Role of endogenous porphyrins in the effects of low-intensity laser radiation of the red region on free radical processes in the blood of rats under experimental endotoxic shock

The role of endogenous porphyrins in the effects of laser radiation of the red region (632.8 nm) on free radical processes in the blood of rats under endotoxic shock induced by the administration of lipopolysaccharide B (25 mg/kg) has been studied. The measurements of the functional activity of polymorphonuclear leukocytes (the method of luminol-dependent chemiluminescence), the superoxide dismutase activity of blood plasma (using nitro blue tetrazolium), and the degree of lipid oxidation of erythrocyte membranes (the method of fluorescence of cis-parinaric acid) have been carried out. It has been found that low-intensity laser radiation strongly affects all processes examined irrespective of the administration of lipopolysaccha-ride B. The effect of radiation was most pronounced in animals injected with the polysaccharide, the changes being dependent on the concentration of endogenous porphyrins in samples.     

The effect of low power laser radiation of blue,green, and red ranges on free radical processes in blood of rats with experimental endotoxic shock

This study was performed to investigate the effects of low power laser radiation in blue (441.2 nm), green (532.5 nm) and red (632.8 nm) wavelength ranges on free radical processes in experimental endotoxic shock in rats. The experimental model was induced by intraperitoneal injection of lipopolysaccharide B (25 mg/kg) (LPS). Functional activity of blood leukocytes was evaluated by the method of luminol-dependent chemiluminescence, plasma superoxide dismutase activity was determined by the nitro blue tetrazolium assay, intensity of lipid peroxidation in erythrocyte membranes was estimated by cis-parinaric acid fluorescence. It was found that the low power laser radiation significantly influenced all investigated processes, in LPS-treated and control animals. The most pronounced effects were observed in all groups of LPS-treated animals, in which the laser radiation increased all investigated parameters. At the radiation dose 0.75 J/cm² green laser was the most effective, while at the dose of 1.5 J/cm² both green and red lasers produced potent effects. Possible mechanisms of the observed phenomena are discussed.     

The role of endogenous porphyrins in laser therapy of experimental skin wounds

The role of endogenous porphyrins in the effect of laser irradiation on the superoxide dismutase (SOD) activity of wound exudate and rat leukocyte activity has been studied on models of aseptic incised skin wounds. Wounds were irradiated with a He-Ne laser (632.8 nm, 1.5 J/cm²) on the 2nd, 3rd, and 4th days after the beginning of the experiment. Irradiation effects were evaluated by the SOD activity (NBT test) and the activity of leukocytes of the wound exudate (as a chemiluminescent response to opsonized zymosan). It was found that in animals subjected to laser irradiation, the SOD activity sharply increased. This effect depended on endogenous porphyrin concentration and was retained throughout the experiment. The SOD activity in unirradiated animals decreased from the 2nd to the 5th day of experiment. The evaluation of the activity of wound exudate leukocytes did not reveal any distinct dependence of the effect on the concentration of endogenous porphyrins.     

Effects of endogenous photosensitizers on the laser-induced priming of leucocytes

The influence of He-Ne (lambda = 632.8 nm) laser irradiation on the functional activity of leucocytes was investigated. The functional activity of leucocytes irradiated in the presence of plasma with phthalocyanine raised. Irradiation of leucocytes without plasma had no influence on the chemiluminescent response of the blood cells. An increase in the concentration of phthalocyanine in plasma first led to an increase and then a decrease in the functional activity of the leucocytes. Similar results were obtained for the case of endogenous porphyrins in plasma. These results make it possible to consider the influence of laser irradiation on the leucocytes' functional activity as the priming. The basic concepts of the free-radical mechanism of laser therapy were formulated.     

Inhibitory effect of porphyrins on the proliferation of mouse spleen lymphocytes in vitro

The influence of various porphyrins (deuteroporphyrin IX, mesoporphyrin IX, protoporphyrin IX, hematoporphyrin) and two related compounds (hemin, biliverdin) on the spontaneous proliferation of mouse spleen lymphocytes has been estimated in vitro by the 3H-thymidine uptake assay. It has been found that porphyrins (endogenous ligands for the mitochondrial benzodiazepine receptor) produce a concentration-dependent inhibition of 3H-thymidine incorporation into the DNA of these cells. Metalloporphyrin-hemin has been observed to evoke a weak inhibitory effect, in a high concentration (10(-4)M), whereas biliverdin, a porphyrins degradation product, was inactive in the same experimental conditions. Those findings indicate that endogenous porphyrins, presumably acting through the mitochondrial benzodiazepine receptor, could regulate the proliferation of mouse spleen lymphocytes in vitro.     

