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.

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 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.

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.

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.

[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.

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.

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%).

Experimental models of heart failure.

Experimental models of heart failure can be used to address specific questions not easily answered in patients, but no single model can reproduce exactly any of the clinical syndromes of heart failure since these are dominated by fatigue and breathlessness. Heart failure may be induced experimentally by pressure loading, volume loading, myocardial infarction, or by the creation of other disease states within the myocardium. Pressure loading may be especially useful in the study of ventricular hypertrophy, cellular derangements and vascular changes. Volume loading may be useful when examining the pathogenesis of hormone and electrolyte disturbances. Models of myocardial infarction or destruction are likely to be the most suitable for assessing novel therapy provided that peripheral reflexes are maintained. Experimental cardiomyopathy can provide an important means of identifying pathological subcellular mechanisms. They may be of use in the evaluation of vasodilator drugs but caution should be exercised in the study of inotropic agents. Any one model may be useful if it permits study of a single factor or variable in isolation or at a time when information is not obtainable from patients. For greatest clinical relevance, studies should be made in conscious animals with intact reflexes.

Radiomimetic property of furazolidone and the caffeine enhancement of its lethal action on the vibrios.

Sensitivities of the strains belonging to four vibrio biotypes to the action of furazolidone were investigated. Vibrio cholerae (classical) was most and Vibrio parahaemolyticus least sensitive to this drug. Statistical analyses revealed significant differences between any two of the four types of vibrio in respect of their sensitivity to furazolidone. The drug was radiomimetic in action, the doses of UV light (DUV) and furazolidone (Df) required for 10% survival of the vibrios being correlated by the equation, Df = 0.28 exp. (0.008 DUV). Caffeine exhibited lethal synergism with furazolidone and the synergistic effect depended on the mode of caffeine treatment, the effect being maximum when caffeine was present along with and also after furazolidone treatment. UV spectrophotometric study revealed that caffeine did not bind with native DNA but did so with denatured DNA resulting in a bathochromic shift and a quenching of the caffeine absorption maximum at 209.4 nm. The binding isotherm (Scatchard plot) indicated the presence of a heterogeneity in the binding sites and that the parameters for the strongest mode of bonding were n = 0.254 and k = 7.5 X 10(5) M-1.

DNA damage, prophage induction and mutation by furazolidone.

Ultraviolet absorption data and thermal chromatography through hydroxyapatite (HAP) column revealed that furazolidone treatment of Vibrio cholerae cells produced more than 80% of DNA reversibly bihelical due to the formation of interstrand cross-links and the reaction obeyed a first order relation. Sensitivities of the Escherichia coli strains to the lethal action of the drug were in the order: AB 2480(uvr- rec-) greater than AB 2463(rec-) greater than AB 1886(uvr-) greater than AB 1157(repair proficient) or AB 4401(wild type). Furazolidone was 'Rec test' positive, produced dose-dependent prophage induction in E. coli cells and also dose-dependent streptomycin-resistance forward mutation in V. cholerae cells. The quantitative aspect and also the mode of furazolidone action on DNA were discussed.

Induction of lambda prophage by furazolidone.

A dose-dependent prophage induction by furazolidone exhibited a gradual rise to a maximum, corresponding to an exposure dose of 1.2 microgram/ml X h and a gradual fall thereafter. A 2-3-fold higher level of induction was achieved when the lysogens were treated with furazolidone in the presence of a metabolizing mixture. A maximum of about 70% efficiency of induction was achieved. Kinetics of prophage induction by any concentration of furazolidone exhibited a common pattern, viz., an initial rise for 15-20 min, then a plateau extending up to about 60 min and a faster rise thereafter. Higher concentrations of the drug (10 micrograms/ml) exhibited a toxic effect. Chloramphenicol at a concentration of 20 micrograms/ml inhibited the furazolidone-induced prophage induction, the plaque-forming units gradually decreasing from several minutes after the chloramphenicol treatment. The burst size of the lysogens was not significantly affected by treatment with 2 micrograms/ml of furazolidone up to a period of about 10 min, but thereafter, decreased faster with the duration of furazolidone treatment. The "latent period' of induction decreased linearly with the duration of furazolidone treatment.

