ABSTRACT
The identification of substances that prevent or minimize the detrimental effects of ionizing radiation is an essential undertaking. The aim of this paper was to evaluate and compare the radioprotective potential of chlorophyllin, protoporphyrin and bilirubin, with amifostine®, an US Food & Drug Administration approved radioprotector Using the somatic mutation and recombination assay in the Drosophila melanogaster wing, it was found that pretreatment (1-9â¯h) with any of the porphyrins or amifostine® alone, did not affect the larva-adult viability or the basal frequency of mutation. However, they were associated with significant reductions in frequency of somatic mutation and recombination compared with the gamma-irradiated (20â¯Gy) control as follows: bilirubin (69.3 %)> chlorophyllin (40.0 %)> protoporphyrin (39.0 %)> amifostine® (19.7 %). Bilirubin also caused a 16 % increase in larva-adult viability with 3â¯h of pretreatment respect to percentage induced in 20â¯Gy control group. Whilst amifostine® was associated with lower genetic damage after pre-treatment of 1 and 3â¯h, this did not attain significance. These findings suggest that the tested porphyrins may have some potential as radioprotectant agents.
Subject(s)
Amifostine/pharmacology , Bilirubin/pharmacology , Chlorophyllides/pharmacology , Drosophila melanogaster/drug effects , Drosophila melanogaster/radiation effects , Protoporphyrins/pharmacology , Radiation-Protective Agents/pharmacology , Animals , Drosophila melanogaster/genetics , Female , Male , Mutagenicity Tests , Mutation/drug effects , Recombination, Genetic/drug effects , Wings, Animal/drug effects , Wings, Animal/radiation effectsABSTRACT
The use of eosin methylene blue according to Giemsa as photosensitizer is presented for the first time in this paper. The present study evaluated the potential application of chlorophyllin sodium copper salt (CuChlNa) and eosin methylene blue according to Giemsa (EMB) as antimicrobial photosensitizers (aPS) for photodynamic inactivation (PDI) of Staphylococcus aureus (gram-positive) and Escherichia coli (gram-negative) bacteria. The experiments were performed using S. aureus stain ATCC 25923 and E. coli ATCC 25922 in which five aPS concentrations (0.0, 1.0, 2.5, 5.0, 10.0, and 20.0 µM for S. aureus and 0.0, 5.0, 10.0, 20.0, 40.0, and 50.0 µM for E. coli) were prepared and added in 2 mL of a saline solution containing the bacterial inoculum. After aPS incubation, the samples were divided into two groups, one kept in the dark and another submitted to the illumination. Then, the bacterial inactivation was determined 18 h after the incubation at 37 °C by counting the colony-forming units (CFU). The results revealed that both EMB and CuChlNa can be used as aPS for the photoinactivation of S. aureus, while only EMB was able to photoinactivate E. coli. Nevertheless, a more complex experimental setup was needed for photoinactivation of E. coli. The data showed that EMB and CuChlNa presented similar photoinactivation effects on S. aureus, in which bacterial growth was completely inhibited at photosensitizer (PS) concentrations over 5 µM, when samples were previously incubated for 30 min and irradiated by a light dose of 30 J cm-2 as a result of an illumination of 1 h at 8.3 mW cm-2 by using a red light at 625 nm with a 1 cm beam diameter and output power of 6.5 mW. In the case of E. coli, bacterial growth was completely inhibited only when combining a PS incubation period of 120 min with concentrations over 20 µM.
