A case of severe benzalkonium chloride intoxication in a cat.

BACKGROUND: Benzalkonium chloride (BAC) is a quaternary ammonium compound (QAC), that can be found in a wide variety of household products-from disinfectants to medicaments and home fragrances-but also professional products. In pets, cats have long been reported as more sensitive than dogs to QACs; in fact, signs of irritation such as oral ulcerations, stomatitis and pharyngitis can be observed after contact with concentrations of 2% or lower. In a review of 245 cases of BAC exposure in cats, reported by the Veterinary Poisons Information Service (United Kingdom) only 1.2% of the cases died or were euthanized. Nevertheless, BAC toxidromes in cats can result in transitory CNS and respiratory distress, as well as severe mucosal and cutaneous lesions. Currently, only a few reports are available concerning BAC poisoning in this species. CASE PRESENTATION: A 4 month-old kitten presented with severe glossitis, lameness in the hindlimbs and episodes of vomiting and diarrhoea. The cause was unknown until the owners reported use of a BAC-containing mould remover (5%) 4 days later. The patient developed severe oral burns requiring a pharyngeal tube for feeding and severe cutaneous chemical burns. The kitten was managed with supportive therapy and required hospitalization for 10 days. The symptoms disappeared completely 3 weeks after exposure. CONCLUSIONS: BAC is a very common compound contained in several household and professional products but, to the best of our knowledge, no previous case had been reported in Italy. We hope that this report will help raise awareness on the hazards of BAC products for cats in both domestic and work contexts.

Benzalkonium chloride greatly deteriorates the biological activities of human corneal stroma fibroblasts in a concentration-dependent manner.

BACKGROUND: Corneal tissues indirectly obtain nutritional needs and oxygen to maintain their homeostasis, and therefore, benzalkonium chloride (BAC) containing ocular instillations for medical therapy may, in turn, induce toxic effects more than expected in corneal tissues, especially the inside stroma layer. METHODS: To evaluate the effects of very low concentrations (10-8%, 10-6%, or 10-4%) of BAC on human corneal stroma, we used two-dimensional (2D) cultures of human corneal stromal fibroblast (HCSF) cells and carried out the following analyses: (1) cell viability measurements, (2) Seahorse cellular bio-metabolism analysis, and (3) the expression of ECM molecules and endoplasmic reticulum (ER) stress-related molecules. RESULTS: In the absence and presence of 10-8%, 10-6%, or 10-4% concentrations of BAC, cell viability deteriorated and this deterioration was dose-dependent. The results showed that maximal mitochondrial respiration was decreased, the mRNA expression of most of ECM proteins was decreased, and ER stress-related molecules were substantially and dose-dependently down-regulated in HCSFs by the BAC treatment. CONCLUSIONS: The findings reported herein indicate that the presence of BAC, even at such low concentrations, is capable of causing the deterioration of cellular metabolic functions and negatively affecting the response to ER stress in HCSF cells resulting in a substantially decreased cellular viability.

Ecotoxicity and rapid degradation of quaternary ammonium compounds (QACs) subjected to combined vacuum UV and UV-C treatment.

