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Volume 37 , Issue 2
April
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Original Article
  • Tang Yan, Li Xiang, Jiang Xuejun, Chen Chengzhi, Qi Youbin, Yu Xuelan, ...
    Volume 37 (2012) Issue 2 Pages 223-233
    Released: April 01, 2012
    JOURNALS FREE ACCESS
    Polybrominated diphenyl ethers (PBDEs), a class of widely used flame retardants, are extensively diffused in the environment. Of particular concern are the reported highly sensitivity of PBDEs in children or developmental animals, however, almost no information is available on their potential effects on adults and the mechanisms are still unknown. In the present study, we investigated the neurotoxic effects of sub-chronic PBDE-47 exposure on adult male Sprague-Dawley rats. Thus, PBDE-47, 0.1, 0.5 and 1 mg/kg per day was administered to rats by gavage for 30 days. The learning and memory function was tested by Morris water maze. Further, in order to explore the potential mechanism, the expression of NMDA-receptors was evaluated by using both immunohistochemistry (IHC) and RT-PCR. Our results showed that sub-chronic exposure to PBDE-47 produced learning and memory deficits in male adult rats. Also, significant decrease in the CA1, CA3 and dentate gyrus areas of hippocampus affected by all three doses of PBDE-47 on the expression of NR1, NR2B and Glu were found by IHC. In addition, the evaluation of expression of the NR1, NR2B and NR2C showed statistically significant decrease in mRNA expression in rats exposed to PBDE-47. These findings showed that sub-chronic exposure to PBDE-47 could also induce behavioral alterations and the neurotoxic effects might due to the down-regulation expression of NMDA receptors. Our data indicated that the possibility of exposure of adults to PBDE-47 warranted further studies to characterize their potential neurotoxicity.
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  • Akin Tekcan, Mehmet Elbistan, Ali Naki Ulusoy
    Volume 37 (2012) Issue 2 Pages 235-243
    Released: April 01, 2012
    JOURNALS FREE ACCESS
    The study aimed to determine the effects of cytostatic and genotoxic drugs used to treat breast cancer on sister chromatid exchange (SCE). SCE values were examined in 25 female patients with breast cancer in pre-treatment, treatment process and remission period as well as in 22 nonsmoker women via peripheral blood culture technique. The SCE values of patient and control group were analyzed via “Mann-Whitney U test”. Whilst SCE values of patient group ​​were 8.25 ± 3.67, 10.19 ± 2.95 and 11.52 ± 3.33 in pre-treatment, treatment process and remission periods respectively, it was 7.01 ± 1.24 in control group. When overall SCE values of patients group in pre-treatment period were compared with those of control group, no significant difference was observed (p > 0.05), whereas highly significant differences were observed between treatment process and remission period of patient groups and control group in terms of SCE values (p < 0.01). If patients are exposed to any cytostatic and clastogenic drugs, the increase in the exchange values was considered remarkable. These findings reinforced the availability of sister chromatid exchange technique in directing of treatment and monitoring the genetic abnormalities caused by genomic instability which may occur due to the drugs used for treatment.
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  • Masaya Uchida, Shota Takumi, Keiko Tachikawa, Ryoko Yamauchi, Yoshiyuk ...
    Volume 37 (2012) Issue 2 Pages 245-254
    Released: April 01, 2012
    JOURNALS FREE ACCESS
    Using glyphosate agrochemical components, we investigated their acute toxicity to juvenile Japanese medaka (Oryzias latipes) as well as their toxic impact at gene expression level on the liver tissues of adult medaka using DNA microarray. In our acute toxicity test, juvenile medaka were exposed for 96 hr to each of the following glyphosate agrochemical components: 10~160 mg/l of glyphosate, 1.25~20 mg/l of fatty acid alkanolamide surfactant (DA), and 12~416 mg/l of a fully formulated glyphosate herbicide. As a result, LC50 values of glyphosate, DA, and the glyphosate herbicide were > 160 mg/l, 8.5 mg/l, and 76.8 mg/l, respectively. On the other hand, adult male medaka fish were exposed to each of the glyphosate agrochemical components for 48 hr at the following concentrations: 16 mg/l of glyphosate, 0.5 mg/l of DA, and 16 mg/l-glyphosate/0.5 mg/l-DA mixture. Interestingly, DNA microarray analysis revealed that there were no significant gene expression changes in the medaka liver after exposure to glyphosate. Nevertheless, 78 and 138 genes were significantly induced by DA and the glyphosate/DA mixture, respectively. Furthermore, we identified five common genes that were affected by DA and glyphosate/DA mixture. These results suggested that glyphosate itself possessed very low toxicity as previously reported by some researchers at least to the small laboratory fish, and the major toxicity of the glyphosate agrochemical resided mainly in DA and perhaps in unintentionally generated byproduct(s) of glyphosate-DA mixture.
