Regulation of carotene synthesis in Phycomyces

Summary Threeindependent mutations of Phycomyces blakesleeanus resulting in overaccumulation of -carotene are recessive and belong to the same complementation group. The corresponding gene has been named carS. Evidence is presented that gene carS is not the same as gene carA, previously defined by mutations blocking carotene production. Vitamin A increases carotenogenesis in wild types and in carS mutants to about the same extent. Intersexual heterokaryosis increases carotenogenesis most prominently in carS genetic backgrounds (up to 300 times the production of the wild type in the same conditions). Vitamin A, intersexual heterokaryosis and carS mutations are thought to stimulate carotenogenesis through different mechanisms. It is suggested that the carS gene product participates in end-product regulation of the pathway.     

Carotene-superproducing mutants ofPhycomyces blakesleeanus

The wild-type mycelia of the fungusPhycomyces blakesleeanus are yellow because they contain small amounts of β-carotene. Some mutations lead to large increases in β-carotene content. These “deep yellow” mutants carry recessive mutations in either of two genes,carD andcarS, not linked to each other or to other genes related to carotenogenesis. ThecarS mutants contain up to 100 times more β-carotene than the wild type; thecarD mutants, up to 20 times.carS mutants are unable to form zygospores and their carotenogenesis is not activated by retinol; on the other hand,carD mutants complete the sexual cycle and respond to retinol.carS mutations are epistatic overcarD mutations. The product of genecarS mediates the end-product regulation of the pathway; it is suggested that thecarD gene product increases the amount or the activity of thecarS gene product.     

Method for the simultaneous determination of retinol and beta-carotene concentrations in human tissues and plasma

To understand differential tissue distribution of retinoids and carotenoids, as it might influence biological processes in humans, we developed and demonstrated a method for measuring them in selected human tissues. The method includes internal standards and a secondary reference standard to eliminate the need for external standard calibration and to minimize sample-handling errors. Tissues were digested (saponified) in ethanolic KOH. Retinol and beta-carotene were extracted with organic solvent containing internal standards. Analytes were separated using isocratic liquid chromatography and quantified at 325 nm for retinol and 450 nm for beta-carotene. Plasma was analyzed in a similar way but without saponification. Retinal-O-ethyloxime and beta-apo-12'-carotenal-O-t-butyloxime served as internal standards. Plasma, breast, and fat from breast surgery patients and colon, liver, muscle, and fat from colon surgery patients were analyzed. Within-day relative standard deviations (RSDs) for plasma were <0.04 for beta-carotene and <0.03 for retinol, between-day RSDs were <0.05 for beta-carotene and <0.04 for retinol. Saponification ensured complete extraction of retinol and beta-carotene and removal of triglycerides that "foul" chromatographic columns. It seems retinol and beta-carotene concentrations in tissues and blood of cancer patients are the same or higher than those in corresponding tissues of patients without these cancers.     

Photoinduced accumulation of carotene in Phycomyces

Blue light stimulates the accumulation of beta-carotene (photocarotenogenesis) in the fungus Phycomyces blakesleeanus. To be effective, light must be given during a defined period of development, which immediately precedes the cessation of mycelial growth and the depletion of the glucose supply. The competence periods for photocarotenogenesis and photomorphogenesis in Phycomyces are the same when they are tested in the same mycelium. Photocarotenogenesis exhibits a two-step dependence on exposure, as if it resulted from the additon of two separate components with different thresholds and amplitudes. The low-exposure component produces a small beta-carotene accumulation, in comparison with that of dark-grown mycelia. The high-exposure component has a threshold of about 100 J· m^–2 blue light and produces a large beta-carotene accumulation, which is not saturated at 2·10⁶ J·m^–2. Exposure-response curves were obtained at 12 wavelengths from 347 to 567 nm. The action spectra of the two components share general similarities with one another and with those of other Phycomyces photoresponses. The small, but significant differences in the action spectra of the two components imply that the respective photosystems are not identical. Light stimulates the carotene pathway in the carB mutants, which contain the colourless precursor phytoene, but not beta-carotene. Carotenogenesis is not photoinducible in carA mutants, independently of their carotene content. This and other observations on various car mutants indicate that light prevents the normal inhibition of the pathway by the carA and carS gene products. The chromophore(s) for photocarotenogenesis are presumably flavins, and not carotenes.We thank Dr. A. Palit, C. Chmielewicz and D. Durant (same address as E.D.L.) and L.M. Corrochano, A. Fernández Estefane, and J. Córdoba López (same address as E.R.B.) for their help. This work was supported by grants from Comisión Asesora para Investigación Científica y Técnica and Comisión Interministerial para Ciencia y Tecnología to E.C.O.; from the National Institutes of Health and the National Science Foundation to E.D.L.; and from the U.S.-Spain Joint Committee for Scientific and Technological Cooperation to E.C.O. and E.D.L.     

