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December 2009

Antimutagenic potency of chlorophyllin in the Salmonella assay and its correlation with binding constants of mutagen-inhibitor complexes.

Chlorophyllin (CHL) is a water-soluble salt of chlorophyll that exhibits antimutagenic activity in short-term genotoxicity assays and inhibits carcinogen-DNA binding in vivo. The antimutagenic potency of CHL was studied against several structurally related heterocyclic amines using the Salmonella assay. The mutagens included 2-amino-3-methylimidazo[4,5,-f]-quinoline (IQ) and seven related IQ-type compounds, and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) and three additional non-IQ-type compounds. No relationship was observed between mutagenic potency (revertants/ng mutagen) and antimutagenic potency when expressed in terms of the CHL dose/plate-inhibiting mutagenicity by 50 percent (I50). However, a correlation was observed between mutagenic potency and the mole ratio of CHL to mutagen giving 50% inhibition (MR50), with most mutagens requiring several hundredfold to several thousandfold molar excess of CHL for inhibition. In spectrophotometric studies, CHL formed noncovalent molecular complexes with the heterocyclic amines, with binding constants in the range 3-13 x 10(3) M-1. Binding constants were inversely correlated with I50 and MR50 values, i.e., with increasing strength of complex formation less CHL/plate and a lower mole ratio of CHL to mutagen was required to inhibit mutagenicity. The results support an inhibitory mechanism in which chlorophylls operate as “interceptor molecules,” interacting with carcinogens and mutagens directly and limiting their bioavailability.

Environ Mol Mutagen. 1993;22(3):164-71

Chlorophyll and chlorophyllin as modifiers of genotoxic effects.

Reports on an inverse relationship between the consumption of fresh vegetables and human gastrointestinal cancer have been followed by screening for the protective activity of a large number of plant extracts, including leafy vegetables. Chlorophyll is ubiquitous in all green plant parts. Chlorophyllins are derivatives of chlorophyll in which the central magnesium atom is replaced by other metals, such as cobalt, copper or iron. An attempt has been made in this article to review the relative efficacy of chlorophyll and chlorophyllin in modifying the genotoxic effects of various known toxicants.

Mutat Res. 1994 Dec;318(3):239-47

Chlorophyllin intervention reduces aflatoxin-DNA adducts in individuals at high risk for liver cancer.

Residents of Qidong, People’s Republic of China, are at high risk for development of hepatocellular carcinoma, in part from consumption of foods contaminated with aflatoxins. Chlorophyllin, a mixture of semisynthetic, water-soluble derivatives of chlorophyll that is used as a food colorant and over-the-counter medicine, has been shown to be an effective inhibitor of aflatoxin hepatocarcinogenesis in animal models by blocking carcinogen bioavailability. In a randomized, double-blind, placebo-controlled chemoprevention trial, we tested whether chlorophyllin could alter the disposition of aflatoxin. One hundred and eighty healthy adults from Qidong were randomly assigned to ingest 100 mg of chlorophyllin or a placebo three times a day for 4 months. The primary endpoint was modulation of levels of aflatoxin-N(7)-guanine adducts in urine samples collected 3 months into the intervention measured by using sequential immunoaffinity chromatography and liquid chromatography-electrospray mass spectrometry. This aflatoxin-DNA adduct excretion product serves as a biomarker of the biologically effective dose of aflatoxin, and elevated levels are associated with increased risk of liver cancer. Adherence to the study protocol was outstanding, and no adverse events were reported. Aflatoxin-N(7)-guanine could be detected in 105 of 169 available samples. Chlorophyllin consumption at each meal led to an overall 55% reduction (P = 0.036) in median urinary levels of this aflatoxin biomarker compared with those taking placebo. Thus, prophylactic interventions with chlorophyllin or supplementation of diets with foods rich in chlorophylls may represent practical means to prevent the development of hepatocellular carcinoma or other environmentally induced cancers.

Proc Natl Acad Sci U S A. 2001 Dec 4;98(25):14601-6

Analysis of the therapeutic effect of sodium copper chlorophyllin tablet in treating 60 cases of leukopenia.

