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

Krishna P. Gupta1 and Jaya Singh

Environmental Carcinogenesis Division, Industrial Toxicology Research Center, Lucknow, India

Received September 8, 2003; accepted January 27, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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.

Key Words: calcium glucarate; skin; carcinogen metabolism; DNA binding; thymidine kinase.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Polycyclic aromatic hydrocarbons (PAHs) are widely distributed in our environment and are implicated in different types of cancer, including skin cancer (Phillips and Sims, 1979Go). 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, 1976Go). Following topical application of [3H] benzo[a]pyrene (BP) to mouse skin, the major DNA adduct formed was (+)-7ß, 8{alpha} dihydroxy-9{alpha}, 10{alpha}-epoxy-7, 8,9,10-tetrahydrobenzo[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, 1978Go). 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., 1986Go). 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 ß- glucuronidase inhibitor called glucarolactone (Abou-Issa et al., 1993Go; Dwivedi et al., 1987Go, 1990Go). 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., 1987Go) and DMBA-induced skin tumorigenesis (Singh and Gupta, 2003Go), 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., 1978Go). There are ample evidences that altered carcinogen metabolism in experimental animal models can have dramatic effects on tumor incidences (Boberg et al., 1987Go). 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, 1982Go). Inhibition of carcinogen-DNA binding generally accompanied the inhibition of tumor initiation (Lesca, 1982Go), in keeping with the view that most inhibitors probably interfere with the metabolic activation of the tumor initiator (Di Giovanni and Slaga, 1981Go).

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, 1998Go) that binds to cellular macromolecules (Hu et al., 1980Go). 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., 1982Go). 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., 1993Go; Saul, 1976Go). 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
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 


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.

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, 1970Go). 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, 1970Go). 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).


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TABLE 1 Experimental Sets

 
In vitro DNA-carcinogen binding.
Animals were divided into groups consisting of three animals each. For in vitro DNA-binding experiments, animals were treated with a single topical application of DMBA (50 µg /0.1 ml acetone) for 24 h. Cag was applied immediately after the application of DMBA on the same area at a dose of 0.5, 1, 3, 6, or 12 mg. Animals were sacrificed, and isolated epidermis was homogenized in 10 volumes of 0.2 M phosphate buffer, pH 7.4. Microsomes were prepared as previously described (Del Tito et al., 1983Go) and were resuspended in homogenization buffer. The incubation mixture consisted of microsomes (equivalent to 2–3 mg protein), 20 mg DNA, 1 µCi [3H] BP, 7 µmol NADPH, 25 µmol MgCl2, 1 µmol EDTA, 100 µmol glucose-6-phosphate, and 3.3 units of glucose-6-phosphate dehydrogenase in a total volume of 4 ml. Reaction mixtures were incubated at 37°C for 1 h. At the end of incubation, the ionic strength of the reaction mixture was raised to 0.15 M by the addition of 1 M NaCl. Reaction mixtures were then extracted with 3 volumes of water-saturated ethyl acetate. The aqueous layer was then used for DNA extraction following phenol chloroform method (Diamond et al., 1967Go; Kates and Beeson, 1970Go). DNA was dissolved in SSC buffer and aliquots were counted for DNA-bound radioactivity using a liquid scintillation counter.

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., 1997Go). Enzyme activity was measured in 105,000 x g supernatant (Gupta and Mehrotra, 1997Go) 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, 1968Go). 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 (100–200 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., 1951Go).

Statistical analysis.
Data were analyzed statistically using Student's t-test; p < 0.05 was considered to be significant.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

The Effect of a Single Application of Cag on In Vivo Carcinogen-DNA Binding
To assess the effect of Cag on [3H] BP binding, animals were treated with 50 µg DMBA/mouse alone or with Cag (1, 3, 6, or 12 mg/0.1 ml DMSO/mouse) for 24 h. [3H] BP was applied for 2 h. A single topical application of 50 µg DMBA caused a 2.6-fold increase in epidermal [3H] BP binding to DNA compared with the vehicle control group treated with acetone.

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).



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FIG. 1. The dose-dependent effect of Cag on DMBA-induced in vivo binding of [3H] BP to epidermal DNA. DMBA and/or Cag were applied once topically in the dose range of 1 to 12 mg. Each value represents the average of triplicate determinations done twice. Values represent mean ± SE. *p < 0.05 compared with the positive control group.

