Production of phytoene, a carotenoid, and induction of connexin 26 in transgenic mice carrying the phytoene synthase gene crtB

Carotenoids have been recognized as chemopreventive agents against human diseases, such as cancer and cardiovascular disease. Mammalians utilize carotenoids supplied from their food since they are unable to perform the de novo synthesis of carotenoids. We previously created mammalian cultured cells producing phytoene, a type of carotenoid, and showed that these cells acquired resistance against oxidative stress and oncogenic transformation. In the present study, we established a transgenic mouse line, carrying the crtB gene encoding phytoene synthase, which could produce phytoene endogenously. It was found that connexin 26 was induced in these phytoene-producing mice. Since it is known that carotenoids enhance gap junctional communication by inducing the expression of connexin genes, the present data suggest that the induction of connexin 26 in phytoene-producing mice may play a role in controlling cell-to-cell communication. Phytoene-producing mice provide a useful system in which to investigate the in vivo function of the carotenoid phytoene.     

Cancer prevention by antioxidants

Various antioxidants in foods, such as phenolic compounds and carotenoids, were proven to have anticarcinogenic activity. In the case of carotenoids, the mixture of them was found to be very effective. In fact, the development of hepatoma in the high risk group of liver cancer, was significantly suppressed by the treatment with natural carotenoids mixture. The role of nitric oxide (NO) in carcinogenesis has been pointed out, since large quantity of NO has been detected in cancer tissues, and the expression of inducible NO synthase (iNOS) was found to correlate with tumor growth and metastasis. Recently, we found that NO possessed tumor initiating activity in mouse skin carcinogenesis. It has been suggested that some parts of pathological effects induced by NO may depend on peroxynitrite, an active metabolite of NO. Thus, we accessed the tumor initiating activity of peroxynitrite, and found that treatment with peroxynitrite (initiator) plus TPA (promoter) resulted in the formation of skin tumors. Under this experimental condition, it has been proven that natural antioxidants, such as curcumin and nobiletin, showed anti-tumor initiating effect. In the case of nobiletin, suppressive effect on iNOS induction has also been demonstrated. It is of interest that suppression of iNOS induction was also observed in phytoene synthase transgenic mouse. After administration of glycerol (a lung tumor promoter), lower induction of iNOS gene was observed in lung of the phytoene producing mice, comparing with that of control mice. Combinational use of various kinds of antioxidants distributed in foods, e.g., mixture of carotenoids and flavonoids, seems to be effective methods for cancer prevention.     

Carotenoids and fatty liver disease: Current knowledge and research gaps

Carotenoids form an important part of the human diet, consumption of which has been associated with many health benefits. With the growing global burden of liver disease, increasing attention has been paid on the possible beneficial role that carotenoids may play in the liver. This review focuses on carotenoid actions in non-alcoholic fatty liver disease (NAFLD), and alcoholic liver disease (ALD). Indeed, many human studies have suggested an association between decreased circulating levels of carotenoids and increased incidence of NAFLD and ALD. The literature describing supplementation of individual carotenoids in rodent models of NAFLD and ALD is reviewed, with particular attention paid to β-carotene and lycopene, but also including β-cryptoxanthin, lutein, zeaxanthin, and astaxanthin. The effect of beta-carotene oxygenase 1 and 2 knock-out mice on hepatic lipid metabolism is also discussed. In general, there is evidence to suggest that carotenoids have beneficial effects in animal models of both NAFLD and ALD. Mechanistically, these benefits may occur via three possible modes of action: 1) improved hepatic antioxidative status broadly attributed to carotenoids in general, 2) the generation of vitamin A from β-carotene and β-cryptoxanthin, leading to improved hepatic retinoid signaling, and 3) the generation of apocarotenoid metabolites from β-carotene and lycopene, that may regulate hepatic signaling pathways. Gaps in our knowledge regarding carotenoid mechanisms of action in the liver are highlighted throughout, and the review ends by emphasizing the importance of dose effects, mode of delivery, and mechanism of action as important areas for further study. This article is part of a Special Issue entitled Carotenoids recent advances in cell and molecular biology edited by Johannes von Lintig and Loredana Quadro.     

