Synergistic interactions of antioxidant nutrients in a biological model system.

OBJECTIVE: The antioxidant activity of fat- and water-soluble antioxidant nutrients and their interactions in physiologic concentrations were determined in an in vitro biological model system. METHODS: Reconstituted human serum consisting of delipidized human serum (DHS) combined with phosphatidylcholine liposomes (PCL) was used to determine antioxidant activities of physiologic concentrations of antioxidant nutrients. Radicals were initiated with 2,2'-azobis(4-methoxy-2,4-dimethylvaleronitrile) (2mmol/L), and oxidation was monitored by 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid. Fat-soluble antioxidant nutrients were incorporated into the PCL prepared by sonication and suspended in DHS to avoid any interference by the endogenous fat-soluble antioxidants. Water-soluble antioxidants were added directly into the DHS. The oxidation kinetics were monitored every 5 min up to 210 min using a microplate reader (excitation wavelength 500 nm, emission wavelength 520 nm). RESULTS: We confirmed the synergistic protective effect of the combination of ascorbic acid (1-5 microM) and alpha-tocopherol (1-5 mircoM) against the oxidation of DHS with PCL. Furthermore, physiologic concentrations of 1) beta-carotene (0.1, 0.5 microM) and alpha-tocopherol (2.5, 5.0 microM), 2) beta-carotene (0.1, 0.5 microM) and ascorbic acid (2.5 microM), and 3) uric acid (10 uM) and alpha-tocopherol (2.5, 5.0 microM) synergistically protected oxidation of reconstituted human serum. CONCLUSION: The present study results suggest a wide antioxidant network between water- and fat-soluble antioxidant nutrients in a biological system, although their actions in vivo warrant further study.

Determination of 9-cis beta-carotene and zeta-carotene in biological samples.

Concentrations of 9-cis beta-carotene (9-cis betaC) and zeta-carotene (zetaC) in biological samples may provide crucial information on the biological activities of these carotenoids. However, in high-performance liquid chromatography (HPLC) these carotenoids are often co-eluted. Therefore, there is an urgent need to develop a method for 9-cis betaC and zetaC quantitation. Both 9-cis betaC and zetaC have peak absorbance at 400 and 450 nm, respectively, whereas only 9-cis betaC has peak absorbance at 475 nm. We developed a HPLC method to quantitate 9-cis betaC and zetaC by using peak absorbance ratios. The 9-cis betaC/zetaC peak area was monitored at 475, 450 and 400 nm. The 9-cis betaC was quantified by using absorbance value at 475 nm; zetaC was then calculated from the 9-cis betaC/zetaC peak at 400 nm by subtracting 9-cis betaC contribution at 400 nm using the 400-nm/475-nm peak absorbance ratio of 9-cis betaC (0.39). This method was applied to determine 9-cis betaC and zetaC concentrations in serum and breast milk samples (n=12) from American lactating women and serum and breast adipose tissue samples (n=16) from Korean women with either benign or malignant breast tumors. 9-cis betaC concentrations in serum and breast milk of American women, and serum and adipose tissue of Korean women were 7.1+/-0.8 and 1.1+/-0.2 nM, and 15.6+/-1.1 nM and 0.2+/-0.1 nmol/g, respectively. zetaC concentrations in the above samples were 54.2+/-7.2 and 8.3+/-1.8 nM, and 49.0+/-3.9 nM and 0.3+/-0.1 nmol/g, respectively.

Doxorubicin as an antioxidant: maintenance of myocardial levels of lycopene under doxorubicin treatment.

