Cancer Letters 120 (1997) 141–147
Antimutagenicity, cytotoxicity and composition of chlorophyllin copper complex Simon Chernomorsky a ,*, Raymond Rancourt a, Kamalpreet Virdi a, Alvin Segelman b,1, Ronald D. Poretz a b
a Departments of Biochemistry and Microbiology, Rutgers University, PO Box 231, New Brunswick, NJ 08903-0231, USA Departments of Chemical Biology and Pharmacognosy, Rutgers University, PO Box 231, New Brunswick, NJ 08903-0231, USA
Received 14 May 1997; received in revised form 5 June 1997; accepted 5 June 1997
Abstract The preparation of chlorophyllin copper complex (CCC), shown to be a tumor promoter in an animal model (Nelson, R.L. (1992) Chlorophyllin, an antimutagen, acts as a tumor promoter in the rat-dimethylhydrazine colon carcinogenesis model. Anticancer Res., 12, 737–740), also inhibits the activities of direct- and indirect-acting mutagens in the Salmonella assay and exhibits cytostatic and cytocidal effects toward myeloma cells. Data from elemental analyses, spectrophotometry and reversed-phase high-performance liquid chromatography indicate that CCC preparations generally used in antimutagenic/ anticarcinogenic experiments are variable, complex mixtures of structurally distinct porphyrins lacking copper in some instances. This variability of the composition may be a cause for the differences reported for the tumor promotion activity of CCC. 1997 Elsevier Science Ireland Ltd. Keywords: Chlorophyllin; N-methyl-N′-nitro-N-nitrosoguanidine; 3-Methylcholanthrene; Antimutagenicity; Cytotoxicity; Composition
1. Introduction Many studies have demonstrated chlorophyllin copper complex (CCC), a commercial derivative of chlorophyll, to be a potent inhibitor of the mutagenic activities exhibited by a large number of chemicals and complex mixtures (see for example Refs. [8,14,22,25,33]). Furthermore, CCC has been * Corresponding author. Tel.: +1 732 9329763, ext. 122; fax: +1 732 9328965. 1 Present address: Nature’s Sunshine Products, Inc., 75 E. 1700 South, Provo, UT 84605, USA.
shown to have anticarcinogenic properties in experimental animal models [10,12,23,24,28,29]. Surprisingly, CCC was found to be a tumor promoter for a dim-ethylhydrazine-induced colorectal cancer in rats [21]. This report linking tumor induction to postinitiation activity of CCC is contradicted by the studies employing rainbow trout exposed to aflatoxin B1 [3] and rats given 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine [11] prior to the CCC treatment. To fully appreciate the heterogeneity and variability of individual CCC preparations obtained from Sigma (St. Louis, MO), the main source for CCC
0304-3835/97/$17.00 1997 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3835 (97 )0 0304-2
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used in the mutagenicity/carcinogenicity tests, we undertook an investigation into the composition of four such preparations. One of these preparations shown to promote tumors [21] was studied also as a potential antimutagen and tumoricidal agent.
CCC only to evaluate their mutagenic and toxic properties. All treatments were performed in triplicate. After 48 h, the plates were inspected for uniform background lawns and the number of his+ revertant colonies, corrected for the spontaneous mutants, was determined.
2. Materials and methods
2.3. Cytotoxicity assay
2.1. Chemicals
Stock cultures of tumor cells (mouse myeloma line P3 × 63Ag8) were routinely grown in Eagle’s minimum essential medium with Hank’s salts supplemented with 10% fetal calf serum, 2 mM glutamine, 100 mg/ml streptomycin and 100 units/ml penicillin at 37°C in a humidified 95% air/5% CO2 atmosphere and were subcultured twice weekly. The stock cultures were diluted with fresh culture medium to afford cell suspensions which contained 3 × 105 cells/ml. Cell suspension (2 ml, 3 × 105 cells/ml) with or without CCC (lot 17F0307) were allowed to incubate as unstirred cultures for 48 h in sterile plastic loosely capped tubes (16 × 125 mm). After incubation, percent cell viability was calculated by comparing the number of viable cells with the number of total cells in the culture. Cell viability was based on Trypan Blue dye exclusion. Cell multiplicity was calculated by comparing the total number of viable cells present after incubation with the test agent to the total number of viable cells in control cultures. Each data point is an average of three tubes.
