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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., (© 2022 John Wiley & Sons Ltd.)

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., (© 2022. The Author(s).)

Mutagenesis and adaptation of the psychrotrophic fungus Chrysosporium pannorum A-1 to the toxic substrate β-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 β-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 β-pinene to trans -pinocarveol (315 mg per g of dry mycelium), a 4.3-fold greater biocatalytic activity, and a higher resistance to H 2 O 2 -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 β-bioconversion of terpenes.

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 1β (IL-1β), 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.

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/β-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 β-catenin, GSK-3β, c-Met and MMP7 in the GES-1 cells were detected by qPCR. The protein expressions of β-catenin, GSK-3β, p-GSK-3β 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 β-catenin, GSK-3β, c-Met and MMP7, and increased the protein expressions of β-catenin, GSK-3β and p-GSK-3β. These results suggest that the damage of GES-1 cells induced by MNNG may be related to the activation of Wnt/β-catenin signaling pathway, which will provide the basis for the study of cell model of gastric mucosal cell injury.

The effects of miR-148a in regulating the expression of TGFβ2 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, TGFβ2, 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 TGFβ2, SMAD2, and SMAD4. A luciferase activity assay was used to monitor the effects of miR-148a on the TGFβ2- and SMAD2-3'UTRs. We identified nude mouse models bearing BGC-823-miR-148a or BGC-823-vector cells. Tumor volumes were detected, and TGFβ2, SMAD2 expression levels were determined in tumor tissues. The in vivo study demonstrated an increase in the mRNA and protein levels of TGFβ2, 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 TGFβ2 and SMAD2 significantly in GC cells. The results of the luciferase activity assay showed that miR-148a could bind to the 3'UTRs of TGFβ2 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 TGFβ2 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.

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., (Copyright © 2013 Elsevier Inc. All rights reserved.)

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.

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., (© 2011 The Authors. Journal of Digestive Diseases © 2011 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and Blackwell Publishing Asia Pty Ltd.)

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.

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.

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.

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., ((c) 2006 Wiley-Liss, Inc.)

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.

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.

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.

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.

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.

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.

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.

The effect of the pH of the medium on the clastogenic activity of several direct-acting and indirect clastogens was evaluated in the in vitro chromosomal aberration test with Chinese hamster ovary K1 cells. Furthermore, the stability of the chemicals in the cell culture medium was measured by HPLC over the pH range of 5.0-11.0. The activity of the direct-acting clastogens mitomycin C (MMC), N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) and 4-nitroquinoline-1-oxide (4NQO) at various pH values depended on their stability. In the case of ENNG, its clastogenic activity decreased to about one-fifth at pH 9 but was about twice as high at acidic pH compared with that at pH 7.4. This is consistent with the observation that ENNG is unstable at basic pH; the residual content of ENNG was 0.5% of the initial amount in cell culture medium at pH 9.0 after a 2-h incubation. 4NQO was unstable at strongly basic pH (pH 10-11), and MMC was unstable at pH 5.0 and pH 11.0. The frequencies of chromosomal aberrations induced by MMC and ENNG were correspondingly decreased at these pH values. On the other hand, the clastogenicities of the indirect clastogens 7,12-dimethylbenz[a]anthracene (DMBA), benzo[a]pyrene (B(a)P) and dimethylnitrosamine (DMN), which require metabolic activation, were reduced at pH 10-11 and pH 5.8. The frequencies of chromosomal aberrations at these pHs were almost equal to negative control values. These chemicals were stable in the medium in the absence of S9 mix over the pH range of 5.0-11.0. Thus clastogenicity of indirect-acting clastogens is reduced under extreme pH conditions, probably because of the instability or nonformation of the active form. The present results indicate that the clastogenic activity of any compound will depend on its stability in the medium irrespective of its direct- or indirect-acting nature. In addition, some of the chemicals that are recognized as clastogens presumably might induce chromosomal aberrations by means of acidic pH itself. It is, therefore, important to take account of the pH of the treatment medium in evaluating the clastogenicity of chemicals.

We have studied the influence of anoxia and respiratory deficiency (RD) in yeast on the cytotoxic and recombinogenic effects of 5 direct-acting alkylating agents, namely N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methylnitrosourea (MNU), ethylnitrosourea (ENU), methyl methanesulphonate (MMS) and ethyl methanesulphonate (EMS). We found that the effects of both conditions parallel each other for MMS, MNNG, MNU and ENU. Both anoxia and RD did not modify the effects of MMS to any significant extent. On the other hand, anoxic and respiratory-deficient cells were found to be more resistant than euoxic and respiratory-proficient cells respectively for MNNG, MNU and ENU. In the case of EMS, which is similar to MMS in its chemical reaction with DNA, the respiratory-deficient cells were found to be more sensitive than the respiratory-proficient ones. These studies indicate that the response of anoxic and respiratory-deficient cells cannot be predicted solely on the basis of the chemical reactivity pattern of the alkylating agents. The physiological state which exists under these conditions may exert considerable influence on the cellular response.

