Calcium D-glucarate Research Articles

Effects of combined phytochemicals on skin tumorigenesis in SENCAR mice

The purpose of our study was to determine the effect of the combined action of phytochemicals on the early stages of skin tumorigenesis, i.e. initiation and promotion. We tested calcium D-glucarate (CG) given in the diet, while resveratrol (RES) and ursolic acid (UA) were applied topically. The 7,12-dimethylbenz[a]anthracene (DMBA)-initiated, 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted multistage skin carcinogenesis model in SENCAR mice was used. Mice received one topical dose of DMBA, then after one month, two weekly doses of TPA for 14 weeks until sacrifice. RES or UA were applied 20 min prior to DMBA or TPA treatment and 2% dietary CG was given from 2 weeks prior to 2 weeks after the DMBA dose or continually beginning 2 weeks prior to the first dose of TPA. UA applied alone and in combination with CG during the promotion stage was the only inhibitor of tumor multiplicity and tumor incidence. A number of combinations reduced epidermal proliferation, but only UA and the combination UA+CG applied during promotion significantly reduced epidermal hyperplasia. DMBA/TPA application resulted in significant increases in c-jun and p50, which were reversed by a number of different treatments. DMBA/TPA treatment also strongly increased mRNA levels of inflammation markers COX-2 and IL-6. All anti-promotion treatments caused a marked decrease in COX-2 and IL-6 expression compared to the DMBA/TPA control. These results show that UA is a potent inhibitor of skin tumor promotion and inflammatory signaling and it may be useful in the prevention of skin cancer and other epithelial cancers in humans.

Dietary D-glucarate effects on the biomarkers of inflammation during early post-initiation stages of benzo[a]pyrene-induced lung tumorigenesis in A/J mice.

Previous studies showed that dietary calcium D-glucarate (CG) inhibited benzo[a]pyrene (B[a]P)-induced A/J mouse lung tumorigenesis, suppressing cell proliferation and chronic inflammation and inducing apoptosis during late post-initiation stages. The present study aimed to investigate changes in the homeostasis of cytokines in blood serum, as well as alterations in biomarkers of inflammation and apoptosis in lung tissue caused by dietary CG during early post-initiation stages of B[a]P-induced lung tumorigenesis. Two doses of 3 mg of B[a]P were given intragastrically to A/J mice 2 weeks apart. CG administration in the AIN-93G diet (2 and 4%, w/w) commenced at 2 weeks following the second dose of B[a]P. The levels of interleukin (IL)-6, IL-10 and tumor necrosis factor α (TNFα) in blood serum were investigated by FCAP array analysis. Two weeks after the second dose of B[a]P, approximately 8- and 28-fold increases of TNFα and IL-6, respectively, occurred in the blood serum and an approximately 16% decrease of IL-10 levels compared to the untreated control group was noted. At 4 weeks after the second dose of B[a]P and after 2 weeks of CG administration in the diet, the 2 and 4% CG diets significantly reduced the levels of IL-6 and TNFα (by 70 and 33%, respectively). In a dose-related manner, the diets also increased the level of anti-inflammatory cytokine IL-10 compared to the B[a]P group. At 6 weeks after the second dose of B[a]P, the cytokine levels in the serum continued to show a decrease in the CG-treated groups. These events are accompanied by an increased level of cleaved caspase-9 product with a molecular weight of 37 kDa. In conclusion, dietary D-glucarate decreases the level of proinflammatory cytokines, increases the level of the anti-inflammatory cytokine IL-10 during early post-initiation stages of B[a]P-induced lung tumorigenesis in A/J mice and affects apoptotic induction.

