Graphite Furnace Atomic Absorption Spectrometric Evaluation of Iron Excretion in Mouse Urine Caused by Whole-Body Gamma Irradiation.

A procedure for the determination of iron in mice urine using graphite furnace atomic absorption spectrometry was developed. The mice urinary samples contain many organic compounds in the matrix, whose concentrations are approximately 20%, and the value is 30-fold higher compared to those found in human urine. Moreover, only 0.2 mL or less of urine was obtained as a sample volume per urination event. It was difficult to decompose the organic materials in the samples by wet digestion using mineral acids and oxidising agents, because of the tiny volumes. In this experiment, raw urinary samples were placed directly into the graphite tube furnace for analysis. The organic contents were simply ashed during the preheating stages. To facilitate ashing in the furnace, air was invaded from the surroundings by interrupting the stream of argon gas. Atomic absorption was measured at 248.3270 nm (wavelength for atomic absorption), with the background monitored at 247.0658 nm (wavelength for background correction). The optimised instrument operating conditions precluded the use of chemical modification technique. The analytical procedures used are quite simple, i.e. an aliquot of raw urine sample was injected directly into the graphite tube furnace and was followed by a suitable heating programme with no chemical modifier. Therefore, this method is useful for scientists who are not familiar with delicate chemical experiments. The proposed analytical method was applied as a kind of biomarker by determining iron concentrations in urinary samples of mice, which were irradiated with 4 Gy of gamma irradiation to their whole body. The time dependence of the iron concentration was determined, and the iron concentrations increased within 1 day of irradiation exposure, then decreased to ordinal values after several days.

Urinary miRNAs as Biomarkers for Noninvasive Evaluation of Radiation-Induced Renal Tubular Injury.

Radiation nephropathy is one of the common late effects in cancer survivors who received radiotherapy as well as in victims of radiation accidents. The clinical manifestations of radiation nephropathy occur months after exposure. To date, there are no known early biomarkers to predict the future development of radiation nephropathy. This study focuses on the development of urinary biomarkers providing readout of acute responses in renal tubular epithelial cells. An amplification-free hybridization-based nCounter assay was used to detect changes in mouse urinary miRNAs after irradiation. After a single LD50 of total-body irradiation (TBI) or clinically relevant fractionated doses (2 Gy twice daily for 3 days), changes in urinary levels of microRNAs followed either an early pattern, peaking at 6-8 h postirradiation and gradually declining, or later pattern, peaking from 24 h to 7 days. Of 600 miRNAs compared, 12 urinary miRNAs showed the acute response and seven showed the late response, common to both irradiation protocols. miR-1224 and miR-21 were of particular interest, since they were the most robust acute and late responders, respectively. The early responding miR-1224 also exhibited good dose response after 2, 4, 6 and 8 Gy TBI, indicating its potential use as a biomarker for radiation exposure. In situ hybridization of irradiated mouse kidney sections and cultured mouse primary renal tubular cells confirmed the tubular origin of miR-1224. A significant upregulation in hsa-miR-1224-3p expression was also observed in human proximal renal tubular cells after irradiation. Consistent with mouse urine data, a similar expression pattern of hsa-miR-1224-3p and hsa-miR-21 were observed in urine samples collected from human leukemia patients preconditioned with TBI. This proof-of-concept study shows the potential translational utility of urinary miRNA biomarkers for radiation damage in renal tubules with possible prediction of late effects.

Quantitative Metabolomic Analysis of Urinary Citrulline and Calcitroic Acid in Mice after Exposure to Various Types of Ionizing Radiation.

