Assessment of the toxicity and carcinogenicity of double-walled carbon nanotubes in the rat lung after intratracheal instillation: a two-year study.

BACKGROUND: Considering the expanding industrial applications of carbon nanotubes (CNTs), safety assessment of these materials is far less than needed. Very few long-term in vivo studies have been carried out. This is the first 2-year in vivo study to assess the effects of double walled carbon nanotubes (DWCNTs) in the lung and pleura of rats after pulmonary exposure. METHODS: Rats were divided into six groups: untreated, Vehicle, 3 DWCNT groups (0.12 mg/rat, 0.25 mg/rat and 0.5 mg/rat), and MWCNT-7 (0.5 mg/rat). The test materials were administrated by intratracheal-intrapulmonary spraying (TIPS) every other day for 15 days. Rats were observed without further treatment until sacrifice. RESULTS: DWCNT were biopersistent in the rat lung and induced marked pulmonary inflammation with a significant increase in macrophage count and levels of the chemotactic cytokines CCL2 and CCL3. In addition, the 0.5 mg DWCNT treated rats had significantly higher pulmonary collagen deposition compared to the vehicle controls. The development of carcinomas in the lungs of rats treated with 0.5 mg DWCNT (4/24) was not quite statistically higher (p = 0.0502) than the vehicle control group (0/25), however, the overall incidence of lung tumor development, bronchiolo-alveolar adenoma and bronchiolo-alveolar carcinoma combined, in the lungs of rats treated with 0.5 mg DWCNT (7/24) was statistically higher (p < 0.05) than the vehicle control group (1/25). Notably, two of the rats treated with DWCNT, one in the 0.25 mg group and one in the 0.5 mg group, developed pleural mesotheliomas. However, both of these lesions developed in the visceral pleura, and unlike the rats administered MWCNT-7, rats administered DWCNT did not have elevated levels of HMGB1 in their pleural lavage fluids. This indicates that the mechanism by which the mesotheliomas that developed in the DWCNT treated rats is not relevant to humans. CONCLUSIONS: Our results demonstrate that the DWCNT fibers we tested are biopersistent in the rat lung and induce chronic inflammation. Rats treated with 0.5 mg DWCNT developed pleural fibrosis and lung tumors. These findings demonstrate that the possibility that at least some types of DWCNTs are fibrogenic and tumorigenic cannot be ignored.

Evaluation of a biomarker for the diagnosis of pancreas cancer using an animal model.

Many approaches have been taken to identify new biomarkers of pancreatic ductal carcinoma (PDC). Since animal models can be sampled under controlled conditions, better standardization is possible compared with heterogeneous human studies. Transgenic rats with conditional activation of oncogenic RAS in pancreatic tissue develop PDC that closely resembles the biological and histopathological features of human PDC. Using this model, we evaluated the usefulness of leucine-rich α2-glycoprotein-1 (LRG-1) as a serum marker. In this study, we found that LRG-1 was overexpressed in rat PDC compared with normal pancreas tissue of the control rats. Serum levels of LRG-1 were also significantly higher in rats bearing PDC than in controls. Importantly, chronic pancreatitis in male Wistar Bonn/Kobori rats, which is a widely accepted as a model of chronic pancreatitis, did not cause serum levels of LRG-1 to become elevated. These results strongly support serum LRG-1 as a candidate biomarker for noninvasive diagnosis of PDC. Our models of pancreas cancer provide a useful strategy for evaluation of candidate markers applicable to human cancer.

Therapeutic and Preventive Effects of Osteoclastogenesis Inhibitory Factor on Osteolysis, Proliferation of Mammary Tumor Cell and Induction of Cancer Stem Cells in the Bone Microenvironment.

BACKGROUND: We examined the effects of recombinant human osteoclastogenesis inhibitory factor (hOCIF) on osteolysis, proliferation of mammary tumor cells, and induction of cancer stem cells (CSCs) in the tumor-bone and tumor-subcutaneous microenvironments (TB- and TS-microE). METHODS: Mouse mammary tumor cells were transplanted onto the calvaria or into a subcutaneous lesion of female mice, creating a TB-microE and a TS-microE, and the mice were then treated with hOCIF. To investigate the preventive effects of hOCIF, mice were treated with hOCIF before tumor cell implantation onto the calvaria (Pre), after (Post), and both before and after (Whole). The number of CSCs and cytokine levels were evaluated by IHC and ELISA assay, respectively. RESULTS: hOCIF suppressed osteolysis, and growth of mammary tumors in the TB-microE, but not in the TS-microE. In the Pre, Post, and Whole groups, hOCIF suppressed osteolysis, and cell proliferation. hOCIF increased mouse osteoprotegrin (mOPG) levels in vivo, which suppressed mammary tumor cell proliferation in vitro. These preventive effects were observed in the dose-dependent. hOCIF did not affect the induction of CSCs in either microenvironment. CONCLUSION: While receptor activator of NF-κB ligand (RANKL) targeting therapy may not affect the induction of CSCs, RANKL is a potential target for prevention as well as treatment of breast cancer bone metastasis.

