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Abstracts

Calcium D-glucarate: 35 Research Abstracts

HUMAN ** 1. Calcium-D-glucarate. [No authors listed] Altern Med Rev. 2002 Aug;7(4):336-9.

Calcium-D-glucarate is the calcium salt of D-glucaric acid, a substance produced naturally in small amounts by mammals, including humans. Glucaric acid is also found in many fruits and vegetables with the highest concentrations to be found in oranges, apples, grapefruit, and cruciferous vegetables. Oral supplementation of calcium-D-glucarate has been shown to inhibit beta-glucuronidase, an enzyme produced by colonic microflora and involved in Phase II liver detoxification. Elevated beta-glucuronidase activity is associated with an increased risk for various cancers, particularly hormone-dependent cancers such as breast, prostate, and colon cancers. Other potential clinical applications of oral calcium-D-glucarate include regulation of estrogen metabolism and as a lipid-lowering agent.

CANCER ** 2. Public Health 2000 May;90(5):777-81 Trends in fruit and vegetable consumption among adults in 16 US states: Behavioral Risk Factor Surveillance System, 1990-1996. Li R, Serdula M, Bland S, Mokdad A, Bowman B, Nelson D Division of Nutrition and Physical Activity, Centers for Disease Control and Prevention (MS K25), Atlanta, GA 30341-3717, USA. ril6@cdc.gov

OBJECTIVES: This study examined trends in fruit and vegetable consumption among adults in 16 US states. METHODS: Data from telephone surveys were used to stratify respondents by sociodemographic and health-related characteristics. RESULTS: The proportion of adults who consumed fruits and vegetables at least 5 times daily was 19%, 22%, and 23% in 1990, 1994, and 1996, respectively. While the proportion increased among those with active leisure-time physical activities and normal weight, it remained almost the same among inactive people and dropped among the obese. CONCLUSIONS: Progress in fruit and vegetable intake from 1990 to 1994 was encouraging, but it changed little between 1994 and 1996.

3. Calcium glucarate as a chemopreventive agent in breast cancer Heerdt A.S.; Young C.W.; Borgen P.I. Breast Service, Memorial Sloan-Kettering Cancer Ctr., 1275 York Avenue,New York, NY 10021 United States Israel Journal of Medical Sciences ( ISR. J. MED. SCI. ) (Israel) 1995, 31/2-3 (101-105)

Although it appears that progress is being made in the treatment of breast cancers of all stages, the etiological agents still remain unclear and render the search for preventive agents extremely difficult. What is clearly required in this situation is a nontoxic compound that can potentially affect various pathways that may be responsible for the rising incidence of breast cancer. In this review, we present the rationale for the use of an agent such as calcium glucarate, which may both change the internal hormonal milieu and also directly detoxify any environmental agents responsible for breast cancer. It is hoped that present and future clinical trials will help to better elucidate the role for this agent in the chemoprevention of breast cancer.

4. Calcium glucarate as a chemopreventive agent in breast cancer.

Heerdt AS, Young CW, Borgen PI. Breast Service, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA. Isr J Med Sci. 1995 Feb-Mar;31(2-3):101-5.

Although it appears that progress is being made in the treatment of breast cancers of all stages, the etiological agents still remain unclear and render the search for preventive agents extremely difficult. What is clearly required in this situation is a nontoxic compound that can potentially affect various pathways that may be responsible for the rising incidence of breast cancer. In this review, we present the rationale for the use of an agent such as calcium glucarate, which may both change the internal hormonal milieu and also directly detoxify any environmental agents responsible for breast cancer. It is hoped that present and future clinical trials will help to better elucidate the role for this agent in the chemoprevention of breast cancer.

5. Activity of D-glucarate analogues: Synergistic antiproliferative effects with retinoid in cultured human mammary tumor cells appear to specifically require the D-glucarate structure Curley Jr. R.W.; Humphries K.A.; Koolemans-Beynan A.; Abou-Issa H.; Webb T.E. Medicinal Chem./Pharmacognosy Div., College of Pharmacy, Ohio State University, 500 W. 12th Ave.,Columbus, OH 43210 United States Life Sciences ( LIFE SCI. ) (United States) 1994, 54/18 (1299-1303)

D-glucarate has shown modest chemopreventive and synergistic chemopreventive effects with retinoids in a number of tumor models as well as a similar antiproliferative effect in MCF-7 human tumor cells in culture. It has been postulated that D-glucarate exerts some of its effects by equilibrium conversion to D-glucarolactone, a potent beta-glucuronidase inhibitor. In the present study, D-glucarate and a number of its analogues, including D-glucarolactone, were evaluated as antiproliferatives in the MCF- 7 model with and without added retinoid. Results suggest that the effects of glucarate are reasonably specific for its structure and may not require conversion to glucarolactone.

6. Antitumour synergism between non-toxic dietary combinations of isotretinoin and glucarate Abou-Issa H.; Koolemans-Beynen A.; Meredith T.A.; Webb T.E. Ohio State Univ. College of Medicine, 320 W. 10th Ave.,Columbus, OH 43210 United States European Journal of Cancer Part A: General Topics ( EUR. J. CANCER PART A GEN. TOP. ) (United Kingdom) 1992, 28/4-5 (784-788)

Dietary calcium glucarate (CGT) increased the activity of non-toxic levels of dietary isotretinoin against preestablished tumors in the chemically-induced rat mammary tumour model. In the range of 1.0-1.5 mmol/kg diet, isotretinoin enhanced tumour growth by 20% over a 4 week course of treatment. Tumour growth inhibition not exceeding 15% was observed only at dosages as high as 2.0 mmol/kg, i.e. in the cumulative toxicity range. Growth inhibition by 64 mmol/kg diet of CGT alone was marginal, varying from zero to 8%. In contrast, the combination of 1.0 mmol/kg of isotretinoin and 64 mmol/kg of CGT caused a reversible inhibition of tumour growth, culminating in a net decrease in tumour volume of 20%. This study documents the marginal enhancement of tumour growth by high sub-optimal concentrations of isotretinoin alone, and describes conditions for inhibition of tumour growth by sub-optimal concentrations of the natural retinoid. Related in vitro studies on retinoid sensitive and insensitive cell lines suggest that the anticancer activity of the combination is dependent on sensitivity of the cells to retinoids.

