Caenorhabditis elegans metallothioneins protect against toxicity induced by depleted uranium.

Depleted uranium (DU) is a dense and heavy metal used in armor, ammunition, radiation shielding, and counterbalances. The military usage has led to growing public concern regarding the health effects of DU. In this study, we used the nematode, Caenorhabditis elegans, to evaluate the toxicity of DU and its effects in knockout strains of metallothioneins (MTs), which are small thiol-rich proteins that have numerous functions, such as metal sequestration, transport, and detoxification. We examined nematode viability, the accumulation of uranium, changes in MT gene expression by quantitative reverse transcription-PCR, and the induction of green fluorescent protein under the control of the MT promoters, following exposure to DU. Our results indicate that (1) DU causes toxicity in a dose-dependent manner; (2) MTs are protective against DU exposure; and (3) nematode death by DU is not solely a reflection of intracellular uranium concentration. (4) Furthermore, only one of the isoforms of MTs, metallothionein-1 (mtl-1), appears to be important for uranium accumulation in C. elegans. These findings suggest that these highly homologous proteins may have subtle functional differences and indicate that MTs mediate the response to DU.

Toxicity studies on depleted uranium in primary rat cortical neurons and in Caenorhabditis elegans: what have we learned?

Depleted uranium (DU) is the major by-product of the uranium enrichment process for its more radioactive isotopes, retaining approximately 60% of its natural radioactivity. Given its properties as a pyrophoric and dense metal, it has been extensively used in armor and ammunitions. Questions have been raised regarding the possible neurotoxic effects of DU in humans based on follow-up studies in Gulf War veterans, where a decrease in neurocognitive behavior in a small population was noted. Additional studies in rodents indicated that DU readily traverses the blood-brain barrier, accumulates in specific brain regions, and results in increased oxidative stress, altered electrophysiological profiles, and sensorimotor deficits. This review summarizes the toxic potential of DU with emphasis on studies on thiol metabolite levels, high-energy phosphate levels, and isoprostane levels in primary rat cortical neurons. Studies in Caenorhabditis elegans detail the role of metallothioneins, small thiol-rich proteins, in protecting against DU exposure. In addition, recent studies also demonstrate that only one of the two forms, metallothionein-1, is important in the accumulation of uranium in worms.

A study of airborne chrysotile concentrations associated with handling, unpacking, and repacking boxes of automobile clutch discs.

Although automotive friction products (brakes and manual clutches) historically contained chrysotile asbestos, industrial hygiene surveys and epidemiologic studies of auto mechanics have consistently shown that these workers are not at an increased risk of developing asbestos-related diseases. Airborne asbestos levels during brake repair and brake parts handling have been well-characterized, but the potential exposure to airborne asbestos fibers during the handling of clutch parts has not been examined. In this study, breathing zone samples on the lapel of a volunteer worker (n=100) and area samples at bystander (n=50), remote area (n=25), and ambient (n=9) locations collected during the stacking, unpacking, and repacking of boxes of asbestos-containing clutches, and the subsequent cleanup and clothes handling, were analyzed by phase contrast microscopy (PCM) and transmission electron microscopy (TEM). In addition, fiber morphology and size distribution was evaluated using X-ray diffraction, polarized light microscopy, and ISO analytical methods. It was observed that the (1) airborne asbestos concentrations increased with the number of boxes unpacked and repacked, (2) repetitive stacking of unopened boxes of clutches resulted in higher asbestos concentrations than unpacking and repacking the boxes of clutches, (3) cleanup and clothes handling tasks yielded very low asbestos concentrations. Fiber size and morphology analyses showed that amphibole fibers were not detected in the clutches and that the vast majority (>95%) of the airborne chrysotile fibers were less than 20 microm in length. Applying the ratio of asbestos fibers:total fibers (including non-asbestos) as determined by TEM to the PCM results, it was found that 30-min average airborne chrysotile concentrations (PCM adjusted) were 0.026+/-0.004 f/cc or 0.100+/-0.017 f/cc for a worker unpacking and repacking 1 or 2 boxes of clutches, respectively. The 30-min PCM adjusted average airborne asbestos concentrations at bystander locations ranged from 0.002+/-0.001 f/cc and 0.004+/-0.002 f/cc when 1 or 2 boxes of clutches were handled, respectively. Estimated 8-h TWA asbestos exposures for a worker handling 1 or 2 boxes of clutches over a workday ranged from 0.002 to 0.006 f/cc. The 30-min PCM adjusted average airborne asbestos concentration for a worker continuously stacking unopened boxes of clutches was 0.212+/-0.014 f/cc; the 8-h TWA was 0.013 f/cc. Additionally, 30-min PCM adjusted average airborne asbestos concentrations following cleanup and clothing handling were 0.002+/-0.001 f/cc and 0.002+/-0.002 f/cc, respectively, both resulting in estimated 8-h TWA asbestos exposures of 0.0001 f/cc. The results of this study indicate that the handling, unpacking, and repacking of clutches, and the subsequent cleanup and clothes handling by a worker within a short-term period or over the entire workday, result in exposures below the historical and current short-term and 8-h occupational exposure limits for asbestos.

