Simultaneous Evaluation of Membrane Permeability and UDP-Glucuronosyltransferase-Mediated Metabolism of Food-Derived Compounds Using Human Induced Pluripotent Stem Cell-Derived Small Intestinal Epithelial Cells.

Pharmacokinetic prediction after oral ingestion is important for quantitative risk assessment of food-derived compounds. To evaluate the utility of human intestinal absorption prediction, we compared the membrane permeability and metabolic activities of human induced pluripotent stem cell-derived small intestinal epithelial cells (hiPSC-SIECs) with Caco-2 cells or human primary enterocytes (hPECs). We found that membrane permeability in hiPSC-SIECs had better predictivity than that in Caco-2 cells against 21 drugs with known human intestinal availability (r = 0.830 and 0.401, respectively). Membrane permeability in hiPSC-SIECs was only 0.019-0.25-fold as compared with that in Caco-2 cells for 7 in 15 food-derived compounds, primarily those that were reported to undergo glucuronidation metabolism. The metabolic rates of the glucuronide conjugate were similar or higher in hiPSC-SIECs as compared with hPECs but lower in Caco-2 cells. Expression levels of UDP-glucuronosyltransferase (UGT) isoform mRNA in hiPSC-SIECs were similar or higher as compared with hPECs. Therefore, hiPSC-SIECs could be a useful tool for predicting human intestinal absorption to simultaneously evaluate membrane permeability and UGT-mediated metabolism. SIGNIFICANCE STATEMENT: Gastrointestinal absorption is an important step for predicting the internal exposure of food-derived compounds. This research revealed that human induced pluripotent stem cell-derived small intestinal cells (hiPSC-SIECs) had better predictivity of intestinal availability than Caco-2 cells; furthermore, the metabolic rates of UDP-glucuronosyltransferase (UGT) substrates of hiPSC-SIECs were closer to those of human primary enterocytes than those of Caco-2 cells. Therefore, hiPSC-SIECs could be a useful tool for predicting human intestinal absorption to simultaneously evaluate membrane permeability and UGT-mediated metabolism.

Simultaneous detection of eight species of tree nut in foods using two tetraplex polymerase chain reaction assays.

Tree nuts comprise a category of food allergens that must be included in the food labels in several countries. We developed a polymerase chain reaction (PCR) method using eight specific primer pairs to detect eight representative tree nuts (almond, Brazil nut, cashew, hazelnut, macadamia nut, pecan, pistachio, and walnut) under the same experimental conditions. The specificity of the eight primer pairs was confirmed by PCR testing against a variety of plant and animal samples. The detection limit of the method ranged from 1 fg to 1 pg DNA of individual tree nuts. The method detected tree nut DNA in processed and unprocessed food. In addition, the primer pairs could be combined into two sets of tetraplex PCR system. The developed method is specific, sensitive, and efficient, making it useful for detecting trace amounts of eight species of tree nut in foods.

Construction of extended and functional bile canaliculi using long-term sandwich-cultured cryopreserved human hepatocytes and the application of hepatocytes for predicting the biliary excretion of pharmaceutical and food-related compounds.

The biliary excretion of pharmaceutical and food-related compounds is an important factor for assessing pharmacokinetics and toxicities in humans, and a highly predictive in vitro method for human biliary excretion is required. We have developed a simple in vitro culture method for generating extended and functional bile canaliculi using cryopreserved human hepatocytes. We evaluated the uptake of compounds by hepatocytes and bile canaliculi, and the biliary excretion index (BEI) was calculated. After 21 days of culture, the presence of extended and functional bile canaliculi was confirmed by the uptake of two fluorescent substrates. Positive BEIs were observed for taurocholic acid-d4, rosuvastatin, pitavastatin, pravastatin, valsartan, olmesartan, and topotecan (reported biliary-excreted compounds in humans), but no difference in BEI was observed for salicylic acid (a nonbiliary-excreted compound). Furthermore, 8 of 21 food-related compounds with specific structures and reported biliary transporter involvement exhibited positive BEIs. The developed in vitro system was characterized by functional bile canaliculus-like structures, and it could be applied to the prediction of the biliary excretion of pharmaceutical and food-related compounds.

In vitro-based prediction of human plasma concentrations of food-related compounds.

Efforts have been made to replace animal experiments in safety evaluations, including in vitro-based predictions of human internal exposures, such as predicting peak plasma concentration (Cmax) values for xenobiotics and comparing these values with in vitro-based toxicity endpoints. Herein, the authors predicted the Cmax values of food-related compounds in humans based on existing and novel in vitro techniques. In this study, 20 food-related compounds, which have been previously reported in human pharmacokinetic or toxicokinetic studies, were evaluated. Human induced pluripotent stem cell-derived small intestinal epithelial cells (hiPSC-SIEC) and Caco-2 cells, HepaRG cells, equilibrium dialysis of human plasma, and LLC-PK1 cell monolayer were used to assess intestinal absorption and availability, hepatic metabolism, unbound plasma fraction, and secretion and reabsorption in renal tubular cells, respectively. After conversion of these parameters into human kinetic parameters, the plasma concentration profiles of these compounds were predicted using in silico methods, and the obtained Cmax values were found to be between 0.017 and 183 times the reported Cmax values. When the in silico-predicted parameters were modified with in vitro data, the predicted Cmax values came within 0.1-10 times the reported values because the metabolic activities of hiPSC-SIECs, such as uridine 5'-diphospho-glucuronosyl transferase, are more similar to those of human primary enterocytes. Thus, combining in vitro test results with the plasma concentration simulations resulted in more accurate and transparent predictions of Cmax values of food-related compounds than those obtained using in silico-derived predictions alone. This method facilitates accurate safety evaluation without the need for animal experiments.

