Synergistic effects of low-dose arsenic and N-methyl-N'-nitro-N-nitrosoguanidine co-exposure by altering gut microbiota and intestinal metabolic profile in rats.

Biological organisms are exposed to low-dose arsenic or N-nitro compounds (NOCs) alone or in combination worldwide, especially in areas with high cancer prevalence through drinking water or food exposure; however, information on their combined exposure effects is limited. Here, we conducted an in-depth study of the effects on the gut microbiota, metabolomics, and signaling pathways using rat models exposed to arsenic or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), one of the most active carcinogenic NOCs, separately or in combination with metabolomics and high-throughput sequencing. Compared to exposure alone, combined exposure to arsenic and MNNG exacerbated damage to gastric tissue morphology, interfered with intestinal microflora and substance metabolism, and exerted a stronger carcinogenic effect. This may be related to intestinal microbiota disorders, including Dyella, Oscillibacter, Myroides, and metabolic pathways such as glycine, serine, and threonine metabolism, arginine biosynthesis, central carbon metabolism in cancer, and purine and pyrimidine metabolism, thereby enhancing the cancer-causing effects of gonadotrophin-releasing hormone (GnRH), P53, and Wnt signaling pathways.

Folic acid ameliorates N-methyl-N'-nitro-N-nitrosoguanidine-induced esophageal inflammation via modulation of the NF-κB pathway.

N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) is a common alkylating agent, which can be experimentally used as a chemical mutagen and carcinogen, extensively existing in the environment. Folic acid (FA), part of the B group of vitamins, plays an important role in defending against inflammation and reducing the risk of cancers. Nevertheless, there is little research on the protective effects of FA against MNNG-induced esophageal inflammation, and its underlying mechanism still remains elusive. Hence, in the present study, we exposed MNNG to SD rats and esophageal cells to establish the esophageal inflammation models. Our research aims to explore the protective roles of FA against esophageal inflammation induced by MNNG via NF-κB pathway by CCK-8, EdU, RT-qPCR, ELISA, H&E, Western blot. Our results revealed that MNNG decreased the viability of esophageal cells, which was restored under FA intervention. Besides, FA relieved the elevation of IL-6, IL-8 and TNF-α in MNNG-induced esophageal inflammation. Moreover, histopathological analysis showed that epithelial spinous cells proliferated in mucous layer, and inflammatory cells were locally infiltrated in the submucosa after MNNG exposure, while the pathological damage of esophageal tissues was gradually alleviated along with increasing FA doses. And Western blot results demonstrated that FA could relieve the rise of phosphorylated IκBα (p-IκBα) and phosphorylated p65 (p-p65) proteins induced by MNNG. Therefore, it is reasonable to believe that FA has a crucial role in preventing MNNG-induced esophageal inflammation through inhibiting the NF-κB pathway, thereby down-regulating the expressions of IL-6, IL-8 and TNF-α.

Modulatory effect of Tagetes erecta flowers essential oils via Nrf2/HO-1/NF-κB/p65 axis mediated suppression of N-methyl-N'nitro-N-nitroguanidine (MNNG) induced gastric cancer in rats.