7. [Study of preventive effect of 1-hexylcarbamoyl-5-fluorouracil (HCFU) or combination of HCFU and 2.5-di-O-acetyl-D-glucaro (1-4) (6-3) dilactone (SLA) after preservative operation against bladder cancer] [Article in Japanese]

Wada S, Yasumoto R, Kashihara N, Kishimoto T, Maekawa M, Hayahara N, Kawakita J, Nishijima T, Morikawa Y, Horii A, et al. Department of Urology, Osaka City University Medical School. Hinyokika Kiyo. 1992 Jan;38(1):19-24.

To treat superficial bladder cancer or invasive bladder cancer presenting as a solitary tumor, conservative therapy such as transurethral resection of bladder tumor or partial cystectomy has long been carried out. We studied the effect of 1-hexylcarbamoyl-5-fluorouracil (HCFU), and the effects of the combination of HCFU and 2.5-di-O-acetyl-D-glucaro-(1-4)(6-3) dilactone (SLA) which is an anti-beta-glucuronidase agent, for preventive therapy. In the patients treated with HCFU, the recurrence rate was 3.6% and 26.6% one year and two years, respectively, after conservative operation. In the patients treated with both SLA and HCFU, the recurrence rate was lower than in those treated with HCFU one year or more after conservative operation, and a long-term preventive effect was expected for nondrinkers.

8. Growth suppression of human breast carcinoma cells in culture by N-(4-hydroxyphenyl)retinamide and its glucuronide and through synergism with glucarate Bhatnagar R.; Abou-Issa H.; Curley Jr. R.W.; Koolemans-Beynen A.; Moeschberger M.L.; Webb T.E. Department of Surgery, College of Medicine, Ohio State University,Columbus, OH 43210 United States Biochemical Pharmacology ( BIOCHEM. PHARMACOL. ) (United Kingdom) 1991, 41/10 (1471-1477)

The inhibitory effects of N-(4-hydroxyphenyl)retinamide (HPR) and its glucuronide derivative on the growth of MCF-7 human breast cancer cells in vitro were compared. The results indicate that the glucuronide had slightly greater potency and much less cytotoxicity than the free retinoid. At a concentration of 10sup -sup 6 M, HPR inhibited MCF-7 cell growth by approximately 25%, whereas an equimolar concentration of the glucuronide caused a 40% growth inhibition. Higher concentrations of HPR were highly cytotoxic. At a 10sup -sup 5 M concentration of the glucuronide, cell viability was 77%, and 65% of the cells were able to resume growth. On the other hand, at 10sup -sup 5 M HPR, cell viability dropped to 49%, and only 15% of the cells were capable of resuming growth. The lower cytotoxicity and higher potency of the retinoid glucuronide compared to the parent retinamide suggest that the conjugate may have a chemotherapeutic advantage over the parent compound. The apparent higher efficacy of HPR in combination with glucarate (GT) compared to the single agents could be due to increased net formation of HPR glucuronide conjugate following conversion of GT to the beta-glucuronidase inhibitor, D-glucaro-1,4-lactone. However, HPLC analysis of the cell metabolites did not show any detectable levels of the retinoid glucuronide upon treatment of MCF-7 cells with HPR and GT.

9. Cancer Lett 1990 Oct 8;54(1-2):1-8 Potential use of D-Glucaric acid derivatives in cancer prevention. Walaszek Z Department of Carcinogenesis, University of Texas M.D. Anderson Cancer Center, Smithville 78957.

There is now growing evidence from animal models for the possible control of different stages of the carcinogenic process by the beta-glucuronidase inhibitor D-glucaro-1,4-lactone and its precursors such as D-Glucaric acid salts, D-glucarates. D-Glucaric acid is a natural, non-toxic compound produced in small amounts by mammals, including humans. It was recently found in some vegetables and fruits. D-Glucaro-1,4-lactone and D-glucarate exhibit potent antiproliferative properties in vivo. Some human subpopulations could have reduced risk of cancer development by ingesting food rich in D-Glucaric acid or self-medication with D-glucarates alone or in combination with other chemopreventive agents.

10. Potential use of D-Glucaric acid derivatives in cancer prevention

Walaszek Z. Department of Carcinogenesis, Science Park-Research Division, The University of Texas M.D. Anderson Cancer Center, P.O. Box 389, Smithville, TX 78957 United States Cancer Letters ( CANCER LETT. ) (Ireland) 1990, 54/1-2 (1-8)

There is now growing evidence from animal models for the possible control of different stages of the carcinogenic process by the beta-glucuronidase inhibitor D-glucaro-1,4-lactone and its precursors such as D-glucaric acid salts, D-glucarates. D-Glucaric acid is a natural, non-toxic compound produced in small amounts by mammals, including humans. It was recently found in some vegetables and fruits. D-glucaro-1,4-lactone and D-glucarate exhibit potent antiproliferative properties in vivo. Some human subpopulations could have reduced risk of cancer development by ingesting food rich in D-Glucaric acid or self-medication with D-glucarates alone or in combination with other chemopreventive agents.

