Safety assessment of L-Arg oral intake in healthy subjects: a systematic review of randomized control trials.

L-Arg is a nonessential amino acid but has many physiological roles. Accordingly, L-Arg has been used in various fields, but there is only limited information available about its safety upon overdose. Generally, the no-observed adverse effect level (NOAEL) is used when setting the upper amount for chemical substances. Recently, systematic reviews have been used to assess the safety as well as the effectiveness and usefulness of them. Therefore, we conducted an assessment of the safety of the oral intake of L-Arg in healthy subjects using gastrointestinal symptoms as an index. We limited the study design to only double-blind randomized controlled trials and searched PubMed, Cochrane Library, EBSCOhost, and Ichushi-Web from inception until May 2021. Assessment of the quality of studies was conducted using the Cochrane Collaboration tool and Jadad score, and the random effects model was used for data analysis. Ultimately, 34 studies were selected for inclusion in this work. The dosage of L-Arg used in the studies ranged from 2000 to 30,000 mg/day (or/one-time dose), and the treatment duration was 1-84 days. The increased risk of gastrointestinal symptoms associated with L-Arg intake from 23 studies (647 participants in total) in which such symptoms were reported was 0.01 (95% confidence interval: - 0.02-0.04), which was not significant difference. NOAEL was estimated as 7531 mg/ one-time dose using a weighted change-point regression model (UMIN000046133).Registration and protocol: Umin.ac.jp as UMIN000046133.

Low-dose radiation induces unstable gene expression in developing human iPSC-derived retinal ganglion organoids.

The effects of low-dose radiation on undifferentiated cells carry important implications. However, the effects on developing retinal cells remain unclear. Here, we analyzed the gene expression characteristics of neuronal organoids containing immature human retinal cells under low-dose radiation and predicted their changes. Developing retinal cells generated from human induced pluripotent stem cells (iPSCs) were irradiated with either 30 or 180 mGy on days 4-5 of development for 24 h. Genome-wide gene expression was observed until day 35. A knowledge-based pathway analysis algorithm revealed fluctuations in Rho signaling and many other pathways. After a month, the levels of an essential transcription factor of eye development, the proportion of paired box 6 (PAX6)-positive cells, and the proportion of retinal ganglion cell (RGC)-specific transcription factor POU class 4 homeobox 2 (POU4F2)-positive cells increased with 30 mGy of irradiation. In contrast, they decreased after 180 mGy of irradiation. Activation of the "development of neurons" pathway after 180 mGy indicated the dedifferentiation and development of other neural cells. Fluctuating effects after low-dose radiation exposure suggest that developing retinal cells employ hormesis and dedifferentiation mechanisms in response to stress.

iGEM as a human iPS cell-based global epigenetic modulation detection assay provides throughput characterization of chemicals affecting DNA methylation.

Chemical-induced dysregulation of DNA methylation during the fetal period is known to contribute to developmental disorders or increase the risk of certain diseases later in life. In this study, we developed an iGEM (iPS cell-based global epigenetic modulation) detection assay using human induced pluripotent stem (hiPS) cells that express a fluorescently labeled methyl-CpG-binding domain (MBD), which enables a high-throughput screening of epigenetic teratogens/mutagens. 135 chemicals with known cardiotoxicity and carcinogenicity were categorized according to the MBD signal intensity, which reflects the degree of nuclear spatial distribution/concentration of DNA methylation. Further biological characterization through machine-learning analysis that integrated genome-wide DNA methylation, gene expression profiling, and knowledge-based pathway analysis revealed that chemicals with hyperactive MBD signals strongly associated their effects on DNA methylation and expression of genes involved in cell cycle and development. These results demonstrated that our MBD-based integrated analytical system is a powerful framework for detecting epigenetic compounds and providing mechanism insights of pharmaceutical development for sustainable human health.

Integrative Approaches of Bioassay and Computational Analysis for Discovering Potential Bioactive Compounds and Predictive Toxicity.

Changes in eating habits are brought about by drastic changes in lifestyle and environment, and, it has been pointed out, are strongly involved in the increase in neurological diseases and onset of cancer in younger adult ages. There is a wide variety of chemical substances in food, and there is a need to analyze the effects of complex exposures on complex mechanisms of action and to develop methods for evaluating and predicting them. The power of molecular nutrition needs to create an integrated approach to human nutrition in line with the grand social challenges of diet-related illnesses. The current article aims to explore some of these areas where integration is appropriate. Therefore, in this symposium, we will introduce the contents of four performers who are conducting cutting-edge research. 1) Chemoprevention by vitamin A and its derivatives, 2) Toxicity prediction of natural compounds from a developing database of bioactive gradients from Kampo medicine, 3) Toxicity prediction of chemicals using pluripotent stem cells. 4) Detection of bioactive compounds in "Aji" or "Umami" in food. By detecting and predicting the biological activity and toxicity of chemical substances such as nutrients in foods, it will be possible to provide better molecular information on dietary components. In addition, we will introduce next-generation health and prevention methods.