Protective effect of Tagetes erecta flowers essential oils was investigated on oxidative stress, immune response, inflammation, and apoptosis against N-methyl-N'nitro-N-nitroguanidine (MNNG) induced gastric cancer in rats. Essential oil were extracted from Tagetes erecta flowers and analyzed using gas chromatography-mass spectrometry (GC-MS). For observing a protective effect against MNNG induced gastric cancer, we divided rats into 4 groups (group A to D) having 10 rats in each group. Performed various experiments and measured a different parameters to investigate antioxidant activity, immune response, anti-inflammatory and anti-apoptotic activity. The levels of malondialdehyde were markedly increased in the presence of N-methyl-N'nitro-N-nitroguanidine, whereas, the antioxidant activities of superoxide dismutase, and catalase were lowered in the treated rats in contrast with the control. Intervention with TEEO to gastric cancer-induced rats upregulated the redox status and the activity of the immune system to decrease cancer risk. The proinflammatory cytokines (IL-6 and TNF-α) secretions that were induced by MNNG were markedly inhibited by TEEO. Administration of TEEO also significantly reduced terminal deoxynucleotidyl transferase dUTP nick end labeling positive gastric cancer cells, expression of mRNA of caspase-3, and Bax. Whereas, the expression of Bcl-2 was increased. Additionally, downregulation of nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) and IκBα degradation and the nuclear factor-κB (NF-κB) p65 expression in tissues of the stomach of MNNG-induced-rats were markedly elevated due to TEEO. This suggested possession of TEEO with a protective shield against MNNG induced gastric cancer by the exertion of antioxidative stress, anti-apoptotic response, the anti-inflammatory response through Nrf2/HO-1, and NF-κB signaling pathways.

[Research progress in establishment of N-methyl-N'-nitro-N-nitroso-guanidine-induced rat model of Precancerous lesion of gastric cancer].

Gastric cancer(GC), one of the most common malignancies worldwide, seriously threatens human health due to its high morbidity and mortality. Precancerous lesion of gastric cancer(PLGC) is a critical stage for preventing the occurrence of gastric cancer, and PLGC therapy has frequently been investigated in clinical research. Exploring the proper animal modeling methods is necessary since animal experiment acts as the main avenue of the research on GC treatment. At present, N-methyl-N'-nitro-N-nitroso-guanidine(MNNG) serves as a common chemical inducer for the rat model of GC and PLGC. In this study, MNNG-based methods for modeling PLGC rats in related papers were summarized, and the applications and effects of these methods were demonstrated by examples. Additionally, the advantages, disadvantages, and precautions of various modeling methods were briefly reviewed, and the experience of this research group in exploring modeling methods was shared. This study is expected to provide a reference for the establishment of MNNG-induced PLGC animal model, and a model support for the following studies on PLGC.

Methylation Tolerance-Based Functional Assay to Assess Variants of Unknown Significance in the MLH1 and MSH2 Genes and Identify Patients With Lynch Syndrome.

BACKGROUND & AIMS: Approximately 75% of patients with suspected Lynch syndrome carry variants in MLH1 or MSH2, proteins encoded by these genes are required for DNA mismatch repair (MMR). However, 30% of these are variants of unknown significance (VUS). A assay that measures cell response to the cytotoxic effects of a methylating agent can determine the effects of VUS in MMR genes and identify patients with constitutional MMR-deficiency syndrome. We adapted this method to test the effects of VUS in MLH1 and MSH2 genes found in patients with suspected Lynch syndrome. METHODS: We transiently expressed MLH1 or MSH2 variants in MLH1- or MSH2-null human colorectal cancer cell lines (HCT116 or LoVo), respectively. The MMR process causes death of cells with methylation-damaged DNA bases, so we measured proportions of cells that undergo death following exposure to the methylating agent; cells that escaped its toxicity were considered to have variants that affect function of the gene product. Using this assay, we analyzed 88 variants (mainly missense variants), comprising a validation set of 40 previously classified variants (19 in MLH1 and 21 in MSH2) and a prospective set of 48 VUS (25 in MLH1 and 23 in MSH2). Prediction scores were calculated for all VUS according to the recommendations of the American College of Medical Genetics and Genomics, based on clinical, somatic, in silico, population, and functional data. RESULTS: The assay correctly classified 39 of 40 variants in the validation set. The assay identified 12 VUS that did alter function of the gene product and 28 VUS that did not; the remaining 8 VUS had intermediate effects on MMR capacity and could not be classified. Comparison of assay results with prediction scores confirmed the ability of the assay to discriminate VUS that affected the function of the gene products from those that did not. CONCLUSIONS: Using an assay that measures the ability of the cells to undergo death following DNA damage induction by a methylating agent, we were able to assess whether variants in MLH1 and MSH2 cause defects in DNA MMR. This assay might be used to help assessing the pathogenicity of VUS in MLH1 and MSH2 found in patients with suspected Lynch syndrome.

