Fenbendazole induces apoptosis of porcine uterine luminal epithelial and trophoblast cells during early pregnancy.

Fenbendazole, is an effective benzimidazole anthelmintic that prevents parasite infection in both human and veterinary health care. Although the well-known and effect of benzimidazole was recently shown to have a broad spectrum of biological abilities, such as anticancer and anti-inflammation activities, the mechanism of benzimidazole's antiproliferative effect via cell signaling pathways and its role in preimplantation has not been studied. Therefore, the purpose of this study was to determine the effects of fenbendazole on porcine trophectoderm and luminal epithelial cells. First, we investigated cell viability in response to a low dose of fenbendazole, which highly inhibited cell proliferation. In addition, we investigated apoptotic molecules in the mitochondria, imbalanced intracellular calcium homeostasis, and the expression of some genes involved in apoptosis to explain the decrease in proliferation. Finally, we examined the intracellular mechanisms of fenbendazole by measuring the extracellular signal-regulated kinase, PI3K/AKT, and c-Jun N-terminal kinase signaling proteins by western blot analysis. Our findings suggest that fenbendazole functions as an effective anti-proliferative molecule that induces critical apoptosis in the porcine trophectoderm and uterine luminal epithelial cells by disrupting the mitochondria membrane potential during early pregnancy.

Swimming activity in zebrafish larvae exposed to veterinary antiparasitic pharmaceuticals.

Veterinary antiparasitic pharmaceuticals have been detected in surface waters and several of these pharmaceuticals act on the nervous system on the target organisms implying that neurological effects also might be of concern in non-target animals such as fish. We tested if exposure to antiparasitic pharmaceuticals affect swimming activity in 6 days old zebrafish larvae. The results revealed that most pharmaceuticals did not cause any effects in swimming activity. However, larvae exposed to 0.58 mg/L doramectin displayed reduced swimming activity even though they were classified as normal, having no morphological abnormalities.

Biotransformation of flubendazole and fenbendazole and their effects in the ribwort plantain (Plantago lanceolata).

Although veterinary anthelmintics represent an important source of environmental pollution, the fate of anthelmintics and their effects in plants has not yet been studied sufficiently. The aim of our work was to identify metabolic pathways of the two benzimidazole anthelmintics fenbendazole (FBZ) and flubendazole (FLU) in the ribwort plantain (Plantago lanceolata L.). Plants cultivated as in vitro regenerants were used for this purpose. The effects of anthelmintics and their biotransformation products on plant oxidative stress parameters were also studied. The obtained results showed that the enzymatic system of the ribwort plantain was able to uptake FLU and FBZ, translocate them in leaves and transform them into several metabolites, particularly glycosides. Overall, 12 FLU and 22 FBZ metabolites were identified in the root, leaf base and leaf top of the plant. Concerning the effects of FLU and FBZ, both anthelmintics in the ribwort plantain cells caused significant increase of proline concentration (up to twice), a well-known stress marker, and significant decrease of superoxide dismutase activity (by 50%). In addition, the activities of four other antioxidant enzymes were significantly changed after either FLU or FBZ exposition. This could indicate a certain risk of oxidative damage in plants influenced by anthelmintics, particularly when they are under other stress conditions.

Mixture toxicity of flubendazole and fenbendazole to Daphnia magna.

Nowadays, residual amounts of many pharmaceuticals can be found in various environmental compartments including surface and ground waters, soils and sediments as well as biota. Even though they undergo degradability, their environmental discharge is relatively continuous, thus they may be regarded as quasi-persistent contaminants, and are also frequently regarded as emerging organic pollutants. Benzimidazoles, especially flubendazole (FLU) and fenbendazole (FEN), represent two anthelmintic drugs belonging to this group. Although their presence in environmental matrices has been reported, there is relatively little data concerning their (eco)toxicological impact. Furthermore, no data is available on their mixture toxicity. FLU and FEN have been found to have a strong impact on an environmentally important non-target organism - Daphnia magna. Moreover, these compounds are usually present in the environment as a part of pharmaceutical mixtures. Therefore, there is a need to evaluate their mixture toxicity, which was the main aim of this study. Single substance toxicity tests were carried out in parallel with mixture studies of FLU and FEN, with the application of two well established concepts of Concentration Addition (CA) and Independent Action (IA). As a result, both models (CA and IA) were found to underestimate the toxicity of mixtures, however CA yielded more accurate predictions.

SUSPECTED FENBENDAZOLE TOXICITY IN AN AMERICAN WHITE PELICAN (PELECANUS ERYTHRORHYNCHOS).

