Kousuke Saoo

Lung Toxicity of 16 Fine Particles on Intratracheal Instillation in a Bioassay Model Using F344 Male Rats

We have developed a bioassay model to estimate toxicity of fine particles in the lungs at an early stage after intratracheal instillation (Yokohira et al. 2005; Yokohira et al. 2007). The present experiment was conducted to improve the model by estimating appropriate doses based on dose-dependent toxicity of instilled quartz (4 mg to 0 mg) as a positive control and assessing the impact of powdered particles without suspension (Experiment 1). In addition, examination of the toxicity of a series of particles was performed with the developed bioassay (Experiments 2A, 2B, and 2C). The materials chosen were sixteen particles, including nanoparticles and diesel powder. Histopathological and immunohistochemical analysis of bromodeoxyuridine (BrdU) incorporation and inducible nitric oxide synthase (iNOS) were performed after exposure of the lungs. A dose of 2 mg quartz suspended in 0.2 mL saline was suggested to be most appropriate for sensitive detection of acute and subchronic inflammatory changes. Although some materials, including nanoparticles, demonstrated toxicity that was too strong for sensitive assessment, the ranking order could be given as follows: CuO > quartz > neutralized Na2PdCl4 > NiO > hydrotalcite > MnO2 > diesel > titanium dioxide (in Experiment 2B) > β-cyclodextrin > diesel standard > titanium dioxide (in Experiment 2A) > CaCO3.

Antioxidant Effects of Flavonoids Used as Food Additives (Purple Corn Color, Enzymatically Modified Isoquercitrin, and Isoquercitrin) on Liver Carcinogenesis in a Rat Medium-Term Bioassay

To clarify the effects of purple corn color, enzymatically modified isoquercitrin (EMIQ), and isoquercitrin (IQ), registered as natural food additives in Japan, on liver carcinogenesis in vivo, a medium-term bioassay was employed. A total of 100 male F344 rats were divided into 5 groups; groups 1 to 4 were given a single intraperitoneal injection of diethylnitrosamine (200 mg/kg b.w.) on day 1. From weeks 2 to 8, they were administered basal diet purple corn color, EMIQ, or IQ as containing test chemicals at doses of 1.0% (groups 1 and 5), 0.1% (group 2), 0.01% (group 3), or 0% (group 4) (experiments 1, 4, and 5). All rats were subjected to two-thirds partial hepatectomy at week 3 and were sacrificed at week 8. Purple corn color exerted no significant modifying effects on GST-P positive foci, preneoplastic foci, development in the liver. However, serum of rats treated with purple corn color provided evidence of antioxidant power significantly by potential antioxidant (PAO) test in vivo (experiment 2). And microarray analyses showed purple corn color to induce RNA expression such as P450 (cytochrome) oxidoreductase, phosphatidylinositol 3-kinase, and phospholipase A2 (experiment 3). Higher doses of EMIQ or IQ with strong antioxidant power in vivo by PAO test treated groups were correlated with smaller numbers of GST-P positive foci, with Spearmans rank correlation coefficients of P= 0.002 and P= 0.049, respectively (experiments 4 and 5). Therefore, the tested food additives may be effective as antioxidants in vivo and have chemopreventive potential against liver preneoplastic lesion development.

Lung Carcinogenic Bioassay of CuO and TiO2 Nanoparticles with Intratracheal Instillation Using F344 Male Rats

Toxicity assessment of nanoparticles, now widespread in our environment, is an important issue. We have focused attention on the carcinogenic potential of copper oxide (CuO) and titanium dioxide (TiO2). In experiment 1, a sequential pilot study, the effectiveness of a carcinogenic bioassay featuring intraperitoneal injection (i.p.) of 20 mg 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) or 0.1% N-bis(2-hydroxypropyl)nitrosamine (DHPN) in drinking water for 2 weeks was examined. Based on the results, DHPN, as the lung carcinogen, and evaluation at week 30 were selected as the most appropriate for our purposes in Experiment 1. In experiment 2, the carcinogenic bioassay was used to assess the carcinogenic potentials of instilled nanoparticles of CuO and TiO2. There were no significant intergroup differences in the lung neoplastic lesions induced by DHPN, although the neoplastic lesions induced by the nanoparticles in the CuO or TiO2 intratracheal instillation (i.t.) groups, demonstrated a tendency to increase compared with the microparticles administration. At the very least, the carcinogenic bioassay with DHPN proved useful for assessment of the modifying effects of instilled particles, and further assessment of the carcinogenic potential of nanoparticles appears warranted.

