Wenli Zheng

Identification of oxidative stress and responsive genes of HepG2 cells exposed to quinocetone, and compared with its metabolites

Quinocetone, a new quinoxaline 1,4-dioxide derivative used in food-producing animals in China, exerts genotoxic effects on HepG2 cells. It triggers significant cytotoxicity and genotoxicity in vitro, but the detailed mechanism by which quinocetone induces adverse biological effects is not yet known. We analyzed the mechanisms behind quinocetone intoxication by investigating oxidative stress based on non-enzymatic and enzymatic antioxidant activities, and by identifying differentially regulated genes of HepG2 cells exposed to quinocetone using polymerase chain reaction (PCR)-based suppression subtractive hybridization to illustrate the toxicity mechanism of quinocetone. Meanwhile, the characteristics of oxidative stress and differentially regulated genes induced by quinocetone metabolites, 1,4-bisdesoxyquinocetone and 3-methylquinoxaline-2-carboxylic acid, were investigated too. Results showed that quinocetone damaged the antioxidant defense abilities of HepG2 cells by reducing the activities of endogenous antioxidant enzymes, lowering glutathione concentration, and elevating malondialdehyde level. We identified 160 quinocetone-responsive genes that were associated with cell proliferation, glucose metabolism, oxidative stress, and apoptosis, such as NAD(P)H dehydrogenase, quinone 1; and prolyl 4-hydroxylase, beta polypeptide. The expressions of some differentially regulated genes were confirmed by real-time reverse transcription-polymerase chain reaction. However, quinocetone metabolites showed little effects on HepG2 cells. These results showed that reactive oxygen species were the key mediators of quinocetone cytotoxicity in HepG2 cells and that c-MYC-dependent activation of the mitochondrial apoptotic pathway may be associated with quinocetone-induced toxicity.

Liquid chromatography–tandem mass spectrometry analysis of nitazoxanide and its major metabolites in goat

A rapid, sensitive and specific liquid chromatography-electrospray ionization (ESI) tandem mass spectrometry (LC-MS-MS) method has been developed for the identification of nitazoxanide metabolites in goat plasma and urine. The purified samples was separated using an XTerra MS C8 column with the mobile phase consisted of acetonitrile and 10-mM ammonium acetate buffer (pH 2.5) followed a linear gradient elution, and detected by MS-MS. Identification and structural elucidation of the metabolites were performed by comparing their retention-times, full scan, product ion scan, precursor ion scan and neutral loss scan MS-MS spectra with those of the parent drug or other available standard. Four metabolites (tizoxanide, tizoxanide glucuronide, tizoxanide sulfate and hydroxylated tizoxanide sulfate) were found and identified in goat after single oral administration of 200mg/kg dose of nitazoxanide. In addition, the possible metabolic pathway was proposed for the first time. The results proved that the established method was simple, reliable and sensitive, revealing that it could be used to rapid screen and identify the structures of active metabolites responsible for pharmacological effects of nitazoxanide and to better understand its in vivo metabolism.

Anticoccidial effects of a novel triazine nitromezuril in broiler chickens.

The anticoccidial efficacy of 2-(3-methyl-4-(4-nitrophenoxy)phenyl)-1,2,4-triazine-3,5(2H,4H)-dione (nitromezuril, NZL), a novel triazine compound, was evaluated in three different studies under experimental conditions. The anticoccidial efficacy was chiefly evaluated using the anticoccidial index (ACI). The resistance level was determined by calculating ACI, percentage optimum anticoccidial activity (POAA), reduction in lesion scores (RLS) and relative oocyst production (ROP). In the dose determination study (study A), NZL was added to the diet at doses of 1, 2, 3, 4, 5 and 6 mg/kg to test its efficacy against coccidiosis caused by Eimeria tenella. Groups treated with NZL 1mg/kg feed could observe the faecal dropping scores and caecal lesions. ACIs of NZL-treated groups reached 179-199. In the study on the anticoccidial efficacy of 3mg/kg NZL in the diet (study B), only a few faecal oocysts and slight lesions were observed. NZL significantly promoted weight gain (WG) and reduced lesion scores (LS) compared to controls receiving diclazuril (DZL) (P<0.05). ACIs of NZL-treated groups were 193, 192, 191 and 163 for E. tenella, Eimeria necatrix, Eimeria acervulina and Eimeria maxima, respectively, whereas those of DZL-treated groups were 185, 176, 176 and 148. In the cross-drug resistance study (study C), ACIs of NZL and toltrazuril (TZL)-treated groups ranged from 188 to 204, which were significantly higher than those of DZL-treated groups (P<0.05). NZL- and TZL-treated groups were sensitive to experimentally induced DZL-resistant E. tenella, whereas DZL-treated groups showed complete resistance. No cross-resistance was observed between DZL and NZL or TZL. Based on the abovementioned studies, it was concluded that diets containing 3 mg/kg NZL had an excellent efficacy in preventing coccidiosis in broiler chickens. The activity of 3mg/kg NZL in the diet was equal or superior to that of 1 mg/kg DZL. These results are of great significance for the future applications of NZL; however, its actual mechanism of action remains unknown. NZL is a potential novel anticoccidial agent suitable for further development.

