Environmental Carcinogenesis Division, Industrial Toxicology Research Center, Lucknow, India
Received September 8, 2003; accepted January 27, 2004
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: calcium glucarate; skin; carcinogen metabolism; DNA binding; thymidine kinase.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Calcium glucarate (Cag), a naturally occurring nontoxic compound, forms a ß- 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 Cag's 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 carcinogen-DNA 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 non-P-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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
Dose selection: DMBA.
In the present study, we used a 5-µg and/or a 50-µg dose of DMBA in 0.1 ml of acetone. The 5-µg dose of DMBA is a subcarcinogenic dose, which has been used in complete carcinogenesis protocol for repeated exposures. However, single application of the 50-µg dose of DMBA has caused genetic changes and mutations and initiated mouse skin tumor development. Hence, this dose was used in single-application experiments. The respective control groups were treated with 0.1 ml acetone and 0.1 ml DMSO.
Dose selection: Cag.
Since there were no data available on the topical application of Cag, we selected 1-, 3-, and 6-mg doses of Cag in 0.1 ml DMSO based on other available reports and our earlier work on natural compounds for the entire study. Apparently, there were no adverse effects at these doses of Cag. Respective control groups were treated with 0.1 ml DMSO and 0.1 ml acetone.
In vivo DNA-carcinogen binding.
Animals were divided into groups consisting of three animals each. Animals were treated with 50 µg DMBA/0.1 ml acetone/mouse. To assess the in vivo effect of Cag, Cag was topically applied to the skin immediately after the DMBA application (on the same area) at a dose of 1, 3, 6, or 12 mg. All animals were then treated with 10-µCi [3H] BP (in 0.1 ml acetone) topically 2 h prior to sacrifice. At 24 h after DMBA application, animals were sacrificed by cervical dislocation. Skin (1 cm2) was removed and epidermis was separated. Epidermal homogenates (10%) was prepared in 0.1 M phosphate buffer containing 10 mM EDTA, pH 7.4.
DNA extraction from epidermis.
DNA from epidermal homogenate was extracted essentially as described previously (Kates and Beeson, 1970). Homogenates were then incubated with proteinase K (1.87 mg/1 ml) for 40 min at 37°C in water bath; DNA was extracted using conventional Phenol chloroform method (Kates and Beeson, 1970
). Next, DNA was precipitated by the addition of 5 M NaCl and chilled 100% ethanol. DNA was then dissolved in sodium chloride sodium citrate (SSC) and digested with ribonuclease A for 40 min at 37°C, extracted again with SEVAG (24:1, chloroform: isoamylalchol), precipitated, and washed with 70% alcohol twice. Extracted DNA was then dissolved in SSC and estimated by measuring its absorption at 260 nm. The purity of the DNA was assessed by the absorbance ratio A 260 nm/A 280 nm, and radioactivity was counted on a scintillation counter.
Multiple applications of Cag on in vivo carcinogen-DNA binding and AHH activity assay.
Two experimental sets consisting of three animals per group were maintained to see the effect of multiple or single application of Cag on [3H] BP binding to epidermal DNA and AHH activity assay. DMBA was used at the dose of 5 µg in 0.1 ml acetone; Cag in 0.1 ml DMSO was used at different doses (Table 1).
|
Assay for TK activity.
In a separate set of experiments for TK assay, animals were divided into groups consisting of three mice per group. Animals were sacrificed at 16 h after DMBA (50 µg in 0.1 ml acetone) application in the presence or absence of Cag (1, 3, or 6 mg). Skin (1 cm2) was separated and epidermal extract was prepared as previously described (Sakamoto et al., 1997). Enzyme activity was measured in 105,000 x g supernatant (Gupta and Mehrotra, 1997
) and was expressed in terms of pmol [3H] thymidine bound/mg protein/hour.
Assay for AHH activity.
