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Nonalcoholic steatohepatitis is a lifestyle-related disease and an increasing threat worldwide. Hepatic fibrosis, which results from chronic hepatic diseases including nonalcoholic steatohepatitis, is closely correlated with mortality among hepatic lesions, such as steatosis and inflammation. Thus, it is important to identify factors that can serve as diagnostic and therapeutic targets for hepatic fibrosis. In this study, we examined the function of CD44 in the development of hepatic fibrosis in choline-deficient, methionine-lowered, L-amino-acid diet-fed rats, especially with respect to the proliferation of bile duct epithelium. Male Fischer 344 rats were fed a choline-deficient, methionine-lowered, L-amino-acid diet for 2, 4, 13, or 26 weeks. This diet decreased the body weight; increased the levels of serum parameters indicating liver injury, such as aspartate and alanine aminotransferase; upregulated inflammation- and fibrosis-related gene expression in the liver; and resulted in the development of hepatic lesions, including fatty changes in hepatocytes, inflammatory cell infiltration, and fibrosis. Hepatic hyaluronan was synthesized and deposited in the liver tissue. The expression of both CD44 mRNA and protein was significantly increased throughout the experimental period. CD44 protein was observed in some of the bile duct epithelium, around which hyaluronic acid was deposited, and these bile duct lesions were concordant with the area of hepatic fibrosis. Thus, CD44 expressed in the bile duct epithelium may be a target for controlling nonalcoholic steatohepatitis-related hepatic fibrosis.

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.

Type 2 diabetes mellitus represents an international health concern with its growing number of patients worldwide. At the same time, excessive salt consumption is also seen as a major cause of diseases such as hypertension and may expedite renal complications in diabetic patients. In this study, we investigated the effects of excessive sodium chloride supplementation on the kidney of the Spontaneously Diabetic Torii-Leprfa (SDT fatty) rat, an obese type 2 diabetes model. Male and female SDT fatty rats and normal Sprague-Dawley (SD) rats at 5 weeks of age were loaded with 0.3% sodium chloride (NaCl) in drinking water for 13 weeks. Blood serum and urinary parameters were observed throughout the experiment and kidney samples were examined in histopathological and genetical analyses. Significant changes on the body weight, blood pressure, urine volume, creatinine clearance, blood urea nitrogen (BUN), relative gene expressions of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), monocyte chemotactic protein-1 (MCP-1) and transforming growth factor-β (TGF-β) were observed in the salt-loaded male SDT fatty rats. Urinary L-type fatty acid-binding protein (L-FABP) and albumin levels were higher observed in the salt-loaded male SDT fatty rats throughout the period, but urinary albumin levels in the female SDT fatty rats remain unchanged. In the kidney, slight Armani-Ebstein changes, tubular degeneration, hyaline cast, and inflammatory cell infiltration were observed in female SDT fatty rats while the levels of some changes were higher in the salt-loaded group. The kidney of the salt-loaded male SDT fatty rats demonstrated a higher degree of lesions compared to the female group and the male unloaded group. Histopathological changes in salt-loaded SDT fatty rats show that excessive salt consumption may act as a diabetic pathology exacerbation factor, but the pathology may be influenced by gender difference. Urinary L-FABP levels may act as a useful biomarker to detect slight tubular damages in the kidney. Excessive salt loading was shown to exacerbate the renal injury in SDT fatty rats.

