Evaluation of the potential in vivo genotoxicity of quercetin

Quercetin, a naturally occurring flavonol commonly detected in apples, cranberries, blueberries, and onions, has been reported to possess antioxidant, anti-carcinogenic, anti-inflammatory, and cardioprotective properties. While positive results have been consistently reported in numerous in vitro mutagenicity and genotoxicity assays of quercetin, tested in vivo, quercetin has generally produced negative results in such studies. Furthermore, no evidence of carcinogenicity related to the oral administration of quercetin was observed in chronic rodent assays. In order to further define the in vivo genotoxic potential of quercetin, a bone marrow micronucleus assay and an unscheduled DNA synthesis (UDS) assay were conducted in Wistar rats. Administered orally to male rats at dose levels of up to 2000 mg/kg body weight, quercetin did not increase the number of micronucleated polychromatic erythrocytes (MN-PCE) 24 or 48 h following dosing in the micronucleus assay. Likewise, orally administered quercetin (up to 2000 mg/kg body weight) did not induce UDS in hepatocytes of male or female rats. While measurable levels of metabolized quercetin were observed in rat plasma samples for up to 48 h after dosing, peaking at 1h following treatment administration, the unmetabolized aglycone was not identified in either plasma or bone marrow. With the exception of only a few rats, the aglycone was also not detected in liver tissue. These results demonstrate that quercetin is not genotoxic under the conditions of these assays and further support the negative results of previously conducted in vivo assays.     

Potentiation of dimethylnitrosamine genotoxicity in rat hepatocytes isolated following ethanol treatment in vivo

Unscheduled DNA synthesis (UDS), following exposure to dimethylnitrosamine (DMN), was potentiated in cultured hepatocytes isolated following treatment of rats for 14 or 28 days with 20% ethanol/5% sucrose solution. Ethanol treatment was associated with increased UDS, a concomitant increase in hepatic microsomal protein concentration and DMN N-demethylase activity. Increased aniline hydroxylase activity of hepatic microsomes from ethanol-treated rats preceded the measured increase in microsomal protein content or DMN metabolism. The increase in metabolism of DMN in vitro and potentiation of DMN-induced UDS associated with ethanol treatment may contribute to a synergistic effect of ethanol on DMN hepatotoxicity and carcinogenicity. In contrast, ethanol pretreatment did not increase the cytotoxicity of DMN as characterized by enzyme release.     

Assessment of the genotoxicity of imidacloprid and metalaxyl in cultured human lymphocytes and rat bone-marrow

Imidacloprid and metalaxyl are two pesticides that are widely used in agriculture, either separately, or in combination. These agents were studied for their possible genotoxic effects with respect to the following cytogenetic end-points: (1) in vitro micronucleus (MN) formation and sister-chromatid exchange (SCE) induction in human lymphocytes and (2) in vivo micronucleus induction in polychromatic erythrocytes (PCEs) of the rat bone-marrow. The results of the MN analysis indicate that MN frequencies after treatment with both pesticides, separately or as a mixture, do not significantly differ from those in the controls except after treatment with metalaxyl alone at 50 μg/ml (p < 0.05). The results of the SCE analysis show that SCE frequencies after treatment with imidacloprid do not differ significantly from those in the controls. A statistically significant increase (p < 0.05) in SCE frequency resulted from treatments with metalaxyl at 5, 10 and 100 μg/ml and with the combination of imidacloprid and metalaxyl at 100 and 200 μg/ml. Finally, the in vivo micronucleus assay with rat bone-marrow polychromatic erythrocytes showed a statistically significant effect upon separate treatments with imidacloprid and metalaxyl at doses of 300 mg/kg body weight (b.w.) (p < 0.01) or upon combined treatment with 200 mg/Kg b.w. (p < 0.001) and 400 mg/kg b.w. (p < 0.05).     

