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.

Iron-56 irradiation diminishes muscarinic but not alpha 1-adrenergic-stimulated low-Km GTPase in rat brain.

Initial findings from our laboratory have indicated that muscarinic enhancement of K(+)-evoked release of dopamine from perifused striatal slices is reduced after exposure to 56Fe-particle irradiation. This finding suggested that there is a radiation-induced deficit in muscarinic receptor sensitivity. Subsequent findings have indicated that at least part of the loss in sensitivity may occur as a result of alterations in the initial steps of the signal transduction process and involve muscarinic receptor-G protein coupling/uncoupling. The present study was carried out to localize this deficit further by determining carbachol-stimulated low-Km guanosine triphosphatase (GTPase) activity in striatal and hippocampal tissue obtained from rats exposed to 0, 0.1 or 1.0 Gy of 56Fe-particle irradiation. In addition, to examine the specificity of the effect of 56Fe-particle irradiation, alpha 1-adrenergic-stimulated low-Km GTPase activity was also examined in these tissues. The results showed that there was a high degree of specificity in the effects of 56Fe particles. Decrements were observed in muscarinic-stimulated low-Km GTPase in striatum but not in hippocampus, and 56Fe-particle irradiation did not affect alpha 1-adrenergic low-Km GTPase activity in either brain tissue.

Exposure to heavy charged particles affects thermoregulation in rats.

Rats exposed to 0.1-5 Gy of heavy particles (56Fe, 40Ar, 20Ne or 4He) showed dose-dependent changes in body temperature. Lower doses of all particles produced hyperthermia, and higher doses of 20Ne and 56Fe produced hypothermia. Of the four HZE particles, 56Fe particles were the most potent and 4He particles were the least potent in producing changes in thermoregulation. The 20Ne and 40Ar particles produced an intermediate level of change in body temperature. Significantly greater hyperthermia was produced by exposure to 1 Gy of 20Ne, 40Ar and 56Fe particles than by exposure to 1 Gy of 60Co gamma rays. Pretreating rats with the cyclo-oxygenase inhibitor indomethacin attenuated the hyperthermia produced by exposure to 1 Gy of 56Fe particles, indicating that prostaglandins mediate 56Fe-particle-induced hyperthermia. The hypothermia produced by exposure to 5 Gy of 56Fe particles is mediated by histamine and can be attenuated by treatment with the antihistamines mepyramine and cimetidine.

Reductions of 56Fe heavy-particle irradiation-induced deficits in striatal muscarinic receptor sensitivity by selective cross-activation/inhibition of second-messenger systems.

Recent experiments have revealed radiation-induced (600 MeV/u 56Fe energetic particles) losses of sensitivity of rodent neostriatal muscarinic receptors to stimulation by cholinergic agonists that appears as reductions in oxotremorine enhancement of K(+)-evoked dopamine release. These losses were postulated to be the result of radiation-induced alterations early in phosphoinositide-mediated signal transduction. Additional findings indicated that if the ligand-receptor-G protein interface was by passed no radiation deficits were seen. In the present study, radiation-induced deficits in K(+)-evoked dopamine release were examined in perifused striatal tissue obtained from rats exposed to 0, 0.1 or 1.0 Gy of 56Fe particles (600 MeV/u). Results showed that these deficits could be reduced by co-applying combinations of various pharmacological agents that were known to have differential effects on various second messengers such as 1,4,5-inositol-trisphosphate (IP3). Combinations included oxotremorine-carbachol, and either oxotremorine or carbachol with arginine vasopressin or arachidonic acid. These results are discussed in terms of putative radiation-induced changes in receptor-containing membranes which alter receptor-G protein coupling/uncoupling.

Deficits in the sensitivity of striatal muscarinic receptors induced by 56Fe heavy-particle irradiation: further "age-radiation" parallels.

We had previously shown that there was a loss of sensitivity of muscarinic receptors (mAChR) to stimulation by cholinergic agonists (as assessed by examining oxotremorine enhancement of K(+)-evoked release of dopamine from neostriatal slices) in animals that had been exposed to energetic particles (56Fe, 600 MeV/n), an important component of cosmic rays. This loss of mAChR sensitivity was postulated to be the result of radiation-induced alterations in phosphoinositide-mediated signal transduction. The present experiments were undertaken as a first step toward determining the locus of these radiation-induced deficits in signal transduction by examining K+ enhancement of release of dopamine in 56Fe-exposed animals (0, 0.1, and 1.0 Gy) with agents [A23187, a potent Ca2+ ionophore, or 1,4,5-inositol trisphosphate (IP3)] that "bypass" the mAChR-G protein interface and by comparing the response to oxotremorine-enhanced K(+)-evoked release of dopamine. Results showed that although oxotremorine-enhanced K(+)-evoked release of dopamine was reduced significantly in the radiation groups, no radiation effects were seen when A23187 or IP3 was used to enhance K(+)-evoked release of dopamine. Since similar findings have been observed in aging, the results are discussed in terms of the parallels between aging and radiation effects in signal transduction that might exist in the neostriatum.

