Continuous infiltration of small peritoneal macrophages in the mouse peritoneum through CCR2-dependent and -independent routes during fibrosis and mesothelioma development induced by a multiwalled carbon nanotube, MWNT-7.

Although toxicities of multiwalled carbon nanotube (MWCNT) have been found to be related with activities of macrophages phagocytosing the fibers, the exact relationship between macrophage population and pathogenesis of fibrosis and mesotheliomas induced by MWCNTs is largely unknown. CCL2-CCR2 axis, a major monocyte/macrophage infiltration route, is thought to be involved in not only acute inflammation but also the formation of tumor microenvironment. We therefore described a time-course of alteration of macrophage population in an attempt to clarify the contribution of the Ccr2 gene to mesotheliomagenesis. Wild-type (WT) C57BL/6 mice and Ccr2-knockout (KO) mice were intraperitoneally administered with MWNT-7 and were sequentially necropsied at 1, 7, 28, 90, and 245 day(s) after the injection. Peritoneal fibrosis was prominent in all MWCNT-treated mice, with a lower severity in the KO mice. No differences were observed in the incidences of neoplastic lesions of mesothelia between WT and KO mice. A flow cytometric analysis revealed that after gross disappearance of macrophages after MWCNT exposure, small peritoneal macrophages (SPMs) were exclusively refurbished by the CCR2-dependent route at day 1 (as Ly-6C+MHC class II- cells), followed by additional CCR2-independent routes (as Ly-6C-MHC class II- cells); i.e., the only route in KO mice; with a delay of 1-7 days. The SPMs derived from both routes appeared to differentiate into maturated cells as Ly-6C-MHC class II+, whose ratio increased in a time-dependent manner among the total SPM population. Additionally, most macrophages expressed M1-like features, but a small fraction of macrophages exhibited an M1/M2 mixed status in MWCNT-treated animals. Our findings demonstrate a long-persistent activation of the CCL2-CCR2 axis after MWCNT exposure and enable a better understanding of the participation and potential roles of SPMs in fibrous material-induced chronic toxicities.

Time-Course of Transcriptomic Change in the Lungs of F344 Rats Repeatedly Exposed to a Multiwalled Carbon Nanotube in a 2-Year Test.

Despite intensive toxicological studies of carbon nanotubes (CNTs) over the last two decades, only a few studies have demonstrated their pulmonary carcinogenicities in chronic animal experiments, and the underlying molecular mechanisms are still unclear. To obtain molecular insights into CNT-induced lung carcinogenicity, we performed a transcriptomic analysis using a set of lung tissues collected from rats in a 2-year study, in which lung tumors were induced by repeated intratracheal instillations of a multiwalled carbon nanotube, MWNT-7. The RNA-seq-based transcriptome identified a large number of significantly differentially expressed genes at Year 0.5, Year 1, and Year 2. Ingenuity Pathway Analysis revealed that macrophage-elicited signaling pathways such as phagocytosis, acute phase response, and Toll-like receptor signaling were activated throughout the experimental period. At Year 2, cancer-related pathways including ERBB signaling and some axonal guidance signaling pathways such as EphB4 signaling were perturbed. qRT-PCR and immunohistochemistry indicated that several key molecules such as Osteopontin/Spp1, Hmox1, Mmp12, and ERBB2 were markedly altered and/or localized in the preneoplastic lesions, suggesting their participation in the induction of lung cancer. Our findings support a scenario of inflammation-induced carcinogenesis and contribute to a better understanding of the molecular mechanism of MWCNT carcinogenicity.

Effects of inhalation of multi-walled carbon nanotube (MWCNT) on respiratory syncytial virus (RSV) infection in mice.