Quaternary ammonium compounds (QACs) are active ingredients in a palette of commercially available disinfectants, sanitizers, and biocides. QACs are widely used because of their broad-spectrum antimicrobial properties but the ubiquitous uses have resulted in frequent detection in aquatic and terrestrial matrices including domestic wastewater, surface waters, urban soils and sediments. An increased domestic QACs consumption has increased the environmental occurrence, and investigation of mitigation methods and effects on non-target organisms are in demand. In this study, we examined the potential ecotoxicity of six QACs and investigated the effect of combined vacuum UV (185 nm) and UV-C (254 nm) irradiation (VUV/UVC) on degradation and mitigation of ecotoxicity of QACs. The study showed that combined VUV/UVC irradiation facilitated rapid degradation of benzalkonium chloride, benzethonium chloride, didecyldimethylammonium chloride, dodecyltrimethylammonium chloride, and hexadecyltrimethylammonium chloride. The estimated half-lives varied between 2 and 7 min, and degradation was affected by the initial QAC concentrations, the UV fluence, and the water matrix. The potential ecotoxicity of QACs and VUV/UVC treated QACs was examined using a battery of test organisms that included the luminescent bacterium Aliivibrio fischeri, the gram-negative and gram-positive bacteria Escherichiacoli and Enterococcus faecalis, the freshwater microalga Raphidocelis subcapitata, and the crustacean Daphia magna. The potential for trophic transfer of QACs was investigated in a simplified aquatic food web. Test organisms from different trophic levels were included to assess adverse effects of bioactive compounds in VUV/UVC treated samples including transformation products. The study showed that several QACs were highly toxic to aquatic test organisms with EC50 and/or EC20 values < 1 µM. VUV/UVC treatment of QACs resulted in substantial photolysis of the parent compounds and comprehensive mitigation of the ecotoxicity potential. VUV/UVC represent an attractive oxidation technology for abatement QACs in contaminated water because the process does not require addition of catalysts or precursors.

Corneal neuroepithelial compartmentalized microfluidic chip model for evaluation of toxicity-induced dry eye.

Part of the lacrimal functional unit, the cornea protects the ocular surface from numerous environmental aggressions and xenobiotics. Toxicological evaluation of compounds remains a challenge due to complex interactions between corneal nerve endings and epithelial cells. To this day, models do not integrate the physiological specificity of corneal nerve endings and are insufficient for the detection of low toxic effects essential to anticipate Toxicity-Induced Dry Eye (TIDE). Using high-content imaging tool, we here characterize toxicity-induced cellular alterations using primary cultures of mouse trigeminal sensory neurons and corneal epithelial cells in a compartmentalized microfluidic chip. We validate this model through the analysis of benzalkonium chloride (BAC) toxicity, a well-known preservative in eyedrops, after a single (6h) or repeated (twice a day for 15 min over 5 days) topical 5.10-4% BAC applications on the corneal epithelial cells and nerve terminals. In combination with high-content image analysis, this advanced microfluidic protocol reveal specific and tiny changes in the epithelial cells and axonal network as well as in trigeminal cells, not directly exposed to BAC, with ATF3/6 stress markers and phospho-p44/42 cell activation marker. Altogether, this corneal neuroepithelial chip enables the evaluation of toxic effects of ocular xenobiotics, distinguishing the impact on corneal sensory innervation and epithelial cells. The combination of compartmentalized co-culture/high-content imaging/multiparameter analysis opens the way for the systematic analysis of toxicants but also neuroprotective compounds.

A fish perspective on SARS-CoV-2: Toxicity of benzalkonium chloride on Danio rerio.

SARS-CoV-2 outbreak led to an increased marketing of disinfectants, creating a potential environmental problem. For instance, pre-pandemic environmental levels of the disinfectant benzalkonium chloride (BAC) ranging from 0.5 to 5 mgL-1 in effluents were expected to further increase threatening aquatic life. Our aim was to characterize potential adverse effects after an acute exposure of zebrafish to different concentrations of BAC. An increase in the overall swimming activity, thigmotaxis behavior, and erratic movements were observed. An increase in CYP1A1 and catalase activities, but inhibitions of CY1A2, GSTs and GPx activities were also noticed. BAC is metabolized by CYP1A1, increasing the production of H2O2, thereby activating the antioxidant enzyme CAT. Data also showed an increase of AChE activity. Our study highlights adverse embryonic, behavioral, and metabolic effects of noteworthy environmental significance, especially considering that the use and release of BAC is most likely to increase in a near future.

Suppression of NLRP3/Caspase-1/GSDMD Mediated Corneal Epithelium Pyroptosis Using Melatonin-Loaded Liposomes to Inhibit Benzalkonium Chloride-Induced Dry Eye Disease.