Subject(s)
Chlorophyllides/pharmacology , Eosine Yellowish-(YS)/pharmacology , Escherichia coli/radiation effects , Light , Methylene Blue/pharmacology , Microbial Viability/drug effects , Microbial Viability/radiation effects , Staphylococcus aureus/radiation effects , Animals , Escherichia coli/drug effects , Escherichia coli/growth & development , Mice , NIH 3T3 Cells , Photosensitizing Agents/pharmacology , Spectrum Analysis , Staphylococcus aureus/drug effects , Staphylococcus aureus/growth & developmentABSTRACT
Fasciolosis is a food borne zoonosis, caused by the digenetic trematode Fasciola. Freshwater lymnaeid snails are the intermediate host of the trematodes. Chlorophyllin, a semi-synthetic derivative of chlorophyll and its formulations obtained from freeze dried cow urine (FCU) had their toxicity tested against redia and cercaria larvae of F. gigantica. The larvicidal activity of chlorophyllin and its formulations were found to depend on both, time and concentration used against the larvae. Toxicity of chlorophyllin + FCU (1:1 ratio) in sunlight against redia larva (8 h LC50: 0.03 mg/mL) was more pronounced than using just chlorophyllin (8 h LC50: 0.06 mg/mL). Toxicity of chlorophyllin + FCU in sunlight against redia (8 h LC50: 0.03 mg/mL) was higher than against cercaria (8 h LC50: 0.06 mg/mL). The larvicidal activity of chlorophyllin in sunlight (redia/cercaria larvae: 8 h LC50: 0.06 mg/mL) was more pronounced than under laboratory conditions (redia: 8 h LC50: 22.21 mg/mL/, cercaria 8 h LC50: 96.21 mg/mL). Toxicity of FCU against both larvae was lower than that of chlorophyllin and chlorophyllin + FCU. Chlorophyllin and its formulations + FCU were 357.4 to 1603.5 times more effective against redia/cercaria larvae in sunlight than under laboratory conditions. The present study has shown that chlorophyllin formulations may be used as potent larvicides against fasciolosis.
Subject(s)
Anthelmintics/pharmacology , Chlorophyllides/pharmacology , Fasciola/drug effects , Animals , Cattle , Larva/drug effects , Lethal Dose 50 , Lymnaea/parasitology , Parasitic Sensitivity Tests , Time Factors , UrineABSTRACT
BACKGROUND AND OBJECTIVE: Chlorophyllin-M is a new chlorophyll-based derivative photosensitive compound developed by our research group with easy laboratorial synthesis and ideal properties for photodynamic therapy (PDT). It is intended for clinical treatments with simple and low cost techniques and reagents. The objective of this study is to evaluate if intravenous chlorophyllin-M is able to deliver a photosensitizer to rabbit retina and rabbit choroid and promote PDT after ocular irradiation with a 660 nm LASER. METHODS: This is a pre-clinical study. Ten eyes of five pigmented Californian rabbits were included in the study. The right eyes served as the treatment group, and the left eyes served as the control group. All eyes had been ophthalmologically evaluated and were considered normal. RESULTS: Ophthalmic exam with anterior biomicroscopy, dilated fundus examination, and fluorescein angiography after the LASER procedure revealed normal anterior segment, retinal and choroid vessels occlusion, lumen narrowing, and capillary non-perfusion in the treated areas, indicating that PDT was successful in the treatment eyes group. CONCLUSION: The results of this pre-clinical study encourage future studies with this new compound. Chlorophyllin-M may become a new cost-effective agent in the retinal therapeutic arsenal.
Subject(s)
Antimutagenic Agents/pharmacology , Chlorophyllides/pharmacology , Choroid/drug effects , Photochemotherapy , Photosensitizing Agents/pharmacology , Retina/drug effects , Animals , Choroid/radiation effects , Fluorescein Angiography , Rabbits , Retina/radiation effectsABSTRACT
Sodium-copper chlorophyllin (SCC), a copper-porphyrin complex, has been shown to act as an inhibitor as well as a promoter of DNA-damage induction by a variety of mutagens in several test systems. In order to investigate the basis of this dual effect, experiments were carried out to compare the influence of pretreatment with intact SCC and that of its constituents, the metal-free protoporphyrin (PP-IX) and copper as CuCl(2). The wing-spot test was employed to monitor mutational events in somatic cells of Drosophila melanogaster. Heterozygous mwh+/+flr(3) larvae were treated for 24h with SCC, PP-IX, CuCl(2) or sucrose. Following this treatment, one group of larvae were immediately allowed to feed on instant medium containing 0.5mM N-nitroso-N-ethylurea (ENU) dissolved in phosphate buffer to reach pH 6. The remaining larvae received treatment with ENU with a delay of 1, 2 or 3days (DTD). Results revealed an (a) overall inhibitory effect for 0-DTD and 1-DTD after pretreatment with SCC, (b) only in 0-DTD after PP-IX, and (c) in all DTDs after treatment with CuCl(2). These results provide evidence that the copper ion plays a central role in the antimutagenic effect of SCC, and for a sustained period of time. Pretreatment with SCC and PP-IX produced a promoter effect at 2-DTD and 3-DTD. The results could be explained as an effect of the accumulation of metal-free porphyrin following the dissociation of the copper-porphyrin complex (SCC), the copper-ion reaching proteins to form complexes and participated in anabolic pathways.