With the safety of existing nuclear power plants being brought into question after the Fukushima disaster and the increased level of concern over terrorism-sponsored use of improvised nuclear devices, it is more crucial to develop well-defined radiation injury markers in easily accessible biofluids to help emergency-responders with injury assessment during patient triage. Here, we focused on utilizing ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to identify and quantitate the unique changes in the urinary excretion of two metabolite markers, calcitroic acid and citrulline, in mice induced by different forms of irradiation; external γ irradiation at a low dose rate (LDR) of 3.0 mGy/min and a high dose rate (HDR) of 1.1 Gy/min, and internal exposure to Cesium-137 ((137)Cs) and Strontium-90 ((90)Sr). The multiple reaction monitoring analysis showed that, while exposure to (137)Cs and (90)Sr induced a statistically significant and persistent decrease, similar doses of external γ beam at the HDR had the opposite effect, and the LDR had no effect on the urinary levels of these two metabolites. This suggests that the source of exposure and the dose rate strongly modulate the in vivo metabolomic injury responses, which may have utility in clinical biodosimetry assays for the assessment of exposure in an affected population. This study complements our previous investigations into the metabolomic profile of urine from mice internally exposed to (90)Sr and (137)Cs and to external γ beam radiation.

[Effect of radiation-induced bystander chemosignals of mice on the humoral immune response in spleen and lymph nodes of intact recipients].

The ability of post-radiation (4 Gy) bystander chemosignals (the volatile components of mouse urine) to distantly modulate the humoral immune response to the sheep red blood cells in the spleen and popliteal lymph nodes of intact recipients has been investigated. It was shown that the exposure of animals to chemosignals before antigen injection resulted in the decrease and increase of the immune response in the spleen and lymph nodes, respectively. When animals were exposed to chemosignals after the antigenic stimulus, an increased immune response was observed in both spleen and lymph nodes. The contribution of radiation-induced bystander signaling in the response of socially organized animals to the effect of ionizing irradiation is discussed.

The metabolomic approach identifies a biological signature of low-dose chronic exposure to cesium 137.

Reports have described apparent biological effects of (137)Cs (the most persistent dispersed radionuclide) irradiation in people living in Chernobyl-contaminated territory. The sensitive analytical technology described here should now help assess the relation of this contamination to the observed effects. A rat model chronically exposed to (137)Cs through drinking water was developed to identify biomarkers of radiation-induced metabolic disorders, and the biological impact was evaluated by a metabolomic approach that allowed us to detect several hundred metabolites in biofluids and assess their association with disease states. After collection of plasma and urine from contaminated and non-contaminated rats at the end of the 9-months contamination period, analysis with a LC-MS system detected 742 features in urine and 1309 in plasma. Biostatistical discriminant analysis extracted a subset of 26 metabolite signals (2 urinary, 4 plasma non-polar, and 19 plasma polar metabolites) that in combination were able to predict from 68 up to 94% of the contaminated rats, depending on the prediction method used, with a misclassification rate as low as 5.3%. The difference in this metabolic score between the contaminated and non-contaminated rats was highly significant (P = 0.019 after ANOVA cross-validation). In conclusion, our proof-of-principle study demonstrated for the first time the usefulness of a metabolomic approach for addressing biological effects of chronic low-dose contamination. We can conclude that a metabolomic signature discriminated (137)Cs-contaminated from control animals in our model. Further validation is nevertheless required together with full annotation of the metabolic indicators.

[Experimental study of the effect of detoxification with enterosorbents on the function of irradiated myocardium].

Increase in intoxication products, such as medium size peptides, indole and myoglobin, in urine was observed in Wistar rats after exposure of their chest to gamma-radiation at a dose of 6 Gy (dose rate 4 Gy/min). The rats exhibited moderate ischemic ECG. Administration of enterosorbents, such as Smekta and Clay of Kaluga deposit, to the irradiated rats resulted in the decrease of the toxicant content in the animals and the recovery of the cardiac function on the 28th day. These sorbents had practically a similar efficacy.

Simplified structure of a new model to describe urinary excretion of plutonium after systemic, liver or pulmonary contamination of rats associated with Ca-DTPA treatments.

