Histological differences between the central and peripheral areas of the testes of busulfan-administered mice.

Busulfan is an anticancer drug known to cause serious damage to seminiferous tubules in the testes and deplete germ cells in human and animal models. The testicular artery is anastomosed with deferential and cremasteric arteries and is divided into capsular arteries, which give rise to the centripetal arteries and then recurrent arteries. The arterial blood in the testicular tissue is supplied by such a consequent system of arterial vessels, in order from the peripheral to the central area. As anticancer drugs are generally distributed throughout the whole body via the bloodstream and the running and distribution of arteries differ among the testicular areas, we hypothesized that the efficacy of busulfan differs in different testicular areas, particularly between the central and peripheral areas. In this study, busulfan was intraperitoneally injected at 40 mg/kg body weight into C57BL/6J male mice. After 28 days, in busulfan-treated mice, the diameters of seminiferous tubules were significantly higher in the central than in the peripheral area of the testes. The seminiferous tubular areas also significantly decreased in the peripheral areas compared with the central areas. The number of germ cells per seminiferous tubule was significantly higher in the central than in the peripheral area. Sertoli cell nuclei were detached into the lumen in the peripheral area. The number of Leydig cells was significantly lower in the peripheral areas. These data suggest that the effects of busulfan differ between the central and peripheral areas of the testis at 4 weeks after busulfan administration.

DTox: A deep neural network-based in visio lens for large scale toxicogenomics data.

With the advancement of large-scale omics technologies, particularly transcriptomics data sets on drug and treatment response repositories available in public domain, toxicogenomics has emerged as a key field in safety pharmacology and chemical risk assessment. Traditional statistics-based bioinformatics analysis poses challenges in its application across multidimensional toxicogenomic data, including administration time, dosage, and gene expression levels. Motivated by the visual inspection workflow of field experts to augment their efficiency of screening significant genes to derive meaningful insights, together with the ability of deep neural architectures to learn the image signals, we developed DTox, a deep neural network-based in visio approach. Using the Percellome toxicogenomics database, instead of utilizing the numerical gene expression values of the transcripts (gene probes of the microarray) for dose-time combinations, DTox learned the image representation of 3D surface plots of distinct time and dosage data points to train the classifier on the experts' labels of gene probe significance. DTox outperformed statistical threshold-based bioinformatics and machine learning approaches based on numerical expression values. This result shows the ability of image-driven neural networks to overcome the limitations of classical numeric value-based approaches. Further, by augmenting the model with explainability modules, our study showed the potential to reveal the visual analysis process of human experts in toxicogenomics through the model weights. While the current work demonstrates the application of the DTox model in toxicogenomic studies, it can be further generalized as an in visio approach for multi-dimensional numeric data with applications in various fields in medical data sciences.

Epichordal vertebral column formation in Xenopus laevis.

Although Xenopus Laevis is the most widely used model amphibian, skeletal development of its vertebral column has not been well illustrated so far. The mode of vertebral column development in anurans has been classified into two modes: perichordal and epichordal. Xenopus vertebral column formation is believed to follow the epichordal mode, but this aspect has been underemphasized, and illustrative examples are currently unavailable to the scientific community. This study documents the entire process of vertebral column formation in X. laevis, from the initial neural arch formation to the completion of metamorphosis. These images reveal that the neural arch arises from the dorsal lamina and lateral pedicle primordia, with no strict adherence to an anteroposterior sequence. Unlike other species, Xenopus centrum primordia exclusively form at the expanded ventral margins of neural arches, rather than from the cartilaginous layer surrounding the notochord. These paired centrum primordia then fuse at the ventral midline, dorsal to the notochord, and subsequently the notochord degenerates. This mode of centrum formation differs from the traditional epichordal mode, indicating that Xenopus might have lost the ability to form a cartilaginous layer around the notochord. Instead, the neural arch's ventral margin appears to have evolved to incorporate centrum precursor cells at its base, thereby forming a centrum-like structure compensating for the absence of a true centrum. It is widely accepted that postsacral vertebrae lack centra, only possessing neural arches, and eventually fuse with the hypochord to form the urostyle. However, we have shown that the paired ventral ends of the postsacral vertebrae also fuse at the midline to form a centrum-like structure. This process might extend to the trunk region during centrum formation. In addition to these findings, we offer evolutionary insights into the reasons why Xenopus retains centrum primordia at the base of neural arches.