In vitro study of the antioxidative properties of the glucose derivatives against oxidation of plasma components

Oxidative stress has been implicated in the pathogenesis of variety of diseases. Since the endogenous antioxidant defense may be not adequate to counteract the enhanced generation of oxidants, a growing interest in research for exogenous nutrients has been observed. The present study was designed to assess in vitro the antioxidative properties of the glucose derivatives: calcium D-glucarate, D-gluconic acid lactone, and sodium D-gluconate (0.5-3 mM) in the protection of plasma proteins and lipids, against the damage caused by 0.1 mM peroxynitrite (ONOO⁻). Exposure of plasma to ONOO⁻ resulted in carbonyl groups increase, 3-nitrotyrosine (3-NT) formation, reduction in thiol groups, and enhanced lipid peroxidation. D-gluconic acid lactone and sodium D-gluconate effectively decreased 3-NT formation; the antinitrative action of calcium D-glucarate was less effective. In plasma samples incubated with ONOO⁻ and tested compounds, the level of carbonyl groups was decreased in comparison to plasma samples treated only with ONOO⁻. The level of protein -SH groups and glutathione was significantly higher in the presence of glucose derivatives than in plasma samples treated with ONOO⁻ only. All the tested compounds had the inhibitory effect on the peroxynitrite-induced plasma lipids peroxidation. The results obtained from our work indicate that calcium D-glucarate, D-gluconic acid lactone, and sodium D-gluconate may partly protect plasma proteins and lipids against peroxynitrite-induced damages.

A comparative study of antioxidative activity of calcium-D-glucarate, sodium-D-gluconate and D-glucono-1,4-lactone in a human blood platelet model

D-glucono-1,4-lactone, sodium D-gluconate and calcium D-glucarate are non-toxic glucose derivatives occurring naturally in fruits and vegetables. Calcium D-glucarate is promoted as an orally bioavailability dietary supplement with potential chemopreventive activity without adverse effects. Despite many commercial applications in pharmaceutical and food industries the potential activity mechanisms of glucarate and gluconate are not clear. The purpose of this study was to investigate and compare the effects of these compounds on blood platelets under oxidative stress conditions and to examine their role in thrombin-induced platelet activation. Platelet activation is essential in haemostasis, tumor progression and allergic and non-allergic inflammation, where reactive oxygen species are involved. The antiplatelet and antioxidative activity was studied in vitro by measuring levels of specific oxidative stress markers: thiobarbituric acid reactive substances, superoxide anion, carbonyl groups, 3-nitrotyrosine, protein and low molecular weight thiols. All tested compounds significantly inhibited thrombin-induced arachidonic peroxidation, production and also platelet protein oxidation/nitration induced by peroxynitrite, which is a strong oxidant formed intravascularly in vivo. Carbonyl group generation, thiol oxidation and nitrotyrosine formation were significantly decreased in the presence of glucose derivatives. The obtained results demonstrate that tested compounds may be helpful in the prevention of excessive platelet activation through the antioxidant mechanisms. Comparative studies indicate the predominant preventive activity of sodium D-gluconate. In general, the consumption of apples or apple juice as well as oranges, grapefruit and cruciferous vegetables, sources of large amounts of tested derivatives, have beneficial effects on platelets under oxidative stress.

Abstract 3470: Effects of combined phytochemicals on skin tumorgenesis in SENCAR mice