Dynamic changes of Sonic Hedgehog signaling pathway in gastric mucosa of rats with MNNG-induced gastric precancerous lesions.

OBJECTIVE: To explore the changes of Sonic Hedgehog (Shh) signaling pathway in the stomach mucosa during the formation of gastric precancerous lesions. METHODS: A total of 72 suckling rats in half genders were randomly and equally divided into the normal group and model group. The rats in the model group were administered with 0.1 ml 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) at the dosage of 800 mg/L for 10 days, whereas the rats in the normal group were similarly administered with normal saline. A total of 12 rats in each group were killed at the end of 10th, 22nd, and 34th weeks in half gender, respectively. Histopathological changes of the gastric mucosa were observed by hematoxylin and eosin (HE) staining; the levels of Shh, Ptch1, Smo, Gli1, Gli2, Gli3, SuFu, Cyclin D1, Cyclin E1, c-Myc, and ß-actin mRNAs in the gastric mucosa were determined by real-time polymerase chain reaction; while the protein expression of Shh, Ptch1, Smo, Gli1, SuFu, Cyclin D1, Cyclin E1, c-Myc, and p-c-Myc was detected by western blot analysis. RESULTS: With the development of atrophy and dysplasia of gastric mucosa, the levels of Shh, Smo, Gli1, Cyclin D1, Cyclin E1, and c-Myc mRNAs increased, while those of Ptch1 and SuFu decreased. The expression of Shh, Smo, Gli1, Cyclin D1, Cyclin E1, and p-c-Myc proteins were elevated, while the expression of Ptch1 and SuFu proteins were decreased, however, without statistical difference. CONCLUSIONS: Shh signaling is activated during the formation of gastric precancerous lesions, which indicates that the Shh signaling pathway participates in the development and progression of gastric precancerous lesions.

Chronic CagA-positive Helicobacter pylori infection with MNNG stimulation synergistically induces mesenchymal and cancer stem cell-like properties in gastric mucosal epithelial cells.

A CagA-positive Helicobacter pylori (H. pylori) infection can cause malignant transformation of human gastric mucosal epithelial cells, and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) is a chemical carcinogen that induces gastric carcinogenesis. Whether this environmental chemocarcinogen may synergistically enhance the risk of H. pylori-infected gastric cancer remains unclear. In this study, we adopted a chronic CagA-positive H. pylori infection with or without MNNG coinduction to establish a cellular model in GES-1 cells and an animal model in C57BL/6J mice. The proliferation, cell phenotype, apoptosis, epithelial-mesenchymal transition (EMT), stemness and tumorigenicity of gastric mucosal epithelial cells were analyzed in vitro and in vivo. The results showed that chronic H. pylori-infected GES-1 cells displayed inhibited apoptosis, abnormal proliferation, enhanced invasion, and migration, increased EMT/mesenchymal phenotype, colony formation and stem cell-like properties, and enhanced tumorsphere-formatting efficiency as well as CD44 expression, a known gastric cancer stem cell (CSC) marker. MNNG synergistically promoted the above actions of chronic H. pylori infection. Further studies in chronic H. pylori-infected C57BL/6J mice models showed that an increased incidence of premalignant lesions in the gastric mucosa tissue of the H. pylori-infected mice had occurred, the mouse gastric mucosa cells exhibited similar mesenchymal and CSC-like properties in the above GES-1 cells, and precancerous lesions and EMT/CSC-like phenotypes were reinforced by the synergistic action of MNNG stimulation. H. pylori infection and/or MNNG induction were capable of causing enhanced expression and activation of Wnt2 and ß-catenin, indicating that the Wnt/ß-catenin pathway is involved in the actions of H. pylori and MNNG. Taken together, these findings suggest that chronic CagA-positive H. pylori infection with MNNG stimulation synergistically induces mesenchymal and CSC-like properties of gastric mucosal epithelial cells.

