Real-Time Monitoring of Pesticide Translocation in Tomato Plants by Surface-Enhanced Raman Spectroscopy.

Understanding the behavior of pesticide translocation is significant for effectively applying pesticides and reducing pesticide exposures from treated plants. Herein, we applied surface enhanced Raman spectroscopy (SERS) for real-time monitoring of pesticide translocation in tomato plant tissues, including leaves and flowers, following root exposure in hydroponic and soil systems. Various concentrations of the systemic pesticide, thiabendazole, was introduced into hydroponic systems used for growing tomato plants. At selected time intervals, tomato leaves and flowers were picked and thiabendazole was measured directly under a Raman microscope after pipetting gold nanoparticle-containing solution onto the plant tissue. We found that the pesticide signals first appeared along the midrib in the lowest leaves and moved distally to the edge of the leaves. As the concentration of pesticide applied to the root was increased, the time necessary to detect the signal was decreased. The SERS surface mapping method was also able to detect thiabendazole in the trichomes of the leaves. In addition, we found a unique SERS peak at 737 cm-1 on both leaves and flowers at 4 and 6 days following the application of 200 mg/L thiabendazole to the hydroponic system. This peak appears to be coming from adenine-containing materials and may be related to the plant's response to pesticide toxicity, which could be used as a potential marker for monitoring plant responses to stresses. These results demonstrate a successful application of SERS as a rapid and effective way to study the real-time translocation behavior of pesticides in a plant system.

Comparison of biotransformation and efficacy of aminoacetonitrile anthelmintics in vitro.

The present in vitro study was designed to test and compare anthelmintic activity, hepatotoxicity, and biotransformation of four selected aminoacetonitrile derivatives (AADs): monepantel (MOP, anthelmintic approved for the treatment), AAD-970, AAD-1154, and AAD-1336. Micro-agar larval development test, MTT test of cytotoxicity, and biotransformation study coupled with Ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) technique were used for this purpose. Larvae of two Haemonchus contortus strains (drug susceptible and multi-drug resistant) and primary cultures of rat and ovine hepatocytes served as model systems. All AADs (including MOP) exhibited significant larvicidal effect in H. contortus susceptible as well as multi-resistant strains, much higher than those of reference anthelmintics thiabendazole and flubendazole. AAD-1154 provides the best results for most tested parameters among all AADs in this study. The cytotoxicity test showed that all AADs can be considered as nontoxic for hepatocytes. In the biotransformation study, Phase I and Phase II metabolites of AADs were identified and schemes of possible metabolic pathways in ovine hepatocytes were proposed. Biotransformation of MOP was much more extensive than biotransformation of other AADs. Based on obtained results, AAD-1154 and AAD-1336 can be considered as promising candidates for further in vivo testing.

Comparisons of metabolic and physiological changes in rats following short term oral dosing with pesticides commonly found in food.

¹H Nuclear Magnetic Resonance spectroscopy has been used to profile urinary metabolites in male Fischer F344 rats in order to assess the metabolic changes induced by oral exposure to two benzimidazole fungicides (carbendazim and thiabendazole) and two bipyridyllium herbicides (chlormequat and mepiquat). Exposure levels were selected to be lower than those expected to cause overt signs of toxicity. We then compared the sensitivity of the metabolomics approach to more traditional methods of toxicity assessment such as the measurement of growth and organ weights. Separate, acute exposure experiments were conducted for each pesticide to identify potential metabolic markers of exposure across four doses (and a control). Growth, organ weights and feeding/drinking rates were not significantly affected by any compounds at any dose levels tested. In contrast, metabolic responses were detected within 8 and 24h for chlormequat and mepiquat, and after 24h for carbendazim and thiabendazole. These results demonstrate the potential for the use of metabolomics in food toxicity testing.

[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.

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.

Factors affecting imazalil and thiabendazole uptake and persistence in citrus fruits following dip treatments.

The effect of concentration, temperature, and length of treatment with imazalil (IMZ) and thiabendazole (TBZ) was studied with application to citrus fruit. The amount of residues retained by fruit after "home" washing was also monitored. IMZ uptake in citrus fruit was related to treatment duration, whereas TBZ residues was not. Residues of IMZ or TBZ fungicides were significantly correlated with dip temperature (r = 0.943 for IMZ; r = 0.911 for TBZ). Treatment at 50 degrees C produced a deposition approximately 8 and approximately 2.5 times higher than when treatments were carried out at 20 degrees C in IMZ and TBZ, respectively. No significant differences in terms of IMZ deposition were detected after treatments carried out alone or in combination. Uptake of the two fungicides was associated with their physicochemical characteristics as well as different formulation types. IMZ residues showed a great persistence during storage when applied separately, and >83% of active ingredient was present after 9 weeks of storage. IMZ residues increased with dip length, doubling when dip time increased from 0.5 to 3 min. In contrast, TBZ residues did not change with the different dip times. Following postharvest dip treatments of citrus fruit at 20 or 50 degrees C, home washing removed approximately 50% of the IMZ and approximately 90% of the TBZ.

Flow cytometry analysis of drug transport mechanisms in Haemonchus contortus susceptible or resistant to anthelmintics.

The role of membrane drug-transport mechanisms in resistance to anthelmintics was examined using a flow cytometry method. This method was adapted from assays developed for the study of similar mechanisms in tumor cells. Rhodamine 123, a P-glycoprotein transport probe, associated with the reversal agent verapamil gave a significantly higher level of green fluorescence in Haemonchus contortus-resistant eggs as compared with that of susceptible eggs. In the same way, verapamilbodipy, a new fluorescent probe for the detection of multidrug resistance in cells, showed a significantly higher degree of binding to resistant eggs. The results confirm those obtained with biological drug assays using both anthelmintics and verapamil and provide a quantitative and effective methodology for the functional study of multidrug resistance in nematodes.