Animals were divided into groups consisting of three mice per group. Animals were treated with DMBA (5 or 50 µg in 0.1 ml acetone) for 24 h in the presence or absence of Cag (0.5, 1, 3, 6, or 12 mg). Animals were sacrificed, and the epidermis was separated from the skin. Then, 10% epidermal homogenate was prepared in 0.1 M phosphate buffer, pH 7.4, containing 0.15 M KCl and centrifuged at 9000 rpm for 20 min at 4°C. Supernatant (S-9 fraction) thus obtained was used for enzyme assay. AHH activity was assayed as previously described (Nebert and Gelboin, 1968). The reaction mixture in a total volume of 1.9 ml contained 0.2 M phosphate buffer (pH 7.5), 1 mM NADP, 10 mM G-6-P, 15 mM MgCl2, 57 mM nicotinamide, and 0.4 ml of enzyme protein (100200 µg). The reaction was started by the addition of 20 µl BP (1 mg/ml in ethanol) and incubated at 37°C for 30 min. The reaction was stopped by the addition of 1 ml of 10% Triton-X 100 in 1 N NaOH. Enzyme activity was estimated spectrophotoflurometrically, with excitation at 465 nm and emission at 520 nm, and expressed in terms of pmole 3-hydroxylated BP/mg protein/hour.
Protein estimation.
Protein estimations were done according to Lowry's method (Lowry et al., 1951).
Statistical analysis.
Data were analyzed statistically using Student's t-test; p < 0.05 was considered to be significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cag caused a marked decrease in DMBA-induced [3H] BP binding to epidermal DNA. All the doses of Cag caused significant decreases in the binding of [3H] BP to epidermal DNA. The inhibition caused by a 1-, 3-, 6-, or 12-mg dose of Cag was 2.2-, 2.7-, 2.9-, or 3.2-fold, respectively. Topical application of Cag resulted in a dose-dependent inhibition of binding of [3H] BP to epidermal DNA. Lower doses of Cag such as 3 mg were also effective in complete inhibition of [3H] BP binding to epidermal DNA (Fig. 1).
|
|
The Effect of Cag on In Vitro Binding of Carcinogen to Calf Thymus DNA
To assess the effect of Cag on in vitro carcinogen-DNA interaction, animals were treated with DMBA (50 µg/0.1 ml of acetone). Immediately after the application of DMBA, Cag was applied at a dose of 0.5, 1, 3, 6, or 12 mg/0.1 ml of DMSO in the same area. Here, [3H] BP was added to the reaction mixture to see the effect of Cag on in vitro binding of [3H] BP to calf thymus DNA. In epidermal microsomes prepared from animals treated with DMBA (50 µg/0.1 ml acetone), DMBA caused a 2.6-fold increase in in vitro binding of [3H] BP to calf thymus DNA compared with microsomes prepared from the vehicle-treated negative control group. Cag treatment caused a marked decrease in the binding of [3H] BP to calf thymus DNA. Cag at a dose of 0.5, 1, 3, 6, or 12 mg caused a 1.6-, 2.1-, 2.6-, 2.3-, or 3.4-fold reduction, respectively, of in vitro binding of [3H] BP to calf thymus DNA. Cag caused a dose-dependent inhibition of [3H] BP binding to calf thymus DNA in DMBA-treated microsomes (Fig. 3). Thus, it can be said that Cag inhibits [3H] BP binding to DNA in vivo as well as in vitro. During the process of chemical carcinogenesis, BP gets converted in BPDE, which is the active moiety that binds covalently with DNA. The enzyme system responsible for this activation is AHH, which is a member of the cytochrome P-450 family. Reduced [3H] BP binding to DNA could be because of the inhibition of the enzyme responsible for the metabolic activation of BP. AHH is the most proffered candidate for BP activation; thus, inhibition of AHH can cause lesser availability of BPDE to bind with DNA. We looked for the effect of Cag on AHH enzyme activity in DMBA-exposed animals.
