key: cord-0760019-fc66mpug authors: Lee, Jivianne T.; Basak, Saroj title: Cytotoxic Effects of N,N-Diethyl-Meta-Toluamide (DEET) on Sinonasal Epithelia date: 2021-05-04 journal: OTO Open DOI: 10.1177/2473974x211009232 sha: d25640bc18a828aa52433569d9da1222f3d5c88a doc_id: 760019 cord_uid: fc66mpug Although the etiology of chronic rhinosinusitis remains unknown, environmental factors including airborne pollutants and toxicants are postulated to contribute to its pathogenesis. However, the precise pathomechanisms with which environmental toxicants may contribute to chronic rhinosinusitis are not fully understood. The purpose of this pilot study is to examine the cytotoxic effects of N,N-diethyl-meta-toluamide (DEET), a commonly used pesticide, on sinonasal epithelial cells (SNECs). Sinus mucosa was obtained from 3 subjects without a history of chronic rhinosinusitis. Cultured SNECs were exposed to various concentrations of DEET (0-5 mM) for 6 days. Cell viability, proliferation, and morphologic changes were assessed using the MTT colorimetric dye assay and the Incucyte Live Cell Monitoring System. Statistically significant dose-dependent reduction in cell viability and proliferation was observed between exposure and control groups (P < .05) at all concentrations tested. Dose-dependent cellular morphological changes were also seen. These findings indicate that DEET exposure induces dose-dependent cytotoxicity in sinonasal epithelia. A lthough the etiology of chronic rhinosinusitis (CRS) remains unknown, environmental factors, including airborne toxicants, are postulated to contribute to its pathogenesis. 1, 2 Occupational and combat zone chemical exposures are associated with increased prevalence of CRS. [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] Farmers with pesticide exposures have a significantly higher incidence of sinusitis compared to matched controls (n = 196). 4 A cross-sectional epidemiologic study of 261 healthy volunteers revealed a dose-response relationship between level of pesticide exposure and reported sinusitis independent of age, sex, and smoking status. 6 N,N-diethyl-meta-toluamide (DEET) is a commonly used insect repellent sprayed on the hands and rubbed onto the skin. It has been extensively used in agriculture, military, and civilian life. 14, 15 However, the impact of DEET exposure on the sinonasal cavity has not previously studied. The purpose of this pilot study is to investigate the cytotoxic effects of DEET on sinonasal epithelial cells (SNECs). We have established a cell culture model system as previously described in the literature with modifications. [16] [17] [18] Sphenoid sinus mucosal specimens were collected from patients undergoing endoscopic transsphenoidal resection of pituitary tumors without a history of sinonasal disease at the Greater Los Angeles VA according to an institutional review boardapproved protocol. SNECs were expanded on tissue culture plates and transferred onto cell culture inserts, and confluent cells were cultured at the air-liquid interface. 17 To determine the effect of DEET on cell growth, we performed the cell viability measurement MTT colorimetric dye assay. 19 SNECs were grown in a 96-well plate and cultured for 6 days in various concentrations of DEET. After the 6-day exposure period, culture plates were treated with MTT dye and incubated overnight. Optical density (OD) were measured at 570 nm. The IncuCyte live-cell analysis system (Essen Bioscience) was used to assess cell proliferation. Live cells were monitored over an extended period of time and data presented as realtime kinetic data. 20-22 SNECs were exposed to different concentrations of DEET, and wells were scanned every 2 hours from 0 hours to 6 days (144 hours) after exposure. Dosing concentrations and exposure schedules were determined based on prior published studies examining the effect of DEET on other cell types. 