Adaptations to cationic biocide exposure differentially influence virulence factors and pathogenicity in Pseudomonas aeruginosa.

Cationic biocides (CBs), which include quaternary ammonium compounds (QACs), are employed to mitigate the spread of infectious bacteria, but resistance to such surface disinfectants is rising. CB exposure can have profound phenotypic implications that extend beyond allowing microorganisms to persist on surfaces. Pseudomonas aeruginosa is a deadly bacterial pathogen that is intrinsically tolerant to a wide variety of antimicrobials and is commonly spread in healthcare settings. In this study, we pursued resistance selection assays to the QAC benzalkonium chloride and quaternary phosphonium compound P6P-10,10 to assess the phenotypic effects of CB exposure in P. aeruginosa PAO1 and four genetically diverse, drug-resistant clinical isolates. In particular, we sought to examine how CB exposure affects defensive strategies and the virulence-associated "offensive" strategies in P. aeruginosa. We demonstrated that development of resistance to BAC is associated with increased production of virulence-associated pigments and alginate as well as pellicle formation. In an in vivo infection model, CB-resistant PAO1 exhibited a decreased level of virulence compared to wild type, potentially due to an observed fitness cost in these strains. Taken together, these results illustrate the significant consequence CB resistance exerts on the virulence-associated phenotypes of P. aeruginosa.

Efficient degradation of benzalkonium chloride (BAC) by zero-valent iron activated persulfate: Kinetics, reaction mechanisms, theoretical calculations and toxicity evolution.

The present study systematically investigated the elimination of benzalkonium chloride (BAC) in the zero valent iron activated persulfate (Fe0/PS) system. The influence of operational parameters, including PS concentration, Fe0 dosage and pH, were investigated through a series of kinetic experiments. When the Fe0 dosage was 5.0 mM, the initial ratio of [PS]: [BAC] was 10:1, the degradation efficiency could achieve 91.7% at pH 7.0 within 60 min. Common inorganic anions and humic acid did not significantly affect BAC degradation, implying that Fe0/PS system had a potential application prospect in the actual wastewater remediation. Based on the electron paramagnetic resonance test and quenching experiments, the BAC degradation was found to be contributed by •OH, SO4•- and Fe(IV). A total of 23 intermediates were identified by the liquid chromatography-mass spectrometry, and the degradation pathways were proposed accordingly, including dealkylation and demethylation, hydroxylation, sulfate substitution and benzyl C-N cleavage reactions. Density functional theory based calculations were conducted to realize the rationality of the proposed reaction mechanisms. The toxicity of transformation products was predicted by ECOSAR program. This work demonstrated the possibility of BAC removal in hospital and municipal wastewater by Fe0/PS treatment, and also provides a safe choice for deep treatment of quaternary ammonium salt wastewater.

Systemic contact dermatitis in a family: Case report.

BACKGROUND: Systemic contact dermatitis (SCD) is an allergic inflammatory skin disease. We report that 3 family members developed SCD after exposing to laundry detergent containing benzalkonium chloride, which is rare. SCD caused by benzalkonium chloride has been reported. However, Similar symptoms in the whole family caused by it have not been reported yet. In our case, a 36-year-old man was diagnosed as SCD, and his symptoms had not controlled after 7 days treatment, until he stopped dressing the clothes washed by the laundry detergent containing benzalkonium chloride. It was interesting that both his wife and the daughter developed SCD successively, and they have not exposed to any haptens besides the benzalkonium chloride in the laundry detergent. METHODS: Dermoscopic examination showed bright-red background, focal branching vessels and white scales. HE staining from the lesion revealed hyperkeratosis and parakeratosis, focal subcorneal microabscess, ocal hyperkeratosis, koilocyte in the epidermis, and erythrocyte extravasation, fibroplasia, hyaline degeneration and scattered aggregates of lymphocytes in the dermis. Then path test was performed 1 month after recovery with benzalkonium chloride 0.05% and 0.1% in petrolatum. RESULTS: Stop the laundry detergent containing benzalkonium chloride. The symptoms had controlled after they stopped the laundry detergent containing benzalkonium chloride. CONCLUSION: The case highlights that benzalkonium chloride with very low concentration and repeated exposure may be an active agent of SCD. It is of the utmost importance to pay close attention to patients presenting with similar symptoms within the family. A thorough examination of the medical history is essential to determine the underlying cause.

