Hazardous ecotoxicological impact of two commonly used nitrofuran-derived antibacterial drugs: Furazolidone and nitrofurantoin.

This paper discusses the impact of two nitrofuran-derived drugs, namely furazolidone and nitrofurantoin on growth of oat and common radish as well as their impact on bacteria Allivibrio fischeri and crustaceans Heterocypris incongruens. Results indicated that both compounds were highly phytotoxic for radish (R. sativus) being simultaneously nearly not harmful for oat (A. sativa). Growing inhibition of shoots, roots, fresh matter and photosynthetic pigments is correlated with growing concentration of drugs in soil. Ecotoxicological impact of both compounds on model luminescence bacteria Aliivibrio fischeri and freshwater crustaceans Heterocypris incongruens as a representative organisms of two different level of food chain, is also reported herein, and the obtained data show significant toxicity against these two organisms. Basing on obtained results, it was concluded that both nitrofuran drugs in case of distribution through environment, by improper utilisation after use or unplanned environmental intoxication with unused drugs may cause serious environmental problems and therefore both should be handled with a reasonable care at any step of their production or utilisation.

Involvement of the activation of Nrf2/HO-1, p38 MAPK signaling pathways and endoplasmic reticulum stress in furazolidone induced cytotoxicity and S phase arrest in human hepatocyte L02 cells: modulation of curcumin.

Furazolidone (FZD) is extensively used as the antiprotozoal and antibacterial drug in clinic. The previous study has shown that curcumin pretreatment could improve FZD induced cytotoxicity by inhibiting oxidative stress and mitochondrial apoptotic pathway. The current study aimed to investigate the potential roles of endoplasmic reticulum (ER) stress, p38 mitogen-activated protein kinases (p38 MAPK) signaling pathway in curcumin against FZD cytotoxicity by using human hepatocyte L02 cells. The results showed that curcumin could markedly attenuate FZD induced cytotoxicity. Compared with FZD alone group, curcumin pretreatment significantly reduced the expression of phospho (p)-p38, cyclin D1, p-checkpoint kinase 1 (ChK1) and breast cancer associated gene 1 (BRCA1) protein, followed to attenuate S phase arrest. Meanwhile, curcumin pretreatment prevented FZD induced ER stress, evidenced by the inhibition of glucose-regulated protein 78 and DNA damage inducible gene 153/C/EBP-homologous protein (GADD153/CHOP) protein expression. Moreover, compared with the control, FZD exposure activated the protein and mRNA expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1), which were further activated by curcumin treatment. These results reveal that curcumin could prevent FZD induced cytotoxicity and S phase arrest, which may involve the activation of Nrf2/HO-1 pathway and the inhibition of p38 MAPK pathway and ER stress.

P21(Waf1/Cip1) plays a critical role in furazolidone-induced apoptosis in HepG2 cells through influencing the caspase-3 activation and ROS generation.

Furazolidone (FZD), a synthetic nitrofuran with a broad spectrum of antimicrobial activities, has been shown to exhibit marked genotoxity and cytotoxicity in vitro, but the proper mechanism was unclear. P21(Waf1/Cip1) (p21), a cyclin-dependent kinase, is critically involved in cell cycle arrest and apoptosis in response to DNA injury. This study was aimed to explore the role of p21 in FZD-induced apoptosis in HepG2 cells and uncover its possible mechanism. Firstly, we demonstrated that FZD (50 µg/mL) treatment increased the mRNA level of p21 but reduced the protein level of p21 by shortening its half-life. Moreover, the degradation of p21 was associated with the inhibition of PI3K/Akt pathway by FZD. Then, the change of p21 protein expression modulated FZD-induced apoptosis. Overexpression of p21 attenuated FZD-induced caspase-3 activation and ROS generation, eventually reduced apoptosis. Conversely, knockdown of p21 by siRNA enhanced FZD-induced those phenomenon. In addition, the influence of p21 on FZD-induced ROS generation might be associated with Nrf2/HO-1 pathway which was a key regulator in defense response against oxidative stress. In conclusion, these findings demonstrated that p21 plays a critical role in FZD-induced apoptosis in HepG2 cells through influencing the caspase-3 activation and ROS generation.

