Mechanisms involved in the photosensitized inactivation of Acanthamoeba palestinensis trophozoites.

AIMS: To advance our understanding of the mechanisms involved in the RLP068 phthalocyanine-photosensitized inactivation of Acanthamoeba palestinensis trophozoites through a precise identification of the targets of the photoprocess in both the cytosolic and mitochondrial compartments. METHODS AND RESULTS: We followed the activities of selected marker enzymes as well as we performed fluorescence and transmission electron microscopy investigations of the alterations induced by the photoprocess in the fine structure of subcellular compartments. RLP068 is preferentially located in the contractile vacuole: the fluorescence in that site is particularly evident in the unirradiated cells and becomes more diffused after irradiation. Electron microscopic analysis of photosensitized A. palestinensis cells clearly shows that the swelling of trophozoites and the appearance of vacuoles spread throughout the cytoplasm after phototreatment. The activity of a typical cytoplasmic enzyme, such as lactate dehydrogenase, underwent a 35% decrease as a consequence of the photoprocess, reflecting the photodamage induced by migrating phthalocyanine molecules in their micro-environment. CONCLUSIONS: The presence of multiple targets for the phthalocyanine-photosensitized process is of utmost importance because this pattern of cell damage makes it unlikely that photoresistant A. palestinensis strains are gradually selected or mutagenic phenomena are developed as a consequence of the photoinduced damage. SIGNIFICANCE AND IMPACT OF THE STUDY: Photosensitization via phthalocyanines appears to represent an efficient and safe approach for achieving a close control of the population of a potentially pathogenic protozoan such as A. palestinensis, opening new perspectives for the disinfection of microbiologically polluted waters.

Sensitivity to copper in a ciliate as a possible component of biological monitoring in the lagoon of venice

The impact of copper was studied in cultures of the microbenthic organism Euplotes vannus (Ciliophora, Hypotrichida). This ciliate was isolated from sediment collected from a particular area in the Lagoon of Venice, which may be considered almost unpolluted by heavy metals. The effects of copper exposure in the laboratory on growth, metal accumulation, total acid-soluble thiol levels, and glutathione levels were examined, together with morphological alterations. E. vannus exhibited tolerance toward copper up to a concentration exposure of 0.2 &mgr;g Cu/ml, at which cell growth rate overlapped that of controls. Copper accumulated up to 239 &mgr;g/g dry wt after exposure to 0.4 &mgr;g Cu/ml, and morphological alterations were evident in cells exposed to concentrations from this value upward. Processes of vegetative reorganization involving nuclear apparatus, membranelles, cirri, and cortex could be observed after 4 days of exposure to 0.4 &mgr;g Cu/ml. Laboratory experiments with cultures of ciliates of cosmopolitan distribution such as Euplotes species in controlled conditions indicated the value of these organisms, which constitute a simple model for a monitoring system suitable for prediction in multicellular organisms.

Porphyrin photosensitised processes in the prevention and treatment of water- and vector-borne diseases.

Water- and vector-borne diseases are a global burden which is estimated to cause several million deaths and innumerable cases of sickness every year. These infectious illnesses are emerging or resurging as a result of several factors, such as changes in climate, in public health and demography policy, as well as the spread of resistance to insecticide and drug, and genetic changes in pathogens. Integrated prevention strategies must be developed and implemented in endemic disease areas to reverse the trend of emergent/resurgent water- and vector-borne diseases. With this perspective porphyrins and their analogues, that have been shown to act as very efficient photosensitising agents against a broad number of microbial pathogens (bacteria, fungi, protozoa) and parasitic animals, could represent an important tool for the prevention and control of these pathologies. The application of photosensitised processes can be exploited to address environmental problems of high significance, including the decontamination of waste waters, the disinfection of fish-farming tanks and the control of populations of noxious insects. Such diversified applications take advantage of the availability of a truly large number of porphyrin derivatives with chemical structures which can be tailored to comply with the physical and chemical properties, as well as the biological features of several milieus. In addition, the property typical of porphyrins to absorb essentially all the wavelengths in the sun emission spectrum allows the promotion of processes largely based on natural resources with significant energy saving and low impact on the ecosystems.

Photosensitized inactivation of Acanthamoeba palestinensis in the cystic stage.

AIMS: To develop alternative approaches for medical and environmental control of pathogenic Acanthamoeba spp. by means of photodynamic treatment with a tetracationic Zn(II)-phthalocyanine (RLP068). METHODS AND RESULTS: Incubation of cyst cultures with RLP068 for 1 h caused an accumulation of readily detectable concentrations of the phthalocyanine, even at doses as low as 0.5 micromol l(-1). RLP068 exhibited no dark toxicity towards cysts up to 5 micromol l(-1) concentration. A decrease of c. 50% in cyst survival in comparison with controls was measured upon incubation of the cysts with 0.5 micromol l(-1) RLP068, followed by exposure to light (600-700 nm) for 20 min at a fluence rate of 50 mW cm(-2) (60 J cm(-2)). After incubation with 3 and 5 micromol l(-1) RLP068 and irradiation, the cysts lost their excystment ability as early as day 5 and up to day 10, and were clearly damaged when observed under an interference contrast microscope. CONCLUSIONS: These data indicate the promising use of RLP068 in phototreatment of diseases caused by pathogenic amoebae and in initial disinfection of wastewaters. SIGNIFICANCE AND IMPACT OF THE STUDY: Rapid and extensive photodamage may be induced in the highly resistant cystic stages by means of 600- to 700-nm light sources.

Chelatins in Euglena gracilis and Ochromonas danica.

Amino acid analysis was performed on low molecular weight copper binding proteins purified from two species of Protozoa after exposure to a high level of this metal. The compound from Ochromonas is similar to Cu-chelatins. The two peptides from Euglena have a different molecular weight and a very dissimilar amino acid composition. Peptide No. 1 has a peculiar composition with a high content of aspartic acid and arginine. Some speculations are made about its detoxification role in comparison with other compounds found in blue-green algae.

Biochemical and ultrastructural data on Tetrahymena pyriformis treated with copper and cadmium.

The addition of copper (10 micrograms ml-1) or cadmium (5 micrograms ml-1) to the medium is well tolerated by Tetrahymena pyriformis GL. Both metals are accumulated by cells, cadmium to a greater extent than copper. The growth rate is not affected and from the micrographs it is evident that the ultrastructure is not altered by the treatments. After 3 days of culture the macronucleus contains dense masses of chromatin and numerous nucleolar fusion bodies. Granules, cytolysomes and many food vacuoles are present in both control and treated cells. Cadmium induces the formation of a chelating protein; the amino acid analyses and the ultraviolet spectrum indicate that it is similar to the metallothionein isolated by higher organisms. The molecular weight of native protein is about 27,000. After treatment by sulphitolysis or oxidation we obtained a peak of molecular weight at about 6,000. The treatment with copper does not appear to induce metallothioneins or other chelatins. The high tolerance of Tetrahymena towards cadmium is believed to be due to the formation of a Cd-Zn metallothionein. The different chelating proteins induced by copper and cadmium in other groups of Protozoa and the different detoxification mechanisms present in these organisms are discussed.