Metabolomic and lipidomic analysis of serum from mice exposed to an internal emitter, cesium-137, using a shotgun LC-MS(E) approach.

In this study ultra performance liquid chromatography (UPLC) coupled to time-of-flight mass spectrometry in the MS(E) mode was used for rapid and comprehensive analysis of metabolites in the serum of mice exposed to internal exposure by Cesium-137 ((137)Cs). The effects of exposure to (137)Cs were studied at several time points after injection of (137)CsCl in mice. Over 1800 spectral features were detected in the serum of mice in positive and negative electrospray ionization modes combined. Detailed statistical analysis revealed that several metabolites associated with amino acid metabolism, fatty acid metabolism, and the TCA cycle were significantly perturbed in the serum of (137)Cs-exposed mice compared with that of control mice. While metabolites associated with the TCA cycle and glycolysis increased in their serum abundances, fatty acids such as linoleic acid and palmitic acid were detected at lower levels in serum after (137)Cs exposure. Furthermore, phosphatidylcholines (PCs) were among the most perturbed ions in the serum of (137)Cs-exposed mice. This is the first study on the effects of exposure by an internal emitter in serum using a UPLC-MS(E) approach. The results have put forth a panel of metabolites, which may serve as potential serum markers to (137)Cs exposure.

Serum Dyslipidemia Is Induced by Internal Exposure to Strontium-90 in Mice, Lipidomic Profiling Using a Data-Independent Liquid Chromatography-Mass Spectrometry Approach.

Despite considerable research into the environmental risks and biological effects of exposure to external beam γ rays, incorporation of radionuclides has largely been understudied. This dosimetry and exposure risk assessment is challenging for first responders in the field during a nuclear or radiological event. Therefore, we have developed a workflow for assessing injury responses in easily obtainable biofluids, such as urine and serum, as the result of exposure to internal emitters cesium-137 ((137)Cs) and strontium-90 ((90)Sr) in mice. Here we report on the results of the untargeted lipidomic profiling of serum from mice exposed to (90)Sr. We also compared these results to those from previously published (137)Cs exposure to determine any differences in cellular responses based on exposure type. The results of this study conclude that there is a gross increase in the serum abundance of triacylglycerides and cholesterol esters, while phostaphatidylcholines and lysophosphatidylcholines displayed decreases in their serum levels postexposure at study days 4, 7, 9, 25, and 30, with corresponding average cumulative skeleton doses ranging from 1.2 ± 0.1 to 5.2 ± 0.73 Gy. The results show significant perturbations in serum lipidome as early as 2 days postexposure persisting until the end of the study (day 30).

Effect of 90Sr internal emitter on gene expression in mouse blood.

BACKGROUND: The radioactive isotope Strontium-90 ((90)Sr) may be released as a component of fallout from nuclear accidents, or in the event of a radiological incident such as detonation of an improvised nuclear device, and if ingested poses a significant health risk to exposed individuals. In order to better understand the response to (90)Sr, using an easily attainable and standard biodosimetry sample fluid, we analyzed the global transcriptomic response of blood cells in an in vivo model system. RESULTS: We injected C57BL/6 mice with a solution of 90SrCl2 and followed them over a 30-day period. At days 4, 7, 9, 25 and 30, we collected blood and isolated RNA for microarray analyses. These days corresponded to target doses in a range from 1-5 Gy. We investigated changes in mRNA levels using microarrays, and changes in specific microRNA (miRNA) predicted to be involved in the response using qRT-PCR. We identified 8082 differentially expressed genes in the blood of mice exposed to (90)Sr compared with controls. Common biological functions were affected throughout the study, including apoptosis of B and T lymphocytes, and atrophy of lymphoid organs. Cellular functions such as RNA degradation and lipid metabolism were also affected during the study. The broad down regulation of genes observed in our study suggested a potential role for miRNA in gene regulation. We tested candidate miRNAs, mmu-miR-16, mmu-miR-124, mmu-miR-125 and mmu-mir-21; and found that all were induced at the earliest time point, day 4. CONCLUSIONS: Our study is the first to report the transcriptomic response of blood cells to the internal emitter (90)Sr in mouse and a possible role for microRNA in gene regulation after (90)Sr exposure. The most dramatic effect was observed on gene expression related to B-cell development and RNA maintenance. These functions were affected by genes that were down regulated throughout the study, suggesting severely compromised antigen response, which may be a result of the deposition of the radioisotope proximal to the hematopoietic compartment in bone.

