The effect of melatonin on the liver of rats exposed to microwave radiation.

OBJECTIVES: We aimed to clarify if melatonin treatment (2 mg/kg i.p.) may favorably impact the liver tissue in rats exposed to microwave radiation. The experiment was performed on 84 six-weeks-old Wistar male rats exposed for 4h a day, for 20, 40 and 60 days, respectively, to microwaves (900 MHz, 100-300 microT, 54-160 V/m). Rats were divided in to four groups: I (control) - rats treated with saline, II (Mel) - rats treated with melatonin, III (MWs) - microwave exposed rats, IV (MWs + Mel) - MWs exposed rats treated with melatonin. We evaluated oxidative stress parameters (malondialdehyde and carbonyl group content), catalase, xanthine oxidase, deoxyribonuclease I and II activity. BACKGROUND: Oxidative stress is the key mechanism of the microwave induced tissue injury. Melatonin, a lipophilic indoleamine primarily synthesized and released from the pineal gland is a powerful antioxidant. RESULTS: Exposure to microwaves caused an increase in malondialdehyde after 40 (p < 0.01), protein carbonyl content after 20 (p < 0.05), catalase (p < 0.05) and xantine oxidase activity (p < 0.05) after 40 days. Increase in deoxyribonuclease I activity was observed after 60 days (p < 0.05), while deoxyribonuclease II activity was unaffected. Melatonin treatment led to malondialdehyde decrease after 40 days (p< 0.05), but surprisingly had no effect on other analyzed parameters. CONCLUSION: Melatonin exerts certain antioxidant effects in the liver of rats exposed to microwaves, by diminishing the intensity of lipid peroxidation(Fig. 6, Ref. 32).

Effects of single-dose ultraviolet radiation on skin superoxide dismutase, catalase, and xanthine oxidase in hairless mice.

The effects of a single exposure to UVB radiation on skin antioxidant enzymes and superoxide-generating xanthine oxidase were examined in Skh:HR-1 hairless mice. Significant decreases in superoxide dismutase (SOD) and catalase (CAT) were observed by 12 h after UV irradiation and remained depressed for up to 72 h. No induction of xanthine dehydrogenase (XD) or xanthine oxidase (XO) occurred with UV treatment, although significant hyperplasia was evident. Ornithine decarboxylase was induced after UV irradiation as has been previously reported. These results demonstrate significant biochemical effects of a single dose of UVB on murine epidermis, especially in terms of antioxidant defenses.

Radiomodfication of xanthine oxidoreductase system in the liver of mice by phenylmethylsulfonyl fluoride and dithiothreitol.

The widely distributed xanthine oxidoreductase (XOR) system has been shown to be modulated upon exposure of animals to ionizing radiation through the conversion of xanthine dehydrogenase (XDH) into xanthine oxidase (XO). In the present work, radiomodification of the XOR system by phenylmethylsulfonyl fluoride (PMSF) and dithiothreitol (DTT) was examined using female Swiss albino mice which were irradiated with gamma rays at a dose rate 0.023 Gy s(-1). PMSF, a serine protease inhibitor, and DTT, the sulfhydryl reagent, were administered intraperitoneally prior to irradiation. The specific activities of XDH and XO as well as the XDH/XO ratio and the total activity (XDH+XO) were determined in the liver of the mice. The inhibition of XO activity, restoration of XDH activity, and increase in the XDH/XO ratio upon administration of PMSF were suggestive of irreversible conversion of XDH into XO mediated through serine proteases. The biochemical events required for the conversion were probably initiated during the early phase of irradiation, as the treatment with PMSF immediately after irradiation did not have a modulatory effect. Interestingly, DTT was not effective in modulating radiation-induced changes in the XOR system or oxidative damage in the liver of mice. The DTT treatment resulted in inhibition of the release of lactate dehydrogenase. However, the protection appears to be unrelated to the formation of TBARS. On the other hand, the presence of PMSF during irradiation inhibited radiation-induced oxidative damage and radiation-induced increases in the specific activity of lactate dehydrogenase. These findings suggest that a major effect of ionizing radiation is irreversible conversion of xanthine to xanthine oxidase.

Effect of radiation on the xanthine oxidoreductase system in the liver of mice.