Benzalkonium chloride induced acute toxicity and its multifaceted implications on growth, hematological metrics, biochemical profiles, and stress-responsive biomarkers in tilapia (Oreochromis mossambicus).

This study aimed to investigate the toxic effects of benzalkonium chloride (BAC) on Oreochromis mossambicus, a freshwater fish species. Probit analysis was used to determine the lethal concentration (LC50) of BAC for different exposure periods (24, 48, 72, and 96 h). The viability of fish exposed to BAC was assessed using the general threshold survival models (GUTS) and confirmed with relevant datasets to evaluate model accuracy. Experimental groups of fish were exposed to BAC concentrations equivalent to 10% and 20% of the 96-h LC50 for 45 days. The study revealed significant alterations in various parameters during sublethal BAC exposure. These effects included decreased specific growth rate (SGR), red blood cell count (RBC), hemoglobin (Hb) concentration, hematocrit (Ht) value, plasma protein, and albumin levels, as well as acetylcholinesterase (AChE) activities in both gills and liver. Additionally, an increase in gastrosomatic index (GSI), feed conversion ratio (FCR), plasma glucose and creatinine concentrations, alanine aminotransferase (ALT), aspartate aminotransferase (AST) enzymatic activities, catalase (CAT), superoxide dismutase (SOD), and malondialdehyde (MDA) levels were observed in the exposed fish's gills and liver. Furthermore, the study found that glutathione S-transferase (GST) and glutathione peroxidase (GPx) levels initially increased and then decreased in both gills and liver after exposure to BAC. Correlation matrix analysis, multivariate multiple regression (MMR), canonical correspondence analysis (CCA), integrated biomarker response (IBR), and biomarker response index (BRI) were utilized to assess the impact of BAC on fish, highlighting significant effects on multiple biomarkers in O. mossambicus following surfactant exposure. Thus, the study provides valuable insights into the toxic effects of BAC on this fish species, emphasizing the importance of monitoring such pollutants in aquatic environments.

Necrostatin-1 protects corneal epithelial cells by inhibiting the RIPK1/RIPK3/MLKL cascade in a benzalkonium chloride-induced model of necroptosis.

PURPOSE: Benzalkonium chloride (BAC) is commonly used as a preservative in ophthalmic medications, despite its potential to induce chemical injury. Extensive research has demonstrated that BAC can lead to adverse effects, including injuries to the ocular surface. Our study aimed to elucidate the underlying mechanism of necroptosis induced by BAC. METHODS: Human corneal epithelial (HCE) cells and mouse corneas were subjected to chemical injury, and the necrostatin-1 (Nec1) group was compared to the dimethylsulfoxide (DMSO) group. The extent of damage to HCE cells was assessed using CCK-8 and flow cytometry. Hematoxylin and eosin staining, as well as fluorescein sodium staining, were used to detect and characterize corneal injury. The activation of inflammatory cytokines and necroptosis-related proteins and genes was evaluated using Western blotting, immunofluorescence staining, and quantitative RT‒PCR. RESULTS: In our study, the induction of necroptosis by a hypertonic solution was not observed. However, necroptosis was observed in HCE cells exposed to NaOH and BAC, which activated the receptor-interacting protein kinase 1 (RIPK1) - receptor-interacting protein kinase 3 (RIPK3) - mixed lineage kinase domain-like protein (MLKL) signaling pathway. In mouse corneal tissues, BAC could induce necroptosis and inflammation. The administration of Nec1 mitigated the inflammatory response and ocular surface damage caused by BAC-induced necroptosis in our experimental models. Furthermore, our in vivo experiments revealed that the severity of necroptosis was greater in the 3-day group than in the 7-day group. CONCLUSIONS: Necroptosis plays a role in the pathological development of ocular surface injury caused by exposure to BAC. Furthermore, our study demonstrated that the administration of Nec1 could mitigate the pathological effects of necroptosis induced by BAC in clinical settings.

