MECHANISM OF GLUCARATE INHIBITION OF MAMMARY CANCER

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The primary objective of this proposal will be to test the hypothesis that formation of the beta-glucuronidase inhibitor, D-glucaro- 1,4-lactone (1,4-GL) from calcium D-glucarate, is of critical importance for the mechanism of D-glucarate inhibition of mammary cancer and that 1,4-GL-mediated modulation of mammotropic hormone levels and DNA synthesis is at least partially responsible for the potent chemopreventive effect of D-glucarate. The well established model of mammary carcinogenesis induced in female Sprague-Dawley rats with a single s.c. dose of N-methylnitrosourea (MNU) will be used. Specific Aims include: (i) Establishment of the theory that in D-glucaric acid/D-glucarate/D-glucarolactones family, 1,4-GL is the ultimate inhibitor of carcinogenesis by testing its efficacy in the MNU-induced rat mammary carcinogenesis model, with a non-inhibitor of beta- glucuronidase, D-glucaro-6,3-lactone (6,3-GL) as negative control. In order to overcome the known instability of 1,4-GL in vivo, some derivatives of 1,4-GL and 6,3GL will be utilized. Different concentrations of these derivatives will be incorporated to the semi- purified AIN-76A diet. The modified diets will be fed to rats continually beginning one week after MNU to avoid any dietary effects on the development of mammary gland prior to initiation. The suggested role of calcium D-glucarate as a precursor of 1,4-GL will be further confirmed by investigation of the metabolism, pharmacokinetics and disposition of calcium D-glucarate in the rat using both non-radioactive and 14C-labeled compounds. (ii) Determination of the systemic and local effects of 1,4-GL, fed continually to young female Sprague-Dawley rats beginning early before MNU treatment, i.e. on mammotropic hormones levels, the estrogen receptor and proliferative status of mammary gland at different time-points, and on the final tumorigenic response. A combination of [3H]thymidine autoradiography and immunohistochemical staining for beta-glucuronidase or estrogen receptor, will be used to investigate a potential correlation between B-glucuronidase and or estrogen receptor expression and DNA synthesis. (iii) Demonstration that hormone replacement has no effect on D-glucaro-1,4-lactone inhibition of rat mammary cancer. The rats with MNU-induced mammary tumors will be hypophysectomized and treated with estradiol and prolactin or vehicle, while fed diets with and without D-glucaro- 1,4-lactone. Changes in the size of tumors will be correlated with the hormone receptor status.