Abstract The purpose of our study was to determine the effect of combined action of phytochemicals on early stages of skin tumorgenesis, i.e., initiation and promotion. We tested calcium D-glucarate (CG) given in the diet, while resveratrol (RES) and ursolic acid (URA) were applied topically. The 7,12-dimethylbenz[a]anthracene (DMBA)-initiated, 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted multistage skin carcinogenesis model in SENCAR mice was used. Mice (6-7 weeks old) with shaved backs received one topical dose of DMBA (20 nmol), then after one month, two weekly doses (2 µg each) of TPA (tumor promoter) for 14 weeks until sacrifice. The phytochemicals RES (2.5 μmol) or URA (1 μmol) were applied topically 20 min prior to DMBA or TPA treatment and 2% dietary CG was given in the AIN-93G diet from 2 weeks prior to 2 weeks after the DMBA dose or continually beginning 2 weeks prior to the first dose of TPA. We determined the tumor incidence (% of mice with tumors) and tumor multiplicity (number of tumors per mouse) and using RT-PCR and immunochistochemical analyses, we checked the expression of some cell proliferation and inflammation related genes. URA applied alone and in combination with CG during the promotion stage was a strong inhibitor of tumor multiplicity and tumor incidence. Combinations of URA with dietary CG appeared to show some synergistic effects. All compounds and their combinations reduced epidermal proliferation, but only URA and a combination CG/URA applied during promotion significantly reduced hyperplasia, as determined by epidermal thickness measurements taken at 14 weeks of TPA treatment. DMBA/TPA treatments resulted in epidermal hyperplasia, characterized by significant increase in transcription factor AP-1 components and nuclear factor kappa B (NFkB) expression levels with a simultaneously increased expression of the inflammation markers COX-2 and IL-6, compared to untreated controls. All anti-promotion treatments (either single compounds or their combinations) caused a marked decrease of inflammation related genes levels compared to the DMBA/TPA control. On the other hand, only URA and URA with dietary CG diminished the induced expression of proto-oncogenes c-jun and c-fos (components of AP-1) and the expression of NFkB protein p50. These results show that URA is a potent inhibitor of skin tumor promotion and it may be useful in the prevention of skin cancer and other epithelial cancers in humans. Supported by NIH grants CA 102747 and P30 CA 54174-16S1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3470.

Synergistic Effects of Combined Phytochemicals and Skin Cancer Prevention in SENCAR Mice

The purpose of our study was to determine the inhibitory effect of combined phytochemicals on chemically induced murine skin tumorigenesis. Our hypothesis was that concurrent topical and dietary treatment with selected compounds would lead to more efficient prevention of skin cancer. We tested ellagic acid and calcium D-glucarate as components of diets, while resveratrol was applied topically; grape seed extract was applied topically or in the diet. The 4-week inflammatory-hyperplasia assay based on the 7,12-dimethylbenz[a]anthracene (DMBA)-induced skin carcinogenesis model in SENCAR mice was used. We have found that all the selected combinations caused a marked decrease of epidermal thickness compared with the DMBA-treated group and also with groups treated with a single compound and DMBA. All combinations of resveratrol with other compounds showed a synergistic effect on hyperplasia and Ha-ras mutations. Skin tissue of mice receiving the combinations showed decreased cell proliferation and Bcl2 expression; decreased p21, a regulator of cell cycle; and decreased marker of inflammation cyclooxygenase-2. All the selected combinations diminished the DMBA-induced mRNA expression of the CYP1B1 level, and also caused a marked decrease of proto-oncogenes c-jun and c-fos, components of transcription factor activator protein. In conclusion, all combinations showed either additive or synergistic effects and their joint actions allowed for decreasing the doses of the compounds. Especially, resveratrol combinations with ellagic acid, grape seed extract, and other phytochemicals are very potent inhibitors of skin tumorgenesis, based on the suppression of epidermal hyperplasia as well as on the modulation of intermediate biomarkers of cell proliferation, cell survival, inflammation, oncogene mutation, and apoptosis.

Convenient Large-Scale Synthesis of d-Glucaro-1,4:6,3-dilactone

Calcium D-glucarate was converted into D-glucaro-1,4:6,3-dilactone on 32-g, 1-kg, and 22-kg scale, using azeotropic distillation with methyl isobutyl ketone to drive the dehydration. The crystalline product was > or = 99.5% pure by GC and NMR, and overall yield was as high as 72%.