11. Effect of calcium glucarate on beta-glucoronidase activity and glucarate content of certain vegetable and fruits Dwivedi C.; Heck W.J.; Downie A.A.; Larroya S.; Webb T.E. College of Pharmacy, South Dakota State University, Brookings, SD 57007 United States Biochemical Medicine and Metabolic Biology ( BIOCHEM. MED. METAB. BIOL. ) (United States) 1990, 43/2 (83-92)

Glucarate is normally present in tissues and body fluids and in equilibrium with D-glucaro-1,4-lactone, a natural inhibitor of beta-glucuronidase activity. Dietary calcium glucarate, a sustained-release from of glucarate, elevates the blood level of D-glucaro-1,4-lactone which suppresses blood and tissue beta-glucuronidase activity. A single dose of CaG (4.5 mmole/kg body weight) inhibited beta-glucuronidase activity in serum and liver, lung, and intestinal microsomes by 57, 44, 37, and 39%, respectively. A chronic administration of calcium glucarate (4% in diet) also decreased beta-glucuronidase activity in intestinal and liver microsomes. Maximal inhibition of beta-glucuronidase activity in serum was observed from 12 noon to 2:00 PM. In contrast, maximum inhibition of beta-glucuronidase activity in intestinal and liver microsomes occurred during mornings, although a secondary depression in intestinal microsomes also occurred around 4 PM. A 4% calcium glucarate supplemented diet also inhibited beta-glucuronidase activity by 70% and 54%, of the bacterial flora obtained from proximal (small intestine) and distal (colon) segments of intestine, respectively. Due to the potential effect of dietary glucarate on net glucuronidation and on other metabolic pathways, glucaric acid levels in various foods were determined. The glucaric acid content varied from a low of 1.12 - 1.73 mg/100 g for broccoli and potatoes to a high of 4.53 mg/100 g for oranges.

12. Synergistic interaction between 13-cis-retinoic acid and glucarate: activity against rat mammary tumor induction and MCF-7 cells.

Abou-Issa H, Koolemans-Beynen A, Minton JP, Webb TE. Department of Surgery, College of Medicine and Comprehensive Cancer Center, Ohio State University, Columbus 43210. Biochem Biophys Res Commun. 1989 Sep 29;163(3):1364-9.

At high dietary levels in vivo, both 13-cis-retinoic acid and calcium glucarate inhibit the induction of rat mammary tumors by 7,12-dimethylbenz(a)anthracene. The present study shows that sub-optimal dietary levels of each, which individually have no effect on tumor induction, when combined together in the diet, significantly increases tumor latency and suppresses tumor frequency in the rat system. Weight gain of animals was similar in control and experimental groups. Furthermore, ineffective sub-optimal dosages of glucarate and 13-cis-retinoic acid interacted synergistically to inhibit the growth in vitro of the MCF-7 human breast cancer cells. By varying the concentrations of glucarate and 13-cis-retinoic acid independently, evidence was obtained that in combination glucarate may play an adjuvant role, with the retinoid as the effector. Thus, the results of this experimental animal study demonstrate for the first time the potential use in synergistic combination of 2 normal metabolites in non-toxic chemoprevention and chemotherapy.

13. [Clinical study on prophylaxis of diacetyl-glucaro-(1-4) (6-3) dilactone for recurrence of bladder cancer] [Article in Japanese]

Isomatsu Y, Kaburagi Y, Jinbo S, Nashimo T, Miki M, Yamanaka H. Gan To Kagaku Ryoho. 1983 Sep;10(9):1958-62.

In order to investigate the effect of Diacetyl-glucaro-(1-4) (6-3) dilactone to prevent post-operative recurrence of bladder cancer, we estimated the recurrent rate of 34 patients who were diagnosed as having bladder cancer and treated by several surgical methods and oral administration of the drug in our hospital during 1971 and 1980. The following results were obtained: The 6, 12, 18, 24, 30, 36 and over 36 months-recurrent rates were 3.1%, 26.4%, 32.5%, 32.5%, 32.5%, 46% and 46%. In control group, they were 10%, 23.3%, 34.8%, 46.2%, 73.1%, 73.1% and 100% In a 24 month follow-up, there was no difference of recurrent rate between the group receiving the drug and control group: a recurrent rate of administrated group was lower than that of the control after 24 months. The recurrent rate of the group receiving Diacetyl-glucaro-(1-4) (6-3) dilactone combined with instillation was lower in a 2 year follow-up. It was anticipated that the combined therapy (Diacetyl-glucaro-(1-4) (6-3) dilactone administration and intravesical instillation of anticancer drugs) was useful in order to prevent recurrence of bladder cancer.

CANCER**

14. Calcium glucarate prevents tumor formation in mouse skin.

Singh J, Gupta KP. Environmental Carcinogenesis Division, Industrial Toxicology Research Center, Post Box No. 80, Mahatma Gandhi Marg, Lucknow-226001, India. Biomed Environ Sci. 2003 Mar;16(1):9-16.

OBJECTIVE: Calcium Glucarate (Cag), Ca salt of D-glucaric acid is a naturally occurring non-toxic compound present in fruits, vegetables and seeds of some plants, and suppress tumor growth in different models. Due to lack of knowledge about its mode of action its uses are limited in cancer chemotherapy thus the objective of the study was to study the mechanism of action of Cag on mouse skin tumorigenesis. METHODS: We have estimated effect of Cag on DMBA induced mouse skin tumor development following complete carcinogenesis protocol. We measured, epidermal transglutaminase activity (TG), a marker of cell differentiation after DMBA and/or Cag treatment and [3H] thymidine incorporation into DNA as a marker for cell proliferation. RESULTS: Topical application of Cag suppressed the DMBA induced mouse skin tumor development. Topical application of Cag significantly modifies the critical events of proliferation and differentiation TG activity was found to be reduced after DMBA treatment. Reduction of the TG activity was dependent on the dose of DMBA and duration of DMBA exposure. Topical application of Cag significantly alleviated DMBA induced inhibition of TG. DMBA also caused stimulation of DNA synthesis in epidermis, which was inhibited by Cag. CONCLUSION: Cag inhibits DMBA induced mouse skin tumor development. Since stimulation of DNA synthesis reflects proliferation and induction of TG represents differentiation, the antitumorigenic effect of Cag is considered to be possibly due to stimulation of differentiation and suppression of proliferation.