Some illegal dietary supplements contain phosphodiesterase type 5 (PDE5) inhibitors, such as sildenafil, for exerting "therapeutic" effects in erectile dysfunction. This is apparently dangerous, and thus, should be appropriately regulated. Identification of descarbonsildenafil was first reported in Singapore in a coffee sample labeled to exert male sexual performance enhancement effects. However, it is unclear whether the compound possesses PDE5 inhibitory activity. We encountered during our survey of dietary supplements, a sexual enhancement product commercially available in Tokyo, in which a peak presumed to be of descarbonsildenafil was detected by LC-UV and electrospray ionization-tandem MS (ESI-MS/MS). The compound was isolated and identified as descarbonsildenafil with liquid chromatography-quadrupole time-of-flight-mass spectrometry (LC-QTOF-MS), NMR, and X-ray crystal structural analysis. In addition, descarbonsildenafil showed PDE5 inhibitory activity in PDE5 inhibition assay, and its IC50 value for PDE5A1 was found to be 30 nmol/L. The results of INADEQUATE NMR and X-ray crystal structural analysis in this study provide information for the identification of descarbonsildenafil. Since this study indicates that this compound is a PDE5 inhibitor having adequate activity, it is regulated as a drug component in Japan.

This study aimed to establish a rat chronic kidney disease (CKD) model by studying the effects of a high-phosphorus diet in rats that had undergone partial ligation of the renal arteries (RL). Separate groups of 10-week-old male Slc:Sprague-Dawley rats underwent RL and were fed diets with varying phosphorous levels for a period of 48 days. A marked suppression of body weight gain necessitating humane euthanization occurred on day 28 in rats that had undergone RL and were given high-phosphorus feed. By contrast, the group of intact animals on a high-phosphorus feed exhibited a slightly decreased body weight gain from day 21 and survived until scheduled euthanization. In rats with RL, hematological, blood biochemical, and histopathological analyses demonstrated the presence of CKD-like conditions, particularly in the group that were fed a high-phosphorus diet. Hyperphosphatemia and hypocalcemia were induced by a high-phosphorus diet in both the RL and intact groups, both of which had high levels of FGF23 and parathyroid hormone in the blood. Rats with RL on a high-phosphorus diet showed decreased hematopoiesis by the hematopoietic cell area being narrower in the medullary cavity, proliferation of mesenchymal cells and osteoblasts/osteoclasts, and expansion of the osteoid area, a furthermore generalized vascular lesions, such as calcification, were observed. These findings demonstrate that the partial ligation of the renal arteries combined with a calcium-phosphorus imbalance induced by a high-phosphorus diet serves as an animal model for CKD-like conditions accompanied by bone lesions, helping to elucidate this clinical condition and its underlying molecular mechanisms.

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.

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.

The Standard for Exchange of Nonclinical Data (SEND), adopted by the US Food and Drug Administration (FDA), is a set of regulations for digitalization and standardization of nonclinical study data; thus, related organizations have begun implementing processes in support of SEND. The Global Editorial and Steering Committee (GESC), which provides oversight of the International Harmonization of Nomenclature and Diagnostic Criteria (INHAND), has prepared the SEND Controlled Terminology (CT) for toxicologic pathology. SEND provides electronic data standards created by the Clinical Data Interchange Standards Consortium (CDISC), and CDISC also collaborates in the implementation of SEND. Furthermore, the Pharmaceutical Users Software Exchange (PhUSE), which includes members of the US FDA, has conducted various activities to promote realistic and effective methods to implement SEND. As we reported in 2015, there is a significant variation in the efficiency and quality of SEND data implementation across pharmaceutical companies and contractors (CROs) globally. To address this problem, the Global SEND Alliance (G-SEND) was established in August 2018 to facilitate the coordination and standardization of SEND datasets across CROs in Asia. This paper reports the first method for organizationally and jointly creating consistent SEND datasets between CROs using G-SEND.

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., We aimed to assess a time course of the development of rat peritoneal mesothelioma by MWCNT and, furthermore, to evaluate a set of lipoprotein‐related molecules as potential mechanism‐based biomarkers for the phenomenon. The histological pattern of the early mesotheliomas was a characteristic structure with a spherical appearance, although that of the advanced mesotheliomas was more versatile. Serum levels of apolipoprotein A‐I and A‐IV, and a ratio of HDL cholesterol to total cholesterol were inversely correlated with the severity of mesothelioma.

