Distribution of the intensity of free radical reactions at different stages of bud growth and in adult hydra

The distribution of free radical reactions was studied by means of grafted copolymerization of 14C-acrylamide monomer in the ectoderm of growing bud and adult Hydra. Several states of regionalization were established in the process of bud growth: absence of local differences, presence of local differences and presence of the peak of free radical reactions in the hypostome region, characteristic of adult Hydra. The initiation of budding is not accompanied by the activation of free radical reactions in this region. No direct relationship between the metabolic regionalization and morphological rearrangements was found.     

Free radical generation by redox cycling of estrogens

Natural and synthetic estrogens elicit normal hormonal responses in concentrations in a clearly defined yet low range. At elevated doses, metabolic reactions of the phenolic moiety, while harmless at low levels, may become the predominant biochemical activity and may exert deleterious effects. These metabolic pathways, such as i) oxidation of estrogens to catechol estrogens and further to their respective quinones, and ii) free radical generation by redox cycling between catechol estrogens or diethylstilbestrol and their quinones, are investigated for their influence in physiological or pathophysiological processes. In this review, the in vitro capacity of various enzymes to oxidize estrogen hydroquinones to quinones or to reduce corresponding quinones to hydroquinones is evaluated. The in vivo activities of enzymes supporting redox cycling of estrogens and free radical generation is correlated with induction of kidney tumors in Syrian hamsters. Concomitant changes in activities in quinone reductase and other detoxifying enzymes in kidneys of hamsters treated with estrogen support a role of free radicals in the induction of tumors by estrogen. Free radical damage to protein and possibly to DNA in kidneys of estrogen-treated hamsters may be used as markers of free radical action in vivo.     

Free radicals and neuronal cell death

Production of oxygen free radicals is a natural consequence of aerobic metabolism and they are constantly generated in vivo by chemical reactions and metabolic processes. Antioxidant defence systems scavenge and minimise the formation of oxygen-radical-derived biochemical products, however, these defences are not completely effective even under normal physiological conditions. In pathologic situations, oxygen free radicals can be generated in excess of a cell's antioxidant capacity resulting in severe damage to cellular constituents including proteins, DNA and lipids. The inherent biochemical and physiological charateristics of the brain, including high lipid concentrations and energy requirements, make it particularly susceptible to free radical mediated insult. Increasing evidence indicates that many neurological disorders may have components of free radical and oxidative stress induce injury.     

Rat brain reactions to acute brain stem lesion on dynamics of parameters of cerebral electric activity

Despite an old history of a question, adaptive brain reactions that develop after an acute brainstem lesion have not been adequately investigated. With the aim to study the central mechanisms of compensation/decompensation and to specify the character of involvement of orbitofrontal cortex and hippocampus into these processes the spatiotemporal organization of brain electric activity was analyzed in 8 rats before and after electrolytic brainstem lesion at the level of the lateral vestibular nucleus Deiters (VND). The electric activity was recorded from symmetric frontal and somatosensory cortical areas, hippocampal areas CA1, and intact VND. Spectralcoherent analysis showed that adaptive reactions are most clearly manifested by changes in the spatiotemporal organization of the theata activity: 1) early brainstemhippocampal synchronization of the electric activity in the frequency range of 6-7 Hz with subsequent involvement of anterior cortical regions is characteristic of survived animals; 2) independent hippocampal-cortical hemispheric system of excitation in the frequency of 4-5 Hz precedes the fatal outcome. On the day before the fatal outcome the interhemispheric coherence in the orbitofrontal cortex dropped, which suggests the involvement of these brain regions into the processes of visceral regulation.     

Free radicals in physiology and pathology.

