Synthesis and biological evaluation of thiabendazole derivatives as anti-angiogenesis and vascular disrupting agents.

Thiabendazole, already approved by FDA for oral use as an anti-fungal and anti-helminthic drug since 1967, has recently been repurposed as a vascular disrupting agent. By optimization of the structure of the lead compound, we successfully identified compound TBZ-19 and the new derivative is over 100-fold more potent than the lead compound against the growth of four different cell lines (A549, HCT-116, HepG2 and HUVECs). The most potent two candidates TBZ-07 and TBZ-19, exhibiting moderate inhibitory cell proliferation activity, were also verified as anti-angiogenesis and vascular disrupting agents. Therefore, TBZ-07 and TBZ-19 would be promising candidates with vasculature targeting activity and merit further development.

Adaptive and specialised transcriptional responses to xenobiotic stress in Caenorhabditis elegans are regulated by nuclear hormone receptors.

Characterisation of the pathways by which xenobiotics are metabolised and excreted in both target and non-target organisms is crucial for the rational design of effective and specific novel bioactive molecules. Consequently, we have investigated the induced responses of the model nematode Caenorhabditis elegans to a variety of xenobiotics which represent a range of putative modes of action. The majority of genes that were specifically induced in preliminary microarray analyses encoded enzymes from Phase I and II metabolism, including cytochrome P450s, short chain dehydrogenases, UDP-glucuronosyl transferases and glutathione transferases. Changes in gene expression were confirmed by quantitative PCR and GFP induction in reporter strains driven by promoters for transcription of twelve induced enzymes was investigated. The particular complement of metabolic genes induced was found to be highly contingent on the xenobiotic applied. The known regulators of responses to applied chemicals ahr-1, hif-1, mdt-15 and nhr-8 were not required for any of these inducible responses and skn-1 regulated GFP expression from only two of the promoters. Reporter strains were used in conjunction with systematic RNAi screens to identify transcription factors which drive expression of these genes under xenobiotic exposure. These transcription factors appeared to regulate specific xenobiotic responses and have no reported phenotypes under standard conditions. Focussing on nhr-176 we demonstrate the role of this transcription factor in mediating the resistance to thiabendazole.

[Distribution and elimination of thiabendazole and its metabolite residue in laying hens].

OBJECTIVE: To explore the distribution and elimination of thiabendazole and its metabolite 5-hydrothiabendazole residues in the hens tissues including liver, muscle, heart, fat, as well as in eggs. METHODS: Laying hens were orally administred thiabendazole for 5 consecutive days (100mg per hen daily) and then the hens were sacrificed at the times of 1 day, 3 days, 5 days and 7 days after the end of treatment. Eggs, liver,muscle,fat and heart tissues were collected and homogenized. The samples were extracted by acetonitrile, further concentrated and purified by an Oasis MCX cartridge, and then the contents of thiabendazole and 5-hydrothiabendazole in tissue homogenates determinated by liquid chromatography electrospray ionisation tandem mass spectrometry (UPLC-ESI-MS/MS). RESULTS: The major residues in the tissue homogenates was 5-hydroxythiabendazole, with more higher concentrations than thiabendazole. Egg samples presented the large majority of both drug residues. For the tissue homogenates,the total concentrations of thiabendazole and 5-hydrothiabendazole residues followed the order of liver > heart > muscle > fat at 1 day after the treatment. The withdrawal period of thiabendazole for eggs was about 7 days. CONCLUSION: Distribution and elimination of thiabendazole and its metabolite residues in laying hens were primarily studied in this study.

Myeloperoxidase-mediated bioactivation of 5-hydroxythiabendazole: a possible mechanism of thiabendazole toxicity.

