Membranes as sensitive targets in thymocyte apoptosis.

The role of cellular membranes in thymocyte apoptosis has been examined. Trolox, a water soluble analogue of vitamin E and inhibitor of membrane damage, inhibits DNA fragmentation in thymocytes exposed to gamma-radiation. Trolox is most effective in inhibiting DNA fragmentation when added to cells within 30 min post-irradiation. Exposure to trolox only during irradiation did not prevent DNA fragmentation, suggesting that it does not work by scavenging free radicals generated during radiation exposure. Incubation of the irradiated cell suspension with trolox for 2 h post-irradiation was sufficient to prevent DNA fragmentation measured at 24 h in irradiated cells. This suggests that trolox irreversibly inhibits a cellular lesion required for apoptosis. The induction of DNA fragmentation appears to be related to a concurrent, pronounced flow of Ca2+ into the cell. At 3 h post-irradiation the amount of Ca2+ in irradiated thymocytes was more than twice that of unirradiated thymocytes. Membrane damage has been shown to affect the transport of Ca2+. Trolox treatment completely blocked the radiation-induced influx of Ca2+ into the thymocytes. These results suggest that membrane damage is a critical lesion that is involved in DNA fragmentation in thymocyte apoptosis.

Radioprotection of hematopoietic tissues in mice by lipoic acid.

Lipoic acid is a lipophilic antioxidant that participates in many enzymatic reactions and is used clinically to treat mushroom poisoning and metal toxicity. In this report the protective effect of lipoic acid (oxidized form) against radiation injury to hematopoietic tissues in mice was assessed by the endogenous and exogenous spleen colony assays and survival (LD50/30) assay. Intraperitoneal administration of lipoic acid at a nonlethal concentration of 200 mg/kg body wt, 30 min before irradiation increased the LD50/30 from 8.67 to 10.93 Gy in male CD2F1 mice. Following a 9-Gy irradiation, the yield of endogenous spleen colony-forming units in mice treated with saline and lipoic acid was 0.75 +/- 0.5 and 8.9 +/- 1.6, respectively. Using the exogenous spleen colony assay, lipoic acid treatment increased the D0 from 0.81 +/- 0.01 to 1.09 +/- 0.01 Gy, yielding a dose modification factor of 1.34 +/- 0.01. Dihydrolipoic acid (reduced form) has no radioprotective effect in CD2F1 mice.

N-(2-mercaptoethyl)-1,3-propanediamine (WR-1065) protects thymocytes from programmed cell death.

Gamma-irradiation, glucocorticoid hormones, and calcium ionophores stimulate a suicide process in thymocytes, known as apoptosis or programmed cell death, that involves internucleosomal DNA fragmentation by a Ca(2+)- and Mg(2+)-dependent nuclear endonuclease. In this study we report that N-(2-mercaptoethyl)-1,3-propanediamine (WR-1065) blocked DNA fragmentation and cell death in thymocytes exposed to gamma-radiation, dexamethasone, or calcium ionophore A23187. WR-1065 protected the thymocytes from radiation-induced apoptosis when incubated with cells after irradiation but not before and/or during irradiation. WR-1065 inhibited Ca(2+)- and Mg(2+)-dependent DNA fragmentation in isolated thymocyte nuclei. Our results suggest that WR-1065 protects thymocytes from apoptosis by inhibiting Ca(2+)- and Mg(2+)-dependent nuclear endonuclease action.

Radioprotective properties of DNA methylation-disrupting agents.

5-Azacytidine and sodium butyrate, two DNA methylation-disrupting agents, were tested for radioprotective properties on V79A03 cells. Both compounds can activate genes not previously expressed (e.g. metallothionein). 5-Azacytidine treatment (3 microM, 24 h) caused a 50% decrease in the 5-methylcytosine content of V79A03 DNA whereas sodium butyrate treatment (1 mM, 24h) resulted in a 700% increase in 5-methylcytosine content. Additionally, 5-azacytidine treatment resulted in the increased survival of V79A03 cells, with treatment 24 h prior to exposure to gamma radiation providing a dose reduction factor of 1.8. Sodium butyrate treatment did not result in a significant increase in survival. These results indicate that the hypomethylation of genomic DNA prior to exposure to gamma radiation correlates with an increase in survival of V79A03 cells, possibly due to the activation of the enzymes involved in repair.

Influence of whole body irradiation and local shielding on matrix-induced endochondral bone differentiation.

