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Carrots, tomatoes and cocoa: Research on dietary antioxidants in Düsseldorf
Archives of Biochemistry and Biophysics 595 (2016) 125e131
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Carrots, tomatoes and cocoa: Research on dietary antioxidants in Düsseldorf Wilhelm Stahl Institute of Biochemistry and Molecular Biology I, Faculty of Medicine, Heinrich-Heine University Düsseldorf, P.O. Box 101007, D-40001, Düsseldorf, Germany
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 June 2015 Accepted 8 June 2015
Dietary antioxidants, their biological effects and underlying mechanisms of action are key topics of research at the Institute of Biochemistry and Molecular Biology I at the Heinrich-Heine University in Düsseldorf where Helmut Sies is active now since more than 35 years. In the present article his research activity on carotenoids is summarized including studies on their bioavailability, antioxidant properties, cellular signaling and dermatological effects. Additionally, comparable studies on cocoa polyphenols are described. © 2015 Elsevier Inc. All rights reserved.
Keywords: Carotenoids Lycopene Polyphenols Flavonoids Antioxidants Skin
1. Introduction
2. Carotenoids
Looking for an interesting and challenging alternative to a current job in pharmaceutical industry I wrote an application to Helmut Sies for a post doc position and he invited me to Düsseldorf in May 1990. I already knew about his major research activities but he offered me to work on a compound I never heard of before. Lycopene was at that time just one of some other 450 carotenoids and research in this area was focused mainly on vitamin A precursors like b-carotene. Just a year before, his group had published an article on the singlet oxygen quenching activities of different carotenoids and it turned out that lycopene is one of the most efficient naturally occurring quenchers of singlet molecular oxygen [1]; by the way the paper is among of the most cited of Helmut Sies. This finding was a result of systematically performed test series on hundreds of natural and non-natural compounds. Their singlet oxygen quenching properties were investigated using naphthalene endoperoxides as generators of singlet molecular oxygen and monomol light emission assessed as a read out [2]. One of my first tasks in the new group was to establish analytical methods for the determination of carotenoids in all kind of samples including human blood, several tissues and various food items identified as major dietary sources of carotenoids.
Plants and other photosynthetic organisms (e.g. algae) as well as bacteria and funghi are capable to synthesize carotenoids de novo. Most of the common carotenoids are composed of a central carbon chain of alternating single and double bonds substituted with different cyclic or acyclic end groups [3]. Carotenoids are responsible for the yellow, orange and red color of many fruits and flowers and are part of the light harvesting complex in higher plants. bCarotene, a-carotene, and lycopene (Fig. 1) are prominent members of the carotene subgroup which includes carotenoids composed only of carbon and hydrogen atoms. Xanthophylls, however, carry at least one oxygen atom. Zeaxanthin, lutein, a- and b-cryptoxanthin, canthaxanthin and astaxanthin are important xanthophylls with hydroxy- and keto groups as structural elements. Due to the extended system of conjugated double bonds, a great number of different cis/trans (E/Z) configurations are possible for a given carotenoid [4] and the compounds tend to isomerize to form a mixture of mono- and poly-cis isomers in addition to the all-trans form. This process is observed in homogenous solutions and tissues. Incorporated into the food matrix carotenoids are more resistant towards isomerization. Usually, the all-trans form is thermodynamically most stable and predominant in nature but several cis isomers of carotenoids are frequently found in most samples. In this context we developed an improved reversed-phase HPLC methodology capable to separate five geometrical isomers of
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Fig. 1. Structures of selected carotenoids and flavonoids.
b-carotene and seven of lycopene in human serum and tissues. In addition to all-trans b-carotene its 13-cis-isomer was the predominant geometrical form in human serum. In tissues, however, considerable amounts of 9-cis- and traces of 15-cis-b-carotene were also detected. In contrast to b-carotene, the pattern of lycopene isomers was quite similar in human blood and selected organs like liver, testes, adrenal gland, or kidney [5]. The newly developed method was reliable and provided stable separation of the major carotene isomers. Thus it could be used in human studies on carotenoid bioavailability with focus on the isomer patterns of single compounds which were initiated at the Institute in Düsseldorf in 1991. 3. Bioavailability of carotenoids Uptake and distribution of carotenoids from the diet is determined by a number of factors including coingestion of dietary lipids, presence of fiber, or food processing [6]. As lipophilic compounds, carotenoids are absorbed via the lymphatic pathway where the formation of micelles from fatty acids, monoglycerides and bile acids is essential and the intestinal uptake is improved by the additional consumption of oil, margarine or butter. Additionally, the particle size of uncooked food modulates carotenoid uptake. The bioavailability of carotenoids from pureed or finely chopped vegetables is considerably higher as compared to whole or sliced fresh vegetables. We could show that the bioavailability of lycopene is high from tomato paste and demonstrate that mild cooking in the presence of lipids increases the absorption of carotenoids [7]. Additional heating, however, augments isomerization of the naturally occurring all-trans double bonds to cis configuration. Lycopene bioavailability from a single dose of fresh tomatoes or tomato paste (23 mg lycopene), ingested together with 15 g corn oil, was compared by analyzing carotenoid concentrations
in the human chylomicron fraction [8]. The lycopene isomer pattern was the same in both fresh tomatoes and tomato paste. Ingestion of tomato paste was found to yield about three-fold higher total and all-trans lycopene peak concentrations and fourfold higher area under curve (AUC) responses than ingestion of fresh tomatoes. The same was calculated for lycopene cis-isomers, but only the AUC response for the cis-isomers was statistically significantly higher after ingestion of tomato paste. No difference was observed in the a- and b-carotene levels. The triacylglycerol response in chylomicrons was comparable after both treatments. Differences in carotenoids bioavailability from various sources (tomato products) might in part explain results from epidemiological studies where protective effects against prostate cancer were associated with specific food items [9]. Here an increased intake of tomato sauce was correlated with a diminished risk, while no association was found with the consumption of tomato juice. For studies on the availability of b-carotene, geometrical isomers we used an extract of the alga Dunaliella salina composed of mainly all-trans and 9-cis b-carotene. In humans the bioavailability of 9-cis b-carotene was far less as compared to its all-trans analog [10]. However, considerable amounts of this isomer were detectable in human tissues [5]. At about the same time it was shown that 9-cis b-carotene is a precursor for 9-cis retinoic acid [11], a high affinity ligand for the RXR-receptor. Selective absorption and or metabolism was suggested to be relevant in the control of liganddependent nuclear receptors. Treatment with carotenoids is a supportive therapy for patients suffering from erythropoietic protoporphyria as evaluated in cooperation with the Clinic of Dermatology of our University. In an intervention study with patients we compared the availability of bcarotene from Dunaliella salina and synthetic origin at high dose levels of 50e150 mg/d [12]. Upon daily ingestion, b-carotene serum level increased from day 0 to day 30; no further elevation was
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present as mono- or di-esters, only free zeaxanthin and b-cryptoxanthin were found in human samples. In contrast the bioavailability of the pepper-specific carotenoids capsanthin and capsorubin from paprika oleoresin was low [16]. Over a long period of time one specific focus of our joint research was on antioxidant properties of dietary constituents and their mechanisms of action. 4. Carotenoids as antioxidants
Helmut Sies and the author at the Gordon Research Conference on Carotenoids in Ventura (CA) 1995.
observed between days 30 and 150. Steady state levels of b-carotene were somewhat lower with the natural isomeric mixture from algae than with synthetic b-carotene. Under all treatment conditions, the level of 9-cis-beta-carotene was very low even when the isomeric mixture containing high amounts of 9-cis-b-carotene was applied. Another provitamin A carotenoid is b-cryptoxanthin present in several fruits such as oranges, tangerines, papaya, mango, and peaches. In tangerines cryptoxanthin is present as carotenol fatty acid esters, predominant are b-cryptoxanthin laurate, myristate and palmitate [13]. Smaller amounts of lutein and zeaxanthin esters are also detectable. Additionally, b-carotene and low levels of free b-cryptoxanthin, lutein and zeaxanthin are found in tangerine juice. Upon ingestion of tangerine juice concentrate, rich in bcryptoxanthin esters we detected increasing amounts of free bcryptoxanthin in human chylomicrons and serum [14]. Peak levels of the carotenoid in chylomicrons were reached at 6 h; the concentration returned to basal levels 9 h after ingestion. No b-cryptoxanthin esters were detected in chylomicrons or serum, indicating efficient cleavage in the intestine before the carotenoid is incorporated into lipoproteins by the liver. Fatty acid esters of zeaxanthin and lutein were present in low amounts in tangerine juice. Like with b-cryptoxanthin, no esters appeared in serum or chylomicrons, suggesting that the cleavage of carotenoid esters prior to release into the lymphatic circulation occurs generally in human oxocarotenoid biokinetics. However, low amounts of carotenol esters, mainly derivatives of lutein, zeaxanthin, 20 ,3ʹ-anhydrolutein, a-cryptoxanthin and b-cryptoxanthin are detectable in human skin [15]. Comparable results were observed for red pepper carotenoids. Although the xanthophylls in paprika oleoresin were mainly
