Comparison of the 1C2 reference filtered cigar to the 1R6F reference filtered cigarette.

Establishment of a human polyclonal oral epithelial cell line.

The presence of regional metastases in patients with head and neck squamous cell carcinoma (HNSCC) is a common and adverse event associated with poor prognosis. The authors' recent work on human HNSCC tissues underlies Snail's role as a molecular prognostic marker for HNSCC. Snail positivity is significantly predictive of poorly differentiated, lymphovascular invasive, and regionally metastatic tumors. Here, the authors investigate the capacity of Snail to drive epithelial-mesenchymal transition (EMT) in human oral epithelial cell lines and its ability to confer drug resistance. Snail was overexpressed in HNSCC and oral epithelial cell lines. Anchorage independent growth assays, wound healing assays, invasion and migration assays, spheroid modeling, and cell survival assays were performed. Academic tertiary medical center. Snail overexpressing HNSCC (OSC, Tu212, Tu686) and oral epithelial cell lines (HOK 16-B, OKF-6) were evaluated using assays for wound healing, invasion and migration, 3-dimensional growth, Western blot, and immunofluorescence. The overexpression of Snail in human HNSCC and oral epithelial cell lines drives EMT. The transfection of Snail confers the expression of a mesenchymal molecular signature, including downregulation of the epithelial adherens, such as E-cadherin and β-catenin, and induction of mesenchymal markers. Snail-overexpressing cell lines demonstrate rapid growth in Anchorage-independent growth assays, a decreased capacity to form tight spheroids, an increased resistance to erlotinib, and an increased capacity for invasion. Snail controls the mesenchymal phenotype and drives erlotinib resistance in HNSCC cells. Snail may prove to be a useful marker in predicting epidermal growth factor receptor inhibitor responsiveness.

Hydrogen cyanide is a toxic compound and plays a critical role in cigarette smoke hazard assessment. Due to its reactivity with other chemicals of cigarette smoke, hydrogen cyanide collection was accomplished by the novel trapping method established in our laboratory using glass-fibre filter pads (GFP) treated by sodium hydroxide. However, the GFP trapping method has not been tested under Health Canada Intense (HCI) smoking regime and the trapping efficiency of GFP versus traditional methods have not been evaluated. The present study employed the two trapping methods to collect hydrogen cyanide in mainstream and sidestream cigarette smoke under ISO and HCI smoking regimes. The causes leading to losses of hydrogen cyanide and effects of smoking parameters used in the traditional trapping method were also investigated in this study. It was found that under both ISO and HCI smoking regimes the amounts of hydrogen cyanide in GFP trapping method were strongly correlated (r>0.99) with those by the traditional trapping method, though the traditional method trapped less hydrogen cyanide in sidestream smoke. Carbonyl compounds, such as formaldehyde, were identified as the contributors for the loss of hydrogen cyanide in the alkaline solution. Puff profile was affected to some extents by the use of impinger. Collectively, the results indicate that the modified trapping method is preferred for the routine analysis of hydrogen cyanide in cigarette smoke under ISO and HCI smoking regimes.

In vitro toxicological evaluation of a tobacco heating product THP COO and 3R4F research reference cigarette on human lung cancer cells

To construct a cell line of oral mucosa epithelial cells that stably express human telomerase reverse transcriptase (hTERT) by lentiviral vectors, approaches for the establishment of stable and efficient immortalized oral mucosa epithelial cell lines were explored. Whole RNA was extracted from 293T cells. The hTERT gene was amplified by polymerase chain reaction (PCR) and cloned into the lentiviral vector as pLVX-puro-hTERT. The lentivirus particles were successfully packaged and used to infect primary oral epithelial cells. The positive cell clones were selected by puromycin. Finally, the expression of hTERT was examined by real-time fluorescent quantitative PCR (qRT-PCR) and Western blot analysis. The sequencing results confirmed the construction of the recombinant lentivirus pLVX-puro-hTERT. The morphology of infected cells was similar to that of normal oral mucosal epithelial cells, with a cobble stone-like appearance. The qRT-PCR and Western blot results showed that hTERT was overexpressed in infected cells compared with the normal group (P<0.05). The oral epithelial cell line with stable expression of hTERT was successfully established by the lentivirus, which provides an experimental basis for the establishment of a highly efficient and stable oral epithelial immortalized cell line.

Summary This study focused on the variation in the yields of constituents in smoke from commercial cigarette brands available on the Japanese market. Nineteen commercial cigarette brands were sampled five times every two months from 2009 to 2010. The target constituents were benzo[a]-pyrene, 1,3-butadiene, benzene, formaldehyde, acetaldehyde, acrolein, N-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), carbon monoxide, “tar”, and nicotine. The results of this study showed that the coefficient of variation (CV) values varied greatly by brands, constituents, and smoking regimes. The yields of NNN and NNK in the smoke were strongly correlated to their yields in the tobacco filler blend for most brands. The yields of benzo[a]pyrene under the International Organization for Standardization (ISO) and the Health Canada Intense (HCI) smoking regimes and 1,3-butadiene under the HCI smoking regime were found to be influenced by the measurement. It was shown that factors for variation were highly varied among constituents. The grand mean of CV values for NNN and formaldehyde associated with cigarette manufacturing over ten months and measurement at the JT laboratory under the HCI smoking regimes were 17.1% and 6.6% respectively. The grand mean of CV values for NNN and formaldehyde associated with both cigarette manufacturing over ten months and measurement at different laboratories under the HCI smoking regimes were 23.7% and 22.9% respectively. This is due to the fact that formaldehyde showed the highest CV values for reproducibility among the constituents. Thus, in order to set realistic and robust confidence intervals, it is very important to take into account the variations associated with cigarette manufacturing and measurement within and between laboratories.

