The Journal of Toxicological Sciences

Description

  • Impact factor
    1.38
  • 5-year impact
    0.00
  • Cited half-life
    4.20
  • Immediacy index
    0.27
  • Eigenfactor
    0.00
  • Article influence
    0.00
  • ISSN
    1880-3989

Publications in this journal

  • [Show abstract] [Hide abstract]
    ABSTRACT: Anti-angiogenic drugs that target Vascular Endothelial Growth Factor (VEGF) signaling pathways caused hypertension as an adverse effect in clinical studies. Since the hypertension may limit the benefit provided for patients, the demand for non-clinical research that predicts the clinical risk of the hypertension has risen greatly. To clarify whether non-clinical research using rats can appropriately estimate the clinical risk of hypertension caused by VEGF signal inhibitors, we investigated the hemodynamic effects and pharmacokinetics (PK) of the VEGF signal inhibitors cediranib (0.1, 3, and 10 mg/kg), sunitinib (5, 10, and 40 mg/kg), and sorafenib (0.1, 1, and 5 mg/kg) in telemetered rats and examined the correlation between the non-clinical and the clinical hypertensive effect. The VEGF signal inhibitors significantly elevated blood pressure (BP) in rats within a few days of the initiation of dosing, and levels recovered after dosing ended. The trend of the hypertension was similar to that in clinical studies. We found that the AUC at which BP significantly increased by approximately 10 mmHg in rats was comparable to the clinical AUC at which moderate to severe hypertension occurred. These results represent correlations between the non-clinical and the clinical hypertensive effect of VEGF signal inhibitors, suggesting that non-clinical research using telemetered rats would be an effective approach to predict the clinical risk of hypertension caused by VEGF signal inhibitors.
    The Journal of Toxicological Sciences 01/2014; 39(2):237-42.
  • [Show abstract] [Hide abstract]
    ABSTRACT: Spontaneous multiple granulomas were present in the animal under the SPF condition and without chemical treatment, in a 19-week-old male Sprague-Dawley control-group rat. Here we describe multiple granulomas and prominent diffuse infiltration by eosinophils in the cecal submucosa, and arteritis in the mesenteric arteries. The multiple granulomas were characterized by central eosinophilic degeneration or necrosis, prominent eosinophils, many multi-nucleated giant cells and abundant fibroblasts. They were restricted to the cecal submucosa. The mesenteric arteritis consisted of fibrinoid necrosis of the intima and media, intense inflammatory cell infiltration and fibrosis in the arterial wall. An affected artery in the cecum was continuous with the mesenteric artery. The foregoing tissue changes in this rat correlate with the high absolute blood eosinophil count found in this animal.
    The Journal of Toxicological Sciences 01/2014; 39(2):243-9.
  • [Show abstract] [Hide abstract]
    ABSTRACT: A widely-used plasticizer di(2-ethylhexyl) phthalate (DEHP) is known to induce apoptosis in neurons, although the mechanisms responsible for DEHP-induced apoptosis is not well explored yet. We recently showed that exposure to DEHP increases the expression of hemeoxygenase (HO)-1, an oxidative stress related enzyme, in the mice brain. In this study, we investigated whether HO-1 is involved in DEHP-induced apoptosis using a mouse neuroblastoma cell line Neuro-2a, which forcibly express SCAT3, a fluorescent indicator of caspase-3 activity. The doses of DEHP at 1, 10 or 100 µM were used in the present study to mimic the level of human exposure to DEHP. Live image analysis of SCAT3-expressing Neuro-2a cells revealed that caspase-3 activity in the cells was significantly increased by DEHP at 100 µM but not 1 or 10 µM. We measured HO-1 mRNA level in Neuro-2a cells exposed to DEHP and found significant increase in HO-1 mRNA level by DEHP at 100 µM but not 1 or 10 µM. Live image analysis of SCAT3-expresisng Neuro-2a cells was further performed to determine the effects of HO-1 siRNA in DEHP-induced apoptosis via caspase-3 activation. We found that knockdown of HO-1 gene nullifies the effects of DEHP to activate caspase-3. These results suggest that HO-1 is involved in DEHP-induced apoptosis. Moreover, this study demonstrates that high-dose DEHP exposure induces caspase-3-dependent apoptosis, which is at least partially mediated by the up-regulation of HO-1 gene, in Neuro-2a cells.