The mechanism of doxorubicin-induced cardiotoxicity remains controversial. Wistar rats (n=96) were randomly assigned to a control (C), lycopene (L), doxorubicin (D), or doxorubicin+lycopene (DL) group. The L and DL groups received lycopene (5 mg/kg body wt/day by gavage) for 7 weeks. The D and DL groups received doxorubicin (4 mg/kg body wt intraperitoneally) at 3, 4, 5, and 6 weeks and were killed at 7 weeks for analyses. Myocardial tissue lycopene levels and total antioxidant performance (TAP) were analyzed by HPLC and fluorometry, respectively. Lycopene metabolism was determined by incubating (2)H(10)-lycopene with intestinal mucosa postmitochondrial fraction and lipoxygenase and analyzed with HPLC and APCI mass spectroscopy. Myocardial tissue lycopene levels in DL and L were similar. TAP adjusted for tissue protein were higher in myocardium of D than those of C (P=0.002). Lycopene metabolism study identified a lower oxidative cleavage of lycopene in D as compared to those of C. Our results showed that lycopene was not depleted in myocardium of lycopene-supplemented rats treated with doxorubicin and that higher antioxidant capacity in myocardium and less oxidative cleavage of lycopene in intestinal mucosa of doxorubicin-treated rats suggest an antioxidant role of doxorubicin rather than acting as a prooxidant.

Asymmetric cleavage of beta-carotene yields a transcriptional repressor of retinoid X receptor and peroxisome proliferator-activated receptor responses.

beta-Carotene and its metabolites exert a broad range of effects, in part by regulating transcriptional responses through specific nuclear receptor activation. Symmetric cleavage of beta-carotene can yield 9-cis retinoic acid (9-cisRA), the natural ligand for the nuclear receptor RXR, the obligate heterodimeric partner for numerous nuclear receptor family members. A significant portion of beta-carotene can also undergo asymmetric cleavage to yield apocarotenals, a series of poorly understood naturally occurring molecules whose biologic role, including their transcriptional effects, remains essentially unknown. We show here that beta-apo-14'-carotenal (apo14), but not other structurally related apocarotenals, represses peroxisome proliferator-activated receptors (PPAR) and RXR activation and biologic responses induced by their respective agonists both in vitro and in vivo. During adipocyte differentiation, apo14 inhibited PPARgamma target gene expression and adipogenesis, even in the presence of the potent PPARgamma agonist BRL49653. Apo14 also suppressed known PPARalpha responses, including target gene expression and its known antiinflammatory effects, but not if PPARalpha agonist stimulation occurred before apo14 exposure and not in PPARalpha-deficient cells or mice. Other apocarotenals tested had none of these effects. These data extend current views of beta-carotene metabolism to include specific apocarotenals as possible biologically active mediators and identify apo14 as a possible template for designing PPAR and RXR modulators and better understanding modulation of nuclear receptor activation. These results also suggest a novel model of molecular endocrinology in which metabolism of a parent compound, beta-carotene, may alternatively activate (9-cisRA) or inhibit (apo14) specific nuclear receptor responses.

Total antioxidant performance: a validated fluorescence assay for the measurement of plasma oxidizability.

The antioxidant capacity of human plasma was determined by following the oxidation kinetics of the lipid-soluble fluorescent marker BODIPY using 2,2'-azobis(4-methoxy-2,4-dimethylvaleronitrile) (MeO-AMVN) as the lipophilic radical initiator. The results are expressed as a total antioxidant performance (TAP) value based on the inhibition of BODIPY oxidation, as determined by the appearance of green fluorescence, with respect to a control sample (phosphatidylcholine with or without delipidized human serum). The suitability of the assay was evaluated on the basis of its precision, reproducibility, and specificity. The intra- and interassay coefficients of variation both were less than 5%. The addition of a representative substrate of plasma peroxidation, phosphatidylcholine, up to 750mug/ml did not induce significant changes in the TAP value. Also, BODIPY photooxidation was not observed during the experimental time course (220min). The TAP values of 6 plasma samples from healthy donors were measured and correlated with the main plasma water- and lipid-soluble antioxidants (uric acid and ascorbic acid, alpha-tocopherol, and carotenoids) and lipid profiles. Significant correlations were found between TAP and uric acid (R=0.97, P<0.05) and cholesterol-adjusted alpha-tocopherol (R=0.93, P<0.01). The results confirm that the TAP assay is suitable to measure the antioxidant activity of plasma antioxidants localized in both the lipophilic and hydrophilic compartments.

