Peter Riesz

Free radical formation induced by ultrasound and its biological implications

The chemical effects of ultrasound in aqueous solutions are due to acoustic cavitation, which refers to the formation, growth, and collapse of small gas bubbles in liquids. The very high temperatures (several thousand K) and pressures (several hundred atmospheres) of collapsing gas bubbles lead to the thermal dissociation of water vapor into .OH radicals and .H atoms. Their formation has been confirmed by electron spin resonance (ESR) and spin trapping. The sonochemistry of aqueous solutions of gases and of volatile and nonvolatile solutes is reviewed. The similarities and differences between sonochemistry and radiation chemistry of aqueous solutions are explained. Some unusual characteristics of aqueous sonochemistry can be understood by considering the properties of supercritical water. By the use of rare gases with different thermal conductivities, it is possible to distinguish between temperature-dependent processes such as redox reactions initiated by .OH radicals and .H atoms and pressure-dependent processes which lead to polymer degradation and cell lysis. The evidence for free radical formation in aqueous solutions by pulsed ultrasound is discussed. This subject is of interest because it is related to the possible deleterious effects of ultrasonic diagnostic devices. The role of free radicals and of mechanical effects induced by ultrasound in DNA degradation, inactivation of enzymes, lipid peroxidation, and cell killing is reviewed.

Chemical effects of ultrasound on aqueous solutions. Evidence for hydroxyl and hydrogen free radicals (.cntdot.OH and .cntdot.H) by spin trapping

Results of spin-trapping and electron spin resonance studies are reported that provide conclusive evidence for the formation of hydroxyl radicals (.OH) and hydrogen atoms (.H) during the sonolysis of aqueous solutions. Nonvolatile nitrone spin traps, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), ..cap alpha..-tert-butyl-..cap alpha..-(1-methyl-4-pyridinio)nitrone (PyBN), and ..cap alpha..-4-pyridyl-N-tert-butylnitrone 1-oxide(POBN), were used. Experiments were carried out in which scavengers competed with these spin traps for the radicals formed. The formate radical and ethanol were used as scavengers since the reactions of HCOO/sup -/ and C/sub 2/H/sub 5/OH with .OH, .H, and e/sub aq//sup -/ are well known. As a final test, aqueous POBN solutions containing CdSO/sub 4/, a well-known e/sub aq//sup -/ scavenger, were sonicated with no decrease in the signal intensity of H-POBN adduct being noted. This was interpreted as indication that no e/sub aq//sup -/ was being formed. (BLM)

A STUDY OF THE PHOTODYNAMIC EFFICIENCIES OF SOME EYE LENS CONSTITUENTS

We have studied the photochemical quantum yields of singlet oxygen production (using the RNO bleaching method) and superoxide production (using the EPR‐spin trapping method and the SOD‐inhibitable ferricytochrome c reduction spectral assay) of kynurenine (Ky), N‐formylkynurenine (NFK), 3‐hydroxykynurenine (3HK), kynurenic acid (KUA), and the flavins, riboflavin (RF) and flavin mononucleotide (FMN). Such a study of the photodynamic efficiencies is important since these compounds appear endogenously in the eye. The singlet oxygen quantum yields of the flavins and KUA are high, while Ky and 3HK generate no detectable amounts of singlet oxygen. The superoxide quantum yields of the sensitizers are low compared to their singlet oxygen, and Ky and 3HK produce no detectable amounts of superoxide. The production of the superoxide radical is enhanced in the presence of electron donor molecules such as EDTA and NADH. These results suggest that the production of oxyradicals in the lens may be modulated by the presence of endogenous electron donor molecules such as the coenzymes NADH and NADPH, which are present in significant amounts in some lenses. They also suggest that Ky and 3HK, which are known to be present in aged lenses, might play a protective rather than a deleterious role in the eye.

Photochemical Generation of Superoxide Radical and the Cytotoxicity of Phthalocyanines

The effect of the central metal atom on the photodynamic activity of phthalocyanine dyes has been estimated by cytotoxicity to cultured Chinese hamster cells. Chloroaluminium phthalocyanine,, followed by the Zn- derivate, were found to be the only active dyes. In parallel it was found that visible light (615 +/- 10 nm) excitation of phthalocyanines dissolved in dimethylsulphoxide in the presence of oxygen generates superoxide radical anion. O2- radicals were spin--trapped with 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) and identified by electron spin resonance. The quantum yields for O2- generation range from 10(-5) (Zn-phthalocyanine) to 4.2 X 10(-4) (Ga-phthalocyanine). The efficiency of generating O2- was apparently uncorrelated with the phototoxicity of the same dyes. Furthermore, the biological photodamage could not be inhibited by the addition of superoxide dismutase. It is concluded that O2- is involved very little, if at all, in the phthalocyanine-induced photo-killing of mammalian cells.

Free radical intermediates in sonodynamic therapy.

Abstract: Current understanding of the mechanism of sonodynamic action (i.e. the ultrasound‐dependent enhancement of the cytotoxic action of certain drugs ‐ sonosensitizers) with potential applications for cancer therapy is presented. The experimental evidence suggests that sonosensitization is due to the chemical activation of sonosensitizers inside or in the close vicinity of hot collapsing cavitation bubbles to form sensitizer‐derived free radicals either by direct pyrolysis or due to reactions with ·H and ·OH radicals, formed by pyrolysis of water. These free radicals (mostly carbon‐centered) react with oxygen to form peroxyl and alkoxyl radicals. Unlike ·OH and ·H, which are also formed by pyrolysis inside cavitation bubbles, the reactivity of alkoxyl and peroxyl radicals with organic components dissolved in biological media is lower and hence have higher probability of reaching critical cellular sites. Sonodynamic therapy appears to be a promising modality for cancer treatment since ultrasound can penetrate deep within the tissue and can be focused in a small region of tumor to chemically activate relatively non‐toxic molecules (e.g. porphyrins) thus minimizing undesirable side effects.