The yields of 16 polycyclic aromatic hydrocarbons (PAHs) were determined from cigarette mainstream smoke condensate extracts using Gas Chromatography- Tandem Mass Spectrometry (GC-MS/MS). The method has been validated for ISO and Health Canada Intense (HCI) smoking protocols. Quantifiable levels (ISO means 0.16 to 365 ng/cig; HCI means 0.33 to 1595 ng/cig; n = 30) of 15 PAHs were found in the Kentucky reference cigarette K3R4F. The coefficient of variance (CV) was derived from ten determinations each run in triplicate. The CV range was 8.7% to 24.8% (ISO) and 6.6% to 24.3% (HCI). The limit of detection (LOD) based on empirical precision was ≤ 0.06 ng/cig (ISO) and ≤ 0.20 ng/cig (HCI) for all components except naphthalene (2.89 and 9.62 ng/cig, respectively). The yields from 5 unspecified branded cigarettes (Samples A-E) and 2 other reference cigarettes, K1R5F and the CORESTA monitor CM7, were determined under ISO smoking conditions. The same 15 PAHs were detected as in the K3R4F; however, cigarettes with lower yields of total particulate matter (TPM) were found to contain significantly less PAHs. One component was measured below the limit of quantification (LOQ) in Sample E and 2 components were &lt; LOQ in the K1R5F.

BackgroundBisphosphonate (BP)-associated osteonecrosis of the jaw (ONJ) has been reported in patients receiving intravenous BP, particularly zoledronic acid (ZA). The purpose of this study was to develop an in vitro model representative of the effects BP has on soft tissue secondary to its release from bone. Human gingival fibroblasts and oral epithelial cell lines were exposed to various concentrations (0-10 μM) of ZA using dentine discs (DDs) as a direct carrier of BP, which were exposed for 24 hours to ZA in normal medium (NM), washed in phosphate-buffered saline (PBS) and placed in a new co-culture with the cells. The cells were allowed to proliferate until they grew over the bone discs and then the discs either were left unchelated, or were chelated using 0.001% EDTA or EGTA to release BP from the discs and to observe the cellular effects. Direct effects were determined using direct and fluorescent imaging. Apoptotic effects were determined by vital stain, terminal dUTP nick-end labeling, and annexin V studies. The effect on cell proliferation was determined by mitochondrial tetrazolium salt assay. The level of BP release was determined based on the effect of BP directly on cells, using the DDs or the supernatant fluids resulting from chelation.ResultsA dose-response effect was seen on imaging, and effects on apoptosis and cell proliferation were observed with increasing ZA concentrations liberated from the DDs, particularly after calcium cleavage and release of ZA from the DDs with a variety of chelating agents. Apoptotic effects were observed microscopically after chelation at 24 hours. Release of ZA was confirmed by extracting medium from non-chelated and chelated cell culture models with DDs and applying this medium to untreated fresh cell cultures, providing appropriate controls.ConclusionsThe results from this study demonstrate that low concentrations of ZA released from bone can rapidly and directly affect the oral mucosal tissues, initially through the induction of apoptosis and long term through the inhibition of cell proliferation. These findings provide an in vitro model for a soft-tissue mechanistic component in the initiation and/or progression of ONJ.

Periodontitis is the inflammatory response in periodontal tissues elicited by bacterial colonization in periodontal pockets. In this response, pocket epithelial cells are the first cells to come into contact with bacteria. To elucidate this mechanism, we determined the adherence of the periodontopathic bacterium Eikenella corrodens 1073, which has a GalNAc-sensitive lectin-like adhesin (EcLS), to a human oral epithelial carcinoma cell line (KB) and the induction of proinflammatory cytokine production in the cells following exposure to this bacterium in vitro. In the adherence assay, EcLS played a role as the adhesin of this bacterium in adherence to KB cells. In a reverse transcriptase PCR, significant interleukin-8 (IL-8) and IL-6 mRNA levels were induced in response to exposure to this bacterium. In an enzyme-linked immunosorbent assay after an 8-h bacterial exposure, the IL-8 and IL-6 protein levels were 13.5- and 8.3-fold higher than those in the nonexposed controls, respectively. These protein responses were time dependent. Interestingly, when E. corrodens was separated from KB cells by cell culture inserts, a slight stimulation of the IL-6 and IL-8 mRNA and secreted protein levels was seen. These results imply that the direct contact of E. corrodens 1073 with oral epithelial cells is not necessarily required for the stimulation of IL-6 and IL-8 secretion. We suggest that E. corrodens induces the epithelial cells to secrete proinflammatory cytokines which serve as an early signaling system to host immune and inflammatory cells in underlying connective tissues.