Carotenoids contribute together with other dietary antioxidants as tocopherols or ascorbate to the antioxidant network which further includes powerful enzymes and secondary defense systems [17,18]. Although the specific needs and amounts of antioxidants provided with the diet or supplements is controversially discussed their activity has been proven in numerous experiments. Various strategies of defense are operative against oxidative damage and carotenoids are involved in the scavenging of two major reactive oxygen species, singlet molecular oxygen (1O2), and peroxyl radicals. Further, they are effective deactivators of electronically excited sensitizer molecules, which are involved in the generation of radicals and singlet oxygen, an important task in the photosynthetic apparatus of plants [19,20]. Singlet oxygen quenching by carotenoids is mediated by physical or chemical quenching [21,22]. In the case of physical quenching excitation energy is transferred from 1O2 to the carotenoid, yielding ground state oxygen and an excited triplet state carotenoid. From the excited carotenoid energy is dissipated to obtain a ground state carotenoid and thermal energy. In this process the carotenoid remains intact and can undergo further cycles of singlet oxygen quenching. The rate constants for the reaction of carotenoids with singlet oxygen are in the range of 109 M1 s1, i.e. near diffusion control. Lycopene, b-carotene and other carotenoids are the most efficient natural 1O2-quenchers and their quenching activity is closely related to the number of conjugated double bonds present in the molecule as we could show in a fruitful cooperation with Hans-Dieter Martin from the Chemical Department of our University [1,23,24]. Carotenoids efficiently scavenge peroxyl radicals, especially at low oxygen tension and contribute to the defense against lipid peroxidation [25]. Under specific conditions carotenoids may also act as prooxidants. Such properties have been determined in vitro and discussed in context with adverse effects observed upon b-carotene supplementation at high levels. Here an increased risk for lung cancer was determined in a risk-population after long-term intake of b-carotene at doses beyond physiological levels [26]. The antioxidant defense system of the organism is a complex network and it has been speculated that interactions between structurally different compounds with variable antioxidant activity provides additional protection against increased oxidative stress. Synergistic interactions against UVA-induced photooxidative stress have been described in cultured human fibroblasts when combinations of antioxidants were applied with b-carotene as main component. In comparison to the individual antioxidants, vitamins E, C and b-carotene exhibited cooperative synergistic effects scavenging reactive nitrogen species. The cooperative interaction between b-carotene and a-tocopherol was also examined in a membrane model. A combination of both lipophilic antioxidants resulted in an inhibition of lipid peroxidation significantly greater than the sum of the individual effects [27e29]. We investigated the antioxidant activity of carotenoid mixtures in multilamellar liposomes, determining the inhibition of the formation of thiobarbituric acid-reactive substances after oxidative challenge [30]. The data provided evidence that mixtures of carotenoids are more effective than the single compounds. Interestingly this synergistic
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effect was most pronounced when lycopene or lutein were present. It was suggested that the effects are related to different physicochemical properties and/or the location of the compounds in the lipid layer. Xanthophylls such as lutein or zeaxanthin span the membrane, while carotenes which lack hydrophilic substituents, remain entirely within the inner part of the membrane and retain a substantial degree of mobility. Therefore, specific orientations of carotenoids might provide shielding effects that account for synergistic properties. In a model with unilamellar liposomes we studied the interaction carotenoids with glutathione (GSH) [31]. In comparison to control, lipid oxidation was decreased when the liposomes were loaded with GSH. Upon additional incorporation of b-carotene, lycopene or lutein, the resistance of unilamellar liposomes towards lipid peroxidation was further modified. The data demonstrated that interactions between GSH and carotenoids may improve resistance of biological membranes towards lipid peroxidation. However, different carotenoids exhibit specific properties, and the level for optimal protection varies between the carotenoids. At high levels prooxidant effects were determined. Lutein and zeaxanthin are the major carotenoids of the human macula lutea. Epidemiological data and experimental studies suggest that they are important for the protection of this tissue against damaging effects of irradiation with blue light. We studied the blue light filter efficacy of carotenoids in unilamellar liposomes loaded in the hydrophilic core space with a fluorescent dye, excitable by blue light [32]. Carotenoids were incorporated into the lipophilic membrane. In this system lutein and zeaxanthin proved to be the most efficient blue light filters. Again it was suggested that the orientation of the compounds in the membrane plays a major role. 5. Carotenoids and gap junctional intercellular communication Carotenoids are efficient antioxidants but additionally exhibit biological activities which are not directly related to their antioxidant properties [33]. The parent compounds or their metabolites (e.g. retinoids or apocarotenals) have impact on cellular signaling pathways, influence the expression of certain genes or act as inhibitors of regulatory enzymes. Thus, they reveal additional biological effects which might be of importance in context with the prevention of degenerative diseases related to the consumption of a diet rich in antioxidants. With respect to carotenoids this implies effects on retinoid-dependent signaling with impact on the regulation of cell growth or induction of detoxifying enzymes such as cytochrome P450-dependent monooxygenases. One focus of the research activities in our group was on the stimulatory effects of carotenoids on gap junctional intercellular communication (GJIC) which has been discussed as one of the possible biochemical mechanisms underlying the cancer-preventive properties of these compounds. This research area was stimulated by first studies from John Bertrams group in Hawaii [34,35]. Gap junctions are specialized microdomaines of the plasma membrane and provide a specific pathway for intercellular signaling required for the coordination of various cellular functions [36]. They consist of an array of cell-to-cell channels connecting the cytosol of neighboring cells which allows the diffusion of small molecules (<1000 Da) within a network of coupled cells. For a functional unit two half-channels (connexons), each provided from one of the coupled cells, are connected where each half channel consists of a hexamer of specific proteins which belong to the gene family of connexins. Connexin proteins exhibit four helical transmembrane domains, two extracellular loops, a cytoplasmic loop, and cytoplasmic N- and C-terminal domains. In the transmembrane and extracellular domains the sequences are most conserved and the extracellular loop contains three cysteines which are required
for channel function. Several subtypes of connexins have been identified and there are differences in the expression of connexin genes in various tissues. GJIC is involved in the regulation of growth, transmission of developmental signals, coordination of muscle contraction, and maintenance of metabolic homeostasis. It has been speculated that disturbances in intercellular communication via gap junctions interfere with the regulation of growth and differentiation in tumor cells and may also be involved in the pathogenesis of cardiac diseases predisposing for arrhythmias. Normal cells are contact-inhibited and have functional GJIC whereas most tumor cells exhibit dysfunctional homologous or heterologous GJIC. Typically, cancer cells lack of growth control and are not able to terminally differentiate which has at least in part been attributed to disturbed GJIC. Among the major carotenoids present in human blood and tissue, b-carotene, cryptoxanthine, zeaxanthin and lutein are efficient inducers of gap junctional communication. a-Carotene and lycopene are less active compounds. Studying structureeactivity relationships we and others have shown that the stimulatory effects of carotenoids on GJIC are not limited to the subgroup of provitamin A compounds [35,37]. Also carotenoids with substituents at the ionone ring are active. However, the position and chemical properties of the substituent at the six-membered ring seem of limited influence on the activity of different carotenoids. Echinenone, canthaxanthin and 4-hydroxy-b-carotene induce GJIC as does retro-dehydro-b-carotene, a structural analog of b-carotene. However, members of the carotenoid family which carry a five-carbon ring system, like the dinorcanthaxanthin are less active. The sixcarbon ring carotenoid canthaxanthin is more active than its fivemembered ring analog. We did not find stimulatory effects for capsorubin or violerythrin (5-ring carotenoids) and methyl-bixin. The mechanism of up-regulated gap junctional intercellular communication in the presence of carotenoids is still not fully understood. Carotenoids are efficient antioxidants but at least two studies demonstrated that the effects of carotenoids on GJIC are independent from their antioxidant activities [35,37]. Neither their singlet oxygen quenching properties nor their inhibitory effects on lipid peroxidation are correlated with the regulatory effects on GJIC. Multiple pathways for the regulation of GJIC are described including effects on the transcription rate of connexin genes or stabilization of connexin mRNA [36]. Connexins may also be modified posttranslationally, and phosphorylation is a common modification of these proteins affecting protein trafficking. Treatment of cells with carotenoids leads to an increased expression of connexin43, one of the most prominent gap junction proteins. There is evidence that metabolites and oxidation products of the carotenoids are the ultimately active compounds activating GJIC and it has been suggested that retinoic acid-dependent pathways are involved in the regulation of connexin expression. All-trans and 13-cis 4-oxo-retinoic acid were isolated as decomposition products of canthaxanthin and shown to be active in the cell communication assay [38]. In cooperation with the group of Prof. Martin we studied the effects of several eccentric cleavage products of canthaxanthin on GJIC. 11-apo-Canthaxanthin-11-oic acid, 13-apo-canthaxanthin13-oic acid, and 14ʹ-apo-canthaxanthin-14ʹ-oic acid) were less active than the central cleavage product 4-oxo-retinoic acid [39]. These data suggest that the major biological effects of canthaxanthin on GJIC are related to activities mediated by compounds related to retinoic acid. Several studies have been published on metabolites and oxidation products of lycopene. We have shown that acycloretinoic acid, a putative metabolite of lycopene, is much less active than retinoic acid and 4-oxo-retinoic acid with respect to inductory effects on GJIC and retinoid-related signaling [40]. Thus, it was discussed that lycopene affects GJIC independent of retinoic