Leptin enhances N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced tumour growth in gastric mucosa of male Sprague-Dawley rats.

Individuals who are obese are at a greater risk of developing gastric cancer. They are however also hyperleptinaemic. Chronic leptin treatment has been shown to upregulate numerous cancer-causing genes in the stomach of male Sprague-Dawley rats. It is however unclear if leptin enhances the effect of gastric carcinogens in vivo. This study was therefore done to investigate the effect of leptin on gastric carcinogenesis in rats treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Twenty-four, 6-week old male Sprague-Dawley rats were divided equally into three groups: G1 served as age-matched controls; G2 was treated with MNNG in drinking water ad libitum (200 mg L-1); G3 was given leptin and MNNG. Rats were euthanized after 40 weeks of treatment and their stomachs were removed for histopathology, microarray, and RT-qPCR analysis. Fisher's exact test and one-way ANOVA were used to analyse the data. Fifty percent of the MNNG-treated rats developed gastric hyperplasia (p < 0.05), but there was no significant change in any carcinogenic genes. Concurrent MNNG and leptin treatment however induced hyperplasia, dysplasia, hypertrophy, and adenocarcinoma in 75% (6/8) of the rats; with upregulation of microRNAs, olfactory receptors, Hey2 (transcription factor), Tmed2 (vesicular trafficking), and Lcn11 (cell proliferation) genes. It appears that leptin enhances MNNG- induced tumour growth in stomachs of Sprague-Dawley rats and its role in gastric cancer requires further scrutiny.

ATR-CHK1-E2F3 signaling transactivates human ribonucleotide reductase small subunit M2 for DNA repair induced by the chemical carcinogen MNNG.

BACKGROUND: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), an alkylating agent and an environmental carcinogen, causes DNA lesions and even carcinomas. DNA damage responses induced by MNNG activate various DNA repair genes and related signaling pathways. The present study aimed to investigate the regulatory mechanisms of human RR small subunit M2 (hRRM2) in response to MNNG. RESULTS: In this study, we demonstrated that the RRM2 gene was transactivated by MNNG exposure more strongly than the other small subunit, p53R2. The upregulated RRM2 translocated to the nucleus for DNA repair. Further study showed that E2F3 transactivated RRM2 expression by directly binding to its promoter after MNNG exposure. The transactivation was enhanced by the upregulation of NFY, which bound to the RRM2 promoter adjacent to the E2F3 binding site and interacted with E2F3. In response to MNNG treatment, E2F3 accumulated mainly through its phosphorylation at S124 and was dependent on ATR-CHK1 signaling. In comparison, p53R2 played a relatively weaker role in the MNNG-induced DNA damage response, and its transcription was regulated by the ATR-CHK2-E2F1/p53 pathway. CONCLUSIONS: We suggest that MNNG-stimulated ATR/CHK1 signaling stabilizes E2F3 by S124 phosphorylation, and then E2F3 together with NFY co-transactivate RRM2 expression for DNA repair. GENERAL SIGNIFICANCE: We propose a new mechanism for RRM2 regulation to maintain genome stability in response to environmental chemical carcinogens.

Down-regulation of PARP1 by miR-891b sensitizes human breast cancer cells to alkylating chemotherapeutic drugs.