Plutonium Systemic Biokinetic Model for Rats.

A baseline compartmental model (relative to modeling decorporation) of the distribution and retention of plutonium (Pu) in the rat for a systemic intake is derived. The model is derived from data obtained from a study designed to evaluate the behavior of plutonium in the first 28 days after incorporation. The model is based on a recently published model of americium (Am) in rats, which incorporated a pharmacokinetic (PK)-front-end modeling approach, which was used to specify transfer to and from the extracellular fluids (ECF) in the various tissues in terms of vascular flow and volumes of ECF. In the americium model, the approach was "cell-membrane limited," meaning that rapid diffusion of americium occurred throughout all the extracellular fluids (i.e., the blood plasma and interstitial fluids), while back-end rates representing transport into and out of the cells were determined empirically. However, this approach was inconsistent with the plutonium dataset. A good fit to the data is obtained by incorporating aspects of the Durbin et al. model structure, with plutonium in plasma separated into "free" and "bound" components. Free plutonium uses a cell-membrane-limited front end as for americium. Bound plutonium uses a capillary-wall-limited front end, where transfer rates from blood plasma into the interstitial fluids are relatively slow, and must be determined either empirically or from a priori knowledge. As in the Durbin et al. model, both free and bound plutonium are available for deposition in bone. In addition, our model has some bound plutonium associated with uptake to the gastrointestinal (GI) tract. Uncertainties in transfer rates were investigated using Markov Chain Monte Carlo (MCMC). It is anticipated that this model structure of plutonium will also be useful in interpreting comparable data from decorporation studies done in experimental animals.

Americium Systemic Biokinetic Model for Rats.

In this work, a baseline compartmental model of the distribution and retention of americium in the rat for a systemic intake was derived. The model was derived from data obtained from a study designed to evaluate the behavior of americium in the first 28 days after incorporation. A pharmacokinetic (PK)-front-end modeling approach was used to specify transfer to and from the extracellular fluids (ECF) in the various tissues in terms of vascular flow and volumes of ECF. Back-end rates representing transport into and out of the cells were determined empirically. Uncertainties in transfer rates were investigated using Markov chain Monte Carlo (MCMC). The combination of PK-front-end model and the back-end model structure used allowed for extrapolation to the earliest times with small uncertainty. This approach clearly demonstrated the rapid transfer of material from ECF to liver and bone. This model provides a baseline for modeling the action of decorporation agents, such as DTPA.

Radiotoxicity of inhaled (239)PuO(2) in dogs.

Beagle dogs inhaled graded exposure levels of insoluble plutonium dioxide ((239)PuO(2)) aerosols in one of three monodisperse particle sizes at the Lovelace Respiratory Research Institute (LRRI) to study the life-span health effects of different degrees of alpha-particle dose non-uniformity in the lung. The primary noncarcinogenic effects seen were lymphopenia, atrophy and fibrosis of the thoracic lymph nodes, and radiation pneumonitis and pulmonary fibrosis. Radiation pneumonitis/ pulmonary fibrosis occurred from 105 days to more than 11 years after exposure, with the lowest associated alpha-particle dose being 5.9 Gy. The primary carcinogenic effects also occurred almost exclusively in the lung because of the short range of the alpha-particle emissions. The earliest lung cancer was observed at 1086 days after the inhalation exposure. The most common type seen was papillary adenocarcinoma followed by bronchioloalveolar carcinoma. These lung cancer results indicate that a more uniform distribution of alpha-particle dose within the lung has an equal or possibly greater risk of neoplasia than less uniform distributions of alpha-particle dose. The results are consistent with a linear relationship between dose and response, but these data do not directly address the response expected at low dose levels. No primary tumors were found in the tracheobronchial and mediastinal lymph nodes despite the high alpha-particle radiation doses to these lymph nodes, and no cases of leukemia were observed.

