Toshinori Yoshida

Rtp801, a suppressor of mTOR signaling, is an essential mediator of cigarette smoke-induced pulmonary injury and emphysema

Rtp801 (also known as Redd1, and encoded by Ddit4), a stress-related protein triggered by adverse environmental conditions, inhibits mammalian target of rapamycin (mTOR) by stabilizing the TSC1-TSC2 inhibitory complex and enhances oxidative stress–dependent cell death. We postulated that Rtp801 acts as a potential amplifying switch in the development of cigarette smoke–induced lung injury, leading to emphysema. Rtp801 mRNA and protein were overexpressed in human emphysematous lungs and in lungs of mice exposed to cigarette smoke. The regulation of Rtp801 expression by cigarette smoke may rely on oxidative stress–dependent activation of the CCAAT response element in its promoter. We also found that Rtp801 was necessary and sufficient for nuclear factor-κB (NF-κB) activation in cultured cells and, when forcefully expressed in mouse lungs, it promoted NF-κB activation, alveolar inflammation, oxidative stress and apoptosis of alveolar septal cells. In contrast, Rtp801 knockout mice were markedly protected against acute cigarette smoke–induced lung injury, partly via increased mTOR signaling, and, when exposed chronically to cigarette smoke, against emphysema. Our data support the notion that Rtp801 may represent a major molecular sensor and mediator of cigarette smoke–induced lung injury.

Acute oral toxicity of microcystin-LR, a cyanobacterial hepatotoxin, in mice

Microcystin-LR (MCLR) is a hepatotoxic peptide produced by Microcystis aeruginosa, an alga found worldwide in reservoirs for drinking supply; however, acute oral toxicity of purified MCLR remains unknown. Therefore, a single dose of MCLR (more than 95% purity) ranging from 8.0 to 20.0 mg/kg body weight was orally given to female 6-week old BALB/c mice, and lethality and pathological changes were observed. Median lethal dose (LD50) of the orally given MCLR estimated by the up and down method was 10.9 mg/kg, which was 167 times higher than the i.p. LD50 value (65.4 microgram/kg by moving average method). Orally administrated toxin caused primarily hepatocellular injuries with characteristics of hemorrhage and necrosis. In situ end-labeling as well as electron microscopic observation revealed an induction of apoptotic cell death to hepatocytes. These results indicate the lethality of MCLR was much lower in oral dosage than by i.p. administration, but toxic effects are similar. In addition, apoptosis is considered one of major components in MCLR-induced hepatotoxicity.

No Chronic oral toxicity of a low dose of microcystin-LR, a cyanobacterial hepatotoxin, in female BALB/c mice

Chronic oral toxicity of a low dose of microcystin‐LR (MCLR) was examined in female BALB/c mice for 18 months. Six‐week‐old female mice received 20 μg/L of the toxin in drinking water, which is about 200‐fold higher than the level in contaminated drinking water. Control mice received water alone. Mortality, clinical signs, body weights, and food and water consumptions were recorded during the study. Examinations on hematology, serum biochemistry, necropsy, organ weights, and histopathology were performed at months, 3, 6, 12, and 18. The immunohistochemical distribution of MCLR was examined in the liver at these time points. Mean cumulative MCLR intake after 18 months was estimated at 35.5 μg per mouse. The present test indicates that administration of a low dose of MCLR in drinking water resulted in neither chronic toxicity nor accumulation of the toxin in the liver. Based on previous epidemiological studies and the present chronic toxicity test, we recommend 0.01 μg/L as an maximum acceptable level for microcystins in drinking water, applying a safety factor of more than 1000. ©1999 John Wiley & Sons, Inc. Environ Toxicol 14: 45–55, 1999

Thymic atrophy induced by methoxychlor in rat pups

The effect of Methoxychlor (MXC) on the thymus was examined in rat pups that were delivered from dams receiving MXC at a dietary concentration of 0 or 1500 ppm for a period from pregnancy to lactation. The pups of both sexes were euthanized on postnatal days (PNDs) 7, 14, and 21. Histologically, the thymus showed marked depletion of cortical lymphocytes on PND 7 and also had an increase in lymphophagocytosis in the cortical area on PNDs 14 and 21. Morphometrical analysis disclosed that both cortex and medulla of the thymus from treated pups were reduced in size, but the reduction was more evident in the cortex. A significant increase in transferase-mediated dUTP nick end labeling-positive cells was detected in the cortex area, corresponding to the presence of lymphophagocytosis. Flow cytometric analysis revealed a significant decrease in the double positive (CD3(int)CD4(+)CD8(+)) immature cells on PND 21. These results have suggested that MXC may impair maturation of thymic lymphocytes in rat pups, which results in enhancement of apoptosis leading to thymic atrophy during the postnatal period.

