Assessment of the molluscicidal impact of extracted chlorophyllin on some biochemical parameters in the nervous tissue and histological changes in Biomphalaria alexandrina and Lymnaea natalensis snails.

Biomphalaria alexandrina and Lymnaea natalensis snails are the intermediate hosts of schistosomiasis and fasciolosis. The aim of the present study is to evaluate the molluscicidal activity of chlorophyll extract as a photodynamic substance against these snails and how it affected its tissues and the biological system. Chlorophyllin was extracted from deep-frozen Moringa oleifera leaves, and then it was transformed into water-soluble chlorophyllin. The present results showed that it had a molluscicidal activity on B. alexandrina snails (LC50 17.6 mg/l; LC90 20.9 mg/l) and L. natalensis snails (LC50 4.3 mg/l; LC90 6.8 mg/l). Exposing B. alexandrina snails to the sublethal concentrations (LC0, LC10, and LC25) resulted in a significant reduction in their survival rates. Regarding its effect on biochemical parameters, chlorophyllin significantly reduced the acetylcholinesterase activity, protein content, and alkaline and acid phosphatase activity in B. alexandrina nervous tissue compared to the control group. Histopathological changes occurred in the digestive gland of treated B. alexandrina snails where cells lost their nuclei, vacuolated, degenerated, and ruptured, and the lumen increased. Photosynthesizing materials like chlorophyllin are new approaches to control schistosomiasis and fasciolosis in developing countries by affecting their intermediate host. These materials were cheap and environmentally safe to replace the synthetic molluscicides for snail control.

Assessment of the impact of chlorophyll derivatives to control parasites in aquatic ecosystems.

Several research groups have studied new biopesticides which are less toxic to the environment and capable of controlling the vectors of parasitic diseases, especially in aquatic ecosystems. Pest control by photodynamic substances is an alternative to chemical or other measures, with chlorophyll and its derivatives as the most studied substances supported by their easy availability and low production costs. The impact of chlorophyll derivatives on four different species, a small crustacean (Daphnia similis), a unicellular alga (Euglena gracilis) and two species of fish (Astyanax bimaculatus and Cyprynus carpio) were tested under short-term conditions. In addition, the effects of long-term exposure were evaluated in D. similis and E. gracilis. In short-term tests, mortality of D. similis (EC50 = 7.75 mg/L) was most strongly affected by chlorophyllin, followed by E. gracilis (EC50 = 12.73 mg/L). The fish species showed a greater resistance documented by their EC50 values of 17.58 and 29.96 mg/L in C. carpio and A. bimaculatus, respectively. A risk quotient is calculated by dividing an estimate of exposure by an estimate of effect. It indicated that chlorophyll derivatives can be applied in nature to control the vectors of parasitic diseases under short-term conditions, but long-term exposure requires new formulations.

Toxicity of chlorophyllin in different wavelengths of visible light against Fasciola gigantica larvae.

Toxicity of chlorophyllin against redia and cercaria larvae of Fasciola gigantica was studied under irradiation of visible light. Highest and lowest toxicity of chlorophyllin against both larvae was noted under red (redia - 8 h LC50 7.88 mg/10 mL and cercaria - 11.99 mg/10 mL) and green (redia - 8 h LC50 32.12 mg/10 mL and cercaria - 8 h LC50 43.80 mg/10 mL) light irradiation respectively. The highest toxicity of chlorophyllin under red light irradiation against redia (8h LC50 7.88 mg/10 mL)/cercaria (8h LC50 11.99 mg/10 mL) was followed by white (8 h LC50 redia - 20.48 mg/10 mL, 8 h LC50 cercaria - 18.0 3mg/10 mL), blue (8 h LC50 redia - 33.10 mg/10 mL/ 8 h LC50 cercaria - 19.98 mg/10 mL) and yellow (8 h LC50 redia - 23.87 mg/10 mL/ 8 h LC50 cercaria - 23.48 mg/10 mL). Chlorophyllin treatment in darkness (control I) and without treatment of chlorophyllin, while all other conditions were same as treatment group (control II) caused no mortality of redia/cercaria larva. Chlorophyllin might be a promising new safe strategy to replace synthetic larvicide in fasciolosis control programme.

A 13-week subchronic toxicity study of sodium iron chlorophyllin in F344 rats.