Optimization of the ICRP 67 and NCRP 156 Transfer Rates Applied to Nonhuman Primates Intramuscularly Injected With 241Am.

Nonhuman primates intramuscularly injected with Am have been investigated using the International Commission on Radiological Protection Report 67 model coupled with National Council on Radiation Protection and Measurements Report 156 model. Default parameters from these models were input into the Integrated Modules for Bioassay Analysis software to predict the intake and skeleton retention in 20 tested nonhuman primates. The predictions generated were compared to the experimental data from the Durbin nonhuman primate studies. A previous study conducted by Alomairy in 2017 indicated that the early behavior of Am(NO3)3 in wound cases can be explained using the default transfer rates. However, these transfer rates were not able to predict the intake and skeleton retention at the time of sacrifice after 100 d postintake due to differences in the amount of activity translocated or deposited in liver tissue and nonliver tissues (primarily skeleton). This is likely due to the physiological differences between the nonhuman primate and human. The objective of this study was to develop new transfer rate parameters for wound and systemic models in an effort to improve biokinetic predictions. Estimates of new transfer rates appropriate for nonhuman primate data were estimated by employing a companion software program called Integrated Modules for Bioassay Analysis Uncertainty Analyzer. During validation of the suggested transfer rates, it was observed that the optimized parameters predicted the intake in 66% of the tested animals used in this investigation. The activity retained in the skeleton improved in almost all cases where the differences between predicted and measured activity is less than 20%.

Application of the ICRP 67 and NCRP 156 Biokinetic Models to 241 Am Wound Data from Nonhuman Primates.

Distribution, retention, and excretion of intramuscularly injected Am citrate have been investigated in cynomolgus and rhesus nonhuman primates (NHP). Bioassay and retention data, obtained from experiments done by Patricia Durbin and her colleagues at Lawrence Berkeley National Laboratory, were evaluated against the International Commission on Radiological Protection (ICRP 67) Am systemic model coupled with to the National Council on Radiation Protection and Measurement wound model (NCRP 156). The default transfer rates suggested in these models were used with the urine and feces excretion data to predict the intake as well as liver and skeleton tissue contents at the time of death. The default models adequately predict the animals' urine bioassay data, but the injected activities were overpredicted by as much 4.41 times and underpredicted by as much as 0.99 times. Poor prediction has been observed in all cases using fecal excretion. The retained activity in the liver and skeleton were investigated using the same approach. It appears that the models predict the amount of the activity retention in the skeleton more accurately than in the liver. The fraction of predicted to measured activity at the time of death in the skeleton was over 1.0 in most cases, and accurate predictions were obtained in seven cases. The predicted activity in skeleton for these cases ranged from 2.7 to 17% overestimated activity and from 9 to 14% underestimated activity. NHPs' urine data and organ retention were compared with data from previously modeled baboons and beagle dogs. About 6% of the injected activity in baboons and beagle dog was excreted in urine and approximately 0.1% in feces in the first 24 h. The results from NHP are not different from excreta analysis in these other species. Urinary excretion in the cynomolgus, rhesus, and baboon NHP is the dominant pathway of Am clearance; however, fecal excretion is considered dominant in beagle dogs. The comparison between NHPs and humans is difficult due to the differences in the number of activities translocated or deposited in the liver tissue and nonliver tissues (primarily skeleton), in addition to the physiological differences between the NHPs and humans.

Second-order Kinetics of DTPA and Plutonium in Rat Plasma.

