Vladimir Misik

Sonodynamic toxicity of gallium-porphyrin analogue ATX-70 in human leukemia cells

Low concentrations (> or = 1 microM) of the gallium-porphyrin analogue ATX-70 significantly enhanced cellular toxicity in human leukemia HL-525 cells exposed to 50 kHz ultrasound. The mechanism of this ATX-70-dependent sonosensitization is unknown, but we have established the requirement of extracellular localization of ATX-70 molecules for sonosensitization. Short-lived toxic intermediates produced from ATX-70 by ultrasound are implicated in the mechanism, since no cytotoxicity was found when medium containing ATX-70 was sonicated and subsequently added to the cells. However, we were unable to demonstrate the existence of radical intermediates by EPR spin trapping with the nitroso spin trap, DBNBS, and ATX-70-dependent sonotoxicity could not be ameliorated by the addition of up to 70 mM POBN and DMPO spin traps during ultrasound exposure.

INCREASED CYTOTOXICITY OF 3-MORPHOLINOSYDNONIMINE TO HEPG2 CELLS IN THE PRESENCE OF SUPEROXIDE DISMUTASE. ROLE OF HYDROGEN PEROXIDE AND IRON

3-Morpholinosydnonimine (SIN-1) is widely used to generate nitric oxide (NO) and superoxide radical (O). The effect of SOD on the toxicity of SIN-1 is complex, depending on what is the ultimate species responsible for toxicity. SIN-1 (<1 mM) was only slightly toxic to HepG2 cells. Copper, zinc superoxide dismutase (Cu,Zn-SOD) or manganese superoxide dismutase (Mn-SOD) increased the toxicity of SIN-1. Catalase abolished, while sodium azide potentiated, this toxicity, suggesting a key role for H2O2 in the overall mechanism. Depletion of GSH from the HepG2 cells also potentiated the toxicity of SIN-1 plus SOD. Although Me2SO, sodium formate, and mannitol had no protective effect, iron chelators, thiourea and urate protected the cells against the SIN-1 plus Cu,Zn-SOD-mediated cytotoxicity. The cytotoxic effect of Cu,Zn-SOD but not Mn-SOD, showed a biphasic dose response being most pronounced at lower concentrations (10-100 units/ml). In the presence of SIN-1, Mn-SOD increased accumulation of H2O2 in a concentration-dependent manner. In contrast, Cu,Zn-SOD increased H2O2 accumulation from SIN-1 at low but not high concentrations of the enzyme, suggesting that high concentrations of the Cu,Zn-SOD interacted with the H2O2. EPR spin trapping studies demonstrated the formation of hydroxyl radical from the decomposition of H2O2 by high concentrations of the Cu,Zn-SOD. The cytotoxic effect of the NO donors SNAP and DEA/NO was only slightly enhanced by SOD; catalase had no effect. Thus, the oxidants responsible for the toxicity of SIN-1 and SNAP or DEA/NO to HepG2 cells under these conditions are different, with H2O2 derived from O dismutation playing a major role with SIN-1. These results suggest that the potentiation of SIN-1 toxicity by SOD is due to enhanced production of H2O2, followed by site-specific damage of critical cellular sites by a transition metal-catalyzed reaction. These results also emphasize that the role of SOD as a protectant against oxidant damage is complex and dependent, in part, on the subsequent fate and reactivity of the generated H2O2.

Effect of gallium-porphyrin analogue ATX-70 on nitroxide formation from a cyclic secondary amine by ultrasound : on the mechanism of sonodynamic activation

Sonodynamic therapy is a promising new modality for cancer treatment based on the synergistic effect on tumor cell killing by combination of a drug (typically a photosensitizer) and ultrasound. The mechanism of sonodynamic action was suggested to involve photoexcitation of the sensitizer by sonoluminescent light, with subsequent formation of singlet oxygen. In this work we studied the aqueous sonochemical reactions of the gallium-porphyrin derivative ATX-70, one of the most active sonodynamic agents found, using 50 kHz ultrasound. The experiments were carried out in the presence of 2,2,6,6-tetramethyl-4-piperidone hydrochloride (TMP), which reacts with singlet oxygen or .OH radicals to give the EPR-detectable nitroxide 2,2,6,6-tetramethyl-4-piperidone-N-oxyl (TMP-NO). Recently it has been suggested that the enhancement of TMP-NO yields in the presence of aqueous solutions of ATX-70 exposed to ultrasound was evidence for the formation of singlet oxygen in the system. Our results show that the surfactant cetyltrimethylammonium bromide (CTAB) can mimic the ATX-70-induced increase in the TMP-NO signal, but it fails to reproduce the behavior of ATX-70 in D2O: while the yields of TMP-NO in the presence of ATX-70 increase in D2O, the opposite effect was found with the surfactant CTAB. However, our data show that the increased TMP-NO yields in D2O are paralleled by an increased concentration of ATX-70 dimer, a form that is inactive in the photochemical generation of singlet oxygen. Our finding that the ATX-70-dependent enhancement of the TMP-NO signal was highest at approximately 20% O2, in both N2/O2 and argon/O2 mixtures, and decreased with increasing oxygen concentration is not compatible with the singlet oxygen mechanism. Finally, our results on the temperature dependence of the ATX-70-induced formation of TMP-NO are not consistent with the photochemical excitation of ATX-70 by sonoluminescent light: the ATX-70-dependent enhancement of TMP-NO signal increased with temperature in the range 10-25 degrees C, while the intensity of sonoluminescence of aqueous solutions both in multiple-bubble fields and in single-bubble experiments is known to decrease with increasing temperature.