A novel alternative method for long-term evaluation of male reproductive toxicity and its recovery using a pre-pubertal mouse testis organ culture system.

Successful treatment of pediatric cancers often results in long-term health complications, including potential effects on fertility. Therefore, assessing the male reproductive toxicity of anti-cancer drug treatments and the potential for recovery is of paramount importance. However, in vivo evaluations are time-intensive and require large numbers of animals. To overcome these constraints, we utilized an innovative organ culture system that supports long-term spermatogenesis by placing the testis tissue between a base agarose gel and a polydimethylsiloxane ceiling, effectively mirroring the in vivo testicular environment. The present study aimed to determine the efficacy of this organ culture system for accurately assessing testicular toxicity induced by cisplatin, using acrosin-green fluorescent protein (GFP) transgenic neonatal mouse testes. The testis fragments were treated with different concentrations of cisplatin-containing medium for 24 h and incubated in fresh medium for up to 70 days. The changes in tissue volume and GFP fluorescence over time were evaluated to monitor the progression of spermatogenesis, in addition to the corresponding histopathology. Cisplatin treatment caused tissue volume shrinkage and reduced GFP fluorescence in a concentration-dependent manner. Recovery from testicular toxicity was also dependent on the concentration of cisplatin received. The results demonstrated that this novel in vitro system can be a faithful replacement for animal experiments to assess the testicular toxicity of anti-cancer drugs and their reversibility, providing a useful method for drug development.

Lethal Phenotype-Based Database Screening Identifies Ceramide as a Negative Regulator of Primitive Streak Formation.

In early embryogenesis, the primitive streak (PrS) generates the mesendoderm and is essential for organogenesis. However, because the PrS is a minute and transient tissue, elucidating the mechanism of its formation has been challenging. We performed comprehensive screening of 2 knockout mouse databases based on the fact that failure of PrS formation is lethal. We identified 812 genes involved in various cellular functions and responses that might be linked to PrS formation, with the category of greatest abundance being "Metabolism." In this study, we focused on genes of sphingolipid metabolism and investigated their roles in PrS formation using an in vitro mouse ES cell differentiation system. We show here that elevated intracellular ceramide negatively regulates gene expression essential for PrS formation and instead induces neurogenesis. In addition, sphingosine-1-phosphate (a ceramide derivative) positively regulates neural maturation. Our results indicate that ceramide regulates both PrS formation and the induction of neural differentiation.

Behavioral effects of adult male mice induced by low-level acetamiprid, imidacloprid, and nicotine exposure in early-life.

Introduction: Acetamiprid (ACE) and imidacloprid (IMI), the neonicotinoid chemicals, are widely used as pesticides because of their rapid insecticidal activity. Although these neonicotinoids exert very low toxicity in mammals, the effects of early, low-level, chronic exposure on the adult central nervous system are largely unclear. This study investigated the effects of low-level, chronic neonicotinoids exposure in early life on the brain functions of adult mice, using environmentally relevant concentrations. Methods: We exposed mice to an acceptable daily intake level of neonicotinoids in drinking water during the prenatal and postnatal periods. Additionally, we also exposed mice to nicotine (NIC) as a positive control. We then examined the effects on the central nervous system in adult male offspring. Results: In the IMI and NIC exposure groups, we detected behavior that displayed impairment in learning and memory. Furthermore, immunohistochemical analysis revealed a decrease in SOX2 (as a neural stem cell marker) and GFAP (as an astrocyte marker) positive cells of the hippocampal dentate gyrus in the IMI and NIC exposure groups compared to the control group. Discussion: These results suggest that exposure to neonicotinoids at low levels in early life affects neural circuit base formation and post-maturation behavior. Therefore, in the central nervous system of male mice, the effects of low-level, chronic neonicotinoids exposure during the perinatal period were different from the expected effects of neonicotinoids exposure in mature animals.

Improving taxonomic classification of marine zooplankton by molecular approach: registration of taxonomically verified 18S and 28S rRNA gene sequences.