Multiwalled carbon nanotubes intratracheally instilled into the rat lung induce development of pleural malignant mesothelioma and lung tumors.

Multiwalled carbon nanotubes (MWCNT) have a fibrous structure and physical properties similar to asbestos and have been shown to induce malignant mesothelioma of the peritoneum after injection into the scrotum or peritoneal cavity in rats and mice. For human cancer risk assessment, however, data after administration of MWCNT via the airway, the exposure route that is most relevant to humans, is required. The present study was undertaken to investigate the carcinogenicity of MWCNT-N (NIKKISO) after administration to the rat lung. MWCNT-N was fractionated by passing it through a sieve with a pore size of 25 µm. The average lengths of the MWCNT were 4.2 µm before filtration and 2.6 µm in the flow-through fraction; the length of the retained MWCNT could not be determined. For the present study, 10-week-old F344/Crj male rats were divided into five groups: no treatment, vehicle control, MWCNT-N before filtration, MWCNT-N flow-through and MWCNT-N retained groups. Administration was by the trans-tracheal intrapulmonary spraying (TIPS) method. Rats were administered a total of 1 mg/rat during the initial 2 weeks of the experiment and then observed up to 109 weeks. The incidences of malignant mesothelioma and lung tumors (bronchiolo-alveolar adenomas and carcinomas) were 6/38 and 14/38, respectively, in the three groups administered MWCNT and 0/28 and 0/28, respectively, in the control groups. All malignant mesotheliomas were localized in the pericardial pleural cavity. The sieve fractions did not have a significant effect on tumor incidence. In conclusion, administration of MWCNT to the lung in the rat induces malignant mesothelioma and lung tumors.

Chemokine (C-C motif) ligand 3 detection in the serum of persons exposed to asbestos: A patient-based study.

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 < 0.001) than for the control group (6.6%). The pleural plaque, benign hydrothorax, asbestosis, and lung cancer groups had serum CCL3 levels and detection rates similar to that of healthy subjects possibly exposed to asbestos. The CCL3 chemokine was detected in the serum of 9 of the 10 patients diagnosed with malignant mesothelioma. Three of the patients with malignant mesothelioma had exceptionally high CCL3 levels. Malignant mesothelioma cells from four biopsy cases and an autopsy case were positive for CCL3, possibly identifying the source of the CCL3 in the three malignant mesothelioma patients with exceptionally high serum CCL3 levels. In conclusion, a significantly higher percentage of healthy persons possibly exposed to asbestos had detectable levels of serum CCL3 compared to healthy unexposed control subjects.

Size- and shape-dependent pleural translocation, deposition, fibrogenesis, and mesothelial proliferation by multiwalled carbon nanotubes.

Multiwalled carbon nanotubes (MWCNT) have a fibrous structure similar to asbestos, raising concern that MWCNT exposure may lead to asbestos-like diseases. Previously we showed that MWCNT translocated from the lung alveoli into the pleural cavity and caused mesothelial proliferation and fibrosis in the visceral pleura. Multiwalled carbon nanotubes were not found in the parietal pleura, the initial site of development of asbestos-caused pleural diseases in humans, probably due to the short exposure period of the study. In the present study, we extended the exposure period to 24 weeks to determine whether the size and shape of MWCNT impact on deposition and lesion development in the pleura and lung. Two different MWCNTs were chosen for this study: a larger sized needle-like MWCNT (MWCNT-L; l = 8 µm, d = 150 nm), and a smaller sized MWCNT (MWCNT-S; l = 3 µm, d = 15 nm), which forms cotton candy-like aggregates. Both MWCNT-L and MWCNT-S suspensions were administered to the rat lung once every 2 weeks for 24 weeks by transtracheal intrapulmonary spraying. It was found that MWCNT-L, but not MWCNT-S, translocated into the pleural cavity, deposited in the parietal pleura, and induced fibrosis and patchy parietal mesothelial proliferation lesions. In addition, MWCNT-L induced stronger inflammatory reactions including increased inflammatory cell number and cytokine/chemokine levels in the pleural cavity lavage than MWCNT-S. In contrast, MWCNT-S induced stronger inflammation and higher 8-hydroxydeoxyguanosine level in the lung tissue than MWCNT-L. These results suggest that MWCNT-L has higher risk of causing asbestos-like pleural lesions relevant to mesothelioma development.

