Xilong Xiao

Investigation of the genotoxicity of quinocetone, carbadox and olaquindox in vitro using Vero cells

Quinoxaline-1,4-dioxides derivatives have been widely used as animal growth promoter. This study was conducted to investigate the cytotoxicity and genotoxicity of quinoxaline-1,4-dioxides derivatives, namely carbadox, olaquindox and quinocetone, in Vero cells. The cell viability results from MTT assay demonstrated the severe inhibitory effects by these chemicals in both dose and time dependent manner. Among these chemicals quinocetone exhibited the highest cytotoxicity followed by olaquindox and carbadox. DNA damage analyses using alkalic comet assay revealed pronounced increase of DNA fragmentation in all three compound treated cells. In contrast, DNA damage was significantly decreased after incubation with S9 mix. These findings suggest that the intermediate metabolites of these compounds exerted lower genotoxicity than their parent drugs. We further described chromosomal damage induced by these drugs employing cytokinesis-block micronucleus assay (MN assays). The micronucleus frequency was significantly higher in these drugs treated cells than that of controls and the nuclear division index was also markedly reduced with increasing drug concentration applied. Similar to the observation in comet assay, incorporation of S9 mix in the MN assays was able to markedly alleviate the chromosome damage. In conclusion, our results strengthened previous reports on the cytotoxicity and genotoxicity of carbadox, olaquindox and quinocetone.

Rapid enzyme-linked immunosorbent assay and colloidal gold immunoassay for kanamycin and tobramycin in Swine tissues.

A monoclonal antibody (Mab) was produced by using the kanamycin-glutaraldehyde-bovine serum albumin (Kan-GDA-BSA) conjugate as the immunogen. The anti-Kan Mab exhibited high cross-reactivity with tobramycin (Tob) and slight or negligible cross-reactivity with other aminoglycosides. The specificity and cross-reactivity of this Mab are discussed regarding the three-dimensional, computer-generated molecular models of the aminoglycosides. Using this Mab, a rapid enzyme-linked immunosorbent assay (ELISA) and a colloidal gold-based strip test for Kan and Tob were developed. The rapid ELISA showed a 50% inhibition value (IC 50) of 0.83 ng/mL for Kan and 0.89 ng/mL for Tob with the analysis time less than 40 min, and the recoveries from spiked swine tissues at levels of 25-200 microg/kg ranged from 52% to 96% for Kan and 61% to 86% for Tob. In contrast, the strip test for Kan or Tob had a visual detection limit of 5 ng/mL in PBS, 50 microg/kg in meat or liver, and 100 microg/kg in kidney, and the results can be judged within 5-10 min. Observed positive samples judged by the strip test can be further quantitated by ELISA, hence the two assays can complement each other for rapid detection of residual Kan and Tob in swine tissues. Moreover, physical-chemical factors that affected the ELISA and strip test performance were also investigated. The effect of pH and antibody amount for gold-antibody conjugation on the strip test sensitivity was determined followed by a theoretical explanation of the effects.

Olaquindox-induced genotoxicity and oxidative DNA damage in human hepatoma G2 (HepG2) cells.

Olaquindox, a synthetic antimicrobial compound, is widely used in China as feed additive for growth promotion. However, it is a mutagen with its functional mechanism yet to be unclear. The purpose of this study was to investigate the genotoxic effects of olaquindox in human hepatoma G2 (HepG2) cells and to determine whether the oxidative DNA damage participated in the mechanism of olaquindox toxicity. The results of cell survival assay revealed that the HepG2 cells viabilities were significantly inhibited by olaquindox in a dose- and time-dependent manner. The cytokinesis-block micronucleus (CBMN) assay demonstrated a clear dose-response relationship between olaquindox treatments and micronucleus (MN) frequencies. Moreover, marked increases of DNA fragment migration were observed in the single cell gel electrophoresis (SCGE) assay. These data suggest that olaquindox treatment produced serious chromosome damage and DNA damage in HepG2 cells. To elucidate the possible oxidative DNA damage mechanism of olaquindox genotoxic activity, the levels of the intracellular reactive oxygen species (ROS) and the formation of 8-hydroxydeoxyguanosine (8-OHdG) were detected. The results showed that olaquindox induced the increased levels of ROS and 8-OHdG in HepG2 cells. Considering all the results, it is inferred that olaquindox exerts genotoxic effects in HepG2 cells probably through the ROS-induced oxidative DNA damage.