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

Yoshimi N, Walaszek Z, Mori H, Hanausek M, Szemraj J, Slaga TJ. Department of Pathology, Gifu University School of Medicine, Gifu 500, Japan. Int J Oncol. 2000 Jan;16(1):43-8.

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.

16. Inhibitory effect of ND2001 on spontaneous multiple metastasis of NC 65 tumors derived from human renal cancer cells intradermally transplanted into nude mice.

Nakatsugawa S, Okuda T, Muramoto H, Koyama K, Ishigaki T, Tsuruoka T, Hosokawa M, Kobayashi H. Department of Radiology, Nagoya University, Japan. shig@med.nagoya-u.ac.jp Anticancer Drugs. 1999 Feb;10(2):229-33.

The NC 65 tumor cell line derived from human renal cell carcinoma was selected from among nine human cell lines by determining the inhibition of invasion by ND2001 (sodium D-glucaro-delta-lactam) in vitro. The efficacy of this agent against these tumor cells was investigated in an experimental metastatic model of human tumors in vivo. Although ND2001 did not inhibit growth of NC 65 cells intradermally transplanted into male KSN mice (nu/nu), this agent inhibited multiple spontaneous metastasis.

17. ND-2001 suppresses lung metastasis of human renal cancer cells in athymic mice.

Kuramitsu Y, Hamada J, Tsuruoka T, Morikawa K, Naito S, Kobayashi H, Hosokawa M. Laboratories of Pathology, Hokkaido University School of Medicine, Sapporo, Japan. Anticancer Drugs. 1998 Sep;9(8):739-41.

Explants of highly metastatic human renal cell carcinoma SN12Cpm6 cells in athymic mice were treated with sodium D-glucaro-delta-lactam (sodium 5-amino-5-deoxy-D-glucosaccharic acid-delta-lactam; ND-2001). ND-2001 (50 micrograms/ml) caused 78% inhibition of lung metastasis of SN12Cpm6 cells (two of five animals remaining metastasis free). The in vitro tumor cell invasion assay showed that ND-2001 (100 micrograms/ml) suppressed the invasive activity of SN12Cpm6 cells to Matrigel matrix at an inhibition rate of 72%. These results suggest that ND-2001 may be a new anti-metastatic drug against human cancer cells.

18. A new anti-metastatic drug, ND-2001, inhibits lung metastases in rat hepatoma cells by suppressing haptotaxis of tumor cells toward laminin.

Kuramitsu Y, Hamada J, Tsuruoka T, Morikawa K, Kobayashi H, Hosokawa M. Laboratory of Pathology, Cancer Institute, Hokkaido University School of Medicine, Kita, Sapporo, Japan. Anticancer Drugs. 1998 Jan;9(1):88-92.

We examined the effects of ex vivo treatment of tumor cells with sodium D-glucaro-delta-lactam (sodium 5-amino-5-deoxy-D-glucosaccharic acid-delta-lactam; ND-2001). The ex vivo treatment of rat hepatoma cKDH-8/11 cells with this new synthetic product of the antibiotic nojirimycin, ND-2001 (50 microg/ml), inhibited the experimentally induced lung metastases of the tumor cells significantly at an inhibition rate of 69.2% (one of 10 animals remained metastasis free). Also, it was elucidated in in vitro tumor cell invasion assays that ND-2001 (50 microg/ml) suppressed the invasion activities of cKDH-8/11 cells to Matrigel Matrix at an inhibition rate of 69.3%. However, phagokinetic track assays revealed that ND-2001 did not suppress the random motility of cKDH-8/11 cells. However, ND-2001 (50 microg/ml) suppressed the haptotaxis, another important role in tumor invasion, of cKDH-8/11 cells toward laminin (inhibition rate of 77.0%). These results suggest that ND-2001 suppressed the haptotaxis of tumor cells toward laminin directly at the step of invading the basement membrane and brought about the inhibition of lung metastases.

19. Metabolism, uptake, and excretion of a D-glucaric acid salt and its potential use in cancer prevention.

Walaszek Z, Szemraj J, Narog M, Adams AK, Kilgore J, Sherman U, Hanausek M. University of Texas M. D. Anderson Cancer Center, Science Park-Research Division, Smithville, USA. Cancer Detect Prev. 1997;21(2):178-90.

D-Glucaric acid (GA) is a nontoxic, natural compound. One of its derivatives is the potent beta-glucuronidase inhibitor D-glucaro-1,4-lactone (1,4-GL). The goal of this study was to demonstrate the in vivo formation of 1,4-GL from a D-glucarate salt and determine its metabolism, uptake by selected organs, and excretion following oral administration of potassium hydrogen D-[14C]glucarate to male and female Sprague-Dawley rats. 1,4-GL increases detoxification of carcinogens and tumor promoters/progressors by inhibiting beta-glucuronidase and preventing hydrolysis of their glucuronides. 1,4-GL and its precursors, such as potassium hydrogen D-glucarate and calcium D-glucarate, may exert their anticancer action, in part, through alterations in steroidogenesis accompanied by changes in the hormonal environment and the proliferative status of the target organ. Thus, GA derivatives may be useful as new or adjuvant cancer preventive and therapeutic agents. In our study, 1,4-GL was found to be formed from the D-glucarate salt in the stomach of rats. It was apparently absorbed from the gastrointestinal tract, transported with the blood to different internal organs, and excreted in the urine and to a lesser extent in bile. There were no significant differences in the metabolism of PHG between male and female rats. Thus, formation of 1,4-GL from D-glucaric acid derivatives may be prerequisite for their inhibition of chemical carcinogenesis in rodents and prevention of breast, prostate, and colon cancer in humans.