Free radicals are molecules with odd number of electrons and a high instability. Free radicals, which can occur in both organic (i.e., quinones) and inorganic molecules (i.e., O2-), are very reactive and their reactions are critical for the normal activity of a wide spectrum of biologic processes. They are also produced in the catalytic action of a variety of cellular enzymes and electron transport processes and are implicated in a number of physiologic and pathologic processes. Organisms can be exposed to free radicals in many ways other than through the processes of normal metabolism. Irradiation of organisms with electromagnetic radiation generates primary radicals (e-aq, OH., and H.), which can then undergo secondary reactions with dissolved O2 or with cellular solutes. In addition, a wide variety of environmental agents (drugs capable of redox cycling, and xenobiotics that can form free radical metabolites) including the aging process cause free radical damage to cells. This review deals with the reactions they can undergo and discusses the free radicals related to toxicology.     

Nicotinamide deficiency in human lymphocytes prevents the [H]thymidine-induced adaptive response for the repair of X-ray-induced chromosomal damage

Human lymphocytes treated with [3H]thymidine ([3H]dThd) become refractory to the induction of chromosomal aberrations by subsequent doses of X rays. This adaptive response to [3H]dThd does not occur in the presence of 3-aminobenzamide (3AB). 3AB inhibits the synthesis of poly(ADP-ribose) by the enzyme adenosine diphosphate ribosyl transferase (ADPRT), which requires NAD as a substrate. 3AB also prevents chromosomal repair, as measured in X-ray dose-fractionation studies. Because 3AB might interfere with metabolic reactions other than those mediated by ADPRT, experiments were carried out to see if the adaptive response was also inhibited in nicotinamide-free medium, which prevents poly(ADP-ribosyl)ation by depleting cellular NAD. The experiments show that the incorporation of [3H]dThd has no effect on the induction of chromosomal aberrations by subsequent doses of X rays if the cells are cultured in nicotinamide-free medium. Nicotinamide deficiency mimics the effects of 3AB on both the adaptive response and chromosome repair. The results indicate that ADPRT activity itself, and not other metabolic processes affected by inhibitors of this enzyme, plays an essential role in the adaptive response.     

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.     

Hyperinsulinemia: the missing link among oxidative stress and age-related diseases?

Mounting evidence supports Harman’s hypothesis that aging is caused by free radicals and oxidative stress. Although it is known that oxidant species are produced during metabolic reactions, it is largely unknown which factor(s), of physiological or pathophysiological significance, modulate their production in vivo. In this hypothesis paper, it is postulated that hyperinsulinemia may have such function and therefore promote aging, independently of elevations of glycemia. Hyperinsulinemia is secondary to impaired insulin stimulated glucose metabolism at the level of skeletal muscle (insulin resistance) and is seen in about one third of glucose tolerant humans following dietary carbohydrate intake. If other insulin-stimulated (or inhibited) pathways retain normal sensitivity to the hormone, hyperinsulinemia could, by its effects on antioxidative enzymes and on free radical generators, enhance oxidative stress. Other proaging effects of insulin involve the inhibition of proteasome and the stimulation of polyunsaturated fatty acid (PUFA) synthesis and of nitric oxide (NO). The hypothesis that hyperinsulinemia accelerates aging also offers a metabolic explanation for the life-prolonging effect of calorie restriction and of mutations decreasing the overall activity of insulin-like receptors in the nematode Caenorhabditis elegans.     

A new nitroxyl-probe with high retention in the brain and its application for brain imaging

In order to estimate free radical reactions and image them in the brain of living animals, a nitroxyl spin-probe, carboxy-PROXYL acetoxymethyl ester (CxP-AM) was newly synthesized. CxP-AM was designed to be hydrolyzed by esterase, but not by lipase, so that it would pass through the blood-brain barrier and be retained in the cytosolic phase of parenchymal cells in the brain after intravenous injection. The pharmacokinetics of CxP-AM was compared with those of carboxy-PROXYL (CxP) and its methyl ester (CxP-M). Carboxyl esterase almost completely hydrolyzed CxP-AM within 3 min. After intravenous injection, the brain retained 1.8 times more CxP-AM than CxP-M, and retained it for more than 30 min. Electron spin resonance computed tomographic (ESR-CT) imaging of CxP-AM in the heads of mice produced marked contrast in the encephalon region, while CxP was distributed only in the extracranial region and CxP-M was distributed in both regions, confirming the pharmacokinetics of CxP-AM. The decay rate of CxP-AM determined with time-resolved ESR-CT imaging was different in the two brain regions, suggesting regional differences in the total reducing capability. CxP-AM should become a powerful probe for the investigation and diagnosis of free radical reactions and their imaging in the brain.     