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  • Katsumi Kobayashi, Kalathil Sadasivan Pillai, Mathews Michael, Kottura ...
    Volume 37 (2012) Issue 2 Pages 255-260
    Released: April 01, 2012
    JOURNALS FREE ACCESS
    Repeated dose toxicity studies with rodents are regulatory requirements for registering chemical substances like drugs and pesticides with the government regulatory agencies. Usually 4 groups of animals, including a control group, are used in repeated dose toxicity studies. Williams’ test, Dunnett’s test and Jonckheere’s trend test are generally used to evaluate the data obtained from these studies. Selection of a statistical tool is relatively easy, when the data obtained from the groups of animals show a dose-dependency. But, occasionally a significance difference, compared to control, is not seen in the mid-dose group alone, thus losing the dose-dependency. We attempted to find the appropriate statistical tool for analyzing the quantitative data obtained from repeated dose toxicity studies, when the data of the mid-dose group alone do not show a significant difference, compared to control. The commonly used Williams’ test to analyse such data has a disadvantage as it assigns an estimated mean value for the mid-dose group, rather than the original mean value, for the analysis. Hence, it is likely that Williams’ test may misjudge in establishing a dose dependency, when in reality it does not exists. Therefore, to analyse such data we suggest the use of Dunnett’s multiple comparison test, to compare each dose group with the control, followed by Jonckheere’s trend test for examining dose dependency.
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  • Sora Park, You-Jin Choi, Byung-Hoon Lee
    Volume 37 (2012) Issue 2 Pages 261-267
    Released: April 01, 2012
    JOURNALS FREE ACCESS
    Intracellular accumulation of phospholipids with lamellar bodies is a hallmark of drug-induced phospholipidosis (PLD) which is caused by impaired phospholipid metabolism of the lysosome. Although it remains uncertain whether PLD is associated with the adverse effects, sponsors generally terminate the development of a candidate drug when PLD is observed in an organ. For drugs that are used without serious adverse events, there should be labels indicating that the drug can induce PLD. We conducted LipidTox and NBD-PE assays for detecting PLD to compare and validate the methods. In the case of contrary results in both assays, electron microscopy was performed to confirm the data. We selected 12 chemicals and divided them into 4 categories: P+S+, PLD and steatosis positive; P+/S-, PLD positive and steatosis negative; P-S+, PLD negative and steatosis positive; P-/S-, PLD and steatosis negative. In general, results showed very good agreement with the known information with some minor discrepancies. LipidTox assay is proven to be a very sensitive method. Considering the contrary results of acetaminophen and menadione in LipidTox and the NBD-PE assay, the combination of two methods using different phospholipids is advantageous to reduce false positives. The finding that acetaminophen was positive in LipidTos assay and increased the frequency of lamellar body implies that acetaminophen is a weak inducer of PLD.
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  • Kazuhiko Hayashi, Taeko Mori, Takayuki Abo, Mirei Koike, Yutaka Takaha ...
    Volume 37 (2012) Issue 2 Pages 269-280
    Released: April 01, 2012
    JOURNALS FREE ACCESS
    For the evaluation of eye irritation, one in vitro alternative test may not completely replace the Draize test. Therefore, a tiered approach combining several in vitro assays, including cytotoxicity assays, is proposed in order to estimate the eye irritation potential of a wide range of chemical classes. The Short Time Exposure (STE) test, a relatively newer alternative eye irritation test, involves exposing Statens seruminstitut rabbit cornea (SIRC) cells for 5 min to two concentrations (5% and 0.05%) of test material. In the present study, we examined the predictive capacity of a tiered approach analyzing the results from the STE test and then the results of the bovine corneal opacity and permeability (BCOP) assay for assessing globally harmonized system (GHS) eye irritation rankings of various chemicals. The accuracy of predicting the GHS rankings was slightly improved when the tiered approach combination of STE test and BCOP assay was used compared to when the STE test irritation rank classification was used alone. Moreover, the under prediction rate was substantially improved when this tiered approach was used. From these results, the tiered approach of combining the data analysis of the STE test and BCOP assay might be a promising alternative eye irritation test strategy.
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  • Nariaki Fujimoto, Kaoru Inoue, Midori Yoshida, Akiyoshi Nishikawa, Sho ...