Introduction: Benzalkonium chloride (BAC) is widely employed as a preservative in eye drops, which will cause the death of corneal epithelial cells due to ROS production, DNA strand breakage, and mitochondrial dysfunction, resulting in dry eye disease (DED)-like changes in ocular surface tissues. In this study, Melatonin (MT) liposomes (TAT-MT-LIPs) designed by loading MT into TAT-modified liposomes have been developed, characterized, and used for inhibiting BAC-induced DED (BAC-DED). Methods: The TAT was chemically grafted onto the Mal-PEG2000-DSPE by Michael's addition between the sulfhydryl group in TAT and the maleimide group in Mal-PEG2000-DSPE. TAT-MT-LIPs were prepared using film dispersion followed by the extrusion method and topically treated in rats once a day. BAC-DED was induced in rats by topical administration with 0.2% BAC twice daily. Defects, edema, and inflammation of the corneas, as well as IOP, were examined. Histologic analyses of corneas were performed to assess the change of mitochondrial DNA oxidation and NLRP3/Caspase-1/GSDMD signaling transduction. Results: After topical administration, TAT-MT-LIPs significantly alleviated DED-clinical symptoms of experimental animals by inhibiting tissue inflammation and preventing the loss of the corneal epithelium and conjunctival goblet cells. Our data suggested continuous ocular surface exposure of BAC-induced NLRP3/Caspase-1/GSDMD mediated corneal epithelium pyroptosis, which was not reported before. BAC caused substantial mt-DNA oxidation, which promoted the transduction of NLRP3/Caspase-1/GSDMD and consequent corneal epithelium pyroptosis. TAT-MT-LIPs could efficiently suppress the BAC-induced corneal epithelium pyroptosis and inflammation by inhibiting mt-DNA oxidation and the subsequent signal transmission. Conclusion: NLRP3/Caspase-1/GSDMD mediated corneal epithelium pyroptosis is involved in the development of BAC-DED. The present study provided new insights into the adverse effects of BAC, which can serve as a new target for protecting corneal epithelium when applying BAC as a preservative in eye drops. The developed TAT-MT-LIPs can efficiently inhibit BAC-DED and give great potential to be developed as a new DED treatment.

Neurosensory abnormalities and stability of a mouse model of dry eye disease.

This study aimed to use a mouse model of dry eye disease (DED) induced by topical administration of benzalkonium chloride (BAK) and assess its stability and the presence of neurosensory abnormalities, including ocular pain. Eight-week-old C57BL6/6 N male mice were used in this study. Mice were treated with 10 µL of 0.2% BAK dissolved in artificial tears (AT), administered twice daily for 7 days. After one week, animals were randomized into two groups: one was administered with 0.2% BAK in AT once per day for 7 days, while the other was not further treated. Corneal epitheliopathy was quantified at days 0, 3, 7, 12, and 14. Moreover, tear secretions, corneal nociception, and corneal nerve integrity were measured after BAK treatment. After sacrifice, corneas were dissected to assess nerve density and leukocyte infiltration by immunofluorescence. Topical BAK instillation for 14 days significantly increased corneal fluorescein staining (p < 0.0001) compared to day 0. On the other hand, interruption of BAK instillation was associated with improvement of corneal epitheliopathy (day 12, p < 0.0001; day 14, p < 0.001). BAK treatment increased ocular pain (p < 0.0001) and resulted in a significant increase in leukocyte infiltration in the cornea (p < 0.01). Moreover, corneal sensitivity was reduced (p < 0.0001), together with corneal nerve density (p < 0.0001) and tear secretion (p < 0.0001). One week twice a day, followed by one additional week once a day, of 0.2% BAK topical administration induces stable clinical and histological signs of DED, which is associated with neurosensory abnormalities, including pain.

Benzalkonium chloride-induced dry eye disease animal models: Current understanding and potential for translational research.

