Page 46 - ITPS-6-2

INNOSC Theranostics and
Pharmacological Sciences Genotoxicity of (4-fluorophenyl) thiazolidin-4-one

1 to 20 µM, were added in triplicates and incubated for from the ratio of the number of dividing cells (prophase
24 h. The media was discarded carefully to avoid cell to telophase) to the total number of cells observed. At
dislodgement, and serum-free media containing MTT was least 2000 cells per test concentration were screened in
added. The plate was then kept in a CO incubator for 2 triplicates.
2
– 3 h to allow drying. Next, 100 µl of dimethylsulfoxide
(DMSO) was added to each well to dissolve the crystals, 2.4.4. MN assay
and OD readings were taken at 570 nm to calculate the MN assay is a method used to assess both the clastogenic and
percentage of cell viability. aneugenic potency of a test chemical. In this study, CHO-
Based on the cytotoxicity results from the MTT K1 cells were treated with three different concentrations of
assay, three sub-lethal concentrations of the (4-TH) 4-TH for 24 h. After treatment, the cells were washed with
(2, 5, and 7.5 µM) were selected for further cytogenetic PBS and treated with cytochalasin-B (3 µg/ml) for another
toxicity studies, including chromosome aberration, mitotic 24 h to obtain bi-nucleated cells. Subsequently, the cells
index, and MN assays. All these tests were performed were collected and incubated with 0.9% (v/v) sodium citrate
following our previously published work [31,33] . for 10 min at 8°C. Following this, the cells were centrifuged
at 1200 rpm for 5 min, and the pellet was collected. To this
2.4.2. Chromosomal aberration assay pellet, 0.5 ml of fresh sodium citrate was added, and smears
Chromosome aberration assay is performed to assess the were prepared on clean, grease-free glass slides. The slides
clastogenic effects of compounds. In the present study, were then kept overnight at 37°C in an incubator under
mitomycin-C and DMSO were used as the positive and 85% relative humidity. Finally, the slides were stained with
vehicle controls, respectively, for comparison. The assay 0.5% of Giemsa stain to visualize and score the presence of
procedure followed our earlier work with some minor MN. At least 2000 cells were scored in triplicate to calculate
modifications, which are described briefly in the following. the percentage of MN for each concentration, which was
Cells were cultured and categorized into five different then compared with the control groups.
test groups. One group was treated with only DMSO as a 2.4.5. Cell cycle analysis
vehicle control, and another with mitomycin-C (2.5 µM) as
a positive control. The remaining three groups were treated The effect of 4-TH on different phases of the cell cycle
with three different concentrations of 4-TH. All the treated was assessed using flow cytometry analysis, following our
[33]
cells were incubated for 24 h in 5% CO at 37°C, then previously reported protocol . Briefly, CHO-K1 cells were
2
transferred to fresh media, and treated with 0.02% (w/v) treated with three different concentrations of 4-TH (2, 5, and
colchicine for 30 – 40 min to arrest the cells in metaphase. 7.5 µM). Following treatment, the cells were trypsinized,
The collected cells were washed with phosphate-buffered collected in ice-cold 1 × PBS, and then washed twice.
saline (PBS) and trypsinized. After hypotonic treatment Subsequently, they were fixed overnight in 70% ethanol
with 0.5% KCl, cells were incubated in a 37°C incubator at −20°C. The following day, the sample was washed with
for 20 – 30 min and then centrifuged at 2000 rpm for 1×PBS, and the cells were stained using a propidium iodide
5 min. The cell pellet was collected and fixed with a fixative (PI) solution supplemented with RNAse and triton-X for
(1:3 v/v methanol and acetic acid). Chromosome slides 45 min at room temperature. After staining, the cells were
were prepared using the flame drying method and stained washed with 1 × PBS to remove any unbound PI, and cell
with 10% Giemsa. At least 100 well-spread chromosome cycle analysis was performed using the BD fluorescence-
spreads in triplicates were scored from each concentration activated cell sorting (FACS) Canto system (USA).
of 4-TH and from both control groups. During the count, 2.5. Statistical analysis
each metaphase spread was screened to determine the
clastogenic effects of the chemical by observing chromatid Each experiment was carried out in triplicate. The results
and chromosome aberrations, such as gaps, breaks, were analyzed using Student’s t-test, followed by one-way
fragments, minutes, pulverization, and translocations. analysis of variance tests (Dennett’s post-test was performed
using GraphPad Prism version 8, GraphPad Software, San
2.4.3. Mitotic index assay Diego, CA). The mean differences were compared with the
Mitotic index assay is performed to assess the effect of corresponding control samples to determine the level of
a compound on the proliferation rate of the cells and significance.
provides information on the rate of cell division in the 3. Results and discussions
presence of a toxicant. In the present study, slides were
prepared following a similar procedure to the chromosome Despite the numerous bioactive properties reported
aberration assay, and the mitotic index was obtained for 4-thiazolidinone derivatives, a comprehensive

Volume 6 Issue 2 (2023) 3 https://doi.org/10.36922/itps.0618