This study validates, by targeted experiments, several modeling hypotheses for interpretation of urinary excretion of plutonium after Ca-DTPA treatments. Different formulations and doses of Ca-DTPA were administered to rats before or after systemic, liver or lung contamination with various chemical forms of plutonium. The biokinetics of plutonium was also characterized after i.v. injection of Pu-DTPA. Once formed, Pu-DTPA complexes are stable in most biological environments. Pu-DTPA present in circulating fluids is rapidly excreted in the urine, but 2-3% is retained, mainly in soft tissues, and is then excreted slowly in the urine after transfer to blood. Potentially, all intracellular monoatomic forms of plutonium could be decorporated after DTPA internalization involving slow urinary excretion of Pu-DTPA with half-lives varying from 2.5 to 6 days as a function of tissue retention. The ratio of fast to slow urinary excretion of Pu-DTPA depends on both plutonium contamination and Ca-DTPA treatment. Fast urinary excretion of Pu-DTPA corresponds to extracellular decorporation that occurs beyond a threshold of the free DTPA concentration in circulating fluids. Slow excretion corresponds mostly to intracellular decorporation and depends on the amount of intracellular DTPA. From these results, the structure of a simplified model is proposed for interpretation of data obtained with Ca-DTPA treatments after systemic, wound or pulmonary contamination by plutonium.

[Postirradiated mice volatile secretion, irradiated in the lethal and superlethal dose].

In the early period after exposure to the radiation in the lethal dose (10 Gy) mice secreted with urine the volatile components, that attract for intact individuals. In the late period appear components with aversive property. In the terminal period after exposure to superlethal dose (70 Gy) dominate the secretions with aversive property. The exposure of intact recipients with the volatile secretions attractive or aversive in equal degree depression result in suppression their ability to the humoral immune response.

[The influence of postradiation chemical signals of mice on the humoral immune response in recipients in different time relative to antigenic stimulus].

The influence of volatile urine chemosignals of irradiated (4 Gy) mice on the primary humoral immune response to sheep red blood cells in intact recipients was investigated. It was demonstrated that the direction of immunomodulatory effect is dependent upon the time at which the postradiation chemosignals was initially applied. The antibody response to antigen was markedly suppressed in mice that were exposed before antigen injection. When chemosignals applied immediately following inoculation of antigen the antibody response was unaffected. The immune response was increased when chemosignals was loadeded for 1-10 days after immunization. The possible mechanisms of immunomodulation are considered.

[Immunomodulation effects volatile secretion of animals at postradiation immunodeficiency conditions].

Was shown, that given off with urine the volatile chemosignals of intact and irradiated (4 Gy) mice and rats restored the ability to humoral immune response and other parameters immunity lowered by ionizing radiation in the dose 1 Gy. The chemosignals off intact individuals have shown higher immunerestoring activity in comparison with the irradiated ones. As opposed to post-radiation signals they activated in the major degree at irradiational mice phagocytic activity peritoneal macrophages and also the number in the blood at rates not only erythrocytes but lymphocytes and granulocytes. It is supposed, that mammals possess the distant immunomodulating chemosignal system, direct on immunoreactivity of individuals with immunodeficiency of state.

[Allelopathic activity of volatile secretions in irradiated animals]. / Allelopaticheskaia aktivnost' letuchikh vydelenii obluchennykh zhivotnykh.

In the period of the most expressed postirradiation (4 Gy) injury, the rats secreted volatile components capable of lowering the blood cells count in intact rats. This ability of the irradiated animals is not strictly specific for species since the depression effect on rats is exerted also by volatile components of irradiated mice. We suggest marking this phenomenon as postirradiation allelopathy.

[Natural excretions of mice in the postradiation period and contact induction of immunodeficiencies]. / Estestvennye vydeleniia myshei v postradiatsionnom periode i kontaktnaia induktsiia immunodefitsitov.

