[Porphyromonas gingivalis promotes the occurrence of esophageal squamous cell carcinoma via an inflammatory microenvironment].

Objective: To investigate the role of an inflammatory microenvironment induced by Porphyromonasgingivalis (P. gingivalis) in the occurrence of esophageal squamous cell carcinoma (ESCC) in mice. Methods: A total of 180 C57BL/6 mice were randomly divided into 6 groups, i.e. control group, P. gingivalis group, 4NQO group, 4NQO + P. gingivalis group, 4NQO + P. gingivalis + celecoxib group, and 4NQO + P. gingivalis + antibiotic cocktail (ABC, including metronidazole, neomycin, ampicillin, and vancomycin) group, with 30 mice in each group, using the random number table. All mice were normalized by treatment with ABC in drinking water for 2 weeks. In the following 2 weeks, the mice in the control group and the P. gingivalis group were given drinking water, while the other 4 groups were treated with 30 µg/ml 4NQO in the drinking water. In weeks 11-12, the mice in the P. gingivalis group, the 4NQO + P. gingivalis group, the 4NQO + P. gingivalis + celecoxib group, and the 4NQO + P. gingivalis + ABC group were subjected to ligation of the second molar in oral cavity followed by oral P. gingivalis infection thrice weekly for 24 weeks in weeks 11-34. In weeks 13-34, the mice in 4NQO + P. gingivalis+celecoxib group and 4NQO + P. gingivalis + ABC group were administered with celecoxib and ABC for 22 weeks, respectively. At the end of 34 weeks, gross and microscopic alterations were examined followed by RT-qPCR and immunohistochemistry to examine the expression profiles of inflammatory- and tumor-molecules in esophagi of mice. Results: At 34 weeks, 4NQO treatment alone did not affect the foci of papillary hyperproliferation, diseased area, and the thickness of the esophageal wall, but significantly enhanced the foci of hyperproliferation (median 1.00, P＜0.05) and mild/moderate dysplasia (median 2.00, P＜0.01). In addition, the expression levels of IL-6 [8.35(3.45,8.99)], IL-1ß [6.90(2.01,9.72)], TNF-α [12.04(3.31,14.08)], c-myc [2.21(1.80,3.04)], pSTAT3, Ki-67, and pH2AX were higher than those in the control group. The pathological changes of the esophageal mucosa were significantly more overt in the 4NQO + P. gingivalis group in terms of the foci of papillary hyperproliferation (median 2.00), diseased area (median 2.51 mm2), the thickness of the esophageal wall (median 172.52 µm), the foci of hyperproliferation (median 1.00, P＜0.05), and mild/moderate dysplasia (median 1.00, P＜0.01). In mice of the 4NQO + P. gingivalis group, the expression levels of IL-6 [12.27(5.35,22.08)], IL-1ß [13.89(10.04,15.96)], TNF-α [19.56(6.07,20.36)], IFN-γ [11.37(8.23,20.07)], c-myc [2.62(1.51,4.25)], cyclin D1 [4.52(2.68,7.83)], nuclear pSTAT3, COX-2, Ki-67, and pH2AX were significantly increased compared with the mice in the control group. In mice of the 4NQO + P. gingivalis group, the diseased area, invasive malignant foci as well as pSTAT3 and pH2AX expression were significantly blunted by celecoxib. Treatment with ABC markedly reduced the papillary hyperproliferative foci, invasive malignant foci, and pSTAT3 expression but not pH2AX. Conclusions: P. gingivalis promotes the occurrence of esophageal squamous cell carcinoma in mice by inducing an inflammatory microenvironment primed with 4NQO induced DNA damage. Clearance of P. gingivalis with ABC or anti-inflammatory intervention holds promise for prevention of esophageal squamous cell malignant pathogenesis via blockage of IL-6/STAT3 signaling and amelioration of inflammation.