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acid related pathways. However, other lycopene metabolites and oxidation products such as 2,6-cyclolycopene-1,5-diol or 2,7,11trimethyl-tetradecahexaen-1,14-dial induce gap junctional communication [41,42]. The latter was obtained by complete in vitro oxidation of lycopene with hydrogen peroxide/osmium tetroxide. All of the major carotenoids present in the human organism, which include a- and b-carotene, lutein, zeaxanthin, b-cryptoxanthin, and lycopene, stimulate GJIC. However, we determined completely different effects with the carotenoid astaxanthin which is used as a food colorant and naturally occurring in algae and crustaceans [43]. In contrast to canthaxanthin which was used for control, astaxanthin strongly suppressed GJIC. Inhibition was reversed when astaxanthin was withdrawn. We could show that upon incubation with astaxanthin a change in the phosphorylation pattern of connexin43 was induced, shifting from higher to lower phosphorylation states. Thus we concluded that astaxanthin affects channel function by changing the phosphorylation pattern of connexin43. Further to carotenoids, retinoids and apocarotenoids other compounds were studied for their modulatory effects on GJIC. Among the molecules with stimulating proporties were vitamin D, thyroid hormones and interstingly thalidomide one of the most active human teratogens [44e46]. 6. Carotenoids: photoprotection Irradiation of skin with light, especially UV light, poses an oxidative challenge and carotenoids as a part of the antioxidant network may help to protect biologically important structures [47]. If not regenerated carotenoids are partially consumed in antioxidative defense reactions and human studies have shown that carotenoid levels in plasma and skin decrease upon UV-irradiation; lycopene was lost preferentially as compared to other carotenoids. Consequently, protective effects of a supplementation with carotenoids against UV-induced photooxidation has been postulated, supported by earlier findings that treatment with carotenoids ameliorates phototoxicity in photosensitivity disorders [48]. Up to date b-carotene is the most popular photoprotective compound sold in various supplements determined for systemic photoprotection and evidence is provided that b-carotene protects against the development of a sunburn reaction. Synthetic b-carotene as well as b-carotene from natural sources is efficient. Dunaliella salina is a saltewater micro-alga that accumulates high amounts of carotenoids (mainly a- and b-carotene) under appropriate growth conditions and we used algal extracts as a natural source for human intervention studies on photoprotection. This was the first study among numerous others that we performed in a fruitful cooperation with Profs Heinrich and Tronnier from the University of Witten to investigate photoprotective properties of dietary constituents and their impact on skin conditions. Under supplementation with carotenoids derived from Dunaliella salina at dose levels of about 24 mg/d, b-carotene levels in skin and serum increase and erythema formation (sunburn) induced with a solar light simulator was significantly diminished [49]. We found that the protection was even more efficient when the algal carotenoids were combined with vitamin E. Interestingly there was a considerable interindividual difference in the bioavailability of carotenoids and protective effects. As mentioned above long-term use of b-carotene may increase the risk of lung cancer in smokers and thus safety concerns have been raised for b-carotene supplements when ingested over several years at non-physiologically high dose levels [26]. In view of this, we performed a study where b-carotene was partially substituted by other carotenoids [50]. The human intervention study showed
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that a carotenoid mixture consisting of b-carotene, lycopene, and lutein, with 8 mg of each compound provides a photoprotective effect comparable to that 24 mg of b-carotene alone. Based on the outcome of seven human intervention studies a meta-analysis was performed estimating the effects of b-carotene in the prevention of UV-induced erythema (sunburn) [51]. This analysis provides evidence that a supplementation with b-carotene is associated with a protection against the development of a sunburn reaction. The protection is moderate compared to the efficacy of a modern sunscreen and has been estimated to be comparable to a sun protection factor of three to four. In contrast to topical sunscreens, protection with systemic b-carotene builds up over period of several weeks which has also been shown as a result of the metaanalysis. Short-term studies showed no effects even when high doses of the carotenoid were applied. Successful intervention studies suggest that minimal doses of about 10e15 mg b-carotene/ d are required for protection and the treatment period must exceed 10 wk. Lycopene is an open-ring carotenoid and has no provitamin A activity. It is found only in a few food items such as watermelon, pink grapefruit, apricots or rosehips. However, the major dietary sources of lycopene are tomatoes and tomato products. We performed an human intervention study for photoprotection with a lycopene-rich tomato paste providing about 16 mg/d of carotenoid over a period of 10 wk. Supplementation was accompanied by an increase of lycopene serum levels [52]. At week 10 of the study, erythema formation induced with a solar light simulator was lower in the group that consumed tomato paste compared to controls, but no significant difference between groups was found at week 4 of treatment. The study demonstrated that it is feasible to achieve protection against UV-light-induced sunburn by ingesting carotenoids from a dietary source. It also showed that the protective compound must be accumulated over a longer period of time (10 wk), before protection is achieved. In following studies several other lycopene containing products were investigated including a lycopene-rich carrot juice from the variety NutriRed, a supplement from tomato extract (Lyco-Mato), a drink with tomato extract (Lyco-Guard), and synthetic lycopene product [53]. All source provided similar lycopene doses per day and treatment was for 12 wk. Photoprotective effects were determined for all of these lycopene sources, however, protection was not statistically significant with synthetic lycopene. Further analyses revealed the presence of high amounts of phytoene and phytofluene in tomato products. Both are precursor compounds for lycopene were found in all of the sources except of the synthetic lycopene. At the end of the study, their plasma levels were increased in the samples of all participants who ingested tomato based products. Phytoene and phytofluene are non-colored carotenoids and carry only 3 and 5 conjugated double bonds in their structure. However, their absorption maxima are at wavelengths of the UVB and UVA range. We speculate that they directly absorb damaging UV light thus contribute to the photoprotection following dietary intervention with tomato products. It should be noted that UV-induced erythema was evaluated as a measure of sunburn in most of these studies which reflects a delayed inflammatory response of cutaneous tissue following UVB irradiation. Only few data from human studies provide evidence for a decrease in UV-induced damage to DNA or other skin constituents. No clear correlation has been shown up till now between the intake of carotenoids or carotenoid-rich food and the risk for skin cancer. We also investigated the prevention of photooxidation by lycopene, b-carotene and lutein in cultured human fibroblasts which were exposed to UVB irradiation [54]. A measure of oxidative damage was the formation of thiobarbituric acid-reactive
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substances (TBARS). Lycopene, b-carotene, and lutein were capable of decreasing UV-induced formation of TBARS significantly. The amounts of carotenoid needed for optimal protection were divergent for lycopene, b-carotene, and lutein, respectively. Doseresponse curves showed a u-formed shape. Beyond the optimum levels, further increases of carotenoid levels led to prooxidant effects. The data suggest that there is an optimum level for a carotenoid to reveal photoprotection. At lower levels photoprotective effects are diminished, whereas at higher levels prooxidative effects may occur. Among other compounds we investigated a carotenoid with an unusual structure. 3,30 -Dihydroxyisorenieratene (DHIR) carries a polyenic backbone substituted with phenolic end groups (Fig. 1). DHIR is present in the bacterium Brevibacterium linens which is used in the dairy industry for the production of various red smear cheeses. The antioxidant activity of the compound exceeds that of other carotenoids like astaxanthin, cryptoxanthin, zeaxanthin or lutein. Due to the presence of a polyenic and phenolic substructure DHIR acts as a bifunctional radical scavenger and quenches singlet molecular oxygen. It also inhibits the formation of UVB-induced thymidine dimers and protects proteins from photoxidative damage [55e57]. In addition to our studies with carotenoids, we examined the question whether high-flavanol cocoa provides photoprotection against UV-induced erythema [58]. In this study, two groups of volunteers consumed either a high flavanol or low flavanol cocoa powder over a period of 12 weeks. Photoprotective effects were measured before and during the intervention. Following exposure of selected skin areas toward 1.25 minimal erythemal dose (MED), UV-induced erythema was significantly decreased in the highflavanol group after 6 and 12 week of treatment, respectively, whereas no change was found in the low-flavanol group. Here we also determined impact on skin parameters not directly related to photoprotection. 7. Skin effects Function and structure of the skin is modulated by dietary factors and several micronutrients and there is evidence that nutrients have impact on skin properties when ingested regularly [59]. In the study mentioned before, ingestion of high flavanol cocoa also led to an increase in blood flow of cutaneous and subcutaneous tissues, and to a significant increase in skin density, thickness, and skin hydration [58]. These parameters were not affected in the low flavanol group. Evaluation of the skin surface showed a significant decrease of skin roughness and scaling in the high flavanol cocoa group, whereas again no change was found in the low flavanol cocoa group, comparing the starting values with week 6 and 12. The data show that ingestion of dietary flavanols from cocoa contributes not only to endogenous photoprotection but also improves dermal blood circulation. Cocoa flavanols further affect cosmetically relevant parameters of skin surface and hydration. In a shortterm study we demonstrated that dermal blood flow is acutely increased and oxygen saturation of dermal hemoglobin is elevated upon intake of high flavanol cocoa [60]. ()-Epicatechin (Fig. 1) is the major dietary flavanol in cocoa and there is evidence that it is responsible for vasodilatory effects observed after cocoa consumption. We have provided direct evidence that the compound increases nitric oxide levels in human endothelial cells. Elevation of NO in endothelial cells via epicatechin may be mediated by the inhibition of NADPH oxidase [61]. 8. Conclusion A lot of interesting, basic research and directly applicable work