Induction of Apoptosis by Calcium D-Glucarate in 7,12-Dimethyl Benz [a] Anthracene-Exposed Mouse Skin

Calcium glucarate (Cag), a naturally occurring nontoxic compound, suppresses the DMBA-induced tumor development in mouse skin. In the process of understanding the mechanisms of tumor suppression by Cag, we investigated the effect of topical application of Cag on selective and critical events of apoptotic pathway in DMBA-exposed mouse epidermis. Varied doses of DMBA or Cag were used for the study. DMBA had an inhibitory effect on proteases in general and on caspases in particular. Cag tried to reverse the inhibitory effect of DMBA on 3, 8, or 9 caspase in a dose-dependent manner. Cag inhibited activity of Poly ADP-ribose polymerase enzyme, a substrate of caspses, after DMBA exposure. As indicated by western blotting, Cag treatment also inhibited PARP expression induced by DMBA at the level of protein. Cag induced the DMBA-inhibited Ca++/Mg++-dependent endonuclease, an enzyme responsible for the DNA fragmentation during apoptosis. DMBA induced the expression of mutant-p53 and Bcl-2. This induced expression of proteins was reversed when Cag was given along with DMBA. Cag showed a dose-dependent inhibition of DMBA-induced mutant-p53 expression. Similarly Bcl-2 overexpression by DMBA was also inhibited by topical treatment of Cag when given along with DMBA. Inhibition of mutant-p53 and Bcl-2 expression by Cag in DMBA-exposed mouse skin might contribute to the apoptogenic effect possibly exerted by Cag while suppressing the tumor development. The study indicates that Cag induces apoptosis in mouse epidermis, a possible mechanism for tumor suppression, and thus could be considered a promising anticancer agent.

Detoxifying cancer causing agents to prevent cancer.

Different vitamins and other micronutrients in vegetables, fruits, and other natural plant products may prevent cancer development (carcinogenesis) by interfering with detrimental actions of mutagens, carcinogens, and tumor promoters. The goal of current studies in cancer prevention is to determine the mechanisms of synergistic action of the natural source compounds known to inhibit one or more stages of carcinogenesis, that is, initiation and promotion/progression. Many natural cancer preventive agents are effective inhibitors of tumor initiation, promotion, and/or progression. The mechanism of action is related to their abilities to prevent critical carcinogen metabolism and to increase detoxification of carcinogens and tumor promoters. The authors review here the potential role of the detoxification system and, in particular, the roles of D-glucaric acid and the enzyme beta-glucuronidase in early detection and prevention of cancer. There is now growing evidence for the possible control of different stages of the cancer induction by inhibiting beta-glucuronidase with D-glucaric acid derivatives, especially with its salts (D-glucarates). D-Glucaric acid has been found in many vegetables and fruits. Therefore, the consumption of fruits and vegetables naturally rich in D-glucaric acid or self-medication with D-glucaric acid derivatives such as calcium D-glucarate offers a promising cancer prevention approach.

Inhibition of azoxymethane-induced rat colon carcinogenesis by potassium hydrogen D-glucarate.

While calcium D-glucarate was shown to inhibit chemical carcinogenesis in various animal models, the effect of potassium hydrogen D-glucarate has not been extensively investigated. In the present study, potassium hydrogen D-glucarate markedly inhibited azoxymethane (AOM)-induced colon carcinogenesis in male F344 rats. Potassium hydrogen D-glucarate (PHG) or potassium hydrogen carbonate (PHC) were administered to rats in a diet (140 mmol/kg). Continual post-initiation treatment with potassium hydrogen D-glucarate reduced both tumor incidence and multiplicity at sacrifice by ca. 60%, while PHC had no effect. amelioration of overexpression of the betaG gene in rat colon carcinomas was observed using RT-PCR and Northern blot analysis. We hypothesize that previously demonstrated conversion of PHG to D-glucaro-1,4-lactone, a potent inhibitor of beta-glucuronidase (betaG), may be responsible for this effect. The mechanism of PHG inhibition of colon carcinogenesis may also involve suppression of cell proliferation and possibly alterations in cholesterol synthesis or cholesterol metabolism to bile acids. In conclusion, PHG possesses excellent potential as a natural, apparently non-toxic inhibitor to prevent colon cancer.

