MNNG Group Research Articles

Resveratrol activates MAPK/ERK pathway to regulate oestrogen metabolism in type I endometrial cancer

ObjectiveEndometrial cancer (EC) is an oestrogen-dependent tumour, the occurrence of which is closely related to an imbalance of oestrogen homeostasis. Our previous studies explored the effects of Resveratrol(Res) on oestrogen metabolism. However, systematic research on the exact mechanism of action of Res is still lacking. Based on network pharmacology, molecular docking and animal experiments, the effects and molecular mechanisms of Res on endometrial cancer were investigated.MethodsThe target of Res was obtained from the high-throughput experiment and reference-guided database of TCM (HERB) and the Encyclopedia of Traditional Chinese Medicine (ETCM) databases, and the target of endometrial cancer was obtained by using the Genecards database. Venny map was used to obtain the intersection target of Res in the treatment of endometrial cancer, and the protein interaction network of the intersection target was constructed by importing the data into the STRING database. Then, the drug–disease–target interaction network was constructed based on Cytoscape 3.9.1 software. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed for intersection targets using the OmicShare cloud platform. Res and core targets were analysed by molecular docking. EC model mice induced by MNNG were randomly divided into the control group, Res group, MNNG group, MNNG + Res group, and MNNG + Res + MAPK/ERKi group. The protein levels of ERK and p-ERK in the mouse uterus were detected by Western blot. The levels of E1, E2, E3, 16-epiE3, 17-epiE3, 2-MeOE1, 4-MeOE1, 2-MeOE2, 4-MeOE2, 3-MeOE1, 2-OHE1, 4-OHE1, 2-OHE2, 4-OHE2, and 16α-OHE1 in the serum and endometrial tissue of mice were measured by LC‒MS/MS.ResultsA total of 174 intersection targets of Res anti-endometrial cancer were obtained. The signalling pathways analysed by KEGG enrichment included the AGE-RAGE signalling pathway in diabetic complications, the PI3K-Akt signalling pathway and the MAPK signalling pathway. The top 10 core targets were MAPK3, JUN, TP53, CASP3, TNF, IL1B, AKT1, FOS, VEGFA and INS. Molecular docking showed that in addition to TNF, other targets had good affinity for Res, and the binding activity with MAPK3 was stable. Western blot results showed that Res increased the phosphorylation level of ERK and that MAPK/ERKi decreased ERK activation. In the LC-MS/MS analysis, the levels of 2-MeOE1, 2-MeOE2 and 4-MeOE1 in serum and uterine tissue showed a significantly decreasing trend in the MNNG group, while that of 4-OHE2 was increased (P < 0.05). The concentrations of 4-MeOE1 in serum and 2-MeOE1 and 2-MeOE2 in the endometrial tissue of mice were significantly increased after Res treatment, and those of 4-OHE2 in the serum and uterus of mice were significantly decreased (P < 0.05). Meanwhile, in the MAPK/ERKi intervention group, the effect of Res on the reversal of oestrogen homeostasis imbalance was obviously weakened.ConclusionRes has multiple targets and multiple approaches in the treatment of endometrial cancer. In this study, it was found that Res regulates oestrogen metabolism by activating the MAPK/ERK pathway. This finding provides a new perspective for subsequent research on the treatment of endometrial cancer.

Hyperbaric oxygen therapy increases the effect of 5-fluorouracil chemotherapy on experimental colorectal cancer in mice.

Tumor hypoxia may compromise the results of chemotherapy for treating colorectal cancer because it stimulates angiogenesis and the release of tumor growth factors. Hyperbaric oxygen (HBO) supplementation may potentiate the effects of chemotherapy in such cases. This study aimed to assess the effect of HBO therapy combined with chemotherapy on the treatment of colorectal cancer in mice. C57BL6 mice were submitted to the intrarectal instillation of N-methyl-N-nitrosoguanidine (MNNG) and treated with 5-fluorouracil (5FU) and/or HBO therapy. The MNNG group presented the highest dysplastic crypt rate. The 5FU + HBO group presented the highest rate of apoptotic cells per dysplastic crypt. The 5FU group presented the highest expression of hypoxia-inducible factor-1 alpha and CD44. HBO therapy increased the effect of 5FU on the treatment of the experimental colorectal neoplasia in mice.

