OBJECTIVEThis study aimed to induce carcinogenesis of lingual mucosa in C57BL/6 mice by feeding them 4-nitroquinoline 1-oxide (4NQO) solution.

METHODSA total of 85 C57BL/6 mice were randomly divided into distilled water control group (DD group, n=5), 1,2-propylene glycol control group (PG group, n=5), and experimental group (EP group, n= 75). The mice in the experimental group were medially fed in 15 cages. By contrast, the mice in DD, EP, and PG groups were watered with distilled water, 50 mg.L-1 4NQO solution, and 1,2-propylene glycol solution. The mice in EP group were executed every two weeks from week 0, and the mice in the control groups were sacrificed at the 28th week. The mice were weighed. Mucosal lesions were measured by macroscopic observation and histopathologic detection.

RESULTSOne mouse in EP group died of unknown reason. The weight of the mice in EP group presented weight loss compared with the mice in DD and PG groups after the 24th week. Seventy-nine macroscopic lesions were observed in the lingual mucosa, oral floor, and upper palatal and buccal mucosa. A total of 70 macroscopic lesions (88.6%) were located in the lingual mucosa. Mucosal lesions changed from simple hyperplasia to squamous cell carcinomas. Well-differentiated squamous cell carcinomas were observed in all mice of EP group by pathological section at the 28th week. No lesion was found in the mice of DD and PG groups.

CONCLUSIONThe animal model of lingual squamous cell carcinomas was successfully established. The periods from 12th to 16th week and 20th to 28th week were the ideal times for the research on pathogenesis of early and medial-advanced stage during carcinogenesis of squamous cell carcinomas.

4-Nitroquinoline-1-oxide ; Animals ; Cell Transformation, Neoplastic ; Disease Models, Animal ; Mice ; Mice, Inbred C57BL ; Mouth Mucosa ; Tongue

OBJECTIVETo study the expression of connexin 43 (Cx43) in various stages of oral carcinogenesis and explore the relation between Cx43 and oral mucous carcinogenesis.

METHODS4-nitroquinoline-1-oxide (4NQO) was used for inducing oral carcinogenesis in SD rats. Tissue samples were obtained from various stages of the disease including normal oral mucosa, precancerous lesions and oral squamous cell carcinoma. Immunohistochemical method was used to determine the expression of Cx43 in various stages of oral carcinogenesis.

RESULTSIn the normal rat lingual mucosa, immunohistochemical staining of Cx43 protein was mainly found in the cell membrane, weakly positive in the basal cell layer, increased in stratum spinosum and stratum granulosum, but was negative in the stratum corneum of normal epithelia. Compared with normal epithelia, was significantly decreased in dysplastic and cancerous oral epithelia the staining. The positive rates of Cx43 were respectively 100.00% (10/10), 85.71% (12/14), 66.67% (8/12), 40.00% (4/10), and 33.33% (4/12) in tongue carcinogenesis (in normal, mild, moderate and severe dysplasia, and squamous cell carcinoma tissues). The differences were statistically significant (P<0.05).

CONCLUSIONExpression level of Cx43 protein was dramatically decreased with the development of rat tongue carcinoma induced by 4NQO, suggesting that abnormal expression of Cx43 protein is involved in oral mucosa carcinogenesis. Decreased Cx43 expression is an early sign of oral mucosa carcinogenesis.

4-Nitroquinoline-1-oxide ; toxicity ; Animals ; Carcinoma, Squamous Cell ; chemically induced ; chemistry ; etiology ; Connexin 43 ; analysis ; genetics ; physiology ; Male ; Rats ; Rats, Sprague-Dawley ; Tongue Neoplasms ; chemically induced ; chemistry ; etiology

OBJECTIVETo evaluate the expression of cytokeratin 19(CK19) and connexin 43(Cx43) in various stages of oral carcinogenesis and investigate the relationship of CK19 and Cx43 in the process of oral cancer.

METHODS4-nitroquinoline-1-oxide(4NQO) was used to induce oral carcinogenesis in the mucosa of SD rats and immunohistoche-mical technique was used to study the expression of CK19 and Cx43 in various stages of oral carcinogenesis.

