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Chrysin is known to exert anti-inflammatory, antioxidant, and anticancer effects. The aim of this study was to investigate the anticancer effects of chrysin in the human melanoma cells A375SM and A375P. The results obtained demonstrated successful inhibition of the viability of these cells by inducing apoptosis and autophagy. This was confirmed by the level of apoptosis-related proteins: Bax and cleaved poly (ADP-ribose) polymerase both increased, and Bcl-2 decreased. Moreover, levels of LC3 and Beclin 1, both autophagy-related proteins, increased in chrysin-treated cells. Autophagic vacuoles and acidic vesicular organelles were observed in both cell lines treated with chrysin. Both cell lines showed different tendencies during chrysin-induced autophagy inhibition, indicating that autophagy has different effects depending on the cell type. In A375SM, the early autophagy inhibitor 3-methyladenine (3-MA) was unaffected; however, cell viability decreased when treated with the late autophagy inhibitor hydroxychloroquine (HCQ). In contrast, HCQ was unaffected in A375P; however, cell viability increased when treated with 3-MA. Chrysin also decreased the phosphorylation of mTOR/S6K pathway proteins, indicating that this pathway is involved in chrysin-induced apoptosis and autophagy for A375SM and A375P. However, studies to elucidate the mechanisms of autophagy and the action of chrysin in vivo are still needed.

Apigenin, an aromatic compound, exhibits antioxidant, anti‑inflammatory and anti‑viral effects. The present study aimed to investigate the effects of apigenin on cell proliferation and apoptosis of human melanoma cells A375P and A375SM. Therefore, melanoma cells were treated with apigenin to determine its anti‑proliferative and survival effects, using wound healing and MTT assays. The results revealed that melanoma cell viability was decreased in a dose‑dependent manner. Furthermore, chromatin condensation, indicating apoptosis, was significantly increased in a dose‑dependent manner, as demonstrated by DAPI staining. In addition, increased apoptosis rate following treatment with apigenin was confirmed by Annexin V‑propidium iodide staining. The changes in the expression levels of apoptosis‑related proteins in A375P and A375SM melanoma cells were subsequently detected using western blot analysis. The results demonstrated that the protein expression levels of Bcl‑2 were decreased, whereas those of Bax, cleaved poly ADP‑ribose polymerase, cleaved caspase‑9 and p53 were upregulated in a dose‑dependent manner in apigenin‑treated cells compared with those noted in untreated cells. In addition, in apigenin‑treated A375P cells, phosphorylated (p)‑p38 was upregulated and p‑extracellular signal‑regulated kinase (ERK), p‑c‑Jun N‑terminal kinase (JNK) and p‑protein kinase B (Akt) were downregulated. However, in A375SM cells, apigenin treatment increased p‑ERK and p‑JNK and decreased p‑p38 and p‑Akt protein expression levels. Subsequently, the inhibitory effect of apigenin on tumor growth was investigated in vivo. Tumor volume was significantly reduced in the 25 and 50 mg/kg apigenin‑treated groups compared with the control group. Additionally, a TUNEL assay was performed to detect apoptotic cells. Immunohistochemical staining also revealed elevated p‑ERK expression in the apigenin‑treated group compared with the control group. Overall, the findings of the present study indicated that apigenin attenuated the growth of A375SM melanoma cells by inducing apoptosis via regulating the Akt and mitogen‑activated protein kinase signaling pathways.

Oleanolic acid (OA) is widely distributed in food and medicinal plants, it reportedly exerts anti-inflammatory and anti-cancer effects. In this study, we investigated the effect of OA on human gastric cancer cells AGS in vitro and in vivo. The OA treatment significantly inhibited the AGS cell viability. Apoptosis was confirmed via annexin V/PI staining and 4′,6-diamidino-2-phenylindole (DAPI) staining. The results from western blotting revealed that treatment with OA affected apoptosis-related protein. Meanwhile, OA induced autophagy, characterized by the formation of autophagic vacuoles and acidic vesicular organelles, also increased autophagy-related protein. Inhibition of autophagy further reduced cell proliferation. Moreover, OA treatment decreased phosphorylation of PI3K/AKT/mTOR pathway proteins. Finally, we found that OA reduced tumor volume and weight in xenograft mice via apoptosis without side effects. Overall, our study provides experimental evidence for the anti-cancer action of OA and suggests the possibility of its use as an adjuvant in gastric cancer therapy.

SummaryThis study was performed to improve production efficiency at the level of recipient pig and donor nuclei of transgenic cloned pigs used for xenotransplantation. To generate transgenic pigs, human endothelial protein C receptor (hEPCR) and human thrombomodulin (hTM) genes were introduced using the F2A expression vector into GalT–/–/hCD55+ porcine neonatal ear fibroblasts used as donor cells and cloned embryos were transferred to the sows and gilts. Cloned fetal kidney cells were also used as donor cells for recloning to increase production efficiency. Pregnancy and parturition rates after embryo transfer and preimplantation developmental competence were compared between cloned embryos derived from adult and fetal cells. Significantly higher parturition rates were shown in the group of sows (50.0 vs. 4.1%), natural oestrus (20.8 vs. 0%), and ovulated ovary (16.7 vs. 5.6%) compared with gilt, induced and non-ovulated, respectively (P < 0.05). When using gilts as recipients, final parturitions occurred in only the fetal cell groups and significantly higher blastocyst rates (15.1% vs. 21.3%) were seen (P < 0.05). Additionally, gene expression levels related to pluripotency were significantly higher in the fetal cell group (P < 0.05). In conclusion, sows can be recommended as recipients due to their higher efficiency in the generation of transgenic cloned pigs and cloned fetal cells also can be recommended as donor cells through correct nuclear reprogramming.