Anti-Inflammatory and Antioxidant Effects of Carvacrol on N-Methyl-N'-Nitro-N-Nitrosoguanidine (MNNG) Induced Gastric Carcinogenesis in Wistar Rats.

Carvacrol is a dietary polyphenol from Lamiaceae plants that has been shown to possess a wide range of biological activities including antioxidant and antitumor effects. This study aimed to investigate its anti-inflammatory and antioxidant effects on N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) induced gastric carcinogenesis in Wistar rats. Forty-nine rats were randomly assigned to four treatment and three control groups. Over 60 days, MNNG (200 mg/kg BW) was orally applied to animals of groups 1–5 while the rats in groups 2–5 also received different doses of carvacrol (10, 25, 50, and 100 mg/kg BW, respectively) until the end of the experiment. Group 6 rats were treated with 100 mg/kg BW carvacrol and no MNNG whereas group 7 was the control group without any treatment. After the euthanasia of all rats, the inflammatory cytokines and oxidative stress parameters were assessed in the blood and tissues. The expression of caspase 9, Bax, and Bcl-2 proteins in the stomach tissues were investigated through histopathological examinations. Statistically significant differences were observed in the body weight, oxidative stress, and inflammation parameters of groups 1 to 6 compared to group 7 (p ≤ 0.001). Animals in MNNG groups 2 and 3 treated with the low dose carvacrol (10 and 25 mg/kg BW) showed significantly reduced oxidative stress, inflammation, and apoptotic effect compared to animals of the MNNG groups receiving increased doses of carvacrol (50 and 100 mg/kg BW) or no carvacrol. Rats exposed to MNNG exhibited gastric cancer cells in several areas. In the MNNG group receiving 100 mg/kg BW carvacrol, the inflammatory cell infiltration was observed in gastric mucosal and submucosal areas whereas MNNG rats supplemented with 10 and 25 mg/kg BW carvacrol showed no pathological alterations of the gastric cells. The results of this study indicate that significant antioxidant and anti-inflammatory effects induced by carvacrol at doses of 10 and 25 mg/kg BW interfered with gastric carcinogenesis induced by MNNG in Wistar rats as well as provide hepatoprotection. However, high doses of carvacrol (50 and 100 mg/kg BW) increased oxidative stress, inflammation, and apoptosis.

Chemoprotective Effect of Scutellarin against Gastric Cancer in Rats: An in vitro and in vivo Study.

This study evaluated the chemoprotective effect of scutellarin (SC) in vitro and in vivo against gastric carcinogenesis in rats and celllines and examined the underlying mechanism. Gastric cancer celllines (AGS) was used for the in vitro study and lactate dehydrogenase (LDH) profile, histone deacetylase (HDAC) assay, cell cycle & apoptosis ratio and antioxidant parameters were measured. N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was used to induce gastric carcinogenesis in rats and the rats received the different doses of SC (10, 20 and 30 mg/kg). The body weight and tumor incidence were measured at regular time intervals. The antioxidant and pro-inflammatory cytokines were estimated. The finding of data showed that the drug was effective against AGS cell line. Supplementation of scutellarin revealed an upregulation in body weight compared with the MNNG group rats. Moreover, it also reduced the incidence of tumor. It also altered the significant DNA density, LDH content, mucus content and acidity. Scutellarin treated rats showed improved activity in enzymatic and non-enzymatic antioxidant profile and reversed the content of cytokines compared with MNNG induced gastric cancer group rats. This research reveals the chemoprotective property of the scutellarin and highlights the promising role of drug by alteration of inflammatory pathway by minimizing its adverse effect.

