Biological effects of embedded depleted uranium (DU): summary of armed forces radiobiology research institute research.

The Persian Gulf War resulted in injuries of US Coalition personnel by fragments of depleted uranium (DU). Fragments not immediately threatening the health of the individuals were allowed to remain in place, based on long-standing treatment protocols designed for other kinds of metal shrapnel injuries. However, questions were soon raised as to whether this approach is appropriate for a metal with the unique radiological and toxicological properties of DU. The Armed Forces Radiobiology Research Institute (AFRRI) is investigating health effects of embedded fragments of DU to determine whether current surgical fragment removal policies remain appropriate for this metal. These studies employ rodents implanted with DU pellets as well as cultured human cells exposed to DU compounds. Results indicate uranium from implanted DU fragments distributed to tissues far-removed from implantation sites, including bone, kidney, muscle, and liver. Despite levels of uranium in the kidney that were nephrotoxic after acute exposure, no histological or functional kidney toxicity was observed. However, results suggest the need for further studies of long-term health impact, since DU was found to be mutagenic, and it transformed human osteoblast cells to a tumorigenic phenotype. It also altered neurophysiological parameters in rat hippocampus, crossed the placental barrier, and entered fetal tissue. This report summarizes AFRRI's depleted uranium research to date.

Detection of depleted uranium in biological samples from Gulf War veterans.

During the Persian Gulf War, soldiers may have inhaled, ingested, and/or experienced wound contamination by depleted uranium (DU), which is used in military projectiles and armor. DU is produced by depleting natural uranium of 234U and 235U during the uranium-enrichment process. Although the long-term effects of significant DU exposures require investigation, many veterans express fears about its impact on health. An assay by which DU exposure can be assessed would not only be a useful research tool, but the information could help mitigate the concerns of exposed individuals. In this study, urine samples from individuals enrolled in the Depleted Uranium Follow-Up Program at the Baltimore Veterans Administration Medical Center were examined for uranium content. Isotopic composition of urine uranium was determined by measuring the 235U/238U ratio, using an inductively coupled plasma mass spectrometer. Using this method, natural and depleted uranium could be readily differentiated. By demonstrating the absence of DU in soldiers who suspect exposure by inhalation or ingestion, the assay should reduce psychological stress in these individuals.

Staining of intracellular deposits of uranium in cultured murine macrophages.

In our studies of the health effects of internalized depleted uranium, we developed a simple and rapid light microscopic method to stain specifically intracellular uranium deposits. Using J774 cells, a mouse macrophage line, treated with uranyl nitrate and the pyridylazo dye 2-(5-bromo-2-pyridylazo)-5-diethylaminophenol, uranium uptake by the cells was followed. Specificity of the stain for uranium was accomplished by using masking agents to prevent the interaction of the stain with other metals. Prestaining wash consisting of a mixture of sodium citrate and ethylenediaminetetraacetic acid eliminated staining of metals other than uranium. The staining solution consisted of the pyridylazo dye in borate buffer along with a quaternary ammonium salt, ethylhexadecyldimethylammonium bromide, and the aforementioned sodium citrate/ethylenediaminetetraacetic acid mixture. The buffer was essential for maintaining the pH within the optimum range of 8 to 12, and the quaternary ammonium salt prevented precipitation of the dye. Staining was conducted at room temperature and was complete in 30 min. Staining intensity correlated with both uranyl nitrate concentration and incubation time. Our method provides a simple procedure for detecting intracellular uranium deposits in macrophages.

4-Hydroxynonenal, an end-product of lipid peroxidation, induces apoptosis in human leukemic T- and B-cell lines.

4-Hydroxynonenal (HNE) is the major aldehydic product resulting from lipid peroxidation and has been implicated as involved in several pathological conditions. In our continuing studies on the role of membranes and lipid peroxidation in the induction of apoptosis, we investigated the effect of HNE on cultured human malignant immune system cells. Two cell lines were utilized; MOLT-4, a human T-cell leukemia cell line, and Reh, a human B-cell lymphoma cell line. A 10 min treatment with 0.01 mM HNE resulted in the apoptotic death, as determined by flow cytometric and morphological analyses, of both cell lines within 24 h. MOLT-4 cells exhibited the manifestations of impending apoptotic death much sooner than did Reh cells, indicating that MOLT-4 cells were more sensitive or not as efficient at detoxifying HNE than were Reh cells. These results suggest that peroxidative damage to cellular membranes resulting in the production of HNE may be a trigger for the induction of apoptosis in immune system cells.