Impact of Juglone, a PIN1 Inhibitor, on Oral Carcinogenesis Induced by 4-Nitroquinoline-1-Oxide (4NQO) in Rat Model.

Background and Objectives: PIN1 is overexpressed in several human cancers, including prostate cancer, breast cancer, and oral squamous carcinomas. Juglone (J), derived from walnut, was reported to selectively inhibit PIN1 by modifying its sulfhydryl groups. In this study, the potential effects of juglone, also known as PIN1 inhibitor, on oral cancer and carcinogenesis were investigated at the molecular level. Materials and Methods: 4-Nitroquinoline N-oxide (4-NQO) was used to create an oral cancer model in animals. Wistar rats were divided into five groups: Control, NQO, Juglone, NQO+J, and NQO+J*. The control group received the basal diet and tap water throughout the experiment. The NQO group received 4-NQO for 8 weeks in drinking water only. The Juglone group was administered intraperitoneally in a juglone solution for 10 weeks (1 mg/kg/day). The NQO+J group received 4-NQO in drinking water for 8 weeks, starting 1 week after the cessation of 4-NQO treatment. They were then administered intraperitoneally in a juglone solution for 10 weeks. (1 mg/kg/day). NQO+J* group: received 4 NQO for 8 weeks in drinking water and administered intraperitoneally in a juglone solution for 10 weeks (1 mg/kg/day). They were sacrificed at the end of the 22-week experimental period. The tongue tissues of the rats were isolated after the experiment, morphological changes were investigated by histological examinations, and the molecular apoptotic process was investigated by rt-qPCR and western blot. Results: Histological results indicate that tumors are formed in the tongue tissue with 4-NQO, and juglone treatment largely corrects the epithelial changes that developed with 4-NQO. It has been determined that apoptotic factors p53, Bax, and caspases are induced by the effect of juglone, while antiapoptotic factors such as Bcl-2 are suppressed. However, it was observed that the positive effects were more pronounced in rats given juglone together with 4-NQO. Conclusions: The use of PIN1 inhibitors such as juglone in place of existing therapeutic approaches might be a promising and novel approach to the preservation and treatment of oral cancer and carcinogenesis. However, further research is required to investigate the practical application of such inhibitors.

An analysis on efficacy of applying ß-elemene intervention on chemically -induced tongue lesions using SAM algorithm.

An artificial intelligence (AI) model was designed to assist pathologists in diagnosing and quantifying structural changes in tongue lesions induced by chemical carcinogens. Using a tongue cancer model induced by 4-nitroquinoline-N-oxide and treated with ß-elemene, a total of 183 digital pathology slides were processed. The Segment Anything Model (SAM) was employed for initial segmentation, followed by conventional algorithms for more detailed segmentation. The epithelial contour area was computed using OpenCV's findcontour method, and the skeletonize method was used to calculate the distance map and skeletonized representation. The AI model demonstrated high accuracy in measuring tongue epithelial thickness and the number of papilla-like protrusions. Results indicated that the model group had significantly higher epithelial thickness and fewer papillae compared with the blank group. Furthermore, the treatment group exhibited reduced epithelial thickness and fewer papilla-like protrusions compared with the model group, though these differences were less pronounced. Overall, the SAM framework algorithm proved effective in quantifying tongue epithelial thickness and the number of papilla-like protrusions, thereby assisting healthcare professionals in understanding pathological changes and assessing treatment outcomes.

Global analysis of DNA methylation changes during experimented lingual carcinogenesis.

