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Stab Wound Injury Model of the Adult Optic Tectum Using Zebrafish and Medaka for the Comparative Analysis of Regenerative Capacity

While zebrafish have a superior capacity to regenerate their central nervous system (CNS), medaka has a lower CNS regenerative capacity. A brain injury ...

National Institute of Advanced Industrial Science and Technology

Repeated-dose toxicity (RDT) studies are one of the critical studies to assess chemical safety. There have been some studies attempting to predict RDT endpoints based on chemical substructures, but it remains very difficult to establish such a method, and a more detailed characterization of chemical compounds seems necessary. Cytochrome P450s (P450s) comprise multiple forms with different substrate specificities and play important roles in both the detoxification and metabolic activation of xenobiotics. In this study, we investigated possible use of P450 reactivity of chemical compounds to classify the compounds. A total of 148 compounds with available rat RDT test data were used as test compounds and subjected to inhibition assays against 18 human and rat P450s. Among the tested compounds, 82 compounds inhibited at least one P450 form. Hierarchical clustering analyses using the P450 inhibitory profiles divided the 82 compounds into nine groups, some of which showed characteristic chemical and biological properties. Principal component analyses of the P450 inhibition data in combination with the calculated chemical descriptors demonstrated that P450 inhibition data were plotted differently than most chemical descriptors in the loading plots. Finally, association analyses between P450 inhibition and RDT endpoints showed that some endpoints related to the liver, kidney and hematology were significantly associated with the inhibition of some P450s. Our present results suggest that the P450 reactivity profiles can be used as novel descriptors for characterizing chemical compounds for the investigation of the toxicity mechanism and/or the establishment of a toxicity prediction model.

Application of cytochrome P450 reactivity on the characterization of chemical compounds and its association with repeated-dose toxicity.

The central nervous system (CNS) of adult zebrafish is capable of recovering from injury, unlike the CNS of mammals such as humans or rodents. Previously, we established a stab wound injury model of the optic tectum (OT) in the adult zebrafish and showed that the radial glial cells (RG) proliferation and neuronal differentiation contributes to OT regeneration. In the present study, we analyzed the function of histone deacetylases (HDACs) as potential regulators of OT regeneration. The expression of both hdac1 and hdac3 was found to be significantly decreased in the injured OT. In order to analyze the roles of HDACs in RG proliferation and differentiation after injury, we performed pharmacological experiments using the HDAC inhibitor trichostatin A. We found that HDAC inhibition after stab wound injury suppressed RG proliferation but promoted neuronal differentiation. Moreover, HDAC inhibition suppressed the injury-induced decline in expression of Notch signaling target genes, her4.1 and her6 after OT injury. These results suggest that HDACs regulate regenerative neurogenesis through changes in Notch target gene expression by histone deacetylation. HDACs and histone acetylation are promising molecular targets for neuronal regeneration and further studies about the molecular mechanisms behind the regulation of regeneration by histone acetylation are necessary.

Histone deacetylase inhibition promotes regenerative neurogenesis after stab wound injury in the adult zebrafish optic tectum.

Drug-induced liver injury (DILI) is one of the major causes for the discontinuation of drug development and withdrawal of drugs from the market. Since it is known that reactive metabolite formation and being substrates or inhibitors of cytochrome P450s (P450s) are associated with DILI, we systematically investigated the association between human P450 inhibition and DILI. The inhibitory activity of 266 DILI-positive drugs (DILI drugs) and 92 DILI-negative drugs (no-DILI drugs), which were selected from Liver Toxicity Knowledge Base (US Food and Drug Administration), against 8 human P450 forms was assessed using recombinant enzymes and luminescent substrates, and the threshold values showing the highest balanced accuracy for DILI discrimination were determined for each P450 enzyme using receiver operating characteristic analyses. The results showed that among the P450s tested, CYP1A1 and CYP1B1 were inhibited by DILI drugs more than no-DILI drugs with a statistical significance. We found that 91% of drugs that showed inhibitory activity greater than the threshold values against CYP1A1 or CYP1B1 were DILI drugs. The results of internal 5-fold cross-validation confirmed the usefulness of CYP1A1 and CYP1B1 inhibition data for the threshold-based discrimination of DILI drugs. Although the contribution of these P450s to drug metabolism in the liver is considered minimal, our present findings suggest that the assessment of CYP1A1 and CYP1B1 inhibition is useful for screening DILI risk of drug candidates at the early stage of drug development.

Association of CYP1A1 and CYP1B1 inhibition in in vitro assays with drug-induced liver injury.