A wild-raised, 5.0-kg male American white pelican ( Pelecanus erythrorhynchos ) of unknown age presented for routine examination at both the start and completion of a 30-day quarantine period at a zoological park. Upon physical examination, the pelican was bright, alert, and responsive and in good body condition. Two complete blood counts and a plasma biochemistry did not reveal any clinically significant abnormalities. Whole-body radiographs were unremarkable. Two fecal flotations (28 days apart) confirmed the presence of ascarid-type eggs. Fenbendazole anthelmintic was prescribed (50 mg/kg p.o. s.i.d. for 5 days). The pelican became lethargic and inappetent on day 3 of fenbendazole treatment and was found dead on day 7. Postmortem examination and histopathology revealed intestinal crypt cell necrosis, stomatitis, and splenic lymphoid depletion consistent with fenbendazole toxicity. To the authors' knowledge, this is the first report to describe fenbendazole toxicity in an American white pelican.

Acute Toxicity and Environmental Risks of Five Veterinary Pharmaceuticals for Aquatic Macroinvertebrates.

Due to the high use of antibiotics and antiparasitics for the treatment of livestock, there is concern about the potential impacts of the release of these compounds into freshwater ecosystems. In this context, the present study quantified the acute toxicity of two antibiotics (sulfadiazine and sulfadimidine), and three antiparasitic agents (flubendazole, fenbendazole, ivermectin) for nine freshwater invertebrate species. These experiments revealed a low degree of toxicity for the sulfonamide antibiotics, with limited implications in the survival of all test species at the highest test concentrations (50 and 100 mg/L). In contrast, all three antiparasitic agents indicated on the basis of their acute toxicity risks for the aquatic environment. Moreover, chronic toxicity data from the literature for antiparasitics, including effects on reproduction in daphnids, support the concern about the integrity of aquatic ecosystems posed by releases of these compounds. Thus, these pharmaceuticals warrant further careful consideration by environmental risk managers.

Toxicity of anthelmintic drugs (fenbendazole and flubendazole) to aquatic organisms.

Flubendazole (FLU) and fenbendazole (FEN) belong to benzimidazoles-pharmaceuticals widely used in veterinary and human medicine for the treatment of intestinal parasites as well as for the treatment of systemic worm infections. In recent years, usage of these drugs increased, which resulted in a larger contamination of the environment and possible negative effects on biota. Hence, in our research, we investigated an aquatic ecotoxicity of these pharmaceuticals towards: marine bacteria (Vibrio fischeri), green algae (Scenedesmus vacuolatus), duckweed (Lemna minor) and crustacean (Daphnia magna). Ecotoxicity tests were combined with chemical analysis in order to investigate the actual exposure concentration of the compounds used in the experiment as well as to stability and adsorption studies. As a result, study evaluating sensitivity of different aquatic organisms to these compounds and new ecotoxicological data is presented. The strongest negative impact of FLU and FEN was observed to D. magna.

Gene expression of ribosomal protein mRNA in Chironomus riparius: effects of endocrine disruptor chemicals and antibiotics.

Ribosomal protein genes are essential for cellular development. To examine the effects of ribosomal protein genes under various cellular stress conditions in chironomids, ribosomal protein S3 (RpS3) and S6 (RpS6) cDNA from Chironomus riparius were characterized and their expression was analyzed during development. A comparative and phylogenetic study among different orders of insects was carried out by analysis of sequence databases. C. riparius RpS3 was highly conserved at the protein level and shared over 85% amino acid identity with homologous sequences from other insects. RpS6 also showed approximately 80% amino acid identity. The RpS3 and S6 transcripts were present during different developmental stages but were most abundant during the embryonic stage. Furthermore, expression of the previously reported ribosomal proteins RpL11, L13, and L15, as well as RpS3 and S6 was analyzed following exposure to various concentrations of three endocrine disruptor chemicals (EDCs), di(2-ethylhexyl) phthalate, bisphenol A, and 4-nonylphenol (4NP), and the veterinary antibiotics (VAs) fenbendazole, sulfathiazole, and lincomycin. Only RpS3 gene expression was up-regulated significantly in response to EDCs and fenbendazole. However, the C. riparius ribosomal proteins showed a limited response to cellular stress, following exposure to EDCs and VAs.

Exacerbation of acetaminophen hepatotoxicity by the anthelmentic drug fenbendazole.