OBJECTIVES: This study aims to assess the role of DNA methylation changes in tongue cancer through a comprehensive analysis of global DNA methylation alterations during experimental lingual carcinogenesis. METHODS: C57BL/6J mice were subjected to 16-week oral administration of 4-nitroquinoline-1-oxide (4NQO, 50 mg/L). Lingual mucosa samples, being representative of normal tissue (week 0) and early (week 12) and advanced (week 28) tumorigenesis, were harvested for microarray and methylated DNA immunoprecipitation sequencing (MeDIP-Seq). The mRNA and promoter methylation of transforming growth factor-beta-signaling protein 1 (SMAD1) were evaluated with real-time quantitative reverse transcription polymerase chain reaction and Massarray in human lingual mucosa and tongue cancer cell lines. RESULTS: The cytosine guanine island (CGI) methylation level observed at 28 weeks surpassed that of both 12 weeks and 0 weeks. The promoter methylation level at 12 weeks exceeded that at 0 weeks. Notably, 208 differentially expressed genes were negatively correlated to differential methylation in promoters among 0, 12, and 28 weeks. The mRNA of SMAD1 was upregulated, concurrent with a decrease in promoter methylation levels in cell lines compared to normal mucosa. CONCLUSIONS: DNA methylation changed during lingual carcinogenesis. Overexpression of SMAD1 was correlated to promoter hypomethylation in tongue cancer cell lines.

Ethanol exposure exacerbates 4-nitroquinoline-1-oxide induced esophageal carcinogenesis and induces invasive carcinoma with muscularis propria infiltration in a mouse model.

Esophageal squamous cell carcinoma (ESCC) is one of the most fatal cancers worldwide. Most ESCC patients are diagnosed at an advanced stage; however, current research on in vivo animal models accurately reflecting their clinical presentation is lacking. Alcohol consumption is a major risk factor for ESCC and has been used in several disease models for disease induction. In this study, we used 4-nitroquinoline-1-oxide in combination with ethanol to induce an in vivo ESCC mouse model. Esophageal tissues were stained with hematoxylin and eosin for histopathological examination and lesion scoring. In cellular experiments, cell adhesion and migration invasion ability were observed using phalloidin staining, cell scratch and transwell assays, respectively, and the expression of epithelial-mesenchymal transition-related markers was detected using quantitative reverse transcription polymerase chain reaction and western blotting. The results showed that ethanol-exposed mice lost more weight and had an increased number of esophageal nodules. Histological examination revealed that the lesion scores of the ethanol-exposed esophageal samples were significantly higher than those of the unexposed esophageal samples. Furthermore, ethanol-exposed esophageal cancer samples had more severe lesions with infiltration of tumor cells into the muscularis propria. In vitro cellular experiments showed that ethanol exposure induced cytoskeletal microfilament formation, promoted cell migration invasion elevated the expression of N-cadherin and Snail, and decreased the expression of E-cadherin. In conclusion, ethanol exposure exacerbates ESCC, promotes tumor cell infiltration into the muscularis propria, and could be an effective agent for establishing innovative models of invasive carcinoma.

Autophagy Contributes to Homeostasis in Esophageal Epithelium Where High Autophagic Vesicle Level Marks Basal Cells With Limited Proliferation and Enhanced Self-Renewal Potential.

BACKGROUND & AIMS: Autophagy plays roles in esophageal pathologies both benign and malignant. Here, we aim to define the role of autophagy in esophageal epithelial homeostasis. METHODS: We generated tamoxifen-inducible, squamous epithelial-specific Atg7 (autophagy related 7) conditional knockout mice to evaluate effects on esophageal homeostasis and response to the carcinogen 4-nitroquinoline 1-oxide (4NQO) using histologic and biochemical analyses. We fluorescence-activated cell sorted esophageal basal cells based on fluorescence of the autophagic vesicle (AV)-identifying dye Cyto-ID and then subjected these cells to transmission electron microscopy, image flow cytometry, three-dimensional organoid assays, RNA sequencing, and cell cycle analysis. Three-dimensional organoids were subjected to passaging, single-cell RNA sequencing, cell cycle analysis, and immunostaining. RESULTS: Genetic autophagy inhibition in squamous epithelium resulted in increased proliferation of esophageal basal cells under homeostatic conditions and also was associated with significant weight loss in mice treated with 4NQO that further displayed perturbed epithelial tissue architecture. Esophageal basal cells with high AV level (Cyto-IDHigh) displayed limited organoid formation capability on initial plating but passaged more efficiently than their counterparts with low AV level (Cyto-IDLow). RNA sequencing suggested increased autophagy in Cyto-IDHigh esophageal basal cells along with decreased cell cycle progression, the latter of which was confirmed by cell cycle analysis. Single-cell RNA sequencing of three-dimensional organoids generated by Cyto-IDLow and Cyto-IDHigh cells identified expansion of 3 cell populations and enrichment of G2/M-associated genes in the Cyto-IDHigh group. Ki67 expression was also increased in organoids generated by Cyto-IDHigh cells, including in basal cells localized beyond the outermost cell layer. CONCLUSIONS: Autophagy contributes to maintenance of the esophageal proliferation-differentiation gradient. Esophageal basal cells with high AV level exhibit limited proliferation and generate three-dimensional organoids with enhanced self-renewal capacity.

