Moshe J. Werman

Fructose and related phosphate derivatives impose DNA damage and apoptosis in L5178Y mouse lymphoma cells

Glycation between reducing sugars and amino groups of long-lived macromolecules results in an array of chemical modifications that may account for several physiological complications. The consequences of the reaction are directly related to the reactivity of the sugars involved, whether aldoses or ketoses, phosphorylated or non-phosphorylated. So far, most studies have been focused on glucose, while fructose, a faster glycating agent, attracted minor attention. We have recently demonstrated that under in vitro conditions fructose and its phosphate derivatives can modify plasmid DNA faster than glucose and its phosphate metabolites. In the present study we provide further evidences suggesting that fructose and its phosphate metabolites, at the tested conditions, are cytotoxic and inflict deleterious DNA modifications to L5178Y cells in culture. Damage was verified by viable cell counts, MTT assay, colony forming ability, induction of mutation in the thymidine kinase gene, internucleosomal DNA cleavage, and single strand breaks. The intensity of the tested sugars to impose damage increased significantly in the following order: sucrose = glucose 1-phosphate < glucose < glucose 6-phosphate < fructose 1-phosphate = fructose < fructose 6-phosphate. Aminoguanidine, an inhibitor of the glycation reaction, inhibited internucleosomal DNA cleavage. Taken together, these results suggest that fructose triggers deleterious modification in cultured cells through the glycation process, and thus should deserve more attention as an agent that may induce physiological complications.

Fructose triggers DNA modification and damage in an Escherichia coli plasmid.

The nonenzymatic reaction between reducing sugars and amino groups of long-lived macromolecules results in an array of chemical modifications that may account for several physiological complications. The characteristics of the reaction are directly related to the type of the reducing sugars involved, whether aldoses or ketoses, phosphorylated or non-phosphorylated, and these in turn determine the consequences of the induced modifications. So far, most studies have been focused on the nonenzymatic reaction between glucose and proteins, while the reaction with fructose, a faster glycating agent, attracted only a minor attention. We have recently demonstrated that long-term fructose consumption induces age-related changes in collagen from skin and cortical bones faster than glucose. In the present study we provide evidence that fructose and its phosphate metabolites can modify DNA faster than glucose and its phosphate metabolites under in vitro conditions. Incubating the plasmid pBR322 with fructose and glucose phosphate metabolites induced DNA modifications and damage that were verified by gel electrophoresis and transformation capacity of the plasmid into an Escherichia coli host. The intensity of the tested sugars to modified and damage DNA after incubation for 15 days increased significantly in the following order: glucose 1-phosphate < glucose < glucose 6-phosphate < fructose 1-phosphate < fructose < fructose 6-phosphate. The data suggest that fructose should deserve more attention as a factor that may influence glycation and induce physiological complications.

Bioavailability of the isomer mixture of phytoene and phytofluene-rich alga Dunaliella bardawil in rat plasma and tissues

Dunaliella bardawil, a beta-carotene-accumulating alga was treated by the bleaching herbicide norflurazon to select sub-species rich with a mixture of 9-cis and all-trans stereoisomers of phytoene and phytofluene. The present study determines the bioavailability of phytoene and phytofluene with their stereoisomers in rats fed on a diet supplemented with Dunaliella phytoene-rich spray dried powder. Three groups of female weanling rats, eight animals each, were fed AIN diets for two weeks. The control consumed the diet as is. The experimental group was supplemented with 50 g Dunaliella powder to give phytoene/phytofluene at a level of 1 g/kg diet, and the placebo was provided with the oxidized algae free of carotenoids at the same amount. Weight gain and tissues weight of rats fed on the control diet, or on the experimental diets were statistically same. Tissue analyses were carried out by liquid chromatography at the end of two weeks feeding for vitamin A, carotenoids, phytoene and phytofluene and theirs stereoisomers. Liver analyses revealed high hepatic storage of phytoene in the experimental group. Analysis of the other tissues, adrenal, brain, heart, kidney, lung, and spleen detected small amounts of phytoene in the adrenal, kidney and spleen and in the plasma. High-pressure liquid chromatography for stereoisomeric composition was performed to all phytoene-containing tissues. The original algal diet content of 9-cis-to-all-trans ratio of 1:1 was maintained in the plasma and adrenal while in the liver, spleen and kidney the ratio was reduced to 1:3. The preferential accumulation of all-trans phytoene over 9-cis phytoene in the liver, spleen and kidney may be interpreted as indicating stronger antioxidative effect of 9-cis phytoene over the all-trans isomer or alternatively, in vivo streoisomerization of 9-cis phytoene to the all-trans structure.

