Erythropoietic protoporphyria: lipid peroxidation and red cell membrane damage associated with photohemolysis.

The mechanism by which long wavelength ultraviolet light hemolyzes red cells obtained from patients with erythropoietic protoporphyria (EPP) was investigated. Previous studies had suggested that irradiation of these red cells with wavelengths of light capable of eliciting dermatological manifestations led to oxygen-dependent colloid osmotic hemolysis through the formation of peroxides. In the present report, lipid peroxidation during in vitro irradiation of EPP red cells with long ultraviolet light was demonstrated by: (a) the formation of 2-thiobarbituric acid reactants; (b) the presence of conjugated diene bonds in red cell lipid; and (c) the selective loss of unsaturated fatty acids proportional to the number of carbon-carbon double bonds in each. Irradiation of EPP red cells was also shown to result in the formation of hydrogen peroxide.Before photohemolysis there was a decline in cell membrane sulfhydryl groups and a loss in activity of the cell membrane enzyme acetylcholinesterase. These parameters provide further evidence suggesting that the cell membrane is a primary site of the photohemolytic effect of long ultraviolet light in EPP red cells. Further evaluation of the radiation-induced inactivation of EPP red cell acetylcholinesterase was performed by radiating mixtures containing bovine erythrocyte acetylcholinesterase and protoporphyrin IX. These studies revealed that the rate of decline in enzyme activity is accelerated by the addition of linoleic acid, an unsaturated fatty acid, but not by palmitic acid, a saturated fatty acid. Partial protection against both photohemolysis and acetylcholinesterase decline is provided by alpha-to-copherol. This lipid antioxidant loses its activity during the irradiation of EPP red cells suggesting that it is utilized in this process.

The inactivation of acetylcholinesterase by alpha-terthienyl and ultraviolet light. Studies in vitro and in larvae of the mosquito Aedes aegypti.

Acetylcholinesterase (EC 3.1.1.7) was inactivated photochemically in solution, in the presence of dissolved terthiophene sensitizers. Alpha-terthienyl (2,2':5,2"-terthiophene) and its isomers 3,2':5',2"- and 3,2':5',3"-terthiophenes showed very similar sensitizing properties. With all three terthiophenes, the photosensitization was completely suppressed under anaerobic conditions, and therefore the inactivation process required the presence of oxygen. The enzyme was inactivated in vivo when fourth instar larvae of the mosquito Aedes aegypti were treated with alpha-terthienyl in the presence of long-wavelength ultraviolet light. No inactivation was observed when the organisms were treated with the ultraviolet light alone, with the chemical alone, or with a previously irradiated sample of the chemical. This paper represents the first example of acetylcholinesterase inactivation in vivo by a photoactive insecticide.

A wavelength dependent mechanism for rose bengal-sensitized photoinhibition of red cell acetylcholinesterase.

A 2-fold enhancement in the efficiency of rose bengal-photosensitized inhibition of red cell acetylcholinesterase activity was observed upon excitation of the dye in the ultraviolet (UV) (313 nm) compared to irradiation in the visible (514 or 550 nm). The measurements of efficiency of photosensitized enzyme inhibition were based on the effect produced when the same number of photons are absorbed by rose bengal (RB) at each wavelength. The mechanism for this unexpected enhancement of RB photosensitization upon UV excitation was investigated. The yield of singlet oxygen (O2(1 delta g], detected by time-resolved luminescence at 1270 nm, was independent of excitation wavelength for RB. Radicals were produced upon irradiation of RB at 313 nm but not at 514 nm as detected by bleaching of N,N-dimethylnitrosoaniline (RNO). Irradiation of RB at 313 nm but not at 514 nm appeared to cause homolytic cleavage of carbon-iodine bonds in the dye because iodine radicals, I, detected as I2 were produced with a quantum yield of 0.0041 +/- 0.0005 upon excitation in the UV. Photolysis of I2 in the presence of RNO caused bleaching of the RNO absorption at 440 nm, apparently resulting from reaction of I with RNO. Thus, the enhanced photosensitization upon UV excitation of RB is attributed to formation of I and/or RB. These results indicate that radicals, produced with low relative yield but having high reactivity compared to O2(1 delta g), can contribute to photosensitized enzyme inhibition and may represent an alternative mechanism for photodynamic therapy.