|
|
In experimental set 1, DMBA caused a 39% induction of AHH activity compared with the vehicle-treated control group. Concomitant application of Cag throughout the treatment with DMBA reversed DMBA-caused induction of AHH activity. Inhibition observed with a 0.5-, 1-, 3-, 6-, or 12-mg dose of Cag was 25, 55, 75, 63.5, or 58.6%, respectively, compared with the DMBA-treated group. In experimental set 2, DMBA caused a 39% induction of AHH activity compared with the vehicle-treated control group. A single Cag application along with DMBA on the last (seventh) day also caused the inhibition of DMBA-induced AHH activity. A 0.5-mg dose of Cag caused a 59% inhibition compared with the positive control. The other Cag doses, 1, 3, 6, or 12 mg, caused a 29, 57, 57, or 63% inhibition of DMBA-induced AHH activity, respectively (Fig. 5). Here again, Cag lowered the AHH activity, though there was not much difference in single and multiple applications at higher doses. But, at lower doses (13 mg), multiple applications of Cag caused greater reductions of AHH activity compared with a single application. The effect of multiple Cag applications was more pronounced than that for the single applications.
|
|
The Effect of Cag on TK Activity
We studied the effect of Cag on DMBA-induced TK activity. TK is the enzyme of the salvage pathway for DNA synthesis and is responsible for the phosphorylation of thymidine. For TK assay, animals were treated with DMBA (50 µg/0.1 ml acetone) for 16 h. Since TK is the enzyme responsible for DNA synthesis that is at its maximum at 16 h after the treatment, TK assay was also done at 16 h after DMBA application. DMBA (50 µg) caused a 47.7% induction of TK activity compared with the vehicle-treated control group. Concomitant application of Cag at a dose of 1, 3, or 6 mg/0.1 ml DMSO reversed this induction; 1 mg Cag caused a 37.6% inhibition of TK activity compared with the DMBA-treated group and 3 mg Cag caused a 39.4% inhibition of DMBA-induced TK activity. The maximum inhibition of TK activity, up to 50.4%, was observed with a 6-mg dose of Cag. All three Cag doses showed an inhibition of DMBA-induced TK activity, but this inhibition was found to be highest with the 6-mg dose (Fig. 7).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our data indicate that Cag inhibits experimental skin tumor formation by disruption of the initiation phase of carcinogenesis. Applications of Cag resulted in marked decreases in the binding of BP to epidermal DNA. There was 2.6-fold increase in the binding of BP to epidermis in animals exposed to DMBA, and Cag caused a dose-dependent inhibition of BP binding to epidermal DNA. Cag also caused a marked decrease in the in vitro binding of BP to calf thymus DNA. There was 2.6-fold increase in the binding of BP to calf thymus DNA in presence of microsomes prepared from animals pretreated with DMBA. Alternatively, microsomes prepared from Cag-treated animals caused a substantial inhibition of the interaction with DNA. In these experiments, we postulated that the inhibitory effect of Cag on DNA-carcinogen interaction could be because of either reduced DNA synthesis or reduced carcinogen metabolism, or both.
Others have shown that ellagic acid, a common plant phenol, inhibits carcinogen-DNA interaction and this contributes to its antitumor activity (Del Tito et al., 1983). Our present findings are in keeping with earlier observations; we showed that Cag inhibited the DNA synthesis as indicated by the inhibition of TK activity and the inhibition of [3H] thymidine incorporation into DNA (Singh and Gupta, 2003
). Inhibition of carcinogen-DNA binding could be due to two factors: (1) a decreased DNA synthesis due to which less deoxyguanosine is available to bind with the BPDE, a reactive metabolite of BP; and (2) an inhibition of BP metabolism so that BP is not converted into BPDE. Thus, it can be said that this inhibition of carcinogen-DNA binding could be due to decreased DNA synthesis and reduced carcinogen metabolites available for DNA binding.