Nuclear translocation and accumulation of glyceraldehyde-3-phosphate dehydrogenase involved in diclazuril-induced apoptosis in Eimeria tenella (E. tenella)

In mammalian cells, GAPDH (glyceraldehyde-3-phosphate dehydrogenase) has recently been shown to be implicated in numerous apoptotic paradigms, especially in neuronal apoptosis, and has been demonstrated to play a vital role in some neurodegenerative disorders. However, this phenomenon has not been reported in protists. In the present study, we report for the first time that such a mechanism is involved in diclazuril-induced apoptosis in Eimeria tenella (E. tenella). We found that upon treatment of parasites with diclazuril, the expression levels of GAPDH transcript and protein were significantly increased in second-generation merozoites. Then, we examined the subcellular localization of GAPDH by fluorescence microscopy and Western blot analysis. The results show that a considerable amount of GAPDH protein appeared in the nucleus within diclazuril-treated second-generation merozoites; in contrast, the control group had very low levels of GAPDH in the nucleus. The glycolytic activity of GAPDH was kinetically analyzed in different subcellular fractions. A substantial decrease (48.5%) in glycolytic activity of GAPDH in the nucleus was displayed. Moreover, the activities of caspases-3, -9, and −8 were measured in cell extracts using specific caspase substrates. The data show significant increases in caspase-3 and caspase-9 activities in the diclazuril-treated group.

Determination of Tetracyclines and Their Epimers in Agricultural Soil Fertilized with Swine Manure by Ultra-High-Performance Liquid Chromatography Tandem Mass Spectrometry

Abstract A rapid, sensitive and specific ultra-high-performance liquid chromatography tandem mass spectrometry (UPLC-MS) method was developed for the analysis of tetracycline antibiotics, including tetracycline (TC), oxytetracycline (OTC), chlortetracycline (CTC) and their 4-epimers (4-epiTCs) in agricultural soil fertilized with swine manure. Soil samples were extracted and cleaned-up with 10 mL EDTA-McIlvaine buffer solution (pH 4.0), then cleaned-up and pre-concentrated using the Oasis MAX cartridge and then eluted with 1 mL solution by mixing formic acid, methanol and water at a ratio of 2:15:83 (v/v/v). The purified samples were separated by an ACQUITY UPLC BEH C18 column using acetonitrile and water containing 0.1% formic acid mobile phase and detected by a single quadrupole MS. The limits of detection for the soil extraction method (LODsoil) ranged from 0.6-2.5 μg kg−1 with recoveries from 23.3-159.2%. Finally, the method was applied to an agricultural field in an area with intensive pig-fattening farming. Tetracyclines were detected in soil from 2.8 to 42.4 μg kg−1 soil. These results demonstrate that soil from swine farms can become severely contaminated with tetracycline antibiotics and their metabolites.

Sulfated glucan can improve the immune efficacy of Newcastle disease vaccine in chicken

To evaluate the immune effect of sulfated glucan from saccharomyces cerevisiae (SGSC) on chickens, two experiments were researched. In vitro experiment, the effects of SGSC on chicken splenic lymphocyte proliferation were determined. The results displayed that SGSC could significantly stimulate chicken splenic lymphocyte proliferation. In vivo experiment, 200 14-day-old chickens were averagely divided into 5 groups. The chickens, except blank control (BC) group, were vaccinated with Newcastle disease (ND) vaccine, repeated vaccination at 28 days old. At the same time of the first vaccination, the chickens in three SGSC groups were injected, respectively, with the SGSC at low, medium and high concentrations, in vaccination control (VC) and BC group, with equal volume of physiological saline, once a day for three successive days. On days 7, 14, 21, 28, 35 and 42 after the first vaccination, the lymphocyte proliferation, serum antibody titer and interleukin-2 (IL-2) and interferon-gamma (IFN-γ) were measured. The results showed that SGSC at suitable dose could significantly promote lymphocyte proliferation, enhance serum antibody titer, and improve serum IL-2 and IFN-γ concentrations. It indicated that SGSC could significantly improve the immune efficacy of Newcastle disease vaccine, and would be as the candidate of a new-type immune adjuvant.

Safety evaluation of a triazine compound nitromezuril by assessing bacterial reverse mutation, sperm abnormalities, micronucleus and chromosomal aberration

Nitromezuril (NZL) is a novel triazine compound that exhibits remarkable anticoccidial activity. However, mutagenicity and genotoxicity of NZL have not been evaluated to date. This study evaluated the potential risks of NZL by testing for bacterial reverse mutation (Ames), mouse sperm abnormality (SA), bone marrow micronucleus (MN) and chromosomal aberration (CA). Mice were orally administered with NZL at 385, 192 and 96 mg/kg, corresponding to 0.5 ×, 0.25 × and 0.125 × the LD50 of NZL, respectively. No significant increases in SA and CA were found in mice treated with NZL for 5d and 3d, respectively (P>0.05). NZL at 96-385 mg/kg did not have significant influence on micronucleated polychromatic erythrocyte counts (P>0.05). These results suggest that NZL is not genotoxic. However, Ames test results were positive both with and without the S9 system for Salmonella typhimurium TA98 and TA100, suggesting that NZL may be mutagenic. The mutagenic effects of NZL were different in in vitro and in vivo assays. Further studies should be conducted to confirm the safety of using and developing NZL as a novel anticoccidial drug.

