Cytotoxicity of eight cigarette smoke condensates in three test systems: Comparisons between assays and condensates

https://doi.org/10.1016/j.yrtph.2010.08.009Get rights and content

Abstract

Cytotoxic properties of tobacco smoke are associated with chronic tobacco-related diseases. The cytotoxicity of tobacco smoke can be tested with short-term predictive assays. In this study, we compare eight mainstream cigarette smoke condensates (CSCs) from commercial and experimental cigarettes in three different cytotoxicity assays with unique and overlapping endpoints. The CSCs demonstrated cytotoxicity in all assays. In the multiple cytotoxicity endpoint (MCE) assay with TK-6 cells, the cigarette varieties that had the highest EC50s for reduced cell growth also showed a positive dose–response relationship for necrotic cells. In the IdMOC multiple cell-type co-culture (MCTCC) system, all CSCs reduced the viability of the cells. Low concentrations of some CSCs had a stimulatory effect in lung microvascular endothelial cells and small airway epithelial cells. In the neutral dye assay (NDA), except for a 100% flue-cured tobacco CSC, there was little consistency between CSCs producing morphological evidence of moderate or greater toxicity and the CSCs with the lowest EC50s in the MCE or MCTCC assays. Overall, cigarettes made with flue-cured tobacco were the most cytotoxic across the assays. When results were expressed on a per-mg of nicotine basis, lower tar cigarettes were the most cytotoxic in primary human respiratory cells.

Introduction

The cytotoxicity of tobacco smoke manifests as several pathologic conditions including irritation and inflammation, cell proliferation and hyperplasia, oxidative stress and damage, and decreased organ function, and these conditions may play a role in tobacco-related chronic diseases including emphysema, carcinogenesis, and atherosclerosis (Andreoli et al., 2003, Bombick et al., 1998). The acute effects of cigarette smoking on inflammation and oxidative stress in human, animal, and in vitro models have been reviewed (van der Vaart et al., 2004).

Cytotoxic agents are present in both the gas and particulate phases of cigarette smoke. Hydrogen cyanide, acrolein, and volatile organic compounds are identified as gas phase cytotoxic agents (Thayer and Kensler, 1964, Battista, 1976, Pouli et al., 2003). In the particulate phase, nonvolatile and semi-volatile fractions (especially semi-volatile acidic and neutral fractions) demonstrate cytotoxic activity (Curvall et al., 1984, Curvall et al., 1985, Matsukura et al., 1991). It has been suggested that particulate phase cytotoxicity is due to formaldehyde and residual hydrogen cyanide and acrolein and possibly cyanohydrins formed from the reaction of hydrogen cyanide and aldehydes such as acetaldehyde (Rickert et al., 2007a). However, results as to the relative cytotoxicity of each phase remain contradictory with some reporting that the particulate phase is more cytotoxic in the neutral red assay in CHO cells (Maertens et al., 2009) while others report that the gas phase is more cytotoxic in the neutral red assay with BALB/c 3T3 cells (Tewes et al., 2003).

In vitro cytotoxicity assays are useful in determining the contribution of different tobacco blends or cigarette design characteristics (e.g., filter) to the toxicity of the smoke and also in identifying causative smoke chemicals and mechanistic pathways. Cigarette smoke in the form of particulate phase smoke condensate (Bombick et al., 1997a), gas phase smoke condensate (Bombick et al., 1997b, Pouli et al., 2003), and whole mainstream smoke (Bombick et al., 1997b) are all cytotoxic in a number of in vitro assays. While whole smoke is the closest approximation of the chemical mixture received by a smoker, particulate phase condensate and gas phase condensate allow an investigation of chemicals that reside primarily in these fractions. Due to the ease of collection, storage, stability and use with standard assays, mainstream cigarette smoke condensate is perhaps the most routinely used test material for in vitro testing; however, it does not provide results equivalent to whole smoke. In one study, the cytotoxicity EC50 for commercial US cigarettes was 8–12 times higher for mainstream smoke condensate than for whole smoke (Bombick et al., 1997a).

