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Background: Mutations in genes encoding spliceosome components result in craniofacial structural defects in humans, referred to as spliceosomopathies. The SF3b complex is a crucial unit of the spliceosome, but model organisms generated through genetic modification of the complex do not perfectly mimic the phenotype of spliceosomopathies. Since the phenotypes are suggested to be determined by the extent of spliceosome dysfunction, an alternative experimental system that can seamlessly control SF3b function is needed., Methods: To establish another experimental system for model organisms elucidating relationship between spliceosome function and human diseases, we administered Pladienolide-B (PB), a SF3b complex inhibitor, to mouse and zebrafish embryos and assessed resulting phenotypes., Results: PB-treated mouse embryos exhibited neural tube defect and exencephaly, accompanied by apoptosis and reduced cell proliferation in the neural tube, but normal structure in the midface and jaw. PB administration to heterozygous knockout mice of Sf3b4, a gene coding for a SF3b component, influenced the formation of cranial neural crest cells (CNCCs). Despite challenges in continuous PB administration and a high death rate in mice, PB was stably administered to zebrafish embryos, resulting in prolonged survival. Brain, cranial nerve, retina, midface, and jaw development were affected, mimicking spliceosomopathy phenotypes. Additionally, alterations in cell proliferation, cell death, and migration of CNCCs were detected., Conclusions: We demonstrated that zebrafish treated with PB exhibited phenotypes similar to those observed in human spliceosomopathies. This experimental system may serve as a valuable research tool for understanding spliceosome function and human diseases., (© 2024 The Author(s). Birth Defects Research published by Wiley Periodicals LLC.)

Background: Embryonic craniofacial development involves several cellular and molecular events that are evolutionarily conserved among vertebrates. Vertebrate models such as mice and zebrafish have been used to investigate the molecular and cellular etiologies underlying human craniofacial disorders, including orofacial clefts. However, the molecular mechanisms underlying embryonic development in these two species are unknown. Therefore, elucidating the shared mechanisms of craniofacial development between disease models is crucial to understanding the underlying mechanisms of phenotypes in individual species., Results: We selected mice and zebrafish as model organisms to compare various events during embryonic craniofacial development. We identified genes (Sox9, Zfhx3 and 4, Cjun, and Six1) exhibiting similar temporal expression patterns between these species through comprehensive and stage-matched gene expression analyses. Expression analysis revealed similar gene expression in hypothetically corresponding tissues, such as the mice palate and zebrafish ethmoid plate. Furthermore, loss-of-function analysis of Zfhx4/zfhx4, a causative gene of human craniofacial anomalies including orofacial cleft, in both species resulted in deformed skeletal elements such as the palatine and ethmoid plate in mice and zebrafish, respectively., Conclusions: These results demonstrate that these disease models share common molecular mechanisms, highlighting their usefulness in modeling craniofacial defects in humans., (© 2024 Kao Corporation and The Author(s). Developmental Dynamics published by Wiley Periodicals LLC on behalf of American Association for Anatomy.)

Recently, it was reported that a testicular organ culture system (TOCS) using polydimethylsiloxane (PDMS) chips with excellent oxygen permeability and biocompatibility, called the PDMS-chip ceiling (PC) method, enables improved spermatogenesis efficiency. We investigated whether this PC method is useful for detecting impaired spermatogenesis caused by busulfan (Bu), a typical testicular toxicant. In this study, testicular tissue fragments from Acro3-EGFP mice, which express the green fluorescent protein (GFP) and reflect the progression of spermatogenesis, were subjected to the PC method. When treated with Bu, cultured tissues shrank in volume, and their GFP-expressing area decreased or disappeared. Histological examination confirmed the regression of spermatogenesis. In addition, immunohistochemical examination revealed that spermatogonia, including spermatogonial stem cells (SSCs), were the primary targets of Bu toxicity. Time-course analysis demonstrated that the recovery of spermatogenesis, dependent on Bu concentration, correlated closely with the severity of damage to these target cells. These results suggest that the PC method is a useful approach for detecting spermatogenesis impairment accurately through faithful recapitulation of spermatogenesis in vivo., (© 2024. The Author(s).)

Physiologically based pharmacokinetic (PBPK) models are considered useful tools in animal-free risk assessment. To utilize PBPK models for risk assessment, it is necessary to compare their reliability with in vivo data. However, obtaining in vivo pharmacokinetics data for cosmetic ingredients is difficult, complicating the utilization of PBPK models for risk assessment. In this study, to utilize PBPK models for risk assessment without accuracy evaluation, we proposed a novel concept-the modeling uncertainty factor (MUF). By calculating the prediction accuracy for 150 compounds, we established that using in vitro data for metabolism-related parameters and limiting the applicability domain increase the prediction accuracy of a PBPK model. Based on the 97.5th percentile of prediction accuracy, MUF was defined at 10 for the area under the plasma concentration curve and 6 for C max . A case study on animal-free risk assessment was conducted for bisphenol A using these MUFs. As this study was conducted mainly on pharmaceuticals, further investigation using cosmetic ingredients is pivotal. However, since internal exposure is essential in realizing animal-free risk assessment, our concept will serve as a useful tool to predict plasma concentrations without using in vivo data., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Inc.)

