Nobiletin, a citrus polymethoxyflavonoid compound, has been considered useful in the development of drugs and functional foods for various diseases, including dementia and diabetes. It is therefore important to understand its toxic effects. We previously reported that nobiletin treatment at a dose of 100 μM induced the expression of DDIT3 and TRIB3 genes and proteins, which are well known to contribute to apoptosis caused by endoplasmic reticulum (ER) stress, commonly in three cell lines, such as SK-N-SH human neuroblastoma cells. Therefore, their increased expression raises concerns that nobiletin might exert a toxic effect by inducing ER stress. In the present study, SK-N-SH cells were treated with 100 μM nobiletin or 1 μg/mL tunicamycin, a potent inducer of ER stress, for 3, 6, 12, and 24 hr. The maximum expression of those proteins appeared later and was much weaker in the nobiletintreated cells than in the tunicamycin-treated cells. The expression level of BiP protein, one of the chaperons, which increases in response to ER stress, was not changed in the nobiletin-treated cells, whereas it was strongly induced 12 and 24 hr after the onset of tunicamycin treatment. In addition, cleavages of caspase-3 and poly (ADP-ribose) polymerase occurred 24 hr after the onset of tunicamycin treatment, whereas cleavage did not occur at any point during nobiletin treatment. Therefore, although nobiletin has the ability to induce the expression of DDIT3 and TRIB3, those increased levels, at doses up to at least 100 μM, cannot be enough to lead to ER stress resulting in apoptosis.

Cadmium (Cd) is a nephrotoxic heavy metal. Several signal transduction pathways have been reported to be associated with Cd toxicity. GPRC5B is a member of the family of G-protein-coupled receptors, which recognize various ligands and can transmit signals from the cell surface into the cell interior. We examined the involvement of GPRC5B in Cd toxicity in HK-2 human proximal tubular cells. Herein, we found that Cd significantly reduced GPRC5B gene expression in HK-2 cells. Moreover, knockdown of GPRC5B by siRNA transfection strengthened Cd toxicity in HK-2 cells. Our findings suggest that Cd partially conferred its toxicity by suppressing GPRC5B gene expression in HK-2 cells.

The ubiquitin-proteasome system is believed to play an important role in the determination of cell sensitivity to methylmercury. The ubiquitin ligase enzyme is involved in the recognition of substrate proteins that are degraded by the ubiquitin-proteasome system. In this study, the ubiquitin ligase species affecting methylmercury sensitivity was investigated by the gene interference method. We found that the inhibition of expression of the gene for Cell division cycle 23 (CDC23), a constitutional component of the ubiquitin ligase anaphase promoting complex/cyclosome, sensitized HEK 293 cells to methylmercury.

The aim of this study was to evaluate the safety of Chlorella vulgaris CK-22 as a food supplement. We examined mutagenicity, acute toxicity and subacute toxicity using Wistar rats administered Chlorella powder (CP). In the mutagenesis test, CP exhibited no mutagenecity in the in vitro assay. In the acute toxicity test, CP was administered orally at 0 mg/kg, 1,000 mg/kg, 2,000 mg/kg and 5,000 mg/kg body weight to Wistar rats (five animals/sex/group). No significance changes were observed test article-related during the 14-day observation period. The LD₅₀ of CP was estimated to be more than 5,000 mg/kg body weight in rats. In the subacute toxicity test, CP was administered at 0%, 2.5%, 5% and 10% in pelleted rodent diet to Wistar rats (ten animals/sex/groups). No mortality or treatment-related clinical signs were observed in any of the groups during the 28-day observation period. In both sexes, renal histopathology was conducted in the control and 10% groups, because absolute and relative renal weights increased in the 10% groups compared to the control groups. Based on the histopathology of the kidneys, the no-observed-adverse-effect level (NOAEL) is greater 8.57 g/kg body weight/day for males and 8.62 g/kg body weight/day for females.

