Nitrovin (difurazone), an antibacterial growth promoter, induces ROS-mediated paraptosis-like cell death by targeting thioredoxin reductase 1 (TrxR1).

Glioblastoma multiforme (GBM) is one of the most lethal malignant tumors in the human brain, with only a few chemotherapeutic drugs available after surgery. Nitrovin (difurazone) is widely used as an antibacterial growth promoter in livestock. Here, we reported that nitrovin might be a potential anticancer lead. Nitrovin showed significant cytotoxicity to a panel of cancer cell lines. Nitrovin induced cytoplasmic vacuolation, reactive oxygen species (ROS) generation, MAPK activation, and Alix inhibition but had no effect on caspase-3 cleavage and activity, suggesting paraptosis activation. Nitrovin-induced cell death of GBM cells was significantly reversed by cycloheximide (CHX), N-acetyl-l-cysteine (NAC), glutathione (GSH), and thioredoxin reductase 1 (TrxR1) overexpression. Vitamins C and E, inhibitors of pan-caspase, MAPKs, and endoplasmic reticulum (ER) stress failed to do so. Nitrovin-triggered cytoplasmic vacuolation was reversed by CHX, NAC, GSH, and TrxR1 overexpression but not by Alix overexpression. Furthermore, nitrovin interacted with TrxR1 and significantly inhibited its activity. In addition, nitrovin showed a significant anticancer effect in a zebrafish xenograft model, which was reversed by NAC. In conclusion, our results showed that nitrovin induced non-apoptotic and paraptosis-like cell death mediated by ROS through targeting TrxR1. Nitrovin might be a promising anticancer lead for further development.

Verification of Bound Aminoguanidine as the Marker Residue for the Banned Antibiotic, Nitrovin, in Pig Tissues.

Nitrovin (NTV) belongs to a class of antibiotics called nitrofurans, which are classified as nonallowed pharmacologically active substances that do not have a maximum residue limit listed in EU legislation. The objectives of this study were to confirm aminoguanidine (AGN) as a suitable marker residue to monitor NTV abuse and to investigate its persistence in porcine tissues. In this work, pigs were fed with NTV-medicated feed (50 mg/kg), and tissues (kidney, muscle, and liver) and plasma were collected on different withdrawal days. All samples were analyzed for bound AGN, total AGN, and the parent drug NTV itself. The highest concentrations of AGN residues were found in the liver, while the lowest were in muscle. Parent NTV was only detected in the kidney at low levels on day 0 of withdrawal. The findings are in support of using AGN as the marker residue for monitoring the illegal use of NTV in animal-derived products.

Residue depletion of nitrovin in chicken after oral administration.

In this study, the residue depletion of nitrovin in chicken was studied after feeding the birds with dietary feeds containing 10 mg/kg of nitrovin for 7 consecutive days. Tissues (muscle, fat, kidney, and liver) and plasma were collected at different withdrawal periods and determined by a high-performance liquid chromatography-ultraviolet (HPLC-UV) method. The limit of detection for nitrovin in tissue and plasma samples was 0.1 ng/(g or mL), and the inter- and intrarecoveries from the blank fortified samples were in the range of 71.1-85.7%. At the withdrawal period of 0 days, the residue concentration of nitrovin in plasma was the highest (average of 84.98 ng/mL) compared to those in muscle, fat, liver, and kidney (average of 21.04, 61.18, 24.04, and 68.28 ng/g, respectively). At the withdrawal period of 28 days, the residue levels of nitrovin in muscle, fat, liver, and plasma were all higher than 1.0 ng/(g or mL) and the highest concentration was in liver (average of 5.8 ng/g). These data are in support of the ban of nitrovin as a feed additive in food-producing animals.

Determination of sodium nifurstyrenate and nitrovin residues in edible food by liquid chromatography-tandem mass spectrometry after ultrasound-assisted extraction.