    Volume 37 (2012) Issue 2 Pages 281-286
    Released: April 01, 2012
    JOURNALS FREE ACCESS
    The present study examined hepatic estrogen receptor (ER) and androgen receptor (AR) levels as well as estrogen-signaling status in a model of rat hepatic hypertrophy induced by phenobarbital (PB), chlofibrate (CF), or piperonyl butoxide (PBO). Male F344 rats were fed with PB at 2,500 ppm, CF at 2,500 ppm, and PBO at 20,000 ppm for 3 days, 4 weeks, and 13 weeks. CF and PBO induced diffuse hypertrophy, while centrilobular hypertrophy was observed with PB administration. The levels of mRNA for ERα, AR and leukemia inhibitory factor receptor (LIFR) which was found to be estrogen responsive in the present study, were determined by quantitative RT-PCR. In the CF and PBO groups, ERα mRNA expression was reduced, and consequently, the expression of a responsive gene, LIFR, was also decreased, while PB had no effect on ER mRNA levels. AR mRNA expression decreased in all the treated groups, but reduction was persistent only in PB group. Recently, LIFR was identified as a tumor suppressor gene in human HCC. Thus, LIFR may be one of the key mediators of hepatic carcinogenesis induced by CF and PBO, but PB appears to act via different mechanisms.
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  • Katsuhiko Warita, Tomoko Mitsuhashi, Yoshiaki Tabuchi, Ken-ichi Ohta, ...
    Volume 37 (2012) Issue 2 Pages 287-295
    Released: April 01, 2012
    JOURNALS FREE ACCESS
    This study investigated the deleterious effects of the synthetic non-steroidal estrogen diethylstilbestrol (DES) on testicular Leydig cells and compared these effects with those of the natural estrogen 17β-estradiol (E2). For that purpose, we performed microarray analysis of a mouse Leydig cell line (TTE1) treated with these estrogens, followed by Gene Ontology (GO) analysis and parametric analysis of gene set enrichment (PAGE). Most notably, GO analysis revealed a significant decrease in the biological processes of the GO categories “DNA repair” and “apoptotic program” in DES-exposed cells. PAGE showed that “cell death,” which is a superior GO category including apoptosis in the GO tree structure, significantly decreased in DES-exposed cells but significantly increased in E2-exposed cells. Interestingly, only 2 genes (Tia1 and Gas1) with altered expression patterns in the “cell death” category were common between DES- and E2-treated cells. The downregulation of apoptotic cell death pathways and DNA repair capability of DES-exposed cells implies that DES promotes carcinogenic processes more strongly than E2 does. These findings suggest that molecular events that occur following DES and E2 treatments differ substantially in Leydig cells, and that the effects of synthetic estrogen and natural estrogen differ more substantially than previously suspected.
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  • Yuri Hojo, Ayako Shiraki, Takuma Tsuchiya, Keisuke Shimamoto, Yuji Ish ...
    Volume 37 (2012) Issue 2 Pages 297-306
    Released: April 01, 2012
    JOURNALS FREE ACCESS
    To investigate the liver tumor-promoting effects of etofenprox (ETF), a pyrethroid-like insecticide, 6 week-old male F344 rats were given an intraperitoneal injection of N-diethylnitrosamine (DEN). After 2 weeks from the DEN treatment, 12 rats per group received a powdered diet containing 0, 0.25, 0.50, or 1.0% ETF for 8 weeks. At the time of 2nd week of ETF administration, all animals were subjected to two-thirds partial hepatectomy (PH). One rat per group except for the 0.25% ETF group died due to surgical operation of PH. The number and area of glutathione S-transferase placental form (GST-P) positive foci significantly increased in the livers of DEN-initiated rats given 0.50% and 1.0% ETF compared with the DEN-alone group. Quantitative real-time RT-PCR analysis revealed that the mRNA expression of phase I enzymes Cyp2b1/2, phase II enzymes such as Akr7a3, Gsta5, Ugt1a6, Nqo1 significantly increased in the DEN+ETF groups. The immunohistochemistry showed the translocation of CAR from the cytoplasm to the nuclei of hepatocytes in the ETF-treated groups. Reactive oxygen species (ROS) production increased in microsomes isolated from the livers of ETF-treated rats, and thiobarbituric acid-reactive substances (TBARS) levels and 8- hydroxy-2-deoxyguanosine (8-OHdG) content significantly increased in all of the ETF-treated groups and DEN+1.0% ETF group, respectively. The results of the present study indicate that ETF has a liver tumor-promoting activity in rats, and suggest that ETF activates the constitutive active/androstane receptor (CAR) and enhances microsomal ROS production, resulting in the upregulation of Nrf2 gene batteries; such an oxidative stress subsequently induces liver tumor-promoting effects by increased cellular proliferation.
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  • Tomoko Shindo, Yukiko Kanazawa, Yoshiaki Saito, Kohichi Kojima, Motoya ...
