Activation of transcription factors in human bronchial epithelial cells exposed to aqueous extracts of mainstream cigarette smoke in vitro.

This study aimed to identify the most sensitive transcription factor activated by cigarette smoke extract (CSE) and to explore cigarette smoke components that have high biological activities in a cell-base assay. Previously, we found evidence that implicated 10 different transcription factors as having a high biological activity to CSE in vitro, based on the results of a comprehensive gene expression profile. For this study, luciferase reporter assays for each transcription factor were developed in two types of human bronchial epithelial cells: NCI-H292 and BEAS-2B cells. The results demonstrated that the nuclear factor erythroid 2-related factor 2 (NRF2)/anti-oxidant response element (ARE) pathway was the most sensitive in response to CSE. Consistently, hemo oxygenase-1 (HO-1), a downstream target gene of NRF2, was effectively up-regulated in BEAS-2B cells exposed to CSE. Moreover, among 1395 cigarette smoke components, naphthoquinones including 9,10-phenaotrenquinone, quinones, benzenediols and α, ß-unsaturated carbonyls, were identified as major smoke components that contribute to activating the NRF2/ARE pathway, as indicated by the ARE-reporter assay in BEAS-2B cells. Taken together, NRF2 appears to be a key molecule in the CSE-induced cellular response, and the employed methodology is helpful for the analysis of molecular and cellular effects by CSE.

Investigation by microarray analysis of effects of cigarette design characteristics on gene expression in human lung mucoepidermoid cancer cells NCI-H292 exposed to cigarette smoke.

The effects of tobacco leaf types and the presence or absence of charcoal in the cigarette filters on gene expression were investigated using cigarette prototypes made of either flue-cured (FC) leaf or burley (BLY) leaf and Kentucky Reference 2R4F as a representative blend cigarette with cellulose acetate filters or charcoal filters. NCI-H292, human lung mucoepidermoid carcinoma cell line, was exposed to the total particulate matter (TPM) and gas/vapor phase (GVP) from each prototype for 8h and then the changes in gene expression from microarray data were analyzed. A number of genes associated with oxidative stress, inflammation, DNA damage and xenobiotic response were modified by the two fractions, TPM and GVP, from the three prototypes with cellulose acetate filters. Both TPM and GVP fractions strongly enhanced the gene expression of HMOX1, which is encoding the limiting enzyme in heme degradation and a key regulator of oxidative stress and inflammatory process. Comparing the effects of TPM and GVP fraction, TPM strongly activated Nrf2 pathway-mediated anti-oxidative stress reaction, whereas GVP caused notable DNA damage response. In comparison of FC and BLY, TPM from FC more strongly induced the expression of histone family proteins than that from BLY. GVP from FC markedly induced gene expression associated with HSP70-mediated inflammation relative to that from BLY. Charcoal included in the filter strongly reduced the effects of GVP from each cigarette on gene expression. However, charcoal did not modified the effects of TPM. As a whole, charcoal is a useful material for reducing the biological effects of GVP.

Detection of the cytotoxicity of water-insoluble fraction of cigarette smoke by direct exposure to cultured cells at an air-liquid interface.

For the biological evaluation of cigarette smoke in vitro, the particulate phase (PP) and the gas vapor phase (GVP) of mainstream smoke have usually been collected individually and exposed to biological material such as cultured cells. Using this traditional method, the GVP is collected by bubbling in an aqueous solution such as phosphate-buffered saline (PBS). In such a way the water-insoluble GVP fraction is excluded from the GVP, meaning that the toxic potential of the water-insoluble GVP fraction has hardly been investigated so far. In our experiments we used a direct exposure method to expose cells at the air-liquid interface (ALI) to the water-insoluble GVP fraction for demonstrating its toxicological/biological activity. In order to isolate the water-insoluble GVP fraction from mainstream smoke, the GVP was passed through 6 impingers connected in series with PBS. After direct exposure of Chinese hamster ovary cells (CHO-K1) with the water-insoluble GVP fraction in the CULTEX(®) system its cytotoxicity was assayed by using the neutral red uptake assay. The water-insoluble GVP fraction was proven to be less cytotoxic than the water-soluble GVP fraction, but showed a significant effect in a dose-dependent manner. The results of this study showed that the direct exposure of cultivated cells at the air-liquid interface offers the possibility to analyze the biological and toxicological activities of all fractions of cigarette smoke including the water-insoluble GVP fraction.

In vitro micronucleus assay for cigarette smoke using a whole smoke exposure system: a comparison of smoking regimens.

Previous studies on the biological assessment of cigarette smoke (CS) mainly focused on the total particulate matter (TPM) collected using a Cambridge filter or gas vapor phase (GVP) bubbled through phosphate-buffered saline (PBS). To study the effects of native CS in vitro, direct exposure methods have been developed. Meanwhile, in vitro micronucleus (MN) assays have been reported to evaluate the mutagenicity of CS. The objective of this research is to investigate the MN-inducing activity of whole smoke (WS) and GVP using a whole smoke exposure system, CULTEX((R)), which allows direct exposure of cultured cells to native CS at the air/liquid interface (ALI). CS was generated according to the International Organization for Standardization (ISO; 35ml, 2s, once per 60s) or the Health Canada Intensive (HCI; 55ml, 2s, once per 30s, with complete ventilation block) regimens and Chinese hamster lung (CHL/IU) cells were then exposed to this smoke. Dosages were adjusted according to the amount of smoke entering the actual exposure position. Under both smoking regimens, WS and GVP from 2R4F reference cigarettes induced MN responses. The concept of the dosage and similar dose-response relationships between theoretical and monitored dosage values under the two regimens enabled us to compare the MN-inducing activities of cigarettes in the direct exposure assay, even in the case of various experimental settings or different TPM amounts. MN-inducing activities of 2R4F under the ISO regimen seemed to be higher than those under HCI estimated by the TPM equivalent calculated values.