Efficacy of Rhodotorula glutinis and Spirulina platensis carotenoids in immunopotentiation of mice infected with Candida albicans SC5314 and Pseudomonas aeruginosa 35

Enhancement of the immune response leading to protection against bacterial and fungal infections was shown using different schedules of immunization with microbial pigments and a polysaccharide. The group of mice given carotenoids of Rhodotorula glutinis (preparation I) and polysaccharide of Spitulina platensis (IV) survived for 2 weeks after Pseudomonas aeruginosa infection. The groups of mice given carotenoids (I), polysaccharide (IV), I+IV and with the crude phycocyanin of S. platensis (III)+IV survived for 2 weeks after Candida albicans infection. All other groups recorded a maximum level of mortality reaching 2 mice per group either after immunization or post-infection. Adding the carotenoids, phycocyanin and polysaccharides to food as additives might therefore enhance the human immune response against microbial infections.     

Carotenoids enhance phosphorylation of Akt and suppress tissue factor activity in human endothelial cells

Enhanced production of tissue factor has been linked to development of cardiovascular disease due to endothelial activation, resulting in thrombosis of blood vessels. Epidemiological studies reported that diet-derived antioxidants might suppress and/or delay progression of cardiovascular disease. Detailed molecular level studies are needed to understand this effect with prevention as a goal. Water-dispersible forms of various carotenoids (beta-carotene, lutein and lycopene) from natural sources in microemulsion were used to study effects of carotenoids on tissue factor activity in human endothelial cells. All carotenoids studied suppressed tissue factor activity (P<.01) and gene expression in human endothelial cells. Our study also demonstrated that addition of Akt-specific inhibitor reversed the inhibitory effect of carotenoids on tissue factor activity, indicating that carotenoids enhanced phosphorylation of Akt and suppressed tissue factor activity in endothelial cells by this mechanism.     

Protection by beta-carotene and related compounds against oxygen-mediated cytotoxicity and genotoxicity: implications for carcinogenesis and anticarcinogenesis.

beta-Carotene protects against photooxidative dermatitis in porphyric humans and mice by quenching of photoactivated species. Other actions of beta-carotene in vivo are explained on the basis of its ability to scavenge free radicals in vitro. For example, in guinea pigs treated with CCl4, beta-carotene decreases pentane and ethane production. Epidemiological studies link low serum beta-carotene levels to elevated risk of lung and other cancers, and in intervention trials, beta-carotene diminishes preneoplastic lesions. However, the dose/response relationships are not well established, and antineoplastic mechanisms await clarification. Given a radical quenching mechanism, beta-carotene should block tumor promotion, but more typically the site of action is progression and an even later role in invasion has not been ruled out. Some antineoplastic actions of carotenoids (such as increased rejection of fibrosarcomas in mice) are attributed to immunoenhancement; others may reflect conversion to retinoids and subsequent gene regulation. Carotenoids other than beta-carotene may act at an earlier stage of carcinogenesis or be more effective as anticarcinogens at certain target sites. As scavengers of hydroxyl radicals, canthaxanthin and astaxanthin are more effective than beta-carotene. Canthaxanthin is sometimes more effective than beta-carotene in chemoprevention, but it is sometimes completely ineffective. Lycopene quenches singlet oxygen more than twice as effectively as beta-carotene. However, the antineoplastic actions of lycopene or astaxanthin remain untested. Explorations of the interactions of carotenoids with other nutrients are just beginning. Dietary fat increases absorption of carotene but decreases antineoplastic effectiveness. Research is hampered by technical problems, including the unavailability of rigorous controls, the instability of carotenoids, and the heterogeneous phase structure induced by hydrophobic compounds in aqueous media. Areas of current controversy and promising approaches for future research are identified.     