Fenbendazole is a broad-spectrum anthelmintic drug widely used to prevent or treat nematode infections in laboratory rodent colonies. Potential interactions between fenbendazole and hepatotoxicants such as acetaminophen are unknown, and this was investigated in this study. Mice were fed a control diet or a diet containing fenbendazole (8-12 mg/kg/day) for 7 days prior to treatment with acetaminophen (300 mg/kg) or phosphate buffered saline. In mice fed a control diet, acetaminophen administration resulted in centrilobular hepatic necrosis and increases in serum transaminases, which were evident within 12 h. Acetaminophen-induced hepatotoxicity was markedly increased in mice fed the fenbendazole-containing diet, as measured histologically and by significant increases in serum transaminase levels. Moreover, in mice fed the fenbendazole-containing diet, but not the control diet, 63% mortality was observed within 24 h of acetaminophen administration. Fenbendazole by itself had no effect on liver histology or serum transaminases. To determine if exaggerated hepatotoxicity was due to alterations in acetaminophen metabolism, we analyzed sera for the presence of free acetaminophen and acetaminophen-glucuronide. We found that there were no differences in acetaminophen turnover. We also measured cytochrome P450 (cyp) 2e1, cyp3a, and cyp1a2 activity. Whereas fenbendazole had no effect on the activity of cyp2e1 or cyp3a, cyp1a2 was suppressed. A prolonged suppression of hepatic glutathione (GSH) was also observed in acetaminophen-treated mice fed the fenbendazole-containing diet when compared with the control diet. These data demonstrate that fenbendazole exacerbates the hepatotoxicity of acetaminophen, an effect that is related to persistent GSH depletion. These findings are novel and suggest a potential drug-drug interaction that should be considered in experimental protocols evaluating mechanisms of hepatotoxicity in rodent colonies treated with fenbendazole.

Ecotoxicological multilevel-evaluation of the effects of fenbendazole exposure to Chironomus riparius larvae.

Veterinary antibiotics may find their way into the aquatic environment through direct or indirect pathways due to their widespread use. Fenbendazole is a benzimidazole anthelmintic that is widely used in veterinary medicine. To evaluate the potential ecological risk of fenbendazole, we examined the molecular and biochemical responses of biomarker genes such as heat shock proteins (HSPs), cytochrome P450 (CYP450), glutathione S-transferases (GSTs) and hemoglobins (Hbs) in Chironomus riparius for long periods. The expression of HSP70, HSP40, HSP90 and CYP450 in C. riparius increased significantly after exposure to all concentrations of fenbendazole evaluated, while the levels of GST and HbA only increased in C. riparius exposed to relatively high concentrations of fenbendazole (30 microg L(-1)). HbB expression did not differ significantly between the control and treatment groups. Exposure to 30 microg L(-1) fenbendazole had significant effects on the survival, growth, sex balance of emergent adults and development of mouthpart deformity in C. riparius. These results should constitute an important contribution to the understanding of the toxicology of fenbendazole in C. riparius. Moreover, the responses of the biomarker genes also provide valuable information that will aid in understanding the effects of fenbendazole in aquatic ecosystems.

The behavioral teratogenic potential of fenbendazole: a medication for pinworm infestation.

Fenbendazole (FBZ) is a benzimidazole currently used for anthelmintic treatment of pinworm populations in numerous animal species although it is not currently approved for laboratory rodents in the U.S. It has received considerable interest for treating rodent populations due to its low toxicity, wide safety margin and apparent absence of gross teratogenic effects. The purpose of this study was to assess the behavioral teratogenic potential of FBZ. Pregnant rats were administered either FBZ-medicated feed at a therapeutic level or normal rat chow throughout pregnancy and gestation. FBZ had no effect on pregnancy indicators such as maternal weight gain or water consumption, number of pups born or pup birth weights. Offspring were examined in a variety of paradigms including righting reflex, negative geotaxis, running wheel activity, Morris water maze (MWM) performance and digging maze performance. FBZ offspring did show delayed righting reflex, some modest changes in locomotor activity in a running wheel and minor alterations in performance during the probe session of the MWM relative to controls. However, the effects of FBZ on behavior were subtle and many of the behaviors examined were unaffected. These results suggest that FBZ may be an effective and relatively safe anthelmintic treatment for use in breeding colonies.

Toxicity evaluation of prophylactic treatments for mites and pinworms in mice.

The detection of external and internal parasites in laboratory mice is a particularly problematic aspect of animal health evaluation. Because these organisms must be detected by direct examination of the feces or hair coat, low-level infestation or sporadic shedding can make them difficult to detect, thereby undermining confidence that negative reports are truly negative. Prophylactic treatment of suspect colonies with anthelminthics and/or insecticides may therefore be indicated under some circumstances. However, when considering the use of prophylactic treatments, the potential for toxicity is an important factor, especially in genetically modified strains of mice. To evaluate the potential toxicity of prophylactic anti- parasitic treatments on strains of mice that are commonly used as experimental models and in genetic engineering in our facility, we surveyed a number of strains and ages of mice for toxic reactions during treatment regimens that combine anthelminthic and anti-acaricidal agents. Three experimental protocols (ivermectin, piperazine, and dichlorvos in combination; ivermectin alone; and fenbendazole/permethrin or fenbendazole/dichlorvos) were evaluated. Our data suggest a potential for toxicity associated with these treatments and indicate to us that prophylactic treatment regimens should be initiated with caution.

