Is Cell Death Responsible for False Positive Results of In Vivo Comet Assay?

Chemicals and Animals

In this study, three non-genotoxic chemicals [carbon tetrachloride (CCl4), allyl alcohol (AA) and D-galactoseamine (D-Gal)] that induce necrosis in the liver and two non-genotoxic chemicals [high concentration sodium chloride solution (NaCl, 250mg/mL w/v)] and dimethylamine (DMA) that induce necrosis in the mucosa of stomach were used. Also, two genotoxic compounds, ethyl methane sulfonate (EMS) and diethylnitrosoamine (DEN) were used. DMA and EMS were obtained from Tokyo Kasei Kogyo, Ltd. (Tokyo) and Sigma Chemical Co., St. Louis, MO (U.S.A.), respectively. Other chemicals were from Wako Pure Chemical Industry, Ltd. (Osaka). Regular (GP- 42) and low melting point (LGT) agarose were obtained from Nacalai Tesque (Kyoto) and diluted, respectively, to 1% and 2% in physiological saline. Male ddY mice were obtained from Japan SLC Co. (Shizuoka, Japan) at 7 weeks of age and used after 1 week of acclimatization. They were fed commercial pellets MF (Oriental Yeast Industries Co., Tokyo, Japan) and tap water ad libitum throughout the acclimatization period and the experiment. The animal room was at 20 - 24 °C with a 12-h light-dark cycle.

The Comet Assay

Four mice per group received intraperitoneally CCl4 at 2000 mg/kg or lower, D-Gal at 2000 mg/kg or lower, AA at 100 mg/kg or lower, and DEN at 160 mg/kg, were sacrificed 12 and 24 h after the administration, and the liver was removed and divided in two; one part of each was fixed in 10% formaldehyde. Another part of each used for the comet and diffusion assays was homogenized in 4mL chilled homogenizing solution (pH 7.5) containing 0.075 M

NaCl and 0.024 M Na2EDTA and homogenized gently using a Potter-Elvehjem type homogenizer at 500 - 800 rpm, in ice [12]. To obtain nuclei, the homogenate was centrifuged at 700g for 10 min at 0°C, and the precipitate was re- suspended in chilled homogenizing buffer [9]. Four mice per group received orally NaCl at 2500 mg/kg, DMA at 150 mg/kg, and EMS at 80 mg/kg were sacrificed 3 h after the administration and the stomach was removed. The stomach was opened and rinsed with physiological saline; it was divided into 3 parts (forestomach, glandula stomach, and ledge) as shown in Figure 2. Glandula stomach, and ledge were divided in two; one part of each was fixed in 10% formaldehyde. Another part of each was used for the comet and diffusion assays; the upper and lower layers of mucosa of glandula stomach were scraped separately into 4 mL chilled homogenizing solution (pH 7.5) containing 0.075 M NaCl and 0.024 M Na2EDTA and homogenized gently using a Potter-Elvehjem type homogenizer at 500 - 800 rpm, in ice. To obtain nuclei, the homogenate was centrifuged at 700_g_ for 10 min at 0°C, and the precipitate was re-suspended in chilled homogenizing buffer [12]. Seventy-five µL agarose GP-42 was quickly layered on a slide (Matsunami Glass Ind., Ltd., Osaka, Japan) coated with agarose GP-42 and covered with another slide. The slide sandwiches were placed horizontally to allow the agarose to solidify. The nucleus suspension was mixed 1:1 (v/v) with 2%, 45oC, agarose-LGT, and 75 µL of the nucleus mixture was quickly layered in the same manner after removal of the covering slide. Finally, 75 µl of agarose GP-42 was quickly layered on again. Three slides were prepared from nuclei isolated by homogenization of each sample and were placed in a chilled lysing solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Trizma, 1% sarkosyl, 10% DMSO, and 1% Triton X-100, pH 10) [9] and kept at 0oC in the dark for about one night.

Three slides were prepared from each sample, one for the neutral comet assay, one for the alkaline comet assay, and one for the diffusion assay. After the lysing, one slide was used for neutral comet assay. The slide was placed in 300 mM sodium acetate and 100 mM Tris (pH 10) at 0oC in the dark and for 20 min followed by the electrophoresis at pH 10 for 15 min at 0oC in the dark at 12 V (0.48 V/cm).

After the electropholysis, the slide was treated with chilled alkaline solution (300 mM NaOH and 1 mM Na2EDTA, pH>13) for 5 min.