In 2008, Serandour et al. reported on their in vitro experiment involving rat plasma samples obtained after an intravenous intake of plutonium citrate. Different amounts of DTPA were added to the plasma samples and the percentage of low-molecular-weight plutonium measured. Only when the DTPA dosage was three orders of magnitude greater than the recommended 30 µmol/kg was 100% of the plutonium apparently in the form of chelate. These data were modeled assuming three competing chemical reactions with other molecules that bind with plutonium. Here, time-dependent second-order kinetics of these reactions are calculated, intended eventually to become part of a complete biokinetic model of DTPA action on actinides in laboratory animals or humans. The probability distribution of the ratio of stability constants for the reactants was calculated using Markov Chain Monte Carlo. These calculations substantiate that the inclusion of more reactions is needed in order to be in agreement with known stability constants.

Dose assessment for inhalation intakes in complex, energetic environments: experience from the US Capstone study.

Because of the lack of existing information needed to evaluate the risks from inhalation exposures to depleted uranium (DU) aerosols of US soldiers during the 1991 Persian Gulf War, the US Department of Defense funded an experimental study to measure the characteristics of DU aerosols created when Abrams tanks and Bradley fighting vehicles are struck with large-caliber DU penetrators, and a dose and risk assessment for individuals present in such vehicles. This paper describes some of the difficulties experienced in dose assessment modelling of the very complex DU aerosols created in the Capstone studies, e.g. high concentrations, heterogeneous aerosol properties, non-lognormal particle size distributions, triphasic in vitro dissolution and rapid time-varying functions of both DU air concentration and particle size. The approaches used to solve these problems along with example results are presented.

Microscopic dose to lung from inhaled alpha emitters in humans.

Because of the short range of alpha particles in tissue, the degree of uniformity of irradiation of the lung varies greatly depending on the form of the inhaled material. Animal studies have shown that the degree of dose uniformity influences the risk of lung cancer. This study investigates the radiation dose distribution of plutonium in human lung. Numerical maps of tissue configuration and target cell locations are obtained from histological sections of human lung tissue stained to enhance the identification of putative cell types for parenchymal lung cancers, i.e. alveolar type II cells and Clara cells. Monte Carlo simulations are used to obtain dose distribution around individual particles, and these distributions are used to compute dose distribution in volumes of lung tissue. Lung dose is characterised both by the degree of non-uniformity of irradiation and the relative degree of irradiation of all tissue versus the special cells of interest.

Uranium dose assessment: a Bayesian approach to the problem of dietary background.

Workers are routinely monitored by urinalysis for exposure to uranium at Los Alamos National Laboratory. Urine samples are analysed by alpha spectroscopy for 234U, 235U and 238U. Interpretation of the data is complicated by the presence of natural uranium in the workers' drinking water and diet. Earlier methods used drinking water samples to estimate the dietary component in urine excretion. However, there proved to be insufficient correlation between drinking water concentration and excretion rate. Instead, an iterative calculation is used to identify a typical excretion rate for each individual in the absence of occupational intakes. Bayesian dose-assessment methods, first developed for plutonium bioassay at Los Alamos, have been adapted for uranium. These methods, coupled with an algorithm for identifying each individual's baseline level of uranium from environmental sources, yield improved reliability in the identification of occupational intakes.

Technical basis for using nose swab bioassay data for early internal dose assessment.

One of the challenges to the dose assessment team in response to an inhalation incident in the workplace is to provide the occupational physicians, operational radiation protection personnel and line managers with early estimates of radionuclide intakes so that appropriate consequence management and mitigation can be done. For radionuclides such as Pu, where in vivo counting is not adequately sensitive, other techniques such as the measurement of removable radionuclide from the nasal airway passages can be used. At Los Alamos National Laboratory (LANL), nose swabs of the ET1 region have been used routinely as a first response to airborne Pu releases in the workplace, as well as for other radionuclides. This paper presents the results of analysing over 15 years of nose swab data, comparing these with dose assessments performed using the Bayesian methods developed at LANL. The results provide empirical support for using nose swab data for early dose assessments. For Pu, a rule of thumb is a dose factor of 0.8 mSv Bq(-1), assuming a linear relationship between nasal swab activity and committed effective dose equivalent. However, this value is specific to the methods and models used at LANL, and should not be applied directly without considering possible differences in measurement and calculation methods.

