Carotenes and Retinal in Phycomyces Mutants

Three different types of beta-carotene mutants of Phycomyces have been studied. In 2 mutants (Type I) beta-carotene is still the principal carotene but scaled down or up relative to wild type. The carotene mixture of 2 mutants (Type II) consists mainly of phytoene and phytofluene. In Type III (2 mutants) beta-carotene is replaced by lycopene.The examination of the mutants reveals that the receptor pigment is very likely neither beta-carotene nor retinal. Transmission spectra through the growing zone of live sporangiophores of 1 of these mutants which contains less than one-thousandth of the beta-carotene content of wild type show that the receptor pigment extinction is less than 0.003 at its maximum.     

Induction of PXR-mediated metabolism by beta-carotene

beta-carotene is the major carotenoid occurring in the human diet and in the human organism. Besides its function as pro-vitamin A, beta-carotene has been shown to be an activator of the human pregnan X receptor (PXR). PXR is mainly expressed in the liver/intestine and an inducer of enzymes involved in phase I, II and III metabolism. This review is focused on the evaluation of physiological and nutritional relevance of beta-carotene as an inducer of phase I enzymes in the human organism via PXR-mediated mechanisms. Beneficial and detrimental effects of beta-carotene on xenobiotica metabolism and metabolism of various other derivatives will be discussed.     

Co-ordinated induction of beta-carotene cleavage enzyme and retinal reductase in the duodenum of the developing chicks

The developmental patterns of expression of beta-carotene cleavage enzyme activity were compared with those of retinal reductase and NAD-dependent retinol dehydrogenase activities in chick duodenum during the perinatal period. The beta-carotene cleavage enzyme activity was not detected in the duodenum before hatching, but it increased rapidly during 24 h after hatching. On the other hand, a considerable level of beta-carotene cleavage enzyme activity was observed in the liver of embryonic stages and its activity gradually rose during the perinatal period. Comparison of kinetic constants for the beta-carotene cleavage enzyme activities in the duodenum and the liver indicated that the enzyme in the duodenum possessed a lower affinity for beta-carotene than that in the liver. The retinal reductase activity was detected in the microsomes of the duodenum at the earliest time examined, i.e. day 16 of embryogenesis and its activity began to rise on the last day of embryogenesis, which was followed by a gradual increase until 1 day of age. The NAD-dependent retinol dehydrogenase activity was also seen in the microsomes of the duodenum in embryonic stages and its activity increased in parallel with the retinal reductase activity around the hatching period. These developmental inductions of beta-carotene cleavage enzyme and retinal reductase activities in the duodenum coincided with those of cellular retinol-binding protein, type II (CRBPII) and lecithin: retinol acyltransferase (LRAT). These results suggest that a co-ordinated induction mechanism should be operative for beta-carotene cleavage enzyme and retinal reductase, both of which are inevitable in the process of beta-carotene absorption and metabolism.     

Redox functions of carotenoids in photosynthesis

Carotenoids are well-known as light-harvesting pigments. They also play important roles in protecting the photosynthetic apparatus from damaging reactions of chlorophyll triplet states and singlet oxygen in both plant and bacterial photosynthesis. Recently, it has been found that beta-carotene functions as a redox intermediate in the secondary pathways of electron transfer within photosystem II and that carotenoid cation radicals are transiently formed after photoexcitation of bacterial light-harvesting complexes. The redox role of beta-carotene in photosystem II is unique among photosynthetic reaction centers and stems from the very strongly oxidizing intermediates that form in the process of water oxidation. Because of the extended pi-electron-conjugated system of carotenoid molecules, the cation radical is delocalized. This enables beta-carotene to function as a "molecular wire", whereby the centrally located oxidizing species is shuttled to peripheral redox centers of photosystem II where it can be dissipated without damaging the system. The physiological significance of carotenoid cation radical formation in bacterial light-harvesting complexes is not yet clear, but may provide a novel mechanism for excitation energy dissipation as a means of photoprotection. In this paper, the redox reactions of carotenoids in photosystem II and bacterial light-harvesting complexes are presented and the possible roles of carotenoid cation radicals in photoprotection are discussed.     

