Electron paramagnetic resonance spectra with g=2.03 of the animal liver tissue treated with butylhydroxytoluene and diethylnitrosamine

ESR spectra g=2.03 intensities of rat liver tissues were studied under the action of cancer agent either diethylnitrosamine (DEN) with butylhydroxytoluene (BHT) injected before DEN or without BHT at 77 K. Combined influence of DEN and BHT upon the intensity change of ESR spectra with g=2.03 at animal disease is explained by modified action of DEN metabolism.     

Comparative study of the ESR spectra of normal and regenerating liver tissue in mice

Low temperature ESR spectra of gamma-irradiated samples of mouse liver regenerating after partial hepatectomy were studied. As compared to ESR spectra of gamma-irradiated samples of health animals liver, in regenerating liver samples new ESR signals from paramagnetic centres S(2.0005) and S(2.0025) were revealed; these signals were earlier recorded by the authors in gamma-irradiated samples of hepatoma 22a. It is concluded that the paramagnetic centres S(2.0005) and S(2.0025) are typical of proliferating tissues of different ethiology.     

Decrease in the level and enzymatic activity of cytochrome P-450 during diethylnitrosamine metabolism in vivo and in vitro

Diethylnitrosamine (DENA) intoxication of rats was accompanied by a reduction of cytochrome P-450 content in the liver, which correlated well with inactivation of cytochrome P-450 during metabolism of DENA in the liver microsomes.     

Local Pharmacokinetic Analysis of a Stable Spin Probe in Mice by In Vivo L-band ESR with Surface-coil-type Resonators

In vivo ESR spectroscopy using a low frequency microwave of approximately 1 GHz has been developed to measure non-invasive ESR spectra in animals given paramagnetic compounds, in which a loop-gap-type resonator was used and ESR spectra were measured at the animal's head or abdomen. Therefore, the concentrations of paramagnetic species in both the blood and organs were compositely contributed to the spectra. When we understand the kinetics of paramagnetic species in detail, it is essentially important to know how these kinetics are expressed in each organ. For this purpose, a surface-coil-type resonator, which enabled local ESR measurement in specific organs, has been developed. By using this method, we studied the real-time pharmacokinetics of spin clearance curves detected in the organs of mice given 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (4-hydroxy-TEMPO) intravenously (i.v.), by monitoring the inferior vena cava, liver and kidney. Quantified clearance curves in the organs were analyzed on the basis of a two-compartment model, and pharmacokinetic parameters were estimated based on the curve-fitting. The obtained pharmacokinetic parameters were found to depend on the measurement site, and the distribution and elimination processes of the spin probe were successfully separated between the blood and organs of mice.     

Local pharmacokinetic analysis of a stable spin probe in mice by in vivo L-band ESR with surface-coil-type resonators

In vivo ESR spectroscopy using a low frequency microwave of approximately 1 GHz has been developed to measure non-invasive ESR spectra in animals given paramagnetic compounds, in which a loop-gap-type resonator was used and ESR spectra were measured at the animal's head or abdomen. Therefore, the concentrations of paramagnetic species in both the blood and organs were compositely contributed to the spectra. When we understand the kinetics of paramagnetic species in detail, it is essentially important to know how these kinetics are expressed in each organ. For this purpose, a surface-coil-type resonator, which enabled local ESR measurement in specific organs, has been developed. By using this method, we studied the real-time pharmacokinetics of spin clearance curves detected in the organs of mice given 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (4-hydroxy-TEMPO) intravenously (i.v.), by monitoring the inferior vena cava, liver and kidney. Quantified clearance curves in the organs were analyzed on the basis of a two-compartment model, and pharmacokinetic parameters were estimated based on the curve-fitting. The obtained pharmacokinetic parameters were found to depend on the measurement site, and the distribution and elimination processes of the spin probe were successfully separated between the blood and organs of mice.     