Calreticulin-STAT3 signaling pathway modulates mitochondrial function in a rat model of furazolidone-induced dilated cardiomyopathy.

BACKGROUND: Calreticulin is a Ca(2+)-binding chaperone of the endoplasmic reticulum which regulates the signal transducer and activator of transcription 3 (STAT3). The effects of the calreticulin-STAT3 signaling pathway on cardiac mitochondria and on the progress of dilated cardiomyopathy (DCM) are still unclear. METHODS AND RESULTS: The DCM model was generated in rats by the daily oral administration of furazolidone. Echocardiographic and hemodynamic studies demonstrated enlarged LV dimensions and reduced systolic and diastolic functions at thirty weeks after the first furazolidone administration. Morphometric analysis showed significant myocardial degeneration, interstitial fibrosis, and mitochondrial swelling with fractured or dissolved cristae in the model group. Compared with the control group, the mitochondrial membrane potential (MMP) level of the freshly isolated cardiac mitochondria and the enzyme activities of cytochrome c oxidase and succinate dehydrogenase in the model group were significantly decreased (P<0.05). Real-time PCR and western-blot revealed the increased expression of calreticulin associated with decreased activity of STAT3 in the model group. When cultured neonatal rat cardiomyocytes were exposed to furazolidone, a dose-dependent decrease in cell viability and MMP, and the increase of apoptosis rate were observed. The mRNA and protein expression of CRT gradually increased with the increase of furazolidone concentration, associated with a gradual decrease of the STAT3 phosphorylation level both in the whole cell and mitochondrial fraction. When calreticulin was knocked down with siRNA in cardiomyocytes, these changes of cardiomyocytes and mitochondria induced by furazolidone were significantly attenuated. CONCLUSIONS: A rat model of DCM induced by furazolidone is successfully established. The calreticulin-STAT3 signaling pathway is involved in cardiac mitochondrial injury and the progress of furazolidone induced DCM.

Involvement of the p38 MAPK signaling pathway in S-phase cell-cycle arrest induced by Furazolidone in human hepatoma G2 cells.

Given the previously described essential role for the p38 mitogen-activation protein kinase (p38 MAPK) signaling pathway in human hepatoma G2 cells (HepG2), we undertook the present study to investigate the role of the p38 MAPK signaling pathway in cell-cycle arrest induced by Furazolidone (FZD). The aim of this study was to determine the effects of FZD on HepG2 cells by activating and inhibiting the p38 MAPK signaling pathway. The cell cycle and proliferation of HepG2 cells treated with FZD were detected by flow cytometry and MTT assay in the presence or absence of p38 MAPK inhibitors (SB203580), respectively. Cyclin D1, cyclin D3 and CDK6 were detected by quantitative real-time PCR and western blot analysis. Our data showed that p38 MAPK became phosphorylated after stimulation with FZD. Activation of p38 MAPK could arise S-phase cell-cycle arrest and suppress cell proliferation. Simultaneously, inhibition of the p38 MAPK signaling pathway significantly prevented S-phase cell-cycle arrest, increased the percentage of cell viability and decreased the expression of cyclin D1, cyclin D3 and CDK6. These results demonstrated that FZD arose S-phase cell-cycle arrest via activating the p38 MAPK signaling pathway in HepG2 cells. Cyclin D1, cyclin D3 and CDK6 are target genes functioning at the downstream of p38 MAPK in HepG2 cells induced by FZD.

Furazolidone induced oxidative DNA damage via up-regulating ROS that caused cell cycle arrest in human hepatoma G2 cells.