Equivocal Colonic Carcinogenicity of Aloe arborescens Miller var. natalensis Berger at High‐Dose Level in a Wistar Hannover Rat 2‐y Study

A 2-y carcinogenicity study of Aloe, Aloe arborescens Miller var. natalensis Berger, a food additive, was conducted for assessment of toxicity and carcinogenic potential in the diet at doses of 4% or 0.8% in groups of male and female Wistar Hannover rats. Both sexes receiving 4% showed diarrhea, with loss of body weight gain. The survival rate in the 4% female group was significantly increased compared with control females after 2 y. Hematological and biochemical examination showed increase of RBC, Hb, and Alb in the 4% males. The cause of these increases could conceivably have been dehydration through diarrhea. AST and Na were significantly decreased in the males receiving 4%, and Cl was significantly decreased in both 4% and 0.8% males. A/G was significantly increased in the 4% females, and Cl was significantly decreased (0.8%) in the female group. Histopathologically, both sexes receiving 4% showed severe sinus dilatation of ileocecal lymph nodes, and yellowish pigmentation of ileocecal lymph nodes and renal tubules. Adenomas or adenocarcinomas in the cecum, colon, and rectum were observed in 4% males but not in the 0.8% and control male groups. Similarly, in females, adenomas in the colon were also observed in the 4% but not 0.8% and control groups. In conclusion, Aloe, used as a food additive, exerted equivocal carcinogenic potential at 4% high-dose level on colon in the 2-y carcinogenicity study in rats. Aloe is not carcinogenic at nontoxic-dose levels and that carcinogenic potential in at 4% high-dose level on colon is probably due to irritation of the intestinal tract by diarrhea.

Intrathyroidal branchial cleft‐like cyst in chronic thyroiditis

An extremely rare case of intrathyroidal branchial cleft‐like cyst is reported. A 71‐year‐old man complained of a growing mass in the right lateral neck. A cystic mass in the upper lobe of the right thyroid was demonstrated by ultrasonography and computed tomography. The surgical specimen revealed a cystic mass with dense fibrous capsule, 22 × 20 × 10 mm in size. Microscopically, the cyst walls and the surrounding thyroid tissue contained severe lymphoid cell infiltration with lymphoid follicle. Squamous epithelium lined the cyst wall. Immunohistochemically, squamous epithelium was positive for keratin, cytokeratin 19, carcinoembryonic antigen, and epithelial membrane antigen, but negative for calcitonin and chromogranin A. The patient is currently well with no evidence or recurrence for 43 months.

Sarcomatoid hepatocellular carcinoma with chondroid variant: Case report with immunohistochemical findings

A case of sarcomatoid hepatocellular carcinoma (HCC) with a chondroid variant in a 72‐year‐old woman is reported. Histologically, the tumor consisted of typical HCC of adult type, with an adenocarcinoma‐like structure and a sarcomatoid component containing multinodular chondroid foci. Transition from carcinoma to sarcomatoid component was observed. The cells containing mucus were extremely few in adenocarcinoma‐like structures. Immunohistochemically, other than typical HCC, some of the chondroid cells, adenocarcinoma‐like cells and sarcomatoid cells were positive for alpha‐fetoprotein.

Molecular analysis of carcinogen-induced rodent lung tumors: Involvement of microRNA expression and Krαs or Egfr mutations.

Genetic and epigenetic alterations play a key role in lung carcinogenesis, and a high frequency of KRAS and epidermal growth factor receptor (EGFR) mutations have been observed in human lung cancers. Recent evidence indicates that the expression of specific microRNAs (miRNAs) may be involved. In rodent lung carcinogenesis models, Kras mutations are frequently observed, whereas genetic alteration of the Egfr gene is generally rare. Since little is known regarding the involvement of miRNAs in rodent lung carcinogenesis, the present study of miRNA expression levels in the liver and lung during 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)- and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx)-induced lung tumorigenesis in A/J mice was conducted. In addition, incidences of Egfr and Kras gene mutations in rat and mouse lung tumors induced by the chemical carcinogens NNK, MeIQx and N-bis(2?hydroxypropyl)nitrosamine (DHPN) were examined. Three miRNAs, let-7a, miR-34c and miR-125a-5p, were selected for attention. In rat lung tumors, one silent mutation was detected in the Egfr gene exon 20 (AAC↷AAT; N772). Activating mutations of the Krαs gene at codon 12 were detected in neoplastic lesions induced by NNK (5/6, 83%), MeIQx (1/1, 100%) and DHPN (7/15, 47%), all resulting in G/C↷A/T transitions. NNK or MeIQx administration reduced the expression of miR-125a-5p (MeIQx alone group, 86.3%; MeIQx + NNK group, 83.6%; p<0.05, at day 15) and let-7a (MeIQx + NNK group, 56.3%; p<0.001, at day 22) in the liver. miR-34c was up-regulated 3.5-fold with NNK treatment as compared to the control group (p<0.001). These findings raise the possibility that aberrant expression of miRNA is involved in lung tumorigenesis, at least in its early stages.