Structural and kinetic effects of mobile phone microwaves on acetylcholinesterase activity.

The present study provides evidence that "in vitro" simple exposure of an aqueous solution of electric eel acetylcholinesterase (EeAChE; EC 3.1.1.7.) to cellular phone emission alters its enzymatic activity. This paper demonstrates, by combining different experimental techniques, that radio frequency (RF) radiations irreversibly affect the structural and biochemical characteristics of an important CNS enzyme. These results were obtained by using a commercial cellular phone to reproduce the reality of the human exposition. This experimental procedure provided surprising effects collected practically without experimental errors because they were obtained comparing native and irradiated sample of the same enzyme solution. Although these results cannot be used to conclude whether exposure to RF during the use of cellular phone can lead to any hazardous health effect, they may be a significant first step towards further verification of these effects on other "ex vivo" or "in vivo" biological systems.

Specific protein dynamics near the solvent glass transition assayed by radiation-induced structural changes.

The nature of the dynamical coupling between a protein and its surrounding solvent is an important, yet open issue. Here we used temperature-dependent protein crystallography to study structural alterations that arise in the enzyme acetylcholinesterase upon X-ray irradiation at two temperatures: below and above the glass transition of the crystal solvent. A buried disulfide bond, a buried cysteine, and solvent exposed methionine residues show drastically increased radiation damage at 155 K, in comparison to 100 K. Additionally, the irradiation-induced unit cell volume increase is linear at 100 K, but not at 155 K, which is attributed to the increased solvent mobility at 155 K. Most importantly, we observed conformational changes in the catalytic triad at the active site at 155 K but not at 100 K. These changes lead to an inactive catalytic triad conformation and represent, therefore, the observation of radiation-inactivation of an enzyme at the atomic level. Our results show that at 155 K, the protein has acquired--at least locally--sufficient conformational flexibility to adapt to irradiation-induced alterations in the conformational energy landscape. The increased protein flexibility may be a direct consequence of the solvent glass transition, which expresses as dynamical changes in the enzyme's environment. Our results reveal the importance of protein and solvent dynamics in specific radiation damage to biological macromolecules, which in turn can serve as a tool to study protein flexibility and its relation to changes in a protein's environment.

Molecular weight estimation of protoheme ferro-lyase by radiation inactivation.

The molecular weight of protoheme ferro-lyase [EC 4.99.1.1], a mitochondrial enzyme, was estimated by the radiation inactivation method. Irradiation in vacuo caused less inactivation than that in air. The approximate molecular weight determined by this method was in the range from 250,000 to 320,000. The significance of the location of the enzyme in connection with its role in heme synthesis is discussed on the basis of Singer's membrane theory.

Brain fixation for acetylcholine measurements.

Brain fixation using a commercially available microwave oven (power output: 750 W) has been investigated as a means for enzyme inactivation preventing post-mortem changes in brain acetylcholine (ACh) and choline (Ch) levels. Rats and mice were decapitated, and the severed heads immediately irradiated for 5.5 and 3 s, respectively, resulting in a complete inactivation of brain acetylcholine esterase (AChE) and choline acetyltransferase (ChAT). The ACh and Ch contents measured in various brain regions of rat and mouse were: (in rat) striatum 60.5 and 32.4 nmol/g, hippocampus 20.4 and 30.9 nmol/g, cortex 24.2 and 19.6 nmol/g; (in mouse) striatum 70 and 47.2 nmol/g, hippocampus 22. 1 and 30.2 nmol/g, cortex 22.9 and 27.9 nmol/g. These values were found in accordance with those reported in the literature by irradiating whole animals in instruments of higher power capabilities. Thus, the procedure described in the present work may be a simple and valuable means of brain fixation for neurochemical studies of brain ACh in small animal species.

