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Adsorption of mutagens to chlorophyllin-chitosan, an insoluble form of chlorophyllin.

Chlorophyllin, a water soluble derivative of chlorophyll is known to suppress the mutagenic and carcinogenic actions of compounds having polycyclic structures, e.g. heterocyclic amines and aflatoxin B1. There is evidence that this suppressing effect arises, at least in part, by a complex formation between the porphyrin-like structure of chlorophyllin and the planar molecular surfaces of these compounds. We report here that chlorophyllin can form an insoluble salt-like material when mixed with chitosan, a polyglucosamine, and that the solid chlorophyllin-chitosan thus prepared can efficiently trap polycyclic mutagenic compounds. The adsorbed polycyclic mutagens were elutable with buffers of acidic pH, but only to small extents. Chlorophyllin-chitosan may be expected to be useful as an intercepting agent against polycyclic mutagens and carcinogens.

Adsorption↗

Chemopreventive properties of chlorophylls towards aflatoxin B1: a review of the antimutagenicity and anticarcinogenicity data in rainbow trout.

The anticarcinogenic activity of chlorophyllin (CHL), a water-soluble derivative of chlorophyll, was first reported in rainbow trout. This review describes certain experiments which set the stage for long-term tumor bioassays, in trout and other species, using CHL and various food-borne carcinogens. Initial work with trout and rat liver enzymes in the Salmonella assay showed that CHL was a potent antimutagen towards heterocyclic amines, polycyclic aromatic hydrocarbons, aflatoxins and other classes of mutagen. Antimutagenic activity was further demonstrated using the corresponding direct-acting mutagens in the absence of an exogenous metabolizing system. Mutagen-inhibitor interaction (molecular complex formation) was identified in spectrophotometry studies, suggesting that CHL acts as an 'interceptor molecule'. In vivo, CHL reduced hepatic AFB1-DNA adducts and hepatocarcinogenesis when the inhibitor and carcinogen were co-administered in the diet. Finally, co-injection of inhibitor and AFB1 into trout embryos established that CHL was more effective than chlorophyll a in reducing AFB1-DNA adducts 2 weeks after injection, and liver tumors after 1 year.

Aflatoxin B1↗

Iron-chlorophyllin-mediated conversion of 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2(NHOH)) into its nitroso derivative.

Early work from our laboratory has shown that the mutagenicity of heterocyclic amines in Salmonella can be inhibited by hemin and chlorophyllins. We have speculated that the inhibition is a result of complex formation between heterocyclic amines and the pigments, and the speculation has been given a line of experimental evidence. We have now found that ferric-chlorophyllin (Fe-chlorophyllin) can modify the mutagenicity of 3-hydroxyamino-1-methyl-5H-pyrido[4, 3-b]indole (Trp-P-2(NHOH)), a metabolically activated form of 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2). The mutagenicity of Trp-P-2(NHOH) in Salmonella typhimurium TA 98 (without S9) was strongly inhibited by an addition of an equimolar Fe-chlorophyllin in the pre-incubation mixture. Fe-chlorophyllin also inhibited the mutagenicity of 2-hydroxyamino-6-methyldipyrido[1,2-a:3',2'-d] imidazole (Glu-P-1(NHOH)). A rapid change in the UV spectrum of a mixture of Trp-P-2(NHOH) and Fe-chlorophyllin was observed. Analysis by high performance liquid chromatography showed that Trp-P-2(NHOH) was converted into 3-nitroso-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2(NO)), the mutagenic potency of which is a quarter of that of Trp-P-2(NHOH). Furthermore, the mutagenicity of Trp-P-2(NO), in turn, was inhibited by Fe-chlorophyllin. We conclude that the suppression of the mutagenicity of Trp-P-2(NHOH) is ascribable to the oxidative function of Fe-chlorophyllin, coupled with its ability to form complex formation with the planar surface of the heterocyclic amine molecules.

Aerobiosis↗

Inhibition of radiation-induced DNA damage in plasmid pBR322 by chlorophyllin and possible mechanism(s) of action.

