Natural-history Characterization of a Murine Partial-body Irradiation Model System: Establishment of a Multiple-Parameter Based GI-ARS Severity-Scoring System.

The purpose of this investigation was to characterize the natural history of a murine total-abdominal-irradiation exposure model to measure gastrointestinal acute radiation injury. Male CD2F1 mice at 12 to 15 weeks old received total-abdominal irradiation using 4-MV linear accelerator X-rays doses of 0, 11, 13.5, 15, 15.75 and 16.5 Gy (2.75 Gy/min). Daily cage-side (i.e., in the animal housing room) observations of clinical signs and symptoms including body weights on all animals were measured up to 10 days after exposure. Jejunum tissues from cohorts of mice were collected at 1, 3, 7 and 10 days after exposure and radiation injury was assessed by histopathological analyses. Results showed time- and dose-dependent loss of body weight [for example at 7 days: 0.66 (±0.80) % loss for 0 Gy, 6.40 (±0.76) % loss at 11 Gy, 9.43 (±2.06) % loss at 13.5 Gy, 23.53 (± 1.91) % loss at 15 Gy, 29.97 (±1.16) % loss at 15.75 Gy, and 31.79 (±0.76) % loss at 16.5 Gy]. Negligible clinical signs and symptoms, except body weight changes, of radiation injury were observed up to 10 days after irradiation with doses of 11 to 15 Gy. Progressive increases in the severity of clinical signs and symptoms were found after irradiation with doses >15 Gy. Jejunum histology showed a progressive dose-dependent increase in injury. For example, at 7 days postirradiation, the percent of crypts, compared to controls, decreased to 82.3 (±9.5), 69.2 (±12.3), 45.4 (±11.9), 18.0 (±3.4), and 11.5 (± 1.8) with increases in doses from 11 to 16.5 Gy. A mucosal injury scoring system was used that mainly focused on changes in villus morphology damage (i.e., subepithelial spaces near the tips of the villi with capillary congestion, significant epithelial lifting along the length of the villi with a few denuded villus tips). Peak levels of total-abdominal irradiation induced effects on the mucosal injury score were seen 7 days after irradiation for doses ≥15 Gy, with a trend to show a decline after 7 days. A murine multiple-parameter gastrointestinal acute-radiation syndrome severity-scoring system was established based on clinical signs and symptoms that included measures of appearance (i.e., hunched and/or fluffed fur), respiratory rate, general (i.e., decreased mobility) and provoked behavior (i.e., subdued response to stimulation), weight loss, and feces/diarrhea score combined with jejunum mucosal-injury grade score. In summary, the natural-history radio-response for murine partial-body irradiation exposures is important for establishing a well-characterized radiation model system; here we established a multiple-parameter gastrointestinal acute-radiation syndrome severity-scoring system that provides a radiation injury gastrointestinal tissue-based assessment utility.

Oxidative damage in metal fragment-embedded Sprague-Dawley rat gastrocnemius muscle.

Injuries suffered in armed conflicts often result in wounds with embedded metal fragments. Standard surgical guidance has been to leave fragments in place except under certain circumstances; meaning that individuals may carry these retained fragments for their lifetime. Because of advancements in weapon design and the use of improvised explosive devices, the list of metals that could be found in a wound is extensive. In most cases the toxicological properties of these metals when embedded in the body are not known. To assess the potential damage embedded metals may cause to surrounding tissue, we utilized a rodent model to investigate the effect of a variety of military-relevant metals on markers of oxidative damage. The metals tested included tungsten, nickel, cobalt, iron, copper, aluminum, lead, and depleted uranium. Herein we report our findings on creatine kinase activity, lipid and protein oxidation, total antioxidant capacity, and glutathione levels in gastrocnemius homogenates from Sprague-Dawley rats surgically implanted with metal pellets for periods up to 12 months. Not all embedded metals affected the measured markers equally. However, metal-associated effects were seen at various times for muscle and serum creatinine levels, protein oxidation, total antioxidant capacity, and glutathione levels. No metal-induced effects on lipid peroxidation were observed. Taken together, these data suggest that subtle oxidative damage may be occurring in the muscle surrounding an embedded metal and indicates the need for medical surveillance of those individuals wounded by metal shrapnel.

Serum Indicators of Oxidative Damage from Embedded Metal Fragments in a Rat Model.