Validation of a genotoxicity test based on p53R2 gene expression in human lymphoblastoid cells.

Genotoxicity assessment is important for predicting the carcinogenicity of chemical substances. p53R2 is a p53-regulated gene that is induced by various genotoxic stresses. We previously developed a p53R2-dependent luciferase reporter gene assay in the MCF-7 human breast adenocarcinoma cell line, and demonstrated its ability to detect genotoxic agents. In this paper, we investigate the applicability of the p53R2-based genotoxicity test in the human lymphoblastoid cell line TK6. TK6 cells that express wild-type p53 have been widely used for genetic toxicology studies. To evaluate the performance of the test system in TK6 cells, we referred to 61 of the chemicals on the list of 20 genotoxic and 42 non-genotoxic chemicals recommended for the evaluation of modified or new mammalian cell genotoxicity tests by the European Centre for the Validation of Alternative Methods. The overall accordance, sensitivity, and specificity of our results with the ECVAM list were 90% (55/61), 85% (17/20), and 93% (38/41), respectively. These results indicate that the p53R2-based genotoxicity test can detect various types of genotoxic chemicals without compromising its specificity. This test will be a valuable tool for rapid screen for identifying chemicals that may be genotoxic to humans.

Identification of tumor promotion marker genes for predicting tumor promoting potential of chemicals in BALB/c 3T3 cells.

Tumor promoters can cause development of tumors in initiated cells and the majority of them are non-genotoxic carcinogens. The detection of tumor promoters is important for the prevention of cancer. The in vitro two-stage transformation assay, using BALB/c 3T3 cells, is a useful system, and benefits from a convenient protocol and high predictability of mammalian carcinogenicity. But these assays are time-consuming and often require expertise for microscopic observation. To construct an in vitro tumor promoting activity test system, we performed large-scale gene expression analyses, using DNA microarrays, of BALB/c 3T3 cells following treatment with nine chemicals that are known to induce tumor promotion: TPA, zinc chloride, sodium orthovanadate, okadaic acid, insulin, lithocolic acid, phenobarbital sodium, sodium saccharide, sodium arsenite. As a result of DNA microarray and real time PCR analyses, 22 marker genes were identified. These consisted of genes related to cell cycle, regulation of transcription, anti-apoptosis, and positive regulation of cell proliferation. There was a correlation between these 22 marker genes and the cell transformation assay results in BALB/c 3T3 cells. These results suggest that this tumor promoting activity test system, based on 22 marker genes, can become a valuable tool for screening potential tumor promoters.

A genotoxicity test system based on p53R2 gene expression in human cells: assessment of its reactivity to various classes of genotoxic chemicals.

The tumor suppressor, p53, plays an important role in DNA damage repair, by regulating the expression of target genes. One p53-target gene, p53R2, which encodes a subunit of ribonucleotide reductase, is activated by DNA damage. We have previously developed a genotoxicity test system, using human cell lines and a p53R2-dependent luciferase reporter gene assay. 80 chemicals have been examined with this system and 40 of 43 Ames-positive chemicals induced luciferase activity. Eight Ames-negative genotoxic chemicals also induced luciferase activity. Although this assay system could, potentially, be applied to the rapid screening of chemicals that are potentially genotoxic to humans, the ability of the assay to detect genotoxic effects was unclear. In this study, to evaluate the performance of this assay system, several different types of DNA damaging agents were screened. 27 chemicals, whose genotoxic mechanisms are well known, were screened. All genotoxic compounds, except for anti-metabolites and histone deacetylase HDAC inhibitors, showed significant luciferase activity with the following rank order of potency: topoisomerase II inhibitors, intercalaters>bleomycin>topoisomerase I inhibitors>alkylating agents=DNA cross-linking agents=polycyclic aromatic hydrocarbons>spindle poisons. This assay showed greater response to those genotoxic agents that induce DNA double strand break damage compared to those agents that cause other forms of DNA damage. DNA double strand breakage initiates genomic instability, a feature of carcinogenicity. These results indicate that this assay system could be a helpful tool for predicting chemical genotoxicity and carcinogenicity in humans.

Re-evaluation of peroxide value as an indicator of the quality of edible oils.

