Single-strand specific nuclease enhances accuracy of error-corrected sequencing and improves rare mutation-detection sensitivity.

Error-corrected sequences (ECSs) that utilize double-stranded DNA sequences are useful in detecting mutagen-induced mutations. However, relatively higher frequencies of G:C > T:A (1 × 10-7 bp) and G:C > C:G (2 × 10-7 bp) errors decrease the accuracy of detection of rare G:C mutations (approximately 10-7 bp). Oxidized guanines in single-strand (SS) overhangs generated after shearing could serve as the source of these errors. To remove these errors, we first computationally discarded up to 20 read bases corresponding to the ends of the DNA fragments. Error frequencies decreased proportionately with trimming length; however, the results indicated that they were not sufficiently removed. To efficiently remove SS overhangs, we evaluated three mechanistically distinct SS-specific nucleases (S1 Nuclease, mung bean nuclease, and RecJf exonuclease) and found that they were more efficient than computational trimming. Consequently, we established Jade-Seq™, an ECS protocol with S1 Nuclease treatment, which reduced G:C > T:A and G:C > C:G errors to 0.50 × 10-7 bp and 0.12 × 10-7 bp, respectively. This was probably because S1 Nuclease removed SS regions, such as gaps and nicks, depending on its wide substrate specificity. Subsequently, we evaluated the mutation-detection sensitivity of Jade-Seq™ using DNA samples from TA100 cells exposed to 3-methylcholanthrene and 7,12-dimethylbenz[a]anthracene, which contained the rare G:C > T:A mutation (i.e., 2 × 10-7 bp). Fold changes of G:C > T:A compared to the vehicle control were 1.2- and 1.3-times higher than those of samples without S1 Nuclease treatment, respectively. These findings indicate the potential of Jade-Seq™ for detecting rare mutations and determining the mutagenicity of environmental mutagens.

Hawk-Seq™ differentiates between various mutations in Salmonella typhimurium TA100 strain caused by exposure to Ames test-positive mutagens.

A precise understanding of differences in genomic mutations according to the mutagenic mechanisms detected in mutagenicity data is required to evaluate the carcinogenicity of environmental mutagens. Recently, we developed a highly accurate genome sequencing method, 'Hawk-Seq™', that enables the detection of mutagen-induced genome-wide mutations. However, its applicability to detect various mutagens and identify differences in mutational profiles is not well understood. Thus, we evaluated DNA samples from Salmonella typhimurium TA100 exposed to 11 mutagens, including alkylating agents, aldehydes, an aromatic nitro compound, epoxides, aromatic amines and polycyclic aromatic hydrocarbons (PAHs). We extensively analysed mutagen-induced mutational profiles and studied their association with the mechanisms of mutagens. Hawk-Seq™ sensitively detected mutations induced by all 11 mutagens, including one that increased the number of revertants by approximately 2-fold in the Ames test. Although the sensitivity for less water-soluble mutagens was relatively low, we increased the sensitivity to obtain high-resolution spectra by modifying the exposure protocol. Moreover, two epoxides indicated similar 6- or 96-dimensional mutational patterns; likewise, three SN1-type alkylating agents indicated similar mutational patterns, suggesting that the mutational patterns are compound category specific. Meanwhile, an SN2 type alkylating agent exhibited unique mutational patterns compared to those of the SN1 type alkylating agents. Although the mutational patterns induced by aldehydes, the aromatic nitro compound, aromatic amines and PAHs did not differ substantially from each other, the maximum total base substitution frequencies (MTSFs) were similar among mutagens in the same structural groups. Furthermore, the MTSF was found to be associated with the carcinogenic potency of some direct-acting mutagens. These results indicate that our method can generate high-resolution mutational profiles to identify characteristic features of each mutagen. The detailed mutational data obtained by Hawk-Seq™ can provide useful information regarding mutagenic mechanisms and help identify its association with the carcinogenicity of mutagens without requiring carcinogenicity data.

Chemical-induced craniofacial anomalies caused by disruption of neural crest cell development in a zebrafish model.