 
The Effect of Multiple Applications of Cag on In Vivo Carcinogen-DNA Binding
To see the effect of multiple applications of Cag, animals were treated with 5 µg DMBA for 7 days with or without Cag (Table 1). In a second set of these experiments, animals were treated with 5 µg DMBA for 7 days and Cag was applied once 24 h prior to killing. Cag was used at a dose of 0.5, 1, 3, 6, or 12 mg/animal. This allowed us to follow the status of metabolism and DNA binding when a chemopreventive agent was present throughout the exposure period (Table 1), before the damage had taken place, and when a chemopreventive agent was given once the damage had already taken place (Table 1 and Fig. 2).



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FIG. 2. The dose-dependent effect of single or multiple applications of Cag (0.5 to 12 mg) on in vivo binding of [3H] BP to epidermal DNA induced by repeated applications of DMBA (5 µg). In one set of experiments, Cag was given repeatedly along with DMBA; in a second set of experiments, Cag was given once along with the last application of DMBA. Each value represents the average of triplicate determinations done twice. Values represent mean ± SE. *p < 0.05 compared with the positive control group.

 
DMBA (5 µg) caused a 2.0-fold induction of [3H] BP binding to epidermal DNA. When Cag was applied along with DMBA (Table 1, set 1), Cag caused the inhibition of [3H] BP binding to DNA. A 0.5-mg dose of Cag caused a 2.5-fold reduction of DMBA-induced [3H] BP binding to epidermal DNA. Higher doses of Cag caused much more inhibition of [3H] BP binding to epidermal DNA. The inhibition caused by 1-, 3-, 6-, or 12-mg doses of Cag was 2.4-, 3.8-, 4.1-, or 4.8-fold, respectively. In a second set of experiments, DMBA again caused the same 2.0-fold increase in [3H] BP binding to epidermal DNA. Cag applied once on the last day along with DMBA also inhibited the [3H] BP binding to epidermal DNA. Cag at a dose of 0.5, 1, 3, 6, or 12 mg caused 1.7-, 1.9-, 1.9-, 2.7-, or 2.6-fold inhibition of DMBA-induced [3H] BP binding to epidermal DNA. These data indicate that, when Cag was given together with DMBA throughout the exposure period (Table 1, set 1), the lower doses of Cag (e.g., 0.5 mg) exerted potential inhibitory effect on [3H] BP binding to epidermal DNA compared with single applications of the same Cag doses after the carcinogenic DNA damage had occurred (Table 1, set 2). From these results, we infer that DMBA with Cag is more effective in reducing carcinogen-DNA interaction when given throughout the carcinogen exposure period.

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.



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FIG. 3. The dose-dependent effect of Cag on in vitro binding of [3H] BP to calf thymus DNA. DMBA and/or Cag were applied once in the dose range of 0.5 to 12 mg. Microsomes were prepared and the assay was done as described in Materials and Methods. Each value represents the average of triplicate determinations done twice. Values represent mean ± SE. *p < 0.05 compared with the positive control group.

 
The Effect of a Single Application of Cag on Epidermal AHH Activity
To see the effect of Cag on epidermal AHH activity, we treated the mice with DMBA (50 µg/0.1 ml acetone) for 24 h. Immediately after the application of DMBA, Cag was applied at a dose of 0.5, 1, 3, 6, or 12 mg/0.1 ml DMSO in the same area. Control animals received vehicle (0.1 ml acetone/0.1 ml DMSO) only. DMBA (5 µg) caused a 57.7% induction of AHH activity compared with vehicle-treated control. Concomitant application of Cag at a dose of 0.5 mg overcame the inducing effect of DMBA on AHH activity by 50%. Also, a 1-, 3-, 6-, or 12-mg dose of Cag caused a 53, 62, 59, or 60% inhibition, respectively, of DMBA-induced AHH activity. All the doses of Cag inhibited the DMBA-induced AHH activity. The effect of Cag on DMBA-induced AHH activity appeared to depend on the dose of Cag used (Fig. 4).



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FIG. 4. The dose-dependent effect of Cag on DMBA-induced epidermal AHH activity. DMBA and/or different concentrations of Cag were applied once. The enzyme was assayed as described in Materials and Methods. Each value represents the average of triplicate determinations done twice. Values represent mean ± SE. *p < 0.05 compared with the positive control group.