    Volume 37 (2012) Issue 2 Pages 307-315
    Released: April 01, 2012
    JOURNALS FREE ACCESS
    It is important to evaluate the ability of novel proteins in food crops and products to elicit potentially harmful immunologic responses, including allergic hypersensitivity. We developed a novel mouse model of food allergy involving an oral challenge of a protein antigen after feeding of the antigen in combination with modulating factors often ingested in daily life, namely, dietary oil emulsion and salicylate. In the model, BALB/c mice were sensitized orally for three weeks with ovalbumin (OVA) in linoleic acid/lecithin emulsion, followed immediately by intraperitoneal injection of sodium salicylate. At the end of the sensitization, the incidence of mice positive for serum OVA-specific IgG1 but not IgE had significantly increased in the combined-sensitization group. After the 3-week sensitization, a single or double oral challenge with OVA effectively and significantly caused severe anaphylaxis, as compared with the groups sensitized with OVA in the emulsion or the vehicle alone. Moderate increase of plasma histamine and intestinal abnormality in histology was found only in the combined-sensitization group. Anaphylaxis symptoms in the sensitized mice were induced more by oral challenge than by intravenous challenge, suggesting a critical role for the mucosal system. This is the first model for successful induction of oral anaphylaxis in mice sensitized by feeding of food protein without adjuvant. It will be useful to elucidate the mechanism of food allergy and to detect modulating factors of oral allergy at sensitization using this model, which simulates real life conditions.
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  • Yoko Sagawa, Mitsuru Futakuchi, Jiegou Xu, Katsumi Fukamachi, Yuto Sak ...
    Volume 37 (2012) Issue 2 Pages 317-327
    Released: April 01, 2012
    JOURNALS FREE ACCESS
    Nano-sized titanium dioxide particles (TiO2) are widely used in cosmetics, sunscreens and food additives. We previously reported that topical application of non-coated rutile type TiO2 did not exhibit a promoting effect on ultraviolet B-initiated skin carcinogenesis in rats, and that this was likely due to lack of penetration of TiO2 into the epidermis. In the present study, we examined the promoting effect of silicone coated TiO2 (sTiO2) suspended in silicone oil and non-coated TiO2 (ncTiO2) suspended in Pentalan 408 on a two-stage skin chemical carcinogenesis model: sTiO2 suspended in silicon oil forms smaller particles than ncTiO2 suspended in Pentalan because of the smaller sizes of aggregates formed. The model used skin carcinogenesis-sensitive human c-Ha-ras proto-oncogene transgenic mice (rasH2) and rats (Hras128) and their wild-type counterparts and CD-1 mice to test the effects of topical application of TiO2. Animals were initially treated with a single dose of 7,12-dimethylbenz[a]anthracene (DMBA) and then with 0, 10, or 20 mg sTiO2 (mice) or 0, 50, or 100 mg ncTiO2 (rats). The incidence and multiplicity of skin tumors (squamous cell papilloma and carcinoma) did not increase over DMBA alone controls in skin carcinogenesis-sensitive mice or rats or wild-type animals. Analysis of rat skin indicated that sTiO2 and ncTiO2 did not penetrate though either healthy or damaged skin. Furthermore sTiO2 did not penetrate an in vitro human epidermis model. Our results indicate that treatment with sTiO2 or ncTiO2 did not promote skin carcinogenesis in mice or rats, probably due to lack of penetration through the epidermis.
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  • Yasutomo Yamada, Takayuki Mokudai, Keisuke Nakamura, Eisei Hayashi, Yo ...
    Volume 37 (2012) Issue 2 Pages 329-335
    Released: April 01, 2012
    JOURNALS FREE ACCESS
    The present study aimed to evaluate the acute locally injurious property of hydroxyl radical generation system by photolysis of H2O2, which is a new disinfection system for the treatment of periodontitis developed in our laboratory. Firstly, generation of the hydroxyl radical by a test device utilizing the photolysis of H2O2 was confirmed by applying an electron spin resonance (ESR)-spin trapping technique. Secondly, the bactericidal effect of the device was examined under a simulant condition in which Staphylococcus aureus suspended in 1 M H2O2 was irradiated with laser light emitted from the test device, resulting in substantial reduction of the colony forming unit of the bacteria within a short time as 2 min. Finally, acute topical effect of the disinfection system on rat oral mucosa and wounded skin was evaluated by histological examination. No abnormal findings were observed in the buccal mucosal region treated three times with 1 M H2O2 and irradiation. Similarly, no abnormal findings were observed during the healing of skin treated with 1 M H2O2 and irradiation immediately after wounding. Since topical treatment with the novel disinfection technique utilizing the photolysis of H2O2 had no detrimental effect on the oral mucosa and the healing of full thickness skin wounds in rats, it is expected that the acute locally injurious property of the disinfection technique is low.
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  • Takashi Mori, Yoshiharu Tanimoto, Mika Ota, Takaomi Masakado, Sachiko ...