The role of molecular oxygen in the photodynamic effect of phthalocyanines

Phthalocyanines are a class of mammalian cell photosensitizers which may be useful in photodynamic therapy for cancer. Chloroaluminum phthalocyanine tetrasulfonate was incubated with Chinese hamster cells in culture and exposed to white light at different concentrations of oxygen. The ability of the cells to form colonies served as an end point for the photobiological effect of the dye. The efficiency of photoinactivation of the sensitized cells decreased with decreasing oxygen concentration. Very little photoinactivation was observed when the atmosphere equilibrated with the cells was oxygen-free nitrogen. At an oxygen partial pressure of 2.5 mm Hg, photoinactivation was reduced by 50% compared to ambient atmosphere. In an attempt to understand the nature of the interaction between excited dyes and oxygen, the ability of several phthalocyanines to photogenerate singlet oxygen was measured. Thus phthalocyanines containing paramagnetic ions (copper, iron, vanadyl) do not generate 1O2 in contradistinction to diamagnetic metals (zinc and aluminum). The latter are efficient photosensitizers, while the former have little if any photobiological activity. In spite of this correlation, singlet oxygen may not be the intermediate involved in cytotoxicity. The reasons are discussed.

Chemical effects of ultrasound on aqueous solutions. Evidence for. OH and. H by spin trapping

Results of spin-trapping and electron spin resonance studies are reported that provide conclusive evidence for the formation of hydroxyl radicals (.OH) and hydrogen atoms (.H) during the sonolysis of aqueous solutions. Nonvolatile nitrone spin traps, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), ..cap alpha..-tert-butyl-..cap alpha..-(1-methyl-4-pyridinio)nitrone (PyBN), and ..cap alpha..-4-pyridyl-N-tert-butylnitrone 1-oxide(POBN), were used. Experiments were carried out in which scavengers competed with these spin traps for the radicals formed. The formate radical and ethanol were used as scavengers since the reactions of HCOO/sup -/ and C/sub 2/H/sub 5/OH with .OH, .H, and e/sub aq//sup -/ are well known. As a final test, aqueous POBN solutions containing CdSO/sub 4/, a well-known e/sub aq//sup -/ scavenger, were sonicated with no decrease in the signal intensity of H-POBN adduct being noted. This was interpreted as indication that no e/sub aq//sup -/ was being formed. (BLM)

Sensitized Photo-oxidation of Thymidine by 2-methyl-1,4-naphthoquinone. Characterization of the Stable Photoproducts

The near ultraviolet photolysis of an aerated aqueous solution of thymidine containing 2-methyl-1,4-naphthoquinone gives rise to two main classes of photoproducts as a result of the initial formation of a pyrimidine radical cation. These photo-oxidation products have been separated by high performance liquid chromatography and further characterized by various spectroscopic techniques including fast atom bombardment mass spectrometry and high field 1H and 13C nuclear magnetic resonance analysis. This photoreaction constitutes an excellent model to study the chemical properties of the thymidine radical cation which is expected to be one of the primary consequences of the direct effects of ionizing radiation.

Sonochemistry of volatile and non-volatile solutes in aqueous solutions : e.p.r. and spin trapping studies

Recent spin trapping studies of the free radical intermediates generated by the sonolysis of aqueous solutions are reviewed. Studies of rare gas saturated solutions of volatile solutes (e.g., methanol and ethanol) and of non-volatile solutes (acetate, amino acids, sugars, pyrimidines, nucleotides and surfactants) are consistent with the theory of three reaction zones in aqueous sonochemistry. The very high temperatures and pressures induced by acoustic cavitation in collapsing gas bubbles in aqueous solutions lead to the thermal dissociation of water vapour into hydrogen atoms and hydroxyl radicals. Reactions take place in the gas phase (pyrolysis reactions), in the region of the gas-liquid interface, and in the bulk of the solution at ambient temperature (similar to radiation chemistry reactions). By use of the rare gases with different thermal conductivities, the contributions of individual reaction steps with widely different energies of activation can be evaluated.

E.S.R. of Spin-trapped Radicals in Aqueous Solutions of Amino Acids: Reactions of the Hydroxyl Radical

The radicals produced by reactions of hydroxyl radicals with amino acids in aqueous solutions have been investigated. Hydroxyl radicals were formed by U.V.-photolysis of hydrogen peroxide and the short-lived amino acid radicals were spin-trapped by tert-nitrosobutane and identified by electron spin resonance spectroscopy. Nineteen amino acids were studied, and several radicals were identified which have not been observed previously by other methods. Only side-chain radicals were identified for alanine, threonine, aspartic acid, asparagine, lysine, phenylalanine, tyrosine, proline and hydroxyproline; whereas for glycine the C(2) carbon radical was spin-trapped. Both C(2) carbon radicals and side-chain radicals were assigned to valine, leucine, isoleucine, serine, glutamic acid, glutamine, arginine and methionine.