EtBr (CAS No. 1239-45-8), MMS (CAS No. 66–27-3), and 4-NQO (CAS No. 56–57-5) were used as test compounds. EtBr was purchased from Nacalai Tesque, Inc. (Kyoto, Japan), and MMS and 4-NQO were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Saline and dimethyl sulfoxide (DMSO) were obtained from Otsuka Pharmaceutical Factory, Inc. (Tokushima, Japan) and Nacalai Tesque, Inc., respectively.
Animals and husbandry
Male Japanese white rabbits (Kbs:JW) of 9–12 weeks of age were purchased from Kitayama Labes, Co., Ltd. (Nagano, Japan), and the test compounds were administered at 11–15 weeks of age (bodyweight 2.0–2.7 kg). The rabbits were individually housed in air-conditioned rooms with 19–25 °C temperature, 40–70% relative humidity, and a 12-h light/dark cycle. Each rabbit was provided with commercial pellet feed (LRC4; Oriental Yeast Co., Ltd., Tokyo, Japan) and tap water ad libitum. The dumbbell made from polypropylene and wood gnawing block (Bio-Serv, Flemington, NJ) were placed in each cage as the environmental enrichment devices. The animals acclimated for at least 6 days before the experiments. Animal care and treatment were provided in accordance with the standard procedures of the facility, which are fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. All experimental procedures were in accordance with the guidelines for animal experimentation at Senju Pharmaceutical Co., Ltd., and the protocol was reviewed by the Institutional Animal Care and Use Committee of Senju Pharmaceutical Co., Ltd.
Procedures for animal treatments
The study schedule is shown in Fig. 1. Experiments were performed per sampling time, and 32 clinically normal rabbits were randomly assigned to each treatment group. To prepare the dosing solutions, EtBr and MMS were dissolved in saline, and 4-NQO was suspended in 5% DMSO in saline. Based on the in vivo corneal comet assay results , the concentrations that do not cause irritative or histopathological changes in the eye were set. Saline was used as a negative control in this study because the value of the % tail DNA in the 5% DMSO-treated cells (7.9%) was within the range of that in the saline-treated cells (7.3–8.8%) in the previously performed corneal comet assay in rabbits .
For comet assay, 50 μL of saline or 3% MMS (1.5 mg/eye) was instilled once per right eye of 3 rabbits per group, and 50 μL of 1% EtBr (0.5 mg/eye) or 1% 4-NQO (0.5 mg/eye) was administered to the left eyes in the same manner (three eyes per group). After ocular instillation, the eyelids were artificially blinked several times. The rabbits were euthanized after 0.5, 2, 4, 6, and 24 h by intravenously administering an overdose (approximately 90 mg/kg) of thiopental solution (Ravonal; Nipro ES Pharma Co. Ltd., Osaka, Japan). The eyeballs of the rabbits were collected after euthanasia and subjected to the comet assay.
For histopathological examination, 50 μL of saline or 3% MMS was instilled once onto the right eyes of one rabbit per group, and 50 μL of 1% EtBr or 1% 4-NQO was administered to the left eye in the same manner as the comet assay (one eye per group). Twenty-four hours after administration, the anterior segment of the eye was observed using a slit-lamp biomicroscope SL-130 (Carl Zeiss Meditec AG, Jena, Germany). The corneal epithelial damage was evaluated by staining with fluorescein (AYUMI Pharmaceutical Corporation, Tokyo, Japan). After anterior ocular observation, the rabbits were euthanized in the same manner as the comet assay, and the eyeballs of the rabbits were collected.
Isolation of corneal epithelial cells for the comet assay
The collected eyes were washed with Ca2+– and Mg2+-free phosphate-buffered saline (PBS). After washing, the corneas were removed from the eyes using scissors. The removed corneas were treated with 1.2 unit/mL Dispase II (Roche Diagnostics K.K., Tokyo, Japan) in minimum essential media (MEM; Thermo Fisher Scientific K. K., Tokyo, Japan) supplemented with 10% bovine serum (10% BS/MEM) overnight at 4 °C. Corneal epithelial cells were isolated using a spatula and placed into fresh 10% BS/MEM. The cells were centrifuged at 200×g for 5 min, and the supernatants were discarded. The cells were resuspended in 1 mL of 0.25% trypsin (Thermo Fisher Scientific K. K.) and incubated for 10 min at 37 °C. Furthermore, 8 mL of 10% BS/MEM was added to the cells, and the cell suspensions were passed through a 70-μm cell strainer. The cells were centrifuged at 200×g for 5 min, and the supernatants were discarded. The cells were then resuspended with PBS to a density of approximately 2 × 105 cells/mL.
Alkaline comet assay
The alkaline comet assay was conducted according to previously published method . A 30 μL of the prepared cell suspension was mixed with 270 μL of melted agarose solution (CometAssay LMAgarose; Trevigen, Inc., Gaithersburg, MD). Then, 30 μL of this mixture was placed in each well of a 20-well slide (CometSlide HT, Trevigen, Inc.), and the slide was left for approximately 10 min at 4 °C to solidify the agarose. The slide was immersed in lysis solution (pH 10) containing 2.5 M sodium chloride, 100 mM di-sodium dihydrogen ethylenediaminetetraacetate dihydrate (EDTA∙2Na), 10 mM tris(hydroxymethyl)aminomethane (Tris), and 1% (v/v) polyethylene glycol mono-p-isooctylphenyl ether for 1 h at 4 °C. Subsequently, the slide was immersed in an alkaline unwinding solution (200 mM sodium hydroxide and 1 mM EDTA, pH > 13) for 20 min at room temperature. Electrophoresis was performed with the same solution at 1 V/cm for 30 min under refrigeration. After electrophoresis, the slide was washed twice with ultrapure water and dehydrated by immersion in ethanol for 10 min. The slide was stained with SYBR Green I Nucleic Acid Gel Stain (excitation maxima at 497 nm, emission maxima at 520 nm; Thermo Fisher Scientific K. K.) diluted 1:1000 with Tris-EDTA buffer (pH 7.5) and mounted using ProLong Gold (Thermo Fisher Scientific K. K.). The slide was observed using a BX51 fluorescence microscope (Olympus Corporation, Tokyo, Japan) with a NIBA filter (excitation at 460–495 nm and emission at 510–550 nm) equipped with a CCD camera (scA1300-32 fm; Basler AG, Ahrensburg, Germany).