A procedure for the rapid detection of depleted uranium in metal shrapnel fragments.

Depleted uranium is now widely used in the armor of military vehicles as well as in kinetic-energy penetrators designed to defeat enemy armor. As a result, the potential that personnel will be wounded by depleted uranium fragments has increased. Because toxicities associated with depleted uranium fragments may ultimately require different treatment protocols than those used for traditional metal fragment injuries, a method to rapidly detect the presence of depleted uranium in surgically excised shrapnel fragments is required. By treating the shrapnel fragment with an extracting agent, such as nitric acid, for 5 minutes in an ultrasonic cleaner, sufficient metal is solubilized to allow for colorimetric detection using a pyridylazo dye. Although several metals are capable of being detected under these conditions, the reaction can be made specific for depleted uranium through the use of masking agents such as sodium citrate and ethylenediaminetetraacetic acid. This procedure allows for the rapid (< 15 minutes) extraction and detection of depleted uranium in metal shrapnel fragments.

N(omega)-nitro-L-arginine decreases resting cytosolic [Ca2+] and enhances heat stress-induced increase in cytosolic [Ca2+] in human colon carcinoma T84 cells.

N(omega)-nitro-L-arginine (LNNA) inhibits the synthesis of heat shock proteins in animals and cultured cells exposed to heat stress. Heat shock protein synthesis is known to be Ca2+-dependent. In this study, we have characterized the effect of LNNA on [Ca2+]i before and after heat stress in human colon carcinoma T84 cells. In untreated cells incubated in the presence of external Ca2+, the resting [Ca2+]i was 201+/-3 nM. If these cells were exposed to 45 degrees C for 10 min, [Ca2+]i increased by 50+/-2%. Preincubation with LNNA (100 microM) without subsequent heating led to a decrease in [Ca2+]i in a LNNA concentration-dependent manner. Preincubation with LNNA followed by heating increased [Ca2+]i to levels 88+/-5% greater than cells heated without LNNA pretreatment. Incubating cells in medium without external Ca2+ (no heating, no LNNA treatment) lowered resting [Ca2+]i to 115+/-2 nM and greatly reduced the increase in [Ca2+]i observed if cells were heated in the presence of Ca2+, indicating that external Ca2+ plays an important role in the maintenance of [Ca2+]i in T84 cells. With external Ca2+ absent, LNNA pretreatment further reduced [Ca2+]i in unheated cells, and heating failed to enhance [Ca2+]i. We determined (with external Ca2+ present) that the heat-stress induced increase in [Ca2+]i in T84 cells was blocked by dichlorobenzamil, a Na+/Ca2+ exchanger inhibitor, suggesting that the exchanger mediates Ca2+ entry. The median inhibitory concentration (IC50) in cells not treated with LNNA was 0.970+/-0.028 microM. With LNNA pretreatment, the IC50 was 5.099+/-0.107 microM. Heat stress of T84 cells did not affect the binding affinity of the Na+/Ca2+ exchanger for external Ca2+, but it increased the maximal velocity of the exchanger. In unheated cells, preincubation with LNNA decreased the binding affinity of the exchanger for Ca2+, but after heat treatment, both the binding affinity and maximal velocity of the exchanger increased. Our data are consistent with the idea that LNNA affects the activity of the Na+/Ca2+ exchanger. We also determined there are intracellular Ca2+ pools in T84 cells sensitive to thapsigargin, monensin, and ionomycin. Treatment with TMB-8, a blocker of Ca2+ sequestration and mobilization, or ionomycin inhibited the LNNA-induced decrease in [Ca2+]i observed in the absence of external Ca2+, suggesting that LNNA promotes Ca2+ sequestration.

Pyruvate prevents hydrogen peroxide-induced apoptosis.

Studies were carried out to investigate the protective effects of pyruvate, a key glycolytic intermediate and alpha-keto-monocarboxylate, against oxidative stress-induced apoptosis. Oxidative stress was induced by treating mouse thymocytes with 25 microM hydrogen peroxide for 15 min at 37 degrees C under 5% CO2 in air. Pre- and post-treatment of cells with 10 mM pyruvate inhibited morphological changes, internucleosomal DNA fragmentation, and translocation of phosphatidylserine to the plasma membrane surface, which are characteristic features of apoptosis. L-lactate (10 mM) and acetate (10 mM) were ineffective in inhibiting apoptosis and appeared to be toxic to the cells under similar conditions. The results suggest that pyruvate has therapeutic potential for use in the treatment of oxidative stress-induced disorders associated with increased apoptosis.