    Volume 37 (2012) Issue 2 Pages 337-343
    Released: April 01, 2012
    JOURNALS FREE ACCESS
    Respiratory sensitization to certain low molecular weight chemicals is a big concern for workers, but unfortunately there are no validated animal models to allow identification of sensitizing chemicals in the environment. In the present study, dermally sensitized and intratracheally challenged mice were used to investigate effective indicators of respiratory sensitizers. Changes in levels of total serum IgE and nine cytokines (G-CSF, IL-4, IL-5, IL-6, IL-12(p70), IL-13, IFN-γ, MCP-1 and TNF-α) in bronchoalveolar lavage fluid (BALF) were analyzed in BALB/c mice exposed to respiratory sensitizers (phthalic anhydride (PA); diphenylmethane-4,4’-diisocyanate (MDI); toluene diisocyanate (TDI); chloramine-T (CH); and piperazine (PI)) or contact sensitizers (2,4-dinitrochlorobenzene (DNCB); and oxazolone (OXA)). Non-sensitized mice were treated dermally with solvents and challenged intratracheally with the respective test chemicals as solvent controls. Increases in total serum IgE levels were observed in all treated mice, with apparent differences in cytokine profiles. PA caused statistically significant increases in Th2 cytokines, IL-4, IL-5 and IL-13, compared with the control. IL-5 was also found to be increased with CH. The other three respiratory sensitizers caused statistically significant increases in IL-13. In contrast, no change was apparent with contact sensitizers, DNCB and OXA, in these Th2 cytokines. Increases in the Th2 cytokines indicate that all five respiratory sensitizers induced immune responses in lungs. Interestingly, elevation of G-CSF levels in BALF appeared with all five respiratory sensitizers but not the two contact sensitizers. The findings suggest that G-CSF could be effective to identify respiratory sensitizers in animal models.
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  • Takumi Ohishi, Liyun Wang, Hirotoshi Akane, Ayako Shiraki, Akira Sato, ...
    Volume 37 (2012) Issue 2 Pages 345-352
    Released: April 01, 2012
    JOURNALS FREE ACCESS
    To clarify the effect of systemic growth retardation on behavior, pregnant rats were fed a synthetic diet with either a normal (20% casein) or low (10% casein) protein concentration from gestational day 10 to postnatal day (PND) 21 at weaning. Offspring were examined for sensory and reflex functions, detailed clinical observations, manipulative test, grip strength, motor activity and water-filled multiple T-maze test. Lowering trend in the air righting reflex index during lactation period and a decrease in grip strength on PND 72 were observed in the low protein diet group showing suppression of systemic growth. However, they were simply the reflection of delayed systemic growth, because parameters on impaired reflex function, disturbance of motor function and paralysis were unaffected. On the other hand, low protein diet resulted in increased motor activity in female offspring. Thus, malnutrition due to maternal protein restriction may cause adolescent hyperactivity.
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  • Mika Takahashi, Hina Kato, Yuko Doi, Akihiro Hagiwara, Mutsuko Hirata- ...
    Volume 37 (2012) Issue 2 Pages 353-361
    Released: April 01, 2012
    JOURNALS FREE ACCESS
    To obtain initial information on the possible repeated-dose oral toxicity of fullerene C60, Crl:CD(SD) rats were administered fullerene C60 by gavage once daily at 0 (vehicle: corn oil), 1, 10, 100, or 1,000 mg/kg/day for 29 days, followed by a 14-day recovery period. No deaths occurred in any groups, and there were no changes from controls in detailed clinical observations, body weights, and food consumption in any treatment groups. Moreover, no treatment-related histopathological changes were found in any organs examined at the end of the administration period and at the end of the recovery period. Blackish feces and black contents of the stomach and large intestine were observed in males and females at 1,000 mg/kg/day in the treatment group. There were no changes from controls in the liver and spleen weights at the end of the administration period, but those weights in males in the 1,000 mg/kg/day group increased at the end of the recovery period. Using liquid chromatography-tandem mass spectrometry, fullerene C60 were not detected in the liver, spleen or kidney at the end of the administration period and also at the end of the recovery period. In conclusion, the present study revealed no toxicological effects of fullerene C60; however, the slight increases in liver and spleen weights after the 14-day recovery period may be because of the influence of fullerene C60 oral administration. In the future, it will be necessary to conduct a long-term examination because the effects of fullerene C60 cannot be ruled out.
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  • Tilak Khanal, Hyung Gyun Kim, Jae Ho Choi, Bong Hwan Park, Minh Truong ...