M2 macrophage-derived extracellular vesicles ameliorate Benzalkonium Chloride-induced dry eye.

Dry eye disease (DED) is a common ocular condition affecting a significant portion of the global population, yet effective treatment options remain elusive. This study investigates the therapeutic potential of M2 macrophage-derived extracellular vesicles (M2-EVs) in a mouse model of DED. The DED model was established using 0.2% benzalkonium chloride (BAC) eye drops, applied twice daily for a week. Post induction, the mice were categorized into 5 groups: PBS, Sodium Hyaluronate (HA, 0.1%), Fluoromethalone (FM, 0.1%), M0-EVs, and M2-EVs. The efficacy of M2-EVs was assessed through tear production, corneal fluorescein staining and HE staining. RNA sequencing (RNA-seq) was employed to investigate the mechanisms underlying the therapeutic effects of M2-EVs in DED. Notably, the M2-EVs treated group exhibited the highest tear secretion, indicating improved tear film stability and reduced corneal surface damage. Histological analysis revealed better corneal structure organization in the M2-EVs group, suggesting enhanced ocular surface repair and corneal preservation. Furthermore, M2-EVs treatment significantly decreased pro-inflammatory cytokine levels and showed unique enrichment of genes related to retinal development. These findings suggest that M2-EVs could serve as a promising noninvasive therapeutic approach for human DED, targeting ocular surface inflammation.

Sex-Specific Effects of Environmental Exposure to the Antimicrobial Agents Benzalkonium Chloride and Triclosan on the Gut Microbiota and Health of Zebrafish (Danio rerio).

The use of disinfectants containing benzalkonium chloride (BAC) has become increasingly widespread in response to triclosan (TCS) restrictions and the COVID-19 pandemic, leading to the increasing presence of BAC in aquatic ecosystems. However, the potential environmental health impacts of BAC on fish remain poorly explored. In this study, we show that BAC and TCS can induce the gut dysbiosis in zebrafish (Danio rerio), with substantial effects on health. Breeding pairs of adult zebrafish were exposed to environmentally relevant concentrations of BAC and TCS (0.4-40 µg/L) for 42 days. Both BAC and TCS exposure perturbed the gut microbiota, triggering the classical NF-κB signaling pathway and resulting in downstream pathological toxicity associated with inflammatory responses, histological damage, inhibited ingestion, and decreased survival. These effects were dose-dependent and sex-specific, as female zebrafish were more susceptible than male zebrafish. Furthermore, we found that BAC induced toxicity to a greater extent than the restricted TCS at environmentally relevant concentrations, which is particularly concerning. Our results suggest that environmental exposure to antimicrobial chemicals can have ecological consequences by perturbing the gut microbiota, a previously underappreciated target of such chemicals. Rigorous ecological analysis should be conducted before widely introducing replacement antimicrobial compounds into disinfecting products.

Developing a Passive Dosing Method for Acute Aquatic Toxicity Tests of Cationic Surfactant Benzalkoniums (BACs).

Benzalkonium chlorides (BACs) have been of environmental concern due to their widespread use and potential harm. However, challenges arise in defining and controlling the exposure concentration (Cw) in aquatic toxicity tests involving BACs with a long alkyl chain (i.e., #C > 14). To address this, a novel passive dosing method was introduced in the 48 h-acute ecotoxicity test on Daphnia magna and compared to the conventional solvent-spiking method in terms of Cw stability and toxicity results. Among 13 sorbent materials tested for their sorption capacity, poly(ether sulfone) (PES) membrane was an optimal passive dosing reservoir, with equilibrium desorption of BACs to water achieved within 24 h. The Cw of BACs remained constant in both applied dosing methods during the test period. However, the Cw in solvent-spiking tests was lower than the nominal concentration for long-chain BACs, particularly at low exposure concentrations. Notably, the solvent-spiking tests indicated that the toxicity of BACs increased with alkyl chain length from C6 to 14, followed by a decline in toxicity from C14 to 18. In contrast, the passive dosing method displayed similar or slightly increasing toxicity levels of BACs from C14 to C18, indicating higher toxicity of C16 and C18-BACs than that inferred by the solvent spiking test. These findings emphasize the potential of applying this innovative passive dosing approach in aquatic toxicity tests to generate reliable and accurate toxicity data and support a comprehensive risk assessment of cationic surfactants.