Dry eye disease (DED) is an emerging health issue affecting people worldwide. There have been rapid advances in the development of novel molecules and targeted therapies for the treatment of DED in the recent past. For testing and optimizing these therapies, it is necessary to have reliable experimental animal models of DED. One such approach is the use of benzalkonium chloride (BAC). Several BAC-induced DED models of rabbits and mice have been described in literature. BAC induces high levels of proinflammatory cytokines in the cornea and conjunctiva, along with epithelial cell apoptosis and reduction of mucins, which leads to tear film instability, thereby successfully simulating human DED. The stability of these models directs whether the treatment is to be applied while BAC is being instilled or after its cessation. In this review, we summarize the previously described BAC animal models of DED and present original data on rabbit DED models created using 0.1%, 0.15%, and 0.2% BAC administration twice daily for two consecutive weeks. The 0.2% BAC model sustained DED signs for 3 weeks, while 0.1% and 0.15% models sustained DED signs for 1-2 weeks after BAC discontinuation. Overall, these models look promising and continue to be used in various studies to investigate the efficacy of therapeutic drugs for DED treatment.

Plasma membrane damage triggered by benzalkonium chloride and cetylpyridinium chloride induces G0/G1 cell cycle arrest via Cdc6 reduction in human lung epithelial cells.

Quaternary ammonium compounds, including benzalkonium chloride (BAC) and cetylpyridinium chloride (CPC), are widely used as disinfectants. Increased use of inhalable products containing BAC or CPC has raised concerns for lung toxicity. This study sought to elucidate the microstructure of plasma membrane damage caused by BAC and CPC and the subsequent mechanism by which the damage is mediated, as assessed using two human pulmonary epithelial cell lines (A549 and BEAS-2B). Scanning electron microscopic observation showed that exposure to BAC or CPC for 3 hr reduced the length and density of microvilli on the plasma membrane in A549 cells. Analysis of cell cycle distribution following plasma membrane damage revealed that BAC and CPC promote G0/G1 cell cycle arrest in both cell lines. The protein levels of Cdc6, an essential regulator of DNA replication during G1/S transition, are decreased significantly and dose dependently by BAC or CPC exposure. CPC and BAC decreased the Cdc6 levels that had been increased by a PI3K agonist in A549 cells, and levels of phosphorylated AKT were reduced in response to BAC or CPC. Conversely, exposure to equivalent concentrations of pyridinium chloride (lacking a hydrocarbon tail) induce no changes. These results suggest that plasma membrane damage triggered by BAC or CPC causes Cdc6-dependent G0/G1 cell cycle arrest in pulmonary cells. These effects are attributable to the long alkyl chains of BAC and CPC. The reduction of Cdc6 following plasma membrane damage may be caused, at least in part, by diminished signaling via the PI3K/AKT pathway.

Pronounced temporal changes in soil microbial community and nitrogen transformation caused by benzalkonium chloride.

As one typical cationic disinfectant, quaternary ammonium compounds (QACs) were approved for surface disinfection in the coronavirus disease 2019 pandemic and then unintentionally or intentionally released into the surrounding environment. Concerningly, it is still unclear how the soil microbial community succession happens and the nitrogen (N) cycling processes alter when exposed to QACs. In this study, one common QAC (benzalkonium chloride (BAC) was selected as the target contaminant, and its effects on the temporal changes in soil microbial community structure and nitrogen transformation processes were determined by qPCR and 16S rRNA sequencing-based methods. The results showed that the aerobic microbial degradation of BAC in the two different soils followed first-order kinetics with a half-life (4.92 vs. 17.33 days) highly dependent on the properties of the soil. BAC activated the abundance of N fixation gene (nifH) and nitrification genes (AOA and AOB) in the soil and inhibited that of denitrification gene (narG). BAC exposure resulted in the decrease of the alpha diversity of soil microbial community and the enrichment of Crenarchaeota and Proteobacteria. This study demonstrates that BAC degradation is accompanied by changes in soil microbial community structure and N transformation capacity.

Ecological Risk Analysis for Benzalkonium Chloride, Benzethonium Chloride, and Chloroxylenol in US Disinfecting and Sanitizing Products.