on the effects of carotenoids and flavonoids was initiated and put forward within the last 30 years in Düsseldorf. Thank you Helmut! Conflict of interest The author declares no conflict of interest. References [1] P. Di Mascio, S. Kaiser, H. Sies, Lycopene as the most efficient biological carotenoid singlet oxygen quencher, Arch. Biochem. Biophys. 274 (1989) 532e538. [2] K. Briviba, H. Sies, Biological singlet oxygen quenchers assessed by monomol light emission, Methods Enzymol. 319 (2000) 222e226. [3] W. Stahl, H. Sies, Carotenoids: occurence, biochemical activities, and bioavailability, in: L. Packer, M. Hiramatsu, T. Yoshikawa (Eds.), Anitoxidant Food Supplements in Human Health, Academic Press, San Diego, 1999, pp. 183e202. [4] G. Britton, Structure and properties of carotenoids in relation to function, FASEB J. 9 (1995) 1551e1558. [5] W. Stahl, W. Schwarz, A.R. Sundquist, H. Sies, cis-trans isomers of lycopene and b-carotene in human serum and tissues, Arch. Biochem. Biophys. 294 (1992) 173e177. [6] K.H. van het Hof, C.E. West, J.A. Weststrate, J.G. Hautvast, Dietary factors that affect the bioavailability of carotenoids, J. Nutr. 130 (2000) 503e506. [7] W. Stahl, H. Sies, Uptake of lycopene and its geometrical isomers is greater from heat-processed than from unprocessed tomato juice in humans, J. Nutr. 122 (1992) 2161e2166. €rtner, W. Stahl, H. Sies, Lycopene is more bioavailable from tomato paste [8] C. Ga than from fresh tomatoes, Am. J. Clin. Nutr. 66 (1997) 116e122. [9] E. Giovannucci, A. Ascherio, E.B. Rimm, M.J. Stampfer, G.A. Colditz, W.C. Willett, Intake of carotenoids and retinol in relation to risk of prostate cancer, J. Natl. Cancer Inst. 87 (1995) 1767e1776. [10] W. Stahl, W. Schwarz, J. von Laar, H. Sies, all-trans-b-Carotene preferentially accumulates in human chylomicrons and very low density lipoproteins compared with the 9-cis geometrical isomer, J. Nutr. 125 (1995) 2128e2133. [11] X. Hebuterne, X.D. Wang, E.J. Johnson, N.I. Krinsky, R.M. Russell, Intestinal absorption and metabolism of 9-cis-beta-carotene in vivo: biosynthesis of 9cis-retinoic acid, J. Lipid Res. 36 (1995) 1264e1273. [12] J. von Laar, W. Stahl, K. Bolsen, G. Goerz, H. Sies, b-Carotene serum levels in patients with erythropoietic protoporphyria on treatment with the synthetic all-trans isomer or a natural isomer mixture of b-carotene, J. Photochem. Photobiol. B Biol. 33 (1996) 157e162. [13] T. Wingerath, W. Stahl, D. Kirsch, R. Kaufmann, H. Sies, Fruit juice carotenol fatty acid esters and carotenoids as identified by matrix-assisted laser desorption ionization (MALDI) mass spectrometry, J. Agric. Food Chem. 44 (1996) 2006e2013. [14] T. Wingerath, W. Stahl, H. Sies, b-Cryptoxanthin selectively increases in human chylomicrons upon ingestion of tangerine concentrate rich in b-cryptoxanthin esters, Arch. Biochem. Biophys. 324 (1995) 385e390. [15] T. Wingerath, H. Sies, W. Stahl, Xanthophyll esters in human skin, Arch. Biochem. Biophys. 355 (1998) 271e274. [16] A. Perez-Galvez, H.D. Martin, H. Sies, W. Stahl, Incorporation of carotenoids from paprika oleoresin into human chylomicrons, Br. J. Nutr. 89 (2003) 787e793. [17] H. Sies, Biochemistry of oxidative stress, Angew. Chem. Int. Ed. Engl. 25 (1986) 1058e1071. [18] H. Sies, Oxidative stress, in: G. Fink (Ed.), Encyclopedia of Stress, Academic Press, 2000, pp. 102e105. [19] A. Telfer, What is beta-carotene doing in the photosystem II reaction centre? Philos. Trans. R. Soc. Lond B Biol. Sci. 357 (2002) 1431e1439. [20] T.G. Truscott, The photophysics and photochemistry of the carotenoids, J. Photochem. Photobiol. B Biol. 6 (1990) 359e371. [21] W. Stahl, H. Sies, Physical quenching of singlet oxygen and cis-trans isomerization of carotenoids, Ann. N. Y. Acad. Sci. 691 (1993) 10e19. [22] W. Stahl, H. Sies, Antioxidant activity of carotenoids, Mol. Asp. Med. 24 (2003) 345e351. € ver, [23] D. Baltschun, S. Beutner, K. Briviba, H.D. Martin, J. Paust, M. Peters, S. Ro H. Sies, W. Stahl, et al., Singlet oxygen quenching abilities of carotenoids, Liebigs Ann. (1997) 1887e1893. [24] S. Beutner, B. Bloedorn, S. Frixel, I. Hern andez Blanco, T. Hoffmann, H.D. Martin, B. Mayer, P. Noack, C. Ruck, et al., Quantitative assessment of antioxidant properties of natural colorants and related compounds: carotenoids, flavonoids and indigoids, in: Application of Natural Colorants for Foods, Nutraceuticals, Confectionery, Beverages, Cosmetics, The Hereld Organization ed., San Diego, 2000, pp. 1e19. [25] G.W. Burton, K.U. Ingold, b-Carotene: an unusual type of lipid antioxidant, Science 224 (1984) 569e573. [26] The ATBC-Study Group, The effect of vitamin E and beta carotene on the incidence of lung cancer and other cancers in male smokers, N. Engl. J. Med. 330 (1994) 1029e1035. [27] F. Bohm, R. Edge, E.J. Land, D.J. McGarvey, T.G. Truscott, Carotenoids enhance
W. Stahl / Archives of Biochemistry and Biophysics 595 (2016) 125e131 vitamin E antioxidant efficiency, J. Am. Chem. Soc. 119 (1997) 621e622. [28] F. Bohm, R. Edge, D.J. McGarvey, T.G. Truscott, Beta-carotene with vitamins E and C offers synergistic cell protection against NOx, FEBS Lett. 436 (1998) 387e389. [29] P. Palozza, N.I. Krinsky, beta-Carotene and alpha-tocopherol are synergistic antioxidants, Arch. Biochem. Biophys. 297 (1992) 184e187. [30] W. Stahl, A. Junghans, B. de Boer, E. Driomina, K. Briviba, H. Sies, Carotenoid mixtures protect multilamellar liposomes against oxidative damage: synergistic effects of lycopene and lutein, FEBS Lett. 427 (1998) 305e308. [31] A. Junghans, H. Sies, W. Stahl, Carotenoid-containing unilamellar liposomes loaded with glutathione: a model to study hydrophobic-hydrophilic antioxidant interaction, Free Rad. Res. 33 (2000) 801e808. [32] A. Junghans, H. Sies, W. Stahl, Macular pigments lutein and zeaxanthin as blue light filters studied in liposomes, Arch. Biochem. Biophys. 391 (2001) 160e164. [33] W. Stahl, N. Ale-Agha, M.C. Polidori, Non-antioxidant properties of carotenoids, Biol. Chem. 383 (2002) 553e558. [34] J.S. Bertram, Dietary carotenoids, connexins and cancer: what is the connection? Biochem. Soc. Trans. 32 (2004) 985e989. [35] L.X. Zhang, R.V. Cooney, J.S. Bertram, Carotenoids up-regulate gap junctional communication: correlation with cancer chemoprevention properties, Carcinogenesis 12 (1991) 2109e2114. [36] M.S. Nielsen, L.N. Axelsen, P.L. Sorgen, V. Verma, M. Delmar, N.H. HolsteinRathlou, Gap junctions, Compr. Physiol. 2 (2012) 1981e2035. [37] W. Stahl, S. Nicolai, K. Briviba, M. Hanusch, G. Broszeit, M. Peters, H.D. Martin, H. Sies, Biological activities of natural and synthetic carotenoids: induction of gap junctional communication and singlet oxygen quenching, Carcinogenesis 18 (1997) 89e92. [38] M. Hanusch, W. Stahl, W.A. Schulz, H. Sies, Induction of gap junctional communication by 4-oxoretinoic acid generated from its precursor canthaxanthin, Arch. Biochem. Biophys. 317 (1995) 423e428. [39] V.B. Teicher, N. Kucharski, H.D. Martin, P. van der Saag, H. Sies, W. Stahl, Biological activities of apo-canthaxanthinois acids related to gap junctional communication, Arch. Biochem. Biophys. 365 (1999) 150e155. [40] W. Stahl, J. von Laar, H.D. Martin, T. Emmerich, H. Sies, Stimulation of gap junctional communication: comparison of acyclo-retinoic acid and lycopene, Arch. Biochem. Biophys. 373 (2000) 271e274. [41] O. Aust, N. Ale-Agha, L. Zhang, H. Wollersen, H. Sies, W. Stahl, Lycopene oxidation product enhances gap junctional communication, Food Chem. Toxicol. 41 (2003) 1399e1407. [42] J.S. Bertram, T. King, L. Fukishima, F. Khachik, Enhanced activity of an oxidation product of lycopene found in tomato products and human serum relevant to cancer prevention, in: C.K. Sen, H. Sies, P.A. Baeuerle (Eds.), Antioxidant and Redox Regulation of Genes, Academic Press, London, 2000, pp. 409e424. [43] F. Daubrawa, H. Sies, W. Stahl, Astaxanthin diminishes gap junctional intercellular communication in primary human fibroblasts, J. Nutr. 135 (2005) 2507e2511. [44] A. Clairmont, D. Tessmann, A. Stock, S. Nicolai, W. Stahl, H. Sies, Induction of gap junctional intercellular communication by vitamin D in human skin fibroblasts is dependent on the nuclear vitamin D receptor, Carcinogenesis 17 (1996) 1389e1391.
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[45] S. Nicolai, H. Sies, W. Stahl, Stimulation of gap junctional intercellular communication by thalidomide and thalidomide analogs in human skin fibroblasts, Biochem. Pharmacol. 53 (1997) 1553e1557. [46] A. Stock, H. Sies, W. Stahl, Enhancement of gap junctional communication and connexin43 expression by thyroid hormones, Biochem. Pharmacol. 55 (1998) 475e479. [47] H. Sies, W. Stahl, Nutritional protection against skin damage from sunlight, Annu. Rev. Nutr. 24 (2004) 173e200. [48] M.M. Mathews-Roth, M.A. Pathak, T.B. Fitzpatrick, L.C. Harber, E.H. Kass, bCarotene as an oral protective agent in erythropoietic protoporphyria, J. Am. Med. Assoc. 228 (1974) 1004e1008. [49] W. Stahl, U. Heinrich, H. Jungmann, H. Sies, H. Tronnier, Carotenoids and carotenoids plus vitamin E protect against ultraviolet light-induced erythema in humans, Am. J. Clin. Nutr. 71 (2000) 795e798. [50] U. Heinrich, C. Gartner, M. Wiebusch, O. Eichler, H. Sies, H. Tronnier, W. Stahl, Supplementation with beta-carotene or a similar amount of mixed carotenoids protects humans from UV-induced erythema, J. Nutr. 133 (2003) 98e101. [51] W. Kopcke, J. Krutmann, Protection from sunburn with beta-caroteneea meta-analysis, Photochem. Photobiol. 84 (2008) 284e288. [52] W. Stahl, U. Heinrich, S. Wiseman, O. Eichler, H. Sies, H. Tronnier, Dietary tomato paste protects against ultraviolet light-induced erythema in humans, J. Nutr. 131 (2001) 1449e1451. [53] O. Aust, W. Stahl, H. Sies, H. Tronnier, U. Heinrich, Supplementation with tomato-based products increases lycopene, phytofluene, and phytoene levels in human serum and protects against UV-light-induced erythema, Intern. J. Vit. Nutr. Res. 75 (2005) 54e60. [54] O. Eichler, H. Sies, W. Stahl, Divergent optimum levels of lycopene, betacarotene and lutein protecting against UVB irradiation in human fibroblastst, Photochem. Photobiol. 75 (2002) 503e506. [55] K. Lutter, S.S. De, S. Kock, K.D. Kroncke, H.D. Martin, T. Wagener, W. Stahl, 3,3ʹDihydroxyisorenieratene prevents UV-induced formation of reactive oxygen species and the release of protein-bound zinc ions in human skin fibroblasts, Mol. Nutr. Food Res. 54 (2010) 285e291. [56] H.D. Martin, S. Kock, R. Scherrers, K. Lutter, T. Wagener, C. Hundsdorfer, S. Frixel, K. Schaper, H. Ernst, et al., 3,3ʹ-Dihydroxyisorenieratene, a natural carotenoid with superior antioxidant and photoprotective properties, Angew. Chem. Int. Ed. Engl. 48 (2009) 400e403. [57] S. Wagener, T. Volker, S.S. De, H. Ernst, W. Stahl, 3,3ʹ-Dihydroxyisorenieratene and isorenieratene prevent UV-induced DNA damage in human skin fibroblasts, Free Radic. Biol. Med. 53 (2012) 457e463. [58] U. Heinrich, K. Neukam, H. Tronnier, H. Sies, W. Stahl, Long-term ingestion of high flavanol cocoa provides photoprotection against UV-induced erythema and improves skin condition in women, J. Nutr. 136 (2006) 1565e1569. [59] G. Nguyen, A. Torres, Systemic antioxidants and skin health, J. Drugs Dermatol. 11 (2012) e1ee4. [60] K. Neukam, W. Stahl, H. Tronnier, H. Sies, U. Heinrich, Consumption of flavanol-rich cocoa acutely increases microcirculation in human skin, Eur. J. Nutr. 46 (2007) 53e56. [61] T. Schewe, Y. Steffen, H. Sies, How do dietary flavanols improve vascular function? A position paper, Arch. Biochem. Biophys. 476 (2008) 102e106.