4-nitroquinoline 1-oxide induces immune cells death to onset early immunosuppression during oral squamous cell carcinoma development.

4-Nitroquinoline N-oxide (4-NQO) and its derivatives react with genomic DNA to form stable quinolone monoadducts, which are highly mutagenic and genotoxic. While the chronic high-dose exposure of epithelial cells to a carcinogen such as 4-NQO leads to tumor development, its effect on other cells has not been explored yet. Since the immunosuppression due to aberrant immunological profile is recognized as a significant cause in tumors, here we determine the interaction between 4-NQO and immune cells both in vivo and in vitro, and its effect on oral squamous cell carcinoma (OSCC) progression in a murine model. Immune cell profiling of the spleen and peripheral blood revealed a significant decrease in the B-cell population in 4-NQO-exposed mice than the untreated group. Additionally, γδ T and CD5+ B lymphocyte populations decreased at both pre- and post-cancerous stages of OSCC. These results suggested that 4-NQO induced tumor transition from pre-malignant lesions to OSCC by altering certain immune cells systemically. Next, to establish the effect of 4-NQO on immune cells, human B- and T-cell lines were subjected to 4-NQO; the reduction in cell viability, increase in DNA damage response marker, and induction of apoptosis were more pronounced in B than T cells. Altogether, our results indicated that in addition to the genotoxicity of oral epithelial cells, 4-NQO potentiates long-range effects on specific immune cells to induce cell death to cause very-early immunosuppressive response during oral carcinogenesis, and thus immunosuppression and tumor development are coevolved.

Spermidine Suppresses Oral Carcinogenesis through Autophagy Induction, DNA Damage Repair, and Oxidative Stress Reduction.

Autophagy has been proposed to play a dual role in cancer-as a tumor suppressor in early stages and oncogenic in late stages of tumorigenesis. This study investigated the role of autophagy in oral carcinogenesis using the model of oral squamous cell carcinoma (OSCC) induced by carcinogen 4-nitroquinoline 1-oxide (4NQO), mimicking molecular and histopathologic aspects of human OSCC. The induction of autophagy by spermidine (SPD) treatment reduced the severity of lesions and the incidence of OSCC in mice exposed to 4NQO. On the other hand, autophagy inhibition by chloroquine treatment had no protection. The comet assay indicated that SPD reduced 4NQO-induced DNA damage, likely related to the activation of DNA repair and the decrease of reactive oxygen species. As sphingolipid alterations have been reported in OSCC, sphingolipids in the tongue and plasma of animals were analyzed and plasma C16 ceramide levels were shown to increase proportionally to lesion severity, indicating its potential as a biomarker. Mice exposed to 4NQO plus SPD had lower levels of C16 ceramide than the 4NQO group, which indicated SPD's ability to prevent the 4NQO-induced carcinogenesis. Together, these data indicate that activation of autophagy has a tumor suppressor role during the early stages of oral carcinogenesis. Because of its ability to induce autophagy accompanied by reduced oxidative stress and DNA damage, SPD may have a protective action against chemically induced oral cancer.