A novel transgenic mouse model carrying human Tribbles related protein 3 (TRB3) gene and its site specific phenotype.

Tribbles related protein 3 (TRB3) pseudokinase plays a crucial role in cell proliferation, migration and morphogenesis during development. In our recent study, an introduction of human TRB3 gene into mouse mammary tumor cells caused an increase of proliferation of tumor cells and their nuclear size. In the current study, to examine whether this gene causes de novo morphological changes in a specific organ site we have developed a novel variation of the transgenic mouse model that conditionally expresses human TRB3 (hTRB3) gene using Cre-recombinase (Cre)/loxP recombination system. By injecting hTRB3 transgene construct into pronuclei of mouse embryo, we eventually obtained four hTRB3 mice. The gene expression was controlled by infection of adenovirus-expressing Cre via the tail vein of hTRB3 mouse. In Cre-mediated hTRB3 mouse, expression of the hTRB3 protein was detected in the cytoplasm of hepatocytes in the liver. Expression of this protein was also seen in lymphocytes in the spleen, glomerular endothelial cells, and epithelial cells of collecting duct of the kidney. In hepatocytes of the hTRB3 mouse, nuclear size was significantly greater than that of the wild type mouse, indicating that hTRB3 can play a role at least in part in hepatic morphogenesis. The present animal model may provide a system for evaluation of de novo morphological changes induced by a specific transgene in a specific organ site.

Nanosized zinc oxide particles do not promote DHPN-induced lung carcinogenesis but cause reversible epithelial hyperplasia of terminal bronchioles.

Zinc oxide (ZnO) is known to induce lung toxicity, including terminal bronchiolar epithelial hyperplasia, which gives rise to concerns that nanosized ZnO (nZnO) might lead to lung carcinogenesis. We studied the tumor promoting activity of nZnO by an initiation-promotion protocol using human c-Ha-ras proto-oncogene transgenic rats (Hras128 rats). The rats were given 0.2 % N-nitrosobis(2-hydroxypropyl)amine (DHPN) in the drinking water for 2 weeks and then treated with 0.5 ml of 250 or 500 µg/ml nZnO suspension by intra-pulmonary spraying once every 2 weeks for a total of 7 times. Treatment with nZnO particles did not promote DHPN-induced lung carcinogenesis. However, nZnO dose-dependently caused epithelial hyperplasia of terminal bronchioles (EHTB) and fibrosis-associated interstitial pneumonitis (FAIP) that were independent of DHPN treatment. Tracing the fate of EHTB lesions in wild-type rats indicated that the hyperplastic lesions almost completely disappeared within 12 weeks after the last nZnO treatment. Since nZnO particles were not found in the lung and ZnCl2 solution induced similar lung lesions and gene expression profiles, the observed lesions were most likely caused by dissolved Zn(2+). In summary, nZnO did not promote carcinogenesis in the lung and induced EHTB and FAIP lesions that regressed rapidly, probably due to clearance of surplus Zn(2+) from the lung.

A novel reporter rat strain that expresses LacZ upon Cre-mediated recombination.

The recent widespread application of Cre/loxP technology has resulted in a new generation of conditional animal models that can better recapitulate many salient features of human disease. These models benefit from the ability to monitor the expression and functionality of Cre protein. We have generated a conditional (Cre/loxP dependent) LacZ reporter rat (termed the LacZ541 rat) to monitor Cre in transgenic rats. When LacZ541 rats were bred with another transgenic rat line expressing Cre recombinase under the control of the CAG promoter, LacZ/Cre double transgenic embryos displayed ubiquitous expression of LacZ, and when LacZ541 rats were bred with transgenic rats expressing Cre/loxP-dependent oncogenic H- or K-ras, LacZ was expressed in the lesions resulting from the activation of the oncogene. The LacZ541 rat enables evaluation of the performance of Cre-expressing systems which are based upon transgenic rats or somatic gene transfer vectors and provides efficient and simple lineage marking.