Impact of benzalkonium chloride on anaerobic granules and its long-term effects on reactor performance.

This study assessed the inhibitory and performance-degrading effects induced by the cationic surfactant benzalkonium chloride (BAC) on anaerobic granules during the long-term operation of a laboratory-scale expanded granular sludge bed (EGSB) reactor. To address the critical scientific problem of how BAC affects the efficiency of EGSB reactors, this research uniquely evaluated the long-term stress response to BAC by systematically comparing continuous and discontinuous inhibitor exposure scenarios. The novel comparison demonstrated that inhibitor concentration is of minor relevance compared to the biomass-specific cumulative inhibitor load in the reactor. After exceeding a critical biomass-specific cumulative inhibitor load of 6.1-6.5 mg BAC/g VS, continuous and discontinuous exposure to BAC caused comparable significant deterioration in reactor performance, including accumulation of volatile fatty acids (VFA), decreased removal efficiency, reduced methane production, as well as the wash-out, flotation, and disintegration of anaerobic granules. BAC exposures had a more detrimental effect on methanogenesis than on acidogenesis. Moreover, long-term stress by BAC led to an inhibition of protein production, resulting in a decreased protein-to-polysaccharide ratio of extracellular polymeric substances (EPS) that promoted destabilizing effects on the granules. Finally, hydrogenotrophic methanogenesis was triggered. Reactor performance could not be restored due to the severe loss of granular sludge.

SPARC overexpression in allogeneic adipose-derived mesenchymal stem cells in dog dry eye model induced by benzalkonium chloride.

BACKGROUND: Nowadays, companion and working dogs hold significant social and economic importance. Dry eye, also known as dry keratoconjunctivitis (KCS), a common disease in ophthalmology, can readily impact a dog's working capacity and lead to economic losses. Although there are several medications available for this disease, all of them only improve the symptoms on the surface of the eye, and they are irritating and not easy to use for long periods of time. Adipose-derived mesenchymal stem cells (ADMSC) are promising candidates for tissue regeneration and disease treatment. However, long-term in vitro passaging leads to stemness loss of ADMSC. Here, we aimed to use ADMSC overexpressing Secreted Protein Acidic and Rich in Cysteine (SPARC) to treat 0.25% benzalkonium chloride-treated dogs with dry eye to verify its efficacy. For in vitro validation, we induced corneal epithelial cell (HCECs) damage using 1 µg/mL benzalkonium chloride. METHODS: Fifteen male crossbred dogs were randomly divided into five groups: normal, dry eye self-healing control, cyclosporine-treated, ADMSC-CMV-treated and ADMSC-OESPARC-treated. HCECs were divided into four groups: normal control group, untreated model group, ADMSC-CMV supernatant culture group and ADMSC-OESRARC supernatant culture group. RESULTS: SPARC-modified ADMSC had the most significant effect on canine ocular surface inflammation, corneal injury, and tear recovery, and the addition of ADMSC-OESPARC cell supernatant also had a salvage effect on HCECs cellular damage, such as cell viability and cell proliferation ability. Moreover, analysis of the co-transcriptome sequencing data showed that SPARC could promote corneal epithelial cell repair by enhancing the in vitro viability, migration and proliferation and immunosuppression of ADMSC. CONCLUSION: The in vitro cell test and in vivo model totally suggest that the combination of SPARC and ADMSC has a promising future in novel dry eye therapy.

Chemical mutagenesis of Listeria monocytogenes for increased tolerance to benzalkonium chloride shows independent genetic underpinnings and off-target antibiotic resistance.

