Ginsenoside Rg3 induces apoptosis and inhibits proliferation by down-regulating TIGAR in rats with gastric precancerous lesions.

BACKGROUND: Ginsenoside Rg3 (GRg3) is one of the main active ingredients in Chinese ginseng extract and has various biological effects, such as immune-enhancing, antitumour, antiangiogenic, immunomodulatory and anti-inflammatory effects. This study aimed to investigate the therapeutic effect of GRg3 on gastric precancerous lesion (GPL) induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and the potential mechanism of action. METHODS: The MNNG-ammonia composite modelling method was used to establish a rat model of GPL. Histopathological changes in the rat gastric mucosa were observed by pathological analysis using haematoxylin-eosin staining to assess the success rate of the composite modelling method. Alcian blue-periodic acid Schiff staining was used to observe intestinal metaplasia in the rat gastric mucosa. Apoptosis was detected in rat gastric mucosal cells by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling staining. The production level of reactive oxygen species (ROS) was determined by the dihydroethidium fluorescent probe method, and that of TP53-induced glycolysis and apoptosis regulator (TIGAR) protein was determined by immunohistochemical staining and western blotting. The production levels of nicotinamide adenine dinucleotide phosphate (NADP) and glucose-6-phosphate dehydrogenase (G6PDH) were determined by an enzyme-linked immunosorbent assay, and that of glutathione (GSH) was determined by microanalysis. RESULTS: GRg3 significantly alleviated the structural disorganization and cellular heteromorphism in the form of epithelial glands in the gastric mucosa of rats with GPL and retarded the progression of the disease. Overexpression of TIGAR and overproduction of NADP, GSH and G6PDH occurred in the gastric mucosal epithelium of rats with GPL, which in turn led to an increase in the ROS concentration. After treatment with GRg3, the expression of TIGAR and production of NADP, GSH G6PDH decreased, causing a further increase in the concentration of ROS in the gastric mucosal epithelium, which in turn induced apoptosis and played a role in inhibiting the abnormal proliferation and differentiation of gastric mucosal epithelial cells. CONCLUSION: Grg3 can induce apoptosis and inhibit cell proliferation in MNNG-induced GPL rats. The mechanism may be related to down-regulating the expression levels of TIGAR and production levels of GSH, NADP and G6PD, and up-regulating the concentration of ROS.

Proteomics analyses of Xiaopi granules in N-methyl-N'-nitro-N-nitrosoguanidine-induced gastric epithelial dysplasia rat model using LC-MS.

Xiaopi granules have been shown to ameliorate gastric epithelial dysplasia in patients. However, the therapeutic mechanism is unclear. Herein, the proteomics method was applied to identify the differentially expressed proteins and related pathways. Sixty male Sprague-Dawley rats were randomly divided into four groups: control (C group, n = 10), model (M group), Xiaopi granules (X group), and vitacoenzyme (V group). The rat gastric epithelial dysplasia model was established by intragastrically administering N-methyl-N'-nitro-N-nitrosoguanidine and ranitidine and by orally administering 0.05% ammonia solution. After 12 weeks, the stomach tissue was analyzed by hematoxylin and eosin staining and proteomics analyses. Western-blot analysis was applied to further validate the proteomics results. Compared to the M group, levels of 326 and 350 proteins were altered significantly in the X and V groups (1.5-fold, p < 0.05), which were significantly enriched in digestion, metabolism, coagulation, and cell apoptosis. CELA2A, GHRL, NDUFB9, and PGC were significantly upregulated (p < 0.0001), whereas CLCA1, PLG, and DAC2 were downregulated (p < 0.001 or 0.0001) in the M group vs. the C group. The change in these proteins could be reversed after the treatment of Xiaopi granules or vitacoenzyme tablets. Xiaopi granules ameliorated gastric epithelial dysplasia by intervening in digestion, metabolism, blood coagulation, cell apoptosis, and other related pathways.

Mutagenesis and Adaptation of the Psychrotrophic Fungus Chrysosporium pannorum A-1 as a Method for Improving ß-pinene Bioconversion.