Subcutaneous implantation of demineralized bone matrix into allogeneic rats induces endochondral bone formation. We have investigated the effects of irradiation on the sequelae of the interaction of collagenous matrix and mesenchymal cells and on cartilage and bone differentiation. Rats were irradiated in a vertical direction with a midline dose of 850 rad. Radiation entered the rats ventrally while a small area of the upper thorax was locally shielded. After irradiation, bone matrix was implanted in shielded and nonshielded sites, and the implants were studied at various stages. On day 3, [3H]thymidine incorporation, an index of cell proliferation, was inhibited by 70% in the nonshielded sites compared to nonirradiated control rats. The degree of inhibition (35%) was less pronounced in shielded sites. Furthermore, there was recovery of cell proliferation in the shielded sites as opposed to the nonshielded contralateral site. A similar pattern was observed on day 7 as assessed by 35SO4 incorporation into proteoglycans during chondrogenesis. Bone formation and mineralization were quantified on day 11 by alkaline phosphatase activity and 45Ca incorporation. In nonshielded sites, there was a 73% inhibition of alkaline phosphatase activity. In conclusion, radiation impaired progenitor cell proliferation which resulted in decreased cartilage and bone differentiation. These findings imply that local mesenchymal cells proliferate and differentiate into bone in response to implanted collagenous matrix.

The effect of gamma radiation on DNA methylation.

The effect of 60Co gamma radiation on DNA methylation was studied in four cultured cell lines. In all cases a dose-dependent decrease in 5-methylcytosine was observed at 24, 48, and 72 h postexposure to 0.5-10 Gy. Nuclear DNA methyltransferase activity decreased while cytoplasmic activity increased in irradiated (10 Gy) V79A03 cells as compared to controls. No DNA demethylase activity was detected in the nuclei of control or irradiated V79A03 cells. Additionally, gamma radiation resulted in the differentiation of C-1300 N1E-115 cells, a mouse neuroblastoma line, in a dose- and time-dependent manner. These results are consistent with the hypothesis that (1) genes may be turned on following radiation via a mechanism involving hypomethylation of cytosine and (2) radiation-induced hypomethylation results from decreased intranuclear levels of DNA methyltransferase.

Elevated cerebrospinal fluid histidine in alcohol withdrawal.

Cerebrospinal fluid (CSF) histidine concentration was significantly elevated in seven patients early in the alcohol withdrawal syndrome (206.3 +/- 74.4 (SEM) nanomols/ml CSF). When these same patients were restudied an average of six days later when alcohol withdrawal was clinically resolved, their mean CSF histidine concentration continued to be significantly elevated (164.7 +/- 24.7) when compared to normal (12.0 +/- 0.5 nanomols/ml CSF). Other amino acids (aspartic acid, serine, alanine, methionine, leucine, tyrosine, phenylalanine, lysine and arginine) showed no definite changes from normal, and no change during the course of alcohol withdrawal. Possible reasons for these high concentrations and the extreme variability (especially early in alcohol withdrawal) are discussed.

Urinary excretion of cyclic nucleotides, creatinine prostaglandin E2 and thromboxane B2 from mice exposed to whole-body irradiation from an enhanced neutron field.

Urine volume and excretion of cyclic AMP, cyclic GMP, prostaglandin E2 (PGE2), thromboxane B2 (TxB2) and creatinine were evaluated as potential indicators of radiation damage in mice given 2-5 Gy to the whole body from an enhanced neutron field. In general, urinary cyclic AMP, cyclic GMP, creatinine and urine volumes were positively correlated across time postexposure, for each radiation dose. TxB2 levels positively correlated with urine volume and cyclic AMP excretion only in animals given 2.0 Gy. None of these parameters suggests their use as a prognostic indicator of the extent of radiation damage. Urinary excretion of PGE2 was negatively correlated with other urinary parameters. Biphasic increases in urinary PGE2 were also observed. The initial transient elevation 2-3 days postexposure was not correlated with the dose (2-5 Gy). The second elevation of PGE2 excretion occurred at 6-10 days. The magnitude of the latter increase suggests that urinary PGE2 excretion may be a useful indicator of whole-body or kidney exposure to neutron fields.

Circadian rhythms in catecholamine metabolites and cyclic nucleotide production.