Subject(s)
Antimutagenic Agents/pharmacology , Chlorophyllides/toxicity , Copper/pharmacology , Ethylnitrosourea/toxicity , Mutagens/pharmacology , Porphyrins/pharmacology , Animals , Chlorophyllides/chemistry , Chlorophyllides/pharmacology , Drosophila/genetics , Mutagenicity Tests , Structure-Activity RelationshipABSTRACT
AIMS: Chlorophyllin (CHLN), a synthetic derivative of chlorophyll, was assayed in the replication of poliovirus (PV-1) and bovine herpesvirus (BoHV-1) in HEp-2 cell cultures. METHODS AND RESULTS: Virucidal activity of CHLN was evaluated and the time-of-addition assay was performed as follows: before the infection (-1 and -2 h), at the time of the infection (0 h) and after the infection (1 and 2 h). Plaque reduction assay (PRA) showed that CHLN inhibited BoHV-1 and PV-1 infection and the 50% inhibitory concentrations (IC(50)) against BoHV-1 and PV-1 infection were 8.6 and 19.8 microg ml(-1), respectively. The time-of-addition study demonstrated that the CHLN was effective inhibiting viral replication in 51% and 66.5% for PV-1 and BoHV-1, respectively, at the highest concentration of 20.0 microg ml(-1), when added during the infection. The directed effect of CHLN on viral strains demonstrated an inhibition of 62% and 66.4% for PV-1 and BoHV-1, respectively, by PRA. CONCLUSIONS: These results demonstrated that CHLN could be used as an antiviral suggesting directed activity on virus particles and on virus-receptor sites to BoHV. For poliovirus, CHLN also demonstrated virucide activity, moreover, showed to inhibit early steps of the replication cycle. SIGNIFICANCE AND IMPACT OF THE STUDY: CHLN demonstrated promising selectivity index for both virus strains; therefore, it can be used for the development of an antiviral agent.
Subject(s)
Antiviral Agents/pharmacology , Chlorophyllides/pharmacology , Herpesviridae/physiology , Poliovirus/physiology , Virus Replication/drug effects , Cell Line, Tumor , Cytopathogenic Effect, Viral/drug effects , Herpesviridae/drug effects , Humans , Inhibitory Concentration 50 , Poliovirus/drug effects , Viral Plaque AssayABSTRACT
It was first demonstrated in Salmonella that higher and lower concentrations of chlorophyllin (CHLN) may have effects in opposite directions, higher doses inhibiting and lower doses promoting the mutagenic activity of certain tobacco-related nitrosamines. Previous work of our group demonstrated that CHLN may have both a promoter and an inhibitory effect on mutagenesis in Drosophila. The present paper reviews the evidence obtained in our laboratory using gamma rays as the mutagenic agent, that higher and lower pretreatment concentrations of CHLN are associated with inhibitory and promoting effects, respectively, as in Salmonella. Employing the wing spot test, 48h larvae were pretreated with various concentrations of CHLN from 0 to 69 mM and then treated with 10 Gy gamma rays. With the highest concentration of CHLN, an approximate 54% reduction in mutagenesis was observed. At 35 mM a remnant of this inhibitory effect was found in that a significant decrease was limited to the twin spot category. Evidence of promotion was first seen at 4.3mM CHLN, an effect which persisted for the remaining five lower concentrations, the most pronounced evidence of promotion being found at the four lowest concentrations, 0.03-1.1 mM CHLN. It should be noted that no evidence of genotoxicity was found for CHLN alone, an observation consistent with the several reports in the literature. The results are taken as strong evidence that pretreatment with low concentrations of CHLN promotes DNA damage induced by gamma rays in somatic cells of Drosophila.