 
The Effect of Multiple Cag Applications on Epidermal AHH Activity
To see the chemopreventive or chemotherapeutic effect of Cag, we assessed AHH activity in animals treated with repeated applications (7 days consecutively) of Cag concomitantly with DMBA or in animals treated with repeated applications of DMBA and a single application of Cag on the seventh day along with DMBA. Two sets of experiments were done. In the first set, animals were treated with DMBA 5 µg/0.1 ml acetone for 7 consecutive days. Cag was applied at a dose of 0.5, 1, 3, 6, or 12 mg/0.1 ml DMSO concomitantly with DMBA in the same area. In the second set, animals were treated with DMBA 5 µg/0.1 ml acetone for 7 consecutive days and Cag was applied once along with DMBA on the last (seventh) day.

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 (1–3 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.



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FIG. 5. The dose-dependent effect of single or multiple applications of Cag on epidermal AHH activity induced by multiple applications of DMBA (5 µg). In one set of experiments, Cag was given repeatedly along with DMBA; in a second set of experiments, Cag was given once along with the last application of DMBA. The enzyme was assayed as described in Materials and Methods. Each value represents the average of triplicate determinations done twice. Values represent mean ± SE. *p < 0.05; **p < 0.2 compared with the positive control group.

 
The In Vitro Effect of Cag on AHH Activity
To see the in vitro effect of Cag on epidermal AHH activity, epidermal microsomes were prepared from vehicle-treated control and DMBA-treated (50 µg/0.1 ml acetone) animals. Cag was added at a concentration of 1, 10, 50, or 100 µM to the reaction mixture for the assay of AHH enzyme activity. AHH activity in epidermal microsomes prepared from DMBA-treated animals was higher compared with control microsomes. DMBA (50 µg) caused a 2.6-fold induction of AHH activity (Fig. 6). The addition of Cag at a concentration of 1, 10, 50, or 100 µM to the reaction mixture reversed DMBA-caused induction of AHH activity. All the doses of Cag were effective in inhibiting AHH activity; a 3.7-, 4.4-, 4.0-, or 5.2-fold inhibition of DMBA-induced AHH activity was observed with Cag at a concentration of 1, 10, 50, or 100 µM, respectively.



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FIG. 6. The dose-dependent effect of in vitro Cag on AHH activity in (A) acetone-treated and (B) DMBA-treated animals. The assay was done as described in Materials and Methods. Cag was added to the reaction mixture in concentrations ranging from 1 to 100 µM. Each value represents the average of triplicate determinations done twice. Values represent mean ± SE. p < 0.05 compared with the positive control group.

 
From these results, we conclude that Cag inhibits [3H] BP binding to DNA in vivo as well as in vitro and suppresses the metabolic conversion of BP to its active metabolites by inhibiting the AHH enzyme.

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).



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FIG. 7. The dose-dependent effect of Cag on DMBA-induced TK activity. Animals were treated topically once with DMBA and/or different doses of Cag for 16 h. The enzyme was assayed as described in Materials and Methods. Each value represents the averages of triplicate determinations done twice. Values represent mean ± SE. *p < 0.05 compared with the positive control group.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cag, a naturally occurring compound present in edibles, suppresses cell growth in different models (Walaszek et al., 1996Go). Most of the studies on the tumor-suppressing effects of Cag have used it as a dietary supplement. Previously, we have shown the effect of topical application of Cag on DMBA-induced tumor development (Singh and Gupta, 2003Go). Here we report the effect of topical application of Cag on DMBA- (a potent environmental carcinogen) caused changes in mouse epidermis.

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., 1983Go). 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, 2003Go). 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
 
We are thankful to Dr. Y. K. Gupta, the Director, Industrial Toxicology Research Center, Lucknow, for providing the facilities to carry out the present study. The Indian Council of Medical Research, (New Delhi, India) provided financial support.


    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
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Abou-Issa, H., Dwivedi, C., Curley, R. W., Kirkpatrick, R., Coolemans-Beynen, A., Engineer, F. N., Humphries, K. A., El-Masry, W., and Webb, T. E. (1993). Basis for the antitumor and chemopreventive activities of glucarate and the glucarate-retinoid combination. Anticancer Res. 13, 395–399.[ISI][Medline]

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, 388–394.[ISI][Medline]

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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, 1223–1226.[ISI][Medline]

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