Mutagenesis and adaptation of the psychrotrophic fungus Chrysosporium pannorum A-1 to the toxic substrate ß-pinene were used to obtain a biocatalyst with increased resistance to this terpene and improved bioconversion properties. Mutants of the parental strain were induced with UV light and N-methyl-N'-nitro-N-nitrosoguanidine. Mutants resistant to ß-pinene were isolated using agar plates with a linear gradient of substrate concentrations. Active mutants were selected based on their general metabolic activity (GMA) expressed as oxygen consumption rate. Compared to the parental strain, the most active mutant showed an enhanced biotransformation ability to convert ß-pinene to trans-pinocarveol (315 mg per g of dry mycelium), a 4.3-fold greater biocatalytic activity, and a higher resistance to H2O2-induced oxidative stress. Biotransformation using adapted mutants yielded twice as much trans-pinocarveol as the reaction catalyzed by non-adapted mutants. The results indicate that mutagenesis and adaptation of C. pannorum A-1 is an effective method of enhancing ß-bioconversion of terpenes.

[Matrine alleviates N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced gastric mucosal damage in rats and its mechanism].

Objective To investigate the effect of matrine on gastric mucosal injury induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in rats and its mechanism. Methods A total of 75 Wister rats were randomly divided into a control group, a model group and three matrine-treated groups (100, 150 and 200 mg/kg). Except for the control group, the other groups were treated with MNNG to establish the models of gastric mucosal injury in the rats. After the models were successfully established, the rats in the three matrine-treated groups were administrated 100, 150, 200 mg/kg matrine, respectively, for successive 45 days. After the last administration, the body mass, daily intake of drinking water and dietary of rats were measured. And then the tissue samples were collected after the rats were sacrificed. The levels of interleukin 1ß (IL-1ß), IL-4, interferon gamma (IFN-γ), and tumor necrosis factor alpha (TNF-α) were measured by ELISA in gastric mucosa. HE staining was used to observe the pathological changes of gastric mucosa tissue. Immunohistochemical staining was performed to evaluate the expression of vascular endothelial growth factor C (VEGF-C) and vascular endothelial growth factor receptor 3 (VEGFR3) in gastric mucosa. The protein levels of Bcl2, BAX, caspase-3, cytochrome C (Cyt-C), Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88) and nuclear factor κB p65 (NF-κB p65) were determined by Western blotting. Results The body mass, daily intake of drinking water and dietary increased in matrine-treated rats in comparison with the model group. In addition, compared with the model group, matrine significantly reduced the expression levels of VEGF-C, VEGFR3, BAX, caspase-3, Cyt-C, TLR4, MyD88 and NF-κB p65, and increase Bcl2 protein level in the gastric mucosa tissues. Conclusion Matrine can reduce gastric mucosal damage induced by MNNG in rats, which is related to the down-regulation of VEGF-C/VEGFR3 and NF-κB/TLR4 signaling pathway.

[Injury of human gastric epithelial GES-1 cells by MNNG and its effects on Wnt/ß-catenin signaling pathway].

The aim of this study was to investigate the mechanisms of mono-functional alkylating agent MNNG to damage human gastric epithelial GES-1 cells and roles of Wnt/ß-catenin signaling pathway in the process. The GES-1 cells were treated with MNNG (2 × 10-5 mol/L) for 24 h. The morphological changes of the GES-1 cells were observed under inverted microscope 2 d after treatment. The cell viability was measured by MTT assay. The apoptosis and cell cycle distribution of the GES-1 cells were analyzed by flow cytometry. The mRNA expressions of ß-catenin, GSK-3ß, c-Met and MMP7 in the GES-1 cells were detected by qPCR. The protein expressions of ß-catenin, GSK-3ß, p-GSK-3ß and c-Met were determined by Western blot. The results showed that MNNG induced the injury of GES-1 cells and changed the normal cell morphology to irregular long spindle shape. MNNG induced the apoptosis of GES-1 cells and blocked the cell cycle progression obviously. MNNG up-regulated the mRNA expressions of ß-catenin, GSK-3ß, c-Met and MMP7, and increased the protein expressions of ß-catenin, GSK-3ß and p-GSK-3ß. These results suggest that the damage of GES-1 cells induced by MNNG may be related to the activation of Wnt/ß-catenin signaling pathway, which will provide the basis for the study of cell model of gastric mucosal cell injury.

miR-148a downregulates the expression of transforming growth factor-ß2 and SMAD2 in gastric cancer.

