How does Benzalkonium Chloride and Prostaglandins Affect Meibomian Gland Epithelial Cells?

Introduction

Long-term use of drops containing benzalkonium chloride (BAC) in the treatment of glaucoma, according to study data, increases the risk of corneal damage and the development of dry eye syndrome. Even very low concentrations of BAC cause functional and morphological changes in the eye, alter the tear film, lead to epithelial apoptosis, and even damage deeper eye structures.

The Meibomian sebaceous glands located in the tarsal plates of the eyelids produce a secretion containing lipids and proteins. This lipid layer prevents the evaporation of the tear film from the surface of the eye. Eye drops used in the treatment of glaucoma also damage Meibomian gland cells based on hyperkeratinization, and this process is a major factor in the pathogenesis of dry eye syndrome. The study described below examined the effect of BAC on the epithelium of Meibomian glands in vitro.

Methodology and Study Objectives

To study the effect of various concentrations of BAC on the proliferation, viability, and function of the cell barrier, an immortalized line of human Meibomian gland epithelial cells (HMGEC) was used. Both proliferating cells and those differentiated by fetal bovine serum (FBS) were chosen. Previous results showed that it is appropriate to use HMGEC as proliferating cells resemble the basal cell layer of the Meibomian glands. However, if FBS is added to the culture medium, the cells resemble mature meibomian tissue.

The second objective of the study was to evaluate whether BAC induces changes similar to hyperkeratinization in HMGEC. The chosen evaluation parameter was the measurement of the rate of gene expression of involucrin and cornulin (using real-time polymerase chain reaction – real-time PCR), two proteins often detected in squamous epithelium and markers of keratinization. The study also compared the differences in the effects of BAC alone, antiglaucomatics without BAC (bimatoprost, travoprost, and latanoprost), and combined solutions containing both BAC and prostaglandin derivatives.

Results

The cytotoxic effect of BAC was evident at a concentration of 0.1 µg/ml, which is 1000 times lower than the concentration used in commercial eye drops. At BAC concentrations > 50 µg/ml, cell viability was reduced after just 10 minutes of exposure, with corresponding changes in cell morphology. The critical BAC concentration was ≥ 10 µg/ml, at which HMGEC cell death occurred in less than 24 hours. The proliferative abilities of the cell line were reduced at all BAC concentrations, most noticeably at higher levels.

Comparisons of BAC products, BAC-free products, and BAC alone revealed that the toxicity of ophthalmic products containing BAC was greater than the toxicity of BAC alone, while alternative preparations without BAC had no significant effect on cell viability. Gene expression of involucrin and cornulin was lower compared to control cells after stimulation with BAC solutions with bimatoprost as well as with BAC alone. Factors with positive effects that facilitated cell line survival included the presence of FBS, cell confluence (forming a continuous layer), and their mutual contact.

Conclusion

The study evaluated the effect of the preservative BAC on the human Meibomian gland cell line. BAC demonstrated its toxicity at concentrations equal to or even lower than those used in registered antiglaucomatics. The combination of BAC and prostaglandin analogs may have a synergistic effect, explaining its higher toxicity compared to BAC alone. Benzalkonium chloride could thus be a significant iatrogenic cause of dry eye syndrome.

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Source: Rath A., Eichhorn M., Träger K. et al. In vitro effects of benzalkonium chloride and prostaglandins on human meibomian gland epithelial cells. Ann Anat 2019; 222: 129–138, doi: 10.1016/j.aanat.2018.12.003.