Oral Epithelial Cultures Research Articles

Assessment of the impact of aerosol from a potential modified risk tobacco product compared with cigarette smoke on human organotypic oral epithelial cultures under different exposure regimens

Cigarette smoke (CS) is affecting considerably the oral mucosa. Heating, instead of burning, tobacco reduces consistently the amount of toxic compounds and may exert a lower impact on oral health than combusted cigarettes. The carbon-heated tobacco product 1.2 (CHTP1.2) is a potential modified risk tobacco product (MRTP) based on heat-not-burn technology. Using a systems toxicology assessment framework, we compared the effects of exposure to CHTP1.2 aerosol with those of CS from a reference cigarette (3R4F). Human organotypic cultures derived from buccal and gingival epithelia were exposed acutely (28-min) or repeatedly (28 min/day for 3 days), respectively, to two matching concentrations of CHTP1.2 aerosol or 3R4F CS, and a non-diluted (100%) CHTP1.2 aerosol. The results showed an absence of cytotoxicity, reduction in pathophysiological alterations, toxicological marker proteins, and inflammatory mediators following exposure to CHTP1.2 aerosol compared with 3R4F CS. Changes in mRNA and miRNA expression were linked by an integrative analysis approach, suggesting a regulatory role of miRNAs in several smoke/disease-relevant biological processes induced by 3R4F CS. The identification of mechanisms by which potential MRTPs can reduce the impact of tobacco use on biological systems is of great importance in understanding the molecular basis of the smoking harm reduction paradigm.

Systems Toxicology Assessment of the Biological Impact of a Candidate Modified Risk Tobacco Product on Human Organotypic Oral Epithelial Cultures.

Cigarette smoke (CS) has been reported to increase predisposition to oral cancer and is also recognized as a risk factor for many conditions including periodontal diseases, gingivitis, and other benign mucosal disorders. Smoking cessation remains the most effective approach for minimizing the risk of smoking-related diseases. However, reduction of harmful constituents by heating rather than combusting tobacco, without modifying the amount of nicotine, is a promising new paradigm in harm reduction. In this study, we compared effects of exposure to aerosol derived from a candidate modified risk tobacco product, the tobacco heating system (THS) 2.2, with those of CS generated from the 3R4F reference cigarette. Human organotypic oral epithelial tissue cultures (EpiOral, MatTek Corporation) were exposed for 28 min to 3R4F CS or THS2.2 aerosol, both diluted with air to comparable nicotine concentrations (0.32 or 0.51 mg nicotine/L aerosol/CS for 3R4F and 0.31 or 0.46 mg/L for THS2.2). We also tested one higher concentration (1.09 mg/L) of THS2.2. A systems toxicology approach was employed combining cellular assays (i.e., cytotoxicity and cytochrome P450 activity assays), comprehensive molecular investigations of the buccal epithelial transcriptome (mRNA and miRNA) by means of computational network biology, measurements of secreted proinflammatory markers, and histopathological analysis. We observed that the impact of 3R4F CS was greater than THS2.2 aerosol in terms of cytotoxicity, morphological tissue alterations, and secretion of inflammatory mediators. Analysis of the transcriptomic changes in the exposed oral cultures revealed significant perturbations in various network models such as apoptosis, necroptosis, senescence, xenobiotic metabolism, oxidative stress, and nuclear factor (erythroid-derived 2)-like 2 (NFE2L2) signaling. The stress responses following THS2.2 aerosol exposure were markedly decreased, and the exposed cultures recovered more completely compared with those exposed to 3R4F CS.

Streptococcus oralis and Candida albicans Synergistically Activate μ-Calpain to Degrade E-cadherin From Oral Epithelial Junctions.

Streptococcus oralis forms robust mucosal biofilms with Candida albicans that have increased pathogenic potential. In this study, using oral epithelial cultures, organotypic oral mucosal constructs, and a mouse model of oral infection, we demonstrated that S. oralis augmented C. albicans invasion through epithelial junctions. C. albicans and S. oralis decreased epithelial E-cadherin levels by synergistically increasing µ-calpain, a proteolytic enzyme that targets E-cadherin. In the mouse coinfection model this was accompanied by increased fungal kidney dissemination. Coinfection with a secreted aspartyl protease (sap) mutant sap2456 and S. oralis increased μ-calpain and triggered mucosal invasion and systemic dissemination, suggesting that fungal protease activity is not required for invasion during coinfection. We conclude that C. albicans and S. oralis synergize to activate host enzymes that cleave epithelial junction proteins and increase fungal invasion.