Metabolomic and transcriptomic profiling of human K-ras oncogene transgenic rats with pancreatic ductal adenocarcinomas.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most debilitating malignancies in humans, and one of the reasons for this is the inability to diagnose this disease early in its development. To search for biomarkers that can be used for early diagnosis of PDAC, we established a rat model of human PDAC in which expression of a human K-ras(G12V) oncogene and induction of PDAC are regulated by the Cre/lox system. In the present study, transgenic rats bearing PDAC and control transgenic rats with normal pancreatic tissues were used for metabolomic analysis of serum and pancreatic tissue by non-targeted and targeted gas chromatography-mass spectrometry and transcriptomic analysis of pancreatic tissue by microarray. Comparison of the metabolic profiles of the serum and pancreatic tissue of PDAC-bearing and control rats identified palmitoleic acid as a metabolite, which was significantly decreased in the serum of PDAC-bearing animals. Transcriptomic analysis indicated that several transcripts involved in anaerobic glycolysis and nucleotide degradation were increased and transcripts involved in the trichloroacetic acid cycle were decreased. Other transcripts that were changed in PDAC-bearing rats were adenosine triphosphate citrate lyase (decreased: fatty acid biosynthesis), fatty acid synthase (increased: fatty acid biosynthesis) and arachidonate 5-lipoxygenase activating protein (increased: arachidonic acid metabolism). Overall, our results suggest that the decreased serum levels of palmitoleic acid in rats with PDAC was likely due to its decrease in pancreatic tissue and that palmitoleic acid should be investigated in human samples to assess its diagnostic significance as a serum biomarker for human PDAC.

Multi-walled carbon nanotubes translocate into the pleural cavity and induce visceral mesothelial proliferation in rats.

Multi-walled carbon nanotubes have a fibrous structure similar to asbestos and induce mesothelioma when injected into the peritoneal cavity. In the present study, we investigated whether carbon nanotubes administered into the lung through the trachea induce mesothelial lesions. Male F344 rats were treated with 0.5 mL of 500 µg/mL suspensions of multi-walled carbon nanotubes or crocidolite five times over a 9-day period by intrapulmonary spraying. Pleural cavity lavage fluid, lung and chest wall were then collected. Multi-walled carbon nanotubes and crocidolite were found mainly in alveolar macrophages and mediastinal lymph nodes. Importantly, the fibers were also found in the cell pellets of the pleural cavity lavage, mostly in macrophages. Both multi-walled carbon nanotube and crocidolite treatment induced hyperplastic proliferative lesions of the visceral mesothelium, with their proliferating cell nuclear antigen indices approximately 10-fold that of the vehicle control. The hyperplastic lesions were associated with inflammatory cell infiltration and inflammation-induced fibrotic lesions of the pleural tissues. The fibers were not found in the mesothelial proliferative lesions themselves. In the pleural cavity, abundant inflammatory cell infiltration, mainly composed of macrophages, was observed. Conditioned cell culture media of macrophages treated with multi-walled carbon nanotubes and crocidolite and the supernatants of pleural cavity lavage fluid from the dosed rats increased mesothelial cell proliferation in vitro, suggesting that mesothelial proliferative lesions were induced by inflammatory events in the lung and pleural cavity and likely mediated by macrophages. In conclusion, intrapulmonary administration of multi-walled carbon nanotubes, like asbestos, induced mesothelial proliferation potentially associated with mesothelioma development.

RAS oncogenic signal upregulates EZH2 in pancreatic cancer.

The neoplastic transformation by mutant RAS is thought to require remodeling of expression of an entire set of genes. However, the underlying mechanism for initiation of gene expression remodeling in tumorigenesis remains elusive. This study was aimed to define the oncogenic role of EZH2, a histone modifier protein that is induced by oncogenic mutant RAS, using pancreatic cancers of transgenic rats exogenously expressing human mutant RAS. Immunohistochemical observation of preneoplastic or cancerous lesions in the animal model suggested that upregulation of Ezh2 protein is an initiating event in pancreatic carcinogenesis. MEK-inhibition or Elk-1-knockdown downregulated EZH2, and MEK-inhibition or EZH2-knockdown restored expression of a tumor suppressor, RUNX3 in human and rat pancreatic cancer cells activated by the oncogenic RAS. Furthermore, Elk-1- or EZH2-knockdown inhibited growth of the cancer cells. These results strongly suggested that the oncogenic RAS upregulates EZH2 through MEK-ERK signaling, resulted in downregulation of tumor suppressors including RUNX3 in pancreatic carcinogenesis.