Use of three topical antiseptic compounds-benzalkonium chloride (BAC), benzethonium chloride (BZT), and chloroxylenol (PCMX)-has recently increased because of the phaseout of other antimicrobial ingredients (such as triclosan) in soaps and other disinfecting and sanitizing products. Further, use of sanitizing products in general increased during the coronavirus (COVID-19) pandemic. We assessed the environmental safety of BAC, BZT, and PCMX based on best available environmental fate and effects data from the scientific literature and privately held sources. The ecological exposure assessment focused on aquatic systems receiving effluent from wastewater-treatment plants (WWTPs) and terrestrial systems receiving land-applied WWTP biosolids. Recent exposure levels were characterized based on environmental monitoring data supplemented by modeling, while future exposures were modeled based on a hypothetical triclosan replacement scenario. Hazard profiles were developed based on acute and chronic studies examining toxicity to aquatic life (fish, invertebrates, algae, vascular plants) and terrestrial endpoints (plants, soil invertebrates, and microbial functions related to soil fertility). Risks to higher trophic levels were not assessed because these compounds are not appreciably bioaccumulative. The risk analysis indicated that neither BZT nor PCMX in any exposure media is likely to cause adverse ecological effects under the exposure scenarios assessed in the present study. Under these scenarios, total BAC exposures are at least three times less than estimated effect thresholds, while margins of safety for freely dissolved BAC are estimated to be greater than an order of magnitude. Because the modeling did not specifically account for COVID-19 pandemic-related usage, further environmental monitoring is anticipated to understand potential changes in environmental exposures as a result of increased antiseptic use. The analysis presented provides a framework to interpret future antiseptic monitoring results, including monitoring parameters and modeling approaches to address bioavailability of the chemicals of interest. Environ Toxicol Chem 2022;41:3095-3115. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Benzalkonium chloride, a common ophthalmic preservative, compromises rat corneal cold sensitive nerve activity.

PURPOSE: Corneal nerves comprise the densest sensory network in the body. Dysfunction of the corneal cold sensitive neurons (CSN) is implicated in ophthalmic disorders, including Dry Eye Disease, the most common ocular surface disorder. The preservative Benzalkonium chloride (BAK) and the mydriatic agent Phenylephrine hydrochloride (PHE) are considered to be inactive at the level of the CSNs. The purpose of this study is to test the impacts of continuous exposures to BAK or PHE at their clinically used concentrations on corneal nerve structure and function. METHODS: In vivo extracellular electrophysiology of the rat trigeminal ganglion was used to monitor CSN functional response to stimuli mimicking physiological states and stressors of the cornea. Corneal nerve structure was evaluated by immunostaining. RESULTS: Among the tested stimuli, cold probe receptive field stimulation and hyperosmolar stress were the most sensitive methods of detecting activity changes. CSN activity was attenuated after 30 min exposure to either PHE or BAK. After an hour-long washout period, BAK-treated neurons failed to recover activity while PHE-treated neurons showed signs of functional recovery. Intraepithelial nerve density was reduced and nerve fragmentation was increased in BAK-treated corneas, while PHE exposure left corneal nerves structurally intact. CONCLUSIONS: Our study suggests that prolonged ocular instillations of BAK or PHE alter CSN activity through two different processes - irreversible neuronal damage in the case of BAK vs. reversible attenuation in the case of PHE.

In vitro neurotoxicity evaluation of biocidal disinfectants in a human neuron-astrocyte co-culture model.

Biocidal disinfectants (BDs) that kill microorganisms or pathogens are widely used in hospitals and other healthcare fields. Recently, the use of BDs has rapidly increased as personal hygiene has become more apparent owing to the pandemic, namely the coronavirus outbreak. Despite frequent exposure to BDs, toxicity data of their potential neurotoxicity (NT) are lacking. In this study, a human-derived SH-SY5Y/astrocyte was used as a co-culture model to evaluate the chemical effects of BDs. Automated high-content screening was used to evaluate the potential NT of BDs through neurite growth analysis. A set of 12 BD substances classified from previous reports were tested. Our study confirms the potential NT of benzalkonium chloride (BKC) and provides the first evidence of the potential NT of poly(hexamethylenebicyanoguanide-hexamethylenediamine) hydrochloride (PHMB). BKC and PHMB showed significant NT at concentrations without cytotoxicity. This test system for analyzing the potential NT of BDs may be useful in early screening studies for NT prior to starting in vivo studies.