Although the cytotoxicity of cigarette smoke has been the subject of published research since at least as early as the 1960s (Thayer and Kensler, 1964, Weiss and Weiss, 1964), and although some have compared a non-commercial reference cigarette in one cell type in several assays (Putnam et al., 2002), none of the previous research has compared the results of a set of diverse cigarette smoke condensates (CSCs) that vary in tobacco blend, novel features associated with claims of reduced smoke emissions, and smoking machine conditions in several in vitro cytotoxicity assays with unique and overlapping endpoints. Here we report on the qualitative and quantitative cytotoxicity of eight CSCs prepared from cigarettes that ranged from “low tar” (>6.5–14.5 mg) to “high tar” (>14.5 mg) as determined using the standard International Organization for Standardization (ISO) smoking regimen, commercial cigarette brands described as “light” or “full flavor” by the manufacturer and a commercial 12 mg tar cigarette with a charcoal-containing filter, a reference cigarette (2R4F) blended to be representative of a US standard ISO-regimen “low tar” cigarette, and experimental cigarettes constructed of single-tobacco types. Depending upon the amount of charcoal and the smoking conditions, charcoal filters can reduce levels of gas phase chemicals in cigarette smoke (Polzin, 2008).

The CSCs were tested in the multiple cytotoxicity endpoint (MCE) assay with cultured human lymphoblastoid (TK-6) cells (endpoints: cell growth, apoptosis and necrosis), in primary human microvascular endothelial cells from the lungs, normal human bronchial epithelial cells, and human small airway epithelial cells co-cultured as physically separate cultures interconnected by an overlying liquid medium (the integrated discrete multiple organ co-culture system (IdMOC™; Li et al., 2004, Li, 2008; endpoint: cell viability), and a comparison of visible morphological evidence of cytotoxicity with two strains of cultured mammalian cells (Chinese hamster ovary (CHO) cells and Balb/c-3T3 cells) with endpoints of cell lysis, intracytoplasmic granulation, and cell rounding. CHO and Balb3/t3 cells were compared in this assay owing to their use in previous neutral dye exclusion studies (Bombick et al., 1997b, Bombick et al., 1998, NTP, 2003, Putnam et al., 2002).

Section snippets

Materials and their sources

Dimethyl sulfoxide (DMSO), >99.9% for molecular biology, was purchased from Sigma–Aldrich (Milwaukee, WI). Cambridge filter pads (CFP) were obtained from Whatman (Maidstone, UK) and used without further purification. Cigarettes for testing were purchased at commercial retail outlets in the Atlanta metropolitan area or were provided by the Massachusetts Department of Public Health. The 2R4F reference cigarettes were purchased from the University of Kentucky Tobacco and Health Research Institute

Relative cell growth

With metabolic activation, BRI had the lowest EC50 (113 μg/mL). EC50s for 2R4F (150 μg/mL) and CHAR (155 μg/mL) were similar. EC50s for LT (176 μg/mL) and LTMAS (173 μg/mL) were also similar. The highest measured EC50 was BUR (187 μg/mL). Condensates FF and REC did not achieve a 50% reduction in cell growth at the tested concentrations. Calculated EC50 values are presented in Table 2.

In the test system without metabolic activation, 2R4F had the lowest EC50 (69 μg/mL), followed by CHAR (77 μg/mL), BRI

Discussion

The cytotoxicity of cigarette smoke has been reported in the published literature (Andreoli et al., 2003, Bombick et al., 1997a, Bombick et al., 1997b, Pouli et al., 2003). This study addresses a variety of CSCs in multiple assays with endpoints that represent different cellular responses to a toxic exposure. In the MCE assay, CSCs reduced cellular growth in TK-6 cells with or without metabolic activation. In the presence of metabolic activation, CSC from cigarettes composed of 100% flue-cured

Conflict of interest statement

None of the authors report a conflict of interest.

Acknowledgments

Dr. David Holiday with the Chronic and Infectious Disease Research Program of RTI International provided guidance in statistical aspects of this manuscript. Dr. David DeMarini with the Environmental Carcinogenesis Division of the US Environmental Protection Agency provided valuable input during the preparation of the manuscript. The study was supported by internal funds of the Centers for Disease Control and Prevention (CDC). Use of trade names is for informational purposes only and in no way

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