Extracts of Musa basjoo induce growth inhibition and changes in the protein expression of cell cycle control molecules in human colorectal cancer cell lines.

Musa basjoo (MB) is a species of the banana plant belonging to the genus Musa that has been used as a folk medicine. However, evidence-based biological activities and the molecular mechanism of action of MB are unknown. Thus, the aim of the present study was to examine whether the crude dried leaf extracts of MB inhibit the growth of colorectal (HT29 and HCT116) and other types (HepG2, MCF-7 and PC-3) of human cancer cell lines. Crude extracts of MB inhibited the growth of cells with IC50 values of 136 µg/ml (acetone extract, HT29), 51 µg/ml (acetone extract, HCT116), 45 µg/ml (acetone extract, HepG2), 40 µg/ml (acetone extract, MCF-7), 29 µg/ml (acetone extract, PC-3), 175 µg/ml (methanol extract, HT29), 137 µg/ml (methanol extract, HCT116), 102 µg/ml (methanol extract, HepG2), 85 µg/ml (methanol extract, MCF-7), and 85 µg/ml (methanol extract, PC-3) in colony formation assays, and 126 µg/ml (acetone extract, HT29), 68 µg/ml (acetone extract, HCT116), 260 µg/ml (methanol extract, HT29), and 216 µg/ml (methanol extract, HCT116) in MTT assays. Thin layer chromatography analysis revealed the potential existence of aromatic compounds in the acetone extract of MB. Flow cytometric analysis indicated that the percentage of cells in G1 increased, and this was associated with a concomitant decrease of cells in the S and/or G2-M phases of the cell cycle. When colorectal cancer cells were treated with acetone extract of MB, there was a marked decrease in the levels of expression of the cyclin D1, cyclin E, cdk2 and cdk4 proteins and a marked increase in the levels of the expression of the p21CIP1, p27KIP1, and p53 proteins, but those of apoptosis-associated protein PARP did not change. There was a tendency for acetone extract of MB to inhibit xenograft tumor growth in mice. Collectively, the crude extracts of MB contain active components that exert growth inhibition of human cancer cells. This is the first systematic study of the anticancer activity of MB and may broaden insights into the possible clinical approach of specific herbal medicines.

Palmitoyl piperidinopiperidine, a novel derivative of 10­hydroxy­2­decenoic acid, as a potent and selective anticancer agent against human colon carcinoma cell lines.

The present study, to the best of our knowledge, is the first systematic study of the inhibitory effects of palmitoyl piperidinopiperidine (PPI; Japan Patent no. 5597427), on colon carcinogenesis. PPI exhibited marked growth inhibitory activity in several human colon carcinoma cell lines, with IC50 values of approximately 0.5­2.2 µM. In silico docking analysis indicated that PPI could bind to the SH2 domain of signal transducer and activator of transcription 3 (STAT3). PPI markedly inhibited the transcriptional activity of the SW837 cell line. Flowcytometric analysis demonstrated that PPI induced an increase in the number of cells in the G1 phase of the cell cycle, and induced sub­G1 fractions of cells at a higher concentration level of PPI. In the HT29 and SW837 cells, western blot analyses exhibited that in whole cell lysates, PPI induced a marked decrease in the expression levels of p­STAT3, but not in the levels of STAT3 in these cells. PPI also induced a marked decrease in the expression levels of both STAT3 and p­STAT3 in the chromatin fraction. In addition, PPI affected the protein expression levels of cyclin D1, p53, Bcl­2, Bcl­xL and vascular endothelial growth factor (VEGF). In the HT29 cells, PPI induced a marked and dose­dependent increase in the expression levels of Bax, cleaved caspase­3, cleaved caspase­7, cleaved caspase­8, cleaved caspase­9 and cleaved poly (ADP­ribose) polymerase (PARP). In animal model systems, PPI inhibited the growth of implanted carcinoma cells, and also induced a significant decrease in the multiplicity of colonic aberrant crypt foci. In addition, a marked and dose­dependent inhibition of angiogenesis of the chick chorioallantoic membrane was observed. As regards the possible molecular mechanisms, it is suggested that the inhibition of STAT3 by PPI may affect the function of molecules that are related to apoptosis, angiogenesis and cell cycle progression, eventually contributing to the PPI­induced growth inhibitory effects.

