The Mechanism Of Liver Injury Induced By Food Additives-sodium Sulfite

Objective:Previous in vivo studies showed that sulfite may cause hepatocytes steatosis and increase the triglyceride (TG) level in hepatocytes. In the present study, we will confirm its liver injury effect and further investigate its possible mechanism of fat deposition in hepatocytes by in vitro experiments. This will provide a theoretically reliable foundation for studying the effect of sodium sulfite additives on health and also provide scientific evidence for the revision of application restriction standard .Methods: Human diploid liver cell line (L-02) were cultured in DMEM/F12 medium containing 10% fetal bovine serum. Sodium sulfite (Na₂SO₃)was added to the L-02 cells with different concentrations, the final concentrations were 0 mmol/L(control group), 0.039mmol/L, 0.156mmol/L, 0.625mmol/L, 2.5mmol/L repectively, and a positive control group (3% ethanol). MTT assay was conducted to observe the effect of Na₂SO₃ on the viability of L-02 hepatic cell. The morphological changes were observed by the inverted microscope. The activities of aspartate aminotransferase (AST), the alanine aminotransferase (ALT), lactate dehydrogenase (LDH), total protein and albumin levels in culture supernatant were determined by biochemical analysis. Zymochemistry colorimetric assay was used for the determination of intracellular triglycerides (TG) levels. The expression of apolipoprotein B100 (apo-B100) and apolipoprotein E (apo-E) which are related closely with the metabolism of triglyceride were detected with immunocytochemical method.Results:1.The effect of sodium sulfite on hepatocytes morphological changes: L-02 liver cells were polygon, which were in single array without overlapping growth. Cell boundaries were clear and cells were adherent well in the control group. Compared with the control group, the cell morphology had no significant difference in 0.039mmol/L treated group. There were few floating round cells in the 0.156mmol/L group, but most cells were healthy. A bit of more round cells were found in the 0.625mmol/L group; In the 2.5mmol/L treated group, cell morphology was obviously abnormal.Cell was present in a single adhesion, the quantities of cells were reduced and the cell volume was diminished.Many adherent cells were floating, the number of dead cells increased and the space between cells was increased.2. The effect of sodium sulfite on the viability of L-02 hepatic cells: According to MTT assay results, the inhibition rates were gradually enhanced with the increasing of Na₂SO₃ concentrations, in a concentration-dependent manner(r=0.813, p<0.01). Compared with the control group, the inhibition rates of the cell viability were increased significantly (p<0.05) in 0.625 mmol/L and 2.5 mmol/L Na₂SO₃ treated groups. The results indicate that Na₂SO₃ can inhibit the viability of hepatocytes within a certain dose range.3.The effect of sodium sulfite on the activity of LDH in the culture medium: L-02 hepatic cells were treated for 24h, 72h and 48h, with increased Na₂SO₃ concentrations, the activities of LDH in the HL-7702 culture supernatants were gradually increased(P<0.05)in a concentration-dependent manner (r24=0.841,p<0.01;r48=0.916,p<0.01; r72 = 0.619, p<0.01),which shows that Na₂SO₃ may have some influence on the hepatocyte membrane permeability.4. The effect of sodium sulfite on the activity of AST in the L-02 culture supernatant:1) When the cells were treated for 24h ,with the increased Na₂SO₃ concentrations, the activities of AST in the culture supernatant were gradually increased in a concentration-dependent manner (r24=0.652, p<0.01).The activities of AST in 0.625mmol/L and 2.5mmol/L groups were significantly increased as compared with the control group (p<0.05 or p<0.01). While exposure time reached 48h and 72h, it showed no statistical differences between the exposed groups and the control group.2) With the extension of treatment time, there was a tendency of decreased AST activities in the cell culture supernatant. Compared with 24h treatment, the activities of AST in 48h and 72h groups were significantly decreased (p< 0.05 or p<0.01).5.The effect of sodium sulfite on the activity of ALT in the L-02 culture supernatant: When exposure time lasted 24h and 48h with increased Na₂SO₃ concentrations, the activities of ALT in the culture supernatant were enhanced significantly, compared with the control group( p<0.05 or p<0.01).The cells exposed 24h to Na₂SO₃, showed a concentration-dependent relationship(r24=0.873, p<0.01).While the exposure time reached 72h, there were no significant differences between the treated groups and the control group.These results (4, 5) suggest that the major damage to the hepatocytes induced by sulfite occurred in the early stage.6.The effect of sodium sulfite on the synthetic ability of albumin and total protein: The contents of albumin and total protein in the culture supernatant were not significantly changed (p>0.05), with the increasing of concentrations of Na₂SO₃. The result shows that Na₂SO₃ doesn't have obvious impact on the synthetic function of albumin and total protein in the hepatocytes.7. The effect of sodium sulfite on the level of hepatocytes intracellular TG: The levels of intracellular TG in the Na₂SO₃ treated groups were not increased by Na₂SO₃ treatment when the time points were in 24h and 48h (p>0.05) as compared with the control group. However, after 72h exposure, the TG level was increased significantly in 0.625mmol/L group as compared with the control group (p< 0.05). 8.The effect of sodium sulfite on the expression of apo-B100 and apo-E in hepatocytes: According to the results of immunocytochemistry, the expression of apo-B100 was not changed significantly in each group at different exposure time(24h, 48h and72 h).After 72h sodium sulfite treatment, the expression of apo-E was significantly reduced (p<0.05) in 2.5mmol/L group, compared with the control group. This result indicates that sulfite may affect the level of apo-E under certain conditions.Conclusions:1. This experiment shows Na₂SO₃ can inhibit the viability of HL-7702 to some degree.2. The activities of LDH , AST, ALT in the hepatocyte cultural supernatant were increased by Na₂SO₃, suggesting that Na₂SO₃ may cause some damages on the HL-7702 hepatocytes membrane.3. Under certain dose condition, sodium sulfite may increase the level of intracellular TG and possibly also has a certain impact on the expression of apo-E, but it had no influence on the content of albumin and total protein in cells.