Subject(s)
Chlorophyllides/pharmacology , Chlorophyllides/toxicity , DNA Damage/drug effects , DNA Damage/radiation effects , Gamma Rays/adverse effects , Mutagens/toxicity , Animals , Chlorophyllides/administration & dosage , Dose-Response Relationship, Drug , Drosophila/genetics , Mutagenicity Tests , Radiation-Protective Agents/pharmacologyABSTRACT
Chlorophyllin (Chln), a sodium-copper salt derivative of chlorophyll, like chlorophyll-a and -b found in green plants, has been studied for its protective action against the carcinogenic effects of various physical and chemical agents and in relation to the mutagenic and clastogenic activities of genotoxic agents. The aim of the present study was to evaluate chlorophyllin in different phases of the cell cycle for clastogenicity and anticlastogenicity, the latter in reversing DNA damage induced by ethyl methane sulfonate (EMS). The test for chromosomal aberrations was performed in cultured mammalian cells (CHO-K1). The three Chln concentrations tested (6.25, 12.5 and 25 microg/ml) were not clastogenic and damage induced by EMS (1240 microg/ml) was reduced in cells treated with Chln as well during S (25-48%) and G2/S (70-80%). The results demonstrate a greater protective effectiveness of Chln against EMS during G2/S.
Subject(s)
Antimutagenic Agents/pharmacology , Chlorophyllides/pharmacology , Animals , CHO Cells , Cell Cycle/drug effects , Cells, Cultured , Cricetinae , DNA Damage , Dose-Response Relationship, DrugABSTRACT
In Drosophila, 48h-old larvae were pretreated for 24h with chlorophyllin (CHLN) or sucrose and then treated with chromium(VI) oxide (CrO(3)) immediately following completion of the pretreatment period (0-day delay) or delayed 1, 2 or 3 days. The effects were scored in the wing spot test. After delays of 0 and 1 day, clear evidence of a protective effect of CHLN was found. Contrarily, after delays of 2 and 3 days, the results showed a reversal, i.e. CHLN-related events appeared more frequently than those in the sucrose control suggesting a promoting effect. It would appear prudent that CHLN be tested in a variety of situations in any given organism before decisions are reached regarding its inhibitor/promoter effects.
Subject(s)
Antimutagenic Agents/pharmacology , Chlorophyllides/pharmacology , Chromium Compounds/pharmacology , Drosophila/genetics , Animals , DNA Damage/genetics , Drosophila/drug effects , Drosophila/growth & development , Drug Antagonism , Drug Synergism , Female , Larva , Male , Sucrose/pharmacology , Wings, Animal/cytology , Wings, Animal/drug effects , Wings, Animal/growth & developmentABSTRACT
Evidence is presented that treating the Drosophila female with chlorophyllin (CHLN) has a marked effect on the yield of dominant lethals induced by the irradiation of sperm. The yield is significantly greater in the embryonic period (between the egg and the first instar) and is significantly reduced in postembryonic stages compared with a sucrose control.
Subject(s)
Chlorophyllides/pharmacology , Drosophila/embryology , Drosophila/genetics , Radiation-Protective Agents/pharmacology , Spermatozoa/radiation effects , Animals , Drosophila/drug effects , Female , Larva/drug effects , Male , Sucrose/pharmacologyABSTRACT
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.