Analysis of the expression of heme oxygenase-1 gene in human alveolar epithelial cells exposed to cigarette smoke condensate.

Airway epithelium is exposed to inhaled exogenous sources. Injury of the alveolar epithelium by cigarette smoking is presumed to be an important process in the pathogenesis of smoking-related pulmonary diseases. Current mechanistic assays that measure the toxicity of cigarette smoke focus on carcinogenesis. However, there is a need to design assays relevant to other disease processes. Oxidative stress is implicated in the pathogenesis of many respiratory diseases including chronic obstructive pulmonary disease. Therefore, we evaluated whether in vitro studies of cigarette smoking are appropriate to examine HO-1 mRNA expression. The human lung epithelial cell line A549 was exposed to the particulate fraction of cigarette smoke (Cigarette Smoke Condensate; CSC) and examined for the induction of HO-1 mRNA. HO-1 gene expression by CSC is increased dose-dependently. In comparison of the induction of HO-1 mRNA by CSC prepared from flue-cured or Burley tobacco, CSC from flue-cured tobacco seems to tend to induce an mRNA of HO-1 higher than CSC from Burley tobacco. The adaptation of HO-1 mRNA expression assay as a biologically relevant indicator of cigarette smoke-induced stress may be exemplified in this study whereby CSC derived from cigarette smoke positively correlated with an increase in HO-1 expression and the difference of the type of tobacco can be detected.

Heme oxygenase-1 gene expression in human alveolar epithelial cells (A549) following exposure to whole cigarette smoke on a direct in vitro exposure system.

Many in vitro studies have employed cigarette smoke condensates or soluble smoke components to investigate the biological effects of cigarette smoke. However, neither of these methods evaluates the biological effects of fresh whole cigarette smoke. It is most desirable to conduct in vitro biological studies under conditions which accommodate the dynamic physicochemical character of fresh cigarette smoke. Previously we reported the development of a whole smoke exposure system to assess the biological effects of mainstream cigarette smoke. The exposure system design was based on a combination of the sedimentation procedure and the CULTEX cultivation technique, which includes a systemized air/liquid interface methodology and exposes the cells to fresh smoke at every puff. The aim of this study was to adopt the other biological endpoint to our whole smoke exposure system. We focused on heme oxygenase (HO)-1 mRNA gene expression, an enzyme which has recently been shown to be highly responsible for oxidative stress. In the present study, a dose-response relationship between the HO-1 mRNA expression based on the reverse transcription real-time PCR method and total exposure to cigarette smoke was observed. When a Cambridge filter pad was placed between the cigarette and exposure module, to ensure the cells were only exposed to the gas/vapor phase, the latter, as well as the whole smoke, induced HO-1 mRNA dose dependently. For the next step, acetate plain and charcoal filters with the same pressure drop were prepared to assess the potential ability of charcoal filters with regard to the vapor phase performance. The results revealed reduced HO-1 mRNA gene expression when a charcoal filter was used. Direct whole smoke exposure is a significant approach and may reflect the conditions of exposure essentially resulting from direct contact between cells and a dynamic mixture of gaseous and particulate constituents. We were able to adopt a gene expression assay for oxidative stress to the whole smoke exposure system, following the adaptation of cytotoxicity assays. This system, which includes several advantages involving the post-exposure washing of cells, by adding the exchanging medium and assuring the exposure of the particulate phase through the sedimentation method, may have potential for further investigations into the molecular basis of smoking-related lung disease.

Modified procedure of a direct in vitro exposure system for mammalian cells to whole cigarette smoke.

In vitro biological studies on cigarette smoke have usually been made using either cigarette smoke condensate--obtained by trapping the particulate phase of smoke on a filter, or soluble smoke components--obtained by trapping cigarette smoke in buffer solution. However, these approaches may not truly reflect the physical and chemical condition of freshly generated smoke. Clearly it is important to be able to evaluate the biological effects of fresh smoke on mammalian cells for a better understanding of the potential effects of smoking. The CULTEX technology is a new experimental system for cultivation and exposure techniques enhanced the efficiency of in vitro studies, and allows direct exposure of cells intermittently at the air/liquid interface with ultrafine particles, gases, or mixtures of both which fixedly flows. The CULTEX technology has therefore been modified to evaluate the biological effects of whole cigarette smoke in an in vitro system. The exposure system design was based on a combination of the sedimentation procedure and the CULTEX cultivation technique. After freshly generated smoke was delivered onto cells, the flow was shut off and the medium was slowly removed. In this manner, cells were exposed to both the vapor and particulate phase of smoke efficiently. Cells were maintained in the liquid medium except during the exposure period to maintain the culture conditions and to protect the cells from both the influence of puff pressure and the airflow, which served to remove residual cigarette smoke. The medium was changed at every puff of smoke and so effectively eliminating the possibility of any effects caused by accumulation of soluble cigarette smoke components into the medium. This cycle was repeated and cells were exposed to freshly generated cigarette smoke intermittently.