The biochemical characterization of ferret carotene-9',10'-monooxygenase catalyzing cleavage of carotenoids in vitro and in vivo.

Previous studies have shown that beta-carotene 15,15'-monooxygenase catalyzes the cleavage of beta-carotene at the central carbon 15,15'-double bond but cleaves lycopene with much lower activity. However, expressing the mouse carotene 9',10'-monooxygenase (CMO2) in beta-carotene/lycopene-synthesizing and -accumulating Escherichia coli strains leads to both a color shift and formation of apo-10'-carotenoids, suggesting the oxidative cleavage of both carotenoids at their 9',10'-double bond. Here we provide information on the biochemical characterization of CMO2 of the ferret, a model for human carotenoid metabolism, in terms of the kinetic analysis of beta-carotene/lycopene cleavage into beta-apo-10'-carotenal/apo-10'-lycopenal in vitro and the formation of apo-10'-lycopenoids in ferrets in vivo. We demonstrate that the recombinant ferret CMO2 catalyzes the excentric cleavage of both all-trans-beta-carotene and the 5-cis- and 13-cis-isomers of lycopene at the 9',10'-double bond but not all-trans-lycopene. The cleavage activity of ferret CMO2 was higher toward lycopene cis-isomers as compared with beta-carotene as substrate. Iron was an essential co-factor for the reaction. Furthermore, all-trans-lycopene supplementation in ferrets resulted in significant accumulation of cis-isomers of lycopene and the formation of apo-10'-lycopenol, as well as up-regulation of the CMO2 expression in lung tissues. In addition, in vitro incubation of apo-10'-lycopenal with the post-nuclear fraction of hepatic homogenates of ferrets resulted in the production of both apo-10'-lycopenoic acid and apo-10'-lycopenol, respectively, depending upon the presence of NAD+ or NADH as cofactors. Our finding of bioconversion of cis-isomers of lycopene into apo-10'-lycopenoids by CMO2 is significant because cis-isomers of lycopene are a predominant form of lycopene in mammalian tissues and apo-lycopenoids may have specific biological activities related to human health.

Modification of lymphocyte DNA damage by carotenoid supplementation in postmenopausal women.

BACKGROUND: Oxidative stress has been implicated in the pathogenesis of chronic diseases related to aging such as cancer and cardiovascular disease. Carotenoids could be a part of a protective strategy to minimize oxidative damage in vulnerable populations, such as the elderly. OBJECTIVE: Our aim was to determine the protective effect of carotenoids against DNA damage. DESIGN: A randomized, double-blind, placebo-controlled intervention study was conducted. Thirty-seven healthy, nonsmoking postmenopausal women aged 50-70 y were randomly assigned to 1 of 5 groups and were instructed to consume a daily dose of mixed carotenoids (beta-carotene, lutein, and lycopene; 4 mg each), 12 mg of a single carotenoid (beta-carotene, lutein, or lycopene), or placebo for 56 d. Plasma carotenoid concentrations were analyzed by using HPLC, and lymphocyte DNA damage was measured by using a single-cell gel electrophoresis (comet) assay. RESULTS: At day 57, all carotenoid-supplemented groups showed significantly lower endogenous DNA damage than at baseline (P < 0.01), whereas the placebo group did not show any significant change. Significantly less (P < 0.05) endogenous DNA damage was found as early as day 15 in the mixed carotenoid (P < 0.01) and beta-carotene (P < 0.05) groups. CONCLUSIONS: The results indicate that carotenoid supplementation decreases DNA damage and that a combination of carotenoids (4 mg each of lutein, beta-carotene, and lycopene), an intake that can be achieved by diet, or a larger dose (12 mg) of individual carotenoids exerts protection against DNA damage.

Carotenoid actions and their relation to health and disease.