Despite a major role of experimental animals in development of biomedical research, there has been historical controversy surrounding animal research. Along with a strategy of 3Rs, various in vitro methods have been suggested to replace potentially painful animal experiments. In this review, we summarize the use of stem cells as an alternative of animal experimentation in predictive toxicology. There have been continuing researches on stem cells and stem cell-derived tissue-specific cells to develop alternative methods/biomarkers for animal toxicity testing including developmental toxicity, genotoxicity, and tissue-specific toxicity. Along with unique abilities of stem cells including self-renewal, infinite proliferation, and differentiation into multiple lineages, human stem cell-based in vitro systems have been proven valuable to increase predictive power of toxicology through providing with better scientific information related to toxic risks in humans without inter-species variability. In particular, stem cells including induced pluripotent stem cell-based system for personalized toxicological assessment could be a better option as an in vitro model system in comparison with immortalized cells with abnormal phenotype or primary cells with small quantity and batch-to-batch variation. This review will be useful for understanding the current status and future direction in using stem cells as an alternative non-animal method for predictive toxicology.

The worldwide restricted use of animal testing makes it challenging to identify the skin sensitizing potentials of newly manufactured products. The HaCaSens assay has shown promise as an in vitro skin sensitizing assay comparable to existing assays, and is currently under pre-validation. However, there is little agreement on how to assess the results of the assay to discriminate sensitizers from non-sensitizers as the stimulation index (SI) cutoff value was arbitrarily chosen without appropriate statistical methods. Here, we investigated the SI cutoff values in identifying sensitizers to obtain the optimal value. Sensitivities and specificities were calculated for a set of 30 test substances, and plotted in receiver operator characteristics (ROC) curves. The SI cutoff values with the highest sum of sensitivity and specificity according to LLNA data were 2.2, 1.8 and 3.0 for interleukin 1α (IL-1α), interleukin 6 (IL-6), and the combination of the two cytokines respectively. Also, the same statistical analysis of human data demonstrated optimal SI cutoff values 2.0, 2.0 and 3.2 for the same respective parameters. When considering the predictive capacity of each possible SI cutoff value determined by ROC curves, the optimal value for HaCaSens is 3.0 for the combination of IL-1α and IL-6 as it had the highest sensitivity (90.9%), specificity (75.0%) and accuracy (86.7%) based on LLNA data. Thus, we recommend the wide use of the SI cutoff value of 3.0 to ensure consistent endpoints.

Gap junctional intercellular communication (GJIC) is considered a key biological mechanism to maintain homeostasis in cell differentiation and growth. In addition, as another major signaling pathway associated with cell proliferation and differentiation, Wnt/β-catenin signaling appears to trigger several cellular responses against injury. The purpose of the present study was to investigate the effects of a known toxic agent, benzo[a]pyrene (BaP), on the regulation and interaction between GJIC and Wnt/β-catenin signaling. BaP treatment resulted in GJIC inhibition and decreases the major GJIC protein connexin 43 (Cx43) in WB-F344 rat liver epithelial cells. We also found BaP-mediated downregulation of Wnt/β-catenin signaling related to the PI3K-Akt pathway. To identify the relationship between GJIC and Wnt/β-catenin signaling, we treated WB-F344 cells with the Wnt agonist CHIR99021 and found that it inhibited GJIC while causing a significant reduction in Cx43 expression at both the mRNA and protein levels, through the repression of promoter activity. This Wnt agonist-mediated GJIC inhibition was confirmed using a small interfering RNA directed against the Wnt antagonist Dact2, indicating that Wnt/β-catenin signaling negatively regulates GJIC. Despite the inverse correlation between Wnt/β-catenin signaling and Cx43 promoter activation as indicated by downregulation of β-catenin nuclear translocation and upregulation of Cx43 promoter activation involving HNF3β, BaP treatment decreased the Cx43 protein expression, which was associated with protein degradation, possibly through protein kinase C activation. In conclusion, our results revealed the mechanism of BaP-induced inhibition of GJIC and Wnt/β-catenin signaling. More importantly, linking Wnt/β-catenin signaling to Cx protein expression will have profound implications in understanding the relationships among different major signaling pathways associated with cell proliferation and differentiation in toxicity.

IntroductionSystemic histaminergic activity is elevated in patients with diabetes mellitus. There are a few studies suggesting that histamine is implicated in the pathogenesis of diabetes, but the exact role of histamine in the development of diabetic retinopathy is unclear. The aim of this study was to investigate the role of histamine receptor H4 (HRH4) in the regulation of retinal pigment epithelium (RPE)-derived pro-angiogenic and anti-angiogenic factors under diabetic conditions.Research design and methodsThe levels of vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), histamine and histidine decarboxylase (HDC) in the serum and vitreous samples of patients with diabetes were compared with those of patients without diabetes. The effect of hyperglycemia on expression levels of HRH4, VEGF, IL-6 and pigment epithelium-derived factor (PEDF) in the RPE was determined. The role of HRH4 in high glucose-induced regulation of VEGF, IL-6 and PEDF in ARPE-19 cells and the underlying regulatory mechanism were verified using an RNA interference-mediated knockdown study.ResultsThe serum and vitreous levels of VEGF, IL-6, histamine and HDC were more increased in patients with diabetic retinopathy than in patients without diabetes. HRH4 was overexpressed in RPE both in vitro and in vivo. Histamine treatment upregulated VEGF and IL-6 and downregulated PEDF expression in ARPE-19 cells cultivated under hyperglycemic conditions. Hyperglycemia-induced phosphorylation of p38 and subsequent upregulation of VEGF and IL-6 and downregulation of PEDF were dampened by small interfering RNA-mediated knockdown of HRH4 in ARPE-19 cells.ConclusionsTaken together, HRH4 was a critical regulator of VEGF, IL-6 and PEDF in the RPE under hyperglycemic conditions and the p38 mitogen-activated protein kinase pathway mediated this regulatory mechanism.

