Institute of Genetics and General Biology, University of Salzburg, Hellbrunnerstrasse 34, A-5020 Salzburg, Austria, 1 Children's Hospital, University of Ulm, Ulm, Germany and 2 Herzog-Julius Hospital for Rheumatology and Orthopedics, Kurhausstrasse 1317, D-38667 Bad Harzburg, Germany
3 To whom correspondence should be addressed. Tel: +43 662 8044 5782; Fax: +43 662 8044 144; Email: peter.eckl{at}sbg.ac.at
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Abstract |
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Abbreviations: BrdU, bromodeoxyuridine; apo8', apo8'-carotenal; CP, ß-carotene cleavage products; EGF, epidermal growth factor; MEM, Minimum essential medium
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Introduction |
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However, prooxidant and co-carcinogenic effects have also been reported (1315). Furthermore, the Alpha-Tocopherol Beta-Carotene Cancer Prevention (ATBC) Study and the Beta-CArotene and RETinol Efficacy Trial (CARET) unexpectedly showed an increased risk of lung cancer in smokers (16,17). Wang and Russell (18) showed that ß-carotene decreases the level of retinoic acid in the lungs and this reduces the inhibitory effect of retinoids on activator protein-1. As a consequence, lung cell proliferation and, potentially, tumor formation is enhanced (18). The same authors suggested that ß-carotene metabolites are responsible for the carcinogenic response in the lungs of cigarette smokers. These data are supported by Leo and Lieber (19), who showed that ß-carotene supplementation in smokers who also consume alcohol promotes pulmonary cancer and possibly also cardiovascular diseases.
In their investigations, Van Poppel et al. (20) demonstrated the lack of a protective effect of ß-carotene on smoking-induced DNA damage in lymphocytes of heavy smokers.
Effects of ß-carotene have been reported to be modified under certain conditions and at certain concentrations. Zhang and Omaye (21) demonstrated that antioxidant and prooxidant effects of ß-carotene are dependent on oxygen tension and the concentration of ß-carotene.
Furthermore, free radical attack on carotenoids results in the formation of numerous breakdown products, ß-carotene cleavage products (CP), which could contribute to the carcinogenic effects (22). In addition, it was demonstrated, that apo8'- carotenal (apo8'), a metabolite of ß-carotene, acts as a strong inducer of liver cytochromes P450 1A1 and 1A2 (23).
Since a genotoxic potential of CP cannot be excluded from these observations, the effects of CP, apo8' and ß-carotene were investigated in primary cultures of rat hepatocytes. These cells proved to be a highly sensitive and reliable test system for the evaluation of the genotoxic potential of mutagens/promutagens (2426). The end-points tested were: the mitotic index, the percentage of necrotic and apoptotic cells, micronucleated cells, chromosomal aberrations and sister chromatid exchanges (SCE).
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Materials and methods |
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Since crystalline ß-carotene is not water-soluble the carotenoid had to be emulsified in a soybean oil carrier to enable physiological activity. The water-dispersable ß-carotene in soybean suspension (2% w/v) and the soybean suspension used as blank matrix in the experiments were a gift of Cognis Australia Pty Ltd (Australia). The carotenoid emulsion was made from a starting material of 30% ß-carotene (derived from an algal extract) in soybean oil which was emulsified into a 30% water, 70% glycerol aqueous phase using a glyceryl mono-oleate emulsifier. The fine emulsion provides the carotenoid in a lipid globule size of 1 µm or less so that interaction can occur at the cellular level.
The generation of CP was performed as described by Siems et al. (22) and Sommerburg et al. (27) by C.-D.Langhans, who is gratefully acknowledged. For degradation, ß-carotene was dissolved in methanol containing 2% (v/v) trichloromethane to achieve sufficient solubility of the carotenoid. Chemical destruction of ß-carotene was done by bleaching with hypochlorite by adding NaOCl in a 100-fold concentration relative to the carotenoid. The samples reacted at room temperature and in daylight for 10 min. After hexane extraction, different CP were identified (HPLC and GCMS) and partially quantified (HPLC) in the aliquots obtained. The CP mixture obtained from a 0.5 mM ß-carotene stock solution contained ß-carotene (0.16 mM), apo15'-carotenal (0.08 mM), apo12'-carotenal (0.12 mM) and apo8'-carotenal (0.006 mM) and a number of products which could not be identified by HPLC. Further products could be identified by GCMS, but not quantified because of the extraordinary technical difficulty. Related to all peaks detected during GCMS analysis was a peak area of 4.8% accounting for ß-cyclocitral, 0.1% for ionene, 9.9% for ß-ionone, 1.9% for ß-ionone-5,6-epoxide and 4.5% for dihydroactinidiolide. Furthermore, 4-oxo-ß-ionone was detected in trace amounts.