Thiabendazole (TBZ), an antihelminthic and antifungal agent, is associated with a host of adverse effects including nephrotoxicity, hepatotoxicity, and teratogenicity. Bioactivation of the primary metabolite of TBZ, 5-hydroxythiabendazole, has been proposed to yield a reactive intermediate. Here we show that this reactive intermediate can be catalyzed by myeloperoxidase (MPO), a neutrophil-bourne peroxidase. Using a cell viability endpoint, we examined the toxicity of TBZ, 5OH-TBZ, and MPO-generated metabolites in cell-based models including primary rat proximal tubule epithelial cells, NRK-52E rat proximal tubule cells, and H9C2 rat myocardial cells. Timecourse experiments with MPO showed complete turnover of 5OH-TBZ within 15 min and a dramatic leftward shift in dose-response curves after 12h. After a 24h exposure in vitro, the LC(50) of this reactive intermediate was 23.3 ± 0.2 µM reduced from greater than 200 µM from 5OH-TBZ alone, an approximately 10-fold decrease. LC(50) values were equal in all cell types used. Comparison of lactate dehydrogenase leakage and caspase 3/7 activity revealed that cell death caused by the reactive intermediate is primarily associated with necrosis rather than apoptosis. This toxicity can be completely rescued via incubation with rutin, an inhibitor of MPO. These results suggest that MPO-mediated biotransformation of 5OH-TBZ yields a reactive intermediate which may play a role in TBZ-induced toxicity.

In vitro and in silico identification and characterization of thiabendazole as a mechanism-based inhibitor of CYP1A2 and simulation of possible pharmacokinetic drug-drug interactions.

Thiabendazole (TBZ) and its major metabolite 5-hydroxythiabendazole (5OH-TBZ) were screened for potential time-dependent inhibition (TDI) against CYP1A2. Screen assays were carried out in the absence and presence of NADPH. TDI was observed with both compounds, with k(inact) and K(I) values of 0.08 and 0.02 min(-1) and 1.4 and 63.3 microM for TBZ and 5OH-TBZ, respectively. Enzyme inactivation was time-, concentration-, and NADPH-dependent. Inactivation by TBZ was irreversible by dialysis and oxidation by potassium ferricyanide, and there was no protection by glutathione. 5OH-TBZ was a weak TDI of CYP1A2, and enzyme activity was recovered by dialysis. IC(50) determination of TBZ and 5OH-TBZ showed both compounds to be potent inhibitors, with IC(50) values of 0.83 and 13.05 microM, respectively. IC(50) shift studies also demonstrated that TBZ was a TDI of CYP1A2. In silico methods identified the thiazole group as a TDI fragment and predicted it as the site of metabolism. The observation pointed to epoxidation of the thiazole and the benzyl rings of TBZ as possible routes of metabolism and mechanisms of TDI. Drug-drug interaction (DDI) simulation studies using SimCyp showed good predictions for competitive inhibition. However, predictions for mechanism-based inhibition (MBI)-based DDI were not in agreement with clinical observations. There was no TBZ accumulation upon chronic administration of the drug. The in vitro MBI findings might therefore not be capturing the in vivo situation in which the proposed bioactivation route is minor. This might be the case for TBZ in which, in vivo, UDP glucuronosyltransferases and sulfanotransferase metabolize and eliminate the 5OH-TBZ.

In vitro metabolic activation of thiabendazole via 5-hydroxythiabendazole: identification of a glutathione conjugate of 5-hydroxythiabendazole.

Thiabendazole (TBZ) is a broad-spectrum antihelmintic used for treatment of parasitic infections in animals and humans and as an agricultural fungicide for postharvest treatment of fruits and vegetables. It is teratogenic and nephrotoxic in mice, and cases of hepatotoxicity have been observed in humans. Recent reports have demonstrated a correlation between 5-hydroxythiabendazole (5-OHTBZ) formation, a major metabolite of TBZ, and covalent binding of [(14)C]TBZ to hepatocytes, suggesting another pathway of activation of TBZ. Current in vitro studies were undertaken to probe the bioactivation of TBZ via 5-OHTBZ by cytochrome P450 (P450) and peroxidases and identify the reactive species by trapping with reduced glutathione (GSH). Microsomal incubation of TBZ or 5-OHTBZ supplemented with NADPH and GSH afforded a GSH adduct of 5-OHTBZ and was consistent with a bioactivation pathway that involved a P450-catalyzed two-electron oxidation of 5-OHTBZ to a quinone imine. The same adduct was detected in GSH-fortified incubations of 5-OHTBZ with peroxidases. The identity of the GSH conjugate suggested that the same reactive intermediate was formed by both these enzyme systems. Characterization of the conjugate by mass spectrometry and NMR revealed the addition of GSH at the 4-position of 5-OHTBZ. In addition, the formation of a dimer of 5-OHTBZ was discernible in peroxidase-mediated incubations. These results were consistent with a one-electron oxidation of 5-OHTBZ to a radical species that could undergo disproportionation or an additional one-electron oxidation to form a quinone imine. Overall, these studies suggest that 5-OHTBZ can also play a role in TBZ-induced toxicity via its bioactivation by P450 and peroxidases.