Dietary habits have been causally implicated in gastric carcinogenesis, whereas minor dietary items may also play a part. Wasabi is a popular pungent spice in Japanese meals. In this study the effect of wasabi on N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced gastric carcinogenesis was studied in rats. Wistar WKY male rats received drinking water containing 50 micrograms/ml of MNNG or tap water alone and a basal diet (PCE-2) or PCE-2 containing 10% (wt/wt) of wasabi powder for 40 weeks. Thus, three groups were completed as MNNG + PCE-2 (n = 30), MNNG + wasabi (n = 30), and tap water + wasabi (n = 30). At autopsy, nine rats (30%) had seven glandular stomach tumors (2 adenocarcinomas, 2 adenomatous polyps, and 3 adenomatous glandular hyperplasias) and three duodenal adenocarcinomas in the MNNG + PCE-2 group, whereas in the MNNG + wasabi group, two rats (7%) had one forestomach epidermoid cyst and one duodenal carcinosarcoma (corrected chi 2 = 4.63, p less than 0.05 for incidences of glandular stomach tumors between 2 groups). In addition, two rats had microscopic atypical glands in the MNNG + PCE-2 group. There was no tumor in the tap water + wasabi group. These results indicated that glandular stomach carcinogenesis induced by MNNG was suppressed by the administration of wasabi.

We have conducted investigations on members of three families with increased predisposition to cancer which appears to be inherited as an autosomal dominant trait. The aim of our studies overall is to provide markers for the mutant genes involved, so that gene carriers may be monitored closely for signs of malignant disease. This paper reports on studies of chromosome breakage in lymphocytes from affected and at-risk family members and control subjects. No increase in spontaneous chromosome breakage was observed in family members compared with controls. An increased sensitivity to chromosome damage induced by the alkylating agent, N-methyl-N1-nitro-N-nitrosoguanidine (MNNG), was observed in three members from two families; one person was affected, the others at risk. These families included cases of osteosarcoma, in addition to various types of cancer of epithelial origin. Two members (one affected, one at-risk) of a third family showed increased sensitivity to the radio-mimetic agent, bleomycin. This family appeared to represent the cancer family syndrome. Whilst not conclusive at present, our results appear to justify investigation of members of cancer families with respect to sensitivity to chromosome breaking agents.

The suitability of using coloscopy as a diagnostic method is investigated with respect to colonic carcinomas induced locally by the administration of N-nitrosoacetoxymethyl-methylamine, N-methyl-N-nitrosourea, and methylnitro-nitrosoguanidine, or systemically by subcutaneous injection of 1,2-dimethylhydrazine in Sprague-Dawley rats. The endoscopic diagnostic examination proved to be clearly superior to methods of animal inspection, palpation, investigation for occult blood and exploratory laparotomy which have so far been employed in animal experiments with small rodents. The relevance of this method is discussed for the early detection of chemically induced colonic tumors, and the observation of tumor development under experimental cytostatic therapy.

Skin tumors chemically induced in mice have provided an important experimental model for studying carcinogenesis and for bioassaying carcinogenic agents. The information obtained from this model suggests that the events leading to tumor formation can be divided into at least two stages, initiation and promotion. A single small dose of carinogen produces initiation which appears to be irreversible. These initiating agents may have to be metabolically activated and can interact with cellular macromolecules. The extent to which they bind to DNA correlates well with their carcinogenicity. Increased DNA replication at the time of or during the first day after these agents have been applied appears to enhance carcinogenesis. Unlike initiation, promotion appears to be reversible and the promoting agents must be applied repeatedly before tumors are formed. Promoters interact with membranes, stimulate and alter genetic expression, and increase the rate of cell proliferation. The knowledge gained from these studies in mouse skin has immeasurably helped the entire field of chemical carcinogenesis. But efforts to determine the cellular and molecular mechanisms involved in the carcinogenic process, particularly in the skin, have been hampered by the difficulties of working on whole animals and by the special problems associated with the biologic and biochemical methods required for this target organ. Such problems, however, can be solved by the use of cell cultures of mouse epidermis which can metabolize and bind carcinogens just as is done in vivo. The fact that epidermal cells in vitro proliferate synchronously should facilitate the study of the relation between the cell cycle and carcinogenesis. These cells repair chemically induced DNA damage by at least two mechanisms, excision repair and base-specific repair. When epidermal cells in vitro are exposed to promoting agents, a proliferative response analogous to that in vivo is elicited, apparently mediated through control of polyamine metabolism. Neoplastic transformation has been induced in these cultures by known skin carcinogens.