Protective Effect of Allyl Isothiocyanate in an Experimentally Induced Rat Model for Dry Eye Syndrome.

PURPOSE: Growing evidence emphasizes the role of inflammation and oxidative stress in the pathogenesis of Dry Eye Syndrome (DES). Concordantly, the importance of agents targeting the inflammatory cascade and oxidative stress in the treatment is also progressively increasing. Herein, the study has investigated the protective effects and underlying mechanism of allyl isothiocyanate (AITC) on the ocular surface in a benzalkonium chloride (BAC)-induced dry eye rat model. METHODS: A total of twenty-one Wistar albino rats were used to form the following three groups: Control, BAC, BAC + AITC. DES was established by topical application of BAC (four times daily for two weeks) in two groups, of which one group was treated with AITC (10 mg/kg BW daily oral dosage) for four weeks. Rats were monitored by dry eye diagnostic tests during the study period, and eventually, corneal tissues were used to evaluate for histopathologic analyzes and inflammatory and oxidative status. RESULTS: A significant improvement was observed in various histopathologic and ophthalmologic findings, including tear volume, tear film integrity, ocular surface damage, ocular inflammatory signs, corneal thickness, and edema through AITC supplementation. AITC prominently balanced the inflammatory status and oxidative stress by lowering key proinflammatory mediators (NF-κB, TNF-α, IL-1ß, IL-6, and IL-8) and increasing the activities of antioxidant enzymes (SOD, GSH-Px). Also, levels of protective tear proteins, including Muc1, Muc4, and Muc5 were recovered with AITC supplementation. CONCLUSION: AITC alleviates clinical and histopathologic signs related to DES. Antioxidative and anti-inflammatory properties of AITC play a significant role in the mechanism of action.

Pulmonary inflammation and cellular responses following exposure to benzalkonium chloride: Potential impact of disrupted pulmonary surfactant homeostasis.

Benzalkonium chloride (BKC) is a prototypical quaternary ammonium disinfectant. Previously, we suggested a no lethal dose level (0.005%) and an LD50 range (0.5-0.05%) of BKC following a single pharyngeal aspiration. Herein, we exposed BKC repeatedly by pharyngeal aspiration for 14 days (0.005 and 0.01%, female mice, total five times with interval of two days, 5 mice/group) and 28 days (0, 0.001, 0.005, and 0.01%, male and female mice, weekly, 16 mice/sex/group). Death following 14 days-repeated exposure did not occur. Meanwhile, chronic pathological lesions were observed in the lung tissues of mice exposed to BKC for 28 days. The total number of bronchial alveolar lavage cells increased, and pulmonary homeostasis of immunologic messenger molecules was disturbed. Following, we investigated BKC-induced cellular responses using human bronchial epithelial cells. The cytotoxicity increased rapidly with concentration. Lysosomal volume, NO production, and lipid peroxidation increased in BKC-treated cells, whereas intracellular ROS level decreased accompanying structural and functional damage of mitochondria. We also found that BKC affected the expression level of immune response, DNA damage, and amino acid biosynthesis-related molecules. More interestingly, lamellar body- and autophagosome-like structures were notably observed in cells exposed to BKC, and necrotic and apoptotic cell death were identified accompanying cell accumulation in the G2/M phase. Therefore, we suggest that repeated respiratory exposure of BKC causes pulmonary inflammation and lung tissue damage and that dead and damaged cells may contribute to the inflammatory response. In addition, the formation process of lamellar body-like structures may function as a key toxicity mechanism.

A novel ophthalmic latanoprost 0.005% nanoemulsion: a cytotoxicity study.

