Two tiered approaches combining alternative test methods and minimizing the use of reconstructed human cornea-like epithelium tests for the evaluation of eye irritation potency of test chemicals.

In order to overcome the limitations of single in vitro eye irritation tests, Integrated Approaches to Testing Assessment strategies have been suggested for evaluating eye irritation. This study developed two tiered approaches combining alternative test methods. They were designed in consideration of the solubility property of test chemicals and to use the RhCE tests at final steps. The tiered approach A is composed of the STE, BCOP, HET-CAM or RhCE tests, whereas the tiered approach B is designed to perform simultaneously two in vitro test methods at the first stage and the RhCE test at the final stage. The predictive capacity of the two tiered approaches was estimated using 47 chemicals. The accuracy, sensitivity, and specificity value of the tiered approach A were 95.7% (45/47), 100% (34/34), and 84.6% (11/13), respectively, whereas those of the tiered approach B were 95.7% (45/47), 97.1% (33/34), and 92.3% (12/13), respectively. The approach A and B were considered to be available methods for distinguishing test chemicals of Category 1 (all 73.3%) and No Category (84.6% and 92.3%), respectively. Especially, the approach B was considered as an efficient method as the Bottom-Up approach, because it predicted correctly test chemicals classified as No Category.

Assessment of the predictive capacity of the optimized in vitro comet assay using HepG2 cells.

Evaluation of DNA damage is critical during the development of new drugs because it is closely associated with genotoxicity and carcinogenicity. The in vivo comet assay to assess DNA damage is globally harmonized as OECD TG 489. However, a comet test guideline that evaluates DNA damage without sacrificing animals does not yet exist. The goal of this study was to select an appropriate cell line for optimization of the in vitro comet assay to assess DNA damage. We then evaluated the predictivity of the in vitro comet assay using the selected cell line. In addition, the effect of adding S9 was evaluated using 12 test chemicals. For cell line selection, HepG2, Chinese hamster lung (CHL/IU), and TK6 cell lines were evaluated. We employed a method for the in vitro comet assay based on that for the in vivo comet assay. The most appropriate cell line was determined by% tail DNA increase after performing in vitro comet assays with 6 test chemicals. The predictivity of the in vitro comet assay using the selected cell line was measured with 10 test chemicals (8 genotoxins and 2 non-genotoxic chemicals). The HepG2 cell line was found to be the most appropriate, and in vitro comet assays using HepG2 cells exhibited a high accuracy of 90% (9/10). This study suggests that HepG2 is an optimal cell line for the in vitro comet assay to assess DNA damage.

Development of a Test Method for the Evaluation of DNA Damage in Mouse Spermatogonial Stem Cells.

Although alternative test methods based on the 3Rs (Replacement, Reduction, Refinement) are being developed to replace animal testing in reproductive and developmental toxicology, they are still in an early stage. Consequently, we aimed to develop alternative test methods in male animals using mouse spermatogonial stem cells (mSSCs). Here, we modified the OECD TG 489 and optimized the in vitro comet assay in our previous study. This study aimed to verify the validity of in vitro tests involving mSSCs by comparing their results with those of in vivo tests using C57BL/6 mice by gavage. We selected hydroxyurea (HU), which is known to chemically induce male reproductive toxicity. The 50% inhibitory concentration (IC50) value of HU was 0.9 mM, as determined by the MTT assay. In the in vitro comet assay, % tail DNA and Olive tail moment (OTM) after HU administration increased significantly, compared to the control. Annexin V, PI staining and TUNEL assays showed that HU caused apoptosis in mSSCs. In order to compare in vitro tests with in vivo tests, the same substances were administered to male C57BL/6 mice. Reproductive toxicity was observed at 25, 50, 100, and 200 mg/kg/day as measured by clinical measures of reduction in sperm motility and testicular weight. The comet assay, DCFH-DA assay, H&E staining, and TUNEL assay were also performed. The results of the test with C57BL/6 mice were similar to those with mSSCs for HU treatment. Finally, linear regression analysis showed a strong positive correlation between results of in vitro tests and those of in vivo. In conclusion, the present study is the first to demonstrate the effect of HU-induced DNA damage, ROS formation, and apoptosis in mSSCs. Further, the results of the current study suggest that mSSCs could be a useful model to predict male reproductive toxicity.

Anti-Inflammatory and Antioxidant Actions of N-Arachidonoyl Serotonin in RAW264.7 Cells.