A specific and sensitive method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed for the determination of nitrovin and sodium nifurstyrenate residues in muscle and liver of swine and chicken and in muscle of fish. Sample preparation procedure includes ultrasound-assisted extraction with acetonitrile, defatting with n-hexane and final clean-up with solid phase extraction (SPE) on Oasis HLB cartridges. The analytes were detected in multiple reaction monitoring (MRM) under negative scan mode acquiring two diagnostic product ions for sodium nifurstyrenate and under positive mode for nitrovin. The averaged decision limits (CCα; α 1%) ranged 0.09-0.26 µg/kg while the detection capability (CCß; ß 5%) was 0.33-0.97 µg/kg in the tissues. Reasonable recoveries (71-110%) spiked in muscle and liver showed excellent relative standard deviation (RSD). The validated method was simple, rapid, sensitive, and complied with the regulations for the determination of nitrovin and sodium nifurstyrenate residues in food matrices.

Identification of a pharmaceutical compound that partially corrects the Niemann-Pick C phenotype in cultured cells.

Niemann-Pick C (NPC) is an autosomal recessive lysosomal lipid storage disease characterized by progressive central nervous system degeneration. In cultured human NPC fibroblasts, LDL-derived cholesterol accumulates in lysosomes and endosomes, LDL-cholesterol transport from endocytic compartments to other cellular compartments is delayed, and LDL does not elicit normal homeostatic responses. Currently, there is no therapy that delays the onset of neurological symptoms or prolongs the life span of NPC children. We have developed and implemented an amphotericin B-mediated cytotoxicity assay to screen for potential therapeutic drugs that induce cholesterol movement in cultured NPC cells. NPC cells are relatively resistant to amphotericin B killing due to intracellular sequestration of cellular cholesterol. The screen was carried out using simian virus 40-transformed ovarian granulosa cells from the npc (nih) mouse model of NPC disease. A library of 44240 compounds was screened and 55 compounds were identified that promote amphotericin B-mediated killing of NPC cells. One compound, NP-27, corrected the NPC phenotype by four different measures of cholesterol homeostasis. In addition to making NPC cells more sensitive to amphotericin B, NP-27 stimulated two separate cholesterol transport pathways and restored LDL stimulation of cholesterol esterification to near normal levels.

High-performance liquid chromatographic determination of carbadox, olaquindox, furazolidone, nitrofurazone, and nitrovin in feed.

A high-performance liquid chromatography with gradient programming method was developed to determine the amount of carbadox (CBX), olaquindox (OLQ), furazolidone (FZ), nitrofurazone (NF), and nitrovin (NTV) in feed simultaneously. Complete separation of the drugs was obtained using a C8 silica gel column with gradients of acetonitrile as mobile phase. The mobile phase used an acetonitrile gradient with an initial hold time of 1 min at 0% acetonitrile, followed by an increase to 50% acetonitrile over 10 min. The correlation coefficients (r) for calibration curves of the five feed additives were greater than 0.999. The relative standard deviations (RSDs) of peak areas from four injections for these drugs at three concentrations were less than 3.0%, although the RSD for NTV at 5 ppm was somewhat large (6.7%). The medicated feeds were extracted by pretreating with water, extracted with 95% dimethylformamide overnight at room temperature, and cleaned up on a column of alumina oxide. Recoveries of CBX, OLQ, FZ, NF, and NTV from low level spiked feed were 102.0, 94.6, 97.4, 110.6, and 66.0%, respectively, and from high level spiked feed, they were 114.09, 99.1, 97.3, 109.9, and 62.7%, respectively.

Tiamulin-nitrovin interaction in pigs: a case report and experimental reproduction.

Tiamulin is a diterpenic veterinary drug widely used in swine for the control of infectious diseases, including swine dysentery and enzootic pneumonia. Tiamulin is well tolerated and only a few drug interactions have been reported with some ionophore antibiotics. A case of tiamulin adverse drug interaction with nitrovin, a nitrofuranic growth promoter, in fattening pigs from a commercial farm is described. To confirm the diagnosis, experimental reproduction was conducted using 20 healthy female pigs. The animals were randomly divided into 4 groups: 1 of them receiving feed medicated with 100 mg tiamulin hydrogen fumarate/kg, another received 20 mg nitrovin/kg to establish the harmlessness of the drugs alone. The other 2 groups received feeds containing both drugs at the previous concentrations, one of which came from the farm. In the last 2 groups clinical signs appeared 72-96 h after initially feeding pigs the tiamulin hydrogen fumarate + nitrovin-containing feed. The animals had uneasiness, anxiety, skin erythema, and rash on snout, vulva and abdomen, and increased body temperatures. No mortality occurred, and there were no characteristic findings during pathological examination. The signs disappeared after 96 h of consuming the medicated feed, leaving a slight skin thickening of the affected regions. All signs disappeared spontaneously 4-5 d after ceasing medication.