Inter-strand cross-linking of Vibrio cholerae DNA induced by furazolidone: a quantitative assay by four simple methods.

Four simple methods, i.e., (i) UV absorption spectrophotometry, (ii) hydroxyapatite chromatography, (iii) fluorescence analysis of ethidium bromide bound to DNA and (iv) assay of S1 endonuclease action, were used in parallel for the estimation of furazolidone-induced inter-strand cross-links in Vibrio cholerae DNA. The data produced by the four methods were in reasonable agreement with each other and provided similar linear dose-response relations, the correlation (between dose and response) coefficient being in any case numerically greater than 0.98. When the data obtained by four independent methods were plotted in a single graph, the resulting dose-response relation could be described by the equation log NR = 1.41 - 0.54 log D, where NR is the % non-reversible DNA remaining in the cells treated by furazolidone at dose D micrograms/ml x h. The correlation coefficient in this plot was -0.98 and significant to a level better than 0.1%. This study thus brings out that any one of these four methods can be used with reasonable confidence for the diagnosis and assay of inter-strand cross-links in DNA.

Studies on the effect of ascorbic acid and selenium on the genotoxicity of nitrofurans: nitrofurazone and furazolidone.

The genotoxic properties of nitrofurazone and furazolidone were studied using the Ames test and SOS-chromotest. Both compounds were found to act as strong mutagens on the TA97 and TA102 strains of S. typhimurium and to induce the SOS-repair system in the PQ37 strain of E. coli. A good concordance was found between the mutagenic activity and the ability to induce the SOS system. Ascorbic acid and sodium selenite only very slightly lowered the genotoxic effect of the 2 nitrofurans studied both in the Ames test and in the SOS-chromotest.

The role of mutagenicity testing in the ecotoxicological evaluation of industrial discharges into the aquatic environment.

An important task of ecotoxicology is to provide data for use by governments as a scientific basis for regulating the discharge of potentially hazardous substances into the environment. The criteria on which such regulations are based include biological and chemical degradability, bioavailability (accumulation) and toxicity, the latter manifesting itself in a reduced ability of organisms to survive and reproduce. There are also arguments for including the mutagenicity of chemicals in these criteria. In the past decade many studies have revealed the contamination of water and the atmosphere by mutagens originating in human activities and having observable effects on organisms in these environments. The extent to which this mutagenic stress can alter ecosystems as a whole is not yet known, but if mutagens are shown to cause major changes, mutagenicity is clearly an important ecotoxicological criterion. In the meantime its use must be based on the actual damage caused to organisms by mutagens and on the rising concern over a novel ecological phenomenon that might be named 'increased mutagenic stress'. A detailed report on the ecotoxicological case history of the discharge from a nitrofuran factory demonstrates the role of mutagenicity tests in the evaluation of effluents. As well as being toxic to algae, the discharge was found to be mutagenic in the Ames test. Toxicity and mutagenicity were used as criteria in studies of the biodegradability of the offending substances in a chemostat, as well as in studies to monitor the success of detoxification attempts. In the chemostat the mutagens did not appear to be very degradable but the algal toxicity decreased by 90%. Detoxification was reasonably successful for the mutagens in contrast to the algal toxicants.

Established cell lines for safety assessment of food contaminants: differing furazolidone toxicity to V 79, HEp-2 and Caco-2 cells.