Construction and characterization of genetically modified synechocystis sp. PCC 6803 photosystem II core complexes containing carotenoids with shorter pi-conjugation than beta-carotene

Beta-carotene has been identified as an intermediate in a secondary electron transfer pathway that oxidizes Chl(Z) and cytochrome b(559) in Photosystem II (PS II) when normal tyrosine oxidation is blocked. To test the redox function of carotenoids in this pathway, we replaced the zeta-carotene desaturase gene (zds) or both the zds and phytoene desaturase (pds) genes of Synechocystis sp. PCC 6803 with the phytoene desaturase gene (crtI) of Rhodobacter capsulatus, producing carotenoids with shorter conjugated pi-electron systems and higher reduction potentials than beta-carotene. The PS II core complexes of both mutant strains contain approximately the same number of chlorophylls and carotenoids as the wild type but have replaced beta-carotene (11 double bonds), with neurosporene (9 conjugated double bonds) and beta-zeacarotene (9 conjugated double bonds and 1 beta-ionylidene ring). The presence of the ring appears necessary for PS II assembly. Visible and near-infrared spectroscopy were used to examine the light-induced formation of chlorophyll and carotenoid radical cations in the mutant PS II core complexes at temperatures from 20 to 160 K. At 20 K, a carotenoid cation radical is formed having an absorption maximum at 898 nm, an 85 nm blue shift relative to the beta-carotene radical cation peak in the WT, and consistent with the formation of the cation radical of a carotenoid with 9 conjugated double bonds. The ratio of Chl(+)/Car(+) is higher in the mutant core complexes, consistent with the higher reduction potential for Car(+). As the temperature increases, other carotenoids become accessible to oxidation by P(680)(+).     

Unique spectroscopic properties of synthetic 15-cis beta-carotene,an important compound in photosynthesis,and a medicine for photoprotective function

The (1(1)B(u)+) energy of synthetic 15-cis beta-carotene exhibits a linear dependence on (n(2)-1)/(n(2)+2) in non-polar and polar solvents; in this it is similar to (that of) all-trans beta-carotene. The point of intersection is at (n(2)-1)/(n(2)+2) = 0.3 for both isomers. The microenvironment of 15-cis beta-carotene in the Photosystem II reaction center was established as having a mean refractive index 1.473. Persistent spectral hole burning with a very broad (approximately 30 nm) hole observed around 500 nm (corresponding to an extremely short excited lifetime tau approximately 9 fs) indicates that 15-cis beta-carotene has/displays very efficient photoprotective quenching.     

Role of beta-carotene in the reaction centres of photosystems I and II of spinach chloroplasts prepared in non-polar solvents.

Spinach chloroplasts have been prepared nonaqueously using non-polar solvents (n-hexane, CCl4, n-hepatane) and the beta-carotene content extracted in a controlled manner. This procedure is reproducible and does not result in large structural or spectral changes of the chloroplasts. The organisation of the chlorophyll-proteins is unaltered, as fragmentation with digitonin results in the appearance of the same fractions as found previously for aqueously-prepared chloroplasts, including the pink zone containing cytochromes f and b6 in the ratio 1 : 2. The chloroplasts possess both Photosystem I activity (P-700 photo-bleaching, and NADP+ photoreduction) and Photosystem II activity (parabenzoquinone reduction with Mn2+ as electron donor, and chlorophyll fluorescence induction). Use of moderate intensity red illumination has allowed a study of the role of beta-carotene in photochemistry separate from its roles in energy transfer and photoprotection. Removal of the fraction of beta-carotene closely associated with the Photosystem I reaction centre caused the rate of NADP+ photoreduction to fall to a low, but significantly non-zero level. Thus, in the complete absence of beta-carotene, photochemistry can still be observed, however the specific association of beta-carotene with the reaction centre is required for maximal rates. We propose that beta-carotene bound at the reaction centre decreases the rate of transfer of excitation energy away from the reaction centre, and increases the rate of photochemistry. It is possible that this occurs via formation of an exciplex between ground state beta-carotene and chlorophyll in the first excited state.     

