Ratio of γ-H2AX level in lymphocytes to that in granulocytes detected using flow cytometry as a potential biodosimeter for radiation exposure

This study aims to assess utilisation of the ratio of γ-H2AX in lymphocytes to that in granulocytes (RL/G of γ-H2AX) in blood as a rapid method for population triage and dose estimation during large-scale radiation emergencies. Blood samples from healthy volunteers exposed to 0–10 Gy of ⁶⁰Co irradiation were collected. The samples were cultured for 0–24 h and then analysed using flow cytometry to measure the levels of γ-H2AX in lymphocytes and granulocytes. The basal RL/G levels of γ-H2AX in healthy human blood, the response of RL/G of γ-H2AX to ionising radiation and its relationship with doses, time intervals after exposure and individual differences were also analysed. The level of γ-H2AX in lymphocytes increased in a dose-dependent manner after irradiation, whereas the level in granulocytes was not affected. A linear dose–effect relationship with low inter-experimental and inter-individual variations was observed. The RL/G of γ-H2AX may be used as a biomarker for population triage and dose estimation during large-scale radiation emergencies if blood samples can be collected within 24 h.     

Comprehensive optimization of LC–MS metabolomics methods using design of experiments (COLMeD)

Introduction

Both reverse-phase and HILIC chemistries are deployed for liquid-chromatography mass spectrometry (LC–MS) metabolomics analyses, however HILIC methods lag behind reverse-phase methods in reproducibility and versatility. Comprehensive metabolomics analysis is additionally complicated by the physiochemical diversity of metabolites and array of tunable analytical parameters.

Objective

Our aim was to rationally and efficiently design complementary HILIC-based polar metabolomics methods on multiple instruments using design of experiments (DoE).

Methods

We iteratively tuned LC and MS conditions on ion-switching triple quadrupole (QqQ) and quadrupole-time-of-flight (qTOF) mass spectrometers through multiple rounds of a workflow we term Comprehensive optimization of LC–MS metabolomics methods using design of experiments (COLMeD). Multivariate statistical analysis guided our decision process in the method optimizations.

Results

LC–MS/MS tuning for the QqQ method on serum metabolites yielded a median response increase of 161.5 % (p < 0.0001) over initial conditions with a 13.3 % increase in metabolite coverage. The COLMeD output was benchmarked against two widely used polar metabolomics methods, demonstrating total ion current increases of 105.8 and 57.3 %, with median metabolite response increases of 106.1 and 10.3 % (p < 0.0001 and p < 0.05 respectively). For our optimized qTOF method, 22 solvent systems were compared on a standard mix of physiochemically diverse metabolites, followed by COLMeD optimization, yielding a median 29.8 % response increase (p < 0.0001) over initial conditions.

Conclusions

The COLMeD process elucidated response tradeoffs, facilitating improved chromatography and MS response without compromising separation of isobars. COLMeD is efficient, requiring no more than 20 injections in a given DoE round, and flexible, capable of class-specific optimization as demonstrated through acylcarnitine optimization within the QqQ method.

     

Serum metabolomic markers for traumatic brain injury: a mouse model

Introduction

Traumatic brain injury (TBI) is physical injury to brain tissue that temporarily or permanently impairs brain function.

Objectives

Evaluate the use of metabolomics for the development of biomarkers of TBI for the diagnosis and timing of injury onset.

Methods

A validated model of closed injury TBI was employed using 10 TBI mice and 8 sham operated controls. Quantitative LC–MS/MS metabolomic analysis was performed on the serum.

Results

Thirty-six (24.0 %) of 150 metabolites were altered with TBI. Principal component analysis (PCA) and Partial least squares discriminant analysis (PLS-DA) analyses revealed clear segregation between TBI versus control sera. The combination of methionine sulfoxide and the lipid PC aa C34:4 accurately diagnosed TBI, AUC (95 % CI) 0.85 (0.644–1.0). A combination of metabolite markers were highly accurate in distinguishing early (4 h post TBI) from late (24 h) TBI: AUC (95 % CI) 1.0 (1.0–1.0). Spermidine, which is known to have an antioxidant effect and which is known to be metabolically disrupted in TBI, was the most discriminating biomarker based on the variable importance ranking in projection (VIP) plot. Several important metabolic pathways were found to be disrupted including: pathways for arginine, proline, glutathione, cysteine, and sphingolipid metabolism.