Metal-mediated inhibition of Escherichia coli methionine aminopeptidase: structure-activity relationships and development of a novel scoring function for metal-ligand interactions.

We report the discovery of thiabendazole as a potent inhibitor (K(i) = 0.4 microM) of Escherichia coli methionine aminopeptidase (ecMetAP) and the synthesis and pharmacological evaluation of thiabendazole congeners with activity in the upper nanomolar range. Elucidation of the X-ray structure of ecMetAP in complex with thiabendazole and an unrelated inhibitor that was independently described by another group showed that that both compounds bind to an additional Co(II) ion at the entrance of the active site. This unexpected finding explains the inactivity of the compounds under in vivo conditions. It also allows us to discuss the structure-activity relationships of this series of compounds in a meaningful way, based upon docking runs with an auxiliary metal ion. We describe a new scoring function for the evaluation of metal-mediated inhibitor binding that, unlike the previously used scoring function implemented in the docking program, allows us to distinguish between active and inactive compounds. Finally, conclusions for the structure-based design of in vivo-active inhibitors of ecMetAP are drawn.

Synthesis, antimicrobial activity and chemotherapeutic potential of inorganic derivatives of 2-(4'-thiazolyl)benzimidazole[thiabendazole]: X-ray crystal structures of [Cu(TBZH)2Cl]Cl.H2O.EtOH and TBZH2NO3 (TBZH=thiabendazole).

Thiabendazole (TBZH) reacts with iron(III) nitrate causing protonation of the ligand to yield the nitrate salt [TBZH(2)NO(3)] (1). Reaction of TBZH with copper(II) acetate results in the deprotonation of the ligand yielding [Cu(TBZ)2.(H2O)2] (2). Reactions of TBZH with the chloride, nitrate and butanedioate salts of copper(II) yields [Cu(TBZH)2Cl]Cl.H2O.EtOH (3), [Cu(TBZH)(2)(NO(3))(2)] (4) and [Cu(TBZH)(O(2)C-CH(2)CH(2)-CO(2))] (5), respectively. The TBZH acts as a neutral chelating ligand in 3-5. Molecular structures of 1 and 3 were determined crystallographically. In 1, the asymmetric unit contains one TBZH(2)(+) cation and one NO(3)(-) anion. The structure of 3 comprises a five coordinate copper centre with the metal bound to two chelating TBZH ligands and one chloride. The geometry is best described as trigonal bipyramidal. Hydrogen bonding connects the complex cation with the uncoordinated chloride anion and the water and ethanol solvate molecules. Compound 1 and the copper complexes 2-5, the metal free ligands and a number of simple copper(II) salts were each tested for their ability to inhibit the growth of Candida albicans. The metal free TBZH and its nitrate salt (1) exhibited very poor activity. Complex 2, in which the TBZH is present as an anionic ligand (TBZ(-)), exhibits moderate activity towards the pathogen. Chelation of the neutral TBZH to copper centres (complexes 3-5) results in potent anti-candida activity. The dimethyl sulphoxide (DMSO) soluble complexes 3 and 4, along with metal free TBZH were assessed for their cancer chemotherapeutic potential towards two human epithelial-derived cancer model cell lines. Complexes 3 and 4 displayed similar dose-dependent cytotoxicity in both cell lines with IC(50) values of approximately 50 microM, which were found to be significantly lower than that for metal free TBZH.

Studies of antimicrobial activity of N-alkyl and N-acyl 2-(4-thiazolyl)-1H-benzimidazoles.

Various N-alkyl and N-acyl derivatives of 2-(4-thiazolyl)-1H-benzimidazole, an anthelmintic and systemic fungicide, were synthesized by polymer-supported reactions and screened for their antifungal and antibacterial potency to establish structure-activity relationships.

Localization and migration of benzimidazole resistant and susceptible adult Cooperia curticei in the small intestine of sheep after treatment with thiabendazole.