Background Citrus sunki Hort. ex Tanaka peel has been traditionally used as an ingredient in folk medicine due to its therapeutic effects on promotion of splenic health and diuresis as well as relief of gastrointestinal symptoms. Although a growing interest in health-promoting natural products and the development of highly concentrated products have facilitated consumption of C. sunki peel, its safety assessment has not been explored, posing a potential health risk. In this study, we carried out a series of systemic and genetic toxicity tests on fermented C. sunki peel extract (FCPE) to provide the essential information required for safe use in human. Methods We conducted acute and 90-day repeated oral toxicity studies in Sprague-Dawley rats to evaluate systemic toxicity, and three genotoxicity assays to measure bacterial mutation reversion, cellular chromosome aberration and in vivo micronucleus formation. Results Single oral administration of FCPE did not cause any clinical signs and lethality in all animals, establishing LD50 to be over 2000 mg/kg BW. Repeated administration of up to 2000 mg/kg BW FCPE for 90 days revealed no test substance-related toxicity as demonstrated in analysis of body weight gain, food/water intake, blood, serum biochemistry, organ weight and histopathology, collectively determining that the no-observable-adverse-effect-level of FCPE is over 2000 mg/kg BW. In addition, we detected no mutagenicity and clastogenicity in FCPE at 5000 μg/plate for the in vitro assays and 2000 mg/kg BW for the in vivo micronucleus test. Conclusion FCPE did not cause systemic and genetic toxicity in our model systems at the tested dose levels. These results suggest a guideline for safe consumption of C. sunki peel in human.

Cinnamomum cassia has been widely used as a natural product to treat diseases in Asia due to its diverse pharmacological functions including anti-inflammatory, anti-oxidant, anti-microbial, anti-diabetic, and anti-tumor effects. Despite its ethnomedicinal benefits, little information regarding its toxicity is currently available. The aim of this study was to evaluate its potential long-term toxicity and genotoxicity in compliance with test guidelines of the Organization for Economic Cooperation and Development. A 13-week repeat-dose oral toxicity study revealed that body weights of rats were normal after receiving cinnamon extract at up to 2000 mg/kg. High-dose intake of cinnamon extract (2000 mg/kg) showed potential nephrotoxicity and hepatotoxicity to both males and females as evidenced by obvious increases of kidney/liver weight along with a small but statistically elevation of total cholesterol level. Overall findings from genetic toxicity testing battery including Ames test, in vitro mammalian cell micronucleus assay, and in vivo bone marrow micronucleus assay indicated that cinnamon extract was not mutagenic or clastogenic. In conclusion, cinnamon extract may possess potential nephrotoxicity and hepatotoxicity at dose higher than its recommended daily safe dose. Further study is needed to clarify the mechanism involved in its induction of liver and kidney injury.

Due to considerable constraints in using animals for risk assessment, much effort has been directed at developing non-animal test methods. Developing assays for skin sensitization, the leading cause of contact dermatitis, is particularly important, but there are currently no in vitro skin sensitization tests that completely replace animal tests. HaCaSens, a simple skin sensitization test using non-transformed HaCaT cells, predicts keratinocyte activation by skin sensitizers with 75% sensitivity, 83% specificity and 77% accuracy in a previous study using 22 coded substances. Although the data show promising results, the number of tested substances is insufficient to prove predictive capacity. Moreover, reproducibility among different laboratories has not been studied. Here, three laboratories participated in a validation in order to assess HaCaSens feasibility for official validation. To examine transferability, intra- and inter-lab reproducibility and predictive capacity, HaCaSens was assessed on a set of 30 test substances coordinated by the Validation Management Team (VMT). The results showed satisfactory transferability as well as intra- and inter-laboratory reproducibility. Further assessment of its predictive capacity on 20 test substances demonstrated a sensitivity of 81.8% (18/22), specificity of 87.5% (7/8), and accuracy of 83.3% (25/30) in identifying skin sensitizers, which is comparable with presently validated assays, KeratinoSens™ and LuSens. This validation study shows that the HaCaSens assay is easily transferable, reproducible and highly predictable for identifying skin sensitizers.

Artemisia capillaris (AC) has been used as an alternative therapy in obesity, atopic dermatitis, and liver diseases through several biological activity including anti-steatotic, antioxidant, and anti-inflammatory activities. Despite its ethnomedicinal benefits, no sufficient background information is available about the long-term safety and genotoxicity of the AC extract. Therefore, the present study was carried out to investigate the 13-week subchronic toxicity and genotoxicity of the AC extract according to the test guidelines published by the Organization for Economic Cooperation and Development. In the 13-week toxicity study using doses of 25, 74, 222, 667, and 2000 mg/kg body weight, oral administration of the AC extract in male and female rats did not result in any significant adverse effects in food/water consumption, body weight, mortality, hematology, serum biochemistry, organ weight and histopathology. Accordingly, the no-observed-adverse-effect level in rats of both genders was established for the AC extract at 2000 mg/kg/day, the highest dose level tested. In addition, the AC extract was not genotoxic in a battery of tests including Ames test, in vitro chromosome aberration assay and in vivo micronucleus assay. In conclusion, we demonstrated that the AC extract is considered as a safe traditional medicine for human consumption.

Clinical cisplatin use is often limited by its drug-induced liver injury (DILI). Particularly, individual differences in susceptibility to DILI can cause life-threatening medical conditions. This study aimed to uncover the inherent genetic factors determining individual variations in hepatotoxicity susceptibility. Rats were subjected to liver biopsy and a 3-week postoperative recovery period before cisplatin administration. At 2 days post-treatment with cisplatin, the rats exhibited histopathological and serum biochemical alterations in the liver, and changes in hydrogen peroxide and cytochrome P450-2E1 levels. Based on these results of liver-related biochemical markers, 32 rats were grouped into the susceptible (top five) and resistant (bottom five) groups. Using RNA-sequencing, we compared gene expressions in the liver pre-biopsied from these two groups before cisplatin treatment and found 161 differently expressed genes between the Susceptible and Resistant groups. Among them, the clock-controlled Ccrn4l responsible for ‘rhythmic process’ was identified as a common gene downregulated inherently prior to drug exposure in both cisplatin- and acetaminophen-sensitive animals. Additionally, low Ccrn4l levels before cisplatin treatment in the Susceptible group were maintained even after treatment, with decreased antioxidants, increased nitration, and apoptosis. The relationship of Ccrn4l with catalase and mitochondrial RNAs in the liver was confirmed by correlation of their hepatic levels among individuals and similar patterns of circadian variation in their mRNA expression. Remarkably, Ccrn4l knockdown promoted cisplatin-induced mitochondrial dysfunction in WB-F344 cells with antioxidant catalase and apoptosis-related Bax changes. Inherent individual hepatic Ccrn4l level might be a novel factor affecting cisplatin-induced hepatotoxicity susceptibility, possibly through regulation of mitochondrial and antioxidant functions.