Multi-walled carbon nanotubes (MWCNTs), a kind of nanomaterial, are widely used in battery electrodes and composite materials, but the adverse effects associated with their accumulation in the living body have not been sufficiently investigated. MWCNTs are a fibrous material with molecules similar to asbestos fibers, and there are concerns about its effects on the respiratory system. In this study, we conducted a risk assessment by exposing mice using a previously developed nanomaterial inhalation exposure method. We quantified the exposure in the lungs by a lung burden test, evaluated the deterioration due to pneumonia using respiratory syncytial virus (RSV) infection, and measured inflammatory cytokines in bronchoalveolar lavage fluid (BALF). As a result, in the lung burden test, the amount of MWCNT in the lung increased according to the inhalation dose. In the RSV infection experiment, CCL3, CCL5, and TGF-ß, which are indicators of inflammation and lung fibrosis, were elevated in the MWCNT-exposed group. Histological examination revealed cells phagocytosing MWCNT fibers. These phagocytic cells were also seen during the recovery period from RSV infection. The present study found that MWCNT remained in the lungs for about a month or more, suggesting that the fibers may continue to exert immunological effects on the respiratory system. Furthermore, the inhalation exposure method enabled the exposure of nanomaterials to the entire lung lobe, allowing a more detailed evaluation of the effects on the respiratory system.

In vivo genotoxicity assessment of a multiwalled carbon nanotube in a mouse ex vivo culture.

BACKGROUND: Multiwalled carbon nanotubes (MWCNTs) are suspected lung carcinogens because their shape and size are similar to asbestos. Various MWCNT types are manufactured; however, only MWNT-7 is classified into Group 2B by The International Agency for Research on Cancer. MWNT-7's carcinogenicity is strongly related to inflammatory reactions. On the other hand, inconsistent results on MWNT-7 genotoxicity have been reported. We previously observed no significant differences in both Pig-a (blood) and gpt (lung) mutant frequencies between MWNT-7-intratracheally treated and negative control rats. In this study, to investigate in vivo MWNT-7 genotoxicity on various endpoints, we attempted to develop a lung micronucleus assay through ex vivo culture targeting the cellular fraction of Clara cells and alveolar Type II (AT-II) cells, known as the initiating cells of lung cancer. Using this system, we analyzed the in vivo MWNT-7 genotoxicity induced by both whole-body inhalation exposure and intratracheal instillation. We also conducted an erythrocyte micronucleus assay using the samples obtained from animals under intratracheal instillation to investigate the tissue specificity of MWNT-7 induced genotoxicities. RESULTS:  We detected a significant increase in the incidence of micronucleated cells derived from the cellular fraction of Clara cells and AT-II cells in both MWNT-7-treated and positive control groups compared to the negative control group under both whole-body inhalation exposures and intratracheal instillation. Additionally, the erythrocyte micronucleus assay detected a significant increase in the incidence of micronucleated reticulocytes only in the positive control group. CONCLUSIONS: Our findings indicated that MWNT-7 was genotoxic in the lungs directly exposed by both the body inhalation and intratracheal instillation but not in the hematopoietic tissue.

Two-year intermittent exposure of a multiwalled carbon nanotube by intratracheal instillation induces lung tumors and pleural mesotheliomas in F344 rats.

BACKGROUND: A mounting number of studies have been documenting the carcinogenic potential of multiwalled carbon nanotubes (MWCNTs); however, only a few studies have evaluated the pulmonary carcinogenicity of MWCNTs in vivo. A 2-year inhalation study demonstrated that MWNT-7, a widely used MWCNT, was a pulmonary carcinogen in rats. In another 2-year study, rats administered MWNT-7 by intratracheal instillation at the beginning of the experimental period developed pleural mesotheliomas but not lung tumors. To obtain data more comparable with rats exposed to MWNT-7 by inhalation, we administered MWNT-7 to F344 rats by intratracheal instillation once every 4-weeks over the course of 2 years at 0, 0.125, and 0.5 mg/kg body weight, allowing lung burdens of MWNT-7 to increase over the entire experimental period, similar to the inhalation study. RESULTS: Absolute and relative lung weights were significantly elevated in both MWNT-7-treated groups. Dose- and time-dependent toxic effects in the lung and pleura, such as inflammatory, fibrotic, and hyperplastic lesions, were found in both treated groups. The incidences of lung carcinomas, lung adenomas, and pleural mesotheliomas were significantly increased in the high-dose group compared with the control group. The pleural mesotheliomas developed mainly at the mediastinum. No MWNT-7-related neoplastic lesions were noted in the other organs. Cytological and biochemical parameters of the bronchoalveolar lavage fluid (BALF) were elevated in both treated groups. The lung burden of MWNT-7 was dose- and time-dependent, and at the terminal necropsy, the average value was 0.9 and 3.6 mg/lung in the low-dose and high-dose groups, respectively. The number of fibers in the pleural cavity was also dose- and time-dependent. CONCLUSIONS: Repeated administration of MWNT-7 by intratracheal instillation over the 2 years indicates that MWNT-7 is carcinogenic to both the lung and pleura of rats, which differs from the results of the 2 carcinogenicity tests by inhalation or intratracheal instillation.

