within the parasite. Unlike the uptake pattern previously observed for albendazole, the parent TCBZ and its sulphoxide and sulphone metabolites showed a similar ability to penetrate into the trematode parasite. Understanding the relationship between TCBZ metabolism, the relative pharmacological potency of its metabolic products and their ability to reach the target parasite may be critical to optimize its flukicidal activity, particularly when TCBZ resistant flukes have been already isolated in the field.

Enantioselective binding of albendazole sulphoxide to cytosolic proteins from helminth parasites.

The pharmacological effect of the active albendazole metabolite, albendazole sulphoxide (ABZSO), depends on its sustained presence at the site of parasite location and its binding to helmith beta-tubulin. ABZSO is found in the plasma and tissues of albendazole-treated animals in two enantiomeric forms: (+)ABZSO and (-)ABZSO. Knowledge of enantioselectivity in drug action is necessary, since any difference in target proteins affinity between enantiomers may have implications on the pharmacological effect of this anthelmintic molecule. The binding of ABZSO to mammalian and helminth parasites cytosolic proteins, as well as the differential binding of both enantiomers, were studied. Cytosolic proteins from Moniezia expansa (cestode), Ascaris suum (nematode), Fasciola hepatica (trematode), rat liver and brain as well as purified porcine brain tubulin were used. Drug analysis was performed by HPLC using both C18 and chiral columns. ABZSO protein binding was quantitatively different between parasite species (4.17, 2.5 and 1.07 ng/mg for cestode, nematode and trematode, respectively); this binding to helminth cytosolic proteins was enantioselective. Enantiomeric ratios of (-)ABZSO/(+)ABZSO as a percentage were: 43/57 (Ascaris), 36/64 (Moniezia) and 91/9 (Fasciola). Conversely, the binding of ABZSO to mammalian cytosolic proteins showed no enantioselectivity. The overall binding affinity of ABZSO for mammalian cytosolic proteins was lower than that observed in helminth proteins. The characterization of the comparative binding pattern of ABZSO enantiomers to cytosolic proteins from helminth parasites and mammalian tissues may contribute to understanding the pharmacological properties of this chiral anthelmintic molecule.

Comparative metabolism of albendazole and albendazole sulphoxide by different helminth parasites.

Albendazole (ABZ) is a broad-spectrum benzimidazole anthelmintic widely used in human and veterinary medicine. The aim of the current work was to characterise the sulphoxidative metabolism of ABZ, and the sulphoreduction of ABZ sulphoxide (ABZSO), by microsomal (Ms) and cytosolic (Cyt) fractions of three different helminth species: Fasciola hepatica, Moniezia expansa and Ascaris suum. After the incubation assays, parasite material was analysed by HPLC to characterise the metabolic product formed. Both the Ms and Cyt fractions of the three parasites studied were able to oxidise ABZ into ABZSO in a non-enantioselective fashion. Oxidation of ABZ was greater in the Ms fraction of the trematode (50%) than in both cestode (19%) and nematode (14%) parasites. Only the incubation of ABZSO with both subcellular fractions of M. expansa generated ABZ as a metabolic product. The results obtained here indicate that helminths have the capacity to biotransform benzimidazole compounds; however, this metabolic activity differs qualitatively and quantitatively among helminth species.

Liver sulphoxidative metabolism of albendazole in rat: enantioselectivity and effect of methimazole.

The aim of the present work was to evaluate the effects of methimazole (MTZ) on the enantioselective sulphoxidation of albendazole (ABZ) by rat liver microsomes and tissue slices. Albendazole sulphoxide (ABZSO) was the metabolite recovered after the incubation with ABZ in both liver preparations. MTZ significantly reduced ABZSO production both in microsomes and slices. ABZSO production decreased as a function of MTZ concentration. The sulphoxidation reaction performed by rat liver explants in the presence of MTZ was 65% lower than that observed in controls. The reduction in the production of ABZSO in the presence of MTZ was mainly due to a lower production of (+) ABZSO. The results reported further contribute to the understanding of the enantioselective metabolism of ABZ. In addition, the work presented provides information on the comparison of two different liver tissue preparations for the evaluation of xenobiotic metabolism.

Histopathology of the digestive gland of an Antarctic limpet exposed to cadmium.

Histopathological alterations induced experimentally with cadmium (Cd) in Antarctic limpets (Nacella concinna), exposed for different times and concentrations were compared to controls. At the light microscope level, samples exposed to the contaminant for short periods (6, 12 and 24 h) at two different concentrations (0.25 and 0.5 mg l(-1)) showed no alterations compared to controls. After 48 h of exposure at a 0.5 mg l(-1) Cd concentration, vacuolisation of the basophilic cells was observed. After 72 h exposure, there was a marked loss of all the digestive gland structure, with cell autolysis and loss of basophilia.

The anthelmintic albendazole affects in vivo the dynamics and the detyrosination-tyrosination cycle of rat brain microtubules.

Albendazole (ABZ) is an anthelmintic benzimidazole drug widely used in human and veterinary medicine. ABZ has binding affinity to both mammalian and helminth parasite tubulin. In the current work, we have performed in vitro assays and in vivo experiments in which rats were given ABZ orally to better characterize the action of the drug on the polymerization of rat brain microtubules and on the detyrosination/tyrosination cycle that occurs on the COOH-terminal end of alpha-tubulin. The results showed that ABZ inhibits brain microtubule polymerization in vitro, and significantly delayed microtubule assembly in vivo. The tyrosination reaction cycle was not affected in vitro; however, in rats to which the drug was administered orally, the levels of in vitro tyrosination were reduced when compared to the controls with mock treatment. These results suggest that this apparent inhibition would be due to a decrease in the amount of substrate caused by the depolymerizing effect of ABZ and the subsequent tyrosination in the intact brain with endogenous tyrosine. In conclusion, ABZ strongly affects tubulin dynamics both in vivo and in vitro. The outcome of these experiments is a contribution to the understanding of the molecular mechanisms involved in the antimicrotubular action of benzimidazole compounds.

The anthelmintic albendazole affects in vivo the dynamics and the detyrosination-tyrosination cycle of rat brain microtubules

Albendazole (ABZ) is an anthelmintic benzimidazole drug widely used in human and veterinary medicine. ABZ has binding affinity to both mammalian and helminth parasite tubulin. In the current work, we have performed in vitro assays and in vivo experiments in which rats were given ABZ orally to better characterize the action of the drug on the polymerization of rat brain microtubules and on the detyrosination/tyrosination cycle that occurs on the COOH-terminal end of alpha-tubulin. The results showed that ABZ inhibits brain microtubule polymerization in vitro, and significantly delayed microtubule assembly in vivo. The tyrosination reaction cycle was not affected in vitro; however, in rats to which the drug was administered orally, the levels of in vitro tyrosination were reduced when compared to the controls with mock treatment. These results suggest that this apparent inhibition would be due to a decrease in the amount of substrate caused by the depolymerizing effect of ABZ and the subsequent tyrosination in the intact brain with endogenous tyrosine. In conclusion, ABZ strongly affects tubulin dynamics both in vivo and in vitro. The outcome of these experiments is a contribution to the understanding of the molecular mechanisms involved in the antimicrotubular action of benzimidazole compounds.