BACKGROUND: Craniofacial anomalies are among the most frequent birth defects worldwide, and are thought to be caused by gene-environment interactions. Genetically manipulated zebrafish simulate human diseases and provide great advantages for investigating the etiology and pathology of craniofacial anomalies. Although substantial advances have been made in understanding genetic factors causing craniofacial disorders, limited information about the etiology by which environmental factors, such as teratogens, induce craniofacial anomalies is available in zebrafish. RESULTS: Zebrafish embryos displayed craniofacial malformations after teratogen treatments. Further observations revealed characteristic disruption of chondrocyte number, shape and stacking. These findings suggested aberrant development of cranial neural crest (CNC) cells, which was confirmed by gene expression analysis of the CNC. Notably, these observations suggested conserved etiological pathways between zebrafish and mammals including human. Furthermore, several of these chemicals caused malformations of the eyes, otic vesicle, and/or heart, representing a phenocopy of neurocristopathy, and these chemicals altered the expression levels of the responsible genes. CONCLUSIONS: Our results demonstrate that chemical-induced craniofacial malformation is caused by aberrant development of neural crest. This study indicates that zebrafish provide a platform for investigating contributions of environmental factors as causative agents of craniofacial anomalies and neurocristopathy.

Genome-wide somatic mutation analysis via Hawk-Seq™ reveals mutation profiles associated with chemical mutagens.

It is difficult to identify mutagen-induced genome-wide somatic mutations using next generation sequencing; hence, mutagenic features of each mutagen and their roles in cancer development require further elucidation. We described Hawk-Seq™, a highly accurate genome sequencing method and the optimal conditions, for using it to construct libraries that would enable the accurate (c.a. 1 error/107-108 bp) and efficient survey of genome-wide mutations. Genomic mutations in gpt delta mice or Salmonella typhimurium TA100 exposed to methylnitrosourea (MNU), ethylnitrosourea (ENU), diethylnitrosamine (DEN), benzo[a]pyrene (BP), and aristolochic acid (AA) were profiled using Hawk-Seq™ to analyse positions, substitution patterns, or frequencies. The resultant vast mutation data provided high-resolution mutational signatures, including for minor mutational fractions (e.g. G:C>A:T by AA), which enabled the clarification of the mutagenic features of all mutagens. The 96-type mutational signatures of MNU, AA, and BP indicate their partial similarity to signature 11, 22, and 4 or 29, respectively. Meanwhile, signatures attributable to ENU and DEN were highly similar to each other, but not to signature 11, suggesting that the mechanisms of these agents differed from those of typical alkylating agents. Thus, Hawk-Seq™ can clarify genome-wide chemical mutagenicity profiles at extraordinary resolutions, thereby providing insight into mutagen mechanisms and their roles in cancer development.

A 90-day repeated-dose toxicity study of dietary alpha linolenic acid-enriched diacylglycerol oil in rats.

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.

Effects of dietary alpha-linolenic acid-enriched diacylglycerol oil on embryo/fetal development in rats.

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.

Central venous catheter-related blood stream infection with pyomyositis due to Stenotrophomonas maltophilia after allogeneic bone marrow transplantation in a patient with aplastic anemia.

Stenotrophomonas maltophilia causes pneumonia and CVC-CRBSI in HSCT. However, there are few reports of pyomyositis due to S. maltophilia. We report a patient with CRBSI and pyomyositis due to S. maltophilia after allogeneic HSCT who was successfully treated by removing the CVC and antibiotics without surgical drainage. Removing the CVC and the combined antibiotics without preventing the neutrophil engraftment could avoid surgical drainage in pyomyositis due to S. maltophilia when detected in an early stage.

Traumatic myopia secondary to ciliary spasm after blunt eye trauma and reconsideration of its pathogenesis.