RESULTSThe CK19 positive staining were distributed in the basal cell layer in the normal rat lingual mucosa. While CK19 positive staining were distributed in cytoplasm of supra-basal layers in the mild dysplasia, moderate dysplasia and severe dysplasia. In oral squamous cell carcinoma(OSCC) tissue, CK19 were expressed in all the stratum of epithelium. The positive rate of CK19 in normal, mild, moderate, severe dysplasia and OSCC tissues were respectively 30.00%, 50.00%, 58.33%, 80.00%, and 91.67%. With the lesions getting worse, the positive rate and the intensity of CK19 raised significantly (P<0.05). In normal tongue mucosa, Cx43 proteins were mainly expressed in the membrane of the epithelial cells of the rat tongue. It was weakly positive in the basal cell layer, increased in the stratum spinosum and stratum granulosum, and negative in the stratum corneum. Compared with normal epithelia, the expression of Cx43 in dysplastic and OSCC epithelia decreased significantly. The positive rate of Cx43 in normal, mild, moderate, severe dysplasia and OSCC tissues were respectively 100.00%, 85.71%, 66.67%, 40.00%, and 33.33%. The expression of Cx43 was significantly decreased with severity increasing (P<0.05).

CONCLUSIONThe expression of CK19 protein significantly increases with the development of rat tongue carcinoma, suggesting that CK19 is associated with carcinogenesis. The expression of Cx43 protein dramatically decrease with the development of rat tongue carcinoma, suggesting that the abnormal expression of Cx43 protein is associated with oral mucosa carcinoma origination. The expression of CK19 and Cx43 has negative correlation. Combined detection of CK19 and Cx43 has an important role in the early diagnosis of OSCC and can help to improve the sensitivity and specificity of the early diagnosis of OSCC.

4-Nitroquinoline-1-oxide ; Animals ; Carcinogenesis ; Carcinoma, Squamous Cell ; Connexin 43 ; Epithelial Cells ; Epithelium ; Keratin-19 ; Keratins ; Male ; Mouth Mucosa ; Mouth Neoplasms ; Oxides ; Rats ; Rats, Sprague-Dawley ; Tongue ; Tongue Neoplasms

OBJECTIVETo establish a monoclone cell line of squamous cell carcinoma (SCC) in rat buccal mucosa and to study its biological characteristics.

METHODSSCC in rat oral mucosa was induced by adding 4-nitroquinoline-1-oxide (4NQO) into the SD rats' drinking water, and the cancer cells were then cultured to obtain mixed cells in vitro. The mixed tumor cells were purified by mono cell cloning method. The biological characteristics of the cells were studied by microscope and electronic microscope observation, chromosome analysis, Methyl thiazolyl tetrazolium (MTT) test, flow cytometry assay and immunohistochemistry staining. Hypodermic inoculations of the cells in nude mice and injection of the cells by nude mice tail veins were performed to observe the tumor formation and long distance metastasis.

RESULTSThe morphology proved that the cell line was squamous cell carcinoma cells, which were cultured from one cell. The population doubling time for passage 65 cells was 25.44 hours. The cells in S-phase accounted for 20.13% of the cell cycle. The chromosome modal number was 84. All the cells expressed the proteins of cytokeratin and vimentin. The xenograft rate and the tumor metastatic rate to the lung were 100% in nu/nu BALB/C mice, but the homograft rate was zero in SD Rats.

CONCLUSIONSRca-B was a typical oral squamous cell carcinoma cell line derived from Sprague-Dawley rat buccal mucosa carcinoma, and the cell line has high metastatic potential and its biological characteristics were well ascertained.

4-Nitroquinoline-1-oxide ; toxicity ; Animals ; Carcinoma, Squamous Cell ; pathology ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Clone Cells ; pathology ; Female ; Mice ; Mice, Nude ; Mouth Mucosa ; pathology ; Mouth Neoplasms ; pathology ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo explore the myeloid-derived suppressor cell (MDSC) expression in the peripheral blood and lesions of 4NQO-induced tongue carcinoma in mouse.

METHODSThe established 4NQO mouse model was used to analyze the distribution of MDSC and T cell subsets in the peripheral blood by flow cytometry. The relations of MDSC with T cell subsets and CD4⁺/CD8⁺ changes were evaluated. The distribution of MDSC in the lesions of tongues was analyzed by immu- nohistochemistry, and the expression of arginase 1 (ARG-1) in tongue tissues was detected by real-time polymerase chain reaction.

