Effects of Acute and Chronic Exposure to Residual Level Erythromycin on Human Intestinal Epithelium Cell Permeability and Cytotoxicity

Abstract

Residual concentrations of erythromycin in food could result in gastrointestinal tract exposure that potentially poses a health-hazard to the consumer, affecting intestinal epithelial permeability, barrier function, microbiota composition, and antimicrobial resistance. We investigated the effects of erythromycin after acute (48 h single treatment with 0.03 μg/mL to 300 μg/mL) or chronic (repeated treatment with 0.3 µg/mL and 300 µg/mL erythromycin for five days) exposures on the permeability of human colonic epithelial cells, a model that mimics a susceptible intestinal surface devoid of commensal microbiota. Transepithelial electrical resistance (TER) measurements indicated that erythromycin above 0.3 µg/mL may compromise the epithelial barrier. Acute exposure increased cytotoxicity, while chronic exposure decreased the cytotoxicity. Quantitative PCR analysis revealed that only ICAM1 (intercellular adhesion molecule 1) was up-regulated during 0.3 μg/mL acute-exposure, while ICAM1, JAM3 (junctional adhesion molecule 3), and ITGA8 (integrin alpha 8), were over-expressed in the 300 μg/mL acute treatment group. However, during chronic exposure, no change in the mRNA expression was observed at 0.3 μg/mL, and only ICAM2 was significantly up-regulated after 300 μg/mL. ICAM1 and ICAM2 are known to be involved in the formation of extracellular matrices. These gene expression changes may be related to the immunoregulatory activity of erythromycin, or a compensatory mechanism of the epithelial cells to overcome the distress caused by erythromycin due to increased permeability.

Keywords: acute or multiple dose exposure; epithelial cell barrier function; erythromycin; gene expression; permeability; single dose exposure.

Figure 1

Effect of acute exposure of erythromycin on intestinal epithelial cell permeability. T84 cells were seeded in transwells and culture media was periodically changed until transepithelial electrical resistance (TER) value reached 800-1000 Ω. The TER value was recorded before and after incubation with five concentrations of erythromycin (Ery 0.03 = 0.03 μg/mL; Ery 0.3 = 0.3 μg/mL; Ery 3 = 3 μg/mL; Ery 30 = 30 μg/mL; and Ery 300 = 300 μg/mL) at 0, 1, 3, 24, and 48 h. The TER value recorded before adding erythromycin (time 0) served as the baseline. Data are presented here as fold increase or decrease in the same well before treatment. Each value represents an average of three independent experiments. * indicates the statistical significance (p < 0.05).

Figure 2

The effect of chronic exposure of erythromycin on the intestinal epithelial cell permeability. T84 cells were seeded in transwells and culture media was periodically changed and cultured until TER value reached 800 to 1000 Ω. The TER value was recorded (at 0, 1, 3, 20, and 22 h) after daily treatment of erythromycin at two concentrations (E0.3 = 0.3 μg/mL and E300 = 300 μg/mL). Data here represent the fold change after daily treatment at each time point (n = 3) for five days. Erythromycin-free media in the control wells was also replaced at the same time.

Figure 3

The effect of acute exposure of erythromycin on cell cytotoxicity. Cell culture supernatants from the transwell apical compartment were assessed to determine the cell cytotoxicity by measuring lactate dehydrogenase (LDH), a stable cytosolic enzyme that is released upon cell lysis. Released LDH in culture supernatants was measured with a 30-minute coupled enzymatic assay, which results in conversion of a tetrazolium salt (INT) into a red formazan product that is quantitated at wavelength absorbance of (OD₄₉₀). Values here depict the fold change in LDH release as a measure of cytotoxicity caused by erythromycin exposure on T84 cells after 48 h single treatment as compared to untreated controls. * indicates the statistical significance (p < 0.05).

Figure 4

The effect of chronic exposure of erythromycin on cell cytotoxicity. Cell culture supernatants from the apical compartment of transwells were collected each day to determine the cell cytotoxicity by measuring released LDH as described in Figure 3. * indicates the statistical significance (p < 0.05).