Morphological and ultrastructural study of the histogenesis of meningeal granular cell tumors in rats

To clarify the cell of origin of granular cell tumors in the rat brain, light and/or electron microscopic examinations were performed on 40 cases of spontaneous meningeal tumors in Wistar (Jcl: Wistar) rats. The meningeal tumors were histologically subclassified into 3 types: meningothelial meningiomas (MMs), 3 cases; granular cell tumors (GCTs), 28 cases; and mixed forms (MIXs) of GCTs and MMs, 9 cases. Of these tumors, 2 MMs, 2 GCTs, and 3 MIXs were examined by transmission electron microscopy. Tumor cells of MMs were characterized by cytoplasmic intermediate filaments and prominent interdigitating cell processes often connected with cellular junctions without surrounding basal laminae. GCTs were composed of 2 cell types: granular cells with many dense bodies and filamentous cells with fine intermediate filaments. MIXs consisted of granular cells, filamentous cells, and intermediate cells. The tumor cells in GCTs and MIXs were apposed to each other and connected with cellular junctions. MIXs displayed a spectrum of cellular differentiation in that 2 MIXs had a close morphological resemblance to GCTs and the other one to MMs. These findings indicate there might be a histogenetic sequence among MMs, GCTs, and MIXs. The present study supports the suggestion that GCTs and MIXs may be variants of MMs from the meningeal arachnoid cell.

Proliferative and Non-Proliferative Lesions of the Rat and Mouse Soft Tissue, Skeletal Muscle and Mesothelium

Abstract The INHAND Project (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) is a joint initiative of the Societies of Toxicologic Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP), and North America (STP) to develop an internationally accepted nomenclature for proliferative and nonproliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature for classifying lesions observed in the soft tissues including skeletal muscle as well as the mesothelium of rats and mice. The standardized nomenclature of lesions presented in this document is also available electronically on the Internet (http://www.goreni.org/). Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous developmental and aging lesions as well as those induced by exposure to test materials. A widely accepted and utilized international harmonization of nomenclature for lesions in soft tissues, skeletal muscle and mesothelium in laboratory animals will decrease confusion among regulatory and scientific research organizations in different countries and provide a common language to increase and enrich international exchanges of information among toxicologists and pathologists. (DOI: 10.1293/tox.26.1S; J Toxicol Pathol 2013; 26: 1S–26S)

Didecyldimethylammonium chloride induces pulmonary inflammation and fibrosis in mice.

Didecyldimethylammonium chloride (DDAC) is used worldwide as a germicide, in antiseptics, and as a wood preservative, and can cause adverse pulmonary disease in humans. However, the pulmonary toxicity of DDAC has not yet been thoroughly investigated. Mice were intratracheally instilled with DDAC to the lung and the bronchoalveolar lavage (BAL) fluid and lung tissues were collected to assess dose- and time-related pulmonary injury. Exposure to 1500 μg/kg of DDAC caused severe morbidity with pulmonary congestive oedema. When the BAL fluid from survivors was examined on day 3 after treatment, exposure to 150 μg/kg of DDAC caused weakly induced inflammation, and exposure to 15μg/kg did not cause any visible effects. Next, we observed pulmonary changes that occurred up to day 20 after 150 μg/kg of DDAC exposure. Pulmonary inflammation peaked on day 7 and was confirmed by expression of interleukin-6, monocyte chemotactic protein-1, macrophage inflammatory protein (MIP)-1α, MIP-1β, and regulated upon activation, normal T-cell expressed and secreted in the BAL fluid; these changes were accompanied by altered gene expression of their chemokine (C-C motif) receptor (Ccr) 1, Ccr2, Ccr3, and Ccr5. Cytotoxicity evoked by DDAC was related to the inflammatory changes and was confirmed by an in vitro study using isolated mouse lung fibroblasts. The inflammatory phase was accompanied or followed by pulmonary remodeling, i.e., fibrosis, which was evident in the mRNA expression of type I procollagen. These results suggest that administering DDAC by intratracheal instillation causes pulmonary injury in mice, and occupational exposure to DDAC might be a potential hazard to human health.

