Piperlongumine induces apoptosis and autophagy via the PI3K/Akt/mTOR pathway in KB human cervical cancer cells.

Natural products are continuously being researched to develop safe and effective treatment options for cervical cancer, the fourth most common cancer in women. Piperlongumine (PL), an amide alkaloid mainly present in long pepper, exhibits neuroprotective and anti-cancer properties. However, the specific effect of PL in cervical cancer and the relationship between the anti-cancer pathway and autophagy remain unclear. Therefore, we aimed to investigate PL-induced apoptosis in KB human cervical cancer cells and the relationship between apoptosis and autophagy therein. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and wound-healing assays showed that PL treatment suppressed KB cell viability and proliferation. Apoptosis was identified through 4',6-diamidino-2-phenylindole and annexin V-propidium iodide staining, increased cleaved-poly (ADP-ribose) polymerase and Bcl-2 associated X levels, and decreased B cell lymphoma 2 levels. Acridine orange staining and increased microtubule-associated protein 1A/1B-light chain 3-II and Beclin-1 levels confirmed autophagy. We determined that KB cell-related autophagy exerted cytoprotective effects using the autophagy inhibitors 3-methyladenine and hydroxychloroquine. PL treatment promoted apoptosis by inhibiting the phosphatidylinositol-3-kinase (PI3K)/protein kinase B/mammalian target of rapamycin pathway in KB cells; inhibiting the pathway using PI3K inhibitors increased autophagy. We suggest that PL is a potential natural anticancer agent for cervical cancer treatment.

Genotoxic carcinogen 7,12-dimethylbenz[a]anthracene inhibits gap junction intercellular communication through post-transcriptional and post-translational processing involved in connexin 43 stability.

Gap junctional intercellular communication (GJIC) is composed of connexin (Cx) and plays an important role in maintaining intracellular homeostasis. Loss of GJIC is involved in the early stages of cancer pathways of non-genotoxic carcinogens; however, the effect of genotoxic carcinogens, including polycyclic aromatic hydrocarbons (PAHs), on GJIC function remains unclear. Therefore, we determined whether and how a representative PAH 7,12-dimethylbenz[a]anthracene (DMBA) suppresses GJIC in WB-F344 cells. First, DMBA significantly inhibited GJIC and dose-dependently reduced Cx43 protein and mRNA expression. In contrast, Cx43 promoter activity was upregulated after DMBA treatment via the induction of specificity protein 1 and hepatocyte nuclear factor 3ß, indicating that the promoter-independent loss of Cx43 mRNA can be associated with the inhibition of mRNA stability, which was verified by actinomycin D assay. In addition to a decrease in mRNA stability involved in human antigen R, we also observed DMBA-induced acceleration of Cx43 protein degradation, which was closely related to the loss of GJIC through Cx43 phosphorylation via MAPK activation. In conclusion, the genotoxic carcinogen DMBA suppresses GJIC by inhibiting post-transcriptional and post-translational processing of Cx43. Our findings suggest that the GJIC assay is an efficient short-term screening test for predicting the carcinogenic potential of genotoxic carcinogens.

Cellular signaling crosstalk between Wnt signaling and gap junctions inbenzo[a]pyrene toxicity.

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/ß-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/ß-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/ß-catenin signaling related to the PI3K-Akt pathway. To identify the relationship between GJIC and Wnt/ß-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/ß-catenin signaling negatively regulates GJIC. Despite the inverse correlation between Wnt/ß-catenin signaling and Cx43 promoter activation as indicated by downregulation of ß-catenin nuclear translocation and upregulation of Cx43 promoter activation involving HNF3ß, 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/ß-catenin signaling. More importantly, linking Wnt/ß-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.

Chrysin Induces Apoptosis and Autophagy in Human Melanoma Cells via the mTOR/S6K Pathway.

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.

Myricetin induces apoptosis and autophagy in human gastric cancer cells through inhibition of the PI3K/Akt/mTOR pathway.

Myricetin, a natural flavonoid present in berries, nuts, and green tea, is well-known for its anticancer properties. Even though several previous studies have reported the anticancer effects induced by myricetin, these effects have not yet been confirmed in the adenocarcinoma gastric cell line (AGS). Moreover, the exact mechanisms of myricetin-induced apoptosis and autophagy have not been clearly identified either. Therefore, in this study, we aimed to examine the role of myricetin in inducing apoptosis and autophagy in AGS gastric cancer cells. First, the survival rate of AGS gastric cancer cells was assessed using the 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) cell viability assay. Thereafter, the rate of apoptosis was analyzed using4',6-diamidino-2-phenylindole (DAPI) staining as well as annexin V and propidium iodide (PI) staining, and the expression of the proteins associated with apoptosis, PI3K/Akt/mTOR pathway, and autophagy was examined by western blotting. We observed that myricetin reduced the survival rate of AGS gastric cancer cells by inhibiting the PI3K/Akt/mTOR pathway, thereby inducing apoptosis and autophagy. Similar results were also obtained in vivo, and tumor growth was inhibited. Therefore, in the AGS gastric cancer cells, myricetin seems to inhibit the PI3K/Akt/mTOR pathway, which in turn leads to apoptosis in vitroand in vivo, cell-protective autophagy, as well as inhibition of cancer cell proliferation. These results indicate the potential of myricetin as a natural anticancer agent.