Saffron and natural carotenoids: Biochemical activities and anti-tumor effects

Saffron, a spice derived from the flower of Crocus sativus, is rich in carotenoids. Two main natural carotenoids of saffron, crocin and crocetin, are responsible for its color. Preclinical studies have shown that dietary intake of some carotenoids have potent anti-tumor effects both in vitro and in vivo, suggesting their potential preventive and/or therapeutic roles in several tissues. The reports represent that the use of carotenoids without the potential for conversion to vitamin A may provide further protection and avoid toxicity. The mechanisms underlying cancer chemo-preventive activities of carotenoids include modulation of carcinogen metabolism, regulation of cell growth and cell cycle progression, inhibition of cell proliferation, anti-oxidant activity, immune modulation, enhancement of cell differentiation, stimulation of cell-to-cell gap junction communication, apoptosis and retinoid-dependent signaling. Taken together, different hypotheses for the antitumor actions of saffron and its components have been proposed such as a) the inhibitory effect on cellular DNA and RNA synthesis, but not on protein synthesis; b) the inhibitory effect on free radical chain reactions; c) the metabolic conversion of naturally occurring carotenoids to retinoids; d) the interaction of carotenoids with topoisomerase II, an enzyme involved in cellular DNA-protein interaction. Furthermore, the immunomodulatory activity of saffron was studied on driving toward Th1 and Th2 limbs of the immune system. In this mini-review, we briefly describe biochemical and immunological activities and chemo-preventive properties of saffron and natural carotenoids as an anticancer drug.     

Carotenoid Formation by Staphylococcus aureus

The carotenoid pigments of Staphylococcus aureus U-71 were identified as phytoene; zeta-carotene; delta-carotene; phytofluenol; a phytofluenol-like carotenoid, rubixanthin; and three rubixanthin-like carotenoids after extraction, saponification, chromatographic separation, and determination of their absorption spectra. There was no evidence of carotenoid esters or glycoside ethers in the extract before saponification. During the aerobic growth cycle the total carotenoids increased from 45 to 1,000 nmoles per g (dry weight), with the greatest increases in the polar, hydroxylated carotenoids. During the anaerobic growth cycle, the total carotenoids increased from 20 nmoles per g (dry weight) to 80 nmoles per g (dry weight), and only traces of the polar carotenoids were formed. Light had no effect on carotenoid synthesis. About 0.14% of the mevalonate-2-(14)C added to the culture was incorporated into the carotenoids during each bacterial doubling. The total carotenoids did not lose radioactivity when grown in the absence of (14)C for 2.5 bacterial doublings. The total carotenoids did not lose radioactivity when grown in the absence of (14)C for 2.5 bacterial doublings. The incorporation and turnover of (14)C indicated the carotenes were sequentially desaturated and hydroxylated to form the polar carotenoids.     

类胡萝卜素产生菌深红酵母(Rhodotorula rubra)的培养条件的优化研究

本文研究深红酵母及其产生的类胡萝卜素的培养优化条件,并对其进行了小型发酵试验,结果表明pH值对深红酵母和类胡萝卜素有影响,初始pH值越低,色素的积累量越高,发酵过程中控制pH值能有效地增加色素积累的速度和初始色素积累量,但随着发酵时间的延长,色素积累量逐渐下降。     

Animal models in carotenoids research and lung cancer prevention

Numerous epidemiological studies have consistently demonstrated that individuals who eat more fruits and vegetables (which are rich in carotenoids) and who have higher serum β-carotene levels have a lower risk of cancer, especially lung cancer. However, two human intervention trials conducted in Finland and in the United States have reported contrasting results with high doses of β-carotene supplementation increasing the risk of lung cancer among smokers. The failure of these trials to demonstrate actual efficacy has resulted in the initiation of animal studies to reproduce the findings of these two studies and to elucidate the mechanisms responsible for the harmful or protective effects of carotenoids in lung carcinogenesis. Although these studies have been limited by a lack of animal models that appropriately represent human lung cancer induced by cigarette smoke, ferrets and A/J mice are currently the most widely used models for these types of studies. There are several proposed mechanisms for the protective effects of carotenoids on cigarette smoke-induced lung carcinogenesis, and these include antioxidant/prooxidant effects, modulation of retinoic acid signaling pathway and metabolism, induction of cytochrome P450, and molecular signaling involved in cell proliferation and/or apoptosis. The technical challenges associated with animal models include strain-specific and diet-specific effects, differences in the absorption and distribution of carotenoids, and differences in the interactions of carotenoids with other antioxidants. Despite the problems associated with extrapolating from animal models to humans, the understanding and development of various animal models may provide useful information regarding the protective effects of carotenoids against lung carcinogenesis.     