Furazolidone (FZD) is an antimicrobial agent that has been shown to have mutagenic, genotoxic and potentially carcinogenic properties when tested in a variety of systems in vitro and in vivo. In this study, we investigated FZD's DNA damaging effect in human hepatoma cells aiming at further defining the molecular mechanism of FZD's cytotoxicity. Addition of FZD resulted in cell growth suppression and cell cycle arrest in S phase accompanied by remarkable DNA strand breaks with increased levels of intracellular reactive oxygen species (ROS) and 8-hydroxydeoxyguanosine. Activities of antioxidases were down-regulated following FZD treatment and antioxidant agent catalase and superoxide dismutase ameliorated FZD's DNA damaging effects. Moreover, FZD caused much more extensive damage to mitochondrial DNA (mtDNA) than to nuclear DNA for which the decrease in mtDNA content correlated with FZD usage in a dose-dependent manner. However, there was no evidence of FZD induced mtDNA mutation in the mitochondrial DNA displacement loop. These results demonstrate that FZD up-regulates the production of intracellular ROS to cause oxidative DNA damage with mtDNA being the most vulnerable targets. Oxidative stress and the injury of mtDNA could be early indicators of FZD-induced cytotoxicity, with the resulting abnormal progression of the cell cycle.

A differential proteomic approach to assess the effects of chemotherapeutics and production management strategy on giant tiger shrimp Penaeus monodon.

The intensification of shrimp farming has been related to the increasing use of chemotherapeutics and potentially suboptimal rearing conditions. For the purpose of assessing the stress level of cultured giant tiger shrimp Penaeus monodon, a proteomic analysis (2D-DIGE) was performed on hemolymph. On the one hand, shrimp were exposed for 7 days to the antibiotics enrofloxacin or furazolidone via feed (4 g kg(-1)) under laboratory conditions. On the other hand, shrimp were submitted to enrofloxacin directly in field conditions in Vietnam, for which two different culture systems were distinguished (intensive and improved extensive). No significant different protein abundance pattern was induced by antibiotics under laboratory conditions, while only one protein spot displayed a 1.53-fold reduction in intensity after exposure to enrofloxacin in improved extensive ponds. When we compared the proteome of shrimp bred either in intensive or in improved extensive system, we observed 9 protein spots displaying significant difference in abundance. Among them, 3 spots of hemocyanin were under-expressed in shrimp from improved extensive ponds. At the opposite 2 spots corresponding to Sarcoplasmic Calcium-binding Protein (SCP) were less abundant in hemolymph of shrimp from intensive ponds. These results demonstrate that the very subtle effects of tested antibiotics on patterns of hemolymph protein expression are overwhelmed by the effects of conditions encountered in different production management systems, such as different oxygen and nitric concentrations.

Furazolidone is a selective in vitro candidate against Leishmania (L.) chagasi: an ultrastructural study.

The current treatment for leishmaniasis is unsatisfactory due to toxic side effects, high cost, and problems with drug resistance. Various approaches have been used to identify novel drug candidates to treat Leishmania sp. parasites including the use of re-purposed drugs. Furazolidone is a nitrofuran derivative with antiprotozoal and antibacterial activity and is used for the treatment of giardiasis. In the present work, we determined the in vitro antileishmanial activity of furazolidone and its ability to induce ultrastructural alterations of parasites. Promastigotes of Leishmania (L.) chagasi, Leishmania (V.) braziliensis, Leishmania (L.) major, and Leishmania (L.) amazonensis were highly susceptible to furazolidone, with IC(50) values ranging between 0.47 and 0.73 microg/mL. Furazolidone was also very effective against L. chagasi intracellular amastigotes, and despite mammalian cytotoxicity, the selectivity index was 8.0 in human monocytes. The drug also had limited toxicity in mice erythrocytes. Furazolidone demonstrated specific activity against Leishmania, a potential consequence of the lack of macrophage nitric oxide activation. As determined by electron transmission microscopy, drug treatment induced severe damage to the parasite mitochondria and nucleus. This older oral drug is an effective agent for the treatment of L. (L.) chagasi in vitro and is a novel candidate for further experimental studies.