Signs of deferasirox genotoxicity

Iron overload is a major health problem for patients who have to have continuous blood transfusions. It brings some metabolic problems together. Various iron chelating agents are being used for treatment of hemochromatosis which arises from excess iron accumulation. This study was conducted with the aim of determining whether deferasirox used as an iron chelator in patients with hemochromatosis has genotoxic effects. Commercial form of deferasirox, Exjade was used as test material. Test material showed a general mutagen character in mutant strains of Salmonella typhimurium. Deferasirox has also led to an increase in mutagenity-related polymorphic band count in random amplification of polymorphic DNA test done with bone marrow cells of rats. Similarly, test material has increased micronucleus formation in cultured in vitro human peripheral lymphocytes particularly in 48 h period. Consistently with the abovementioned findings, deferasirox reduced nuclear division index (NDI) compared to controls and some part of these reductions are statistically significant. NDI reductions were found at positive control levels at high concentrations.     

In vivo studies on genotoxicity of pure and commercial linuron

The ureic herbicide linuron [3-(3,4-dichlorophenyl)-1-methoxy-1-methylurea] (CAS 330-55-2) was investigated for genotoxicity in a series of in vivo experiments. Since human exposure to herbicides is not only to the active principles, but also to all the chemicals present in the commercial formulation, we tested both pure and commercial linuron. Groups of rats were treated with gavage containing different doses of the herbicide (pure compound or commercial formulation) for 14 days. The doses were 150, 300 and 450 mg/kg b.wt. for the pure compound and 315.8, 631.6 and 947.4 mg/kg b.wt. for the commercial formulation (47.5% of linuron). Faeces and urine were collected at regular intervals. Urine specimens were analysed for their mutagenic metabolites, thioethers and d-glucaric acid content. Faeces extracts were tested for mutagenicity. Linuron's ability to cause DNA damage and cytogenetic effects was also investigated after treating groups of rats once with different doses of pure or commercial linuron. DNA single-strand breaks were assessed in rat liver using the alkaline elution technique and the single-cell microgel electrophoresis assay (SCGE: `comet' assay), and in rat testes cells with the SCGE assay. Micronuclei induction was analysed in rat bone marrow erythrocytes. Results obtained were mainly negative when the excretion of mutagenic metabolites in urine and faeces of animals treated with the pure compound or with the linuron-based commercial formulation were monitored, whereas an increase in the urinary excretion of thioethers and d-glucaric acid was observed in rats treated with the commercial formulation. No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the treated animals. However, linuron affected the viability of hepatocytes isolated from animals treated with higher doses. This cytotoxicity was accompanied by the induction of DNA single-strand breaks in the liver, as seen by the alkaline elution assay. The potential of pure linuron to induce in vivo DNA damage was confirmed with the microgel electrophoresis technique (`comet' assay). Cytotoxicity was also seen in rat testes cells. However, no indication of DNA damage was visible.     

槲皮素药理作用研究进展

本文概括了国内外近20年来有关槲皮素的药理研究,综述了槲皮素抗肿瘤,抗炎,抗血小板聚集,扩张冠脉等作用,并从分子生物学角度初步探讨了槲皮素抗病毒的机理。     

In vitro genotoxicity of polycyclic musk fragrances in the micronucleus test

The synthetic polycyclic musk fragrance compounds galaxolide (1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-(g)-2-benzopyrane), tonalide (7-acetyl-1,1,3,4,4,6-hexamerthyltetraline), celestolide (4-acetyl-1,1-dimethyl-6-tert-butylindane), phantolide (6-acetyl-1,1,2,3,3,5-hexamethylindane), cashmeran (6,7-dihydro-1,1,2,3,3-pentamethyl-4-(5H) indanone) and traseolide (5-acetyl-1,1,2,6-tetramethyl-3-isopropylindane) were examined for their genotoxicity in the micronucleus test (MNT) with human lymphocytes in vitro in the presence and absence of an exogenous metabolizing system containing rat liver S9 and the metabolically competent human hepatoma cell line Hep G2. Compound concentrations were employed up to cytotoxic doses. Galaxolide, tonalide, celestolide, phantolide, cashmeran and traseolide revealed no genotoxicity in the micronucleus test with human lymphocytes and with the human hepatoma cell line Hep G2.     