Sodium taurodeoxycholate (TDCA) has been investigated for various inflammatory disorders such as sepsis. We recently evaluated nonclinical safety profile of TDCA using rats infused intravenously. As a series of preclinical safety investigations, we further conducted toxicity studies with TDCA delivered to dogs

Background/aim Piperine is a major pungent alkaloid present in black pepper (Piper nigrum L). This study investigated the potential anticancer effects of piperine on human melanoma cells and explored the potential pharmacological mechanisms in vitro and in vivo. Materials and methods Studies were performed using the MTT assay, 4',6-diamidino-2-phenylindole (DAPI) staining, western blotting, a xenograft model, the terminal deoxynucleotidyl transferase dUTP nick end labeling assay, and immunohistochemistry. Results Piperine inhibited the growth of melanoma cells. Several apoptotic events were observed following treatment, as revealed by DAPI staining. Piperine increased the expression of BCL2-associated X, apoptosis regulator (BAX), cleaved poly(ADP-ribose)polymerase, cleaved caspase-9, phospho-c-Jun N-terminal kinase and phospho-p38, and reduced that of B-cell lymphoma 2 (BCL2), X-chromosome-linked inhibitor of apoptosis, and phospho-extracellular signal-regulated protein kinase (ERK1/2) in a concentration-dependent manner. Treatment of mice for 4 weeks with piperine inhibited tumor growth without apparent toxicity. Piperine increased the expression of apoptotic cells and cleaved-caspase-3 protein and reduced the expression of phospho-ERK1/2 protein in melanoma tumors. Conclusion Piperine suppressed the growth of human melanoma cells by the induction of apoptosis via the inhibition of tumor growth of human melanoma cells and tumor xenograft models.

Taurodeoxycholate (TDCA) inhibits various inflammatory responses suggesting potential clinical application. However, the toxicity of TDCA has not been evaluated in detail in vivo. We investigated the acute toxicity and 4-week repeated-dose toxicity of TDCA following intravenous infusion under Good Laboratory Practice regulations. In the sighting study of acute toxicity, one of two rats (one male and one female) treated with 300 mg/kg TDCA died with hepatotoxicity, suggesting that the approximate 50% lethal dose of TDCA is 300 mg/kg. Edema and discoloration were observed at the injection sites of tails when rats were infused with 150 mg/kg or higher amount of TDCA once. In 4-week repeated-dose toxicity study, no treatment-related mortality or systemic changes in hematology and serum biochemistry, organ weights, gross pathology, or histopathology were observed. However, the tail injection site showed redness, discharge, hardening, and crust formation along with histopathological changes such as ulceration, edema, fibrosis, and thrombosis when rats were infused with 20 mg/kg TDCA. Taken together, TDCA induced no systemic toxicity or macroscopic lesions at the injection site at a dose of 10 mg/kg/day, which is 33 times higher than the median effective dose observed in a mouse sepsis model. These findings suggest that TDCA might have a favorable therapeutic index in clinical applications.

In vitro testing methods for classifying sensitizers could be valuable alternatives to in vivo sensitization testing using animal models, such as the murine local lymph node assay (LLNA) and the guinea pig maximization test (GMT), but there remains a need for in vitro methods that are more accurate and simpler to distinguish skin sensitizers from non-sensitizers. Thus, the aim of our study was to establish an in vitro assay as a screening tool for detecting skin sensitizers using the human keratinocyte cell line, HaCaT. HaCaT cells were exposed to 16 relevant skin sensitizers and 6 skin non-sensitizers. The highest dose used was the dose causing 75% cell viability (CV75) that we determined by an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. The levels of extracellular production of interleukin-1α (IL-1α) and IL-6 were measured. The sensitivity of IL-1α was 63%, specificity was 83% and accuracy was 68%. In the case of IL-6, sensitivity: 69%, specificity: 83% and accuracy: 73%. Thus, this study suggests that measuring extracellular production of pro-inflammatory cytokines IL-1α and IL-6 by human HaCaT cells may potentially classify skin sensitizers from non-sensitizers. Copyright © 2015 John Wiley & Sons, Ltd.

In vivo animal studies are required by regulatory agencies to investigate drug safety before clinical trials. In this review, we summarize the process of selecting a relevant non-rodent species for preclinical studies. The dog is the primary, default non-rodent used in toxicology studies with multiple scientific advantages, including adequate background data and availability. Rabbit has many regulatory advantages as the first non-rodent for the evaluation of reproductive and developmental as well as local toxicity. Recently, minipigs have increasingly replaced dogs and rabbits in toxicology studies due to ethical and scientific advantages including similarity to humans and breeding habits. When these species are not relevant, nonhuman primates (NHPs) can be used as the available animal models, especially in toxicology studies investigating biotherapeutics. Particularly, based on the phylogenetic relationships, the use of New-World marmosets can be considered before Old-World monkeys, especially cynomolgus with robust historical data. Importantly, the use of NHPs should be justified in terms of scientific benefits considering target affinity, expression pattern, and pharmacological cross-reactivity. Strict standards are required for the use of animals. Therefore, this review is helpful for the selection of appropriate non-rodent in regulatory toxicology studies by providing sufficient regulatory, ethical, and scientific data for each species.

Background Seed of mature Croton tiglium Linne, also known as Tiglium seed (TS), has been widely used as a natural product due to its several health beneficial properties including anti-tumor and antifungal activities. Despite its ethnomedicinal beneficial properties, toxicological information regarding TS extract, especially its long-term toxicity, is currently limited. Therefore, the objective of the present study was to evaluate acute and subchronic toxicity of TS extract in rats after oral administration following test guidelines of the Organization for Economic Cooperation and Development (OECD). Methods Toxicological properties of TS extract were evaluated by toxicity assays to determine its single-dose acute toxicity (125, 250, 500, 1000, or 2000 mg/kg), 14-day repeated-dose toxicity (125, 250, 500, 1000, or 2000 mg/kg) and 13-week repeated-dose toxicity (31.25, 62.5, 125, 250, and 500 mg/kg) in Sprague-Dawley rats and F344 rats. Hematological, serum biochemical, and histopathological parameters were analyzed to determine its median lethal dose (LD50) and no-observed-adverse-effect-level (NOAEL). Results Oral single dose up to 2000 mg/kg of TS extract resulted in no mortalities or abnormal clinical signs. In 13-week toxicity study, TS extract exhibited no dose-related changes (mortality, body weight, food/water consumption, hematology, clinical biochemistry, organ weight, or histopathology) at dose up to 500 mg/kg, the highest dosage level suggested based on 14-day repeat-dose oral toxicity study. Conclusion Acute oral LD50 of TS extract in rats was estimated to be greater than 2000 mg/kg. NOAEL of TS extract administered orally was determined to be 500 mg/kg/day in both male and female rats. Results from these acute and subchronic toxicity assessments of TS extract under Good Laboratory Practice regulations indicate that TS extract appears to be safe for human consumption. Electronic supplementary material The online version of this article (10.1186/s12906-018-2315-5) contains supplementary material, which is available to authorized users.

