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.

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.

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.

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.

Periodontal disease affects oral cancer progression in a surrogate animal model for tobacco exposure.

For decades, the link between poor oral hygiene and the increased prevalence of oral cancer has been suggested. Most recently, emerging evidence has suggested that chronic inflammatory diseases from the oral cavity (e.g., periodontal disease), to some extent, play a role in the development of oral squamous cell carcinoma (OSCC). The present study aimed to explore the direct impact of biofilm­induced periodontitis in the carcinogenesis process using a tobacco surrogate animal model for oral cancer. A total of 42 Wistar rats were distributed into four experimental groups: Control group, periodontitis (Perio) group, 4­nitroquinoline 1­oxide (4­NQO) group and 4NQO/Perio group. Periodontitis was stimulated by placing a ligature subgingivally, while oral carcinogenesis was induced by systemic administration of 4NQO in the drinking water for 20 weeks. It was observed that the Perio, 4NQO and 4NQO/Perio groups presented with significantly higher alveolar bone loss compared with that in the control group. Furthermore, all groups receiving 4NQO developed lesions on the dorsal surface of the tongue; however, the 4NQO/Perio group presented larger lesions compared with the 4NQO group. There was also a modest overall increase in the number of epithelial dysplasia and OSCC lesions in the 4NQO/Perio group. Notably, abnormal focal activation of cellular differentiation (cytokeratin 10­positive cells) that extended near the basal cell layer of the mucosa was observed in rats receiving 4NQO alone, but was absent in rats receiving 4NQO and presenting with periodontal disease. Altogether, the presence of periodontitis combined with 4NQO administration augmented tumor size in the current rat model and tampered with the protective mechanisms of the cellular differentiation of epithelial cells.

Fat and exposure to 4-nitroquinoline-1-oxide causes histologic and inflammatory changes in murine livers.

Risk factors for liver cancer include tobacco use, alcohol consumption, obesity, and male sex. Administration of 4-nitroquinonline-1-oxide (4NQO) in drinking water mimics the effects of tobacco and leads to oral carcinoma in mice. This study compared the effects of diets high and low in saturated fat (HF and LF, respectively), and sex, on liver histopathology in 4NQO-treated mice and controls. We hypothesized that 4NQO would cause histopathological changes in liver, and that a HF diet would increase hepatic pathology when compared to the LF diet. Mice (C57Bl/6, 36/sex), were divided into a low fat (10 kcal% fat; LF) or high fat (60 kcal% fat, HF) diet. Mice were further subdivided into one of 3 water treatment groups for 17 weeks: water (control), vehicle (1.25% propylene glycol in water [PG]), or 4NQO in (50 µg/ml; 4NQO). All mice were subsequently given water alone for 6 more weeks. Upon euthanasia, livers were harvested, fixed, sectioned, and stained with hematoxylin and eosin (H&E). H&E slides were graded for histopathology; frozen liver samples were analyzed for triglyceride content. Trichrome stained sections were graded for fibrosis. CD3+ T cells, CD68+ macrophages, and Ly6+ neutrophils were detected by immunohistochemistry. Compared to water controls, 4NQO-treatment caused mouse liver histopathological changes such as fibrosis, and increases in hepatic neutrophils, T cells, and macrophages. HF diet exacerbated pathological changes compared to LF diet. Male controls, but not females, demonstrated severe steatosis and increased triglyceride content. 4NQO treatment decreased hepatic fat accumulation, even in animals on a HF diet. In conclusion, this murine model of oral cancer may serve as a model to study the effects of tobacco and diet on liver.

Early antitumor activity of oral Langerhans cells is compromised by a carcinogen.

Early diagnosis of oral squamous cell carcinoma (OSCC) remains an unmet clinical need. Therefore, elucidating the initial events of OSCC preceding tumor development could benefit OSCC prognosis. Here, we define the Langerhans cells (LCs) of the tongue and demonstrate that LCs protect the epithelium from carcinogen-induced OSCC by rapidly priming αßT cells capable of eliminating γH2AX+ epithelial cells, whereas γδT and natural killer cells are dispensable. The carcinogen, however, dysregulates the epithelial resident mononuclear phagocytes, reducing LC frequencies, while dendritic cells (DCs), macrophages, and plasmacytoid DCs (pDCs) populate the epithelium. Single-cell RNA-sequencing analysis indicates that these newly differentiated cells display an immunosuppressive phenotype accompanied by an expansion of T regulatory (Treg) cells. Accumulation of the Treg cells was regulated, in part, by pDCs and precedes the formation of visible tumors. This suggests LCs play an early protective role during OSCC, yet the capacity of the carcinogen to dysregulate the differentiation of mononuclear phagocytes facilitates oral carcinogenesis.

Assessment of genotoxic chemicals using chemogenomic profiling based on gene-knockout library in Saccharomyces cerevisiae.

Understanding the adverse effects of genotoxic chemicals and identifying them effectively from non-genotoxic chemicals are of great worldwide concerns. Here, Saccharomyces cerevisiae (yeast) genome-wide single-gene knockout screening approach was conducted to assess two genotoxic chemicals (4-nitroquinoline-1-oxide (4-NQO) and formaldehyde (FA)) and environmental pollutant dichloroacetic acid (DCA, genotoxicity is controversial). DNA repair was significant enriched in the gene ontology (GO) biology process (BP) terms and KEGG pathways when exposed to low concentrations of 4-NQO and FA. Higher concentrations of 4-NQO and FA influenced some RNA metabolic and biosynthesis pathways. Moreover, replication and repair associated pathways were top ranked KEGG pathways with high fold-change for low concentrations of 4-NQO and FA. The similar gene profiles perturbed by DCA with three test concentrations identified, the common GO BP terms associated with aromatic amino acid family biosynthetic process and ubiquitin-dependent protein catabolic process via the multivesicular body sorting pathway. DCA has no obvious genotoxicity as there was no enriched DNA damage and repair pathways and fold-change of replication and repair KEGG pathways were very low. Five genes (RAD18, RAD59, MUS81, MMS4, and BEM4) could serve as candidate genes for genotoxic chemicals. Overall, the yeast functional genomic profiling showed great performance for assessing the signatures and potential molecular mechanisms of genotoxic chemicals.