Effects of Low Molecular Weight Yeast β-Glucan on Antioxidant and Immunological Activities in Mice

To evaluate the antioxidant and immune effects of low molecular yeast β-glucan on mice, three sulfated glucans from Saccharomyces cerevisiae (sGSCs) with different molecular weight (MW) and degrees of sulfation (DS) were prepared. The structures of the sGSCs were analyzed through high performance liquid chromatography-gel permeation chromatography (HPLC-GPC) and Fourier transform infrared spectroscopy (FTIR). sGSC1, sGSC2, and sGSC3 had MW of 12.9, 16.5 and 19.2 kDa, respectively, and DS of 0.16, 0.24 and 0.27, respectively. In vitro and in vivo experiments were conducted to evaluate the antioxidant and immunological activities of the sGSCs. In vitro experiment, the reactive oxygen species (ROS) scavenging activities were determined. In vivo experiment, 50 male BALB/c mice were divided into five groups. The sGSC1, sGSC2 and sGSC3 treatment groups received the corresponding sGSCs at 50 mg/kg/day each. The GSC (glucans from Saccharomyces cerevisiae) treatment group received 50 mg/kg/day GSC. The normal control group received equal volume of physiological saline solution. All treatments were administered intragastrically for 14 day. Results showed that sGSC1, sGSC2 and sGSC3 can scavenge 1,1-diphenyl-2-picryl-hydrazyl (DPPH), superoxide, and hydroxyl radicals in vitro. The strength of the radical scavenging effects of the sGSCs was in the order of sGSC1 > sGSC2 > sGSC3. Oral administration of sGSC1 significantly improved serum catalase (CAT) and glutathione peroxidase (GSH-Px) activities and decreased malondialdehyde (MDA) level in mice. sGSC1 significantly improved the spleen and thymus indexes and the lymphocyte proliferation, effectively enhanced the percentage of CD4+ T cells, decreased the percentage of CD8+ T cells, and elevated the CD4+/CD8+ ratio. sGSC1 significantly promoted the secretion of IL-2 and IFN-γ. These results indicate that sGSC1 with low MW and DS has better antioxidant and immunological activities than the other sGSCs, and sGSC1 could be used as a new antioxidant and immune-enhancing agent.

Effect of the diclazuril on Hsp90 in the second-generation merozoites of Eimeria tenella

Eimeria tenella (E. tenella) is one of the most virulent pathogens of coccidiosis. In apicomplexan parasites, Hsp90 (Heat shock protein 90) is essential for the invasion and survival in host cells. In this study, the effect of diclazuril, an effective benzeneacetonitrile anticoccidial agent, on the expression of Hsp90 in the second-generation merozoites of E. tenella was investigated. We inoculated 8 × 10(4) oocysts/chicken suspended in 1 ml of distilled water, and chickens were challenged with E. tenella oocysts and provided with normal feed as Control group; chickens challenged with E. tenella oocysts and provided with 1mg/kg diclazuril in feed from 96 h to 120 h after inoculation as treatment group. Then the second-generation merozoites were obtained after 120 h from the infected caeca. Our results showed that the transcription level of mzHsp90 was reduced by 29.7% in the diclazuril treatment group, accompanied by reduced level of mzHsp90 protein in second-generation merozoites prepared from infected chickens. We also found that the subcellular localization of mzHsp90 was more dispersed in these merozoites. Moreover, we demonstrated that the effects of diclazuril on mzHsp90 expression were direct by in vitro experiments. Taken together, our data provide insights into the molecular mechanisms of diclazuril in the chemotherapy of E. tenella, and suggest that mzHsp90 represents a promising target for the intervention with E. tenella infection.

Simultaneous determination of triclabendazole and its metabolites in bovine and goat tissues by liquid chromatography-tandem mass spectrometry.

A sensitive liquid chromatography-tandem mass spectrometry method for the simultaneous determination of triclabendazole, its main metabolites (triclabendazole sulphone and triclabendazole sulphoxide) and a marker residue (ketotriclabendazole) in bovine and goat muscle, liver, and kidney samples is developed and validated. Analyte extraction from samples is effectively performed using liquid-liquid extraction by acetonitrile. Chromatographic separation is performed on a C₁₈ reversed-phase column with gradient elution. The analytes are detected by tandem quadrupole mass spectrometry after positive electrospray ionization by multiple reaction monitoring. The limits of detection for analytes are found to be 0.25-2.5 μg/kg in muscle tissues and 1-10 μg/kg in liver and kidney tissues, respectively. The recoveries of edible bovine and goat tissues range from 84.9% to 109.5% when spiked at different levels with analytes, with relative standard deviations generally below 12.8%.