Cag was also capable of inhibiting the epidermal metabolism of BP. The effect of in vivo application of Cag resulted in a marked inhibition of DMBA-induced epidermal AHH activity. All the doses of Cag were found to be effective in inhibiting the AHH enzyme activity. Increasing concentrations of Cag added to incubation mixtures containing carcinogen-induced epidermal microsomes caused a dose-dependent inhibition of epidermal AHH activity. Similar to the binding experiments, we also showed the inhibitory effect of multiple applications of Cag on AHH activity. The present study indicates that Cag exerts its tumor-suppressing effect probably by inhibiting the carcinogen-DNA interaction, an event almost mandatory for the tumor development. This inhibition appears to be the culmination of the inhibition of DNA-synthesizing and PAH-metabolizing enzymes. These results could be of importance in manipulating tumor management by calcium glucarate and its use in combination with other anticancer drugs.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
NOTES |
---|
1 To whom correspondence should be addressed at Environmental Carcinogenesis Division, Industrial Toxicology Research Center, P.O. Box 80, Mahatma Gandhi Marg, Lucknow 226001, India. Fax: +91 522-2228227. E-mail: krishnag522{at}yahoo.co.in
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Del Tito, B. J., Jr., Mukhtar, H., and Bickers, D. R. (1983). Inhibition of epidermal metabolism and DNA binding of benzo[a]pyrene by ellagic acid. Biochem. Biophys. Res. Commun. 114, 388394.[ISI][Medline]
Bickers, D. R., Dutta, C. T., and Mukhtar, H. (1982). Epidermis: A site for drug metabolism in neonatal rat skin. Studies on cytochrome P-450 content and mixed function oxidase and epoxide hydrolase activity. Mol. Pharmacol. 21, 239247.[Abstract]
Boberg, E. W., Liem, A., Miller, E. C., and Miller, J. A. (1987). Inhibition by pentachlorophenol of the initiating and promoting activities of 1 hydroxysafrole for the formation of enzyme altered foci and tumors in rat liver. Carcinogenesis 8, 537539.
Brookes, P., and Osborne, M. R. (1982). Mutation in mammalian cells by sterioisomers of the antibenzo[a]pyrene diol epoxide in relation to the extent and nature of the DNA reaction products. Carcinogenesis 3, 12231226.[ISI][Medline]
Cavalieri, E. L., and Rogan, E. G. (1998). Central role of radical cation in metabolic activation of polycyclic aromatic hydrocarbon. Xenobiotica 25, 677688.
Diamond, L., Defendi, V., and Brookes, P. (1967). The interaction of 7,12 dimethyl benz(a)anthracene with cells sensitive and resistant to toxicity induced by this carcinogen. Cancer Res. 27, 890897.[ISI][Medline]
Di Giovanni, J., and Slaga, T. J. (1981). Modification of polycyclic aromatic hydrocarbon carcinogenesis. In Polycyclic Hydrocarbons and Cancer (H. V. Gelboin and Tso, POP, Eds.). Academic Press, New York. 3, 259292.
Dwivedi, C., Downie, A., and Webb, T. E. (1987). Net glucuronidation in different rat strains: Importance of microsomal ß glucuronidase. FASEB J. 4, 303307.
Dwivedi, C., Heck, J. W., Downie, A., Laroya, S., and Webb, T. E. (1990). Effect of calcium glucarate on ß glucuronidase activity and glucarate content of vegetables and fruits. Biochem. Med. Metab. Biol. 43, 8393.[CrossRef][ISI][Medline]
Eastman, A., Sweetenham, J., and Bresnick, E. (1978). Comparison of in vivo and in vitro binding of polycyclic hydrocarbons to DNA. Chem. Biol. Interact. 23, 345353.[CrossRef][ISI][Medline]
Gupta, K. P., and Mehrotra, N. K. (1997). Differential effects of butyric acid on mouse skin tumorigenesis. Biomed. Environ. Sci. 10, 436441.[Medline]
Hannigam, B. M., Barnett, Y. A., Armstrong, D. B., Mckelvey-Martin, V. J., and McKenna, P. G. (1993). Thymidine kinase: The enzymes and their clinical usefulness. Cancer Biother. 8, 189197.[ISI][Medline]
Hu, X., Benson, P. J., Srivastava, A., Xia, H., Bleicher, R. J., Zaren, H. A., Awasthi, S., and Gelboin, H. V. (1980). Benzopyrene metabolism, activation, and carcinogenesis: Role of mixed function oxidases and related enzymes. Physiol. Rev. 60, 11071166.
