Long-Term Chronic Toxicity and Mesothelial Cell Reactions Induced by Potassium Octatitanate Fibers (TISMO) in the Left Thoracic Cavity in A/J Female Mice.

The present study was conducted to examine the chronic effects of potassium octatitanate fibers (trade name TISMO; chemical formula K2O·6TiO2) on the mouse lung and thoracic cavity. This method of infusion was employed to examine the direct effects of the fibers to the pleura. In the present study, 52- and 65-week experiments were employed to examine the long-term chronic effects after infusion of fiber-shaped TISMO into the thoracic cavities of A/J mice. Following this infusion, TISMO fibers were observed in the alveoli, indicating penetration through the visceral pleura. The additional histopathological detection of TISMO fibers in the liver, spleen, kidneys, ovary, heart, bone marrow, and brain of TISMO-infused mice indicated migration of the fibers out from the thoracic cavity. Atypical mesothelial cells with severe pleural proliferation were observed, but malignant mesotheliomas were not detected. This study demonstrated that intrathoracic infusion of TISMO fiber did not cause malignant mesothelioma but did cause severe chronic inflammation and proliferation of pleural mesothelial cells.

Immunohistochemical characteristics of surfactant proteins a, B, C and d in inflammatory and tumorigenic lung lesions of f344 rats.

Surfactant proteins (SPs), originally known as human lung surfactants, are essential to respiratory structure and function. There are 4 subtypes, SP-A, SP-B, SP-C and SP-D, with SP-A and SP-D having immunological functions, and SP-B and SP-C having physicochemical properties that reduce the surface tension at biological interfaces. In this experiment, the expressions of SP-A, SP-B, SP-C and SP-D in lung neoplastic lesions induced by N-bis (2-hydroxypropyl) nitrosamine (DHPN) and inflammatory lesions due to quartz instillation were examined and compared immunohistochemically. Formalin fixed paraffin embedded (FFPE) lung samples featuring inflammation were obtained with a rat quartz instillation model, and neoplastic lesions, hyperplasias and adenomas, were obtained with the rat DHPN-induced lung carcinogenesis model. In the rat quartz instillation model, male 10-week old F344 rats were exposed by intratracheal instillation (IT) to quartz at a dose of 2 mg/rat suspended in saline (0.2 ml) on day 0, and sacrificed on day 28. Lung tumorigenesis in F344 male rats was initiated by DHPN in drinking water for 2 weeks, and the animals were then sacrificed in week 30. Lung proliferative lesions, hyperplasias and adenomas, were observed with DHPN, and inflammation was observed with quartz. The expressions of SP-A, SP-B, SP-C and SP-D were examined immunohistochemically. SP-B and SP-C showed strong expression in lung hyperplasias and adenomas, while SP-A and SP-D were observed in mucus or exudates in inflammatory alveoli. These results suggest the possibility that SP-B and SP-C are related to lung tumorigenesis.

[A case of advanced gastric cancer with multiple liver metastases treated with preoperative TS-1/CDDP chemotherapy and resection, with a complete response and survival for 7 years].

CASE: An 82-year-old man died because of squamous cell carcinoma of the right lung with metastasis to the left femoral bone. At the age of 75 years, he was admitted to our hospital because of hematemesis. Widespread type 3 gastric cancer was detected in the lesser curvature. Computed tomography(CT)showed multiple liver metastases. Preoperative chemotherapy with TS-1/cisplatin(CDDP)was administered. TS-1 was orally administered at 80mg/body/day and CDDP was administered by intravenous infusion at 20mg/body/day every week for 3 weeks and this was followed by a drug-free 2-week period as the first course. After the fourth course, gastrectomy was performed for the primary lesion and radiofrequency ablation(RFA)was performed for the liver metastases. The patient survived for more than 7 years with a complete response (CR)and died thereafter because of squamous cell carcinoma of the lung.

Association of longer telomere length in cancer cells and cancer-associated fibroblasts with worse prognosis.