StemPanTox: A fast and wide-target drug assessment system for tailor-made safety evaluations using personalized iPS cells.

An alternative model that reliably predicts human-specific toxicity is necessary because the translatability of effects on animal models for human disease is limited to context. Previously, we developed a method that accurately predicts developmental toxicity based on the gene networks of undifferentiated human embryonic stem (ES) cells. Here, we advanced this method to predict adult toxicities of 24 chemicals in six categories (neurotoxins, cardiotoxins, hepatotoxins, two types of nephrotoxins, and non-genotoxic carcinogens) and achieved high predictability (AUC = 0.90-1.00) in all categories. Moreover, we screened for an induced pluripotent stem (iPS) cell line to predict the toxicities based on the gene networks of iPS cells using transfer learning of the gene networks of ES cells, and predicted toxicities in four categories (neurotoxins, hepatotoxins, glomerular nephrotoxins, and non-genotoxic carcinogens) with high performance (AUC = 0.82-0.99). This method holds promise for tailor-made safety evaluations using personalized iPS cells.

Molecular profiling of ginsenoside metabolites to identify estrogen receptor alpha activity.

20(S)-Protopanaxadiol (PPD) and 20(S)-Protopanaxatriol (PPT) are major metabolites of ginseng in humans and are considered to have estrogenic activity in cellular bioassays. In this study, we conducted in silico analyses to determine whether PPD and PPT interact with estrogen receptor alpha (ERα) and compared them with ERα agonists, partial agonists, and antagonists to identify their ERα activity. The transcriptome profile of 17ß-estradiol (E2), PPD, and PPT in MCF-7 cells expressing ERα was further compared to understand the ERα activity of ginsenoside metabolites. The results showed that PPD and PPT interacted with the 1ERE, 1GWR, and 3UUD ERα proteins in the E2 interaction model, the 3ERD protein in the diethylstilbestrol (DES) interaction model, and the 1X7R protein in the genistein (GEN) interaction model. Conversely, neither the 4PP6 protein of the interaction model with the antagonist resveratrol (RES) nor the 1ERR protein of the interaction model with the antagonist raloxifene (RAL) showed the conformation of amino acid residues. When E2, PPD, and PPT were exposed to MCF-7 cells, cell proliferation and gene expression were observed. The transcriptomic profiles of E2, PPD, and PPT were compared using a knowledge-based pathway. PPD-induced transcription profiling was similar to that of E2, and the neural transmission pathway was detected in both compounds. In contrast, PPT-induced transcription profiling displayed characteristics of gene expression associated with systemic lupus erythematosus. These results suggest that ginsenoside metabolites have ERα agonist activity and exhibit neuroprotective effects and anti-inflammatory actions. However, a meta-analysis using public microarray data showed that the mother compounds GRb1 and GRg1 of PPD and PPT showed metabolic functions in insulin signaling pathways, condensed DNA repair and cell cycle pathways, and immune response and synaptogenesis. These results suggest that the ginsenoside metabolites have potent ERα agonist activity; however, their gene expression profiles may differ from those of E2.

Oxidative Stress as a Common Key Event in Developmental Neurotoxicity.

The developing brain is extremely sensitive to many chemicals. Perinatal exposure to neurotoxicants has been implicated in several neurodevelopmental disorders, including autism spectrum disorder, attention-deficit hyperactive disorder, and schizophrenia. Studies of the molecular and cellular events related to developmental neurotoxicity have identified a number of "adverse outcome pathways," many of which share oxidative stress as a key event. Oxidative stress occurs when the balance between the production of free oxygen radicals and the activity of the cellular antioxidant system is dysregulated. In this review, we describe some of the developmental neurotoxins that target the antioxidant system and the mechanisms by which they elicit stress, including oxidative phosphorylation in mitochondria and plasma membrane redox system in rodent models. We also discuss future directions for identifying adverse outcome pathways related to oxidative stress and developmental neurotoxicity, with the goal of improving our ability to quickly and accurately screen chemicals for their potential developmental neurotoxicity.