Photosensitizing effect of protoporphyrin IX in pigmented melanoma of mice

No fluorescence of protoporphyrin IX (PpIX) was measured using a fiber optic probe in pigmented B16F10 melanoma in mice after topical application of 5-aminolevulinic acid methylester (ALA-Me). However, chemical extraction of tissues excised from mice after intratumoral administration of ALA-Me or its parent compound ALA revealed that this tumor had the capability to produce PpIX. Small amounts of endogenous porphyrins, mainly PpIX, were found in the melanoma not treated with these drugs. Topical application of ALA-Me followed by exposure with laser light (633nm) delayed the growth of the tumors slightly. Light alone also had a significant effect on the tumor growth.     

The role of tetrapyrrol photosensitizers in photoinduced variation of free radical characteristics of rat blood in endotoxic shock

The main goal of the present study was to evaluate the comparative effectiveness of tetrapyrrol photosensitizers (protoporphyrine IX and chlorine e6) in red (632.8 nm) and green (532.5) spectrum bands on rat blood free radical status, using the experimental model of endotoxic shock. Endotoxic shock was produced by intraperitoneal injection of lipopolysaccharide B. Irradiation effectiveness was estimated by leukocyte activation (measured with luminol-dependent chemiluminescence), superoxide dismutase activity of blood plasma (nitro blue tetrasolium assay) and lipid peroxidation (assay with cis-parinaric acid). It was found that laser irradiation has multidirectional effects on leukocyte activation, membrane lipid peroxidation and plasma SOD activity and all these effects were more pronounced in the case of endotoxic shock. Protoporphyrin was more effective in leukocyte activation and chlorine e6 demonstrated maximal effects on blood SOD activity.     

The role of tetrapyrrol photosensitizers in photoinduced variation of free radical characteristics of rat blood in endotoxic shock

The main goal of the present study was to evaluate the comparative effectiveness of tetrapyrrol photosensitizers (protoporphyrine IX and chlorine e ₆) in red (632.8 nm) and green (532.5) spectrum bands on rat blood free radical status, using the experimental model of endotoxic shock. Endotoxic shock was produced by intraperitoneal injection of lipopolysaccharide B. Irradiation effectiveness was estimated by leukocyte activation (measured with luminol-dependent chemiluminescence), superoxide dismutase activity of blood plasma (nitro blue tetrasolium assay) and lipid peroxidation (assay with cis-parinaric acid). It was found that laser irradiation has multidirectional effects on leukocyte activation, membrane lipid peroxidation and plasma SOD activity and all these effects were more pronounced in the case of endotoxic shock. Protoporphyrin was more effective in leukocyte activation and chlorine e ₆ demonstrated maximal effects on blood SOD activity.     

Effect of allylisopropylacetamide on glutathione metabolism in the rat liver. The possible role of glutathione in the induction of 5-aminolaevulinate synthase

Administration of allylisopropylacetamide to rats caused a marked decline in the concentrations of reduced and oxidized glutathione in the liver. However, this decrease occurred in the presence of uninhibited activities of gamma-glutamylcysteine synthase and glutathione reductase, and unaltered activities of glutathione transferases A, B and C. The administration of cysteine, the rate-limiting precursor of glutathione formation, to rats treated with allylisopropylacetamide potentiated the inductive effects of the agent on 5-aminolaevulinate synthase, and markedly decreased the extent of decrease in glutathione concentrations by the agent. Conversely, the administration of diethyl maleate, which depletes the hepatic glutathione concentrations, to allylisopropylacetamide-pretreated rats (1h) diminished the extent of 5-aminolaevulinate synthase induction and the production of porphyrins by nearly 50%, when measured at 16h. This treatment did not alter the extent of non-enzymic degradation of liver haem by allylisopropylacetamide. When diethyl maleate was administered to the animals possessing high 5-aminolaevulinate synthase activity (at 3, 7 and 15h after allylisopropylacetamide), in 1h the enzyme activity was markedly decreased. Diethyl maleate had no effect on induction of 5-aminolaevulinate synthase by 3,5-diethoxycarbonyl-1,4-dihydrocollidine, also a potent porphyrinogenic agent. Diethyl maleate alone neither inhibited 5-aminolaevulinate synthase activity nor decreased the cellular content of porphyrins and haem. The data suggest that the decreases observed in the glutathione concentrations after allylisopropylacetamide administration are not the result of decreased production of the tripeptide. Rather, they most likely reflect the increased utilization of glutathione. The findings further suggest that the inhibition by diethyl maleate of allylisopropylacetamide-stimulated 5-aminolaevulinate synthase involves the inhibition of induction processes.     