Oral EBV and KSHV Infection in HIV

The gamma herpesviruses, Kaposi's-sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), are tightly associated with the development of AIDS-associated oral disease and malignancy during immune suppression. The objective of this investigation was to characterize oral infection and pathogenesis in healthy and immune-suppressed individuals. To characterize oral EBV and KSHV infection, we examined throat washings and oral epithelial cells from HIV-positive and HIV-negative individuals. Quantitative/real-time polymerase-chain-reaction (PCR) assays, transmission electronmicroscopy, immunostaining, and sequence analysis were used to identify viral infection. Virus was isolated from throat-wash samples and was used to infect epithelial and lymphoid cell lines. We detected EBV and KSHV in the oral cavity in healthy and immune-suppressed individuals. Viral strain analysis of KSHV K1 in multiple clones from the oral cavities of healthy persons and immunosuppressed patients detected several strains previously detected in KS lesions, with minor strain variation within individuals. Immunoelectron microscopy for multiple viral antigens detected consistent expression of viral proteins and oral epithelial specimens. In oral epithelial cells infected with wild-type KSHV in vitro, the K8.1 glycoprotein associated with lytic KSHV infection was detected in both primary and telomerase immortalized oral epithelial cultures by 24 hours post-infection. Virions were detected, subsequent to infection, by scanning electron microscopy. Oral epithelial cells were also infected in vitro with wild-type EBV originating from throat washes. Analysis of these data suggests that, like EBV, KSHV infection is present in the oropharynx of healthy individuals, is transmissible in vitro, and may be transmitted by saliva.

Expression of cytochrome P450 and microsomal epoxide hydrolase in cervical and oral epithelial cells immortalized by human papillomavirus type 16 E6/E7 genes.

Epidemiological evidence suggests that the presence of human papillomaviruses (HPV), when combined with smoking behaviors, considerably enhances the risk of developing oral, cervical, vulvar, and/or anal carcinomas. It is well established that the cytochrome P450 (CYP), microsomal epoxide hydrolase (mEH), and other biotransformation enzymes are important modulators of the bioactivation and detoxification of many environmental chemicals, including constituents of tobacco smoke such as certain nitrosamines and polycyclic aromatic hydrocarbons (PAH). Since there is little information regarding oral and cervical epithelial-specific expression of these genes, established primary and HPV-immortalized oral and cervical epithelial cell lines were analyzed for morphology, mRNA and protein expression patterns of specific CYPs and mEH. Primary human oral and cervical epithelial cells were immortalized using retroviral infection with HPV-16 E6/E7 genes. Primary human keratinocyte cells were immortalized by transfection of HPV-18 and made tumorigenic with nitrosomethylurea treatment. Expression profiles for mEH, CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2D6, CYP3A, and CYP2E1 were evaluated in these cultures in the presence or absence of a PAH inducer, using reverse transcriptase-coupled polymerase chain reaction analysis. mEH gene expression was evident in all cultures, while CYP2A6 mRNA was not detected in any of the cell lines, regardless of culture conditions. CYP2E1 mRNA expression was greatest in the oral epithelial cultures and detectable in all other epithelial cultures except for the HPV-18 immortalized keratinocyte cell line. Elevated levels of CYP2D6 mRNA existed in both oral epithelial cell lines and the HPV-16 immortalized cervical epithelial cells when compared to the other cell lines examined. CYP1A1 and CYP1A2 mRNAs were detected in all the cells and several cultures were inducible by PAH exposure. To corroborate the RT/PCR data, Western immunoblotting experiments were conducted on selected samples. Using these methods, CYP1A1 and CYP2E1 proteins were detected in primary and HPV-immortalized oral and cervical epithelial cultures. These data indicate that both primary and HPV immortalized cells appear to express certain biotransformation enzymes necessary for the activation of tobacco-specific nitrosamines and PAHs. Although the overall impact of HPV gene infection on expression of these systems remains to be fully elucidated, as in vitro system is characterized which should prove useful in examining interactive mechanisms of HPV with xenobiotic activation in the etiology of squamous cell carcinomas.