RESULTSDuring tumor progression, a significant increase was observed in the frequency of MDSC in the peripheral blood of 4NQO treated mice (P < 0.01). The frequency of MDSC was positively correlated with systemic CD3⁺CD8+T cells but negatively correlated with the CD4⁺/CD8⁺ ratio. Squamous cell carcinomas were extensively infiltrated with MDSC, whereas dysplastic area and normal tongue mucosa had only sparse MDSC infiltration. The majority of MDSCs were located in the stroma, particularly along the tumor invasive front. Moreover, 4NQO-treated mice showed significantly higher ARG-1 mRNA levels in the tumor site (P<0.01).

CONCLUSIONMDSC may contribute to oral tumor progression and represents a potential target for immunotherapy of oral cancer.

4-Nitroquinoline-1-oxide ; Animals ; Arginase ; Cell Count ; Flow Cytometry ; Mice ; Models, Animal ; Myeloid-Derived Suppressor Cells ; immunology ; Real-Time Polymerase Chain Reaction ; T-Lymphocyte Subsets ; immunology ; Tongue Neoplasms ; immunology

OBJECTIVETo observe the influence of 1.8 GHz microwave (MW) specific absorption rate (SAR, 3 W/kg) on human lymphocytes DNA damage induced by 4 chemical mutagens [mitomycin C (MMC), bleomycin (BLM), methyl methanesulfonate (MMS), and 4-nitroquinoline 1-oxide (4NQO)].

METHODSComet assay in vitro was used to detect human lymphocyte DNA damage induced by 1.8 GHz MW, 4 chemical mutagens, and MW plus 4 chemicals 0 h and 21 h respectively after exposure. The time exposed to MW or mutagens was 2 h or 3 h respectively. The results were showed by tail length (TL) and tail moment (TM).

RESULTSThe difference of DNA damage between MW group and control group was not statistically significant (P > 0.05). DNA damages in MW plus MMC groups and MW plus 4NQO groups were significantly greater than those in the corresponding concentrations of MMC groups and 4NQO groups (P < 0.01 or P < 0.05). However, MW did not enhance DNA damage induced by MMS and BLM (P > 0.05).

CONCLUSIONExposure to 1.8 GHz (SAR, 3 W/kg) microwave may not induce human lymphocyte DNA damage, but could enhance DNA damage induced by MMC and 4NQO.

4-Nitroquinoline-1-oxide ; toxicity ; Adult ; Bleomycin ; toxicity ; Cells, Cultured ; Comet Assay ; DNA ; drug effects ; DNA Damage ; Humans ; Lymphocytes ; drug effects ; radiation effects ; Male ; Methyl Methanesulfonate ; toxicity ; Microwaves ; adverse effects ; Mitomycin ; toxicity ; Mutagens ; toxicity

OBJECTIVETo investigate the expression of survivin during oral carcinogenesis and its relationship with the expression of Bcl-2 and p53.

METHODSTwo-step immunohistochemical method was employed to detect the expression of survivin, Bcl-2 and p53 in 60 rat tongue carcinogenesis specimens induced by 4-nitro-quinoline 1-oxide (4NQO).

RESULTSSurvivin was present in 1/36 cases of normal mucosa, 6/11 cases of oral epithelial dysplasia and 10/11 cases of oral carcinoma. There were significant difference in the expression of survivin among normal mucosa, oral epithelial dysplasia and carcinoma (P < 0.001). While in the 17 cases of positive expression of survivin, Bcl-2 was present in 12 cases and p53 was present in 8 cases. The expression of survivin in oral epithelial dysplasia and carcinoma were significantly higher than that in normal oral mucosa (P < 0.01). The expression of survivin was positively correlated to the expression of Bcl-2 and p53 in rat tongue (P < 0.01).

CONCLUSIONSThe expression of survivin may play an important role in oral carcinogenesis. Survivin, Bcl-2 and p53 may be synergetic in the carcinogenesis of oral cancer.

4-Nitroquinoline-1-oxide ; adverse effects ; Animals ; Apoptosis ; Cell Transformation, Neoplastic ; chemically induced ; metabolism ; pathology ; Genes, bcl-2 ; Male ; Microtubule-Associated Proteins ; metabolism ; Mouth Mucosa ; metabolism ; pathology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; Tongue Neoplasms ; chemically induced ; metabolism ; pathology ; Tumor Suppressor Protein p53 ; metabolism