Based on extensive epidemiological observation, fruits and vegetables that are a rich source of carotenoids are thought to provide health benefits by decreasing the risk of various diseases, particularly certain cancers and eye diseases. The carotenoids that have been most studied in this regard are beta-carotene, lycopene, lutein and zeaxanthin. In part, the beneficial effects of carotenoids are thought to be due to their role as antioxidants. beta-Carotene may have added benefits due its ability to be converted to vitamin A. Additionally, lutein and zeaxanthin may be protective in eye disease because they absorb damaging blue light that enters the eye. Food sources of these compounds include a variety of fruits and vegetables, although the primary sources of lycopene are tomato and tomato products. Additionally, egg yolk is a highly bioavailable source of lutein and zeaxanthin. These carotenoids are available in supplement form. However, intervention trials with large doses of beta-carotene found an adverse effect on the incidence of lung cancer in smokers and workers exposed to asbestos. Until the efficacy and safety of taking supplements containing these nutrients can be determined, current dietary recommendations of diets high in fruits and vegetables are advised.

Bioavailability of synthetic and biosynthetic deuterated lycopene in humans.

Current knowledge of the bioavailability of lycopene in humans is limited due to the inability to distinguish newly administered lycopene from the body reserves of lycopene. A quantitative method to assess the absorption and relative bioavailability of newly absorbed synthetic or natural lycopene was developed using two deuterated lycopene sources, in conjunction with an advanced LC/APCI-MS (liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry) to analyze newly absorbed lycopene in blood samples of study subjects. Two subjects (1 male and 1 female) consumed hydroponically grown tomatoes containing deuterium-enriched lycopene (80-84 g wet weight tomato containing 16.3 and 17.4 micromol lycopene, respectively) and two subjects (1 male, and 1 female) consumed 11 micromol synthetic (2)H(10) lycopene in 6 g of corn oil. Tomatoes were steamed and pureed. The doses were given together with a liquid formulated drink with 25% energy from fat. Our results showed that up to 34 days after taking an oral (2)H(10) lycopene dose (synthetic or from tomato) with a liquid formula drink, the area under the curve of the average serum percent enrichment response of synthetic lycopene reached 33.9 (+/-1.7) nmol-day/micromol lycopene in the dose, whereas that of lycopene from the tomato dose was 11.8 (+/-0.3) nmol-day/mumol lycopene in the dose. Our study provides evidence that the absorption of physiological levels of lycopene in intrinsically labeled tomatoes can be studied in humans. From these preliminary investigations, we find that the bioavailability of synthetic lycopene in oil appears to be about three times higher than that of lycopene from steamed and pureed tomatoes.

Enzymatic and oxidative metabolites of lycopene.

Using the post-mitochondrial fraction of rat intestinal mucosa, we have investigated lycopene metabolism. The incubation media was composed of NAD+, KCI, and DTT with or without added lipoxygenase. The addition of lipoxygenase into the incubation significantly increased the production of lycopene metabolites. The enzymatic incubation products of 2H10 lycopene were separated using high-performance liquid chromatography and analyzed by UV/Vis spectrophotometer and atmospheric pressure chemical ionization-mass spectroscopy. We have identified two types of products: cleavage products and oxidation products. The cleavage products are likely: (1) 3-keto-apo-13-lycopenone (C18H24O2 or 6,10,14-trimethyl-12-one-3,5,7,9,13-pentadecapentaen-2-one) with lambdamax = 365 nm and m/z =272 and (2) 3,4-dehydro-5,6-dihydro-15-apo-lycopenal (C20H28O or 3,7,11,15-tetramethyl-2,4,6,8,12,14-hexadecahexaen-l-al) with lambdamax= 380 nm and m/z = 284. The oxidative metabolites are likely: (3) 2-ene-5,8-lycopenal-furanoxide (C37H50O) with lambdamax = 415 nm, 435 nm, and 470 nm, and m/z = 510; (4) lycopene-5, 6, 5', 6'-diepoxide (C40H56O2) with lambdamax = 415 nm, 440 nm, and 470 nm, and m/z =568; (5) lycopene-5,8-furanoxide isomer (I) (C40H56O2) with lambdamax = 410 nm, 440 nm, and 470 nm, and m/z = 552; (6) lycopene-5,8-epoxide isomer (II) (C40H56O) with lambdamax = 410, 440, 470 nm, and m/z = 552; and (7) 3-keto-lycopene-5',8'-furanoxide (C40H54O2) with lambdamax = 400 nm, 420 nm, and 450 nm, and m/z = 566. These results demonstrate that both central and excentric cleavage of lycopene occurs in the rat intestinal mucosa in the presence of soy lipoxygenase.