Room temperature photooxidation of β-carotene and peripheral chlorophyll in photosystem II reaction centre

Differential kinetic absorption spectra were measured during actinic illumination of photosystem II reaction centres and core complexes in the presence of electron acceptors silicomolybdate and ferricyanide. The spectra of samples with ferricyanide differ from those with both ferricyanide and silicomolybdate. Near-infrared spectra show temporary beta-carotene and peripheral chlorophyll oxidation during room temperature actinic illumination. Peripheral chlorophyll is photooxidized even after decay of beta-carotene oxidation activity and significant reduction of beta-carotene content in both reaction centres and photosystem II core complexes. Besides, new carotenoid cation is observed after about 1 s of actinic illumination in the reaction centres when silicomolybdate is present. Similar result was observed in PSII core complexes. HPLC analyses of illuminated reaction centres reveal several novel carotenoids, whereas no new carotenoid species were observed in HPLC of illuminated core complexes. Our data support the proposal that pigments of inner antenna are a sink of cations originating in the photosystem II reaction centre.     

What is beta-carotene doing in the photosystem II reaction centre?

During photosynthesis carotenoids normally serve as antenna pigments, transferring singlet excitation energy to chlorophyll, and preventing singlet oxygen production from chlorophyll triplet states, by rapid spin exchange and decay of the carotenoid triplet to the ground state. The presence of two beta-carotene molecules in the photosystem II reaction centre (RC) now seems well established, but they do not quench the triplet state of the primary electron-donor chlorophylls, which are known as P(680). The beta-carotenes cannot be close enough to P(680) for triplet quenching because that would also allow extremely fast electron transfer from beta-carotene to P(+)(680), preventing the oxidation of water. Their transfer of excitation energy to chlorophyll, though not very efficient, indicates close proximity to the chlorophylls ligated by histidine 118 towards the periphery of the two main RC polypeptides. The primary function of the beta-carotenes is probably the quenching of singlet oxygen produced after charge recombination to the triplet state of P(680). Only when electron donation from water is disturbed does beta-carotene become oxidized. One beta-carotene can mediate cyclic electron transfer via cytochrome b559. The other is probably destroyed upon oxidation, which might trigger a breakdown of the polypeptide that binds the cofactors that carry out charge separation.     

Requirement of carotene isomerization for the assembly of photosystem II in Synechocystis sp. PCC 6803

Carotene isomerase mutant (crtH mutant) cells of Synechocystis sp. PCC 6803 can accumulate beta-carotene under light conditions. However, the mutant cells grown under a light-activated heterotrophic growth condition contained detectable levels of neither beta-carotene nor D1 protein of the photosystem (PS) II reaction center, and no oxygen-evolving activity of PSII was detected. beta-Carotene and D1 protein appeared and a high level of PSII activity was detected after the cells were transferred to a continuous light condition. The PSI activities of thylakoid membranes from mutant cells were almost the same as those of thylakoid membranes from wild-type cells, both before and after transfer to the continuous light condition. These results suggest that beta-carotene is required for the assembly of PSII but not for that of PSI.     

Isolation and spectral characterization of Photosystem II reaction center from Synechocystis sp. PCC 6803

We isolated highly-purified photochemically active photosystem (PS) II reaction center (RC) complexes from the cyanobacterium Synechocystis sp. PCC 6803 using a histidine-tag introduced to the 47 kDa chlorophyll protein, and characterized their spectroscopic properties. Purification was carried out in a one-step procedure after isolation of PS II core complex. The RC complexes consist of five polypeptides, the same as in spinach. The pigment contents per two molecules of pheophytin a were 5.8 +/- 0.3 chlorophyll (Chl) a and 1.8 +/- 0.1 beta-carotene; one cytochrome b(559) was found per 6.0 Chl a molecules. Overall absorption and fluorescence properties were very similar to those of spinach PS II RCs; our preparation retains the best properties so far isolated from cyanobacteria. However, a clear band-shift of pheophytin a and beta-carotene was observed. Reasons for these differences, and RC composition, are discussed on the basis of the three-dimensional structure of complexes.     