Differential effects of carotenoids on lipid peroxidation due to membrane interactions: X-ray diffraction analysis

The biological benefits of certain carotenoids may be due to their potent antioxidant properties attributed to specific physico-chemical interactions with membranes. To test this hypothesis, we measured the effects of various carotenoids on rates of lipid peroxidation and correlated these findings with their membrane interactions, as determined by small angle X-ray diffraction approaches. The effects of the homochiral carotenoids (astaxanthin, zeaxanthin, lutein, beta-carotene, lycopene) on lipid hydroperoxide (LOOH) generation were evaluated in membranes enriched with polyunsaturated fatty acids. Apolar carotenoids, such as lycopene and beta-carotene, disordered the membrane bilayer and showed a potent pro-oxidant effect (>85% increase in LOOH levels) while astaxanthin preserved membrane structure and exhibited significant antioxidant activity (40% decrease in LOOH levels). These findings indicate distinct effects of carotenoids on lipid peroxidation due to membrane structure changes. These contrasting effects of carotenoids on lipid peroxidation may explain differences in their biological activity.     

The effect of an acute phase response on tissue carotenoid levels of growing chickens (Gallus gallus domesticus)

Plasma, liver and skin carotenoids decrease following infectious disease challenges. Since these challenges often involve substantial host pathology and chronic immune responses, the mechanism underlying altered carotenoid deposition is unclear. Therefore, changes in tissue carotenoid levels were examined during an acute phase response induced by lipopolysaccharide (LPS) or interleukin-1 (IL-1). In two experiments, chicks were hatched from carotenoid-deplete eggs (n=28, n=64, respectively) and fed 0, 8 or 38 mg carotenoids (lutein+canthaxanthin)/kg diet. For chicks fed 38 mg carotenoids, but not those fed 0 or 8 mg, LPS generally reduced plasma lutein, canthaxanthin and total carotenoids (P<0.05), and liver lutein, zeaxanthin, canthaxanthin and total carotenoids (P<0.05). Additionally, LPS reduced thymic total carotenoids (P=0.05) and increased thymocyte lutein (P=0.07), zeaxanthin (P=0.07) and total carotenoids (P=0.07). Finally, LPS increased bursal canthaxanthin (P<0.01), but had no effect on shank carotenoids (P>0.5). In chicks hatched from carotenoid-replete eggs (n=36) and fed dietary lutein (38 mg/kg diet), LPS reduced plasma and liver zeaxanthin and liver total carotenoids (P<0.05); IL-1 reduced plasma and liver lutein, zeaxanthin and total carotenoids (P<0.05). Therefore, an acute phase response plays a role in reduced tissue carotenoids during infectious disease.     

Evaluation of the antioxidant effects of carotenoids from Deinococcus radiodurans through targeted mutagenesis, chemiluminescence, and DNA damage analyses

Deinococcus radiodurans is highly resistant to reactive oxygen species (ROS). The antioxidant effect of carotenoids in D. radiodurans was investigated by using a targeted mutation of the phytoene synthase gene to block the carotenoid synthesis pathway and by evaluating the survival of cells under environmental stresses. The colorless mutant R1DeltacrtB of D. radiodurans failed to synthesize carotenoids, and was more sensitive to ionizing radiation, hydrogen peroxide, and desiccation than the wild type, suggesting that carotenoids in D. radiodurans help in combating environmental stresses. Chemiluminescence analyses showed that deinoxanthin, a major product in the carotenoid synthesis pathway, had significantly stronger scavenging ability on H2O2 and singlet oxygen than two carotenes (lycopene and beta-carotene) and two xanthophylls (zeaxanthin and lutein). Deinoxanthin also exhibited protective effect on DNA. Our findings suggest that the stronger antioxidant effect of deinoxanthin contribute to the resistance of D. radiodurans. The higher antioxidant effect of deinoxanthin may be attributed to its distinct chemical structure which has an extended conjugated double bonds and the presence of a hydroxyl group at C-1' position, compared with other tested carotenoids.     