Epigallocatechin-3-gallate (EGCG), a major green tea polyphenol, has beneficial effects on human health. This study aimed to elucidate the detailed EGCG sulfation process to better understand its phase II metabolism, a process required to maximize its health benefits. Results show that kinetic activity of sulfation in the human liver and intestinal cytosol is 2-fold and 60- to 300-fold higher than that of methylation and glucuronidation, respectively, suggesting sulfation as the key metabolic pathway. Moreover, SULT1A1 and SULT1A3 are responsible for sulfation in the liver and intestine, respectively. Additionally, our human ingestion study revealed that the concentration of EGCG-4″-sulfate in human plasma ( C max : 177.9 nmol·L -1 , AUC: 715.2 nmol·h·L -1 ) is equivalent to free EGCG ( C max : 233.5 nmol·L -1 , AUC: 664.1 nmol·h·L -1 ), suggesting that EGCG-4″-sulfate is the key metabolite. These findings indicate that sulfation is a crucial factor for improving EGCG bioavailability, while also advancing the understanding of the bioactivity and toxicity of EGCG.

Read-across based on structural and biological similarities is expected to be a promising alternative method for assessing systemic toxicity. A concrete strategy for quantitative chemical risk assessment would be to stack read-across case studies and extract key considerations from them. Thus, we developed a read-across case study by comparing the toxicological effects based on adverse outcome pathways and exposure levels of different structurally similar chemicals for a target organ. In this study, we selected the hepatotoxicity of triclosan and its structurally similar chemicals including diclosan and 1-chloro-3-(4-chlorophenoxy)benzene. The results of in vitro toxicogenomics showed that disorders of cholesterol synthesis were commonly detected with both triclosan and diclosan. The decrease in hepatocellular cholesterol levels was similar in the cells treated with triclosan and diclosan. Furthermore, the exposure levels of triclosan and diclosan for the liver were similar. Collectively, these results suggest that triclosan and diclosan show similar toxicological effects and severity of hepatotoxicity. Considering the existing repeated dose toxicity data, our prediction results are reasonable regarding the toxicological effect and its severity. Thus, the present study demonstrated the usability of comparing toxicological effects and exposure levels using read-across for quantitative chemical risk assessment., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Recently, a new sustainable anionic surfactant called bio-based internal olefin sulfonate (Bio IOS) has been developed. This surfactant enables excellent water solubility and high surface activity. It has a unique structure of long hydrophobic alkyl chains (C16 to C18) with two types of hydrophilic groups in its midsection, which distinguish it from other conventional anionic surfactants. However, the effects of the specific structural features of the surfactant on its environmental properties and the consequent effects on the environment remain unclear. In this study, we investigated the environmental fate and ecotoxicity of Bio IOS and the effects of the types and positions of hydrophilic groups on biodegradability and ecotoxicity. Biodegradation studies demonstrated that Bio IOS was readily biodegradable with >99.5% removal in wastewater treatment activated sludge (test concentration: 1 mg/L) and a fast half-life of 5.8 h in river water (test concentration: 10 μg/L); the excellent biodegradability was likely due to the high water solubility attributed to the internal hydrophilic groups. Meanwhile, moderately toxic effects were observed, whereby the 50% lethal and effect concentrations of the three freshwater species were above 1 mg/L. Ecotoxicity studies with different types and positions of hydrophilic groups revealed that hydroxyalkane sulfonate was less toxic and that toxicity was reduced in the presence of more internally located hydrophilic groups. These findings suggest that the hydroxyl group and the internal positions of hydrophilic groups that constitute the molecular configuration resembling two separate shorter alkyl chains may reduce the adverse effects on organisms despite the long alkyl chains., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Babesia odocoilei-like parasites were first reported in 2003, and their virulence and hosts remain unknown. We report three cases of dogs with canine babesiosis in Iwate Prefecture. Since Iwate Prefecture area is an area of Japan where canine babesiosis is not endemic, we suspected that these cases of canine babesiosis were caused by B. odocoilei-like parasites. In the present study, we tried to identify the Babesia species that caused these cases of canine babesiosis. To classify Babesia parasites, the heat shock protein 70 (HSP70) gene was examined. Accordingly, we cloned and analyzed the HSP70 gene sequences of B. odocoilei-like parasites from three Ixodes ovatus ticks. It was determined that the nucleotide sequence of the HSP70 gene of the B. odocoilei-like parasites was not consistent with that of B. odocoilei, which suggests that these parasites were from a different species than B. odocoilei. Second, we identified the Babesia species that infected the three dogs by using the HSP70 gene and 18S rRNA. A partial HSP70 gene of B. odocoilei-like parasites was detected in the three dogs, but that of B. gibsoni was not detected. Additionally, a partial sequence of 18S rRNA of B. odocoilei-like parasites was detected in two dogs. These results demonstrated that two dogs were certainly infected with B. odocoilei-like parasites and that one dog was probably infected with B. odocoilei-like parasites. Therefore, these dogs were diagnosed with canine babesiosis due to the presence of B. odocoilei-like parasites. As there were only three cases, additional cases are needed to confirm our findings., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Integrated Approaches to Testing and Assessment provides a framework to improve the reliability of read-across for chemical risk assessment of systemic toxicity without animal testing. However, the availability of only a few case studies hinders the use of this concept for regulatory purposes. Thus, we compared the biological similarity of structurally similar chemicals using in vitro testing to demonstrate the validity of this concept for grouping chemicals and to extract key considerations in read-across. We analyzed the hepatotoxicity of naphthalene and three chemicals structurally similar to naphthalene (2,7-naphthalenediol, 1,5-naphthalenediol, and 1-naphthol) for which 90-day repeated dose toxicity data are available. To elucidate and compare their potential mechanisms, we conducted in vitro microarray analysis using rat primary hepatocytes and validated the results using a biomarker and metabolic activation analysis. We observed that 2,7-naphthalenediol, 1,5-naphthalenediol, and 1-naphthol had similar potential mechanisms, namely, induction of oxidative stress by their metabolic activation. Conversely, naphthalene did not show a similar toxicity effect. The existing in vivo data confirmed our grouping of chemicals based on this potential mechanism. Thus, our findings suggest that in vitro toxicogenomics and related biochemical assays are useful for comparing biological similarities and grouping chemicals based on their toxicodynamics for read-across., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Read-across based on only structural similarity is considered to have a risk of error in chemical risk assessment. Under these circumstances, considering biological similarity based on adverse outcome pathways using in vitro omics technologies is expected to enhance the accuracy and robustness of conclusions in read-across. However, due to a lack of practical case studies, key considerations and use of these technologies for data gap filling are not well discussed. Here we extracted and compared the potential mechanisms for hepatotoxicity for structural analogs of p-dialkoxy chlorobenzenes including 1,4-dichloro-2,5-dimethoxybenzene (DDMB), 2,5-dichloro-1,4-diethoxybenzene (DDEB), 2-chloro-1,4-dimethoxybenzene (CDMB), and 1-chloro-2,5-diethoxybenzene (CDEB) using in vitro omics technologies for read-across. To reveal the potential mechanisms for hepatotoxicity, we conducted microarray analysis with rat primary hepatocytes. The results showed that three (DDMB, DDEB, CDEB) of the four chemicals affected similar biological pathways such as peroxisome proliferation, oxidative stress, and mitochondrial dysfunction. Furthermore, these biological pathways are consistent with in vivo hepatotoxicity in the source chemical, DDMB. In contrast, CDMB did not affect a specific toxicological pathway. Taken together, these data show the potential mechanisms for hepatotoxicity for three chemicals (DDMB, DDEB, CDEB) and provide novel insights into grouping chemicals using in vitro toxicogenomics for read-across., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

