Chemicals
Common trade name of the chemotherapeutic drug is vinblastine sulfate, velban and others. Vinblastine was obtained from Sigma Aldrich (MO,USA). Drug Bank Accession Number: DB00570. All other chemicals were purchased from ADWICC (Cairo, Egypt).
Animals
Male white Swiss mice (Mus musculus), aged 9 to 12 weeks, were used in all experiments. Animals were obtained from a closed random-bred colony at the National Research Centre (Giza, Egypt). Mice used for each experiment were of similar age (±1 week) and weight (± 2 g). Animals were housed in polycarbonate boxes with steel wire tops and bedded with wood shavings. Ambient temperature was controlled at 22°C ± 3°C with a relative humidity of 50% ± 15% and a 12 h light/dark photoperiod. Food and water were provided ad libitum. The experiments were conducted according to the Animal Research Ethical Committee Guidelines of the National Research Centre, Egypt. The Approval Certificate is under number: 19 163.
Experimental design
In these experiments a total of 85 mice were taken as follows: 40 mice for chromosomal aberrations in bone marrow and mouse spermatocytes, 25 mice for DNA fragmentation and alkaline comet assay and 20 for morphological sperm abnormalities. In each of these testes, mice were subdivided into groups (5 /group).
The main groups represented chromosomal aberration analysis: Group I: Negative control, Groups II-V, mice were i.p injected with a single dose of VB (3, 4.5, 6 and 10 mg/kg), Groups VI-VIII in which mice received repeated i.p injections (three successive days) of VB at the dose levels 3, 4.5 and 6 mg/kg. In all experiments samples were taken 24h after the last treatment.
Analysis for DNA damage, showed five groups: Animals were treated with a single i.p injection of VB at the dose levels 3, 4. 5, 6 and 10 mg/kg, in addition to the control group. Samples were taken 24h after injection.
Analysis of sperm abnormalities showed four groups as follows: Negative control group and three treated groups with VB (3, 4.5 and 6 mg/kg, 3 injections) and samples were taken 35 days after the 1st injection. Different doses were taken to cover all doses required for different types of human cancerous.
Cytogenetic analysis
Chromosomal aberration assay in mouse bone marrow and spermatocytes
Mitotic and meiotic chromosomes were prepared from bone marrow and testis of the same animal, respectively. Bone marrow chromosomes were prepared according to the technique described by Diab et al. [11]. In brief, mouse bone- marrow cells were collected from both femurs, cells were incubated in hypotonic solution (KCL 0.075 M) for 20 min at 37°C, and then centrifuged. The cell pellets were suspended in a fixative (methanol/glacial acetic acid 3:1). This step was repeated at least twice, then the cells were suspended in a few drops of fixative and spread onto frozen slides, air-dried, stained with 10% Giemsa for 30 min, washed, and air dried again.
Spermatocyte chromosomes were prepared from the testes according to the protocol described by Evans et al.[12]with some modifications [13]. Briefly, the testis was removed and squashed into a petri dish containing an isotonic solution of 2.2% trisodium citrate. Then the cell suspension was centrifuged for 5 minutes at 1500 rpm. The cell pellet was incubated in a hypotonic solution of 1.1% trisodium citrate for 20 minutes at 37C◦ followed by centrifugation. The cell pellet was washed twice by a freshly prepared fixative. A few drops of the fixative cell suspension were dropped in clean microscopic slides, air dried and stained with 10% Giemsa stain. One hundred well-spread metaphases were analyzed per mouse describing different kinds of chromosome abnormalities (CAs) in bone marrow and mouse spermatocytes. Scoring for CAs was performed under 2000× magnification with a light microscope.
DNA fragmentation assay in mouse spleen cells
1. DNA gel electrophoresis laddering assay
Apoptotic DNA fragmentation was qualitatively analyzed by detecting the laddering pattern of nuclear DNA as described according to Lu et al. [14]. Briefly, spleen tissues were homogenized, washed in PBS, and lysed in 0.5 ml of DNA extraction buffer (50 mM Tris–HCl, 10 mM EDTA. 0.5% Triton, and 100 μg/ml proteinase K, pH 8.0) for overnight at 37 °C. The lysate was then incubated with100 μg/ml DNase-free RNase for 2h at 37 °C, followed by three extractions of an equal volume of phenol/chloroform (1:1 v/v) and a subsequent re-extraction with chloroform by centrifuging at 15,000 rpm for 5 min at 4 °C. The extracted DNA was precipitated in two volume of ice-cold 100% ethanol with 1/10 volume of 3 M sodium acetate, pH 5.2 at −20 °C for 1h, followed by centrifuging at 15,000 rpm for 15 min at 4 °C. After washing with 70% ethanol, the DNA pellet was air-dried and dissolved in 10 mM Tris–HCl/1 mM EDTA, pH 8.0. The DNA was then electrophoresed on 1.5% agarose gel and stained with ethidium bromide in Tris/acetate/EDTA (TAE) buffer (pH 8.5, 2 mM EDTA, and 40 mM Tris–acetate). A 100-bp DNA ladder (Invitrogen, USA) was included as a molecular size marker and DNA fragments were visualized and photographed by exposing the gels to ultraviolet trans-illumination.