Effect of amplitude-modulated radio frequency radiation on cholinergic system of developing rats.

We examined the effect of long-term exposure to radio frequency radiation 147 MHz and its sub-harmonics 73.5 and 36.75 MHz amplitude modulated at 16 and 76 Hz (30-35 days, 3 h per day) on cholinergic systems in developing rat brain. A significant decrease in acetylcholine esterase activity was found in exposed rats as compared to the control. Decrease in acetylcholine esterase (AChE) activity was independent of carrier wave frequencies. A short-term exposure did not have any significant effect on AChE activity.

Effects of X-irradiation on nerve growth factor in the developing mouse brain.

The involvement of neurotrophins after radiation injury during brain development were studied in pregnant mice (C 57/B1) exposed on gestation day 15 to X-ray doses of 0.02-2 Gy. Nerve growth factor protein (NGF) and different cholinergic markers were investigated on postnatal day 1 (P1) and day 21 (P21); in situ hybridization with brain-derived neurotrophic factor (BDNF) and trkC (receptor serving to bind neurotrophin-3) probes was investigated on P21 in cortex, hippocampus, septum and cerebellum. The level of NGF protein was increased in irradiated forebrain on P1 in a dose-related manner. However, on P21 the NGF protein dropped down below the control levels in irradiated hippocampus and cerebellum. The response of acetylcholine esterase (AChE) activity in cerebellum at P21 was correlated with the changes in the amount of NGF. The intensity of cell labelling with trkC probe decreased after irradiation in the region of the hippocampus at P21, especially in dentate gyrus. The expression of BDNF mRNA was increased at P21 by low doses of irradiation (0.02-1 Gy) but was decreased by a high dose (2 Gy) in the same area. Thus, the radiation induced an alteration of neurotrophins, and the changes varied depending on the dose or time after irradiation. Such alterations in the pattern of growth factor production may modulate the response of cells to radiation. Furthermore, NGF protein levels and the expression of BDNF and trkC mRNA were affected by radiation doses as low as 0.02 Gy, indicating that during development the neurotrophins and their receptors are very sensitive to radiation.

Oxidation, photosensitized by certain diketones, of enzymes and protection against such oxidation by histidine derivatives.

Bovine milk lactoperoxidase, eel acetylcholinesterase, and Aeromonas aminopeptidase were photooxidized and inactivated in broad-spectrum visible light in the presence of 2,3-butanedione and 1-phenyl-1,2-propanedione. Methylglyoxal caused similar effects at 254 nm. 2-Thiol-L-histidine and 3-methyl-L-histidine protected the enzymes against photoinactivation more effectively then N3-, even at a molar ratio of 2:1 (protector to enzyme). These compounds also delayed the photoinactivation of acetylcholinesterase, induced by ultraviolet light.

[Influence of UV-light on erythrocyte membrane structure and catalytic behaviour of membrane acetylcholine esterase]. / Vliianie UF-sveta na strukturu membran zritrotsitov i na kataliticheskie svoistva membrannoi atsetilkholinzsterazy.

UV-light is shown to induce the structural transitions in the erythrocyte membrane described by S-shape curves in plots of the structural response versus the irradiation dose. In contrast to the free acetylcholine esterase (AChE) UV-light acts on the membrane enzyme as a mixed inhibitor (simultaneous change in Vmax and Km). The modification of the environment structure of residual enzyme is suggested to be the main reason of this phenomenon. The effect is under the control of membrane integrity and disappears after its desintegration. Membrane AChE treated ultrasonically both prior to and after irradiation is inactivated without a Km change. The data obtained show the influence of erythrocyte membrane structure on the catalytic behaviour of membrane-bound AChE.