Reduction of retinaldehyde bound to cellular retinol-binding protein (type II) by microsomes from rat small intestine

Cellular retinol-binding protein, type II (CRBP (II], an abundant protein of the rat small intestine, has recently been shown to be able to bind retinaldehyde in addition to retinol (MacDonald, P.N., and Ong, D. E. (1987) J. Biol. Chem. 262, 10550-10556). Retinaldehyde is produced in the intestine by oxidative cleavage of beta-carotene. The next step in the intestinal metabolism of vitamin A is the reduction of retinaldehyde to retinol which is then esterified for incorporation into chylomicrons. In the present study retinaldehyde bound to CRBP(II) was found to be available for reduction by microsomal preparations from rat small intestinal mucosa. The microsomal activity was about 8 times greater than the activity observed for an equal amount of cytosolic protein. Retinaldehyde reduction utilized either NADH or NADPH as cofactor, with NADH being slightly more effective. The apparent Km for retinaldehyde-CRBP(II) was 0.5 microM, and the Vmax was approximately 300 pmol/min/mg protein, a rate more than sufficient for the needs of the animal. The product retinol remained complexed to CRBP(II). The microsomal enzyme activity reduced free and bound retinaldehyde to approximately the same extent, although the aldehyde function of retinaldehyde bound to CRBP(II) was less accessible to chemical reducing agents than that of free retinaldehyde. Retinol bound to CRBP(II) could not be oxidized by the microsomal activity in the presence of NAD+, while free retinol or retinol bound to bovine serum albumin was oxidized to retinaldehyde. The more favorable reduction versus oxidation of retinoid bound to CRBP(II) consequently favored the reaction known to be required for the ultimate conversion of beta-carotene to retinyl esters for export from the gut.     

Co-oxidation of carotenes requires one soybean lipoxygenase isoenzyme.

The type II lipoxygenase (optimum pH 6.5) from soybeans was purified and separated into two fractions either by chromatography on DEAE-Sephadex or by isoelectric focusing. In the presence of linoleic acid and oxygen both fractions co-oxidise canthaxanthine or beta-carotene as effectively as a combination of these fractions. Oxygenation of linoleic acid and co-oxidation of canthaxanthine by type II lipoxygenase is stimulated by 13-hydroperoxy-cis-9,trans-11-octadecadienoic acid but not by 13-hydroxy-cis-9,trans-11-octadecadienoic acid or 9-hydroperoxy-trans-10,cis-12-octadecadienoic acid.     

Two redox-active beta-carotene molecules in photosystem II

Photosystem II (PS II) contains secondary electron-transfer paths involving cytochrome b(559) (Cyt b(559)), chlorophyll (Chl), and beta-carotene (Car) that are active under conditions when oxygen evolution is blocked such as in inhibited samples or at low temperature. Intermediates of the secondary electron-transfer pathways of PS II core complexes from Synechocystis PCC 6803 and Synechococcus sp. and spinach PS II membranes have been investigated using low temperature near-IR spectroscopy and electron paramagnetic resonance (EPR) spectroscopy. We present evidence that two spectroscopically distinct redox-active carotenoids are formed upon low-temperature illumination. The Car(+) near-IR absorption peak varies in wavelength and width as a function of illumination temperature. Also, the rate of decay during dark incubation of the Car(+) peak varies as a function of wavelength. Factor analysis indicates that there are two spectral forms of Car(+) (Car(A)(+) has an absorbance maximum of 982 nm, and Car(B)(+) has an absorbance maximum of 1027 nm) that decay at different rates. In Synechocystis PS II, we observe a shift of the Car(+) peak to shorter wavelength when oxidized tyrosine D (Y(D)*) is present in the sample that is explained by an electrostatic interaction between Y(D)* and a nearby beta-carotene that disfavors oxidation of Car(B). The sequence of electron-transfer reactions in the secondary electron-transfer pathways of PS II is discussed in terms of a hole-hopping mechanism to attain the equilibrated state of the charge separation at low temperatures.     