Possible "accelerated striatal aging" induced by 56Fe heavy-particle irradiation: implications for manned space flights.

The present experiments were carried out to determine the effects of energy deposition from energetic iron (56Fe particles, an important component of cosmic rays) on motor behavioral performance and to determine if the observed deficits were caused by alterations in the neostriatum (an important motor control area). Neostriatal function was assessed with two correlated parameters, i.e., motor behavioral performance (wire suspension task), and oxotremorine-enhanced K(+)-evoked release of dopamine from perifused striatal slices. Rats were exposed to one of several doses of 56Fe-particle irradiation (0.10-1.0 Gy) and tested on a wire suspension task at 3-180 days postirradiation. Results indicated that profound decrements occurred in both of these indices. The effects on K(+)-evoked release of dopamine were evident for as long as 180 days after irradiation, and a subsequent experiment indicated that these effects appeared as early as 12 h postirradiation. Since similar findings have been observed in aged rats, the results are discussed in terms of these particles producing a possible accelerated striatal aging effect.

Relationship between linear energy transfer and behavioral toxicity in rats following exposure to protons and heavy particles.

Rats were exposed to protons (155 MeV) or to helium (165 MeV/amu), neon (522 MeV/amu) or argon (670 MeV/amu) particles to evaluate the behavioral toxicity of these types of radiations. Behavioral toxicity was assessed using the conditioned taste aversion paradigm. Exposure to all types of radiation produced dose-dependent increases in the intensity of the acquired taste aversion. However, the intensity of the aversions, measured as the dose that produced a 50% decrease in the intake of the sucrose-conditioned stimulus, did not show significant variation as a function of the linear energy transfer (LET) of the radiation. The results are discussed in terms of the relationship between LET and behavioral toxicity.

Reduction of 3-methoxytyramine concentrations in the caudate nucleus of rats after exposure to high-energy iron particles: evidence for deficits in dopaminergic neurons.

Exposure to low doses of high-energy iron particles can alter motor behavior. The ability of rats to hang from a wire has been reported to be significantly degraded after exposure to doses as low as 0.5 Gy. In addition, deficits in the ability of acetylcholine to regulate dopamine release in the caudate nucleus (an area in the brain important for motor function) have been found. The concentrations of 3-methoxytyramine (3-MT), a metabolite of dopamine whose concentrations reflect dopamine release in vivo, were measured after rats were exposed to different doses of high-energy iron particles to gain further information about the effect of radiation on the dopaminergic system. Concentrations of 3-MT were significantly reduced 3 days after exposure to 5 Gy but returned to control values by 8 days. After 6 months, concentrations were again less than control values. Exposure to 5 Gy of high-energy electrons or gamma photons had no effect 3 days after exposure. Very high doses of electrons were needed to alter 3-MT concentrations. One hundred grays of electrons decreased 3-MT 30 min after irradiation but levels returned to control values by 60 min. Gamma photons had no effect after doses up to 200 Gy. These results provide further evidence that exposure to heavy particles can degrade motor behavior through an action on dopaminergic mechanisms and that this can occur after doses much lower than those needed for low-LET radiation.

Age-related decrements in the muscarinic enhancement of K+-evoked release of endogenous striatal dopamine: an indicator of altered cholinergic-dopaminergic reciprocal inhibitory control in senescence.

Previous experiments have indicated that the release of striatal dopamine (DA) is controlled by inhibitory DA autoreceptors which are mediated by inhibitory cholinergic heteroreceptors (HTRs). Activation of the HTRs by muscarinic or nicotine agonists potentiates the K+-evoked release of DA from the striatum. Present experiments were carried out to determine if this relationship is altered as a function of aging. Cross-cut striatal tissue slices obtained from 3 age-groups (6, 12-18 and 24 months) Wistar rats were superfused with a modified Krebs-Ringer basal release medium containing 2.5 mM KCl. After a 30-min equilibration period, a 5-min baseline fraction was collected from each chamber. The medium was then switched to one containing 30 mM KCl, and depending upon the experiment, 1 of 4 concentrations of a particular muscarinic (oxotremorine, pilocarpine, carbachol or bethanecol) or nicotinic (nicotine) agonist. In some experiments DA autoreceptor function was assessed directly with haloperidol. Six 5-min fractions were taken during depolarization. DA release was assessed using high performance liquid chromatography coupled to electrochemical detection. Results indicated that the efficacy of the muscarinic agonists was reduced in an age-dependent manner with the oldest age groups showing the smallest enhancement. The age at which the decline was seen was dependent on the muscarinic agonist that was applied. Deficits were seen as early as 12 months when full agonists (e.g. carbachol) were applied, but did not appear until 18 months when partial agonists (e.g. oxotremorine) were applied. These age-related alterations were not seen when haloperidol or nicotine were used to enhance the K+-evoked release of DA.(ABSTRACT TRUNCATED AT 250 WORDS)

Alterations in muscarinic control of striatal dopamine autoreceptors in senescence: a deficit at the ligand-muscarinic receptor interface?