Epigenetic effects of insecticides on early differentiation of mouse embryonic stem cells.

Increasing evidence indicates that many insecticides produce significant epigenetic changes during embryogenesis, leading to developmental toxicities. However, the effects of insecticides on DNA methylation status during early development have not been well studied. We developed a novel nuclear phenotypic approach using mouse embryonic stem cells harboring enhanced green fluorescent protein fused with methyl CpG-binding protein to evaluate global DNA methylation changes via high-content imaging analysis. Exposure to imidacloprid, carbaryl, and o,p'-DDT increased the fluorescent intensity of granules in the nuclei, indicating global DNA methylating effects. However, DNA methylation profiling in cell-cycle-related genes, such as Cdkn2a, Dapk1, Cdh1, Mlh1, Timp3, and Rarb, decreased in imidacloprid treatments, suggesting the potential influence of DNA methylation patterns on cell differentiation. We developed a rapid method for evaluating global DNA methylation and used this approach to show that insecticides pose risks of developmental toxicity through DNA methylation.

Human ES and iPS cells display less drug resistance than differentiated cells, and naïve-state induction further decreases drug resistance.

Pluripotent stem cells (PSCs) possess unique characteristics that distinguish them from other cell types. Human embryonic stem (ES) cells are recently gaining attention as a powerful tool for human toxicity assessment without the use of experimental animals, and an embryonic stem cell test (EST) was introduced for this purpose. However, human PSCs have not been thoroughly investigated in terms of drug resistance or compared with other cell types or cell states, such as naïve state, to date. Aiming to close this gap in research knowledge, we assessed and compared several human PSC lines for their resistance to drug exposure. Firstly, we report that RIKEN-2A human induced pluripotent stem (iPS) cells possessed approximately the same sensitivity to selected drugs as KhES-3 human ES cells. Secondly, both ES and iPS cells were several times less resistant to drug exposure than other non-pluripotent cell types. Finally, we showed that iPS cells subjected to naïve-state induction procedures exhibited a sharp increase in drug sensitivity. Upon passage of these naïve-like cells in non-naïve PSC culture medium, their sensitivity to drug exposure decreased. We thus revealed differences in sensitivity to drug exposure among different types or states of PSCs and, importantly, indicated that naïve-state induction could increase this sensitivity.

Endocrine Disrupting Chemicals: Current Understanding, New Testing Strategies and Future Research Needs.

Endocrine disrupting chemicals (EDCs) are exogenous chemicals which can disrupt any action of the endocrine system, and are an important class of substances which play a role in the Developmental Origins of Health and Disease (DOHaD) [...].

Early Transcriptomic Changes upon Thalidomide Exposure Influence the Later Neuronal Development in Human Embryonic Stem Cell-Derived Spheres.

Stress in early life has been linked with the development of late-life neurological disorders. Early developmental age is potentially sensitive to several environmental chemicals such as alcohol, drugs, food contaminants, or air pollutants. The recent advances using three-dimensional neural sphere cultures derived from pluripotent stem cells have provided insights into the etiology of neurological diseases and new therapeutic strategies for assessing chemical safety. In this study, we investigated the neurodevelopmental effects of exposure to thalidomide (TMD); 2,2',4,4'-tetrabromodiphenyl ether; bisphenol A; and 4-hydroxy-2,2',3,4',5,5',6-heptachlorobiphenyl using a human embryonic stem cell (hESC)-derived sphere model. We exposed each chemical to the spheres and conducted a combinational analysis of global gene expression profiling using microarray at the early stage and morphological examination of neural differentiation at the later stage to understand the molecular events underlying the development of hESC-derived spheres. Among the four chemicals, TMD exposure especially influenced the differentiation of spheres into neuronal cells. Transcriptomic analysis and functional annotation identified specific genes that are TMD-induced and associated with ERK and synaptic signaling pathways. Computational network analysis predicted that TMD induced the expression of DNA-binding protein inhibitor ID2, which plays an important role in neuronal development. These findings provide direct evidence that early transcriptomic changes during differentiation of hESCs upon exposure to TMD influence neuronal development in the later stages.

The Key Characteristics of Carcinogens: Relationship to the Hallmarks of Cancer, Relevant Biomarkers, and Assays to Measure Them.