The chlorophyll derivatives, isochlorin e4, chlorin e6 and pheophorbide a, obtained from Porphyrin Products (Logan, UT), were employed for the preparation of authentic standard Cu complexes as described in Ref. [15]. The CCC preparation (lot 17F0307), found to be a tumor promoter [21], other CCC preparations, N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and 3-methylcholanthrene (3-MC) were purchased from Sigma (St. Louis, MO). Cell culture media, analytical grade reagents for reversed-phase high-performance liquid chromatography (RP-HPLC) and all other chemicals were obtained from Fisher Scientific (Pittsburgh, PA). 2.2. Mutagenicity assay Salmonella typhimurium strain TA100 was employed with or without activation by liver microsomes (S9) isolated from Aroclor 1254-pretreated Wistar rats [18]. The S. typhimurium cells were diluted 1:10 with sterile phosphate buffer (pH 7.4) and mixed in tubes with various concentrations of CCC (lot 17F0307) in sterile distilled water and appropriate mutagen. The direct-acting mutagen MNNG (5 mg/plate) was used as an aqueous solution, whereas the indirect-acting mutagen 3-MC (50 mg/ plate) was dissolved in dimethylsulfoxide (DMSO). In experiments involving the indirect-acting mutagen, S9 and glucose-6-phosphate dehydrogenase were also introduced. The tubes were placed in a 37°C water bath for 20 min, mixed with melted top layer agar and evenly distributed on previously prepared plates of minimal glucose agar. Following solidification of the agar, the plates were inverted and placed in a 37°C incubator. Controls included plates with DMSO or water lacking mutagen and CCC to determine spontaneous mutation and plates containing mutagen or
2.4. Analytical procedures Copper content in CCC was determined by the supplier and the nitrogen content of the preparations was determined by the Kjeldahl method [32]. The purity of the preparations and concentrations used in the biological experiments were estimated based on their Cu content. The actual percent weight of Cu in the preparations was compared with the value expected for fully coppered chlorophyll-derived components [5]. To evaluate the coordination of these compounds with Cu, the Cu/N index calculated as a ratio of these elements [4] along with spectrophotometric data were employed. Absorption spectra of CCC preparations were obtained and analyzed using a computer controlled Perkin-Elmer Lambda 3B UV-VIS spectrophotometer.
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2.5. Chromatographic analysis RP-HPLC was performed with a Waters system containing two model 510 pumps, a Rheodyne model 7125 sample injector with a 20 ml injector loop and the Millennium 2010 Chromatography Manager software run on an NEC image 466es model computer. A reverse-phase column (3.9 × 150 mm ID) packed with C18-bonded silica was used. The preparations were dissolved in acetone, filtered and applied to the column. The mobile phase was a linear gradient from methanol/1 M ammonium acetate (80:20 v/v) to methanol/acetone (80:20 v/v), similar to that described in Ref. [36]. The flow-rate was 1 ml/ min. Spectra of individual chlorophyll-derived components of the CCC preparations were obtained in the range of 400–700 nm with a photodiode array detector (Waters model 991) incorporated in the RP-HPLC system.
3. Results and discussion 3.1. Antimutagenic activity In the control studies, CCC (lot 17F0307) itself does not exhibit any mutagenic or toxic effects on
Fig. 2. Effect of CCC on the growth of murine tumor cells. Myeloma cells were incubated in medium containing different concentrations of CCC for 48 h. After incubation the cells were counted and evaluated for viability.
the S. typhimurium when added in concentrations used in experiments with MNNG and 3-MC (data not shown). When mixed with either compound, however, CCC greatly reduces or completely eliminates their mutagenic properties (Fig. 1). The effect of CCC is dose-dependent. It follows a normal sigmoid doseresponse profile, though the inhibitory activity of the sample is approximately 10-times greater when analyzed against 3-MC as compared to MNNG. Thus, 50% inhibition of the indirect-acting mutagen requires a CCC concentration of 0.22 mM as opposed to 2.4 mM for the direct-acting mutagen. These results may be indicative of potential differences in the mechanisms by which CCC operates regarding direct- and indirect-acting mutagens. However, the variance in activity is difficult to attribute to a single cause, since in addition to the specific mode of action the concentration and potency of the two mutagens employed differed. 3.2. Toxicity toward tumor cells in vitro
Fig. 1. Antimutagenic activity of CCC against 3-MC and MNNG in Salmonella typhimurium strain TA100. The method [18] was employed using 50 mg/plate of 3-MC or 5 mg/plate of MNNG. Various concentrations of CCC were added to the melted agar.
The effect of various concentrations of CCC (lot 17F0307) on murine myeloma cells is presented in Fig. 2. The dose-response curves for the activity of CCC for cell multiplicity and cell viability, with respect to control cultures not exposed to CCC, are
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depicted. The shapes of the curves are noticeably different. Relatively low concentrations of CCC appear to be predominantly cytostatic to the tumor cells, though at higher concentrations CCC is definitely cytocidal. That is, at 50 mM CCC the relative total number of viable cells (multiplicity) is reduced by 53% with virtually no loss of relative cellular viability. However, the higher concentration of CCC (300 mM) results in 100% loss of cell viability. 3.3. Analytical studies The results of elemental and spectrophotometric analyses are shown in Table 1. The approximate purity of all preparations studied, calculated on the basis of the Cu content, yielded values between 23 and 60%. Apparently, these preparations either contain uncoppered porphyrins and/or non-porphyrin substances at levels as high as 77% of the total weight. The two CCC preparations examined have Cu/N indices of 0.7, as opposed to a value of 1.1 calculated for completely coppered compounds [3]. Such a difference indicates that approximately 33% of chlorophyll-derived constituents in CCC are lacking the metal and/or that these samples contain significant quantities of non-chlorophyll nitrogenous substances. Although the positions of absorption maxima (Soret and Q bands) for the aqueous solutions of all the CCC preparations studied are similar, the ratio of absorbances at the maxima varies from 3.7 to 4.6. Variability of these values may be taken as additional evidence that some of the chlorophyll-derived constituents in CCC are Cu-free. The latter compounds have higher ratios than those for the Cu-containing counterparts [27].
