DANCE In Mouse Liver Cells And The Mechanisms

Objective: To study DANCE (Density Alteration in Non-physiological Cells) in mouse livercells caused by all-trans retinoic acid (RA), ethanol, and the pathological changes in thedevelopment of S180tumor in mice, and thereby to explore the mechanisms of DANCE inmouse liver cells in vivo.Methods:1. Discontinuous Sucrose Gradient Centrifugation (DSGC) of the liver tissue homogenates ofKuming (KM) mice was carried out: mice weighing between20-25g, with half of each sex,were euthanized and the liver tissues were collected. About0.15g of mouse liver tissue wasadded with0.6ml saline and then homogenized.0.2ml of the homogenate was applied onto adiscontinuous sucrose gradient, with sucrose concentrations as30%,40%,50%,60%and70%（w/v）from the top through the bottom layers. Centrifugation was carried out at3500RPM（about1650g）for5min. Three bands were separated as labeled as S40, S50, S60(“S”for sucrose), which stop accordingly on the layers of sucrose. The bands were separatelystained with HE (Hematoxylin-eosin) to identify the components.2. Mice were gavaged with different doses of RA. The liver tissues were collected anddetermined for DANCE as described previously. Analysis was carried out for the dose andtime dependence of RA causing DANCE in the liver cells.3. Mice were gavaged with different doses of75%ethyl alcohol, and then determined forDANCE in the liver cells as described previously. The dose and time dependence of the drugcausing DANCE in the liver was analyzed. Serum AST (Aspartate transaminase) and ALT(Alanine transaminase) activity levels in the mice were determined by using a clinicalautomatic biochemical analyzer (carried out by the Clinical Test Center of Over-sea ChineseHospital of Guangzhou), and the relationship between mouse serum AST/ALT levels andDANCE in the liver was analyzed respectively. 4. A mouse model of S180-transplanted tumor was constructed, and DANCE in the liver wasdetermined at the time of every5days after tumor transplantation. The relationship betweenDANCE in the mouse liver and the time course of tumor formation in mice was analyzed.5. ELISA and Automatic Biochemical Analyzer were employed to determine the serum CRPand ALT levels in the tumor-baring mice.Results：1. KM mouse liver homogenates were separated by DSGC into three bands, that is, S60, S50and S40, respectively. It was found by HE staining that the three bands were mainly the livertissue fragments, consisting of liver cells with clear cell morphology under light microscope.2. RA induced DANCE-UP (bands moved up in DSGC) in KM mouse liver cells, and theDANCE reaction was found to be dependent on RA dose and the time of RA action.3. Ethanol alcohol was found to be able to cause DANCE in the mouse liver, and DANCEreaction in the liver was observed to be dependent on the ethanol dose and time of the drug’saction. The results of automatic biochemical analyzer showed that, compared to the controlgroup, serum ALT and AST had significantly higher levels.4. Results of S180tumor-bearing mice showed that DANCE occurred in the mouse liver withdifferent rates at different time points after the tumor transplantation. It was found thatDANCE in the mouse liver reached two peaks at the time of tenth and twenty-fifth day afterthe tumor-transplantation, respectively.5. Serum CRP of S180tumor-bearing mice was detected by ELISA and ALT by automaticbiochemical analyzer. It was found that the serum levels of ALT and CRP in tumor-bearingmice were higher than those in the control group.Conclusion: Oral administration of RA and ethanol alcohol, as well as transplantation of S180tumor, may result in DANCE in mouse liver cells. DANCE reaction in mouse liver is dependenton the dose and action time of the drugs, and is also involved in the genesis and development oftransplanted tumor, with increase of serum CRP, ALT and AST, indicating increased hepaticapoptosis or immune/inflammation reactions in, at least part of, the liver cells, which may be oneof the mechanisms for DANCE in the mouse liver cells.