Animals
Female Fischer 344 rats weighing 100 g were obtained from Harlan (Winkelman, Germany). They were kept in a temperature and humidity controlled room with a 12 h lightdark cycle. Water was provided ad libitum. The animals were allowed to acclimatize for at least 2 weeks prior to hepatocyte isolation.
Hepatocyte isolation and culture
Hepatocytes were isolated from female Fischer 344 rats by the in situ two-step collagenase perfusion technique as described by Michalopoulos et al. (28). The isolated hepatocytes were plated at a density of 20 000 viable cells/cm2 on collagen-coated 60 mm diameter plastic culture dishes. According to Eckl et al. (29), the hepatocytes were plated in 5 ml of serum-free MEM containing 1.8 mM calcium supplemented with non-essential amino acids, pyruvate (1 mM), aspartate (0.2 mM), serine (0.2 mM) and penicillin (100 U)/streptomocin (100 µg/ml). The cultures were incubated at 37°C, 5% CO2 and 95% relative humidity. After an incubation period of 3 h, the medium was exchanged for fresh MEM and the cultures were returned to the incubator.
Treatment
Approximately 20 h after the first exchange of the medium, the test substances were added to the cultures at concentrations of 0.01, 0.1, 1, 5 and 10 µM CP, apo8' and ß-carotene and incubated for 3 h. Then the medium was aspirated and the plates were washed twice with fresh medium to completely remove the applied substances. Finally, fresh MEM containing 0.4 mM Ca2+, supplemented as described above with the further addition of insulin (0.1 µM) and EGF (40 ng/ml), was added. To determine SCE induction, bromodeoxyuridine (BrdU) (10 µM) was added to three dishes of each concentration. Thereafter, cells were incubated for an additional 48 h.
Fixation, staining and cytogenetic analysis
Cytogenetic studies were performed as described by Eckl et al. (24). After 48 h colcemid (0.4 µg/ml) was added to three dishes (where BrdU was added) per concentration and the cultures were incubated for a further 3 h. No colcemid was added to the cultures for the micronucleus assay.
For the micronucleus assay, cells were fixed in the dishes with methanol: glacial acetic acid (3:1) for 15 min, briefly rinsed with distilled water and air dried.
For chromosome preparations cells were harvested by replacing the medium with 2 ml of collagenase solution (0.5 mg collagenase/ml) and incubation for 10 min. The detached cells were collected by centrifugation (44 g), treated with hypotonic KCl solution (0.02 M) for 10 min at 37°C and fixed in freshly prepared methanol:glacial acetic acid (3:1) overnight. Preparations were made by dropping the cell suspension on precleaned frosted slides.
For micronucleus determination the fixed cells were stained with the fluorescent dye DAPI (4,6',6-diamidino-2-phenylindole) in McIlvaine buffer (0.2 M Na2HPO4 buffer adjusted with 0.1 M citric acid to pH 7.0) for 30 min in the dark at room temperature. After washing with McIlvaine buffer, the dishes were rinsed with distilled water followed by air drying. For microscopic observation, fixed and stained cells were mounted in glycerol. To determine the mitotic index, the frequencies of apoptotic and necrotic cells and the number of cells with micronuclei per 1000 cells per dish were analysed under a fluorescence microscope (Leitz Aristoplan).
The slides for studying chromosomal aberrations and SCE induction were stained with Hoechst 33258 (4.5 µg/ml) in Sörensen phosphate buffer pH 6.8 for 20 min, rinsed with Sörensen phosphate buffer and exposed to black light (General Electric F 40 BLB Black light) for 15 min on a warming plate kept at 50°C. After removal of the coverslips the slides were briefly washed with distilled water and stained in 5% Giemsa solution. Twenty well-spread first division metaphases were analysed for chromosomal aberrations under a phase contrast microscope (Leitz Laborlux 11). Twenty well-spread second division metaphases were analysed for SCE. The number of aberrations is given per diploid cell, i.e. 42 chromosomes. The SCEs are reported per chromosome.