Biomarkers of antioxidant capacity in the hydrophilic and lipophilic compartments of human plasma.

Antioxidants located in both the hydrophilic and lipophilic compartments of plasma are actively involved as a defense system against reactive oxygen species (ROS), which are continuously generated in the body due to both normal metabolism and disease. However, when the production of ROS is not controlled, it leads to cellular lipid, protein, and DNA damage in biological systems. Several assays to measure 'total' antioxidant capacity of plasma have been developed to study the involvement of oxidative stress in pathological conditions and to evaluate the functional bioavailability of dietary antioxidants. Conventional assays to determine antioxidant capacity primarily measure the antioxidant capacity in the aqueous compartment of plasma. Consequently, water-soluble antioxidants such as ascorbic acid, uric acid and protein thiols mainly influence these assays, whereas fat-soluble antioxidants such as tocopherols and carotenoids play only a minor role. However, there are active interactions among antioxidants located in the hydrophilic and lipophilic compartments of plasma. Therefore, new approaches to define the 'true' total antioxidant capacity of plasma should reflect the antioxidant network between water- and fat-soluble antioxidants in plasma. Revelation of the mechanism of action of antioxidants and their true antioxidant potential will help us to optimize the antioxidant defenses in the body.

Beta-carotene and beta-apo-14'-carotenoic acid prevent the reduction of retinoic acid receptor beta in benzo[a]pyrene-treated normal human bronchial epithelial cells.

Low-dose beta-carotene (BC) supplementation, such as would be provided by daily consumption of approximately 5-9 servings of fruits and vegetables, has no apparent detrimental effects, but rather appears to have a protective effect against cigarette smoke-induced lung lesions in ferrets. In the present study, we investigated the effects of BC, beta-apo-14'-carotenoic acid (14'CA), or benzo[a]pyrene (BP; a primary lung carcinogen from cigarette smoke) treatments, either alone or in combination, on cell growth and expression of the retinoic acid receptor (RAR) of normal human bronchial epithelial (NHBE) cells. We found that both BC and 14'CA inhibited the growth of NHBE cells (P < 0.05) with or without BP. The level of RARbeta, a tumor suppressor, but not RARalpha or RARgamma, was reduced by 50% in the NHBE cells treated with BP. However, treatment with either BC or 14'CA significantly induced the expression of RARbeta in the NHBE cells, and prevented the reduction of RARbeta by BP. Furthermore, 14'CA transactivated the RARbeta promoter primarily via its conversion to retinoic acid (RA). In the presence of 3-mercaptopropionic acid, an inhibitor of fatty acid oxidation, both RA formation and transactivation activity from 14'CA were decreased. These observations indicate that the growth inhibitory effects of BC and beta-apo-carotenoic acid are through their conversion to RA and upregulation of RARbeta.

Enzymatic and oxidative metabolites of lycopene.