Iron oxide nanoparticles exert inhibitory effects on N-Bis(2-hydroxypropyl)nitrosamine (DHPN)-induced lung tumorigenesis in rats.

Iron oxide nanoparticles (magnetite) have been widely used in industry and medicine. However, the safety assessment of magnetite has not been fully completed. The present study was conducted to assess effects of magnetite on carcinogenic activity, using a medium-term bioassay protocol. A total of 100 male Fischer 344 rats, 6 weeks old, were randomly divided into 5 groups of 20 animals each, and given a basal diet and drinking water containing 0 or 0.1% of N-bis(2-hydroxypropyl)nitrosamine (DHPN) for 2 weeks. Two weeks later, the rats were intratracheally instilled magnetite 7 times at an interval of 4 weeks, at the doses of 0, 1.0 or 5.0 mg/kg body weight, and sacrificed at the end of the experimental period of 30 weeks. The multiplicities of macroscopic lung nodules and histopathologically diagnosed bronchiolo-alveolar hyperplasia, induced by DHPN, were both significantly decreased by the high dose of magnetite. The expression of minichromosome maintenance (MCM) protein 7 in non-tumoral alveolar epithelial cells, and the number of CD163-positive macrophages in tumor nodules were both significantly reduced by magnetite. It is suggested that magnetite exerts inhibitory effects against DHPN-induced lung tumorigenesis, by the reduction of alveolar epithelial proliferation and the M2 polarization of tumor-associated macrophages.

A case of spontaneous Zymbal's gland carcinoma with lung metastasis in an aged Fischer 344 rat.

Zymbal's gland neoplasms are induced in rats through the administration of various carcinogens, but spontaneous neoplasia is rare. This report describes a spontaneous Zymbal's gland carcinoma with lung metastasis found in an aged male Fischer 344 rat. Macroscopically, the dome-like tumor nodule, approximately 30 mm in diameter with ulceration, was located near the ear canal of the rat. No healthy tissue or structure of Zymbal's gland was identified on the corresponding side, while the normal salivary glands and a lacrimal gland were observed. Histologically, a large part of the tumor mass was occupied by poorly differentiated neoplastic cells, the shapes of which were oval to polygonal or fusiform. Additionally, clusters of sebaceous-like foamy cells and squamous metaplasia with prominent keratinization were observed. Tumor cells were found to metastasize to the lung; these cells displayed histological similarities, including a sebaceous gland-like pattern, to those in the primary site. The tumor cells were immunohistochemically positive for cytokeratin AE1/AE3 or vimentin but negative for CD68, S100, α-smooth muscle actin, von Willebrand factor, and desmin. Our results indicate that the tumor was a poorly differentiated Zymbal's gland carcinoma with lung metastasis.

Histological sequence of the development of rat mesothelioma by MWCNT, with the involvement of apolipoproteins.

A rat model of mesothelioma development by peritoneal injection of multiwalled carbon nanotube (MWCNT) has been established and found to be useful to understand the mechanisms underlying fibrous particles-associated carcinogenesis. Its detailed histological sequence, however, remains largely obscure. We therefore aimed to assess the time-course of mesothelioma development by MWCNT and evaluate a set of lipoprotein-related molecules as potential mechanism-based biomarkers for the phenomenon. Male Fischer 344 rats were injected intraperitoneally (ip) with MWCNT (MWNT-7) at 1 mg/kg body weight, and necropsied at 8, 16, 24, 32, or 42 wk after injection. For biochemical analyses of the lipoprotein-related molecules, more samples, including severe mesothelioma cases, were obtained from 2 other carcinogenicity tests. Histologically, in association with chronic inflammation, mesothelial proliferative lesions appeared at c. Wk-24. Before and at the beginning of the tumor development, a prominent infiltration of CD163-positive cells was seen near mesothelial cells. The histological pattern of early mesothelioma was not a papillary structure, but was a characteristic structure with a spherical appearance, composed of the mesothelioma cells in the surface area that were underlain by connective tissue-like cells. Along with the progression, mesotheliomas started to show versatile histological subtypes. Serum levels of apolipoprotein A-I and A-IV, and a ratio of HDL cholesterol to total cholesterol were inversely correlated with mesothelioma severity. Overall, the detailed histological sequence of mesotheliomagenesis by MWCNT is demonstrated, and indicated that the altered profile of apolipoproteins may be involved in its underlying mechanisms.