Background: Zooplankton plays an important role in the marine ecosystem. A high level of taxonomic expertise is necessary for accurate species identification based on morphological characteristics. As an alternative method to morphological classification, we focused on a molecular approach using 18S and 28S ribosomal RNA (rRNA) gene sequences. This study investigates how the accuracy of species identification by metabarcoding improves when taxonomically verified sequences of dominant zooplankton species are added to the public database. The improvement was tested by using natural zooplankton samples. Methods: rRNA gene sequences were obtained from dominant zooplankton species from six sea areas around Japan and registered in the public database for improving the accuracy of taxonomic classifications. Two reference databases with and without newly registered sequences were created. Comparison of detected OTUs associated with single species between the two references was done using field-collected zooplankton samples from the Sea of Okhotsk for metabarcoding analysis to verify whether or not the newly registered sequences improved the accuracy of taxonomic classifications. Results: A total of 166 sequences in 96 species based on the 18S marker and 165 sequences in 95 species based on the 28S marker belonging to Arthropoda (mostly Copepoda) and Chaetognatha were registered in the public database. The newly registered sequences were mainly composed of small non-calanoid copepods, such as species belonging to Oithona and Oncaea. Based on the metabarcoding analysis of field samples, a total of 18 out of 92 OTUs were identified at the species level based on newly registered sequences in the data obtained by the 18S marker. Based on the 28S marker, 42 out of 89 OTUs were classified at the species level based on taxonomically verified sequences. Thanks to the newly registered sequences, the number of OTUs associated with a single species based on the 18S marker increased by 16% in total and by 10% per sample. Based on the 28S marker, the number of OTUs associated with a single species increased by 39% in total and by 15% per sample. The improved accuracy of species identification was confirmed by comparing different sequences obtained from the same species. The newly registered sequences had higher similarity values (mean >0.003) than the pre-existing sequences based on both rRNA genes. These OTUs were identified at the species level based on sequences not only present in the Sea of Okhotsk but also in other areas. Discussion: The results of the registration of new taxonomically verified sequences and the subsequent comparison of databases based on metabarcoding data of natural zooplankton samples clearly showed an increase in accuracy in species identification. Continuous registration of sequence data covering various environmental conditions is necessary for further improvement of metabarcoding analysis of zooplankton for monitoring marine ecosystems.

Immunohistochemical analysis of the vimentin filaments in Sertoli cells is a powerful tool for the prediction of spermatogenic dysfunction.

The close interaction between male germ cells and Sertoli cells, a type of somatic cell found in the seminiferous tubules of mammalian testis, is essential for the normal progression of spermatogenesis in mammals. Vimentin is an intermediate filament protein that primarily provides mechanical support, preserves cell shape, and maintains the nuclear position, and it is often used as a marker to identify Sertoli cells. Vimentin is known to be involved in many diseases and aging processes; however, how vimentin is related to spermatogenic dysfunction and the associated functional changes is still unclear. In a previous study, we reported that vitamin E deficiency affected the testes, epididymis, and spermatozoa of mice, accelerating the progression of senescence. In this study, we focused on the Sertoli cell marker vimentin and explored the relationship between the cytoskeletal system of Sertoli cells and spermatogenic dysfunction using testis tissue sections that caused male reproductive dysfunction with vitamin E deficiency. The immunohistochemical analysis showed that the proportion of the vimentin-positive area in seminiferous tubule cross-sections was significantly increased in testis tissue sections of the vitamin E-deficient group compared with the proportion in the control group. The histological analysis of testis tissue sections from the vitamin E-deficient group showed that vimentin-positive Sertoli cells were greatly extended from the basement membrane, along with an increased abundance of vimentin. These findings suggest that vimentin may be a potential indicator for detecting spermatogenic dysfunction.

Behavioral effects induced by the oral administration of acetamiprid in male mice during the postnatal lactation period or adulthood.

Acetamiprid (ACE), a neonicotinoid chemical, is widely used as a pesticide due to its rapid insecticidal activity. Although neonicotinoids exert very low toxicity in mammals, the effects of early exposure to neonicotinoids on the adult central nervous system are poorly understood. This study investigated the effects of ACE exposure in early life on brain function in adult mice. We exposed male C57BL/6N mice to ACE (10 mg/kg) orally when they were two (postnatal lactation) or 11 weeks old (adult). We examined the effects of ACE on the central nervous system using the mouse behavioral test battery, consisting of the open field test, light/dark transition test, elevated plus-maze test, contextual/cued fear conditioning test, and pre-pulse inhibition test at 12-13 weeks old. In the mouse behavioral test battery, learning memory abnormalities were detected in the mature treatment group. In addition, learning memory and emotional abnormalities were detected in the postnatal lactation treatment group. These results suggest that the behavioral effects of postnatal lactation treatment with ACE were qualitatively different from the behavioral abnormalities in the mature treatment group.