Glomerular hyperfiltration is observed in an early stage of kidney diseases including diabetic nephropathy. A better understanding of pathophysiological changes in glomerular hyperfiltration is essential for development of new therapies to prevent kidney disease progression. In this study, we investigated glomerular changes including glomerular filtration rate (GFR) and glomerular size in the Spontaneously Diabetic Torii (SDT) fatty rat, an obese type 2 diabetic model, and we also evaluated pharmacological effects of the sodium glucose cotransporter 2 inhibitor dapagliflozin on the renal lesions. Dapagliflozin was administered to SDT fatty rats from 5 to 17 weeks of age. Blood and urinary biochemical parameters were periodically measured. GFR was determined by transdermal GFR monitor at 16 weeks of age and histopathological analysis was performed at 17 weeks of age. SDT fatty rat developed severe hyperglycemia and exhibited pathophysiological abnormalities in the kidney, such as an increased GFR, glomerular hypertrophy and tissue lesions. Dapagliflozin achieved good glycemic control during the experimental period, inhibited the increase in GFR, and improved histopathological abnormalities in tubules. These results suggest that the SDT fatty rat is a useful model for analyzing the pathogenesis of diabetic nephropathy during its early stage and dapagliflozin improves not only hyperglycemia but also glomerular hyperfiltration and tubule lesions in SDT fatty rat.

Background Nonalcoholic steatohepatitis (NASH) is a form of liver disease characterized by steatosis, necroinflammation, and fibrosis, resulting in cirrhosis and cancer. Efforts have focused on reducing the intake of trans fatty acids (TFAs) because of potential hazards to human health and the increased risk for NASH. However, the health benefits of reducing dietary TFAs have not been fully elucidated. Here, the effects of TFAs vs. a substitute on NASH induced in mice by feeding a choline-deficient, methionine-lowered, L-amino acid-defined, high-fat diet (CDAA-HF) were investigated. Methods Mice were fed CDAA-HF containing shortening with TFAs (CDAA-HF-T(+)), CDAA-HF containing shortening without TFAs (CDAA-HF-T(−)), or a control chow for 13 or 26 weeks. Results At week 13, NASH was induced in mice by feeding CDAA-HF-T(+) containing TFAs or CDAA-HF-T(−) containing no TFAs, but rather mostly saturated fatty acids (FAs), as evidenced by elevated serum transaminase activity and liver changes, including steatosis, inflammation, and fibrosis. CDAA-HF-T(−) induced a greater extent of hepatocellular apoptosis at week 13. At week 26, proliferative (preneoplastic and non-neoplastic) nodular lesions were more pronounced in mice fed CDAA-HF-T(−) than CDAA-HF-T(+). Conclusions Replacement of dietary TFAs with a substitute promoted the development of proliferation lesions in the liver of a mouse NASH model, at least under the present conditions. Attention should be paid regarding use of TFA substitutes in foods for human consumption, and a balance of FAs is likely more important than the particular types of FAs.

Background: Nonalcoholic steatohepatitis (NASH) is a form of liver disease characterized by steatosis, necroinflammation, and fibrosis, resulting in cirrhosis and cancer. Trans fatty acid (TFA) is hazardous for human health and a risk factor of NASH; thus, efforts have focused on reducing its intake. However, the health benefits of reducing dietary TFA are not fully elucidated. We investigated effects of TFA and its substitute on NASH induced in mice by feeding a choline-deficient, methionine-lowered, L-amino acid-defined, high-fat diet (CDAA-HF). Methods: Mice were fed CDAA-HF containing shortening with TFA (CDAA-HF-T(+)), CDAA-HF containing shortening with a TFA substitute (CDAA-HF-T(−)), or a control chow for 13/26 weeks. Results: CDAA-HF-T(+) contained TFA, whereas CDAA-HF-T(−) contained no TFA and much saturated fatty acids. CDAA-HF-T(+) and CDAA-HF-T(−) induced NASH in mice, evidenced by elevated serum transaminase activity and liver changes, including steatosis, inflammation, and fibrosis. CDAA-HF-T(−) induced more hepatocellular apoptosis and proliferative (preneoplastic and non-neoplastic) nodular lesions than CDAA-HF-T(+). Conclusions: Thus, replacement of dietary TFA with its substitute does not prevent but aggravates nutritionally induced NASH in mice, at least under the present conditions. Attention should be paid regarding future TFA substitute use in humans, and a fatty acid balance is likely more important than the particular types of fatty acids.