Differential effects of carotenoids on lipid peroxidation due to membrane interactions: X-ray diffraction analysis

The biological benefits of certain carotenoids may be due to their potent antioxidant properties attributed to specific physico-chemical interactions with membranes. To test this hypothesis, we measured the effects of various carotenoids on rates of lipid peroxidation and correlated these findings with their membrane interactions, as determined by small angle X-ray diffraction approaches. The effects of the homochiral carotenoids (astaxanthin, zeaxanthin, lutein, β-carotene, lycopene) on lipid hydroperoxide (LOOH) generation were evaluated in membranes enriched with polyunsaturated fatty acids. Apolar carotenoids, such as lycopene and β-carotene, disordered the membrane bilayer and showed a potent pro-oxidant effect (> 85% increase in LOOH levels) while astaxanthin preserved membrane structure and exhibited significant antioxidant activity (40% decrease in LOOH levels). These findings indicate distinct effects of carotenoids on lipid peroxidation due to membrane structure changes. These contrasting effects of carotenoids on lipid peroxidation may explain differences in their biological activity.     

Addition of lutein, lycopene, or β-carotene to LDL or serum in vitro: Effects on carotenoid distribution, LDL composition, and LDL oxidation

Carotenoids are dietary antioxidants transported with plasma lipoproteins, primarily low-density lipoprotein (LDL). In this study in vitro methods were used to increase the amounts of specific, individual carotenoids in LDL. By addition of carotenoid to isolated LDL or to serum, followed by (re)isolation of the lipoproteins, samples of LDL were enriched 4- to 150-fold with lutein, 2- to 15-fold with lycopene, or 3- to 25-fold with β-carotene. Enrichment with specific carotenoids was achieved without affecting the electrophoretic mobility of the lipoprotein, its cholesterol to protein ratio, or the levels of other cartenoids or -tocopherol. The distributions among lipoproteins of carotenoid added to serum were similar, but not identical, to the distributions of the endogenous carotenoids. In particular, for added lutein, a greater proportion was found in HDL, and for added β-carotene, more was found in very low-density lipoprotein (VLDL). We then studied the effect of enriching LDL with specific carotenoids on its susceptibility to oxidation by copper ions. Lutein, β-cryptoxanthin, lycopene, and β-carotene, the four major plasma carotenoids, and -tocopherol were destroyed before the formation of lipid peroxidation products. The rates of destruction of the individual carotenoids differed; lycopene was destroyed most rapidly and lutein most slowly. Upon oxidation of β-carotene-enriched LDL, the rates of destruction of β-carotene, lycopene, and lutein were slowed and the lag times before the initiation of lipid peroxidation increased from 19 to 65 min. Neither effect was observed in LDL enriched with lutein or lycopene. Thus, β-carotene was unique among the carotenoids studied in having a small, but significant effect on LDL oxidation in vitro.     