Sodium iron chlorophyllin (SIC), a water-soluble chlorophyll derivative, has been used as a food additive for green coloration. In the present study, a subchronic toxicity study of SIC was performed in male and female F344 rats with oral administration in diet at concentrations of 0%, 0.2%, 1.0%, and 5.0% for 13 weeks. No mortalities, abnormal clinical signs, and hematological changes were observed in any of the groups during the experiment. Significant reduction of body weight gain was noted in 5.0% males. In serum biochemistry, serum transferrin levels were significantly increased in 5.0% males and females. Relative spleen weights of both sexes were markedly reduced with 5.0% SIC as compared to the controls, and absolute weights of spleen were also significantly decreased in males. On histopathological assessment, diffuse hypertrophy of acinar cells in the parotid gland was observed in all examined 5.0% males and females, but not in the other groups. Based on the histopathology of the parotid glands, the no-observed-adverse-effect level (NOAEL) of SIC in the present study was estimated to be 1.0% (609 mg/kg bw/day for males and 678 mg/kg bw/day for females).

Sodium copper chlorophyllin (SCC) induces genetic damage in postmeiotic and somatic wing cells of Drosophila melanogaster.

There is no apparent evidence to indicate that sodium copper chlorophyllin (SCC) is mutagenic. The aim of the present study was thus to determine the mutagenic effect of SCC, in postmeiotic germ cells of the adult male Drosophila. This investigation was based on the ability to examine whether SCC induced sex-linked recessive lethal mutations (SLRL), as well as the somatic mutation and recombination test (SMART). Four different SCC concentrations were used: 0, 45, 69, 80, and 100 mM. For SLRL, two broods were generated to test sperm and primarily spermatids. Results showed a significant frequency of recessive lethal mutations compared with control sperm cells with SCC at 69, 80, and 100 mM. In contrast, the frequency of somatic mutations rose by 0.21 only with 100 mM of SCC. These findings provide evidence that SCC is a weak mutagen in both cell lines. The differential response may be attributed to repair mechanisms that are active in somatic cells but almost absent in germ cells.

Chlorophyllin e4 is a novel photosensitizer against human bladder cancer cells.

The aim of the study was to investigate the photodynamic effect of the novel photosensitizer chlorophyllin e4 against human bladder cancer cells. T24 and 5637 bladder cancer cell lines were incubated with chlorophyllin e4 and irradiated with a 650-nm laser light. The controls included cells treated with chlorophyllin e4 but without light as well as cells exposed to laser light without chlorophyllin e4. Photocytotoxicity was monitored with MTT assay and apoptosis was measured by flow cytometry. In addition, confocal laser scanning microscopy was used to assess the subcellular localization of chlorophyllin e4. Chlorophyllin e4 exhibited significant photocytotoxicity in both T24 and 5637 cells, which resulted in a maximum of 82.43 and 85.06% cell death, respectively. Treatment with chlorophyllin e4 or laser light alone did not induce cytotoxicity. In addition, chlorophyllin e4-mediated PDT induced a significantly higher percentage of apoptosis in T24 and 5637 cells compared to the control groups (p<0.01). Moreover, confocal laser scanning microscopy revealed that chlorophyllin e4 co-localized with mitochondria in both cell lines. In conclusion, the remarkable photocytotoxicity, natural abundance and inexpensive composition of chlorophyllin e4 suggest that this compound may be a novel, effective photosensitizer for the treatment of human superficial bladder cancer.

A study of the inhibition/promotion effects of sodium-copper chlorophyllin (SCC)-mediated mutagenesis in somatic cells of Drosophila.

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.

Evidence that low concentrations of chlorophyllin (CHLN) increase the genetic damage induced by gamma rays in somatic cells of Drosophila.

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.

Chlorophyllin protects HEp-2 cells from nuclear fragmentation induced by poliovirus.

AIMS: Chlorophyllin (CHLN) is a synthetic derivative of chlorophyll that possesses antimutagenic activity against several environmental contaminants. In the present study, CHLN was assayed for its capacity to prevent nuclear fragmentation (NF) in HEp-2 cells infected with poliovirus. METHODS AND RESULTS: CHLN was assayed at concentrations of 0.5 and 2.5 microg ml(-1), and NF was monitored using the comet assay and acridine orange staining. We demonstrated that CHLN reduced the percentage of NF in poliovirus-infected HEp-2 cells, when cells were treated with drug before infection or exposed continuously to drug. However, the highest degree of protection was achieved when the virus was exposed to CHLN before infection followed by protocol where infected cultures were continuously exposed to the drug after infection. CONCLUSIONS: It is suggested that CHLN primarily binds to the virus which inhibits cell penetration, thereby maintaining nuclear integrity. SIGNIFICANCE AND IMPACT OF THE STUDY: Considering that CHLN has several beneficial properties and no significant toxic effects in humans and animals, it would be an ideal candidate drug to test for antiviral activity.

The mutagenic potentiator effect of chlorophyllin by the HGPRT assay.

Chlorophyllin, a sodium-copper salt derivative of chlorophyll-a and -b, was evaluated for antimutagenic activity against ethyl methane sulfonate by the hypoxanthin-guanine-phosphoribosyl transferase (HGPRT) assay. The results obtained suggest that this chlorophyllin can potentiate the mutagenicity of an alkylating agent which induces DNA damage.