p27Kip1 is overexpressed in very early stages of hepatocarcinogenesis

Hepatocellular carcinoma (HCC) associated with chronic liver disease evolves from precancerous lesions and early HCC to more malignant forms. Despite the demonstrated importance of cell‐cycle regulators in tumor biology, there have been few studies of their role in multistep hepatocarcinogenesis. Expression of p27Kip1 and a degradation pathway associated protein, S‐phase kinase‐interacting protein 2 (Skp2), was therefore evaluated in surgically resected specimens of eight adenomatous hyperplasias, 16 early HCC and 126 classical HCC. Immunohistochemistry revealed no p27Kip1 expression in the majority of hepatocytes from normal and cirrhotic liver, whereas positive staining for p27Kip1 protein was found in 75.0% and 93.8% of adenomatous hyperplasias and early HCC, respectively. The average p27Kip1 labeling indices (LI) for adenomatous hyperplasias, early HCC, well differentiated HCC, moderately differentiated HCC and poorly differentiated HCC were 36.99, 43.59, 47.73, 49.24, and 30.21, respectively. Real‐time quantitative reverse transcription–polymerase chain reaction (RT‐PCR) analyses confirmed the increases. Skp2 LI were also significantly elevated in accordance with stepwise progression of hepatocarcinogenesis. Increased expression of Skp2 mRNA was observed most frequently in less differentiated tumors and Kaplan–Meier survival analysis showed a significantly association with a poor prognosis (P = 0.0496). In conclusion, a high level of p27Kip1 expression is evident from early stages of hepatocarcinogenesis, indicating that this parameter could be a useful diagnostic marker for precancerous lesions and early HCC. In addition, Skp2 expression correlates with tumor dedifferentiation and may contribute to biological aggression in HCC. (Cancer Sci 2008; 99: 2152–2159)

An Intratracheal Instillation Bioassay System for Detection of Lung Toxicity Due to Fine Particles in F344 Rats

It is an urgent priority to establish in vivo bioassays for detection of hazards related to fine particles, which can be inhaled into deep lung tissue by humans. In order to establish an appropriate bioassay for detection of lung damage after particle inhalation, several experiments were performed in rats using quartz as a typical lung toxic particle. The results of pilot experiments suggest that Days 1 and 28 after intratracheal instillation of 2 mg of fine test particles in vehicle are most appropriate for detection of acute and subacute inflammatory changes, respectively. Furthermore, the BrdU incorporation on Day 1 and the iNOS level on Day 28 proved to be suitable end-point markers for this purpose. An examination of the toxicity of a series of particles was performed with the developed bioassay. Although some materials, including nanoparticles, demonstrated toxicity that was too strong for sensitive assessment, a ranking order could be clarified. The bioassay thus appears suitable for rapid hazard identification with a possible ranking of the toxicity of various particles at single concentrations.

Establishment of a bioassay model for lung cancer chemoprevention initiated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in female A/J mice

AIMS In order to prevent lung cancer development in people at high risk, identification of chemopreventive agents may be important. The present study was conducted to establish a bioassay model for this purpose. In particular, the time course of 4-(methylnitrosamno)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumor development was examined to determine the most appropriate shortest period to assess effects of test agents, with 8-methoxypsoralen (8-MOP) as a typical example. METHODS A total of 124 mice were separated into two groups (Group A: 60 mice, Group B: 64 mice), pretreated with 100ppm 8-MOP (Group A) or basal diet (Group B) for 3 days before receiving single doses of NNK (2mg/0.1ml saline/mouse i.p.) on days 0 and 7. Subgroups of 15 mice of each group were then sacrificed after 8, 10, 12, and 16 weeks. RESULTS Microscopically, the earliest time point when significant differences in data for hyperplasia, adenoma and hyperplasia and adenoma could be detected was 12 weeks. A trend was noted for 8-MOP to reduce adenomas to a greater extent than hyperplasia. DISCUSSION In conclusion, the results of this study showed that the double i.p. treatment with NNK and 12 weeks duration are effective for detection of lung cancer chemoprevention in our A/J mouse lung tumorigenesis model.