Effects of Beta-carotene on primary lung cells

ß-Carotene, a precursor of vitamin A, is a widely used antioxidant due to its known radical scavenging properties. Additionally, its use is further supported by the demonstrated antigenotoxic effects. The epidemiological evidence for such a beneficial role of ß-carotene could, however, not be supported by several large scale, randomized supplementation trials: the Alpha-Tocopherol Beta-carotene-Cancer prevention (ATBC) study and the Beta- CArotene and RETinol Efficacy (CARET) Trial, which demonstrated an increase of lung cancer incidence in smokers and asbestos workers supplemented with ß-carotene. It was therefore hypothesised that heavy oxidative stress leads to degradation of -carotene giving rise to the formation of high amounts of cleavage products (CP) such as 5,6-epoxi-ß-ionone, ionene, ß-cyclocitral, ß-ionone, dihydroactinidiolide and apo-carotenals with prooxidant properties. This hypothesis was tested with metabolically competent primary rat hepatocytes by applying both a ß-carotene cleavage product mixture and apo8`-carotenal, one of the major products formed. The results of this study clearly indicate that supplementation of ß-carotene to the culture medium does not induce any cyto- or genotoxic effects in primary rat hepatocytes at concentrations ranging from 0.01 up to 10 M. However, both CP and apo8`-carotenal induce significant genotoxic effects, even at nanomolar concentrations. Furthermore, it has been shown in previous experiments that ß-carotene under conditions of oxidative stress, i.e. hypoxia/reoxygenation, causes a ß-carotene concentration dependent increase of genotoxicity. Although these observations indicate a clear association between ß-carotene cleavage products and mutagenicity, it has to be tested how the putative target cells in the lung, namely alveolar type II cells, respond to ß-carotene in the presence and absence of oxidative stress. Therefore, the aim of this project is to establish primary cultures of both rat and human pneumocytes, and to determine eventual adverse effects of ß-carotene in the presence of oxidative stress. In parallel the formation of CPs will be determined by chemical analytical methods. In addition, the protective effect of antioxidants will be evaluated, as well as eventual differences of sensitivity with respect to age and sex.

ß-Carotene, a precursor of vitamin A, is a widely used antioxidant due to its known radical scavenging properties. Additionally, its application is further justified by the demonstrated antigenotoxic effects. The epidemiological evidence for such a beneficial role of ß-carotene could, however, not be supported by several large scale, randomized supplementation trials: the Alpha-Tocopherol Beta-carotene-Cancer prevention (ATBC) study and the Beta- CArotene and RETinol Efficacy (CARET) Trial, which demonstrated an increase of lung cancer incidence in smokers and asbestos workers supplemented with ß-carotene. Further investigations revealed that pronounced oxidative stress leads to degradation of -carotene giving rise to the formation of high amounts of cleavage products (CPs), such as apo- carotenals, with prooxidant properties. Cleavage product mixtures prepared by hypochlorite bleaching of ß-carotene and apo-8-carotenal (apo-8), one of the major products formed, proved to be genotoxic to primary hepatocytes. In addition, ß-carotene also caused genotoxicity under oxidative stress indicating that related cleavage products might be formed. To further elaborate the toxicological properties of ß-carotene under oxidative stress, primary rat pneumocytes were treated with mixtures of CPs, apo-8 and ß-carotene under oxidative stress (induced by dimethoxynaphthoquinone (DMNQ) or hypoxia/reoxygenation). While the COMET assay revealed significant dose-dependent increases of DNA strand breaks after treatment with CPs and apo-8 as well as increased reoxygenation damage in the presence of ß-carotene, no direct evidence for mutagenicity was obtained in the micronucleus assay. Instead, at the highest concentration of ß-carotene (50 M) applied under DMNQ-induced oxidative stress significantly elevated levels of apoptosis were found as well as significantly reduced cell densities and mitotic indices, indicating that micronucleus formation is likely to be masked by the increasing cytotoxicity. Based on these observations the following mechanism of action during carcinogenesis can be envisaged: The particles inhaled during smoking lead to an activation of alveolar macrophages and neutrophils in the lung causing the formation of reactive oxygen radicals (oxidative burst), which together with the radicals contained in tobacco smoke (approximately 1015 per puff) can lead to a chronic damage of the lung. The consequences are chronic inflammatory processes, which contribute to lung cancer development. In the presence of ß- carotene generated oxygen radicals can further cause the formation of toxic cleavage products causing an aggravation of the inflammatory processes and thus the cancer risk. Preliminary analytical investigations with activated alveolar macrophages confirmed the formation of ß-carotene cleavage products.