Investigation of quinocetone-induced genotoxicity in HepG2 cells using the comet assay, cytokinesis-block micronucleus test and RAPD analysis.

Quinocetone, a new quinoxaline 1,4-dioxide derivative, has been approved as an animal growth promoter in China since 2003. To investigate the genotoxicity of quinocetone in vitro, its effects on the extent of DNA injury in human hepatoma (HepG2) cells accompanied by chromosomal damage and genomic DNA alterations were tested. The cell viability test indicated that quinocetone inhibited cell proliferation as a function of dose and time. In the comet assay, significant DNA fragment migration was observed in a dose-dependent manner. A dose-dependent increase of the micronucleated (MN) cell frequency was shown in cytokinesis-block micronucleus (CBMN) test. The gain/loss of randomly amplified polymorphic DNA (RAPD) bands and the change of band intensity in RAPD profiles were obtained after HepG2 cells were exposed to quinocetone at concentrations of 1.25, 2.5 and 5 microg/mL. The results demonstrated that quinocetone exerted genotoxic effects on HepG2 cells. Thus, the use of quinocetone as a growth promoter in animal feed should be seriously considered.

Olaquindox induces apoptosis through the mitochondrial pathway in HepG2 cells

Olaquindox is used in China as feed additive for growth promotion in pigs. Recently, we have demonstrated that olaquindox induced genome DNA damage and oxidative stress in HepG2 cells. The aim of this study was to explore the molecular mechanism of cell cycle arrest and apoptosis induced by olaquindox in HepG2 cells. In the present study olaquindox induced cell cycle arrest to the S phase and dose-dependent apoptotic cell death in HepG2 cells, indicated by accumulation of sub-G1 cell population, nuclear condenstion, DNA fragmentation, caspases activation and PARP cleavage. Meanwhile, the data showed that olaquindox triggered ROS-mediated apoptosis in HepG2 cells correlated with both the mitochondrial DNA damage and nuclear DNA damage, collapse of Δψ(m), opening of mPTP, down-regulation of Bcl-2 and up-regulation of Bax. Furthermore, we also found that olaquindox increased the expression of p53 protein and induced the release of cytochrome C from mitochondria to cytosol. In conclusion, olaquindox induced apoptosis of HepG2 cells through a caspase-9 and -3 dependent mitochondrial pathway, involving p53, Bcl-2 family protein expression, Δψ(m) disruption and mPTP opening.

Development of an immunochromatographic strip test for rapid detection of melamine in raw milk, milk products and animal feed.

A simple, rapid and sensitive immunogold chromatographic strip test based on a monoclonal antibody was developed for the detection of melamine (MEL) residues in raw milk, milk products and animal feed. The limit of detection was estimated to be 0.05 μg/mL in raw milk, since the detection test line on the strip test completely disappeared at this concentration. The limit of detection was 2 μg/mL (or 2 μg/g) for milk drinks, yogurt, condensed milk, cheese, and animal feed and 1 μg/g for milk powder. Sample pretreatment was simple and rapid, and the results can be obtained within 3-10 min. A parallel analysis of MEL in 52 blind raw milk samples conducted by gas chromatography-mass spectrometry showed comparable results to those obtained from the strip test. The results demonstrate that the developed method is suitable for the onsite determination of MEL residues in a large number of samples.

Sample Preparation for Determination of Neomycin in Swine Tissues by Liquid Chromatography-Fluorescence Detection

A liquid chromatography method was developed for the determination of neomycin in swine tissues. Sample clean-up was especially focused on in this research. After neomycin was extracted from tissue with 10% trichloroacetic acid (w/v) solution, several clean-up procedures using MCX, CBA, WCX, and C18 solid-phase extraction (SPE) columns were compared for the purification effects and recoveries. The best result was obtained using MCX SPE column. The purified extract was derivatized with 9-fluorenylmethyl chloroformate and then separated on a C18 column and detected by a fluorescence detector. The spiked recoveries at 0.2–10 mg/kg ranged from 80.8–95.3% with coefficient of variations less than 12.1%.