3.4. Individual chlorophyll-derived constituents RP-HPLC chromatograms of the four CCC preparations supplied by Sigma are shown in Fig. 3. They demonstrate that preparations are variable in composition and include many Soret band (405 nm) absorbing components. The retention times for the peaks representing the majority of the total absorptivity in the blue region do not exceeded 15 min. By the comparison of the spectral properties and retention times with those of authentic samples, peaks 2 and 5 are assigned to Cu complexes of chlorin e6 and isochlorin e4, respectively. No chlorophyll derivatives in the CCC preparations studied exhibit properties similar to those of authentic Cu complexed pheophorbide a. In general, these results are in agreement with the data reported in Refs. [15,34]. Chlorophyll-derived components corresponding to peaks 1, 3, 4 and 6 were not specifically identified. However, the spectral characteristics of peaks 1, 3 and 4 indicate that they are Cu complexes of chlorins, while those for peak 6 suggest that it is a non-chlorin type of porphyrin (data not shown). The present study demonstrates that the same CCC preparation (lot 17F0307), found to be a tumor promoter when introduced after carcinogen treatment [21], inhibits direct- and indirect-acting mutagens. These findings on the antimutagenic activity of CCC are consistent with the results obtained by others using various CCC preparations and a broad range of mutagens. We also report here that in experiments with myeloma cells, the same preparation of CCC which acts as a tumor promoter and antimutagen also exhibits cytostatic and cytocidal effects toward myeloma cells depending upon the concentrations used. Both
Table 1 Analytical characteristics of some commercial CCC preparations Sample lot number
35F0890 17F0307 125H0624 74H0067
Percent Cu
N
2.1 2.4 4.5 5.5
2.9 3.3 ND ND
Percent purity
Ratio Cu/N
Soret band (nm)a
Q band (nm)a
Soret/ Q ratiob
23 26 49 60
0.7 0.7 – –
404 404 405 404
627 628 627 628
4.6 4.6 3.7 3.7
ND, not determined. Wavelength of maximum absorption, nm. b Ratio of the absorbances at the maxima of the Soret and Q bands. a
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antimutagenic and cytotoxic activities have been observed for chlorin e6, a Cu-free counterpart of one chlorophyll-derived component in CCC, when employed individually in the Salmonella assay [2] and in a tissue culture model [7]. The proposed mechanisms for the antimutagenic
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activity of CCC in a number of studies have included deactivation of the mutagens through molecular complexation [1,3,9,20], modulation of the enzymatic transformation of the mutagens [16,22,31,35] and acting as a scavenger for toxic radicals [16,22,26]. With respect to the cytotoxicity exhibited by CCC against myeloma cells, the underlying mechanism remains unclear. Porphyrins are found to preferentially accumulate in tumor cells as compared to normal cells [19]. Depending on the localization, different intracellular structures (mitochondria, lysosomes, membranes and nucleus) can be affected. The ability of CCC or its chlorophyll-derived constituents, in general, to interfere with enzymes has been shown in vitro [17,30,35] and in vivo [13]. This mode of action may be the cause of the cytotoxic effect of CCC toward myeloma cells. While differences in animal models and the carcinogens used may explain a discrepancy in the results regarding the postinitiation effect of CCC on tumor promotion [3,11,21], the variability of the CCC composition also has to be taken into consideration [6]. Results from elemental analysis, spectrophotometry and RP-HPLC indicate that CCC is a complex mixture of structurally distinct porphyrins of chlorin and nonchlorin types with variable numbers of carboxyl groups which lack copper in some instances. Nonchlorophyll-derived components may constitute more than 40% of the total samples examined. Although CCC preparations of unknown characteristics are still employed in a majority of studies, reports have appeared on the antimutagenic/anticarcinogenic activities of individual chlorophyll derivatives [2,30]. Only complete identification, isolation and testing of individual purified components of CCC will allow for the sorting of the possible and proposed modes of action and for the full appreciation of the practical application of CCC in cancer prevention.
References Fig. 3. RP-HPLC chromatograms of the CCC preparations supplied by Sigma. (A) Lot 17F0307; (B) lot 35F0890; (C) lot 125H0624; (D) lot 74H0067. The preparations were dissolved in acetone and subjected to RP-HPLC employing a linear gradient from methanol/ 1 M ammonium acetate (80:20 v/v) to methanol/acetone (80:20 v/ v). Detection was performed with a photodiode array detector in the range of 400–700 nm.
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