    The Journal of Toxicological Sciences 01/2014; 39(2):217-29.
  • [Show abstract] [Hide abstract]
    ABSTRACT: We compared the cadmium (Cd) concentration in the liver and kidney of different strains of mice after exposure to 50 ppm Cd for 30 days via drinking water. Cd concentration in the liver and kidney of C57BL/6J mice were higher than those of 129/Sv and DBA/2 mice. Since orally ingested heavy metals are absorbed in the small intestine and then widely distributed to target tissues, microarray analyses were performed to compare the expression levels of transport-related genes in the duodenum between C57BL/6J mice and 129/Sv or DBA/2 mice. The expression levels of 9 and 11 genes were elevated more than 2.0-fold and 13 and 12 genes were reduced less than 0.5-fold in 129/Sv mice and DBA/2 mice, respectively. Among these low expressed genes, 10 genes (Slc2a2, Slc5a1, Slc16a2, Slc22a13, Slc22a18, Slc25a11, Slc36a1, Slco6c1, Abca3 and Abcd1) were common between the two types of strains. These results suggest that some of those genes might be involved in Cd absorption and its toxicity.
    The Journal of Toxicological Sciences 01/2014; 39(1):173-7.
  • [Show abstract] [Hide abstract]
    ABSTRACT: Sodium iron chlorophyllin (SIC), a water-soluble chlorophyll derivative, has been used as a food additive for green coloration. In the present study, a subchronic toxicity study of SIC was performed in male and female F344 rats with oral administration in diet at concentrations of 0%, 0.2%, 1.0%, and 5.0% for 13 weeks. No mortalities, abnormal clinical signs, and hematological changes were observed in any of the groups during the experiment. Significant reduction of body weight gain was noted in 5.0% males. In serum biochemistry, serum transferrin levels were significantly increased in 5.0% males and females. Relative spleen weights of both sexes were markedly reduced with 5.0% SIC as compared to the controls, and absolute weights of spleen were also significantly decreased in males. On histopathological assessment, diffuse hypertrophy of acinar cells in the parotid gland was observed in all examined 5.0% males and females, but not in the other groups. Based on the histopathology of the parotid glands, the no-observed-adverse-effect level (NOAEL) of SIC in the present study was estimated to be 1.0% (609 mg/kg bw/day for males and 678 mg/kg bw/day for females).
    The Journal of Toxicological Sciences 01/2014; 39(1):109-19.
  • [Show abstract] [Hide abstract]
    ABSTRACT: Heat shock protein 90 (Hsp90) is a constitutively expressed molecular chaperone and plays an important role in the folding of client proteins with key regulatory roles in growth, survival, differentiation and metastasis. Because inhibition of Hsp90 degrades multiple oncogenic client proteins, it is considered to be an attractive anticancer therapy, and clinical trials of several Hsp90 inhibitors have been carried out. In the present study, two structurally distinct Hsp90 inhibitors, CH5164840 and CH5449302, were orally administered to beagle dogs to evaluate systemic toxicity. CH5164840 induced symptoms that suggest visual disorder, and ophthalmological observation and electroretinography (ERG) revealed loss of pupillary light reflex and abnormal waveforms, respectively. Histopathological examination showed changes in the photoreceptor cell layer and the outer nuclear layer of retina. On the other hand, while there were no clinical symptoms related to visual disorder, animals treated with CH5449302 showed similar abnormalities of ERG responses and histopathological changes in the photoreceptor cell layer and the outer nuclear layer of retina. The visual symptoms and abnormalities of ERG responses were noted at an earlier stage or lower dose than other toxicities in both compounds. Considering that two structurally distinct Hsp90 inhibitors induced a retinal toxicity in dogs after repeated administration, and that visual disorders were also reported in some clinical trials of Hsp90 inhibitors, it would seem highly likely that Hsp90 inhibition induces retinal toxicity. Also, our study indicated that a detailed ocular examination to evaluate the safety of Hsp90 inhibitors would be useful in both preclinical and clinical studies.
    The Journal of Toxicological Sciences 01/2014; 39(1):59-69.
