| 1. ObjectivesAntineoplastic drugs constitute a heterogenous group of chemicals that share an ability to inhibit cancer growth by killing actively growing cells and disrupting cell division. However, many antineoplastic drugs have been shown to be carcinogenic, mutagenic and teratogenic in experimental animals and in vitro test systems. The commonly used cytogenetic methods to detect cytogenetic damage are chromosomal aberrations (CAs) assay, sister chromatid exchanges (SCEs) assay, micronucleus (MN) assay, comet assay and gene mutation assays and so on.Vincristine (VCR) is an antineoplastic drug with the antimitotic action mode, also vincristine is one of ten anticancer agents which have been classified into group 1 (carcinogenic to humans) by IARC (1987, 1990). VCR is a spindle poison, which can impact mitosis and meiosis and induce the polyploidy, chromosomal aberrations and micronelei. Even VCR at lower dose has teratogenic effect. In order to study the characteristics of the genetic damage induced by VCR, in this investigation 4 cytogentic assays (micronucleus assay, comet assay, hprt gene mutation assay and TCR gene mutation assay) were used to assess the genetic damage of human lymphocytes exposed to VCR in vitro at several different genetic endpoints (chromosomal structure aberration, chromosomal number aberration, DNA damage and gene mutation).2. Materials and methods2.1 SamplesThe peripheral blood samples were collected from two healthy donors without occupational exposure to harmful agents. The blood samples were exposed to VCR at final doses of 0.00, 0.01, 0.02, 0.04, 0.08μg ml-1 for 24 h, then washed twice by normal solution after exposure.2.2 MethodsComet assay: Human lymphocytes were isolated from whole blood and resuspended in PBS. Then cells were mixed with trypan blue solution and checked for viability. The following steps: slide preparation, alkaline lysis, DNA unwinding, electrophoresis and stain were performed. The comet lengths and moment were measured under a fluorescence microscope (OLYMPUS-BX51).Cytokinesis-block micronucleus (CBMN) assay: The cultures were incubated at 37℃for 72 h. 4.5μg/ml cytochalasin B was added to each culture at 28 h before harvesting cells. Then slides were prepared by cyto-centrifugation, fixed with methanol and stain. Number of micronucleated cells (i.e. micronucleated cell rate, MCR) and number of micronuclei (i.e. micronucleus rate, MNR) per 1000 binucleated lymphocytes served as the indicators were scored at 400×magnification under a light-microscope. Micronucleus rate (MNR), micronucleated cell rate (MCR), nuclear buds (Buds), nucleoplasmic bridges (NPBs) and numbers of apoptotic cells were utilized as indexes in MN assay. Meanwhile, Nuclear Division Index (NDI) was calculated in another 500 cells.;The hprt gene mutation assay: The basic method is similar to Cytokinesis-block micronucleus (CBMN) assay. 0.2mM 6-thioguanine was added into one set of culture. The cultures were incubated at 37℃for 72 h. The binucleated and multinucleated cells per 6000 lymphocytes in two sets of cultures were scored under light microscopy (magnification 400×). Mutant frequency of hprt gene (Mf-hprt) was ??calculated.The TCR gene mutation assay: Human lymphocytes were isolated and resuspended in PBS. Then cells were mixed with trypan blue solution and checked for viability, and the cell number was counted. The peripheral blood mononuclear cells were stained with fluorescein isothiocyanate (FITC)-labeled anti-CD4 MoAb and phycoerythrin (PE)-labeled anti-CD3 MoAb obtained from Becton Dickinson Immunocytometry System, mixed well and incubated for 30 min on ice. TCR mutant CD4+T cells (CD3-CD4+) were analyzed by Coulter EPICS XL(Coulter Co, Hialeah, Florida, U.S.A.) with SystemⅡsoftware. Mutant frequency of TCR gene (Mf-TCR) was calculated: the number of events in the mutant cell window was divided by the total number of CD4+ T cells in the flow cytogram.3. ResultsThe results of present investigation indicated that the cytogentic damage of human lymphocytes induced by VCR could be detected with comet assay, CBMN assay, hprt gene mutation assay and TCR gene mutation assay in vitro.