In silico models for predicting chemical-induced side effects have become increasingly important for the development of pharmaceuticals and functional food products. However, existing predictive models have difficulty in estimating the mechanisms of side effects in terms of molecular targets or they do not cover the wide range of pharmacological targets. In the present study, we constructed novel in silico models to predict chemical-induced side effects and estimate the underlying mechanisms with high general versatility by integrating the comprehensive prediction of potential chemical-protein interactions (CPIs) with machine learning. First, the potential CPIs were comprehensively estimated by chemometrics based on the known CPI data (1,179,848 interactions involving 3,905 proteins and 824,143 chemicals). Second, the predictive models for 61 side effects in the cardiovascular system (CVS), gastrointestinal system (GIS), and central nervous system (CNS) were constructed by sparsity-induced classifiers based on the known and potential CPI data. The cross validation experiments showed that the proposed CPI-based models had a higher or comparable performance than the traditional chemical structure-based models. Moreover, our enrichment analysis indicated that the highly weighted proteins derived from predictive models could be involved in the corresponding functions of the side effects. For example, in CVS, the carcinogenesis-related pathways (e.g., prostate cancer, PI3K-Akt signal pathway), which were recently reported to be involved in cardiovascular side effects, were enriched. Therefore, our predictive models are biologically valid and would be useful for predicting side effects and novel potential underlying mechanisms of chemical-induced side effects.