Listeria monocytogenes, a potentially fatal foodborne pathogen commonly found in food processing facilities, creates a significant economic burden that totals more than $2 billion annually in the United States due to outbreaks. Quaternary ammonium compounds (QACs), including benzalkonium chloride (BAC), are among the most widely used sanitizers to inhibit the growth and spread of L. monocytogenes from food processing facilities. However, resistance to QACs has been increasing in L. monocytogenes and different genetic mechanisms conferring resistance have been discovered. Here, we used ethyl methanesulfonate (EMS) to chemically mutagenize the BAC-susceptible strain, L. monocytogenes FSL-N1-304. We isolated two mutants with increased tolerance to BAC compared to the parental strain. Next, we assessed the off-target effect of increased tolerance to BAC by measuring the minimum inhibitory concentrations (MICs) of a diverse set of antibiotics, revealing that mut-1 and mut-2 displayed significantly increased resistance to fluoroquinolone antibiotics compared to the parental strain. A hemolysis assay was then used to investigate a potential correlation between BAC tolerance and virulence. Interestingly, mut-1 and mut-2 both exhibited significantly higher hemolysis percentage than the parental strain. We then sequenced the genomes of the parental strain and both mutants to identify mutations that may be involved in the increased resistance to BAC. We identified 3 and 29 mutations in mut-1 and mut-2, respectively. mut-1 contained nonsynonymous mutations in dagK (a diacylglycerol kinase), lmo2768 (a permease-encoding gene), and lmo0186 (resuscitation promoting factor). mut-2 contained a nonsense mutation in the nucleotide excision repair enzyme UvrABC system protein B encoding gene, uvrB, which likely accounts for the higher number of mutations observed. Transcriptome analysis in the presence of BAC revealed that genes related to the phosphotransferase system and internalins were up-regulated in both mutants, suggesting their significance in the BAC stress response. These two mutants provide insights into alternative mechanisms for increased BAC tolerance and could further our understanding of how L. monocytogenes persists in the food processing environment.

Exploring the defense strategies of benzalkonium chloride exposures on the antioxidant system, photosynthesis and ROS accumulation in Lemna minor.

With the advent of technological advancements post the industrial revolution, thousands of chemicals are introduced into the market annually to enhance different facets of human life. Among these, pharmaceutical and personal care products (PPCPs), including antibiotics and disinfectants, such as benzalkonium chlorides (BACs), are prominent. BACs, often used for surface and hand disinfection in high concentrations or as preservatives in health products such as nasal sprays and eye drops, may present environmental risks if they seep into irrigation water through prolonged exposure or improper application. The primary objective of this study is to elucidate the tolerance mechanisms that may arise in Lemna minor plants, known for their remarkable capability to accumulate substances efficiently, in response to exogenously applied BACs at varying concentrations. The study applied six different concentrations of BACs, ranging from 0.25 to 10 mg L-1. The experimental period spanned seven days, during which the treatments were conducted in triplicate to ensure reliability and reproducibility of the results. It was observed that low concentrations of BACs (0.25, 0.5 and 1 mg L-1) did not elicit any statistically significant changes in growth parameters. However, higher concentrations of BACs (2.5, 5, and 10 mg L-1) resulted in a reduction in RGR by 20%, 28%, and 36%, respectively. Chlorophyll fluorescence declined significantly at BAC doses of 5 and 10 mg L-1, with Fv/Fm ratios decreasing by 9% and 15%, and Fv/Fo ratios by 40% and 39%, respectively. Proline content decreased in all treatment groups, with a 46% reduction at 10 mg L-1 BAC. TBARS and H2O2 contents increased proportionally with BAC dosage, showing the highest increases of 30% and 40% at 10 mg L-1, respectively. The noticeable increase in SOD enzyme activity at BAC concentrations of 0.5, 1, and 2.5 mg L-1, with increases of 2.7-fold, 2.2-fold, and 1.7-fold respectively, along with minimal accumulation of H2O2, suggests that L. minor plants have a strong tolerance to BAC. This is supported by the efficient functioning of the CAT and GST enzymes, especially evident at the same concentrations, where increased activities effectively reduce the buildup of H2O2. In the AsA-GSH cycle, although variations were observed between groups, the contribution of the GR enzyme to the preservation of GSH content by recycling GSSG likely maintained redox homeostasis in the plant, especially at low concentrations of BACs. The study revealed that L. minor effectively accumulates BAC alongside its tolerance mechanisms and high antioxidant activity. These results underscore the potential for environmental cleanup efforts through phytoremediation.

