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OBJECTIVE To evaluate the genotoxicity of Descarboxyl Levofloxacin by In silico and In vitro methods.METHODS Assessment of genotoxicity using Derek for Windows We used DEREK for Windows(DfW,Lhasa Ltd.,Leeds,UK) version 11.0 for toxicity assessment.Molecular structures are entered into the program in mdl.mol format.The program applies structure-activity relationships 〔(Q) SARs〕and other expert knowledge rules to derive a reasoned prediction about the potential toxicity of the query chemical.Bacterial reverse mutation assay(Ames test) The Salmonella histidine point mutation assay proposed by Maron and Ameswas followed.Four S.typhimurium histidine autotrophic strains(TA97,TA98,TA100 and TA102) used in this test were obtained from Shandong center for disease control.Normal saline(NS) was used as a vehicle control.2-Aminofluorene(2-AF,10 μg /plate),1,8-dihydroxyanthraquinone(50 μg /plate),Sodium azide(NaN3,1.5 μg /plate),and Dexon(50 μg /plate) were used as positive controls.Descarboxyl levofloxacin was assayed at concentrations of 31.25,62.5,125,250,and 500 μg /plate.Briefly,0.1 ml of test substance(or control solution),0.1 ml of bacterial culture 〔(1-2) × 1012 L-1 〕and 0.5 ml of S9 mix(NS for treatment without activation) were mixed and poured onto the agar plates after addition of 1.8 ml of top agar(0.6% agar Difco,0.5% NaCl,50 μmol·L-1 L-histidine,50 μmol·L-1 biotin,45℃) at 45℃.Plates were inverted and incubated for 48h at 37℃ in the dark.Revertant colonies were counted and the background lawn was inspected for signs of toxicity or compound precipitation.Each assay was performed two times in triple and included negative and positive controls.Chromosom al aberrations in Chinese ham ster lung cells Chinese Hamster Lung(CHL) cells were obtained from Shandong center for disease control.The cells were grown in DMEM medium supplemented with 10% heat-inactivated calf serum at 37℃ in an atmosphere containing 5% of CO2.The test for Chromosomal aberrations in CHL cells was conducted according to published procedures.The cells were seeded at 2.5 × 105 cells in 10 ml medium per 50ml culture dish and incubated overnight.Then the cells were incubated for 6 h with medium containing 1000,500,250 μg.ml-1 of descarboxyl levofloxacin both in presence and absence of metabolic activation(S9 mix).Cells were washed for 3 times and then the medium was changed to one without descarboxyl levofloxacin.Concurrent solvent controls were also conducted.For the chromosome aberration study,Colchicine was added to accumulate metaphase cells 4 h before the end of culture.After incubation,cells cultured for cytogenetic studies were trypsinized and harvested.Normal saline,Cyclophosphamide(CP),and mitomycin c(MMC) were used for solvent control,positive control with metabolic activation,and positive control without metabolic activation,respectively.The harvested cells were separated by centrifugation,and then resuspended in dilute potassium chloride solution(KCl 75 mmol·L-1) for 20 min.And the cells again centrifuged after addition of fixative(methanol: acetic acid D 3∶1,V/V).This fixation procedure was carried out at least twice.For observation of metaphase cells,a few drops of cell suspension were spread onto clean slides,air-dried and stained with 5% Giemsa solution.Then the slides were mounted with cover slips and examined by light microscopy by trained personal.Light microscope assessment was performed with a 100 × objective in an oil immersion system.RESULTS Assessment of genotoxicity using DfW The prediction results showed that quinoline was listed as an alert(alert number: 016).The endpoints of mutagenicity were plausible in some spicies,including bacterium,Escherichia coli and Salmonella typhimurium in vitro,and dog,guinea pig,hamster human,mammal,monkey,mouse,primate,rabbit,rat,rodent in vivo.Then we considered the in silico genotoxicity of descarboxyl levofloxacin as positive.Am es test Mutagenicity of Descarboxyl levofloxacin was evaluated in a bacterial reverse mutation assay using histidine requiring S.typhimurium(TA97,TA98,TA100,TA102).Descarboxyl levofloxacin was assayed at concentrations of 31.25,62.5,125,250,and 500μg /plate.Regardless of presence of S9 mix,the number of reverting colonies in all the level of treatment was not significantly higer than that in vehicle control.Remarkable increase in the number of revertant colonies was founded in all positive control compared to vehicle control.From these data,we can consider that descarboxyl levofloxacin has no mutagenic activity,at least under our conditions of testing.Chromosomal aberrations in CHL The positive controls resulted in significant increases in chromosomal aberrations with or without the activation system,which indicate a valid assay.The percent of cells with aberrations is more than 5% but less than 10% at the dose of 1000 μg.ml-1 and less than 5% at the dose of 500 μg.ml-1 or 250 μg.ml-1.The mutagenic potential of descarboxyl levofloxacin in this assay was doubt at the high dose.CONCLUSION Based on these assays,descarboxyl levofloxacin could be controlled as a non-genotoxic impurity.The quantitative limitation of the impurity could be set as 0.1%,which is equal to the identification threshold but less than the qualification threshold(0.15%).