Engineered nanomaterials (ENM) are now used in a wide variety of fields, and, thus, their safety should urgently be assessed and secured. It has been suggested that inflammatory responses via the phagocytosis of ENM by macrophages is a key mechanism for their genotoxicity. The present study was conducted to establish a mechanism‐based assay to evaluate the genotoxicity of ENM under conditions simulating an in vivo situation, featuring a co‐culture system of murine lung resident cells (GDL1) and immune cells (RAW264.7). GDL1 were cultured with or without RAW264.7, exposed to a multi‐walled carbon nanotube (MWCNT), and then analyzed for mutagenicity and underlying mechanisms. Mutation frequencies induced in GDL1 by the MWCNT were significantly greater with the co‐existence of RAW264.7 than in its absence. Mutation spectra observed in GDL1 co‐cultured with RAW264.7 were different from those seen in GDL1 cultured alone, but similar to those observed in the lungs of mice exposed to the MWCNT in vivo. Inflammatory cytokines, such as IL‐1β and TNF‐α, were produced from RAW264.7 cells treated with the MWCNT. The generation of reactive oxygen species and the formation of 8‐oxodeoxyguanosine in GDL1 exposed to the MWCNT were greater in the co‐culture conditions than in the single culture conditions. Based on these findings, it is indicated that inflammatory responses are involved in the genotoxicity of MWCNT, and that the presently established, novel in vitro assay featuring a co‐culture system of tissue resident cells with immune cells is suitable to evaluate the genotoxicity of ENM.

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.

The Standard for Exchange of Nonclinical Data (SEND), introduced by the US Food and Drug Administration (FDA), is a scheme for the computerization, electronic application, and screening of preclinical data. Since its establishment, related organizations have been working together to implement SEND. However, it is difficult for individual pharmaceutical companies that often outsource to achieve complete compliance with SEND; hence, the cooperation of contract research organizations (CROs) and SEND Registered Solution Providers (RSPs) is indispensable. In SEND, most data, including those on pathology findings, are converted into controlled terminology (CT), but it is not a simple process to convert findings or levels of severity in the field of pathology, which is a descriptive science. The authors have successfully completed an FDA trial submission for a toxicology test conducted at a CRO and in doing so acquired important knowledge. This article presents a clear picture of such important knowledge from a pathologist’s viewpoint.

Rice powder extract (RPE) from black and brown rice (Oryza sativa L. indica) improves hepatic lipid accumulation in obese and diabetic model mice via peroxisomal fatty acid oxidation. RPE showed PPARα agonistic activity which did not differ between black and brown RPE despite a higher anthocyanin content in black RPE.

We encountered during our investigation a case of herbal drug products commercially available in the Tokyo metropolitan area in 2014, in which a small unknown peak was detected, along with the intense peak of FUB-144, by liquid chromatography–ultraviolet detection. The present study was conducted to identify and clarify the pharmacological characteristics of the compound present in this small peak. We isolated a compound using a silica gel column from the peak, which was then identified to have a molecular weight of 241 Da by liquid chromatography–mass spectrometry and gas chromatography–mass spectrometry. The accurate mass measurement suggested an elementary composition of C16H19NO. Using these mass data together with those obtained by the nuclear magnetic resonance analysis, the compound was finally identified as (1H-indol-3-yl)(2,2,3,3-tetramethylcyclopropyl)methanone (despentyl-UR-144; DP-UR-144). In addition, this compound was revealed to have affinities for cannabinoid receptors CB1 and CB2 with EC50s of 2.36 × 10−6 and 2.79 × 10−8 M, respectively. To our knowledge, there is no information in the scientific literature on structural or pharmacological properties of this chemical. These results suggest that the components present in small amounts can contribute to the effects of a major component in their mother product, if they have sufficient pharmacological activities, and, therefore, even such small amounts of components should be precisely characterized and well evaluated to control illegal and potentially illegal drug products.