Liver tumor promoting effects of fenbendazole in rats.

In order to examine whether fenbendazole has tumor-promoting activity, a total of 70 male Fischer 344 rats were initiated with a single intraperitoneal injection of 100 mg/kg of diethylnitrosamine (DEN) or were given the saline vehicle alone; beginning 1 wk later, rats were given a diet containing 3,600; 1,800; 600; 200; 70; or 0 ppm of fenbendazole for 8 wk. Subgroups of 5 rats each from the DEN+ 1,800; DEN+0; 1,800; and 0 ppm groups were euthanatized after 1 wk of fenbendazole treatment, and the remaining animals were euthanatized at 8 wk. After 1 wk, relative liver weights (ratios to body weights) were significantly increased in the DEN+ 1,800 and 1,800 ppm groups, and based on light microscopy, periportal hepatocellular hypertrophy was evident in these groups. After 8 wk, relative liver weights were significantly increased in the groups given > or =600 ppm with or without DEN initiation. Periportal hepatocellular hypertrophy, characterized by a marked increase in smooth endoplasmic reticulum, was observed in the groups given > or =600 ppm with or without DEN initiation. Induction of cytochrome P-450 (CYP) 1A2, 2B1, or 4A1 was noted in the fenbendazole-treated groups with or without DEN initiation; that associated with CYP 1A2 was most marked. Positive immunostaining for anti-CYP 1A1/2 or CYP 2B1/2 was observed diffusely in the livers of animals in the DEN+1,800 and DEN+3,600 ppm groups. The numbers and areas of connexin 32 (Cx32)-positive spots per square centimeter in centrilobular hepatocytes were significantly decreased in an almost dose-dependent manner with fenbendazole treatment after DEN initiation. In situ hybridization for Cx32 mRNA revealed a remarkable decrease in its expression in the centrilobular hepatocytes in the DEN+70 ppm group. The numbers of glutathione S-transferase placental-form positive single cells (plus mini foci) were significantly increased in the DEN+ 1,800 and DEN+3,600 ppm groups. Since those agents that induce CYP 2B1/2 isozymes and reduce Cx32 in centrilobular hepatocytes have been suggested to be liver tumor promoters, the present results indicate that fenbendazole may be a liver tumor promoter.

Anthelmintic resistance of trichostrongylids in sheep in the highveld of Zimbabwe.

A survey was conducted on the occurrence of anthelmintic resistance of trichostrongylids on commercial sheep farms in the highveld of Zimbabwe. On ten farms the efficacy of fenbendazole, levamisole and rafoxanide was tested by a faecal egg count reduction test. Benzimidazole resistance was additionally examined using an egg hatch assay with thiabendazole. Results of the faecal egg count reduction tests and larval differentiations showed fenbendazole resistance of Haemonchus sp. on all investigated farms. Resistance of Haemonchus sp. against rafoxanide was demonstrated on all farms with reliable egg counts. Levamisole resistance of Haemonchus sp. was found on most farms but 2 farms showed an efficacy of 100%.

[Intensity of liver tumor promotion effects in rats given repeated oral administrations of benzimidazole compounds].

Liver tumor-promoting effects of anthelminthic agents, febantel (Feb), fenbendazole (Fen) or oxfendazole (Oxf), were investigated in a rodent 2-stage carcinogenesis model. Five-week-old male F344 rats were initiated with or without diethylnitrosamine (DEN) and one week later given diet containing Fen (3600, 1800, 600, 200 or 70 ppm), Feb (2000, 1000, 500 or 100 ppm) or Oxf (500, 250, 100 or 10 ppm) for 8 weeks. Induction of CYP1A1/2 was observed in treated groups of DEN + Feb and DEN + Oxf groups, and its induction was most marked in DEN + Oxf groups. CYP2B1 and CYP4AI were also induced in these treated groups. The number or area of Cx32 positive spots per hepatocyte was significantly decreased in treated groups except for DEN + Oxf 100 ppm group, as compared to DEN alone group. GST-P positive foci was significantly increased in DEN + Fen groups treated with 1800 ppm or more, DEN + Feb groups treated with 1000 ppm Feb or more and DEN + Oxf groups treated with 250 ppm Oxf or more. These results suggest that these three compounds have liver tumor promotion effects and the promoting action in Oxf is most strong among them.