Triiodothyronine stimulates CMO1 gene expression in human intestinal Caco-2 BBe cells

Vitamin A is derived from provitamin A carotenoids, mainly beta-carotene, by beta-carotene 15,15'-monooxygenase (CMO1; EC 1.13.11.21). We previously found that enhancement of CMO1 mRNA expression was related to the levels of hormones, such as thyroid hormones, in chick duodenum. We investigated whether CMO1 expression was increased by triiodothyronine (T3), a thyroid hormone, using human intestinal Caco-2 BBe cells. Treatment of 7 days post-confluent Caco-2 BBe cells with T3 significantly enhanced CMO1 mRNA levels in both dose- and time-dependent manners. This T3-inducing effect on CMO1 mRNA level was blocked by actinomycin D. The levels of mRNAs for the thyroid hormone receptors TRalpha1 and TRbeta1 were significantly increased in 7 days post-confluent Caco-2 BBe cells. CMO1 enzyme activity was also significantly increased by T3 treatment in medium supplemented with fetal bovine serum. Furthermore, T3 treatment also increased the level of mRNA for lecithin:retinol acyltransferase (LRAT), but not those for cellular retinol-binding protein, type II (CRBPII) and retinal dehydrogenase 1 (RALDH1), in Caco-2 BBe cells. These results indicate that T3 is an important hormone for the regulation of vitamin A and beta-carotene metabolism-related gene expression in human small intestinal cells.     

Beta-carotene inhibits growth of human colon carcinoma cells in vitro by induction of apoptosis

Epidemiological studies suggest that beta-carotene is able to modulate the risk of cancer. A number of in vitro studies reported that beta-carotene inhibits the growth of cancer cells; however, so far little is known about the molecular mechanisms of the antiproliferative effect of beta-carotene. Here we have investigated the effects of two beta-carotene preparations, (i) beta-carotene dissolved in tetrahydrofuran (final concentration in cell culture medium: 0.5%) and (ii) beta-carotene incorporated in a water dispersible bead form, on cultured human colon carcinoma cells HT29. The treatment of cells with beta-carotene up to 30 microM for 72 h led to a significant increase in the cellular beta-carotene concentration and formation of retinol. Beta-Carotene showed only low cytotoxicity for confluent cells tested up to 30 microM, but at dietary relevant concentrations for the intestinal tract (10, 30 microM) beta-carotene was strongly cytotoxic for growing cells and induced apoptosis in HT29 cells as assessed by the Annexin-V assay (the maximal effect was observed 15 h after treatment with beta-carotene). Exposure of cells to retinol at concentrations yielding cellular retinol levels similar to those observed by beta-carotene treatment had no antiproliferative or cytotoxic effect. Furthermore, beta-carotene did not affect the activation of the extracellular signal-regulated kinases (ERK1 and ERK2) that are essential for cellular growth. In summary, beta-carotene can inhibit growth of human colon carcinoma cells in vitro by induction of apoptosis in proliferating cells.     

The effect of alpha-tocopherol on the oxidative cleavage of beta-carotene

Two cleavage pathways of beta-carotene have been proposed, one by central cleavage and the other by random (excentric) cleavage. The central cleavage pathway involves the metabolism of beta-carotene at the central double bond (15, 15') to produce retinal by beta-carotene 15, 15'-dioxygenase (E.C.888990988). The random cleavage of beta-carotene produces beta-apo-carotenoids, but the mechanism is not clear. To understand the various mechanisms of beta-carotene cleavage, beta-carotene was incubated with the intestinal postmitochondrial fractions of 10-week-old male rats for 1 h, and cleavage products of beta-carotene were analyzed using reverse-phase, high-performance liquid chromatography (HPLC). We also studied the effects of alpha-tocopherol and NAD(+)/NADH on beta-carotene cleavage. In addition to beta-carotene, we used retinal and beta-apo-14'-carotenoic acid as substrates in these incubations. Beta-apo-14'-carotenoic acid is the two-carbon longer homologue of retinoic acid. In the presence of alpha-tocopherol, beta-carotene was converted exclusively to retinal, whereas in the absence of alpha-tocopherol, both retinal and beta-apo-carotenoids were formed. Retinoic acid was produced from both retinal and beta-apo-14'-carotenoic acid incubations only in the presence of NAD(+). Our data suggest that in the presence of an antioxidant such as alpha-tocopherol, beta-carotene is converted exclusively to retinal by central cleavage. In the absence of an antioxidant, beta-carotene is cleaved randomly by enzyme-related radicals to produce beta-apo-carotenoids, and these beta-apo-carotenoids can be oxidized further to retinoic acid via retinal.     