Effects of chlorophyllin on mouse embryonic and fetal development in vivo.

Chlorophyllin (CHL) has proven that there is antimutagenic and anticarcinogenic activity in several organisms without causing lethal effects. However, there is no information about its effects when it is administered in gestation. In the present study, we assessed possible effects of CHL when it was administered to CD-1 mice on the 8th day of gestation using the same doses and administration route used in ours previous antimutagenic and antigenotoxic studies. Females were exposed to a single dose of CHL by i.p. injection (20, 40, 50, or 100mg/kg b.w.). On day 18 all dams were subjected to cesarean section and the fetuses were examined with common teratological methods. Results show that CHL-treatment induced total litter loss and is dose-dependent, probably due to either the interaction between CHL and some general control mechanisms of embryo development or by an impairment of maternal-fetal interactions. The analysis of uterine horns of the CHL-treated females with total litter loss revealed the presence of green rings in the uterus. Results show the inverse relationship between the number of live implants and the frequency of green rings, indicating implantation sites where embryo death and early resorptions occurred. Although CHL was given in a single dose on day 8 in this study, the results indicate that CHL is associated with significant embryo lethality.

Investigation of genotoxic and antigenotoxic activities of chlorophylls and chlorophyllin in cultured V79 cells.

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.

Evidence that chlorophyllin (CHLN) may behave as an inhibitor or a promoter of radiation-induced genetic damage in somatic cells of drosophila.

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.

Validation of single cell gel assay in human leukocytes with 18 reference compounds.

To validate the alkaline single cell gel (SCG) assay as a tool for the detection of DNA damage in human leukocytes, we investigated the in vitro activity of 18 chemicals. Thirteen of these chemicals (pyrene (PY), benzo(a)pyrene (BaP), cyclophosphamide (CP), 4-nitroquinoline-1-oxide (4NQO), bleomycin (BLM), methylmercury chloride (MMC), mitomycin C (MTC), hydrogen peroxide (HP), diepoxybutane (DEB), glutaraldehyde (GA), formaldehyde (FA), griseofulvin (GF), sodium azide (NA)) are genotoxic in at least one cell system, while five compounds (ascorbic acid (AA), glucose (GL), D-mannitol (MAN), O-vanillin (VAN), chlorophyllin (CHL)) are classified as non-genotoxic. In this in vitro SCG assay, PY, BaP and CP were positive with exogeneous metabolic activation (rat S9 mix) while 4NQO, BLM, MMC, MTC, hydrogen peroxide, and diepoxbutane were positive in the absence of metabolic activation. CHL and VAN were unexpectedly found to induce a dose-dependent increase in DNA migration. AA, GL, and MAN were negative in a non-toxic range of doses. GF gave equivocal results, while FA and GA increased DNA migration at low doses and decreased DNA migration at higher doses. This behaviour is consistent with the known DNA damaging and crosslinking properties of these compounds. These data support the sensitivity and specificity of this assay for identifying genotoxic agents.

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.

Chlorophyllin, an antimutagen, acts as a tumor promoter in the rat-dimethylhydrazine colon carcinogenesis model.

Chlorophyllin (CHL), the water soluble sodium/copper salt of chlorophyll, was investigated for its effect on colorectal cancer risk in the rat-dimethyldrazine colon carcinogenesis model. Ninety weanling Fisher 344 male rats were treated with five weekly injections of 1,2 dimethylhydrazine (DMH), 20 mg base/kg body weight. Rats had been previously divided into three groups, consuming either rat chow and water (Group I), rat chow and CHL 1.5 mM in water throughout the experiment (Group II), or water and rat chow during DMH injection, adding CHL 1.5 mM to the drinking water after completion of the DMH treatments. At sarcifice, the incidence and yield of colorectal tumors were as follows: Group I 10% and 0.1; Group II, 23% and 0.27; and Group III, 47% and 0.53 (p less than 0.005 for incidence and = 0.003 for yield). These data demonstrate that, though it is well established that CHL is an antimutagen, CHL in this colorectal carcinogenesis model acted as a tumor promoter.

Photodynamic toxicity of porphyrins and chlorins for a human tumor cell line: combined light and concentration dose responses for the retained fraction.

In recent years porphyrins and related materials have been tested as antitumor agents. A technique was devised to obtain dose-response curves for the sensitizer fraction that resists one day of elution by tissue culture medium--the retained fraction. We found a steep "threshold" dose response relationship that helps to explain tumor destruction without damage to normal tissues. The family of dose-response curves produced by a wide range of light exposures suggests that chlorins and porphyrins do not act by identical mechanisms. Moreover, they suggest that chlorins will prove superior in practical use.