A histopathological analysis of spontaneous neoplastic and non-neoplastic lesions in aged male RccHan:WIST rats.

Histopathological information about spontaneous lesions in aged Hannover Wistar rats is limited. In this study, we describe spontaneous lesions found in 39 male RccHan:WIST rats used as a control in a carcinogenicity study. Neoplastic lesions were frequently seen in the endocrine system, such as pituitary adenomas in the pars distalis. This strain exhibited a high incidence of thymoma (10.3%), compared to other strains. We encountered an oligodendroglioma, a pituitary adenoma of the pars intermedia, and a prostate adenocarcinoma, which are comparatively rare in rats. While the variety and incidence of non-neoplastic lesions were similar to those in other strains, several interesting lesions occurred with relatively high incidence, including "harderianization" of the extraorbital lacrimal gland, common bile duct ectasia, and hyperplasia of pulmonary endocrine cells in the lung. Furthermore, comparative analyses demonstrated that the severity of chronic progressive nephropathy and murine progressive cardiomyopathy in RccHan:WIST rats was less than that in F344 rats.

Comparative study for carcinogenicity of 7 different multi-wall carbon nanotubes with different physicochemical characteristics by a single intraperitoneal injection in male Fischer 344 rats.

The present study comparatively examined carcinogenicity of 7 different multi-wall carbon nanotubes (MWCNTs) with different physicochemical characteristics. Physicochemical characteristics of MWCNTs (referred to as M-, N-, WL-, SD1-, WS-, SD2- and T-CNTs in the present study) were determined using scanning electron and light microscopes and a collision type inductively coupled plasma mass spectrometer. Male Fischer 344 rats (10 weeks old, 15 animals per group) were administered MWCNTs at a single intraperitoneal dose of 1 mg/kg body weight, and sacrificed up to 52 weeks after the commencement. Fibers of M-, N-, WL- and SD1-CNTs were straight and acicular in shape, and contained few agglomerates. They were relatively long (38-59% of fibers were longer than 5 µm) and thick (33% to more than 70% of fibers were thicker than 60 nm). All of these 4 MWCNTs induced mesotheliomas at absolute incidences of 100%. Fibers of WS-, SD2- and T-CNTs were curled and tightly tangled to form frequent agglomerates. They were relatively short and thin (more than 90% of measured fibers were thinner than 50 nm). WS- CNT did not induce mesothelioma, and only one of 15 rat given SD2- or T-CNT developed tumor. Any correlations existed between the metal content and neither the size or form of fibers, nor the carcinogenicity. It is thus indicated that the physicochemical characteristics of MWCNTs are critical for their carcinogenicity. The straight and acicular shape without frequent agglomerates, and the relatively long and thick size, but not the iron content, may be critical factors. The present data can contribute to the risk management, practical use and social acceptance of MWCNTs.

Unexpected link between polyketide synthase and calcium carbonate biomineralization.

INTRODUCTION: Calcium carbonate biominerals participate in diverse physiological functions. Despite intensive studies, little is known about how mineralization is initiated in organisms. RESULTS: We analyzed the medaka spontaneous mutant, ha, defective in otolith (calcareous ear stone) formation. ha lacks a trigger for otolith mineralization, and the causative gene was found to encode polyketide synthase (pks), a multifunctional enzyme mainly found in bacteria, fungi, and plant. Subsequent experiments demonstrate that the products of medaka PKS, most likely polyketides or their derivatives, act as nucleation facilitators in otolith mineralization. The generality of this novel PKS function is supported by the essential role of echinoderm PKS in calcareous skeleton formation together with the presence of PKSs in a much wider range of animals from coral to vertebrates. CONCLUSION: The present study first links PKS to biomineralization and provides a genetic cue for biogeochemistry of carbon and calcium cycles.

