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.

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.

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.

Significance of the progesterone receptor and epidermal growth factor receptor, but not the estrogen receptor, in chemically induced lung carcinogenesis in female A/J mice.

In the present study, the expression levels of female hormone receptors, estrogen receptor (ER) and progesterone receptor (PR) and the epidermal growth factor receptor, (EGFR), as well as proliferating cell nuclear antigen (PCNA) were examined in lung tumors that were induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in female A/J mice. Each seven-week-old mouse was administered with 2 mg NNK via intraperitoneal injection and the mice were subsequently euthanized at week 52. Lung tumors, including adenomas, carcinomas in adenomas and adenocarcinomas, were obtained and analyzed by immunohistochemistry for the expression levels of the receptors, ER, PR and EGFR, and PCNA. The results were as follows: i) In mouse lung adenomas, a significant correlation was identified between the size of the tumor and PCNA expression, although not with the expression of the receptors (ER, PR and EGFR); ii) in the carcinoma components of the carcinomas in adenomas, the size of the tumor and PCNA expression were correlated, while EGFR expression demonstrated a significant correlation with PR expression; iii) in adenocarcinomas, the tumor size significantly correlated with PCNA, EGFR and PR expression; and iv) EGFR and PR expression was identified to be significantly correlated in adenocarcinomas, and to a certain extent in the carcinoma components of the carcinomas in adenomas, although not in the adenomas. Notably, ER expression was not associated with tumor growth or the other factors, particularly EGFR expression, and no significant differences were identified between the three types of lesion. These results indicate that PR, like EGFR, may be significant in lung carcinogenesis.

[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.

Rat strain differences in levels and effects of chronic inflammation due to intratracheal instillation of quartz on lung tumorigenesis induced by DHPN.

Chronic inflammatory effects of single intratracheal instillation (i.t.) of quartz on rat lung tumorigenesis were examined using 4 different animal models. At first, in order to determine an appropriate dose of quartz i.t. to promote lung tumorigenesis, F344 male rats were administrated single 0, 0.5, 1, 2 or 4 mg quartz/rat after initiation by N-bis(2-hydroxypropyl) nitrosamine (DHPN). Further studies were performed to examine strain differences of the effects of chronic inflammation caused by quartz i.t. in 3 strains of rat, i.e. F344, Wistar-Hannover and SD. Each was instilled with 2mg quartz/rat after DHPN administration and sacrificed in week 24. In addition, strain differences in generation of inflammation were determined at days 1 and 28. Finally, for determination of long-term effects period, F344 and Wistar-Hannover rats were similarly treated, but the experiment was terminated at week 52. In F344 rats, the tumor areas in DHPN treated groups showed a tendency to increase along with the dose of quartz. F344 rats demonstrated the highest and Wistar-Hannover rats the lowest sensitivity to quartz in acute and chronic phases in the 3 strains. In 52 week, in F344 rats, the multiplicity of tumors and the serum concentration of IL-6 in the group treated with DHPN and quartz were significantly increased. The present experiments indicated that chronic inflammation due to quartz instillation exerted promoting effects on lung carcinogenesis in F344, SD and Wistar-Hannover rats. The strain differences in tumor promotion appeared to correlate with inflammatory reactions to quartz and increase of IL-6.

Napsin A is possibly useful marker to predict the tumorigenic potential of lung bronchiolo-alveolar hyperplasia in F344 rats.