Organ weights of male and female F344 rats orally administered with Tiglium seed extract for 14Â days. (DOCX 30 kb)

Summary incidence of gross findings of F344 rats orally administered with Tiglium seed extract for 13Â weeks. (DOCX 18 kb)

Summary incidence of gross findings of F344 rats orally administered with Tiglium seed extract for 14Â days. (DOCX 17 kb)

Flavonols are compounds that have been shown to possess potent anti‑inflammatory effects in cellular and animal models of inflammation. In the present study, the anti‑inflammatory effects and mechanisms of two natural flavonols, quercetin and galangin, in lipopolysaccharide (LPS)‑stimulated RAW264.7 macrophages were investigated. It was identified that quercetin and galangin markedly reduced the production of nitric oxide (NO), inducible NO synthase and interleukin‑6, and the nuclear translocation of nuclear factor‑κB (NF‑κB). In addition, LPS‑induced activation of extracellular signal‑regulated kinase 1/2 (Erk1/2) and c‑Jun N‑terminal kinase (JNK) was suppressed by quercetin and galangin. Taken together, these data implied that NF‑κB, Erk1/2 and JNK may be potential molecular targets of quercetin and galangin in an LPS‑induced inflammatory response. Subsequently, the effects of oral administration of quercetin or galangin, either alone or in combination, in a 2,4‑dinitrochlorobenzene‑induced atopic dermatitis (AD) mouse model were investigated. As a result, measurements of ear thickness and the levels of serum immunoglobulin E, and histological analysis revealed that the two flavonols led to a decrease in inflammation, whereas, in combination, they were even more effective. These results suggested that quercetin and galangin may be promising therapeutic agents for AD. Additionally, their combination may be a novel therapeutic strategy for the prevention of AD.

Human adipose tissue-derived mesenchymal stem cells (hATMSCs) have great potential as a therapy for various diseases. However, emerging evidence shows that there are conflicting results concerning effects of hATMSCs on tumor progression. Our objective was to determine whether and how hATMSCs modulate tumor growth. After cancer cell lines were subcutaneously inoculated into BALB/c-nude and hairless severe combined immunodeficient mice, hATMSCs were intratumorally injected into the mice. The growth of the A549 tumors was inhibited by hATMSCs, yet that of the HT-29 tumors was significantly promoted by hATMSCs in the in vivo xenograft models. In vitro study using a co-culture system of cancer cells and hATMSCs was consistent with the in vivo experiments. To reveal the molecular events induced by hATMSCs in the xenograft models, global gene expression profiles of the A549 and HT-29 tumors in the absence or presence of hATMSCs were determined. Significant numbers of genes involved in biological processes were altered in the hATMSC-treated A549 tumors, whereas no biological process was regulated by treatment with hATMSCs in the HT-29 tumors, reflecting the different effects of hATMSCs in the different types of cancer. Notably, histone cluster 1, H2aj and neuropeptide Y receptor Y4 were found to be expressed in direct or inverse proportion to tumor size in both xenograft models. In addition, nuclear factor κB (NF-κB) p65 was differentially phosphorylated by the hATMSCs dependent on the source of the cancer cells. In conclusion, the identified gene profiling and NF-κB signaling provide molecular evidence to explain the conflicting findings in tumor‑MSC studies, although further study is needed to confirm these findings using various types of cancer.

As a well-known traditional medicine, Angelica gigas (AG) and its active constituents, including decursin and decursinol, have been shown to possess several health beneficial properties such as anti-bacterial, immunostimulating, anti-tumor, neuroprotective, anti-nociceptive and anti-amnestic activities. However, there is lack of toxicity studies to assess potential toxicological concerns, especially long-term toxicity and genotoxicity, regarding the AG extract. Therefore, the safety of AG extract was assessed in subchronic toxicity and genotoxicity assays in accordance with the test guidelines published by the Organization for Economic Cooperation and Development. In a subchronic toxicity study for 13 weeks (125, 250, 500, 1000 and 2000 mg/kg body weight, delivered by gavage), data revealed no significant adverse effects of the AG extract in food consumption, body weight, mortality, hematology, biochemistry, necropsy, organ weight and histopathology throughout the study in male and female rats. These results suggest that no observed adverse effect level of the AG extract administered orally was determined to be greater than 2000 mg/kg/day, the highest dose tested. In addition, a battery of tests including Ames test, in vitro chromosome aberration assay and in vivo micronucleus assay suggested that the AG extract was not genotoxic. In conclusion, the AG extract appears to be safe as a traditional medicine for oral consumption.

