Smoking attenuates the negative association between carotenoids consumption and colorectal cancer risk.

OBJECTIVES: Consumption of vegetables and fruits, physical activity, obesity and caloric intake are all strongly related to the risk of colorectal cancer (CRC). The association between dietary intake of carotenoids from vegetables/fruits and risk of CRC in the context of cigarette smoking was studied in a nutritionally diverse population. METHODS: The study included 1,817 age sex residence-matched case-control pairs from a population-based study in Northern Israel. Data were acquired by food-frequency questionnaire. Individual intake of carotenoid isomers was calculated using an Israeli food content database. Odds ratios (ORs) were calculated using conditional logistic regression models adjusted for known risk factors. RESULTS: Strong inverse associations were found with consumption of 9-cis-beta-carotene (OR = 0.35, 0.26-0.47), all-trans-beta-carotene (OR = 0.58, 0.44-0.76), cis-beta-cryptoxanthin (OR = 0.67, 0.50-0.90), all-trans-zeaxanthin (OR = 0.64, 0.48-0.86), and lutein (OR = 0.74, 0.57-0.96). Lycopene (OR = 2.22, 1.71-2.89) and all-trans-beta-cryptoxanthin (OR = 2.01, 1.48-2.73) were associated with increased risk of CRC. Inverse associations of most carotenoids with CRC, demonstrated in non-smokers, were much attenuated or reversed in past or current smokers with a highly significant interaction term. CONCLUSIONS: Consumption of most dietary carotenoids was found to be strongly associated with reduced risk of CRC. However, smoking significantly attenuated or reversed this observed protective effect on CRC occurrence. Smokers should be advised that smoking also hampers the potential health promoting effects of high fruit and vegetable consumption.

Bioavailability of a natural isomer mixture compared with synthetic all-trans beta-carotene in human serum.

The unicellular alga Dunaliella bardawil was shown previously to contain very high concentrations of beta-carotene composed of about equal amounts of the all-trans and 9-cis isomers, which differ in their physicochemical features and antioxidative activity. The uptake of alpha- and beta-carotenes, oxycarotenoids, and other lipophilic substances from a basal diet supplemented with synthetic beta-carotene or dry D. bardawil power was studied in humans. Subjects were given a basal diet supplemented daily with 40 mg beta-carotene, synthetic or natural, for a relatively short period of 14 d. Serum analyses at the end of this period detected mainly oxycarotenoids, and to a lesser extent all-trans beta-carotene and alpha-carotene, but not 9-cis beta-carotene. Retinol was increased by the all-trans beta-carotene diet. A high amount of oxidized dienic, lipophilic polar products was exhibited in HPLC predominantly in sera from the placebo and synthetic all-trans beta-carotene groups by strong, short ultraviolet absorbance peaks of 232 nm. The preferential serum absorption of all-trans beta-carotene over 9-cis beta-carotene, in parallel with the appearance of a high concentration of oxidized dienic products with supplementation of the basal diet with all-trans beta-carotene compared with the low concentration of serum-oxidized dienic products with supplementation with a natural beta-carotene source, suggests that 9-cis beta-carotene acts as an in vivo lipophilic antioxidant more efficiently than does all-trans beta-carotene.

Enhanced in vitro oxidation of plasma lipoproteins derived from hypercholesterolemic patients.

In vitro oxidation of plasma lipoproteins, derived from either normolipidemic or hypercholesterolemic subjects, was performed in the presence of copper ions. Following this procedure, hypercholesterolemic low-density lipoprotein (LDL), very-low-density lipoprotein (VLDL), and high-density lipoprotein (HDL) demonstrated greater propensity for oxidation than the corresponding normocholesterolemic lipoproteins. The oxidation was determined by the concentration of thiobarbituric acid-reactive substances (TBARS), which was 44%, 71%, and 54% greater in the patients' VLDL, LDL, and HDL in comparison to the normocholesterolemic lipoproteins, respectively. An associated reduction in trinitrobenzensulfonic acid (TNBS) reactivity in the patients' lipoproteins was noted. These changes were consistent whether expressed per lipoprotein protein or per concentration. Macrophage cholesterol esterification induced by oxidized LDL was substantially increased (up to 59%) when patients' lipoproteins were used, in comparison to control lipoproteins. A positive correlation was present between the LDL cholesterol to protein ratio, the extent of lipoprotein oxidation, and macrophage uptake of the oxidized lipoproteins. The lipoprotein content of pro-oxidant and antioxidant constituents was also analyzed. No measurable ferric or copper ions could be found in association with any of the lipoproteins. However, arachidonic acid content of the patients' LDL was 10.1% +/- 1.0% in comparison to 6.2% +/- 0.8% of total lipoprotein fatty acids in the control group (n = 5). Antioxidants such as vitamin E and carotenoids were significantly reduced in all patients' lipoproteins compared with those of controls. Thus, we suggest that increased cholesterol and arachidonic acid content and reduced concentration of antioxidants in lipoproteins of hypercholesterolemic patients may be responsible for the enhanced propensity for oxidation observed in these lipoproteins.