Research has indicated that the release of striatal dopamine (DA) is controlled by inhibitory DA autoreceptors which are in turn regulated by inhibitory muscarinic inhibitory cholinergic heteroreceptors (HTRs) located in close vicinity to the autoreceptors. Muscarinic activation enhances K+-evoked release of DA from striatal slices from mature but not senescent rats. Since it has been shown that age-dependent declines in Ca2+ mediated acetylcholine release can be restored by the ionophore A23187, it was of interest to determine if age-related decrements in Ca2+ mobilization might contribute to the alterations in muscarinic control of the striatal DA autoreceptors seen in senescence. Cross-cut striatal tissue slices obtained from two age-groups (6 and 24 months) of Wistar rats were superfused with a modified Krebs-Ringer medium containing 2.5 mM KCl. After a 30-min equilibration period, a 5-min baseline fraction was collected. The medium was then switched to one which contained 30 mM KCl and, depending upon the experiment, the muscarinic agonists carbachol, or oxotremorine or the Ca2+ mobilizing agents A23187 or inositoltrisphosphate (IP3) and enhancement of K+-evoked release of DA was examined. Six 5-min fractions were collected. DA release was determined by HPLC coupled to electrochemical detection. Results indicated that although deficits were seen in oxotremorine and carbachol enhancement of K+-evoked release of DA, these decrements were not observed when either A23187 or IP3 were utilized to enhance the K+-evoked release of DA.(ABSTRACT TRUNCATED AT 250 WORDS)

Radiation-induced increases in sensitivity of cataleptic behavior to haloperidol: possible involvement of prostaglandins.

The effects of radiation exposure on haloperidol-induced catalepsy were examined in order to determine whether elevated prostaglandins, through an action on dopaminergic autoreceptors, could be involved in the radiation-induced increase in the potency of this neuroleptic. Cataleptic behavior was examined in animals irradiated with various doses of gamma photons (1-150 Gy) and pretreated with a subthreshold dose of haloperidol (0.1 mg/kg). This approach was chosen to maximize any synergistic effects of radiation and haloperidol. After irradiation with doses less than or equal to 30 Gy, the combined treatment of haloperidol and radiation produced catalepsy, whereas neither treatment alone had an effect. This observed catalepsy could be blocked with prior administration of indomethacin, a prostaglandin synthesis inhibitor. Animals exposed to doses of radiation less than or equal to 50 Gy and no haloperidol, however, displayed apparent catalepsy. This effect was also antagonized by indomethacin. Prostaglandins can induce catalepsy and when administered in subthreshold doses along with subthreshold doses of haloperidol, catalepsy was observed. In order to assess a possible action of prostaglandins and radiation on dopaminergic activity, the functioning of striatal dopaminergic autoreceptors was examined by determining the effects of varying concentrations of haloperidol on the K+-evoked release of dopamine from striatal slices obtained from parallel groups of animals treated as above. Results indicated that sensitivity to haloperidol increased (higher K+-evoked dopamine release) in slices from irradiated or prostaglandin-treated animals and that this increase in sensitivity was blocked by indomethacin. Results from both experiments suggest that radiation-induced increases in endogenous neuronal mediators, such as prostaglandins, can induce catalepsy through an action on dopaminergic autoreceptors.

Actions of ethanol on voltage-sensitive sodium channels: effects of acute and chronic ethanol treatment.

The effects of acute and chronic ethanol treatment on neurotoxin-stimulated 22Na+ uptake and [3H]batrachotoxinin A20-alpha-benzoate binding to neuronal sodium channels were studied in rat forebrain synaptosomes. Fluorescence measurements were used to assess the intrinsic order or fluidity and the sensitivity to ethanol of rat forebrain synaptic plasma membranes at various intervals during and after chronic ethanol treatment. Acute ethanol administration had no significant effect on neurotoxin binding in the absence or presence of ethanol in vitro or on sodium uptake in the absence of ethanol in vitro. However, a single dose of ethanol produced a dose and time-dependent attenuation of the inhibitory effect of ethanol on sodium uptake, suggestive of acute tolerance. Chronic ethanol treatment reduced the influx of 22Na+ in the presence of batrachotoxin and diminished the inhibitory effect of ethanol in vitro on sodium uptake for up to 20 days after withdrawal, but the specific binding of the neurotoxin in the presence or absence of ethanol was unchanged. Synaptic plasma membranes from chronic ethanol-treated rats showed no change in intrinsic order but the disordering effect of ethanol was significantly smaller for up to 20 days after withdrawal. Results of this study demonstrate that brain tissue from ethanol-treated rats can adapt rapidly to some effects of ethanol and that chronic ethanol administration can reduce the effects of ethanol on physical and functional properties of neurons for a prolonged period of time.