Although coffee components have gained interest for use as pharmaceuticals, little is known about their safety pharmacological effects. Hence, we aimed to evaluate the safety pharmacological effects of a chlorogenic acid (CGA)-related compound contained in coffee, 5- O -caffeoylquinic acid (5-CQA), and its metabolites, 5- O -feruloylquinic acid (5-FQA), caffeic acid (CA), and ferulic acid (FA). Langendorff perfused heart assay, electrophysiological assay of acute rat hippocampal slices, and in vitro Magnus assay of gastrointestinal tracts were conducted at 1-100 µM. Moreover, in vitro profiling assays against 38 major targets were conducted. In the Langendorff assay, no significant adverse effects were observed. In the electrophysiological assay, although epileptiform discharge rates were increased at 10 µM CA with 4-aminopyridine, and area under the curve (AUC) and number of population spike were increased at 10 µM FA with bicuculline, dose dependency was not confirmed, and no significant changes were observed at 1 µM and by CGAs alone. In the Magnus assay, a slight increase in contraction activity was observed at >1 µM FA in the stomach fundi and 100 µM 5-CQA in the ileum, suggesting enterokinesis promotion. No significant interactions were observed in the in vitro profiling assays. Therefore, CGAs could have a fundamental function as safe pharmaceuticals.

Recent studies suggest that diets supplemented with alpha-linolenic acid (ALA)-enriched diacylglycerol (DAG) oil provide potential health benefits in preventing or managing obesity. However, available safety information about reproductive and developmental toxicities of ALA-DAG oil is limited. This study was conducted to clarify the effect, if any, of ALA-DAG oil on embryo-fetal development, following maternal exposure during the critical period of major organogenesis. ALA-DAG oil was administered via gavage to pre-mated female Sprague Dawley rats from gestation day 6 through 19, at dose levels of 0, 1.25, 2.5, and 5.0 mL/kg/day (equivalent to 0, 1149, 2325, and 4715 mg/kg/day, respectively), with total volume adjusted to 5 mL/kg/day with rapeseed oil. All females survived to the scheduled necropsy. There were no treatment-related changes in clinical or internal findings, maternal body weights, feed consumption, intrauterine growth, survival, and number of implantations. No ALA-DAG oil-related fetal malformations or developmental variations were noted. A maternal maximum tolerated dose for ALA-DAG oil could not be achieved in this study. Based on these results, a dose level of 5.0 mL/kg (4715 mg/kg/day), the highest dose tested, was considered as the no-observed-adverse-effect level (NOAEL) for both maternal and developmental toxicity., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Diets supplemented with alpha-linolenic acid (ALA)-enriched diacylglycerol (DAG) oil-which mainly consists of oleic and linolenic, linoleic acids-have potential health benefits in terms of preventing or managing obesity. Although safety of DAG oil has been extensively investigated, toxicity of ALA-DAG oil has not been well understood. Hence, the present study was conducted to clarify the potential adverse effects, if any, of ALA-DAG oil in rats (10/sex/group) fed diets containing 1.375%, 2.75%, or 5.5% ALA-DAG oil for 90 days. Compared to control rats fed rapeseed oil or ALA-triacylglycerol oil (flaxseed oil), rats receiving ALA-DAG oil did not reveal any toxicologically significant treatment-related changes as evaluated by clinical signs, functional observational battery, body weight, food consumption, ophthalmology, urinalysis, hematology, clinical chemistry, organ weight, necropsy and histopathology. The no observed adverse effect levels for dietary exposure to ALA-DAG oil for male and female rats were 2916 and 3326 mg/kg body weight/day, respectively, the highest dose tested. The findings from this study suggest that consumption of ALA-DAG oil is unlikely to cause adverse effects., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The Short Time Exposure (STE) test method is an alternative method for assessing eye irritation potential using Statens Seruminstitut Rabbit Cornea cells and has been adopted as test guideline 491 by the Organisation for Economic Co-operation and Development. Its good predictive performance in identifying the Globally Harmonized System (GHS) No Category (NC) or Irritant Category has been demonstrated in evaluations of water-soluble substances, oil-soluble substances, and water-soluble mixtures. However, the predictive performance for oil-soluble mixtures was not evaluated. Twenty-four oil-soluble mixtures were evaluated using the STE test method. The GHS NC or Irritant Category of 22 oil-soluble mixtures were consistent with that of a Reconstructed human Cornea-like Epithelium (RhCE) test method. Inter-laboratory reproducibility was then confirmed using 20 water- and oil-soluble mixtures blind-coded. The concordance in GHS NC or Irritant Category among four laboratories was 90%-100%. In conclusion, the concordance in comparison with the results of RhCE test method using 24 oil-soluble mixtures and inter-laboratory reproducibility using 20 water- and oil-soluble mixtures blind-coded were good, indicating that the STE test method is a suitable alternative for predicting the eye irritation potential of both substances and mixtures., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