Experimental Model of Rectal Carcinogenesis Induced by N-Methyl-N-Nitrosoguanidine in Mice with Endoscopic Evaluation.

Background and purpose: The discovery of chemical substances with carcinogenic properties has allowed the development of several experimental models of colorectal cancer (CRC). Classically, experimental models of CRC in mice have been evaluated through clinical or serial euthanasia. The present study aims to investigate the role of low endoscopy in the analysis of carcinogenesis induced by N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG).Methods: Thirty C57BL6 mice were divided into two groups: a control group with fifteen animals that underwent rectal instillation of saline solution on day 0 and a carcinogen group with fifteen animals that underwent a 100 mg/kg MNNG rectal instillation on day 0. In both groups, low endoscopies were performed on weeks 4 and 8. We used a validated endoscopic scoring system to evaluate the severity of colitis and colorectal tumor. Euthanasia was carried out at week 12.Results: We observed higher inflammation scores (p <0.001) and a higher number of tumors (p <0.05) in the MNNG group than the control group, both at weeks 4 and 8. A worsening of inflammation scores from the first to the second endoscopy was also noticeable in the MNNG group. There were no bowel perforations related to the procedure, and there was one death in the control group.Conclusion: Low endoscopy in experimental animals allows safe macroscopic evaluation of colorectal carcinogenesis without the need for euthanasia.

Avicennia alba mangrove derived squalene compound inhibited the MNNG induced gastric cancer in Albino rats

ABSTRACTThe chemo-preventive effects of Squalene compound from Avicennia alba leaves on N-methyl-N-nitro-N-nitrosoguanidine (MNNG)-induced gastric carcinogenesis (intragastrically) in albino rats were analyzed. The animals were divided into four groups with six animals in each group. Group 1 was the control, Group 2 was administrated 150mg bw−1 MNNG for two times at 3-week interval. Group 3 was given MNNG and Squalene 50µg bw−1 and group 4 was given only Squalene at the end of the experiment (25 weeks). Squalene prolonged the survival of MNNG-induced cancer bearing animals and delayed the onset of tumor in animals administrated with Squalene and MNNG simultaneously. The incidence of gastric tumors in group 2 was 91.5% (5/6 animals) with a mean tumor burden of 117.14mm3. The lipid peroxidation in the stomach tumor tissue was associated with enhanced antioxidant levels. The level of antioxidant effects increased in Squalene group, while it decreased in MNNG groups. Data suggested that Squalene from A. alba extract may have a beneficial effect on patients with gastric cancer.

Omeprazole promotes carcinogenesis of fore-stomach in mice with co-stimulation of nitrosamine.

ObjectivesTo investigate if oral omeprazole application induces cancers of fore and glandular stomach in mice.MethodsA total of 66 eligible male mice were randomly divided into 6 groups, which were treated with control reagent, low (6 mg/kg) and high dose omeprazole (30 mg/kg), N-methyl-N’-nitro-N-nitrosoguanidine (MNNG, 100 mg/L water), and MNNG plus low and high dose omeprazole, respectively. After 24 weeks, concentrations of acid phosphatase (ACP) and N-acetyl-β-D-glucosaminidase(NAG) in serum and spleen was examined, and p21 and mTOR levels in stomach were detected.ResultsThe mouse spleen weight index was smaller in the omeprazole group than the control group, and in the MNNG plus omeprazole groups than the MNNG group. In the fore-stomach, more carcinomas were observed in the MNNG plus omeprazole groups than in the MNNG group. In the glandular stomach, there existed more atypical hyperplasia cases in the MNNG plus omeprazole groups than the MNNG-treated group, and one carcinoma was induced in the MNNG plus high dose omeprazole group. Omeprazole alone caused minor gastric pathological changes. Omeprazole treatment lowered both serum and spleen ACP and NAG levels in both the non-MNNG-treated and MNNG-treated subgroups. In fore-stomach, there existed decreased p21 and mTOR levels in the omeprazole-treated groups than in the control group, and in the MNNG plus omeprazole groups than the MNNG-treated group.ConclusionOmeprazole promotes carcinogenesis of the mouse fore-stomach but not the glandular stomach following treatment with MNNG. Lysosomal hydrolase activity was inhibited and some cancer-associated proteins was dysregulated, which requires further explorations.