Teriparatide, a drug used in the treatment of osteoporosis, was administered to rats subcutaneously for the duration of 3 months, at a frequency of either once weekly or once daily to demonstrate the varying levels of anabolic action the drug can have on bone depending on the dosing frequency. The levels of biomarkers in the blood were compared and found to vary in osteocalcin (OC), a biomarker of bone formation, and cross-linked N-telopeptide of type 1 collagen (NTx), a biomarker of bone resorption, according to the dosing frequency. In the once-weekly regimen, teriparatide did not affect NTx levels at any of the doses studied, while OC levels increased with dose, peaking at 72 hr, then returning to normal before the next injection (after 1 week). Bone mineral density (BMD) levels increased moderately with no difference between doses. This was thought to result from the steady state achieved following increases in bone formation and bone absorption. In the once-daily dosing regimen, meanwhile, NTx levels increased with dose, and OC levels were markedly higher when compared to those with the once-weekly dosing. BMD levels were higher than those with the once-weekly dosing, but with no difference between doses. This was considered a result of unlimited, excessive increases in bone formation due to daily administration of the drug. These results suggest that teriparatide promotes normal bone metabolism ("stationary mini-modeling") when administered once weekly, and has an anabolic action with high metabolic turnover ("high-turnover remodeling") when administered once daily.

Exposure to asbestos results in serious risk of developing lung and mesothelial diseases. Currently, there are no biomarkers that can be used to diagnose asbestos exposure. The purpose of the present study was to determine whether the levels or detection rate of chemokine (C-C motif) ligand 3 (CCL3) in the serum are elevated in persons exposed to asbestos. The primary study group consisted of 76 healthy subjects not exposed to asbestos and 172 healthy subjects possibly exposed to asbestos. The secondary study group consisted of 535 subjects possibly exposed to asbestos and diagnosed with pleural plaque (412), benign hydrothorax (10), asbestosis (86), lung cancer (17), and malignant mesothelioma (10). All study subjects who were possibly exposed to asbestos had a certificate of asbestos exposure issued by the Japanese Ministry of Health, Labour and Welfare. For the primary study group, levels of serum CCL3 did not differ between the two groups. However, the detection rate of CCL3 in the serum of healthy subjects possibly exposed to asbestos (30.2%) was significantly higher (P

DNA damage and cytotoxicity induced by a hydroxylated fullerene [C60(OH)24], which is a spherical nanomaterial and/or a water-soluble fullerene derivative, and their protection by sulfhydryl compounds were studied in freshly isolated rat hepatocytes. The exposure of hepatocytes to C60(OH)24 at a concentration of 50 μM caused time (0 to 3 h)-dependent cell death accompanied by the formation of cell surface blebs, the loss of cellular levels of ATP and reduced glutathione, accumulation of glutathione disulfide, and induction of DNA fragmentation assayed using alkali single-cell agarose-gel electrophoresis. C60(OH)24-induced cytotoxicity was effectively prevented by pretreatment with sulfhydryl compounds. N-acetyl-L-cysteine (NAC), L-cysteine and L-methionine, at a concentration of 2.5 mM, ameliorated cell death, accompanied by a decrease in cellular ATP levels, formation of cell surface blebs, induction of reactive oxygen species (ROS) and loss of mitochondrial membrane potential caused by C60(OH)24. In addition, DNA fragmentation caused by C60(OH)24 was also inhibited by NAC, whereas an antioxidant ascorbic acid did not affect C60(OH)24-induced cell death and DNA damage in rat hepatocytes. Taken collectively, these results indicate that incubation of rat hepatocytes with C60(OH)24 elicits DNA damage, suggesting that nuclei as well as mitochondria are target sites of the hydroxylated fullerene; and induction of DNA damage and oxidative stress is ameliorated by an increase in cellular GSH levels, suggesting that the onset of toxic effects may be partially attributable to a thiol redox-state imbalance caused by C60(OH)24. Copyright © 2015 John Wiley & Sons, Ltd.

