DNA Damage Induced by Satraplatin - Lab Report Example

Experiment 1. DNA damage induced by satraplatin as measured by alkaline Comet assay Human lymphocytes treated with different concentrations of satraplatin were subjected to a modified Comet assay to determine the formation of DNA crosslinks. In the comet assay, DNA damage is evaluated by the Olive Tail Moment (i.e., % DNA x distance of centre of gravity of DNA). Following a 30-min incubation of the human lymphocytes with 50μM H2O2, substantial DNA damage was evident as a sharp increase in the mean Olive Tail Moment (OTM) values compared to the control sample (Fig. 1). In contrast, a reduction in the mean Olive Tail Moment induced by H2O2 was observed in cells that had been subjected to a one-hour pre-incubation with satraplatin (0.02μM - 200μM), indicating the formation crosslinks to rejoin DNA strand breaks. Figure 1 shows the results expressed as mean OTM obtained after H2O2 treatment of human lymphocytes pre-incubated with 0.02μM, 0.2μM, 2μM, 20μM and 200μM satraplatin in comparison with appropriate controls. All the concentrations of satraplatin tested produced statistically significant reduction in mean OTM. Besides, the rate of decrease was inversely proportional to the concentration of satraplatin used. Thus, while the least reduction in mean OTM of ~30% was observed with 0.02μM satraplatin, the maximum reduction of ~75% was noted in the case of human lymphocytes incubated with 200μM satraplatin. 0.2μM satraplatin produced 50% reduction in mean OTM, 2μM satraplatin produced 55% and 20μM satraplatin yielded 60% reduction in mean OTM. 2. The cytokinesis-block micronucleus (CBMN) assay The cytokinesis-block micronucleus (CBMN) assay is another well-established approach to measure the genotoxic potential of chemicals. The technique detects modification of chromosome structure that leads to the formation of micronuclei in interphase cells, and also corresponds to initial genetic changes. The formation of micronuclei by satraplatin was studied using the CBMN assay. Positive control consisted of mitomycin C (MMC) which was run in parallel with satraplatin. As shown in Table 1 and depicted in Fig. 2, the mononucleted cells frequencies in lymphocytes treated with 0.02μM, 0.2μM and 2μM satraplatin were 482, 711 and 867, respectively. These values were considerably higher than those obtained for either the control (251) or MMC (312) indicating greater genomic instability in satraplatin treated lymphocytes. In opposition to this trend, the binucleation frequency produced by satraplatin showed an inverse relationship to satraplatin concentration (Table 1). Also, the values ranged from 12% to 48% as compared to 73% and 66% shown by the control and MMC amended lymphocytes. In comparison to the control with no added test chemicals, a significant 51% increase in binucleated cells with micronuclei (Bi MN) was observed when MMC was used. With satraplatin used at a concentration of 2μM, significantly higher (61%, P=0.01) Bi MN were observed whereas 0.02μM and 0.2μM satraplatin accounted for ~24% and 48% Bi MN, respectively. In contrast, the number of multinuclei cells produced by different concentrations of satraplatin was not significantly different from those observed with either the control or MMC treated lymphocytes (Table 1, Fig. 2). As seen from Table 1, the values of NDI decreased in a dose-dependent manner in the presence of satraplatin. Also, the values were 12 to 36% lower than the NDI of control. Incidentally, the NDI values obtained in control and MMC treated lymphocytes were very similar (i.e., 1.77 and 1.73). 3. Sister Chromatid Exchange induction. Sister chromatid exchanges (SCEs) are extremely sensitive indicators of chromosome damage. Also, SCE is a mechanism that resolves replication-dependent double-strand breaks and is thus an indicator of DNA damage and repair. Thus, analysis of SCEs facilitates a highly sensitive detection/monitoring of damage to DNA caused by chemical agents since SCEs are produced by chemicals that damage DNA. Human lymphocytes were incubated with satraplatin or without any treatment (control) for 48 hr. In vitro challenge with MMC was used as a positive control. The plot of SCEs measured per metaphase in relation to satraplatin concentration is depicted in Fig.3. The number of SCEs formed per metaphase was seen to be significantly higher in the case of MMC (0.4 μM) as also satraplatin ( 0.02 μM - 2μM) treatments than in the control (Fig. 3). The rate of SCE occurrence in satraplatin treatment was found to increase linearly with satraplatin