Overexpression of HSP-70 attenuates increases in [Ca2+]i and protects human epidermoid A-431 cells after chemical hypoxia.

This laboratory previously reported that thermotolerance diminishes the NaCN-induced increase in intracellular free calcium concentrations ([Ca2+]i) in human epidermoid A-431 cells and that blocking this increase protects the cells from NaCN toxicity. In this study, we report that cell viability after exposure to NaCN (10 mM, 1 h) is enhanced by the overexpression of HSP-70 resulting from heat shock (45 degrees C, 10 min), treatment with a protein kinase C activator phorbol 12 myristate 13-acetate (PMA; 1 microM, 4 h), or HSP-70 cDNA transfection. Because the toxicity of NaCN is mediated by increases in [Ca2+]i, we sought to determine whether the overexpression of HSP-70 might protect the cells by altering the [Ca2+]i response induced by NaCN. Basal [Ca2+]i in vector-, HSF1 cDNA-, and HSP-70 cDNA-transfected cells was 114 +/- 11 (n = 11), 95 +/- 5 (n = 6), and 151 +/- 11 (n = 15) nM, respectively, suggesting that HSP-70 metabolism is associated with maintenance of resting [Ca2+]i. Removal of external Ca2+ reduced the resting [Ca2+]i in all of these cells. With external Ca2+ reduced the resting [Ca2+]i by 97 +/- 21% in vector-transfected cells and 111 +/- 5% in HSF1 vector-transfected cells but by only 27 +/- 8% in HSP-70 cDNA-transfected cells. Heat shock or PMA treatment of vector- or HSF1 cDNA-transfected cells to induce HSP-70 also attenuated the NaCN-induced increase in [Ca2+]i, perhaps because of a decrease in Vmax for the uptake of external Ca2+. Removal of external Ca2+ or treatment with inhibitors of Na+/Ca2+ exchangers eliminated the NaCN-induced increase in [Ca2+]i in HSP-70 cDNA-transfected cells, but ryanodine treatment did not. HSP-70 cDNA transfection also reduced Ca2+ mobilization stimulated by various Ca(2+)-mobilizing agents. The results suggest that HSP-70 overexpression protects cells from NaCN cytotoxicity, perhaps by attenuating the [Ca2+]i response.

The antioxidant Trolox enhances the oxidation of 2',7'-dichlorofluorescin to 2',7'-dichlorofluorescein.

The use of antioxidants to prevent intracellular free radical damage is an area currently attracting considerable research interest. The compound 2',7'-dichlorofluorescin diacetate (DCFH-DA) is a probe for intracellular peroxide formation commonly used in such studies. During our studies we unexpectedly found that incubation of Trolox, a water soluble vitamin E analog, with DCFH-DA in cell-free physiological buffers resulted in the deacetylation and oxidation of DCFH-DA to form the fluorescent compound, 2',7'-dichlorofluorescein (DCF). The reaction was time-, temperature-, and pH-dependent. Fluorescence intensity increased with an increase in either Trolox or DCFH-DA concentration. These results indicate that even at physiological pH, DCFH-DA can be deacetylated to form 2',7'-dichlorofluororescin (DCFH), DCFH can then be oxidized to DCF by abstraction of a hydrogen atom by the phenoxyl radical of Trolox. Exposure of the reaction mixture to 10 Gy of 60Co gamma radiation greatly increased production of DCF. Antioxidant compounds reported to "repair" the Trolox phenoxyl radical (e.g., ascorbic acid, salicylate) can also prevent the Trolox-induced DCFH-DA fluorescence. However, radical (e.g., catechin) or can themselves form a radical (e.g., uric acid, TEMPOL) either have no effect or can increase levels of DCF. These results demonstrate that experimental design must be carefully considered when using DCFH-DA to measure peroxide formation in combination with certain antioxidants.

Ebselen inhibition of apoptosis by reduction of peroxides.