The fetal toxicities of multi-wall carbon nanotubes (MWCNTs) with various sizes were compared in CD1(ICR) mice. MWCNTs were suspended in 2% sodium carboxymethyl cellulose solution in phosphate-buffered saline. On day 9 of gestation, dams were administered a single intraperitoneal dose of MWCNTs (4mg/kg body weight), while dams in the control group were administered vehicle (10mL/kg body weight). The rectal temperatures of the dams were monitored 2h after administaration to asses statuses of the dams. The dams and fetuses were examined on day 18 of gestation. The number of live fetus per dam decreased in some MWCNTs-administered groups. The mean percentages of live fetuses in total implantations in the MWCNTs-administered groups markedly varied from 0% to 95%, and the highest mean percentage of live fetuses in the MWCNTs-administered group was equivalent to that of the control group. The decrease in live fetuses depended on an increased number of early dead fetuses. In the groups with markedly lowered rectal temperature after administration, the fetal loss were evident. The blood levels of interleukin-6 and/or monocyte chemoattractant protein-1 in dam 2h after administration of MWCNTs markedlyr increased, especially in the goups with significant decrease in live fetuses. These results indicated a relationship between inflammation in the dam, which probabely depended on the particular length of the MWCNTs, and the fetal toxicioty of MWCNTs in mice.

The Standard for the Exchange of Nonclinical Data (SEND), adopted by the US FDA, is part of a set of regulations and guidances requiring the submission of standardized electronic study data for nonclinical and clinical data submissions. SEND is the nonclinical implementation of SDTM (Study Data Tabulation Model), the standard electronic format for clinical regulatory submissions to FDA. SEND, SDTM, and the associated Controlled Terminology have been developed by CDISC (Clinical Data Interchange Standards Consortium). In order to successfully implement SEND, interdisciplinary contributions between sponsors and CROs, need a model for task allocation. This is being undertaken by the Pharmaceutical Users Software Exchange (PhUSE). Because SEND is currently the preferred submission format of the US FDA only and will become required by it starting in December 2016, only American academic societies and companies are actively involved. An exception to this is the INHAND initiative, which leads the way in standardizing terminology for toxicological pathology. On the other hand, international globalization of other clinical and nonclinical practices is not feasible because there are substantial differences between the US and non-US countries in CRO involvement in drug development. Thus, non-US countries must consider and develop approaches to SEND that meet their needs. This paper summarizes the activities of the major organizations involved in SEND development and implementation, discusses the effective use of SEND, and details a compliance scheme (research material of the Showa University School of Medicine) illustrating how pharmaceutical companies can complete a large amount of work up to an FDA application with the effective utilization of CROs and solution providers.