Multiple anthelmintic resistance in Haemonchus contortus on a sheep farm in India.

Multiple resistance to benzimidazoles (fenbendazole, albendazole and mebendazole) in a strain of Haemonchus contortus in sheep was detected on a farm where fenbendazole resistance had already been identified. Following a faecal egg count reduction test, this was confirmed by both critical and controlled anthelmintic tests. Different groups of sheep infected naturally or given an experimental infection with the fenbendazole-resistant strain were treated with the recommended doses of various anthelmintics. Compared to the control group, percentage reductions in faecal egg counts of sheep treated with fenbendazole, albendazole, mebendazole, levamisole and morantel varied between 56% and 81% and worm counts between 71% and 86%. The results indicate the presence of multiple anthelmintic resistance in this strain of H. contortus on this farm. Sheep treated with ivermectin and closantel showed 100% reductions in faecal egg and worm counts, suggesting high efficacy of these drugs against the population of H. contortus on this farm.

Potential carcinogenicity assessment of alpha-aescin and phenbendasol.

Potential carcinogenic activity of alpha-aescin and phenbendasole made by "Polfa" (Poland) as well as phenbendasole produced by "Hoechst" (Germany) was studied using Salmonella/microsome test, DNA repair test and micronucleus assay. None of tested preparations were mutagenic or genotoxic what suggest that none of them possess potential carcinogenic activity. Besides, it was established that alpha-aescin exhibits strong and phenbendasol weak acute systemic toxicity for mice. Alpha-aescin and phenbendasole produced in Poland have been found to be toxic for bone marrow cells of mice but only when administered at a high dose of 80% LD50.

Subchronic toxicity studies of fenbendazole in rats.

The effects of prolonged exposure of rats to fenbendazole were investigated. Fenbendazole was given daily by gavage for 14 consecutive days. These dosages (3000, 500 or 50 mg/kg/d) produced reductions in body weight gains. Renal tubular hyperemia or hemorrhage and glomerular capsule dilation, increased serum creatinine and hepatocellular granular degeneration occurred at dosages of 500 and 3000 mg/kg/d. Renal tubular epithelial cell granular degeneration and tubular dilation, increased serum glutamic pyruvic transaminase, cardiac hemorrhage and granular degeneration also occurred at 3000 mg/kg/d.

[Embryotoxicity of fenbendazole in Paracentrotus lividus]. / Embriotossicità del fenbendazolo in Paracentrotus lividus.

The aim of the present work was to evaluate the embryotoxicity of Fenbendazole, a benzimidazole carbamate-derived anthelmintic drug widely employed in Veterinary Medicine, by using the embryonal development of Paracentrotus lividus (sea urchin) as a experimental model. Embryos were obtained by in vitro eggs fertilization and cultured in seawater. Five embryo suspensions were added by Fenbendazole reaching a final concentration of 5 micrograms/l, 7.5 micrograms/l, 10 micrograms/l, 12.5 micrograms/l and 25 micrograms/l; a suspension was kept drug-free as a control. Embryo development was evaluated by microscopical examination of suspensions at 3 and 40 hours. Our results show that a concentration of 5 micrograms/l of the drug determines a considerable delay of the embryonal development in the 95 percent of the elements observed, and a concentration of 25 micrograms/l produces a block of the embryogenesis at the phase of morula and blastula in all embryos. Results confirm that the effects observed are probably due to an extended inhibition of several enzyme complexes of the embryo cells.

Comparative studies on the effect of fenbendazole on the liver and liver microsomal enzymes in goats, quail and rats.

To compare the effect of fenbendazole on the liver and liver microsomal mono-oxygenases of goats, quail and rats, an oral dose of 25 mg/kg was administered to the animals daily for 9 consecutive days. On the tenth day, blood samples and livers were collected from both the control and the treated animals for preparation of serum and microsomes respectively. Determination of the activities of sorbitol dehydrogenase (SDH, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum samples showed that there was no significant increase in the activities of these enzymes in the treated animals as compared to their corresponding controls, suggesting no liver damage. Similarly, no significant difference in the amount of microsomal cytochrome P-450 was found between the control and the treated animals of the same species. Compared to their respective controls, the activities of microsomal benzphetamine N-demethylase and aniline hydroxylase were almost unchanged in the treated goats and rats. However, fenbendazole treatment appeared to enhance the activity of these two microsomal enzymes in quail. The results indicate that fenbendazole is not liver toxic to goats, quail or rats at a dose rate of 25 mg/kg.