Dynamics of DNA-demethylation in early mouse and rat embryos developed in vivo and in vitro

Virtually all mammalian species including mouse, rat, pig, cow, and human, but not sheep and rabbit, undergo genome-wide epigenetic reprogramming by demethylation of the male pronucleus in early preimplantation development. In this study, we have investigated and compared the dynamics of DNA demethylation in preimplantation mouse and rat embryos by immunofluorescence staining with an antibody against 5-methylcytosine. We performed for the first time a detailed analysis of demethylation kinetics of early rat preimplantation embryos and have shown that active demethylation of the male pronucleus in rat zygotes proceeds with a slower kinetic than that in mouse embryos. Using dated mating we found that equally methylated male and female pronuclei were observed at 3 hr after copulation for mouse and 6 hr for rat embryos. However, a difference in methylation levels between male and female pronuclei could be observed already at 8 hr after copulation in mouse and 10 hr in rat. At 10 hr after copulation, mouse male pronuclei were completely demethylated, whereas rat zygotes at 16 hr after copulation still exhibited detectable methylation of the male pronucleus. In addition in both species, a higher DNA methylation level was found in embryos developed in vitro compared to in vivo, which may be one of the possible reasons for the described aberrations in embryonic gene expression after in vitro embryo manipulation and culture.     

A synthetic approach to the generation of quercetin sulfates and the detection of quercetin 3'-O-sulfate as a urinary metabolite in the rat

To study the biological effects of quercetin, authentic products of quercetin metabolism are required as standards. The synthesis of quercetin sulfate standards is thus described. Quercetin was reacted with a 10-fold molar excess of sulfur trioxide-N-triethylamine, and the products were analyzed by HPLC and mass spectrometry. Four monosulfates and three disulfates were identified, and structural inferences were drawn by ¹H NMR spectrometry of HPLC peak isolates. Analysis of the urine of rats that had received quercetin (1.9 g/kg po) yielded a single peak, which by comparison with the products of the reaction between quercetin and sulfur trioxide-N-triethylamine was identified as quercetin 3′-O-sulfate.     

Evaluation of the anti-Toxoplasma gondii Activity of Hederagenin in vitro and in vivo

Toxoplasma gondii infection is widespread worldwide, not only posing a serious threat to human food safety and animal husbandry, but also endangering human health. The selectivity index was employed to measure anti-T. gondii activity. Hederagenin (HE) exhibited potent anti-T. gondii activity and low cytotoxicity. For this reason, HE was selected for in vivo experiments. HE showed 64.8%±13.1% inhibition for peritoneal tachyzoites in mice, higher than spiramycin 56.8%±6.0%. Biochemical parameters such as alanine aminotransferase, aspartate aminotransferase, glutathione, and malondialdehyde, illustrated that HE was a good inhibitor of T. gondii in vivo. This compound was also effective in relieving T. gondii-induced liver damage. Collectively, it was demonstrated that HE had potential as an anti-T. gondii agent.     

克隆鸭乙型肝炎病毒DNA双体体内转染的研究

用一种含头尾相连DHBVDNA双体的质粒体内转染2日龄芙蓉鸭,大多数鸭（86％）产生了短暂病毒血症。血清DHBs／preSAg和DHBVDNA于转染后第9天出现,第12～14天达峰值,第28天时多数转阴;少数鸭的病毒血症可持续50天以上。转染鸭肝组织中也检测到复制中间型DHBVDNA的存在。用转染鸭病毒血症期的血清作磷钨酸负染电镜观察,找到了完整的DHBV病毒颗粒,并且用此血清腹腔注射1日龄鸭,60％的鸭被感染成功,证明体内转染后有生物活性的DHBV病毒颗粒的产生。该研究方法的建立．对于研究DHBV变异株．DHBV基因结构与功能的关系等,均有一定理论意义及应用价值。     

Evaluation of the micronucleus assay in bone marrow and peripheral blood of rats for the determination of cigarette mainstream-smoke activity