Moringa oleifera Lamk. (M. oleifera) is an edible plant and used for traditional medicine formulation. Some bioactive phytochemicals found in M. oleifera leaves thus far were identified as quercetin, chlorogenic acid, astragalin, and kaempferol. The flavonoid kaempferol was reported to induce apoptosis in human HCT116 colon cancer cells. Here, we investigated the anti-proliferative activity present in the methanol extract from M. oleifera leaves toward human HCT116 colon cancer cells. Fractionation of the methanol extract from M. oleifera leaves by gel filtration chromatography on Sephadex LH-20 enabled us to find anti-proliferative and apoptosis-inducing activities. Treatment of HCT116 cells with each pooled fraction (pf1, pf2, or pf3) inhibited the cell proliferation in a dose-dependent manner, and the inhibitory activities contained in pf2 and pf3 were more potent than that in pf1. Compared with kaempferol, pf1, pf2, and pf3 were found to exhibit strong anti-proliferative effects on HCT116 cells. Futhermore, treatment with pf1 induced much larger numbers of cleaved caspase-3-positive cells than that with pf2 or pf3. The apoptosis-inducing activity found in each pooled fraction was higher than that of kaempferol. Cells treated with pf2 displayed the typical characteristics of apoptosis, such as membrane blebbing, nuclear condensation and apoptotic bodies, whereas cells treated with pf1 showed early apoptotic morphologies. In contrast, pf3 barely induced apoptosis despite its strong inhibition of cell proliferation. Taken together, these results suggest that, in addition to kaempferol, M. oleifera leaves may contain new substances having anti-proliferative and apotosis-inducing activities on HCT116 cells.

We examined whether repeated injections with low-doses lithium chloride (LiCl) as unconditioned stimulus (US) established conditioning as applied conditioned taste aversion (CTA) experiment, using xylene solution as a conditioned stimulus (CS). In the conditioning procedure, water-deprived male rats were exposed to xylene solution for 30 min, followed by LiCl or saline injection. As a two-bottle test, xylene solution and usual drink water were simultaneously provided to rats on the next day of the conditioning and measured each consumption volume. Conditioning and two-bottle test were repeated eight times respectively by turns. Groups of no treatment and sham injection after xylene ingestion were added to verify the effects of external contexts on establishment of CTA. Results indicate that the CTA was gradually established when the US was repeatedly presented even if the US was very low concentration, and the organic solvent functioned as CS even if it was not so desirable for animals. External contexts, such as handling and the ‘pain’ induced by injection, did not affect the establishment of the CTA in the present study. Although xylene was used as solution in the present study and defined as flavor stimulus, gas should be used to examine the effects of odor stimulus.

Cadmium (Cd) is a toxic heavy metal, particularly in the kidney. Zinc transporters have been reported to be responsible for the absorption of Cd in the kidney. Interestingly, we previously found in a DNA microarray that exposure to Cd suppressed the expression of the gene coding for the zinc transporter ZIP1 (SLC39A1) in HK-2 human kidney proximal tubular cells. In this study, we validated by realtime RT-PCR that SLC39A1 gene expression was indeed decreased upon treatment with 40 μM Cd. We also demonstrated that knockdown of SLC39A1 by siRNA transfection conferred resistance to Cd in HK-2 cells. Together, this suggests that gene suppression of SLC39A1 by Cd is involved in the defense mechanism against the Cd toxicity in HK-2 cells.

1,4-Naphthoquinone (NAPH) is found in diesel exhaust particles and it is an active metabolite of naphthalene, a fumigant insecticide. This compound is known to cause oxidative stress. Therefore, it is plausible to suggest that NAPH increases cell vulnerability to oxidative stress in an additive or synergistic manner. We tested this possibility using rat thymocytes with flow-cytometric techniques and appropriate fluorescent probes. NAPH attenuated the increase in cell lethality induced by hydrogen peroxide (H₂O₂). The combination of NAPH and H₂O₂ promoted the transition from normal cells to apoptotic living cells, but attenuated further transition to cell death. Thus, the process of cell death induced by H₂O₂ was not completed in the presence of NAPH. However, NAPH did not attenuate certain lethal cellular events such as decrease in the cellular content of non-protein thiols and increases in intracellular Ca²⁺ and Zn²⁺ levels, induced by H₂O₂. The inhibitory effect of NAPH on the increase in cell lethality induced by H₂O₂ was also observed when caspase activity was suppressed. In the present study, the mechanism underlying the NAPH-induced attenuation of cell death in cells affected by H₂O₂-generated oxidative stress was, however, not fully elucidated. Since both H₂O₂ and NAPH elevated intracellular Ca²⁺ and Zn²⁺ levels, and since Zn²⁺ is known to partly attenuate Ca²⁺-dependent cell death, the intracellular interaction between Ca²⁺ and Zn²⁺ may complicate the process of cell death induced by oxidative stress.

