ASSESSMENT OF GENOTOXICITY OF VINCRISTINE, VINBLASTINE AND VINORELBINE IN HUMAN CULTURED LYMPHOCYTES: A COMPARATIVE STUDY
Mhaidat NM, Alzoubi KH, Khabour OF, Alawneh KZ, Raffee LA, Alsatari ES, Hussein EI, Bani-Hani KE
*Corresponding Author: Nizar M. Mhaidat, Ph.D., Department of Clinical Pharmacy, Faculty of Pharmacy, Jordan University of Science and Technology, Amman-Ramtha Hwy, Irbid 22110, Jordan. Tel: +962- 2-720-100. Fax: +962-2-720-1075. E-mail: nizarm@just.edu.jo
page: 13

MATERIALS AND METHODS

Subjects. Five healthy male nonsmoking volunteers, with an age range of 20-25 years old, were the blood donors. Exclusion criteria were alcohol, cigarette smoking, medications and vitamin use. A sample of whole venous blood (15 mL) was collected in heparin tubes from each donor under sterile conditions. Whole blood cells were cultured within 1 hour of sampling. Informed consent was obtained from each volunteer. This study was approved by the Institutional Review Board of Jordan University of Science and Technology, Irbid, Jordan. Drugs and Treatment. Vincristine, VBL and VRL were purchased from Sigma-Aldrich Produktions GmbH (Steinheim am Albuch, Germany). To evaluate the effect of VCR, VBL and VRL on DNA, seven groups were used: a control group and drug-treated groups (VCR, VBL and VRL; each at concentrations of 0.01 and 0.1 μg/mL). The control group was treated with distilled water. Drug-treated groups were treated with the corresponding drug 4 hours prior to harvesting. This was based on previous studies [21,22]. Lymphocytes Culture. Blood cultures were set up by inoculating 1 mL of freshly drawn blood into 75 mL tissue-culture flasks containing 9 mL of peripheral blood (PB)-Max medium composed of Roswell Park Memorial Institute (RPMI) 1640 medium with 15.0% fetal bovine serum (FBS), 1.0% penicillinstreptomycin and 3.0% of phytohaemagglutinin (Gibco- Invitrogen Ltd., Paisley, Ren-frewshire, UK). To collect metaphase cells, cultures were exposed to 100 μL of 10 μg/mL colcemid (Gibco-Invitrogen Ltd.) 2 hours prior to cell harvesting [23,24]. The 8-Hydroxy-2-Deoxy Guanosine Assay. The 8-OHdG assay was performed as previously described [25]. In brief, blood cultures were set up by inoculating 0.5 mL of freshly drawn blood into 50 mL culture flasks containing 4.5 mL of PB-Max medium. Then, the cultures were incubated for 72 hours at 37 °C. Treatment was as described above. Cultures were then centrifuged at 1000 × g and 200 μL from each was used for the 8-OHdG assay. Competitive enzyme-linked-immunosorbent serologic assay (ELISA) for 8-OHdG were performed according to the manufacturer’s protocol protocol (Abcam Inc., Cambridge, MA, USA). Plates were read at 405 nm using an Epoch Biotek microplate reader (BioTek, Winooski, VT, USA). Levels of 8-OHdG were calculated from the blotted standard curve. Sister Chromatid Exchange Assay. A 25 μL of 0.01 g/L mL bromodeoxyridine (BrdU; Sigma- Aldrich, St. Louis, MO, USA), was added to the culture media prior to incubation and throughout the experiment [23,24]. All cultures were maintained in total darkness to minimize photolysis of BrdU [26-28]. The culture initiation and slide preparation were similar to that described for the chromosomal aberration assay. Air-dried slides were differentially stained by 10 μg/mL of Hoechst 32285 dye solution (Fluka, Münich, Germany) for 15 min., followed by rinsing in water and mounted in McILvian buffer (Fisher Scientific, Waltham, MA, USA) (pH 8.0). The slides were then irradiated with two UV lamps (350 nm and 15 W each) at a distance of 7 cm for 35 min. at 40 °C [22]. Slides were then rinsed with distilled water, restained for 6-8 min. with 5.0% Giemsa in phosphate buffer (pH 6.8) and then air-dried [27,29,30]. Sister chromatid exchanges were scored using second division cells (M2, 50 cells per donor) that contained 42-46 chromosomes and high resolution (Nikon, Shinagawa, Japan) light microscopy [25,31]. Cells in M2 phase have two differentially stained chromatids, one lightly stained and one darkly stained, while M1 phase cells are uniformly stained (two chromatids are darkly stained), and M3 phase cells contain a mixture of lightly stained, darkly stained and differentially stained chromatids [26]. Cell Kinetics Analysis. The mitotic index was calculated by analyzing at least 1000 cells from each subject and scoring the cells that were in metaphase as previously described [26]. For the cell proliferation index, 100 metaphase cells from each donor were scored. The proliferation index was calculated using the following formula: (1 X M1 + 2 X M2 + 3 X > M3)/100, where M1, M2 and M3 are the number of cells at the first, second and third metaphase, respectively [24,32]. Depending on the proliferation index, the average generation time was calculated as the number of hours for the cells in BrdU (Sigma- Aldrich), divided by the proliferation index [28]. Statistical Analyses. Statistical analysis was performed using Graphpad Prism statistical software version 4 (GraphPad Software, Inc., La Jolla, CA, USA). Data were expressed as mean ± standard error Figure 1. The level of 8-OHdG values in controls, VCR, VBL and VRL groups; each at concentrations of 0.01 and 0.1 μg/mL. The levels of 8-OHdG in all drug-treated groups at concentrations of 0.01 and 0.1 μg/mL were higher than control group. The asterisks (*) indicate significant differences (p <0.05) from the control group. The hash (#) indicates significant difference from all other groups. (SE). The comparisons of parameters were performed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. Differences were regarded as significant at p <0.05.



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