The mammalian in vivo micronucleus assay is widely used as part of the genotoxicity testing battery required during the development of new drugs. As such, the in vivo micronucleus assay has been used in a battery of assays for the assessment of cigarette ingredients or design modifications to help ensure that there is no increase in risk or any new risk introduced by these additions or modifications. The present series of studies was conducted to optimize and evaluate this assay for the assessment of the effects of mainstream smoke on the micronucleus frequency in the bone marrow and peripheral blood of rats. In a first experiment, the optimal conditions for performing the micronucleus assay in these tissues were determined. This was done by use of two compounds known for their micronucleus-inducing activity, i.e., the clastogen cyclophosphamide and the aneugen colchicine. In a second experiment, the effects of tube restraint on untreated control rats were investigated. In a third experiment, the optimal conditions were used to assess the clastogenic/aneugenic activity of cigarette smoke in Sprague-Dawley rats. The rat micronucleus assay in both bone marrow and peripheral blood is able to detect clastogenic and aneugenic activity. The flow cytometric determination of micronucleated cells in rat blood is at least as sensitive as determinations in bone marrow. No statistically significant differences were observed in micronucleus frequencies between rats with and without the additional stress of tube restraint; however, the cautious approach would be to use a fresh-air-exposed group (with tube restraint) as the negative control in inhalation experiments. Using the conditions identified as optimal in the above-mentioned experiments, the micronucleus assay was not able to detect effects induced by smoke from conventional cigarettes. Nevertheless, the micronucleus assay will remain a valuable tool as part of a testing battery used to investigate possible adverse effects related to product modifications.     

Glial metabolism of quercetin reduces its neurotoxic potential

The neuroprotective effects of flavonoids will ultimately depend on their interaction with both neuronal and glial cells. In this study, we show that the potential neurotoxic effects of quercetin are modified by glial cell interactions. Specifically, quercetin is rapidly conjugated to glutathione within glial cells to yield 2′-glutathionyl-quercetin, which is exported from cells but has significantly reduced neurotoxicity. In addition, quercetin underwent intracellular O-methylation to yield 3′-O-methyl-quercetin and 4′-O-methyl-quercetin, although these were not exported from glia at the same rate as the glutathionyl adduct. The neurotoxic potential of both quercetin and 2′-glutathionyl-quercetin paralleled their ability to modulate the pro-survival Akt/PKB and extracellular signal-regulated kinase (ERK) signalling pathways. These data were supported by co-culture investigation, where the neurotoxic effects of quercetin were significantly reduced when they were cultured alongside glial cells. We propose that glial cells act to protect neurons against the neurotoxic effects of quercetin and that 2′-glutathionyl-quercetin represents a novel quercetin metabolite.     

体内连续进化技术的研究进展

实验室进化是遗传育种、提高微生物性能的重要方式.近几十年来,实验室进化的方法快速发展,应用也越来越广泛,但是常见的菌株进化策略以及针对特定蛋白的进化存在突变过程不连续,需要多轮操作、工作量大等缺点.微生物突变和筛选技术的进步促进了体内连续进化的发展,大大提高了实验室进化的效率.体内连续进化技术实现了体内突变,完美地把突...     

Novel self-emulsifying formulation of quercetin for improved in vivo antioxidant potential: Implications for drug-induced cardiotoxicity and nephrotoxicity

Quercetin (QT) was formulated into a novel self-emulsifying drug delivery system (SEDDS) to improve its oral bioavailability and antioxidant potential compared to free drug. Capmul MCM was selected as the oily phase on the basis of optimum solubility of QT in oil. Tween 20 and ethanol were selected as surfactant and cosurfactant from a large pool of excipients, depending upon their spontaneous self-emulsifying ability with the selected oily phase. Pseudoternary-phase diagrams were constructed to identify the efficient self-emulsification regions in various dilution media, viz., water, pH 1.2, and pH 6.8. The ratio of 40:40:20 w/w, Capmul MCM:QT (19:1)/Tween 20/ethanol was optimized based on its ability to form a spontaneous submicrometer emulsion in simulated gastrointestinal fluids. DPPH scavenging assay showed comparable antioxidant activity of QT-SEDDS to free QT. QT-SEDDS was robust in terms of stability against short-term excursion of freeze/thaw cycles and accelerated stability for 6 months as per International Conference on Harmonisation guidelines. A fluorescent dye-loaded SEDDS formulation showed rapid internalization within 1 h of incubation with Caco-2 cells as evident by confocal laser scanning microscopy. QT-SEDDS showed a significant increase in cellular uptake by 23.75-fold in comparison with free QT cultured with Caco-2 cells. The SEDDS demonstrated ~5-fold enhancement in oral bioavailability compared to free QT suspension. The in vitro–in vivo relation between in vitro Caco-2 cell uptake and in vivo pharmacokinetics of QT-SEDDS showed a correlation coefficient of ~0.9961, as evident from a Levy plot. Finally, QT-SEDDS showed a significantly higher in vivo antioxidant potential compared to free QT when evaluated as a function of ability to combat doxorubicin- and cyclosporin A-induced cardiotoxicity and nephrotoxicity, respectively.     