Naturally occurring compounds capable of protecting DNA against ionizing radiation and chemical mutagens have considerable potential for prevention of mutation-based health impairment including cancer and other degenerative diseases. Chlorophyllin (CHL), a water-soluble derivative of chlorophyll, has been examined for its ability to protect DNA against radiation induced strand breaks using an in vitro plasmid DNA system. Gamma-radiation, up to a dose of 6 Gy (dose rate 1.25 Gy/min), induced a dose-dependent increase in single-strand breaks (ssbs) in plasmid pBR322 DNA. CHL per se did not induce, but inhibited radiation-induced ssbs in a concentration-dependent manner; 500 microM giving about 90% protection. The protection afforded by CHL was comparatively less than that of trolox, a water-soluble analogue of alpha-tocopherol. To elucidate the underlying mechanism(s), reaction of CHL with the radiation-derived hydroxyl radical (.OH) and deoxyribose peroxyl radical (ROO.) was studied by pulse radiolysis. CHL exhibited a rate constant of 6.1+/-0.4x109 M-1 s-1 with.OH and 5.0+/-1.3x107 M-1 s-1 with ROO. To our knowledge, this is the first report providing direct evidence of free radical-scavenging properties of CHL. The results showed that CHL, effectively protects plasmid DNA against ionizing radiation, in an in vitro system independent of DNA repair or other cellular defense mechanisms. The ability of CHL to scavenge. OH and ROO., may contribute to its protective effects against radiation induced DNA damage in the pBR322 system.

Antimutagenic Agents↗

Chlorophyll and chromosome breakage.

Increased consumption of green vegetables in the diet has been associated with protection against carcinogenic effects and related mutagenic and clastogenic (chromosome breaking) activity of genotoxic agents. Chlorophyll, present in all green plant parts, has been suggested to be a major protective factor in the process. We have, however, observed that while a crude aqueous extract of Indian spinach leaf significantly reduced genotoxic effects, chlorophyll alone was ineffective. On the other hand, chlorophyll, both as an aqueous extract from the leaf and in a purified commercial form, induced a significantly high frequency of chromosome breaks in bone marrow cells of mice on oral administration. The crude aqueous extract of the leaf was non-toxic. The protective activity of the crude leaf extract may be attributed to the total effect of the interaction between different components, in which the clastogenicity of chlorophyll has been neutralized.

Administration, Oral↗

Suppressive effects of chlorophyllin on mutagen-induced umu C gene expression in Salmonella typhimurium (TA 1535/pSK 1002) and tumor promoter-dependent ornithine decarboxylase induction in BALB/c 3T3 fibroblast cells.

The potentially protective role of chlorophyllin, the sodium and copper salt of chlorophyll a against the initiation and promotion stages in carcinogenesis was studied by in vitro short-term assays. Chlorophyllin showed a dose-dependent suppressive effect on 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indol (Trp-P-1)-induced umu C gene expression of Salmonella typhimurium (TA 1535/pSK 1002) in the presence of metabolizing enzyme mixture. The similar inhibitory effect of chlorophyllin was detected in mitomycin C (MMC)-dependent umu C gene expression in the absence of metabolizing enzyme mixture. Furthermore chlorophyllin also exhibited a dose-dependent inhibition on 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced ornithine decarboxylase (ODC) activity of 3T3 fibroblast cells at the same concentrations. However, when chlorophyll a isolated from Japanese tea leaves was applied on the same assay systems as a comparative experiment, chlorophyll a showed much weaker activity compared with that of chlorophyllin. The significance of this finding is discussed from the viewpoint of the protective role of chlorophyllin against carcinogenesis.

3T3 Cells↗

Effects of chlorophyllin on acetaldehyde: lack of modulation of the rate of sister-chromatid exchanges in mouse bone marrow, and of complex formation in aqueous solution.

Acetaldehyde (Ace) is a reactive compound widely found in natural and industrialized products. On the other hand, chlorophyllin (Chl) is a chloropyll derivative which has shown DNA modulatory effects in several models. The first aim of the present study was to determine the capacity of Ace to increase the rate of sister-chromatid exchanges (SCEs) in mouse bone marrow cells in vivo, as well as to determine its capacity to modify the mitotic index (MI) and the average generation time (AGT). For this experiment we tested four dosages of Ace by the i.p. route (0.4, 4.0, 40.0 and 400 mg/kg), and found a genotoxic effect with the two highest dosages (more than double the basal level was observed with 400 mg/kg). We also found that none of the doses tested modified the MI or the AGT. A second objective was to explore the potential of Chl to modulate the genotoxicity of Ace in the same model. We evaluated whether an oral administration of Chl (2.0, 6.0 and 10.0 mg/kg), given 1 h before an i.p. administration of Ace (100 mg/kg), could modulate the SCEs produced by the mutagen. The result showed a similar SCE rate in both, the Ace-treated mice and those administered with the two chemicals, indicating that Chl was not a modulatory chemical on the genotoxicity of Ace. No modifications were observed concerning the MI or the AGT either. A third objective was to determine whether the two compounds (Ace and Chl) may form a molecular complex in aqueous solution. In agreement with the lack of modulatory effect by Chl, a reversed HPLC and a spectrophotometric analysis showed that the two compounds were unable to form a complex. This report confirms the importance of the specificity concerning the interaction mutagen/antimutagen.