The effects of miR-148a in regulating the expression of TGFß2 and SMAD2 in MNNG-initiated gastric cancer rats and the mechanism of action in GC cells were determined. Effects of miR-148a on the proliferation, migration, and invasion of GC cell lines were demonstrated. We used Wistar rats, Balb/c nude mice, and GC cell lines. Rats were treated with MNNG to establish a GC rat model. Levels of miR-148a, TGFα, TGFß2, SMAD2, SMAD3, and SMAD4 were tested in gastric tissues from different groups. In GC cell lines, we constructed and transfected a primary miR-148a plasmid to determine the expression patterns of TGFß2, SMAD2, and SMAD4. A luciferase activity assay was used to monitor the effects of miR-148a on the TGFß2- and SMAD2-3'UTRs. We identified nude mouse models bearing BGC-823-miR-148a or BGC-823-vector cells. Tumor volumes were detected, and TGFß2, SMAD2 expression levels were determined in tumor tissues. The in vivo study demonstrated an increase in the mRNA and protein levels of TGFß2, SMAD2, and SMAD4 in the MNNG-treated group compared with the control group. However, there were no differences in the mRNA and protein levels in either TGFα or SMAD3. The in vitro study demonstrated that overexpression of miR-148a reduced TGFß2 and SMAD2 significantly in GC cells. The results of the luciferase activity assay showed that miR-148a could bind to the 3'UTRs of TGFß2 and SMAD2 and inhibited their activity. Overexpression of miR-148a inhibited proliferation, migration, and invasion significantly in GC cell lines. In vivo, tumor volume of BGC-823-miR-148a was smaller than that of BGC-823-vector. Overall, miR-148a inhibited the proliferation, migration, invasion, and expression of TGFß2 and SMAD2 in GC cells. It was concluded that miR-148a might play an important role in gastric cancer, and is a potential candidate for GC treatment.

Suppression of alkylating agent induced cell transformation and gastric ulceration by low-dose alkylating agent pretreatment.

Exposure to mild stress by chemicals and radiation causes DNA damage and leads to acquired stress resistance. Although the linear no-threshold (LNT) model of safety assessment assumes risk from any dose, evidence from radiological research demonstrates a conflicting hormetic phenomenon known as the hormesis effect. However, the mechanisms underlying radiation hormesis have not yet been clarified, and little is known about the effects of low doses of chemical carcinogens. We analyzed the efficacy of pretreatment with low doses of the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) on the subsequent induction of cell transformation and gastric ulceration by high-dose MNNG. We used an in vitro Balb/3T3 A31-1-1 cell transformation test and monitored the formation of gastric ulcers in 5-week-old male ICR mice that were administered MNNG in drinking water. The treatment concentrations of MNNG were determined by the cell survival rate and past reports. For low-dose in vitro and in vivo experiments, MNNG was used at 0.028 µM, and 2.8 µg/mL, respectively. The frequency of cell transformation induced by 10 µm MNNG was decreased by low-dose MNNG pretreatment to levels similar to that of spontaneous transformation. In addition, reactive oxygen species (ROS) and mutation frequencies induced by 10 µm MNNG were decreased by low-dose MNNG pretreatment. Importantly, low-dose MNNG pretreatment had no effect on cell proliferation. In vivo studies showed that the number of gastric ulcers induced by 1 mg/mL MNNG decreased after low-dose MNNG pretreatment. These data indicate that low-dose pretreatment with carcinogens may play a beneficial role in the prevention of chemical toxicity under specified conditions.