Statistical analysis
Student's double-sided t-test for independent samples was used to calculate the levels of significance.
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Results |
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Discussion |
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Based on the observation of an increased risk of lung cancer in smokers (16,17) in the ATBC Study and CARET Siems et al. (22) hypothesized that degradation of ß-carotene leads to the formation of high amounts of cleavage products with prooxidant properties (CP) under heavy oxidative stress, while under conditions of moderate oxidative stress, antioxidant effects of ß-carotene are dominant. The results of our study do not directly support this assumption, but demonstrate a genotoxic effect which could either be due to the prooxidant properties of CP or to a direct or indirect (involving metabolism in hepatocytes or other metabolically competent cells such as those in the lung) action on DNA. Such an action has not been described before and appears to have relevance for the in vivo situation, since the effects are already seen at nanomolar concentrations which can be produced in vivo under defined conditions.
Apo8' has been reported to be a strong inducer of liver cytochromes P450 1A1 and 1A2 (23). Our data obtained with apo8' revealed a significant mutagenic potential with respect to micronucleus induction, induction of chromosomal aberrations and SCE. Whether these observations are interrelated or not cannot be answered. However, the doseresponse effects obtained in this study could indicate a link between these observations:
Induction of micronuclei and chromosomal aberrations by apo8' follow saturation-type doseresponse characteristics, whereas the doseresponse effect obtained with CP appears to be bell-shaped. For both CP and apo8' the efficiency of micronucleus induction decreases at concentrations >0.1 µM, with the induction of chromosomal aberrations decreasing at concentrations >0.1 and >1 µM, respectively. Treatment with either CP or apo8' did not influence the mitotic index and the frequencies of necrotic and apoptotic cells remained unchanged at all concentrations tested, thus both substances showed no cytotoxic effects. Therefore, the shapes of the doseresponse curves cannot be explained by an increased toxicity of CP. Since it has been demonstrated that apo8' acts as a strong inducer of liver cytochromes P450 1A1 and 1A2 (23), the plateau in the doseresponse curve may reflect these changes. In other words, the induced drug-metabolizing or other enzymes could reduce the genotoxic action of apo8'. The doseresponse curve for CP treatment may also include such a component, due to the presence of apo8'. In addition, one can also expect effects from other breakdown products, the combined effect of which is a decrease at higher CP concentrations. This behaviour is not uncommon and has been observed in the hepatocyte test after treatment with complex environmental mixtures (30,31).
Since micronuclei are the result of either chromosome breaks or disturbances of the mitotic spindle (32) and chromosome aberrations result from clastogenic events with and without chromosomal rearrangements (33), these parameters are usually considered to be clear evidence for mutagenicity. On the other hand, SCE may not represent actual damage to chromosomes, but could instead be considered a result of damage repair. Thus, the different doseresponse effects obtained for the different end-points tested indicate that both CP and apo8' primarily induce clastogenic events while the SCE-inducing activity becomes significant at high concentrations. This observation could eventually be linked to the shapes of the doseresponse curves for chromosomal aberrations and micronuclei, and could indicate the generation of differently acting metabolites.
During the perfusion, washing steps and medium changes hepatocytes are exposed to increased oxygen tension and it has been shown that elevated oxygen concentrations may lead to an increase in the formation of free radicals, in other words may exert oxidative stress which particularly influences the frequencies of micronucleated cells and the occurrence of SCEs (32). This effect is taken into consideration with the appropriate controls, however, it cannot be excluded that the genotoxic effect of ß-carotene breakdown products (CP and apo8') is influenced to some extent. Further investigations under hyperoxic conditions and the use of antioxidants will help to clarify a potential combinational effect.
Summarizing, the results obtained in this study indicate that ß-carotene breakdown products are capable of inducing genotoxic effects at concentrations which may occur under conditions of intense oxidative stress, e.g. induced by heavy smoking. Therefore, our findings could be helpful in explaining the adverse side effects reported in the ATBC study and CARET (16,17).
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Acknowledgments |
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References |
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