Acetylcholinesterase activity as a biomarker of exposure to antibiotics and pesticides in the black tiger shrimp (Penaeus monodon).

This study aimed to assess the potentiality to use cholinesterase activity (ChE) in black tiger shrimp (Penaeus monodon) as a biomarker of exposure to 2 antibiotics (enrofloxacin, furazolidone) and 2 pesticides (endosulfan, deltamethrin), commonly used in Vietnamese farms. ChE from muscle and gills was first characterised using three different substrates and specific inhibitors. Results showed that both tissues possess only one ChE which displays the typical properties of an acetylcholinesterase (AChE). In a second part, shrimp (average weight of 8.8-10 g) were fed with medicated-feed containing 4g enrofloxacin (quinolone) or furazolidone (nitrofuran)/kg for 7 days, or exposed to 3 actual concentrations of endosulfan (0, 0.009, 0.09, 0.9 microg/L) or deltamethrin (0, 0.0007, 0.007, 0.07 microg/L) for 4 days. After treatment, animals were decontaminated during 7 days. We observed that AChE activity in muscle was not significantly affected in shrimp fed with enrofloxacin or furazolidone, while it significantly decreased (up to 28%) in gills of shrimp fed with furazolidone. Following endosulfan and deltamethrin exposure, no significant changes in AChE activity were observed in gills. However, a significant decrease occurred in muscle after 4 days exposure (inhibition of 30% and 49% at 0.9 microg/L endosulfan and 0.07 microg/L deltamethrin, respectively). While muscle AChE activity should be assessed to point out endosulfan or deltamethrin exposure, gill AChE activity impairment could indicate an exposure to furazolidone. The present study underlines the benefits to use AChE as a biomarker of chemotherapeutics as part of an integrated aquaculture management to reach industry sustainability.

Chemical fate and changes in mutagenic activity of antibiotics nitrofurazone and furazolidone during aqueous chlorination.

Reactions of nitrofuran antibiotics (nitrofurazone (NFZ) and frazolidone (FZD)) with hypochlorite in aqueous solution were investigated under the conditions that simulate wastewater disinfection. The chlorination byproducts were determined by high performance liquid chromatography. At the levels of 5 microM, NFZ reacted rapidly with free chlorine in neutral pH (7.0), while the FZD-hypochlorite reaction was reasonably slow under the same pH. Nevertheless, the strong mutagenic parents disappeared completely after the hypochlorite reactions, and the chlorination byproducts were observed to exert a weak mutagenic effect on Salmonella typhimurium TA100 without S9-mix. The extent of the reactions depended on the chlorine dose, solution pH and compound structures.

[A study on toxicity of aquaculture drugs to luminescent bacteria].

OBJECTIVE: To study the dose-response relation of the fish drugs and their joint toxicity on the luminescent bacteria. METHODS: The single and joint toxicity of four kinds of fish drugs were inspected and evaluated by applying luminescent bacteria toxicity test. RESULTS; Four chemical componds were negatively correlated with luminescence and all correlation coefficient were greater than 0.99. From the dose response curve 50% effective concentration calculated for luminescent bacteria were 0.61 mg/L for Pb2+, 0.79 mg/L for Cu2+, 0.81 mg/L for furazolidone and 489.2 mg/L for chloramphenicol. K value of joint toxicity were 1.65 between Pb2+ and furazolidone, 1.09 between Pb2+ and chLoramphenicol and 1.58 between furazolidone and chloramphenicol. CONCLUSION: Pb2+, Cu2+, furazolidone and chLoramphenicol inhibited bacterial luminescence under certain concentration. Pb2+ and furazolidone, Pb2+ and chloramphenicol, furazolidone and chloramphenicol showed an addition effect to luminescent bacteria.

Potential role of cysteine and methionine in the protection against hormonal imbalance and mutagenicity induced by furazolidone in female rats.