PURPOSE: Breast cancer is the most common invasive type of cancer among women. Role of different microRNAs (miRNAs) and poly(ADP-ribose) polymerases (PARPs) in breast cancer has been well established. This study aimed to explore the effects of miR-891b on sensitizing breast cancer cells to alkylating chemotherapeutic drugs through PARPs. METHODS: The expression of miR-891b and PARP1 in human breast cancer cells HCC1806 was overexpressed by transfection with their mimics or expressing vector. Then, the transfected cells were exposed to 40 µM N-methyl-N-nitro-N-nitrosoguanidine (MNNG) for 1 h. The correlation between miR-891b and PARP1 was detected by RT-qPCR, western blot, and dual-luciferase reporter assay. Besides, MTT assay and Annexin V assay were done to measure cell proliferation and apoptosis, respectively. RESULTS: PARP1 was a target of miR-891b, and it was negatively regulated by miR-891b. MiR-891b increased the sensitivity of the HCC1806 cells to the cytotoxic effects of MNNG through suppressing cell proliferation and increasing the percentage of apoptotic cells. Restoration of PARP1 activity in the HCC1806 cells led to loss of miR-891b mediated sensitivity of the HCC1806 cells to MNNG. CONCLUSION: MiR-891b increases the sensitivity of the breast cancer cells (HCC1806) to the cytotoxic effects of the chemotherapeutic agent MNNG by suppressing the expression of PARP1.

Preventive Efficacy of Vanillic Acid on Regulation of Redox Homeostasis, Matrix Metalloproteinases and Cyclin D1 in Rats Bearing Endometrial Carcinoma.

Endometrial carcinoma is a malignant tumor of the female genital tract. This study has been performed to evaluate the chemopreventive efficacy of vanillic acid (a bio flavonoid) on endometrial carcinoma (EC) by assessing the levels of thiobarbituric acid reactive substances (TBARS), lipid hydroperoxides (LOOH), cytochrome P450, antioxidants-superoxide dismutase (SOD), catalase (CAT), glutathione peroxides (GPx), reduced glutathione (GSH), vitamins C and E, matrix metalloproteinases (MMP-2 and 9) and cell cycle check point protein (cyclin D1) in N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) induced carcinogenic rats. EC provoked by intravaginal detention of MNNG (150 mg/kg b.w. for 90 days), lead to enhancement of the levels of TBARS, LOOH, cytochrome P450, and decrement in the levels of antioxidants (SOD, CAT, GPx, GSH, vitamins C and E) and upregulated expression of MMP-2 and 9 and cyclin D1 (by western blot analysis). The treatment of vanillic acid (100 mg/kg b.w.) to MNNG treated rats (1) normalized the histopathological alterations, (2) reduced the levels of TBARS, LOOH and cytochrome P450 (3) increased the levels of antioxidants (SOD, CAT, GPx, GSH, vitamins C and E) in plasma and uterus and (4) down regulated the expression of MMP-2, 9 and cyclin D1. The effect of vanillic acid is more predominant in pre-treatment group than co-treated rats. Our results designate that vanillic acid inhibits the EC by elevating antioxidants and by regulating the levels of metalloproteinase and cell cycle check point protein.

Cellular response to alkylating agent MNNG is impaired in STAT1-deficients cells.

The SN 1 alkylating agents activate the mismatch repair system leading to delayed G2 /M cell cycle arrest and DNA repair with subsequent survival or cell death. STAT1, an anti-proliferative and pro-apoptotic transcription factor is known to potentiate p53 and to affect DNA-damage cellular response. We studied whether STAT1 may modulate cell fate following activation of the mismatch repair system upon exposure to the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Using STAT1-proficient or -deficient cell lines, we found that STAT1 is required for: (i) reduction in the extent of DNA lesions, (ii) rapid phosphorylation of T68-CHK2 and of S15-p53, (iii) progression through the G2 /M checkpoint and (iv) long-term survival following treatment with MNNG. Presence of STAT1 is critical for the formation of a p53-DNA complex comprising: STAT1, c-Abl and MLH1 following exposure to MNNG. Importantly, presence of STAT1 allows recruitment of c-Abl to p53-DNA complex and links c-Abl tyrosine kinase activity to MNNG-toxicity. Thus, our data highlight the important modulatory role of STAT1 in the signalling pathway activated by the mismatch repair system. This ability of STAT1 to favour resistance to MNNG indicates the targeting of STAT1 pathway as a therapeutic option for enhancing the efficacy of SN1 alkylating agent-based chemotherapy.