Using the post-mitochondrial fraction of rat intestinal mucosa, we have investigated lycopene metabolism. The incubation media was composed of NAD(+), KCl, and DTT with or without added lipoxygenase. The addition of lipoxygenase into the incubation significantly increased the production of lycopene metabolites. The enzymatic incubation products of (2)H(10) lycopene were separated using high-performance liquid chromatography and analyzed by UV/Vis spectrophotometer and atmospheric pressure chemical ionization-mass spectroscopy. We have identified two types of products: cleavage products and oxidation products. The cleavage products are likely: (1) 3-keto-apo-13-lycopenone (C(18)H(24)O(2) or 6,10,14-trimethyl-12-one-3,5,7,9,13-pentadecapentaen-2-one) with lambdamax = 365 nm and m/z = 272 and (2) 3,4-dehydro-5,6-dihydro-15,15'-apo-lycopenal (C(20)H(28)O or 3,7,11,15-tetramethyl-2,4,6,8,12,14-hexadecahexaen-1-al) with lambdamax = 380 nm and m/z = 284. The oxidative metabolites are likely: (3) 2-apo-5,8-lycopenal-furanoxide (C(37)H(50)O) with lambdamax = 415 nm, 435 nm, and 470 nm, and m/z = 510; (4) lycopene-5, 6, 5', 6'-diepoxide (C(40)H(56)O(2)) with lambdamax = 415 nm, 440 nm, and 470 nm, and m/z = 568; (5) lycopene-5,8-furanoxide isomer (I) (C(40)H(56)O) with lambdamax = 410 nm, 440nm, and 470 nm, and m/z = 552; (6) lycopene-5,8-epoxide isomer (II) (C(40)H(56)O) with lambdamax = 410, 440, 470 nm, and m/z = 552; and (7) 3-keto-lycopene-5',8'-furanoxide (C(40)H(54)O(2)) with lambdamax = 400 nm, 420 nm, and 450 nm, and m/z = 566. These results demonstrate that both central and excentric cleavage of lycopene occurs in the rat intestinal mucosa in the presence of soy lipoxygenase.

The activities of antioxidant nutrients in human plasma depend on the localization of attacking radical species.

The oxidation of endogenous antioxidant nutrients in human plasma was determined to examine their activities against free radicals generated in the aqueous and lipid compartments of plasma. Free radicals were induced at a constant rate in the aqueous compartment by the hydrophilic radical generator, 2,2'-azobis-(2-amidinopropane)dihydrochloride (AAPH; 10-20 mmol/L) and in the lipid compartment by the lipophilic radical generator, 2,2'-azobis(4-methoxy-2,4-dimethylvaleronitrile) (MeO-AMVN; 1-2 mmol/L). The depletion of endogenous plasma antioxidant nutrients (lutein, cryptoxanthin, beta-carotene, lycopene, alpha-tocopherol, ascorbic acid, uric acid) was determined after incubation with either AAPH or MeO-AMVN at 37 degrees C using HPLC. The oxidation of the aqueous and lipid compartments of plasma was selectively monitored by a fluorimetric method using either the hydrophilic probe, 2',7'-dichlorodihydrofluorescein (DCFH) or the lipophilic probe, 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a, 4a-diaza-s-indacene-3-undecanoic acid (BODIPY 581/591). When plasma was incubated with AAPH, the rates of consumption of the antioxidant nutrients were as follows: ascorbic acid > alpha-tocopherol > uric acid > lycopene > lutein > cryptoxanthin > beta-carotene. When plasma was incubated with MeO-AMVN, alpha-tocopherol and carotenoids were depleted at similar rates and ahead of the major water-soluble antioxidants. Our study indicates that the antioxidant nutrients present in both the lipid and aqueous compartments can remove free radicals generated in plasma, and their activity depends on the localization of the attacking radical species.

Carotenoid-radical interactions.

Carotenoids have been reported to react with virtually any radical species likely to be encountered in a biological system. The products of such reactions are frequently short-lived radical species that can decay to more stable products. In some cases, stable adducts can be observed, but in the majority of interactions with radicals, carotenoids break down to degradation products very similar to what is seen with oxidative degradation. It is only recently that the biological activity of these breakdown products has begun to be investigated.

Biologic mechanisms of the protective role of lutein and zeaxanthin in the eye.

The macular region of the primate retina is yellow in color due to the presence of the macular pigment, composed of two dietary xanthophylls, lutein and zeaxanthin, and another xanthophyll, meso-zeaxanthin. The latter is presumably formed from either lutein or zeaxanthin in the retina. By absorbing blue-light, the macular pigment protects the underlying photoreceptor cell layer from light damage, possibly initiated by the formation of reactive oxygen species during a photosensitized reaction. There is ample epidemiological evidence that the amount of macular pigment is inversely associated with the incidence of age-related macular degeneration, an irreversible process that is the major cause of blindness in the elderly. The macular pigment can be increased in primates by either increasing the intake of foods that are rich in lutein and zeaxanthin, such as dark-green leafy vegetables, or by supplementation with lutein or zeaxanthin. Although increasing the intake of lutein or zeaxanthin might prove to be protective against the development of age-related macular degeneration, a causative relationship has yet to be experimentally demonstrated.