Pulse radiolysis studies of beta-carotene in oxygenated DMSO solution. Formation of beta-carotene radical cation

The spectroscopic and kinetic characteristics of beta-carotene radical cation (beta-carotene(.+)) were studied by pulse radiolysis in aerated DMSO solution. The buildup of beta-carotene(.+) with k(1) = (4.8 +/- 0.2) x 10(8) dm(3) mol(-1) s(-1) [lambda(max) = 942 nm, epsilon = (1.6 +/- 0.1) x 10(4) dm(3) mol(-1) cm(-1)] results from an electron transfer from beta-carotene to DMSO(.+). The beta-carotene(.+) species decays exclusively by first-order reaction, k = (2.1 +/- 0.1) x 10(3) s(-1), probably by two processes: (1) at low substrate concentration by hydrolysis and (2) at high concentrations also by formation of dimer radical cation (beta-carotene)(2)(.+). Under the experimental conditions, a small additional beta-carotene triplet-state absorption ((3)beta-carotene) in the range of 525 to 660 nm was observed. This triplet absorption is quenched by oxygen (k = 7 x 10(4) s(-1)), resulting in singlet oxygen ((1)O(2)), whose reactions can also lead to additional formation of beta-carotene(.+).     

Singlet oxygen production in photosynthesis

A photosynthetic organism is subjected to photo-oxidative stress when more light energy is absorbed than is used in photosynthesis. In the light, highly reactive singlet oxygen can be produced via triplet chlorophyll formation in the reaction centre of photosystem II and in the antenna system. In the antenna, triplet chlorophyll is produced directly by excited singlet chlorophyll, while in the reaction centre it is formed via charge recombination of the light-induced charge pair. Changes of the mid-point potential of the primary quinone acceptor in photosystem II modulate the pathway of charge recombination in photosystem II and influence the yield of singlet oxygen production. Singlet oxygen can be quenched by beta-carotene, alpha-tocopherol or can react with the D1 protein of photosystem II as target. If not completely quenched, it can specifically trigger the up-regulation of the expression of genes which are involved in the molecular defence response of plants against photo-oxidative stress.     

Molecular characteristics of astaxanthin and beta-carotene in the phospholipid monolayer and their distributions in the phospholipid bilayer.

The molecular characteristics of the monolayers of astaxanthin with polar group on the beta-ionone ring in the molecule and beta-carotene without polar group and their interactions in mixed carotenoid-phospholipid monolayers and the effects of carotenoids on the phase behavior of the phospholipid bilayers were examined by the monolayer technique and differential scanning calorimetry (DSC). We found from the monolayer study that beta-carotene had an amphiphilic nature. The molecular assembly of astaxanthin in the monolayer at the hydrophobic/hydrophilic interface was more stable than that of beta-carotene. Dimyristoylphosphatidylcholine (DMPC) in the monolayer was miscible with astaxanthin in the range of 0-0.4 mol fractions of astaxanthin, but not fully miscible with beta-carotene even at low concentrations below 0.1 mol fraction of beta-carotene. Surface potential and compression/expansion cycles of beta-carotene monolayer indicated the formation of molecular aggregates by itself. DSC study showed that when small amount of astaxanthin was added, the transition temperature of dipalmitoylphosphatidylcholine (DPPC) was markedly shifted to lower temperatures and that the transition peak was asymmetrically broadened, indicative of a significant depression in cooperativity of the gel to liquid-crystalline transition. The asymmetric DSC endothermic bands of DPPC incorporating small amounts of astaxanthin were well fit by deconvolution into two to three domains containing different concentrations of astaxanthin. On the contrary, the incorporation of beta-carotene resulted in a small depression of the main transition temperature with a slight broadening of the transition peak, suggesting a small miscibility of beta-carotene with the phospholipid bilayer or a formation of aggregates of beta-carotene in the membranes. These results suggest that there would be a high localized concentration in the phase separated membrane for astaxanthin or beta-carotene to function effectively as scavenger.