Chronic Alcohol Exposure of Cells Using Controlled Alcohol-Releasing Capillaries

Abstract

Alcohol is one of the main causes of liver diseases such as fatty liver, alcoholic hepatitis, and chronic hepatitis with liver fibrosis or cirrhosis. To reproduce the conditions of alcohol-induced liver diseases and to identify the disease-causing mechanisms at the cellular level, several methods have been used to expose the cells to ethanol. As ethanol evaporates easily, it is difficult to mimic chronic alcohol exposure conditions at the cellular level. In this study, we developed a glass capillary system containing ethanol, which could steadily release ethanol from the polyethylene tubing and hydrogel portion at both sides of the capillary. The ethanol-containing capillary could release ethanol in the cell culture medium for up to 144 h, and the concentration of ethanol in the cell culture medium could be adjusted by controlling the number of capillaries. A long-term exposure to ethanol by the capillary system led to an increased toxicity of cells and altered the cellular physiologies, such as increasing the lipid accumulation and hepatic transaminase release in cells, as compared to the traditional direct ethanol addition method. Ethanol capillaries showed different gene expression patterns of lipid accumulation- or chronic alcoholism-related genes. Our results suggest that our ethanol-containing capillary system can be used as a valuable tool for studying the mechanism of chronic alcohol-mediated hepatic diseases at the cellular level.

Keywords: alcohol; capillary; chronic; liver.

Figure 1

Glass capillary device. (A) Schematic diagram of the device containing a glass capillary tube connected to a polyethylene (PE) tubing (0.2 cm) on both sides. Half of the PE tubing was filled with polyethylene glycol (PEG) hydrogel (0.1 cm). (B) Real image of the 20-mm long glass capillary device. (C,D) The glass capillary was inserted into the culture medium containing HepG2 or L-02 cells, and it was incubated for 24 h. Following this, cytotoxicity was measured and the control was set to 100. n = 4, non-significant (ns), as determined by the unpaired t-test.

Figure 2

Controlled and long-term ethanol release by the capillary device. (A) Schematic diagram of ethanol-laden glass capillary. Ethanol is released through the PEG hydrogel (pore size: 1 nm) portions when the device is immersed in the media. (B) Photograph of capillaries floating on top of the culture medium. (C) The indicated final concentration of ethanol was directly added to 3 mL of phosphate-buffered saline (PBS) in 6-well plates. The concentration of ethanol in PBS was measured at the indicated time points. (D) 0 or 4 capillaries containing PBS or ethanol were immersed in 3 mL of PBS in one well of 6-well plates, and the concentration of ethanol in PBS was measured at the indicated time points. (E) Control, which means non-treated, or direct and repeated addition of ethanol every 24 h or 10 capillaries containing PBS or ethanol were immersed in the culture medium in 6-well plates, and the culture dishes were kept in a humidified atmosphere at 37 °C under 5% CO2. Subsequently, the concentration of ethanol in the culture medium was measured at the indicated time points. All schematics were created with BioRender.com.

Figure 3

Comparison of cytotoxicities using direct addition and capillary device. (A) Ethanol was directly added into culture medium of HepG2 cells for the indicated concentrations. Subsequently, the cells were incubated for 24 or 72 h to measure cytotoxicity. n = 6, ** p < 0.01, *** p < 0.001, **** p < 0.0001, as determined by two-way ANOVA and Tukey’s multiple comparisons test. (B,C) Ethanol-harboring capillaries were placed in the culture medium, four capillaries containing 86 mM ethanol in 4 mL of 35 mm culture dish or 10 capillaries containing 171 mM ethanol, and this was followed by incubation for 24 or 72 h. Cells were subjected to cytotoxicity measurements, and the viability of the control capillary harboring PBS was set to 100. n = 8, **** p < 0.0001. (D,E) Ethanol was added using the direct addition or capillary system up to a concentration of 86 or 171 mM. After 24 or 72 h of incubation, the cell viability was determined. The viability of each direct addition batch was set to 100. n = 6, * p < 0.05, *** p < 0.001, **** p < 0.0001, as determined by two-way ANOVA and Tukey’s multiple comparisons test. (F) Ethanol was added using the direct addition or capillary system, and live and dead cells were counted at indicated time points. n = 4, **** p < 0.0001, (two-way ANOVA and Tukey’s multiple comparisons test).

Figure 4

Effect of long-term exposure of ethanol to HepG2 cells. (A,B) Cells were incubated for 120 h with 514 mM ethanol using direct addition or capillaries up to 171 mM ethanol concentration. The amount of alanine transaminase and aspartate aminotransferase leakage was measured in the culture medium. n = 3, * p < 0.05, *** p < 0.001, **** p < 0.0001 (one-way ANOVA and Tukey’s multiple comparisons test). (C–E) Cells were treated with PBS for control and 171 or 514 mM ethanol by direct addition or capillaries up to 171 mM ethanol concentration, and they were incubated for 24 or 120 h. Subsequently, the cells were subjected to total RNA preparation, and the mRNA levels were quantified using the indicated gene-specific primers. The relative mRNA levels were normalized using RPL32 mRNA levels, and the mRNA value of control batches was set to 1. n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (two-way ANOVA and Tukey’s multiple comparisons test).

Figure 5

Chronic exposure of ethanol induces lipid accumulation. (A) HepG2 cells were treated with PBS and 514 mM ethanol using the direct addition method or ethanol-harboring capillaries up to 171 mM ethanol concentration. Subsequently, the cells were cultivated for 120 h. Lipid accumulation was determined using Oil Red O staining. (B) The absorbance of samples from panel A was measured at 450 nm, and the value of control was set to 1. n = 4, * p < 0.05, ** p < 0.01, **** p < 0.0001. (C,D) Control of PBS and 514 mM ethanol using direct addition or ethanol capillaries up to 171 mM ethanol concentration was added to HepG2 cells. After 120 h of incubation, the relative mRNA levels were measured using the indicated gene-specific primers, FASN, SREBP1C, and RPL32. Relative FASN or SREBP1c mRNA levels were quantified using endogenous RPL32. The relative mRNA levels of control were set to 1. n = 3, ** p < 0.01, *** p < 0.001.