Protective effect of Lactobacillus rhamnosus 231 against N-Methyl-N'-nitro-N-nitrosoguanidine in animal model.

The protective effect of Lactobacillus rhamnosus 231 (Lr 231) against potent carcinogen N-Methyl-N'-Nitro-N-Nitrosoguanidine (MNNG) in the rat model is studied. Daily feeding with Lr 231 improved the body weight of male Wistar rats compared with control groups. Fecal azoreductase (p < 0.001) and nitroreductase (p < 0.01) enzyme activity decreased significantly in Lr 231 group in comparison with control groups that received only phosphate buffer or MNNG. Oral administration of MNNG led to a significant increase in Glutathione transferase (GST) while Glutathione reductase (GSH) showed decreased activity. Conversely, feeding Lr 231 showed significantly increased GSH and decreased GST activity in comparison to the MNNG group, emphasizing the protection provided by Lr 231 against MNNG. Histopathological analysis of liver, spleen and colon showed decreased signs of inflammation in the Lr 231 group. The present study highlights that inclusion of active Lr 231 in regular diets could be used to prevent MNNG induced colon carcinoma.

Chemopreventive effect of epigallocatechin-3-gallate (EGCG) and folic acid on the N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced gastrointestinal cancer in rat model.

OBJECTIVE: To investigate the chemopreventive effect and mechanisms of epigallocatechin-3-gallate (EGCG) and folic acid on N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced gastrointestinal cancer in rats, and to investigate and compare the combinatorial effects of EGCG and folic acid on the chemoprevention of gastrointestinal carcinogenesis. METHODS: A total of 159 healthy male Wistar rats were randomly divided into seven groups to have the MNNG in drink (group M), MNNG in drink and EGCG in the feed (group ME), MNNG in drink and folic acid in the feed (group MF), MNNG in drink and EGCG+folic acid in the feed (group MEF), EGCG in the feed (group E), folic acid in the feed (group F) or normal feed (group C), respectively. At 44 weeks, all the rats were killed and assessed for the presence of gastrointestinal tumor. The occurrence of cancer was evaluated by histology. Ki-67 in cancerous tissues and in situ apoptosis were determined by immunohistochemical staining or terminal deoxyribonucleotide transferase-mediated nick-end labeling (TUNEL) assay, respectively. RESULTS: The experiment was completed in 157 rats (98.74%). As compared with group M, the tumor incidence of group MEF decreased significantly (P=0.011). Ki-67 expression in cancerous tissues of group ME and MEF also decreased significantly (P=0.038, P=0.009), while apoptosis of group ME, MF and MEF increased significantly (P=0.000, P=0.003, P=0.000). CONCLUSION: EGCG combined with folic acid has an obvious chemopreventive effect on gastrointestinal carcinogenesis induced by MNNG in rats.

Altered expression of a putative progenitor cell marker DCAMKL1 in the rat gastric mucosa in regeneration, metaplasia and dysplasia.