Crosstalk between apoptosis, necrosis and autophagy.

Apoptosis and necrosis are the two major modes of cell death, the molecular mechanisms of which have been extensively studied. Although initially thought to constitute mutually exclusive cellular states, recent findings reveal cellular contexts that require a balanced interplay between these two modes of cellular demise. Several death initiator and effector molecules, signaling pathways and subcellular sites have been identified as key mediators in both processes, either by constituting common modules or alternatively by functioning as a switch allowing cells to decide which route to take, depending on the specific situation. Importantly, autophagy, which is a predominantly cytoprotective process, has been linked to both types of cell death, serving either a pro-survival or pro-death function. Here we review the recent literature that highlights the intricate interplay between apoptosis, necrosis and autophagy, focusing on the relevance and impact of this crosstalk in normal development and in pathology. This article is part of a Special Section entitled: Cell Death Pathways.

PARP-1 activation causes neuronal death in the hippocampal CA1 region by increasing the expression of Ca(2+)-permeable AMPA receptors.

An excessive activation of poly(ADP-ribose) polymerases (PARPs) may trigger a form of neuronal death similar to that occurring in neurodegenerative disorders. To investigate this process, we exposed organotypic hippocampal slices to N-methyl-N'-nitro-N'-nitrosoguanidine (MNNG, 100µM for 5min), an alkylating agent widely used to activate PARP-1. MNNG induced a pattern of degeneration of the CA1 pyramidal cells morphologically similar to that observed after a brief period of oxygen and glucose deprivation (OGD). MNNG exposure was also associated with a dramatic increase in PARP-activity and a robust decrease in NAD(+) and ATP content. These effects were prevented by PARP-1 but not PARP-2 inhibitors. In our experimental conditions, cell death was not mediated by AIF translocation (parthanatos) or caspase-dependent apoptotic processes. Furthermore, we found that PARP activation was followed by a significant deterioration of neuronal membrane properties. Using electrophysiological recordings we firstly investigated the suggested ability of ADP-ribose to open TRPM2 channels in MNNG-induced cells death, but the results we obtained showed that TRPM2 channels are not involved. We then studied the involvement of glutamate receptor-ion channel complex and we found that NBQX, a selective AMPA receptor antagonist, was able to effectively prevent CA1 neuronal loss while MK801, a NMDA antagonist, was not active. Moreover, we observed that MNNG treatment increased the ratio of GluA1/GluA2 AMPAR subunit expression, which was associated with an inward rectification of the IV relationship of AMPA sEPSCs in the CA1 but not in the CA3 subfield. Accordingly, 1-naphthyl acetyl spermine (NASPM), a selective blocker of Ca(2+)-permeable GluA2-lacking AMPA receptors, reduced MNNG-induced CA1 pyramidal cell death. In conclusion, our results show that activation of the nuclear enzyme PARP-1 may change the expression of membrane proteins and Ca(2+) permeability of AMPA channels, thus affecting the function and survival of CA1 pyramidal cells.

Tumor-associated mutations in O6 -methylguanine DNA-methyltransferase (MGMT) reduce DNA repair functionality.

MGMT is the primary vehicle for cellular removal of alkyl lesions from the O-6 position of guanine and the O-4 position of thymine. While key to the maintenance of genomic integrity, MGMT also removes damage induced by alkylating chemotherapies, inhibiting the efficacy of cancer treatment. Germline variants of human MGMT are well-characterized, but somatic variants found in tumors were, prior to this work, uncharacterized. We found that MGMT G132R, from a human esophageal tumor, and MGMT G156C, from a human colorectal cancer cell line, are unable to rescue methyltransferase-deficient Escherichia coli as well as wild type (WT) human MGMT after treatment with a methylating agent. Using pre-steady state kinetics, we biochemically characterized these variants as having a reduced rate constant. G132R binds DNA containing an O6 -methylguanine lesion half as tightly as WT MGMT, while G156C has a 40-fold decrease in binding affinity for the same damaged DNA versus WT. Mammalian cells expressing either G132R or G156C are more sensitive to methylating agents than mammalian cells expressing WT MGMT. G132R is slightly resistant to O6 -benzylguanine, an inhibitor of MGMT in clinical trials, while G156C is almost completely resistant to this inhibitor. The impared functionality of expressed variants G132R and G156C suggests that the presence of somatic variants of MGMT in a tumor could impact chemotherapeutic outcomes.