  • [Show abstract] [Hide abstract]
    ABSTRACT: The development of hepatitis is associated with the infiltration and activation of immune cells in liver. N-3 polyunsaturated fatty acids (n-3 PUFAs) rich fish oil (FO) is used to prevent and treat inflammatory diseases. But, the effects of dietary FO on autoimmune hepatitis remain largely unknown. In this study, Concanavalin A (Con A) induced hepatitis was used to evaluate the actions of dietary FO. Unexpectedly, 2-week FO treatment had not shown any protection, on the contrary, exacerbated liver injury in this hepatitis model. The levels of alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) statistically increased from 10,501 ± 2,154 and 30,394 ± 2,420 in low fat diet (LFD)/Con A group to 17,579 ± 693 and 49,439 ± 4,628 in FO/Con A group. Simultaneously, FO diet induced more necrotic liver tissues and apoptotic hepatocytes, and up-regulated the hepatic expression of TNF-α and IFN-γ after Con A challenge. Interestingly, FO promoted severe liver injury was accompanied by decreasing the percentage of CD4(+) T cell, NK1.1(+) cells and CD8(+) T cells in CD45(+) liver non-parenchymal hepatic cells (NPCs) through inducing apoptosis. Further experiments declared 2-week FO diet intake firstly increased the proportion of CD11b(+)Gr-1(hi) neutrophils in liver, but then dramatically expanded CD11b(+)Gr-1(int) inflammatory monocytes population after Con A administration. Collectively, our study indicated that high FO intake not only aggravated liver injury, but also altered the population of immune cells in liver. Thus, these results indicated that when dietary FO was used to benefit health in autoimmune diseases, its potential risks of side effect also need paying close attention.
    The Journal of Toxicological Sciences 01/2014; 39(2):179-90.
  • [Show abstract] [Hide abstract]
    ABSTRACT: Steroids are treated for most inflammatory diseases but cause serious side effects such as diabetes and osteoporosis after their long-term usage. Recently, we identified novel roles of Substance-P (SP) in the suppression of the injury-mediated inflammation and also in stem cell mobilization. In this study, for clinical application of SP as an anti-inflammatory agent, its safety in long-term usage was evaluated with regard to diabetes and osteoporosis. Dexamethasone (DEX) and methylprednisolone (MP) were used as comparative drugs. While DEX-injection for 24 weeks developed severe weight loss, unstable blood glucose, and bone loss, SP-injection did not affect blood glucose and bone mass. MP-injection for 24 weeks also influenced blood glucose and body weight much milder than DEX-injection. After 66 weeks, MP-injection caused unstable blood glucose, alleviation in the age-related increase of body weight, and bone weakness, which was featured by reduction in collagen deposition and trabecular bone volume based on histological and micro CT analysis. However, SP-injection for 66 weeks rather increased collagen deposition, bone volume, and bone density. Therefore, this comparative study suggests that SP, even after long-term usage of effective dose, may not cause side effects such as osteoporosis in comparison to that of DEX and MP and can be developed as an anti-inflammatory agent and/or stem cell mobilizer for long-term treatment.
    The Journal of Toxicological Sciences 01/2014; 39(3):391-9.
  • [Show abstract] [Hide abstract]
    ABSTRACT: The zebrafish has been considered as a suitable animal model for drug discovery, especially for evaluation of the teratogenicity, due to their small size, rapid development, transparency, and developmental similarities to mammalian development. These features of zebrafish make it possible to maintain them in culture plates, evaluate the teratogenicity in short term, conduct morphological assessment of each organ without any autopsy operation. The purpose of the present study was to improve an evaluation method for the teratogenicity of test compounds with high throughput ability and prediction rateusing zebrafish embryos. In this study, we established a modified evaluation method as using non-dechorionated embryos and observation a limited number of parameters without grading. Zebrafish embryos were exposed to test compounds from 5-6 to 144 hr post-fertilization, (hpf) corresponding to the organogenesis period. Morphological changes or functional abnormalities induced by test compound treatments were assessed and scored at 11 endpoints, and the potential of teratogenicity was judged based on the score. As a validation assay of the system, the potentials of 59 known teratogenic or non-teratogenic test compounds were evaluated using the present standard zebrafish assay, and the teratogenicity was correctly predicted in 90% (53/59) of all compounds with low false negative and false positive rates. These results indicated that the evaluation method using zebrafish for the teratogenicity we have improved was a valuable tool for early stage screening in drug discovery.