Kakefuda, T., and Yamamoto, H. A. (1978). Modification of DNA by the benzo[a]pyrene metabolite diol epoxide r-7, t-8-dihydroxy-t-9-7, 8,9,10-tetrahydrobenzo[a]pyrene. Proc. Natl. Acad. Sci. USA 75, 415417.[Abstract]
Kates, J., and Beeson, J. (1970). Ribonucleic acid synthesis in vaccinia virus. I. The mechanism of synthesis and release of RNA in vaccinia cores. J. Mol. Biol. 50, 118.[ISI][Medline]
Lesca, P. (1982). Influence of the rate of 7,12-dimethyl benz anthracene metabolic activation, in vitro, on its binding to epidermal DNA and skin carcinogenesis. Carcinogenesis 2, 199204.[ISI]
Lowry, O. W., Rosebrough, M. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement by folin phenol reagent. J. Biol. Chem. 193, 265275.
Nebert, D. W., and Gelboin, H. V. (1968). Substrate inducible microsomal aryl hydrocarbon hydroxylase in mammalian cell culture. Assay and properties of induced enzymes. J. Biol. Chem. 243, 62426249.
Newbold, R. F., and Brookes, P. (1976). Exceptional mutagenecity of a benzo[a]pyrene diol epoxide in cultured mammalian cells. Nature 261, 5254.[Medline]
Phillips, D. H., and Sims, P. (1979). Polycyclic aromatic hydrocarbon metabolites: Their reactions with nucleic acids. In Chemical Carcinogens and DNA (P. L. Grover, Ed.), Vol. 2, pp. 2957. CRC Press, Boca Raton, FL.
Sakamoto, S., Hirai, H., Taga, H., Uchikoshi, T., Sunaga, T., Endo, Y., Kuwa, K., Kudo, H., Kawachi, Y., Kasahara, N., et al. (1997). Thymidine kinase and -fetoprotein as biochemical markers of hepatocarcinogenesis induced by 3' methyl-4-dimethylaminobenzene treatment in rats. Carcinogenesis 11, 145150.
Saul, K. (1976). Thymidine kinase, DNA synthesis, and cancer. Mol. Cell. Biochem. 11, 161181.[ISI][Medline]
Singh, J., and Gupta, K. P. (2003). Calcium glucarate protects tumor formation in mouse skin. Biomed. Environ. Sci. 16, 916.[CrossRef][ISI][Medline]
Stoner, G. D., Schut, H. A., Daniel, F. B., and Dixit, R. A. (1986). Comparison of covalent DNA binding of benzo[a]pyrene and 7,12-dimethylbenzanthracene in respiratory tissues from human, rat and mouse. Cancer Lett. 30, 231241.[CrossRef][ISI][Medline]
Walaszek, Z., Hanausek-Walaszek, M., Minton, J. P., and Webb, T. E. (1987). Dietary Cag as antipromoter of 7,12-dimethyl benz anthracene induced mammary tumorigenesis. Carcinogenesis 7, 14631466.[ISI]
Walaszek, Z., Szemraz, J., Hanausek, M., Adams, A. K., and Sherman, V. D. (1996). D-glucaric acid content of different fruits and vegetables and cholesterol lowering effects of dietary D-glucarate in the rats. Nutr. Res. 16, 673681.[CrossRef][ISI]
|