concentration. Fig. 3 shows that, compared to MMC, enrichment of SCEs per metaphase in satraplatin treatment was nearly 18% higher when 0.2μM satraplatin was used, and registering a further 60% increase when satraplatin concentration was increased to 2μM. Experiment 2 1. The cytokinesis-block micronucleus (CBMN) assay A second set of experiments was conducted to study the formation of micronuclei in human lymphocytes treated with vinflunine using the cytokinesis-block micronucleus (CBMN) assay. Again, MMC (0.4μM) was used as a positive control. The mononuclei (MN) frequencies observed in lymphocytes treated with 0.06μM, 0.6μM and 6μM vinflunine are recorded in Table 2 and also plotted as a bar graph (Fig. 4). The formation of mono-, bi- and multinucleated cells in MMC-treated lymphocytes was not significantly different from the control cells with no treatment (Fig. 4). However, compared to the MMC treatment, treatment with 0.6μM and 6μM vinflunine resulted in statistically significant 1.8-fold (P = 0.01) and 2.3-fold (P=0.001) increases in MN formation in the human lymphocytes. Furthermore, with all the 3 concentrations of viflunine tested significant decrease in the binucleated cells was obvious (Fig. 4). In contrast, only the lowest concentration of viflunine tested i.e., 0.06μM, gave rise to significantly high multinucleated cells; while the result was insignificant in all other treatments, including MMC treatment. Treatment with vinflunine did not induce any increase in MN within binucleated cells. But it increased MN within mononucleated cells as shown in Fig. 5. As compared to the MMC-treated lymphocytes, NDI values were found to be decreased only in cells treated with 0.6μM (1.2-fold decrease) and 6μM (1.3-fold decrease) vinflunine. The evaluated decrease corresponded to 1.2-fold (0.6μM) and 1.3-fold (6μM) (Table 2). 2. Sister Chromatid Exchange assay The results of the Sister Chromatid Exchange assay performed with human lymphocytes incubated with vinflunine or without any treatment (control) for 48 hr and with MMC used as positive control are tabulated as Table 3. SCE rate/cell was found to go up to 24 ± 0.73 in MMC-treated cells from 2.3 ±0.66 in control cells without treatment. In vinflunine-exposed cells, the observed SCE rates were 2.89 ± 0.60, 2.66 ± 0.33 and 2.5 ± 0.40, respectively when the exposure concentrations were 0.06μM, 0.6μM and 6μM. Also, in the presence of 0.6μM and 6μM vinflunine, the proliferative rate index (PRI) decreased from the control value of 2.54 to 1.56 and 1.32, respectively (Table 3). An important observation was that vinflunine at a concentration of 0.06 led to an increase in the number of chromosomes per cell to 92 (Table 3). Figures and Tables Fig. 1. Effect of pre-treatment with satraplatin (0.02 µM - 200 µM) in the Comet assay measured as Olive Tail Moment. Statistical significance: **P < 0.01; ***P < 0.001. Bars indicate the standard error of the mean. Fig. 2. Cytokinesis-block micronucleus (CBMN) assay of human lymphocytes treated with 0.02μM to 2μM satraplatin. Mitomycin C (MMC, 0.4 μM) was used as a positive control. Fig. 3. Rate of sister chromatid exchanges per metaphase in control (without any treatment) and satraplatin-treated human lymphocytes. Mitomycin C (MMC, 0.4μM) formed the positive control. Statistical significance: *P < 0.05; **P < 0.01; ***P < 0.001. Bars indicate the standard error of the mean. Fig. 4. Cytokinesis-block micronucleus (CBMN) assay of human lymphocytes treated with 0.06μM to 6μM vinflunine. Mitomycin C (MMC, 0.4μM) was used as a positive control. Statistical significance: *P < 0.05; **P < 0.01; ***P < 0.001. Fig. 5. Formation of mononuclei within mononucleated human lymphocyte cells generated by vinflunine (0.06μM, 0.6μM and 6μM concentration). Control cells were without any treatment. Bars indicate the standard error of the mean. Table 1. Treatment Mono Bi %Bi MN Multi %Bi NDI P Value Control 251 728 3 12 72.8 1.77 MMC 312 659 51 15 65.9 1.73 0.03 Satraplatin(0.02µM) 482 483 23.61 11 48.3 1.56 0.05 Satraplatin(0.2µM) 711 248 48.3 10 24.8 1.3 0.01 Satraplatin(2µM) 867 123 67.2 13 12.3 1.14 0.01 Table 2. Column1 Mono BI Multi MNMono %Bi MN NDI P Value control 251 728 12 2 3 1.77 MMC 0.4µM 312 659.33 15 8 51 1.73 Vin 0.06µM 233 600 165 82 4 1.92 0.05 Vin 0.6µM 566 418 23 125 3 1.44 0.01 Vin 6µM 690 289 18.66 129 4 1.32 0.001 Table 3. Treatment Concentration SCE/Cell±S.E PRI Chromosome No. Control 0 2.3 ±0.66 2.54 46 MMC 0.4µM 24 ± 0.73 2.13 46 Vinflunine 0.06µM 2.89 ± 0.60 2.48 92** Vinflunine 0.6µM 2.66 ± 0.33 1.56 46 Vinflunine 6µM 2.5 ± 0.40 1.32 46 Read More