Photodynamic Effect. Experience of Application of Photosensibility Series for Monitoring Microbiological Water Pollution

Water used for food purposes requires special water treatment procedures that ensure the death of microorganisms. One of the new promising ways to combat microbiological water pollution along with chlorination and ozonation is considered a photodynamic method involving the use of light and photosensitizers. The method is based on photo induced by the sensitizer the formation of active forms of oxygen, which due to the activation of free radical processes cause the death of microorganisms.

The beginning of studies of the photodynamic effect is connected with the works of O. Raab and G. von Thappeyner [1, 2], who in 1897 discovered that infusoria and other protozoans, stained with acridine derivatives, stop their growth and die under illumination. This phenomenon was called the photodynamic effect (action) (FD), which denoted the influence of light on the dynamics of cell growth, their mobility and death. It was soon shown that. For photodynamic damage of cells, in addition to the dye and light, oxygen is needed. The photodynamic effect is found in all living organisms. During the 20th century, primary mechanisms of photodynamic cell death were studied [3, 4]. It is shown that multiple lesions are induced in procaryotes as a result of photodynamic action: loss of ability to form colonies, damage to DNA, proteins, cell membranes. For the manifestation of the photodynamic effect, the presence of a photosensitizer is necessary, which increases the sensitivity of tissues and cells to light. The critical effect of the photosensitizer is the formation of active forms of oxygen in the body, the action of which as a result of photooxidation of most biologically significant structures: amino acids (methionine, histidine, tryptophan, etc.), nucleosides, lipids, polysaccharides leads to damage and cell death. There are two types of photodynamic processes. In the photodynamic effect of type I, the photoexcited molecules of the sensitizers of S pass into the excited singlet state of 1S * and then into the long-lived triplet state of 3T * and react with the substrate RHand the molecules of the medium, in particular, with water. Intermediate free-radical intermediates are formed, which then interact with oxygen and give a complex mixture of highly active products of a radical nature that continue reactions of free radical oxidation and damage biostructures. One of the damaging factors is singlet oxygen 1O2, which can destroy cells in the immediate vicinity of the photosensitizer molecules. Oxidizing ability of singlet oxygen is 2 orders of magnitude higher than that of normal oxygen. It can damage all the major components of cells. In nucleic acids, it attacks mainly a pair of thymine and uracil, and also causes cross-linking of DNA-DNA or DNA-protein, single-strand breaks of DNA. These effects are exacerbated by the fact that enzymes that repair DNA are particularly sensitive to singlet oxygen. However, in interphase cells, DNA is not a primary target for PDeffects, since photosensitizers usually localize in the cytoplasm and do not penetrate the nucleus [3, 4]. In proteins, disulfide bonds, cysteine, histidine, tyrosine, tryptophan and phenylalanine are most easily photocontained, especially if they are located on the surface of globules and are accessible to the photosensitizer. They usually playa key role in enzymatic activity, and therefore proteins are very sensitive to photodynamic effects. Proteins lose activity as a result of photoinduced disruption of the structure of the active site, internal cross-links or intermolecular cross-links with other proteins, lipids, RNA and DNA. In type I photodynamic reactions, the radical pairs formed during electron transfer are relatively stable in an aqueous medium, where the reverse electron transport is difficult. In nonpolar lipid media, the lifetime and solubility of IO2 are higher. Consequently, type I photodynamic reactions are easier in the cytosol, and type II in the lipid phase of biomembranes. Thus, the photodynamic reactions with the participation of hydrophilic photosensitizers predominantly proceed according to the first type, and the hydrophobic photosensitizers according to the second type. Type II reactions dominate the damaging effects of most photosensitizers, including porphyrins, chlorins, phthalocyanines, and so on.

The development of oxidative stress, the disruption of the functions of cells and, as a result, their death are due to the intense generation of reactive oxygen species: superoxide radical anion (O2-), hydroxyl, hydroperoxyl radicals (OH •, HO2 •), hydrogen peroxide (H2O2), singlet oxygen (IO2). The photodynamic effect manifests itself both with UVirradiation, but especially light acts in the red wavelength range (620-780) nm. Since 1903, the study of the potential therapeutic value of the photodynamic effect began, the skin cancer was first cured with eosin staining and bright sunlight [5], since 1970 photodynamic therapy has been widely used to treat tumors [6, 7], in treatment of periodontal diseases [8-15]. Methylene green, acridine orange and proflavine, methylene blue and toluidine blue, indocyanine green, Bengal pink, eosin, curcumin, chlorine, porphyrins, phthalocyanines, chalcogen-containing benzophenoxazinium dyes, conjugates of nanoparticles with methylene blue, porphyrin or chlorine are used as photosensitizers. Cationic photosensitizers are most effective, since a positive charge enhances the interaction of the dye with the negatively charged surface of the microorganism.

In recent years, the prospects for using the photodynamic effect for purifying drinking water, controlling microbial contamination of water in aquariums and basins have been extensively studied [16-21].

This paper presents the experience of inactivation of a number of microorganisms in water due to the photodynamic effect using red light and a number of non-toxic photosensitizers in the process of water conditioning for food purposes. The dynamics of growth and death of standard museum strains of microorganisms of different types: prokaryotic cells of Escherichia coli ATCC 35218, and also cells of eukaryotes Candida albicans ATCC 24433 were studied.

The bacterium Escherichia coli (E. coli) is found in the intestines of humans and warm-blooded animals. Most strains of E. coli are harmless, but some strains, for example, O157: H7, O121, O104: H4 and O104: H21, synthesize potentially deadly toxins that can contribute to human infection by nutritional methods with low food hygiene. The ability of virulent strains to survive for sometime in the environment makes them an important indicator for investigating the presence of traces of fecal contamination in water. Representatives of the genus Candida (primarily Candida albicans) are classified as conditionally pathogenic varieties of fungal infection. Microorganisms of the genus Candida are part of the normal microflora of the mouth, esophagus, vagina and large intestine of most healthy people. The disease is caused not only by the presence of fungi of the genus Candida, but by their multiplication in large numbers or by the entry of more pathogenic strains of the fungus. Most often, candidiasis occurs in people with a decrease in general and local immunity.

Methylene blue (I), eosin (II), sodium fluorescein (III) and riboflavin (vitamin B2) (IV) were used as sensitizers. The compound formulas are shown in Scheme 1.

Methylene blue (I) Eosin (II)

Fluorescein sodium (III) Riboflavin (vitamin B ) (IV) 2 SCHEME