Antimutagenicity of milk fermented by Enterococcus faecium.

The diethyl ether extracts isolated from unfermented milk and milk fermented by Enterococcus faecium exhibited dose-dependent inhibition of mutagenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), nitrovin (NIT), 5-nitro-2-furylacrylic acid (NFA) and UV-irradiation on the Ames bacterial test (Salmonella typhimurium strains TA97 and TA100) and the unicellular flagellate Euglena gracilis. Overall, the fermented milk extract was the most active against UV-irradiation, less active against NIT and MNNG, and the least active against NFA on bacteria. The highest antibleaching effects were observed against MNNG. The differences between antimutagenic effects from fermented and unfermented milk extracts were determined to be statistically significant at the 0.95 CI level.

Antimutagenic properties of fresh-water blue-green algae.

The antimutagenic properties of whole fresh-water blue-green algae Aphanisomenon flos-aquae, marketed under the commercial name "Alpha Sun" were tested using the Ames test. Simultaneous addition of both algae and Nitrovin (a mutagen) to the test medium did not reduce the mutagenic activity. On the other hand, addition of freeze-dried blue-green algae to the test medium 2-24 h before the application of mutagen reduced its mutagenic activity.

Antimicrobials and competitive exclusion.

The compatibility of commonly used antimicrobial feed additives with CE treatment are examined. It appears that additives permitted in the U.K. are without effect on CE, with the possible exception of avoparcin. However, a sharp difference has been found with the effect of antimicrobials on CE treatment cultures in vitro, and the in vivo effect; and it is postulated that these differences may be accounted for by the fact that the substances may not be 'caecum available' in the bird. When effects are observed in the bird they appear to be largely due to dose variation. In the case of adult birds already infected with Salmonella, the time lapse between completion of antimicrobial treatment and the administration of Broilact will be examined; as well as the selection of appropriate antimicrobial treatment in infected birds prior to the administration of replacement microflora.

Determination of nitrovin in medicated animal feeds by high-performance liquid chromatography. Analytical Methods Committee.

Nitrovin is extracted from a medicated feed by means of a solvent mixture consisting of dichloromethane, methanol and ammonia solution and then the nitrovin is determined in the extract by high-performance liquid chromatography using a cyano-column.

[Use of nitrovin in feed and its effect on the development of Marek's disease in chickens]. / Pouzitie nitrovinu vo výkrme a jeho vplyv na rozvoj Markovej choroby u kurciat.

The effect of the Czechoslovak product Nitrovin, administered at a rate of 15 mg per kg of feed mixture, was studied as exerted on the growth activity, meat quality, and the development and intensity of occurrence of patho-morphological changes ascribable to Marek's disease. An increase of weight gains and dressing percentage was recorded in the group of birds given Nitrovin, as well as in those which had Nitrovin in their feed and were infected with a highly virulent virus of Marek's disease. No significant changes occurred in the contents of dry matter and protein, but there was a much lower fat content in breast muscle in the groups of chickens infected with the Marek disease virus. Internal organs were subjected to biometrical measurements. The birds treated with Nitrovin and infected with the highly virulent virus of Marek's disease had significantly lighter hearts and heavier spleens. Liver, nervus ischiadicus, gonads and spleen were affected most intensively by the changes testifying to Marek's disease. The results are just preliminary and the investigation continues.

[The effect of growth stimulators on the levels of various trace elements in chickens]. / Vplyv stimulátorov rastu na obsah niektorých mikroprvkov v organizme kurciat.

Chickens, fattened for 56 days, were treated with nitrovin at the dose of 15 mg per kg of feed, cyadox at the dose of 20 mg per kg of feed, and virginiamycin at the dose of 10 mg per kg of feed. After the fattening, the chickens were studied for the contents of copper, zinc, and manganese in the blood serum, thigh muscle, breast muscle, and feathers. The contents of copper, zinc and manganese in the serum ranged within the limits of physiological values. As for the breast muscle, the content of copper increased but the content of manganese declined in both breast and thigh muscle, as compared with the control group. The feathers contained more manganese in all the groups, as compared with the controls.