The feasibility of non-viral gene transfer to the diaphragm in vivo

Gene transfer using electroporation is an essential method for the study of developmental biology, especially to understand the internal control of degeneration and apoptosis of the muscle cells that occurs earlier and quicker than the usual degeneration process occurring by aging. Such experimental studies may have a role in developing new strategies for treating patients suffering from inherited primary myopathies such as Duchenne muscular dystrophy (DMD). The present study was designed to evaluate the feasibility of electroporation mediated transfer of reporter genes to the diaphragm in vivo. This is the first report of gene transfer of naked plasmid DNA into the diaphragm muscle in vivo using electroporation. Our results showed that in vivo gene transfer of naked plasmid DNA into the diaphragm muscle using electroporation is feasible.     

Autoradiographic Study of Gamma-Ray Induced Unscheduled DNA Synthesis in Bean Root Meristem Cells

The gamma-ray induced unscheduled DNA synthesis in root meristem cells of Vica faba was studied autoradiographically by calculating the number of cells with different 3H-thymidine labelling degree. It was found that the level of unscheduled synthesis in cells with intermediate dose (500 R) irradiation was higher than that in cells with lower dose (250 R) irradiation; however, higher dose (1000 R) irradiation would inhibit the reparative replication.     

Effect of different exposed lights on quercetin and quercetin glucoside content in onion (Allium cepa L.)

Quercetin and quercetin glucosides are the major flavonols present in onion (Allium cepa L.) and are predominantly present as quercetin, quercetin-3,4′-diglucoside and quercetin-4′-glucoside. Effect of different light wavelengths on onion after harvest and storage, with fluorescent, blue, red and ultra violet light influenced the quercetin and quercetin glucosides profile. In a peeled onion, all the light treatments elevated quercetin content in bulb. Among them, particularly fluorescent light effect was more eminent which stimulates the maximum synthesis of quercetin in onion. In case of whole onion bulb, skin and pulp showed different responses to light treatment, respectively. The pulp had the highest quercetin glucosides under blue light, whereas the lowest under fluorescent light. Onion skin showed nearly opposite pattern as compared to the pulp. In particular, light treatment proved to be a better way to increase the level of quercetin content in onions which might be utilized for industrial production of bioactive compounds from onion and onion waste products.     

DNA binding and oxidative DNA damage induced by a quercetin copper(II) complex: potential mechanism of its antitumor properties

The interaction of a quercetin copper(II) complex with DNA was investigated using UV–vis spectra, fluorescence measurement, viscosity measurement, agarose gel electrophoresis, and thiobarbituric acid reactive substances assay. The results indicate that the quercetin copper(II) complex can promote the cleavage of plasmid DNA, producing single and double DNA strand breaks, and intercalate into the stacked base pairs of DNA. Moreover, the complex can induce oxidative DNA damage involving generation of reactive oxygen species such as H₂O₂ and Cu(I)OOH. In addition, the cytotoxicity experiments carried out with A549 cells confirmed its apoptosis-inducing activity. And we also demonstrate that the levels of survivin protein expression in A549 cells decreased, and that relative activity of caspase-3 increased significantly after treatment with the complex. So our results suggest that the antitumor mechanism of the quercetin copper(II) complex involves not only its oxidative DNA damage with generation of reactive oxygen species but also its specific interaction with DNA. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.