BRCA1/Trp53 heterozygosity and replication stress drive esophageal cancer development in a mouse model.

BRCA1 germline mutations are associated with an increased risk of breast and ovarian cancer. Recent findings of others suggest that BRCA1 mutation carriers also bear an increased risk of esophageal and gastric cancer. Here, we employ a Brca1/Trp53 mouse model to show that unresolved replication stress (RS) in BRCA1 heterozygous cells drives esophageal tumorigenesis in a model of the human equivalent. This model employs 4-nitroquinoline-1-oxide (4NQO) as an RS-inducing agent. Upon drinking 4NQO-containing water, Brca1 heterozygous mice formed squamous cell carcinomas of the distal esophagus and forestomach at a much higher frequency and speed (∼90 to 120 d) than did wild-type (WT) mice, which remained largely tumor free. Their esophageal tissue, but not that of WT control mice, revealed evidence of overt RS as reflected by intracellular CHK1 phosphorylation and 53BP1 staining. These Brca1 mutant tumors also revealed higher genome mutation rates than those of control animals; the mutational signature SBS4, which is associated with tobacco-induced tumorigenesis; and a loss of Brca1 heterozygosity (LOH). This uniquely accelerated Brca1 tumor model is also relevant to human esophageal squamous cell carcinoma, an often lethal tumor.

Application of HepG2/C3A liver spheroids as a model system for genotoxicity studies.

HepG2 cells continue to be a valuable tool in early drug discovery and pharmaceutical development. In the current study we develop a 3D in vitro liver model, using HepG2/C3A cells that is predictive of human genotoxic exposure. HepG2/C3A cells cultured for 7-days in agarose-coated microplates formed spheroids which were uniform in shape and had well defined outer perimeters and no evidence of a hypoxic core. Quantitative real-time-PCR analysis showed statistically significant transcriptional upregulation of xenobiotic metabolising genes (CYP1A1, CYP1A2, UG1A1, UGT1A3, UGT1A6, EPHX, NAT2) and genes linked to liver function (ALB, CAR) in 3D cultures. In response to three model pro-genotoxicants: benzo[a]pyrene, amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-aminoanthracene (2-AA), we observed further transcriptional upregulation of xenobiotic metabolising genes (CYP1A1, CYP1A2, NAT1/2, SULT1A2, UGT1A1, UGT1A3) compared to untreated spheroids. Consistent with this, spheroids were more sensitive than 2D monolayers to compound induced single- and double- stranded DNA-damage as assessed by the comet assay and γH2AX phosphorylation respectively. In contrast, levels of DNA-damage induced by the direct acting mutagen 4-nitroquinoline N-oxide (4NQO) was the same in spheroids and monolayers. In support of the enhanced genotoxic response in spheroids we also observed transcriptional upregulation of genes relating to DNA-damage and cellular stress response (e.g. GADD45A and CDKN1A) in spheroids. In conclusion, HepG2/C3A 3D spheroids are a sensitive model for in vitro genotoxicity assessment with potential applications in early stage drug development.

The 4-NQO mouse model: An update on a well-established in vivo model of oral carcinogenesis.

The early detection and management of oral premalignant lesions (OPMDs) improve their outcomes. Animal models that mimic histological and biological processes of human oral carcinogenesis may help to improve the identification of OPMD at-risk of progression into oral squamous cell carcinoma and to develop preventive strategies for the entire field of cancerization. No animal model is perfectly applicable for investigating human oral carcinogenesis. However, the 4-nitroquinoline 1-oxide (4-NQO) mouse model is well established and mimics several morphological, histological, genomic and molecular features of human oral carcinogenesis. Some of the reasons for the success of this model include its reproducible experimental conditions with limited variation, the possibility of realizing longitudinal studies with invasive intervention or gene manipulation, and sample availability for all stages of oral carcinogenesis, especially premalignant lesions. Moreover, the role of histological and molecular alterations in the field of cancerization (i.e., macroscopically healthy mucosa exposed to a carcinogen) during oral carcinogenesis can be easily explored using this model. In this review, we discuss the advantages and drawbacks of this model for studying human oral carcinogenesis. In summary, the 4-NQO-induced murine oral cancer model is relevant for investigating human oral carcinogenesis, including the immune microenvironment, and for evaluating therapeutic and chemoprevention agents.

4NQO induced carcinogenesis: A mouse model for oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is the most common subsite of head and neck cancer, with a 5-year survival rate of only 50%. There is a pressing need for animal models that recapitulate the human disease to understand the factors driving OSCC carcinogenesis. Many laboratories have used the chemical carcinogen 4-nitroquinoline-1-oxide (4NQO) to investigate OSCC formation. The importance of the 4NQO mouse model is that it mimics the stepwise progression observed in OSCC patients. The 4NQO carcinogen model has the advantage that it can be used with transgenic mice with genetic modification in specific tissue types to investigate their role in driving cancer progression. Herein, we describe the basic approach for administering 4NQO to mice to induce OSCC and methods for assessing the tissue and disease progression.