The ABCG2 protein in vitro transports the xenobiotic thiabendazole and increases the appearance of its residues in milk.

Thiabendazole (TBZ) is a broad-spectrum anthelmintic and fungicide used in humans, animals, and agricultural commodities. TBZ residues are present in crops and animal products, including milk, posing a risk to food safety and public health. ABCG2 is a membrane transporter which affects bioavailability and milk secretion of xenobiotics. Therefore, the aim of this work was to characterize the role of ABCG2 in the in vitro transport and secretion into milk of 5-hydroxythiabendazole (5OH-TBZ), the main TBZ metabolite. Using MDCK-II polarized cells transduced with several species variants of ABCG2, we first demonstrated that 5OH-TBZ is efficiently in vitro transported by ABCG2. Subsequently, using Abcg2 knockout mice, we demonstrated that 5OH-TBZ secretion into milk was affected by Abcg2, with a more than 2-fold higher milk concentration and milk to plasma ratio in wild-type mice compared to their Abcg2-/- counterpart.

Developmental defects induced by thiabendazole are mediated via apoptosis, oxidative stress and alteration in PI3K/Akt and MAPK pathways in zebrafish.

Thiabendazole, a benzimidazole fungicide, is widely used to prevent yield loss in agricultural land by inhibiting plant diseases derived from fungi. As thiabendazole has a stable benzimidazole ring structure, it remains in the environment for an extended period, and its toxic effects on non-target organisms have been reported, indicating the possibility that it could threaten public health. However, little research has been conducted to elucidate the comprehensive mechanisms of its developmental toxicity. Therefore, we used zebrafish, a representative toxicological model that can predict toxicity in aquatic organisms and mammals, to demonstrate the developmental toxicity of thiabendazole. Various morphological malformations were observed, including decreased body length, eye size, and increased heart and yolk sac edema. Apoptosis, reactive oxygen species (ROS) production, and inflammatory response were also triggered by thiabendazole exposure in zebrafish larvae. Furthermore, PI3K/Akt and MAPK signaling pathways important for appropriate organogenesis were significantly changed by thiabendazole. These results led to toxicity in various organs and a reduction in the expression of related genes, including cardiovascular toxicity, neurotoxicity, and hepatic and pancreatic toxicity, which were detected in flk1:eGFP, olig2:dsRED, and L-fabp:dsRed;elastase:GFP transgenic zebrafish models, respectively. Overall, this study partly determined the developmental toxicity of thiabendazole in zebrafish and provided evidence of the environmental hazards of this fungicide.

Recovery from spindle checkpoint-mediated arrest requires a novel Dnt1-dependent APC/C activation mechanism.

The activated spindle assembly checkpoint (SAC) potently inhibits the anaphase-promoting complex/cyclosome (APC/C) to ensure accurate chromosome segregation at anaphase. Early studies have recognized that the SAC should be silenced within minutes to enable rapid APC/C activation and synchronous segregation of chromosomes once all kinetochores are properly attached, but the underlying silencers are still being elucidated. Here, we report that the timely silencing of SAC in fission yeast requires dnt1+, which causes severe thiabendazole (TBZ) sensitivity and increased rate of lagging chromosomes when deleted. The absence of Dnt1 results in prolonged inhibitory binding of mitotic checkpoint complex (MCC) to APC/C and attenuated protein levels of Slp1Cdc20, consequently slows the degradation of cyclin B and securin, and eventually delays anaphase entry in cells released from SAC activation. Interestingly, Dnt1 physically associates with APC/C upon SAC activation. We propose that this association may fend off excessive and prolonged MCC binding to APC/C and help to maintain Slp1Cdc20 stability. This may allow a subset of APC/C to retain activity, which ensures rapid anaphase onset and mitotic exit once SAC is inactivated. Therefore, our study uncovered a new player in dictating the timing and efficacy of APC/C activation, which is actively required for maintaining cell viability upon recovery from the inhibition of APC/C by spindle checkpoint.

Characterization of the biodegradation, bioremediation and detoxification capacity of a bacterial consortium able to degrade the fungicide thiabendazole.

Thiabendazole (TBZ) is a persistent fungicide used in the post-harvest treatment of fruits. Its application results in the production of contaminated effluents which should be treated before their environmental discharge. In the absence of efficient treatment methods in place, biological systems based on microbial inocula with specialized degrading capacities against TBZ could be a feasible treatment approach. Only recently the first bacterial consortium able to rapidly transform TBZ was isolated. This study aimed to characterize its biodegradation, bioremediation and detoxification potential. The capacity of the consortium to mineralize 14C-benzyl-ring labelled TBZ was initially assessed. Subsequent tests evaluated its degradation capacity under various conditions (range of pH, temperatures and TBZ concentration levels) and relevant practical scenarios (simultaneous presence of other postharvest compounds) and its bioaugmentation potential in soils contaminated with increasing TBZ levels. Finally cytotoxicity assays explored its detoxification potential. The consortium effectively mineralized the benzoyl ring of the benzimidazole moiety of TBZ and degraded spillage level concentrations of the fungicide in aqueous cultures (750 mg L-1) and in soil (500 mg kg-1). It maintained its high degradation capacity in a wide range of pH (4.5-7.5) and temperatures (15-37 °C) and in the presence of other pesticides (ortho-phenylphenol and diphenylamine). Toxicity assays using the human liver cancer cell line HepG2 showed a progressive decrease in cytotoxicity, concomitantly with the biodegradation of TBZ, pointing to a detoxification process. Overall, the bacterial consortium showed high potential for future implementation in bioremediation and biodepuration applications.