A Standardized Extract of Green-Cotyledon Small Black Soybean (EYESOY®) Ameliorates Dry Eye Syndrome in an Animal Model.

PURPOSE: Dry eye syndrome is a common ocular disease that causes morbidity, high healthcare burden, and decreased quality of life. In this study, we evaluated the beneficial effects of a standardized extract of small black soybean (EYESOY®) in a benzalkonium chloride (BAC)-induced murine model of dry eye. METHODS: Experimental dry eye was induced by instillation of 0.02% BAC on the right eye of the Sprague-Dawley rats. Saline solution or EYESOY were administered orally every day for 8 weeks. RESULTS: EYESOY significantly improved tear volume in the cornea compared with that in the BAC group. Moreover, EYESOY inhibited damage to the corneal epithelial cells and lacrimal glands by suppressing the oxidative and inflammatory responses in a mouse dry eye model. It also increased the goblet cell density and mucin integrity in the conjunctiva. CONCLUSIONS: Our results suggest that EYESOY has the potential to alleviate dry eye syndrome.

Bacterial dealkylation of benzalkonium chlorides in wastewater produces benzyldimethylamine, a potent N-nitrosodimethylamine precursor.

N-nitrosodimethylamine (NDMA) is a carcinogenic disinfection byproduct that forms during chloramine disinfection of municipal wastewater effluents which are increasingly used to augment drinking water supplies due to growing water scarcity. Knowledge of wastewater NDMA precursors is limited and the known pool of NDMA precursors has not closed the mass balance between precursor loading, precursor NDMA yield, and formed NDMA. Benzalkonium chlorides (BACs) are the most prevalent quaternary ammonium surfactants and have antimicrobial properties. The extensive utilization of BACs in household, commercial and industrial products has resulted in their detection in wastewater at elevated concentrations. We report the formation of a potent NDMA precursor, benzyldimethylamine (BDMA) from the biodegradation of BACs during activated sludge treatment. BDMA formation and NDMA formation potential (FP) were functions of BAC and mixed liquor suspended solids concentration at circumneutral pH, and the microbial community source. Sustained exposure to microorganisms reduced NDMA FP through successive dealkylation of BDMA to less potent precursors. BAC alkyl chain length (C8 - C16) had little impact on NDMA FP and BDMA formation because chain cleavage occurred at the C-N bond. Wastewater effluents collected from three facilities contained BDMA from 15 to 106 ng/L, accounting for an estimated 4 to 38 % of the NDMA precursor pool.

Melatonin mediated tolerance to benzalkonium chloride phytotoxicity through improved growth, photochemical reactions, and antioxidant system in wild-type and snat2 mutant Arabidopsis lines.

Melatonin (Mel) is a phytohormone that plays a crucial role in various plant processes, including stress response. Despite numerous studies on the role of Mel in stress resistance, its significance in plants exposed to benzalkonium chloride (BAC) pollution remains unexplored. BAC, a common antiseptic, poses a threat to terrestrial plants due to its widespread use and inefficient removal, leading to elevated concentrations in the environment. This study investigated the impact of BAC (0.5 mg L-1) pollution on wild-type Col-0 and snat2 knockout mutant Arabidopsis lines, revealing reduced growth, altered water relations, and gas exchange parameters. On the other hand, exogenous Mel (100 µM) treatments mitigated BAC-induced phytotoxicity and increased the growth rate by 1.8-fold in Col-0 and 2-fold in snat2 plants. snat2 mutant seedlings had a suppressed carbon assimilation rate (A) under normal conditions, but BAC contamination led to further A repression by 71% and 48% in Col-0 and snat2 leaves, respectively. However, Mel treatment on stressed plants was successful in improving Fv/Fm and increased the total photosynthesis efficiency by regulating photochemical reactions. Excessive H2O2 accumulation in the guard cells of plants exposed to BAC pollution was detected by confocal microscopy. Mel treatments triggered almost all antioxidant enzyme activities (except POX) in both Arabidopsis lines under stress. This enhanced antioxidant activity, facilitated by foliar Mel application, contributed to the alleviation of oxidative damage, regulation of photosynthesis reactions, and promotion of plant growth in Arabidopsis. In addition to corroborating results observed in many agricultural plants regarding the development of tolerance to environmental stresses, this study provides novel insights into the action mechanisms of Mel under the emerging pollutant benzalkonium chloride.