An HPLC-tandem mass spectrometry method for simultaneous detection of alkylated base excision repair products.

DNA glycosylases excise a broad spectrum of alkylated, oxidized, and deaminated nucleobases from DNA as the initial step in base excision repair. Substrate specificity and base excision activity are typically characterized by monitoring the release of modified nucleobases either from a genomic DNA substrate that has been treated with a modifying agent or from a synthetic oligonucleotide containing a defined lesion of interest. Detection of nucleobases from genomic DNA has traditionally involved HPLC separation and scintillation detection of radiolabeled nucleobases, which in the case of alkylation adducts can be laborious and costly. Here, we describe a mass spectrometry method to simultaneously detect and quantify multiple alkylpurine adducts released from genomic DNA that has been treated with N-methyl-N-nitrosourea (MNU). We illustrate the utility of this method by monitoring the excision of N3-methyladenine (3 mA) and N7-methylguanine (7 mG) by a panel of previously characterized prokaryotic and eukaryotic alkylpurine DNA glycosylases, enabling a comparison of substrate specificity and enzyme activity by various methods. Detailed protocols for these methods, along with preparation of genomic and oligonucleotide alkyl-DNA substrates, are also described.

Genotoxic and antigenotoxic assessment of four newly synthesized dihydropyridine derivatives.

The current study aims to determine the genotoxic and antigenotoxic potential of four newly synthesized dihydropyridine derivatives using Escherichia coli WP2 and Ames/Salmonella bacterial reversion assay systems. The bacterial mutant tester strains, E. coli WP2uvrA with a point mutation and Salmonella typhimurium TA1537 with a frameshift mutation, were used to determine genotoxic potentials of the test compounds. To determine antigenotoxic potentials of the test compounds, the same strains were also used together with positive mutagens N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) for E. coli WP2uvrA and 9-aminoacridine (9-AA) for S. typhimurium TA1537. According to the results, neither of the test compounds showed significant genotoxic activity on both tester strains at the tested concentrations. However, except compound 4, all the test compounds showed significant antigenotoxic activity on MNNG- or/and 9-AA-induced mutations. The inhibition rates of mutagenesis ranged from 27.0% (compound 2: 2.5 mM/plate) to 65.0% (compound 2: 0.5 mM/plate) for MNNG and from 30.6% (compound 2: 2 mM/plate) to 58.5% (compound 1: 1 mM/plate) for 9-AA genotoxicity. According to these results, it is concluded that all the test compounds do not have a mutagenic potential on the bacterial strains at the tested concentrations, and some of them have antigenotoxic potentials against MNNG- and 9-AA-induced mutagenesis.

The demethylase ALKBH5 mediates ZKSCAN3 expression through the m6A modification to activate VEGFA transcription and thus participates in MNNG-induced gastric cancer progression.