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Carrots, tomatoes and cocoa: Research on dietary antioxidants in Düsseldorf Wilhelm Stahl Institute of Biochemistry and Molecular Biology I, Faculty of Medicine, Heinrich-Heine University Düsseldorf, P.O. Box 101007, D-40001, Düsseldorf, Germany
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 June 2015 Accepted 8 June 2015
Dietary antioxidants, their biological effects and underlying mechanisms of action are key topics of research at the Institute of Biochemistry and Molecular Biology I at the Heinrich-Heine University in Düsseldorf where Helmut Sies is active now since more than 35 years. In the present article his research activity on carotenoids is summarized including studies on their bioavailability, antioxidant properties, cellular signaling and dermatological effects. Additionally, comparable studies on cocoa polyphenols are described. © 2015 Elsevier Inc. All rights reserved.
Keywords: Carotenoids Lycopene Polyphenols Flavonoids Antioxidants Skin
1. Introduction
2. Carotenoids
Looking for an interesting and challenging alternative to a current job in pharmaceutical industry I wrote an application to Helmut Sies for a post doc position and he invited me to Düsseldorf in May 1990. I already knew about his major research activities but he offered me to work on a compound I never heard of before. Lycopene was at that time just one of some other 450 carotenoids and research in this area was focused mainly on vitamin A precursors like b-carotene. Just a year before, his group had published an article on the singlet oxygen quenching activities of different carotenoids and it turned out that lycopene is one of the most efficient naturally occurring quenchers of singlet molecular oxygen [1]; by the way the paper is among of the most cited of Helmut Sies. This finding was a result of systematically performed test series on hundreds of natural and non-natural compounds. Their singlet oxygen quenching properties were investigated using naphthalene endoperoxides as generators of singlet molecular oxygen and monomol light emission assessed as a read out [2]. One of my first tasks in the new group was to establish analytical methods for the determination of carotenoids in all kind of samples including human blood, several tissues and various food items identified as major dietary sources of carotenoids.
Plants and other photosynthetic organisms (e.g. algae) as well as bacteria and funghi are capable to synthesize carotenoids de novo. Most of the common carotenoids are composed of a central carbon chain of alternating single and double bonds substituted with different cyclic or acyclic end groups [3]. Carotenoids are responsible for the yellow, orange and red color of many fruits and flowers and are part of the light harvesting complex in higher plants. bCarotene, a-carotene, and lycopene (Fig. 1) are prominent members of the carotene subgroup which includes carotenoids composed only of carbon and hydrogen atoms. Xanthophylls, however, carry at least one oxygen atom. Zeaxanthin, lutein, a- and b-cryptoxanthin, canthaxanthin and astaxanthin are important xanthophylls with hydroxy- and keto groups as structural elements. Due to the extended system of conjugated double bonds, a great number of different cis/trans (E/Z) configurations are possible for a given carotenoid [4] and the compounds tend to isomerize to form a mixture of mono- and poly-cis isomers in addition to the all-trans form. This process is observed in homogenous solutions and tissues. Incorporated into the food matrix carotenoids are more resistant towards isomerization. Usually, the all-trans form is thermodynamically most stable and predominant in nature but several cis isomers of carotenoids are frequently found in most samples. In this context we developed an improved reversed-phase HPLC methodology capable to separate five geometrical isomers of
E-mail address: [email protected]. http://dx.doi.org/10.1016/j.abb.2015.06.023 0003-9861/© 2015 Elsevier Inc. All rights reserved.
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Fig. 1. Structures of selected carotenoids and flavonoids.
b-carotene and seven of lycopene in human serum and tissues. In addition to all-trans b-carotene its 13-cis-isomer was the predominant geometrical form in human serum. In tissues, however, considerable amounts of 9-cis- and traces of 15-cis-b-carotene were also detected. In contrast to b-carotene, the pattern of lycopene isomers was quite similar in human blood and selected organs like liver, testes, adrenal gland, or kidney [5]. The newly developed method was reliable and provided stable separation of the major carotene isomers. Thus it could be used in human studies on carotenoid bioavailability with focus on the isomer patterns of single compounds which were initiated at the Institute in Düsseldorf in 1991. 3. Bioavailability of carotenoids Uptake and distribution of carotenoids from the diet is determined by a number of factors including coingestion of dietary lipids, presence of fiber, or food processing [6]. As lipophilic compounds, carotenoids are absorbed via the lymphatic pathway where the formation of micelles from fatty acids, monoglycerides and bile acids is essential and the intestinal uptake is improved by the additional consumption of oil, margarine or butter. Additionally, the particle size of uncooked food modulates carotenoid uptake. The bioavailability of carotenoids from pureed or finely chopped vegetables is considerably higher as compared to whole or sliced fresh vegetables. We could show that the bioavailability of lycopene is high from tomato paste and demonstrate that mild cooking in the presence of lipids increases the absorption of carotenoids [7]. Additional heating, however, augments isomerization of the naturally occurring all-trans double bonds to cis configuration. Lycopene bioavailability from a single dose of fresh tomatoes or tomato paste (23 mg lycopene), ingested together with 15 g corn oil, was compared by analyzing carotenoid concentrations
in the human chylomicron fraction [8]. The lycopene isomer pattern was the same in both fresh tomatoes and tomato paste. Ingestion of tomato paste was found to yield about three-fold higher total and all-trans lycopene peak concentrations and fourfold higher area under curve (AUC) responses than ingestion of fresh tomatoes. The same was calculated for lycopene cis-isomers, but only the AUC response for the cis-isomers was statistically significantly higher after ingestion of tomato paste. No difference was observed in the a- and b-carotene levels. The triacylglycerol response in chylomicrons was comparable after both treatments. Differences in carotenoids bioavailability from various sources (tomato products) might in part explain results from epidemiological studies where protective effects against prostate cancer were associated with specific food items [9]. Here an increased intake of tomato sauce was correlated with a diminished risk, while no association was found with the consumption of tomato juice. For studies on the availability of b-carotene, geometrical isomers we used an extract of the alga Dunaliella salina composed of mainly all-trans and 9-cis b-carotene. In humans the bioavailability of 9-cis b-carotene was far less as compared to its all-trans analog [10]. However, considerable amounts of this isomer were detectable in human tissues [5]. At about the same time it was shown that 9-cis b-carotene is a precursor for 9-cis retinoic acid [11], a high affinity ligand for the RXR-receptor. Selective absorption and or metabolism was suggested to be relevant in the control of liganddependent nuclear receptors. Treatment with carotenoids is a supportive therapy for patients suffering from erythropoietic protoporphyria as evaluated in cooperation with the Clinic of Dermatology of our University. In an intervention study with patients we compared the availability of bcarotene from Dunaliella salina and synthetic origin at high dose levels of 50e150 mg/d [12]. Upon daily ingestion, b-carotene serum level increased from day 0 to day 30; no further elevation was
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present as mono- or di-esters, only free zeaxanthin and b-cryptoxanthin were found in human samples. In contrast the bioavailability of the pepper-specific carotenoids capsanthin and capsorubin from paprika oleoresin was low [16]. Over a long period of time one specific focus of our joint research was on antioxidant properties of dietary constituents and their mechanisms of action. 4. Carotenoids as antioxidants
Helmut Sies and the author at the Gordon Research Conference on Carotenoids in Ventura (CA) 1995.
observed between days 30 and 150. Steady state levels of b-carotene were somewhat lower with the natural isomeric mixture from algae than with synthetic b-carotene. Under all treatment conditions, the level of 9-cis-beta-carotene was very low even when the isomeric mixture containing high amounts of 9-cis-b-carotene was applied. Another provitamin A carotenoid is b-cryptoxanthin present in several fruits such as oranges, tangerines, papaya, mango, and peaches. In tangerines cryptoxanthin is present as carotenol fatty acid esters, predominant are b-cryptoxanthin laurate, myristate and palmitate [13]. Smaller amounts of lutein and zeaxanthin esters are also detectable. Additionally, b-carotene and low levels of free b-cryptoxanthin, lutein and zeaxanthin are found in tangerine juice. Upon ingestion of tangerine juice concentrate, rich in bcryptoxanthin esters we detected increasing amounts of free bcryptoxanthin in human chylomicrons and serum [14]. Peak levels of the carotenoid in chylomicrons were reached at 6 h; the concentration returned to basal levels 9 h after ingestion. No b-cryptoxanthin esters were detected in chylomicrons or serum, indicating efficient cleavage in the intestine before the carotenoid is incorporated into lipoproteins by the liver. Fatty acid esters of zeaxanthin and lutein were present in low amounts in tangerine juice. Like with b-cryptoxanthin, no esters appeared in serum or chylomicrons, suggesting that the cleavage of carotenoid esters prior to release into the lymphatic circulation occurs generally in human oxocarotenoid biokinetics. However, low amounts of carotenol esters, mainly derivatives of lutein, zeaxanthin, 20 ,3ʹ-anhydrolutein, a-cryptoxanthin and b-cryptoxanthin are detectable in human skin [15]. Comparable results were observed for red pepper carotenoids. Although the xanthophylls in paprika oleoresin were mainly