    Volume 37 (2012) Issue 2 Pages 363-371
    Released: April 01, 2012
    JOURNALS FREE ACCESS
    Baicalin, a glycoside present in Scutellaria baicalensis Georgi, is metabolized to its aglycone, baicalein, in intestine. In the present study, possible role of metabolism of baicalin by intestinal bacteria to baicalein in baicalin-induced toxicity was investigated in HepG2 cell cultures. As an intestinal bacterial metabolic system for baicalin, human fecal preparation containing intestinal microflora (fecalase) was employed. Initially, when cytotoxic effects of baicalin and baicalein were compared, baicalin was more cytotoxic than baicalein in HepG2 cells. When baicalin was incubated with fecalase, it was metabolized to baicalein. In addition, baicalin-incubated with fecalase reduced cytotoxicity of HepG2 cells in a concentration-dependent manner. Moreover, baicalin-incubated with fecalase significantly caused an increase in Bcl-2 expression together with a decrease in Bax expression and cleaved Caspase-3. Furthermore, anti-apoptotic effect by the incubation of baicalin with fecalase was also confirmed by the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end labeling assay. Taken all together, the findings suggested that metabolism of baicalin by human fecalase to baicalein might have protective effects against baicalin-induced toxicity in HepG2 cells.
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  • Katsuhide Igarashi, Satoshi Kitajima, Ken-ichi Aisaki, Kentaro Tanemur ...
    Volume 37 (2012) Issue 2 Pages 373-380
    Released: April 01, 2012
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    The human steroid and xenobiotic receptor (SXR), (also known as pregnane X receptor PXR, and NR1I2) is a low affinity sensor that responds to a variety of endobiotic, nutritional and xenobiotic ligands. SXR activates transcription of Cytochrome P450, family 3, subfamily A (CYP3A) and other important metabolic enzymes to up-regulate catabolic pathways mediating xenobiotic elimination. One key feature that demarcates SXR from other nuclear receptors is that the human and rodent orthologues exhibit different ligand preference for a subset of toxicologically important chemicals. This difference leads to a profound problem for rodent studies to predict toxicity in humans. The objective of this study is to generate a new humanized mouse line, which responds systemically to human-specific ligands in order to better predict systemic toxicity in humans. For this purpose, the ligand binding domain (LBD) of the human SXR was homologously knocked-in to the murine gene replacing the endogenous LBD. The LBD-humanized chimeric gene was expressed in all ten organs examined, including liver, small intestine, stomach, kidney and lung in a pattern similar to the endogenous gene expressed in the wild-type (WT) mouse. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that the human-selective ligand, rifampicin induced Cyp3a11 and Carboxylesterase 6 (Ces6) mRNA expression in liver and intestine, whereas the murine-selective ligand, pregnenolone-16-carbonitrile did not. This new humanized mouse line should provide a useful tool for assessing whole body toxicity, whether acute, chronic or developmental, induced by human selective ligands themselves and subsequently generated metabolites that can trigger further toxic responses mediated secondarily by other receptors distributed body-wide.
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  • Ryo Kobayashi, Tsuyoshi Nakanishi, Hisamitsu Nagase
    Volume 37 (2012) Issue 2 Pages 381-387
    Released: April 01, 2012
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    Trichloroethylene (TCE) is suspected as a potent immunomodulator that accelerates the development of allergic diseases. We previously reported that TCE promotes ovalbumin (OVA)-induced active cutaneous anaphylaxis, including enhancing antigen-specific serum IgE levels and splenic lymphocyte proliferation. However, the target cells and molecular mechanism through which TCE modulates antigen-specific immune responses remain unclear. To identify a potential underlying mechanism, we investigated whether TCE modulates T cell receptor (TCR)-induced T cell activation and proliferation in vitro. TCE enhanced T cell proliferation primed by anti-CD3 antibody, but not concanavalin A, in a dose-dependent fashion. In addition, TCE enhanced anti-CD3-primed proliferation of CD8+ rather than CD4+ T cells. Consistent with this result, TCE markedly enhanced the Lck phosphorylation mediated by anti-CD3 antibody in CD8+ but not CD4+ T cells. Furthermore, we analyzed the effect of TCE exposure via drinking water for 2 weeks on splenocyte populations in non-immunized and OVA-immunized mice. In OVA-immunized mice, TCE (3 mg/l) significantly expanded CD3+, CD8+ and CD4+ cell populations, however the effect at the lower concentration was significant only in the CD8+ populations, whereas TCE had no effect on these cells population in non-immunized mice. These findings suggest that TCE enhances TCR-CD3-induced proliferation of CD8+ rather than CD4+ cells and disrupts various activities of peripheral T cells.
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  • Ayako Kanno, Tsuyoshi Kawakami, Yasuo Takahashi, Sukeo Onodera
    Volume 37 (2012) Issue 2 Pages 389-400
    Released: April 01, 2012
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    A reproducible method for monitoring traces of cholinesterase (ChE) inhibitors in aqueous samples is described: the method is based on chemical oxidation and a ChE inhibition assay. Chlorine was tested as an oxidizing reagent for conversion of various thiophosphorus pesticides (P=S compounds) into their P=O analogs, which have higher ChE-inhibiting activity. After treating buffered pesticide solutions (pH 6.0) with chlorine (final concentration less than 10 mg/l) of at 25°C for 15 min, the ChE-inhibiting activities and detection limits for each pesticide were determined. Greater ChE-inhibiting activities, leading to lower detection limits (ppb levels), were observed for the chlorine-treated solutions fortified azinphos methyl, diazinon, isoxathion and ronnel etc. No changes in the ChE-inhibiting activities were observed for carbamate pesticide solutions tested before and after chlorination, but an additive effect showed against ChE when these compounds were mixed with paraoxon in water. This combination of oxidative derivatization and ChE inhibition assay was applied successfully to the detection and determination of ChE inhibitors in natural and drinking water samples.