Neurotoxic potential of depleted uranium effects in primary cortical neuron cultures and in Caenorhabditis elegans.

Depleted uranium (DU) is an extremely dense metal that is used in radiation shielding, counterbalances, armor, and ammunition. In light of the public concerns about exposure to DU and its potential role in Gulf War Syndrome (GWS), this study evaluated the neurotoxic potential of DU using focused studies on primary rat cortical neurons and the nematode Caenorhabditis elegans. We examined cell viability, cellular energy metabolism, thiol metabolite oxidation, and lipid peroxidation following exposure of cultured neurons to DU, in the form of uranyl acetate. We concurrently evaluated the neurotoxicity of uranyl acetate in C. elegans using various neuronal-green fluourescent protein reporter strains to visualize neurodegeneration. Our studies indicate that uranyl acetate has low cytotoxic potential, and uranium exposure does not result in significant changes in cellular energy metabolism, thiol metabolite oxidation, or lipid peroxidation. Furthermore, our C. elegans studies do not show any significant neurodegeneration following uranyl acetate exposure. Together, these studies suggest that DU, in the form of uranyl acetate, has low neurotoxic potential. These findings should alleviate the some of public concerns regarding DU as an etiologic agent of neurodegenerative conditions associated with GWS.

Down-regulation of early ionotrophic glutamate receptor subunit developmental expression as a mechanism for observed plasticity deficits following gestational exposure to benzo(a)pyrene.

The focus of this study was to characterize the impact of gestational exposure to benzo(a)pyrene [B(a)P] on modulation of glutamate receptor subunit expression that is critical for the maintenance of synaptic plasticity mechanisms during hippocampal or cortical development in offspring. Previous studies have demonstrated that hippocampal and/or cortical synaptic plasticity (as measured by long-term potentiation and S1-cortex spontaneous/evoked neuronal activity) and learning behavior (as measured by fixed-ratio performance operant testing) is significantly impaired in polycyclic aromatic or halogenated aromatic hydrocarbon-exposed offspring as compared to controls. These previous studies have also revealed that brain to body weight ratios are greater in exposed offspring relative to controls indicative of intrauterine growth retardation which has been shown to manifest as low birth weight in offspring. Recent epidemiological studies have identified an effect of prenatal exposure to airborne polycyclic aromatic hydrocarbons on neurodevelopment in the first 3 years of life among inner-city children [Perera FP, Rauh V, Whyatt RM, Tsai WY, Tang D, Diaz D, et al. Effect of prenatal exposure to airborne polycyclic aromatic hydrocarbons on neurodevelopment in the first 3 years of life among inner-city children. Environ Health Perspect 2006;114:1287-92]. The present study utilizes a well-characterized animal model to test the hypothesis that gestational exposure to B(a)P causes dysregulation of developmental ionotropic glutamate receptor subunit expression, namely the N-methyl-d-aspartate receptor (NMDAR) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate receptor (AMPAR) both critical to the expression of synaptic plasticity mechanisms. To mechanistically ascertain the basis of B(a)P-induced plasticity perturbations, timed pregnant Long-Evans rats were exposed in an oral subacute exposure regimen to 0, 25 and 150mug/kg BW B(a)P on gestation days 14-17. The first sub-hypothesis tested whether gestational exposure to B(a)P would result in significant disposition in offspring. The second sub-hypothesis tested whether gestational exposure to B(a)P would result in down-regulation of early developmental expression of NMDA and AMPA receptor subunits in the hippocampus of offspring as well as in primary neuronal cultures. The results of these studies revealed significant: (1) disposition to the hippocampus and cortex, (2) down-regulation of developmental glutamate receptor mRNA and protein subunit expression and (3) voltage-dependent decreases in the amplitude of inward currents at negative potentials in B(a)P-treated cortical neuronal membranes. These results suggest that plasticity and behavioral deficits produced as a result of gestational B(a)P exposure are at least, in part, a result of down-regulation of early developmental glutamate receptor subunit expression and function at a time when excitatory synapses are being formed for the first time in the developing central nervous system. The results also predict that in B(a)P-exposed offspring with reduced early glutamate receptor subunit expression, a parallel deficit in behaviors that depend on normal hippocampal or cortical functioning will be observed and that these deficits will be present throughout life.