Carotenoids contribute together with other dietary antioxidants as tocopherols or ascorbate to the antioxidant network which further includes powerful enzymes and secondary defense systems [17,18]. Although the specific needs and amounts of antioxidants provided with the diet or supplements is controversially discussed their activity has been proven in numerous experiments. Various strategies of defense are operative against oxidative damage and carotenoids are involved in the scavenging of two major reactive oxygen species, singlet molecular oxygen (1O2), and peroxyl radicals. Further, they are effective deactivators of electronically excited sensitizer molecules, which are involved in the generation of radicals and singlet oxygen, an important task in the photosynthetic apparatus of plants [19,20]. Singlet oxygen quenching by carotenoids is mediated by physical or chemical quenching [21,22]. In the case of physical quenching excitation energy is transferred from 1O2 to the carotenoid, yielding ground state oxygen and an excited triplet state carotenoid. From the excited carotenoid energy is dissipated to obtain a ground state carotenoid and thermal energy. In this process the carotenoid remains intact and can undergo further cycles of singlet oxygen quenching. The rate constants for the reaction of carotenoids with singlet oxygen are in the range of 109 M1 s1, i.e. near diffusion control. Lycopene, b-carotene and other carotenoids are the most efficient natural 1O2-quenchers and their quenching activity is closely related to the number of conjugated double bonds present in the molecule as we could show in a fruitful cooperation with Hans-Dieter Martin from the Chemical Department of our University [1,23,24]. Carotenoids efficiently scavenge peroxyl radicals, especially at low oxygen tension and contribute to the defense against lipid peroxidation [25]. Under specific conditions carotenoids may also act as prooxidants. Such properties have been determined in vitro and discussed in context with adverse effects observed upon b-carotene supplementation at high levels. Here an increased risk for lung cancer was determined in a risk-population after long-term intake of b-carotene at doses beyond physiological levels [26]. The antioxidant defense system of the organism is a complex network and it has been speculated that interactions between structurally different compounds with variable antioxidant activity provides additional protection against increased oxidative stress. Synergistic interactions against UVA-induced photooxidative stress have been described in cultured human fibroblasts when combinations of antioxidants were applied with b-carotene as main component. In comparison to the individual antioxidants, vitamins E, C and b-carotene exhibited cooperative synergistic effects scavenging reactive nitrogen species. The cooperative interaction between b-carotene and a-tocopherol was also examined in a membrane model. A combination of both lipophilic antioxidants resulted in an inhibition of lipid peroxidation significantly greater than the sum of the individual effects [27e29]. We investigated the antioxidant activity of carotenoid mixtures in multilamellar liposomes, determining the inhibition of the formation of thiobarbituric acid-reactive substances after oxidative challenge [30]. The data provided evidence that mixtures of carotenoids are more effective than the single compounds. Interestingly this synergistic
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effect was most pronounced when lycopene or lutein were present. It was suggested that the effects are related to different physicochemical properties and/or the location of the compounds in the lipid layer. Xanthophylls such as lutein or zeaxanthin span the membrane, while carotenes which lack hydrophilic substituents, remain entirely within the inner part of the membrane and retain a substantial degree of mobility. Therefore, specific orientations of carotenoids might provide shielding effects that account for synergistic properties. In a model with unilamellar liposomes we studied the interaction carotenoids with glutathione (GSH) [31]. In comparison to control, lipid oxidation was decreased when the liposomes were loaded with GSH. Upon additional incorporation of b-carotene, lycopene or lutein, the resistance of unilamellar liposomes towards lipid peroxidation was further modified. The data demonstrated that interactions between GSH and carotenoids may improve resistance of biological membranes towards lipid peroxidation. However, different carotenoids exhibit specific properties, and the level for optimal protection varies between the carotenoids. At high levels prooxidant effects were determined. Lutein and zeaxanthin are the major carotenoids of the human macula lutea. Epidemiological data and experimental studies suggest that they are important for the protection of this tissue against damaging effects of irradiation with blue light. We studied the blue light filter efficacy of carotenoids in unilamellar liposomes loaded in the hydrophilic core space with a fluorescent dye, excitable by blue light [32]. Carotenoids were incorporated into the lipophilic membrane. In this system lutein and zeaxanthin proved to be the most efficient blue light filters. Again it was suggested that the orientation of the compounds in the membrane plays a major role. 5. Carotenoids and gap junctional intercellular communication Carotenoids are efficient antioxidants but additionally exhibit biological activities which are not directly related to their antioxidant properties [33]. The parent compounds or their metabolites (e.g. retinoids or apocarotenals) have impact on cellular signaling pathways, influence the expression of certain genes or act as inhibitors of regulatory enzymes. Thus, they reveal additional biological effects which might be of importance in context with the prevention of degenerative diseases related to the consumption of a diet rich in antioxidants. With respect to carotenoids this implies effects on retinoid-dependent signaling with impact on the regulation of cell growth or induction of detoxifying enzymes such as cytochrome P450-dependent monooxygenases. One focus of the research activities in our group was on the stimulatory effects of carotenoids on gap junctional intercellular communication (GJIC) which has been discussed as one of the possible biochemical mechanisms underlying the cancer-preventive properties of these compounds. This research area was stimulated by first studies from John Bertrams group in Hawaii [34,35]. Gap junctions are specialized microdomaines of the plasma membrane and provide a specific pathway for intercellular signaling required for the coordination of various cellular functions [36]. They consist of an array of cell-to-cell channels connecting the cytosol of neighboring cells which allows the diffusion of small molecules (<1000 Da) within a network of coupled cells. For a functional unit two half-channels (connexons), each provided from one of the coupled cells, are connected where each half channel consists of a hexamer of specific proteins which belong to the gene family of connexins. Connexin proteins exhibit four helical transmembrane domains, two extracellular loops, a cytoplasmic loop, and cytoplasmic N- and C-terminal domains. In the transmembrane and extracellular domains the sequences are most conserved and the extracellular loop contains three cysteines which are required
for channel function. Several subtypes of connexins have been identified and there are differences in the expression of connexin genes in various tissues. GJIC is involved in the regulation of growth, transmission of developmental signals, coordination of muscle contraction, and maintenance of metabolic homeostasis. It has been speculated that disturbances in intercellular communication via gap junctions interfere with the regulation of growth and differentiation in tumor cells and may also be involved in the pathogenesis of cardiac diseases predisposing for arrhythmias. Normal cells are contact-inhibited and have functional GJIC whereas most tumor cells exhibit dysfunctional homologous or heterologous GJIC. Typically, cancer cells lack of growth control and are not able to terminally differentiate which has at least in part been attributed to disturbed GJIC. Among the major carotenoids present in human blood and tissue, b-carotene, cryptoxanthine, zeaxanthin and lutein are efficient inducers of gap junctional communication. a-Carotene and lycopene are less active compounds. Studying structureeactivity relationships we and others have shown that the stimulatory effects of carotenoids on GJIC are not limited to the subgroup of provitamin A compounds [35,37]. Also carotenoids with substituents at the ionone ring are active. However, the position and chemical properties of the substituent at the six-membered ring seem of limited influence on the activity of different carotenoids. Echinenone, canthaxanthin and 4-hydroxy-b-carotene induce GJIC as does retro-dehydro-b-carotene, a structural analog of b-carotene. However, members of the carotenoid family which carry a five-carbon ring system, like the dinorcanthaxanthin are less active. The sixcarbon ring carotenoid canthaxanthin is more active than its fivemembered ring analog. We did not find stimulatory effects for capsorubin or violerythrin (5-ring carotenoids) and methyl-bixin. The mechanism of up-regulated gap junctional intercellular communication in the presence of carotenoids is still not fully understood. Carotenoids are efficient antioxidants but at least two studies demonstrated that the effects of carotenoids on GJIC are independent from their antioxidant activities [35,37]. Neither their singlet oxygen quenching properties nor their inhibitory effects on lipid peroxidation are correlated with the regulatory effects on GJIC. Multiple pathways for the regulation of GJIC are described including effects on the transcription rate of connexin genes or stabilization of connexin mRNA [36]. Connexins may also be modified posttranslationally, and phosphorylation is a common modification of these proteins affecting protein trafficking. Treatment of cells with carotenoids leads to an increased expression of connexin43, one of the most prominent gap junction proteins. There is evidence that metabolites and oxidation products of the carotenoids are the ultimately active compounds activating GJIC and it has been suggested that retinoic acid-dependent pathways are involved in the regulation of connexin expression. All-trans and 13-cis 4-oxo-retinoic acid were isolated as decomposition products of canthaxanthin and shown to be active in the cell communication assay [38]. In cooperation with the group of Prof. Martin we studied the effects of several eccentric cleavage products of canthaxanthin on GJIC. 11-apo-Canthaxanthin-11-oic acid, 13-apo-canthaxanthin13-oic acid, and 14ʹ-apo-canthaxanthin-14ʹ-oic acid) were less active than the central cleavage product 4-oxo-retinoic acid [39]. These data suggest that the major biological effects of canthaxanthin on GJIC are related to activities mediated by compounds related to retinoic acid. Several studies have been published on metabolites and oxidation products of lycopene. We have shown that acycloretinoic acid, a putative metabolite of lycopene, is much less active than retinoic acid and 4-oxo-retinoic acid with respect to inductory effects on GJIC and retinoid-related signaling [40]. Thus, it was discussed that lycopene affects GJIC independent of retinoic