We investigated the capacity of ebselen [2-phenyl-1,2-benzisoselenazol-3(2H)-one], a glutathione peroxidase mimic, to protect cells from radiation-induced apoptosis. Incubating mouse thymocytes with 25 microM ebselen immediately after 60Co gamma-radiation exposure (5 Gy) inhibited morphological changes associated with apoptosis. Treatment of thymocytes with ebselen before, during, or after irradiation completely blocked internucleosomal DNA fragmentation, a biochemical marker for apoptosis. We measured peroxides formed in cells during and after irradiation, using the oxidation-sensitive fluorescent probe 2',7'-dichlorofluorescin diacetate. By 2 min postirradiation, levels of peroxides in irradiated thymocytes were approximately 10-11 times greater than those in the same cells before irradiation, and levels continued to increase with time. We also measured membrane lipid peroxidation using cis-parinaric acid, a naturally fluorescent polyunsaturated fatty acid that readily incorporates into cell membranes. The oxidation of cis-parinaric acid also began soon after irradiation and increased with time. Peroxide generation and membrane lipid peroxidation preceded both internucleosomal DNA fragmentation and morphological changes characteristic of apoptosis. Treatment of cells with ebselen reduced peroxide levels and appeared to protect thymocytes from radiation-induced apoptosis by scavenging peroxides generated during and after irradiation. The results suggest that peroxide generation and membrane lipid peroxidation may be important signaling events that trigger apoptosis in irradiated cells.

Thermotolerance attenuates heat-induced increases in [Ca2+]i and HSP-72 synthesis but not heat-induced intracellular acidification in human A-431 cells.

BACKGROUND: Thermotolerance affects cell viability, retards translation of heat shock proteins, and protects RNA slicing mechanisms. We reported previously that heat shocking nonthermotolerant cells causes an intracellular acidification and an increase in cytosolic free Ca2+ ([Ca2+]i) in addition to an induction of heat shock protein 72kDa (HSP-72) production. This study characterized heat-induced changes in cytosolic Ca2+, H+, and HSP-72 synthesis in thermotolerant A-431 cells. METHODS: We studied heat-induced changes in pH(i), [Ca2+]i, and HSP-72 using thermotolerant A-431 cell monolayers. pH(i) and [Ca2+]i were determined using fluorescence probes, and HSP-72 was measured by SDS-PAGE. The mRNA encoding HSP-72 was measured by Northern blots probed with a [32P]-labeled 2.3 kb fragment of an HSP-70 cDNA insert. RESULTS: Heat shocking thermotolerant cells induced the same degree of intracellular acidification as that induced in nonthermotolerant cells, but the heat-induced increase in [Ca2+]i was less in thermotolerant cells. This diminished response was characterized by an increase in Km for external Ca2+ and was blocked by pretreatment with cycloheximide, indicating a newly synthesized protein is involved. Similar to what was seen in nonthermotolerant cells, the heat-induced increase in [Ca2+]i in thermotolerant cells depended on external Na+ concentration and was blocked by dichlorobenzamil, though thermotolerant cells were more sensitive to the inhibitor (IC50 = 0.21 mumol/L for nonthermotolerant, 0.025 mumol/Lm for thermotolerant). Thermotolerant cells contained high resting levels of HSP-72. Heat shocking these cells attenuated the HSF translocation from cytosol to nucleus and did not induce a further synthesis of HSP-72 mRNA and protein. CONCLUSIONS: The results suggest that thermotolerance desensitizes the machinery required for Ca2+ entry. Low [Ca2+]i levels probably result in diminished HSP-72 mRNA production and less HSP-72 synthesis.

Trolox inhibits apoptosis in irradiated MOLT-4 lymphocytes.

MOLT-4 cells, a human lymphocytic leukemia line, undergo apoptosis in response to a variety of stimuli, including exposure to ionizing radiation. Very little is known of the molecular mechanisms by which radiation induces apoptosis. Morphology changes and chromatin cleavage at internucleosomal sites accompany apoptosis in these cells. We found that trolox, a water-soluble derivative of vitamin E that penetrates biomembranes and protects mammalian cells from oxidative damage, blocks DNA fragmentation in irradiated MOLT-4 cells. Levels of DNA fragmentation in cells not treated with trolox were directly related to both radiation dose and time postirradiation. Preincubation of cells with trolox or incubation with trolox only during irradiation did not protect cells. A 4 h postirradiation incubation with trolox was sufficient to completely block fragmentation measured at 24 h, indicating the processes triggered by radiation to induce DNA fragmentation occur early after irradiation. Removal of cells from trolox earlier than 4 h resulted in progressively less inhibition. Trolox preserves the integrity of irradiated cells as judged by increased viability and thymidine incorporation. Radiation induces an uptake of extracellular Ca2+ into MOLT-4 cells that was blocked by a postirradiation incubation with trolox. These results suggest that membrane-associated oxidations triggered by radiation are responsible for radiation-induced apoptosis in MOLT-4 cells.

Rapid isolation of nuclear transport-competent Xenopus nucleoplasmin produced in Escherichia coli strain BL21(DE3).