Teratogenicity of asbestos in mice.

Possible teratogenicity of 3 different asbestos (crocidolite, chrysotile and amosite) was assessed in CD1(ICR) mice. Dams on day 9 of gestation were given a single intraperitoneal administration at dose of 40 mg/kg body weight of asbestos suspended in 2% sodium carboxymethyl cellulose solution in phosphate buffered saline, while dams in the control group were given vehicle (10 ml/kg body weight). Dams and fetuses were examined on day 18 of gestation. To compare with the control group, the mean percentage of live fetuses in implantations in the group given crocidolite and the incidence of dams with early dead fetuses in the groups given chrysotile or amosite were increased. While no external or skeletal malformation was observed in the control group, the incidence of external malformation (mainly reduction deformity of limb) in the group given amosite, and the incidences of skeletal malformation (mainly fusion of vertebrae) in the all dosed groups were significantly increased. The result indicated that asbestos (crocidolite, chrysotile and amosite) have fetotoxicity and teratogenicity in mice.

Phenotypic analysis of a novel chordin mutant in medaka.

We have isolated and characterized a ventralized mutant in medaka (the Japanese killifish; Oryzias latipes), which turned out to have a mutation in the chordin gene. The mutant exhibits ventralization of the body axis, malformation of axial bones, over-bifurcation of yolk sac blood vessels, and laterality defects in internal organs. The mutant exhibits variability of phenotypes, depending on the culture temperature, from embryos with a slightly ventralized phenotype to those without any head and trunk structures. Taking advantages of these variable and severe phenotypes, we analyzed the role of Chordin-dependent tissues such as the notochord and Kupffer's vesicle (KV) in the establishment of left-right axis in fish. The results demonstrate that, in the absence of the notochord and KV, the medaka lateral plate mesoderm autonomously and bilaterally expresses spaw gene in a default state.

Right-elevated expression of charon is regulated by fluid flow in medaka Kupffer's vesicle.

Recent studies have revealed that a cilium-generated liquid flow in the node has a crucial role in the establishment of the left-right (LR) axis in the mouse. In fish, Kupffer's vesicle (KV), a teleost-specific spherical organ attached to the tail region, is known to have an equivalent role to the mouse node during LR axis formation. However, at present, there has been no report of an asymmetric gene expressed in KV under the control of fluid flow. Here we report the earliest asymmetric gene in teleost KV, medaka charon, and its regulation. Charon is a member of the Cerberus/DAN family of proteins, first identified in zebrafish. Although zebrafish charon was reported to be symmetrically expressed in KV, medaka charon displays asymmetric expression with more intense expression on the right side. This asymmetric expression was found to be regulated by KV flow because symmetric and up-regulated charon expression was observed in flow-defective embryos with immotile cilia or disrupted KV. Taken together, medaka charon is a reliable gene marker for LR asymmetry in KV and thus, will be useful for the analysis of the early steps downstream of the fluid flow.

Expression patterns of the chicken peroxisome proliferator-activated receptors (PPARs) during the development of the digestive organs.

Peroxisome proliferator-activated receptors (PPARs) play very important roles in various biological phenomena such as regulation of lipid metabolism, homeostasis, cell differentiation and proliferation, in a variety of organs and tissues. However, their functions in the development of the digestive organs have not been studied yet, although it has been supposed that they are involved in the tumor development and regression of digestive organs. To provide fundamental data to analyze functions of PPARs in the developing digestive organs in the chicken embryos, we performed thorough analysis of expression of PPARalpha, beta (delta) and gamma in the esophagus, proventriculus (glandular stomach), gizzard (muscular stomach), small and large intestines from early developmental stages to post hatch stages. The results showed that each PPAR is expressed in spatio-temporally regulated manner. In general, PPARbeta is widely expressed among digestive organs whereas PPARalpha and gamma showed restricted expression. In the intestine, all PPARs are expressed after hatch, indicating that they play important roles in the physiology of the adult intestine.