    The Journal of Toxicological Sciences 01/2014; 39(3):453-64.
  • [Show abstract] [Hide abstract]
    ABSTRACT: To clarify the relationship between arginine metabolism and hepatic injury, metabolomic analysis was performed in rats treated with 3 representative hepatotoxicants, monocrotaline (MCT), concanavalin A (ConA), and α-naphthyl isothiocyanate (ANIT); or a myotoxicant, tetramethyl-p-phenylenediamine (TMPD). A single dose of MCT, ConA, or ANIT dose-dependently induced hepatocellular necrosis accompanied by decreased blood arginine and increased blood alanine aminotransferase (ALT) and arginase. A close correlation was detected between arginine and ALT (r = -0.746, -0.795, -0.787 for MCT, ConA, ANIT, respectively) or between arginine and arginase (r = -0.605, -0.808, -0.672 for MCT, ConA, ANIT, respectively) in all three hepatic injury models. In contrast, neither hepatocellular necrosis nor alterations in arginine were found in the skeletal muscle injury model, although ALT was slightly increased. An in vitro assay revealed that blood samples obtained from ConA-treated rats transformed external arginine to ornithine, and the reaction was totally inhibited by an arginase inhibitor. These results suggest that blood arginase plays a crucial role in arginine metabolism associated with hepatic injury. In metabolomic analysis, nearly 450 endogenous metabolites were identified in blood obtained from all the models. Among the 13 metabolites involved in arginine metabolism, decreased arginine and increased ornithine occurred in common in the hepatic injury models, whereas citrulline and other metabolites were not altered. These results indicate that arginine metabolism, especially the arginine-to-ornithine pathway, is altered in association with acute hepatic injury. Furthermore, blood arginine and ornithine are possibly specific biomarkers for hepatic injury.
    The Journal of Toxicological Sciences 01/2014; 39(1):41-50.
  • [Show abstract] [Hide abstract]
    ABSTRACT: Pau d'arco is a plant-derived traditional medicine that acts by poorly understood molecular mechanisms. Here, we studied the effect of pau d'arco on the cytoprotective transcription factor Nrf2. An aqueous extract of pau d'arco stimulated Nrf2-dependent gene expression and led to nuclear localization of Nrf2 in vitro. Chromatographic separation and mass spectrometry of the extract identified benzene trioles or benzene tetraoles within the active fractions. The extract stimulated the mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase (MEK)/extracellular-signal-regulated kinase (ERK1/2) pathway. The pharmacological inhibition of MEK, but not of p38 mitogen-activated protein kinase, glycogen synthase kinase-3 or phosphoinositide 3-kinase was required for the activation of Nrf2-dependent gene expression by pau d'arco, but not for the nuclear translocation of Nrf2. In vivo pau d'arco increased the expression of Nrf2-target genes in the intestine. The results suggest that the activation of Nrf2 could mediate beneficial effects of pau d'arco, in particular in the intestine.
    The Journal of Toxicological Sciences 01/2014; 39(2):353-61.
  • [Show abstract] [Hide abstract]
    ABSTRACT: The psychoactive recreational drug 3,4-methylenedioxymethamphetamine (MDMA) is widely abused. The fact that MDMA induces neurotoxic damage in serotonergic nerve endings is well known. However, the effects of MDMA on pregnant and neonatal animals remain unknown. Therefore, we studied the effects of gestational exposure to MDMA on birth, growth, and behavior of pups. Female BALB/c mice were orally administered either water (10 ml/kg) or MDMA (20 mg/10 ml/kg) from gestational day 1 to postnatal day (P) 21. MDMA did not affect the birth rate, but the survival rate of the pups significantly decreased. A significant reduction in body weight gain was observed in pups from MDMA-administered dams during P3-P21. Maternal MDMA treatment caused an attenuated cliff avoidance reaction and decreased motor function in the pups, as determined by the wire hanging test. These results suggest that MDMA treatment during pregnancy and lactation causes growth retardation and dysfunction of motor neurons in mouse pups.
    The Journal of Toxicological Sciences 01/2014; 39(1):33-9.