First, the number of “hedgehogs” was counted among 150 comets per eye (450 comets per group). According to the Atlas of Comet Assay Images, hedgehogs are highly fragmented cells presenting as a small or non-existent comet head and large diffuse comet tail . Second, 150 scorable comets (i.e., with a clearly defined head and tail with no interference from neighboring cells) without hedgehogs were measured per eye (450 comets per group). The % tail DNA (DNA fluorescence intensity in the tail/total DNA fluorescence intensity × 100) was measured as a DNA damage indicator using the Comet Assay IV software, version 4.3.2 (Perceptive Instruments, Haverhill, UK).
Microscopic examination of the corneal tissue
The collected eyes (one eye per group) were fixed with 1% formaldehyde/2.5% glutaraldehyde in 0.1 M phosphate buffer fixative overnight at 4 °C and post-fixed with 10% neutral-buffered formalin solution. The tissues were dehydrated using a graded alcohol series and embedded in paraffin. Approximately 3-μm-thick corneal tissue sections were prepared and stained with hematoxylin and eosin. All microscopic images were obtained with a BX53 microscope fitted with a DP74 digital camera (Olympus Corporation) and analyzed using the cellSens Standard imaging software, version 2.3 (Olympus Corporation).
The mean and standard deviation (SD) of the % tail DNA and the number of hedgehogs were calculated for all experimental groups. The median % tail DNA was also calculated according to the OECD TG 489 . The data were assumed to have a normal distribution and homogeneous variance. For each corneal sampling time, Student’s t-test (one-tailed) was used to compare the mean of each test compound group with that of the negative control group for % tail DNA and hedgehog. JMP version 15.1.0 (SAS Institute Japan, Ltd., Tokyo, Japan) was used for all statistical analyses. Probability (p) values of < 0.05 were considered statistically significant.
Measurement of DNA damage and cell viability in vitro
To assess time-course changes in DNA damage after short-time exposure to the genotoxic compounds, the in vitro comet assay was performed using HCE-T cells with reference to our previous study . Briefly, the HCE-T cells were seeded in a 12-well culture plate at a density of 5 × 104 cells/well (1 mL/well) and incubated for approximately 24 h. After a 24-h incubation, the cells were washed with PBS. The cells were then exposed to a mixture of 500 μL of serum-free medium and an equal volume of the test compounds which were dissolved in distilled water (Otsuka Pharmaceutical Factory, Inc.) at twice concentration of final concentration (final concentration: 0.5% EtBr, 0.5% MMS, and 0.001% 4-NQO) for 1 min at room temperature. After the treatment, the cells were washed with PBS, and 1 mL of fresh culture medium was added. The cells were further incubated for 2, 4, 6, and 24 h at 37 °C. After incubation, the cells were trypsinized (TrypLE™ Express, Thermo Fisher Scientific K.K.). In the same manner, the cells were exposed to the test compounds or distilled water as a negative control for 1 min to evaluate DNA damage immediately after exposure. Immediately after the treatment, the cells were washed and trypsinized. The collected cells were centrifuged and re-suspended in PBS at a density of 1–2 × 105 cells/mL. Comet assay was performed as described previously herein (n = 3/group).
For cytotoxicity assay, the HCE-T cells were seeded in a 96-well culture plate at a density of 5 × 103 cells/well (100 μL/well) and incubated for approximately 24 h. After the 24-h incubation, the cells were washed with PBS. The cells were then exposed to a mixture of 50 μL of serum-free medium and an equal volume of test compounds at twice concentration of final concentration for 1 min at room temperature. After the treatment, the cells were washed with PBS, and 100 μL of fresh culture medium was added. The cells were further incubated for 2, 4, 6, and 24 h at 37 °C. After that, the cells were incubated with 100 μL of Cell Counting Kit-8 (Dojindo Laboratories Co., Ltd., Kumamoto, Japan) diluted to one-tenth with a serum-free medium for 2 h at 37 °C. Absorbance was measured at 450 nm using a microplate reader (Synergy HTX, BioTek Instruments Inc., Winooski, VT). Cell viability was calculated as follows: the absorbance of compound-treated cells/the absorbance of non-treated cells, expressed as a percentage (n = 3/group).
The mean and SD of the % tail DNA and cell viability (%) were calculated for all experimental groups. For % tail DNA, Dunnett’s multiple comparison test (one-tailed) was used to compare the mean of each test compound group with that of the negative control group. For cell viability (%), Dunnett’s multiple comparison test (one-tailed) was used to compare the mean of each test compound group with that of the non-treated group. JMP version 15.1.0 was used for all statistical analyses. Probability (p) values of < 0.05 were considered statistically significant.
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