Improved efficiencies in the generation of multigene-modified pigs by recloning and using sows as the recipient.

This 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.

Comparison of Genetically Engineered Immunodeficient Animal Models for Nonclinical Testing of Stem Cell Therapies.

For the recovery or replacement of dysfunctional cells and tissue-the goal of stem cell research-successful engraftment of transplanted cells and tissues are essential events. The event is largely dependent on the immune rejection of the recipient; therefore, the immunogenic evaluation of candidate cells or tissues in immunodeficient animals is important. Understanding the immunodeficient system can provide insights into the generation and use of immunodeficient animal models, presenting a unique system to explore the capabilities of the innate immune system. In this review, we summarize various immunodeficient animal model systems with different target genes as valuable tools for biomedical research. There have been numerous immunodeficient models developed by different gene defects, resulting in many different features in phenotype. More important, mice, rats, and other large animals exhibit very different immunological and physiological features in tissue and organs, including genetic background and a representation of human disease conditions. Therefore, the findings from this review may guide researchers to select the most appropriate immunodeficient strain, target gene, and animal species based on the research type, mutant gene effects, and similarity to human immunological features for stem cell research.

Nonclinical toxicology studies with sodium taurodeoxycholate: acute and subacute toxicity in dogs.

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 via intravenous administration under Good Laboratory Practice regulation in this study. In dose range-finding study (dose escalation study), dogs given with TDCA at a dose of 150 mg/kg showed marked changes in clinical signs, hematology, and serum biochemistry. And biochemical markers of liver damage and local skin lesions were observed following intravenous infusion of 100 mg/kg TDCA, suggesting that 100 mg/kg was chosen as the highest dose of TDCA for 4-week repeated-dose toxicity study using dogs. Despite no treatment-related significant changes in body weight, food consumption, ophthalmoscopy, and urinalysis, skin lesions were observed at the injection site of animals administered with higher than 50 mg/kg of TDCA along with biochemical and histopathological changes associated with liver injury. However, most of off-target effects were found to be reversible since these were recovered after stopping TDCA infusion. These findings indicate that the no-observed-adverse-effect-level (NOAEL) for TDCA in dogs was considered to be 5 mg/kg/d. Taken together, our results provide important toxicological profiles regarding the safe dose of TDCA for drug development or clinical application.

Evaluation of acute and subacute toxicity of sodium taurodeoxycholate in rats.

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.

Histamine causes an imbalance between pro-angiogenic and anti-angiogenic factors in the retinal pigment epithelium of diabetic retina via H4 receptor/p38 MAPK axis.

INTRODUCTION: Systemic 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 METHODS: The 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. RESULTS: The 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. CONCLUSIONS: Taken 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.

Apigenin induces apoptosis by regulating Akt and MAPK pathways in human melanoma cell A375SM.

Apigenin, an aromatic compound, exhibits antioxidant, anti­inflammatory and anti­viral 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 anti­proliferative and survival effects, using wound healing and MTT assays. The results revealed that melanoma cell viability was decreased in a dose­dependent manner. Furthermore, chromatin condensation, indicating apoptosis, was significantly increased in a dose­dependent manner, as demonstrated by DAPI staining. In addition, increased apoptosis rate following treatment with apigenin was confirmed by Annexin V­propidium iodide staining. The changes in the expression levels of apoptosis­related proteins in A375P and A375SM melanoma cells were subsequently detected using western blot analysis. The results demonstrated that the protein expression levels of Bcl­2 were decreased, whereas those of Bax, cleaved poly ADP­ribose polymerase, cleaved caspase­9 and p53 were upregulated in a dose­dependent manner in apigenin­treated cells compared with those noted in untreated cells. In addition, in apigenin­treated A375P cells, phosphorylated (p)­p38 was upregulated and p­extracellular signal­regulated kinase (ERK), p­c­Jun N­terminal kinase (JNK) and p­protein kinase B (Akt) were downregulated. However, in A375SM cells, apigenin treatment increased p­ERK and p­JNK and decreased p­p38 and p­Akt 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 apigenin­treated groups compared with the control group. Additionally, a TUNEL assay was performed to detect apoptotic cells. Immunohistochemical staining also revealed elevated p­ERK expression in the apigenin­treated 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 mitogen­activated protein kinase signaling pathways.

In vivo and in vitro safety evaluation of fermented Citrus sunki peel extract: acute and 90-day repeated oral toxicity studies with genotoxicity assessment.

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.

Advances in selecting appropriate non-rodent species for regulatory toxicology research: Policy, ethical, and experimental considerations.

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.

Ccrn4l as a pre-dose marker for prediction of cisplatin-induced hepatotoxicity susceptibility.

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.

Antitumor and Apoptosis-inducing Effects of Piperine on Human Melanoma Cells.

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.

Preclinical study of safety of Dendropanax morbifera Leveille leaf extract: General and genetic toxicology.

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.

Use of stem cells as alternative methods to animal experimentation in predictive toxicology.

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.

Optimizing the cutoff for the identification of skin sensitizers by the HaCaSens assay: Introducing an ROC-analysis-based cutoff approach.

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.

Assessment of acute, 14-day, and 13-week repeated oral dose toxicity of Tiglium seed extract in rats.

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.

Long-term safety outcome of systemic immunosuppression in pig-to-nonhuman primate corneal xenotransplantation.

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.