A morphometric study of the midgut in resistant and non-resistant Anticarsia gemmatalis (Hübner) (Lepidoptera: Noctuidae) larvae to its nucleopolyhedrovirus (AgMNPV)

In this investigation, the anterior and posterior regions of the midgut of resistant (RL) and non-resistant (SL) Anticarsia gemmatalis larvae were analyzed morphometrically to characterize different regions along their length. Also, this investigation compares the results between SL and RL to improve the understanding of the resistance mechanisms to the virus. Histological sections were analyzed in a computerized system and the data were statistically analyzed by the Kruskal-Wallis test and by multivariate analysis. The midguts are morphometrically different in the two larval populations; we observed higher values in RL. The morphometric analysis of the epithelial cells showed that only columnar and goblet cells were distinct along the midgut, in both larvae, with the higher values found in the anterior region. Comparing the results between the two larval populations, all the epithelial cells presented significant differences, with RL showing the higher morphometric values. We concluded that there are regional differences along the length of midgut in SL and RL that confirm the idea of two morpho-functional distinct regions. The consistently morphometric superior values in RL indicate that this variability can be related with the resistance of A. gemmatalis to its AgMNPV.     

Effect of the level of free-radical lipid peroxidation on the ribonucleotide reductase activity of liver tissue

The dynamics of RNA and DNA synthesis as well as the activity of free radical processes in rat liver during the first 40 hours after partial hepatectomy was studied. It was shown that RNA synthesis activation follows the activation of lipid peroxidation (LPO), whereas the DNA synthesis activation follows the decrease of the LPO level. These facts are suggestive of the dependence of the ribonucleotide reductase (RNR) activity on free radical processes. This observation was confirmed by a RNR activity analysis of regenerating liver homogenates. The activity peak was shown to precede the peak of DNA synthesis. Evidence for free radical RNR suppression was also obtained in direct experiments, using intact animal liver homogenates, to which a natural antioxidant (tocopherol) was added.     

The role of taurine in adaptation of visceral systems under psycho-emotional stress in rats

In the recent years, an identification of regulatory mechanisms underlying the general adaptation syndrome as an organism’s response to drastic emotional stress-evoking environmental changes is gaining in its importance. The ability to control over visceral functions plays a crucial role in stress reactions due to a threat of neurodynamic imbalance in sympathetic-parasympathetic relationships with the heart as their most vulnerable element. Fast stress adaptation promotes restoration not only of the sympathetic-parasympathetic balance, but also of energy metabolism. Taurine is one of the major regulatory molecules that activate metabolic processes. The present work addresses the following issues: (1) the descending influence of the paraventricular nucleus (PVN) on neural reaction of the solitary tract nucleus (STN), which is the first link in the visceral sensitivity pathway, (2) the mechanisms of central control over visceral reactions as investigated by mathematical modelling and analysis of the heart rate variability (HRV), and (3) morphofunctional changes in brain structures, integrating and regulating the visceral sphere (hypothalamic PVN, amygdala), under psycho-emotional stress with and without intraperitoneal injection of taurine (50 mg/kg). Acute and semichronic experiments were conducted on white nonlinear rats under 5-h immobilization stress. An extremely strong centralization of the vegetative HRV parameters (HR, VBI, SSTI) was revealed, with these parameters normalized on days 7 and 14 post taurine injection. An interaction and interdependence of the central regulatory mechanisms of cardiovascular reactions as well as a considerable protective role of taurine, promoting fast restoration of adaptive properties of the central and peripheral visceral sensitivity components under the development of long-term psycho-emotional stress, were shown.     