BACKGROUND: Telomere dysfunction has been reported to be directly involved in carcinogenesis owing to chromosomal instability and immortalization; however, the clinicopathological significance of telomeres remains controversial. We have shown that telomere shortening occurs in normal-appearing duct cells at initiation and then continues during the progression of pancreatic cancer. In this study, we determined the clinicopathological and prognostic value of telomere length (TL) in cancer progression. METHODS: TL in both cancer cells and cancer-associated fibroblasts (CAFs) was analyzed by high-throughput quantitative fluorescence in situ hybridization using a previously reported cohort comprising 1434 cases of adenocarcinoma (ADC), squamous cell carcinoma (SCC), adenosquamous carcinoma, hepatocellular carcinoma, and renal cell carcinoma (RCC), which are known cancers with a statistically significantly low incidence of alternative lengthening of telomeres. Cases were divided into 2 groups as follows: longer and shorter telomeres, according to the median TL of cancer cells and CAFs. The statistical significance of TL in cancer cells and CAFs on clinicopathological characteristics and prognosis was analyzed. RESULTS: There was a close association between TL in cancer cells and CAFs. Longer telomeres in cancer cells and CAFs were associated with aggressive features such as advanced stage, high mitosis score and nuclear score, poorly differentiated cancer, and desmoplastic stroma in ADC. Furthermore, a longer TL was an independent prognostic factor for ADC, SCC, and RCC. CONCLUSIONS: Longer telomeres are associated with worse prognosis in ADC, SCC, and RCC. Thus, TL is a novel biomarker for the diagnosis of aggressive cancers with poor prognoses.

Toxicity of nicotine by repeated intratracheal instillation to f344 rats.

In vivo, nicotine in cigarette smoke induces various effects not only on the respiratory system but also the central and peripheral nerve systems, circulatory organs and digestive organs, and there is a possibility of promotion of lung tumorigenesis. The present experiment was conducted to examine histopathological changes caused by nicotine in the lung with repeated intratracheal instillation (i.t.). Six-week-old male F344 rats were administered nicotine by i.t. at doses of 0.05, 0.1 and 0.2 mg nicotine/rat every 3 weeks beginning at week 4, for up to a total of 9 times and were then sacrificed at week 30. The total number of administrations, total dose of nicotine and effective number of rats were 9 times, 0.45 mg and 5 rats and 4 times, 0.20 mg and 5 rats for the 0.05 mg nicotine/rat group; 3 times, 0.30 mg and 5 rats and 4 times, 0.40 mg and 3 rats for the 0.1 mg group; and 3 times, 0.60 mg and 3 rats for the 0.2 mg group, respectively. As a control group, 5 rats were administered 0.2 ml saline/rat 9 times. Some rats administered 0.1 and 0.2 mg nicotine suffered convulsions just after administration. Histopathologically, though proliferative changes were not observed, neutrophil infiltration, edema and fibrosis in the lung were induced by nicotine. In conclusion, repeated treatment of nicotine promoted neurologic symptoms in the acute phase, and strong inflammation in the lungs in the chronic phase, even at a low dose. Toxicity of nicotine is suggested to depend not on total dose of nicotine in the experiment but rather on repeated injury with consecutive administration.

Single Intratracheal Quartz Instillation Induced Chronic Inflammation and Tumourigenesis in Rat Lungs.

Crystalline silica (quartz) is known to induce silicosis and cancer in the lungs. In the present study, we investigated the relationship between quartz-induced chronic inflammation and lung carcinogenesis in rat lungs after a single exposure to quartz. F344 rats were treated with a single intratracheal instillation (i.t.) of quartz (4 mg/rat), and control rats were treated with a single i.t. of saline. After 52 or 96 weeks, the animals were sacrificed, and the lungs and other organs were used for analyses. Quartz particles were observed in the lungs of all quartz-treated rats. According to our scoring system, the lungs of rats treated with quartz had higher scores for infiltration of lymphocytes, macrophages and neutrophils, oedema, fibrosis, and granuloma than the lungs of control rats. After 96 weeks, the quartz-treated rats had higher incidences of adenoma (85.7%) and adenocarcinoma (81.0%) than control rats (20% and 20%, respectively). Quartz-treated and control rats did not show lung neoplastic lesions at 52 weeks after treatment. The number of lung neoplastic lesions per rat positively correlated with the degree of macrophage and lymphocyte infiltration, oedema, fibrosis, and lymph follicle formation around the bronchioles. In conclusion, single i.t. of quartz may induce lung cancer in rat along with chronic inflammation.

