Understanding the impact of environmentally relevant alkyl C12-16 dimethylbenzyl ammonium chloride concentrations on zebrafish health.

Alkyldimethylbenzylammonium chlorides (ADBACs), classified as second-generation quaternary ammonium compounds, are extensively employed across various sectors, encompassing veterinary medicine, food production, pharmaceuticals, cosmetics, ophthalmology, and agriculture. Consequently, significant volumes of ADBAC C12-C16 are discharged into the environment, posing a threat to aquatic organisms. Regrettably, comprehensive data regarding the toxicological characteristics of these compounds remain scarce. This research aimed to determine whether or not ADBAC C12-C16, at environmentally relevant concentrations (0.4, 0.8, and 1.6 µg/L), may instigate oxidative stress and alter the expression of apoptosis-related genes in the liver, brain, gut, and gills of Danio rerio adults (5-6 months). The findings revealed that ADBAC C12-C16 elicited an oxidative stress response across all examined organs following 96 h of exposure. Nonetheless, the magnitude of this response varied among organs, with the gills exhibiting the highest degree of susceptibility, followed by the gut, liver, and brain, in descending order. Only the gut and gills of the examined organs displayed a concentration-dependent reduction in the activity of superoxide dismutase (SOD) and catalase (CAT). Akin to the oxidative stress response, all organs exhibited a marked increase in bax, blc2, casp3, and p53 expression levels. However, the gills and gut manifested a distinctive suppression in the expression of nrf1 and nrf2. Our Principal Component Analysis (PCA) confirmed that SOD, CAT, nrf1, and nrf2 were negatively correlated to oxidative damage biomarkers and apoptosis-related genes in the gills and gut; meanwhile, in the remaining organs, all biomarkers were extensively correlated. From the above, it can be concluded that ADBAC C12-C16 in low and environmental concentrations may threaten the health of freshwater fish.

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.

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.

Lacrimal osmolarity and ocular surface in experimental model of dry eye caused by toxicity / Osmolaridade lacrimal e superfície ocular em modelo de olho seco por toxicidade

Purpose: Describe an animal model of dry induced by topical instillation of BAK and evaluate ocular surface biomarkers and histological findings. Methods: Male Wistar rats were used.Topical instillation of 0.2% BAK eyedrops twice a day during 7 days, in the right eye of each animal, while the other eye was taken as control. After 7 days treatment, we performed evaluation of tear film osmolarity, the red phenol thread and ocular surface staining with fluorescein and lissamine green. Afterwards, the animals were sacrificed for tissue extraction and histological evaluation under optical microscopy and H&E staining. Results: Compared with untreated controls, the BAK-group presented tear secretion significantly decreased, increased ocular surface staining by fluorescein and lissamine green and tear film hyperosmolarity (p <0,05). Histological evaluation revealed epithelial thinning and estromal oedema. Conclusions: A toxicity animal model of dry eye induced by topical instillation of benzalkonium chloride, which presents corneal and ocular surface alterations, decreased tear film volume and tear hyperosmolarity as seen in dry eye condition.

Objetivo: Descrever um modelo animal de olho seco induzido pela aplicação tópica de cloreto de benzalcônio (BAC) e avaliar marcadores de integridade da superfície ocular e os achados histológicos. Métodos: Foram utilizados ratos wistar machos adultos. Foi realizada a administração tópica de colírio de BAC 0,2% no olho direito de cada animal duas vezes por dia, durante 7 dias, sendo o olho contralateral tido como controle. Após o tratamento foi realizada a avaliação da osmolaridade do filme lacrimal, o teste de fenol vermelho e a coloração com fluoresceína e lisamina verde. Os animais foram sacrificados e os tecidos extraídos para o estudo histológico da córnea, por microscopia óptica, corada com hematoxilina eosina (H&E). Resultados: Comparados com os controles não tratados o grupo BAC apresentou diminuição significativa na secreção lacrimal, defeitos na integridade epitelial da superfície ocular marcada por corantes vitais, fluoresceína e lisamina verde além do aumento da osmolaridade do filme lacrimal (p < 0,05). À avaliação histológica observou-se diminuição da espessura do epitélio e edema estromal induzidos pela aplicação de BAC. Conclusão: O modelo animal de olho seco por toxicidade induzido pela aplicação tópica de cloreto de benzalcônio apresentou alterações estruturais da córnea e da superfície ocular, diminuição do volume lacrimal e hiperosmolaridade da lágrima características dessa condição.

Restoring conjunctival tolerance by topical nuclear factor-κB inhibitors reduces preservative-facilitated allergic conjunctivitis in mice.