20. J Natl Cancer Inst 1996 Jul 3;88(13):899-907 Wheat bran and psyllium diets: effects on N-methylnitrosourea-induced mammary tumorigenesis in F344 rats. Cohen LA, Zhao Z, Zang EA, Wynn TT, Simi B, Rivenson A Division of Nutrition and Endocrinology, American Health Foundation, Valhalla, NY 10595, USA.

BACKGROUND. Experimental and epidemiologic evidence suggests that increased dietary fiber is associated with decreased breast cancer risk. Little is known about the role played by different types of fiber and, particularly, mixtures of soluble and insoluble fibers similar to those consumed by human populations in reducing breast cancer risk. High intake of fiber may suppress bacterial hydrolysis of biliary estrogen conjugates to free (absorbable) estrogens in the colon and thus may decrease the availability of circulating estrogens necessary for the development and growth of breast cancers. PURPOSE. The purpose of this study was to evaluate the effect of wheat bran (an insoluble fiber) and psyllium (a soluble fiber) alone and in combination on overall + status, on fecal bacterial beta-D-glucuronidase (a key diet-responsive estrogen- econjugating enzyme) activity, and on the induction of mammary tumors in rats treated with N-methylnitrosourea (MNU). METHODS. One hundred fifty virgin female F344 rats were fed the NIH-07 diet from 28 days of age until 50 days of age; they were then given a single dose (40 mg/kg of body weight) of MNU by tail vein injection. Three days later, they were randomly assigned to one of five experimental dietary groups (30 animals per group). Soft, white wheat bran (45% dietary fiber content) and psyllium (80% dietary fiber content) were added to a modified (high-fat) American Institute of Nutrition (AIN)-76A diet at the following percents, respectively: 12% + 0% (group 1), 8% + 2% (group 2), 6% + 3% (group 3), 4% + 4% (group 4), and 0% + 6% (group 5). Blood, urine, and feces were collected and analyzed by radioimmunoassay techniques for estrogens. Cecal contents were analyzed for bacterial beta-D-glucuronidase activity. After 19 weeks on the experimental diets, the rats were killed, and mammary tumors were counted and classified by histologic type. Cumulative tumor incidence was evaluated by the Kaplan-Meier life-table method and the logrank test. Tumor number was evaluated by the chi-squared test of association, and tumor multiplicity was evaluated by the Mantel-Haenszel chi-squared test. All statistical tests were two-tailed. RESULTS. As the level of psyllium relative to that of wheat bran increased, the total tumor number and multiplicity of mammary adenocarcinomas in rats decreased as a statistically significant linear trend across groups 1-5 (P < .05). Compared with the group given wheat bran alone, the group given the 1:1 (wheat bran:psyllium) combination had maximum protection against mammary tumorigenesis, while the groups given the 4:1 or 2:1 (wheat bran:psyllium) combination or psyllium alone had intermediate protection. No statistically significant differences in circulating estrogens or urinary estrogen excretion patterns were observed among the five experimental groups. Fecal estrogen excretion, however, decreased with increasing levels of psyllium (P < .01), and cecal beta-D-glucuronidase activity exhibited a decreasing trend with respect to the increasing psyllium content of the diet across groups 1-5 (P < .01). CONCLUSIONS. The addition of a 4%:4% mixture of an insoluble (wheat bran) fiber and a soluble (psyllium) fiber to a high-fat diet provided the maximum tumor-inhibiting effects in this mammary tumor model. Although increasing levels of dietary psyllium were associated with decreased cecal bacterial beta-D-glucuronidase activity, these changes were not reflected in decreased circulating levels of tumor-promoting estrogens. Therefore, the mechanism(s) by which mixtures of soluble and insoluble dietary fibers protect against mammary tumorigenesis remains to be clarified.

21. Inhibition of tumor cell haptotaxis by sodium D-glucaro-delta-lactam (ND2001).

Tsuruoka T, Azetaka M, Iizuka Y, Saito K, Inouye S, Hosokawa M, Kobayashi H. Pharmaceutical Research Center, Meiji Seika Kaisha, Ltd., Yokohama. Jpn J Cancer Res. 1995 Nov;86(11):1080-5.

We used the Boyden chamber system to investigate the mechanism by which the antimetastatic agent sodium D-glucaro-delta-lactam (ND2001) inhibits tumor cell invasion, and by establishing what ND2001 did not achieve, we were able to pinpoint the areas in which it was successful as an inhibitor. ND2001 did not inhibit cell adhesion of a highly metastatic B16 melanoma variant (the B16 variant) to the reconstituted basal membrane Matrigel, nor did it affect the production or activity of basal membrane-degrading type i.v. collagenase, but, in the Boyden chamber, ND2001 inhibited cell migration of the B16 variant toward a chemoattractant, laminin, on the lower surface of a Matrigel-free filter set (haptotaxis). Lewis lung carcinoma (3LL) cells that had been treated with ND2001 also exhibited hardly any haptotaxis, although the cells showed no alteration in behavior during cell adhesion to Matrigel. Since ND2001 did succeed in inhibiting the pulmonary metastases of the B16 variant and 3LL, we infer that inhibition of the metastases by ND2001 in these tumors is likely to be due to the inhibition of haptotactic migration.

