Beta-carotene breakdown products enhance genotoxic effects of oxidative stress in primary rat hepatocytes.

Since it has to be expected that individuals exposed to oxidative stress who take supplements of beta-carotene are simultaneously exposed to both beta-carotene cleavage products (CPs) and oxidative stress, and both exposures have been demonstrated to cause genotoxic effects in primary rat hepatocytes, cyto- and genotoxic effects on primary rat hepatocytes after supplementation of the medium with increasing concentrations of a CP mixture during exposure to oxidative stress by treatment with either DMNQ (2,3-dimethoxy-1,4-naphthoquinone) or hypoxia/reoxygenation (Hy/Reox) was investigated. The cytological endpoints analysed were the mitotic indices, the percentages of apoptotic and necrotic cells, the percentages of micronucleated (MN) cells and the number of chromosomal aberrations (CAs) and sister chromatid exchanges (SCE). The results obtained clearly demonstrate that the CP mixture enhances the genotoxic effects of oxidative stress exposure, whereas it had no effect at all on the endpoints of cytotoxicity studied. These results further support the hypothesis that CP might be responsible for the reported carcinogenic response in the beta-CArotene and Retinol Efficacy Trial (CARET) and Alpha-Tocopherol Beta-carotene Cancer prevention (ATBC) chemoprevention trials.

Cyto- and genotoxic potential of beta-carotene and cleavage products under oxidative stress.

Free radical attack on beta-carotene results in the formation of high amounts of cleavage products with prooxidant activities towards subcellular organelles such as mitochondria, a finding which could provide an explanation for the contradictory results obtained with beta-carotene in clinical efficacy and cancer prevention trials. Since primary hepatocytes proved to be very sensitive indicators for the genotoxic action of suspect mutagens/carcinogens we therefore investigated a beta-carotene cleavage products mixture (CP), apo-8'-beta-carotenal (apo-8') and beta-carotene in the primary rat hepatocyte assay in the presence and absence of oxidative stress provided by hypoxia/reoxygenation (Hy/re). The endpoints tested were: the mitotic indices, the percentages of necrotic and apoptotic cells, micronucleated cells (MN), chromosomal aberrations (CA) and sister chromatid exchanges (SCE). The results obtained indicate a genotoxic potential of both CP and apo-8' already in the concentration range of 100 nM and 1 microM, i.e. at physiologically relevant levels of beta-carotene and beta-carotene breakdown products. In contrast, no significant cytotoxic effects of these substances were observed, nor did beta-carotene induce significant cytotoxic or genotoxic effects at concentrations ranging from 0.01 up to 10 microM. However, when beta-carotene is supplemented during oxidative stress induced by hypoxia/reoxygenation, a dose-dependent increase of CP is observed accompanied by increasing genotoxicity. Furthermore, when beta-carotene cleavage products were supplied during oxidative stress significant additional increases of genotoxic effects were observed, the additional increases indicating an additive effect of both exposures. Summarizing, these results provide strong evidence that beta-carotene breakdown products are responsible for the occurrence of carcinogenic effects found in the Alpha-Tocopherol Beta-carotene-Cancer prevention (ATBC) study and the beta-CArotene and RETinol Efficacy (CARET) Trial.

Cytotoxic and genotoxic effects of beta-carotene breakdown products on primary rat hepatocytes.

According to Siems and colleagues, free radical attack on beta-carotene results in the formation of high amounts of cleavage products with prooxidant activities towards subcellular organelles such as mitochondria. This finding may be an explanation for the contradictory results obtained with beta-carotene in clinical efficacy and cancer prevention trials. Since primary hepatocytes proved to be very sensitive indicators of the genotoxic action of suspect mutagens/carcinogens we therefore investigated a beta-carotene cleavage products mixture (CP), apo8'- carotenal (apo8') and beta-carotene utilizing primary cultures of rat hepatocytes. The end-points tested were: the mitotic index, the percentage of necrotic and apoptotic cells, micronucleated cells, chromosomal aberrations and sister chromatid exchanges (SCE). Our results indicate a genotoxic potential of both CP and apo8' already at the concentrations 100 nM and 1 microM, i.e. at pathophysiologically relevant levels of beta-carotene and beta-carotene breakdown products. A 3 h treatment with CP induced statistically significant levels of micronuclei at concentrations of 0.1, 1 and 10 microM and chromosomal aberrations at concentrations of 1, 5 and 10 microM. Apo8' induced statistically significant levels of micronuclei at concentrations of 0.1, 1 and 5 microM and chromosomal aberrations at concentrations of 0.1, 1 and 10 microM. Statistically significant increases in SCE induction were only observed at a concentration of 10 microM CP and apo8'. In contrast, no significant cytotoxic effects of these substances were observed. Since beta-carotene induced neither significant cytotoxic nor genotoxic effects at concentrations ranging from 0.01 up to 10 microM, these observations indicate that most likely beta-carotene breakdown products are responsible for the occurrence of carcinogenic effects found in the Alpha-Tocopherol Beta-Carotene Cancer Prevention (ATBC) Study and the Beta-CArotene and RETinol Efficacy Trial (CARET).