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 effects of rofecoxib and folic acid on gastric carcinogenesis induced by N‐methyl‐N′‐nitro‐N‐nitrosoguanidine in rats

Epidemiological and experimental studies indicate that non-steroidal anti-inflammatory drugs (NSAIDs) are chemopreventive agents of gastrointestinal cancers, but few studies on gastric cancer have been carried out. A decrease in folic acid supplement and subsequent DNA hypomethylation are related to gastrointestinal cancers, and it has been shown that high-dose folic acid may interfere with gastric carcinogenesis in dogs. The objective of this study was to investigate the effects of rofecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, and folic acid on the chemoprevention of gastric cancer induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in Wistar rats, and to evaluate the cell proliferation of gastric mucosa in different experimental groups. Eighty male Wistar rats were randomly divided into five groups (16 rats in each group). In the control group, the rats were given pure water and basal diet. In the MNNG group, the rats received MNNG in drinking water (100 mg/L) and basal diet. In the MNNG + low-dose rofecoxib group, the rats were given MNNG and rofecoxib 5 mg/kg per day with basal diet. In the MNNG + high-dose rofecoxib group, the rats were given MNNG and rofecoxib 15 mg/kg per day with basal diet. In the MNNG + folic acid group, the rats were given MNNG and folic acid 5 mg/kg per day with basal diet. The experiment was terminated at 50 weeks, and all rats were killed. Blood samples of 3 mL were obtained for measurement of serum folic acid concentrations in the control group, the MNNG group and the MNNG + folic acid group by using chemiluminescent method. The stomach was removed from all rats for histopathological examination and immunohistochemical study. Proliferating cell nuclear antigen (PCNA) expression in gastric epithelial cells was also determined. In the MNNG group, five of 11 rats (45.5%) developed gastric cancer, while in all other four groups no gastric cancer was found (P < 0.05). The positivity rate of PCNA expression in the cancerous tissues was significantly higher than that in the non-cancerous tissues (80.0%vs 14.1%, P < 0.05). The positivity rate of PCNA expression in the gastric mucosal cells of the MNNG group was significantly higher than that in the other four groups. The mean serum folic acid concentration of rats was significantly higher in the MNNG + folic acid group (193.70 +/- 60.73 ng/mL) than those in the control group (84.21 +/- 25.26 ng/mL) and the MNNG group (72.27 +/- 16.70 ng/mL, P < 0.05). It was shown that both low- and high-dose rofecoxib as well as folic acid interfered with the development of gastric cancer induced by MNNG in Wistar rats. The results indicate that rofecoxib as well as folic acid interferes with gastric carcinogenesis induced by MNNG in Wistar rats, and the suppression of gastric cell proliferation may play a crucial role in the chemoprevention of gastric cancer by rofecoxib and folic acid. The higher serum folic acid concentration of rats may play an important role in the prevention of gastric cancer.

Cadmium affects genes involved in growth regulation during two-stage transformation of Balb/3T3 cells