N-Nitroso compounds (NOCs) are recognized as important factors that promote gastric cancer development, but the specific effects and potential mechanisms by which NOC exposure promotes gastric cancer are still poorly understood. In this study, we explored the effects and potential molecular mechanisms of NOCs on the promotion of gastric cancer using methylnitronitrosoguanidine (MNNG), a classical direct carcinogen of NOC. The results of in vivo and in vitro experiments showed that chronic and low-concentration MNNG exposure significantly promoted the malignant progression of tumors, including cell migration, cell invasion, vasculogenic mimicry (VM) formation, cell spheroid formation, stem cell-like marker expression, and gastric cancer growth and metastasis. Mechanistically, we revealed that demethylase ALKBH5 regulated the level of the N6­methyladenosine (m6A) modification in the 3'UTR and CDS region of the ZKSCAN3 mRNA to promote ZKSCAN3 expression, mediated the binding of ZKSCAN3 to the VEGFA promoter region to regulate VEGFA transcription, and participated in MNNG-induced gastric cancer cell migration, invasion, VM formation, cell spheroid formation, stem cell-like marker expression and ultimately gastric cancer progression. In addition, our study revealed that ALKBH5-ZKSCAN3-VEGFA signaling was significantly activated during MNNG-induced gastric carcinogenesis, and further studies in gastric cancer patients showed that ALKBH5, ZKSCAN3, and VEGFA expression were upregulated in cancers compared with paired gastric mucosal tissues, that ALKBH5, ZKSCAN3, and VEGFA could serve as important biomarkers for determining patient prognosis, and that the molecular combination showed greater prognostic value. These findings provide a theoretical basis for developing gastric cancer interventions for NOCs and for determining gastric cancer progression.

Modeling gastric intestinal metaplasia in 3D organoids using nitrosoguanidine.

Gastric intestinal metaplasia (GIM) represents a precancerous stage characterized by morphological and pathophysiological changes in the gastric mucosa, where gastric epithelial cells transform into a phenotype resembling that of intestinal cells. Previous studies have demonstrated that the intragastric administration of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) induces both gastric carcinoma and intestinal metaplasia in mice. Here, we show that MNNG induces GIM in three-dimensional (3D) mouse organoids. Our histological analyses reveal that MNNG-induced gastric organoids undergo classical morphological alterations, exhibiting a distinct up-regulation of CDX2 and MUC2, along with a down-regulation of ATP4B and MUC6. Importantly, metaplastic cells observed in MNNG-treated organoids originate from MIST1+ cells, indicating their gastric chief cell lineage. Functional analyses show that activation of the RAS signaling pathway drives MNNG-induced metaplasia in 3D organoids, mirroring the characteristics observed in human GIM. Consequently, modeling intestinal metaplasia using 3D organoids offers valuable insights into the molecular mechanisms and spatiotemporal dynamics of the gastric epithelial lineage during the development of intestinal metaplasia within the gastric mucosa. We conclude that the MNNG-induced metaplasia model utilizing 3D organoids provides a robust platform for developing preventive and therapeutic strategies to mitigate the risk of gastric cancer before precancerous lesions occur.

miR-520f-3p blocks MNNG-induced gastric precancerous lesions via the KLF7/NFκB pathway.

Studying the regulatory mechanism of gastric disease progression to gastric cancer (GC) is essential. miR-520f expression is down-regulated in GC and inhibits the proliferation of gastric cancer cells, suggesting that it is associated with the development of GC, but whether it plays a role in the gastric precancerous lesion (GPL) is unclear. This study aimed to investigate the effect of miR-520f-3p in the N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced GPL model and to elucidate the role of its downstream target gene Kruppel-like factor 7 (KLF7) in it. The experimental results showed that miR-520f-3p expression was down-regulated in the MNNG-induced GES-1 cell model, and overexpression of miR-520f-3p reversed the effects of MNNG on cell migration, invasion and epithelial-mesenchymal transition (EMT) -related protein expression. Meanwhile, overexpression of KLF7 attenuated the effect of miR-520f-3p on GPL. In a mouse GPL model, it was observed that MNNG elicited inflammation and EMT processes in mouse gastric tissues through the KLF7/ Nuclear Factor Kappa B (NFκB) pathway, and silencing KLF7 alleviated MNNG-induced gastric epithelial cell injury and gastric atrophy symptoms. These results provide a new perspective for understanding the development of GPL, and the development of new therapies targeting miR-520f-3p and KLF7 may provide new ideas for the prevention and treatment of gastric cancer.