Four groups of three lambs per group were experimentally infected with Cooperia curticei susceptible (two groups) or resistant (two groups) to benzimidazoles, and distributions of adult worms in the small intestine were studied. For each Cooperia isolate, one group was treated with thiabendazole (TBZ) (5 or 50mg/kg bodyweight) 28 days after infection. In the two untreated groups, the population of C. curticei were present from the second to the tenth meter of intestine from the pylorus with a maximum in the sixth meter for both isolates. After treatment with TBZ, the size of the resistant worm population did not significantly decrease but a large number of worms were found towards the proximal sections of the intestine. In contrast, the susceptible population was reduced by about 40% but the surviving worms remained at this same site of predilection after treatment. Measurements of the concentration of TBZ and 5OH-thiabendazole (5OH-TBZ) in the intestinal segments do not indicate a clear relationship between the localization of worms and TBZ or 5OH-TBZ concentrations at least 12h after the anthelmintic treatment. The hypothesis of an enhanced expression of the mechanisms of resistance in the first few meters of small intestine is suggested.

Evidence for cytochrome P4501A2-mediated protein covalent binding of thiabendazole and for its passive intestinal transport: use of human and rabbit derived cells.

Thiabendazole (TBZ), an anthelmintic and fungicide benzimidazole, was recently demonstrated to be extensively metabolized by cytochrome P450 (CYP) 1A2 in man and rabbit, yielding 5-hydroxythiabendazole (5OH-TBZ), the major metabolite furtherly conjugated, and two minor unidentified metabolites (M1 and M2). In this study, exposure of rabbit and human cells to 14C-TBZ was also shown to be associated with the appearance of radioactivity irreversibly bound to proteins. The nature of CYP isoforms involved in this covalent binding was investigated by using cultured rabbit hepatocytes treated or not with various CYP inducers (CYP1A1/2 by beta-naphthoflavone, CYP2B4 by phenobarbital, CYP3A6 by rifampicine, CYP4A by clofibrate) and human liver and bronchial CYP-expressing cells. The covalent binding to proteins was particularly increased in beta-naphthoflavone-treated rabbit cells (2- to 4-fold over control) and human cells expressing CYP1A2 (22- to 42-fold over control). Thus, CYP1A2 is a major isoenzyme involved in the formation of TBZ-derived residues bound to protein. Furthermore, according to the good correlation between covalent binding and M1 or 5OH-TBZ production, TBZ would be firstly metabolized to 5OH-TBZ and subsequently converted to a chemically reactive metabolic intermediate binding to proteins. This metabolic activation could take place preferentially in liver and lung, the main biotransformation organs, rather than in intestines where TBZ was shown to be not metabolized. Moreover, TBZ was rapidly transported by passive diffusion through the human intestinal cells by comparison with the protein-bound residues which were not able to cross the intestinal barrier. Consequently, the absence of toxicity measured in intestines could be related to the low degree of TBZ metabolism and the lack of absorption of protein adducts. Nevertheless, caution is necessary in the use of TBZ concurrently with other drugs able to regulate CYP1A2, particularly in respect to liver and lung tissues, recognised as sites of covalent-binding.

Simultaneous determination of thiabendazole and its major metabolite, 5-hydroxythiabendazole, in bovine tissues using gradient liquid chromatography with thermospray and atmospheric pressure chemical ionisation mass spectrometry.

A novel method is presented for the determination of thiabendazole and 5-hydroxythiabendazole in animal tissues. Samples are homogenised in buffer at pH=7.0, extracted with ethyl acetate and cleaned up using CN solid-phase extraction columns. Thiabendazole and 5-hydroxythiabendazole are separated chromatographically using gradient elution and analysed by liquid chromatography-mass spectrometry. Deuterated thiabendazole is employed as an internal standard for thiabendazole determination; 5-hydroxythiabendazole is quantified via external standards. Samples are screened by monitoring the protonated molecular ions at m/z=202 for thiabendazole, 206 for deuterated thiabendazole and 218 for 5-hydroxythiabendazole using thermospray LC-MS. Positives are confirmed by multiple ion monitoring using APCI LC-MS. Validation of the method was carried out at 50, 100 and 200 microg kg(-1). Recoveries for thiabendazole in bovine muscle, liver and kidney ranged from 96-103% with C.V.s between 0.7 and 4.8% and for 5-hydroxythiabendazole recoveries ranged from 70-85% with C.V.s between 3.1 and 11.5%.

Identification of human and rabbit cytochromes P450 1A2 as major isoforms involved in thiabendazole 5-hydroxylation.