The present study reports on the occurrence and chiral behavior of the anti-inflammatory drug (S)-naproxen (NAP)—(S)-2-(6-methoxynaphthalen-2-yl)propionic acid—in an aquatic environment under both field and laboratory conditions. In influents and effluents of sewage treatment plants (STPs) in the Tama River basin (Tokyo), (S)-NAP was detected at concentrations of 0.03 µg L−1 to 0.43 µg L−1 and 0.01 µg L−1 to 0.11 µg L−1, respectively. The concentrations of a major metabolite, 6-O-desmethyl NAP (DM-NAP) were up to 0.47 µg L−1 and 0.56 µg L−1 in influents and effluents, respectively. (R)-naproxen was not detected in STP influents, although it was present in effluents, and the enantiomeric faction (= S/[S + R]) of NAP ranged from 0.88 to 0.91. Under laboratory conditions with activated sludge from STPs, rapid degradation of (S)-NAP to DM-NAP and chiral inversion of (S)-NAP to (R)-NAP were observed. During river die-away experiments, degradation and chiral inversion of NAP were extremely slow. In addition, chiral inversion of (S)-NAP to (R)-NAP was not observed during photodegradation experiments. In the river receiving STP discharge, NAP and DM-NAP concentrations reached 0.08 µg L−1 and 0.16 µg L−1, respectively. The enantiomeric faction of NAP in the river ranged from 0.84 to 0.98 and remained almost unchanged with the increasing contribution of rainfall to the river water. These results suggest that the absence and decrease of (R)-NAP in river waters could indicate the inflow of untreated sewage. Environ Toxicol Chem 2014;33:2671–2678. © 2014 SETAC

Male and female mice were housed in cages, containing different types of bedding materials (wood flakes or pulp chips), from 4 weeks of age in the F0 generation to 11 weeks of age in the F1 generation; selected reproductive and neurobehavioral parameters were measured in the F1 generation. There were no adverse effects of bedding materials on litter size, litter weight, or sex ratios at the time of birth. With regard to behavioral development parameters, bedding materials did not influence any variables (p > 0.05) in both sexes. Regarding exploratory behavior in the F1 generation, number of defecations significantly varied (p = 0.0203) with bedding materials in males at 3 weeks of age. The number of horizontal activities also significantly varied (p = 0.0342) with bedding materials in males at 8 weeks of age. Multiple-T water maze performance data indicated that the time required was significantly shortened across trials in pulp chips group than wood flakes group in males (p = 0.0211). Moreover, all spontaneous behavior variables in males significantly varied with bedding materials, particularly the average time of movement was significantly different (p = 0.0037) in distance between parallel lines of types of bedding materials in the F1 generation. The present study shows that bedding materials influence the neurobehavioral development in mice.

Introduction It is known that fibrous particles of micrometer length, such as carbon nanotubes, which have same dimensions as asbestos, are carcinogenic. Carcinogenicity of nanomaterials is strongly related to inflammatory reactions; however, the genotoxicity mechanism(s) is unclear. Indeed, inconsistent results on genotoxicity of multi-walled carbon nanotubes (MWCNTs) have been shown in several reports. Therefore, we analyzed the in vivo genotoxicity induced by an intratracheal instillation of straight MWCNTs in rats using a different test system—the Pig-a gene mutation assay—that can reflect the genotoxicity occurring in the bone marrow. Since lungs were directly exposed to MWCNTs upon intratracheal instillation, we also performed the gpt assay using the lungs. Findings We detected no significant differences in Pig-a mutant frequencies (MFs) between the MWCNT-treated and control rats. Additionally, we detected no significant differences in gpt MFs in the lung between the MWCNT-treated and control rats. Conclusions Our findings indicated that a single intratracheal instillation of MWCNTs was non-mutagenic to both the bone marrow and lung of rats.

Because multi-wall carbon nanotubes (MWCNTs) have asbestos-like shape and size, concerns about their pathogenicity have been raised. Contaminated metals of MWCNTs may also be responsible for their toxicity. In this study, we employed high-temperature calcined fullerene nanowhiskers (HTCFNWs), which are needle-like nanofibers composed of amorphous carbon having similar sizes to MWCNTs but neither metal impurities nor tubular structures, and investigated their ability to induce production a major proinflammatory cytokine IL-1β via the Nod-like receptor pyrin domain containing 3 (NLRP3)-containing flammasome-mediated mechanism. When exposed to THP-1 macrophages, long-HTCFNW exhibited robust IL-1β production as long and needle-like MWCNTs did, but short-HTCFNW caused very small effect. IL-1β release induced by long-HTCFNW as well as by long, needle-like MWCNTs was abolished by a caspase-1 inhibitor or siRNA-knockdown of NLRP3, indicating that NLRP3-inflammasome-mediated IL-1β production by these carbon nanofibers. Our findings indicate that the needle-like shape and length, but neither metal impurities nor tubular structures of MWCNTs were critical to robust NLRP3 activation.