Porphyrin, chlorin, and bacteriochlorin isothiocyanates: useful reagents for the synthesis of photoactive bioconjugates

A novel method for conjugating porphyrins and related molecules to proteins has been developed. The method, which involves synthesizing porphyrins, chlorins, and bacteriochlorins bearing a single amine-reactive isothiocyanate group represents a facile system for protein labeling with these photoactive species. Problems associated with the noncovalent binding of porphyrins to proteins are highlighted, and a method for purifying conjugates to yield exclusively covalently bound porphyrin protein species is demonstrated. Biological activity of porphyrin-bovine serum albumin conjugates formed and purified by these methods is demonstrated using laser scanning confocal microscopy.     

Cytokines as agents for the early pathogenetic therapy of radiation injuries. Their efficacy and mechanism of action

It has been shown in experiments with three species of laboratory animals that an early administration (during the first hours following irradiation) of human recombinant interleukins 1 alpha and 1 beta separately (to mice, rats) or in combination with antibiotic therapy (dogs) substantially increases survivability, favours a more rapid regeneration of the cellular content of the bone marrow and peripheral blood, intensifies the processes of endogenous colony formation and DNA synthesis in the bone marrow and liver and lowers the expressivity of radiation-induced endotoxemia. The significance of using cytokines in the system of remedial measures in radiation pathology is discussed.     

Study of the effects of laser irradiation (540 nm) on some components of blood enzyme antioxidant system

The influence of blood laser irradiation (lambda = 540 nm) in doses range 0.3-1.2 J/cm2 on the neutrophyles superoxide-dismutase and katalase activity has been studied. Correcting effect of laser light (0.6; 1.2 J/cm2) on the functional activity of studied enzymes has been found. The high degree of correlation (r = 0.6) between neutrophil superoxide dismutase and neutrophil catalase activity of the irradiated blood has been discovered. The close correlation between components of the antioxidant system and processes of active oxygen metabolite generation has been revealed. The scheme of free radical mechanisms of the laser irradiation influence on the several parts of the enzyme antioxidant system has been suggested.     

Chronic hypoxia state as one of the factors of increased fluorescence of endogenous porphyrins in alive biological tissues

The hypothesis about the influence of chronic hypoxia in soft alive tissues on the intensity of He-Ne laser-induced fluorescence of endogenous porphyrins in tissues in the red region of the optical spectrum is discussed. Some theoretical prerequisites for enormous accumulation of porphyrins in soft tissues in a number of pathological cases and diseases are presented. A method for conducting clinical experimental trials to verify the hypothesis with the use of up-to-date laser noninvasive fluorescent diagnostics is proposed. Both the theoretical reasoning and new clinical experimental data show that chronic hypoxia can be one of the factors responsible for the appearance of a strong and abnormal laser-induced fluorescent signal from biotissues in the spectrum range 600-800 nm, which is associated with abnormally high accumulation of endogenous porphyrins in tissues.     

A study of the effect of low-intensity laser radiation of the blue, green, and red spectral regions on the healing of experimental skin wounds in rats

The effect of low-intensity laser radiation of the blue (441.2 nm), green (532 nm), and red (632.8 nm) spectral regions on the healing of experimental skin wounds in rats has been studied. The effect of the traditionally applied laser radiation in the red region has been compared with the effect of laser radiation in the other spectral regions, assuming that, upon irradiation of wounds by lasers emitting in the blue and green regions, a similar effect can be achieved at lower doses. The following parameters characterizing the healing of experimental wounds were used: the functional activity of phagocytes of wound exudates, which was determined by luminol-dependent chemiluminescence, and their number; the antioxidant activity of wound exudates; and the rate of healing, which was determined as a change in the wound area. It was shown that irradiation with laser accelerated the healing of wounds in all cases. The exposure to laser radiations in the red (1.5 J/cm), blue, and green (0.75 J/cm2) spectral regions shortened the time of wound healing from 22 to 17 and 19 days, respectively. The functional activity of leukocytes after the exposure increased on day 5 after the infliction of the wound, whereas in the control it decreased. The superoxide dismutase activity increased in all experimental groups by day 5 after the operation. A maximum increase in the superoxide dismutase activity occurred after the exposure to laser radiation in the red region at a dose of 1.5 J/cm and in the blue and green spectral regions at a dose of 0.75 J/cm2.     