Involvement of macrophage inflammatory protein 1alpha (MIP1alpha) in promotion of rat lung and mammary carcinogenic activity of nanoscale titanium dioxide particles administered by intra-pulmonary spraying.

Titanium dioxide (TiO(2)) is evaluated by World Health Organization/International Agency for Research on Cancer as a Group 2B carcinogen. The present study was conducted to detect carcinogenic activity of nanoscale TiO(2) administered by a novel intrapulmonary spraying (IPS)-initiation-promotion protocol in the rat lung. Female human c-Ha-ras proto-oncogene transgenic rat (Hras128) transgenic rats were treated first with N-nitrosobis(2-hydroxypropyl)amine (DHPN) in the drinking water and then with TiO(2) (rutile type, mean diameter 20 nm, without coating) by IPS. TiO(2) treatment significantly increased the multiplicity of DHPN-induced alveolar cell hyperplasias and adenomas in the lung, and the multiplicity of mammary adenocarcinomas, confirming the effectiveness of the IPS-initiation-promotion protocol. TiO(2) aggregates were localized exclusively in alveolar macrophages and had a mean diameter of 107.4 nm. To investigate the underlying mechanism of its carcinogenic effects, TiO(2) was administered to wild-type rats by IPS five times over 9 days. TiO(2) treatment significantly increased 8-hydroxydeoxy guanosine level, superoxide dismutase activity and macrophage inflammatory protein 1alpha (MIP1alpha) expression in the lung. MIP1alpha, detected in the cytoplasm of TiO(2)-laden alveolar macrophages in vivo and in the media of rat primary alveolar macrophages treated with TiO(2) in vitro, enhanced proliferation of human lung cancer cells. Furthermore, MIP1alpha, also detected in the sera and mammary adenocarcinomas of TiO(2)-treated Hras128 rats, enhanced proliferation of rat mammary carcinoma cells. These data indicate that secreted MIP1alpha from TiO(2)-laden alveolar macrophages can cause cell proliferation in the alveoli and mammary gland and suggest that TiO(2) tumor promotion is mediated by MIP1alpha acting locally in the alveoli and distantly in the mammary gland after transport via the circulation.

Mature acinar cells are refractory to carcinoma development by targeted activation of Ras oncogene in adult rats.

Pancreatic ductal adenocarcinoma (PDA) is one of the most debilitating malignancies in humans. A thorough understanding of the cytogenesis of this disease will aid in establishing successful treatments. We have developed an animal model which uses adult Hras(G12V) and Kras(G12V) transgenic rats in which oncogene expression is regulated by the Cre/loxP system and neoplastic lesions are induced by injection of adenovirus-expressing Cre recombinase. When adenovirus with Cre recombinase under the control of the CMV enhancer/chicken beta-actin (CAG) promoter (Ad-CAG-Cre) is injected into the pancreatic duct of these animals, pancreatic neoplasias develop. Pathologically, the origin of these lesions is duct, intercalated duct, and centroacinar cells, but not acinar cells. The present study was undertaken to test the effect of acinar cell-specific oncogenic ras expression. Adult transgenic rats were injected with adenovirus with Cre recombinase under the control of the acinar cell-specific promoters amylase (Ad-Amy-Cre) and elastase-1 (Ad-Ela-Cre) or under the control of the non-specific CAG promoter. Injection of either Ad-Amy-Cre or Ad-Ela-Cre into the pancreatic ducts of transgenic animals in which oncogenic Kras is tagged with hemagglutinin (HA), HA-Kras(G12V) rats resulted in expression of oncogenic ras in acinar cells but not in duct, intercalated duct, or centroacinar cells. Notably, injected animals did not develop any observable proliferative or neoplastic lesions. In marked contrast, injection of Ad-CAG-Cre resulted in pancreatic cancer development within 4 weeks. These results indicate that adult acinar cells are refractory to Ras oncogene activation and do not develop neoplasia in this model.

A medium-term, rapid rat bioassay model for the detection of carcinogenic potential of chemicals.