The use of nitrofurans as veterinary drugs has been banned in the EU since 1993 due to doubts on the safety of the protein-bound residues of these drugs in edible products. Furazolidone (FUZ) is a nitrofuran drug, which has been used for many years as an antibacterial drug in veterinary practice. The aim of the current study is to investigate the role of L-cysteine and L-methionine in the protection against hormonal imbalance and the genotoxicity induced by FUZ using the micronucleus (MN) assay and random amplified polymorphism DNA (RAPD-PCR) analysis in female rats. Forty female Sprague-Dawley rats were divided into four groups included the untreated control group; a group treated with FUZ (300 mg/kg b.w.); a group treated with a mixture of L-cysteine (300 mg/kg b.w.) and L-methionine (42.8 mg/kg b.w.) and a group treated with FUZ plus the mixture of L-cysteine and L-methionine for 10 days. The results indicated that FUZ induced hormonal disturbances involving thyroid, ovarian and adrenal hormones. Moreover, FUZ increased the micronucleus formation and induced changes in polymorphic band patterns. The combined treatment with FUZ and the mixture of L-cysteine and L-methionine succeeded to prevent or diminish the endocrine disturbance and the clastogenic effects of FUZ. The current study is casting new light on the complex mechanisms underlying the ameliorating action of dietary L-cysteine and L-methionine against FUZ toxicity in experimental animals.

The effect of probiotics on the genotoxicity of furazolidone.

Antigenotoxic activity of probiotic bacteria against furazolidone was studied using the short-term bacterial assay SOS chromotest, with Escherichia coli PQ37 as the test organism. The supernatants from probiotic and furazolidone co-incubation exhibited rather strong suppression on SOS induction produced by furazolidone on E. coli PQ 37 (sfiA: lacZ). Genotoxicity inhibition was found for all strains of the examined bacteria belonging to three genera. The highest genotoxicity inhibition was detected for Bifidobacterium lactis Bb-12 (92.0%) and for Lactobacillus acidophilus T20 (81.9%).

Evaluation of the carcinogenic effects of furazolidone and its metabolites in two fish species.

The major metabolite from the use of furazolidone (FZD) in mammals, birds and fish is 2,3-dihydro-3-cyanomethyl-2-hydroxy-5-nitro-1alpha, 2-di(2-oxo-oxazolidin-3-yl)iminomethyl-furo[2,3-beta]furan, also called 3-amine-2-oxazolidone (AOZ). A minor metabolite was identified as N-(5-amine-2-furfuryliden)-3-amine-2-oxazolidone (FOZ). To assess the potential carcinogenicity of FZD and the metabolic mixture of AOZ/FOZ, 11 mg FZD/kg feed/day was fed for 12 weeks to mollies (Poecilia formosa), an ornamental fish species prone to develop tumors. The rate of tumors was quantified and defined both in mollies and their offspring. Then, some fish was made into fishmeal and incorporated into fish food at 500 g of meal/kg of food and fed to other mollies for 12 weeks. The rate of tumors was assessed. A similar trial design was carried out in tilapia fish (Oreochromis niloticus) by adding 50 mg FZD/kg to the feed for 90 days. All animals were placed in glass fishponds under controlled laboratory conditions. Each week, a significant biomass was collected from both groups to assess the macroscopic and histopathological changes. All mollies developed melanohistiocytomic tumors in the liver and other organs. Offspring from surviving mollie females stimulated to breed showed no changes compared to control animals. None of the mollies fed with the mollie-meal food contaminated with AOZ/FOZ developed tumors. Neither tilapia medicated with FZD nor tilapia fed with tilapia-meal contaminated with AOZ/FOZ developed tumors. These results do not support the established viewpoint that FZD must be banned from trophic chains based on its potential carcinogenic properties.

Development of a bioassay to test the possible role of thiamine disturbance as a mechanism behind pollution-induced reproductive failures in birds.