The oxidation of oils has important effects on the quality of oily foods, such as instant noodles. In particular, the generation of aldehydes, which accompanies the oxidation of oils, is one of the first factors to reduce food quality. We examined various indicators of oil quality during temperature-accelerated storage and found that peroxide value (POV) was highly correlated with the total concentration of major odorants. Moreover, the correlation of POV with the total concentration of five unsaturated aldehydes (t-2-heptenal, t-2-octenal, t-2-decenal, t-2-undecenal and t,t-2,4-decadienal) that show strong cytotoxicity was greater than the correlation of POV with the total concentration of major odorants. The maximum allowable concentration of the five aldehydes was calculated based on the 'no observed adverse-effect level' of the aldehyde that showed the highest cytotoxicity, t,t-2,4-decadienal, along with the human daily oil intake. We showed that it is useful to utilize POV as an indicator to control food quality and safety.

Genotoxicity test system based on p53R2 gene expression in human cells: examination with 80 chemicals.

p53R2, which encodes a subunit of ribonucleotide reductase, is activated by DNA damage induced by gamma-ray and ultraviolet irradiation, and also by genotoxic chemicals such as adriamycin. For the purpose of constructing an easy-operating genotoxicity test system using human cell lines, we developed a p53R2-dependent luciferase reporter gene assay, and demonstrated dose-dependent luminescence caused by adriamycin in two human cell lines that express wild-type p53, MCF-7 and HepG2. The performance of this assay system was evaluated with 80 chemicals including those known in the Ames test as genotoxic or non-genotoxic. When the luciferase activity of cells treated with the test sample was over 200% to that of control cells in a dose-dependent increasing manner, the sample was judged positive as a genotoxic chemical. Forty of 43 Ames-positive chemicals induced luciferase activity in this assay system. Eight Ames-negative chemicals also induced luciferase activity. These eight chemicals are genotoxic in other in vitro test systems using mammalian cells. It is suggested that this assay system can be applied to rapid screening of chemicals for potential human genotoxicity.

Structural analysis of the sheath of a sheathed bacterium, Leptothrix cholodnii.

Leptothrix cholodnii is an aerobic sheath-forming bacterium often found in oligotrophic and metal-rich aquatic environments. The sheath of this bacterium was isolated by selectively lysing the cells. Glycine and cysteine were the major amino acids of the sheath. The sheath was readily dissolved in hydrazine, and a polysaccharide substituted with cysteine was recovered from the solution. Galactosamine, glucosamine and galacturonic acid were detected in the hydrazinolysate by gas liquid chromatography analysis. FAB-MS analysis of the hydrazinolysate suggested a sugar sequence of HexN-GalA-HexN-HexN. Methylation linkage analysis revealed the presence of 4-linked GalA, 3-linked HexN and 4-linked HexN. The sulfhydryl groups of the sheath were used for labeling with the fluorogenic reagent, 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F). The labeled sheath (ABD-sheath) was partially hydrolyzed and three fluorescent fragments were purified by HPLC. One of them was identified as ABD-cysteine. The second one was found to be the ABD-cysteine tetramer. Another fragment was indicated to be a pentasaccharide substituted with ABD-cysteine by nuclear magnetic resonance (NMR) analysis. It can be assumed that the polysaccharide and peptide moieties of the sheath are connected by a cysteine residue. NMR analysis of the hydrazinolysate revealed that the polysaccharide moiety of the sheath was constructed from a pentasaccharide repeating unit containing 2-amino-2-deoxygalacturonic acid (GalNA), as shown below. -->4)-alpha-GalNA-(1-->4)-alpha-D-GalN(p)-(1-->4)-alpha-D-GalA(p)-(1-->4)-beta-D-GlcN(p)-(1-->3)-beta-D-GalN(p)-(1-->.

A PCR assay based on a sequence-characterized amplified region marker for detection of emetic Bacillus cereus.

A PCR assay for the detection of Bacillus cereus strains able to produce an emetic toxin (cereulide) was developed in this study based on a sequence-characterized amplified region (SCAR) derived from a random amplified polymorphic DNA (RAPD) fragment. One of the RAPD fragments generated was selected, cloned, and sequenced. A set of PCR primers was newly designed from the SCAR obtained (the sequence of the cloned RAPD fragment) and used in this assay. To determine the specificity of the assay, 30 different B. cereus strains, 8 other Bacillus strains (of six species), and 16 other non-Bacillus strains (from 16 genera) were tested. Results were positive for every emetic B. cereus strain and for only one nonemetic B. cereus strain. For all other bacterial strains, results were negative. Bacterial DNA for PCR was prepared by a simple procedure using Chelex 100 resin from the bacterial colony on the agar plate or from culture after growth in brain heart infusion medium. This PCR assay enabled us to detect the bacteria of emetic B. cereus grown on agar plates but not the bacteria of nonemetic B. cereus. To test this PCR assay for the monitoring of the emetic bacteria, 10 to 70 CFU of B. cereus DSM 4312 (emetic) per g of food was inoculated into several foods as an indicator, followed by a 7-h enrichment culture step. Because this PCR assay based on the SCAR derived from the RAPD fragment was able to detect bacterial cells, this assay should be useful for rapid and specific detection of emetic B. cereus.