There are 2 types of bronchiolo-alveolar hyperplasia found in rat lungs. One is 'inflammatory hyperplasia' with a potential to recover in future with removal of the stimulating insult and the other is 'latent tumorigenic hyperplasia' as an independent preneoplastic lesion for adenocarcinoma. In the present experiment, we focused on rat lung bronchiolo-alveolar hyperplasia induced by 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), which decreases with time after induction and reverts to normal, or by N-bis(2-hydroxypropyl)nitrosamine (DHPN), with tumorigenic potential to progress to adenoma and adenocarcinoma. Though NNK is a typical carcinogen inducing lung adenocarcinoma in female A/J mice, the tumorigenic potential by NNK in rats is weak. Differences between hyperplasias induced by DHPN and by NNK were here examined immunohistochemically. Formalin fixed paraffin embedded lung samples with hyperplastic and inflammatory lesions were obtained from rats exposed to DHPN or NNK and from lung inflammation models induced with fine particles like CuO, NiO and quartz. The 19 markers were examined immunohistochemically. Napsin A, in the inflammatory lesions and hyperplasia induced by NNK, was positive for macrophages and secretions in the alveoli spaces but less so in the walls of the alveoli. In the proliferative lesions including hyperplasia induced by DHPN, strong positive staining for napsin A was observed in the walls of the alveoli. Thus high expression was suggested to be possibly useful for detecting tumorigenic potential of rat lung hyperplasia.

Strain differences in pleural mesothelial cell reactions induced by potassium octatitanate fibers (TISMO) infused directly into the thoracic cavity.

Although we have previously reported that the fiber-shaped TISMO, morphologically similar to asbestos, can induce a severe mesothelial reaction in A/J mice, it is important to clarify any strain differences. In the present study, female A/J, C3H/HeN, ICR and C57BL/6 mice were therefore employed as test strains. At the beginning of the experiment, all mice underwent a left thoracotomy and direct administration of 3mg of TISMO particles suspended in 0.2 ml saline into the left thorax. The experiment was terminated after 21 weeks and all groups were sacrificed and the mesothelium and main organs were examined histopathologically. To contribute to mechanistic analysis, iron staining with Berlin blue and Turnbull's blue, and immunostaining for calretinin were also performed. The present experiment demonstrated only minor strain differences in the degree of pleural reaction to TISMO. However, there was clear variation in the iron and lymphocyte accumulation in the pleura and in the liver. This difference in response to TISMO fibers in vivo is important information when considering the development of mesothelioma as an animal model and the extrapolation to human risk from such animal studies.

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.

Lack of promoting effects from physical pulmonary collapse in a female A/J mouse lung tumor initiated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) with remarkable mesothelial cell reactions in the thoracic cavity by the polymer.

Experimental identification of potential chemopreventive or tumor promotive agents in the lung is important. Establishment of short-term bioassay models is therefore a high priority. In an attempt to induce strong promotion effects, in Experiment 1, left thoracotomy was performed on A/J mice at week 3 after initiation with 4-(methylnitrosamno)-1-(3-pyridyl)-1-butanone (NNK) (2mg/0.1 ml saline/mouse i.p.) at weeks 0 and 1. In Experiment 2, at week 3, 0.2 ml of polymer gel was infused directly into the left cavity of the thorax with thoracotomy to occupy certain thoracic cavity volume and to examine the influence of physical pulmonary collapse. The experiments were terminated after 8, 10, 12 and 16 weeks in Experiment 1, and 12 weeks in Experiment 2 but no clear promotion effects in either experiment or pulmonary collapse due to infused polymer were apparent. However, a pronounced mesothelial cell reaction to the infused polymer was evident on the left lung surfaces and parietal pleura in Experiment 2. In conclusion, the present experiments did not demonstrate any clear lung tumor promotion effects of thoracotomy or physical left lung collapse. It remains possible, however, that alternative approaches might have greater efficacy and these need more consideration. In addition, mesothelial cells reaction was observed with the infused polymer.

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.

Different threshold levels for 2-amino-3,8 dimethylimidazo[4,5-f]quinoxaline (MelQx) initiation of lung and colon carcinogenesis and the effects of an additional initiation by 4 (methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in A/J mice.