OBJECTIVE: To evaluate the efficacy and safety of sodium copper chlorophyllin (trademarked as “Yebaike Tablet which is abbreviated as YBK in treating leukopenia. METHODS: One hundred and five patients with leukopenia caused by various factors were randomized into 3 groups. The 60 patients in the YBK group took orally YBK Tablets at the dose of 40 mg, three times per day, the 30 patients in the leucogen group were treated with Leucogen Tablets at the dose of 20 mg, three times per day, and the 15 patients in the placebo group were administered with vitamin C tablets 100 mg, three times per day. All the subjects were treated for 1 month. The change of peripheral leucocytes count after treatment and adverse drug reaction that occurred in patients were studied. RESULTS: In the 60 patients treated with YBK, the treatment proved to be markedly effective in 34 cases, effective in 17 and ineffective in 9, the total effective rate being 85%, which was significantly higher than that in the placebo group (26.7%, P < 0.01) and similar to that in the leucogen group (83.3%, P > 0.05). No adverse reaction was found in the treatment course. CONCLUSION: YBK can be used in the treatment of leukopenia caused by various factors, satisfactory in efficacy and safe in use.

Chin J Integr Med. 2005 Dec;11(4):279-82

The importance of carcinogen dose in chemoprevention studies: quantitative interrelationships between, dibenzo[a,l]pyrene dose, chlorophyllin dose, target organ DNA adduct biomarkers and final tumor outcome.

Chlorophyllin (CHL) is a potent antimutagen in vitro, an effective anti-carcinogen in several animal models, and significantly reduced urinary biomarkers of aflatoxin B(1) (AFB(1)) exposure in a human population. Here we report an expanded analysis of CHL chemoprevention using the potent environmental hydrocarbon dibenzo[a,l]pyrene (DBP). A dose-dose matrix design employed over 12,000 rainbow trout to evaluate the interrelationships among dietary carcinogen dose, anti-carcinogen dose, carcinogen-DNA adduct levels at exposure and eventual tumor outcome in two target organs. Included was an evaluation of the pharmaceutical CHL preparation (Derifil), used previously in a study of individuals chronically exposed to AFB(1). CHL was pre-, co- and post-fed at doses of 0-6000 p.p.m. and co-fed with DBP at doses of 0-371.5 p.p.m. for 4 weeks. This protocol generated a total of 21 dose-dose treatment groups, each evaluated with three or more replicates of 100 animals. The DBP-only treatment produced dose-responsive increases in liver and stomach DBP-DNA adducts, whereas increasing CHL co-treatment doses produced successive inhibition in liver (49-83%) and stomach (47-75%) adduct levels at each DBP dose examined. The remaining 8,711 trout were necropsied, 10 months later. DBP treatment alone produced a logit incidence versus log [DBP] dose-response curve in stomach that was linear; CHL co-treatment provided dose-dependent tumor inhibition which ranged from 30 to 68% and was predictable from the adduct response. The Derifil CHL preparation was also found to effectively reduce DNA adduction and final tumor incidence in stomach (as well as liver), with a potency compatible with its total chlorin content. Liver tumor incidence in the DBP-only groups appeared to plateau near 60%. At DBP doses of <or=80 p.p.m., increasing CHL doses generally reduced tumor incidence and multiplicity consistent with early DNA adducts as biomarkers. At 225 p.p.m. DBP, however, very high CHL doses were required to reduce tumor incidence below the 60% plateau. Apparent tumor multiplicity in liver was neither linear nor monotonic with DBP dose, but peaked at 80 p.p.m. DBP and declined at 225 p.p.m., where it was increased by all but one CHL dose. Consequently, the effects of a given CHL dose and the predictivity of DNA adducts as biomarkers were highly dependent on carcinogen dose. These results underscore the critical importance of establishing carcinogen-end point dose-response relationships in chemoprevention studies, and the potential otherwise for misleading interpretations in chemoprevention studies carried out solely at high-carcinogen dose.

Carcinogenesis. 2007 Mar;28(3):611-24

Chlorophyllin significantly reduces benzo[a]pyrene-DNA adduct formation and alters cytochrome P450 1A1 and 1B1 expression and EROD activity in normal human mammary epithelial cells.