Diallyl disulfide (DADS), the major sulfur compound in garlic, reduces the number of circulating T-lymphocytes, B-lymphocytes, and monocytes via activation of the hypothalamus-pituitaryadrenal axis. However, the translocation of those cells that migrate in response to DADS administration is still unclear. Therefore, in this study, we examined the effects of DADS administration on a number of lymphocyte subsets and monocyte-derived cells including macrophages (monocytes/macrophages) in spleen, the largest secondary lymphoid organ. Ten-wk-old male Sprague-Dawley rats were orally administered with DADS (dose = 20 mg/kg body weight) or equivalent volume of vehicle. The spleen was harvested 4 hr after administration, and then the splenic cells were isolated and the total number of cells was counted. T-lymphocytes, B-lymphocytes, natural killer (NK) cells, and monocytes/macrophages were fractionated by flow-cytometry and the total number of these cells was calculated. The total number of splenic cells was significantly increased by 1.18-fold after DADS administration. Among the lymphocyte subsets in the spleen, the number of B-lymphocytes significantly increased by 1.28-fold after DADS administration. The number of T-lymphocytes also showed a tendency to increase. However, the number of NK cells and monocytes/macrophages did not change after DADS administration. These results suggest that B-lymphocytes migrate from the circulation and translocate to the spleen in response to DADS administration.

Using the yeast strain library we established by overexpressing each gene essential for yeast growth, we comprehensively searched for genes affecting the sensitivity of yeast to paraquat. As a result, seven novel genes, UTP4, UTP25, SEC65, NDD1, TFB2, TIM23, and CCT6, were identified as conferring paraquat resistance in yeast via overexpression.

We screened for genes associated with cadmium resistance among genes essential for cell growth in yeast. Four novel genes, RPN8, SKP1, MIA40 and MES1, were identified as genes providing cadmium resistance to yeast via overexpression.

Chlorinated polycyclic aromatic hydrocarbons (Cl-PAHs) have recently been found in the environment at relatively high concentrations. However, their toxicological information has not been well documented. In this study, a 24 hr in vivo experiment was conducted to evaluate the sex-dependent difference of the acute toxicological effects of 7-chlorinated benz[a]anthracene (7-ClBaA) as a model Cl-PAH. 7-ClBaA or its parent chemical, BaA, was once orally administered to male or female ICR mice at concentrations of 1, 10, and 100 mg/kg body weight. The relative liver weights of the males were significantly increased at the highest dose of both chemicals compared to the vehicle controls, but the weights were comparable among all groups in the females. The plasma 7-ClBaA level was similar in both sexes, but significantly higher than that of BaA. 7-ClBaA dose-dependently induced expression of the genes Cyp1a1, 1a2, and 1b1 in the liver and lung, and these stimulations were significantly higher in both organs and genders at a dose of 100 mg/kg 7-ClBaA compared with an equivalent amount of BaA, except in the case of hepatic Cyp1a2 and 1b1 and pulmonary Cyp1a2 in the female mice. The results suggest that acute toxicity of 7-ClBaA is gender- and organ-specific, and female mice might be less sensitive to acute toxicity of both 7-ClBaA and BaA than the males.

When considering mechanisms of toxicity development to chemical substances, one potentially important mechanism is the selective inhibition of proteins essential for cell growth (target molecules). In this study, to detect the target molecules of chemical substances, we established a method for comprehensively screening for essential proteins that confer resistance against chemical substances via overexpression in yeast. We used budding yeast, a common eukaryotic model organism, to produce yeast strains showing overexpression of different genes encoding essential proteins. This method was used to search for overexpressed genes conferring arsenite resistance in yeast, and as a result, we successfully identified ten types of new genes correlated with arsenite resistance.