Acetaldehyde↗

Chlorophyllin as a protector of mitochondrial membranes against gamma-radiation and photosensitization.

Ionizing radiation and photosensitization are highly damaging events and they generate oxygen-derived free radicals as well as excited species. However, the types as well as extent of reactive oxygen species (ROS) differ. They have been linked to various pathological conditions. Hence natural compounds capable of preventing oxidative damage induced by these agents may have potential applications. Chlorophyllin (CHL), the water-soluble analogue of chlorophyll, has been examined for its ability to inhibit membrane damage induced by y-radiation and photosensitization involving methylene blue plus visible light. Using rat liver mitochondria as model systems the mechanisms of damage induced by these two agents as well as its possible prevention by CHL have been examined. The parameters used were lipid peroxidation as assessed by formation of thiobarbituric acid reactive substances (TBARS) and 4-hydroxynonenal (4-HNE), protein oxidation besides glutathione (GSH) and superoxide dismutase (SOD). Peroxidation increases with radiation dose, in the range of 75-600 Gy. A similar observation also was observed with photosensitization, as a function of time. CHL, at a concentration of 10 microM offered a high degree of protection against radiation and photosensitization as indicated by decreased peroxidation, protein oxidation as well as the restoration of GSH and SOD. When compared with the established antioxidants, ascorbic acid and GSH, CHL offered a much higher degree of protection. Pulse radiolysis studies show that this compound has a relatively high rate constant with hydroxyl radical (*OH), a crucial species generated during y-radiation. Hence the studies show that CHL is a potent antioxidant in mitochondrial membranes.

Aldehydes↗

Interactions of chlorophyllin with acridine orange, quinacrine mustard and doxorubicin analyzed by light absorption and fluorescence spectroscopy.

The present study was designed to estimate the ability of chlorophyllin (CHL) to interact with two acridine mutagens, quinacrine mustard (QM) and acridine orange (AO), and with the antitumor anthracycline doxorubicin (Dox). To this end, aqueous solutions of QM, AO or Dox during titration with CHL were subjected to spectrophotometry and spectrofluorimetry to detect possible interactions between these reagents. The data indicate that CHL forms complexes with AO, QM or Dox in these solutions. The presence of the complexes was manifested by a bathochromic shift of the absorption spectra, as well as by strong quenching of the fluorescence of each of these mutagens in the presence of CHL. CHL, thus, may serve as an interceptor of these mutagenic acridines in different in vivo or in vitro applications. Its ability to interact with Dox may potentially be utilized to detoxify patients overdosed with this or similar drugs.

Acridine Orange↗

Effects of n-tritriacontane-16,18-dione, curcumin, chlorphyllin, dihydroguaiaretic acid, tannic acid and phytic acid on the initiation stage in a rat multi-organ carcinogenesis model.

The modifying effects of the naturally occurring antioxidants n-tritriacontane-16,18-dione (TTAD), curcumin, dihydroguaiaretic acid (DHGA), chlorophyllin, tannic acid and phytic acid on the initiation stage in a rat multi-organ carcinogenesis model were examined in male F344 rats. Animals were initiated with two i.p. injections of 2,2'-dihydroxy-di-n-propylnitrosamine (DHPN), followed by two i.g. administrations of N-ethyl-N-hydroxyethylnitrosamine (EHEN), and then three s.c. injections of 3,2'-methyl-4-aminobiphenyl (DMAB) during the first 3 weeks. Starting 1 day before the first carcinogen application, groups of rats received diet containing one of the antioxidants (0.2% TTAD, the others at 1% each) until 1 week after the last carcinogen exposure. Surviving animals were killed and complete autopsies were performed at the end of week 36. Histological examination revealed no inhibitory effects in terms of the multiplicities and/or incidences of neoplastic lesions in any of the organs examined, other than a significant increase in seminal vesicle atypical hyperplasia observed in rats treated with tannic acid. Thus, the antioxidants, with the exception of tannic acid, did not show any modifying effects on the initiation stage in the present multi-organ carcinogenesis model and at the present dose levels applied.

Aminobiphenyl Compounds↗

Antimutagenicity, cytotoxicity and composition of chlorophyllin copper complex.

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.

Animals↗

Chemoprevention studies of heterocyclic amine-induced colon carcinogenesis.