Subject(s)
Acetaldehyde/antagonists & inhibitors , Acetaldehyde/toxicity , Antimutagenic Agents/pharmacology , Bone Marrow/ultrastructure , Chlorophyllides/pharmacology , Sister Chromatid Exchange/drug effects , Acetaldehyde/chemistry , Animals , Cell Division/drug effects , Chlorophyllides/chemistry , Chromatography, High Pressure Liquid , Male , Mice , Mitotic Index , Mutagens/toxicity , Spectrophotometry, UltravioletABSTRACT
The effect of chlorophyllin on micronucleated polychromatic erythrocytes (MN-PCE) induction by chromium trioxide (CrO(3)) exposure in peripheral blood of mice was studied. Animals were treated with a single intraperitoneal dose of chlorophyllin (CHL) (20mg/kg), CrO(3) (20mg/kg), and CHL (20mg/kg) 4h before (CHL-CrO(3)) or 4h before and 20h after chromium treatments (20mg/kg; CHL-CrO(3)-CHL). Peripheral blood samples were drawn from the caudal vein at 0, 12 and 48h, and analyzed by the acridine orange (AO) technique. The results obtained in present study shown that CHL injection did not modify the number of MN-PCE. CrO(3) treatment resulted in a significantly increases 12 and 48h after the injection, reaching a four-fold increase 48h after CrO(3) administration. Whereas treatment with 20mg/kg of CHL prior to chromium, decreased the MN frequency induced by chromium in the 12h samples. When the samples were analyzed 48h after CrO(3) injection, no significant differences between CHL-CrO(3) and CHL-CrO(3)-CHL in comparison with CrO(3) treatment, were observed. These results indicate that increase of MN-PCE by CrO(3) is CHL-blocked in both protocols used (CHL-CrO(3) and CHL-CrO(3)-CHL) at 12h after treatment, but it was unable to modify the frequency of MN-PCE measured 48h after CrO(3) injection. The absence of a protective effect by CHL in the MN-PCE induction by CrO(3) at 48h, show that CHL has action only on one of the times of MN induction and suggests the possible action of CrO(3) by two different mechanisms, and not by CHL time-limited in vivo.
Subject(s)
Antimutagenic Agents/pharmacology , Chlorophyllides/pharmacology , Chromium Compounds/toxicity , DNA Damage/drug effects , Mutagens/toxicity , Animals , Cell Count , Female , Mice , Micronucleus Tests , Reticulocytes/cytology , Reticulocytes/drug effects , Time FactorsABSTRACT
Chlorophyll and its derivatives are examples of plant compounds (purified and/or extracted) which appear to protect DNA from damage caused by chemical or physical agents, although some studies have identified clastogenic activity of these compounds. This study was carried out to assess the genotoxic activity of chlorophyll-a (Chl-a), -b (Chl-b) and chlorophyllin (Chl) and their antigenotoxic activity against the DNA damage induced by methyl methanesulphonate (MMS) under conditions of simultaneous, pre-, post-treatment, and simultaneous treatment after pre-incubation of the chemical with MMS. The micronucleus (MN) test was used in binucleated cells (induced by cytochalasin-B) of a mammalian cell line (V79). The three concentrations of Chl-a, Chl-b or Chl (0.1375, 0.275, 0.55microM) were not genotoxic and the genotoxic action of MMS (400microM) decreased (74-117%) under all treatment conditions. The results showed that there was no significant difference among the treatment types, the concentration or the nature of chlorophyll used. The data obtained suggest that Chl-a, Chl-b and Chl when associated with the DNA damaging agent, MMS, may protect the DNA by desgenotoxic action and/or by bio-antigenotoxic mechanisms, with the similar efficiency.