Exposure to bisphenol A or its phenolic analogs during early life induces different types of anxiety-like behaviors after maturity in male mice.

Products used in daily life contain multiple chemicals capable of inducing endocrine disruption in animals, including humans. One such typical substance is bisphenol A (BPA). BPA has been widely used in epoxy resins and polycarbonate plastics and can exert several adverse effects. Furthermore, given their structural similarity to BPA, phenolic analogs of BPA, i.e., synthetic phenolic antioxidants (SPAs), are considered to exhibit similar toxicity; however, the effects of early SPA exposure on the adult central nervous system remain poorly clarified. In the present study, we aimed to evaluate and compare the neurobehavioral effects of early life exposure to BPA and two selected SPAs, 4,4'-butylidenebis (6-tert-butyl-m-cresol) (BB) and 2,2'-methylenebis (6-tert-butyl-p-cresol) (MB). We exposed mice to low levels of these chemicals through drinking water during prenatal and postnatal periods. Subsequently, we examined the adverse effects of these chemicals on the central nervous system using a mouse behavioral test battery, comprising the open field test, light/dark transition test, elevated plus-maze test, contextual/cued fear conditioning test, and prepulse inhibition test, at 12-13 weeks old. Based on the behavioral analysis, SPAs, like BPA, may cause affective disorders even at low doses, although qualitative differences were noted in anxiety-related behaviors. In conclusion, our findings could be valuable for clarifying the potential adverse developmental risks of SPA exposure in early life.

Development of a non-invasive method for testicular toxicity evaluation using a novel compact magnetic resonance imaging system.

In non-clinical animal studies for drug discovery, histopathological evaluation is the most powerful tool to assess testicular toxicity. However, histological analysis is extremely invasive; many experimental animals are needed to evaluate changes in the pathology and anatomy of the testes over time. As an alternative, small animal magnetic resonance imaging (MRI) offers a non-invasive methodology to examine testicular toxicity without radiation. The present study demonstrated the suitability of a new, ready-to-use compact MRI platform using a high-field permanent magnet to assist with the evaluation of testicular toxicity. To validate the utility of the MRI platform, male mice were treated with busulfan (40 mg/kg, intraperitoneal injection). Twenty-eight days after treatment, both testes in busulfan-treated and control mice (n = 6/group) were non-invasively scanned in situ by MRI at 1 tesla. On a T1-weighted 3D gradient-echo MRI sequences (voxel size: 0.23 × 0.23 × 0.50 mm), the total testicular volume in busulfan-treated mice was significantly smaller than in controls. On T1-weighted images, the signal intensity of the testes was significantly higher in busulfan-treated mice than in controls. The mice were sacrificed, and the testes were isolated for histopathological analysis. The weight of the testes in busulfan-treated mice significantly decreased, similar to the results of the non-invasive analysis. Additionally, periodic acid-Schiff stain-positive effusions were observed in the interstitium of the busulfan-treated mouse testes, potentially explaining T1 shortening due to a high concentration of glycoproteinaceous content. The present data demonstrated a rapid evaluation of testicular toxicity in vivo by compact MRI.

Crocosphaera as a Major Consumer of Fixed Nitrogen.