BACKGROUND: Benzalkonium chloride (BAK), the most commonly used preservative in anti-glaucoma eye drops, inflicts damage to the ocular surface. A novel anti-glaucoma formulation that avoids the use of BAK has been developed. The aim of this study was to evaluate the cytotoxicity of this formulation and to compare it with an ophthalmic solution containing BAK. METHODS: Two different latanoprost eye drops were used: one ophthalmic solution (LSc) containing BAK 0.02% and one ophthalmic nanoemulsion (LNe) with a soft preservative (potassium sorbate 0.18%). Human epithelial conjunctival cells were incubated for 15, 30, and 60 min with either LSc or LNe. The cytotoxicity was determined by MTT assay. Cell death was measured by flow cytometry using annexin V-FITC and propidium iodide. RESULTS: The values of cell viability and proliferation obtained from cells exposed to LNe were between 80 and 90% relative to the control group, whereas values obtained from cells exposed to LSc were around 30% at all study times (p < 0.05 at 15 and 30 min; p < 0.01 at 60 min). The percentage of viable cells decreased significantly when cells were incubated with LSc compared with cells incubated with LNe at all the study times, while the percentage of cells in late apoptosis/necrosis increased significantly in cells exposed to LSc compared to LNe. CONCLUSIONS: The new latanoprost nanoemulsion is significantly less cytotoxic on human conjunctival cells than LSc. These results suggest that the new formulation might be gentler on the eye surface than currently available BAK-preserved latanoprost solutions.

Exosomes Derived from Mouse Adipose-Derived Mesenchymal Stem Cells Alleviate Benzalkonium Chloride-Induced Mouse Dry Eye Model via Inhibiting NLRP3 Inflammasome.

PURPOSE: The objective of the study was to investigate efficacy and mechanisms of mouse adipose-derived mesenchymal stem cell-derived exosomes (mADSC-Exos) in the benzalkonium chloride (BAC)-induced mouse dry eye model. METHODS: Exosomes in the mADSC culture supernatant were isolated by ultracentrifugation. Western blotting, nanoparticle tracking analysis, and transmission electron microscopy were used to characterize mADSC-Exos. An experimental mouse model of dry eye was established by instillation of 0.2% BAC. mADSC-Exos were administered following BAC treatment. The positive control group was treated with commercial eye drops (0.1% pranoprofen). Corneal fluorescein staining, tear secretion, and tear film break-up time (BUT) were evaluated, and histologic analysis of the cornea and conjunctiva was performed by hematoxylin and eosin and periodic acid-Schiff staining. Apoptosis in the corneal epithelium was detected with the terminal deoxynucleotidyl transferase dUTP nick-end labeling assay and by Western blotting. Levels of pro-inflammatory cytokines in the cornea and conjunctiva were evaluated by flow cytometry, and mRNA and protein levels of NLR family pyrin domain-containing 3 (NLRP3) pathway components were assessed by quantitative real-time PCR and Western blotting, respectively. RESULTS: mADSC-Exos were characterized as vesicles with a bilayer membrane. The particle size distribution peak was at 134 nm. mADSC-Exos specifically expressed cluster of differentiation (CD)9, CD63, and CD81. mADSC-Exos treatment repaired ocular surface damage. Additionally, mADSC-Exos inhibited cell apoptosis, decreased the levels of interleukin (IL)-1ß, IL-6, IL-1α, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α, and increased levels of the anti-inflammatory cytokine IL-10. Meanwhile, NLRP3 inflammasome activation and upregulation of caspase-1, IL-1ß, and IL-18 were reversed by mADSC-Exos. CONCLUSIONS: mADSC-Exos alleviate ocular surface inflammation, suggesting that it is a promising treatment for dry eye.

Adsorption of benzalkonium chlorides onto polyethylene microplastics: Mechanism and toxicity evaluation.