BACKGROUND: Doublecortin and calcium/calmodulin-dependent protein kinase-like-1 (DCAMKL1) is a candidate marker for progenitor cells in the gastrointestinal mucosa. Lineage cells in the gastric mucosa are derived from progenitor cells, but this process can be altered after injury. Therefore, we explored DCAMKL1 expression under pathological conditions. METHODS: An immunohistochemical analysis was performed in rat stomach with acute superficial injury, chronic ulcer, intestinal metaplasia and dysplasia. RESULTS: DCAMKL1 was exclusively expressed in immature quiescent cells in the isthmus of normal fundic glands, where putative progenitor cells are thought to reside. DCAMKL1-positive cells and proliferating cells shed into the lumen after superficial injury and re-appeared during the regenerative process, mainly in the superficial mucosa. In the marginal mucosa around the active ulcer, parietal and chief cells diminished, foveolar hyperplasia was evident, and trefoil factor family 2 (TFF2)/spasmolytic polypeptide-expressing metaplasia (SPEM) emerged at the gland base. DCAMKL1 cells re-emerged in the deep mucosa juxtaposed with SPEM and proliferating cells. In the healing ulcer, the TFF2 cell population expanded and seemed to redifferentiate to chief cells, while proliferating cells and DCAMKL1 cells appeared above and below the TFF2 cells to promote healing. SPEM appeared and PCNA cells increased in the intestinalized mucosa, and DCAMKL1 was expressed in the proximity of the PCNA cells in the deep mucosa. DCAMKL1, PCNA and TFF2 were expressed in different dysplastic cells lining dilated glands near SPEM. CONCLUSION: The ultrastructural appearance of DCAMKL1-positive cells and the expression patterns of DCAMKL1 in normal and pathological states indicate that the cells belong to a progenitor cell population. DCAMKL1 expression is closely associated with TFF2/SPEM cells after injury. DCAMKL1 cells repopulate close to proliferating, hyperplastic, metaplastic and dysplastic cells, and the progenitor zone shifts according to the pathological circumstances.

Endoscopic observation of N-methyl-N'-nitro-N-nitrosoguanidine-induced gastric carcinogenesis in rat using a newly-developed flexible endoscope.

Endoscopy can be used for sequential observation of gastric carcinogenesis in animal models. In the present study, we applied endoscopic examination and biopsy technique on N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced stomach cancer in rats using a newly-developed flexible 2.5 mm endoscope. A total of 36 rats were divided into MNNG-treated and non-treated groups, observed on gastric endoscopy every 5 weeks, and sacrificed at week 10, 25, 35, and 50. The sequential growth process of MNNG-induced gastric tumor was clearly found by the endoscopic examination. Endoscopic appearances including incidence and size of tumor were well consistent with histological findings. In addition, biopsy specimens could be extracted from gastric mucosa in living rats using a biopsy forceps. These results indicate that the endoscopic technique can be a useful tool for investigating gastric carcinogenesis by sequential observation and collection of biopsy specimens.

Proliferation, angiogenesis and apoptosis-associated proteins are molecular targets for chemoprevention of MNNG-induced gastric carcinogenesis by ethanolic Ocimum sanctum leaf extract.

INTRODUCTION: This study was designed to evaluate the chemopreventive effects of ethanolic Ocimum sanctum (OS) leaf extract on cell proliferation, apoptosis and angiogenesis during N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced gastric carcinogenesis. METHODS: The rats were divided into four groups of ten each. Rats in group one were given MNNG (150 mg/kg body weight) by intragastric intubation three times, with a two-week interval between treatments. Rats in group two were administered MNNG as in group one, and in addition, they received intragastric intubation of ethanolic OS extract (300 mg/kg body weight) three times per week, starting on the day following the first exposure to MNNG. The intubation of ethanolic OS extract continued until the end of the experimental period. Rats in group three were given ethanolic OS leaf extract only. Group four served as controls. All the rats were killed after an experimental period of 26 weeks. RESULTS: Intragastric administration of MNNG-induced well-differentiated squamous cell carcinomas that showed increased cell proliferation, and angiogenesis with evasion of apoptosis, as revealed by the upregulation of proliferating cell nuclear antigen (PCNA), glutathione S-transferase-pi (GST-pi), Bcl-2, cytokeratin (CK) and vascular endothelial growth factor (VEGF) and with downregulation of Bax, cytochrome C and caspase 3 protein expression. Administration of ethanolic OS leaf extract reduced the incidence of MNNG-induced gastric carcinomas. This was accompanied by decreased expression of PCNA, GST-pi, Bcl-2, CK and VEGF, and overexpression of Bax, cytochrome C, and caspase 3. CONCLUSION: This study provides evidence that, in MNNG-induced gastric carcinogenesis, the key proteins involved in the proliferation, invasion, angiogenesis and apoptosis, are viable molecular targets for chemoprevention using ethanolic OS leaf extract.

Urinary concentrations of bisphenol A in relation to biomarkers of sensitivity and effect and endocrine-related health effects.