Cadmium (Cd), a carcinogenic metal in human and rodents, has been shown to transform cells in vitro. However, the carcinogenic mechanisms of Cd as a mutagen and/or promoter are not well clarified. We already reported that CdCl 2 in a range of 1.5∼360 ng/ml enhanced transformation of Balb/3T3 A31 cells induced by N-methyl- N′-nitro- N-nitrosoguanidine (MNNG, 0.1 μg/ml) in a dose-dependent manner (Fang et al., Toxicol. In Vitro 15(3) (2001a) 51–7). In previous study, we observed that Cd stimulated cell proliferation on MNNG-initiated cells through inactivation of p53 and p27 and increase of proliferating cell nuclear antigen (PCNA) expression after 24 h treatment (Fang et al., Toxicology 163 (2001b) 175–84). The aim of this study is to further elucidate the long-term effect of Cd in terms of cell cycle control gene expressions during the promotion stage of in vitro two-stage transformation. For the purpose, we determined the expression levels of the genes involved in growth regulation, such as p53, p27, c- myc, mdm2, cyclins D1 and B1, CDK4, and PCNA in the cells treated with Cd for 14 days after MNNG-initiation. In MNNG+CdCl 2 group, cells apparently expressed cellular tumor antigen p53 mRNA, but did not express the wild-type p53 protein; the protein and mRNA levels of p27 were reduced apparently in the cells of MNNG+CdCl 2 group compared to the cells of control and MNNG group. In addition, the protein levels of cyclin D1, CDK4, PCNA, c- myc, and mdm2, and cyclin B1 mRNA level were higher in MNNG+CdCl 2 group than control and MNNG group. Together with previous data (Fang et al., Toxicology 163 (2001b) 175–84), our results indicated that during the transformation process of MNNG-treated cells, Cd may activate oncogenes such as c- myc, mdm2, and cellular tumor antigen p53, inhibit the tumor suppressor genes such as wild-type p53 and p27, and consequently accelerate the proliferation of initiated cells. This work firstly demonstrates that Cd affects the genes involved in growth regulation on initiated cells during the promotion stage of in vitro cell transformation.

Effects of Helicobacter pylori and bile on N-methyl-N'-nitro-N'-nitrosoguanidine exposed antral mucosa of C57BU6 mice.

The aim of this study was to evaluate the early influence of Helicobacter pylori infection on cell kinetics in the antral mucosa of mice exposed to N-methyl-N'-nitro-N'-nitrosoguanidine (MNNG) and bile alone or in combinations. Four hundred and one C57BL/6 male and female mice were assigned into seven treatment groups and one non-treated control group. The gastric antrums were assessed by histology and immunohistochemistry for studies of cell proliferation and apoptosis at 32 and 44 weeks. One female and one male mouse had developed dysplastic adenomas in the pylorus mucosa and one male animal had dysplastic proliferation in the antrum. Only one of these lesions occurred in a H. pylori colonized animal. H. pylori infection significantly increased the cell proliferation at 32 weeks and promoted the cell proliferation in the MNNG and bile group at 44 weeks. Female mice showed less increase in cell proliferation than did the males. No change in apoptosis was seen in any of the groups. Bile had no promotional effect on cell proliferation. These results indicate that H. pylori infection has the potential to alter epithelial cell kinetics as well as antrum mucosa of an animal species that is regarded as resistant to MNNG. However, this change is not sufficient to promote the early development of neoplastic lesions.

Effect of Helicobacter pylori infection on the N-methyl-N'-nitro-N-nitrosoguanidine-induced gastric carcinogenesis in mongolian gerbils.

The effect of Helicobacter pylori infection on N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced gastric cancer was studied using a Mongolian gerbil model. Five-week-old male Mongolian gerbils were divided into four groups of 25-30 animals each and challenged for 20 weeks with H.pylori, MNNG, a combination of H.pylori and MNNG, or neither of them. Four to 20 animals from each group were killed at 16, 24 and 52 weeks after H.pylori inoculation, and histopathological changes in their stomachs were examined. A well-differentiated adenocarcinoma was first observed 24 weeks after inoculation in the combination group. At 52 weeks, only six of 15 animals were colonized with H.pylori persistently, and four of them showed well-differentiated adenocarcinomas; on the other hand, neither of the animals with disappearance of H.pylori from the combination group showed adenocarcinoma. At the same observation time, three of 17 animals from MNNG group showed poorly differentiated adenocarcinomas. The incidence of gastric carcinoma in the combination group was significantly higher than that in the MNNG group (P < 0.05). However, no tumors were seen in the control and H.pylori groups. The present findings demonstrate that H.pylori infection enhances the carcinogenic action of MNNG.