The NCRP wound model: development and application.

The US National Council on Radiation Protection and Measurements, in collaboration with the International Commission on Radiological Protection, has been developing a biokinetic and dosimetric model for radionuclide-contaminated wounds. The finalised model is described briefly in this paper, together with the scientific basis and application. The multicompartment model uses first-order linear biokinetics to describe the retention and clearance of a radionuclide deposited in a wound site using seven default retention categories. Examples using plutonium nitrate in colloidal form and uranium in metal fragments show the behaviour of the less soluble forms of radionuclides in wounds, in which long-term retention is predicted. Using uranium as an example, the wound model is coupled to a uranium International Commission on Radiological Protection systemic model to predict urinary excretion patterns for different physicochemical forms of uranium. The latter application is needed for bioassay interpretation.

Use of air monitoring and experimental aerosol data for intake assessment for Mayak plutonium workers.

One of the major uncertainties in reconstructing doses to Mayak Plutonium (Pu) workers is the unknown exposure patterns experienced by individuals. These uncertainties include the amounts of Pu inhaled, the temporal exposure pattern of Pu air concentration, the particle-size distribution and solubility of the inhaled aerosols. To date, little individual and workplace-specific information has been used to assess these parameters for the Mayak workforce. However, extensive workplace-specific alpha activity air monitoring data set has been collated, which, if coupled with individual occupational histories, can potentially provide customised intake scenarios for individual Mayak workers. The most available Pu air concentration data are annual averages, which exist for over 100 defined work stations at radiochemical and chemical-metallurgical manufacturing facilities and basically for the whole period of Mayak production operations. Much sparser but more accurate data on Pu concentrations in workers' breathing zone are available for some major workplaces and occupations. The latter demonstrate that within a working shift, Pu concentrations varied over a range of several orders of magnitude depending on the nature of the operations performed. An approach to use the collated data set for individual intake reconstruction is formulated and its practical application is demonstrated. Initial results of ongoing experimental study on historic particle size at Mayak PA and their implications for intake estimation are presented.

ICRP 67 Biokinetic Models for AM-241 Applied to Nonhuman Primates.

Between 1960 and 1985, Patricia Durbin and colleagues performed studies on the distribution of intravenously and intramuscularly injected Am citrate with dosages ranging from 16 to 32 kBq kg in 30 male and female non-human primates (NHP). Dr. Durbin died unexpectedly in March of 2009, leaving much of the extensive serial blood, bioassay, and autopsy data from these NHP studies unanalyzed. As part of the experimental design, serial blood samples were taken, and urine and feces samples were collected separately for the duration of the study. The measurements of urine, fecal excretion, blood samples, and organ burden data obtained from the animals were used to evaluate the transfer rates of the ICRP 67 biokinetic model for Am. Seven cases, in which the primates were administered Am citrate by intravenous injection, were evaluated using the ICRP 67 systemic model. There were differences ranging from 51.4% underestimated to 102.7% overestimated activity between the predicted intake, which was calculated using IMBA Professional Plus software and based upon the urine bioassay data and the actual activity. The difference between the predicted activity at the time of death in the liver and skeleton using IMBA professional software and the value of the measured activity at the time of death were also compared. Generally, the ratios of predicted activity in the liver and skeleton at the time of death to the measured activity were consistently more than 1. However, the ratios were less than 1 in the skeleton for animals that were sacrificed 2,199 and 973 d post injection. The posterior probability distributions for model parameters derived using WeLMoS method were inconsistent with the ICRP 67 default parameters. The prediction made based on the posterior probability distributions for model parameters derived using WeLMoS gave the best fit to these data; however, the modified parameters overestimated the activity in almost all cases. The difference between the predicted Am activity and the value of the measured activity may be due to the physiological age-related characteristics relative to the age of the animal at the time of the injection and early and long scarified time.

Application of NCRP 156 Wound Models for the Analysis of Bioassay Data from Plutonium Wound Cases.