The remained two slides were used for alkaline comet and diffusion assays. After the lysing, two slides were placed in chilled alkaline solution (300 mM NaOH and 1 mM Na2EDTA, pH>13) in the dark and for 10 min. For the alkaline comet assay, one slide was undertaken the electrophoresis at 12 V (0.48 V/cm, pH>13) in the dark and for 15 min. For the diffusion assay, the slide was not undertaken the electrophoresis. And then, the slides for three assays were neutralized by 400 mM Tris buffer followed by the staining by 50 µL of 20 µg/mL ethidium bromide. In the diffusion and alkaline comet assays, the frequencies of nuclei with diffused DNA (Halo image, Figure 1) and Ghost images (Figure 1) among 1000 nuclei were scored, respectively, using a fluorescence microscopy at 200 x magnification. In the neutral and alkaline comet assays, the length of the whole comet (“length”) and the diameter of the head (“diameter”) were measured for 50 comet images. The differences between the averages of four treated animals and the untreated control animals were compared with the Dunnett test after one-way ANOVA. A p-value less than 0.05 was considered statistically significant (Figure 2).

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Figure 2: Sampling sites of stomach mucosa. The upper layer was obtained by scraping the surface of the glandular gastric mucosa and the lower layer was obtained by scraping the remained glandular gastric mucosa after scrapping the upper layer. The ridge was obtained by scraping the mucosa of bump protrusion forming the boundary between the forestomach and the glandular stomach.

Histopathological Examination

Fixed portion of glandular stomach, ledge, and liver were dehydrated and embedded in paraffin. Tissue sections stained by the hematoxylin-eosin and TUNEL methods were observed histopathologically. In the TUNEL method, tissue sections were incubated with 20 mg/mL proteinase K (Sigma Chemical Co., St. Louis, MO) for 20 min at 37 oC after deparaffinization and hydration. Slides were immersed in Terminal Transferase (TdT, Gibco, BRL, MD) buffer (10U TdT in 100mM HEPES buffer pH7.2, 8mM MgCl2, 0.05mM dTTP, and 0.5mM biotinylated dUTP) at 37°C for 90 min. The reaction was terminated with a TB buffer for 30 min, and the sections were covered with 10% rabbit serum for 10 min and avidin-biotin-peroxidase complex for 30 min at room temperature. Finally, the slides were incubated with 0.02% 3,3’-diaminobenzidine tetrahydrochloride (Dojindo Laboratories, Kumamoto, Japan) in 0.05M Tris- HCl buffered solution (pH7.6) containing 0.005% hydrogen peroxide for 1-5 min at room temperature. Tissue sections were counterstained with hematoxylin, dehydrated and coverslipped. The frequency of TUNNEL stained (TUNNEL- positive) cells per microscope field (0.02 mm2) was scored, using a microscopy at 400x magnification. The differences between treated and the untreated control mice were compared with the Dunnett test after one-way ANOVA. A p-value less than 0.05 was considered statistically significant.

Stomach Mucosa

In mice received NaCl and DMA, necrosis was observed in the upper and ridge layers of the gastric mucosa in all mice, and in the mucosa of the lower layer of mucosa in most mice. On the other hand, necrosis was not observed in any of the areas of the gastric mucosa (upper, lower, or ridge layers) in mice received EMS and those in the control group. In mice received NaCl and DMA, the frequencies of Halo increased significantly in the upper layers and ridge of the gastric mucosa, the frequencies of TUNNEL-positive cells increased in the ridge and upper layer but not lower layer of the gastric mucosa. In the gastric mucosa of the in mice received EMS, tail length increased in the alkaline comet assay but not neutral comet assay. Any increases in TUNNEL-positive-frequency, the frequencies of Halo and Ghost were not observed in mice received EMS and those in the control group.

Liver

In mice received CCl4 at 500–2000 mg/kg, hepatic necrosis was observed in all mice 24 hours after the administration, and the frequency of TUNNEL-positive cells and Halo but not Ghost images significantly increased. The increases in the tail length in the neutral but not alkaline comet assay were observed in mice received the CCl4.

In mice received D-Gal at 500–2000 mg/kg, hepatic necrosis was observed in all mice 12 hours after its administration, Also, the frequency of Halo but not Ghost images increased significantly. At 24 hours after its administration, while Halo-frequency increased, any deaths were not observed in D-Gal received mice. In mice receive AA at 50–100 mg/kg, hepatic necrosis of was observed in all mice 12 hours after its administration, and the frequencies of TUNNEL-positive cells and ˙Halo but not Ghost images significantly increased. The tail length increased in neutral but not alkaline comet assays at 100 mg/kg. All mice died within 24 hours after the administration of AA at 100 mg/kg.

On the other hand, hepatic necrosis was not observed in mice received DEN and in the control group, and any significant increase in the frequency of TUNNEL-positive cells, Halo images, and Ghost images did not observed in mice received DEN and mice in the control group, a significant increase in the tail length increased significantly in the alkaline but not neutral comet assays. Tail length did not increase in neither in alkaline nor neutral comet assays in mice in the control group.