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acid related pathways. However, other lycopene metabolites and oxidation products such as 2,6-cyclolycopene-1,5-diol or 2,7,11trimethyl-tetradecahexaen-1,14-dial induce gap junctional communication [41,42]. The latter was obtained by complete in vitro oxidation of lycopene with hydrogen peroxide/osmium tetroxide. All of the major carotenoids present in the human organism, which include a- and b-carotene, lutein, zeaxanthin, b-cryptoxanthin, and lycopene, stimulate GJIC. However, we determined completely different effects with the carotenoid astaxanthin which is used as a food colorant and naturally occurring in algae and crustaceans [43]. In contrast to canthaxanthin which was used for control, astaxanthin strongly suppressed GJIC. Inhibition was reversed when astaxanthin was withdrawn. We could show that upon incubation with astaxanthin a change in the phosphorylation pattern of connexin43 was induced, shifting from higher to lower phosphorylation states. Thus we concluded that astaxanthin affects channel function by changing the phosphorylation pattern of connexin43. Further to carotenoids, retinoids and apocarotenoids other compounds were studied for their modulatory effects on GJIC. Among the molecules with stimulating proporties were vitamin D, thyroid hormones and interstingly thalidomide one of the most active human teratogens [44e46]. 6. Carotenoids: photoprotection Irradiation of skin with light, especially UV light, poses an oxidative challenge and carotenoids as a part of the antioxidant network may help to protect biologically important structures [47]. If not regenerated carotenoids are partially consumed in antioxidative defense reactions and human studies have shown that carotenoid levels in plasma and skin decrease upon UV-irradiation; lycopene was lost preferentially as compared to other carotenoids. Consequently, protective effects of a supplementation with carotenoids against UV-induced photooxidation has been postulated, supported by earlier findings that treatment with carotenoids ameliorates phototoxicity in photosensitivity disorders [48]. Up to date b-carotene is the most popular photoprotective compound sold in various supplements determined for systemic photoprotection and evidence is provided that b-carotene protects against the development of a sunburn reaction. Synthetic b-carotene as well as b-carotene from natural sources is efficient. Dunaliella salina is a saltewater micro-alga that accumulates high amounts of carotenoids (mainly a- and b-carotene) under appropriate growth conditions and we used algal extracts as a natural source for human intervention studies on photoprotection. This was the first study among numerous others that we performed in a fruitful cooperation with Profs Heinrich and Tronnier from the University of Witten to investigate photoprotective properties of dietary constituents and their impact on skin conditions. Under supplementation with carotenoids derived from Dunaliella salina at dose levels of about 24 mg/d, b-carotene levels in skin and serum increase and erythema formation (sunburn) induced with a solar light simulator was significantly diminished [49]. We found that the protection was even more efficient when the algal carotenoids were combined with vitamin E. Interestingly there was a considerable interindividual difference in the bioavailability of carotenoids and protective effects. As mentioned above long-term use of b-carotene may increase the risk of lung cancer in smokers and thus safety concerns have been raised for b-carotene supplements when ingested over several years at non-physiologically high dose levels [26]. In view of this, we performed a study where b-carotene was partially substituted by other carotenoids [50]. The human intervention study showed
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that a carotenoid mixture consisting of b-carotene, lycopene, and lutein, with 8 mg of each compound provides a photoprotective effect comparable to that 24 mg of b-carotene alone. Based on the outcome of seven human intervention studies a meta-analysis was performed estimating the effects of b-carotene in the prevention of UV-induced erythema (sunburn) [51]. This analysis provides evidence that a supplementation with b-carotene is associated with a protection against the development of a sunburn reaction. The protection is moderate compared to the efficacy of a modern sunscreen and has been estimated to be comparable to a sun protection factor of three to four. In contrast to topical sunscreens, protection with systemic b-carotene builds up over period of several weeks which has also been shown as a result of the metaanalysis. Short-term studies showed no effects even when high doses of the carotenoid were applied. Successful intervention studies suggest that minimal doses of about 10e15 mg b-carotene/ d are required for protection and the treatment period must exceed 10 wk. Lycopene is an open-ring carotenoid and has no provitamin A activity. It is found only in a few food items such as watermelon, pink grapefruit, apricots or rosehips. However, the major dietary sources of lycopene are tomatoes and tomato products. We performed an human intervention study for photoprotection with a lycopene-rich tomato paste providing about 16 mg/d of carotenoid over a period of 10 wk. Supplementation was accompanied by an increase of lycopene serum levels [52]. At week 10 of the study, erythema formation induced with a solar light simulator was lower in the group that consumed tomato paste compared to controls, but no significant difference between groups was found at week 4 of treatment. The study demonstrated that it is feasible to achieve protection against UV-light-induced sunburn by ingesting carotenoids from a dietary source. It also showed that the protective compound must be accumulated over a longer period of time (10 wk), before protection is achieved. In following studies several other lycopene containing products were investigated including a lycopene-rich carrot juice from the variety NutriRed, a supplement from tomato extract (Lyco-Mato), a drink with tomato extract (Lyco-Guard), and synthetic lycopene product [53]. All source provided similar lycopene doses per day and treatment was for 12 wk. Photoprotective effects were determined for all of these lycopene sources, however, protection was not statistically significant with synthetic lycopene. Further analyses revealed the presence of high amounts of phytoene and phytofluene in tomato products. Both are precursor compounds for lycopene were found in all of the sources except of the synthetic lycopene. At the end of the study, their plasma levels were increased in the samples of all participants who ingested tomato based products. Phytoene and phytofluene are non-colored carotenoids and carry only 3 and 5 conjugated double bonds in their structure. However, their absorption maxima are at wavelengths of the UVB and UVA range. We speculate that they directly absorb damaging UV light thus contribute to the photoprotection following dietary intervention with tomato products. It should be noted that UV-induced erythema was evaluated as a measure of sunburn in most of these studies which reflects a delayed inflammatory response of cutaneous tissue following UVB irradiation. Only few data from human studies provide evidence for a decrease in UV-induced damage to DNA or other skin constituents. No clear correlation has been shown up till now between the intake of carotenoids or carotenoid-rich food and the risk for skin cancer. We also investigated the prevention of photooxidation by lycopene, b-carotene and lutein in cultured human fibroblasts which were exposed to UVB irradiation [54]. A measure of oxidative damage was the formation of thiobarbituric acid-reactive
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substances (TBARS). Lycopene, b-carotene, and lutein were capable of decreasing UV-induced formation of TBARS significantly. The amounts of carotenoid needed for optimal protection were divergent for lycopene, b-carotene, and lutein, respectively. Doseresponse curves showed a u-formed shape. Beyond the optimum levels, further increases of carotenoid levels led to prooxidant effects. The data suggest that there is an optimum level for a carotenoid to reveal photoprotection. At lower levels photoprotective effects are diminished, whereas at higher levels prooxidative effects may occur. Among other compounds we investigated a carotenoid with an unusual structure. 3,30 -Dihydroxyisorenieratene (DHIR) carries a polyenic backbone substituted with phenolic end groups (Fig. 1). DHIR is present in the bacterium Brevibacterium linens which is used in the dairy industry for the production of various red smear cheeses. The antioxidant activity of the compound exceeds that of other carotenoids like astaxanthin, cryptoxanthin, zeaxanthin or lutein. Due to the presence of a polyenic and phenolic substructure DHIR acts as a bifunctional radical scavenger and quenches singlet molecular oxygen. It also inhibits the formation of UVB-induced thymidine dimers and protects proteins from photoxidative damage [55e57]. In addition to our studies with carotenoids, we examined the question whether high-flavanol cocoa provides photoprotection against UV-induced erythema [58]. In this study, two groups of volunteers consumed either a high flavanol or low flavanol cocoa powder over a period of 12 weeks. Photoprotective effects were measured before and during the intervention. Following exposure of selected skin areas toward 1.25 minimal erythemal dose (MED), UV-induced erythema was significantly decreased in the highflavanol group after 6 and 12 week of treatment, respectively, whereas no change was found in the low-flavanol group. Here we also determined impact on skin parameters not directly related to photoprotection. 7. Skin effects Function and structure of the skin is modulated by dietary factors and several micronutrients and there is evidence that nutrients have impact on skin properties when ingested regularly [59]. In the study mentioned before, ingestion of high flavanol cocoa also led to an increase in blood flow of cutaneous and subcutaneous tissues, and to a significant increase in skin density, thickness, and skin hydration [58]. These parameters were not affected in the low flavanol group. Evaluation of the skin surface showed a significant decrease of skin roughness and scaling in the high flavanol cocoa group, whereas again no change was found in the low flavanol cocoa group, comparing the starting values with week 6 and 12. The data show that ingestion of dietary flavanols from cocoa contributes not only to endogenous photoprotection but also improves dermal blood circulation. Cocoa flavanols further affect cosmetically relevant parameters of skin surface and hydration. In a shortterm study we demonstrated that dermal blood flow is acutely increased and oxygen saturation of dermal hemoglobin is elevated upon intake of high flavanol cocoa [60]. ()-Epicatechin (Fig. 1) is the major dietary flavanol in cocoa and there is evidence that it is responsible for vasodilatory effects observed after cocoa consumption. We have provided direct evidence that the compound increases nitric oxide levels in human endothelial cells. Elevation of NO in endothelial cells via epicatechin may be mediated by the inhibition of NADPH oxidase [61]. 8. Conclusion A lot of interesting, basic research and directly applicable work