This report characterized one of the major cytochrome P450 isozyme involved in thiabendazole metabolism. This study was undertaken by using both cultured rabbit hepatocytes treated or not with drugs known to specifically induced various cytochromes P450 isoenzymes (i.e., P450 1A1/2 by beta-naphthoflavone, P450 2B4 by phenobarbital, P450 3A6 by rifampicine and P450 4A by clofibrate) and human liver (THLE-5) and bronchial (BEAS-2B) epithelial cells expressing or not the major constitutive human cytochromes P450 (i.e., CYP1A2, 2A6, 2B6, 2C9, 2D6, 2E1 or 3A4). Only hepatocytes exposed to beta-naphthoflavone and clofibrate significantly metabolized thiabendazole to 5-hydroxythiabendazole. Extensive biotransformation of this anthelmintic only occurred in human cells expressing CYP1A2. Moreover, experiments performed on rabbit preparations showed good correlations between thiabendazole 5-hydroxylase activity and both ethoxyresorufin and methoxyresorufin O-dealkylase activities. Thus, CYP1A2 is a major isoenzyme involved in thiabendazole 5-hydroxylation.

Tiempo de normalización de los niveles de gammaglutamil transpeptidasa en ovinos con fasciolosis experimental tratados con triclabendazol / Normalization period of gammaglutamyl transpeptidase serum levels in sheep with fasciolosis treated with triclabendazole

Se determinaron los niveles séricos de gammaglutamil transpoptidasa (GGT) en ganado ovino infectado experimentalmente con Fasciola hepatica, con el objetivo de valorar el tiempo en que se normalizan después del tratamiento con triclabendazol. Se empleó un lote de corderos adultos y se conformaron 3 lotes de 5 animales cada uno. Los lotes 1 y 2 fueron infectados con 60 metacarcarias de Fasciola hepatica, los días 1, 30 y 75 del experimento. A los 90 días de la infección, se trató el lote 1 con triclabendazol a una dosis de 10 mg/kg; el lote 3 no fue infectado ni tratado, fue el testigo negativo. Los niveles séricos de GGT fueron cuantificados 8 días antes de la infección y posteriormente cada 30 días durante 165. Se encontró un incremento significativo (P< 0.05) de GGT en los lotes 1 y 2 en relación con el 3 posinfección, con una x de 35.4 y de 44.7 UY, y un pico a los 90 días con una x de 71.7 y 57.3 unidades internacionales, respectivamante. Los niveles séricos de GGT se normalizaron en los ovinos a los 75 días de haber sido tratados con triclabendazol

Structural requirements for the induction of cytochromes P450 by benzimidazole anthelmintic derivatives in cultured rabbit hepatocytes.

The effect of sulfur-containing benzimidazoles (thiabendazole, 5-hydroxy-thiabendazole, cambendazole) and sulfur-free derivatives (benzimidazole, carbendazim and 5-hydroxycarbendazim) on cytochrome P450 enzymes was investigated in primary cultures of rabbit hepatocytes considered 72 h after plating. Thiabendazole, cambendazole and carbendazim led to a significant dose-dependent increase in both EROD activity and cytochrome P4501A1/2 proteins and mRNA expression. Experiments using actinomycin D strongly suggest that these compounds have a transcriptional control on both CYP1A1 and CYP1A2 genes in primary hepatocytes. Thiabendazole increased both COH activity and P4502A protein levels. We conclude that sulfur is not a prerequisite to the P450 induction potential of benzimidazoles, while 5-hydroxylation leads to inefficient metabolites in terms of inducibility.

Liquid chromatographic fluorescence method for multiresidue determination of thiabendazole and 5-hydroxythiabendazole in milk.

A novel liquid chromatographic multiresidue method has been developed for quantitation of thiabendazole (TBZ), the metabolite 5-hydroxythiabendazole (5-OH-TBZ), and the sulfate conjugate of 5-hydroxythiabendazole (5-HSO4-TBZ) in raw cow's milk. The 5-HSO4-TBZ is hydrolyzed quantitatively under acidic conditions to 5-OH-TBZ. TBZ and 5-OH-TBZ are extracted from milk at pH 8.0 with ethyl acetate followed by cleanup of the extract on a cation-exchange solid-phase extraction column. Analytes are quantitated by liquid chromatography with a cation-exchange stationary phase and fluorescence detection. Recoveries from raw cow's milk samples fortified with TBZ and 5-OH-TBZ or TBZ and 5-HSO4-TBZ at 0.05 to 2 ppm ranged from 87 to 103% for TBZ, 98-109% for 5-OH-TBZ, and 96-115% for 5-HSO4-TBZ (quantitated as 5-OH-TBZ). The assay provides a simple, rapid, and sensitive multiresidue method for monitoring total residues of TBZ, 5-OH-TBZ, and 5-HSO4-TBZ in milk.