The cooking of meat and fish produces heterocyclic amine mutagens, including 2-amino-1-methyl-6-phenylimidazo[4,5b]pyridine (PhIP) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). Chronic administration of PhIP or IQ to the F344 rat induces tumors at several sites, including adenocarcinomas of the colon, and short-term treatment leads to the formation of colonic aberrant crypt foci (ACF). We have used these end-points to identify potential chemopreventive agents that might be effective against heterocyclic amine colon carcinogens. Typically, IQ or PhIP were administered to groups of 10-15 rats by oral gavage on alternating days in weeks 3 and 4, and ACF were scored after 8, 12, or 16 weeks or tumors were detected at 52 weeks. To distinguish between 'blocking' and 'suppressing' agents, potential inhibitors were administered during the initiation or post-initiation phases, respectively, and subsequent studies focused on the inhibitory mechanisms. Among the most effective inhibitors identified to date, and their major mechanisms, were the following: chlorophyllin (molecular complex formation); indole-3-carbinol (inhibition and induction of cytochromes P450 and phase II enzymes); green and black tea catechins (induction of UDP-glucuronosyl transferase, inhibition of NADPH-cytochrome P450 reductase, scavenging of reactive intermediates); and conjugated linoleic acids (inhibition of cytochrome P450 and prostaglandin H synthase).

Animals↗

Inhibitory effects of chlorophyllin on 7,12-dimethylbenz[a]anthracene-induced bacterial mutagenesis and mouse skin carcinogenesis.

Chlorophyllin (CHL), a water-soluble derivative of chlorophyll, has been used for the treatment of several abnormal human conditions without apparent toxicity. Recent studies have revealed that CHL has the excellent chemopreventive potential. In the present investigation, we have found the inhibitory activities of CHL against 7,12-dimethylbenz[a]anthracene (DMBA)-induced mutagenesis in Salmonella typhimurium TA100 and also on DMBA-initiated and 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-promoted mouse skin tumor formation. The incidence and the multiplicity of skin tumors were not significantly decreased in mice by a single topical application of CHL prior to the DMBA treatment, but there was a marked suppression of papillomagenesis in mice treated with CHL during the promotional stage. Furthermore, the formation of DMBA-induced papillomagenesis was reduced in all mice that had received CHL for 6 weeks following treatment with TPA for 6, 18 and 24 weeks. These results indicate that CHL can inhibit both tumor promotion and the progression of papillomagenesis in the two-stage mouse skin carcinogenesis induced by DMBA and TPA.

9,10-Dimethyl-1,2-benzanthracene↗

Photodynamic treatment has chondroprotective effects on articular cartilage.

Osteoarthritis (OA) is a disabling joint disease for which there is currently no cure. It is characterized by the destruction of articular cartilage. One strategy that is being explored for protecting the cartilage in OA is the administration of transforming growth factor-beta, which in vitro antagonizes cartilage degradation initiated by catabolic stimulants such as interleukin-1 (IL-1). The problems associated with selective delivery of the growth factor to chondrocytes, undesirable side-effects on joint tissues, and short biological half-life have led us to explore modalities aimed at activating transforming growth factor-beta that is stored in the cartilage as latent complexes. Photodynamic therapy is a two-step protocol of tissue sensitization with a light-activatable chemical called a photosensitizer followed at some interval by irradiation with the appropriate wavelength visible light. Biological effects are typically elicited through oxygen-dependent photochemistry without heat generation. Transforming growth factor-beta1 can be activated by oxidative mechanism(s), prompting us to explore whether photodynamic technology can be harnessed to modulate cartilage metabolism. Disks of bovine articular cartilage were photosensitized by incubation with a chlorin(e6)-succinylated polylysine conjugate and irradiated with 1-2 J/cm2 red light (lambdamax = 671 nm). This two-step regimen dramatically inhibited IL-1-stimulated proteoglycan degradation and concomitantly increased latent and active transforming growth factor-beta1 in culture medium. This research may lead to the development of minimally invasive photodynamic therapy in which light is delivered to locally activate a chondroprotective program in photosensitized cartilage in the context of OA.

Animals↗

Antitumor agents. Part 209: Pheophorbide-a derivatives as photo-Independent cytotoxic agents.

A methanolic crude extract of the plant Garuga pinnata Roxb. (Burseraceae) showed promising cytotoxic activity against a panel of human tumor cell lines in vitro, including KB and its drug-resistant sublines (Ferguson et al. Cancer Res. 1988, 48, 5956). Pheophorbide-a and-b methyl esters (3,4) were isolated as active principles with broad photo-dependent cytotoxic activities in the micromolar range. These findings prompted SAR studies of known and novel pheophorbide-a derivatives as photo-dependent and photo-independent cytotoxic agents. The results showed that zinc-protoporphyrin IX (10), zinc 13(R)-hydroxypheophorbide-a methyl ester (22), and zinc chlorin-e6 trimethyl ester (13) possessed photo-independent cytotoxic activity. Compounds 13 and 22 were the most active cytotoxic agents of the series (mean ED(50) 4.6 +/- 1.0 microM and 5.7 +/- 0.7 microM, respectively) against KB cells incubated in the dark.

Antineoplastic Agents↗