The xanthine oxidoreductase system is one of the major sources of free radicals in many pathophysiological conditions. Since ionizing radiations cause cell damage and death, the xanthine oxidoreductase system may contribute to the detrimental effects in irradiated systems. Therefore, modulation of the xanthine oxidoreductase system by radiation has been examined in the present study. Female Swiss albino mice (7-8 weeks old) were irradiated with gamma rays (1-9 Gy) at a dose rate of 0.023 Gy s(-1) and the specific activities of xanthine oxidase (XO) and xanthine dehydrogenase (XDH) were determined in the liver of the animals. The mode and magnitude of change in the specific activities of XO and XDH were found to depend on radiation dose. At doses above 3 Gy, the specific activity of XO increased rapidly and continued to increase with increasing dose. However, the specific activity of XDH was decreased. These findings are suggestive of an inverse relationship between the activity of XO and XDH. The ratio of the activity of XDH to that of XO decreased with radiation dose. However, the total activity (XDH + XO) remained constant at all doses. These results indicate that XDH may be converted into XO. An intermediate form, D/O, appears to be transient in the process of conversion. The enhanced specific activity of XO may cause oxidative stress that contributes to the radiation damage and its persistence in the postirradiation period. Radiation-induced peroxidative damage determined in terms of the formation of TBARS and the change in the specific activity of lactate dehydrogenase support this possibility.

Reactive oxygen species (ROS)-generating oxidases in the normal rabbit cornea and their involvement in the corneal damage evoked by UVB rays.

The corneas of albino rabbits were irradiated (5 min exposure once a day) with UVB rays (312 nm) for 4 days (shorter procedure) or 8 days (longer procedure). The eyes were examined microbiologically and only the corneas of sterile eyes or eyes with non-pathogenic microbes were employed. Histochemically, the activities of reactive oxygen species (ROS)-generating oxidases (xanthine oxidase, D-amino acid oxidase and alpha-hydroxy acid oxidase) were examined in cryostat sections of the whole corneas. Biochemically, the activity of xanthine oxidoreductase/xanthine oxidase was investigated in the scraped corneal epithelium. UVB rays significantly changed enzyme activities in the corneas. In comparison to the normal cornea, where of ROS-generating oxidases only xanthine oxidase showed significant activity in the corneal epithelium and endothelium, D-amino acid oxidase was very low and alpha-hydroxy acid oxidase could not be detected at all, in the cornea repeatedly irradiated with UVB rays, increased activities of xanthine oxidase and D-amino acid oxidase were observed in all corneal layers. Only after the longer procedure the xanthine oxidase and D-amino acid oxidase activities were decreased in the thinned epithelium in parallel with its morphological disturbances. Further results show that the xanthine oxidase/xanthine oxidoreductase ratio increased in the epithelium together with the repeated irradiation with UVB rays. This might suggest that xanthine dehydrogenase is converted to xanthine oxidase. However, in comparison to the normal corneal epithelium, the total amount of xanthine oxidoredutase was decreased in the irradiated epithelium. It is presumed that xanthine oxidoreductase might be released extracellularly (into tears) or the enzyme molecules were denatured due to UVB rays (particulary after the longer procedure). Comparative histochemical and biochemical findings suggest that reactive oxygen species-generating oxidases (xanthine oxidase, D-amino acid oxidase) contribute to the corneal damage evoked by UVB rays.

Photoreactivation of alloxanthine-inhibited xanthine oxidase.

Alloxanthine-inhibited xanthine oxidase (XOD) was found to be photoreactivated by irradiation of light of wavelengths in the range of 340-430 nm. The enzyme activity can be fully controlled to be on or off by many dark-light cycles. Electron spin resonance measurement shows the appearance of the molybdenum (V) ion and the reduced form of flavin adenine dinucleotide (FADH.) radical signals after irradiation of the alloxanthine-XOD complex. Electronic-absorption spectrum also shows the bleaching of Fe/S and flavin adenine dinucleotide chromophores at 375 and 450 nm as well as broad-band absorption of FADH. in the range of 500-700 nm. The quantum yield of photoreactivation of the enzyme activity is approximately 0.06. A photoinduced intraenzyme electron-transfer model is proposed to rationalize the photoreactivation process.

Inactivation of xanthine oxidase by oxidative radical attack.