Proposal of a secure and efficient protocol for a murine oral carcinogenesis model induced by 4-nitroquinoline-1-oxide (4NQO).

An important rat model using the chemical carcinogen 4-nitroquinoline-1-oxide (4NQO) has been described for the study of the process of oral carcinogenesis. This model replicates the gradual progression seen in oral carcinoma patients. However, due to its high level of toxicity, its use in fundamental research is challenging. Here, we propose a secure and efficient modified protocol based on a lower dose of 4NQO concentration as well as an increased water supply and hypercaloric diet, in order to reduce the damage caused to the animals during the process of oral carcinogenesis. Twenty-two male Wistar rats were exposed to 4NQO, evaluated clinically once a week and euthanized at 12 and 20 weeks for histopathological analysis. The protocol involves a staggered dose of 4NQO up to a concentration of 25 ppm, associated with two days of pure water, a 5% glucose solution once a week and a hypercaloric diet. This modified protocol prevents the immediate consequences of the carcinogen. At week 7, all animals displayed clinically evident tongue lesions. From a histological perspective, after 12 weeks of 4NQO exposure, 72.7% of the animals developed epithelial dysplasia and 27.3% developed in situ carcinoma. In the group exposed for 20 weeks, epithelial dysplasia and in situ carcinoma were diagnosed in one case each, whereas invasive carcinoma was diagnosed in 81.8% of the cases. Nonsignificant modification of animal's behavior and weight was observed. This new proposed 4NQO protocol was secure and effective for studying oral carcinogenesis and can be used to conduct lengthy investigations.

A method for scoring 4-nitroquinoline 1-oxide-induced murine esophageal squamous neoplasia.

A mouse model for esophageal squamous cell carcinoma (ESCC) is induced by oral administration of the carcinogen 4-nitroquinoline 1-oxide (4-NQO). There is not an objective method for determining histopathologic severity of disease in this model. We aim to create a clearly defined and easily applied scoring system that can quantify the severity of 4-NQO-induced murine ESCC. Fifteen wild-type C57BL/6J mice were treated with 4-NQO for 8 (n = 8) or 16 (n = 7) weeks, while the rest (n = 9) were treated with vehicle, as 8 weeks of 4-NQO typically results in dysplasia and 16 weeks in carcinoma. We identified histologic abnormalities of the esophagus in this model and developed metrics to grade severity of dysplasia, papillomas, and invasion. Scores were then calculated using quantitative digitized image analysis for measuring depth and extent of each feature within the entire sample. Each feature was also assigned a weight based on its relation to cancer severity. Histology scores were significantly different in the three groups, suggesting that this method can discriminate dysplasia from carcinoma. This model can be applied to any mouse treated with 4-NQO.

GPR68 limits the severity of chemical-induced oral epithelial dysplasia.

Head and neck cancer is the sixth most common malignancy, and there is an urgent need to identify physiological processes contributing to tumorigenesis. Extracellular acidification caused by aerobic glycolysis within tumor microenvironments can stimulate proton-sensing receptors. GPR68, or ovarian cancer G protein-coupled receptor 1, responds to extracellular acidity and is highly expressed in head and neck squamous cell carcinoma (HNSCC) as well as normal esophageal tissue. To study the role of GPR68 in oral dysplasia, wild-type and GPR68-/- mice were treated with 4-Nitroquinoline N-oxide (4NQO) in drinking water for 11-13 weeks, followed by normal water for 11-12 weeks. 4NQO treatment resulted in 45 percent of GPR68-/- mice developing severe dysplasia or squamous cell carcinoma compared to only 10.5 percent of GPR68+/+ mice. This correlated with increased frequencies of regulatory T cells in the spleens of male GPR68-/- mice. Dysplastic regions of the tongue had increased CD31 staining compared to normal regions in both GPR68-/- and GPR68+/+ mice, suggesting that angiogenesis was GPR68-independent. RNA knockdown studies using HNSCC cell lines demonstrated no direct effect of GPR68 on survival or growth. Overall, we demonstrate that GPR68-deficiency worsens the severity of chemical-induced oral dysplasia, suggesting a protective role for this gene in tumorigenesis.