We have previously identified four human astrocytoma cell strains as defective in the repair of N-methyl-N' -nitro-N-nitrosoguanidine (MNNG) damaged adenovirus 5. We now show that two of these strains (the only two tested), in comparison to other tumor strains or normal human skin fibroblasts, are very sensitive to MNNG-produced killing as measured by colony forming ability, but are normally sensitive to ultraviolet light. Further, such repair deficient cells may be cultured from tumors of the colon, lung, skin, and neck. The phenotype of deficient repair of MNNG-treated adenovirus 5 has now been found in a subgroup of 9 of the 39 human tumor strains tested. We propose to call this phenotype the Mer(-) phenotype. None of the 22 strains of normal human skin fibroblasts tested showed deficient repair of MNNG damage. MNNG treatment (80 microM) causes a decrease in semi-conservative DNA synthesis from which Mer(-) tumor cells do not recover, but from which cells capable of normal repair of MNNG damage (Mer(+)) do. Somewhat paradoxically, Mer(-) cells show more MNNG-stimulated DNA synthesis ('repair synthesis') than do Mer(+) cells. Besides being deficient in the repair of MNNG-damaged adenoviruses Mer(-) cells also have difficulty in repairing viruses damaged either by other N-alkyl-N'-nitro-N-nitrosoguanidines, or by N-methyl- or N-ethyl-N-nitrosoureas.

Cimetidine is a histamine H2-antagonist. Since in vitro formed N-nitrosocimetidine (NC) has been shown to be genotoxic, we investigated it for carcinogenicity. Two groups of 40 Sprague-Dawley rats each received 500 and 50 mg/kg NC, respectively, by gavage twice weekly for one year with subsequent lifetime follow-up. An additional 40 rats were given the structurally related carcinogen methylnitro-nitrosoguanidine (MNNG) orally at weekly doses of 80 mg/kg for 3 months. 100 animals served as untreated controls. MNNG-treated rats died with carcinomas of the forestomach (median induction time: 226 days). No evidently treatment-related tumors were seen in animals receiving NC. The median survival time in both NC-treated groups was reduced (400 and 393 vs. 630 days in the control). No evidence of carcinogenicity of NC was seen in this bioassay. It is concluded that NC is not carcinogenic, or at least significantly less so than MNNG.

We have studied the chromosomal sensitivity to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) of human diploid skin fibroblasts derived from individuals with adenomatosis coli (AC), which is a dominantly inherited disorder associated with multiple adenomas of the colon and rectum. Spontaneous frequencies of chromosome aberrations in the cell strains from the AC patients were similar to those in the control cells from normal individuals. However, the AC cells exhibited elevated chromosome instabilities when cells were expected to MNNG, with aberration frequencies approximately twice as high as in similarly treated control cells. The present results, together with findings by others, suggest that the AC cells are defective in a function which regulates cellular condition and are in a state more susceptible to the action of agents that react with chromosomal DNA. These findings also raise the possibility of developing a diagnostic procedure for early detection of abnormal gene carriers of AC.

The study was carried out on 72 rats receiving during 7 weeks rectal infusions of N-methyl-N'nitro-N-nitrosoguanidine (MNNG), a carcinogenic agent. During the follow-up of 52 weeks the changes were investigated in the quantitative composition of the aerobic and anaerobic bacterial flora of the large intestine, and the basic composition of the aerobic flora was established. It was found that MNNG modified the quantity and composition of the bacterial flora of the colon not only with a reduction in the number of bacteria in the faeces but also with a transient change in the composition and proportions of various bacterial species. Attention is called to the observation that the appearance and development of malignant tumours in the large intestine was connected with a rise in the number of micro-organisms correlated with the histological transformation of the colonic mucosa and the number of tumours in it.

Tumours of the glandular stomach and upper small intestine were induced in rats by oral administration of MNNG. In most cases the lesions were identified histologically as adenocarcinomas and their prestages, such as polypeous and downward growing adenomatous hyperplasias. Out of 48 adenomatous hyperplasias and adenocarcinomas of the stomach and 24 well differentiated adenocarcinomas of the small intestine, we observed argyrophilic cells in nearly the half of the cases. Endocrine cells were also identified by electron microscopy. The frequency of endocrine cells was reduced with decreasing degree of tissue differentiation. In poorly differentiated carcinomas, including signet ring cell carcinomas, no argyrophilic cells were found. Out of 10 adenomatous hyperplasias and tumours of the stomach investigated immunohistochemically, 5 cases showed gastrin producing cells. Most of these animals were radioimmunologically characterized by strongly elevated serum gastrin levels. Derivation and potential relevance of the endocrine cells in tumours are discussed.