Effect of garlic on micronuclei frequency in peripheral blood lymphocytes of rats with N-methyl-N'nitro-N-nitrosoguanidine-induced gastric carcinoma and precancerous lesions.

To investigate the effects of garlic on micronuclei frequency (MNF) in peripheral blood lymphocytes (PBLs) of Wistar rats with N-methyl-N'nitro-N-nitrosoguanidine (MNNG)-induced gastric carcinoma (GC) and precancerous lesions (PLs). Wistar rats were exposed to MNNG at 1.25 mg/5 mL per day for 10 mo to induce GC and PLs (MNNG group, n = 30); rats not exposed to MNNG served as unmodeled controls (control group, n = 16). The MNNG rats were randomly divided into a prevention treatment group (n = 30; receiving 10 mL of a 10% garlic solution per day) and an untreated model control group (n = 20; receiving tap water). MNF in PBLs were detected at experiment months 10 and 16 mo by the microculture technique. The MNNG group had similar MNF levels at months 10 and 16. Compared to the control group, the MNNG, prevention and untreated model control groups had remarkably higher MNF levels (P < 0.01). The level of PLs was significantly lower in the prevention treatment group than in the untreated model control group (P < 0.01). The prevention group showed significantly lower MNF than the MNNG group (P < 0.01), and the MNF level was reduced in month 16 compared to month 10 (P < 0.01). However, the difference in MNF levels in groups given prevention or treatment was not significant. MNNG exposure exerted continuous mutagenicity and carcinogenicity properties on PBLs, and garlic exerted a remarkable anti-mutagenic and anti-carcinogenic effect. MNF in PBL may be a novel marker of early-stage GC.

Rosiglitazone suppresses gastric carcinogenesis by up-regulating HCaRG expression

Our previous study demonstrated that PPARgamma ligand rosiglitazone prevents gastric carcinogenesis in rats induced by chemical carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). In this study, we attempted to identify novel anti-cancer mechanisms of rosiglitazone. By examining the gene expression profiles of MNNG-induced and rosiglitazone-treated gastric cancer with Uniset Rat I Bioarray microarray, we identified a gene that showed prominent responses in the rosiglitazone-treated group. The hypertension-related, calcium-regulated gene (HCaRG) was significantly up-regulated in rat gastric carcinoma of the rosiglitazone-treated group when compared with the MNNG group. We further examined HCaRG expression in human gastric cancer and found that the expression of HCaRG was down-regulated in human gastric cancerous tissue. Rosiglitazone markedly induced the expression of HCaRG in the AGS cell line. The up-regulation of HCaRG may be one of the mechanisms underlying the chemopreventive effect of rosiglitazone in gastric cancer.

Enhanced Growth Potential of Cultured Rahbit Tracheal Epithelial Cells Following Exposure to N‐Methyl‐N′‐nitro‐N‐nitrosoguanidine

To establish a standardized model for the transformation of rabbit airway epithelial cells, we attempted to transform rabbit tracheal epithelial (RbTE) cells in culture with N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG). RbTE cells, harvested by enzymatic digestion from male New Zealand white rabbits, were plated onto feeder layers of irradiated 3T3 cells. Control cells proliferated exponentially during the 2nd week of culture and reached the plateau phase by the 3rd week. Cells exposed to MNNG (0.1 γg/ml) proliferated in a fashion similar to the control cells, except that there was some delay before proliferation began. The clonogenic activity of RbTE cells rapidly decreased in parallel with the increase in cell population equally in the control and MNNG groups. During the late plateau phase, cells exposed to MNNG regained clonogenic activity, and this compartment size expanded with time, whereas the clonogenic activity in control cultures remained below the detectable level. In RbTE cell cultures exposed three times to 0.1 γg/ml MNNG, large, persistent and proliferating colonies emerged at a frequency of 1–3 × 10 x−2 among the surviving clones, whereas all the control cultures eventually became senescent. The MNNG‐indnced alteration in the growth potential of RbTE cells, i.e., the extended lifespan, and the maintenance and even expansion of clonogenic activity, was similar to that of transformed rat tracheal epithelial cells. However, no immortal cell line could be established from these growth‐altered RbTE cells. We therefore concluded that the growth‐altered RbTE cells were partially transformed.