Laser photobiomodulation does not alter clinical and histological characteristics of 4-NQO-induced oral carcinomas and leukoplakia in mice.

OBJECTIVE: This study evaluated the effect of laser photobiomodulation (PBM) on oral leukoplakia and squamous cell carcinomas (OSCC) in a model of oral carcinogenesis. MATERIALS AND METHODS: Forty-one C57Bl/6 female mice were distributed in control group, 4-NQO group, Laser group 1.5 J and Laser group 9 J. Oral cancer was induced on the tongue by nitroquinoline oxide (4-NQO), diluted in the water for 16 weeks. In the 18th and 19th weeks, PBM with a diode laser, 0.028 cm2 spot size, continuous emission mode, 660 nm wavelength was applied on the tongue of animals for seven sessions. Laser group 1.5 J received 30 mW power and 1.5 J energy. In the Laser group 9 J, 100 mW power, and 9 J energy were applied. In the 20th week the animals were euthanized. RESULTS: All animals exposed to carcinogen developed clinical and histological alterations such as leukoplakia and OSCC on the tongue. There was no significant difference among Laser groups 1.5 and 9 J and 4-NQO group (not irradiated) regarding the area of leukoplakia and carcinomas (P > 0.05) or thickness of epithelial tissue and keratin (P > 0.05). There were also no association between PBM and histologic classification of the lesions (P = 0.87), frequency of OSCC (P = 0.57), grade of tumor differentiation (P = 0.88) or depth of invasion (P = 0.45). CONCLUSION: Laser PBM, in both parameters used, does not influence on clinical and histological characteristics of oral leukoplakia and OSCC. CLINICAL RELEVANCE: Results suggest that PBM may be a safe treatment for adverse effects of antineoplastic therapies in patients with leukoplakia and OSCC.

Sulfolobus islandicus Employs Orc1-2-Mediated DNA Damage Response in Defense against Infection by SSV2.

All living organisms have evolved DNA damage response (DDR) strategies in coping with threats to the integrity of their genome. In response to DNA damage, Sulfolobus islandicus activates its DDR network in which Orc1-2, an ortholog of the archaeal Orc1/Cdc6 superfamily proteins, plays a central regulatory role. Here, we show that pretreatment with UV irradiation reduced virus genome replication in S. islandicus infected with the fusellovirus SSV2. Like treatment with UV or the DNA-damaging agent 4-nitroquinoline-1-oxide (NQO), infection with SSV2 facilitated the expression of orc1-2 and significantly raised the cellular level of Orc1-2. The inhibitory effect of UV irradiation on the virus DNA level was no longer apparent in the infected culture of an S. islandicus orc1-2 deletion mutant strain. On the other hand, the overexpression of orc1-2 decreased virus genomic DNA by ~102-fold compared to that in the parent strain. Furthermore, as part of the Orc1-2-mediated DDR response genes for homologous recombination repair (HRR), cell aggregation and intercellular DNA transfer were upregulated, whereas genes for cell division were downregulated. However, the HRR pathway remained functional in host inhibition of SSV2 genome replication in the absence of UpsA, a subunit of pili essential for intercellular DNA transfer. In agreement with this finding, lack of the general transcriptional activator TFB3, which controls the expression of the ups genes, only moderately affected SSV2 genome replication. Our results demonstrate that infection of S. islandicus by SSV2 triggers the host DDR pathway that, in return, suppresses virus genome replication. IMPORTANCE Extremophiles thrive in harsh habitats and thus often face a daunting challenge to the integrity of their genome. How these organisms respond to virus infection when their genome is damaged remains unclear. We found that the thermophilic archaeon Sulfolobus islandicus became more inhibitory to genome replication of the virus SSV2 after preinfection UV irradiation than without the pretreatment. On the other hand, like treatment with UV or other DNA-damaging agents, infection of S. islandicus by SSV2 triggers the activation of Orc1-2-mediated DNA damage response, including the activation of homologous recombination repair, cell aggregation and DNA import, and the repression of cell division. The inhibitory effect of pretreatment with UV irradiation on SSV2 genome replication was no longer observed in an S. islandicus mutant lacking Orc1-2. Our results suggest that DNA damage response is employed by S. islandicus as a strategy to defend against virus infection.