The existence of possible threshold dose levels of genotoxic carcinogens for carcinogenesis is of particular interest for human risk assessment. Recently, no observed effect levels (NOELs) for various hepatocarcinogens have been reported. However, reports on threshold levels for lung carcinogenesis have hitherto been lacking. In the present study, we first investigated low dose response lung and colon carcinogenesis with a food-derived genotoxic carcinogen, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) (0, 0.01, 0.1, 1, 10 and 100 ppm in the diet) alone for 32 weeks using female A/J mice. The endpoints were histopathologically diagnosed hyperplasias and adenomas in the lung, and aberrant crypt foci (ACF) in the colon. The results showed NOELs of 100 and 1 ppm, respectively. We next investigated the effect of additional pre-treatment with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) (2 mg/mouse, single dose, intraperitoneal injection) prior to the low-dose application of MeIQx (0, 0.01, 0.1, 1, 10, 100 and 600 ppm in the diet) for 32 weeks. Lung lesions were significantly increased in the NNK + MeIQx (1 ppm) group, but not in the NNK + MeIQx (≥10 ppm) groups. Since the dose-response curve was not of so-called 'hockey stick type', it was not possible to determine a NOEL for lung tumorigenesis. Significant increase in the mRNA expression of CYP2A5, a major metabolic enzyme for NNK, was also observed in the NNK + MeIQx (1 ppm) group, and a similar pattern was noted for O6-methylguanine DNA methyltransferase (MGMT). By contrast, the formation of colon ACF showed a dose-dependent increase. The NOEL for the formation of colon ACF was considered to be 10 ppm MeIQx with NNK. These results suggest that MeIQx may have different threshold dose levels for the induction of lung tumorigenic lesions and ACF formation in the colon. Pre-treatment with NNK, a potent lung carcinogen, concealed the effects of MeIQx in the lung, but exerted minimal influence in the colon. CYP2A5 and MGMT expression may be of importance, particularly in the lung. The present study provides critical suggestions for the human risk assessment of genotoxic carcinogens.

Tumorigenesis of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), but not enhancing effects of concomitant high-fat diet, on lung carcinogenesis in female A/J mice.

It has been reported that 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) induces liver tumors and to a lesser extent lung lesions, lymphomas and leukemias in CDF(1) mice. Since a number of case control studies have pointed to a positive association between fat consumption and lung cancer, we examined the lung carcinogenic potential of MeIQx treatment concomitant with a high-fat diet using female A/J mice. Groups 1 and 2 were fed a diet supplemented with MeIQx at a concentration of 600 ppm. Groups 1 and 3 received a diet containing 20% corn oil and group 4 was fed the basal diet alone. After 1 week, 10 mice in each group were sacrificed for measurement of cytochrome P450 (CYP)1A2 mRNA in the liver and lung. The remaining mice were maintained on their respective diets until termination, 32 weeks after the initial MeIQx treatment, when lung proliferative lesions were analyzed. The incidences and multiplicities of hyperplasias and adenomas in MeIQx-treated groups (groups 1 and 2) were significantly higher than in the groups without MeIQx treatment, with a significant increase in the incidences and multiplicities of adenomas + carcinomas, as well as hyperplasia + adenomas + carcinomas (lung proliferative lesions). Lung carcinomas were observed in 1 mouse in each of the MeIQx-treated groups. However, the high-fat diet (groups 1 and 3) did not affect the incidences or multiplicities of lung proliferative lesions. Expression levels of CYP1A2 mRNA after MeIQx treatment significantly increased >3-fold in livers, but no significant change was noted in the lungs, where levels were very low at 1/210 and 1/923 the values for livers. In conclusion, following a 32-week period, we confirmed the lung tumorigenic potential of MeIQx which possibly occurs due to proximate carcinogens activated by CYP1A2 in the liver. However, we failed to detect any influence of a high-fat diet.

Potassium octatitanate fibers (TISMO) induce pleural mesothelial cell reactions with iron accumulation in female A/J mice.

It is crucial to develop therapeutic approaches for malignant mesothelioma, as well as to obtain information involving the possible mechanism involved in the development of mesothelioma. Subsequently, thoracotomy was performed to infuse test particles directly into the thoracic cavity of A/J mice. Fiber-shaped particles of potassium octatitanate (TISMO) and granular-shaped micro- and nano-size order particles of titanium dioxide (TiO(2)) were employed (1.5 mg in 0.2 ml saline/mouse). The experiment was terminated after 21 weeks to assess responses. Only the fiber-shaped TISMO, morphologically similar to asbestos, induced a severe reaction of the pleura. A number of TISMO fibers were observed in the alveoli, indicating penetration through the pleura. Following Berlin blue staining, positive spots were observed around the TISMO fibers, indicative of iron. These positive spots corresponded with cells that immunostained positively for calretinin, a marker of mesothelial cells. Similar observations were reported for asbestos-induced mesothelioma. The present study showed that only fiber-shaped TISMO induced severe reactions of the mesothelium in the pleura, and these involved iron accumulation derived from endogenous sources. The results indicate that the risk of mesothelial cell reaction does not depend on particle size, but may depend on shape.