The role of visceral receptors in the mechanisms of neuroimmune interactions in mammalian small intestine

Exogenous administration of mast cell degranulation products into blood as well as their endogenous release induced by compound 48/80 or repeated egg albumin administration to sensitized rats proved to increase activity of mesenteric afferent nerve fibers in the small intestine. In addition, the primary contact with a foreign protein triggered a cascade of chemical processes mobilizing antioxidant systems and increasing nitric oxide production in the small intestinal tissues. Interleukin-1β or Escherichia coli lipopolysaccharide injections also had a long-term stimulating effect on intestinal receptors. Indometacin inhibited the effects induced by the bacterial endotoxin and interleukin-1β. The obtained data suggest that small intestinal interoceptors can monitor chemical environment and modulate body’s responses to foreign antigens. Interleukins-1β acts as a signal transmitter between immune cells and interoceptors, which is mediated by the production of secondary signal substances, in particular, prostaglandin E2 and histamine.     

Molecular and cellular mechanisms of the low intensity laser radiation effect

The main aspects of the free radical conception of the molecular and cellular mechanisms of the stimulating action of low-intensity radiation in the red region of the spectrum were considered. These are: (1) Primary acceptors of incident radiation are endogenous porphyrins, which may act as photosensitizers giving initiator-radicals for secondary free radical reactions. (2) Target cells for light irradiation during quantum therapy may be blood leukocytes, fibroblasts, keratinocytes, endotheliocytes, etc. (3) The initiation of the secondary free radical reactions due to lipid peroxidation of cell membranes (in particular, of leukocytes) brings about an increase in ion permeability including that for calcium. The increase in intracellular calcium concentration leads to phagocytes priming, i.e., to increased production of reactive oxygen species (ROS) under subsequent stimulation of the cell. (4) Photosensitized generation of ROS in the cytoplasm of some cells induces a free-radical activation of synthesis of proteins, the most significant in the light of the present concept being the de novo synthesis of inducible NO-synthase, superoxide dismutase, and various cytokines. The experimental evidence for the basic statements of the conception of free radical mechanisms for the stimulating action of low-intensity laser and noncoherent radiations is presented. A relation between the primary mechanisms of the stimulating action of light and the secondary effects that determine the sanative effect of quantum therapy in the process of wound healing (bactericidity, cell proliferation, and improved microcirculation) was established. Moreover, it was shown that nitrosyl complexes of heme proteins, such as hemoglobin and cytochrome c, are the primary chromophores of laser radiation. Upon irradiation, they can easily dissociate to produce free nitric oxide. In turn, released nitric oxide may be responsible for blood vessel relaxation and activation of mitochondrial respiration. This phenomenon is just observed during phototherapy by means of low-intensity laser radiation.     

Effects of AANAT overexpression on the inflammatory responses and autophagy activity in the cellular and transgenic animal levels

To explore the anti-inflammatory activity of endogenous produced melatonin, a melatonin-enriched animal model (goat) with AANAT transfer was successfully generated with somatic cell nuclear transfer (SCNT) technology. Basically, a pIRES2-EGFP-AANAT expression vector was constructed and was transferred into the female fetal fibroblast cells (FFCs) via electrotransfection and then the nuclear of the transgenic FFC was transferred to the eggs of the donor goats. The peripheral blood mononuclear cells (PBMCs) of the transgenic offspring expressed significantly higher levels of AANAT and melatonin synthetic function than those PBMCs from the wild-type (WT) animals. After challenge with lipopolysaccharide (LPS), the transgenic PBMCs had increased autophagosomes and LC3B expression while they exhibited suppressed production of the proinflammatory cytokines, IL1B and IL12 (IL12A-IL12B/p70), compared to their WT. The mechanistic analysis indicated that the anti-inflammatory activity of endogenous melatonin was mediated by MTNR1B (melatonin receptor 1B). MTNR1B stimulation activated the MAPK14 signaling pathway to promote cellular macroautophagy/autophagy, thus, suppressing the excessive inflammatory response of cellular. However, when the intact animals challenged with LPS, the serum proinflammatory cytokines were significantly higher in the transgenic goats than that in the WT. The results indicated that endogenous melatonin inhibited the MAPK1/3 signaling pathway and ROS production, subsequently downregulated gene expression of BECN1, ATG5 in PMBCs and then suppressed the autophagy activity of PBMCs and finally elevated levels of serum proinflammatory cytokines in transgenic animals, Herein we provided a novel melatonin-enriched animal model to study the potential effects of endogenously produced melatonin on inflammatory responses and autophagy activity.     