  • [Show abstract] [Hide abstract]
    ABSTRACT: Aflatoxin B1 (AFB1) airway inhalation represents an additional route of exposure to this toxin. However, the association between AFB1 inhalation and serum AFB1 albumin adducts remains unclear. The aim of this study was to explore the association between airway exposure to AFB1 and serum AFB1 albumin adduct concentrations via an epidemiological study, as well as in an AFB1 airway exposure animal model. Our epidemiological study was conducted in a sugar factory in the Guangxi Autonomous Region of China. In order to examine fungal contamination, air samples were obtained in the workshop and areas outside the workshop, such as the office and nearby store. Dust samples were also collected from the bagasse warehouse and presser workshop, and were analyzed using an indirect competitive enzyme-linked immunosorbent assay (ELISA). Additionally, blood samples were collected from a total of 121 workshop workers, and a control group (n = 80) was comprised of workers who undertook administrative tasks or other work outside the workshop. The animal experiment was conducted in the laboratory animal center of Guangxi Medical University, where a total of 60 adult male rabbits were involved in this study. By intubation, AFB1 was administered in three groups of rabbits daily, at dose rates of 0.075, 0.05 and 0.025 mg/kg/day for a period of 7 days. Blood samples were collected on day 1, day 3, day 7 and day 21, and the measurements of the AFB1 albumin adducts in the serum were performed by a double antibody sandwich ELISA. The epidemiological study showed that serum albumin adducts were detected in 67 workshop workers (55.37%), and the values ranged 6.4 pg/mg albumin to 212 pg/mg albumin (mean value: 51 ± 4.62 pg/mg albumin). In contrast, serum albumin adducts were detected in only 7 control group participants, with the values ranging from 9 pg AFB1/mg albumin to 59 pg/mg albumin (mean value: 20 ± 13.72 pg/mg albumin). The animal experiment revealed that the rabbits had detectable levels of AFB1 in their serum with a minimum effective dose of 0.05 mg/kg/day; while 11 of 17 (64.71%) rabbits had detectable levels of AFB1 albumin adducts in the high exposure group (0.075 mg/kg/day), and only 5 rabbits (26.32%) had detectable levels of AFB1 albumin adducts in the moderate exposure group (0.05 mg/kg/day). No rabbits had detectable levels of AFB1 albumin adducts in the low exposure group (0.025 mg/kg/day). Our results demonstrated that only exposure to a certain level of AFB1 would result in detectable levels of serum AFB1 albumin adducts. Interventional programs aimed at reducing exposure to AFB1 by inhalation are urgently needed in high-risk populations. Additional large-sample, well-designed randomized controlled trials are needed to further confirm our results.
    The Journal of Toxicological Sciences 01/2014; 39(4):645-53.
  • [Show abstract] [Hide abstract]
    ABSTRACT: Dihydropyrazines (DHPs) are glycation products that are nonenzymatically generated in vivo and in food. In this study, we compared the effects of 2,3-dihydro-5,6-dimethylpyrazine (DHP-1), a low toxicity DHP, and 3-hydro-2,2,5,6-tetramethylpyrazine (DHP-3), a high toxicity DHP on the redox indices in HepG2 cells. An apparent increase in intracellular hydrogen peroxide concentration was observed at 24 hr after 1 mM DHP-3 treatment. In addition, DHP-3 exposure significantly increased the mRNA levels of heme oxygenase-1 (HO-1) and glutamate cysteine ligase catalytic subunit (GCLC), which are stress-responsive genes, at 6 hr (HO-1 and GCLC), 12 hr (HO-1 and GCLC) and 24 hr (GCLC) after exposure. These indices, with the exception of the increase in GCLC mRNA after a 6 hr exposure, were not affected by treatment with 1 mM DHP-1. HO-1, GCLC, and nuclear factor erythroid 2-related factor 2 (Nrf2) protein levels also increased at 6 hr (Nrf2), 12 hr (Nrf2, HO-1 and GCLC) and 24 hr (GCLC) after DHP-3 treatment. The increase in HO-1 and Nrf2 protein levels were observed with lower concentration (0.5 mM) of DHP-3, and in agreement with this, antioxidant responsive element-luciferase reporter activity was significantly increased with exposure to at least 0.5 mM DHP-3. These results support our previous report establishing that oxidative stress is in part involved in the effects of DHP on mammalian cells. Additionally, our results suggest that the cell response to DHP-3 exposure was exerted via the activation of the Nrf2-ARE signal pathway.