22. Inhibition of pulmonary metastases and tumor cell invasion in experimental tumors by sodium D-glucaro-delta-lactam (ND2001).

Tsuruoka T, Fukuyasu H, Azetaka M, Iizuka Y, Inouye S, Hosokawa M, Kobayashi H. Pharmaceutical Research Center, Meiji Seika Kaisha, Ltd., Yokohama. Jpn J Cancer Res. 1995 Jan;86(1):41-7.

Sodium D-glucaro-delta-lactam (ND2001) inhibited spontaneous pulmonary metastases of the highly metastatic B16 melanoma variant with a maximal inhibition rate of 99.5%, and 6 of 7 animals remained metastasis-free. Likewise, ND2001 inhibited the spontaneous pulmonary metastases of both Lewis lung carcinoma (3LL) with a rate of 98.0% (3 of 5 animals remaining metastasis-free) and rat KDH-8 liver carcinoma with a rate of 82.5% (3 of 7 animals remaining metastasis-free), although it was unable to inhibit the metastases of mouse BMT-11 fibrosarcoma and rat SST-2 breast carcinoma. Pretreatment with ND2001 in vitro inhibited the pulmonary metastases of the B16 variant and 3LL cells, which indicates direct action upon the cancer cells. When the invasive activity of cancer cells was measured by the Boyden chamber method, the number of invading B16 variant or 3LL cells was reduced with maximal inhibition rates of 93.0% or 89.9%, respectively, but pretreatment with ND2001 failed to reduce the invasive activity of BMT-11 or SST-2 cells. ND2001 showed neither cytocidal nor antitumor activity. These results suggest that ND2001 inhibited pulmonary metastases at the invasive step into the basement membrane by directly changing some property of the tumor cells.

23. Relative efficacy of glucarate on the initiation and promotion phases of rat mammary carcinogenesis.

Abou-Issa H, Moeschberger M, el-Masry W, Tejwani S, Curley RW Jr, Webb TE Department of Surgery, College of Medicine, Ohio State University, Columbus 43210, USA. Anticancer Res 1995 May-Jun;15(3):805-10

The independent effects of the potential cancer chemopreventive agent calcium glucarate (CGT) when fed (128 mmol/kg diet) during the initiation (I), promotion (P) or (I+P) phases of 7,12-dimethylbenzanthracene-induced rat mammary carcinogenesis, was compared to that of the known chemopreventive agent N-(4-hydroxyphenyl) retinamide (4-HPR) fed (2.0 mmol/kg diet) during these same phases. CGT and especially 4-HPR both significantly increased tumor latency when fed during the P-phase. When fed during I, P or I+P phases mammary tumor incidence was reduced compared to the controls 33%, 42% and 67% by 4-HPR and 18%, 42% and 50% by CGT. Similarly, tumor multiplicity was significantly reduced by either agent. For example, as compared to the corresponding control, when fed during the I, P or I+P phases 4-HPR reduced tumor multiplicity 63, 34 and 63%, while CGT reduced tumor multiplicity 28, 42 and 63% respectively. CGT, like 4-HPR, acts on both the I and P phases with the effect being maximal when fed during P and I+P phases.

24. Mechanism of growth inhibition of mammary carcinomas by glucarate and the glucarate: retinoid combination.

Webb TE, Abou-Issa H, Stromberg PC, Curley RC Jr, Nguyen MH. Department of Medical Biochemistry, College of Medicine, Ohio State University, Columbus 43210. Anticancer Res. 1993 Nov-Dec;13(6A):2095-9.

In synergistic combination 0.75 mmol/kg diet of N-(4-hydroxyphenyl) retinamide and 32 mmol/kg diet of glucarate inhibits the growth of primary rat mammary tumors, but are equally effective as single agents at 1.5 and 128 mmol/kg diet, respectively. Dose-response studies suggest that like retinoids, glucarate acts directly on tumor cells, rather than having an adjuvant effect. Although synergism is maintained down to at least 0.38 mmol/kg diet of the retinoid, experiments using Vitamin A-deficient diets indicates 128 mmol/kg glucarate acts independent of retinoid. Both alone and in combination, glucarate and retinoid inhibited the growth of human mammary tumor cells grown in the athymic mouse, the growth of rat mammary tumors in germfree rats and the hormone-independent MTW 9a/R rat mammary tumor. Like retinoids, glucarate suppresses protein kinase C and induces transforming growth factor-beta, in the mammary tumor cells.

25. Basis for the anti-tumor and chemopreventive activities of glucarate and the glucarate:retinoid combination.

Abou-Issa H, Dwivedi C, Curley RW Jr, Kirkpatrick R, Koolemans-Beynen A, Engineer FN, Humphries KA, el-Masry W, Webb TE. Department of Surgery, Ohio State University, Columbus 43210.

Anticancer Res. 1993 Mar-Apr;13(2):395-9.

The biochemical basis for the cancer chemopreventive and anti-cancer activities of glucarate, retinoids (13-cis-retinoic acid, hydroxyphenyl retinamide) and their synergistic combination, has been evaluated. Neither alone nor in combination did these agents affect the level in the rat, of enzymes which are (a) known to correlate with reduced risk of carcinogenesis (detoxification enzyme, catalase, glutathione reductase) nor (b) enzymes which correlate with increased risk of carcinogenesis (beta-glucuronidase, xanthine oxidase, glucose-6-phosphate dehydrogenase). Retinoids, but neither glucarate nor its lactone inhibited free radical-induced lipid peroxidation. Both agents alone and synergistically in combination, raise cellular cAMP levels, repress protein kinase C and more generally inhibited DNA synthesis.

