Liver Disease & Nutrition Section, Veterans Affairs Medical Center & Mount Sinai School of Medicine, Bronx, NY 10468-3992, USA
Received 20 September 2000; in revised form 18 December 2000; accepted 15 January 2001
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ABSTRACT |
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INTRODUCTION |
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Retinol supplementation has been recommended for correction of vitamin A deficiency in alcoholics. However, vitamin A supplementation is complicated by its intrinsic hepatotoxicity, which is potentiated by concomitant alcohol consumption (Leo et al., 1982). Unlike retinoids, carotenoids (ß-carotene) are not known to produce toxic manifestations. Furthermore, as ß-carotene was shown to be an antioxidant, it was viewed as an effective, but less toxic, substitute for retinol. Nevertheless, in baboons, consumption of ethanol together with ß-carotene resulted in striking hepatic injury (Leo et al., 1992
). The well-known hepatotoxicity of ethanol was potentiated by large amounts of ß-carotene and, in rats, the concomitant administration of both ß-carotene and alcohol also resulted in striking liver lesions (Leo et al., 1997
).
Thus far, no reports have been published on the effect of the combination of ß-carotene and ethanol on liver cells in vitro. Accordingly, we investigated the effects of ß-carotene and acetaldehyde (the toxic metabolite of ethanol) on liver cells in culture. HepG2 cells have served as a good model to study the hepatotoxicity of different chemicals or drugs. Although these cells have lost most of their ability to express some enzymes related to ethanol metabolism, such as alcohol dehydrogenase (ADH) (Clemens et al., 1995) and cytochrome P4502E1 (Dai et al., 1993
), which are needed to metabolize ethanol to acetaldehyde, they still retain aldehyde dehydrogenase (ALDH) activity (Clemens et al., 1995
). Acetaldehyde has been shown to mediate many of the biological effects of ethanol (Lieber, 1992
) and plays an important role in the pathogenesis of alcoholic liver injury. In the present experiment, acetaldehyde was added directly to the HepG2 cell culture medium in view of the deficient conversion of ethanol to acetaldehyde in these cells.
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MATERIALS AND METHODS |
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ß-Carotene-liposome preparation
ß-Carotene liposomes were prepared according to Liebler et al. (1997) with the following modifications: ß-carotene (type II, synthetic; Sigma, St Louis, MO, USA) was dissolved in hexane (0.147 mg/ml, as measured at 450 nm with a spectrophotometer); it was mixed with 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC) dissolved in ethanol (2.5 mg/ml) in a glass tube and evaporated to dryness under a stream of nitrogen. The residue was dissolved in cyclohexane and evaporated again. It was then suspended in MEM and sonicated for 8 s. The ß-carotene-liposome mixture was added directly to the culture medium. DLPC liposomes alone, processed as described above, were used as liposomal control.
Measurement of ß-carotene and acetaldehyde in cells and medium
ß-Carotene in the cells and medium was measured by high-performance liquid chromatography (HPLC) as previously described (Leo et al., 1992), with minor modifications: an aliquot of medium or cell suspension was mixed with 2 volumes of ethanol and extracted twice with 3 volumes of n-hexane. The hexane was removed under a stream of nitrogen, and the extract was dissolved in the mobile phase and injected onto a Zorbax C18 column. All HPLC analyses were carried out with an HP-1090 liquid chromatograph equipped with a photo diode-array spectrophotometric detector and HPLC chemstation (Hewlett Packard, Palo Alto, CA, USA). Chromatograms at 295, 325 and 450 nm were recorded to assess tocopherols, retinols and carotenoids, respectively. ß-Carotene was quantified by comparing the peak height in the unknown with the peak height in the known amount of the standard (Leo et al., 1992
). Acetaldehyde was measured by head-space gas chromatography as described before (Hernandez-Munoz et al., 1992
).
Mitochondrial activity
The effects of acetaldehyde and ß-carotene on mitochondrial reductive activity were measured by the MTT test (Scudiero et al., 1988) modified as follows: before each treatment, the media were removed from the flasks, and the flasks were washed twice with Hanks' solution; MEM with different concentrations of ß-carotene and/or acetaldehyde was added, and the flasks were sealed with rubber stoppers; 24 h thereafter, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution was added to each flask at a final concentration of 0.2 mg/ml. The MTT was reduced to formazan (a product with a blue colour) by the active mitochondria. After a 2-h incubation at 37°C, the medium was removed, and 1 ml 100% dimethyl sulphoxide was added to lyse the cells and dissolve the reduced MTT. The absorbance by the reduced MTT was determined at 570 nm. The A570 was taken as an index of the activity of mitochondria. The net absorbance from the flasks of cells cultured with the control medium was considered as 100% of the mitochondrial activity.
Cell membrane damage
Leakage of LDH was measured as an index of cell-membrane damage (Thomas et al., 1993; Yildiz et al., 1999
). HepG2 cells were sub-cultured in 12.5 cm2 flasks and grown for 3 days before treatment with acetaldehyde and ß-carotene. At the end of the experiment, the media were collected, and the cells were harvested by scraping and suspended in 0.5 ml of MEM, then sonicated with an ultrasonicator (Branson Sonifier 450) for 8 s on ice. The LDH activities in the medium and in the cells were measured with an LDH assay kit (Boehringer Mannheim, Indianapolis, IN, USA). To that effect, 200 µl aliquots of culture medium or 10 µl of cell lysate were added to the LDH assay system, and the absorbance at 492 nm was recorded.