Chemoprevention of 4NQO-Induced Mouse Tongue Carcinogenesis by AKT Inhibitor through the MMP-9/RhoC Signaling Pathway and Autophagy.

Oral cancer (OC), the most common cancer in the head and neck, which has a poor prognosis, histopathologically follows a stepwise pattern of hyperplasia, dysplasia, and cancer. Blocking the progression of OC in the precancer stage could greatly improve the survival and cure rates. AKT protein plays a critical role in the signal transduction of cancer cells, and we found that AKT was overexpressed in human OC samples through analysis of TCGA database. Therefore, this study is aimed at investigating the chemopreventive effect of an AKT inhibitor (MK2206 2HCl) on OC. In vivo, we established a 4-nitroquinoline-1-oxide- (4NQO-) induced mouse tongue carcinogenesis model to investigate the potential chemopreventive effect of MK2206 2HCl on mouse OC resulting from 4NQO. The results showed that MK2206 2HCl could significantly reduce the incidence rate and growth of OC, inhibit the transformation of dysplasia to cancer in the 4NQO-induced mouse tongue carcinogenesis model, and simultaneously markedly suppress cell proliferation, angiogenesis, and mast cell (MC) infiltration in 4NQO-induced mouse tongue cancers. In vitro, our results revealed that MK2206 2HCl could also inhibit oral squamous cell carcinoma (OSCC) cell malignant biological behaviors, including cell proliferation, colony formation, cell invasion, and migration, while promoting apoptosis. Mechanistic studies revealed that MK2206 2HCl suppressed matrix metalloproteinase 9 (MMP-9) and RhoC expression and promoted autophagy gene LC3 II expression. In summary, our findings demonstrated the chemopreventive effect of MK2206 2HCl on the 4NQO-induced mouse tongue carcinogenesis model, which likely has an underlying mechanism mediated by the MMP-9/RhoC signaling pathway and autophagy.

Negative terpinen-4-ol modulate potentially malignant and malignant lingual lesions induced by 4-nitroquinoline-1-oxide in rat model.