26. Dietary glucarate-mediated inhibition of initiation of diethylnitrosamine-induced hepatocarcinogenesis.

Oredipe OA; Barth RF; Dwivedi C; Webb TE Ohio State Univ., Dep. Pathol., 165 Hamilton Hall, 1645 Neil Ave., Columbus, Ohio 43210. Toxicology; 74 (2-3). 1992. 209-222

Previously, it has been reported that calcium glucarate is a potent inhibitor of chemical carcinogenesis, including phenobarbital-promoted diethylnitrosamine-initiated hepatic toxicity expressed as altered hepatic foci in rats. The purpose of the present study was to determine whether calcium glucarate could inhibit the immediate and delayed appearance of altered hepatic foci when fed to rats during the initiation phase of diethylnitrosamine-induced hepatocarcinogenesis. The effects of dietary mode of administration of carbon glucarate on the initiation phase of hepatocarcinogenesis were also examined. Since diethylnitrosamine is not known to undergo glucuronidation and calcium lucarate has been shown to enhance clearance of circulating estrogens, an indirect mechanism of action of calcium glucarate was also evaluated by pretreating rats an anti-estrogen, tamoxifen, prior to partial hepatectomy and administration of diethylnitrosamine. Calcium glucarate significantly inhibited both the early and delayed appearance of altered hepatic foci and exerted maximal inhibition when administered by gavage prior to diethylnitrosamine. Maximal inhibition was obtained when calcium glucarate was provided continuously in the diet of animals up to 5 and 7 months. Pretreatment of animals with tamoxifen before partial hepatectomy and diethylnitrosamine resulted in maximal inhibition of the initiation phase of hepatocarcinogenesis. This suggests but does not prove that the anti-carcinogenic activity of calcium glucarate was due to decreased liver proliferation. In the present study, the proliferation of ductal epithelial and oval cells appeared to be associated with the administration of diethylnitrosamine. Collectively, our data suggest that calcium glucarate inhibited the initiation phase of diethylnitrosamine-induced hepatocarcinogenesis.

27. Potential use of D-glucaric acid derivatives in cancer prevention.

Walaszek Z. Department of Carcinogenesis, University of Texas M.D. Anderson Cancer Center, Smithville 78957. Cancer Lett. 1990 Oct 8;54(1-2):1-8.

There is now growing evidence from animal models for the possible control of different stages of the carcinogenic process by the beta-glucuronidase inhibitor D-glucaro-1,4-lactone and its precursors such as D-glucaric acid salts, D-glucarates. D-Glucaric acid is a natural, non-toxic compound produced in small amounts by mammals, including humans. It was recently found in some vegetables and fruits. D-Glucaro-1,4-lactone and D-glucarate exhibit potent antiproliferative properties in vivo. Some human subpopulations could have reduced risk of cancer development by ingesting food rich in D-glucaric acid or self-medication with D-glucarates alone or in combination with other chemopreventive agents.

28. Antiproliferative effect of dietary glucarate on the Sprague-Dawley rat mammary gland.

Walaszek Z, Hanausek M, Sherman U, Adams AK. Department of Carcinogenesis, University of Texas M.D. Anderson Cancer Center, Smithville 78957. Cancer Lett. 1990 Jan;49(1):51-7.

Dietary glucarate has previously been shown to inhibit chemical carcinogen-induced rat mammary tumorigenesis. It is demonstrated in this paper that in the mammary gland of the female Sprague-Dawley rat, feeding glucarate at a dose of 70 mmol/kg AIN76A diet for 2 weeks beginning at 35 days of age, markedly reduces [3H]thymidine labeling. Specific histochemical staining for beta-glucuronidase is used to show that the glucarate diet fed to rats for 2-4 weeks inhibits beta-glucuronidase activity in the mammary gland and has a marked antiproliferative effect on mammary epithelium. Glucarate may inhibit rat mammary carcinogenesis, in part, by changing the proliferative status of the target organ.

29. Cancer Lett 1990 Jan;49(1):51-7 Antiproliferative effect of dietary glucarate on the Sprague-Dawley rat mammary gland. Walaszek Z, Hanausek M, Sherman U, Adams AK Department of Carcinogenesis, University of Texas M.D. Anderson Cancer Center, Smithville 78957.

Dietary glucarate has previously been shown to inhibit chemical carcinogen-induced rat mammary tumorigenesis. It is demonstrated in this paper that in the mammary gland of the female Sprague-Dawley rat, feeding glucarate at a dose of 70 mmol/kg AIN76A diet for 2 weeks beginning at 35 days of age, markedly reduces [3H]thymidine labeling. Specific histochemical staining for beta-glucuronidase is used to show that the glucarate diet fed to rats for 2-4 weeks inhibits beta-glucuronidase activity in the mammary gland and has a marked antiproliferative effect on mammary epithelium. Glucarate may inhibit rat mammary carcinogenesis, in part, by changing the proliferative status of the target organ.

30. Carcinogenesis 1989 Aug;10(8):1539-41 Effects of the experimental chemopreventative agent, glucarate, on intestinal carcinogenesis in rats. Dwivedi C, Oredipe OA, Barth RF, Downie AA, Webb TE Department of Physiological Chemistry, College of Medicine, Ohio State University, Columbus 43210.