We hypothesized that chlorophyllin (CHLN) would reduce benzo[a]pyrene-DNA (BP-DNA) adduct levels. Using normal human mammary epithelial cells (NHMECs) exposed to 4 microM BP for 24 hr in the presence or absence of 5 microM CHLN, we measured BP-DNA adducts by chemiluminescence immunoassay (CIA). The protocol included the following experimental groups: BP alone, BP given simultaneously with CHLN (BP+CHLN) for 24 hr, CHLN given for 24 hr followed by BP for 24 hr (preCHLN, postBP), and CHLN given for 48 hr with BP added for the last 24 hr (preCHLN, postBP+CHLN). Incubation with CHLN decreased BPdG levels in all groups, with 87% inhibition in the preCHLN, postBP+CHLN group. To examine metabolic mechanisms, we monitored expression by Affymetrix microarray (U133A), and found BP-induced up-regulation of CYP1A1 and CYP1B1 expression, as well as up-regulation of groups of interferon-inducible, inflammation and signal transduction genes. Incubation of cells with CHLN and BP in any combination decreased expression of many of these genes. Using reverse transcription real time PCR (RT-PCR) the maximal inhibition of BP-induced gene expression, >85% for CYP1A1 and >70% for CYP1B1, was observed in the preCHLN, postBP+CHLN group. To explore the relationship between transcription and enzyme activity, the ethoxyresorufin-O-deethylase (EROD) assay was used to measure the combined CYP1A1 and CYP1B1 activities. BP exposure caused the EROD levels to double, when compared with the unexposed controls. The CHLN-exposed groups all showed EROD levels similar to the unexposed controls. Therefore, the addition of CHLN to BP-exposed cells reduced BPdG formation and CYP1A1 and CYP1B1 expression, but EROD activity was not significantly reduced.

Environ Mol Mutagen. 2009 Mar;50(2):134-44

Mechanisms of the in vitro antimutagenic action of chlorophyllin against benzo[a]pyrene: studies of enzyme inhibition, molecular complex formation and degradation of the ultimate carcinogen.

Mechanisms of the antimutagenic action of chlorophyllin (CHL) towards benzo[a]pyrene (BP) were studied in vitro. In the Salmonella assay, CHL inhibited the mutagenic activity of BP in the presence of an S9 activation system and was particularly effective against the direct-acting ultimate carcinogen, benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE). Spectral studies indicated that the time-dependent hydrolysis of BPDE to tetrols was augmented in the presence of CHL concentrations on the order of 5 microM. Dose-related inhibition of several cytochrome P450-dependent enzyme activities was observed upon addition of CHL to in vitro incubations. Spectral changes for the interaction between CHL and cytochrome P450 indicated that CHL does not bind to the active site of the enzyme, but exerts its inhibitory effect indirectly. This was achieved by inhibiting NADPH-cytochrome P450 reductase (Ki approximately 120 microM with cytochrome c as substrate), and did not involve lowering of the effective substrate concentration by complex formation with the procarcinogen. It is concluded that the in vitro antimutagenic activity of CHL towards BP involves accelerated degradation of the ultimate carcinogen, with inhibition of carcinogen activation occurring only at high CHL concentrations. The latter mechanism is unlikely to occur in vivo following p.o. administration due to the limited uptake of CHL from the gut, but tissue concentrations may be sufficiently high to cause degradation of BPDE.

Mutat Res. 1994 Jul 16;308(2):191-203

Chlorophyll, chlorophyllin and related tetrapyrroles are significant inducers of mammalian phase 2 cytoprotective genes.

Plant chlorophylls and carotenoids are highly colored, conjugated polyenes that play central roles in photosynthesis. Other porphyrins (tetrapyrroles), such as cytochromes, which are structurally related to chlorophyll, participate in redox reactions in many living systems. An unexpected new property of tetrapyrroles, including tetramethyl coproporphyrin III, tetrabenzoporphine, copper chlorin e4 ethyl ester, and of carotenoids including zeaxanthin and alpha-cryptoxanthin is their ability to induce mammalian phase 2 proteins that protect cells against oxidants and electrophiles. The capacity of these compounds to induce the phase 2 response depends upon their ability or that of their metabolites to react with thiol groups, a property shared with all other classes of phase 2 inducers, which show few other structural similarities. Pseudo second-order rate constants of these inducers are correlated with their potency in inducing the phase 2 enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1) in murine hepatoma cells. One of the most potent inducers was isolated from chlorophyllin, a semisynthetic water-soluble chlorophyll derivative. Although chlorophyll itself is low in inducer potency, it may nevertheless account for some of the disease-protective effects attributed to diets rich in green vegetables because it occurs in much higher concentrations in those plants than the widely studied ‘phytochemicals’.

