The role of chloride in tobacco-smoke-induced cancer
Abstracts
Initial Award Abstract |
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Tobacco smoke contains a large number of carcinogenic components, all participating to different degrees and with different potencies to initiate the development of cancer. Polycyclic aromatic hydrocarbons (PAHs) are a class of chemical carcinogens that occur as byproducts of organic combustion. Several members of this class are potent carcinogens and considerable amounts are found in tobacco smoke. Benzo[a]pyrene (BaP) is a well-studied tobacco component which causes tumors in animals and may cause lung tumors in humans. Over the past two decades studies have revealed how BaP and other PAHs cause DNA damage.
In the process of eliminating these foreign chemicals from the body, cellular metabolism results in the formation of an intermediate compound, the BaP diol epoxide (BPDE), that can bind to DNA (i.e., form DNA adducts). These DNA addicts can cause mutations in genes whose products control cell growth (e.g., proto-oncogenes and tumor suppressor genes). To date, this metabolic activation and DNA binding process is incompletely understood. Recent evidence from our laboratory that suggests the existence of a previously unrecognized cellular pathway in the formation of DNA adducts. This pathway is catalyzed by one of the ions that form common table salt (sodium chloride). The chloride ion is present at high levels in lung cells, and test tube studies have shown that it alters the structure of cancer-causing adducts to form certain types of DNA adducts that may be more mutagenic than those formed without chloride present. Thus, chloride could play a role in mutagenesis and carcinogenesis. An additional indirect influence of chloride on tumor initiation in lung cells can be exerted by two different ways.
First, changes in intracellular chloride concentrations can lead to differences in mutations at sensitive sites in DNA (such as within important genes). Second, chloride can interfere with DNA repair enzymes and thus increase the probability that these adducts will induce a mutation. To test this hypothesis, we must determine if chloride, which plays a role in numerous normal cell functions, could indirectly influence the mutagenicity of PAHs in vivo. For this purpose, cultured lung epithelial cells seem to be an appropriate model, since they (1) represent possible target cells in humans, (2) possess metabolic activation enzymes, (3) are able to proliferate in vivo, and (4) are involved in chloride transport in vivo. These cells will be exposed to drugs which cause either an increase or decrease in intracellular chloride concentration. Then these cells will be treated with BPDE and tested at different time points. The aim is to elucidate the possible synergistic effects between DNA adduct formation by BPDE and chemicals stimulating or inhibiting chloride channels (particularly tobacco smoke components) and to find a significant biological cancer risk marker for tobacco-induced tumors in humans. |
Final Report |
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Tobacco smoke contains a large number of carcinogenic components, all participating to different degrees and with different potencies to initiate the development of cancer. Polycyclic aromatic hydrocarbons (PAHs) are one of the major components of smoke. Benzo[a]pyrene (BaP) is the best studied PAH which causes tumors in animals and may be responsible for or contribute to lung tumors in humans. Over the past two decades studies have revealed how BaP and other PAHs cause DNA damage. Metabolic activation results in the formation of an electrophilic intermediate, the BaP diol epoxide (BPDE) that binds to DNA. These DNA adducts can result in mutations in genes that control cell growth eg protooncogenes and tumor suppressor genes. Despite extensive investigation the relation between PAH-induced DNA damage and mutation and cell transformation leading to carcinogenesis is not clearly defined. The relative amount of stereochemically distinct DNA adducts is the critical first step in defining how the efficacy of repairs, mutation, transformation frequency influence. Therefore, it is important to evaluate DNA adduct stereochemistry (cis- or trans configuration) and the relative numbers of stereochemically distinct adducts. Chemical analysis has shown that sodium chloride can catalyze the binding reaction of BPDE to form more cis DNA adducts. In this study the proportion of cis- and trans DNA adducts formed in mouse and rat skin and as well as in human lung epithelial cells was determined. It was found that in mouse and rat skin, BPDE forms primarily (+)-traps-dGuo adducts thus confirming previous studies. However, when mouse and rat skin was treated with traps-chlorohydrin, an intermediate of BPDE and chloride, more. cis-adducts were formed [(+)-cis-BPDE-N Z-dGuo adduct > (+)-traps-BPDE-N2-dGuo = (-)-traps-BPDE-N2-dGuo > (-)-cis-BPDE-N Z-dGuo]. This ex vivo observation could be of practical importance for establishing the role of stereochemistry on tumor formation using the two-stage mouse skin carcinogenesis model. Weinvestigated whether chloride could drive the alkylation reaction to form more cis-oriented BPDE-DNA adducts in human lung epithelial cells. A549 human lung tumor cells were treated with BPDE while the cells were kept in buffer which varied only in chloride concentration and which were supplemented with ionophores to equilibrate the intra- to the extracellular chloride concentration. It was observed that the proportion of (+)-cis-dAdo adduct increased when the intracellular chloride concentration was increased to about 100 mM. Exposing the cells to the same buffers described above but without the ionophores actually caused a slight reduction of the intracellular chloride concentration from the normal 22 mM to 12, 15 and 5 mM chloride for 0, 50 and 100 mM extracellular chloride concentrations, respectively. Although the total BPDE-DNA adduct concentration remained the same, the proportion (+)trans-dGuo significantly decreased and (+)-cis-dAdo significantly increased (Student t-test p <_ 0.017). Therefore, chloride can drive the alkylation reaction to form more cis-oriented BPDE-DNA adducts but only at a minor extent. Thus, the treatment buffer in which the cells are kept can significantly influence the proportion of cis to trans BPDE-DNA adducts, probably by changing the conformation of the DNA double helix and may play a role in cell toxicity. |