The purpose of this study is to evaluate the effect of N-arachidonoyl serotonin (NA-5HT) on inflammatory response or oxidative stress in RAW264.7 cells exposed to lipopolysaccharide (LPS). When RAW264.7 cells were pre-incubated with NA-5HT before LPS treatment, NA-5HT was found to suppress LPS-induced formation of nitric oxide (NO), tumor necrosis factor-α or interleukins as well as expression of inducible NO synthase and cyclooxygenase-2 at non-cytotoxic concentrations. Consistent with this, NA-5HT efficiently reversed LPS-induced phosphorylative activation of nuclear factor-κB pathway probably through the suppression of mitogen-activated protein kinases (MAPKs) pathway or phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway. Separately, NA-5HT enhanced the antioxidant capacity accompanied by nuclear translocation of nuclear factor-E2-related factor-2 (Nrf2) in RAW264.7 cells. Additionally, NA-5HT-induced nuclear translocation of Nrf2 was suppressed significantly by the inhibition of c-Jun N-terminal kinase1/2 or PI3K/Akt pathways, although NA-5HT phosphorylated signal molecules in MAPKs and PI3K/Akt pathways. Taken together, NA-5HT is proposed to exert anti-inflammatory and antioxidant actions in RAW264.7 cells.

Neuroprotective action of deer bone extract against glutamate or Aß1₋42-induced oxidative stress in mouse hippocampal cells.

Water extracts of deer bone, called nokgol in Korean, and deer antlers have been traditionally used as anti-aging medicines. Deer antler extract is known to possess various activities, including anti-aging or anti-amnesic activity. However, there are no reports about the neuroprotective effect of deer bone extract (DBE). The objective of this study was to examine the neuroprotective effect of DBE on glutamate-induced cell death of mouse hippocampal cells (HT-22 cells) and to elucidate the mode of neuroprotective action of DBE. In this study, HT-22 cells was pretreated with DBE before stimulation with glutamate, and then, the effects of DBE on cell viability, oxidative stress markers, and MAP kinases were determined. Separately, the effect of DBE on H2O2 or amyloid beta peptide (1-42) (Aß1₋42)-induced cytotoxicity of HT-22 cells was evaluated. DBE protected HT-22 cells from glutamate-induced cell death and prevented the increase in lactate dehydrogenase leakage in HT-22 cells. DBE also prevented glutamate-induced oxidative stress, as indicated by increased reactive oxygen species and lipid peroxidation as well as by decreases in glutathione (GSH) levels and GSH peroxidase activity. In addition, DBE inhibited glutamate-induced activation of c-Jun N-terminal kinases (JNK), p38, and extracellular signal-regulated kinase, indicators of oxidative stress-induced cell death. Furthermore, DBE also protected against H2O2 and Aß1₋42-induced cytotoxicity. These results suggest that DBE may be a useful functional agent for the prevention against neurodegenerative disorders involving oxidative stress.

Evaluation of the Antioxidant Capacity and Phenolic Content of Agriophyllum pungens Seed Extracts from Mongolia.

Antioxidants are an important group of medicinal preventive compounds as well as being food additives inhibiting detrimental changes of easily oxidizable nutrients. The present investigation has been carried out to evaluate the antioxidant properties of different solvent extracts of Agriophyllum pungens seeds by various in vitro systems. The anti-oxidative activities of these samples were determined using four methods: 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activity, ferric-reducing antioxidant potential (FRAP), and hydroxyl (OH) radical scavenging activities. Additionally, total flavonoids and phenolic contents (TPC) were also determined. Yield of extracts varied widely among solvents and was the highest for water extract (5.642% based on dry weight basis), while ethyl acetate extract exhibited the highest total phenolic content (0.149 mg/mL), total flavonoid content (0.111 mg/mL), and antioxidant activities (P<0.05). The ABTS radical scavenging activity of A. pungens seeds occurred in the following order: ascorbic acid (92.9157%)>BHA (90.1503%)>α-tocopherol (87.7527%)>APEA (83.9887%) >APWR (75.5633%); the antioxidant activity of the extracts might be attributed to the presence of these phenolics. This suggests that A. pungens seed extract is a potential source of natural antioxidants, which could be added to dietary supplements to help prevent oxidative stress.

Antioxidant Activities of Ribes diacanthum Pall Extracts in the Northern Region of Mongolia.

Ribes diacanthum Pall (RDP) is a member of the Saxifragaceae family. The plant is traditionally used in Mongolia for the treatment of various ailments associated with kidney and bladder's diseases, cystitis, kidney stone, and edema. This study was aimed to investigate antioxidant activities of different solvent extracts of whole Pall plants, based on ferric-reducing antioxidant potential (FRAP), 2,2'-azinobis(3-ethybenzothiazoline-6-sulfonic acid) (ABTS· +) radical scavenging activity, 1,1-diphenyl-2-picrydrazyl (DPPH·), and hydroxyl (·OH) radical scavenging activities. Additionally, total flavonoids and phenolic contents (TPC) were also determined. The ethyl acetate extract of RDP (EARDP) had a remarkable radical scavenging capacity with an IC50 value of 0.1482 mg/mL. In addition, EARDP was shown to be higher in total phenolic and flavonoid contents than the methanol extract of RDP (MRDP). Moreover, the EARDP had the predominant antioxidant capacity, DPPH, hydroxyl, and ABTS radical scavenging activities and ferric reducing power. These results suggest a potential for R. diacanthum Pall extract as a functional medicinal material against free-radical-associated oxidative damage.