The intestinal epithelium barrier holds a strong defense against foreign substances while at the same time regulating the absorption of nutrients, water, and electrolytes.
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This passage of molecules is controlled by intercellular tight junction (TJ) proteins located at the apical domain of the epithelial cells. TJs form a multiprotein complex including transmembrane proteins (eg, occludin [OCLN], claudins [CLDNs], junctional adhesion molecule, and tricellulin) and intracellular plaque proteins (eg, zonula occludens [ZOs], ie, ZO-1, ZO-2, and ZO-3).
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Disruption of the gut epithelial TJ barrier has been proposed as a central pathogenic factor leading to the systemic inflammation observed in obesity, inflammatory bowel disease, and a long list of other chronic noninfectious inflammatory diseases of distant organs.
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The recently postulated “Epithelial Barrier Hypothesis” associates the rise in allergic, autoimmune, and metabolic diseases observed in the last decades to the increased exposure to epithelial barrier–damaging agents in industrialized and urbanized society.
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Does the epithelial barrier hypothesis explain the increase in allergy, autoimmunity and other chronic conditions?.
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These epithelial barrier–damaging agents include toxic substances present in laundry and household detergents, as well as surfactants, enzymes, and emulsifiers in processed food, cigarette smoke, particulate matter, diesel exhaust, ozone, nanoparticles, microplastics, and toothpaste.
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Does the epithelial barrier hypothesis explain the increase in allergy, autoimmunity and other chronic conditions?.
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Increased intestinal barrier permeability allows greater subepithelial exposure of the innate and acquired immune system to proinflammatory components and microbiota in the intestinal lumen.
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IL-1beta causes an increase in intestinal epithelial tight junction permeability.
These inflammatory stimuli and proinflammatory cytokines can directly suppress TJ protein expression in the intestinal barrier.
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Bacterial translocation to subepithelial area accompanies this damage as demonstrated in many systemic diseases.
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Professional dishwashers have gained popularity since the 2000s,
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because they reduce the workforce and ensure the dishware is clean and sanitized. They are currently used on a regular basis, especially in public food consumption areas including restaurants, schools, military barracks, and hotels. During professional dishwashing, 3 to 4 L of water is heated to at least 65°C and a detergent is added for a maximum of a 60-second wash cycle at 2 to 4 atmospheres of pressure. A rinse/dry cycle follows with an additional 3 to 4 L of water and rinse aid heated to 82°C. It should be noted that there are no washes to remove the rinse aid residue. These 2 cycles are completed in 60 to 150 seconds, depending on the type of professional dishwasher used.
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The residue remaining after the rinse cycle varies between 1:250 to 1:667 for the dishwasher detergent and 1:2,000 to 1:10,000 dilutions for the rinse aid. The detergents and rinse aid used in professional dishwashers contain several potentially hazardous substances; however, their toxicity on gastrointestinal epithelial cells has not been fully investigated. In the present study, we hypothesized that if the detergent and rinse aid residues are not completely removed, once dry, they may remain on the surface of the dishware. Accordingly, we investigated the health hazard of exposure to professional and household dishwasher detergent and rinse aid, and its individual components. Liquid-liquid interface cultures, organoids and gut-on-a-chip studies, and transcriptome and targeted proteomics analysis of the intestinal epithelial cells provided insights into the doses and molecular mechanisms by which the detergents and rinse aid induce apoptosis and inflammation that result in gut epithelial barrier damage. These findings may have an important impact on public health, which mark the beginning of intestinal epithelial disruption and proinflammatory responses in many chronic diseases.