Porphyrin-mediated boron neutron capture therapy: a preclinical evaluation of the response of the oral mucosa

Preclinical studies are in progress to determine the potential of boron neutron capture therapy (BNCT) for the treatment of carcinomas of the head and neck. Recently, it has been demonstrated that various boronated porphyrins can target a variety of tumor types. Of the porphyrins evaluated so far, copper tetracarboranylphenyl porphyrin (CuTCPH) is potentially a strong candidate for clinical use. In the present investigation, the response of the oral mucosa to CuTCPH-mediated boron neutron capture (BNC) irradiation was assessed using the ventral surface of the tongue of adult male Fischer 344 rats, a standard rodent model. CuTCPH was administered by intravenous infusion, at a dose of 200 mg/kg body weight, over a 48-h period. Three days after the end of the administration of CuTCPH, biodistribution studies indicated very low levels of boron (<2 microg/g) in the blood. Levels of boron in tongue tissue were 39.0 +/- 3.8 microg/g at this time. This was the time selected for irradiation with single doses of thermal neutrons from the Brookhaven Medical Research Reactor. The estimated level of boron-10 in the oral mucosa was used in the calculation of the physical radiation doses from the 10B(n,alpha)7Li reaction. This differs from the approach using the present generation of clinical boron carriers, where boron levels in blood at the time of irradiation are used for this calculation. Dose-response curves for the incidence of mucosal ulceration were fitted using probit analysis, and the doses required to produce a 50% incidence of the effect (ED50 +/- SE) were calculated. Analysis of the dose-effect data for CuTCPH-mediated BNC irradiation, compared with those for X rays and thermal neutrons alone, gave a compound biological effectiveness (CBE) factor of approximately 0.04. This very low CBE factor would suggest that there was relatively low accumulation of boron in the key target epithelial stem cells of the oral mucosa. As a consequence, with low levels of boron (<2 microg/g) in the blood, the response of the oral mucosa to CuTCPH-mediated BNCT will be governed primarily by the radiation effects of the thermal neutron beam and not from the boron neutron capture reaction [10B(n,alpha)7Li].     

Mitogenic action of the liquid phase of a microbial suspension of Bordetella pertussis

The suspension of B. pertussis cells in 0.15 M NaC1 solution, used for the preparation of corpuscular pertussis vaccine contains components loosely bound to microbial cells and producing pronounced mitogenic effect on mouse splenocytes at a concentration of 10 micrograms/ml. The mitogenic activity of B. pertussis is due to complex substances (lipopolysaccharide, protein, nucleic acids) with a wide range of molecular weights (70,000 to greater than 400,000). The mitogenic factor showing no leukocyte-stimulating and protective activity has been isolated by sedimentation with ammonium sulfate and gel filtration on Sephadex G-200. The mitogenic activity of B. pertussis lipopolysaccharide in the blast transformation test has been confirmed.     

A comparative study of the effects of laser and light-emitting diode radiation on superoxide dismutase activity and nitric oxide production in rat wound fluid

The effect of laser and light-emitting diode radiation in the visible region of the spectrum on the content of reactive nitrogen species and superoxide dismutase activity in rat wound fluid was studied. The efficiency of action of coherent laser radiation and incoherent light-emitting diode radiation in the red region of the spectrum on the parameters analyzed was compared. The study was performed using the model of cut aseptic wounds proposed by L.I. Slutskii. A He-Ne laser (632 nm) or an U-332B light-emitting diode (630 nm) was used as the source of radiation. It was shown that (1) exposure of wounds to visible light of both laser and light-emitting diode causes dose-dependent changes in superoxide dismutase activity and nitrite production and that (2) radiation coherence does not play a significant role in the changes in superoxide dismutase activity or nitric oxide production by wound fluid phagocytes.