Carcinogenesis. 2005 Jul;26(7):1247-55

The chlorophyllin-induced cell cycle arrest and apoptosis in human breast cancer MCF-7 cells is associated with ERK deactivation and Cyclin D1 depletion.

Targeting the mitogen-activated protein kinases (MAPKs) has been suggested as a novel strategy to treat cancer. Chlorophyllin (CHL) is the sodium-copper salt of chlorophyll derivative and is a commonly used food dye for green coloration; CHL was found previously to retard growth of the human breast carcinoma MCF-7 cells. Extracellular signal-regulated kinases (ERKs) constitute a subfamily of MAPKs, participating in cell survival, proliferation and differentiation. We report here the first evidence that CHL deactivates ERKs to inhibit the breast cancer cell proliferation. The results from flow cytometry showed that 200 microg/ml CHL reduced the phosphorylated and activated ERK-positive cells in different cell cycle phases from the control of >96 to <38% at 24 h of incubation; the ERK deactivations occurred in both dose- and time-dependent manner, so that nearly all ERKs were de-activated by 400 microg/ml CHL at 72 h of treatment. Immunoblot studies, however, illustrated that the levels of total ERKs were not significantly affected by the CHL treatments, suggesting that the phytochemical retards the enzyme activation rather than its expression. Cyclin D1, but not its enzyme Cdk6, was also depleted after the CHL treatments; the depletions were associated with elevations of G0/G1 cells. Apoptosis occurred time-dependently with the ERK deactivations by 400 microg/ml CHL; the apoptotic cells elevated from 2.7-fold of the control level at 24 h, to 4.7-fold at 48 h and to 16.6-fold at 72 h of treatment. Bcl-2 was also depleted at 72 h when there was the most prominent elevation of the apoptotic cells, suggesting that it participates during the exacerbation rather than the initiation phases of the CHL-induced apoptosis. Results from this study support further research on CHL for preventing and treating those tumors with deregulated ERK activations.

Int J Mol Med. 2005 Oct;16(4):735-40

Protection by chlorophyllin and indole-3-carbinol against 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced DNA adducts and colonic aberrant crypts in the F344 rat.

The most abundant heterocyclic amine in fried ground beef, 2-amino-1-methyl-6-pheny-limidazo[4,5-b]pyridine (PhIP), induces colon carcinomas in the male F344 rat. The potential chemopreventive effects of two compounds, namely, the ‘interceptor molecule’ chlorophyllin (CHL) and a modulator of carcinogen activation, indole-3-carbinol (I3C), were examined in a PhIP colon carcinogenesis model. During weeks 3 and 4 of a 16-week study, F344 rats were given PhIP by oral gavage (50 mg/kg body weight, alternating days). Inhibitors were given either before and during PhIP exposure, after PhIP treatment, or continuously for 16 weeks. Treatment of rats with 0.1% CHL in the drinking water inhibited the formation of aberrant crypt foci (ACF) with > or = 4 crypts/focus, from 1.4 +/- 0.9 in controls to 0.7 +/- 0.3 following post-initiation CHL treatment, and to 0.3 +/- 0.5 in rats given CHL continuously for 16 weeks (mean +/- SD; P < 0.05). Potent inhibition of PhIP-induced ACF occurred following initiation, post-initiation and continuous exposure to 0.1% I3C in the diet. Using the initiation protocol, I3C completely inhibited the induction of the ACF with > or = 4 crypts/focus. In a separate experiment, rats were given 0.1% CHL in the drinking water or 0.1% I3C in the diet for 4 weeks. At the end of week 3, animals received 50 mg PhIP/kg body weight by single oral gavage and PhIP-DNA adducts were quantified in the colon and several other tissues by 32P-postlabeling analysis. In addition, the urine and feces were collected to study the effects of inhibitor treatment on PhIP metabolism and excretion. No significant protection against PhIP-DNA adduct formation was detected in the colon after CHL dosing, nor was a consistent pattern of CHL inhibition observed in several other tissues. In contrast, I3C shifted the time-course of adducts in all tissue; compared with controls, adducts were increased by I3C at 6 h but decreased at 24 h and 7 days following PhIP treatment. Analysis of urine metabolites revealed that I3C and CHL decreased the excretion of unmetabolized PhIP and 4’-hydroxy- << PhIP but increased the phase II detoxification products PhIP-4’-O-glucuronide and PhIP-4’- sulfate. In the feces, the elimination of unmetabolized PhIP was increased from 54.5% in controls to approximately 67% in CHL-treated rats and decreased to 28% in rats given I3C (P < 0.05). These results support a protective role for CHL and I3C against PhIP-induced colon carcinogenesis through mechanisms which alter the uptake or metabolism of the carcinogen, and by suppression in the post-initiation phase.