The Ito Liver Model and the Ito Multi-organ Model are used in conjunction and constitute an efficient and rapid bioassay for the identification of both genotoxic and nongenotoxic carcinogenic chemicals. The Ito Liver Model is an 8-week bioassay system that uses the number and size of foci of hepatocytes positive for glutathione S-transferase placental form (GST-P) as the end-point marker. One hundred fifty-nine compounds were tested using the Ito Liver Model: 61 of 66 hepatocarcinogens tested positive, and 10 of 43 nonliver carcinogens were also positive. The false-positive detection of noncarcinogens was low; a single false-positive result was obtained from the 50 noncarcinogens tested. Since more than half of all known carcinogens are hepatocarcinogens in rodents, the initial 8-week bioassay is able to detect most carcinogens. The Ito Multi-organ Model is a 28-week bioassay system for the detection of carcinogens that were not identified by the Ito Liver Model. Results are evaluated by preneoplastic and neoplastic lesions in major organs. Forty-four compounds were tested using the Ito Multi-organ Model: 17 out of 17 liver carcinogens were positive, and 19 out of 22 (86%) nonliver carcinogens were positive. None of the five noncarcinogens tested positive.

Cancer prevention by bovine lactoferrin: from animal studies to human trial.

Colorectal cancer (CRC) is one of the most frequently diagnosed cancers and, despite improved colonoscopic screening, CRC is a leading cause of death from cancer. Administration of bovine lactoferrin (bLF) suppresses carcinogenesis in the colon and other organs of test animals, and recently it was shown that ingestion of bLF inhibits the growth of adenomatous polyps in human patients. Here we review work which established bLF as an anti-carcinogenic agent in laboratory animals and the results of a clinical trial which demonstrated that bLF can reduce the risk of colon carcinogenesis in humans.

An animal model of preclinical diagnosis of pancreatic ductal adenocarcinomas.

Pancreatic ductal adenocarcinoma (PDA) is a highly lethal disease, which is usually diagnosed in an advanced stage. Animal PDA models which reflect the human condition are clearly necessary to develop early diagnostic tools and explore new therapeutic approaches. We have established transgenic rats carrying a mutated H- or K-ras gene (Hras250 and Kras327) controlled by Cre/loxP activation. These animals develop PDA which are histopathologically similar to that in humans. We utilized this model to identify biomarkers to detect early PDA. We report here that serum levels of Erc/Mesothelin are significantly higher in rats bearing PDA than in controls. Importantly, the levels are significantly elevated in rats before grossly visible carcinomas develop. Even in rats with very small microscopic ductal carcinoma lesions, elevated serum Erc/Mesothelin can be detected. We believe this is the first report of a pancreas tumor animal model in which pre-symptomatic lesions can be diagnosed.

Induction of a novel histone deacetylase 1/c-Myc/Mnt/Max complex formation is implicated in parity-induced refractoriness to mammary carcinogenesis.

Refractoriness to carcinogen-induced increases in epithelial cell proliferation is a very important characteristic of parous mammary glands. We found that N-methyl-N-nitrosourea (MNU)-induced proliferative burst in the mammary ductal epithelium was blocked in parous glands but not in age-matched virgin (AMV) glands. The inhibition of the proliferative burst in MNU-treated parous mammary glands coincided with the upregulation of Mnt, a Myc-suppressor, and the formation of histone deacetylase 1/Mnt/Max complexes that unexpectedly contained c-Myc. These complexes formed on the promoters of Myc targets, such as ornithine decarboxylase, cyclin D2, and transforming growth factor beta1 genes, in quiescent fibroblasts, and were disassembled in serum-stimulated cells. These results suggest that the complexes also function as transcription repressors of the growth-related Myc targets in MNU-treated parous mammary glands. Using the chemical mammary carcinogenesis model of human c-Ha-ras transgenic (Tg) rats, we confirmed that parity protected the mammary glands at the postinitiation phase of tumorigenesis. Although the incidence of 7,12-dimethylbenz[alpha]anthracene-induced palpable tumors was reduced from 61.5% in the AMV Tg rats to 28.5% in the parous animals, the incidence of early neoplastic lesions in the parous rats was the same as that in the AMV rats. Restriction fragment length polymorphism analysis detected mutations in the human c-Ha-ras gene in most of the normal-appearing parous Tg glands, as well as in the virgin glands. We propose that accelerated formation of HDAC1/c-Myc/Mnt/Max complexes in response to carcinogen exposure results in down-regulation of growth-related genes, leading to the refractoriness of parous mammary glands at the postinitiation phase of carcinogenesis.