Intracellular free iron in liver tissue and liver homogenate: studies with electron paramagnetic resonance on the formation of paramagnetic complexes with desferal and nitric oxide.

Treatment of intact liver and liver homogenate with sodium nitrite, or desferal, brings about the appearance of g = 2.03 and g = 4.3 electron paramagnetic resonance spectroscopy (EPR) signals, respectively. The g = 2.03 signal is conditioned by the formation of dinitrosyl complexes of Fe(II); the g = 4.3 signal is related to the appearance of paramagnetic desferal-Fe(III) complexes. Desferal and sodium nitrite were administered successively into liver homogenate, resulting in only a g = 4.3 EPR signal. And, vice versa, if desferal was administered after sodium nitrite, there appeared only the signal with g = 2.03. These data testify to the fact that one and the same endogenous free iron is included in both paramagnetic centers. The concentration of iron ions was measured in intact tissue according to the formation of dinitrosyl-iron complexes and desferal-iron complexes. It was 33.2 +/- 4.6 and 20.3 +/- 4.0 nmol/g of tissue weight, respectively. The data obtained testify to the fact that free endogenous iron is present in intact tissue. Possibilities of the EPR method for estimation of the content of intracellular free iron are discussed.     

EPR study of flavo- and ubisemiquinones in liver tissue

Several types of flavins and quinones having different parameters of magnetic relaxation have been detected in the mouse liver tissue by means EPR technique. The values of these parameters depend on the redox state of the respiratory chain meal-containing centres. This means that there is an interaction between the free radicals and the metal-containing paramagnetic centres.     

Coordination behaviour of antithyroid drugs against the Fe(I)(NO)2 group in solution: ESR and FT-NMR study.

The binding sites of some types of antithyroid drugs in the presence of the Fe(I)(NO)2 paramagnetic probe were investigated. Coordination behaviour in solution of different structured ligands was determined by means of ESR parameters and 13C FT-NMR spectra. Selective isotopic substitution with 15NO combined with computer simultation was used to elucidate overlapping ESR patterns. A correlation between chemical structure and antithyroid activity of the pharmacological bases is suggested.     

Electron spin resonance of phosphorylating and non-phosphorylating submitochondrial particles

Paramagnetic centres related with the work of ATP-synthetase, found earlier on mitochondria were studied on phosphorylating and non-phosphorylating submitochondrial particles (SMP). A complex doublet signal with half-width 38 . 10(-4) T was shown to be recorded only on actively phosphorylating and oligomycin-inhibited SMP. It was found that the signal components were conditioned by different paramagnetic centres, one of which (g = 2.03) seemed to be the metal centre, more probable non-hemeiron. The nature of the second signal characterized by evenly saturating doublet components with g = 2.03 and g = 2.00 is unclear. Studies of flavin signal saturation have also shown that it consists of two components: one of them--saturating, specific for non-phosphorylating SMP, another--non-saturating, mostly observed in phosphorylating SMP, probably conditioned by flavin--the constituent of ATP-synthetase.     

Photolysis of N-methyl and N-acetyl substituted amino acids

Summary Experiments on the production of free radicals in aqueous solutions of N-methyl and N-acetyl substituted amino acids by UV light are reported. The ESR spectra observed at 77 K show that the bond of the CH_3–group of these substances is ruptured to a great deal compared with other bond ruptures producing paramagnetic centres. But in N-methyl substituted amino acids the methyl radical is less predominant than in N-acetyl substituted ones. At temperatures higher than 200 K the ESR spectra of all substances studied in our experiments are similar. It is supposed that a radical is formed with an unpaired spin near an oxygen atom.A part of this paper was reported at a Congress in Freiburg i. Br. (Germany) organized by the Deutsche Gesellschaft für Biophysik, October 2–4, 1974.     