The key characteristics (KC) of human carcinogens provide a uniform approach to evaluating mechanistic evidence in cancer hazard identification. Refinements to the approach were requested by organizations and individuals applying the KCs. We assembled an expert committee with knowledge of carcinogenesis and experience in applying the KCs in cancer hazard identification. We leveraged this expertise and examined the literature to more clearly describe each KC, identify current and emerging assays and in vivo biomarkers that can be used to measure them, and make recommendations for future assay development. We found that the KCs are clearly distinct from the Hallmarks of Cancer, that interrelationships among the KCs can be leveraged to strengthen the KC approach (and an understanding of environmental carcinogenesis), and that the KC approach is applicable to the systematic evaluation of a broad range of potential cancer hazards in vivo and in vitro We identified gaps in coverage of the KCs by current assays. Future efforts should expand the breadth, specificity, and sensitivity of validated assays and biomarkers that can measure the 10 KCs. Refinement of the KC approach will enhance and accelerate carcinogen identification, a first step in cancer prevention.See all articles in this CEBP Focus section, "Environmental Carcinogenesis: Pathways to Prevention."

The IARC Monographs: Updated Procedures for Modern and Transparent Evidence Synthesis in Cancer Hazard Identification.

The Monographs produced by the International Agency for Research on Cancer (IARC) apply rigorous procedures for the scientific review and evaluation of carcinogenic hazards by independent experts. The Preamble to the IARC Monographs, which outlines these procedures, was updated in 2019, following recommendations of a 2018 expert advisory group. This article presents the key features of the updated Preamble, a major milestone that will enable IARC to take advantage of recent scientific and procedural advances made during the 12 years since the last Preamble amendments. The updated Preamble formalizes important developments already being pioneered in the Monographs program. These developments were taken forward in a clarified and strengthened process for identifying, reviewing, evaluating, and integrating evidence to identify causes of human cancer. The advancements adopted include the strengthening of systematic review methodologies; greater emphasis on mechanistic evidence, based on key characteristics of carcinogens; greater consideration of quality and informativeness in the critical evaluation of epidemiological studies, including their exposure assessment methods; improved harmonization of evaluation criteria for the different evidence streams; and a single-step process of integrating evidence on cancer in humans, cancer in experimental animals, and mechanisms for reaching overall evaluations. In all, the updated Preamble underpins a stronger and more transparent method for the identification of carcinogenic hazards, the essential first step in cancer prevention.

Consensus on the key characteristics of endocrine-disrupting chemicals as a basis for hazard identification.

Endocrine-disrupting chemicals (EDCs) are exogenous chemicals that interfere with hormone action, thereby increasing the risk of adverse health outcomes, including cancer, reproductive impairment, cognitive deficits and obesity. A complex literature of mechanistic studies provides evidence on the hazards of EDC exposure, yet there is no widely accepted systematic method to integrate these data to help identify EDC hazards. Inspired by work to improve hazard identification of carcinogens using key characteristics (KCs), we have developed ten KCs of EDCs based on our knowledge of hormone actions and EDC effects. In this Expert Consensus Statement, we describe the logic by which these KCs are identified and the assays that could be used to assess several of these KCs. We reflect on how these ten KCs can be used to identify, organize and utilize mechanistic data when evaluating chemicals as EDCs, and we use diethylstilbestrol, bisphenol A and perchlorate as examples to illustrate this approach.

Multiparameter Phenotypic Profiling in MCF-7 Cells for Assessing the Toxicity and Estrogenic Activity of Whole Environmental Water.

Multi-parameter phenotypic profiling of small molecules is a powerful approach to their toxicity assessment and identifying potential mechanisms of actions. The present study demonstrates the application of image-based multi-parameter phenotypic profiling in MCF-7 cells to assess the overall toxicity and estrogenic activity of whole environmental water. Phenotypic profiling of 30 reference compounds and their complex mixtures was evaluated to investigate the cellular morphological outcomes to targeted biological pathways. Overall toxicity and estrogenic activity of environmental water samples were then evaluated by phenotypic analysis comparing with conventional bioassays and chemical analysis by multivariate analysis. The phenotypic analysis for reference compounds demonstrated that size and structure of cells related to biological processes like cell growth, death, and communication. The phenotypic alteration and nuclei intensity were selected as potential biomarkers to evaluate overall toxicity and estrogenic activities, respectively. The phenotypic profiles were associated with the chemical structure profiles in environmental water samples. Since the phenotypic parameters revealed multiple toxicity endpoints, it could provide more information that is relevant to assessing the toxicity of environmental water samples in compare with conventional bioassays. In conclusion, the image-based multi-parameters phenotypic analysis with MCF-7 cells provides a rapid and information-rich tool for toxicity evaluation and identification in whole water samples.