Beta-carotene and CNS oxygen toxicity in rats.

Beta-carotenes are reported to be potent free radical quenchers, singlet oxygen scavengers, and lipid antioxidants. Oxygen free radicals that are produced in excess during exposure to oxygen at high pressures and overwhelm the body's normal antioxidant defense systems seem to mediate the hyperoxic insult. We decided to test the possible protective effect against central nervous system oxygen toxicity of a natural beta-carotene composed of equal amounts of the all-trans and 9-cis isomers obtained from the unicellular halotolerant alga Dunaliella bardawil. Rats implanted with chronic cortical electrodes for continuous electroencephalogram monitoring were fed on ground commercial food enriched with natural beta-carotene (1 g/kg diet). On completion of 1 wk of the diet, the rats were exposed to 0.5 MPa oxygen and then their livers were removed for beta-carotene and vitamin A analysis. A significant increase was noted in the latent period preceding oxygen seizures in the group of rats in which the diet was supplemented by natural beta-carotene compared with rats given a normal diet (38.5 +/- 3.4 vs. 16.8 +/- 1.8 min; P < 0.05). Further experiments are required to evaluate the potential benefit of supplementing the diet of divers and patients exposed to high pressures of oxygen with the beta-carotene-rich D. bardawil.

Availability and antiperoxidative effects of beta-carotene from Dunaliella bardawil in alcohol-drinking rats.

The present study demonstrated the high bioavailability and antiperoxidative capacity of the natural beta-carotene isomer mixture of Dunaliella bardawil compared with synthetic beta-carotene under alcohol-induced oxidative stress. Weanling rats were adapted to ethanol by increasing ethanol levels in their drinking water to 30% at 5% intervals per week; other rats received water with no added ethanol. One water-drinking group and one alcohol-drinking group with no dietary carotene were used as controls. Two water-drinking groups were supplemented with 1 g/kg diet beta-carotene either from Dunaliella or a synthetic source, and due to reduced food intake, two ethanol-fed groups received 2 g beta-carotene per kilogram of diet from each source. Following 3 months of ethanol consumption, both carotene sources were found to prevent ethanol-induced lipid peroxidation as expressed by the hepatic conjugated oxidized dienes level. However, in the algal-fed rats, hepatic carotene and vitamin A levels were higher. In addition to a lower performance of the group fed ethanol and synthetic beta-carotene, there were three deaths in this group.

Suppression by beta-carotene-rich algae Dunaliella bardawil of the progression, but not the development, of spontaneous mammary tumours in SHN virgin mice.

We previously found that beta-carotene-rich algae Dunaliella bardawil markedly inhibited spontaneous mammary tumourigenesis of mice. This study was carried out to clarify whether D. bardawil inhibits the development or the progression or both of mammary tumours. A high mammary tumour strain of SHN virgin mice were given vitamin A deficient AIN-76TM diet supplemented with D. bardawil during the limited period of 3 months between 1-4 months of age (Experiment I: the stage of tumour development), 4-7 months (Experiment II: the stage of initiation and progression) or 7-10 months (Experiment III: the stage of progression). The concentration of beta-carotene in the diet was 5.1 x 10(-5)%. The respective controls received AIN-76TM diet containing retinyl palmitate (2.2 x 10(-4)%) during the same periods as in the experimental groups. Both the experimental and the control mice were fed a commercial diet during all other periods. The diets and tap water were provided ad libitum. In Experiment I, mammary tumour incidence was higher in the experimental group than in the control at all months examined except at 5 months of age, while the cause is not clear at present. Meanwhile, mammary tumourigenesis was significantly suppressed in the experimental mice compared to the controls in Experiments II and III. Whereas tumorous mice were higher than non-tumorous mice in blood levels of lactic acid and glucose in the control, mice given D. bardawil maintained the levels of non-tumorous mice even after the development of tumours. D. bardawil feeding also induced a higher glucose tolerance. All results strongly suggest that D. bardawil can inhibit the progression of spontaneous mammary tumours of mice by increasing the homeostatic potential of the host animals as well as by the well-known antioxidant function of beta-carotene in D. bardawil.