A Possible Antineoplastic Potential of Selective,Irreversible Proteasome Inhibitor,Carfilzomib on Chemically Induced Hepatocarcinogenesis in Rats

The antineoplastic effect of carfilzomib (CFZ) against chemically induced hepatocarcinogenesis was studied. A total of 60 male Wistar albino rats were divided into six groups with 10 animals in each group. Rats in group 1 (control group) were given dimethylsulphoxide (DMSO) (0.4 mL/kg i.p) twice a week for 3 weeks from week 8 to week 10. Animals in groups 2 and 3 were given CFZ (2 and 4 mg/kg i.p) twice a week from week 8 to week 10, respectively. Rats in group 4 were given diethylnitrosamine (DENA) at a dose of 0.01% in drinking water for 10 weeks and received a DMSO (0.4 mL/kg i.p) twice a week from week 8 to week 10. Animals in groups 5 and 6 were given DENA at a dose of 0.01% in drinking water for 10 weeks and treated with CFZ (2 and 4 mg/kg i.p) twice a week from week 8 to week 10, respectively. CFZ succeeded in suppressing the elevated serum tumor marker α‐fetoprotein and carcinoembryonic antigen. The antineoplastic effect of CFZ was also accompanied by normalization of elevated hepatic tissue growth factors, matrix metalloproteinase‐2 and tissue inhibitor of metalloproteinase‐1, and augmentation of hepatic endostatin and metallothionein. A histopathological examination of liver samples treated with CFZ after DENA intoxication correlated with the biochemical observation. Treatment with CFZ confers an antineoplastic activity against chemically induced hepatocarcinogenesis. These findings suggest that CFZ plays a pivotal role in the treatment of hepatocarcinogenesis.     

Spin-trapping studies of hepatic free radicals formed following the acute administration of ethanol to rats: in vivo detection of 1-hydroxyethyl radicals with PBN.

The generation of free radicals in rat liver following the acute oral administration of ethanol was studied with the spin-trapping method, using a deuterated derivative of phenyl-N-tert-butylnitrone (PBN-d14) as the spin-trapping agent. After administration of ethanol and PBN-d14 to rats, organic extracts of the liver were prepared and subjected to ESR spectroscopy. In the case of ethanol-treated rats, the ESR spectra indicated that mixtures of radicals had been trapped, while spectra from control rats were essentially negative. The predominant spin adduct detected after ethanol treatment is proposed to be from a carbon-centered, primary alkyl radical, based on gamma-hydrogen hyperfine splitting patterns observed with PBN-d14. Oxygen-centered radicals also contributed to the ESR spectra. Liver extracts also contained low concentrations of the 1-hydroxyethyl radical spin adduct, which was indicated by weak spectral lines corresponding to those of the 1-13C-ethanol adduct. These data confirm previous suggestions that ethanol is metabolized to a free radical metabolite in rat liver. In addition, some information on types of lipid radicals generated during alcohol intoxication has been obtained.     

The decomposition of the EPR spectra of multicomponent systems irradiated at 77 K into paramagnetic center signals of different natures]

Main principles of the way to decompose an EPR spectrum of a multicomponent system, irradiated at 77 K, into separate radiation-induced paramagnetic centre signals are given. The decomposition is possible due to the computer assistant spectra processing, and is based on different properties of different paramagnetic centres, namely, on different thermostability of the centres, on different rate of relaxation, and on different photosensitivity. Concrete examples of the EPR spectrum decomposition into different free radical signals are given for cases of murine liver and spleen irradiated at 77 K. Radiochemical yields of different free radicals, induced by gamma radiation at 77 K in whole biological tissues, were defined. The data on nature and properties of the paramagnetic centres induced by radiation in biological tissues are shortly reviewed.     