It was showed that the daily transfer of natural secrets litter from irradiated mice to intact mice decreased parameters of intact mice immune reactivity in 14 days. Presumedly this effect, conditioned of pathological odours, discovered on one from mechanism of contact induction second immunodeficient, also caged intact with irradiated animals.

In-vivo measurements of early and late effects in normal tissue after abdominal X-irradiation--development of a clinically orientated score for the determination of side-effects and the individual radiation response in a mouse model.

Heiligenberger mice received abdominal single X-ray doses of either 10 or 12 Gy. Early (up to one month) and late effects (two to eight months) were measured in vivo with ten parameters (nitrite, albumin, glucose, urobilinogen, bilirubin, haemoglobine, ketone, pH, weight development, general clinical conditions). These parameters permit the assessment of the condition of the essential organ systems. Thus, their use as screening parameters appears meaningful. By creating indices and integrating these in the animal index (range 1 to 3) it is possible to obtain an impression of the health condition of the individual animal. Early and late damage can be determined with certainty despite large individual variances. Radiation-induced chronic but not lethal changes can be measured. A large early damage is also predictive for a large late morbidity; not the individual parameters allow conclusions to be drawn but instead only the total number of the parameters used here are capable of this. The results have tended to show that the systematic listing of early responses also allows for very early indication of late morbidity or chronification events to be expected. The results have shown that the individual analysis of single parameters can give rise to important indications for a decisive diagnosis. This corresponds to the usual clinical procedure and can be applied to animal experiments without restriction.

[Evaluation and correction of the vitamin C status in relation to the problem of the experimental modification of long-term radiation effects]. / Otsenka i korrektsiia C-vitaminnogo statusa v probleme éksperimental'noi modifikatsii otdalennykh posledstvii radiatsionnykh vozdeistvii.

A lognormal double-peak pattern of ascorbic acid content distribution within rat daily urine and the presence in the population of vitamin C hypo-, hyper-, and normally producing individuals have been established. Radiation-induced changes in the C-vitamin status of rats during their lifetime were assessed by average geometrical levels of acid excretion. A 1.5-3-fold decrease in providing vitamin C producing animals with vitamin C was demonstrated a year after intrapulmonary incorporation of 239PuO2.

Urinary excretion of iohexol after enteral administration in rats with radiation injury of the small intestine.

Gut membrane dysfunction after acute and subacute irradiation injury to 10-cm-long small-bowel segments was assessed in 85 rats receiving doses of 17 and 21 Gy. Four and 14 days after irradiation 2 ml iohexol was administered via orogastric tubes, and hourly radiographs were taken. After 8 h, blood and urine were sampled for testing, and the intestine biopsied for light and scanning electron microscopy. Dense opacification of the urinary bladder was seen on abdominal films, and increased serum and urinary levels of iodine were demonstrated by X-ray fluorescence analysis in irradiated animals. Urinary levels were up to 20 times higher than in controls 4 days after irradiation, subsiding after 14 days. The effects were prolonged in the 21-Gy group. Our results indicate that measurement of iodine levels in serum and urine may be helpful in assessment of bowel injury in the course of irradiation treatment to pelvic or abdominal organs.

[The effect of local x-irradiation on the excretion of oxyproline]. / Izuchenie vliianiia lokal'nogo rentgenovskogo oblucheniia na ékskretsiiu oksiprolina.

The dependence of excretion of oxyproline in urine on dosage and damaged area was estimated after local exposure of rat skin to low-energy X-radiation. The pronounced hyperoxyprolineuria was observed throughout the periods of latency and formation of ulceronecrotic skin defects.

[The diagnostic value of the status of urinary taurine in the early stages following the irradiation of animals]. / Izuchenie diagnosticheskoi tsennosti sostoianiia taurinurii v rannye sroki posle oblucheniia zhivotnykh.