Hepatic fructose-metabolizing enzymes and related metabolites: role of dietary copper and gender

The purpose of this study was to further examine the hypothesis that variations in hepatic fructose-metabolizing enzymes between males and females might account for the differences in the severity of copper (Cu) deficiency observed in fructose-fed male rats. Weanling rats of both sexes were fed high-fructose diets either adequate or deficient in copper for 45 days. Cu deficiency decreased sorbitol dehydrogenase activity and dihydroxyacetone phosphate levels and increased glyceraldehyde levels in both sexes. Gender effects were expressed by higher activities of glycerol 3-phosphate dehydrogenase and aldehyde dehydrogenase in male than in female rats and higher levels of dihydroxyacetone phosphate and fructose 1,6-diphosphate (F1,6DP) in female than in male rats. The interactions between dietary Cu and gender were as follows: alcohol dehydrogenase activities were higher in female rats and were further increased by Cu deficiency in both sexes; aldehyde dehydrogenase activities were decreased by Cu deficiency only in male rats; sorbitol levels were higher in male rats and were further increased by Cu deficiency in male rats; fructose 1-phosphate (F1P) levels were increased by Cu deficiency in both sexes, but to a greater extent in male rats; glyceraldehyde 3-phosphate levels were higher in female rats, but were decreased by Cu deficiency in female and increased in male rats. Though most of the examined hepatic fructose-metabolizing enzymes and metabolites showed great differences between rats fed diets either adequate or deficient in Cu, it is the activity of fructokinase and aldolase-B, and the concentrations of their common metabolites, F1P and notably F1,6DP, that could be in part responsible for differences in the severity of pathologies associated with Cu deficiency observed between female and male rats.

Dietary Dunaliella bardawil, a β‐carotene–rich alga, protects against acetic acid–induced small bowel inflammation in rats

BackgroundReactive oxygen species mediate tissue injury in inflammatory bowel disease. &bgr;-Carotene is known as a potent free radical quencher and antioxidant. AimThe authors evaluated the efficacy of prefeeding Dunaliella bardawil, rich in &bgr;-carotene, to ameliorate acid-induced enteritis in a rat model. MethodsEnteritis was induced in female Sprague-Dawley rats by injection of 2 mL acetic acid (0.67 mol/L) to a ligated duodenal loop following 10 weeks of feeding diets containing &bgr;-carotene and compared with various controls. The effects of &bgr;-carotene were evaluated by changes in myeloperoxidase activity, histology, and histomorphometry. ResultsFeeding &bgr;-carotene resulted in suppressed mucosal myeloperoxidase activity, both basal and that induced by acetic acid injection. Acetic acid treatment induced major histopathologic changes in the duodenal mucosa, including small, irregular, and distorted villi; damage to the epithelium; edema of the lamina propria; accumulation of inflammatory cells; and hemorrhage. &bgr;-Carotene treatment prevented these acid-induced histopathologic changes, and this was confirmed by histomorphometry of the villi. ConclusionsThese results demonstrate the effectiveness of &bgr;-carotene in a rat model as a prophylactic dietary measure in reducing the effects of acid-induced enteritis and raise the possibility that patients with Crohns disease may benefit from the consumption of natural &bgr;-carotene.

The effect of various avocado oils on skin collagen metabolism

The effects of various avocado oils on collagen metabolism in skin were studied in growing rats fed diets containing 10% (w/w) of the tested oils. Rats fed the unrefined avocado oil extracted with hexane from the intact fruit, its unsaponifiables or the avocado seed oil, showed significant increases in soluble collagen content in skin, though total collagen content was not affected. The increased soluble collagen content appears to be a consequence of the inhibition of lysyl oxidase activity. The active factor was found to be present in the unrefined avocado oil and probably originated from the avocado seed, since collagen metabolism was affected only by fractions which contained lipids fraction from the seed. In comparison rats fed the refined or unrefined soybean oils showed no effects.