Although silicon dioxide (SiO2), silver (Ag) and iron oxide (Fe2O3) nanoparticles are widely used in diverse applications from food to biomedicine, in vivo toxicities of these nanoparticles exposed via the oral route remain highly controversial. To examine the systemic toxicity of these nanoparticles, well-dispersed nanoparticles were orally administered to Sprague-Dawley rats daily over a 13-week period. Based on the results of an acute toxicity and a 14-day repeated toxicity study, 975.9, 1030.5 and 1000 mg kg(-1) were selected as the highest dose of the SiO2 , Ag and Fe2O3 nanoparticles, respectively, for the 13-week repeated oral toxicity study. The SiO2 and Fe2O3 nanoparticles did not induce dose-related changes in a number of parameters associated with the systemic toxicity up to 975.9 and 1000 mg kg(-1) , respectively, whereas the Ag nanoparticles resulted in increases in serum alkaline phosphatase and calcium as well as lymphocyte infiltration in liver and kidney, raising the possibility of liver and kidney toxicity induced by the Ag nanoparticles. Compared with the SiO2 and Fe2O3 nanoparticles showing no systemic distribution in all tissues tested, the Ag concentration in sampled blood and organs in the Ag nanoparticle-treated group significantly increased with a positive and/or dose-related trend, meaning that the systemic toxicity of the Ag nanoparticles, including liver and kidney toxicity, might be explained by extensive systemic distribution of Ag originating from the Ag nanoparticles. Our current results suggest that further study is required to identify that Ag detected outside the gastrointestinal tract were indeed a nanoparticle form or ionized form.

Therapeutic cancer vaccines promote immune responses by delivering tumour-specific antigens. Recently, we developed iron oxide (Fe3O4)–zinc oxide (ZnO) core-shell nanoparticles (CSNPs) as carriers for antigen delivery into dendritic cells (DCs), and the CSNPs were injected subcutaneously into C57BL/6 mice to examine the systemic toxicity, tissue distribution and excretion of the CSNPs. The doses injected were 0, 4, 20 and 200 mg kg–1 weekly for 4 weeks. No significant changes were observed after the CSNPs administration with respect to mortality, clinical observations, body weight, food intake, water consumption, urinalysis, haematology, serum biochemistry,and organ weights. A dose-dependent increase in granulomatous inflammation was observed at the injection site of the CSNP-treated animals, but no other histopathological lesions in other organs could be attributed to the CSNPs. The Zn concentration, which is an indicator for CSNPs, was not significantly higher in the sampled tissues, urine, or faeces after the CSNP injection. In contrast, the Zn concentration at the subcutaneous skin of the site injected with the CSNPs increased in a dose-dependent manner, along with a macroscopic deposition of the CSNPs. The CSNP residue at the injection site resulted in a foreign body response with the appearance of macrophage infiltration, but otherwise did not show any systemic distribution or toxicity at up to 200 mg kg–1 during this study. In conclusion, CSNPs could be used as good antigen carriers for DC-based immunotherapy, although further study is needed to completely clear the residue of the CSNPs at the injection site. Copyright © 2015 John Wiley & Sons, Ltd.

Paecilomyces tenuipes is entomogenous fungus that is called snow-flake Dongchunghacho in Korea. Although it is widely used in traditional medicines, its safety has not yet been comprehensively investigated. Therefore, the aim of this study was to evaluate the genotoxicity, acute and subchronic toxicity of P. tenuipes . The acute oral LD 50 of P. tenuipes extract in rats was estimated to be greater than 2000 mg/kg of body weight. In the subchronic study, the oral treatment of rats with 500, 1000 or 2000 mg/kg P. tenuipes extract daily for 13 weeks did not induce any dose-related changes (body weight, food consumption, clinical observation, urinalysis, hematology, clinical chemistry and organ weight). In contrast, histopathological observation revealed that P. tenuipes extract induced karyomegaly in outer medulla of kidney in all treated rats. Importantly, P. tenuipes extract exerted the mutagenic potential in Ames assay. Since karyomegalic alterations have been known to be associated with carcinogenicity, our finding on the mutagenicity of P. tenuipes extract supports the possibility on the potential involvement of P. tenuipes in carcinogenicity at least partially. In conclusion, the subchronic oral exposure of P. tenuipes may induce kidney abnormality at the concentration higher than 500 mg/kg body weight, although further studies using other animal models are needed to identify the toxicity of P. tenuipes .

Ecklonia cava (EC) is known to have antioxidant, anti-inflammatory, antidiabetic, and anticancer properties. Despite its wide use and beneficial properties, comprehensive toxicological information regarding EC extract is currently limited. Therefore, the purpose of this study was to investigate acute toxicity, subchronic toxicity, and genotoxicity of enzymatic EC extract according to test guidelines published by Organization for Economic Cooperation and Development. The acute oral LD50 values of this EC extract administered to rats and dogs were estimated to be more than 3000 mg/kg BW. In an oral 13-week toxicity study, changes in body weights of rats exposed to the EC extract up to 3000 mg/kg BW were found to be normal. In addition, repeated doses of EC extract failed to influence any systematic parameters of treatment-related toxic symptoms such as food/water consumption, mortality, urinalysis, hematology, serum biochemistry, organ weight, or histopathology. These results indicated that the no-observed-adverse-effect level for the EC extract was 3000 mg/kg/day for male and female rats. Data obtained from Ames test, chromosome aberration assay, and micronucleus assay indicated that EC extract was not mutagenic or clastogenic. Taken together, these results support the safety of enzymatic EC extract as a potential therapeutic for human consumption against various diseases.

Among three representative species of Angelica found in Asian countries, including Korea, China, and Japan, Angelica acutiloba (AA) has been used as traditional herbal medicine with antitumor, anti-inflammatory, anti-obesity, and anti-diabetes activities. In this study, the potential genotoxicity and mutagenicity of the AA extract were examined in a battery of in vitro and in vivo tests (bacterial reverse mutation assay, in vitro chromosomal aberrations assay, and in vivo micronucleus assay) in accordance with the test guidelines for toxicity testing developed by the Organization for Economic Cooperation and Development. Upon testing in the bacterial mutation assay (Ames test) using five Salmonella typhimurium TA98, TA100, TA102, TA1535 and TA1537, no significant increase the number of revertant colonies in the metabolic activation system and non-activation system was noted in the AA extract groups. Also, in the chromosome aberration test, the AA extract did not cause chromosomal aberration with or without metabolic activation by S9 mix. A bone marrow micronucleus test of mice demonstrated that the incidence of micronucleated polychromatic erythrocytes in the AA extract groups (500, 1000 and 2000 mg/kg BW) was equivalent to that of the negative control group. Based on these results from a standard battery of assays, the AA extract was concluded to have no genotoxic at the proper dose.