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  • Yoshimasa Kurata, Nobuaki Shimamura, Masanobu Katoh
    Volume 37 (2012) Issue 2 Pages 401-414
    Released: April 01, 2012
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    Di(2-ethylhexyl)phthalate (DEHP) is known to be a reproductive toxicant in male rodents, and its primary or secondary metabolites seem to be causative agents. To identify and quantify urinary metabolites of DEHP in humans, urine samples collected from healthy male and female volunteers following oral administration of deuterium labeled DEHP (d4-DEHP) at 3 mg/person were analyzed by a high performance liquid chromatograph/mass spectrometer (LC-MS), and the excretion behavior of orally taken DEHP was evaluated. Mono(2-ethylhexyl)phthalate (MEHP), OH-MEHP, oxo-MEHP, COOH-MEHP, and their glucuronides were identified as metabolites in the male and female urine. The ratios of the conjugate forms in the total urinary metabolites were remarkably high from immediately after administration (0 to 4-hr post-dose), which were approximately 69% to 86% (male) and 80% to 89% (female) until 36-hr post-dose. It was suggested that DEHP taken orally was promptly converted to MEHP and its oxidative metabolites such as OH-MEHP, oxo-MEHP, and COOH-MEHP, and most of these metabolites received the conjugation reaction and were excreted as glucuronides. Remarkable differences from rodents, in which most of these metabolites were excreted as free forms, were demonstrated.
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  • Pin Gong, Fuxin Chen, Xiaoying Liu, Xing Gong, Jing Wang, Yangmin Ma
    Volume 37 (2012) Issue 2 Pages 415-425
    Released: April 01, 2012
    JOURNALS FREE ACCESS
    Cadmium (Cd) is classified as an environmental pollutant and human carcinogen. Caffeic acid phenethyl ester (CAPE), a biological active component of honeybee propolis extracts, has been used as a folk medicine with no harmful effects on normal cells. Here we investigated the beneficial effect of CAPE on Cd-induced renal damage in mice. Since renal damage induced by Cd (II) is related to oxidative stress, lipid peroxidation (LPO), protein carbonyl (PCO), superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) were evaluated. Moreover, the concentrations of Cd and zinc (Zn) in the kidney were analyzed. The intoxication of Cd (II) leads to the enhanced production of LPO and PCO, and the decrease of SOD activity and GSH level, probably due to the serious oxidative stress. However, the activities of CAT in the Cd (II)-induced group showed an elevated tendency, probably relating to an adaptive-response to the oxidative damage. The co-administration of CAPE can attenuate the oxidative stress caused by the intoxication of Cd and restore the altered antioxidant defense system. Based on our data, it is proposed that CAPE may involve in the protection of renal damage induced by Cd (II) owing to its antioxidant capacity and anti-inflammatory effect.
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Letter
  • Keiichi Itoh, Shoji Masumori, Madoka Nakajima, Makoto Hayashi, Hiroyuk ...
    Volume 37 (2012) Issue 2 Pages 427-430
    Released: April 01, 2012
    JOURNALS FREE ACCESS
    Mammals, including human beings, have a circadian clock system to regulate behavioral and physiological processes. In this study, we investigated the effect of dosing time on micronucleus induction in the bone marrow by evaluating the frequencies of micronucleated peripheral reticulocytes (MNRETs) in mice exposed to N-ethyl-N-nitrosourea (ENU) to assess any difference in genotoxic sensitivity to chemicals between light and dark periods (inactive phase for rodents and active phase for rodents). Male C3H/He mice were treated intraperitoneally with ENU (12.5 or 25 mg/kg body weight) at zeitgeber time (ZT) 3 in the light period or ZT15 in the dark period, and then the time courses of the frequencies of the MNRETs were determined. The frequencies of the MNRETs induced by ENU increased time-dependently and peaked at 48 hr after treatment for ZT3 and ZT15, and were obviously higher in the ZT15 treatment group than the ZT3 treatment group. The MNRETs were measured at 48 hr after treatment with ENU (25 mg/kg body weight) at various dosing times (ZT0, 3, 6, 12, 15 and 18). The frequencies of the MNRETs in mice treated at ZT0, 15 and 18 were significantly higher than those in mice treated at ZT3, 6 and 12. These results suggest that genotoxic sensitivity to chemicals in mouse bone marrow is different between light and dark periods maybe due to different biological responses (detoxification, cell cycle, DNA repair, etc.) related to circadian rhythms.