Crocosphaera watsonii (hereafter referred to as Crocosphaera) is a key nitrogen (N) fixer in the ocean, but its ability to consume combined-N sources is still unclear. Using in situ microcosm incubations with an ecological model, we show that Crocosphaera has high competitive capability both under low and moderately high combined-N concentrations. In field incubations, Crocosphaera accounted for the highest consumption of ammonium and nitrate, followed by picoeukaryotes. The model analysis shows that cells have a high ammonium uptake rate (~7 mol N [mol N]-1 d-1 at the maximum), which allows them to compete against picoeukaryotes and nondiazotrophic cyanobacteria when combined N is sufficiently available. Even when combined N is depleted, their capability of nitrogen fixation allows higher growth rates compared to potential competitors. These results suggest the high fitness of Crocosphaera in combined-N limiting, oligotrophic oceans heightening its potential significance in its ecosystem and in biogeochemical cycling. IMPORTANCE Crocosphaera watsonii is as a key nitrogen (N) supplier in marine ecosystems, and it has been estimated to contribute up to half of oceanic N2 fixation. Conversely, a recent study reported that Crocosphaera can assimilate combined N and proposed that unicellular diazotrophs can be competitors with non-N2 fixing phytoplankton for combined N. Despite its importance in nitrogen cycling, the methods by which Crocosphaera compete are not currently fully understood. Here, we present a new role of Crocosphaera as a combined-N consumer: a competitor against nondiazotrophic phytoplankton for combined N. In this study, we combined in situ microcosm experiments and an ecosystem model to quantitatively evaluate the combined-N consumption by Crocosphaera and other non-N2 fixing phytoplankton. Our results suggest the high fitness of Crocosphaera in combined-N limiting, oligotrophic oceans and, thus, heightens its potential significance in its ecosystem and in biogeochemical cycling.

[Percellome Project: research on molecular mechanisms of toxicological responses based on transcriptomics and epigenetics].

We are constructing the "Percellome Database" containing many transcriptomes of mice exposed to a series of chemicals to elucidate the molecular mechanism of toxicity and to develop toxicity prediction technology. Acute toxicity of a chemical can be predicted to a certain extent by searching the similarity of the transcriptomes obtained by the single-dose exposure experiments. In addition, we are analyzing the relation between the transcriptome and the epigenome i.e. histone modification and genomic DNA methylation to understand the molecular mechanism of the repeated dose toxicity. We are attempting to expand the scale and improve the efficiency of the analysis by introducing artificial intelligence technologies. This approach should maximize the use of toxicogenomics technology for optimizing the experimental protocols for repeated dose toxicity studies towards 3Rs principle, and optimizing the process of in silico toxicity prediction by combining the available big data.

Establishment of a developmental toxicity assay based on human iPSC reporter to detect FGF signal disruption.

The number of man-made chemicals has increased exponentially recently, and exposure to some of them can induce fetal malformations. Because complex and precisely programmed signaling pathways play important roles in developmental processes, their disruption by external chemicals often triggers developmental toxicity. However, highly accurate and high-throughput screening assays for potential developmental toxicants are currently lacking. In this study, we propose a reporter assay that utilizes human-induced pluripotent stem cells (iPSCs) to detect changes in fibroblast growth factor signaling, which is essential for limb morphogenesis. The dynamics of this signaling after exposure to a chemical were integrated to estimate the degree of signaling disruption, which afforded a good prediction of the capacity of chemicals listed in the ECVAM International Validation Study that induce limb malformations. This study presents an initial report of a human iPSC-based signaling disruption assay, which could be useful for the screening of potential developmental toxicants.

Pharmacokinetics of primary metabolites 5-hydroxythalidomide and 5'-hydroxythalidomide formed after oral administration of thalidomide in the rabbit, a thalidomide-sensitive species.

The teratogenicity of the chemotherapeutic drug thalidomide is species-specific and affects humans, non-human primates, and rabbits. The primary oxidation of thalidomide in previously investigated rodents predominantly resulted in the formation of deactivated 5'-hydroxythalidomide. In the current study, similar in vivo biotransformations to 5-hydroxythalidomide and 5'-hydroxythalidomide were confirmed by the analysis of blood plasma from male rabbits, a thalidomide-sensitive species, after oral administration of thalidomide (2.0 mg/kg). Similar levels of thalidomide in seminal plasma and in blood plasma were detected using liquid chromatography-tandem mass spectrometry at 4 hr and 7 hr after oral doses in male rabbits. Seminal plasma concentrations of 5-hydroxythalidomide and 5'-hydroxythalidomide were also seen in male rabbits in a roughly similar time-dependent manner to those in the blood plasma after oral doses of thalidomide (2.0 mg/kg). Furthermore, the values generated by a simplified physiologically based pharmacokinetic rabbit model were in agreement with the measured in vivo blood plasma data under metabolic ratios of 0.01 for the hepatic intrinsic clearance of thalidomide to both unconjugated 5-hydroxythalidomide and 5'-hydroxythalidomide. These results suggest that metabolic activation of thalidomide may be dependent on rabbit liver enzymes just it was for cytochrome P450 enzymes in humanized-liver mice; in contrast, rodent livers predominantly mediate biotransformation of thalidomide to 5'-hydroxythalidomide. A developmental toxicity test system with experimental animals that involves intravaginal exposures to the chemotherapeutic drug thalidomide via semen should be considered in the future.