In vitro models, preferentially derived from human tissues, may be valuable tools to study the biotransformation and toxicity of compounds that may be present as residues in food products. Such residues may represent a risk to human health, and therefore call for increased testing. Three established cell lines were used to study the toxic effect of furazolidone (FZ), a widely used veterinary drug: HEp-2 cells, derived from a human larynx carcinoma, previously used in toxicity screening of several compounds; Caco-2 cells, derived from a human colon adenocarcinoma, able to differentiate partially in culture, and V 79, a fibroblast cell line derived from Chinese hamster lung, widely used to assess direct toxicants. Various toxicity parameters were used, primarily dealing with cell death and cell proliferation. In all cell lines FZ at a concentration of 5 micrograms/ml caused a marked decrease in cell viability and especially in cell proliferation. Inhibition of DNA synthesis has also been observed, even if at higher concentrations. However, only in V 79 cells was the decrease in cell number accompanied by a marked increase in lactate dehydrogenase leakage due to membrane damage. Moreover, the surviving V 79 cells, after removal of FZ, fully recovered from the effect of the drug, as shown by their full capacity to attach to dishes and to form colonies. Surviving cells of the other two cell lines showed much poorer colony-forming ability. Exposure of Caco-2 cells and, to a lesser extent, HEp-2 cells, caused a marked increase in oxygen consumption, that possibly was due to redox cycling of the initially formed radical nitro anion. Biotransformation of the drug by all three cell lines was accompanied by the formation of protein-bound metabolites, HEp-2 being the most active cells. The toxic effects recorded show that cell lines provide a sensitive system in toxicity assessment. Moreover, it may be suggested that a battery of cell lines, including some of human origin, as well as a battery of endpoints, may be of help in addressing further specific mechanistic investigations.

Differential response in cardiomyopathy of chicks and turkeys to furazolidone toxicity.

Dietary furazolidone (500 ppm or higher) significantly slowed the growth of chicks fed either a conventional or a purified diet and of turkeys fed a conventional diet. Deaths were significantly increased in turkeys fed 500 ppm and in chicks fed 700 ppm in the purified diet. Ratio of heart to body weight was significantly increased by furazolidone in turkeys, but not in chicks. Furazolidone did not induce anemia in either species. Turkeys and chicks differed markedly in cardiac response to toxic levels of furazolidone.

The production of cardiomyopathy in turkey poults by the oral administration of furazolidone.

Furazolidone (FZ) was given to turkey poults two weeks post-hatching by stomach tube in doses of 60 and 80 mg/kg per day, and via the feed at a concentration of 700 ppm. Oral doses were given either as one dose at 8 a.m. or as two equal doses given at 8 a.m. and 4 p.m. Electrocardiographic (ECG) recordings were used to monitor the development of round-heart disease (RHD) and necropsies were performed on all poults to corroborate the ECG findings. All poults receiving FZ gained significantly less weight (p less than or equal to .05) than control poults. At oral doses of 80 mg/kg per day, the apparent acute toxicity of FZ resulted in high mortality during the first week of the experiment. At necropsy there were no signs of RHD in these poults. Most of the poults given FZ orally in doses of 60 mg/kg per day or at 700 ppm in the feed survived to the termination of the experiments. The majority of these poults showed ECG patterns that are typical of RHD. Myocardial lesions indicative of RHD were present at necropsy.

Ultrastructural alterations in furazolidone-induced cystic testicular degeneration in ducklings.

Cystic testicular degeneration was induced in groups of ducklings by feeding furazolidone at 250, 400, 550, or 750 mg per kilogram of feed (ppm) for 4 weeks. In normal ducklings, tall, columnar Sertoli cells predominated in the seminiferous epithelium. In treated ducklings, the mildest ultrastructural alteration was cytoplasmic vacuolation of Sertoli cells. In birds with more severely affected tubules, cytoplasmic vacuolation was more severe and the cells were cuboidal or rounded. The rounded cells desquamated into the tubular lumens, eventually undergoing cytolysis. In birds with the most severely affected tubules, only extensively flattened epithelial cells lined the extremely dilated seminiferous tubules. The interstitium was edematous and compacted between the expanded tubules. Many of the testicular ultrastructural alterations in furazolidone-intoxicated ducklings were similar to those described in the testicles of sodium-intoxicated cockerels.