Subject(s)
Antimutagenic Agents/pharmacology , Chlorophyll/pharmacology , Chlorophyll/toxicity , Chlorophyllides/pharmacology , Chlorophyllides/toxicity , Mutagens/toxicity , Animals , Cell Line , Chlorophyll/administration & dosage , Chlorophyll A , Chlorophyllides/administration & dosage , Cricetinae , DNA Damage , Methyl Methanesulfonate/antagonists & inhibitors , Methyl Methanesulfonate/toxicity , Micronucleus TestsABSTRACT
Irradiation of 96h old Drosophila following a 24h pretreatment with 5% chlorophyllin (CHLN) was delayed 0-4 days. The antimutagenic effect of CHLN in somatic cells monitored by the wing spot test persisted for 3 days after completion of the pretreatment and appeared to terminate at a time corresponding to the cessation of mitotic divisions of wing anlagen cells. Within the same population of cells, CHLN demonstrated both an inhibitory effect as measured in mwh single spot classes, and contrarily, a promoting effect in the class of mwh/flr twin spots and to an extent in the class of large flr spots. The reason for the contrasting effects of CHLN remains to be determined.
Subject(s)
Antimutagenic Agents , Chlorophyllides/pharmacology , Drosophila/genetics , Mutagens , Wings, Animal/radiation effects , Animals , Chlorophyllides/toxicity , Crosses, Genetic , Drosophila/growth & development , Female , Larva/drug effects , Larva/radiation effects , Male , Wings, Animal/drug effectsABSTRACT
The effect of chlorophyllin (CHLN) on the mutagenicity of four monofunctional alkylating agents (MFAAs) was evaluated in the wing spot test in Drosophila. Three of the compounds are direct-acting (ethylnitrosamine (ENU), methylnitrosourea (MNU), and methylmethanesulfonate (MMS)) and one indirect-acting (diethylnitrosamine, DEN). Results indicate that the mutagenicity of all four compounds is strongly inhibited by CHLN. The findings are not in agreement with the conclusion of Romert et al. (1992) that CHLN has no effect on the mutagenicity of direct acting MFFAs inferred from their work with MNU and ethylmethanesulfonate (EMS) in the V79 and Salmonella in vitro test systems. The results suggest the possibility that the action of CHLN need not include an inhibiting effect on metabolic activation.
Subject(s)
Alkylating Agents/toxicity , Antimutagenic Agents/pharmacology , Chlorophyllides/pharmacology , Drosophila/drug effects , Animals , Diethylnitrosamine/toxicity , Dose-Response Relationship, Drug , Drosophila/genetics , Drosophila/metabolism , Ethylnitrosourea/toxicity , Female , Male , Methyl Methanesulfonate/toxicity , Methylnitrosourea/toxicity , Mutagenesis , Mutagenicity Tests , Wings, Animal/drug effectsABSTRACT
By delaying the time of gamma irradiation of 72 h larvae, pretreated at 48 h with 5% chlorophyllin (CHLN), it was established that the overall inhibiting effect of CHLN in somatic cells of Drosophila, as measured in the wing spot test, persists for about 4 days or until the time of cessation of the proliferation of wing anlagen. In the same population of cells, some spot classes gave evidence of an inhibitory effect whereas others did not arguing against the suggestion that the radioprotective effect of CHLN is a consequence of an induced delay in development, shrinking of the potential radiation target and lowering the probability of induced events. Other observations of interest are described.
Subject(s)
Chlorophyllides/pharmacology , Drosophila/drug effects , Drosophila/radiation effects , Radiation-Protective Agents/pharmacology , Animals , Drosophila/genetics , Female , Gamma Rays , Larva/drug effects , Larva/genetics , Larva/radiation effects , Male , Mutation , Wings, Animal/drug effects , Wings, Animal/physiology , Wings, Animal/radiation effectsABSTRACT
The radioprotective capacity of chlorophyllin was determined by measuring the reduction of gamma-ray-induced sister chromatid exchange (SCE) in murine spermatogonia in vivo. The results obtained in experiments using 100 and 200 micrograms of chlorophyllin per gram of body weight (bw) and irradiated either before or after BrdU incorporation, indicate that a chlorophyllin dose of 200 micrograms/g bw protects 100% against the induction of SCE by 0.75 Gy of gamma-rays and 100 micrograms/g bw protects less than 50%. Chlorophyllin per se did not have any effect on the SCE frequency.