Pingping Shang

Evaluation method for the cytotoxicity of cigarette smoke by in vitro whole smoke exposure

An in vitro whole smoke (WS) exposure method was established to evaluate the toxicological effects of fresh cigarette smoke using the VITROCELL(®) system associated with the neutral red uptake (NRU) cytotoxicity assay. The VITROCELL(®) system is a newly representative culture and exposure system for in vitro studies of gases or complex mixtures. The impacts of two factors on cytotoxicity measurements of cigarette smoke were investigated using this WS exposure system. The factors include synthetic air exposure and optimal time to perform the NRU assay after smoke exposure. Results showed that synthetic air exposure used in the system did not significantly alter cell survival; 24h after smoke exposure appeared to be an optimal time-point to assess the cytotoxicity of cigarette smoke. A clear dose-response relationship between smoke exposure and cell viability was demonstrated using this system, and the evaluation method was sensitive to distinguish the differences in smoke-induced cytotoxic effects from different cigarettes. In addition, we tried converting the values of EC50 from WS exposure testing into the values in unit used in total particulate matter (TPM) testing for a purpose of comparison, and the data indicate that the cytotoxicity of smoke measured by WS exposure is greater than that measured by TPM exposure.

Evaluation of the cytotoxicity of cigarette smoke total particulate matter using three in vitro assays and two types of cells

Abstract In vitro cytotoxicity assays can be used to evaluate potential toxicological effects of tobacco products. Total particulate matter (TPM) from mainstream cigarette smoke trapped by a Cambridge filter is used widely for biological evaluation of smoke. This study compared neutral red uptake (NRU), lactate dehydrogenase (LDH) activity and WST-1 assays for assessing the cytotoxicity of TPM, and evaluated the sensitivity of Chinese hamster ovary (CHO) cells and human lung adenocarcinoma epithelial cell line (A549 cells) to TPM-induced cytotoxic effects. The results indicate that NRU and WST-1 assays are preferable to LDH activity assay for assessing the TPM-induced cytotoxicity, and NRU assay might be more sensitive than WST-1 assay. The cytotoxicity of 3R4F reference cigarettes and two commercial brands of cigarettes were tested by NRU assay in CHO and A549 cells. The results showed that EC50 values in CHO cells treated with TPM were lower than EC50 values in A549 cells, indicating CHO cells are more sensitive to TPM-induced cytotoxic effects than A549 cells.

Effects of smoking regimens and test material format on the cytotoxicity of mainstream cigarette smoke.

The purpose of this study was to evaluate the effects of test material format and smoking regimens on comparative toxicity testing of cigarette smoke. Total particulate matter (TPM) or whole smoke (WS) generated from three test cigarettes under International Organization for Standardization (ISO) or Health Canada Intensive (HCI) regimens were assessed for cytotoxicity using the neutral red uptake (NRU) cytotoxicity assay. Under both ISO and HCI regimens, the relative differences of cytotoxicity among the test cigarettes indicated by the EC50 values in WS were significantly higher than those in TPM. For TPM testing, cytotoxicity was decreased going from ISO regimen to HCI regimen, consistent with the reported reductions of toxicant output on a per unit of TPM basis under the HCI regimen. For WS, cytotoxicity increased for the two lower TPM cigarettes, and decreased for the higher TPM cigarette going from HCI regimen to ISO regimen. Results from this study demonstrated WS should be the preferable test material format for smoke toxicity testing whenever possible. Intensive smoking regimens, such as HCI, are less likely to underestimate smoke toxicant intakes by smokers, and should be included in the comparative toxicological testing strategy.

Immunomodulatory effects of cigarette smoke condensate in mouse macrophage cell line

Increasing evidence has demonstrated that the secretion of cytokines may be associated with cigarette smoke–induced immunomodulatory effects, but a comprehensive analysis of the cytokine profile for cigarette smoke condensate (CSC) exposure is lacking. The aims of this study were to (1) examine the release of 20 cytokines induced by CSC from 12 brands of cigarettes in macrophages cells (Ana-1) and (2) to investigate the general characteristics of the immunomodulatory effects of CSC. Luminex technology was used to simultaneously determine the levels of 20 cytokines (interleukin (IL)-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IL-17, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-γ (IFN-γ), keratinocyte-derived Chemokine (KC), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1α (MIP-1α), induced protein 10 (IP-10), tumor necrosis factor α (TNF-α), vascular endothelial growth factor (VEGF), monkine inducible by γ interferon (MIG), and fibroblast growth factor (FGF)-basic) in the supernatants from Ana-1 cells treated with the CSC. The results showed that the release of eight cytokines was altered (IL-5, IL-6, IL-12, TNF-α, VEGF, IP-10, MCP-1, and MIP-1α) compared with the control. These cytokines fall into two major subtypes: proinflammatory cytokines, including IL-5, IL-6, IL-12, TNF-α, and VEGF, and chemokines, including IP-10, MCP-1, and MIP-1α. Compared with control, the remaining 12 cytokines were not significantly affected by CSC from the 12 brands of cigarettes. As a general characteristic, CSC exerts potently suppressive immunomodulatory effects on cytokine production of Ana-1 cells. Proinflammatory cytokines and chemokines may account for or contribute to the immunosuppressive properties of CSC.