[Effect of the state of the lipid phase of the membrane on the effectiveness of photochemical modification of erythrocyte acetylcholinesterase]. / Vliianie sostoianiia lipidnoi fazymembrany na éffektivnost' fotokhimicheskoi modifikatsii éritrotsitarnoi atsetilkholinésterazy.

Modification of the lipid phase structure of the erythrocyte membrane by phospholipases A2, C and D as well as the partial depletion of cholesterol was shown to be accompanied by the change of the acetylcholinesterase (AChE) UV-sensitivity. The ability of UV-light to change the catalytic properties (Km) of the membrane-bound AChE not observed for free AChE (constant value of Km) and known as the phenomenon of photochemical allotopy, is retained in the cholesterol depleted membranes and disappears after an enzymatic treatment of the membranes by phospholipases. The possible non-photochemical influence of the membrane lipid phase in response to UV-damage of membrane-bound AChE is discussed.

[The effect of local ionizing irradiation on the presence of acetylcholinesterase-positive nerve fibers in the spleen of rats]. / Vplyv lokálneho pôsobenia ionizacného ziarenia na prítomnost ACHE-pozitívnych nervových vlákien v slezine potkana.

Changes in the distribution of ACHE-positive nerve fibres in the spleen of the rat after local irradiation have been studied by light microscopy using direct tiocholin method of El-Badawi and Schenk (1967). ACHE-positive nerve fibers enter the spleen in the vicinity of splenic artery branches and they are gradually distributed in form of perivascular plexuses in the white pulp to the periarterial lymphatic sheath (PALS). One dose of local irradiation of the head of the rat causes changes in the presence and distribution of the ACHE-positive nerve fibres in the spleen of the rat. After an intense decrease or the absence of these fibres during the period of the first days after irradiation, there is an evident restoration of ACHE-positive nerve fibres in the white pulp of the organ. The distribution and topography of these nerve structures is the same as in non-irradiated rats on the 35th day after irradiation. During the following days, there is another evident decrease in ACHE-positive nerve fibres in all described localizations of the spleen. Our findings suggest that the indirect effect of irradiation can affect some changes in innervation of the organ which was not exposed to its direct influence. (Fig. 3, Ref. 13.)

[Morphological and cytogenetic disorders in rats following chronic exposure to elevated radiation]. / Morfologicheskie i tsitogeneticheskie narusheniia u krys, nakhodivshikhsia v usloviiakh povyshennogo radiatsionnogo fona na protiazhenii dlitel'nogo vremeni.

Object of the investigation were effects of elevated radiation background on specific body systems in two generations of rats that had been chronically exposed to radiation in the most badly polluted areas of the Kaluga district and eaten local food. Findings included some compensatory deviations in the white blood count, decrease in karyocytes and increase in chromosomal aberration rate in nucleus-containing cells from the femoral bone marrow in younger animals of several groups, morphologic and histochemical shifts in mucous membrane of the esophagus and the stomach, and morphofunctional changes in neurons and glia of the cerebral cortex. These changes are compensatory-adaptive by nature and indicative of destabilization of homeostasis in experimental animals.

Effect of ultraviolet radiation on acetylcholinesterase activity in freshwater copepods.

We analyzed the effects of UV radiation (UVR) effects on acetylcholinesterase (AChE) activity in two calanoid copepods, Boeckella gibbosa and Parabroteas sarsi that inhabit Patagonian shallow lakes. We studied the effect of experimental UVR (UV-B and UV-A) exposure on AChE activity in relation to basal antioxidant capacities of both copepods. Our experiments showed that UVR can effectively depress AChE activity, although with differences between species. In both copepods AChE was affected by UV-B, whereas UV-A only affected AChE in B. gibbosa. Both copepods also differed in body elemental composition (C:N:P), photoprotecting compound content (carotenoids and mycosporine-like amino acids) and enzymatic antioxidant capacity (glutathione S-transferase [GST]). Our results suggest that when exposed to UVR, AChE activity would depend more on the antioxidant capacity (GST) and P availability for enzyme synthesis than on the photoprotective compounds.