    The Journal of Toxicological Sciences 01/2014; 39(4):601-8.
  • [Show abstract] [Hide abstract]
    ABSTRACT: Advances in the synthesis and utilization of new chemical compounds have led to improvements in our daily lives. However, new chemicals may be both beneficial and toxic. Thus, exposure to these new compounds should be restricted in an attempt to limit their potential toxicities. We predicted the safety of three biocides (p-cresol, diazinon and resmethrin) by comparing their skin permeability coefficients and desquamation rate (the counter flux of permeability coefficient for chemical compounds induced by skin turnover) following skin exposure. In vitro skin permeation experiments revealed that the permeability coefficients of diazinon and resmethrin were smaller than the desquamation rate; therefore, these biocides could not permeate the skin, which resulted in very low skin concentrations of these compounds. On the other hand, the skin concentration of p-cresol was high because of its higher permeability coefficient than the desquamation rate. Furthermore, low in vitro cell viability was reported for skin exposed to p-cresol. These results in the present study indicate that the method described herein is useful for predicting the toxicities of chemicals following their topical exposure.
    The Journal of Toxicological Sciences 01/2014; 39(3):475-85.
  • [Show abstract] [Hide abstract]
    ABSTRACT: Ca(2+) overload is one of the mechanisms for H2O2-induced cell death in rat pancreatic β-cell line RIN-5F cells. RIN-5F cells express TRPM2, which is a Ca(2+)-permeable channel activated by H2O2, and voltage-dependent L-type Ca(2+) channels, both of which induce Ca(2+) entry by H2O2. This study examined the contribution of these channels to H2O2-induced Ca(2+) entry and cell death in RIN-5F cells. Cytosolic Ca(2+) concentration was measured using fura-2 as a Ca(2+) indicator. Cell death was estimated by trypan blue exclusion. Pre-treatment with poly(ADP-ribose) polymerase (PARP) inhibitors, which inhibit TRPM2 activation, strongly reduced Ca(2+) entry by H2O2. The PARP inhibitors used had no effect on the Ca(2+) elevation by voltage-dependent L-type Ca(2+) channels. On the other hand, pre-treatment with L-type Ca(2+) channel blockers, which did not affect TRPM2 activation, partly reduced H2O2-induced Ca(2+) entry. Treatment with PARP inhibitors but not L-type Ca(2+) channel blockers, around the early phase in H2O2-induced Ca(2+) elevation, also reduced the late phase. Moreover, H2O2-induced RIN-5F cell death was strongly attenuated by PARP inhibitors, in compared to L-type Ca(2+) channel blockers. Our results suggest that TRPM2 channels rather than L-type Ca(2+) channels primarily contribute to H2O2-induced Ca(2+) entry and cell death.
    The Journal of Toxicological Sciences 01/2014; 39(2):199-209.
  • [Show abstract] [Hide abstract]
    ABSTRACT: The Pig-a mutation assay is becoming one of the major experimental procedures used to assess in vivo genotoxicity. The assay allows simple in vivo analysis and enables gene mutations in the hematopoietic system to be measured using high throughput flow cytometry. Previously, we demonstrated that X-irradiation increased the Pig-a mutant frequencies in red blood cells (RBCs) of mice in a radiation dose-dependent manner. In this study, to understand how RBCs with Pig-a mutation induced by X-irradiation persist, we compared Pig-a mutant frequencies between irradiated C57BL/6J (p53(+/+)) mice and irradiated p53 homozygous knockout (p53(-/-)) mice by using the RBC Pig-a assay. After the peak in radiation-induced Pig-a mutant frequencies, a gradual decrease in mutant frequencies in irradiated p53(-/-) mice was observed, while irradiated p53(+/+) mice had a rapid decrease, which suggests that RBCs with Pig-a mutation are eliminated normally in irradiated p53(+/+) mice but not in irradiated p53(-/-) mice due to lack of p53 function. In addition, we also found that the p53 function affected the regulation of Pig-a mutagenesis in aging mice. Our results suggest that p53 function, distinct types of mutation, and the life span of RBCs play key roles in the persistence of Pig-a mutation in the hematopoietic system of RBCs after irradiation.
    The Journal of Toxicological Sciences 01/2014; 39(1):7-14.