PURPOSE: To reconsider the pathogenesis of traumatic myopia by describing two cases and literature review. METHODS: Refraction was measured, A-scan ultrasonography was performed, and ultrasound biomicroscopy (UBM) was used to observe the ciliary body in the acute and recovery stages. RESULTS: The first patient had a myopic shift of -6.12 diopters (D) on the initial examination (day 2) compared with the recovered stage. UBM showed ciliochoroidal effusion in one half of the circumference and severe edema in three eighths of the ciliary body, which led to an anterior rotation of the ciliary body. Immediately after the cycloplegia, the myopic shift decreased by -3.9 D. On day 11, the refraction was -0.63 D, and the UBM findings were normalized. The second patient had a myopic shift of -4.38 D on the first examination compared with the recovered stage. UBM showed an annular ciliochoroidal effusion leading to anterior rotation of the ciliary body. Immediately after the cycloplegia, there was a decrease of -2.75 D. Biometric measurements showed an increased anterior chamber depth and a decreased thickness of the lens. On day 16, the refraction was -1.0 D, and the UBM findings were normalized. In both cases, biometric measurements in the acute phase suggested a anterior chamber shallowing, a thickening of the lens, and an anterior shift of the lens-iris diaphragm CONCLUSIONS: Cycloplegia decreased the myopic shift by more than 60 %. These findings indicate a significant contribution of ciliary spasm combined with ciliochoroidal effusion, anterior shift of the lens-iris diaphragm, and thickening of the lens.

Application of testicular organ culture system for the evaluation of spermatogenesis impairment.

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.

Traumatic brain injury inducing swift transition from syndrome of inappropriate antidiuretic hormone secretion to central diabetes insipidus: a case report.

Heavy traumatic brain injury (TBI) may lead to the manifestation of either syndrome of inappropriate secretion of antidiuretic hormones (SIADH) or central diabetes insipidus (CDI). We present a case of TBI where SIADH transformed into CDI within a remarkably short timeframe. A previously healthy 4-yr-old boy was admitted to our hospital with hyponatremia and elevated urinary sodium level on the day following a traumatic head injury. Within 150 min after initiating SIADH treatment, a significant increase in urine volume and a decrease in urinary sodium levels were observed. Therefore, the treatment plan was modified to include desmopressin. By the 5th day of admission, the urine volume gradually stabilized and normalized without the need for further desmopressin treatment. Mild TBI can give rise to various conditions that may undergo rapid changes. Closely monitoring serum and urine electrolytes, along with urine volume, is imperative for the administration of appropriate and timely treatment.

The reconstructed skin micronucleus assay in EpiDerm™: reduction of false-positive results - a mechanistic study with epigallocatechin gallate.

The high rate of false-positive or misleading results in in vitro mammalian genotoxicity testing is a hurdle in the development of valuable chemicals, especially those used in cosmetics, for which in vivo testing is banned in the European Union. The reconstructed skin micronucleus (RSMN) assay in EpiDerm™ (MatTek Corporation, USA) has shown promise as a follow-up for positive in vitro mammalian genotoxicity tests. However, few studies have explored its better predictive performance compared with existing in vitro assays. In the present study, we followed the protocol of the RSMN assay and used eight chemicals to compare micronucleus (MN) induction with EpiDerm™ with that in normal human epidermal keratinocytes (NHEKs), both derived from human skin. The assessments of EpiDerm™ conformed to those of in vivo MN assay, whereas those of NHEKs did not. The effect of cell differentiation status on MN induction was further addressed using a model compound, epigallocatechin gallate (EGCG), which is a major component of green tea extract that shows positive results in in vitro mammalian genotoxicity assays via oxidative stress and negative results in in vivo MN studies. RSMN assay in an underdeveloped epidermal model, EpiDerm-201™ (MatTek Corporation), showed a negative result identical to that in EpiDerm™, indicating that the barrier function of keratinocytes has limited impact. Analysis of the gene expression profile of both EpiDerm™ and NHEKs after EGCG treatment for 12h revealed that the expression of genes related to genotoxic response was significantly induced only in NHEKs. Conversely, antioxidative enzyme activities (catalase and glutathione peroxidase) in EpiDerm™ were higher than those in NHEKs. These results indicate that EpiDerm™ has antioxidant properties similar to those of a living body and is capable of eliminating oxidative stress that may be caused by EGCG under in vitro experimental conditions.

A case with transient refractive change after removal of pituitary tumor.