To evaluate chemicals (e.g. lipophilic chemicals, pre/pro-haptens) that are difficult to correctly evaluate using in vitro skin sensitization tests (e.g. DPRA, KeratinoSens or h-CLAT), we developed a novel in vitro test termed "Epidermal Sensitization Assay: EpiSensA" that uses reconstructed human epidermis. This assay is based on the induction of multiple marker genes (ATF3, IL-8, DNAJB4 and GCLM) related to two keratinocyte responses (inflammatory or cytoprotective) in the induction of skin sensitization. Here, we first confirmed the mechanistic relevance of these marker genes by focusing on key molecules that regulate keratinocyte responses in vivo (P2X 7 for inflammatory and Nrf2 for cytoprotective responses). The up-regulation of ATF3 and IL-8, or DNAJB4 and GCLM induced by the representative sensitizer 2,4-dinitrochlorobenzene in human keratinocytes was significantly suppressed by a P2X 7 specific antagonist KN-62, or by Nrf2 siRNA, respectively, which supported mechanistic relevance of marker genes. Moreover, the EpiSensA had sensitivity, specificity and accuracy of 93%, 100% and 93% for 29 lipophilic chemicals (logKow≥3.5), and of 96%, 75% and 88% for 43 hydrophilic chemicals including 11 pre/pro-haptens, compared with the LLNA. These results suggested that the EpiSensA could be a mechanism-based test applicable to broad sets of chemicals including lipophilic chemicals and pre/pro-haptens., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Dynamic changes of cytoplasmic and cortical actin filaments drive various cellular and developmental processes. Although real-time imaging of actin filaments in living cells has been developed, imaging of actin filaments in specific cells of living organisms remains limited, particularly for the analysis of gamete formation and early embryonic development. Here, we report the production of transgenic zebrafish expressing the C-terminus of Moesin, an actin filament-binding protein, fused with green fluorescent protein or red fluorescent protein (GFP/RFP-MoeC), under the control of a cyclin B1 promoter. GFP/RFP-MoeC was expressed maternally, which labels the cortical actin cytoskeleton of blastula-stage cells. High levels of GFP/RFP fluorescence were detected in the adult ovary and testis. In the ovaries, GFP/RFP-MoeC was expressed in oocytes but not in follicle cells, which allows us to clearly visualize the organization of actin filaments in different stages of the oocyte. Using full-grown oocytes, we revealed the dynamic changes of actin columns assembled in the cortical cytoplasm during oocyte maturation. The number of columns slightly decreased in the early period before germinal vesicle breakdown (GVBD) and then significantly decreased at GVBD, followed by recovery after GVBD. Our transgenic fish are useful for analyzing the dynamics of actin filaments in oogenesis and early embryogenesis., (© 2015 Wiley Periodicals, Inc.)

The Short Time Exposure (STE) test is an in vitro eye irritation test based on the cytotoxicity in SIRC cells (rabbit corneal cell line) following a 5 min treatment of chemicals. This study evaluated the predictive performance of the STE test to identify the globally harmonized system (GHS) Not Classified category and other irritant categories (i.e., GHS Category 1 or 2) when used to test 40 chemical mixtures that included irritants. The STE test correctly identified 30 tested mixtures classified as GHS irritant categories and 5 out of 10 tested mixtures classified as GHS Not Classified. The sensitivity, specificity, positive predictivity, negative predictivity, and overall accuracy of the STE test were 100% (30/30), 50% (5/10), 86% (25/30), 100% (5/5), and 88% (35/40), respectively. These predictive performances were comparative to or greater than those in other in vitro eye irritation tests that have been accepted as test guideline by the Organisation for Economic Co-operation and Development. This suggests that the STE test has sufficient predictivity for identifying the eye irritation potential of chemical mixtures. Since no false negatives in this study were found, this indicates that the STE test is applicable as a part of the bottom-up approach., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Recent changes in regulatory requirements and social views on animal testing have accelerated the development of reliable alternative tests for predicting skin sensitizing potential of chemicals. In this study, we aimed to develop a new in vitro skin sensitization assay using reconstructed human epidermis, RhE model, which is expected to have broader applicability domain rather than existing in vitro assays. Microarray analysis revealed that the expression of five genes (ATF3, DNAJB4, GCLM, HSPA6 and HSPH1) related to cellular stress response were significantly up-regulated in RhE model after 6h treatment with representative skin sensitizers, 1-fluoro-2,4-dinitrobenzene and oxazolone, but not a non-sensitizer, benzalkonium chloride. The predictive performance of five genes was examined with eight skin sensitizers (e.g., cinnamic aldehyde), four non-sensitizers (e.g., sodium lauryl sulfate) and four pre-/pro-haptens (e.g., p-phenylenediamine, isoeugenol). When the positive criteria were set to obtain the highest accuracy with the animal testing (LLNA), ATF3, DNAJB4 and GCLM exhibited a high predictive accuracy (100%, 93.8% and 87.5%, respectively). All tested pre-/pro-haptens were correctly predicted by both ATF3 and DNAJB4. These results suggested that the RhE-based assay, termed epidermal sensitization assay (EpiSensA), could be an useful skin sensitization assay with a broad applicability domain including pre-/pro-haptens., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The Short Time Exposure (STE) test is a simple and easy-to-perform in vitro eye irritation test, that uses the viability of SIRC cells (a rabbit corneal cell line) treated for five minutes as the endpoint. In this study, our goal was to define the applicability domain of the STE test, based on the results obtained with a set of 113 substances. To achieve this goal, chemicals were selected to represent both different chemical classes and different chemical properties, as well as to cover, in a balanced manner, the categories of eye irritation potential according to the Globally Harmonised System (GHS). Accuracy analysis indicated that the rates of false negatives for organic/inorganic salts (75.0%), hydrocarbons (33.3%) and alcohols (23.5%) were high. Many of the false negative results were for solid substances. It is noteworthy that no surfactant resulted in a false negative result in the STE test. Further examination of the physical property data and performance showed a significant improvement in the predictive accuracy, when substances with vapour pressures over 6kPa were excluded from the analyses. Our results indicate that several substances - i.e. certain solids such as salts, alcohols, hydrocarbons, and volatile substances with a vapour pressure over 6kPa - do not fall within the applicability domain of the STE test. Overall, we are encouraged by the performance and improved accuracy of the STE test., (2013 FRAME.)