Steady-state radiolysis, pulse radiolysis and epr studies, combined with enzyme activity measurements, were carried out on the mechanism by which radical attack, through one-electron oxidation, inactivates xanthine oxidase. Electron transfer to both the N3 and Br2- radical species was used to initiate oxidative damage on the enzyme. Inactivation was found to occur to a greater extent at low than at high pH and is associated with the initial formation of a tryptophanyl radical which converts by a known intramolecular pathway to a tyrosyl radical with a rate constant of 5 x 10(3) S-1. The tyrosyl radical in turn slowly loses around half of its absorbance at an intramolecular rate constant of 350S-1 and is consistent with the establishment of a radical equilibrium with cysteine residue(s). The sequence of reactions could be repeated several times on the same irradiated sample implying that restitution of the implied cysteinyl radical occurs leading to other damage in the protein. N3+Trp/N-->Trp/N-->Tyr/O<-->Cys/S-->?. Epr evidence implies that inactivation of the enzyme from the above sequence of reactions arises in part from alternations to Fe/S center I in the enzyme.

Influence of gamma-rays on the mouse liver cytochrome P450 system and its modulation by phenothiazine drugs.

PURPOSE: Phenothiazine drugs have been found to sensitize hypoxic cancer cells while offering protection to normal cells. Since phenothiazines are known to induce the cytochrome P450 system, its radiomodulation by phenothiazines has been examined. MATERIALS AND METHODS: Mice were administered phenothiazines intraperitoneally and irradiated with different doses of gamma-rays at 1.38 Gy/min. The activities of NADPH-cytochrome P450 reductase and NADH-cytochrome b5 reductase, the content of cytochrome P450 and b5, the extent of lipid peroxidation as well as the activities of LDH, XO, SOD, GST and DTD were determined in the liver. RESULTS: The levels of different components of the cytochrome P450 system and antioxidant enzymes were enhanced up to 5 Gy and decreased thereafter. However, a progressive increase was noticed in peroxidative damage and the activities of LDH and XO. Administration of phenothiazines enhanced the radiation effect on components of the cytochrome P450 system (except NADH-cytochrome b5 reductase) and the activities of SOD, GST and DTD. Concomitantly, phenothiazines inhibited lipid peroxidation, LDH and XO. CONCLUSIONS: Activation of the cytochrome P450 system by phenothiazines leading to the enhancement of antioxidant potential of animals and free-radical scavenging are attributes of the radioprotective action of phenothiazines.

Histochemical study on xanthine oxidase activity in the normal rabbit cornea and lens and after repeated irradiation of the eye with UVB rays.

In the normal rabbit cornea and lens the activity of xanthine oxidase, an enzyme belonging to oxidases generating reactive oxygen species (ROS), is present in the corneal epithelium as well as endothelium and lens epithelium. Repeated irradiation of the eyes with UVB rays (5 min 1 x daily, for 1 to 4 days) caused a gradual increase of xanthine oxidase activity, particularly in the corneal epithelium. Application of catalase, a scavenger of hydrogen peroxide, to the eye surface during the irradiation diminished the increase of xanthine oxidase activity. On the contrary, the pretreatment of the rabbit eyes with 3-aminotriazole, an inhibitor of catalase, for 3 days before the irradiation enhanced the increase of xanthine oxidase activity. In comparison to untreated eyes, protracted irradiation of the eyes with UVB rays (up to 10 days) caused a decrease of xanthine oxidase activity in the same cell layers of the cornea and lens. It is suggested that xanthine oxidase is involved in the generation of ROS in the anterior eye segment during early irradiation of the eyes with UVB rays and participates in its damage. Prolonged repeated irradiation of the eye (5 min 1 x daily for 5 to 10 days) caused a decrease of xanthine oxidase activity in the cornea and lens which is attributed to profound damage of the whole anterior eye segment.

Light product of photoreactive 6-azido-FAD bound to deflavo-milk xanthine oxidase.