23, 24 The data are presented as fold changes in cell density from initiation (0 hours) to specific time points during the assay. Cell density was calculated with IncuCyte software and phase contrast images. When SNECs (4000/well in quadruplicates) were treated with DEET ( Figure 1 ), a dose-response result was obtained, such that a 5-mM concentration eliminated all viable cells, and correspondingly lower concentrations (0.625-2.5 mM) had reduced effects on cell viability. When cells were treated with DEET (4000 cells/well, in quadruplicate) (Figure 2) , cell proliferation was inhibited in a concentration-dependent manner (0.625-5 mM), similar to the MTT assay. Treatment with 2.5 mM DEET was most effective for inhibition of primary cell growth. Morphological changes and cytotoxic effects were observed with DEET when SNECs were treated for 6 days. High doses of DEET ( Figure 3A ) disintegrated SNECs at a 5-mM concentration. At lower concentrations ( Figure 3B ,C; 1.25 mM and 0.625 mM, respectively), few live cells were observed. Remaining cells lost their morphology and were in the process of disintegration. These results indicate that DEET exerts deleterious effects on SNECs in a dose-dependent manner. The precise role of environmental pollutant and chemical exposure in the pathogenesis of CRS is not fully understood. Prolonged combined pyridostigmine bromide/DEET exposure has been shown to increase serum levels of proinflammatory cytokines in a mouse model and cause neuroinflammation. 24 DEET has also been found to potentiate persistence of chronic pain in a rat model. 25, 26 However, the deleterious effects of DEET on sinonasal epithelia have never been previously explored. To our knowledge, this study is the first to illustrate the cytotoxic effects of DEET on SNECs. DEET exposure significantly reduced SNEC growth and viability in a dosedependent manner, with a 5-mM concentration eliminating all viable cells and correspondingly lower concentrations (0.625-2.5 mM) with reduced effects on cell viability. IncuCyte livecell monitoring showed inhibition of SNEC proliferation in a concentration-dependent manner (0.625-5 mM), with 2.5 mM DEET being the most effective for inhibition of SNEC growth. SNECs exposed to DEET also exhibited morphologic changes, with lower concentrations resulting in fewer live cells and higher doses (5 mM) causing complete disintegration. A prior in vitro study demonstrated dose-dependent genotoxic effects of DEET when administered to primary nasal mucosal epithelial cells. However, no significant cytotoxic effects were observed at the concentrations studied (0.5-1 mM). This may be due to the fact that cells were only exposed for 60 minutes, whereas previous studies administered exposures over several days. 27 In vivo, DEET toxicity is dependent upon the route and duration of exposure as well as the dosage administered. Commercially available products contain anywhere from 10% (100 mg/mL) to 100% DEET. Neurotoxicity including seizures and respiratory arrest have been reported with ingestion (ie, spraying into the oral cavity unintentionally) and excessive dermal application (25 mL of 50% DEET). 28 However, minimum toxicity thresholds for upper airway toxicity from inhalant exposure have not been established. The concentrations used in this in vitro study (0-5 mM: .956 mg/mL) are significantly less than the reported dosages associated with neurotoxicity. Cytotoxicity as a driver of inflammation has been extensively reported in the lower airway and implicated as a contributing factor in multiple pulmonary inflammatory diseases. [29] [30] [31] Chemical exposures have been shown to induce lytic cell death, generation of cellular debris, and reactive oxygen species, leading to release of proinflammatory cytokines in lung tissue. 30 Primary bronchial epithelial cells exposed to diesel exhaust and aldehydes demonstrate increased interleukin (IL)-8 secretion and gene expression of inflammatory (IL-6, IL-8) and oxidative stress (hemeoxygenase 1) biomarkers. 30, 31 Caspase 1-dependent IL-1b release has been reported as the underlying pathomechanism for pneumonia, respiratory syncytial virus bronchiolitis, and COVID-19-mediated lung injury. 31, 32 Similar to the lower airway, the cytotoxic effects induced by DEET exposure may lead to a proinflammatory response in sinonasal tissue, potentially contributing to CRS pathophysiology. Previous studies have also demonstrated that DEET induces CYP isoforms, adenylate kinase, and caspase 3/7, leading to cytotoxicity in human hepatocytes. 