Visualizing the spatial localization of ciclesonide and its metabolites in rat lungs after inhalation of 1-µm aerosol of ciclesonide by desorption electrospray ionization-time of flight mass spectrometry imaging.

Inhaled ciclesonide (CIC), a corticosteroid used to treat asthma that is also being investigated for the treatment of corona virus disease 2019, hydrolyzes to desisobutyryl-ciclesonide (des-CIC) followed by reversible esterification when exposed to fatty acids in lungs. While previous studies have described the distribution and metabolism of the compounds after inhalation, spatial localization in the lungs remains unclear. We visualized two-dimensional spatial localization of CIC and its metabolites in rat lungs after administration of a single dose of a CIC aerosol (with the mass median aerodynamic diameter of 0.918-1.168 µm) using desorption electrospray ionization-time of flight mass spectrometry imaging (DESI-MSI). In the analysis, CIC, des-CIC, and des-CIC-oleate were imaged in frozen lung sections at high spatial and mass resolutions in negative-ion mode. MSI revealed the coexistence of CIC, des-CIC, and des-CIC-oleate on the airway epithelium, and the distribution of des-CIC and des-CIC-oleate in peripheral lung regions. In addition, a part of CIC independently localized on the airway epithelium. These results suggest that distribution of CIC and its metabolites in lungs is related to both the intended delivery of aerosols to pulmonary alveoli and peripheral regions, and the potential deposition of CIC particles on the airway epithelium.

Effect of vitamin E deficiency on spermatogenesis in mice and its similarity to aging.

Vitamin E (VE) plays numerous important roles in mammals because of its antioxidant activity. As a result, VE deficiency (VED) leads to the dysfunction of central nervous, reproductive, and immune systems. However, few studies have reported the effects of VED on the male reproductive system. In this study, we investigated the effects of VED on male reproductive function and examined its relationship to involution in the male reproductive system with aging. We fed a VED or control diet to 4-week-old mice for 12 or 24 weeks. Following the histopathological analysis of reproductive organs, we found seminiferous tubules with exfoliation in the VED groups, and its frequency was significantly increased compared with the controls. Additionally, in the epididymis, a decrease in spermatozoa and an increase in apoptotic germ cells were observed in the VED groups compared with the controls. By Papanicolaou staining, we also found an increase in the proportion of sperm with abnormal morphology in the VED groups compared with the controls. These reproductive effects induced by VED were highly similar to one aspect of those observed in aged mice. Our findings demonstrate that the aging of the male reproductive system may be accelerated because of the impaired in vivo antioxidant capacity induced by VED.

A Geometric Clustering Tool (AGCT) to robustly unravel the inner cluster structures of time-series gene expressions.

Systems biology aims at holistically understanding the complexity of biological systems. In particular, nowadays with the broad availability of gene expression measurements, systems biology challenges the deciphering of the genetic cell machinery from them. In order to help researchers, reverse engineer the genetic cell machinery from these noisy datasets, interactive exploratory clustering methods, pipelines and gene clustering tools have to be specifically developed. Prior methods/tools for time series data, however, do not have the following four major ingredients in analytic and methodological view point: (i) principled time-series feature extraction methods, (ii) variety of manifold learning methods for capturing high-level view of the dataset, (iii) high-end automatic structure extraction, and (iv) friendliness to the biological user community. With a view to meet the requirements, we present AGCT (A Geometric Clustering Tool), a software package used to unravel the complex architecture of large-scale, non-necessarily synchronized time-series gene expression data. AGCT capture signals on exhaustive wavelet expansions of the data, which are then embedded on a low-dimensional non-linear map using manifold learning algorithms, where geometric proximity captures potential interactions. Post-processing techniques, including hard and soft information geometric clustering algorithms, facilitate the summarizing of the complete map as a smaller number of principal factors which can then be formally identified using embedded statistical inference techniques. Three-dimension interactive visualization and scenario recording over the processing helps to reproduce data analysis results without additional time. Analysis of the whole-cell Yeast Metabolic Cycle (YMC) moreover, Yeast Cell Cycle (YCC) datasets demonstrate AGCT's ability to accurately dissect all stages of metabolism and the cell cycle progression, independently of the time course and the number of patterns related to the signal. Analysis of Pentachlorophenol iduced dataset demonstrat how AGCT dissects data to identify two networks: Interferon signaling and NRF2-signaling networks.