Methods
Cell cultures
Caco-2 and HT-29 cells were purchased from the American Type Culture Collection (ATCC HTB-37, Manassas, Va) and were used between passages 20 and 45 in all experiments. Cells were cultured as a monolayer in Dulbecco modified Eagle medium (Gibco-BRL, Invitrogen, Carlsbad, Calif) supplemented with 10% FBS (Sigma-Aldrich, St Louis, Mo), 1% penicillin/streptomycin (Sigma-Aldrich), 1% nonessential amino acids (Sigma-Aldrich), and 1% sodium pyruvate (Sigma-Aldrich) in a 75-cm2 T-flask in a humidified incubator at 37°C in 5% CO2. For liquid-liquid interface cultures, Caco-2 cells were seeded in 24-well Transwell culture plates (0.4 μm pore size; Corning, Inc, Corning, NY) at a density of 8 × 104 cells per well, replacing the media every 2 days. Our preliminary results showed similar effects with 3 different commercial detergents and rinse aids. Afterwards, the detailed experiments were performed with the same detergent and rinse aid throughout the study. Detergent and rinse aid exposure studies were performed between days 14 and 18 at the liquid-liquid interface. OrganoReady Caco-2 plates (Mimetas BV, Leiden, The Netherlands) were purchased and used for investigating the effects of professional dishwasher detergent and rinse aid on gut-on-a-chips. Induced pluripotent stem cell (iPSC)-derived intestinal organoids were purchased from DefiniGEN (Cambridge, UK) and were used between passages 2 and 3 in the experiments. Cells were cultured in intestinal recovery and maintenance medium supplemented with intestinal supplement Definitive A (DefiniGEN) in 24-well/plates in a humidified incubator at 37oC in 5% CO2. Organoid cultures were exposed to professional dishwasher detergent or rinse aid with adding of fluorescein isothiocyanate (FITC)-labeled dextran 4 kD at the same concentrations. Then, the paracellular flux (PF) was assessed with confocal microscopy at the end of 24-hour incubation.
Dilution factor for household and professional dishwasher detergents and rinse aid
A professional dishwasher completes 1 or 2 wash and rinse cycles using 3.5 L of water per cycle. The detergent and rinse aid are automatically dispensed into the water at a concentration of 1.5 to 4 mL/L and 0.1 to 0.5 mL/L, respectively. At these concentrations, the residual dilution factor after rinse ranges from 1:250 to 1:667 for detergents and 1:2,000 to 1:10,000 for rinse aids. The chemicals and dilution factors used in household and professional dishwasher detergents and rinse aid are listed in Table E1 in this article’s Online Repository at www.jacionline.org.
Household dishwasher detergents in a normal cup and plate washing program typically consume a total of 12 L of water: 4.8 L during the washing cycle, 3.6 L of water for the intermediate rinse cycle, and 3.6 L of water for the final rinse cycle. Between the washing and rinsing cycles, 200 mL of water remains inside the dishwasher. Accordingly, the dilution factor for one 20-g tablet of detergent is 1:80,000 (w/v).
Exposure to hazardous chemicals in household and professional dishwasher detergents and rinse aid
Professional dishwasher detergent or rinse aid was added to the apical compartment of the liquid-liquid interface and organ-on-a-chip cultures and diluted as follows (v/v): detergent, 1:250, 1:500, 1:1,000, 1:2,000, 1:4,000; rinse aid, 1:2,500, 1:5,000, 1:10,000, 1:20,000, 1:40,000. To study the effects of different detergents on gut epithelial cells, Triton X-100 (Sigma-Aldrich) and sodium dodecyl sulphate (SDS) (Sigma-Aldrich) were added to the apical compartment of the Caco-2 monolayers and liquid-liquid interface cultures and diluted as follows: 1:5,000, 1:10,000, 1:20,000, 1:40,000, and 1:80,000. In the control group, only the culture media was added to the apical compartment.
To study whether there is a synergistic toxic effect of the professional dishwasher detergent and rinse aid, a mixture of the 2 was applied to the apical compartment of the cells for 3 days or more with 0.3 mL Dulbecco modified Eagle medium at different dilutions. In addition, 0.6 mL of Dulbecco modified Eagle medium was pipetted to the basolateral compartment.
To study the chemicals in the residue remaining on the surface of dishware after washing, 10 porcelain cups (190 mL size) were subjected to 1 full washing and drying cycle in professional (Winterhalter, GS501, Germany) and household (AEG, GS60AV, Poland) dishwashers and then 1 mL of culture medium was added into them. After shaking and waiting for 5 minutes, the culture medium was collected from the cups, filter-sterilized, and added to the monolayer and differentiated cell cultures directly or in 1:2, 1:5, and 1:10 dilutions.
To examine the chemicals in the rinse aid, citric acid (Fluka, St Louis, Mo), sodium cumenesulfonate (Angene, Montlucon, France), or alcohol ethoxylates (C12-15) (BOC Sciences, Shirley, NY) were diluted in PBS 10% (w/v), 3% (w/v), or 15% (v/v), respectively. These were then applied alone, or as a mixture, to the apical compartment at a final dilution of 1:10,000 in the culture medium.
To exclude the endotoxin influence on barrier function, endotoxin assays were performed at different dilutions of professional dishwasher detergent and rinse aid. Endotoxins were not found at detectable levels (<0.01 EU/mL) in all of the detergent and rinse aid mixtures.