23 Further study is needed to investigate this concept of chemically induced cytotoxicity as a potential factor in CRS. This pilot study is the first to demonstrate the cytotoxic effects of the commonly used pesticide, DEET, on sinonasal epithelia. DEET exposure significantly reduced SNEC viability and proliferation in a dose-dependent manner in vitro. Additional studies are necessary to determine if such cytotoxicity is associated with chronic inflammation and establish minimum toxicity thresholds that could predict sinonasal morbidity. Jivianne T. Lee, conception and design, data acquisition, analysis and interpretation, drafting and revising work critically for intellectual content, approval, agreement of accountability; Saroj Basak, conception and design, data acquisition, analysis and interpretation, drafting and revising work critically for intellectual content, approval, agreement of accountability. Competing interests: None. Clinical practice guideline (update): adult sinusitis executive summary The association of air pollutants and allergic and nonallergic rhinitis in chronic rhinosinusitis Health risks associated with inhaled nasal toxicants Respiratory symptoms, skin disorders and serum IgE levels in farm workers Combat zone exposure and respiratory tract disease A preliminary study of potential human health effects in private residences following chordane applications for termite control The effects of air pollutants on the prevalence of common ear, nose, and throat diseases in South Korea: a national population-based study Increased prevalence of upper and lower respiratory disease in Operation Enduring Freedom and Operation Iraqi Freedom US veterans Lifetime prevalence of respiratory diseases and exposures among veterans of Operation Enduring Freedom and Operation Iraqi Freedom Veterans: results from the National Health Study for a New Generation of US Veterans Prevalence of respiratory diseases among veterans of Operation Enduring Freedom and Operation Iraqi Freedom: results from the National Health Study for a New Generation of US Veterans Experience of first deployed otolaryngology team in Operation Iraqi Freedom: the changing face of combat injuries A histopathological study of head and neck specimens from a cohort of Persian Gulf War military veterans Symptoms and medical conditions in Australian veterans of the 1991 Gulf War: relation to immunisations and other Gulf War exposures Impact of environmental chemicals on the transcriptome of primary human hepatocytes: potential for health effects Differential expression profile of lncRNAs from primary human hepatocytes following DEET and fipronil exposure Sinonasal epithelial expression of Toll-like receptor 9 is decreased in chronic rhinosinusitis with polyps Culturing of human nasal epithelial cells at the air liquid interface Bactericidal antibiotics promote oxidative damage and programmed cell death in sinonasal epithelial cells Liposome encapsulated curcumin-difluorinated (CDF) inhibits the growth of cisplatin resistant head and neck cancer stem cells Incucyte Systems: empower your research with IncucyteÒ livecell analysis Novel agent DMAMCL suppresses osteosarcoma growth and decreases the stemness of osteosarcoma stem cell A novel real time imaging platform to quantify macrophage phagocytosis Enzyme induction and cytotoxicity in human hepatocytes by chlorpyrifos and N,N-diethyl-m-toluamide (DEET) Corticosterone and pyridostigmine/DEET exposure attenuate peripheral cytokine expression: supporting a dominant role for neuroinflammation in a mouse model of Gulf War illness Corticosterone primes the neuroinflammatory response to DFP in mice: potential animal model of Gulf War illness Corticosterone potentiates DFP-induced neuroinflammation and affects highorder diffusion imaging in a rat model of Gulf War illness Genotoxicity studies on permethrin, DEET and diazinon in primary human nasal mucosal cells DEET-insect repellant toxicity Inflammatory effects of acrolein, crotonaldehyde and hexanal vapors on human primary bronchial epithelial cells cultured at air-liquid interface Inflammatory response of lung cells exposed to whole, filtered, and hydrocarbon denuded diesel exhaust Lytic cell death mechanisms in human respiratory syncytial virus-infected macrophages: roles of pyroptosis and necroptosis Inflammasomes are activated in response to SARS-CoV-2 infection and are associated with COVID-19 severity in patients Funding source: American Rhinologic Society Friends in Research Grant.