An automated method for the determination of biogenic amines and their metabolites by high-performance liquid chromatography.

An automated high-performance liquid chromatographic method has been developed that allows for the determination of a number of compounds related to catechol- and indoleamine metabolism. The compounds that can be measured include L-DOPA, dopamine, dihydroxyphenylacetic acid, homovanillic acid, 3-methoxytyramine, norepinephrine, 3-methoxy-4-hydroxyphenylglycol, dihydroxyphenylglycol, vanilmandelic acid, epinephrine, 5-hydroxytryptophan, serotonin, 5-hydroxyindoleacetic acid, and 5-hydroxytryptophol. Dihydroxybenzylamine is used as an internal standard. Although dihydroxyphenylglycol and vanilmandelic acid could be detected and quantified, they could not be separated from each other. The method is completely automated and is sensitive enough to detect amounts as low as 500 fmol. Up to 200 samples a week can be analyzed in the automated mode. Using this method, analyses of brain tissue can be accomplished with no need for a cleanup procedure. The value of this procedure lies in its ability to simultaneously determine various amines and metabolites from small tissue samples in the same animals and with automation to analyze a relatively large number of samples a day with little attention by a technician.

Neurotransmitter-receptor binding in various brain regions in ethanol-dependent rats.

Since chronic ethanol administration has been demonstrated to induce a number of alterations in neurotransmitter utilization, the possibility was investigated that the receptors, on which these transmitters act, are altered because of a modified synaptic input. Male Sprague-Dawley rats were rendered physically-dependent on ethanol by the oral administration of 9-13 g/kg of ethanol each day over a 4 day period. The binding of radioligands specific for alpha-adrenergic, beta-adrenergic, dopaminergic, serotonergic, muscarinic cholinergic, and GABAergic receptors was assessed at various intervals after withdrawal in several areas of the brain. No alteration in receptor binding was observed at any point under the conditions studied. The data suggest that the signs of an ethanol withdrawal syndrome are not mediated through changes in the ability of neurotransmitters to interact with their receptors.

Alterations in neurotransmitter activity after acute and chronic ethanol treatment: studies of transmitter interactions.

Acute and chronic ethanol treatment has multiple effects on the neurotransmitter systems in the nigrostriatal complex. A single dose of ethanol increases striatal dopamine release at low doses, but depresses it at high doses. In ethanol-dependent rats, dopamine release is accelerated during intoxication, but is reduced during a withdrawal syndrome. Concomitantly, high-affinity choline uptake, an index of cholinergic activity, is elevated at times when dopamine release is depressed. Changes in dopaminergic or cholinergic receptor activity do not induce or result from these effects. Neither has a role for GABA or substance P yet been implicated. The data suggest that interactions between at least two trasmitters in the caudate nucleus may occur after acute and chronic ethanol treatment.

Alterations in brain cyclic guanosine 3':5'-monophosphate levels after acute and chronic treatment with ethanol.

A previous finding from our laboratory, that a single dose of ethanol depletes cerebellar cyclic guanosine 3':5'-monophosphate (cGMP), has now been extended to an investigation of the effects of acute and chronic ethanol treatment of cGMP levels in six areas of the rat brain. Rats were either gavaged with a single dose of ethanol (6g/kg) or rendered ethanol-dependent with 11 to 15 g/kg/day in 3 to 5 fractions over a 4-day period. A single dose of ethanol depleted cGMP levels in only five areas of the brain studied. In the two areas in which a time course of the response was determined, the caudate nucleus and cerebral cortex, cGMP depletion was maximal 1 hour after ethanol administration when blood ethanol concentrations were highest. cGMP levels returned to control values as blood ethanol was eliminated. Inethanol-dependent animals still intoxicated, cGMP was reduced but not to the same magnitude in the cerebellum and brain stem when compared with the response obtained after a single dose at equivalent blood ethanol concentrations. During the ethanol withdrawal syndrome cGMP levels had returned to control vlaues. The data suggest that cBMP depletion may play a role in ethanol-induced intoxication and that tolerance to this effect develops concurrently with behavioral tolerance.