E171 (HOMBITAN® FG; Purity 99.5%) was purchased from Venator Germany GmbH (Duisburg, Germany) and suspended in DW (stock concentration of 100 mg/mL). The suspension was added to AGF and cell culture medium to evaluate its stability in biological systems. Particle morphology was observed by TEM (JEM-3000F, 200 kV, JEOL Ltd., Tokyo, Japan), and particle size distribution and surface charge were measured with a zeta-potential and particle size analyzer (ELSZ-1000 Photal; Otsuka Electronics, Osaka, Japan).
Animals and housing
Five-week-old male and female specific pathogen-free SD rats (fifty rats per sex) were obtained from Orient Bio Inc. (Seongnam-si, Gyeongi-do, Korea) and maintained in a controlled environment (stainless wire cages 255 mm W × 465 mm L × 200 mm H (five rats/cage), 12 h/12 h light/dark cycle, temperature 23 ± 3 °C, relative humidity 50 ± 10 %, air ventilation 10–20×/h, light intensity 150–300 lx, and ad libitum access to food (PMI Nutrition International, St. Louis, MO, USA) and tap water. The rats were randomly assigned to one of four groups (0, 10, 100, or 1,000 mg/kg) via Pristima v. 7.4 (Xybion Medical Systems Corporation, Lawrenceville, NJ, USA). E171 (10 rats/sex/dose) was administered daily by oral gavage for 13 consecutive weeks. The control group received equal volumes of DW. The experimental design was reviewed and assessed by the Association for the Assessment and Accreditation of Laboratory Animal Care International (AAALAC) and the Institutional Animal Care and Use Committee (IACUC) of the Korea Institute of Toxicology.
Clinical observations and assessments
The health status of all rats was observed daily according to a predetermined schedule during the study period. The type, time of occurrence, and severity of abnormal symptoms were recorded with the Pristima v. 7.4 system. The rats were weighed upon arrival, before randomization, weekly during pretreatment, before dosing during treatment, and before necropsy. Food consumption was recorded once weekly during the pretreatment and treatment periods and calculated as g/rat/day. Urinalysis was performed during the treatment period on all surviving animals being administered E171. The urine was collected for ~17 h before necropsy and its volume, specific gravity (SG), color, pH, and protein (PRO), ketone body (KET), occult blood (BLD), glucose (GLU), bilirubin (BIL), nitrite (NIT), and urobilinogen (URO) levels were measured with a Cobas U411 urine analyzer (Roche, Basel, Switzerland) and a urine chemical analyzer (TBA 120FR; Toshiba Corp., Tokyo, Japan). The urine was centrifuged (1500 rpm, 5 min) and its sediment casts (epithelial cells (EPI), erythrocytes (RBC), leucocytes (WBC), and blood (BLO) were stained and microscopically observed (Nikon Eclipse Ci, Nikon, Japan). Upon necropsy, blood was drawn from the venae cavae of all rats and stored in tubes coated with EDTA-2K and heparin. Hematological and clinical chemistry analyses were performed in an ADVIA 2102i hematology system (Siemens, Washington, DC, USA) and an automatic analyzer (TBA 120FR; Toshiba Corp., Tokyo, Japan), respectively.
Macroscopic and microscopic findings
At necropsy, 42 tissue samples were taken from all rats. The eyes with optic nerves intact were weighed and fixed in Davidson’s fixative solution. All other tissues were weighed and preserved in 10% (v/v) neutral buffered formalin. All preserved tissues were embedded in paraffin, sectioned, stained with hematoxylin and eosin (H&E), and examined under microscope (Olympus BX53, Olympus America, USA). Relative organ weights were calculated using the body weights measured at necropsy.
The colons obtained from rats administered the maximum dose were chopped, and AGS cells were incubated with 40 μg/mL E171 for 24 h. The stomach tissues and the AGS cells were put in Karnovesky’s fixative solution (Electron Microscopy Sciences, Hatfield, PA, USA) overnight at 4 ºC. The cells were then fixed in a mixture of 2% (v/v) glutaraldehyde and 0.1 M sodium cacodylate buffer for 2 h, stained with 0.5% (w/v) uranyl acetate, dehydrated in graded ethanol solutions and propylene oxide, and embedded in Spurr's resin (Electron Microscopy Sciences, Hatfield, PA, USA). The colon tissues and AGS cells were sectioned with an ultramicrotome (MT-X; RMC, Tucson, AZ, USA), stained with 2% (w/v) uranyl acetate and Reynolds's lead citrate, and imaged with a transmission electron microscope (TEM) at 120 kV (Talos L120C, FEI, Hillsboro, OR, USA) as well as an 80-kV TEM (JEM1010, JEOL, Tokyo, Japan).