Peroxyl radical formation in aqueous solutions of N,N-dimethylformamide, N-ethylformamide, and dimethylsulfoxide by ultrasound: Implications for sonosensitized cell killing

Sonodynamic therapy, which refers to a synergistic effect of drugs and ultrasound, is a promising new modality for cancer treatment. The sonodynamic effect was found for a number of structurally unrelated compounds, and the underlying mechanisms are still unknown. Recently, Jeffers et al. (J. Acoust. Soc. Am. 97:669-676; 1995) have shown that the sonodynamic action of nontoxic concentrations of N,N-dimethylformamide (DMF), N-methyl formamide (MMF), and dimethylsulfoxide (DMSO) combined with ultrasound, on killing of cultured HL-60 human promyelocytic leukemia cells, and attributed this toxic effect to unknown short lived reactive species produced from these solutes by ultrasonic cavitation. Using the spin trap 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS) in nitrogen-saturated aqueous solutions of DMF, MMF, or DMSO exposed to 50 kHz ultrasound, we detected formation of .CH3 and .CH2N(CH3)CHO radical adducts for DMF, mostly .CH2NHCHO adducts for MMF, and .CH3 adducts for DMSO. These radicals were formed either by reactions of the solutes with ultrasound-generated .H and .OH radicals (such as .CH2R-type radicals in DMF and MMF, and .CH3 radicals in DMSO), or by direct pyrolysis of the weak bonds in the solute molecules (e.g., .CH3 radicals from DMF). In air-saturated sonicated solutions these carbon centered radicals were converted to the corresponding peroxyl radicals and spin trapped with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO); .OOCH2N(CH3)CHO radicals were identified in DMF, .OOCH2NHCHO radicals in MMF, and .OOCH3 radicals in DMSO solutions. We suggest that these radical species by virtue of their longer lifetimes and higher selectivity, compared to .OH radicals, which are also formed in sonicated solutions, are the species responsible for sonodynamic cell killing by the combined effect of ultrasound with DMF, MMF, or DMSO.

EPR study of free radicals induced by ultrasound in organic liquids. II. Probing the temperatures of cavitation regions

Abstract The spin trap nitrosodurene was used for the detection of radical intermediates formed by 50kHz ultrasound in argon-saturated n-alcohols, n-alkanes, cyclic ethers, toluene, N,N-dimethylformamide (DMF) and dimethylacetamide (DMA). Radicals, produced by pyrolysis in collapsing cavitation bubbles, such as .CH2R in n-alkanes, .CH2R and .CH2OH in n-alcohols, .CH2-phenyl in toluene, .CH2OR and .CH2R in cyclic ethers and .CH3 and .N(CH3)R in DMF and DMA were spin trapped. Secondary radicals formed by hydrogen abstraction from organic liquids (such as .CHRR′ radicals in n-alcohols and n-alkanes and .CH2N-type radicals in DMF and DMA) by the primary pyrolysis radicals were also spin trapped. For a series of n-alcohols, the logarithm of the rate of radical formation decreases linearly with the vapour pressures of the n-alcohols. The kinetic isotope effect, kH/kD, for the ultrasound-induced production of .CHRR′ and .CDRR′ radicals in mixtures of n-dodecane and n-dodecane-d26 was found to be 2.6. For the .CH2-phenyl and . CD 2 - phenyl -d 5 radicals from toluene-toluene-d8, the ratio kH/kD = 1.09 was obtained. From the temperature dependence of the kinetic isotope effect, the temperature region of hydrogen abstraction radical formation (.CHRR′) in n-dodecane was estimated to be 750±150 K; the effective temperature of the region where benzyl radicals are formed from toluene by sonochemical pyrolysis was estimated to be about 6000 K. This method appears to be a promising tool for probing the temperatures to different sonochemical regions.