Carotene-superproducing strains of Phycomyces.

Production of beta-carotene by wild-type Phycomyces blakesleeanus can be stimulated by light, chemicals, regulatory mutations, and sexual interaction between mycelia of opposite sex. Through genetic manipulations, we have isolated strains which have simultaneously and constitutively incorporated several of these stimulatory effects. In the dark and in a simple medium, some of the strains produce up to 25 mg of beta-carotene per g (dry weight), or about 500 times the wild-type production under the same conditions. High lycopene-producing strains have also been isolated by using carR mutants, which are blocked in the conversion of lycopene to beta-carotene. These strains should be useful in both industrial production of these pigments and basic research related to carotenogenesis.     

Antimutagenicity profiles of some natural substances.

Selected antimutagenicity listings and profiles have been prepared from the literature on the antimutagenicity of retinoids and the carotenoid beta-carotene. The antimutagenicity profiles show: (1) a single antimutagen (e.g., retinol) tested in combination with various mutagens or (2) antimutagens tested against a single mutagen (e.g., aflatoxin B1). Data are presented in the profiles showing a dose range for a given antimutagen and a single dose for the corresponding mutagen; inhibition as well as enhancement of mutagenic activity is indicated. Information was found in the literature on the testing of selected combinations of 16 retinoids and carotenoids vs. 33 mutagens. Of 528 possible antimutagen-mutagen combinations, only 82 (16%) have been evaluated. The most completely evaluated retinoids are retinol (28 mutagens), retinoic acid and retinol acetate (7 mutagens each), and retinal and retinol palmitate (6 mutagens each). beta-Carotene is the most frequently tested carotenoid (15 mutagens). Of the remaining retinoids and carotenoids, 8 were evaluated in combination with a single mutagen and the other 2 were tested against only 2 or 3 mutagens. Most of the data on antimutagenicity in vitro are available for S. typhimurium strains TA98 and TA100. Substantial data also are available for sister-chromatid exchanges in vitro and chromosome aberrations in vitro and in vivo. This report emphasizes the metabolic as well as the antimutagenic effects of retinoids in vitro and in vivo.     

Effect of renal and liver failure on blood levels of vitamin A, its precursor (beta-carotene) and its carrier proteins (prealbumin and retinol binding protein)

Plasma beta-carotene and retinol assay was performed by high pressure liquid chromatography (HPLC) in subjects with chronic renal failure or liver cirrhosis. In the same subjects blood prealbumin (PA) and retinol binding protein (RBP) were determined by immunological technique. A considerable increase of retinol and in a lesser extent of beta-carotene was noted in the blood of patients with renal insufficiency. In cirrhotic patients it was shown a marked decrease both of beta-carotene and retinol plasma concentrations. PA and RBP there were greatly increased in renal failure and decreased in liver cirrhosis. This results suggest that kidney and liver chronic failure interfere with vitamin A metabolism throughout their action on metabolic processes of synthesis and elimination of PA and RBP.     

Effect of organic acids on the biosynthesis of carotenes by an Actinomyces chrysomallus strain

Synthesis of carotenes by Actinomyces chrysomallus var. carotenoides was stimulated by citric, acetic, oxalacetic, fumaric, succinic, malic, alpha-ketoglutaric, tartaric, pyruvic, and propionic acids. Acetic acid acts as a precursor of carotene synthesis and also has another stimulating mechanism of action on carotenogenesis of the actinomycete. Acetic, furmaric, malic, succinic, and alpha-ketoglutaric acids stimulate cyclization of lycopene yielding beta-carotene.     

Biphasic action of retinoids on gonadotropin receptor induction in rat granulosa cells in vitro

Vitamin A (retinol) has been held to be uniquely essential for normal vision and reproduction, all other functions being served by its metabolite retinoic acid. The inability of retinoic acid to maintain adequate serum progesterone is implicated as the cause of fetal resorption. The availability of lipoproteins is a major limiting factor in progesterone production and the ovarian expression of lipoprotein receptors is dependent on the action of luteinizing hormone (LH). Therefore, we investigated the effects of retinol and retinoic acid on LH receptor induction by ovarian cells in an attempt to determine the basis for the reported differences in the gonadal action of these two retinoids. Our results indicate that retinoic acid (10(-10) M) and retinol (10(-8) M) each synergistically enhance the ability of follicle stimulating hormone (FSH) to induce LH-receptors and to stimulate the formation of cyclic adenosine 3',5'-monophosphate (cAMP) and progesterone. However, at higher concentrations, both retinoids inhibited these effects of FSH. For every measured effect, retinoic acid was more potent than retinol. Since retinol is metabolized to retinoic acid in other tissues, these results suggest that retinoic acid may be the mediator of the action of retinol on the ovary and that retinol's unique effect on reproduction needs to be investigated further.     