OBJECTIVE To evaluate the genotoxicity of Descarboxyl Levofloxacin by In silico and In vitro methods. METHODS Assessment of genotoxicity using Derek for Windows We used DEREK for Windows (DfW, Lhasa Ltd., Leeds, UK) version 11.0 for toxicity assessment. Molecular structures entered are into the program applies to mdl.mol format. The program applies structure-activity relationships [(Q) SARs] and other expert knowledge rules to derive a reasoned prediction about the potential toxicity of the query chemical. Bacterial reverse mutation assay (Ames test) The Salmonella histidine point mutation assay by Maron and Ameswas followed. Flow S. typhimurium histidine autotrophic strain (TA97, TA98, TA100 and TA102) used in this test were obtained from Shandong center for disease control. Normal saline (NS) was used as a vehicle control.2 -Aminofluorene (2-AF, 10 μg / plate), 1,8-dihydroxyanthraquinone (50 μg / plate), Sodium azide (NaN3,1.5 μg / plate), and Dexon as positive controls.Descarb oxyl levofloxacin was assayed at concentrations of 31.25, 62.5, 125, 250, and 500 μg / plate.Briefly, 0.1 ml of test substance (or control solution), 0.1 ml of bacterial culture [(1-2) × 1012 L-1] and 0.5 ml of S9 mix (NS for treatment without activation) were mixed and poured onto the agar plates after addition of 1.8 ml of top agar (0.6% agar Difco, 0.5% NaCl, 50 μmol·L- L-1 biotin, 45 ° C) at 45 ° C. Plates were inverted and incubated for 48 h at 37 ° C in the dark. Revertant colonies were counted and the background lawn was inspected for signs of toxicity or compound precipitation. Each assay was performed two times in triple and included negative and positive controls. Chromosom al aberrations in Chinese ham ster lung cells Chinese Hamster Lung (CHL) cells were obtained from Shandong center for disease control. The cells were grown in DMEM medium supplemented with 10% heat-inactivated calf serum at 37 ° C in an atmosphere containing 5% of CO2. The test for Chromosomal aberra tions in CHL cells was conducted according to published procedures. The cells were seeded at 2.5 × 105 cells in 10 ml medium per 50 ml culture dish and incubated overnight. These the cells were incubated for 6 h with medium containing 1000, 500, 250 μg.ml-1 of descarboxyl levofloxacin both in presence and absence of metabolic activation (S9 mix) .Cells were washed for 3 times and then the medium was changed to one without descarboxyl levofloxacin. Concurrent solvent controls were also conducted. For the chromosome aberration study, Colchicine was added to accumulate metaphase cells After 4 h before the end of culture. After incubation, cells cultured for cytogenetic studies were trypsinized and harvested. Normal saline, Cyclophosphamide (CP), and mitomycin c (MMC) were used for solvent control, positive control with metabolic activation, and positive control without metabolic activation, respectively. The harvested cells were separated by centrifugation, and then resuspended in dilute potassium chloride solution (KCl 75 mmol·L-1) for 20 min. End the cells again centrifuged after addition of fixative (methanol: acetic acid D 3: 1, V / V). This fixation procedure was carried out at least twice. For observation of metaphase cells, a few drops of cell suspension were spread onto clean slides, air-dried and stained with 5% Giemsa solution.. the slides were mounted with cover slips and examined by light microscopy by trained personal. Light microscope assessment was performed with a 100 × objective in an oil immersion system. of genotoxicity using DfW The prediction results showed that quinoline was listed as an alert (alert number: 016). The endpoints of mutagenicity were plausible in some spicies, including bacterium, Escherichia coli and Salmonella typhimurium in vitro, and dog, guinea pig, hamster human, mammal, monkey, mouse, primate, rabbit, rat, rodent in vivo. Shen we considered the in silico genotoxicity of descarboxyl levofloxacin as positive. Am es test Mutagenicity of Descarboxyl levofloxacin was evaluated in a bac terialreverse mutation assay using histidine requiring S. typhimurium (TA97, TA98, TA100, TA102) .Descarboxyl levofloxacin was assayed at concentrations of 31.25, 62.5, 125, 250, and 500 μg / plate.Regardless of presence of S9 mix, the number of reverting colonies in all the level of treatment was not significantly higer than that in vehicle control. Remarkable increase in the number of revertant colonies was founded in all positive control compared to vehicle control. These data, we can consider that descarboxyl levofloxacin has no mutagenic activity, at least under our conditions of testing. Chromosomal aberrations in CHL The positive controls resulted in significant increases in chromosomal aberrations with or without the activation system, which indicate a valid assay. percent of cells with aberrations is more than 5% but less than 10% at the dose of 1000 μg.ml-1 and less than 5% at the dose of 500 μg.ml-1 or 250 μg.ml-1. The mutagenic potential of descarboxyl levofloxacin in this assay was doub t at the high dose. CONCLUSION Based on these assays, descarboxyl levofloxacin could be controlled as a non-genotoxic impurity. The quantitative limitation of the impurity could be set as 0.1%, which is equal to the identification threshold but less than the qualification threshold (0.15%).