on the effects of carotenoids and flavonoids was initiated and put forward within the last 30 years in Düsseldorf. Thank you Helmut! Conflict of interest The author declares no conflict of interest. References [1] P. Di Mascio, S. Kaiser, H. Sies, Lycopene as the most efficient biological carotenoid singlet oxygen quencher, Arch. Biochem. Biophys. 274 (1989) 532e538. [2] K. Briviba, H. Sies, Biological singlet oxygen quenchers assessed by monomol light emission, Methods Enzymol. 319 (2000) 222e226. [3] W. Stahl, H. Sies, Carotenoids: occurence, biochemical activities, and bioavailability, in: L. Packer, M. Hiramatsu, T. Yoshikawa (Eds.), Anitoxidant Food Supplements in Human Health, Academic Press, San Diego, 1999, pp. 183e202. [4] G. Britton, Structure and properties of carotenoids in relation to function, FASEB J. 9 (1995) 1551e1558. [5] W. Stahl, W. Schwarz, A.R. Sundquist, H. Sies, cis-trans isomers of lycopene and b-carotene in human serum and tissues, Arch. Biochem. Biophys. 294 (1992) 173e177. [6] K.H. van het Hof, C.E. West, J.A. Weststrate, J.G. Hautvast, Dietary factors that affect the bioavailability of carotenoids, J. Nutr. 130 (2000) 503e506. [7] W. Stahl, H. Sies, Uptake of lycopene and its geometrical isomers is greater from heat-processed than from unprocessed tomato juice in humans, J. Nutr. 122 (1992) 2161e2166. €rtner, W. Stahl, H. Sies, Lycopene is more bioavailable from tomato paste [8] C. Ga than from fresh tomatoes, Am. J. Clin. Nutr. 66 (1997) 116e122. [9] E. Giovannucci, A. Ascherio, E.B. Rimm, M.J. Stampfer, G.A. Colditz, W.C. Willett, Intake of carotenoids and retinol in relation to risk of prostate cancer, J. Natl. Cancer Inst. 87 (1995) 1767e1776. [10] W. Stahl, W. Schwarz, J. von Laar, H. Sies, all-trans-b-Carotene preferentially accumulates in human chylomicrons and very low density lipoproteins compared with the 9-cis geometrical isomer, J. Nutr. 125 (1995) 2128e2133. [11] X. Hebuterne, X.D. Wang, E.J. Johnson, N.I. Krinsky, R.M. Russell, Intestinal absorption and metabolism of 9-cis-beta-carotene in vivo: biosynthesis of 9cis-retinoic acid, J. Lipid Res. 36 (1995) 1264e1273. [12] J. von Laar, W. Stahl, K. Bolsen, G. Goerz, H. Sies, b-Carotene serum levels in patients with erythropoietic protoporphyria on treatment with the synthetic all-trans isomer or a natural isomer mixture of b-carotene, J. Photochem. Photobiol. B Biol. 33 (1996) 157e162. [13] T. Wingerath, W. Stahl, D. Kirsch, R. Kaufmann, H. Sies, Fruit juice carotenol fatty acid esters and carotenoids as identified by matrix-assisted laser desorption ionization (MALDI) mass spectrometry, J. Agric. Food Chem. 44 (1996) 2006e2013. [14] T. Wingerath, W. Stahl, H. Sies, b-Cryptoxanthin selectively increases in human chylomicrons upon ingestion of tangerine concentrate rich in b-cryptoxanthin esters, Arch. Biochem. Biophys. 324 (1995) 385e390. [15] T. Wingerath, H. Sies, W. Stahl, Xanthophyll esters in human skin, Arch. Biochem. Biophys. 355 (1998) 271e274. [16] A. Perez-Galvez, H.D. Martin, H. Sies, W. Stahl, Incorporation of carotenoids from paprika oleoresin into human chylomicrons, Br. J. Nutr. 89 (2003) 787e793. [17] H. Sies, Biochemistry of oxidative stress, Angew. Chem. Int. Ed. Engl. 25 (1986) 1058e1071. [18] H. Sies, Oxidative stress, in: G. Fink (Ed.), Encyclopedia of Stress, Academic Press, 2000, pp. 102e105. [19] A. Telfer, What is beta-carotene doing in the photosystem II reaction centre? Philos. Trans. R. Soc. Lond B Biol. Sci. 357 (2002) 1431e1439. [20] T.G. Truscott, The photophysics and photochemistry of the carotenoids, J. Photochem. Photobiol. B Biol. 6 (1990) 359e371. [21] W. Stahl, H. Sies, Physical quenching of singlet oxygen and cis-trans isomerization of carotenoids, Ann. N. Y. Acad. Sci. 691 (1993) 10e19. [22] W. Stahl, H. Sies, Antioxidant activity of carotenoids, Mol. Asp. Med. 24 (2003) 345e351. € ver, [23] D. Baltschun, S. Beutner, K. Briviba, H.D. Martin, J. Paust, M. Peters, S. Ro H. Sies, W. Stahl, et al., Singlet oxygen quenching abilities of carotenoids, Liebigs Ann. (1997) 1887e1893. [24] S. Beutner, B. Bloedorn, S. Frixel, I. Hern andez Blanco, T. Hoffmann, H.D. Martin, B. Mayer, P. Noack, C. Ruck, et al., Quantitative assessment of antioxidant properties of natural colorants and related compounds: carotenoids, flavonoids and indigoids, in: Application of Natural Colorants for Foods, Nutraceuticals, Confectionery, Beverages, Cosmetics, The Hereld Organization ed., San Diego, 2000, pp. 1e19. [25] G.W. Burton, K.U. Ingold, b-Carotene: an unusual type of lipid antioxidant, Science 224 (1984) 569e573. [26] The ATBC-Study Group, The effect of vitamin E and beta carotene on the incidence of lung cancer and other cancers in male smokers, N. Engl. J. Med. 330 (1994) 1029e1035. [27] F. Bohm, R. Edge, E.J. Land, D.J. McGarvey, T.G. Truscott, Carotenoids enhance
W. Stahl / Archives of Biochemistry and Biophysics 595 (2016) 125e131 vitamin E antioxidant efficiency, J. Am. Chem. Soc. 119 (1997) 621e622. [28] F. Bohm, R. Edge, D.J. McGarvey, T.G. Truscott, Beta-carotene with vitamins E and C offers synergistic cell protection against NOx, FEBS Lett. 436 (1998) 387e389. [29] P. Palozza, N.I. Krinsky, beta-Carotene and alpha-tocopherol are synergistic antioxidants, Arch. Biochem. Biophys. 297 (1992) 184e187. [30] W. Stahl, A. Junghans, B. de Boer, E. Driomina, K. Briviba, H. Sies, Carotenoid mixtures protect multilamellar liposomes against oxidative damage: synergistic effects of lycopene and lutein, FEBS Lett. 427 (1998) 305e308. [31] A. Junghans, H. Sies, W. Stahl, Carotenoid-containing unilamellar liposomes loaded with glutathione: a model to study hydrophobic-hydrophilic antioxidant interaction, Free Rad. Res. 33 (2000) 801e808. [32] A. Junghans, H. Sies, W. Stahl, Macular pigments lutein and zeaxanthin as blue light filters studied in liposomes, Arch. Biochem. Biophys. 391 (2001) 160e164. [33] W. Stahl, N. Ale-Agha, M.C. Polidori, Non-antioxidant properties of carotenoids, Biol. Chem. 383 (2002) 553e558. [34] J.S. Bertram, Dietary carotenoids, connexins and cancer: what is the connection? Biochem. Soc. Trans. 32 (2004) 985e989. [35] L.X. Zhang, R.V. Cooney, J.S. Bertram, Carotenoids up-regulate gap junctional communication: correlation with cancer chemoprevention properties, Carcinogenesis 12 (1991) 2109e2114. [36] M.S. Nielsen, L.N. Axelsen, P.L. Sorgen, V. Verma, M. Delmar, N.H. HolsteinRathlou, Gap junctions, Compr. Physiol. 2 (2012) 1981e2035. [37] W. Stahl, S. Nicolai, K. Briviba, M. Hanusch, G. Broszeit, M. Peters, H.D. Martin, H. Sies, Biological activities of natural and synthetic carotenoids: induction of gap junctional communication and singlet oxygen quenching, Carcinogenesis 18 (1997) 89e92. [38] M. Hanusch, W. Stahl, W.A. Schulz, H. Sies, Induction of gap junctional communication by 4-oxoretinoic acid generated from its precursor canthaxanthin, Arch. Biochem. Biophys. 317 (1995) 423e428. [39] V.B. Teicher, N. Kucharski, H.D. Martin, P. van der Saag, H. Sies, W. Stahl, Biological activities of apo-canthaxanthinois acids related to gap junctional communication, Arch. Biochem. Biophys. 365 (1999) 150e155. [40] W. Stahl, J. von Laar, H.D. Martin, T. Emmerich, H. Sies, Stimulation of gap junctional communication: comparison of acyclo-retinoic acid and lycopene, Arch. Biochem. Biophys. 373 (2000) 271e274. [41] O. Aust, N. Ale-Agha, L. Zhang, H. Wollersen, H. Sies, W. Stahl, Lycopene oxidation product enhances gap junctional communication, Food Chem. Toxicol. 41 (2003) 1399e1407. [42] J.S. Bertram, T. King, L. Fukishima, F. Khachik, Enhanced activity of an oxidation product of lycopene found in tomato products and human serum relevant to cancer prevention, in: C.K. Sen, H. Sies, P.A. Baeuerle (Eds.), Antioxidant and Redox Regulation of Genes, Academic Press, London, 2000, pp. 409e424. [43] F. Daubrawa, H. Sies, W. Stahl, Astaxanthin diminishes gap junctional intercellular communication in primary human fibroblasts, J. Nutr. 135 (2005) 2507e2511. [44] A. Clairmont, D. Tessmann, A. Stock, S. Nicolai, W. Stahl, H. Sies, Induction of gap junctional intercellular communication by vitamin D in human skin fibroblasts is dependent on the nuclear vitamin D receptor, Carcinogenesis 17 (1996) 1389e1391.
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[45] S. Nicolai, H. Sies, W. Stahl, Stimulation of gap junctional intercellular communication by thalidomide and thalidomide analogs in human skin fibroblasts, Biochem. Pharmacol. 53 (1997) 1553e1557. [46] A. Stock, H. Sies, W. Stahl, Enhancement of gap junctional communication and connexin43 expression by thyroid hormones, Biochem. Pharmacol. 55 (1998) 475e479. [47] H. Sies, W. Stahl, Nutritional protection against skin damage from sunlight, Annu. Rev. Nutr. 24 (2004) 173e200. [48] M.M. Mathews-Roth, M.A. Pathak, T.B. Fitzpatrick, L.C. Harber, E.H. Kass, bCarotene as an oral protective agent in erythropoietic protoporphyria, J. Am. Med. Assoc. 228 (1974) 1004e1008. [49] W. Stahl, U. Heinrich, H. Jungmann, H. Sies, H. Tronnier, Carotenoids and carotenoids plus vitamin E protect against ultraviolet light-induced erythema in humans, Am. J. Clin. Nutr. 71 (2000) 795e798. [50] U. Heinrich, C. Gartner, M. Wiebusch, O. Eichler, H. Sies, H. Tronnier, W. Stahl, Supplementation with beta-carotene or a similar amount of mixed carotenoids protects humans from UV-induced erythema, J. Nutr. 133 (2003) 98e101. [51] W. Kopcke, J. Krutmann, Protection from sunburn with beta-caroteneea meta-analysis, Photochem. Photobiol. 84 (2008) 284e288. [52] W. Stahl, U. Heinrich, S. Wiseman, O. Eichler, H. Sies, H. Tronnier, Dietary tomato paste protects against ultraviolet light-induced erythema in humans, J. Nutr. 131 (2001) 1449e1451. [53] O. Aust, W. Stahl, H. Sies, H. Tronnier, U. Heinrich, Supplementation with tomato-based products increases lycopene, phytofluene, and phytoene levels in human serum and protects against UV-light-induced erythema, Intern. J. Vit. Nutr. Res. 75 (2005) 54e60. [54] O. Eichler, H. Sies, W. Stahl, Divergent optimum levels of lycopene, betacarotene and lutein protecting against UVB irradiation in human fibroblastst, Photochem. Photobiol. 75 (2002) 503e506. [55] K. Lutter, S.S. De, S. Kock, K.D. Kroncke, H.D. Martin, T. Wagener, W. Stahl, 3,3ʹDihydroxyisorenieratene prevents UV-induced formation of reactive oxygen species and the release of protein-bound zinc ions in human skin fibroblasts, Mol. Nutr. Food Res. 54 (2010) 285e291. [56] H.D. Martin, S. Kock, R. Scherrers, K. Lutter, T. Wagener, C. Hundsdorfer, S. Frixel, K. Schaper, H. Ernst, et al., 3,3ʹ-Dihydroxyisorenieratene, a natural carotenoid with superior antioxidant and photoprotective properties, Angew. Chem. Int. Ed. Engl. 48 (2009) 400e403. [57] S. Wagener, T. Volker, S.S. De, H. Ernst, W. Stahl, 3,3ʹ-Dihydroxyisorenieratene and isorenieratene prevent UV-induced DNA damage in human skin fibroblasts, Free Radic. Biol. Med. 53 (2012) 457e463. [58] U. Heinrich, K. Neukam, H. Tronnier, H. Sies, W. Stahl, Long-term ingestion of high flavanol cocoa provides photoprotection against UV-induced erythema and improves skin condition in women, J. Nutr. 136 (2006) 1565e1569. [59] G. Nguyen, A. Torres, Systemic antioxidants and skin health, J. Drugs Dermatol. 11 (2012) e1ee4. [60] K. Neukam, W. Stahl, H. Tronnier, H. Sies, U. Heinrich, Consumption of flavanol-rich cocoa acutely increases microcirculation in human skin, Eur. J. Nutr. 46 (2007) 53e56. [61] T. Schewe, Y. Steffen, H. Sies, How do dietary flavanols improve vascular function? A position paper, Arch. Biochem. Biophys. 476 (2008) 102e106.