Functional studies of newly synthesized benzoic acid derivatives: identification of highly potent retinoid-like activity

Three newly synthesized benzoic acid derivatives (terephthalic acid anilides, chalcone carboxylic acid, and azobenzene carboxylic acid), with a certain structural similarity to retinoic acid, were examined for their retinoid-like bioactivity and their capacity to bind to cellular retinoid binding proteins. Two in vitro systems were used to evaluate their retinoid-like bioactivity: inhibition of adipose conversion of ST 13 murine preadipose cells and growth promotion of murine sarcoma virus (MSV)-transformed 3T3 cells in serum-free culture. All three compounds tested inhibited ST 13 adipose conversion at nanomolar concentrations in a manner similar to classical retinoids such as retinoic acid. The growth-stimulating activity of these compounds on MSV-transformed 3T3 cells was one to two orders of magnitude greater than that of retinoic acid. Simultaneous treatment with these compounds and retinoic acid produced only a barely detectable additive effect, suggesting a common mechanism of action, whereas unrelated mitogens, thrombin, and insulin worked synergistically in combination with retinoic acid. None of the compounds competed with retinol for binding to cellular retinol binding protein. However, two of the three competed with retinoic acid for binding to cellular retinoic acid binding protein. This study provides evidence that the newly synthesized compounds should be included among the retinoids and that their strong biological activity will undoubtedly contribute to the biological and medical application of retinoids.     

Retinoid modulation of plasminogen activator production in rat Sertoli cells

Tissue type (t) and urokinase type (u) plasminogen activators (PAs) have been shown to be secreted by Sertoli cells in the seminiferous tubules in a cyclic fashion and to be dependent upon FSH stimulation or upon the presence of adjacent spermatogenic cells. In the present study we have analyzed the production of PAs by retinoid-treated rat Sertoli cells. In addition, because retinoids modulate the response of Sertoli cells to FSH either potentiating or antagonizing its action, we have investigated a possible modulation of FSH-stimulated PA production. Under basal conditions, Sertoli cells, isolated from prepubertal rats, secrete predominantly uPA. A significant dose-dependent inhibition of uPA activity was observed after treatment with retinol, while no significant effect was detected upon tPA secretion. When Sertoli cells were cultured in the presence of 0.25 microM retinol, a significant inhibition of uPA activity was evident after 16 h of treatment and reached approximately 80% after 48 h of treatment. The analysis of the mRNA levels revealed that retinol induces an inhibition of the steady-state levels of uPA mRNA without affecting those of tPA. Moreover, retinol affected uPA mRNA levels by increasing mRNA turnover. The effect of retinoids on Sertoli cells isolated from older animals was less evident, possibly due to the reduced production of uPA with the increase of age of the donor animals. Our results on the effect of retinoids upon Sertoli cell uPA production reinforce the importance of retinoids in the control of postnatal testis development.     

Binding of retinoids and beta-carotene to beta-lactoglobulin. Influence of protein modifications

The binding of retinol, retinyl acetate, retinoic acid and beta-carotene to native, esterified and alkylated beta-lactoglobulin was followed by quenching of tryptophan fluorescence. Three studied retinoids bind to native or modified beta-lactoglobulin in 1:1 molar ratios, with apparent dissociation constants in the range of 10(-8) M. The maximum tryptophan fluorescence quenching of unmodified beta-lactoglobulin by beta-carotene is observed at the ligand/protein ratio of 1:2. Esterification and alkylation of beta-lactoglobulin shift the ratio of beta-carotene/protein to 1:1. In all the cases, except for retinoic acid binding to N-ethyllysyl-BLG, the performed chemical modifications of beta-lactoglobulin enhance protein binding affinity. Measured apparent dissociation constants of beta-carotene complexes with native and modified beta-lactoglobulin are an order of magnitude lower from binding constants of other studied retinoids.     

Effect of retinoids and carotenoids on prostaglandin formation by oral squamous carcinoma cells.