Cyclin A is essential for the p53-modulated inhibition from benzo(a)pyrene toxicity in A549 cells

Benzo(a)pyrene (B(a)P) is an environment carcinogen that can enhance cell proliferation by disturbing the signal transduction pathways in cell cycle regulation. The p53 tumor suppressor as a cell cycle check-point determinant plays a critical role in cell proliferation. However, the mechanism of p53 that accounts for the remarkable toxicity of B(a)P remains elusive. Here we reported that exposure of B(a)P to A549 cells caused G1 to S and G2/M phase transition along with increased expression of p53, cyclin D1, Cdk2, Cdk4, p21 and decreased expression of cyclin E, but no change in cyclin A and p27 expression. Up-regulation of p53 expression via transfection caused G1 phase arrest with decreased expression of cyclin A, E, Cdk2 and Cdk4, and increased expression of p21, when the expression of cyclin D1 and p27 were not significant changed. While B(a)P exposure to A549 cells following p53 transfection, up-regulation of p53 significantly attenuated the B(a)P-induced enhancement of cell proliferation and cell arrest, with increased expression of cyclin D1, Cdk2 and Cdk4, and with declined expression of cyclin A, cyclin E and p21, and p27 were not significant changed. Compared to the untreated cells, cylin A expression reduced in p53-transfected cells and in the B(a)P-treated cells following p53 transfection, but showed no change in the only B(a)P-treated cells. These results indicated that cyclin A is regulated by p53, not by B(a)P, and it is essential in the p53-modulated inhibition from benzo(a)pyrene toxicity in A549 cells, cyclin E and p21 also as downstream genes of p53 involved it, which is p27-independent.

Preparation of a newly formulated long-acting ceftiofur hydrochloride suspension and evaluation of its pharmacokinetics in pigs

A new long-acting ceftiofur hydrochloride preparation was formulated and its physical properties, stability, and pharmacokinetics were investigated in this study. The prepared ceftiofur hydrochloride suspension demonstrated a milk white consistency, was easy to re-disperse and was stable in light, heat and humidity stability tests. Its other physical properties such as flowability, syringeability, settling volume ratio, particle size and distribution were perfectly consistent with the standard of Ministry of Agriculture of the Peoples Republic of China. After intramuscular administration of a single dose in swine (5 mg/kg B.W.), the drug concentration-time data in plasma were well fitted using the two-compartment open model. Compared with the ceftiofur hydrochloride preparation (EXCENEL) from Pfizer, the peak concentration (C(max)) in plasma was decreased by 2.34 times (P < 0.001), the half-life of elimination phase (T(1/2beta)) was 1.65 times longer (P < 0.001), and the therapeutic level of ceftiofur above the lowest effective plasma concentration of 0.2 microg/mL (T > 0.2) was prolonged from 87.20 h to 135.36 h (P < 0.001). The ceftiofur hydrochloride suspension prepared in this study provides therapeutically effective plasma concentrations for a longer duration, which make it more effective and more convenient to use in the treatment of respiratory diseases that require the maintenance of therapeutic plasma concentrations over a long duration.

LC method for the analysis of kanamycin residue in swine tissues using derivatization with 9-fluorenylmethyl chloroformate.

A sensitive and robust HPLC method was developed for the determination of kanamycin (Kan) in swine tissues. Kan was extracted from tissue with 10% TCA (w/v) solution and cleaned up with MCX SPE column. The purified extract was derivatized with 9-fluorenylmethyl chloroformate, and then separated on a C18 column and detected by a fluorescence detector. The linear range of the calibration curve was 0.025-1 microg/mL with R(2) of 0.9998. The limits of quantification were determined to be 0.1 mg/kg in muscle, 0.2 mg/kg in liver, and 0.6 mg/kg in kidney, and the average recoveries of Kan from swine tissues at different fortification levels (0.1-0.4 mg/kg for muscle, 0.2-1.2 mg/kg for liver, and 0.6-5.0 mg/kg for kidney) ranged from 80.7 to 91.3% with intraday and interday coefficient of variations less than 12.1%. The validated method was successfully applied to determine Kan in incurred samples, indicating that it can be used as a routine tool for the surveillance of Kan residue in swine tissues.