Usage of disposable plastic products and disinfectants has been skyrocketing due to the COVID-19 pandemic. The random disposal of plastic products may result in greater microplastic pollution. Benzalkonium chloride is known as one of the most common ingredients of disinfectants. In this study, the adsorption behavior of benzalkonium chlorides (BAC12, BAC14, BAC16) on polyethylene microplastics (PE-MPs) and the combined toxic effects were investigated using batch adsorption experiment and Daphnia magna. The results showed that PE-MPs had strong adsorption capacity for BACs and the adsorption capacity increased (11.03-22.77 mg g-1) with their octanol-water distribution coefficients. The effect of pH was negligible while dissolved organic matter inhibited the adsorption. A slightly inverse relationship between particle size of PE-MPs and adsorption was observed. Additionally, the MP aging with UV/H2O2 increased the adsorption of BAC12 but decreased that of relatively hydrophobic BAC14 and BAC16. The survival rate of Daphnia magna increased up to 100% in the presence of PE-MPs depending upon their adsorption capacities, suggesting that PE-MPs do not act as a carrier but rather as a scavenger for BACs. This study provides important information necessary for environmental risk assessment with regard to the combined pollution of MPs and toxic chemicals.

Protective effects of low-molecular-weight components of adipose stem cell-derived conditioned medium on dry eye syndrome in mice.

The present study demonstrated the protective effects of low-molecular-weight adipose-derived stem cell-conditioned medium (LADSC-CM) in a mouse model of dry eye syndrome. Mice subjected to desiccating stress and benzalkonium chloride had decreased tear secretion, impaired corneal epithelial tight junction with microvilli, and decreased conjunctival goblet cells. Topical application of adipose-derived stem cell-conditioned medium (ADSC-CM) stimulated lacrimal tear secretion, preserved tight junction and microvilli of the corneal epithelium, and increased the density of goblet cells and MUC16 expression in the conjunctiva. The low-molecular-weight fractions (< 10 kDa and < 3 kDa) of ADSC-CM (LADSC-CM) provided better protections than the > 10 kDa or > 3 kDa fractions of ADSC-CM. In the in vitro study, desiccation for 10 min or hyperosmolarity (490 osmols) for 24 h caused decreased viability of human corneal epithelial cells, which were reversed by LADSC-CM. The active ingredients in the LADSC-CM were lipophobic and stable after heating and lyophilization. Our study demonstrated that LADSC-CM had beneficial effects on experimental dry eye. It is worthy of further exploration for the active ingredient(s) and the mechanism.

Lacrimal Gland as a Target Organ for Adenovirus Gene Therapy Encoding Erythropoietin for Dry Eye Induced by Benzalkonium Chloride.

Purpose: The aims of this work were a) to describe the histology of the lacrimal gland (LG) and cornea induced by an adenovirus (Ad) vector encoding the human erythropoietin (Epo) gene delivered to the LG and b) to evaluate the therapeutic potential of this strategy to prevent benzalkonium chloride (BAK) corneal toxicity.Methods: Structure and function of male Wistar rats LG were compared in the groups: 1) naïve control and 2) Ad-hEpo in the right LG (RLG). The protective response against BAK eye drops was compared among the groups 1) naïve control, 2) BAK in the right eye, 3) Ad-hEpo RLG + BAK and 4) Ad-hEpo in the right salivary gland (RSG)+BAK. Ad-hEpo groups received an injection of AdLTR2EF1a-hEPO (25 ul, 1010 particles/ml) in the right LG or SG (positive control). The BAK groups received 0.2% BAK in the right cornea twice a day. The tests applied after 7 days, included tear secretion, hEPO mRNA detection by qRT-PCR, LG and cornea histology, LG ELISA for cytokines and hematocrit.Results: hEPO mRNA was present in the Ad-hEpo RLG and RSG, but not kidney or liver samples (negative controls). TNF-α and IL-1ß increased in the LG exposed to Ad-hEpo compared to naïve control (p = .0115 and p = .0397, respectively). BAK reduced tear secretion, but this reduction was prevented by Ad-hEpo RLG+BAK and Ad-hEpo RSG+BAK (p = .017). The corneal epithelia were thinner in the BAK-treated groups independent of Ad-hEpo (p = .0009). Hematocrit increased only in the Ad-hEpo RSG group (p = .01).Conclusions: Ad-hEpo infection of rat LG and SG induces local, but only the SG infection induced systemic changes in rats. Importantly, Ad-hEpo attenuated the BAK-mediated toxic reduction in tear flow. Future studies must consider viral vector tissue tropism, biodistribution and effective therapeutic gene products for ocular surface diseases.