Effect of gas-containing microspheres and echo contrast agents on free radical formation by ultrasound

Stabilized microbubbles (microspheres) are widely used to enhance the contrast of ultrasound imaging. Our data provide direct evidence that the contrast agents, Levovist, PVC-AN (polyvinylidene chloride-acrylonitryl copolymer), and Albunex (compared to 5% human albumin), at concentrations comparable to those used for ultrasound imaging, enhance H2O2 production (through the superoxide-dependent pathway) in air-saturated aqueous solutions exposed to 47 kHz ultrasound above the cavitation threshold. These agents also act as scavengers of .H atoms and .OH radicals, thus lowering H2O2 formation (by recombination of .OH radicals) in argon-saturated solutions. EPR spin trapping also reveals that secondary radicals derived from the contrast agents are produced by reactions with .H and .OH which are formed by pyrolysis of water inside cavitation bubbles. In addition, the contrast agents themselves undergo pyrolysis reactions in the cavitation bubbles as demonstrated by formation of methyl radicals. Possible deleterious consequences of the formation of sonochemical intermediates may have to be assessed, particularly since some of the echo contrast agents have been shown to lower the cavitation threshold of diagnostic ultrasound. Unlike the microspheres formed from organic molecules, inorganic microspheres, Eccospheres, because of their stability and inert nature with respect to participation in free radical processes, appear to be suitable tools for enhancing the yields of aqueous sonochemical reactions.

EPR characterization of free radical intermediates formed during ultrasound exposure of cell culture media.

Free radicals and/or hydrogen peroxide produced by exposure of cells to ultrasound are potentially cytotoxic and mutagenic. The formation and type of free radical species can be substantially modulated by the chemical composition of the media in which the ultrasound exposures of cells are carried out. In the current study, we examined the free radical intermediates formed during ultrasound exposure of a typical cell culture medium (RPMI-1640); the dominant free radicals that were identified by spin trapping were derived from the hydrophobic amino acids Trp, Leu, and Phe, and were formed by hydrogen abstraction from these amino acids. Compared to exposures in phosphate-buffered saline, the yield of *OH radicals and H2O2 was significantly reduced in the cell culture medium, glucose (the main organic component in the medium), and the hydrophobic amino acids (Trp, Phe, Tyr, Leu, Val, Met) being chiefly responsible for this effect. In contrast, other nonhydrophobic amino acids did not contribute significantly to the *OH or H2O2 decrease. These findings are consistent with the accumulation of hydrophobic solutes at the liquid-gas interface of the collapsing cavitation bubbles resulting in increased efficiency of radical scavenging.

Effects of cysteamine and cystamine on the sonochemical accumulation of hydrogen peroxide—implications for their mechanisms of action in ultrasound-exposed cells

Based on the observed cytoprotective effect of the intracellularly permeable radical scavenger cysteamine (+NH3CH2CH2SH) in cells exposed to ultrasound and the lack of protection by its oxidized cell-nonpermeable form, cystamine (+NH3CH2CH2S-SCH2CH2NH3+), it was suggested that inertial cavitation (the growth of small gas bubbles present in the liquid exposed to ultrasound and their subsequent violent collapse) and associated free radical production may occur intracellularly (Radiat. Res. 89:369; 1982). Here we demonstrate that high concentrations (> 10 mM) of the thiol cysteamine effectively lower H2O2 yields following ultrasound exposure in argon- and air-saturated phosphate buffered saline (PBS), while cystamine is less effective under argon and practically without effect in air-saturated PBS. Direct removal of H2O2 by cysteamine is the dominant mechanism while scavenging of the H2O2 precursors .OH and superoxide plays a lesser role. Since H2O2 is a known cytotoxic species capable of penetrating cells if produced extracellularly, these results offer an alternative hypothesis for the protective effect of cysteamine and the lack of protection by cystamine, based on their differential ability to lower ultrasound-dependent H2O2 yields, without the necessity of invoking intracellular cavitation.

Nitric oxide formation from hydroxylamine by myoglobin and hydrogen peroxide

Hydroxylamine (HA), which is a natural product of mammalian cells, has been shown to possess vasodilatory properties in several model systems. In this study, HA and methyl-substituted hydroxylamines, N-methylhydroxylamine (NMHA) and N,N-dimethylhydroxylamine (NDMHA), have been tested for their ability to generate free diffusible nitric oxide (NO) in the presence of myoglobin (Mb) and hydrogen peroxide. A NO-specific conversion of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO) to 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl (carboxy-PTI), measured by electron spin resonance (ESR) spectroscopy, along with nitrite and nitrate production, was observed for HA but not for NMHA and NDMHA. ESR measurements at 77 K showed the formation of the ferrous nitrosyl myoglobin, Mb-NO, in the reaction mixtures containing Mb, H2O2 and HA. Our data also demonstrate that Mb-NO is an end product of the reaction pathway involving Mb, H2O2 and HA, rather than a reaction intermediate in the formation of NO. In summary, our results demonstrate a possible pathway of NO formation from HA, however, the significance of this mechanism for bioactivation of HA in vivo is unknown at the present time.

Recent applications of EPR and spin trapping to sonochemical studies of organic liquids and aqueous solutions

Abstract In this paper we review some of our recent applications of the EPR spin trapping technique to sonochemical studies which include identification of radicals formed in organic liquids and aqueous mixtures of organic liquids, estimation of temperatures of sonochemical regions in mixtures of deuterated and non-deuterated solvents, and the identification of reactive radical intermediates which may play a role in synergistic cell killing by ultrasound and drugs (sonodynamic interactions).