Integrated high-throughput drug screening microfluidic system for comprehensive ocular toxicity assessment.

Traditional experimental methodologies suffer from a few limitations in the toxicological evaluation of the preservatives added to eye drops. In this study, we overcame these limitations by using a microfluidic device. We developed a microfluidic system featuring a gradient concentration generator for preservative dosage control with microvalves and micropumps, automatically regulated by a programmable Arduino board. This system facilitated the simultaneous toxicological evaluation of human corneal epithelial cells against eight different concentrations of preservatives, allowing for quadruplicate experiments in a single run. In our study, the IC50 values for healthy eyes and those affected with dry eyes syndrome showed an approximately twofold difference. This variation is likely attributable to the duration for which the preservative remained in contact with corneal cells before being washed off by the medium, suggesting the significance of exposure time in the cytotoxic effect of preservatives. Our microfluidic system, automated by Arduino, simulated healthy and dry eye environments to study benzalkonium chloride toxicity and revealed significant differences in cell viability, with IC50 values of 0.0033% for healthy eyes and 0.0017% for dry eyes. In summary, we implemented the pinch-to-zoom feature of an electronic tablet in our microfluidic system, offering innovative alternatives for eye research.

A water-soluble preparation for intravenous administration of isorhamnetin and its pharmacokinetics in rats.

Flavonoids are considered as health-protecting food constituents. The testing of their biological effects is however hampered by their low oral absorption and complex metabolism. In order to investigate the direct effect(s) of unmetabolized flavonoid, a preparation in a biologically friendly solvent for intravenous administration is needed. Isorhamnetin, a natural flavonoid and a human metabolite of the most frequently tested flavonoid quercetin, has very low water solubility (<3.5 µg/mL). The aim of this study was to improve its solubility to enable intravenous administration and to test its pharmacokinetics in an animal model. By using polyvinylpyrrolidone (PVP10) and benzalkonium chloride, we were able to improve the solubility approximately 600 times to 2.1 mg/mL. This solution was then administered intravenously at a dose of 0.5 mg/kg of isorhamnetin to rats and its pharmacokinetics was analyzed. The pharmacokinetics of isorhamnetin corresponded to two compartmental model with a rapid initial distribution phase (t1/2α: 5.7 ± 4.3 min) and a slower elimination phase (t1/2ß: 61 ± 47.5 min). Two sulfate metabolites were also identified. PVP10 and benzalkonium did not modify the properties of isorhamnetin (iron chelation and reduction, and cell penetration) substantially. In conclusion, the novel preparation reported in this study is suitable for future testing of isorhamnetin effects under in vivo conditions.

Experimental testing of two urban stressors on freshwater biofilms.