Preexisting diabetes mellitus had no effect on the no-observed-adverse-effect-level of acetaminophen in rats.

Information on the safety of chemical substances in patients with various preexisting conditions remains limited. Acetaminophen was added to the basal diet at 0, 80, 253, 800, 2530, or 8000 ppm and administered to type 2 diabetes mellitus rats (GK/Jcl) and the control male rats (Wistar) for 13 weeks. Both strains treated with 8000 ppm acetaminophen (561.4 and 567.7 mg/kg body weight/day, GK/Jcl and Wistar rats, respectively) showed decreased levels of red blood cell counts, blood urea nitrogen, creatinine, and total bilirubin compared to those of non-treated rats. Treatment with 8000 ppm of acetaminophen reduced the blood glucose and hemoglobin A1c levels of GK/Jcl rats. An increase in the relative weights of the kidneys and liver, and a decrease in the weight of the salivary glands were observed in both GK/Jcl and Wistar rats treated with 8000 ppm acetaminophen relative to those of non-treated control rats. Microscopically, both strains treated with 2530 (174.3 and 164.2 mg/kg body weight/day, GK/Jcl and Wistar rats, respectively) or 8000 ppm acetaminophen showed hepatocellular hypertrophy and degenerative lesions in the salivary glands, whereas similar lesions were not observed in non-treated rats. In conclusion, the no-observed-adverse-effect-level of acetaminophen was 800 ppm in both diabetic and control rats.

Lung Carcinogenic Bioassay of CuO and TiO(2) 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 (TiO(2)). 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 TiO(2). 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 TiO(2) 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.

Lack of Modifying Effects of Intratracheal Instillation of Quartz or Dextran Sulfate Sodium (DSS) in Drinking Water on Lung Tumor Development Initiated with 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in Female A/J Mice.

The purpose of the present study was to investigate the effects of inflammation, induced by intratracheal instillation (i.t.) of quartz as an environmental factor in the lung or drinking of dextran sulfate sodium (DSS) as an environmental factor in the colon on lung tumors in female A/J mice initiated with NNK. For comparison, colonic preneoplastic lesions, aberrant crypt foci (ACF), were also assessed. A/J mice at 6 weeks of age were divided into 5 groups, and Groups 1, 2 and 3 were pretreated with NNK (2 mg / 0.1 ml saline / mouse, intraperitoneal injection) at week 0. For a week, 2% DSS in drinking water was administered to the mice in Groups 2 and 4 beginning in week 1. In week 2, the mice of Groups 3 and 5 were exposed to intratracheal instillation of quartz (0.1 mg/rat) suspended in 25 µl saline. The experiment was terminated after 16 weeks. The results for the lung tumors and colonic ACFs showed a lack of modifying effects of the inflammation in either site. Hematologically and histopathologically, the inflammation induced by 0.1 mg quartz in the lung and 2% DSS in the colon was lacking or only mild at the end of 16 weeks. These results suggest that there may be differences in sensitivity to inflammation that determine tumor promoting potential.

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.

p27(Kip1)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 p27(Kip1) 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 p27(Kip1) expression in the majority of hepatocytes from normal and cirrhotic liver, whereas positive staining for p27(Kip1) protein was found in 75.0% and 93.8% of adenomatous hyperplasias and early HCC, respectively. The average p27(Kip1) 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 p27(Kip1) 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.

Lack of modifying potential of 8-methoxypsoralen in the promotion or progression stages of lung carcinogenesis in A/J female mice.