Injuries suffered in armed conflicts often result in embedded metal fragments. Standard surgical guidance recommends leaving embedded fragments in place except under certain circumstances in an attempt to avoid the potential morbidity that extensive surgery often brings. However, technological advances in weapon systems and insurgent use of improvised explosive devices now mean that practically any metal can be found in these types of wounds. Unfortunately, in many cases, the long-term toxicological properties of embedded metals are not known, further complicating treatment decisions. Because of concerns over embedded metal fragment injuries, the U.S. Departments of Defense and Veterans' Affairs developed a list of "metals of concern" for these types of injuries. In this study, we selected eight of these metals including tungsten, nickel, cobalt, iron, copper, aluminum, lead, and depleted uranium to investigate the long-term health effects using a rodent model developed in our Institute to study embedded fragment injuries. In this report, we show that metals surgically implanted into the gastrocnemius muscle of laboratory rats to simulate a shrapnel wound induce a variety of cytokines including IFN-γ, IL-4, IL-5, IL-6, IL-10, and IL-13. TNF-α and KC/GRO were not affected, and IL-1ß was below the limit of detection. Serum levels of C-reactive protein were also affected, increasing with some metals and decreasing with others. The TBARS assay, an assessment of lipid peroxidation, demonstrated that implanted aluminum and lead increased markers of lipid peroxidation in serum. Taken together, the results suggest that serum cytokine levels, as well as other indicators of oxidative damage, may prove useful in identifying potential adverse health effects of embedded metals.

Protein Expression in the Gastrocnemius Muscle of a Rodent Shrapnel-Injury Model.

With shrapnel injuries, the metal fragment is usually left in place to reduce the risk of morbidity extensive surgery might bring. This means the individual may retain those metals for the remainder of their life. Often the long-term health effects of the embedded metal are not known, especially with respect to protein damage and perturbations of muscle repair pathways. In this study, using homogenates of rat gastrocnemius muscle implanted with pellets of military-relevant metals, we investigated expression of iNOS and eNOS, enzymes involved in nitric oxide production, as well as MMP-2 and MMP-9, matrix metalloproteinases associated with muscle repair. In addition, hydroxynonenal-modified proteins were investigated to assess metal-induced oxidative damage and metal levels in the gastrocnemius determined. Metals were implanted for up to 12 months in order to determine the long-term effects on the expression of muscle-associated proteins. With the exception of iron and cobalt at 1-month post-implantation, there were no significant differences in metal levels in the gastrocnemius in any of the cohorts. Protein expression analysis showed significant decreases in iNOS and eNOS in the 6-month and 12-month lead and depleted uranium groups. Hydroxynonenal-modified proteins were also significantly increased in the iron, copper, lead, and depleted uranium groups. These results suggest that some embedded metals can induce long-term oxidative damage, as well as affect enzyme systems involved in signal transduction.

Urine miRNAs as potential biomarkers for systemic reactions induced by exposure to embedded metal.

Aim: Explore the potential of urine microRNAs as biomarkers that may reflect the biological responses to pure metals embedded in skeletal muscle over time. Materials & methods: We tested a panel of military-relevant metals embedded in the gastrocnemius muscles of 3-month-old, male, Sprague-Dawley rats (n = 8/group) for a duration of 1, 3, 6 and 12 months, and performed small RNA-sequencing on the urine samples. Results: Results provide potential tissue targets affected by metal exposure and a list of unique or common urine microRNA biomarkers indicative of exposure to various metals, highlighting a complex systemic response. Conclusion: We have identified a panel of miRNAs as potential urine biomarkers to reflect the complex systemic response to embedded metal exposure.

Nutraceuticals as Potential Radionuclide Decorporation Agents.

Exposure of individuals to radioactive material as a result of ingestion of contaminated food and water is an increasing public health concern. Unfortunately, there are limited treatment modalities for dealing with these types of potentially toxic exposures. Recent research suggests that many plant-based nutraceuticals may possess metal-binding properties. This preliminary study investigated the ability of genistein, curcumin, quercetin, and lentinan to bind metals considered internal contamination risks, namely cesium, uranium, cobalt, and strontium, in a variety of matrices. The efficacy of these nutraceuticals in protecting cultured cells from metal-induced toxicity was also explored. Results showed that none of the compounds bound cesium or strontium. However, genistein, curcumin, and quercetin could bind uranium. Curcumin and quercetin also bound cobalt and could also protect cultured cells from metal-induced cytotoxicity. Lentinan did not bind any of the metals tested. Metal binding was also pH dependent, with no binding observed at lower pH values. This project showed that nutraceuticals could function as chelators for metals considered internal radionuclide contamination hazards. Further investigations are required in order to determine whether these compounds will become a new nontoxic arsenal of pharmaceutical compounds with which to treat radionuclide contamination.

Effect of embedded metal fragments on urinary metal levels and kidney biomarkers in the Sprague-Dawley rat.