8-Methoxypsoralen, a potent human CYP2A6 inhibitor, inhibits lung adenocarcinoma development induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in female A/J mice.

Previously, we demonstrated that 8-methoxypsoralen (methoxsalen), a potent human cytochrome P450 2A6 (CYP2A6) inhibitor, strongly suppresses lung adenoma induction by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in female A/J mice. In the present study, we examined the inhibitory effects of methoxsalen on the development of lung adenocarcinomas, as well as on adenomas and alveolar hyperplasia. Female A/J mice were treated with methoxsalen at doses of 12.5 or 1.25 mg/kg body weight, administered by stomach tube once daily for 3 days. One hour after the final treatment, NNK was injected i.p. at a dose of 2 mg/mouse. The experiments were terminated 52 weeks after the first methoxsalen treatment, and lung adenomas and adenocarcinomas were analyzed histopathologically. Pretreatment with methoxsalen significantly reduced the incidence of adenocarcinomas from 94.7 to 46.7% (12.5 mg/kg) and 44.4% (1.25 mg/kg), and their tumor multiplicity from 4.68 to 0.87 (12.5 mg/kg) and 0.61 (1.25 mg/kg) tumors/mouse. The tumor multiplicity of adenomas and adenocarcinomas in the methoxsalen-treated groups was significantly reduced from 12.47 to 5.67 (12.5 mg/kg) and 4.28 (1.25 mg/kg) tumors/mouse. Approximately 60% of the adenocarcinomas arose within adenomas. In comparing the methoxsalen + NNK and NNK alone groups, there was no significant difference in the frequency of such compound lesions, indicating that pretreatment with methoxsalen did not suppress the eventual progression of adenomas to adenocarcinomas. These results clearly demonstrate that methoxsalen, a potent human CYP2A6 inhibitor, inhibits not only lung adenoma but also adenocarcinoma development.

Enhancing effects of a high fat diet on 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline-induced lung tumorigenesis in female A/J mice.

Both heterocyclic amines and a high fat diet are associated with an increased risk of cancer in many organs. Female A/J mice were fed a diet supplemented with 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and a high fat diet to test for the development of lung tumors. In experiment 1, the mice were divided into 6 groups. Groups 1, 2, 3 and 4 were fed a diet supplemented with MeIQx at a concentration of 600?ppm for 0-12 weeks. A high fat diet containing 20% corn oil was given to Groups 1 and 5 for 0-32 weeks, Group?2 for 12-32 weeks and Group 3 for 0-12 weeks. Group 6 was fed a basal diet without supplements. MeIQx-treated groups (Groups?1, 2, 3 and 4) showed a significant increase in macroscopic and microscopic lung nodules compared with the control (Group 6). Areas of adenomas were increased dependent on the duration of exposure to the high fat diet. In experiment 2, Group 1 mice were fed MeIQx and a high fat diet, Group?2 a MeIQx alone diet, Group 3 a high fat alone diet, and Group?4 a basal diet without supplements. CYP1A2 mRNA in the liver was significantly decreased by a high fat diet (Group?3). The MeIQx alone group (Group 2) showed a tendency towards increased CYP1A2 expression, which was partially reduced in the MeIQx + high fat-treated group (Group 1). In the lungs, CYP1A2 mRNA expression was at an extremely low level, with no intergroup differences. In conclusion, MeIQx exerts tumorigenic potential in the lungs, and a high fat diet increases the size of induced lesions. The expression level of CYP1A2 in relation to MeIQx and a high fat diet may be associated with lung carcinogenesis.

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.

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.