Modulation of Carcinogen Metabolism and DNA Interaction by Calcium Glucarate in Mouse Skin

Advance Access publication February Modulation of Carcinogen Metabolism and DNA Interaction by Calcium Glucarate in Mouse Skin Krishna P. Gupta 0 1 Jaya Singh 0 1 0 Toxicological Sciences vol. 79 no. 1 Society of Toxicology 2004; all rights reserved 1 Environmental Carcinogenesis Division, Industrial Toxicology Research Center , Lucknow , India Almost all the polycyclic aromatic hydrocarbons (PAHs) require metabolic activation to exert their carcinogenic activity. Environmental carcinogen [3H] benzo[a]pyrene (BP) is carcinogenic only after its metabolic transformation to a reactive intermediate, which can then bind to cellular macromolecules. Inhibition of dimethylbenz anthracene- (DMBA-) DNA binding generally accompanied inhibition of tumor initiation as most inhibitors of initiation interfere with the metabolic activation of the initiator. The importance of carcinogen-DNA interaction and the enzymes involved in the metabolism of carcinogenic polycyclic hydrocarbons has led to a search for inhibitors that would be useful in modifying the cancer-causing effects of the PAHs. We tested the effect of calcium glucarate (Cag), a naturally occurring nontoxic compound, on carcinogen metabolism and DNA interaction. Cag inhibited [3H] BP binding to both calf thymus DNA in vitro and to epidermal DNA in vivo. Application of Cag to mouse skin caused a dose-dependent inhibition of [3H] BP binding to epidermal DNA. To establish the relevance of the in vivo results to the in vitro situation, we followed the in vitro effect of Cag on [3H] BP binding to calf thymus DNA and observed that Cag inhibited the [3H] BP binding to calf thymus DNA in the presence of microsomes prepared from animals treated with DMBA. We also studied related events like DNA synthesis and carcinogen metabolism. For assessing the DNA synthesis, thymidine kinase was used as marker. Cag caused a dose-dependent inhibition of DMBA-induced thymidine kinase activity. At the same time, Cag caused a marked inhibition of DMBA-induced aryl hydrocarbon hydroxylase (AHH) activity, an enzyme responsible for the metabolism of PAHs like BP, both in vivo and in vitro. Our study indicates that Cag exerted its antitumor effect possibly by inhibiting the carcinogen-DNA binding, which appears to be due to reduced DNA synthesis and AHH activity. - Polycyclic aromatic hydrocarbons (PAHs) are widely distributed in our environment and are implicated in different types of cancer, including skin cancer (Phillips and Sims, 1979). There have been extensive studies to show that PAHs must be metabolically activated to reactive intermediates that bind covalently to DNA to exert their mutagenic, cell-transforming, and tumorigenic or carcinogenic effects (Newbold and Brookes, 1976). Following topical application of [3H] benzo[a]pyrene (BP) to mouse skin, the major DNA adduct formed was ( )-7 , 8 dihydroxy-9 , 10 -epoxy-7, 8,9,10tetrahydrobenzo[a]pyrene diol epoxide [( ) anti-BPDE] bound through a transaddition to the exocyclic amino group of deoxyguanosine (7R-anti BPDE-d Guo; Kakefuda and Yamamoto, 1978). The metabolic activation of PAH depends on a large number of factors ranging from the geometry of the PAH to the enzymes that metabolize it and the stereochemistry of that metabolism (Stoner et al., 1986). The proportion of PAH activated to ultimate carcinogenic metabolites that interact with DNA in a particular tissue depends mainly on the cytochrome P-450 dependent microsomal carcinogen-metabolizing enzyme, aryl hydrocarbon hydroxylase (AHH). Calcium glucarate (Cag), a naturally occurring nontoxic compound, forms a - glucuronidase inhibitor called glucarolactone (Abou-Issa et al., 1993; Dwivedi et al., 1987, 1990). Dietary Cag has been shown to be an effective chemopreventive agent in dimethylbenz anthracene (DMBA-) and N-methyl-N-nitrosourea (MNU-) induced rat mammary carcinogenesis (Walaszek et al., 1987) and DMBA-induced skin tumorigenesis (Singh and Gupta, 2003), but Cags mechanism of action is not known. The covalent binding of carcinogenic PAHs to nuclear DNA is considered to be the most probable tumor-initiating event in the neoplastic transformation (Eastman et al., 1978). There are ample evidences that altered carcinogen metabolism in experimental animal models can have dramatic effects on tumor incidences (Boberg et al., 1987). The covalent binding of organic chemicals to DNA in vivo is considered to be a quantitative indicator of the initiation of chemical carcinogenesis (Brookes and Osborne, 1982). Inhibition of carcinogenDNA binding generally accompanied the inhibition of tumor initiation (Lesca, 1982), in keeping with the view that most inhibitors probably interfere with the metabolic activation of the tumor initiator (Di Giovanni and Slaga, 1981). One of the metabolites of BP, environmental pollutant benzopyrene 7, 8 diol 9,10 epoxide (BPDE), is a known mutagen and is believed to be the ultimate carcinogenic metabolite of BP (Cavalieri and Rogan, 1998) that binds to cellular macromolecules (Hu et al., 1980). It is formed by the action of cytochrome P-450 dependent microsomal enzyme AHH and the nonP-450 dependent microsomal enzyme epoxide hydrolase (Bickers et al., 1982). Thymidine kinase (TK) is a marker for DNA synthesis. TK and deoxy ribonucleoside triphosphate pools increase substantially in proliferating cells and tumors (Hannigam et al., 1993; Saul, 1976). AHH is largely responsible for the metabolism of many PAHs, chemicals, drugs, pesticides, and so on. AHH converts many PAHs to hydroxylated derivatives, which are carcinogenic. Our report covers the inhibition of carcinogen metabolism in terms of AHH activity, the binding of carcinogen to DNA, and DNA synthesis in terms of TK activity. MATERIALS AND METHODS Animals. Female Swiss albino mice from the inbred colony of Industrial Toxicology Research Center (ITRC, Lucknow, India) were used throughout the study. Animals were 4 6 weeks old and weighed between 10 12 g. They were fed synthetic pellet diet and water ad libitum. Each animal was shaved on the back in the interscapular region using surgical clippers 2 days before the start of the experiment and only the animals with resting phase of hair growth were selected for the study. Chemicals. Glucaric acid (Ca salt), 7,12 dimethyl benzanthracene, calf thymus DNA, proteinase K, and ribonuclease A were obtained from Sigma Chemical Co. (St. Louis, MO). [3H] BP (specific activity 8000 mCi/mmol) and [3H] thymidine (specific activity 17,000 mCi/mmol) were obtained from Bhabha Atomic Research Center (BARC, Mumbai, India). Nicotinamide adenine dinucleotide phosphate (NADP), glucose 6 phosphate (G6P), glucose 6 phosphate dehydrogenase (G6PD), and nicotinamide adenine dinucleotide phosphate reduced (NADPH) were also purchased from Sigma Chemical Co. DE 81 filters were from Whatman International Co. (Clifton, NJ). The rest of the chemicals and reagents were obtained from local commercial sources and were of high purity. (...truncated)