Adult zebrafish have many neurogenic niches and a high capacity for central nervous system regeneration compared to mammals, including humans and rodents. The majority of radial glia (RG) in the zebrafish optic tectum are quiescent under physiological conditions; however, stab wound injury induces their proliferation and differentiation into newborn neurons. Although previous studies have functionally analyzed the molecular mechanisms of RG proliferation and differentiation and have performed single-cell transcriptomic analyses around the peak of RG proliferation, the cellular response and changes in global gene expression during the early stages of tectum regeneration remain poorly understood. In this study, we performed histological analyses which revealed an increase in isolectin B4+ macrophages prior to the induction of RG proliferation. Moreover, transcriptome and pathway analyses based on differentially expressed genes identified various enriched pathways, including apoptosis, the innate immune system, cell proliferation, cytokine signaling, p53 signaling, and IL6/Jak-Stat signaling. In particular, we found that Stat3 inhibition suppressed RG proliferation after stab wound injury and that IL6 administration into cerebroventricular fluid activates RG proliferation without causing injury. Together, the findings of these transcriptomic and functional analyses reveal that IL6/Stat3 signaling is an initial trigger of RG activation during optic tectum regeneration.

Transcriptome Analyses Reveal IL6/Stat3 Signaling Involvement in Radial Glia Proliferation After Stab Wound Injury in the Adult Zebrafish Optic Tectum.

Zebrafish have a greater capacity for adult neurogenesis and brain regeneration than mammals. In the adult zebrafish optic tectum (OT), neuroepithelial-like stem cells (NE) contribute to adult neurogenesis, whereas radial glia (RG) contribute to neuronal regeneration after the stab wound injury. The molecular mechanisms regulated by acetylated histone play important roles in these events; however, the functions of histone acetyltransferase (HAT) require further elucidation. The aim of this study was to study the proliferation and differentiation of neural stem cells (NSCs) following treatment with C646, a HAT EP300 inhibitor, to identify the functions of HAT in adult neurogenesis and neuronal regeneration. C646 treatment decreased acetylation of histone 3 lysine 9 in the adult OT. Under physiological conditions, C646 promoted NE proliferation and generation of newborn neurons. EP300 inhibition promoted RG proliferation but suppressed the generation of newborn neurons after the injury. EP300 inhibition downregulated the Notch target genes her4 and her6, which was correlated with NE and RG proliferation in the adult OT. EP300 inhibition regulates the proliferation and differentiation of NSCs by inhibiting histone acetylation and Notch target genes expression, suggesting that the functions of HAT in neurogenesis are opposite to those of histone deacetylase.

Histone acetyltransferase EP300 regulates the proliferation and differentiation of neural stem cells during adult neurogenesis and regenerative neurogenesis in the zebrafish optic tectum.

Drug-induced liver injury (DILI) is one of major causes of discontinuing drug development and withdrawing drugs from the market. In this study, we investigated chemical properties associated with DILI using in silico methods, to identify a physicochemical property useful for DILI screening at the early stages of drug development. Total of 652 drugs, including 432 DILI-positive drugs (DILI drugs) and 220 DILI-negative drugs (no-DILI drugs) were selected from Liver Toxicity Knowledge Base of US Food and Drug Administration. Decision tree models were constructed using 2,473 descriptors as explanatory variables. In the final model, the descriptor AMW, representing average molecular weight, was found to be at the first node and showed the highest importance value. With AMW alone, 276 DILI drugs (64%) and 156 no-DILI drugs (71%) were correctly classified. Discrimination with AMW was then performed using therapeutic category information. The performance of discrimination depended on the category and significantly high performance (>0.8 balanced accuracy) was obtained in some categories. Taken together, the present results suggest AMW as a novel descriptor useful for detecting drugs with DILI risk. The information presented may be valuable for the safety assessment of drug candidates at the early stage of drug development.

Identification of average molecular weight (AMW) as a useful chemical descriptor to discriminate liver injury-inducing drugs.

Zebrafish have superior regenerative capacity in the central nervous system (CNS) compared to mammals. In contrast, medaka were shown to have low regenerative capacity in the adult heart and larval retina, despite the well-documented high tissue regenerative ability of teleosts. Nevertheless, medaka and zebrafish share similar brain structures and biological features to those of mammals. Hence, this study aimed to compare the neural stem cell (NSC) responses and regenerative capacity in the optic tectum of adult medaka and zebrafish after stab wound injury. Limited neuronal differentiation was observed in the injured medaka, though the proliferation of radial glia (RG) was induced in response to tectum injury. Moreover, the expression of the pro-regenerative transcriptional factors ascl1a and oct4 was not enhanced in the injured medaka, unlike in zebrafish, whereas expression of sox2 and stat3 was upregulated in both fish models. Of note, glial scar-like structures composed of GFAP radial fibers were observed in the injured area of medaka at 14 days post injury (dpi). Altogether, these findings suggest that the adult medaka brain has low regenerative capacity with limited neuronal generation and scar formation. Hence, medaka represent an attractive model for investigating and evaluating critical factors for brain regeneration.

Differential Regenerative Capacity of the Optic Tectum of Adult Medaka and Zebrafish.