Neurotoxicity of depleted uranium: reasons for increased concern.

Depleted uranium (DU) is a byproduct of the enrichment process of uranium for its more radioactive isotopes to be used in nuclear energy. Because DU is pyrophoric and a dense metal with unique features when combined in alloys, it is used by the military in armor and ammunitions. There has been significant public concern regarding the use of DU by such armed forces, and it has been hypothesized to play a role in Gulf War syndrome. In light of experimental evidence from cell cultures, rats, and humans, there is justification for such concern. However, there are limited data on the neurotoxicity of DU. This review reports on uranium uses and its published health effects, with a major focus on in vitro and in vivo studies that escalate concerns that exposure to DU might be associated with neurotoxic health sequelae.

Analysis of cellular, transgenic and human models of Huntington's disease reveals tyrosine hydroxylase alterations and substantia nigra neuropathology.

Huntington's disease (HD) is a progressive, autosomal dominant neurodegenerative disorder that is pathologically characterized by a striatal-specific degeneration. Aberrant dopamine neurotransmission has been proposed as a mechanism underlying the movement disorder of HD. We report that the enzymatic activity of tyrosine hydroxylase (TH), the rate-limiting enzyme for dopamine biosynthesis, is decreased in a transgenic mouse model of HD. In addition, mutant huntingtin was found to disrupt transcription of TH and dopamine beta-hydroxylase (DbetaH) promoter reporter constructs. In situ hybridization revealed extensive loss of TH mRNA and decreased dopaminergic cell size in human HD substantia nigra. TH-immunoreactive protein was reduced in human grade 4 HD substantia nigra by 32% compared to age-matched controls. These findings implicate abnormalities in dopamine neurotransmission in HD and may provide new insights into targets for pharmacotherapy.

Inhibition of tryptophan hydroxylase activity and decreased 5-HT1A receptor binding in a mouse model of Huntington's disease.

The pathogenic mechanisms of the mutant huntingtin protein that cause Huntington's disease (HD) are unknown. Previous studies have reported significant decreases in the levels of serotonin (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the brains of the R6/2 transgenic mouse model of HD. In an attempt to elucidate the cause of these neurochemical perturbations in HD, the protein levels and enzymatic activity of tryptophan hydroxylase (TPH), the rate-limiting enzyme in 5-HT biosynthesis, were determined. Enzyme activity was measured in brainstem homogenates from 4-, 8-, and 12-week-old R6/2 mice and compared with aged-matched wild-type control mice. We observed a 62% decrease in brainstem TPH activity (p = 0.009) in 4-week-old R6/2 mice, well before the onset of behavioral symptoms. In addition, significant decreases in TPH activity were also observed at 8 and 12 weeks of age (61%, p = 0.02 and 86%, p = 0.005, respectively). In the 12-week-old-mice, no change in immunoreactive TPH was observed. In vitro binding showed that TPH does not bind to exon 1 of huntingtin in a polyglutamine-dependent manner. Specifically, glutathione-S-transferase huntingtin exon 1 proteins with 20, 32 or 53 polyglutamines did not interact with radiolabeled tryptophan hydroxylase. Therefore, the inhibition of TPH activity does not appear to result from a direct huntingtin/TPH interaction. Receptor binding analyses for the 5-HT1A receptor in 12-week-old R6/2 mice revealed significant reductions in 8-OH-[3H]DPAT binding in several hippocampal and cortical regions. These results demonstrate that the serotonergic system in the R6/2 mice is severely disrupted in both presymptomatic and symptomatic mice. The presymptomatic inhibition of TPH activity in the R6/2 mice may help explain the functional consequences of HD and provide insights into new targets for pharmacotherapy.