Circadian rhythms in noradrenergic (NE) and dopaminergic (DA) metabolites and in cyclic nucleotide production were measured in discrete regions of rat brain. A circadian rhythm was found in the concentration of the NE metabolite, 3-methoxy-4-hydroxyphenylglycol (MHPG), in the hippocampus. No MHPG rhythm was found in frontal, cingulate, parietal, piriform, insular or temporal cortex, or in hypothalamus. Circadian rhythms in the concentration of the NE metabolite, 3,4-dihydroxyphenylglycol (DHPG), occurred in occipital and parietal cortex and hypothalamus, with no rhythm observable in temporal or insular cortex, hippocampus, pons-medulla or cerebellum. The 24-hr mean concentration of MHPG varied 3.5-fold, highest in cingulate and lowest in parietal, temporal and occipital cortex. The 24-hr mean concentration of DHPG varied 6-fold, highest in hypothalamus and lowest in parietal cortex. Circadian rhythms in the concentration of the DA metabolite, homovanillic acid (HVA), were found in olfactory tubercle, amygdala and caudate-putamen, but not in nucleus accumbens. A circadian rhythm in the concentration of the DA metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), occurred in nucleus accumbens, but not in olfactory tubercle or caudate-putamen. The mean 24-hr concentration of HVA was highest in caudate-putamen, intermediate in nucleus accumbens, and lowest in olfactory tubercle and amygdala. The mean 24-hr concentration of DOPAC was highest in nucleus accumbens and lower in olfactory tubercle and caudate-putamen. Circadian rhythms were found in the concentration of cyclic GMP (cGMP) in all regions measured except parietal cortex. The mean 24-hr concentration varied 128-fold, highest in nucleus accumbens, frontal poles, and hypothalamus and lowest in cingulate cortex. Circadian rhythms in cyclic AMP (cAMP) concentration were found in piriform, temporal, occipital, cingulate, and parietal cortex, amygdala and nucleus accumbens. No rhythms were found in frontal or insular cortex, hypothalamus, hippocampus, caudate-putamen or olfactory tubercle. The 24-hr mean cAMP concentration varied 4-fold, highest in parietal cortex and lowest in caudate-putamen and amygdala. Norepinephrine metabolites and dopamine metabolites were rhythmic in few regions. It is, therefore, unlikely that the rhythmicity measured in adrenergic receptors is, in general, a response to rhythmic changes in adrenergic transmitter release. The putative second messenger response systems, especially cGMP, were more often rhythmic. The rhythms in cGMP are parallel in form and region to those in the alpha 1-adrenergic receptor and may act as 2nd messenger for that receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Radiation effects on diamine oxidase activities in intestine and plasma of the rat.

Diamine oxidase (DAO; EC 1.4.3.6) activity was measured in plasma and in ileal tissue homogenates prepared from male Sprague-Dawley rats euthanized at 1-15 days after acute whole-body irradiation with 14.5-MeV electrons. Animals irradiated with 1 Gy showed no diminution in plasma and ileal DAO activities through Day 13 relative to nonirradiated controls. Animals irradiated with 5, 10, and 12 Gy displayed marked declines in ileal DAO activity, with levels reaching a nadir on Day 3. This was paralleled by a decrease in plasma DAO activity in all three dose groups. Recovery of ileal and plasma DAO levels was later seen as early as Day 4 in animals irradiated with 5- and 10-Gy doses, but animals receiving 12 Gy did not survive beyond Day 3. The relationship between radiation dose and levels of plasma and ileal DAO on Day 3, the time of maximum decrease at all doses, was also investigated. Ileal DAO activity decreased almost linearly between 2 and 8 Gy. Plasma DAO activity closely paralleled the dose dependency of the ileal levels. These data suggest that plasma DAO activity might be useful as a biologic marker of intestinal epithelial injury and recovery after acute radiation exposure.

WR-2721 inhibition of radiation-induced prostaglandin excretion in rats.

Pre-irradiation administration of the radioprotectant drug WR-2721 to rats resulted in a significant reduction in radiation-induced increases in excretion rates of prostaglandins (PGE and PGF2 alpha) and thromboxane (TxB2). In animals not irradiated. WR-2721 did not significantly alter these excretion rates. Dramatic reductions in the levels of urinary PGE and TxB2 were observed following exposure to 9.0 Gy of whole-body, unilateral gamma-radiation in WR-2721-treated animals, whereas changes in PGF2 alpha levels were less pronounced. Radiation-induced diuresis was also significantly depressed in animals given WR-2721 before irradiation. Reduced prostaglandin excretion rates may reflect the general radioprotective capacity of the chemoprotector WR-2721 on the release of prostaglandins from radiation-damaged tissue. The decrease in diuresis may be related to the observed prostaglandin decreases.