The impact of endocrine-disrupting chemicals (EDCs) on human health is not yet clear because of difficulties in ascertaining their biological effects. In the present study, we evaluated exposure to the EDC, bisphenol A (BPA), in 172 Koreans in relation to biomarkers of susceptibility and effect. The subjects completed questionnaires, which documented occupation, education, lifestyle factors, potential sources of BPA-exposure, and the occurrence of self-diagnosed endocrine disorders. None of the subjects were occupationally exposed to BPA; however, urinary levels of conjugated BPA, determined by HPLC/FD, ranged from 0.03-62.4 microg/l (median, 7.86). The frequencies of potential susceptibility biomarkers, the UGT1A6-Arg184Ser and the SULT1A1-Arg213His polymorphisms, were not associated with urinary BPA levels, either as single genes or in combination. Indirect effects of BPA exposure on the susceptibility to mutagens were evaluated by comparing urinary BPA concentrations with MNNG-induced sister-chromatid exchange (SCE) in lymphocytes cultured from the subjects. BPA exposure showed marginal or significant associations with theSCEs induced by the low doses of MNNG (0-0.4 mM). However, there was no overall association between urinary BPA levels and MNNG-induced frequency at doses ranging from 0.2-0.6 mM. Finally, we did not detect an association between urinary BPA concentration and endocrine-related disorders. Even though we were unable to find a strong association between BPA exposure and a biological response, possibly because of the limited number of subjects, we observed that most of the subjects were exposed to BPA. Therefore, continuous biological monitoring of BPA is a prudent measure to prevent possible BPA-related health risks.

Characterization of gene expression changes associated with MNNG, arsenic, or metal mixture treatment in human keratinocytes: application of cDNA microarray technology.

The identification of molecular markers related to critical biological processes during carcinogenesis may aid in the evaluation of carcinogenic potentials of chemicals and chemical mixtures. Work from our laboratory demonstrated that a single treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) enhanced spontaneous malignant transformation of the human keratinocyte cell line RHEK-1. In contrast, chronic low-level exposure of cells to arsenic alone or in a mixture containing arsenic, cadmium, chromium, and lead inhibited malignant conversion. To identify changes in gene expression that influence these different outcomes, cDNA microarray technology was used. Analysis of multiple human arrays in MNNG-transformed RHEK-1 cells, designated OM3, and those treated with arsenic or the arsenic-containing metal mixture showed unique patterns of gene expression. Genes that were overexpressed in OM3 included oncogenes, cell cycle regulators, and those involved in signal transduction, whereas genes for DNA repair enzymes and inhibitors of transformation and metastasis were suppressed. In arsenic-treated cells, multiple DNA repair proteins were overexpressed. Mixture-treated cells showed increased expression of a variety of genes including metallothioneins and integrin 4. These cells showed decreased expression of oncogenes, DNA repair proteins, and genes involved in the mitogen-activated protein kinase pathway. For comparison we are currently analyzing gene expression changes in RHEK-1 cells transformed by other means. The goal of these studies is to identify common batteries of genes affected by chemical modulators of the carcinogenic process. Mechanistic studies may allow us to correlate alterations in their expression with sequential stages in the carcinogenic process and may aid in the risk assessment of other xenobiotics.

Effect of selenomethionine on N-methylnitronitrosoguanidine-induced colonic aberrant crypt foci in rats.

An association between low selenium intake and the incidence or prevalence of cancers is well known. Selenium in the form of selenomethionine supplemented in drinking water has been found to be highly effective in reducing tumour incidence and preneoplastic foci during the development of hepatocarcinogenesis in rats in our previous studies. Here, an attempt has been made to investigate whether the dose and form of selenium found to be effective during hepatocarcinogenesis is equally effective in N-methylnitronitrosoguanidine-induced colorectal carcinogenesis in terms of antioxidant defence enzyme systems, DNA chain breaks and incidences of aberrant crypt foci. Treatment with selenomethionine either on initiation or on selection/promotion, or during the entire experiment showed that selenomethionine was most effective in regulating the cellular antioxidant defence systems, DNA chain break control and reducing aberrant crypt foci in the colorectal tissues of rats. Our results also confirm that selenium is particularly effective in limiting the action of the carcinogen during the initiation phase of this colorectal carcinogenesis, just as we found with hepatocarcinogenesis in our previous studies.