[The effect of growth stimulators on the levels of various macroelements in chickens]. / Vplyv stimulátorov rastu na obsah niektorých makroprvkov v organizme kurciat.

Nitrovin administered at the dose of 15 mg per 1 kg of diet, cyadox (20 mg) and virgiamycin (10 mg) were studied as to their effect on the contents of calcium, sodium, potassium and magnesium in the blood serum, breast and thigh muscles, and feathers of chickens. The contents of calcium, sodium, potassium and magnesium in the blood serum ranged within the physiological limits. An increased content of calcium and potassium and a decreased content of sodium were recorded in the breast and thigh muscles, as compared with the control group. The contents of these macroelements in the feathers of the experimental group were significantly different from those in the birds of the control group.

The effect of growth-promoting antibiotics on the faecal enterococci of healthy young chickens.

Small groups of chickens were given feed containing either avoparcin, nitrovin, virginiamycin or zinc bacitracin from the day of their purchase as day-olds. Differences between the birds receiving growth promoters and the untreated controls were observed during the last third of the 23 d survey period. The enterococcal population of the 'dosed' birds contained a greater proportion of Enterococcus faecium than did that of the control birds while the converse was true for Ent. gallinarum. This apparent selection of Ent. faecium by the growth-promoting antibiotics had an influence on the incidence of resistance to therapeutic antibiotics among the enterococcal population as a whole. This was because this species was generally more resistant than Ent. gallinarum to cephalothin, the MLS antibiotics (erythromycin, lincomycin and tylosin) and tetracycline.

Influence of ascorbic acid on the mutagenicity of N-methyl-N-nitrosoguanidine and nitrofurans studied by SOS chromotest.

Mutagenicity inhibition of MNNG*, NFAA* as well as of nitrovin by ascorbic acid was observed. The influence of ascorbic acid on these compounds was parallelly studied also by spectroscopy. Mutagenicity inhibition was characterized by SOS chromotest. Inhibition degree was evaluated quantitatively introducing the coefficient of inhibition. The influence of ascorbic acid was expressed most markedly in MNNG. It has been found that the decrease of mutagenicity should be mainly caused by acceleration of MNNG decomposition by ascorbic acid.

[Effects of growth-stimulating agents and furazolidone in broiler chicks]. / Effecten van groeibevorderaars en furazolidon bij slachtkuikens.

The effect of growth-stimulating agents (15 ppm of virginiamycin, 10 ppm of avoparcin and 12 ppm of nitrovin) and that of seven days' treatment with 300 ppm of furazolidone on the performance of broilers was studied in an experimental study of 9,600 animals. Furazolidone was fed in either the fourth or the fifth week of life and combined with virginiamycin or nitrovin. Non-significant improvements in growth were observed when virginiamycin (1.5 per cent), avoparcin (0.7 per cent) and nitrovin (0.7 per cent) were administered. Treatment with furazolidone for seven days resulted in substantial retardation of growth. When broilers were treated during the fourth week of life, the retardation of growth was largely compensated for during the other two weeks. Treatment in the fifth week of life resulted in lower weights prior to slaughter. Therefore, care should be taken in recommending treatment with furazolidone at the end of the fattening period of broilers.

Feed additives to chick starter mixtures under tropical conditions.

In two short-term experiments (28 and 31 days, respectively), the ergotropic effect was tested of the growth promoters nitrovin (25 mg/kg mixture, and a chinoxalin-1,4-N-dioxide derivate (25 and 50 mg/kg mixture). The experiments were carried out on a total of 1,952 unsorted one-day-old White Leghorn chicks under tropical conditions in Eastern Ethiopia (College of Agriculture at Alemaya). On addition of 50 mg chinoxalin-1,4-N-dioxide, the final weight of the chicks was 105.6 and 104.6%, respectively, in the first and second experiments as compared with the unsupplemented groups. The amount of feed required for each weight gain was reduced to 94.0 and 91.4%, respectively. Nitrovin and smaller amounts of chinoxalin-1,4-N-dioxide increased the body weight to 104.2 and 104.0%, respectively, as compared with the control. The feed requirement (94.6 and 99.9%, respectively) was not reduced significantly (alpha greater than 0.05). Further experiments will be necessary, using other feed additives, more chicks, and longer experimental periods.