Five-year yield variation in N-nitrosonornicotine and (4-methylnitrosoamino)-1-(3-pyridyl)-1-butanone from the smoke of commercial cigarette brands on the Japanese market

A new reference cigarette, the 3R4F, has been developed to replace the depleting supply of the 2R4F cigarette. The present study was designed to compare mainstream smoke chemistry and toxicity of the two reference cigarettes under the International Organization for Standardization (ISO) machine smoking conditions, and to further compare mainstream smoke chemistry and toxicological activity of the 3R4F cigarette by two different smoking regimens, i.e., the machine smoking conditions specified by ISO and the Health Canada intensive (HCI) smoking conditions. The in vitro cytotoxicity and mutagenicity was determined in the neutral red uptake assay, the Salmonella reverse mutation assay, and the mouse lymphoma thymidine kinase assay. Additionally, a 90-day nose-only inhalation study in rats was conducted to assess the in vivo toxicity. The comparison of smoke chemistry between the two reference cigarettes found practically the same yields of total particulate matter (TPM), ‘tar’, nicotine, carbon monoxide, and most other smoke constituents. For both cigarettes, the in vitro cytotoxicity, mutagenicity, and in vivo toxicity showed the expected smoke-related effects compared to controls without smoke exposure. There were no meaningful differences between the 2R4F and 3R4F regarding these toxicological endpoints. The assessments for the 3R4F cigarette by smoking regimen found as a trivial effect, due to the higher amount of smoke generated per cigarette under HCI conditions, an increased yield of toxicant and higher toxicological activity per cigarette. However, per mg TPM, ‘tar’, or nicotine, the amounts of toxicants and the in vitro toxicity were generally lower under HCI conditions, but the in vivo activity was not different between the two machine smoking conditions. Overall, as the main result, the present study suggests equivalent smoke chemistry and in vitro and in vivo toxicity for the 2R4F and 3R4F reference cigarettes.

Design and chemical evaluation of reduced machine-yield cigarettes

Differences in cadmium transfer from tobacco to cigarette smoke, compared to arsenic or lead

SUMMARY Heated tobacco products (HTPs) are a recent category of tobacco products, with their relative safety compared to cigarette smoking and potential to help smokers to quit being two reasons why regulators may consider their market approval. Designed to heat tobacco rather than to burn in order to produce aerosol, different heating techniques are applied to commercial HTPs, which may result in differing aerosol formation. Therefore, each product requires separate assessment. This work focuses on a closed-end HTP (coded as HTP-A), which is electrically heated and designed to allow puffing air flow to bypass its tobacco section, resulting in reduced oxygen concentration within the tobacco section during heating and aerosol forming. To provide a preliminary aerosol chemistry and in vitro toxicological screening, this study assessed HTP-A against a commercial electrically heated HTP (IQOSTM, coded as HTP-B) and a 3R4F reference cigarette. Under Health Canada Intense (HCI) smoking regime, the levels of 9 regulatory priority toxicants in the aerosol of HTP-A were either reduced or comparable to those in HTP-B on a per-stick basis. Additionally, both HTPs showed significant reduction (greater than 90%) in comparison to those measured in mainstream smoke of 3R4F cigarette for these toxicants. Using a set of standard in vitro toxicological assays (Ames, Micronucleus and Neutral Red Uptake), the two HTPs showed no observable responses while significant toxicity responses were recorded for 3R4F’s total particulate matter. Based on these preliminary results, the novel closed-end HTP-A design may provide similar toxicological profiles to the comparator HTP-B. Further toxicological and clinical assessments are warranted to evaluate HTP-A’s potential for exposure or disease risk reduction. [Contrib. Tob. Nicotine Res. 32 (2023) 146–156]

ObjectiveSnail positivity is predictive of poorly differentiated, invasive, and regionally metastatic HNSCC tumors. We have demonstrated the role of Snail in the inflammation‐induced promotion of EMT. Here, we investigate the capacity of Snail to drive EMT in human oral epithelial cell lines, and its ability to confer drug resistance.MethodSnail was overexpressed in HOK and OKF oral epithelial cell lines. AIG assays, wound healing assays, invasion and migration assays, spheroid modeling, and drug resistance assays were performed. Differential gene expression between Snail‐overexpressing and control epithelial and tumor cell lines was evaluated using gene expression microarray analysis.ResultsThe overexpression of Snail in human oral epithelial cell lines (HOK, OKF) drives EMT. OKF‐Snail and HOK‐Snail lines demonstrate growth in anchorage‐independent growth assays; a decreased capacity to form tight spheroids; increased resistance to erlotinib; and a highly invasive and migratory nature. Gene expression analysis also revealed Snail‐associated differential gene expression with the potential to affect inflammatory cytokine regulation, migration, invasion, and diverse aspects of HNSCC progression.ConclusionSnail controls the mesenchymal phenotype and drives erlotinib resistance in HNSCC cells. Snail may prove to be a useful marker in predicting EGFR inhibitor responsiveness.