Anemia is a well-observed toxicity of chemical substances, and aniline is a typical anemia-inducing substance. However, it remains unclear whether all aniline-like substances with various substituents could induce anemia. We thus investigated the physicochemical characteristics of anemia-inducing substances by decision tree analyses. Training and validation substances were selected from a publicly available database of rat repeated-dose toxicity studies, and discrimination models were constructed by decision tree and bootstrapping methods with molecular descriptors as explanatory variables. To improve the accuracy of discrimination, we individually evaluated the explanatory variables to modify them, established "prerules" that were applied before subjecting a substance to a decision tree by considering metabolism, such as azo reduction and N-dealkylation, and introduced the idea of "partly negative" evaluation for substances having multiple aniline-like substructures. The final model obtained showed 79.2% and 77.5% accuracy for the training and validation dataset, respectively. In addition, we identified some chemical properties that reduce the anemia inducibility of aniline-like substances, including the addition of a sulfonate or carboxy functional group and/or a bulky multiring structure to anilines. In conclusion, the present findings will provide a novel insight into the mechanistic understanding of chemically induced anemia and help to develop a prediction system.

Chemical characterization of anemia-inducing aniline-related substances and their application to the construction of a decision tree-based anemia prediction model.

Experimental animals are indispensable in life science-related research, including cancer studies. After rats and mice, small fishes, such as zebrafish and medaka, are the second most frequently used model species. Fish models have some advantageous physical characteristics that make them suitable for research, including their small size, some transparency, genetic manipulability, ease of handling, and highly ortholog correspondence with humans. This review introduces technological advances in carcinogenesis model production using small fish. Carcinogenesis model production begins with chemical carcinogenesis, followed by mutagenesis. Gene transfer technology has made it possible to incorporate various mechanisms that act on cancer-related genes in individuals. For example, scientists may now spatiotemporally control gene expression in a single fish through methods including the localization of an expression site via a tissue-specific promoter and expression control using light, heat, or a chemical substance. In addition, genome editing technology is realizing more specific and more efficient gene disruption than conventional mutagenesis, in which the disruption of the gene of interest depends on chance. These technological advances have improved animal models and will soon create carcinogenesis models that better mimic human pathology. We conclude by discussing future expectations for cancer research using small fish.

Carcinogenesis Models Using Small Fish.

Radial glial cells (RGCs) play an essential role in developing, maintaining, and repairing the central nervous system (CNS). However, a specific reporter line of RGCs is limited in medaka. Glial fibrillary acid protein (GFAP) is abundant in teleost CNS, including the brain and spinal cord, and is a possible candidate for a marker for RGCs in medaka CNS. We generated a transgenic medaka in which enhanced green fluorescent protein (EGFP) expression is regulated under putative medaka gfap regulatory elements. We observed EGFP expression in the CNS of live larval and juvenile medaka through the transparent body of the See-through medaka strain. Histological analysis for juvenile and adult Tg(gfap:EGFP) medaka showed that EGFP was expressed in GFAP-positive cells in the telencephalon, optic tectum, retina, and spinal cord. We further found another EGFP expressing cells in the optic tectum and retina. These cells are possibly neuroepithelial-like stem cells, deducing from the distribution of these EGFP-positive cells. We concluded that this reporter line would be valuable in the investigation of neural stem cell function during the development and regeneration of medaka CNS visualizing two types of neural stem cells, RGCs and neuroepithelial-like stem cells.

Article	Year
Application of cytochrome P450 reactivity on the characterization of chemical compounds and its association with repeated-dose toxicity. Toxicology and applied pharmacology\| PubMed ID: 31836524	2020
Histone deacetylase inhibition promotes regenerative neurogenesis after stab wound injury in the adult zebrafish optic tectum. Biochemical and biophysical research communications\| PubMed ID: 32703437	2020
Association of CYP1A1 and CYP1B1 inhibition in in vitro assays with drug-induced liver injury. The Journal of toxicological sciences\| PubMed ID: 33814510	2021
Transcriptome Analyses Reveal IL6/Stat3 Signaling Involvement in Radial Glia Proliferation After Stab Wound Injury in the Adult Zebrafish Optic Tectum. Frontiers in cell and developmental biology\| PubMed ID: 33996824	2021
Histone acetyltransferase EP300 regulates the proliferation and differentiation of neural stem cells during adult neurogenesis and regenerative neurogenesis in the zebrafish optic tectum. Neuroscience letters\| PubMed ID: 34023416	2021
Identification of average molecular weight (AMW) as a useful chemical descriptor to discriminate liver injury-inducing drugs. PloS one\| PubMed ID: 34170966	2021
Differential Regenerative Capacity of the Optic Tectum of Adult Medaka and Zebrafish. Frontiers in cell and developmental biology\| PubMed ID: 34268310	2021
Chemical characterization of anemia-inducing aniline-related substances and their application to the construction of a decision tree-based anemia prediction model. Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association\| PubMed ID: 34509582	2021
Carcinogenesis Models Using Small Fish. Chemical & pharmaceutical bulletin\| PubMed ID: 34602577	2021
Gfap transgenic medaka as a novel reporter line for neural stem cells. Gene\| PubMed ID: 35104578	2022

Yuki Shimizu

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