Dietary copper intake influences skin lysyl oxidase in young men

The effect of low dietary copper on copper status and the copper-containing enzyme lysyl oxidase was studied in young men. The study was divided into three dietary periods. During the first period, subjects were fed 0.66 mg/day Cu for 24 days (marginal copper). The level of copper was dropped to 0.38 mg/day for the next 42 days (low copper) and they were repleted with 2.49 mg/day Cu for next 24 days. Skin biopsies were taken at the beginning of the study and at the end of each dietary period and lysyl oxidase was measured enzymatically. There was a 24% drop in activity when the dietary copper level was reduced from 0.66 to 0.38 mg/day. When the subjects were repleted with copper, there was a significant increase in the activity of lysyl oxidase. The activity reached the level observed before the subjects were fed the restricted copper diet. These data show that, in humans, lysyl oxidase activity declines when dietary copper intake is inadequate and suggests that the cross-linking of collagen may be modulated by dietary copper. Lysyl oxidase in healthy young men can serve as a useful indicator of copper status.

Lysyl oxidase activity, collagen cross-links and connective tissue ultrastructure in the heart of copper-deficient male rats

Abstract Copper deficiency is characterized by multiple connective tissue manifestations, such as skeletal and joint abnormalities, and vascular lesions that lead to aneurysms and aortic rupture. However, the rupture of the heart observed in cooper-deficient male rats is not fully understood. We demonstrated the effect of copper deficiency on cardiac collagen content and solubility, regional differences in cardiac lysyl oxidase activity, collagen cross-links, and on the ultrastructural morphology of collagen in the myocardium. Weaned male rats fed copper-deficient or copper-adequate diets were examined. Copper-deficient rats died prematurely of heart rupture at the apex, and those who survived exhibited heart enlargement, decreased cardiac lysyl oxidase activity with increasing heart soluble collagen content. Mature collagen cross-links, as assessed by the concentrations of pyridinoline and deoxypyridinoline, were lower both in the apex and in the right and left ventricles of copperdeficient rats as compared with copper-adequate controls. However, in copper-deficient rats, cross-links levels were significantly lower at the apex than at the left and right ventricles. In addition, severe ultrastructural abnormalities in the size and shape of endo-and epimysium collagen fibers were observed in the apex of copper-deficient rats. Transmission electron microscopy showed giant, spiralled or frayed, and spiny collagen fibers. These findings allow us to postulate that the reduced cardiac lysyl oxidase activity accompanied by altered collagen cross-links and an abnormal connective tissue ultrastructure play a significant role in the manifestation of copper deficiency in the male rat.

The effect of avocado oils on some liver characteristics in growing rats

The effects of various avocado oils on some liver characteristics were studied in growing rats. The rats were fed diets containing 10% (w/w) avocado oil for 4 wk. In comparison with rats fed refined oil obtained from cored fruit by centrifugal separation, rats fed unrefined avocado oil obtained by solvent extraction from the intact fruit, or refined avocado oil containing avocado-seed oil, showed significant growth inhibition, an increase in the amount of hepatic lipids (identified as steatosis by histopathological examination), and a decrease in levels of triglycerides in blood. Rats fed the refined oil containing unsaponifiable material prepared from unrefined oil from the intact fruit showed similar responses. Fatty livers were not induced by feeding rats unrefined avocado oil obtained from intact fruit by centrifugal separation, although a significant decrease in blood triglycerides was observed. There were no significant differences between groups in serum total protein, albumin or bilirubin content or in alanine aminotransferase activity. However, serum alkaline phosphatase activity was increased in rats fed the seed oil, the unrefined solvent-extracted oil from intact fruit, or the unsaponifiables, and aspartate aminotransferase activity was significantly increased in the group fed avocado-seed oil. These data suggest that consumption of avocado oil extracted from intact fruit may cause changes in liver metabolism.

Effects of the marine unicellular alga Nannochloropsis sp. to reduce the plasma and liver cholesterol levels in male rats fed on diets with cholesterol.

The effects of Nannochloropsis were studied on rats consuming hypercholesterolemic diets. The whole biomass and the hexane/ethanol extract increased the plasma and hepatic eicosapentaenoic and docosahexaenoic acids levels, and reduced the cholesterol levels. We also observed a higher level of propionate, and a lower ratio between acetate and propionate. These data suggest the efficacy of Nannochloropsis in reducing cholesterol levels.