Mitochondrial dysfunction in patients with severe sepsis: an EPR interrogation of individual respiratory chain components

Electron paramagnetic resonance (EPR) spectra of complex biological systems contain information about the paramagnetic centres present. Retrieving such information is important since paramagnetic species are common intermediates of all redox reactions in both normal and abnormal metabolism. However, it is often difficult to determine the nature and content of all paramagnetic species present because the EPR signals from individual centres overlap. Here, we apply our deconvolution method based on spectra subtraction with variable coefficient to quantify individual paramagnetic components of human muscle biopsies taken from critically ill patients with severe sepsis. We use low temperature EPR spectroscopy to identify and quantify nine different paramagnetic species in the tissue. These include the majority of the mitochondrial iron-sulfur centres and the first in vivo report of a mitochondrial radical assigned to a spin-coupled pair of semiquinones (SQ*-SQ*). We have previously demonstrated in these same muscle biopsies that biochemical assays of mitochondrial dysfunction correlate with clinical outcomes (D. Brealey, M. Brand, I. Hargreaves, S. Heales, J. Land, R. Smolenski, N.A. Davies, C.E. Cooper, M. Singer, Association between mitochondrial dysfunction and severity and outcome of septic shock. Lancet 360 (2002) 219-223.). Analysis of the paramagnetic centres in the muscle confirms and extends these findings: the (SQ*-SQ*) radical species negatively correlates with the illness severity of the patient (APACHE II score) and a decreased concentration of mitochondrial Complex I iron-sulfur redox centres is linked to mortality.     

On the detection of paramagnetic species in the adriamycin-perfused rat heart: a reappraisal

Recently, L. Costa et al. reported the direct detection of the superoxide anion and other paramagnetic species in the isolated, adriamycin-perfused rat heart [L. Costa et al. (1988) Biochem. Biophys. Res. Commun. 153, 275-280]. We have reevaluated the results of their study and concluded that the ESR parameters of the spectrum obtained from the adriamycin-perfused heart are consistent with that of the peroxyl radical and not with that of the superoxide anion. In addition, the ESR spectrum of the peroxyl radical is very likely produced as an artifact caused by the grinding of myocardial tissue. This artifact may mask the ESR spectra of the adriamycin-derived semiquinone radical and the iron-sulfur protein components of myocardium.     

Iron sources forming nitrosyl complexes in animal tissues

Treatment of perfused mouse liver with nitric oxide does not change the intensities of ESR signals of iron-sulfur proteins characteristic of this tissue. Proceeding from this evidence and also from the ratio between the iron content in these proteins and dinitrosyl iron complexes (complexes 2.03) formed in the liver when it contacts with NO, it is concluded that iron-sulfur proteins are not involved in the formation of complexes 2.03. It seems that only the loosely bound form of non-heme iron-free iron is involved in this process.     

Mitochondrial dysfunction in patients with severe sepsis: An EPR interrogation of individual respiratory chain components

Electron paramagnetic resonance (EPR) spectra of complex biological systems contain information about the paramagnetic centres present. Retrieving such information is important since paramagnetic species are common intermediates of all redox reactions in both normal and abnormal metabolism. However, it is often difficult to determine the nature and content of all paramagnetic species present because the EPR signals from individual centres overlap. Here, we apply our deconvolution method based on spectra subtraction with variable coefficient to quantify individual paramagnetic components of human muscle biopsies taken from critically ill patients with severe sepsis. We use low temperature EPR spectroscopy to identify and quantify nine different paramagnetic species in the tissue. These include the majority of the mitochondrial iron-sulfur centres and the first in vivo report of a mitochondrial radical assigned to a spin-coupled pair of semiquinones (SQ^·-SQ^·). We have previously demonstrated in these same muscle biopsies that biochemical assays of mitochondrial dysfunction correlate with clinical outcomes (D. Brealey, M. Brand, I. Hargreaves, S. Heales, J. Land, R. Smolenski, N.A. Davies, C.E. Cooper, M. Singer, Association between mitochondrial dysfunction and severity and outcome of septic shock. Lancet 360 (2002) 219-223.). Analysis of the paramagnetic centres in the muscle confirms and extends these findings: the (SQ^·-SQ^·) radical species negatively correlates with the illness severity of the patient (APACHE II score) and a decreased concentration of mitochondrial Complex I iron-sulfur redox centres is linked to mortality.     