Nephrotoxicity of thiazoles structurally related to thiabendazole in mice depleted of glutathione by treatment with buthionine sulfoximine.

In mice depleted of glutathione (GSH) by treatment with DL-buthionine sulfoximine (BSO), thiabendazole [2-(4'-thiazolyl)benzimidazole, TBZ] produces renal damage characterized by increases in relative kidney weight and serum urea nitrogen (SUN) concentration. Several thiazole and benzimidazole compounds related to TBZ were examined for the ability to cause nephrotoxicity in mice pretreated with BSO. 4-Methyl- and 4-phenylthiazoles were highly effective compounds. In the absence of BSO, 4-methylthiazole resulted in no nephrotoxicity; inhibitors of hepatic and renal cytochrome P-450 enzymes such as methoxsalen and piperonyl butoxide prevented the nephrotoxicity of 4-methylthiazole given in combination with BSO. In addition, there was a sex difference in the nephrotoxicity of 4-methylthiazole in combination with BSO; the nephrotoxicity was observed only in males. These features of nephrotoxicity of 4-methylthiazole are well in accord with those of TBZ previously reported. This suggests that TBZ and 4-methylthiazole share a common mechanism of nephrotoxicity.

Determination of thiabendazole, 5-hydroxythiabendazole, fenbendazole, and oxfendazole in milk.

The liquid chromatographic determination previously developed for benzimidazoles in cattle liver has been slightly modified and applied to the determination of 4 benzimidazoles in milk. Recoveries of fenbendazole (FBZ), oxfendazole (OFZ), and thiabendazole (TBZ) from milk fortified at the 10 ppb level were 80% or greater with an intralaboratory coefficient of variation of 11% or less. Recovery of 5-hydroxythiabendazole (5-OH-TBZ) at the 30 ppb level averaged 56% with an intralaboratory coefficient of variation of 5%. Limited data on the depletion of FBZ, OFZ, TBZ, and 5-OH-TBZ in milk were also generated.

HPLC determination and pharmacokinetics of thiabendazole and its major metabolite 5-OH thiabendazole in equine plasma.

Separate high performance liquid chromatographic methods were developed for thiabendazole (TBZ) and 5-hydroxy thiabendazole (5-OH-TBZ) determination in horse plasma using 1-methyl-2-phenyl benzimidazole (MPBZ) as an internal standard. In both methods TBZ and 5-OH-TBZ were extracted from plasma using organic solvents, injected on to a C-18 column, and eluents monitored by a fluorescence detector. However, mobile phase composition, extraction solvent as well as detector wavelength differed in the two methods. The linear range for TBZ was 0.02 to 0.77 microgram ml-1 while that for 5-OH-TBZ was 0.96 to 8.0 micrograms ml-1. A commercially available TBZ oral suspension was administered to four thoroughbred horses in the following manner: days 1 and 2, 44 mg kg-1; days 4 and 5, 440 mg kg-1. Blood samples were collected during the 24 hours after administration and then analysed for TBZ and 5-OH-TBZ. Half-lives (t1/2), maximum plasma concentrations (Cmax), area under plasma concentration time curves (AUC O-alpha), and relative apparent bioavailability (F), were determined using pharmacokinetic equations. The pharmacokinetic parameters varied in the following manner: 1.16 to 13.63 hours (t1/2), 12 to 131 micrograms ml-1 X hours (AUC O-alpha), 3.33 to 8.90 micrograms ml-1 (Cmax), 1.38 to 0.12 (F) after 44 mg kg-1 and 440 mg kg-1 doses, respectively. The ratios of concentrations of TBZ to 5-OH-TBZ after oral administration of TBZ, were significantly lower for 44 mg kg-1 than 440 mg kg-1 doses.(ABSTRACT TRUNCATED AT 250 WORDS)