Inhibition by beta-carotene-rich algae Dunaliella of spontaneous mammary tumourigenesis in mice.

Effects of beta-carotene-rich algae Dunaliella on spontaneous mammary tumourigenesis were studied in SHN mice. In both breeder and virgin mice, mammary tumourigenesis was significantly inhibited in the experimental groups fed diets supplemented with spray dried powder of D. bardawil and oily solution of D. salina Teod. extract compared with the control. There was little difference between the experimental and the control groups in plasma prolactin level, endocrine organ weights and pattern of oestrous cycles. Moreover, mammary gland susceptibility to mammotrophic hormones was affected little by Dunaliella. These results suggest that the inhibition by Dunaliella of spontaneous mammary tumourigenesis of mice may not be through their modulation of endocrine systems.

Effects of beta-carotene-rich algae Dunaliella bardawil on the dynamic changes of normal and neoplastic mammary cells and general metabolism in mice.

As a possible step to estimate the mechanism of the inhibitory role of Dunaliella bardawil (Db) in the initial progression of spontaneous mammary tumours of SHN virgin mice, the DNA synthesizing enzyme activities and the in vivo 31P-NMR spectroscopy of normal and neoplastic mammary cells were examined in mice fed Db. The blood levels of glucose and free fatty acids and 1H-NMR spectroscopy of serum or urine were also studied in these animals to check the general metabolic background. Beginning at 4 months of age, the experimental and the control mice were given a vitamin A deficient AIN-76TM diet supplemented with Db (the concentration of beta-carotene was 5.1 x 10(-5)%) and AIN-76TM diet containing retinyl palmitate (2.2 x 10(-4)%), respectively, until 3 weeks after mammary tumour appearance (tumourous) or 9 months of age (non-tumourous). An increasing rate of thymidine kinase activity in mammary tumour was decreased by Db, while the thymidylate synthetase activity was affected little by the treatment. Lipid synthesis was accelerated by Db in normal mammary cells; however, no difference was observed between the experimental and the control groups in energy and lipid metabolism of mammary tumour cells. The growth of preneoplastic mammary hyperplastic alveolar nodules was also enhanced by Db. Db prevented an increase of blood levels of glucose and free fatty acids and a decrease of cholin plus phosphorylcholin level after tumour appearance. Liver weights of mice given Db were greater than those of the control in both the tumourous and non-tumourous groups. Db had little effect on the urinary component levels before and after the appearance of mammary tumours. These results indicate that Db promotes the growth of normal mammary gland cells, but inhibits that of neoplastic cells, and that the inhibitory effect of Db on mammary tumour progression is due to its normalizing both the organ specific and the general metabolism.

Effects of beta-carotene-rich algae Dunaliella on reproduction and body growth in mice.

Effects of beta-carotene-rich algae Dunaliella on reproduction and body growth were investigated in SHN mice. At 2-3 months of age, female mice were placed with males and fed different diets simultaneously throughout the experiment; Group I was provided with AIN-76TM diet containing 2.2 X 10(-4)% retinyl palmitate. Group II or III was given vitamin A deficient AIN-76TM diet supplemented with spray dried powder of Dunaliella or its oily solution. The final concentrations of beta-carotene in the diets were 5.1 X 10(-5)% and 3.0 X 10(-2)% in groups II and III respectively. Groups II and III were markedly lower and higher than group I in still-birth rate and litter size or rearing rate, respectively. Body growth was also enhanced in male offspring fed supplemental beta-carotene continuously (groups II and III). These results have demonstrated that, in mice, reproduction and body growth are improved by beta-carotene from Dunaliella.

[Effect of an extract containing esterol on plasma cholesterol and oxidants].

16 patients with hypercholesterolemia were treated with an extract of alpha-alpha leaves (esterol) while on a low-fat, low-cholesterol diet. Esterol is believed to inhibit the absorption of cholesterol and bile acids and may interfere with the absorption of essential nutrients. As oxidative modification of lipoproteins is required for the process of atherosclerosis, plasma antioxidant vitamins were followed. After 4 months of treatment, plasma cholesterol decreased by 10% from 282 to 250 mg/dl (p < 0.001) and LDL cholesterol by 13%, from 203 to 177 mg/dl (p < 0.001). Plasma antioxidant vitamins E, A and beta-carotene were unchanged. Thus, esterol has a cholesterol-lowering effect but apparently does not lower fat-soluble, plasma antioxidant vitamins. Both cholesterol-lowering and plasma antioxidant vitamins are important for the primary prevention of coronary artery disease in hypercholesterolemia.

