PLEIOTROPIC EFFECTS OF CALCIUM D-GLUCARATE ON CHEMICALLY-INDUCED LUNG CARCINOGENESIS IN A/J MICE

Zoltaszek R, ^* Walaszek Z ,^ Hanausek M,^ Slaga TJ,^ Kilianska ZM*

ABSTRACT

Calcium D-glucarate (CG) reportedly suppresses benzo[a]pyrene (B[a]P)-induced lung carcinogenesis in A/J mice, by inhibiting the enzyme ß-glucuronidase (ß-G). The protective mechanism conferred by ß-G inhibition operates, in part, through a reduction in oncogene mutations and suppression of cell proliferation and chronic inflammation. The aim of the current study was to provide evidence on the ability of CG not only to suppress cell proliferation and inflammation but also to induce apoptosis during post-initiation stages of B[a]P-induced lung tumorigenesis in A/J mice. Two doses of 3 mg of B[a]P were given intragastrically to A/J mice two weeks apart. CG administration in the AIN-93G diet (2.0% and 4.0%, w/w) started at 2 weeks after the second dose of B[a]P. There was no change in the calcium content of CG diets in comparison with the control AIN-93G diet. Mice were scarified at week 20 and estimation on serum ß-G level and proliferation, inflammation, and also chosen apoptosis marker analyses were performed. The proapoptotic effects of CG were investigated immunohistochemically using antibodies against active fragments of caspase 9 and poly(ADP-ribose) polymerase-1 (PARP-1), respectively. Both 2% and 4% diets reduced significantly not only the number of lung adenomas and hyperplasia 4 months after B[a]P gavaging. These diets also increased, in a dose related fashion, levels of active fragments of caspase 9 as well as PARP-1 in the lung tissue. The activity of above apoptosis-related proteins increased up to over 99% and 152%, respectively in the lung tissues of A/J mice treated with B[a]P and dietary D-glucarate compared to mice treated with B[a]P. At the same time, approximately 47% inhibition of cyclooxygenase-2 (COX-2) was estimated in the epithelial cells lining the bronchioli and bronchus as well as type 2 alveolar cells in the group of mice on the 4% CG diet. The BrdU-labeling indices for the alveolar/bronchiolar hyperplasia and adenoma cells were also determined. Post-initiation dietary CG caused significant and dose related decrease in the number of proliferating pneumocyte type 2 cells (i.e., up to 37%) and macrophages (i.e., up to 44%) compared to those in alveoli of the B[a]P-treated A/J mice. We conclude that post-initiation D-glucarate may inhibit B[a]P-induced carcinogenesis in A/J mice not only by suppressing cell proliferation and inflammation but also by programmed cell death induction.

Keywords: Mouse lung carcinogenesis, calcium glucarate, ß-glucuronidase, proliferation, inflammation, apoptosis.