A test system was developed to examine the effects of environmental contaminants on thiamine homeostasis in bird embryos. This system employs fresh chicken egg yolk lipids as a vehicle for use in egg injection studies. Furazolidone, an antibiotic suspected to interfere with thiamine metabolism, was used as a positive control to evaluate the utility of the test system. It was determined that fresh chicken egg yolk lipids were preferable over chemical vehicles as it resulted in lower mortality rates (16% versus 23-62%) and did not induce any observable effects in the embryo. Injection of 1 mg/egg of furazolidone at day 0 of development resulted in decreased respiration followed by death, with mortality rates being twice as high as in carrier controls. In addition, transketolase activity, which was measured as an indicator of thiamine availability in the body, was decreased 25% in brains of 19-day-old embryos. This mechanism may be of importance for effects of environmental contaminants in wild bird populations.

Metabolism of furazolidone: alternative pathways and modes of toxicity in different cell lines.

1. The metabolism and cytotoxicity of the antimicrobial nitrofuran drug furazolidone have been studied in Caco-2, HEp-2 and V79 cell lines. Free radical production, metabolite pattern, formation of bound residues, inhibition of cellular replication and protection by the antioxidant glutathione were compared for the three cell lines. 2. All three cell lines produced the same nitro-anion radical with similar kinetics. Little further metabolic breakdown was observed in V79 cells, whereas Caco-2 and HEp-2 cells showed extensive degradation of furazolidone, but with different end patterns. 3. Under hypoxic conditions, the colony-forming ability was extensively impaired in HEp-2 cells whereas the other two cell lines were less affected, suggesting that irreversible damage to DNA occurred prevalently in HEp-2 cells. In V79 cells the absence of oxygen caused a 25-fold increase in the formation of protein-bound residues. 4. Brief exposure to furazolidone caused a 50% loss of endogenous glutathione in Caco-2 cells, but no loss could be detected in V79 and HEp-2 cells. Consistently, when glutathione was depleted by buthionine-[S,R]-sulphoximine (BSO) and diethylmaleate (DEM) treatment, the viability of V79 and HEp-2 cells was minimally affected by furazolidone, whereas that of Caco-2 cells was substantially reduced. 5. It is concluded that the cytotoxicity of furazolidone in these cell lines can be exerted by a number of different mechanisms, possibly related to different metabolic pathways. The cytotoxicity of nitrofuran drugs, therefore, cannot be ascribed to a single toxic intermediate, but in Caco-2 cells furazolidone is extensively metabolized and detoxified by GSH, in V79 is only partially activated and then bound to proteins, whereas in HEp-2, once activated, may react with DNA.

Pharmacological, therapeutic and toxicological properties of furazolidone: some recent research.

Some of the recent publications on the pharmacological, therapeutic and toxicological properties of the antimicrobial agent furazolidone (FZ) are briefly reviewed. In animals, most of the recently published papers focus on (1) the methodology of measuring the residues of the drug and its metabolites in edible tissues; (2) the carcinogenicity and genotoxicity of FZ; (3) the cellular and molecular basis of FZ-induced cardiomyopthy, and the action of different cardioprotectant drugs thereon; and (4) hormonal effects. In humans, the use of FZ as an anti-ulcer drug and in controlling infectious diseases, especially opportunistic infections in AIDS patients, is described.

A contribution to safety assessment of veterinary drug residues: in vitro/ex vivo studies on the intestinal toxicity and transport of covalently bound residues.

1. The gastrointestinal fate of protein-bound residues of the model compound furazolidone (FZD) was investigated in vitro and ex vivo. Protein-bound residues were generated in rat liver microsomes, isolated by solvent extraction and digested with 0.5% hydrochloric acid and Pronase E. 2. During digestion, 3-amino-2-oxazolidinone (AOZ), the side chain of furazolidone, was partly released from bound residues. 3. The absorption of free AOZ and digested protein-bound residues was tested in isolated perfused rat gut segments (IPGS) and in the intestinal cell line Caco-2. Free AOZ was transfered both in the IPGS model and in Caco-2 monolayer cultures, while no indications for passage of bound residues were obtained. 4. No acute toxicity of AOZ or digested food residues respectively was observed in gut segments and Caco-2 cells at concentrations that were substantially above maximum residue levels to be expected in food of animal origin after administration of therapeutic doses. 5. The results demonstrate that digestive processes can alter the chemical nature of drug residues and yield degradation products that may be bioavailable for the consumer. Thus, the covalent binding of xenobiotics to macromolecular tissue constituents cannot necessarily be regarded as an irreversible endpoint of residue bioavailability and toxicity.