Statistics
Results were expressed as means ± SEM, and the significance of differences among individual groups was obtained by using one-way analysis of variance followed by the Student-Newman-Keuls test. For two-group comparison, the unpaired Student's group t-test was used. P < 0.05 was considered significant (Snedecor and Cochran, 1980).
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RESULTS |
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DISCUSSION |
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The MTT test has been used extensively to assess the toxicity of cancer chemotherapeutic drugs and to examine their impact on the mitochondrial activity of cells. In this test, soluble tetrazolium salt is metabolically reduced to a coloured formazan. To reduce MTT to formazan, dehydrogenase activity is needed to produce NADH + H+ (Scudiero et al., 1988). Any factor that inhibits dehydrogenase activity will affect the associated colour reaction. ß-Carotene inhibited the reducing function of mitochondria (Fig. 1
). This inhibition of mitochondrial activity appeared already at a concentration (50 nM) (Fig. 2
) lower than that observed in human plasma (Leo et al., 1995
). The effect was exaggerated by combination with acetaldehyde (Fig. 1
). Its mechanism needs to be further investigated. The effect of ß-carotene alone was not accompanied by LDH leakage (Fig. 3
). The metabolism of ß-carotene involves the activities of a dioxygenase, ALDHs (Duester, 2000
), cytosolic retinol dehydrogenase (CRD), microsomal retinol dehydrogenase (MRD), and CYP450 enzymes (Leo and Lieber, 1999
). ALDHs have wide substrate specificity, metabolizing acetaldehyde, retinoids, and many other physiologically important aldehydes to the corresponding carboxylic acids. These enzymes are shared by the metabolism of retinol and acetaldehyde, and thus might be the site of acetaldehyde and ß-carotene interactions, such as competitive inhibition. Indeed, the effect by ß-carotene or acetaldehyde on each other's disappearance from the medium suggests that these two chemicals share a common metabolic pathway and that a competitive interaction occurs. This decrease of each other's metabolism may contribute to the exaggerated toxicity when the two are combined.
In the Alpha-Tocopherol, Beta-Carotene and Cancer Prevention Study (ATBC) (The Alpha-Tocopherol, Beta-Carotene and Cancer Prevention Study Group, 1994), it was noted that, in smokers, ß-carotene supplementation increased death from coronary artery disease and the incidence of pulmonary cancer. It has previously been shown that while promoting deficiency of vitamin A (Sato and Lieber, 1981; Leo and Lieber, 1982
), ethanol also enhances its toxicity (Mak et al., 1984
, 1987
) as well as that of ß-carotene (Leo et al., 1992
). Because heavy smokers are commonly heavy drinkers, we raised the possibility that alcohol abuse was contributory (Leo and Lieber, 1994
), in part because alcohol is known to act as a co-carcinogen and to exacerbate the carcinogenicity of other xenobiotics, especially those of tobacco smoke (Garro et al., 1992
), and also because of the toxic interaction between ß-carotene and alcohol we had observed in the liver (Leo et al., 1992
). Subsequently, analysis of the data of the ATBC (Albanes et al., 1997
) and the Carotene and Retinol Efficacy Trial (CARET) (Omenn et al., 1996a
,b
) studies showed that the increased incidence of pulmonary cancer was related to the alcohol consumed by the participants.
Many observations show that ß-carotene is not a conventional antioxidant. Under certain conditions, such as low oxygen pressure, ß-carotene can behave as a very effective antioxidant, but under other conditions, such as at elevated oxygen pressures, ß-carotene and related compounds may act as prooxidants (Burton and Ingold, 1984). Independent of oxygen pressure, at high concentrations, ß-carotene can act as a prooxidant, but at low concentrations, it becomes an antioxidant (Woods et al., 1999
). There are two broad classes of antioxidants in living organisms, referred to as preventive antioxidants and chain-breaking antioxidants (McBrien and Slater, 1983
). Previous experimental results indicate that ß-carotene is neither a peroxide-decomposing preventive antioxidant nor a conventional chain-breaking antioxidant (McBrien and Slater, 1983
). Thus, ß-carotene belongs to a previously unrecognized class of biological antioxidants.
Our present study supports the hypothesis that ß-carotene exerts toxicity to liver cells, especially when combined with acetaldehyde. Oxidative stress and lipid peroxidation have been reported to be one of the main aspects of acetaldehyde-mediated toxicity in the liver (Lieber, 1992; Olivares et al., 1997
; Ni et al., 2000
). The mechanism by which ß-carotene enhances the acetaldehyde-induced damage in vitro could be explained by ß-carotene acting as a prooxidant and potentiating the corresponding effect of acetaldehyde. The mechanism by which ß-carotene directly inhibits the reducing activity of mitochondria is presently not understood, but will be the subject of further study.
In conclusion, the experimental results of this preliminary study demonstrate that ß-carotene, even at concentrations lower than those observed in vivo, inhibits the activity of mitochondria in human liver tumour cells, and that acetaldehyde enhances this effect. The toxicity of acetaldehyde to cell membranes was also exaggerated by ß-carotene. Exacerbation of each other's toxicity by these two chemicals might involve a mechanism of competition in partially common metabolic pathways and their prooxidant property. This potentiation might be responsible for the exaggeration of the toxicity of ß-carotene by ethanol observed in vivo in baboons (Leo et al., 1992) and in rats (Leo et al., 1997
) as well as in man in clinical trials (Albanes et al., 1997
).
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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