Aquatic ecosystems and their communities are exposed to numerous stressors of various natures (chemical and physical), whose impacts are often poorly documented. In urban areas, the use of biocides such as dodecyldimethylbenzylammonium chloride (DDBAC) and their subsequent release in wastewater result in their transfer to urban aquatic ecosystems. DDBAC is known to be toxic to most aquatic organisms. Artificial light at night (ALAN) is another stressor that is increasing globally, especially in urban areas. ALAN may have a negative impact on photosynthetic cycles of periphytic biofilms, which in turn may result in changes in their metabolic functioning. Moreover, studies suggest that exposure to artificial light could increase the biocidal effect of DDBAC on biofilms. The present study investigates the individual and combined effects of DDBAC and/or ALAN on the functioning and structure of photosynthetic biofilms. We exposed biofilms in artificial channels to a nominal concentration of 30 mg.L-1 of DDBAC and/or ALAN for 10 days. ALAN modified DDBAC exposure, decreasing concentrations in the water but not accumulation in biofilms. DDBAC had negative impacts on biofilm functioning and structure. Photosynthetic activity was inhibited by > 90% after 2 days of exposure, compared to the controls, and did not recover over the duration of the experiment. Biofilm composition was also impacted, with a marked decrease in green algae and the disappearance of microfauna under DDBAC exposure. The integrity of algal cells was compromised where DDBAC exposure altered the chloroplasts and chlorophyll content. Impacts on autotrophs were also observed through a shift in lipid profiles, in particular a strong decrease in glycolipid content was noted. We found no significant interactive effect of ALAN and DDBAC on the studied endpoints.

Antioxidant Carbon Dots Nanozyme Loaded in Thermosensitive in situ Hydrogel System for Efficient Dry Eye Disease Treatment.

Purpose: Dry eye disease (DED) is a multifactorial ocular surface disease with a rising incidence. Therefore, it is urgent to construct a reliable and efficient drug delivery system for DED treatment. Methods: In this work, we loaded C-dots nanozyme into a thermosensitive in situ gel to create C-dots@Gel, presenting a promising composite ocular drug delivery system to manage DED. Results: This composite ocular drug delivery system (C-dots@Gel) demonstrated the ability to enhance adherence to the corneal surface and extend the ocular surface retention time, thereby enhancing bioavailability. Furthermore, no discernible ocular surface irritation or systemic toxicity was observed. In the DED mouse model induced by benzalkonium chloride (BAC), it was verified that C-dots@Gel effectively mitigated DED by stabilizing the tear film, prolonging tear secretion, repairing corneal surface damage, and augmenting the population of conjunctival goblet cells. Conclusion: Compared to conventional dosage forms (C-dots), the C-dots@Gel could prolong exhibited enhanced retention time on the ocular surface and increased bioavailability, resulting in a satisfactory therapeutic outcome for DED.

Co-culture of benzalkonium chloride promotes the biofilm formation and decreases the antibiotic susceptibility of a Pseudomonas aeruginosa strain.

Benzalkonium chloride (BAC) is a disinfectant with broad-spectrum antibacterial properties, yet despite its widespread use and detection in the environment, the effects of BAC exposure on microorganisms remain poorly documented. Herein, the impacts of BAC on a Pseudomonas aeruginosa strain Jade-X were systematically investigated. The results demonstrated that the minimum inhibitory concentration (MIC) of BAC against strain Jade-X was 64 mg L-1. Exposure to BAC concentrations of 8, 16, 32, and 64 mg L-1 significantly augmented biofilm formation by 2.03-, 2.43-, 2.96-, and 2.56-fold respectively. The swimming and twitching abilities, along with the virulence factor production, were inhibited. Consistently, quantitative reverse transcription PCR assays revealed significant downregulation of genes related to flagellate- and pili-mediated motilities (flgD, flgE, pilB, pilQ, and motB), as well as phzA and phzB genes involved in pyocyanin production. The results of disk diffusion and MIC assays demonstrated that BAC decreased the antibiotic susceptibility of ciprofloxacin, levofloxacin, norfloxacin, and tetracycline. Conversely, an opposite trend was observed for polymyxin B and ceftriaxone. Genomic analysis revealed that strain Jade-X harbored eleven resistance-nodulation-cell division efflux pumps, with mexCD-oprJ exhibiting significant upregulation while mexEF-oprN and mexGHI-opmD were downregulated. In addition, the quorum sensing-related regulators LasR and RhlR were also suppressed, implying that BAC might modulate the physiological and biochemical behaviors of strain Jade-X by attenuating the quorum sensing system. This study enhances our understanding of interactions between BAC and P. aeruginosa, providing valuable insights to guide the regulation and rational use of BAC.