Pretreatment with 8-methoxypsoralen (8-MOP), a potent human CYP2A6 inhibitor, strongly suppresses lung tumorigenesis by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in female A/J mice. Since it has been reported that CYP2A protein is highly expressed in NNK-induced lung adenomas and adenocarcinomas, potential anticancer properties of 8-MOP in female A/J mice were examined subsequent to initiation. The agent was administered at 100 ppm levels in the diet during the promotion phase (experimental weeks 1-16) and progression phase (experimental weeks 16-32) and mice were sacrificed for histopathological examination of lung tumor development at 16 and 32 weeks after the initiation of NNK treatment (2 mg/0.1 ml saline/mouse, i.p.), respectively. Chemopreventive effects of 8-MOP were not observed in either experiment. In addition, no modifying effects on hepatic mRNA levels for CYP2A5, considered to be the mouse ortholog of human CYP2A6, were evident. On immunohistochemical analysis, the CYP2A protein was found to be overexpressed in all lung adenomas and adenocarcinomas, with or without 8-MOP. It can be concluded from the present data that 8-MOP at 100 ppm in the diet does not prevent mouse lung carcinogenesis when administered in the post-initiation phase.

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) > beta-cyclodextrin > diesel standard > titanium dioxide (in Experiment 2A) > CaCO3.

A 90-day toxicity study of L-asparagine, a food additive, in F344 rats.

L-asparagine is an amino acid listed as an existing food additive in Japan. The present 90-day toxicity study in F344/DuCrlCrj rats was conducted for safety assessment and to determine a no observed adverse effect level (NOAEL) of L-asparagine. Groups of 10 males and 10 females were given the material at dose levels of 0%, 1.25%, 2.5% or 5% in diet for 90 days. During the experiment, there were no remarkable changes in general conditions and no deaths occurred in any group. Final body weights of male 5% and 1.25% groups were significantly decreased. There were also significant increases in relative organ weights of the brain, kidney and testis in 5% males. On serological examination, GLU, PL, K and ALT were increased significantly in 5% females, and GLU was increased significantly and CRN was decreased significantly in the female 1.25% group. However, histopathological examination did not reveal any significant variation in development of lesions among the groups. Changes in body and organ weights, as well as other parameters, were concluded to be due to treatment with 5% L-asparagine. The NOAEL was determined to be 2.5% in the diet (males, 1.65 g/kg body weight/day; females, 1.73 g/kg body weight/day).

Induction of multiple granulomas in the liver with severe hepatocyte damage by montan wax, a natural food additive, in a 90-day toxicity study in F344 rats.

Montan wax is a mineral wax extracted from lignite type coal. It has been registered as a food additive in Japan though there have been no reports of toxicological evaluation, mainly due to the fact that it is considered a natural product. As part of a general safety assessment of montan wax, we have performed a 90-day toxicity study in Fisher 344 (F344) rats. Groups of 10 males and 10 females were given the material at dose levels of 0 (Group 1), 0.56 (Group 2), 1.67 (Group 3), or 5% (Group 4) in the diet for 90 days. During the experiment, there were no remarkable changes in general conditions and no deaths occurred in any group. On hematological examination, Hb, Ht, MCV and MCH were significantly decreased and WBC was significantly increased in all treated rats. On serum biochemical examination, AST and ALT were found to be elevated more than four fold in all treated groups as compared to the respective control group values in both sexes. Furthermore, relative organ weights for the liver, spleen, lung and kidneys were increased in all treated groups of both sexes. Histopathological examination revealed diffuse multiple granulomas in the livers with severe hepatocyte damage and lymphocytic infiltration. Granulomatous lesions were also apparent in the mesenteric lymph nodes in all treated males and females. These findings clearly demonstrate that montan wax, at doses of more than 0.56% in the diet, induces multiple granulomas with severe inflammation in the liver. Because pathological, hematological and serum biochemical changes were observed in the lowest dose group, a no-observed-adverse-effect level (NOAEL) could not be determined in the present study.

One-year chronic toxicity study of Aloe arborescens Miller var. natalensis Berger in Wistar Hannover rats. A pilot study.