Expression of human mutant cyclin dependent kinase 4, Cyclin D and telomerase extends the life span but does not immortalize fibroblasts derived from loggerhead sea turtle (Caretta caretta).

Conservation of the genetic resources of endangered animals is crucial for future generations. The loggerhead sea turtle (Caretta caretta) is a critically endangered species, because of human hunting, hybridisation with other sea turtle species, and infectious diseases. In the present study, we established primary fibroblast cell lines from the loggerhead sea turtle, and showed its species specific chromosome number is 2n = 56, which is identical to that of the hawksbill and olive ridley sea turtles. We first showed that intensive hybridization among multiple sea turtle species caused due to the identical chromosome number, which allows existence of stable hybridization among the multiple sea turtle species. Expressions of human-derived mutant Cyclin-dependent kinase 4 (CDK4) and Cyclin D dramatically extended the cell culture period, when it was compared with the cell culture period of wild type cells. The recombinant fibroblast cell lines maintained the normal chromosome condition and morphology, indicating that, at the G1/S phase, the machinery to control the cellular proliferation is evolutionally conserved among various vertebrates. To our knowledge, this study is the first to demonstrate the functional conservation to overcome the negative feedback system to limit the turn over of the cell cycle between mammalian and reptiles. Our cell culture method will enable the sharing of cells from critically endangered animals as research materials.

[Construction of a High-precision Chemical Prediction System Using Human ESCs].

　Toxicity prediction based on stem cells and tissue derived from stem cells plays a very important role in the fields of biomedicine and pharmacology. Here we report on qRT-PCR data obtained by exposing 20 compounds to human embryonic stem (ES) cells. The data are intended to improve toxicity prediction, per category, of various compounds through the use of support vector machines, and by applying gene networks. The accuracy of our system was 97.5-100% in three toxicity categories: neurotoxins (NTs), genotoxic carcinogens (GCs), and non-genotoxic carcinogens (NGCs). We predicted that two uncategorized compounds (bisphenol-A and permethrin) should be classified as follows: bisphenol-A as a non-genotoxic carcinogen, and permethrin as a neurotoxin. These predictions are supported by recent reports, and as such constitute a good outcome. Our results include two important features: 1) The accuracy of prediction was higher when machine learning was carried out using gene networks and activity, rather than the normal quantitative structure-activity relationship (QSAR); and 2) By using undifferentiated ES cells, the late effect of chemical substances was predicted. From these results, we succeeded in constructing a highly effective and highly accurate system to predict the toxicity of compounds using stem cells.

Stem Cell-Based Methods to Predict Developmental Chemical Toxicity.

Human pluripotent stem cells such as embryonic stem (ES) and induced pluripotent stem (iPS) cells, combined with sophisticated bioinformatics methods, are powerful tools to predict developmental chemical toxicity. Because cell differentiation is not necessary, these cells can facilitate cost-effective assays, thus providing a practical system for the toxicity assessment of various types of chemicals. Here we describe how to apply machine learning techniques to different types of data, such as qRT-PCRs, gene networks, and molecular descriptors, for toxic chemicals, as well as how to integrate these data to predict toxicity categories. Interestingly, our results using 20 chemical data for neurotoxins (NTs), genotoxic carcinogens (GCs), and nongenotoxic carcinogens (NGCs) demonstrated that the highest and most robust prediction performance was obtained by using gene networks as the input. We also observed that qRT-PCR and molecular descriptors tend to contribute to specific toxicity categories.

Transcriptome Analysis Uncovers a Growth-Promoting Activity of Orosomucoid-1 on Hepatocytes.

The acute phase protein orosomucoid-1 (Orm1) is mainly expressed by hepatocytes (HPCs) under stress conditions. However, its specific function is not fully understood. Here, we report a role of Orm1 as an executer of HPC proliferation. Increases in serum levels of Orm1 were observed in patients after surgical resection for liver cancer and in mice undergone partial hepatectomy (PH). Transcriptome study showed that Orm1 became the most abundant in HPCs isolated from regenerating mouse liver tissues after PH. Both in vitro and in vivo siRNA-induced knockdown of Orm1 suppressed proliferation of mouse regenerating HPCs and human hepatic cells. Microarray analysis in regenerating mouse livers revealed that the signaling pathways controlling chromatin replication, especially the minichromosome maintenance protein complex genes were uniformly down-regulated following Orm1 knockdown. These data suggest that Orm1 is induced in response to hepatic injury and executes liver regeneration by activating cell cycle progression in HPCs.