Recent changes in regulatory restrictions and social views against animal testing have accelerated development of reliable alternative tests for predicting skin sensitizing potential and potency of many chemicals. Lately, a test battery integrated with different in vitro tests has been suggested as a better approach than just one in vitro test for replacing animal tests. In this study, we created a dataset of 101 test chemicals with LLNA, human cell line activation test (h-CLAT), direct peptide reactivity assay (DPRA) and in silico prediction system. The results of these tests were converted into scores of 0-2 and the sum of individual scores provided the accuracy of 85% and 71% for the potential and potency prediction, compared with LLNA. Likewise, the straightforward tiered system of h-CLAT and DPRA provided the accuracy of 86% and 73%. Additionally, the tiered system showed a higher sensitivity (96%) compared with h-CLAT alone, indicating that sensitizers would be detected with higher reliability in the tiered system. Our data not only demonstrates that h-CLAT can be part of a test battery with other methods but also supports the practical utility of a tiered system when h-CLAT and DPRA are the first screening methods for skin sensitization., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Recently, it has been reported that reactive oxygen species (ROS) produced by contact allergens can affect dendritic cell migration and contact hypersensitivity. The aim of the present study was to develop a new in vitro assay that could predict the skin sensitizing potential of chemicals by measuring ROS production in THP-1 (human monocytic leukemia cell line) cells. THP-1 cells were pre-loaded with a ROS sensitive fluorescent dye, 5-(and 6-)-chloromethyl-2', 7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA), for 15min, then incubated with test chemicals for 30min. The fluorescence intensity was measured by flow cytometry. For the skin sensitizers, 25 out of 30 induced over a 2-fold ROS production at more than 90% of cell viability. In contrast, increases were only seen in 4 out of 20 non-sensitizers. The overall accuracy for the local lymph node assay (LLNA) was 82% for 50 chemicals tested. A correlation was found between the estimated concentration showing 2-fold ROS production in the ROS assay and the EC3 values (estimated concentration required to induce positive response) of the LLNA. These results indicated that the THP-1 cell-based ROS assay was a rapid and highly sensitive detection system able to predict skin sensitizing potentials and potency of chemicals., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The human Cell Line Activation Test (h-CLAT), an in vitro skin sensitization test, is based on the augmentation of CD86 and CD54 expression in THP-1 cells following exposure to chemicals. The h-CLAT was found to be capable of determining the hazard of skin sensitization. In contrast, the local lymph node assay (LLNA), widely used as a stand-alone method in Europe and US, identifies the same hazard, but also classifies the potency by using the estimated concentration of SI=3 (EC3). In this study, several values calculated from the h-CLAT data were evaluated for its correlation to the LLNA EC3 determination. A statistically significant correlation was observed between h-CLAT concentration providing a cell viability of 75% (CV75), h-CLAT estimated concentration of RFI=150 for CD86 (EC150), and for CD54 (EC200) with LLNA's EC3. From EC150 and EC200, a minimum induction threshold (MIT) was determined as the smaller of either EC150 or EC200. MIT showed a correlation with EC3 (R=0.638). Also, MIT had an approximate 80% accuracy for sub-categories of the globally harmonized system (GHS) when a tentative threshold of 13 μg/mL was used. From these data, the h-CLAT values may be one of the useful tools to predict the allergic potency of chemicals., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: Recent changes in regulatory restrictions and social opposition to animal toxicology experiments have driven the need for reliable in vitro tests for predicting the skin sensitizing potentials of a wide variety of industrial chemicals. Previously, we developed the human cell line activation test (h-CLAT) as a cell-based assay to predict the skin sensitizing potential of chemicals, and showed the correspondence between the h-CLAT and the murine local lymph node assay results., Objectives: This study was conducted to investigate the predictive performance of the h-CLAT for human skin sensitizing potential., Materials/methods: We selected a total of 66 test chemicals with known human sensitizing potential, and tested all chemicals with the h-CLAT. We then evaluated the performance of the h-CLAT in predicting human sensitizing potential., Results and Conclusion: Forty-five of 51 tested sensitizers were positive in the h-CLAT, indicating relatively high sensitivity. Also, 10 of 15 non-sensitizers were correctly detected as negative. The overall agreement between human data and h-CLAT outcome was 83%. Furthermore, the h-CLAT could accurately predict the human sensitizing potential of 23 tested chemicals that were amines, heterocyclic compounds, or sulfur compounds. Our data indicate the utility of the h-CLAT for predicting the human skin sensitizing potential of a variety of chemicals., (© 2011 John Wiley & Sons A/S.)

