Fundamental Toxicological Sciences

2014 - Vol. 1 No. 4

2014 - Vol. 1

Estimation of endoplasmic reticulum stress-inducing ability of nobiletin, a citrus polymethoxyflavonoid, in SK-N-SH human neuroblastoma cells Vol.1, No.4, p.169-172
Ayaka Ikeda , Shingo Miyata , Akihito Yokosuka , Yoshihiro Mimaki , Yasushi Ohizumi , Masakuni Degawa , Kiyomitsu Nemoto
Released: December 25, 2014
Abstract Full Text PDF[363K]

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.

Toxicomics Report
The involvement of GPRC5B in cadmium toxicity in HK-2 cells Vol.1, No.4, p.165-167
Jin-Yong Lee , Maki Tokumoto , Yasuyuki Fujiwara , Moo-Yeol Lee , Masahiko Satoh
Released: December 25, 2014
Abstract Full Text PDF[754K]

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.

Toxicomics Report
CDC23 knockdown reinforces methylmercury sensitivity in HEK293 cells Vol.1, No.4, p.161-164
Ke Du , Tsutomu Takahashi , Miyuki Iwai-Shimada , Nobuhiko Miura , Akira Naganuma , Gi-Wook Hwang
Released: December 24, 2014
Abstract Full Text PDF[1M]

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.

Original Article
Safety evaluation of mutagenecity, acute and subacute toxicity study of Chlorella vulgaris CK-22 in rats Vol.1, No.4, p.151-159
Sayaka Himuro , Sugi Ueno , Naoto Noguchi , Takuya Uchikawa , Koji Watanabe
Released: December 22, 2014
Abstract Full Text PDF[333K]

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.

Original Article
Apoptotic activities of the extract from Moringa oleifera leaves on human HCT116 colon cancer cells Vol.1, No.4, p.143-149
Jintana Tragulpakseerojn , Ryuzaburo Yuki , Takuya Honda , Mariko Morii , Auayporn Apirakaramwong , Noritaka Yamaguchi , Perayot Pamonsinlapatham , Naoto Yamaguchi
Released: December 16, 2014
Abstract Full Text PDF[664K]

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.

Effects of a repeated low dose of LiCl injection under conditioned taste/flavor aversion using xylene Vol.1, No.4, p.135-142
Rieko Hojo , Mitsutoshi Takaya , Yukie Yanagiba , Akinori Yasuda , Masao Tsuchiya , Yasutaka Ogawa
Released: December 08, 2014
Abstract Full Text PDF[276K]

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.

Toxicomics Report
Effects of cadmium on the gene expression of SLC39A1 coding for ZIP1 protein Vol.1, No.4, p.131-133
Jin-Yong Lee , Maki Tokumoto , Yasuyuki Fujiwara , Moo-Yeol Lee , Masahiko Satoh
Released: December 04, 2014
Abstract Full Text PDF[413K]

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.

Original Article
The paradoxical effect of 1,4-naphthoquinone on the process of cell death induced by hydrogen peroxide in rat thymocytes Vol.1, No.4, p.123-129
Hana Ohtani , Eri Fukunaga , Yasuo Oyama , Shiro Ishida , Norio Akaike
Released: November 21, 2014
Abstract Full Text PDF[937K]

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.

Original Article
Diallyl disulfide administration increases the number of B-lymphocytes in the rat spleen Vol.1, No.4, p.115-121
Yoko Hashizume , Ken Shirato , Kaoru Tachiyashiki , Kazuhiko Imaizumi
Released: November 19, 2014
Abstract Full Text PDF[395K]

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.