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  • Hae Kyung Lee, Tae Sung Kim, Chang Yeong Kim, Il Hyun Kang, Mi Gyeong ...
    Volume 37 (2012) Issue 2 Pages 431-437
    Released: April 01, 2012
    JOURNALS FREE ACCESS
    The estrogenic activity of industrial chemicals, di(2-ethylhexyl) phthalate (DEHP), di(n-butyl) phthalate (DBP), benzylbutyl phthalate (BBP), diethyl phthalate (DEP), tetrabromobisphenol A (TBBPA), bisphenol A (BPA), and nonylphenol (NP), was compared using OECD test guideline 455(TG455), stably transfected transcriptional activation (STTA) and estrogen receptor (ER) binding assays. The estrogenic activity of BBP, BPA and NP were approximately 180,000-fold (PC50, 4.32 x 10-6 M), 5,000-fold (PC50, 1.26 x 10-7 M) and 120,000-fold (PC50, 2.92 x 10-6 M) less than 17β-estradiol (PC50, 2.43 x 10-11M), whereas DEHP, DBP and DEP did not show any estrogenicity activity in the STTA assay. Moreover, binding affinities to human ERα of BBP, BPA, and NP were approximately 200,000-fold (IC50, 4.91 x 10-4 M), 8000-fold (IC50, 1.92 x 10-5 M) and 1400-fold (IC50, 3.34 x 10-6 M) less than 17β-estradiol (IC50, 2.45 x 10-9 M) in competitive human ERα binding assay. The relative potencies of STTA assay were very similar to ER binding, E-screen, and Yeast screening assays. Therefore, our results suggested that OECD test guideline TG455 may be useful as a screening test for potential endocrine disruptors.
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  • Makoto Seo, Ryo Kobayashi, Tetsunori Okamura, Koji Ikeda, Masahiko Sat ...
    Volume 37 (2012) Issue 2 Pages 439-445
    Released: April 01, 2012
    JOURNALS FREE ACCESS
    Trichloroethylene (TCE) and tetrachloroethylene (perchloroethylene; PCE) are commonly identified as environmental contaminants of groundwater. Previously, we investigated the enhancing effects of TCE and PCE on antigen-induced histamine release and inflammatory mediator production in rat mast cells. In this study, to examine the potential effect of TCE and PCE on antigen-induced histamine release from mouse mast cells, mouse bone marrow-derived mast cells (BMMC) were sensitized with anti-dinitrophenol (DNP) monoclonal IgE antibody and then stimulated with DNP-BSA containing with TCE or PCE. Both TCE and PCE significantly enhanced antigen-induced histamine release from BMMC. Next we investigated the effects of TCE and PCE on the passive cutaneous anaphylaxis (PCA) reaction in vivo using ICR mice. TCE and PCE significantly enhanced the PCA reaction in a dose-dependent manner. In addition, we examined the enhancing effects of ingesting small amount of TCE and PCE in drinking water on antigen-stimulated allergic responses. After the ICR mice had ingested TCE or PCE in their drinking water for 2 or 4 weeks, we performed the PCA reaction. Both TCE and PCE ingestion enhanced the PCA reaction in a dose-dependent manner for 4 weeks. These results suggest that exposure to TCE and PCE leads to the augmentation of type I allergic responses in many species.
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  • Lillian Zhang, Anthony Rowe, Filip Braet, Iqbal Ramzan
    Volume 37 (2012) Issue 2 Pages 447-453
    Released: April 01, 2012
    JOURNALS FREE ACCESS
    Liver toxicity is a side effect observed with some herbal treatments, including Piper methysticum. The possible mechanisms responsible include inflammation subsequent to activation of liver macrophages and oxidative damage. Hepatotoxicity of the pharmacologically active component of Piper methysticum (kavalactones) was tested in isolated, perfused livers from rats which were pretreated with the macrophage intoxicant gadolinium chloride. Perfusions without kavalactones in gadolinium chloride pretreated and untreated livers were included as negative controls. Serial liver lobe biopsies were collected to measure temporal changes in available (reduced) hepatic glutathione. There were no statistically significant changes in reduced glutathione over the course of perfusion in any experimental group. Liver damage was observed using electron microscopy. Hepatic sinusoids displayed extensive damage to the endothelium in kavalactone-perfused, rat livers. This damage was significantly reduced by pre-treatment with gadolinium chloride. Hence liver macrophages may be a factor in liver injury induced by Piper methysticum. Characterisation and modulation of the liver macrophage response may enable the development of strategies to avoid these hepatic side effects.
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