Several studies have correlated the excessive production of prostaglandins (PGs) with tumor promotion and the suppression of the immune response. Inhibition of PGs by pharmacological agents has been demonstrated to enhance immunocompetence, and to suppress growth of tumors in animals and humans. In this study we examined the effect of retinol (I), all-trans-retinoic acid (II), N-(4-Hydroxyphenyl) retinamide (N-4-HPR) (III), canthaxanthin (CTX) (IV), and beta-carotene (beta-CT) (V) on the bioconversion of 14C-arachidonic acid (AA) to PGE2 by squamous carcinoma cells of the tongue, SCC-25. Agents (I), (II), (III), (IV) inhibited while (V) stimulated PGE2 formation in a dose related manner. N-4-HPR was the most potent inhibitor of PGE2 synthesis. The data suggest that certain retinoids and carotenoids have the potential of inhibition of PG synthesis by oral squamous carcinoma cells. Inhibitory effects such as those described here and antioxidant properties might in part contribute to the antiinflammatory and anticarcinogenic activity of retinoids in vivo.     

An in vitro cell model system to study the action of retinoids on Leydig cell steroidogenesis

In this study we examined the effects of retinol and retinoic acid on steroid production in MA-10 mouse Leydig tumor cells. Results showed that both retinol and retinoic acid greatly increased progesterone production in this cloned cell line. The stimulatory effect of retinoids is not inhibited by cycloheximide suggesting that de novo protein synthesis is not required. The presence of the retinoid binding proteins CRBP and CRABP could not be detected in MA-10 Leydig cell cytosol indicating that the stimulatory action of retinoids on progesterone production is not mediated through these cellular binding proteins. Both previous and present findings suggest that retinoids play an important role in the regulation of Leydig cell steroidogenesis and that MA-10 Leydig tumor cells may represent an ideal in vitro cell system to study the mechanism of action of retinoids in Leydig cell steroidogenesis.     

Effect of retinoids on growth factor-induced anchorage independent growth of human fibroblasts

Summary We have studied the effects of all-trans retinol, all-trans retinoic acid, and anhydroretinol, a biologically inactive retinoid, on anchorage-independent growth of human fibroblasts induced by purified growth factors. The anchorage-independence assay was, conducted in medium supplemented with serum that had had its peptide growth factors inactivated by treatment with dithiothreitol and iodoacetamide. Physiologic concentrations of either all-trans retinol (0.5 μM) or all-trans retinoic acid (1.0 nM) but not anhydroretinol (0.5 μM) reduced the frequency of anchorage-independent growth of normal human fibroblasts induced by platelet-derived growth fator (PDGF). All-trans retinol was also tested for its effect on the frequency of anchorage-independent growth induced by basic fibroblast growth factor (bFGF) and was found to decrease this growth. All-trans retinol also reduced the frequency of anchorage-independent growth of the human fibrosarcoma-derived cell, line, HT1080, which grew in semisolid medium without added growth factors. Inasmuch as these retinoids reduced the frequency of anchorage-independent growth induced by either PDGF or bFGF and because PDGF and bFGF bind to independent cell membrane receptors and are known to stimulate different pathways leading to DNA synthesis, the data suggest that physiologically active retinoids, have an effect on a step that is common to both signal pathways. This research was supported in part by Department of Energy, Washington, DC, grant DE-F602-87ER-60524 and by DHHS grants CA21289 and, CA32490 from the National Cancer Institute, Bethesda, MD.     

New Mutants of Phycomyces blakesleeanus for (beta)-Carotene Production

The accumulation of (beta)-carotene by the zygomycete Phycomyces blakesleeanus is increased by mutations in the carS gene. The treatment of spores of carS mutants with N-methyl-N(prm1)-nitro-N-nitrosoguanidine led to the isolation, at very low frequencies, of mutants that produced higher levels of (beta)-carotene. Strain S556 produced about 9 mg of (beta)-carotene per g of dry mass when it was grown on minimal agar. Crosses involving strain S556 separated the original carS mutation from a new, unlinked mutation, carF. The carF segregants produced approximately as much carotene as did carS mutants, but they were unique in their ability to produce zygospores on mating and in their response to agents that increase carotenogenesis in the wild type. The carotene contents of carF segregants and carF carS double mutants were increased by sexual interaction and by dimethyl phthalate but were not increased by light or retinol. Mixed opposite-sex cultures of carF carS mutants contained up to 33 mg of (beta)-carotene per g of dry mass. Another strain, S444, produced more (beta)-carotene than did S556 but was marred by slow growth, defective morphology, and bizarre genetic behavior. In all the strains tested, the carotene concentration was minimal during the early growth phase and became higher and constant for several days in older mycelia.