The Eye Drop Preservative Benzalkonium Chloride Potently Induces Mitochondrial Dysfunction and Preferentially Affects LHON Mutant Cells

Abstract

PurposeBenzalkonium chloride (BAK) is the most commonly used eye drop preservative. Benzalkonium chloride has been associated with toxic effects such as “dry eye” and trabecular meshwork degeneration, but the underlying biochemical mechanism of ocular toxicity by BAK is unclear. In this study, we propose a mechanistic basis for BAK's adverse effects.MethodMitochondrial O2 consumption rates of human corneal epithelial primary cells (HCEP), osteosarcoma cybrid cells carrying healthy (control) or Leber hereditary optic neuropathy (LHON) mutant mtDNA [11778(G>A)], were measured before and after acute treatment with BAK. Mitochondrial adenosine triphosphate (ATP) synthesis and cell viability were also measured in the BAK-treated control: LHON mutant and human-derived trabecular meshwork cells (HTM3).ResultsBenzalkonium chloride inhibited mitochondrial ATP (IC50, 5.3 μM) and O2 consumption (IC50, 10.9 μM) in a concentration-dependent manner, by directly targeting mitochondrial complex I. At its pharmaceutical concentrations (107–667 μM), BAK inhibited mitochondrial function >90%. In addition, BAK elicited concentration-dependent cytotoxicity to cybrid cells (IC50, 22.8 μM) and induced apoptosis in HTM3 cells at similar concentrations. Furthermore, we show that BAK directly inhibits mitochondrial O2 consumption in HCEP cells (IC50, 3.8 μM) at 50-fold lower concentrations than used in eye drops, and that cells bearing mitochondrial blindness (LHON) mutations are further sensitized to BAK's mitotoxic effect.ConclusionsBenzalkonium chloride inhibits mitochondria of human corneal epithelial cells and cells bearing LHON mutations at pharmacologically relevant concentrations, and we suggest this is the basis of BAK's ocular toxicity. Prescribing BAK-containing eye drops should be avoided in patients with mitochondrial deficiency, including LHON patients, LHON carriers, and possibly primary open-angle glaucoma patients.

Mitochondrial Dysfunctions May Be One of the Major Causative Factors Underlying Detrimental Effects of Benzalkonium Chloride

“…Here, we showed that BAC, even at low concentrations, induced dysfunction of mitochondria, inhibiting respiration in state 3, decreasing the membrane potential, promoting opening of the mPTP, potentiating hydrogen peroxide production by mitochondria, and severely blocking synthesis of ATP. Previously, it was reported that BAC inhibited mitochondrial ATP production and oxygen consumption in human corneal epithelial cells and cells bearing LHON mutations possibly by directly targeting mitochondrial complex I [48]. However, closer examination of this paper showed that the authors used permeabilized cells as models and, more importantly, inadequate, from the point of view of bioenergetics, "mitochondrial buffer" causing high-amplitude swelling of mitochondria with all the consequences.…”

“…Moreover, mitochondrial oxidative stress can influence pathogenesis and progression of age-related corneal diseases, as well as generalized corneal aging [47]. BAC at pharmacologically relevant concentrations inhibited mitochondrial ATP production and oxygen consumption in human corneal epithelial cells and cells bearing LHON mutations and osteosarcoma cybrid cells, possibly by directly targeting mitochondrial complex I [48,49]. The authors concluded that the findings obtained support the urgent need for additional research on the mitochondriarelated inhibitory effects of BAC [49].…”

The Beast of Beauty: Environmental and Health Concerns of Toxic Components in Cosmetics

“…[66]. In addition, BAC is the most active ingredient and widely used as a preservative in ophthalmic solutions [67]. Nevertheless, the usage of BAC has been linked to inducing considerable adverse consequences in humans.…”

Open-angle glaucoma: therapeutically targeting the extracellular matrix of the conventional outflow pathway

“…Strategies for enhanced delivery include drug reformulation into nanocapsules or nanospheres, reducing the amount of preservatives (which may illicit side effects [180]), or modulation of solution viscosity by incorporation of polymers, collagen shields, gels, or liposomes. Such engineering allows greater cell specificity and retention of stability.…”