Koji products have been considered as an effective fermented food consumed in East Asia with many health benefits. Particularly, rice koji with Aspergillus terreus (RAT) has been reported to be able to prevent hyperlipidemia and hepatic steatosis through regulating cholesterol synthesis. Despite its biological activities, there is a lack of comprehensive information to give an assurance of its safety. Therefore, the objective of this study was to perform a series of toxicological studies (repeated dose oral toxicity and genotoxicity) according to test guidelines published by the Organization for Economic Cooperation and Development. Along with acute toxicity study using rats and beagle dogs, a 13-week toxicity study revealed no clear RAT-related toxic changes, including body weight, mortality, hematology, serum biochemistry, organ weight, and histopathology after oral administration at doses of 500, 1000, and 2000 mg/kg BW. The no-observed-adverse-effect level of RAT was considered to be more than 2000 mg/kg BW/day in rats of both genders. In addition, potential genotoxicity was evaluated using a standard battery of tests (Ames test, chromosome aberration assay, and micronucleus assay) which revealed that RAT showed no genotoxicity. Accordingly, these results suggest that RAT is a safe and non-toxic functional food for human consumption at proper dose.

Ixeris dentata (Thunb. Ex Thunb.) Nakai (ID) exhibits various physiological activities, and its related plant derived-products are expected to represent promising cancer therapeutic agents. However, the anticancer effects of ID extract on breast cancer cells classified as estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) are still unknown. In this study, we investigated the anti-cancer effects and analyzed the molecular mechanism of ID extract in T47D, MCF-7 (ER-, PR-positive, HER2-negative), SK-BR-3(ER-, PR-negative, HER2-positive), and MDA-MB-231 (Triple-negative) through in vitro studies. Additionally, we examined its anti-tumor effects through in vivo studies. Our findings indicated that ID extract-induced apoptosis was mediated via various survival pathways on four breast cancer cells by identifying the factors including Bcl-2 family, phospho-Akt and phospho-nuclear factor-κB (NF-κB). Based on in vitro findings that induced apoptosis via Akt-NF-κB signaling, we investigated the effects of ID extract on mice bearing MDA-MB-231 cells. The results showed that ID extract significantly decreased MDA-MB-231 tumor volume and weight via inducing apoptosis by suppressing phospho-Akt. Overall, these results indicate that ID extract induces apoptosis through the Akt-NFκB signaling pathway in MDA-MB-231 breast cancer cells and tumors, and it may serve as a therapeutic agent for triple-negative human breast cancer.Academic Editor: Terrence Piva

Ethnopharmacology relevance Dendropanax morbifera Leveille (DM) has been used in traditional medicines for infectious and skin diseases, and dysmenorrhea. It exhibits a diverse therapeutic potential including anti-cancer, anti-thrombotic, anti-diabetic, anti-oxidant, and anti-inflammatory activities. Aim of the study Despite promising health benefits of DM, knowledge of its potential adverse effects is very limited. The current study focused on the investigation of subchronic toxicity and genotoxicity of extract obtained from DM according to the test guidelines published by the Organization for Economic Cooperation and Development. Materials and methods We conducted a toxicological evaluation of DM extracts using 14-day repeated-dose toxicity study and 13-week repeated-dose toxicity study in Sprague-Dawley rats administered orally at doses of 500, 1000, or 2000 mg/kg/day. The clastogenicity of DM extract was also evaluated by in vitro chromosome aberration assay and in vivo micronucleus assay. Results Assessment of subchronic toxicity of DM extract by oral administration in rats revealed unremarkable treatment-related findings with respect to food/water consumption, body weight, mortality, urinalysis, hematology, serum biochemistry, necropsy, organ weight and histopathology at doses of 500, 1000, and 2000 mg/kg. Accordingly, the level of no-observed-adverse-effect for DM extract in 13-week subchronic toxicity study was considered to be 2000 mg/kg/day in rats. The data observed from in vitro chromosome aberration assay and in vivo micronucleus assay exclude any clastogenicity of DM extract. Conclusion The results suggest that the oral consumption of DM extract has no adverse effects in humans and represents a safe traditional medicine.

In polymeric nanoparticle preparation, despite similar conditions, large fluctuations in particle size distributions are usually observed. Herein, we demonstrate that the intermittent addition of a desolvating agent can improve reproducibility in the preparation of polymeric bovine serum albumin (BSA) nanoparticles. Using this modification, BSA nanoparticles of controlled size can be manufactured with narrow particle size distributions. In our study, ethanol as a desolvating agent was added intermittently to 1% BSA solutions at different pHs with stirring at 700rpm. The effect of the preparation parameters on size and optical density of the fabricated nanoparticles were studied. The average particles sizes of BSA nanoparticles prepared at pH values of 6, 7 and 9 were approximately 100, 200 and 300nm, respectively. As ethanol addition increased, desolvation of BSA molecules resulted in formation of loose-structured particles with pH-dependent size. Beyond that, only particle density increased, but size remained unchanged with further addition of ethanol. Consistently uniform particle size distribution was achieved by adding ethanol intermittently.

Zinc oxide (ZnO) nanoparticles (NPs) are used in diverse applications ranging from paints and cosmetics to biomedicine and food. Although micron-sized ZnO is a traditional food supplement, ZnO NPs are an unknown public health risk because of their unique physicochemical properties. Herein, we studied the 13-week subchronic toxicity of ZnO NPs administered via the oral route according to Organization for Economic Cooperation and Development (OECD) test guideline 408. Well-dispersed ZnO NPs were administered to Sprague–Dawley (SD) rats (11/sex/group) at doses of 67.1, 134.2, 268.4 or 536.8 mg kg–1 per body weight over a 13-week period. The mean body weight gain in males given 536.8 mg kg–1 ZnO NPs was significantly lower than that of control male rats, whereas no significant differences were observed between the other treatment groups and the controls. Male and female rats dosed at 536.8 mg kg–1 ZnO NPs had significant changes in anemia-related hematologic parameters. Mild to moderate pancreatitis also developed in both sexes dosed at 536.8 mg kg–1, whereas no histological changes were observed in the other treatment groups. To evaluate the mechanism of toxicity, we performed a bio-persistence study and evaluated the effects of the ZnO NPs on cell proliferation. The treatment of a human gastric adenocarcinoma cell line with ZnO NPs resulted in a significant inhibition of cellular proliferation. The anti-proliferative effect of ZnO NPs or Zn2+ was effectively blocked by treatment with chelators. These results indicate that the bio-persistence of ZnO NPs after ingestion is key to their toxicity; the no-observed-adverse effect level (NOAEL) of ZnO NPs was found to be 268.4 mg kg–1 per day for both sexes. Copyright © 2013 John Wiley & Sons, Ltd.