Nucleoplasmin is a thermostable karyophilic protein widely used in nuclear transport studies. An expression vector was constructed that contains a string of 10 histidine residues ligated, in frame, to the amino terminal end of the Xenopus nucleoplasmin gene. The vector was then transformed into Escherichia coli strain BL21(DE3). This strain possesses the gene for T7 RNA polymerase under control of the lacUV5 promoter. The induction of the RNA polymerase and subsequent production of nucleoplasmin occurs after exposure to isopropyl-beta-D-thiogalactopyranoside. The nucleoplasmin, produced in milligram quantities per liter of culture, is then isolated by a rapid purification method that includes metal chelation chromatography to purify the oligohistidine-linked nucleoplasmin. Nuclear transport studies indicate that fluorescently labeled nucleoplasmin is translocated to the nuclear interior of permeabilized V79A03 cells, while nucleoplasmin that lacks a nuclear localization signal (core nucleoplasmin) is not imported. The use of this method to produce nuclear transport-competent nucleoplasmin avoids the lengthy purification procedure used to isolate nucleoplasmin from Xenopus laevis oocytes as well as the cost of purchasing and maintaining a toad colony.

HSP-72 synthesis is promoted by increase in [Ca2+]i or activation of G proteins but not pHi or cAMP.

The family of 70-kDa heat-shock proteins (HSP-70) is evolutionarily highly conserved and has been shown to enhance cell survival from thermal injury. This study characterized HSP-72 induction in human epidermoid A-431 cells exposed to 45 degrees C for 10 min and determined the relationship between HSP-72, intracellular pH (pHi), adenosine 3',5'-cyclic monophosphate (cAMP), G proteins, and intracellular cytosolic free Ca2+ concentration ([Ca2+]i). Heat shock induced HSP-72 production, which was dependent on both temperature and the duration of heating. This HSP-72 induction was confirmed by Western blot analysis. HSP-72 levels in cells that had been heated then returned to 37 degrees C were elevated at 2 h (1.5 +/- 0.1 x control), reached a maximum at 8 h (2.7 +/- 0.1 x control), and remained above baseline for up to 4 days. Levels of HSP-72 mRNA, determined by dot-blot analysis, reached a maximum at 2 h and returned to baseline within 8 h. Both actinomycin D and cycloheximide blocked HSP-72 induction. Because heating causes intracellular acidification and increases in cAMP and [Ca2+]i, we studied the effect of pHi, cellular cAMP, and [Ca2+]i on HSP-72 induction. The reduction of pHi to 6.9 by acid loading did not affect the basal level of HSP-72 in unheated cells. Treatment with pertussis toxin, cholera toxin, or forskolin, but not 8-bromo-cAMP, 3-isobutyl-1-methylxanthine, or N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide potentiated heat-induced HSP-72 production. Inhibition of the heat-induced increase in [Ca2+]i attenuated, but failed to completely block, heat-induced HSP-72 production, mRNA synthesis, and the heat-shock transcriptional factor-heat-shock element binding complex formation, which suggests there are Ca(2+)-dependent and -independent processes involved in HSP-72 synthesis. Our results show that an increase in [Ca2+]i or activation of G proteins, but not pHi and cAMP, enhances HSP-72 induction.

Oxidative stress-induced apoptosis prevented by Trolox.

The ability of oxidative stress to induce apoptosis (programmed cell death), and the effect of Trolox, a water soluble vitamin E analog, on this induction were studied in vitro in mouse thymocytes. Cells were exposed to oxidative stress by treating them with 0.5-10 microM hydrogen peroxide (H2O2) for 10 min, in phosphate-buffered saline supplemented with 0.1 mM ferrous sulfate. Cells were resuspended in RPMI 1640 medium with 10% serum and incubated at 37 degrees C under 5% CO2 in air. Electron microscopic studies revealed morphological changes characteristic of apoptosis in H2O2-treated cells. H2O2 treatment fragmented the DNA in a manner typical of apoptotic cells, producing a ladder pattern of 200 base pair increments upon agarose gel electrophoresis. The percentage of DNA fragmentation (determined fluorometrically) increased with increasing doses of H2O2 and postexposure incubation times. Pre- or posttreatment of cells with Trolox reduced H2O2-induced DNA fragmentation to control levels and below. The results indicate that oxidative stress induces apoptosis in thymocytes, and this induction can be prevented by Trolox, a powerful inhibitor of membrane damage.

Corticotropin-releasing factor increases protein kinase C activity by elevating membrane-bound alpha and beta isoforms.

