Acrolein-induced cytotoxicity in cultured human bronchial epithelial cells. Modulation by alpha-tocopherol and ascorbic acid

doi:10.1016/S0300-483X(01)00540-6

Toxicology

Volume 170, Issue 3, 25 January 2002, Pages 173-185

https://doi.org/10.1016/S0300-483X(01)00540-6 Get rights and content

Abstract

Acrolein is a highly reactive unsaturated hazardous air pollutant of human health concern, particularly as a component of cigarette smoke. In this study, the mechanisms of acrolein-induced cytotoxicity in human bronchial epithelial cells (HBE1) and the modulating effects of antioxidants were examined. Our results show that acrolein induces a cell death pathway in human bronchial epithelial cells, which retain key features of apoptosis, as indicated by phosphatidylserine (PS) externalization and DNA fragmentation. Acrolein-induced apoptosis was associated with depletion of cellular GSH and intracellular generation of oxidants. Supplementation of cells with either alpha-tocopherol or ascorbic acid was found to strongly inhibit acrolein-induced apoptosis and to prevent the increase in the generation of intracellular oxidants, although GSH depletion was unaffected. Moreover, recovery of cellular GSH levels after acrolein exposure was enhanced following either alpha-tocopherol or ascorbic acid supplementation. The intracellular generation of oxidants following acrolein exposure seems to be an important event triggering the apoptotic response in this model system.

Introduction

Acrolein is a highly reactive, volatile α,β-unsaturated aldehyde that is generated by incomplete combustion or pyrolysis of organic materials such as wood, fuels, food and tobacco (Astry and Jakab, 1983, World Health Organization, 1991). It has been identified as an hazardous air pollutant of important human concern, particularly as a component of photochemical smog and cigarette smoke (Caldwell et al., 1998, Ayer and Yeager, 1982, Altschuler and Mc Pherson, 1963). Toxic and genotoxic effects of this chemical have been extensively described (International Agency for Research on Cancer, 1995). Cigarette smoke causes widespread respiratory epithelial cell damage and apoptosis (Lannan et al., 1994), GSH depletion and free radical species generation. Smoking one cigarette per m³ air of room-space in 10–13 min (10 puffs) generates acrolein levels up to 0.84 mg/m³ (Li and Holian, 1998). Moreover, acrolein is estimated to reach concentrations up to 80 μM in respiratory tract lining fluids as a result of smoking (Eiserich et al., 1995).

Acrolein can also be generated endogeneously as a metabolic product of the anticancer-drug cyclophosphamide, during conditions of lipid oxidation (Fraiser et al., 1991, Uchida et al., 1998a, Uchida, 1999a), and during oxidation of threonine by neutrophil myeloperoxidase at sites of inflammation (Anderson et al., 1997). Indeed, acrolein-protein adducts have been demonstrated in patients with chronic disease believed to be, at least in part, related to oxidative stress, such as diabetic nephropathy (Suzuki and Miyata, 1999), Alzheimer's disease (Calingasan et al., 1999), and in plaque deposits in atherosclerosis (Uchida et al., 1998b). When inhaled, acrolein is a highly selective respiratory tract toxicant (Astry and Jakab, 1983, World Health Organization, 1991, Aranyi et al., 1986) inducing both upper and lower respiratory tract lesions (Beauchamp et al. 1985). Acrolein exposure has also been reported to affect inflammatory-immune processes (Li et al., 1997, Li et al., 1999, Nguyen et al., 2001, Finkelstein et al., 2001) contributing to the reported deficiencies in lung defense against infections in smokers (Green, 1985, Jakab, 1977).

Acrolein reacts rapidly with cellular nucleophiles, particularly GSH (Kehrer and Biswal, 2000). In addition, acrolein has been found to induce the generation of oxygen radicals, such as superoxide anion and hydroxyl radical, and initiates lipid peroxidation (Adams and Klaidman, 1993, Patel, 1987). Administration of acrolein to rats decreased lung and liver levels of glutathione, ascorbic acid (AA) and alpha-tocopherol (Arumugam et al., 1999a, Arumugam et al., 1999b).

In spite of extensive toxicology literature on acrolein, there is an incomplete understanding of the molecular basis of acrolein-induced cytotoxicity and the ability of different antioxidants to counteract the adverse effects of this compound. In this study we investigate the mechanisms of acrolein-induced cell death in human bronchial epithelial cells and the modulatory effect of alpha-tocopherol and AA.

Section snippets

Materials

The following materials were obtained from the indicated sources: dexamethasone, apo-transferrin, insulin, bromobimane, l-AA, CHAPS, 2′,7′-dichlorofluorescin diacetate (DCFH-DA), bovine erythrocytes superoxide dismutase (3940 U/mg), bovine liver catalase (17 000 U/mg), horseradish peroxidase type 1 (120 U/mg) (HRP), N-acetyl cysteine, deferoxamine and d-mannitol from Sigma (St. Louis, MO, USA); F12 medium, penicillin, streptomycin and gentamicin from Gibco BRL (Gaithersburg, MD); epidermal

Acrolein induces apoptotic cell death in HBE1 cells

Apoptosis is characterized by a variety of morphological features. Changes in the plasma membrane are one of the earliest of these features. In apoptotic cells, the membrane phospholipid PS is translocated from the inner to the outer leaflet of the plasma membrane, thereby exposing PS to the external cellular environment. Annexin V, a phospholipid-binding protein with high affinity for PS, binds to cells with exposed PS. Annexin V conjugated to the fluorochrome FITC was used to detect apoptosis

Discussion

In the present study, we report that acrolein induces a cell death pathway in human bronchial epithelial cells which displays several features of apoptotic cell death. Moreover, our results illustrate that this response seems to be related to the intracellular generation of oxidants, and can be attenuated by preloading cells with antioxidants. Indeed, we found that cell supplementation with alpha-tocopherol and AA at concentrations close to those in human plasma, inhibited both the generation

Acknowledgements

The authors thank Dr Carol Oxford (Department of Medical Pathology, UC Davis, CA) for FACScan analyses. This work was supported by grants from NIH (HL47528; HL07013 and HL60812) and the University of California Tobacco-Related Disease Research Program (7RT-0160 and 7RT-0167).

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Acrolein-induced cytotoxicity in cultured human bronchial epithelial cells. Modulation by alpha-tocopherol and ascorbic acid

Abstract

Introduction

Section snippets

Materials

Acrolein induces apoptotic cell death in HBE1 cells

Discussion

Acknowledgements

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