This study was conducted to evaluate the chronic toxicity of Aloe arborescens Miller var. natalensis Berger (ALOE) in the diet at doses of 4.0%, 0.8% or 0.16% to groups of male and female Wistar Hannover rats. No deaths occurred at any dose level throughout the treatment period. Both sexes receiving 4.0% showed diarrhea, with a reduced body weight gain. Increase of WBCs in the male 4.0% group, decrease of Hb in the female 4.0% and 0.8% groups, decrease of IP in the male 4.0% and 0.8% groups and female 4.0% group, and decrease of Ca and ALT in the female 4.0% group were observed. Relative kidney weight showed increase in the female 4.0% group and relative heart and brain weights were decreased in the female 4.0% and 0.8% groups. Histopathologically, both sexes receiving 4.0% showed severe sinus dilatation of ileocecal lymph nodes, and yellowish pigmentation of ileocecal lymph nodes and renal tubules. In conclusion, the no observed adverse effect level (NOAEL) for ALOE was the 0.16% in diet, which is equivalent to 87.7 and 109.7 mg/kg/day in males and females, respectively.

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.

Post-initiation chemopreventive effects of dietary bovine lactoferrin on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced lung tumorigenesis in female A/J mice.

We investigated the effects of bovine LF (bLF) on different phases of NNK-induced lung tumorigenesis in A/J mice. Mice were orally administered 0.02, 0.2 and 2% bLF during the initiation phase, and 2% bLF during the whole tumorigenesis phase or post-initiation phase. Administered bLF during the post-initiation phase showed significant reduction of macroscopical lung nodules, and immunohistochemically decreased expression levels of cell proliferation marker and increased expression levels of apoptosis marker in lung proliferative lesions. bLF might inhibit NNK-induced mouse lung tumorigenesis, only when given limited to the post-initiation phase, through modification of cell proliferation and/or apoptosis.

CYP2A6 overexpression in human lung cancers correlates with a high malignant status.

CYP2A6 is a major phase I enzyme metabolizing tobacco-specific nitrosamines, implicated as risk factors for lung cancer. In this study, immunohistochemistry and in situ hybridization (ISH) for CYP2A6 with human lung cancer tissues (n=31) obtained by surgical resection showed significantly higher immunoreactivity in the cases with lymph node metastasis. The adenocarcinoma cases (n=23) with lymph node metastasis or large tumor size showed a high immunoreactivity for CYP2A6. The squamous cell carcinoma cases (n=6) with large tumor size showed a tendency for low CYP2A6 immunoreactivity. ISH for CYP2A6 revealed mRNA expression in both adenocarcinoma and squamous cell carcinoma cells. The data suggest that CYP2A6 could have an important role in the development and proliferation of lung carcinomas. With adenocarcinomas, CYP2A6 could be a target candidate for therapeutic and chemopreventive intervention.

Bioassay by intratracheal instillation for detection of lung toxicity due to fine particles in F344 male rats.

We have established and documented an in vivo bioassay for detection of hazards with intratracheally instilled fine particles, which can be used for risk assessment of toxicity of materials inhaled into deep lung tissue of humans (Yokohira et al. Establishment of a bioassay system for detection of lung toxicity due to fine particle instillation: sequential histopathological changes with acute and subacute lung damage due to intratracheal instillation of quartz in F344 male rats. J Toxicol Pathol 2005;18:13-8). For validation we here examined toxicity of fine particles from quartz, hydrotalcite, potassium octatitanate, palladium oxide and carbon black with this bioassay. A total of 108, 10-week-old F344/DuCrj male rats were randomly divided into 8 groups. Groups 1 to 5 underwent intratracheal instillation of the 5 test particles (4 mg/rat) suspended in 0.2 ml vehicle (saline or 10% propylene glycol and 1% sodium carboxymethyl cellulose in saline: PG-CMC) with a specially designed aerolizer, and subgroups of 7 rats were killed on Days 1 and 28 thereafter. Groups 6 and 7 similarly were exposed to saline and PG-CMC, respectively, as vehicle controls, while group 8 was maintained untreated. Using histopathological changes and immunohistochemically assessed bromodeoxyuridine (BrdU) labeling indices, inducible nitric oxide synthase (iNOS) and matrix metalloproteinase-3 (MMP-3) levels as end points, the quartz treated group exhibited high toxicity, while the values for the other particle-treated groups pointed to only slight effects. Although additional efforts are needed to establish advantages and disadvantages with our bioassay, models featuring intratracheal instillation clearly can be useful for detection of acute or subacute lung toxicity due to inhaled fine particles by using histopathological scoring and markers like BrdU and iNOS for screening purposes in short-term studies.