Carbon nanotubes (CNTs) are used in many fields; however, little is known about the effects of CNTs on the central nervous system (CNS). In this study, we found that extracts of sonicated CNTs suppressed the proliferation of neural stem cells (NSCs). Single-walled CNTs (SWCNTs) and multiple-walled CNTs (MWCNTs) were suspended in PBS (1 mg/mL) and sonicated for 5 hr using a water bath sonicator. Supernatants from both types of CNTs suppressed NSC proliferation. The effects weakened in a dilution-ratio-dependent manner and strengthened in a sonication time-dependent manner. Metal concentrations extracted from SCNTs and MCNTs after 5-hr of sonication were determined using inductively coupled plasma mass spectrometry. Mn, Rb, Cs, Tl, and Fe were detected in the SWCNT supernatant, and Mn, Cs, W, and Tl were detected in the MWCNT supernatant. The concentration of Mn, Rb, and Fe eluted from the SWCNTs and Rb eluted from MWCNTs following sonication were sufficient to suppress NSC proliferation alone. N-acetyl cysteine (NAC) and ascorbic acid (AA) reversed the effects of Mn and Fe and restored NSC proliferation. The effects of Rb and Tl were not affected by the antioxidants. Both antioxidants largely restored the suppression of NSC proliferation induced by the SWCNT and MWCNT supernatants. These results suggest that metals extracted from CNTs via a strong vibration energy can suppress NSC proliferation through ROS production by the extracted metals.

It has been reported that titanium dioxide nanoparticles (TiO₂ NPs) show toxicity in organs such as liver, lung, and intestine. There is, however, only a limited number of reports regarding the effect of TiO₂ NPs on the male reproductive system. We examined the effect of TiO₂ NPs on testicular function using mouse model. TiO₂ NPs (Aeroxide P25) were evenly dispersed in disodium phosphate solution by sonication. Mice were treated intravenously with TiO₂ NPs (0, 2, or 10 mg/kg body weight) once per week for four weeks followed by sacrificing nine days after the last injection. The sperm head numbers and two sperm motion parameters, motile percent (MP) and progressive percent (PP), in the cauda epididymis were evaluated. TiO₂ NPs significantly reduced the sperm head numbers in the cauda epididymis and testis, further, the motility ratios of both MP and PP. These results indicated that TiO₂ NPs may possess hazardous effects on testicular function in mice.

The effects of organic and inorganic mercury ions on gene expression were studied by in vitro cell-free assays for transcription and translation. While organic mercury (methylmercury chloride, MeHgCl) showed no effects, treatment of template DNA with inorganic mercury (HgCl₂) inhibited mRNA synthesis in a bacteriophage T7 RNA polymerase transcription system. The inhibited transcription resulted in reduced protein synthesis after the subsequent application of the transcripts to a translation system consisting of Sf21 insect cell lysate. Treatment of mRNA with inorganic mercury also reduced translation, although this inhibitory effect was weaker than the effect produced by DNA exposure. Treatment of DNA and RNA with mercury did not increase oxidative damage such as strand cleavage and base oxidation. Instead, circular dichroism spectrometry demonstrated that mercury ions, not methyl mercury, drastically changed strand conformation of DNA and RNA. Therefore, the gene expression inhibition observed in this study was thought to be caused by crossbridging of DNA bases with mercury ions, which blocked the transcriptional machinery. Taken together with reports on biological conversion of organic mercury to inorganic forms in animals, our results show that transcriptional inhibition via conformational changes in DNA could be a toxic mechanism involved in mercury poisoning.

To investigate the safety of L-alanine, male and female Sprague-Dawley strain SPF rats [Crj:CD(SD)IGS] were administered L-alanine at 2,000 mg/kg/day by gavage for 4 weeks. After the end of the dosing period, reversibility was assessed following a 2-week recovery period. In the results, there were no toxicologically significant changes caused by L-alanine in general condition, body weight, food consumption, ophthalmology, hematology, blood chemistry, organ weight or at necropsy. In urinalysis, increased number of animals showing urine protein-positive or phosphate salt was observed in males and females. In addition, urine volume was significantly increased in males. In histopathological examination, squamous cell hyperplasia in the limiting ridge in the stomach was observed in males and females. These changes were reduced or no longer observed after the 2-week recovery period and thus were reversible changes. These results suggest that repeated oral administration of L-alanine at 2,000 mg/kg/day for 4 weeks is well tolerated in male and female rats.