Early kinetics of Ca2+ fluxes and histamine release in rat mast cells stimulated with compound 48/80.

The kinetics of Ca2+ uptake and efflux have been measured in rat peritoneal mast cells stimulated with compound 48/80 using rapid mixing and a silicone oil centrifugation technique. Responses at one-second time intervals were resolved beginning as early as three seconds after initial stimulation. The results clearly demonstrate that Ca2+ uptake occurs after the initiation of histamine release. Ca2+ efflux occurs simultaneously with histamine release. The implications of these findings are discussed and the technique is described.

Effect of ionizing radiation on physiological function in the anesthetized rat.

Exposure of pentobarbital-anesthetized rats to 14.5-MeV electrons results in radiation-induced physiological dysfunction. Responses include transient hypotension, a transient decrease in heart rate, respiratory dysrhythmias, and a prolonged increase in pulse pressure. Magnitudes of these responses are dose related, and maximal responses can be elicited by either whole- or partial-body (head or abdominal) exposure to 10,000 rad. These responses were associated with a fivefold increase in arterial plasma concentration of epinephrine, whereas histamine, norepinephrine, and beta-endorphin did not change during the first minute after the onset of exposure. Administration of diphenhydramine, a histamine receptor antagonist, resulted in a significant decline of baseline cardiovascular function and inhibited radiation-induced cardiovascular dysfunction. The diphenhydramine-induced decrease in preexposure blood pressure was reversed by angiotensin infusion, but this procedure failed to restore the mechanism(s) responsible for the cardiovascular responses to radiation. Results of these experiments and information available in the literature support the hypothesis that these responses are due to an interference in the autonomic pathways that modulate cardiovascular function.

In vitro prostaglandin release from guinea pig parenchymal lung tissues is not stimulated by thymic peptides.

Prostaglandins (PGs) have been demonstrated to both enhance and inhibit immune responses. As several chemically distinct serum and thymic polypeptide preparations have been shown to stimulate immunologic reactivity in several cell populations, animal models, or clinical patient trials, we have investigated the capacity of these hormone-like products from the thymus and blood to modulate PGs generation/release in normal parenchymal lung tissues of the guinea pig. Several concentrations of thymosin fraction 5, serum thymic factor, tuftsin or thymopentin, as well as histamine or A23187 (as positive controls) were exogenously applied to parenchymal lung fragments in vitro, and supernatants analyzed for PG content by radioimmunoassay. No alteration in PG levels (enhancement or suppression) from basal (spontaneous) release was found. These findings suggest that during a 30-min incubation, all four polypeptide immunomodulators were ineffective in eliciting an immediate response in the arachidonic acid cascade via the cyclooxygenase pathway.

The effect of calmodulin on histamine release in the rat peritoneal mast cell.

To investigate the role of the Ca2+-binding protein calmodulin on histamine release in the rat peritoneal mast cell, we exposed cells to exogenous calmodulin in the presence of a variety of histamine secretagogues. Histamine release stimulated by compound 48/80, polymyxin B and ionophore A23187 was inhibited while concanavalin A-stimulated release was not affected. Calmodulin in the presence of the secretagogues did not affect cell viability and calmodulin alone had no effect on histamine release. No direct interaction between calmodulin and the secretagogues was observed. Exogenous calmodulin does not appear to be incorporated into the cell. The inhibition of histamine release by calmodulin can be explained as a labile interaction between the protein and the cell that requires externally-bound Ca2+. These experiments demonstrate the use of exogenous calmodulin as a probe in the study of the mechanism of histamine release.

Radiation-induced alterations in prostaglandin excretion in the rat.

Dose-related alterations in the levels of prostaglandins (PGF2 alpha and PGE) and thromboxane B2 (TxB2) were observed in the urine of unanesthetized rats following whole-body gamma radiation. Exposure doses of 100 and 900 rads resulted in significant changes in urinary levels of these cyclooxygenase products. These findings suggest the potential use of radioimmunoassay measurement of urinary prostaglandins and thromboxane as a noninvasive indicator of radiation exposure.

Effect of radioprotectant WR 2721 on cyclic nucleotides, prostaglandins, and lysosomes.