Our aim was to verify the modulative TP-4-ol capacity in 4-nitroquinoline-1-oxide induced oral rat cancer. The stereoisomers of TP-4-ol were used against the human tongue squamous cell line and the negative stereoisomer showed lower IC50. Thirty-one Holtzman rats (120-130 g) were cancer-induced by 4-nitroquinoline-1-oxide (4-NQO/8 weeks/25 ppm) and 32 Holtzman rats (120-130 g) were used to healthy and TP-4-ol toxicity experiments. Six groups were used, healthy, 0.1nL/g of TP-4-ol, 8nL/g of TP-4-ol, 4-NQO, 4-NQO + 0.1nL/g of TP-4-ol, and 4-NQO + 8nL/g of TP-4-ol. We performed the toxicity analysis by biochemical and histopathological analysis. The biochemistry analysis includes alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate transaminase (AST), urea, and creatinine and the histopathology analysis includes the liver, kidney, lung, and spleen. Specifically, for malign modulation, we performed a macroscopic and microscopic analysis. The group exposed to 0.1nL/g of TP-4-ol demonstrated a reduced risk of malignancy in dysplasia considering the criteria of architecture and cytology. Similarly, a drop of percentual rats with SCC diagnosis was observed in 4-NQO + 0.1nL/g (41.6%) when compared to 4-NQO (87.5%). Moreover, the 4-NQO group presented a median of 2.62 SCC/rat and the 4-NQO + 0.1nL/g demonstrated a median of 0.75 SCC/rat. For toxicity analysis, 4-NQO + 0.1nL/g showed focal necrosis in the kidney and 4-NQO showed lung hemorrhagic areas. The concentration of 0.1nL/g was more effective in reducing the tongue induction of potentially malignant and malignant lesions by 4-NQO. A kidney toxicity was observed in healthy animals exposed to 0.1nL/g of TP-4-ol. The negative isoform of terpinen-4-ol negatively modulates the development of potentially malignant and malignant lesions in rats (Rattus nonverdicts albinos, Holtzman) exposed to 4-NQO. (-)-Terpinen-4-ol reduced the mice percentual with squamous cell carcinoma, 87.5 to 41.6%, and decreased the cancer/rat ratio of 2.62 in 4-NQO to 0.75 in 4-NQO + 0.1nL/g. This represents 52.4% by group and 71.3% in the cancer/rat ratio.

Genotoxicity of 12 Mycotoxins by the SOS/umu Test: Comparison of Liver and Kidney S9 Fraction.

Liver S9 fraction is usually employed in mutagenicity/genotoxicity in vitro assays, but some genotoxic compounds may need another type of bioactivation. In the present work, an alternative S9 fraction from the kidneys was used for the genotoxicity assessment of 12 mycotoxins with the SOS/umu test. The results were compared with liver S9 fraction, and 2-4 independent experiments were performed with each mycotoxin. The expected results were obtained with positive controls (4-nitroquinoline-N-oxide and 2-aminoanthracene) without metabolic activation or with liver S9, but a potent dose-dependent effect with 4-nitroquinoline-N-oxide and no activity of 2-aminoanthracene with kidney S9 were noticed. Aflatoxin B1 was genotoxic with metabolic activation, the effect being greater with liver S9. Sterigmatocystin was clearly genotoxic with liver S9 but equivocal with kidney S9. Ochratoxin A, zearalenone and fumonisin B1 were negative in all conditions. Trichothecenes were negative, except for nivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, T-2 and HT-2 toxins, which showed equivocal results with kidney S9 because a clear dose-response effect was not observed. Most of the mycotoxins have been assessed with kidney S9 and the SOS/umu test for the first time here. The results with the positive controls and the mycotoxins confirm that the organ used for the S9 fraction preparation has an influence on the genotoxic activity of some compounds.

4-nitroquinoline-1-oxide (4NQO) induced oral carcinogenesis: A systematic literature review.

OBJECTIVE: Based on a critical review of published studies, we aimed to develop a good practice guide for using 4-nitroquinoline-1-oxide (4NQO) as an inducer of oral carcinogenesis in Wistar rats. DESIGN: A systematic search was performed on Medline Ovid, PubMed, Embase, Web of Science, and Scopus databases. The SYRCLE's risk of bias tool was used to assess the quality of the studies. RESULTS: Thirty-five articles met the selection criteria; 22 (62.9%) of them administered 4NQO systemically in drinking water, with a mean concentration of 30.2 ppm (SD±15.9) and during a mean period of 20.8 (SD±7.8) weeks. The other 13 (37.1%) studies performed topical applications of 4NQO painting the oral mucosa of the animals three times a week (100%) with a mean period of administration of 16.8 (SD±7.0) weeks. Different 4NQO concentrations used for other periods achieved significant tumor development. Most studies didn't perform quantitative clinical analysis, and the histopathological diagnosis/grading criteria varied considerably. CONCLUSIONS: A poor description of solution care, adverse effects, and the number of losses were observed, and the reporting of these features needs to be improved. Suggestions to guide the development of future research are provided.