2. Diphenylamine reaction procedure
Animal spleen tissues were used to determine the quantitative profile of the DNA fragmentation. Spleen samples were collected immediately after sacrificing the animals. The tissues were lysed in 0.5 ml of lysis buffer containing, 10 mM tris-HCl (pH 8), 1 mM EDTA, 0.2% triton X-100, centrifuged at 10 000 rpm (Eppendorf) for 20 min at 4°C. The pellets were re-suspended in 0.5 ml of lysis buffer. To the pellets (P) and the supernatants (S), 0.5 ml of 25% tri-chloroacetic acid (TCA) was added and incubated at 4°C for 24 h. The samples were then centrifuged for 20 min at 10 000 rpm (Eppendorf) at 4°C and the pellets were suspended in 80 ml of 5% TCA, followed by incubation at 83°C for 20 min. Subsequently, to each sample 160 ml of Diphenyl Amine (DPA) solution [150 mg DPA in 10 ml glacial acetic acid, 150 ml of sulfuric acid and 50 ml acetaldehyde (16 mg:ml)] was added and incubated at room temperature for 24h [15]. The proportion of fragmented DNA was calculated from absorbance reading at 600 nm wavelength using the formula:
% Fragmented DNA = [OD(S)/[OD(S) + OD(P)] X 100
(OD: optical density, S: supernatants, P: pellets)
Comet Assay in the testes
Comet assay was performed referring to the protocol developed by Blasiak et al. [16] with minor modifications. Cells from testes of each treatment were mixed with low-melting-point agarose (ratio of1:10v/v), then pipetted to precoated slides with normal-melting-point agarose. The slides were kept flat at 4°C for 30 min in dark environment. The third layer of low melting point agarose was then pipetted on slides, left to solidify for 30 min at 4°C. The slides were transferred to pre-chilled lysis solution, kept for 60min at 4°C. After that, slides were immersed in freshly prepared alkaline unwinding solution at room temperature in the dark for 60 min. Slides were subjected to an electrophoresis run at 0.8 V/cm, 300mAmps at 4°C for 30 min. The slides were rinsed in neutralizing solution followed by immersion in 70% ethanol and then air-dried. Ethidium bromide was used for slides stain and then visualized by using Zeiss epifluorescence microscope (510–560 nm, barrier filter 590 nm) with a magnification of ×400. 100 cells per animal were scored then analyzed with DNA damage analysis software (Comet Score, TriTek corp., Sumerduck, VA22742). The nonoverlapping cells were randomly selected and were visually assigned a score on an arbitrary scale of 0–3 (i.e., class 0 = no detectable DNA damage and no tail; class 1 = tail with a length less than the diameter of the nucleus; class 2 = tail with length between 1× and 2× the nuclear diameter; and class 3 = tail longer than 2× the diameter of the nucleus) based on perceived comet tail length migration and relative proportion of DNA in the nucleus [17, 18].
Sperm shape abnormalities
Sperm were prepared according to the recommended method of Wyrobek and Bruce [19] with some modifications recorded by Fahmy et al. [20] and smears were stained with 1% Eosin Y. A total of 1000 sperm were counted per animal (5000/each treatment), and different types of sperm abnormalities were scored (Head & Tail abnormalities). Sperm preparations were examined by light microscopy at 1000× magnification.
Data analysis
Data were analyzed using computerized software SPSS (Statistical Package of Social Science, version 20, Armonk, New York: IBM Corp). The data were checked for normality and the homogeneity of the variance using the Kolmogorov-Smirnov's test and Levene's test, respectively. The differences among groups with normal distribution were analyzed by one-way analysis of variance (ANOVA) followed by the Tukey HSD test. The results were regarded as significant when the P-value was less than or equal to 0.05.