Increased pathology incidence in the forestomach of rats maintained on a diet containing ivermectin and given a single dose of N-methyl-N1-nitro-N-nitrosoguanidine.

Ivermectin is widely used against parasitic infections in veterinary and human medicine and was found to promote the growth of lesions leading to neoplasia when given continuously in the diet to Wistar rats receiving a single low dose of N-methyl-N1-nitro-N-nitrosoguanidine (MNNG). No tumors or pathological lesions were observed in the forestomach of the control animals or those given ivermectin alone. However, compared to animals receiving MNNG alone, rats maintained on a diet containing ivermectin (2 ppm) and given MNNG (12.5 mg/kg) by gavage showed an increased number of neoplasms (9/26 vs 3/18; p = 0.30) and a statistically significant fourfold increase in the number of pathological lesions (18/26 vs 3/18; p = 0.002), which include preneoplasia in the forestomach. In all cases, the pathological lesions were more severe in the animals receiving ivermectin and MNNG, compared to those receiving MNNG alone.

A rat model of pancreatic ductal adenocarcinoma: targeting chemical carcinogens.

BACKGROUND: Current experimental models of pancreatic cancer either fail to reproduce the ductal phenotype or cause simultaneous cancers in other organs also. To develop an animal of pancreatic cancer that accurately mimics the human condition, we restricted carcinogenic exposure to the pancreas and specifically targeted ductal epithelial cells. Three different carcinogens were either implanted directly into the pancreas or infused into the pancreatic duct, with or without near-total pancreatectomy (as a means of inducing pancreatic ductal cell proliferation). METHODS: Groups of male Sprague-Dawley rats were exposed to varying doses of dimethylbenzanthracine (DMBA), methynitronitrosoguanidine, or ethylnitronitrosoguanidine either through direct implantation into the pancreas or infusion into the pancreatic duct. Near-total pancreatectomy was added in all groups except two DMBA implantation groups. Surviving rats were killed at 3, 6, 9, or 12 months, and the pancreata were evaluated histologically. RESULTS: All three carcinogens caused pancreatic inflammation, ductal hyperplasia, atypia, and dysplasia beginning by 3 months and becoming more prominent at later time points. Only DMBA caused frequent invasive pancreatic ductal adenocarcinoma, which was first evident by 6 months. The prevalence of pancreatic cancer among DMBA-treated rats evaluated after 10 months was 39% (19 of 49). The addition of pancreatic resection did not enhance pancreatic cancer development. CONCLUSIONS: Of the strategies tested, only direct implantation of DMBA into the rat pancreas frequently produces pancreatic cancer histologically similar to human ductal adenocarcinoma. The development of hyperplastic, atypical, and dysplastic changes preceding and accompanying carcinomas suggests that these lesions are preneoplastic. This model recapitulates the progression from normal to neoplastic epithelium and is likely to be useful for the study of morphologic and molecular mechanisms underlying the early stages of pancreatic carcinogenesis and for the investigation of novel diagnostic and therapeutic techniques.

Reduced recovery of a Cryptococcus neoformans adherence mutant from a rat model of cryptococcosis.

Stable mutants of Cryptococcus neoformans (strain CSF-1) induced by treatment with ultraviolet light and nitrosoguanidine were isolated that demonstrated reduced adherence to glial cells in culture. Adherence of the mutants, as measured by a radiometric assay, was reduced by 50-70% of that attained for the parent CSF-1 strain. The adherence mutants appeared to be phenotypically similar to the CSF-1 strain. However, all but one mutant (designated as CSF-23) demonstrated slightly slower growth rates than the wild-type strain. The CSF-1 and CSF-23 strains were injected intravenously and intratracheally into normal rats and rats immunosuppressed by cyclophosphamide treatment, and the organ distribution and recovery of viable yeasts determined over 2-96 h. During this relatively short period of observation the majority of the yeasts were localized in the lungs. By either route of injection, the recovery of the CSF-23 adherence mutant was reduced by as much as 90% of that obtained for the wild-type strain. The results indicated that host cell adherence may be important for the persistence of cryptococci in tissue and that further studies with the adherence mutants are warranted.