As hydrophilic pigments, anthocyanins reduce the incidence of cancer and cardiovascular diseases. In this study, the anthocyanin content and composition following steaming and baking of the roots of the Korean purple-fleshed sweet potato variety “Shinzami” were evaluated using liquid chromatography with diode array detection and electrospray ionisation/mass spectrometry (LC-DAD-ESI/MS). Anthocyanins of Shinzami were composed of mono- or di-acylated forms of p-hydroxybenzoic acid, caffeic acid and ferulic acid with the basic structure of cyanidin 3-sophoroside-5-glucoside or peonidin 3-sophoroside-5-glucoside. A total of 15 individual anthocyanins were isolated and confirmed, one of which was presumed to be a newly identified compound, peonidin 3-feruloyl-p-hydroxybenzoyl sophoroside-5-glucoside. Additionally, the amounts of di-acylated cyanidin 3-caffeoyl-p-hydroxybenzoyl sophoroside-5-glucoside and peonidin 3-caffeoyl-p-hydroxybenzoyl sophoroside-5-glucoside were the highest (137.0 and 565.9 mg/100 g DW, respectively) among cyanidin and peonidin compounds. After steaming, the total anthocyanin content was reduced by nearly a half, while roasting only slightly reduced the total anthocyanin content.

Endothelin (ET)-1 and its receptors (ETA and ETB receptor) are present in the central nervous system. ET exerts biological effects on gliogenesis and glial cell functions. In order to define a possible mechanism of ETA receptor signaling, the distribution of the ETA receptor in developing oligodendrocytes and the effects of ET-1 on the myelination of oligodendrocytes were examined. ETA receptor immunoreactivity was confined to the perivascular elements of the blood vessels during early postnatal development. However later in development, ETA receptor immunoreactivity was no longer observed in the vessels but became localized to the myelinating oligodendrocytes of the primitive corpus callosum of the white matter, apart from the vessels. ET-1 induced myelin basic protein (MBP) in primary oligodendrocyte precursor cell culture though the ETA receptor and was blocked by an ETA receptor antagonist. In addition, ET-1 evoked the release of Ca(2+) which is a central regulator of oligodendrocyte differentiation. Our results provide a link between ET-1 and its ETA receptor and myelination during oligodendrocyte differentiation.

Purpose Lactic acid, the end product of glycolysis, has emerged as an immune-modulating metabolite in various diseases. In this study, we aimed to examine whether lactic acid contributes to the disease pathogenesis of choroidal neovascularization (CNV) and to investigate the role of macrophages in CNV pathogenesis. Methods CNV was induced by laser photocoagulation in C57BL/6J mice. Lactic acid concentration was measured in the RPE-choroid region. Macrophage infiltration and VEGF were quantified by flow cytometry. VEGF-positive areas and CNV lesions were measured by flat-mount immunofluorescence staining. To inhibit lactic acid uptake in vivo, alpha-cyano-4-hydroxycinnamic acid (α-CHC), a monocarboxylate transporter (MCT) blocker, was injected intravitreally 1 day after laser. VEGF productions were measured in ARPE-19, THP-1 cells, and human umbilical vein endothelial cells (HUVECs) by quantitative PCR and ELISA. Angiogenic activity of lactic acid-treated macrophages was assessed by HUVEC tube formation assay. Results Lactic acid was significantly increased in the RPE-choroid region of CNV-induced mice. Lactic acid upregulated VEGFA mRNA and VEGF protein expressions in THP-1 macrophages, but did not in ARPE-19 or HUVECs. THP-1 macrophages treated with lactic acid increased the angiogenesis of endothelial cells independent of MCT activity. Intravitreal injection of α-CHC substantially reduced the VEGF-positive area that colocalized with F4/80-positive macrophages. CNV lesions were also significantly reduced following α-CHC injection compared with vehicle-injected controls. Conclusions To our knowledge, these results show for the first time the role of lactic acid in facilitating neovascularization through macrophage-induced angiogenesis. We suggest that targeting macrophage metabolism can be a promising strategy for CNV treatment.

Background Safety concerns exist for corneal recipients under immunosuppression. We report long-term safety results of porcine corneal xenotransplantation under immunosuppression in nonhuman primates. Methods Systemic monitoring data from 49 Chinese rhesus macaques that received pig corneal transplant between 2009 and 2018 were retrospectively reviewed. The recipients were divided into 4 groups depending on the systemic immunosuppressants used: (a) conventional steroid group; costimulation blockade groups ([b] anti-CD154 antibody, [c] anti-CD40 antibody); and (d) commercially available immunosuppressants (anti-CD20 antibody, tacrolimus, basiliximab) group. We compared results of general condition monitoring; hematologic, biochemical, and electrolyte tests; and Rhesus Cytomegalovirus infection monitoring. Results All recipients recovered from early weight loss. White blood cell counts significantly decreased at 6 months in the steroid and anti-CD154 groups. Abnormal liver and kidney function and electrolyte imbalance were not observed in all groups. The mean value of Rhesus Cytomegalovirus DNA copies was consistently lower than 200 copies/mL, and antibody titers did not change over time in all groups. Tacrolimus-associated thrombotic microangiopathy was developed in one case, which resolved after discontinuation of tacrolimus. In 2017, a simian varicella virus outbreak led to clinical signs in 5 that received immunosuppressive therapies, of which 3 died. Conclusion Costimulatory blockade-based and anti-CD20 antibody/tacrolimus-based immunosuppressive therapies seem to be comparably safe with steroid therapy in nonhuman primates receiving corneal xenotransplantation, as they did not reactivate Rhesus Cytomegalovirus and maintained manageable systemic status. Although reactivation is rare, antiviral prophylaxis for simian varicella virus should be considered in immunocompromised hosts.