Pulmonary injury and antioxidant response in mice exposed to arsenate and hexavalent chromium and their combination

Chromated copper arsenate, which is used worldwide as a wood preservative, can adversely affect human health. Accumulating evidence suggests that chromium (Cr) and arsenic (As) can potentially disrupt the redox balance and cause respiratory diseases and cancer in humans. The present study was designed to determine the combined toxic effects of these metals in the lungs and to clarify the specific molecules that are stimulated by combined exposure to both metals. Male C57BL/6J mice were intratracheally instilled with arsenate [As(V)], hexavalent chromium [Cr(VI)], or a combination of both metals. Mice were sacrificed 2 days after treatment to collect bronchoalveolar lavage fluid and lung tissue samples. Inflammation, cytotoxicity, apoptosis, and oxidative stress markers were measured. Our results indicated that administration of Cr(VI) alone or in combination with As(V) induced neutrophil-dominant inflammation as well as phosphorylation of mitogen-activated protein kinases; effects of treatment with As(V) alone were comparatively less potent. By analyzing the production of interleukin-6 and activity of lactate dehydrogenase and caspase, we confirmed that co-treatment intensified pulmonary injury and that it was accompanied by oxidative stress, as confirmed by marked increases in the production of reactive oxygen species, reduced glutathione content, and thioredoxin reductase (TRXRD) activity. Expressed mRNA levels of heme oxygenase-1, glutamylcysteine ligase, glutathione peroxidase 2, thioredoxin (TRX) 1, and TRXRD1 were also enhanced by co-treatment, whereas treatment with As(V) alone reduced the mRNA expression level of TRX2. Our data suggest that co-treatment with As(V) exacerbated Cr(VI)-induced pulmonary injury and that this effect may be exerted through a disruption in the balance among several antioxidant genes.

Human T-Cell Leukemia Virus Type 1 (HTLV-1) Infection of Mice: Proliferation of Cell Clones with Integrated HTLV-1 Provirus in Lymphoid Organs

ABSTRACT Human T-cell leukemia virus type 1 (HTLV-1) is suggested to cause adult T-cell leukemia after 40 to 50 years of latency in a small percentage of carriers. However, little is known about the pathophysiology of the latent period and the reservoir organs where polyclonal proliferation of cells harboring integrated provirus occurs. The availability of animal models would be useful to analyze the latent period of HTLV-1 infection. At 18 months after HTLV-1 infection of C3H/HeJ mice inoculated with the MT-2 cell line, which is an HTLV-1-producing human T-cell line, HTLV-1 provirus was detected in spleen DNA from eight of nine mice. No more than around 100 proviruses were found per 105 spleen cells. Cellular sequences flanking the 3′ long terminal repeat (LTR) and the clonalities of the cells which harbor integrated HTLV-1 provirus were analyzed by linker-mediated PCR. The results showed that the flanking sequences are of mouse genome origin and that polyclonal proliferation of the spleen cells harboring integrated HTLV-1 provirus had occurred in three mice. A sequence flanking the 5′ LTR was isolated from one of the mice and revealed the presence of a 6-nucleotide duplication of cellular sequences, consistent with typical retroviral integration. Moreover, PCR was performed on DNA from infected tissues, with LTR primers and primers derived from seven novel flanking sequences of the three mice. Data revealed that the expected PCR products were found from lymphatic tissues of the same mouse, suggesting that the lymphatic tissues were the reservoir organs for the infected and proliferating cell clones. The mouse model described here should be useful for analysis of the carrier state of HTLV-1 infection in humans.

Phenotypic alteration of hepatocellular foci in rats treated with clofibrate and phenobarbital

In male F344 rats pretreated with diethylnitrosamine (DEN), subsequent administration of clofibrate increased the proportion of eosinophilic foci, to become the most abundant type, and reduced numbers of basophilic, clear and vacuolated foci, the total not being changed. A similar shift towards eosinophilia was also observed in phenobarbital-treated animals, but in this case clear increases in total number and area were apparent. Expression of the glutathione S-transferase placental form (GST-P) in foci was much lowered by clofibrate treatment, while the proportion of positive foci was very high in both phenobarbital and control groups. A marked contrast was found with eosinophilic foci, with 74% positive after phenobarbital as compared to only 15% for clofibrate. Thus, the decrease in GST-P positive foci by clofibrate was mainly due to increased negativity in the most abundant eosinophilic type foci. In a long-term feeding study without DEN initiation, similar negativity of foci was observed and, furthermore, only minimal effects of clofibrate on foci development was revealed in both young and old animals.