Furazolidone treatment suppresses pubertal testosterone secretion in male broiler breeder birds (Gallus domesticus).

We determined the effect of chronic administration of furazolidone (Fz) on sexual maturation of male broiler breeder birds (Ross 308; Gallus domesticus). A total of 20 15-w-old birds were randomly assigned to receive 0, 150, 250 or 350 mg Fz/kg feed daily for 5-w. Blood samples were taken at weekly intervals. The birds were challenged with 500 IU human chorionic gonadotropin (hCG) i.v. at the age of 24 w before slaughtering. Concentration of testosterone in the plasma was measured by a specific radioimmunoassay. Testicular tissue was processed for morphometric studies. Testicular weights of the groups fed 250 or 350 mg Fz/kg feed/d were decreased (P < 0.05). Plasma testosterone levels were affected by age (P < 0.001) and dose of Fz (P < 0.001). Mean plasma testosterone levels during and after drug administration were reduced (P < 0.05) by all Fz doses. Human chorionic gonadotropin administration led to poor testosterone response (P < 0.05) in all Fz-dosed groups, but not the control group (P < 0.05). As compared to the control, Fz-dosing reduced the seminiferous tubule diameter (P < 0.05) at the 350 mg/kg feed dose, seminiferous epithelial height (P < 0.05) at the 250 and 350 mg/kg feed doses, and Leydig cell nuclear diameter (P < 0.05) at the 350 mg/kg feed dose. This data suggest that sexual maturation in male broiler breeder birds is adversely affected by chronic Fz-administration. Actions of Fz on sexual maturation probably involve a direct effect at the testicular level.

Characterization of furazolidone apical-related effects to human polarized intestinal cells.

In studying the effects of furazolidone (FZ) on the human intestinal Caco-2 cell line grown on microporous membrane, we have previously demonstrated a higher toxicity when the compound was administered at the apical (AP) side than at the basolateral (BL) side. Moreover, we have also shown the production, in the intact cells, of a nitroanion radical from FZ by a cytochrome c P450 reductase. The aim of the present study was to investigate which specific cell structures and functions are involved in the observed domain-related toxicity. The relevance of alterations in integrity and selective properties of the intestinal barrier as first-pass site for ingested molecules is also discussed. We have confirmed that, as expected, the Caco-2 cells are protected from FZ injury by a specific inhibitor of the cytochrome c P450 reductase, and we have shown that this protection is more active on the apical side of the cells. In sublethal conditions, FZ causes increased permeability to 3H-mannitol and, to a different extent, to 3H-inulin. Again the effect is higher when the cells are apically exposed. We have thus examined the tight junctions morphology: a disruption of the apical perijunctional actin-bound cytoskeleton was detected by rhodamine-phalloidin staining and microtubule disorganization by antitubulin fluoresceinated antibodies. Again, the effect was more evident when the cells were apically treated with FZ. Preferential transport and accumulation of the compound by active transport mechanisms could be excluded, since transport of FZ was linear and no intracellular accumulation was detected either from the AP and or the BL sides. All together these results may suggest that the AP formation of the active metabolite and its possible reactivity with SH groups of perijunctional microfilaments could be responsible of the higher FZ apical toxicity. This study shows that polarized differentiated cells are very interesting in vitro models to investigate specific cellular domains as targets of toxic effects and to detect subtle changes that may be induced, in absence of cell death, in specialized epithelial layers.