(-)-Epigallocatechin-3-gallate prevents oxidative damage in both the aqueous and lipid compartments of human plasma.

When human plasma was exposed to the hydrophilic radical initiator, AAPH, (-)-epigallocatechin-(3)-gallate (EGCG) dose-dependently inhibited the aqueous compartment oxidation (IC(50)=0.72 microM) (monitored by DCFH oxidation) and spared the lipophilic antioxidants, alpha-tocopherol, and carotenoids, but not ascorbic acid. When radicals were selectively induced in the lipid compartment by the lipophilic radical initiator, MeO-AMVN, EGCG spared alpha-tocopherol, but not carotenoids and inhibited the lipid compartment oxidation (monitored by BODIPY 581/591) with a potency lower than that found in the aqueous compartment (IC(50)=4.37 microM). Our results indicate that EGCG, mainly localized in the aqueous compartment, effectively quenches aqueous radical species, thus limiting their diffusion into the lipid compartment and preventing lipid-soluble antioxidant depletion. Further, ESR experiments confirmed that EGCG recycled alpha-tocopherol through a H-transfer mechanism at the aqueous/lipid interface affording an additional protective mechanism to the lipid compartment of plasma.

Excentric cleavage products of beta-carotene inhibit estrogen receptor positive and negative breast tumor cell growth in vitro and inhibit activator protein-1-mediated transcriptional activation.

Both retinoids and carotenoids are potentially useful chemopreventive agents. In this study we tested the effect of synthetic excentric cleavage products of beta-carotene on the growth of the MCF-7, Hs578T and MDA-MB-231 human breast cancer cells. The apo-beta-carotenoic acids (beta-apo-CA) beta-apo-14'-, beta-apo-12'-, beta-apo-10'- and beta-apo-8'-CA are structurally similar to all-trans-retinoic acid (atRA) but have different side chain lengths. Nine days of treatment with atRA inhibited MCF-7 and Hs578T cell proliferation in a dose-dependent manner. beta-apo-14'-CA and beta-apo-12'-CA significantly inhibited MCF-7 growth, whereas only beta-apo-14'-CA inhibited Hs578T growth. None of these treatments inhibited the growth of MDA-MB-231 cells. Potential mechanisms of growth inhibition, i.e., regulation of the cell cycle control proteins E2F1 and retinoblastoma protein (RB), and effect on activator protein-1 (AP-1)-mediated gene regulation were examined. beta-apo-14'-CA and atRA inhibited the expression of E2F1 protein in MCF-7 and Hs578T cells. beta-apo-14'-CA, beta-apo-12'-CA and atRA down-regulated RB protein expression in MCF-7 but not in Hs578T cells. The effect of phorbol ester-induced transcriptional activation of a collagenase promoter-reporter gene construct was strongly inhibited by 1 micromol/L beta-apo-14'-CA, atRA (MCF-7, Hs578T) or beta-apo-12'-CA (MCF-7). These effects were due neither to cellular conversion of beta-apo-CA to atRA nor to high affinity binding to the retinoid acid receptors. Thus, beta-apo-CAs were effective inhibitors of breast tumor cell proliferation, possibly mediated through down-regulation of cell cycle regulatory proteins and/or inhibition of AP-1 transcriptional activity. The ability of beta-apo-CA to regulate breast tumor cell growth independently of conversion to atRA suggests that these compounds may have fewer side effects than retinoids and, therefore, have a potential chemotherapeutic value that deserves further examination.

Possible biologic mechanisms for a protective role of xanthophylls.

This contribution surveys the evidence linking the presence of the two xanthophylls, lutein and zeaxanthin, to a protective role in the macular region of the retina. Although the evidence is still associative in nature, it is biologically plausible, and may be resolved with additional intervention trials.