A study of the features of taurine excretion early after irradiation of animals in comparison with the peripheral blood level of leukocytes is necessary for the development of methods of early diagnosis and prediction of an outcome of radiation disease. The paper is concerned with a study of the correlation between a dose of ionizing radiation and enhanced taurine excretion with urine in rats and dogs as compared to the time course of the blood level of leukocytes, an indicator used for the diagnosis of radiation disease. The doses were 2-10 Gy for rats and 5-15 Gy for dogs. Intensive taurine excretion was shown to be an early (recorded in the first hours after radiation exposure), stable and dose-dependent body reaction. The authors discussed the problem of the appropriateness of the use of this test for the diagnosis and prognosis of an outcome of acute radiation disease in combination with common indices.

Urinary excretion of cyclic nucleotides, creatinine prostaglandin E2 and thromboxane B2 from mice exposed to whole-body irradiation from an enhanced neutron field.

Urine volume and excretion of cyclic AMP, cyclic GMP, prostaglandin E2 (PGE2), thromboxane B2 (TxB2) and creatinine were evaluated as potential indicators of radiation damage in mice given 2-5 Gy to the whole body from an enhanced neutron field. In general, urinary cyclic AMP, cyclic GMP, creatinine and urine volumes were positively correlated across time postexposure, for each radiation dose. TxB2 levels positively correlated with urine volume and cyclic AMP excretion only in animals given 2.0 Gy. None of these parameters suggests their use as a prognostic indicator of the extent of radiation damage. Urinary excretion of PGE2 was negatively correlated with other urinary parameters. Biphasic increases in urinary PGE2 were also observed. The initial transient elevation 2-3 days postexposure was not correlated with the dose (2-5 Gy). The second elevation of PGE2 excretion occurred at 6-10 days. The magnitude of the latter increase suggests that urinary PGE2 excretion may be a useful indicator of whole-body or kidney exposure to neutron fields.

Evaluation of two compounds as possible radioprotectors.

Radioprotecting effectiveness of S-[2-(diethylamino)ethyl] 4-methylbenzothiohydroximate hydrochloride (Dev-B-9) and S-[2-(diethylamino)ethyl]alpha-keto 4-methyl benzothiohydroximate hydrochloride (Dev-B 43) were evaluated by both survival studies in mice exposed to 10.5 Gy whole body gamma-irradiation and 24 hours deoxycytidine (CdR) excretion in urine of rats following 5 Gy irradiation. The drugs were tested after intraperitoneal (i.p.) administration. The drugs rendered significant protection in sublethal (5 Gy) irradiation but could not protect against 10.5 Gy exposure.

The influence of overall treatment time on renal injury after multifraction irradiation.

The influence of overall treatment time on the radiation response of the mouse kidney was studied in an experiment in which 16 fractions were administered either evenly distributed over 20, 40 or 80 days, or as a split course (8 F/3 days; 74 days rest; 8 F/3 days). Urine output and an isotope assay of glomerular filtration were used to test the mice sequentially. The data were used both to obtain dose-response curves and also to determine the latent period before a chosen level of injury was expressed functionally. Prolonging the overall time from 20 to 80 days increased the isoeffect dose by 2-5 Gy (4-9%) for the isotope assay, and by 4-9 Gy (7-18%) for the urine output assay. This additional recovery as the interval between fractions was prolonged from 1 to 5 days is consistent with slow repair and can be expressed as a small "T" exponent of 0.02-0.12. (One analysis gave a result consistent with negative repair, but the errors on this result were unusually wide.) When the radiation was given as a split course, at the rate of 2 fractions per day, with a large gap of 10.5 weeks between courses, there was no additional sparing compared with 16 fractions over 20 days. This indicates that any sparing that might have resulted from slow repair or stimulated repopulation in the gap has been counterbalanced by having less time for repair of sublethal injury when intervals of 6-12 h are used instead of 24-48 h. Clearly no great increase in the tolerance dose for mouse kidney resulted from the split course.