Paraffin-embedded stomach tissues were dewaxed with xylene and a graded alcohol series (100%, 95%, 70%, and 50%). After washing with phosphate-buffered saline (PBS), the tissues were placed in an antigen retrieval solution (ENZO; Seoul, Korea) and permeabilized with PBS containing Tween-20 (PBST, 1%). After blocking with 5% (v/v) bovine serum albumin (BSA) in PBST (0.01%), the tissues were incubated overnight with rabbit polyclonal antibody against superoxide dismutase (SOD)-1 and SOD-2 (Santa Cruz Biotechnology; Dallas, TX, USA) and cytochrome C (Cell Signaling Technology, Danvers, MA, USA) at 4 °C. Following, the tissues were reacted with affinity-purified Alexa Fluor 488-conjugated goat anti-rabbit IgG (Invitrogen, Carlsbad, CA, USA) and mounted with 4’,6-diamidino-2-phenylindole (DAPI) mounting medium. Lastly, the images were then captured with an inverted phase-contrast fluorescence microscope (IX51, Olympus, Tokyo, Japan).
Trace element determination by ICP-MS
The tissues (colons, spleens, and kidneys) were digested in 70% (v/v) nitric acid solution using a microwave digestion system (Milestone; Sorisole, Italy). Finally, concentrations of trace elements (Al, Cu, Zn, Mn and Fe) in tissues were measured by inductively coupled plasma mass spectrometry (ICP‐MS) at the Korean Basic Science Institute (Seoul, Korea).
AGS cells (70 - 80 % of confluence) were incubated with E171 (0, 10, 20, 40 μg/mL) for 24 h. As described previously, proteins in the cell lysates were quantified by bicinchoninic acid assay (Sigma-Aldrich, St. Louis, MO, USA), and the same amounts of proteins were electrophoretically separated on SDS polyacrylamide gel. Then, the proteins were transferred to nitrocellulose membranes (0.45 μm pore, GE Healthcare Life Sciences, Freiburg, Baden-Württemberg, Germany) and blocked with 5% (v/v) skim milk in PBST (0.05%). The membranes were reacted overnight at 4 °C with primary mouse monoclonal antibodies against lysosome-associated membrane protein (LAMP)-1, β-actin, ER oxidoreductin (ERO)-1alpha, ferritin (HC), phospho-JNK, protein disulfide isomerase (PDI), eukaryotic translation initiation factor (eIF)2-alpha, catalase, caspase-1 (Santa Cruz Biotechnology, Dallas, TX, USA), C/EBP homologous protein (CHOP) (Cell Signaling Technology, Danvers, MA, USA), p62 (Abcam, Cambridge, UK), and rabbit monoclonal antibody against protein kinase RNA-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme (IRE)1-alpha (Cell Signaling Technology, Danvers, MA, USA), and rabbit polyclonal antibody against superoxide dismutase (SOD)-1, SOD-2, interleukin (IL)-18 (Santa Cruz Biotechnology, Dallas, TX, USA), binding immunoglobulin protein (Bip), calnexin, microtubule-associated proteins 1A/1B light chain (LC)3B (Cell Signaling Technology, Danvers, MA, USA), and goat polyclonal antibody against NACHT, LRR and PYD domains-containing protein (NALP)3 (Abcam, Cambridge, UK). The proteins were then reacted with HRP-conjugated mouse and rabbit or goat secondary antibodies (Santa Cruz Biotechnology, Dallas, TX, USA) and blotted in a ChemiDoc XRS+ system (Bio-Rad Laboratories, Hercules, CA, USA).
Gene profiling analysis
AGS cells were incubated with or without E171 (40 μg/mL) for 24 h. The effects of E171 on the gene profile were evaluated by microarray analysis. Briefly, the mRNA was prepared and the microarray analysis was conducted at Macrogen (Seoul, Korea) using an Affymetrix® uman 2.0ST gene chip according to the manufacturer's instructions (Illumina, San Diego, CA, USA). The data were summarized and normalized by the robust multi-average (RMA) method in Affymetrix® Power Tools (APT). The results of the gene-level RMA analysis were exported and a differentially expressed gene (DEG) analysis was performed. Statistical significance of the expression data was determined by fold change. For each DEG set, a hierarchical cluster analysis was conducted using complete linkage and Euclidean distance as measures of similarity. Gene enrichment and functional annotation analyses of significant probe lists were performed via GO (http://geneontology.org) and KEGG (www.genome.jp/kegg/). All data analyses and DEG visualizations were performed in R v. 3.3.3 (www.r-project.org).
Data were statistically analyzed by multiple comparison methods. When Bartlett’s test revealed no significant deviations from variance homogeneity, ANOVA was used to determine whether any group means differed at the P < 0.05 level. Dunnett’s test was used to identify differences in the means for the control and treatment groups when the data were found to be significant according to ANOVA. When significant deviations from variance homogeneity were identified by Bartlett’s test, the nonparametric Kruskal-Wallis (H) test was conducted to establish whether any of the group means differed at the P < 0.05 level. When significant differences were observed by the Kruskal-Wallis (H) test, Dunn’s Rank Sum test was conducted to identify the pairs of group data that significantly differed from the mean. Fisher’s exact test was run to compare data pairs including prevalence and percentage. The probability level was 1% or 5%. Statistical analyses were performed using Prisitima v. 7.4 by comparing the data of the various treatment groups with those of the control.