Novel hepatotoxicity biomarkers of extracellular vesicle (EV)-associated miRNAs induced by CCl4.

Recent findings have revealed that extracellular vesicles (EVs) are secreted from cells and circulate in the blood. EVs are classified as exosomes (40-100 nm), microvesicles (50-1,000 nm) or apoptotic bodies (500-2,000 nm). EVs contain mRNAs, microRNAs, and DNAs and have the ability to transfer them from cell to cell. Recently, especially in humans, the diagnostic accuracy of tumor cell type-specific EV-associated miRNAs as biomarkers has been found to be more than 90 %. In addition, microRNAs contained in EVs in blood are being identified as specific biomarkers of chemical-induced inflammation and organ damage. Therefore, microRNAs contained in the EVs released into the blood from tissues and organs in response to adverse events such as exposure to chemical substances and drugs are expected to be useful as novel biomarkers for toxicity assessment. In this study, C57BL/6 J male mice orally dosed with carbon tetrachloride (CCl4) were used as a hepatotoxicity animal model. Here, we report that not only the known hepatotoxicity biomarkers miR-122 and miR-192 but also 42 novel EV-associated biomarkers were upregulated in mice dosed with CCl4. Some of these novel biomarkers may be expected to be able to use for better understanding the mechanism of toxicity. These results suggest that our newly developed protocol using EV-associated miRNAs as a biomarker would accelerate the rapid evaluation of toxicity caused by chemical substances and/or drugs.

CYP3A4 Induction in the Liver and Intestine of Pregnane X Receptor/CYP3A-Humanized Mice: Approaches by Mass Spectrometry Imaging and Portal Blood Analysis.

Induction of cytochrome P450 enzyme 3A (CYP3A) in response to pregnane X receptor (PXR) activators shows species-specific differences. To study the induction of human CYP3A in response to human PXR activators, we generated a double-humanized mouse model of PXR and CYP3A. CYP3A-humanized mice generated by using a mouse artificial chromosome (MAC) vector containing the entire genomic human CYP3A locus (hCYP3A-MAC mouse line) were bred with PXR-humanized mice in which the ligand-binding domain of mouse PXR was replaced with that of human PXR, resulting in double-humanized mice (hCYP3A-MAC/hPXR mouse line). Oral administration of the human PXR activator rifampicin increased hepatic expression of CYP3A4 mRNA and triazolam (TRZ) 1'- and 4-hydroxylation activities, CYP3A probe activities, in the liver and intestine microsomes of hCYP3A-MAC/hPXR mice. The plasma concentration of TRZ after oral dosing was significantly decreased by rifampicin treatment in hCYP3A-MAC/hPXR mice but not in hCYP3A-MAC mice. In addition, mass spectrometry imaging analysis showed that rifampicin treatment increased the formation of hydroxy TRZ in the intestine of hCYP3A-MAC/hPXR mice after oral dosing of TRZ. The plasma concentration of 1'- and 4-hydroxy TRZ in portal blood was also increased by rifampicin treatment in hCYP3A-MAC/hPXR mice. These results suggest that the hCYP3A-MAC/hPXR mouse line may be a useful model to predict human PXR-dependent induction of metabolism of CYP3A4 substrates in the liver and intestine. SIGNIFICANCE STATEMENT: We generated a double-humanized mouse line for CYP3A and PXR. Briefly, CYP3A-humanized mice generated by using a mouse artificial chromosome vector containing the entire genomic human CYP3A locus were bred with PXR-humanized mice in which the ligand-binding domain of mouse PXR was replaced with that of human PXR. Expression of CYP3A4 and metabolism of triazolam, a typical CYP3A substrate, in the liver of CYP3A/PXR-humanized mice were enhanced in response to rifampicin, a typical human PXR activator. Enhancement of triazolam metabolism in the intestine of CYP3A/PXR-humanized mice was firstly shown by combination of mass spectrometry imaging of sliced intestine and liquid chromatography with tandem mass spectrometry analysis of metabolite concentration in portal blood after oral dosing of triazolam.