Induction of preneoplastic lesions by sodium arsenite in human fetal respiratory epithelia in organ culture.

The effects of sodium arsenite (As) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) on epithelia of human fetal trachea and bronchiolar epithelia of human fetal lung were studied by using organ-cultured explants. In epithelium of human fetal trachea, 34 microM MNNG induced hyperplasia, metaplasia, and dysplasia; 1 microM As induced hyperplasia; and 3-9 microM As induced hyperplasia and cellular atypia. In glandular epithelium of human fetal trachea, 34 microM MNNG induced hyperplasia and metaplasia; 1 microM As did not induce obvious changes; and 3-9 microM As induced hyperplasia and epidermoid metaplasia with nuclear atypia. In bronchiolar epithelium of human fetal lung, the induction of dysplasia was observed for 1 microM As. Arsenic-induced preneoplastic lesions support the conclusion of epidemiological studies that arsenic is carcinogenic to human lung.

Transfection and expression of human O6-methylguanine-DNA methyltransferase (MGMT) cDNA in Chinese hamster cells: the role of MGMT in protection against the genotoxic effects of alkylating agents.

O6-Methylguanine-DNA methyltransferase (MGMT) is responsible for removal of O6-alkylguanine from DNA induced by alkylating mutagens/carcinogens. To analyze the involvement of O6-alkylguanine in the generation and MGMT in avoidance of various genotoxic effects of alkylating agents, we transfected Chinese hamster ovary (CHO) cells that lack MGMT activity with human MGMT cDNA cloned into a mammalian expression vector (pSV2MGMT). A high proportion (60-80%) of transfectants selected for a cotransfected neo gene survived treatment with high doses of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and N-hydroxyethyl-N-chloroethylnitrosourea (HeCNU). Parallel transfections with an expression vector containing the bacterial ada gene (pSV2ada) showed the human MGMT to be more effective than the ada expression vector in mediating alkylation resistance. Various clonal CHO cell lines have been established stably transfected with the human MGMT cDNA. The transfectants expressed human MGMT at levels ranging from 8600 to 210,000 molecules per cell. The high MGMT expressors became strongly resistant to the killing effects of MNNG, HeCNU, N-methyl-N-nitrosourea (MNU) and, to a significant lesser degree, methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS). No killing resistance was observed to N-ethyl-N-nitrosourea (ENU), though the MGMT and ada transfectants showed reduction in mutation frequency induced by this agent. Protection from mutation induction by MGMT (and ada) expression was also demonstrated for MNNG. The transfectants were also protected from the sister chromatid exchange (SCE) inducing and, to a lesser degree, clastogenic effect of MNNG and MNU, and slightly to EMS and MMS. Again no protection was observed towards ENU. Correlations between MGMT activity and resistance to a given end point suggest that, for MNNG, O6-methylguanine is the preponderant toxic, mutagenic and SCE inducing lesion. About 90% of MNNG (and MNU) induced SCEs and nearly all of the MNNG-induced gene mutations seem to be due to this adduct. For alkylation-induced chromosomal aberrations, however, and for cell killing and SCEs induced by MMS, EMS and ENU, other lesions than O6-alkylguanine appear to be of major importance. The data strongly support the view that O6-methylguanine is a genotoxic lesion and MGMT a function decisively involved in avoidance of genotoxic effects in cells exposed to MNNG and related compounds. They indicate also that it is important to take into account the property and mode of action of any given alkylating agent in assessing the protective role of MGMT against alkylation-induced genotoxicity.