(1) Comet assayFor donor 1, MTL and MTM increased from 1.87±0.09μm and 0.77±0.04 to 4.44±0.28μm and 2.22±0.21 at the dose of 0.01μg ml-1 and 0.08μg ml-1 . Exposure to VCR resulted in significant increases in MTL and MTM (P< 0.05 or P< 0.01) compared to untreated control (1.14±0.03μm and 0.51±0.01).For donor 2, MTL and MTM increased from 1.76±0.07μm and 0.65±0.03 to 4.05±0.18μm and 1.86±0.10 at the dose of 0.01μg ml-1 and 0.08±μg ml-1 .Statistically significant differences were observed in the assay from 0.01±μg ml-1 compared to untreated control (1.08±0.04μm and 0.49±0.02).(2) CBMN assayChromosome damage was detected by the CBMN assay and the results are expressed as the frequencies of MN, MNC, Buds, and NPBs per 1000 cells along with NDI: there were significant differentces when the cells were treated with VCR at a dose of 0.01μg ml-1. Also apoptosis are presented: numbers of apoptotic cells were significantly higher than those in control from the dose of 0.01μg ml-1.For donor 1: The frequency of MN, MNC , Buds, NPBs and apoptotic cells increased in a dose dependent manner from 16.00±2.52,13.00±1.00,6.33±0.33,1.33±0.33,0.67±0.33 to 67.00±1.15,47.33±3.53,14.00±1.53,4.67±0.43,17.33±1.20 at the dose of 0.01μg ml-1 and 0.08μg ml-1 . There is significant differences (P< 0.05 or P< 0.01) from 0.01μg ml-1 (0.02μg ml-1 for AC) compared to untreated control 2.00±0.58,2.00±0.58,0.00±0.0,00.00±0.00,00.00±0.00. Treatment with VCR produced a dose-dependent decrease in NDI: There was a significant decrease when the cells were treated with VCR at a dose of 0.01μg ml-1 i.e. 1.82±0.04 for treated cells versus 2.34±0.01 for untreated cells.For donor 2: The frequency of MN, MNC , Buds, NPBs and apoptotic cells increased in a dose dependent manner from 19.00±2.65,17.00±2.08,5.33±0.33,1.67±0.67,0.33±0.33 to 74.67±1.33,60.00±0.58,17.00±1.53,5.00±0.58,12.67±0.88 at the dose of 0.01μg ml-1 and 0.08μg ml-1 . There is significant differences (P< 0.05 or P< 0.01) from 0.01μg ml-1 (0.02μg ml-1 for AC) compared to untreated control 6.00±1.53,5.67±1.20,1.67±0.33,0.00±0.00,0.00±0.00,0.00±0.00. In the NDI there was a significant decrease when the cells were treated with VCR at a dose of 0.01μg ml-1 i.e. 1.83±0.05 for treated cells versus 2.27±0.01 for untreated cells.(3) hprt gene mutation assayThere was a significant increase (P<0.05) in Mf-hprt when the cells were treated with VCR at a dose of 0.02μg ml-1VCR, i.e. 1.01±0.04, 1.10±0.04 for treated cells versus 0.85±0.02, 0.80±0.02 for untreated cells in two donors. Significant increases (P<0.01) were found at doses above 0.02μg ml-1VCR.(4) TCR gene mutation assayAfter exposure to VCR for 24h, the TCR gene mutation frequencies were 2.65±0.34×10-4,3.66±0.65×10-4 for 0.01μg ml-1, 3.81±0.55×10-4,4.15±0.67×10-4 for 0.02μg ml-1, 7.68±1.13×10-4,6.51±0.30×10-4 for 0.08μg ml-1 and 1.97±0.12×10-4,2.46±0.41×10-4 for the control in two donors, respectively. There was a significant increase (P<0.05 or P< 0.01) when the cells were treated with VCR at a dose of 0.04μg ml-1 and 0.02μg ml-1 VCR in donor 1 and 2. An ascending trend was observed in the TCR gene mutation test when doses of VCR increased.4. ConclusionThe results of this experiment indicated that VCR could induce the genetic damage of human lymphocytes at different genetic endpoints in vitro.5. Novelty5.1 Four cytogenetic assays were utilized to assess the genetic damage of human lymphocytes induced by VCR at different genetic endpoints in vitro.5.2 In CBMN assay several indexes (MN, Buds, NPBs, apoptotic cells and NDI) were simultaneously observed to understand the characteristics of cytogentic damage in human lymphocytes induced by VCR in vitro.5.3 It was firstly reported that gene mutation assays were used to detect genetic damage of human lymphocytes induced by VCR in vitro.
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