Dietary calcium glucarate was previously shown to protect effectively against chemically-induced mammary, lung, liver and skin carcinogenesis in rodents, whereas the negative dietary calcium control, calcium gluconate, had no effect. In the present study the chemopreventative activity of dietary calcium glucarate was evaluated in the azoxymethane intestinal carcinogenesis model using the Fischer strain rat. The protocol limited the duration of azoxymethane treatment to 3 weeks to permit the evaluation of the separate effects of glucarate on the initiation and promotion phases. Control rats, treated with azoxymethane and maintained on a low fat chow diet throughout the 32-week experiment had an intestinal adenocarcinoma incidence of 55%, with an equal incidence of 27.7% in the small and large intestines. There was no significant difference between this control group and a negative calcium control group fed 128 mmol/kg chow of calcium as calcium gluconate. In contrast to these two control groups, supplementation of the diet of azoxymethane-treated rats with 128 mmol/kg diet of calcium glucarate during both the initiation and promotion phases significantly inhibited the overall induction of adenocarcinomas in the intestine, the incidence in the entire intestine and in the small and large intestines being 11.8, 5.8 and 5.8%, respectively. When fed only during the initiation phase, the inhibition again was statistically significant, the corresponding values being 11.8%, 5.8 and 5.8%. When calcium glucarate was fed during the promotion phase, a statistically significant inhibition of adenocarcinoma induction was observed only in the colon where the incidence was 5.5%. Weight gain was similar in all groups. These and related data indicate that dietary glucarate exerts a significant inhibitory effect on azoxymethane-induced intestinal and in particular colon carcinogenesis in the rat, decreasing their incidence and size and reducing their metastic potential.

31. Chemopreventative activity of dietary glucarate on azoxymethane-induced altered hepatic foci in rats Oredipe O.A.; Barth R.F.; Dwivedi C.; Webb T.E. Department of Pathology, The Ohio State University, Columbus, OH 43210 United States Research Communications in Chemical Pathology and Pharmacology ( RES. COMMUN. CHEM. PATHOL. PHARMACOL. ) (United States) 1989, 65/3 (345-359)

Previous studies have shown that dietary calcium glucarate, an inhibitor of beta-glucuronidase, is a potent inhibitor of promotion of diethylnitrosamine-induced altered hepatic foci, 7,12-dimethylbenzanthracene-induced mammary tumorigenesis and benzo(a)pyrene-induced lung carcinogenesis. The present study was undertaken to test the chemopreventive activity of calcium glucarate on azoxymethane-induced hepatocarcinogenesis in female Fischer 344 rats. A series of experiments were carried out over 36 weeks to evaluate the effects of calcium glucarate on the initiation and promotion phases separately and also in combination with each other. A calcium gluconate group was included and used as a negative calcium control. Histopathologic evaluation of H and E stained liver sections of all animals in this study showed that a statistically significant inhibition of hepatocarcinogenesis only occurred when dietary calcium glucarate supplementation was provided throughout the combined initiation and promotion phases. This inhibitory effect approximately equaled the summation of that obtained when calcium glucarate was fed only during initiation phase and only during promotion phase.

32. Putative metabolites derived from dietary combinations of calcium glucarate and N-(4-hydroxyphenyl)retinamide act synergistically to inhibit the induction of rat mammary tumors by 7,12-dimethylbenz[a]anthracene.

Abou-Issa HM, Duruibe VA, Minton JP, Larroya S, Dwivedi C, Webb TE. Comprehensive Cancer Center, Ohio State University, Columbus 43210. Proc Natl Acad Sci U S A. 1988 Jun;85(12):4181-4.

Calcium glucarate and N-(4-hydroxyphenyl)retinamide were evaluated individually and in combination in the diet as preventative chemical agents, by using the induction of rat mammary tumors by 7,12-dimethylbenz[a]anthracene as the test system. When tested separately over 18 weeks, optimal doses of calcium glucarate (128 mmol/kg of diet) or N-(4-hydroxyphenyl)retinamide (1.5 mmol/kg of diet) administered daily inhibited tumor incidence by 50% or 57% and tumor multiplicity by 50% or 65%, respectively. Suboptimal doses of calcium glucarate (32 mmol/kg) and of N-(4-hydroxyphenyl)retinamide (0.75 mmol/kg) inhibited tumor incidence by 15% and 5% but had no inhibitory effect on tumor multiplicity. In contrast, the combination of calcium glucarate (32 mmol/kg) and N-(4-hydroxyphenyl)retinamide (0.75 mmol/kg) inhibited tumor incidence and tumor multiplicity by 50%. Similar synergism was observed with the combination of calcium glucarate (64 mmol/kg) and N-(4-hydroxyphenyl)retinamide (0.75 mmol/kg), the inhibition being 55-60%. HPLC analysis of the bile of female rats injected intraperitoneally with a single dose of the retinamide [60 mg/kg (body weight)] showed that the excretion of the retinamide and its glucuronide were markedly suppressed by pretreatment with an oral dose of calcium glucarate [4.5 mmol/kg (body weight)].

33. Carcinogenesis 1986 Sep;7(9):1463-6 Dietary glucarate as anti-promoter of 7,12-dimethylbenz[a]anthracene-induced mammary tumorigenesis. Walaszek Z, Hanausek-Walaszek M, Minton JP, Webb TE

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.

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

Walaszek Z, Hanausek-Walaszek M, Webb TE. Cancer Lett. 1986 Oct;33(1):25-32.

Serum beta-glucuronidase activity is shown to differ quantitatively in the following strains of mice, listed in order of increasing activity: C3H, C57BL/6 less than BALB/c, DBA/2, ICR less than 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 beta-glucuronidase inhibitor, reduced the steady state level of both beta-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.

CHOLESTEROL**

35. D-Glucaric acid content of various fruits and vegetables and cholesterol- lowering effects of dietary D-glucarate in the rat Walaszek Z.; Szemraj J.; Hanausek M.; Adams A.K.; Sherman U. Bio-Organic/Nat. Product Chem. Lab., Biomedical Horizons Institute, P.O. Box 695,Smithville, TX 78957 United States Nutrition Research ( NUTR. RES. ) (United States) 1996, 16/4 (673-681)

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- glucaronic 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.