DNA replication in mammalian cells after the action of physical, chemical or biological factors. VI. The recovery of DNA synthesis in a culture of irradiated cells

The kinetics of DNA synthesis restoration in cultured HeLa cells and in L-929 mouse fibroblasts irradiated by gamma-rays of 60Co with a dose of 10 Gy was studied. Early after irradiation the rate of DNA synthesis in HeLa cells measured with 3H-thymidine incorporation was seen to decrease. Two hours later the incorporation starts to increase to reach the control level 4 hours after irradiation and then becomes even higher than this level. The distribution of cells among phases of the cell cycle measured with flow cytometry undergoes changes. 4-6 hours after irradiation part of S-phase cells increased contributing presumably to the elevating of 3H-thymidine incorporation observed at this time. The restoration of the incorporation was suppressed by inhibitors of protein and RNA synthesis--cycloheximide and actinomycin D. It is suggested that the processes of restoration of DNA synthesis in irradiated cells can be of inducible nature. In irradiated HeLa and L-929 cells the restoration of DNA synthesis is resistant to novobiocin, an inhibitor of DNA replication.     

DNA damage, repair, and antioxidant systems in brain regions: a correlative study

8-Hydroxy-2'-deoxyguanosine (oxo(8)dG) has been used as a marker of free radical damage to DNA and has been shown to accumulate during aging. Oxidative stress affects some brain regions more than others as demonstrated by regional differences in steady state oxo(8)dG levels in mouse brain. In our study, we have shown that regions such as the midbrain, caudate putamen, and hippocampus show high levels of oxo(8)dG in total DNA, although regions such as the cerebellum, cortex, and pons and medulla have lower levels. These regional differences in basal levels of DNA damage inversely correlate with the regional capacity to remove oxo(8)dG from DNA. Additionally, the activities of antioxidant enzymes (Cu/Zn superoxide dismutase, mitochondrial superoxide dismutase, and glutathione peroxidase) and the levels of the endogenous antioxidant glutathione are not predictors of the degree of free radical induced damage to DNA in different brain regions. Although each brain region has significant differences in antioxidant defenses, the capacity to excise the oxidized base from DNA seems to be the major determinant of the steady state levels of oxo(8)dG in each brain region.     

Age-dependent characteristics of oxidative stress formation in the liver of rats with hypothyroidism during intensive physical exercise

The objective of the present experiment was to study free radical protein oxidation and lipid peroxidation in the liver of 1.5-month-old and 12-month-old rats with drug-induced hypothyroidism caused by exercises. The results of the present study suggest that intensive exercises are accompanied by an increase of intensity of free radical processes in the liver. Hypothyroidism and exercises do not greatly affect free radical processes in the liver of 12-month-old rats but result in additional stimulation of free radical oxidation in subcellular liver fractions of 1.5-month-old rats. An increase of free radical processes in the liver of 1.5-month-old rats with hypothyroidism caused by exercises is associated neither with changes in the first level antioxidant defense system enzymes function, nor with modulation of hepatocytes subcellular sensitivity to prooxidants. Such change is due largely to an increase of free radical production in the liver cells.     

Compensatory growth of coronary arterioles in postinfarcted heart: regional differences in DNA synthesis and growth factor/receptor expression patterns