[Relationship between plasma antioxidants and coronary artery disease].

Plasma concentrations of the antioxidants, vitamin E, beta-carotene and vitamin A were studied. 29 patients in whom at least 70% obstruction of 1 coronary artery was found, and 73 who were normal according to noninvasive tests were compared using multivariable, logistic, regression analysis. The group with coronary disease had significantly lower plasma vitamin E and beta-carotene concentrations as compared with the normal group (means +/- SD: 44.2 +/- 18.3 micrograms/ml vs 50.6 +/- 22.4 and 1.0 +/- 0.5 micrograms/ml vs 1.1, respectively) but plasma vitamin A concentration was not reduced. Decrease in plasma antioxidant vitamin concentrations may be linked to atherogenesis by LDL oxidative modification, which explains the inverse relationship found between the risk of coronary artery disease and plasma antioxidant vitamins.

Osmoregulation in the Halotolerant Alga Asteromonas gracilis.

Asteromonas gracilis, a green wall-less halotolerant alga, grows on salt concentrations from 0.5 molar NaCl (seawater) to saturation (4.5 molar NaCl). The specific growth rate was maximal at concentrations between 0.5 and 2.5 molar and only gradually decreased above 2.5 molar. Photosynthetic oxygen evolution was maximal over a range of salinities around 2.5 molar and the photosynthesis to respiration ratio showed a maximum at 1.5 molar NaCl. The alga accumulates large amounts of intracellular glycerol in response to saline conditions. The glycerol content of the cells varied in direct proportion to the extracellular salt concentration, being about 50 and 400 picograms glycerol per cell in algae grown at 0.5 and 4.5 molar NaCl, respectively. In salt concentrations lower than 3.5 molar and at growth temperatures below 40 C, essentially all the glycerol was intracellular. Above 3.5 molar NaCl, about 25 per cent of the total glycerol leaked slowly from the cells to the medium. Treating the algae for several minutes at temperatures exceeding 47 C caused 50 per cent release of the internal glycerol. At 60 C, 100 per cent of the glycerol was released. When the extracellular salt concentration was increased or decreased, the intracellular glycerol varied accordingly, reaching its new intracellular level after a few hours. Both photosynthesis and respiration were inhibited on transfer of the cells from 1.5 to 3.5 molar NaCl but were not inhibited on transfer of the cells from 3.5 to 1.5 molar NaCl. The kinetics of photosynthetic resumption preceded the kinetics of glycerol biosynthesis. The above results indicate the existence of osmotic regulations in Asteromonas gracilis via the accumulation of intracellular glycerol.

Dietary lipid level and the availability of beta-carotene of Dunaliella-bardawil in rats.

High plasma and tissue levels of beta-carotene have been implicated to be inversely related to the incidence of some types of cancer. Previous studies indicated that the availability of the natural beta-carotene isomer mixture of Dunaliella bardawil was severalfold higher than that of synthetic all-trans beta-carotene. This study illustrated that this higher availability, as expressed by hepatic and plasma beta-carotene, can be further enhanced by increased dietary lipid level. For 12 days, weanling male and female rats were fed diets containing either 0.1% all-trans beta-carotene or spray-dried Dunaliella bardawil having an equal level of the beta-carotene isomer mixture. The diets contained 5 or 15% soybean oil. The rise in dietary fat resulted in a marked increase in plasma and liver beta-carotene, with the Dunaliella isomer mixture being more effective. Higher levels were found in female than in male animals. These findings may be relevant when a high dose of beta-carotene is considered as a potential anticancer agent.

Isolation, Characterization, and Partial Purification of a Reduced Nicotinamide Adenine Dinucleotide Phosphate-dependent Dihydroxyacetone Reductase from the Halophilic Alga Dunaliella parva.

An NADP(+)-dependent dihydroxyacetone reductase, which catalyzes specifically the reduction of dihydroxyacetone to glycerol, has been isolated from the halophilic alga Dunaliella parva. The enzyme has been purified about 220-fold. It has a molecular weight of about 65,000 and is highly specific for NADPH. The pH optima for dihydroxyacetone reduction and for glycerol oxidation are 7.5 and 9.2, respectively. The enzyme has a very narrow substrate specificity and will not catalyze the reduction of glyceraldehyde or dihydroxyacetone phosphate. It is suggested that this enzyme functions physiologically as a dihydroxyacetone reductase in the path of glycerol synthesis and accumulation in Dunaliella.