COLONIC PROSTAGLANDIN-E2 IN CARCINOGENESIS IN RATS WITH N-METHYL-N'-NITROSOGUANIDINE

The effects of prostaglandin E2 and indomethacin on carcinogenesis were studied in male Wistar rats. Of the rats, those treated with MNNG (MNNG group) were given 1 ml of 0.1% solution of MNNG in distilled water by intrarectal instillation every day for 14 days. The diet for the rats treated with Indomethacin and MNNG (Indo-MNNG group) was supplemented with 0.005% indomethacin. No tumor was found in a group of controls. Tumor incidence was 43.8% at week 20 and 81.8% at week 40 of treatment for the MNNG group whereas, it was 5.3% at week 20 and 27.8% at week 40 for the Indo-MNNG group. These differences were of statistical significance (p<0.01). Tumor incidence per animal was lower for the Indo-MNNG group: a mean of 0.50 tumors/animal, compared with 2.18 tumors/animal for the MNNG group (p<0.01). PGE2 levels in colonic mucosa were: 23.9 (pg/mg total protein) at week 5: 27.8 at week 10; 33.2 at Week 20, and 34.8 at week 40 for the control group. It was 44.7 at week 5; 43.1 at week 10; 70.1 at week 20, and 79.7 at week 40 for the MNNG group. Intrinsic PGE2 levels in noncancerous mucosa were significantly higher for the MNNG group than for the control group at all stages of observation. PGE2 significantly decreased in the Indo-MNNG group. with mean values of 22.0 at week 5; 29.0 at week 10; 43.1 at week 20, and 40.6 at week 40, compared with the MNNG group. These findings demonstrated that PGE2 of colonic mucosa promoted the development and proliferation of carcinoma in MNNG-induced large bowel carcinogenesis in rats.

Determination of carcinogenic potency of alkytoxynol-741 (AP-741) by rat peritoneal cell cultures

The carcinogenic potency of AP-741 was tested in rats using the Nashed method of rat peritoneal cell short-term carcinogenic test. N-methyl-N-nitro-N′-nitrosoguanidian (MNNG) was used as positive control; saline and nonoxynol-9 (NP-9) were used as negative control. Two doses of AP-741 (4 mg/kg and 40 mg/kg equivalent to 4 and 40 times human doses) were tested. The result showed that no colonies of more than 9 cells were seen in the saline and NP-9 groups, and the two dose groups of AP-741. However, 12 ± 8.3 colonies (9–29 cell/colony) and 4.5 ± 4.2 colonies (30–300 cell/colony) were seen in the MNNG group. According to the Nashed criterion, we can say MNNG has potential carcinogenesis, while AP-741 does not have any potential carcinogenesis and its use as a vaginal contraceptive drug is safe.

Time-related interference of misoprostol with experimental gastric cancer formation induced by N-methyl-N'-nitro-N-nitrosoguanidine in the rat.