Within 1 hr after ip injection of the radioprotectant WR 2721 into rats, splenic cGMP levels dropped and remained suppressed for 6 hr before returning to normal. However, if rats were exposed to ionizing radiation 30-40 min after WR 2721 treatment, they had higher cGMP levels at 3 hr postirradiation than the nonirradiated, drug-treated controls, but the cGMP content was still found to be lower than that of the irradiated nondrug-treated controls. Radiation exposure of animals pretreated with WR 2721 also resulted in higher liver and spleen levels of cAMP and additional elevations in spleen prostaglandin content, compared with irradiated controls at 3-6 hr after radiation treatment. The secondary fluctuations of lysosomal enzyme activities, prostaglandin content, and cyclic nucleotide levels were also altered in irradiated rats pretreated with WR 2721 when compared with irradiated controls. Liver and spleen lysosomal beta-glucuronidase activities, spleen cAMP and cGMP levels, and spleen prostaglandin concentrations were closer to physiological levels at 3 days postirradiation in rats given WR 2721 before the radiation treatment.

Effects of 60Co radiation on synthesis of prostaglandins F2 alpha, E, and thromboxane B2 in lung airways of guinea pigs.

At 1 hr to 14 days after total-body exposure of guinea pigs to 3.0 Gy 60Co, changes were detected in prostaglandin concentrations in bronchial airway tissues. At 3 hr postexposure, tissue levels of PGE were significantly elevated, while at 48 hr transiently elevated levels of PGF2 alpha were observed. By 72 hr, levels returned to control values. Airway synthesis of thromboxane B2 in irradiated animals did not differ from that in controls. Also assessed were the capacities of bronchial airway preparations to respond to H-1 receptor stimulation by the exogenous addition of histamine or transmembrane divalent cation transport stimulation with ionophore. Tissues from irradiated animals demonstrated alterations in the amount and type of prostaglandins generated, varying with time postirradiation.

Radiation-induced changes in production of prostaglandins F2 alpha, E, and thromboxane B2 in guinea pig parenchymal lung tissues.

At 1 hour to 4 days after unilateral exposure of guinea pigs to a single dose (0 X 5, 1 X 5, or 3 X 0 Gy) of gamma-radiation, changes were detected in prostaglandin and thromboxane concentrations in parenchymal lung tissues. At 1-3 hours after exposure, tissue levels of PGF2 alpha, PGE, and thromboxane B2 were significantly elevated in animals receiving 3 X 0 Gy, with the magnitude of alteration revealing a radiation dose effect. By 24 hours, tissue prostaglandin and thromboxane levels returned to near control values. Lung tissue synthesis of prostaglandins in response to H-1 receptor stimulation by the exogenous addition of histamine revealed similar radiation dose effects. The carboxylic acid ionophore A23187, exogenously applied to lung tissues, revealed a transient peak of increased sensitivity to ionophore stimulation for TxB2 synthesis at 24 hours and for PGF2 alpha at 72 hours post-irradiation. The data suggest that significant alterations in prostaglandin and thromboxane concentrations in parenchymal lung tissues occur following irradiation, in a dose-dependent manner, and that altered responsiveness to H-1 receptor stimulation and divalent cation transport also occur.

A characterization of beta-adrenergic receptors on cellular and perigranular membranes of rat peritoneal mast cells.

Beta-adrenergic receptors were characterized by measuring the specific binding of 3H-dihydroalprenolol (DHA) on intact isolated rat peritoneal mast cells (RPMC) and on perigranular membranes derived from purified RPMC granules. The specific binding of 3H-DHA reaches an equilibrium within 30 min at 5 degrees C and is linear with cell number. Scatchard analysis reveals two populations of binding sites on intact cells: with KD = 10.6 +/- 2.6 and 129 +/- 4.7 nM and Bmax of 186 +/- 38 and 1200 +/- 415 fmol/10(6) cells, respectively. Each cell contains 120 X 10(3) high-affinity binding sites and 720 X 10(3) low-affinity binding sites. There appears to be neither alpha-adrenergic nor muscarinic cholinergic receptors on the RPMC. Specific binding of 3H-DHA also occurred to isolated granules with perigranular membranes. The binding was saturable with a single population of binding sites with an affinity (KD) of 7.0 +/- 0.45 nM. Maximum binding (Bmax) was calculated at 56.6 +/- 1.9 fmol/10(9) granules. Subfractionation of granule components demonstrated that the specific binding sites appear to be localized exclusively on the perigranular membrane.