Study of the binding of substrates and paramagnetic Mn2+ ions with phosphoglycerate kinase by saturating ESR spectra of a spin-labelled protein]

A single SH-group of phosphoglycerate kinase from yeast was modified by mercury-containing spin label. The saturation curves of ESR spectra of the spin-labeled enzyme were studied. The paramagnetic ions of Mn2+ bound to the centre of ion nonspecific binding or active centre in the complex with ATP can influence the saturation of the spin-labeled enzyme. The saturation curves of the ESR signal of the spin-labeled enzyme in the presence of paramagnetic complex of CrATP were studied. It has been demonstrated that the second nonspecific centre of ATP binding is located at the active site of the enzyme (3-phosphoglycerate binding centre).     

Generation and recycling of radicals from phenolic antioxidants

Hindered phenols are widely used food preservatives. Their pharmacological properties are usually attributed to high antioxidant activity due to efficient scavenging of free radicals. Butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) also cause tissue damage. Their toxic effects could be due to the production of phenoxyl radicals. If phenoxyl radicals can be recycled by reductants or electron transport, their potentially harmful side reactions would be minimized. A simple and convenient method to follow phenoxyl radical reactions in liposomes and rat liver microsomes based on an enzymatic (lipoxygenase + linolenic acid) oxidation system was used to generate phenoxyl radicals from BHT and its homologues with substitutents in m- and p-positions. Different BHT-homologues display characteristic ESR signals of their radical species. In a few instances the absence of phenoxyl radical ESR signals was found to be due to inhibition of lipoxygenase by BHT-homologues. In liposome or microsome suspensions addition of ascorbyl palmitate resulted in disappearance of the ESR signal of phenoxyl radicals with concomittant appearance of the ascorbyl radical signal. After exhaustion of ascorbate, the phenoxyl radical signal reappears. Comparison of the rates of ascorbyl radical decay in the presence or absence of BHT-homologues showed that temporary elimination of the phenoxyl radical ESR signal was due to their reduction by ascorbate. Similarly, NADPH or NADH caused temporary elimination of ESR signals as a result of reduction of phenoxyl radicals in microsomes. Since ascorbate and NADPH might generate superoxide in the incubation system used, SOD was tested. SOD shortened the period, during which the phenoxyl radicals ESR signal could not be observed. Both ascorbyl palmitate and NADPH exerted sparing effects on the loss of BHT-homologues during oxidation. These effects were partly diminished by SOD. These data indicate that reduction of phenoxyl radicals was partly superoxide-dependent. It is concluded that redox recycling of phenoxyl radicals can occur by intracellular reductants like ascorbate and microsomal electron transport.     

Spectroscopic characterization of the nickel and iron-sulphur clusters of hydrogenase from the purple photosynthetic bacterium Thiocapsa roseopersicina. 1. Electron spin resonance spectroscopy

The thermostable hydrogenase from Thiocapsa roseopersicina was examined by low-temperature ESR spectroscopy. Two types of signals were detected, from an oxidized iron-sulphur cluster and a nickel centre (Ni-A). In the oxidized protein additional signals were observed due to spin-spin interaction between the two paramagnetic centres. This interaction could be reversibly abolished by reduction to a redox potential below 105 mV. This implies that an additional redox centre is involved in the interaction, for which an Fe3+ ion is suggested. Reduction with hydrogen induced a second type of nickel ESR signal (Ni-C), corresponding to an intermediate redox state seen in other nickel hydrogenases. The Ni-C species was light-sensitive at cryogenic temperatures. At temperatures near to 4.2 K the Ni-C signal showed evidence of interaction with another paramagnetic centre, presumably a second iron-sulphur cluster. On reoxidation a signal due to a third Ni(III) species, Ni-B, increased in amplitude. These results establish that metal centres in the hydrogenase from T. roseopersicina are closely similar to those of the well-studied hydrogenase from Chromatium vinosum.