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.

Release of protein-bound residues of thiabendazole from liver.

Tissue-bound residues of thiabendazole (TBZ), a veterinary anthelmintic and postharvest fungicide, are formed when this compound is incubated with rabbit hepatocytes or administered to mice or pigs. Several pretreatment steps were investigated for removing free TBZ and metabolites prior to the release of bound residues, and three procedures were evaluated for the release of bound residues from solvent-extracted rabbit hepatocytes: incubation under acidic conditions, enzymatic action using cystathionine beta-lyase, and Raney nickel desulfurization. Immunoaffinity chromatography utilizes monoclonal antibodies capable of binding TBZ or its 5-hydroxy metabolite enabled isolation of crossreactive residue fractions. Residues released from incurred pig liver and isolated by immunoaffinity included TBZ, as determined by HPLC with photodiode array detection. The methodology described should facilitate food safety assessments of TBZ.

Thiabendazole induces urinary tract toxicity in male ICR mice.

Male ICR mice were administered thiabendazole (TBZ) in the diet at concentration of 0 (control), 0.8, 1.2 and 1.6% for 44 weeks. The mortality was 10, 6, 40 or 90% in control, 0.8, 1.2 or 1.6% TBZ group, respectively. In dead mice, the gross findings included the abnormalities of kidney such as atrophy, hydronephrosis or swelling in 2, 67, 95 or 96% of the 0, 0.8, 1.2 or 1.6% TBZ group, respectively. In surviving mice at the end of study, the right kidney weight of treated groups was significantly lower than that of control group. The urinary bladder weight of treated groups was significantly higher than that of control group. Gross findings in treated mice included the renal atrophy, hydronephrosis, calculi in renal pelvis or urinary bladder and thickening of the bladder wall. Microscopic findings in the kidneys of treated mice included nephrosis, hydronephrosis and hyperplasia of transitional epithelium of renal pelvis and/or papilla. In the urinary bladder, hyperplasia or squamous metaplasia of transitional epithelium were found in treated mice. Administration of TBZ in the diet for 44 weeks results in nephrosis and calculus formation in the renal pelvis and urinary bladder of male ICR mice, and is associated with hyperplasia of transitional epithelium of renal pelvis or urinary bladder.

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.

Unexpected increased thiabendazole tolerance in Haemonchus contortus resistant to anthelmintics by modulation of glutathione activity.

The effect of several modulators of the synthesis or activity of glutathione (GSH) on the susceptibility of Haemonchus contortus eggs susceptible or resistant to anthelmintics was investigated using in vitro egg-hatch assays. Diethylmaleate, D,L-buthionine-[S,R]-sulfoximine, and patulin induced an unexpected decrease in the susceptibility of resistant eggs to thiabendazole, which was chosen as a reference for resistance to benzimidazole compounds. The results demonstrate that the level of GSH or SH analog plays an important role in the toxicity of thiabendazole to nematode eggs. Comparison with changes observed in the cytotoxicity of antitumor or antiprotozoal drugs after GSH modulation suggests that in H. contortus eggs this increased thiabendazole tolerance might depend on different factors, whether associated or not, including the ability of thiabendazole to conjugate with parasitic GSH or analog, the potential toxicity of such conjugates, their cellular distribution, and their role in the expression of glutathione S-transferase activities, and, perhaps, in the regulation of apoptosis.

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.

Glutathione and cysteine enhance and diethylmaleate reduces thiabendazole teratogenicity in mice.

The effects of cysteine (CYS), glutathione (GSH) and diethylmaleate (DM) on the teratogenicity of thiabendazole (TBZ) were investigated. On day 9 of gestation mice were given ip a dose of 0, 50 or 100 mg CYS/kg body weight, or 0, 400 or 800 mg GSH/kg, or 0, 0.05, 0.10, 0.15 or 0.60 DM/kg. One hr later they were dosed orally with 0, 250, 500 or 1000 mg TBZ/kg. All foetuses were removed from the uterus on day 18 of gestation, and were examined for external and skeletal anomalies. The number of malformed foetuses was increased in mice pretreated with CYS or GSH and was decreased in those pretreated with DM, in comparison with numbers in the corresponding group treated with TBZ alone GSH pretreatment enlarged the area under the curve (AUC) of TBZ and 5-hydroxyTBZ, a representative metabolite, in foetal tissue. DM pretreatment reduced the AUC of TBZ and 5-hydroxyTBZ.