BACKGROUND: Wounds with embedded metal fragments are an unfortunate consequence of armed conflicts. In many cases the exact identity of the metal(s) and their long-term health effects, especially on the kidney, are not known. AIM OF STUDY: The aim of this study was to quantitate the urinary levels of metals solubilized from surgically implanted metal pellets and to assess the effect of these metals on the kidney using a battery of biomarker assays. MATERIALS AND METHODS: Using a rodent model system developed in our Institute to simulate embedded fragment injuries, eight metals considered likely components of an embedded fragment wound were individually implanted into the gastrocnemius muscle of male Sprague-Dawley rats. The rats were followed for 12 months post-implantation with urine collected prior to surgery then at 1-, 3-, 6-, 9-, and 12-months post-implantation to provide a within-subjects cohort for examination. Urinary metal levels were determined using inductively coupled plasma-mass spectrometry and urinary biomarkers assessed using commercially available kits to determine metal-induced kidney effects. RESULTS: With few exceptions, most of the implanted metals rapidly solubilized and were found in the urine at significantly higher levels than in control animals as early as 1-month post-implantation. Surprisingly, many of the biomarkers measured were decreased compared to control at 1-month post-implantation before returning to normal at the later time points. However, two metals, iron and depleted uranium, showed increased levels of several markers at later time points, yet these levels also returned to normal as time progressed. CONCLUSION: This study showed that metal pellets surgically implanted into the leg muscle of Sprague-Dawley rats rapidly solubilized with significant levels of the implanted metal found in the urine. Although kidney biomarker results were inconsistent, the changes observed along with the relatively low amounts of metal implanted, suggest that metal-induced renal effects need to be considered when caring for individuals with embedded metal fragment wounds.

Brain region- and metal-specific effects of embedded metals in a shrapnel wound model in the rat.

The health effects of prolonged exposure to embedded metal fragments, such as those found in shrapnel wounds sustained by an increasing number of military personnel, are not well known. As part of a large collaborative effort to expand this knowledge, we use an animal model of shrapnel wounds originally developed to investigate effects of embedded depleted uranium to investigate effects of military-relevant metals tungsten, nickel, cobalt, iron, copper, aluminum, lead, and depleted uranium compared to an inert control, tantalum. Rats are surgically implanted with pellets of one of the metals of interest in the gastrocnemius (leg) muscle and tracked until 1 month, 3 months, 6 months, or 12 months from the time of implant, at which point they are euthanized and multiple organs and tissue samples are collected for inspection. Here we focus on four regions of the brain: frontal cortex, hippocampus, amygdala, and cerebellum. We examined changes in accumulated metal concentration in each region as well as changes in expression of proteins related to blood brain barrier tight junction formation, occludin and ZO-1, and synapse function, PSD95, spinophilin, and synaptotagmin. We report few changes in metal accumulation or blood brain barrier protein expression, but a large number of synapse proteins have reduced expression levels, particularly within the first 6 months of exposure, but there are regional and metal-specific differences in effects.

Time-course analysis of the effect of embedded metal on skeletal muscle gene expression.

As a consequence of military operations, many veterans suffer from penetrating wounds and long-term retention of military-grade heavy metal fragments. Fragments vary in size and location, and complete surgical removal may not be feasible or beneficial in all cases. Increasing evidence suggests retention of heavy metal fragments may have serious biological implications, including increased risks for malignant transformation. Previous studies assessed the tumorigenic effects of metal alloys in rats, demonstrating combinations of metals are sufficient to induce tumor formation after prolonged retention in skeletal muscle tissue. In this study, we analyzed transcriptional changes in skeletal muscle tissue in response to eight different military-relevant pure metals over 12 mo. We found that most transcriptional changes occur at 1 and 3 mo after metal pellets are embedded in skeletal muscle and these effects resolve at 6 and 12 mo. We also report significant immunogenic effects of nickel and cobalt and suppressive effects of lead and depleted uranium on gene expression. Overall, skeletal muscle exhibits a remarkable capacity to adapt to and recover from internalized metal fragments; however, the cellular response to chronic exposure may be restricted to the metal-tissue interface. These data suggest that unless affected regions are specifically captured by biopsy, it would be difficult to reliably detect changes in muscle gene expression that would be indicative of long-term adverse health outcomes.

Hydrophobic Sand Versus Metabolic Cages: A Comparison of Urine Collection Methods for Rats (Rattus norvegicus).