PURPOSE: To evaluate the role of nuclear factor-κB (NF-κB) activation in eye drop preservative toxicity and the effect of topical NF-κB inhibitors on preservative-facilitated allergic conjunctivitis. METHODS: Balb/c mice were instilled ovalbumin (OVA) combined with benzalkonium chloride (BAK) and/or NF-κB inhibitors in both eyes. After immunization, T-cell responses and antigen-induced ocular inflammation were evaluated. Nuclear factor-κB activation and associated inflammatory changes also were assessed in murine eyes and in an epithelial cell line after BAK exposure. RESULTS: Benzalkonium chloride promoted allergic inflammation and leukocyte infiltration of the conjunctiva. Topical NF-κB inhibitors blocked the disruptive effect of BAK on conjunctival immunological tolerance and ameliorated subsequent ocular allergic reactions. In line with these findings, BAK induced NF-κB activation and the secretion of IL-6 and granulocyte-monocyte colony-stimulating factor in an epithelial cell line and in the conjunctiva of instilled mice. In addition, BAK favored major histocompatibility complex (MHC) II expression in cultured epithelial cells in an NF-κB-dependent fashion after interaction with T cells. CONCLUSIONS: Benzalkonium chloride triggers conjunctival epithelial NF-κB activation, which seems to mediate some of its immune side effects, such as proinflammatory cytokine release and increased MHC II expression. Breakdown of conjunctival tolerance by BAK favors allergic inflammation, and this effect can be prevented in mice by topical NF-κB inhibitors. These results suggest a new pharmacological target for preservative toxicity and highlight the importance of conjunctival tolerance in ocular surface homeostasis.

Benzalkonium chloride breaks down conjunctival immunological tolerance in a murine model.

The impact of topical eye drops with benzalkonium chloride (BAK) as a preservative could involve more than the reported toxic effects on the ocular surface epithelium and ultimately affect the immune balance of the conjunctiva. We found that BAK not only impairs tolerance induction in a murine model, but leads to mild systemic immunization. Contrasting with antigen only-treated mice, there was no induction of interleukin 10-producing antigen-specific CD4(+) cells in BAK-treated animals. Moreover, the tolerogenic capacity of migrating dendritic cells (DCs) was reduced, apparently involving differential conditioning by soluble epithelial factors. Accordingly, epithelial cells exposed in vitro to BAK were less suppressive and failed to induce tolerogenic DCs in culture. As this effect of BAK was dependent on epithelial nuclear factor κB pathway activation, our findings may provide new therapeutic targets. Thus, tolerance breakdown by BAK should be considered an important factor in the management of glaucoma and immune-mediated ocular surface disorders.

Experimental megaileum.

Typical megaileum occurred in young male Wistar rats three months after ileum myenteric plexus denervation. An average of 58.4% denervation of the Auerbach plexus was obtained by serosal application of benzalkonium chloride (0.2% v/v). Denervation was assessed by ganglion cell counts in an 8 nm ring-shaped histological sectfrom the midportion of the treated segment. A morphometric study showed that the increased thickness of the megaileum wall was due to muscle hypertrophy and mucosal hyperplasia. The potential usefulness of this model of megaileum is emphasized.

Efeitos tóxicos de cloreto de banzalcônio em ratos / Toxic effects of benzalkonium chloride in rats

A inervação do coração é de fundamental importância para o ritmo, condução e repolarização cardíacos (JAMES 1967). Por este motivo, a aparente ausência de anormalidades estruturais cardíacas detectáveis em pacientes portadores de e ritmias ou com sindrome do QT alongado ou notadamente nos que têm morte súbita, serviu como estímulo para a investigação de nervos e gân glios cardíacos (JAMES e cols 1978 e 1979; JAMES 1980). Assim surgiu o conceito de cardioneuropatia, termo criado por JAMES, em 1979, para caracterizar toda cardiopatia associada è alterações histopatológicas significativas ou não, do sistema excito- condutor e da inervação intrinseca do coração. Tais alterações estão representadas por processo inflamatório, edema, todos os estágios de degeneração e fibrose dos componentes celulares nervosos (JAMES 1983) e, são também frequentemente vistas no coração de portadores de cardiopatia chagásica (ANDRADE E ANDRADE (1979). Em virtude deste fato, a cardiopatia chagásica foi reconhecida por JAMES (1983), como uma verdadeira cardioneuropatia e mais recentemente, rotulada por OLIVEIRA (1985) como um modelo natural humano de desnervação intrínseca parassimpática.