Carcinogenesis. 1995 Dec;16(12):2931-7

Effects of tea and chlorophyllin on the mutagenicity of N-hydroxy-IQ: studies of enzyme inhibition, molecular complex formation, and degradation/scavenging of the active metabolites.

Green tea and black tea inhibit the formation of carcinogen-DNA adducts and colonic aberrant crypts in rats given 2-amino-3-methylimidazo[4, 5-f]quinoline (IQ), a mutagen from cooked meat. The Salmonella mutagenicity assay was used in the present study to test individual constituents of tea as inhibitors of 2-hydroxyamino-3-methylimidazo[4, 5-f]quinoline (N-hydroxy-IQ), a direct-acting metabolite of IQ. Testing of pure compounds at doses relevant to their levels in tea identified epigallocatechin (EGC) and epigalocatechin-3-gallate (EGCG) as the primary antimutagens. Studies of the inhibitory mechanisms established that the rate of degradation of N-hydroxy-IQ under aqueous conditions was not increased significantly in the presence of tea, in contrast to the results obtained with the complexing agent chlorophyllin (CHL), which rapidly degraded the mutagen. Interaction between N-hydroxy-IQ and several tea constituents was detected in spectrophotometric studies, but the binding constants were only on the order of 1 x 10(3) M-1, suggesting that mechanisms other than complex formation might prevail under the conditions of the Salmonella assay. Comparison of the results in two different strains of Salmonella typhimurium, TA98 and TA98/1,8-DNP6, indicated that the antimutagenic activity of EGCG was dependent, at least in part, on a functional O-acetyltransferase activity in the bacteria. These studies suggest that tea constituents inhibit the enzyme(s) which generate the aryl nitrenium ion and directly scavenge the reactive electrophile, whereas CHL complexes with heterocyclic amines and facilitates the degradation of active metabolites.

Environ Mol Mutagen. 1997;30(4):468-74

Antiapoptotic and immunomodulatory effects of chlorophyllin.

Chlorophyllin (CHL) was earlier shown to reduce the level of intracellular ROS and apoptosis induced by ionizing radiation and 2,2’-azobis(2-propionimidinedihydrochloride) (AAPH). In the present studies, the effect of CHL on radiation-induced immunosuppression and modulation of immune responses in mice was examined. Chlorophyllin inhibited the in vitro lymphocyte proliferation induced by concanavalin A (Con A) in a dose dependent manner at doses>or=50 microM. At lower doses (10 microM) CHL significantly inhibited activation induced cell death (AICD) in Con A stimulated spleen cells. Spleen cells obtained from CHL treated mice showed an inhibition of response to Con A depending on dose of CHL and the time after its administration. Spleen cells obtained from CHL treated mice (24 h) showed lower inhibition of response to Con A following in vitro (5 Gy) as well as whole body irradiation (2 Gy). The expression of antiapoptotic genes bcl-2 and bcl-xL was up-regulated in these cells. Chlorophyllin treatment of mice led to splenomegaly and increase in the number of peritoneal exudate cells (PEC). The numbers of T cells, B cells and macrophages in the spleen were also increased. Increased phagocytic activity was seen in PEC obtained from CHL treated mice. Most importantly, CHL administration to mice immunized with sheep red blood cells (SRBC) augmented both humoral and cell-mediated immune responses.

Mol Immunol. 2007 Jan;44(4):347-59

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.

Toxicology. 2000 Nov 30;155(1-3):63-71