BACKGROUND: Refractive change can be caused by systemic illnesses such as Lupus erythematosus, thyroid deficiency, and diabetes mellitus. However, refractive change after pituitary tumor removal has so far not been reported. CASE PRESENTATION: A 62-year-old woman presented with blurred near vision 10 days after trans-sphenoidal surgery (TSS) for a pituitary tumor. Around the same time, she experienced intercurrent hyponatremia. The corrected visual acuity of both eyes was 20/20, the spherical equivalent of the right eye was -2.125 diopters, and of the left eye was -2.0 diopters before TSS. However, 11 days after TSS, the spherical equivalent of the right eye changed to -0.75 diopters, and that of left eye changed to -1.125 diopters without hyperglycemia. There were no changes in the corrected visual acuity during the follow-up. CONCLUSION: We demonstrated a case with transient refractive change after TSS. The following mechanism is proposed: Hyponatremia induced by the pituitary tumor removal causes an osmotic change in the aqueous humor with lens swelling. This case report is a reminder to both ophthalmologists and neurosurgeons that ophthalmological factors such as lens thickness and axial length should be taken into account when conducting preoperative examinations especially for patients undergoing TSS.

Identification of an adverse outcome pathway (AOP) for chemical-induced craniofacial anomalies using the transgenic zebrafish model.

Craniofacial anomalies are one of the most frequent birth defects worldwide and are often caused by genetic and environmental factors such as pharmaceuticals and chemical agents. Although identifying adverse outcome pathways (AOPs) is a central issue for evaluating the teratogenicity, the AOP causing craniofacial anomalies has not been identified. Recently, zebrafish has gained interest as an emerging model for predicting teratogenicity because of high throughput, cost-effectiveness and availability of various tools for examining teratogenic mechanisms. Here, we established zebrafish sox10-EGFP reporter lines to visualize cranial neural crest cells (CNCCs) and have identified the AOPs for craniofacial anomalies. When we exposed the transgenic embryos to teratogens that were reported to cause craniofacial anomalies in mammals, CNCC migration and subsequent morphogenesis of the first pharyngeal arch were impaired at 24 hours post-fertilization. We also found that cell proliferation and apoptosis of the migratory CNCCs were disturbed, which would be key events of the AOP. From these results, we propose that our sox10-EGFP reporter lines serve as a valuable model for detecting craniofacial skeletal abnormalities, from early to late developmental stages. Given that the developmental process of CNCCs around this stage is highly conserved between zebrafish and mammals, our findings can be extrapolated to mammalian craniofacial development and thus help in predicting craniofacial anomalies in human.

Multidrug resistance-associated protein 2 (MRP2) is an efflux transporter of EGCG and its metabolites in the human small intestine.

Green tea polyphenols have various beneficial effects on human health, such as antiobesity and anti-carcinogenesis. (-)-Epigallocatechin-gallate (EGCG) is one of the major potent green tea catechins; however, detailed mechanisms of EGCG transport and metabolism in the human small intestine remain unknown due to lack of a suitable model. We investigated metabolite profiles of EGCG in the fresh human duodenal biopsy, cryopreserved human duodenal mucosal enterocytes and Caco-2 cells, and found that EGCG was readily metabolized into methylated and sulphate conjugates, which are major metabolites in these models. Next, we examined possible efflux transporters of EGCG and its metabolites using specific inhibitors of MRP2, P-gp and BCRP in Caco-2 cell monolayers. MRP2 was thereby identified as an efflux transporter, and further analysis using MRP2-knockout Caco-2 cells and vesicular transport assays confirmed that MRP2 is a selective efflux transporter of EGCG and its metabolites. Assuming that functional inhibition of MRP2 would result in efficient uptake of EGCG, we screened for MRP2 functional blockade and identified quercetin, which led to increased intracellular accumulation and basal transport of EGCG in Caco-2 cells. This result suggested that co-administration of quercetin and EGCG would enable efficient transport of EGCG in the human intestine. Therefore, we performed co-oral administration of quercetin and EGCG in human subjects to examine whether this occurred in humans. These studies demonstrated that MRP2 is a selective transporter of EGCG and conjugates and Caco-2 is a model to examine transport mechanisms and metabolites of polyphenols in the human small intestine.