D-Glucaric acid content of various fruits and vegetables and cholesterol-lowering effects of dietary d-glucarate in the rat

The beneficial properties of different vitamins, minerals, and other micronutrients have been studied for quite some time. But only recently has the potential usefulness of d-glucaric acid and its derivatives in disease prevention been demonstrated. d-Glucaric acid is an end product of the d-glucuronic acid pathway in mammals. Its dietary sources include different fruits and vegetables. In the present study, d-glucaric acid content in various fruits and vegetables was found to range from about 0.1 g/kg in grapes and lettuce to about 3.5 g/kg in apples and broccoli. It was also shown that purified diets containing calcium d-glucarate or potassium hydrogen d-glucarate markedly lowered serum levels of cholesterol in female Sprague-Dawley rats. The d-glucarates reduced total serum cholesterol in rats by up to 14% (P<0.05) and lowered LDL-cholesterol by up to 35% (P<0.05), but had no effect on HDL cholesterol. These results provide a starting point for further studies of the mechanism by which d-glucaric acid salts lower serum cholesterol.

Dietary glucarate-mediated reduction of sensitivity of murine strains to chemical carcinogenesis

Serum β-glucuronidase activity is shown to differ quantitatively in the following strains of mice, listed in order of increasing activity: C3H, C57BL 6 < BALB c , DBA 2 , ICR < SENCAR, A He . The level of the enzyme in the murine strains is shown to correlate with the urinary excretion of 17-ketosteroids, which in turn reflects the endogenous level of androgens. Dietary calcium d-glucarate, an in vivo β-glucuronidase inhibitor, reduced the steady state level of both β-glucuronidase and 17-ketosteroid excretion in the highly susceptible A He and SENCAR strains to that of strains known to be resistant to chemical carcinogenesis. Sensitivity of the A He strain is significantly reduced by dietary calcium glucarate, which is shown to inhibit DNA binding and the induction of pulmonary adenomas by benzo[ a]pyrene.

Dietary glucarate as anti-promoter of 7,12-dimethylbenz[a]anthracene-induced mammary tumorigenesis.

Using as a criterion the inhibition of serum beta-glucuronidase activity, dietary calcium D-glucarate is shown to serve as an efficient slow-release source in vivo of D-glucaro-1,4-lactone, the potent endogenous inhibitor of this enzyme. Using the 7,12-dimethylbenz[a]anthracene model of mammary tumor induction in rats it is shown for the first time that feeding the rats calcium D-glucarate-supplemented diet after treatment with the carcinogen, inhibits tumor development by over 70%. Supportive evidence is presented for the theory that calcium D-glucarate inhibits or delays the promotion phase of mammary carcinogenesis by lowering endogenous levels of estradiol and precursors of 17-ketosteroids. Therefore, dietary glucarate can be used to lower blood and tissue levels of beta-glucuronidase, and in turn of those carcinogens and promoting agents which are excreted, at least in part, as glucuronide conjugates.

Interactions of Calcium Ions with Carbohydrates: Crystal Structure of Calcium d-Glucarate Tetrahydrate

Abstract The crystal structure of calcium d-glucarate tetrahydrate has been determined from three dimensional X-ray data, and refined by the least squares method including an anisotropic temperature factor. The crystals are orthorhombic, space group P212121 with a=16.09, b=11.36 and c=6.68Å. The calcium ion is bound to two glucarate molecules and three water molecules. The eight oxygen atoms form the calcium coordination shell of a distorted square-antiprism geometry. The carboxyl group including C(1) of d-glucarate has an eclipsed conformation relative to C(2) and O(2), and is coordinated with one calcium ion through the carboxyl oxygen O(1) and the α-hydroxyl oxygen O(2), and with the other calcium ion through the carboxyl oxygen O′(1) and the hydroxyl oxygens O(3) and O(4). The other carboxyl group including C(6) has an unusual staggered conformation relative to C(5) and O(5), and is not coordinated with calcium ions. However the oxygen atoms of the latter carboxyl group are attached by hydrogen bonds to a number of hydroxyl groups and water molecules, all of which are coordinated with calcium ions.