The purpose of this study was to investigate the effect of long-term misoprostol administration, at non-antisecretory doses, on N-methyl-N'-nitro-N-nitrosoguanidine(MNNG)-induced gastric carcinogenesis. The incidence of gastric carcinomas and precancerous lesions was evaluated in 50 male 250-g Sprague-Dawley rats after 52 weeks of continuous oral administration of MNNG (120 mg/l; n = 20), MNNG plus misoprostol (2 mg kg-1 day-1; n = 20) or tap water (n = 10) (experiment 1), and in 30 rats treated with MNNG for 30 weeks followed by tap water (n = 15) or by misoprostol (n = 15) for 22 weeks; a third group (n = 10) received tap water only for 52 weeks (experiment 2). After sacrifice, gastric mucosal lesions were macroscopically evaluated and their histology obtained. MNNG consumption was comparable in all groups (6.5 +/- 1.1 mg rat-1 day-1). Misoprostol consumption was 180 +/- 0.25 mg kg-1 day-1 rat-1. In experiment 1 the incidence of gastric carcinomas was 60% in the MNNG group and 25% in the group treated with MNNG plus misoprostol (P less than 0.05). Cytotoxic and hyperplastic gastric mucosal lesions were also significantly reduced by misoprostol. In experiment 2 the incidence of carcinomas was 31% and 38.6% respectively. Misoprostol significantly decreased the incidence of gastric cancer formation when given from the beginning of the experiment. By contrast, when administered after 30 weeks of MNNG treatment it did not interfere with experimental gastric cancer formation. Exogenous prostaglandins are able to prevent the early MNNG-induced gastric mucosal lesions, thus interfering with gastric carcinogenesis.

Investigation of colonic prostaglandins in carcinogenesis in the rat colon

Large bowel carcinoma was induced in rats by injecting MNNG intrarectally, and changes in the large bowel mucosa prostaglandin (PG) with time were determined. The PGE2 levels of the colonic mucosa in a control group were 20.9 +/- 9.7 (pg/mg total protein) at 5 weeks, 25.5 +/- 9.7 at 10 weeks, 26.5 +/- 18.1 at 20 weeks, and 34.8 +/- 12.7 at 40 weeks. The PGE2 levels in the MNNG-treated group were 44.7 +/- 6.2 at 5 weeks, 43.1 +/- 14.9 at 10 weeks, 70.1 +/- 23.4 at 20 weeks, and 79.7 +/- 54.1 at 40 weeks. The intrinsic PGE2 levels of the noncancerous mucosa were thus significantly higher for the MNNG group than for the control group at all weeks. At 40 weeks, the PGE2 levels of cancer lesions were significantly high compared with those of the noncancerous area. In the cancerous lesions, 6-keto-PGF1 alpha and PGF2 alpha decreased and TXB2 increased significantly at 40 weeks. The observations demonstrated that PGE2 was implicated as a promoter in the development and proliferation of carcinoma in MNNG-induced large bowel carcinogenesis in rats.

Anticarcinogenic effect of refined amorphophallus konjac on MNNG-induced lung cancer in LACA mice

Five hundreds and fifty LACA mice were used in 3 batches for studying the anticarcinogenic effect of Konjaku powder on MNNG-induced lung cancers. These mice (within each batch) were randomly allocated to four groups, namely, positive control (MNNG), Amorphophallus konjac (A.K.), complex (MNNG+A.K.), and blank control (C) group. In MNNG group, MNNG (250 µg) was injected intravenously once five days for seven times in each mouse, the total dosage of MNNG being 1.75 mk. In A.K. group, according to w/w, 8% A.K. was well mixed into 92% common diet for long-term breeding. In complex group, MNNG was given as that in MNNG group and the mice were reared as those in A.K. group. The mice in MNNG group and in C group were all reared by common diet. The results showed different degrees of anticarcinogenic and preventive effect of refined A.K. on MNNG-induced lung cancers in LACA mice. A.K. not only exerted effect on the number of induced cancer and precancer, causing a drop of the cancerous rate from 70.87% to 19.38% and the mean number of cancer and precancer in each animal, but also postponed the incubation period. The proportional distribution of the kinds of tumor denoted a decrease in malignancy (adenoma with malignant change), absence of adenocarcinoma, relative increase in benign adenoma, and prolonged survival time of animals. The results of experiments in 3 batches also exhibited good duplication.