Previous studies have not addressed regional differences in adaptive arteriolar growth in the surviving left ventricular (LV) myocardium after infarction in appropriately aged animals, namely middle-aged or older. Accordingly, we examined the adaptive postinfarction growth of arterioles in two distinct regions, i.e., the LV free wall (LVFW) and septum, of middle-aged rats. We induced a myocardial infarction (MI) in 12-mo-old rats to analyze 1) protein expression in VEGF/Flt-1/Flk-1 and angiopoietin (Ang)-1/Ang-2/Tie-2 systems, 2) the arteriolar DNA synthesis, 3) the extent of the arteriolar bed, and 4) the alteration in minimal coronary vascular resistance. In both regions, arteriolar DNA synthesis was activated between days 4 and 7 after MI. Whereas in the LVFW the degree of DNA synthesis declined between days 11 and 14 post-MI, it continued to rise in the septum, and at day 14, the percentage of the arterioles undergoing DNA synthesis was comparable in the LVFW and the septum (9.7 +/- 1.6 and 7 +/- 2.1%, respectively). Arteriolar DNA synthesis was mainly associated with upregulation of Ang-2 and Tie-2 in both LV regions. Although 4 wk after MI the arteriolar beds in the LVFW and the septum expanded to the size of sham-operated rats, this growth did not compensate for the greater minimal coronary vascular resistance in the former. Thus our findings suggest that 1) the dynamics in adaptive arteriolar growth were similar between the two regions, despite a delay in the septum; and 2) the perfusion deficit in post-MI rats cannot be accounted for by inadequate adaptive growth of arterioles.     

Tracheotomy in a case of tracheobronchitis secondary to influenza

Five to 10 per cent of cretinism in the United States is due to some congenital enzymatic defect in thyroid hormone synthesis. The clinical signs of hypothyroidism appear in early infancy. Differentiation from athyreotic cretinism is important because the metabolic defect tends to be familial and its presence in the patient's infant relatives should be diagnosed as early as possible. The differentiation is easily made if a goiter is discernible, but if it is not, radioiodine uptake should be measured, for in this condition the uptake is normal or greater. Thyroid replacement is the treatment in either the athyreotic state or the metabolic deficiency. The three known defects in thyroid hormone synthesis are (1) failure to oxidize iodine to elemental iodine resulting in failure of all subsequent processes; (2) failure to deiodinate free iodotyrosine, and (3) failure to form iodothyronine although the previous steps are accomplished.     

Gene Activity Patterns and Cellular Differentiation

Puffing in giant chromosomes ot Diptera is considered to reflectthe pattern of active gene loci in these chromosomes. In anyone tissue only a relatively small portion of the total bands(about 10 to 20%) have been observed to form a puff at sometime or another in larval development. These patterns of "potentiallyactive" loci are tissue specific, though greatly overlapping.The actual rate of activity at these loci is controlled independentlyof each other and independently in each tissue by factors ofthe extranuclear metabolism. Puffing at some loci seems to berelated to specific cellular functions, such as secretion ofthe salivary glands. The activity of others may be related tomore basic metabolic processes. In relation to larval development,puffing patterns may change with changing cell functions orwith developmental processes in the cells themselves. In salivaryglands ofChironomus activity of DNAase and of acid phosphataseseems to change in relation to cell breakdown at the end ofthe pupal molt. Changes of acid phosphatase activity begin earlyin the last larval ins tar, but the enzyme is bound to lysosomesuntil metamorphosis. This suggests that the genes specificallyactive during metamorphosis have to interact with a longtermcontrol-system of development. The induction of metamorphosisis a sequential process, gene activations being among the firststeps in this sequence. The activation of these genes by ecdysoneis independent of protein synthesis. It is only the reactionof these genes that leads to the subsequent events in the cell,including the subsequent puff activations. This is shown bythe fact that they depend on early RNA synthesis as well ason protein synthesis. These results on puffing are discussedwith regard to the general problem of the relationships betweenpatterns of gene activity and differentiation.     

Aging: a shift from redox regulation to oxidative damage

Proteins, nucleic acids, and lipids can undergo various forms of oxidative modification. In numerous instances, these modifications result in irreversible loss of function. The age-dependent accumulation of oxidatively modified and dysfunctional macromolecules provides the basis for the free radical theory of aging. Pro-oxidants, however, are also capable of catalyzing fully reversible modifications to protein. It is increasingly apparent that these reactions participate in redox-dependent regulation of cell metabolism and response to stress. The adventitious use of free radical species adds complexity to the experimental and theoretical manner in which the free radical theory is to be tested and considered. Elucidation of mechanisms by which reversible oxidative processes are controlled, the components involved, and the metabolic consequences and how they are altered with age will provide new insight on the aging process and attempts to delay the inevitable.