Corticotropin-releasing factor (CRF) has been shown to attenuate vascular leakage in injured skin, mucous membrane, muscle, lung, and brain. We previously reported that CRF increases cytosolic free calcium concentrations ([Ca2+]i), cellular cAMP, and inositol trisphosphates in human epidermoid A-431 cells. This study identified protein kinase C isoforms in A-431 cells and investigated the effect of CRF on PKC activity and isoform translocation. PKC alpha, beta, gamma, delta, and zeta isoforms were present in cytosolic and membrane fractions, but the epsilon isoform was detected only in the membrane fraction. Exposure of cells to CRF at 420 pM for 1 min increased PKC activity and led to the translocation of PKC alpha and beta isoforms from cytosol to membrane. The translocation was dependent on an increase in [Ca2+]i, because cells treated with 100 microM BAPTA-am (an intracellular Ca2+ chelator), then exposed to CRF, showed neither increases in PKC activity nor translocation of PKC isoforms. This suggests that a CRF-induced increase in [Ca2+]i mediates the increase in PKC activity.

Effect of heat shock, [Ca2+]i, and cAMP on inositol trisphosphate in human epidermoid A-431 cells.

The basal levels of inositol monophosphate, inositol bisphosphate, and inositol trisphosphate (InsP3) in A-431 cells incubated in Na(+)-Hanks' solution were, respectively, 1.23 +/- 0.18, 0.17 +/- 0.03, and 0.69 +/- 0.07% of the total radioactivity in the cell. When cells were heated, InsP3 increased in a temperature-dependent manner related to the duration of heating. The active form of InsP3, inositol 1,4,5-trisphosphate, increased 237 +/- 17% after heating (45 degrees C, 20 min) then returned to baseline within 15 min after the return to 37 degrees C. The heat-induced increase in InsP3 was not observed in the absence of extracellular Ca2+ or with amiloride treatment. Treatment with the nonhydrolyzable GTP analogue 5'-guanylylimidodiphosphate stimulated that component of the InsP3 increase due to G proteins. U-73122, an inhibitor of phospholipase C-mediated processes, blocked the increase in InsP3 resulting from heat exposure. Both pertussis toxin (30 ng/ml, 24 h), an inhibitor of G inhibitory protein, and cholera toxin (1 microgram/ml, 1 h), a stimulator of G stimulatory protein, increased InsP3 in unheated cells, and heating failed to induce a further increase, suggesting that heat activates G proteins. Likewise, 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP), 3-isobutyl-1-methylxanthine, Ro 20-1724, or forskolin increased InsP3 in unheated cells, and heat did not cause an additional increase. The InsP3 increase induced by 8-BrcAMP was inhibited by removal of extracellular Ca2+ or treatment with verapamil, suggesting that an influx of extracellular Ca2+ stimulates InsP3 production.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

An in vitro method for radiolabeling proteins with 35S.

The radiolytic decomposition products of [35S]-methionine have been used to radiolabel proteins in vitro. The process occurs in a time-, temperature-, and pH-dependent manner. Maximum labeling of bovine serum albumin occurs after a 24 h incubation at 37 degrees C and pH 8.5. Once incorporated, the radiolabel cannot be removed by extended incubation at various temperatures, multiple freeze/thaw cycles, or boiling, indicating that the 35S moiety is covalently attached to the protein. A wide variety of proteins have been radiolabeled. The method is simple to perform and yields radiolabeled proteins of high specific activity.

Effect of gamma radiation on membrane fluidity of MOLT-4 nuclei.

These experiments measured the effect of gamma radiation on the nuclear envelope using doxyl-fatty acid spin-label probes. Nuclei were isolated from cultured MOLT-4 cells, a radiation-sensitive human T-cell lymphocyte. Membrane fluidity was measured from the electron paramagnetic resonance spectra of the probes. MOLT-4 cells were grown under standard conditions, and suspensions were exposed to 60Co gamma radiation at room temperature. The spectra of 5-doxylstearic acid in the nuclei were those of a strongly immobilized label. A difference in the membrane fluidity was detected in a series of experiments comparing labeled irradiated and nonirradiated nuclei. The change in fluidity was measured by comparing the changes in the order parameter, S, of the spin label in irradiated nuclei with those in control nuclei. The change in the S ratio is dependent on radiation dose, increasing with doses up to 15 Gy. The maximum change of the order parameter with time after irradiation occurs 16-20 h after radiation exposure. These observations are correlated with changes in cell viabilities.