Xanthine oxidase from milk was reconstituted with the photoreactive flavin, 6-azido-FAD. While irradiation of the reconstituted enzyme under anaerobic conditions yielded 6-amino-FAD as a light product, aerobic irradiation resulted in formation of an unknown product, which gave the enzyme almost the same activity as that of the native enzyme. The light product could be extracted from the enzyme without breakdown and was found to be highly fluorescent. Upon treatment with phosphodiesterase, this light product was converted to the FMN form. The absorption spectrum of the FMN form has a peak at 464 nm, a shoulder at 450 nm in the visible region, and two peaks at 260 and 298 nm in the UV. Irradiation of free 6-azido-3-methyllumiflavin in the presence of a saturating concentration of oxygen yielded a light product whose absorbance and fluorescence spectra were very similar to those of the light product extracted from the enzyme, suggesting that the two had undergone some common photochemical change at the same place in the isoalloxazine ring. Analysis of the light product of 6-azido-3-methyllumiflavin with 1H NMR and FAB mass spectrometry suggested its possible structure with a new five-membered ring, C(6) = N-O-CH = C(7), adjacent to the benzene ring of the flavin.

Activation of flavin-containing oxidases underlies light-induced production of H2O2 in mammalian cells.

Violet-blue light is toxic to mammalian cells, and this toxicity has been linked with cellular production of H2O2. In this report, we show that violet-blue light, as well as UVA, stimulated H2O2 production in cultured mouse, monkey, and human cells. We found that H2O2 originated in peroxisomes and mitochondria, and it was enhanced in cells overexpressing flavin-containing oxidases. These results support the hypothesis that photoreduction of flavoproteins underlies light-induced production of H2O2 in cells. Because H2O2 and its metabolite, hydroxyl radicals, can cause cellular damage, these reactive oxygen species may contribute to pathologies associated with exposure to UVA, violet, and blue light. They may also contribute to phototoxicity often encountered during light microscopy. Because multiphoton excitation imaging with 1,047-nm wavelength prevented light-induced H2O2 production in cells, possibly by minimizing photoreduction of flavoproteins, this technique may be useful for decreasing phototoxicity during fluorescence microscopy.

NO synthase and xanthine oxidase activities of rabbit brain synaptosomes: peroxynitrite formation as a causative factor of neurotoxicity.

In the present study we demonstrated that synaptosomes isolated from rabbit brain cortex contain NO synthase and xanthine oxidase that can be activated by ultraviolet B radiation and Ca2+ accumulation to produce nitric oxide and superoxide which react together to form peroxynitrite. Irradiation of synaptosomes with ultraviolet B (up to 100 mJ/cm2), or increase the intrasynaptosomal calcium concentration using various doses (up to 100 mu M) of the calcium ionophore A 23187, a gradual increase in both nitric oxide and peroxynitrite release that was inhibited by N-monomethyl-L-arginine (100 mu M) was observed. The rate of nitric oxide release and cyclic GMP production by NO synthase and soluble guanylate cyclase, both located in the soluble fraction of synaptosomes (synaptosol), were increased approximately eight fold after treatment of synaptosomes with Ultraviolet B radiation (100 mJ/cm2). In reconstitution experiments, when purified NO synthase isolated from synaptosol was added to xanthine oxidase, in the presence of the appropriate cofactors and substrates, a ten fold increase in peroxynitrite production at various doses (up to 20 mJ/cm2) of UVB radiation was observed. Ultraviolet B irradiated synaptosomes promptly increased malondialdehyde production with subsequent decrease of synaptosomal plasma membrane fluidity estimated by fluorescence anisotropy of 1-4-(trimethyl-amino-phenyl)-6-phenyl-hexa-1 ,3,5-triene. Desferrioxamine (100 mu M) tested in Ultraviolet B-irradiated synaptosomes showed a decrease (approximately 80%) in malondialdehyde production with subsequent restoration of the membrane fluidity to that of non-irradiated (control) synaptosomes. Ca(2+)-stimulated ATPase activity was decreased after Ultraviolet B (100 mJ/cm2) radiation of synaptosomes indicating that the subsequent increase of intrasynaptosomal calcium promoted peroxynitrite production by a calmodulin-dependent increase of NO synthase and xanthine oxidase activities. Furthermore, it was shown that UVB-irradiated synaptosomes were subjected to higher oxidative stress by exogenous peroxynitrite (100 mu M) compared to non-irradiated (control) synaptosomes. In summary, the present results indicate that activation of NO synthase and xanthine oxidase of brain cells lead to the formation of peroxynitrite providing important clues in the role of peroxynitrite as a causative factor in neurotoxicity.