A common method for urine collection from rats requires the use of a metabolic cage, thus exposing animals to extended periods of isolation in an unfamiliar cage with a wire-mesh floor. A new method involving hydrophobic sand, a material more similar to bedding, has become available recently but has not been extensively compared with metabolic cages in regard to collection efficiency or stress. Using a within-subjects crossover design, we examined differences in stress markers, urinary markers, and urine volume of clinically healthy male Sprague-Dawley rats during 2-, 4-, and 6-h collection sessions in hydrophobic sand and metabolic cages. Stress response markers of weight loss, fecal pellet output, or corticosterone did not differ between hydrophobic sand and metabolic cages, and observed behavior suggested that sand may be less stressful than metabolic cages. All clinically relevant urinary markers examined were normal, with no differences between collection methods. Total urine volume collected was greater from the metabolic cage than sand in 3 of the 5 sessions, but the volume collected during the shortest session (2 h) did not differ between methods and accounted for 62% of the total volume collected during the longest session (6 h). Our results suggest that hydrophobic sand is a refinement of urine collection methods for rats that decreases isolation time, risk of injury, and stress and maintains the integrity of urine samples.

Hydrophobic Sand Is a Non-Toxic Method of Urine Collection, Appropriate for Urinary Metal Analysis in the Rat.

Hydrophobic sand is a relatively new method of urine collection in the rodent, comparable to the established method using a metabolic cage. Urine samples are often used in rodent research, especially for biomarkers of health changes after internal contamination from embedded metals, such as in a model of a military shrapnel wound. However, little research has been done on the potential interference of hydrophobic sand with urine metal concentrations either by contamination from the sand particulate, or adsorption of metals from the urine. We compare urine collected from rats using the metabolic cage method and the hydrophobic sand method for differences in metal concentration of common urinary metals, and examine physical properties of the sand material for potential sources of contamination. We found minimal risk of internal contamination of the rat by hydrophobic sand, and no interference of the sand with several common metals of interest (cobalt, strontium, copper, and manganese), although we advise caution in studies of aluminum in urine.

The Role of the Component Metals in the Toxicity of Military-Grade Tungsten Alloy.

Tungsten-based composites have been recommended as a suitable replacement for depleted uranium. Unfortunately, one of these mixtures composed of tungsten (W), nickel (Ni) and cobalt (Co) induced rhabdomyosarcomas when implanted into the leg muscle of laboratory rats and mice to simulate a shrapnel wound. The question arose as to whether the neoplastic effect of the mixture could be solely attributed to one or more of the metal components. To investigate this possibility, pellets with one or two of the component metals replaced with an identical amount of the biologically-inert metal tantalum (Ta) were manufactured and implanted into the quadriceps of B6C3F1 mice. The mice were followed for two years to assess potential adverse health effects. Implantation with WTa, CoTa or WNiTa resulted in decreased survival, but not to the level reported for WNiCo. Sarcomas in the implanted muscle were found in 20% of the CoTa-implanted mice and 5% of the WTa- and WCoTa-implanted rats and mice, far below the 80% reported for WNiCo-implanted mice. The data obtained from this study suggested that no single metal is solely responsible for the neoplastic effects of WNiCo and that a synergistic effect of the three metals in tumor development was likely.

Induction of rhabdomyosarcoma by embedded military-grade tungsten/nickel/cobalt not by tungsten/nickel/iron in the B6C3F1 mouse.

Continued improvements in the ballistic properties of military munitions have led to metal formulations for which little are known about the long-term health effects. Previously we have shown that a military-grade tungsten alloy comprised of tungsten, nickel, and cobalt, when embedded into the leg muscle of F344 rats to simulate a fragment wound, induces highly aggressive metastatic rhabdomyosarcomas. An important follow-up when assessing a compound's carcinogenic potential is to test it in a second rodent species. In this study, we assessed the health effects of embedded fragments of 2 military-grade tungsten alloys, tungsten/nickel/cobalt and tungsten/nickel/iron, in the B6C3F1 mouse. Implantation of tungsten/nickel/cobalt pellets into the quadriceps muscle resulted in the formation of a rhabdomyosarcoma around the pellet. Conversely, implantation of tungsten/nickel/iron did not result in tumor formation. Unlike what was seen in the rat model, the tumors induced by the tungsten/nickel/cobalt did not exhibit aggressive growth patterns and did not metastasize.

Urinary and serum metal levels as indicators of embedded tungsten alloy fragments.

Novel metal formulations are being used with increasing frequency on the modern battlefield. In many cases the health effects of these materials are not known, especially when they are embedded as fragments. Imaging techniques, although useful for determining location, provide no information regarding the composition of embedded fragments. In this report, we show that laboratory rats implanted with weapons-grade tungsten alloy (tungsten, nickel, and cobalt) pellets demonstrate significant increases in both urinary and serum levels of tungsten, nickel, and cobalt, which indicates that such measurements can provide information on the composition of embedded fragments. We also propose that, in addition to the requirements promulgated by the recent directive on analysis of metal fragments removed from Department of Defense personnel (Health Affairs policy 07-029), urine and blood/serum samples should be collected from personnel and analyzed for metal content. Such measurements could yield information on the composition of retained fragments and provide the basis for further treatment options.