The Role of Glycerol in the Osmotic Regulation of the Halophilic Alga Dunaliella parva.

Dunaliella parva, a green halophilic alga, was found to accumulate very large amounts of intracellular glycerol. Through measurements of the intracellular volume the internal concentration of glycerol was calculated and found to be around 2.1 m in cells cultured in 1.5 m NaCl. When the extracellular salt concentration of an algal suspension was increased or decreased, the intracellular glycerol varied accordingly, reaching its new osmotic equilibrium after about 90 minutes. Since no leakage of intracellular glycerol was observed above 0.6 m NaCl, these alterations in glycerol content are interpreted as due to metabolic formation and degradation of intracellular glycerol. The above results indicate the existence of a new type of algal osmoregulation, in which the osmotic balance depends on the synthesis or degradation of intracellular glycerol in response to the external salt concentration.

Photosynthetic Activities of the Halophilic Alga Dunaliella parva.

Dunaliella parva, a unicellular halophilic alga, was found to evolve oxygen photosynthetically only in the presence of a high osmolar concentration. Cell free preparations were obtained by placing the cells in a medium of low osmolarity. The fragments obtained showed a high photoreducing and photophosphorylating activity except for their inability to catalyze all ferredoxin dependent photoreactions. Placing the cells in a medium of intermediate osmolarity produced a "chloroplast" preparation which maintained some capacity for O(2) evolution and CO(2) fixation, while possessing the ability to catalyze the photoinduced reduction of ferricyanide. Enzymic and photosynthetic reactions of cell-free preparations from D. parva were inhibited, rather than stimulated, by the salt concentration optimal for growth. These results were interpreted as indicating the existence of a steep NaCl gradient in vivo between the medium and the cell compartments which are not permeable to salt.

Is nicotinamide adenine dinucleotide phosphate an obligatory intermediate in photosynthesis?

The site of action of the inhibitors disalicylidenepropanediamine and pyrophosphate was more closely defined as acting on ferredoxin. Three inhibitors which act on the electron transport path between ferredoxin and NADP: disalicylidenepropanediamine, pyrophosphate, and phosphoadenosinediphosphate ribose, had no effect on photosynthesis in cell free preparations of Dunaliela parva at concentrations which completely inhibited the enzymic activity on which each inhibitor acts. The addition of disalicylidenepropanediamine to dark-grown Euglena gracilis cells prevented the light-induced formation of NADP-dependent glyceraldehyde-3-phosphate dehydrogenase, but not of photosynthesis, chlorophyll synthesis, or NAD-dependent glyceraldehyde-3-phosphate dehydrogenase.The above results are interpreted as indicating that, at least under some conditions, a reduced product of photosystem I preceding ferredoxin in the electron transport path can serve as the reductant of CO(2) in photosynthesis.

On the Factors Which Determine Massive beta-Carotene Accumulation in the Halotolerant Alga Dunaliella bardawil.

Dunaliella bardawil, a beta-carotene-accumulating halotolerant alga, has been analyzed for the effect of various growth conditions on its pigment content, and compared with Dunaliella salina, a beta-carotene nonaccumulating species. In D. bardawil, increasing light intensity and light period or inhibiting growth by various stress conditions such as nutrient deficiency or high salt concentration caused a decrease in the content of chlorophyll per cell and an increase in the amount of beta-carotene per cell. As a result, the beta-carotene-to-chlorophyll ratio increased from about 0.4 to 13 grams per gram and the alga changed its visual appearance from green to deep orange. D. salina grown similarly decreased in content of both chlorophyll and beta-carotene per cell and the culture turned from green to yellowish. Low chlorophyll-containing cells of D. bardawil or D. salina exhibit very high photosynthetic rates when expressed on a chlorophyll basis ( approximately 600 micromoles O(2) evolved per milligram chlorophyll per hour).Variation of pigment content in D. bardawil by a large variety of environmental agents has been correlated with the integral irradiance received by the algal culture during a division cycle. The higher the integral irradiance per division cycle, the lower the chlorophyll content per cell; the higher the beta-carotene content per cell, and therefore the higher the beta-carotene-to-chlorophyll ratio. The results are interpreted as indicating a protecting effect of beta-carotene against injury by high irradiance under conditions of impairment in chlorophyll content per cell.