Interaction of peroxynitrite with carotenoids in human low density lipoproteins

Interaction of peroxynitrite, the product of the reaction between nitric oxide and superoxide, with carotenes (lycopene, alpha-carotene, and beta-carotene) and oxocarotenoids (beta-cryptoxanthin, zeaxanthin, and lutein) was studied both in homogeneous solution and in human low-density lipoproteins (LDL). All carotenoids prevented the formation of rhodamine 123 from dihydrorhodamine 123 caused by peroxynitrite, suggesting that the carotenoids react with peroxynitrite. Oxocarotenoids were as effective as biothiols, known scavengers of peroxynitrite, whereas lycopene, alpha-carotene, and beta-carotene exhibited a considerably more pronounced effect. Moreover, peroxynitrite caused a loss of carotenoids in LDL as was revealed by HPLC. The concentration of peroxynitrite causing half-maximal loss of carotenoids in LDL ranged from 13 +/- 3 to 68 +/- 3 microM for lycopene and lutein, respectively. Again, oxocarotenoids were less reactive in this system. A correlation between efficiency of carotenoids in the competitive assay with dihydrorhodamine 123 and the concentration of peroxynitrite causing half-maximal loss of carotenoids in LDL was observed (r(2) = 0.91). These findings suggest that carotenoids can efficiently react with peroxynitrite and perform the role of scavengers of peroxynitrite in vivo.     

Carotenoids as protective response against oxidative damage in Dunaliella bardawil

In the present work the relation between carotenoids production and cell response mechanisms to oxidative damage was studied. High light intensity and nitrogen starvation, both conditions, which may increase the oxidative damage in microalgae, significantly increased total carotenoids content in Dunaliella bardawil, the effect of N-starvation being more noticeable when acting synergetically with light on carotenoid production. S-starvation stimulated carotenoids production as much as N-starvation. The use of norflurazon, inhibitor of phytoene desaturase that blocks formation of epsilon-carotene from phytoene, caused a decrease of carotenoid content down to 5% that of the control cells incubated without the inhibitor. The decrease in the oxygen consumption rate of D. bardawil cells exposed to norflurazon suggests a connection between carotenoids desaturation and chloroplastic oxygen species dissipation processes reported in the literature for other algae. It is an indication of the carotenoids involvement in chloroplastic response mechanisms to oxidative damage.     

Effect of polar and non-polar carotenoids on Xanthophylomyces dendrorhous membranes by EPR

The red yeast Xanthophyllomyces dendrorhous is one of the microbiological production systems for natural carotenoids. High-performance liquid chromatography (HPLC) and electron paramagnetic resonance spectroscopy (EPR) experiments were performed on X. dendrorhous membranes in order to study the effect of incorporation rates of different type of carotenoids. In the case of fluid-phase membranes, it was found that polar carotenoids, such as astaxanthin and cis-astaxanthin, increased the EPR order parameter and decreased the motional freedom and phase-transition temperature. In contrast the non-polar carotenoids beta-cryptoxanthin and beta-carotene decreased the EPR order parameter and increased motional freedom and phase-transition temperature. A noteworthy coherence was observed between the polarities of the strains and the phase-transition temperatures.     

Effects of carotenoids, immune activation and immune suppression on the intensity of chronic coccidiosis in greenfinches

Allocation trade-offs of carotenoids between their use in the immune system and production of integumentary colouration have been suggested as a proximate mechanism maintaining honesty of signal traits. We tested how dietary carotenoid supplementation, immune activation and immune suppression affect intensity of coccidian infection in captive greenfinches Carduelis chloris, a passerine with carotenoid-based plumage. Immune activation with phytohaemagglutinin (PHA) decreased body mass among birds not supplemented with lutein, while among the carotenoid-fed birds, PHA had no effect on mass dynamics. Immune suppression with dexamethasone (DEX) induced loss of body mass and reduced the swelling response to PHA. DEX and PHA increased the concentration of circulating heterophils. Lutein supplementation increased plasma carotenoid levels but had no effect on the swelling response induced by PHA. PHA and DEX treatments did not affect plasma carotenoids. Immune stimulation by PHA suppressed the infection, but only among carotenoid-supplemented birds. Priming of the immune system can thus aid in suppressing chronic infection but only when sufficient amount of carotenoids is available. Our experiment shows the importance of carotenoids in immune response, but also the complicated nature of this impact, which could be the reason for inconsistent results in studies investigating the immunomodulatory effects of carotenoids. The findings about involvement of carotenoids in modulation of an immune response against coccidiosis suggest that carotenoid-based ornaments may honestly signal individuals’ ability to manage chronic infections.