Contact allergens induce the augmentation of cell surface molecules on and release of cytokines from Langerhans cells (LC) in skin sensitization. THP-1 and U937 cell lines, surrogates of LC, were used as analytical tools of this phenomenon recently. In THP-1 cells, contact allergens are reported to induce the phenotypic alteration including the production of pro-inflammatory cytokines and augmentation of cell surface molecules especially at sub-toxic doses. However, the relationship between phenotypic alteration and cytotoxicity is not clear yet. The purpose of this study is to understand the relationship between the protein expression and cytotoxicity induced by contact allergens. First, we observed that the cytotoxicity induced by contact allergens is caused by both apoptosis and necrosis. Apoptosis was preferentially confirmed in stimulation with contact allergens, but non-allergen sodium lauryl sulfate (SLS) hardly induced apoptosis. Moreover, there was no effect to augmentation of protein expression when apoptosis induction pathways were inhibited. Based on these findings, we proposed that the protein expression and cytotoxicity were controlled independently. Next, oxidative stress was found to be generated by contact allergens at the early phase, and this regulated the protein expression and cytotoxicity at least partially. Finally, the humoral factors from dead cells induced by dinitrochlorobenzene (DNCB) were exposed to fresh THP-1 cells to confirm whether protein expression depended on cytotoxicity. The protein expression was not induced. Altogether, these results suggest that cytotoxicity induced by contact allergens may result in apoptosis and may also be stimulated in parallel with protein expression through an intracellular signal or signals.

We previously developed the human cell-line activation test (h-CLAT) in vitro skin sensitisation test, based on our reported finding that a 24-hour exposure of THP-1 cells (a human monocytic leukaemia cell line) to sensitisers is sufficient to induce the augmented expression of CD86 and CD54. The aim of this study is to confirm the predictive value of h-CLAT for skin sensitisation activity by employing a larger number of test chemicals. One hundred chemicals were selected, according to their categorisation in the local lymph node assay (LLNA), as being: extreme, strong, moderate and weak sensitisers, and non-sensitisers. The correlation of the h-CLAT results with the LLNA results was 84%. There were some false negatives (e.g. benzoyl peroxide, hexyl cinnamic aldehyde) and some false positives (e.g. 1-bromobutane, diethylphthalate). Eight out of the 9 false negatives (89%) were water-insoluble chemicals. The h-CLAT could positively predict not only extreme and strong sensitisers, but also moderate and weak sensitisers, though the detection rates of weak sensitisers and non-sensitisers were comparatively low. Some sensitisers enhanced both CD86 and CD54 levels, and some enhanced the level of only one of them. The use of the combination of CD86 and CD54 induction as a positive indicator, improved the accuracy of the test. In conclusion, the h-CLAT is expected to be a useful cell-based in vitro method for predicting skin sensitisation potential., (2010 FRAME.)

Contact allergens induce in vitro and in vivo the activation of dendritic cells (DC) and Langerhans cells (LC), which includes the up-regulation of surface marker expression (e.g. CD86, CD54) and cytokine production (e.g. TNF-alpha, IL-1beta, IL-8). The mitogen-activated protein kinase (MAPK) pathway also has a crucial role in this activation. However, the extent of MAPK involvement in the IL-8 production during DC/LC activation is not well understood. Earlier, we reported that contact allergens activated THP-1 cells, human monocytic cell line, like LC/DC in vitro. In this study, we further characterize the mechanism of IL-8 production using THP-1 cells as surrogate DCs. First, we evaluated the potential of 23 chemicals with different skin sensitization potencies to predominantly induce IL-8 production in vitro. Next we investigated the role of MAPK signaling and TNF-alpha, which is known to have autocrine effects on DC activation (e.g., IL-8 production). Inhibition of extracellular signal-regulated kinase (ERK), one of the MAPK pathways, suppressed the IL-8 production induced by both 2,4-dinitrochlorobenzene (DNCB) and nickel sulfate (NiSO(4)), and inhibition of p38 MAPK, a second MAPK pathway, significantly suppressed IL-8 production induced by only DNCB. Additionally, neutralization of TNF-alpha activity suppressed IL-8 production in THP-1 cells exposed to DNCB and NiSO(4). In conclusion, IL-8 production was predominantly induced in THP-1 cells following allergen stimulation, and MAPK pathways and TNF-alpha were involved in the IL-8 production induced by DNCB and NiSO(4). A better understanding of the mechanism of DC activation in vitro might lead to the clarification of the in vivo skin sensitization mechanism.