Formation of toxic metabolites from thiabendazole and other thiazoles in mice. Identification of thioamides as ring cleavage products.

The metabolism of three nephro- or hepatotoxic thiazoles--2-(thiazol-4-yl)benzimidazole (thiabendazole) (1a), 4-tert-butyl-2-methyl-thiazole (1b), and 2-(p-methoxyphenyl)-4-methylthiazole (1c)--was examined in mice with special regard to the formation of ring cleavage products. By GC/MS analyses of derivatized metabolites and comparison with authentic samples, thioformamide and benzimidazol-2-ylglyoxal as the accompanying fragment were identified as urinary metabolites in mice dosed with 1a. Similarly, 1b produced thioacetamide and tert-butylglyoxal, and 1c produced p-methoxy-thiobenzamide (and its S-oxide) and methylglyoxal. These results could be explained by the postulated metabolic pathways where thiazoles would undergo microsomal epoxidation of the C = C double bond and, after being hydrolyzed, the resulting epoxide would then be decomposed to form the corresponding thioamides and alpha-dicarbonyl fragments.

Fasciola hepatica: mebendazole and thiabendazole pharmacokinetics in sheep.

Pharmacokinetics of the two anthelmintic drugs mebendazole and thiabendazole were determined in sheep before and 4, 8, 13, 19, and 25 weeks after an infestation of animals by an oral administration of 150 metacercariae of Fasciola hepatica. The parasitic pathology was ascertained by the increase in plasma enzyme activities of glutamate dehydrogenase and gamma-glutamyltransferase. After oral administration of mebendazole (25 mg.kg-1), the parent drug and especially its reduced metabolite were present in plasma of animals. A significant 1.5- to 2.7-fold increase in the mean residence time occurred by Weeks 13 to 25 postinfection. This change was related to decreases in both the elimination from the pharmacokinetic compartment representing the reduced metabolite and the area under the curve of plasma metabolite concentration versus time. A 59% decrease in MBZ reduction was demonstrated in liver microsomes prepared from 12-week-infected sheep. This reductase activity was characterized by NADPH dependency and a pH peak activity of 6.0 and was competitively inhibited by daunomycin. In sheep receiving a 50 mg.kg-1 oral dose of thiabendazole, fascioliasis provoked only decreased plasma concentrations of the metabolite 5-hydroxythiabendazole by Weeks 4 to 25 postinfection. This change parallels an increase in urinary excretion of free metabolite but this is of minor significance in the general fate of the drug because of the prevalence of excretion as conjugates. In summary, fascioliasis appears to have more of an effect on the pharmacokinetics of mebendazole, a drug intensively metabolized by the liver into a metabolite present at high concentrations in the plasma of animals and humans.

Treatment of Strongyloides stercoralis hyperinfection syndrome with thiabendazole administered per rectum.

There is a rising interest in Strongyloides stercoralis infection due to the expanding population of immunosuppressed patients. Currently the drug of choice for both enteric and tissue forms of infection with this organism is oral thiabendazole. We report a patient with a small bowel obstruction due to S. stercoralis hyperinfection who was unable to take thiabendazole orally. Thiabendazole was administered rectally, and the hyperinfection syndrome resolved. Peak serum concentrations of thiabendazole were achieved 4 hours after rectal administration, and drug levels were sustained longer than previously reported with oral dosing. In addition, elevated levels of thiabendazole metabolites in the patient's urine further confirmed significant absorption. Rectal administration of thiabendazole should be considered for patients unable to take the medication orally.

Prodrugs of thiabendazole with increased water-solubility.

The poor peroral absorption of benzimidazole anthelmintics limits their usefulness for the treatment of systemic infections such as alveolar or cystic echinococcosis. The low bioavailability has mainly been attributed to the low aqueous solubility of the benzimidazoles. Using thiabendazole as a model compound the prodrug approach was investigated as a mean to obtain derivatives with improved water-solubilities. Bioreversible derivatization of thiabendazole was performed by N-acylation of the benzimidazole moiety with various chloroformates as well as by N-acyloxymethylation. Both the N-alkoxycarbonyl and the N-acyloxymethyl derivatives were readily hydrolyzed to thiabendazole in human plasma and in rat and pig liver homogenates. The pH-rate profiles for the hydrolysis of the derivatives were determined and the lipophilicity of the compounds was assessed by partition experiments. The water-solubility of the N-alkoxycarbonyl derivatives was up to 12 times higher than that of the parent drug. An N-(4-amino-methylbenzoyl)oxymethyl derivative possessed a 300-fold higher water-solubility. The improved aqueous solubility, adequate lipophilicity and chemical stability combined with a facile enzymatic hydrolysis make such derivatives promising prodrugs for benzimidazole anthelmintics with the aim of improving the peroral bioavailability.

[The hygienic validation of the MPEL for the fungicide tekto in reservoir water]. / Gigienicheskoe obosnovanie PDK fungitsida tekto v vode vodoemov.

Fungicide tecto (thiabendazol) is a low-toxic compound, produces a weak skin-resorptive and local irritating effect. Cumulation coefficient is 3.96. Threshold and subthreshold doses for tecto were determined as a result of 12-months chronic experiment on white rats--3.7 and 0.37 mg/kg, respectively. The allowable 24-hours dose for human exposure to the chemical is 0.6 mg. The threshold tecto concentration according to the effect on the general sanitary regimen of water reservoirs appeared to be 1 mg/l. According to the limiting toxicological index 0.05 mg/l was recommended as tecto MAC for the water of reservoirs.