Nanomaterials are useful for their characteristic properties and are commonly used in various fields. Nanosized-magnetite (MGT) is widely utilized in medicinal and industrial fields, whereas their toxicological properties are not well documented. A safety assessment is thus urgently required for MGT, and genotoxicity is one of the most serious concerns. In the present study, we examined genotoxic effects of MGT using mice and revealed that DNA damage analyzed by a comet assay in the lungs of imprinting control region (ICR) mice intratracheally instilled with a single dose of 0.05 or 0.2 mg/animal of MGT was approximately two- to three-fold higher than that of vehicle-control animals. Furthermore, in gpt delta transgenic mice, gpt mutant frequency (MF) in the lungs of the group exposed to four consecutive doses of 0.2 mg MGT was significantly higher than in the control group. Mutation spectrum analysis showed that base substitutions were predominantly induced by MGT, among which G:C to A:T transition and G:C to T:A transversion were the most significant. To clarify the mechanism of mutation caused by MGT, we analyzed the formation of DNA adducts in the lungs of mice exposed to MGT. DNA was extracted from lungs of mice 3, 24, 72 and 168 h after intratracheal instillation of 0.2 mg/body of MGT, and digested enzymatically. 8-Oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) and lipid peroxide-related DNA adducts were quantified by stable isotope dilution liquid chromatography-mass spectrometry (LC-MS/MS). Compared with vehicle control, these DNA adduct levels were significantly increased in the MGT-treated mice. In addition to oxidative stress- and inflammation related-DNA adduct formations, inflammatory cell infiltration and focal granulomatous formations were also observed in the lungs of MGT-treated mice. Based on these findings, it is suggested that inflammatory responses are probably involved in the genotoxicity induced by MGT in the lungs of mice.

There are no reported spontaneous cases of pancreatic ductal adenocarcinoma (PDAC), and there are few reports about chemically-induced PDAC in rats. We encountered a PDAC in a Wistar Hannover GALAS rat that had been subjected to a medium-term multiorgan carcinogenicity bioassay. This article describes the histological and histochemical findings of the tumor. The tumor was located in the pancreatic tissue and had not invaded the liver parenchyma or the mucosal layer of the alimentary tract. The tumor cells were atypical and were mainly arranged in small tubules. In addition, abundant stroma and mucus production were observed in the tumor. In an immunohistochemical examination, the tumor cells were positive for cytokeratin, Sox9 and pancreas duodenum homeobox 1 and negative for amylase 2A and insulin. Therefore, the tumor was diagnosed as a PDAC based on its histological and histochemical findings. We considered that the tumor was caused by the carcinogens administered during the abovementioned bioassay.

The present study examined the effects of formic acid and acetic acid on human adenocarcinoma-derived alveolar basal epithelial A549 cells. The organic acids were administered either individually or in combination, into either the culture medium (aqueous phase) or the gaseous phase of an air-liquid interface. When either of the acids was administered into the aqueous phase, cell proliferation was inhibited at doses of 1-10 mg/mL. In contrast, when the acids were administered either individually or in combination, into the gaseous phase of the air-liquid interface, cell proliferation was not altered. Under the gaseous phase administration, acetic acid and mixed acids caused a slight increase, decrease and increase on the interleukin-8 production, the mRNA expression of the heme oxygenase-1 (HO-1) gene and the HO-1 production, respectively, at one or more time points. The results therefore indicated that organic acids might be less reactive in the gaseous phase than in the aqueous phase. However, acetic acid in the gaseous phase either individually or in combination with formic acid exerts some effects on A549 cells.