Rats were administered L-threonine in the diet at concentrations of 0 (basal diet control), 1.25%, 2.5%, or 5.0% for 13 weeks. Animals were sacrificed following the treatment period or after a 5-week recovery period (for animals receiving the control or 5.0% L-threonine diet). The mean achieved doses of L-threonine during the treatment period were 0, 811.5, 1615.3, and 3266.9 mg/kg body weight/ day in males, and 0, 909.9, 1850.0, and 3673.3 mg/kg body weight/day in females. No toxicologically significant changes in general condition, body weight, food consumption, feed efficiency, water intake, ophthalmoscopy, urinalysis, hematology, blood chemistry or pathology were observed. Based on the results of the study, no-observed-adverse effect levels (NOAEL) of 3266.9 and 3673.3 mg/kg body weight/day can be established for male and female rats, respectively, under the present experimental conditions.

The present study was designed to evaluate the morphological changes in corneal endothelial cells (CECs), induced by K-115 ophthalmic solution in the ophthalmology, using a specular microscope. Unclear borders between CECs and slightly decreasing trends in corneal thicknesses were noted following ocular instillation of the K-115 ophthalmic solutions into cynomolgus monkeys. These changes were transient and disappeared within 24 hr after instillation. In addition, no significant differences were noted in the degrees or frequencies of these lesions, between the single and 10-day repeated instillations, and no appreciable changes were noted in the number of CECs at 24 hr after instillation. No significant structural changes were noted by histopathological examinations using light, transmission electron, and scanning electron microscopy. Similar changes were also noted following ocular instillation of Y-39983 ophthalmic solution at 0.25%, with Rho-Associated, Coiled-Coil Containing Protein Kinase (ROCK) inhibitory effects, similar to the K-115 ophthalmic solution 2.0%. Therefore, the changes were due to pharmacological effects of the K-115 ophthalmic solution. In conclusion, some morphological changes in CECs, following instillation with K-115, were considered to be of minimal toxicological significance because, 1) they were noted transiently after instillation, and CECs recovered by 24 hr after instillation, 2) no enhancement by repeated instillation was noted, 3) no appreciable changes were noted in the corneal thickness or the number of CECs at 24 hr after instillation, and 4) no significant structural changes were noted.

The purpose of this study was to evaluate the toxicity of L-phenylalanine when administered daily in the diet to rats for at least 28 days. Male and female Crl:CD®(SD)IGS BR rats were assigned to four groups. Each group received diets containing basal diet or 0.5, 1.5, or 5.0% (w/w) L-phenylalanine. There were no clinical or ophthalmic observations that were considered to be related to L-phenylalanine. Effects of L-phenylalanine administration were noted in mean body weights and mean body weight gains in females fed 0.5% and in males and females fed 5.0% (w/w) L-phenylalanine diets. Effects were also noted in mean food consumption in males and females fed the 5.0% (w/w) L-phenylalanine diet. The lower food consumption and body weights of the males and females fed the 5.0% L-phenylalanine diet were considered to be signs of mild toxicity. Administration of L-phenylalanine at a dose of 5.0% of the diet was associated with mildly increased red blood cell count and mildly decreased mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, and glucose in females. There were no L-phenylalanine-related toxic changes for organ weight, or macroscopic or microscopic findings. In conclusion, the no-observed-effect level (NOEL) of dietary exposure of male rats to L-phenylalanine is 1.5% (w/w) L-phenylalanine. The NOEL of dietary exposure of female rats to L-phenylalanine is less than 0.5% (w/w) L-phenylalanine. However, the no-observed-adverse-effect level (NOAEL) for males and females is 1.5% (w/w) L-phenylalanine.