The activation of dendritic cells (DC), including Langerhans cells (LC) that reside within the epidermis, is a critical event in the induction phase of allergic contact hypersensitivity. Although recently, p38 mitogen-activated protein kinase (MAPK) has been reported to play a role in the activation of DC induced by allergens, the signaling pathways involved in this process have yet to be determined. We previously found that THP-1 cells have a high capacity to induce TNF-alpha release and CD86, CD54, and CD40 expression following allergen treatment; reflecting in vitro allergen-induced DC activation during skin sensitization. In this study, we investigated the signaling pathways in THP-1 cells activated by two representative allergens, DNCB and NiSO(4). We found that DNCB and NiSO(4) induced phosphorylation of p38 MAPK and extracellular signal-regulated kinase (ERK). Inhibition of p38 MAPK activation selectively blocked DNCB-induced TNF-alpha release, but not NiSO(4)-induced release. In contrast, inhibition of ERK pathways selectively suppressed NiSO(4)-induced TNF-alpha release but not DNCB-induced release. In addition, we found that the inhibition of p38 MAPK and ERK pathways caused a selective inhibition of CD86, CD54, and/or CD40 expression following treatment with DNCB or NiSO(4). In particular, inhibition of p38 MAPK suppressed CD86, CD54, and CD40 expression induced by DNCB and CD86 expression induced by NiSO(4) while inhibition of ERK pathways suppressed CD86, CD54 and CD40 expression induced by DNCB and NiSO(4). These data indicate that both DNCB and NiSO(4) activate p38 MAPK and ERK, and thereby stimulate TNF-alpha release and phenotypic changes through the different signal transduction pathways.

Dendritic cells (DCs), including Langerhans cells (LCs), play a critical role in the induction phase of allergic contact hypersensitivity. Following exposure to chemical allergens in the skin, LCs undergo a maturation process leading to the up-regulation of expression of co-stimulatory molecules, such as CD86, CD54 and CD40. Our previous study revealed that chemical allergens induce phenotype alterations (e.g., CD86, CD54 and CD40) and cytokine production (TNF-alpha and IL-8) in THP-1 cells that possibly reflect the maturation of dendritic cells during skin sensitization. However, the physiological signals for phenotypic alterations by chemical allergens are still not fully understood. Therefore, in this study, we investigated the effect of TNF-alpha and extracellular ATP on THP-1 cell activation induced by chemical allergens. Kinetic studies revealed that TNF-alpha and IL-8 release occurred in a time-dependent manner with release of two cytokines beginning at 3 hr post-exposure to well-known haptens, DNCB and NiSO(4). While recombinant human TNF-alpha augmented CD54 and CD40 expression in a dose-dependent manner, rhTNF-alpha did not increase CD86 expression. Furthermore, neutralization of TNF-alpha activity strongly inhibited CD54 and CD40 expression induced by allergens. On the contrary, extracellular ATP induced the up-regulation of both CD86 and CD54 expression. In the presence of the P2 receptor antagonist suramin, the up-regulation of CD86 and CD54 expression by allergens was in part suppressed. Therefore, we postulate that not only TNF-alpha but also extracellular ATP may contribute to cell activation following allergen stimulation, which might reflect the mechanism by which DCs respond to allergens.

Adult T cell leukemia/lymphoma (ATLL) has been characterized as one of the most aggressive human neoplasias and its incidence is thought to be caused by both genetic and epigenetic alterations to the host cellular genes of T cells infected with human T cell leukemia virus type I (HTLV-I). A multilobulated nuclear appearance is an important diagnostic marker of ATLL, and we have now identified that the molecular mechanisms underlying these formations occur through microtubule rearrangement via phosphatidylinositol 3-kinase (PI3-kinase) activation by AILIM/ICOS signaling. We also show that PTEN and/or SHIP-1, which are PIP3 inositol phosphatases that inhibit the activation of downstream effectors of the PI3-kinase cascade, are disrupted in both ATLL neoplasias and in multilobulated nuclei-forming Jurkat cells. This down-regulation of PTEN was found to be essential for the formation of ATLL-type nuclear lobules. Furthermore, PI3-kinase and PTEN activities were observed to be closely associated with cellular proliferation. Thus, our results suggest that alteration of PI3-kinase signaling cascades, as a result of the down-regulation of inositol phosphatases, induces ATLL-type multilobulated nuclear formation and is also associated with the cellular proliferation of malignant T cell leukemias/lymphomas.