The NCRP 156 wound model was heavily based on data from animal experiments. The authors of the report acknowledged this limitation and encouraged validation of the models using data from human wound exposures. The objective of this paper was to apply the NCRP 156 wound models to the bioassay data from four plutonium-contaminated wound cases reported in the literature. Because a wide variety of forms of plutonium can be expected at a nuclear facility, a combination of the wound models-rather than a single model-was used to successfully explain both the urinary excretion data and wound retention data in three cases. The data for the fourth case could not be explained by any combination of the default wound models. While this may possibly be attributed to the existence of a category of plutonium whose solubility and chemistry are different than those described by the NCRP 156 default categories, the differences may also be the result of differences in systemic biokinetics. The concept of using a combination of biokinetic models may be extended to inhalation exposures as well, where more than one form of radionuclide-particles of different solubility or different sizes-may exist in a workplace.

Behavior of Americium in Simulated Wounds in Nonhuman Primates.

An americium solution injected intramuscularly into several nonhuman primates (NHPs) was found to behave differently than predicted by the wound models described in the NCRP Report 156. This was because the injection was made along with a citrate solution, which is known to be more soluble than chlorides, oxides, or nitrates on which the NCRP Report was based. A multi-exponential wound model specific to the injected americium solution was developed based on the retention in the intramuscular sites. The model was coupled with the americium systemic model to interpret the urinary excretion data and assess the intake, and it was determined that the models were adequate to predict early urinary excretion in most cases but unable to predict late urinary excretion. This was attributed to the differences in the systemic handling of americium between humans and nonhuman primates. Information on the type of wounds, solubility, particle size, mass, chemical form, etc., should always be considered when performing wound dosimetry.

Plasma Retention and Systemic Kinetics of 90Sr Intramuscularly Injected in Female Nonhuman Primates.

Thirteen female Rhesus macaques were intramuscularly injected with Sr(NO3)2 diluted in sodium citrate solution. The biokinetic data from these animals were compared against the predictions of the NCRP 156 wound models combined with the ICRP systemic models. It was observed that the activities measured in plasma of these nonhuman primates (NHPs) were consistently lower than those predicted by the default human biokinetic models. The urinary excretion from the NHPs at times immediately after injection was much greater than that in humans. The fecal excretion rates were found to be in relatively better agreement with humans. Similarly, the activities retained in the skeleton of the NHPs were lower than those in humans. These differences were attributed to the higher calcium diet of the NHPs (0.03 to 0.12 g d kg body weight) compared to that of humans. These observations were consistent with the early animal and human studies that showed the effect of calcium on strontium metabolism, specifically urinary excretion. Strontium is preferentially filtered at a much higher rate in kidneys than calcium because it is less completely bound to protein than is calcium. These differences, along with large inter-animal variability, should be considered when estimating the behavior of strontium in humans from the metabolic data in animals or vice versa.

SPECIFIC BLOOD ABSORPTION PARAMETERS FOR 239PUO2 AND 238PUO2 NANOPARTICLES AND IMPACTS ON BIOASSAY INTERPRETATION.

Specific absorption parameters for 239PuO2 and 238PuO2 have been determined based on available biokinetic data from studies in rodents, and the impacts of these parameters on bioassay interpretation and dosimetry after inhalation of nanoPuO2 materials have been evaluated. Calculations of activities after an acute intake of nanoparticles of 239PuO2 and 238PuO2 are compared with the corresponding calculations using standard default absorption parameters using the International Commission on Radiological Protection (ICRP) 66 respiratory tract model. Committed effective doses are also evaluated and compared. In this case, it was found that interpretation of bioassay measurements with the assumption that the biokinetic behaviour of PuO2 nanoparticles is the same as that of micrometre-sized particles can result in an overprediction of the committed effective dose by two orders of magnitude. Although in this case the use of the default assumptions (5 µm AMAD, Type S) for assessing dose following inhalation exposure to airborne PuO2 nanoparticles appears to be conservative, the evaluation of situations involving PuO2 nanoparticles that may have different particle size and solubility properties should prudently follow the ICRP recommendation to obtain and use additional, material-specific information whenever possible.