K-115, a strong and selective inhibitor of Rho-associated coiled coil-forming protein kinase (ROCK), was developed as a therapeutic drug for glaucoma. In long-term phase 3 clinical studies, blepharitis and allergic conjunctivitis were observed. Nonclinical studies were conducted to determine if these adverse effects were caused by the sensitizing potential of K-115. In mouse local lymph node assays (LLNA), sensitizing reactions were not observed at 8.17% w/v of K-115, but weak positive skin sensitivity with a 2.0% K-115 ophthalmic solution was observed. K-115 ophthalmic solution was classified as having slight sensitizing potential. No photosensitization potential was observed with UV-LLNA studies and skin photosensitization studies. In a guinea pig conjunctivitis model study, K-115 had no immediate allergic effect on conjunctivitis, and did not function as an antigen. Furthermore, in ocular toxicity studies in rabbits (26 weeks) and monkeys (52 weeks), no abnormalities were observed in ophthalmic and pathological examinations. Considering the results of nonclinical sensitization and ocular toxicity studies, the clinical adverse effects were possibly not caused by K-115 sensitization potential.

Philodryas nattereri is distributed in arid and semiarid regions of South America and is most common in northeastern Brazil. The aims of the work were to investigate the effects of the venom from P. nattereri in cultured of MDCK and RAW cells and abdominal writhes in mice. Based on oxidative metabolism, it was possible to observe that the venom was capable of significantly reducing cell viability only at higher concentrations of venom at 50 and 100 μg/mL for MDCK cells, while in 200 μg/mL to RAW cells, with an IC₅₀ of 169.5 μg/mL. Regarding writhing in mice promoted by the poison and acetic acid, it held a greater number of writhes when compared to promoted by saline. The venom of P. nattereri has a cytotoxic effect in MDCK and RAW cells and abdominal writhes, which appears to be similar to those caused by acetic acid.

Philodryas nattereri is distributed in arid and semiarid regions of South America and is most common in northeastern Brazil. The aims of the work were to investigate the edematogenic and myotoxic effects promoted by P. nattereri venom. In this work, mice weighing 20-30 g (n = 4 for each experimental group) were used. For the edematogenic activity mice were injected in the subplantar region of the right foot pad with 50 μL of solutions containing different amounts of venom (3 and 10 μg) measured by plethysmometry at 0.5, 1, 2, 3, 6, 12 and 24 hr and pretreated with indomethacin, dexamethasone and antibothropic serum, whereas the left foot pad was injected with 50 μL of NaCl 0.15 M. Two hours after injection mice were killed by cervical dislocation and both feet were cut off and weighed individually. For the myotoxic activity mice were injected i.m. with 100 mL of solutions containing 50 μg of venom. Blood samples were extracted after 2, 4, 8, 12, 24 and 48 hr of venom injection to determinate serum CK activity and mice were sacrificed at the same time intervals to obtain the inoculated gastrocnemius muscle. They were fixed with formalin solution and stained with Hematoxylin-Eosin. Results showed that P. nattereri venom exhibits a high edematogenic and myotoxic activities. Myonecrosis reached its highest level after 2 hr of venom injection as shown by plasmatic CK levels (364 ± 92 U/L) and microscopic assay. It demonstrates the potential toxicity of the venom of P. nattereri, who inhabits the North-East region of Brazil.

In risk assessment of chemicals, we often use default assessment factors to compensate for lack of knowledge. Such assessment factors can be especially useful for regulatory decisions. The present study focuses on assessment factors related to exposure duration, especially under sub-acute and sub-chronic conditions; and discussions attempt to utilize chemical-specific toxicological data. Most previous studies have not focused on target organs, but recent reports such as Malkiewicz et al. (2009) suggest that assessment factors may be target organ-dependent. Therefore, we addressed selected target organs (liver, kidney, blood, and body weight) by investigating assessment factors for these target organs. Using existing data, we calculated the ratio of the no-observed-effect level (NOEL) derived from sub-acute studies to the NOEL derived from sub-chronic studies, to assess for effects involving individual target organs (liver, kidney, blood, or body weight). Then, we compared these ratios with the ratio derived from the substances’ sub-acute and sub-chronic NOELs (the minimum values among all four target organs’ NOELs) by using the Dunnett’s multiple comparison test. Our analysis indicates that effects involving liver, kidney, and body weight need not be treated independently, although the effect on blood should be treated separately. Based on our results, we discuss potential refinement of assessment factors to reflect exposure duration.