Unexpectedly Large Contribution of Oxygen to Charge Compensation Triggered by Structural Disordering: Detailed Experimental and Theoretical Study on a Li3NbO4-NiO Binary System.

Dependence on lithium-ion batteries for automobile applications is rapidly increasing. The emerging use of anionic redox can boost the energy density of batteries, but the fundamental origin of anionic redox is still under debate. Moreover, to realize anionic redox, many reported electrode materials rely on manganese ions through π-type interactions with oxygen. Here, through a systematic experimental and theoretical study on a binary system of Li3NbO4-NiO, we demonstrate for the first time the unexpectedly large contribution of oxygen to charge compensation for electrochemical oxidation in Ni-based materials. In general, for Ni-based materials, e.g., LiNiO2, charge compensation is achieved mainly by Ni oxidation, with a lower contribution from oxygen. In contrast, for Li3NbO4-NiO, oxygen-based charge compensation is triggered by structural disordering and σ-type interactions with nickel ions, which are associated with a unique environment for oxygen, i.e., a linear Ni-O-Ni configuration in the disordered system. Reversible anionic redox with a small hysteretic behavior was achieved for LiNi2/3Nb1/3O2 with a cation-disordered Li/Ni arrangement. Further Li enrichment in the structure destabilizes anionic redox and leads to irreversible oxygen loss due to the disappearance of the linear Ni-O-Ni configuration and the formation of unstable Ni ions with high oxidation states. On the basis of these results, we discuss the possibility of using σ-type interactions for anionic redox to design advanced electrode materials for high-energy lithium-ion batteries.

Chemical-Induced Cleft Palate Is Caused and Rescued by Pharmacological Modulation of the Canonical Wnt Signaling Pathway in a Zebrafish Model.

Cleft palate is one of the most frequent birth defects worldwide. It causes severe problems regarding eating and speaking and requires long-term treatment. Effective prenatal treatment would contribute to reducing the risk of cleft palate. The canonical Wnt signaling pathway is critically involved in palatogenesis, and genetic or chemical disturbance of this signaling pathway leads to cleft palate. Presently, preventative treatment for cleft palate during prenatal development has limited efficacy, but we expect that zebrafish will provide a useful high-throughput chemical screening model for effective prevention. To achieve this, the zebrafish model should recapitulate cleft palate development and its rescue by chemical modulation of the Wnt pathway. Here, we provide proof of concept for a zebrafish chemical screening model. Zebrafish embryos were treated with 12 chemical reagents known to induce cleft palate in mammals, and all 12 chemicals induced cleft palate characterized by decreased proliferation and increased apoptosis of palatal cells. The cleft phenotype was enhanced by combinatorial treatment with Wnt inhibitor and teratogens. Furthermore, the expression of tcf7 and lef1 as a readout of the pathway was decreased. Conversely, cleft palate was prevented by Wnt agonist and the cellular defects were also prevented. In conclusion, we provide evidence that chemical-induced cleft palate is caused by inhibition of the canonical Wnt pathway. Our results indicate that this zebrafish model is promising for chemical screening for prevention of cleft palate as well as modulation of the Wnt pathway as a therapeutic target.

Progression of glaucomatous optic neuropathy associated with chorioretinal microvascular embolism after intranasal injection of a corticosteroid suspension.

A patient with glaucoma developed sudden blurred vision immediately after the nasal mucosal injection of a betamethasone acetate solution into the inferior turbinate. The fundus examination revealed several white emboli in the choroidal vessels of the temporal region of the optic disc. After vigorous massage, her visual acuity recovered from counting fingers to 20/32. Six days after the initial examination, Goldmann perimetry showed expansion of the superior and inferior arcuate scotomas. In this case, temporary ischemia of the central retinal and short posterior ciliary arteries involving the arterial circle of Zinn-Haller led to the deterioration of the preexisting glaucomatous optic neuropathy.

In silico systems for predicting chemical-induced side effects using known and potential chemical protein interactions, enabling mechanism estimation.

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.

Safety Pharmacological Evaluation of the Coffee Component, Caffeoylquinic Acid, and Its Metabolites, Using Ex Vivo and In Vitro Profiling Assays.

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.