Conclusion

Using serum metabolomic analysis we were able to identify novel putative serum biomarkers of TBI. They were accurate for detecting and determining the timing of TBI. In addition, pathway analysis provided important insights into the biochemical mechanisms of brain injury. Potential clinical implications for diagnosis, timing, and monitoring brain injury are discussed.

     

Metabolite profiling of small cerebrospinal fluid sample volumes with gas chromatography–mass spectrometry: application to a rat model of multiple sclerosis

Analysis of metabolites in biofluids by gas chromatography–mass spectrometry (GC–MS) after oximation and silylation is a key method in metabolomics. The GC–MS method was modified by a modified vial design and sample work-up procedure in order to make the method applicable to small volumes of cerebrospinal fluid (CSF), i.e. 10 μL, with similar coverage compared to the standard procedure using ≥100 μL of CSF. The data quality of the modified GC–MS method was assessed by analyzing a study sample set in an animal model for multiple sclerosis, including repetitively analysed quality control rat CSF samples. Automated normalization and intra- and inter-batch correction significantly improved the data quality with the majority of metabolites showing a relative standard deviation <20 %. The modified GC–MS method was successfully applied in rat model of multiple sclerosis where statistical analysis of 93 metabolites, of which 73 were (tentatively) identified, in 10 μL of rat CSF showed statistically significant differences in metabolite profiles of rats at the onset and peak of experimental autoimmune encephalomyelitis compared to rats in the control group. The modified GC–MS method presented proved to be a valid and valuable metabolomics method when only limited sample volumes are available.     

Ischemic injury of the liver in a porcine model of cardiac death assessed by in vivo microdialysis

This study aims to evaluate the ischemic injury of the liver in a porcine model of cardiac death assessed by in vivo microdialysis. A porcine model of cardiac death was established by the suffocation method. Metabolic indicators were monitored using the microdialysis technique during warm ischemia time (WIT) and cold ischemia time (CIT). Pathological changes in ischemic-injured livers were observed by haematoxylin–eosin staining. The predictive values of biochemical parameters regarding the liver donor were evaluated by receiver operating characteristic curve analysis. All statistical analyses were conducted using the SPSS 18.0 software (SPSS Inc, Chicago, Illinois, USA). The degree of warm ischemic injury of the livers increased with prolonged WIT. Serum glucose, glycerol, pyruvate, lactic acid levels and lactate-to-pyruvate (L/P) ratio increased gradually during WIT. Results from Pearson correlation analyses indicated that serum lactate level and L/P ratio were positively associated with the degree of warm ischemic injury of the livers. The degree of cold ischemic injury of the livers gradually increased after 12 h CIT. Serum glucose, lactic acid and L/P ratio achieved a peak after 6–8 h of CIT, but gradually decreased with prolonged CIT. The peak of glycerol occurred after 8 h of CIT, while no changes were found with prolonged CIT. Serum pyruvate level exhibited an increasing trend after 12 h CIT. Our results confirmed that serum glucose and lactate levels were negatively correlated with cold ischemic injury of the liver. However, serum glycerol and pyruvate levels showed positive correlations with cold ischemic injury of the liver. The liver donor was unavailable after 30 min WIT and 24 h CIT. The cut-off value of serum lactate level for warm ischemic injury of the livers was 2.374 with a sensitivity (Sen) of 90 % and specificity (Spe) of 95 %; while the L/P radio was 0.026 (Sen = 80 %, Spe = 83 %). In addition, the cut-off values of serum glucose, lactate, glycerol and pyruvate levels for cold ischemic injury of the livers were 0.339 (Sen = 100 %, Spe = 77 %), 1.172 (Sen = 100 %, Spe = 61 %), 56.359 (Sen = 100 %, Spe = 65 %) and 0.020 (Sen = 100 %, Spe = 67 %), respectively. Our findings provide empirical evidences that serum glucose, lactate levels and L/P ratio may be good indicators for the degree of warm ischemic injury of the livers after cardiac death; while serum glucose, lactate, glycerol and pyruvate levels may be important in predicting cold ischemic injury.     

A lipidomic and metabolomic serum signature from nonhuman primates exposed to ionizing radiation

Introduction

Due to dangers associated with potential accidents from nuclear energy and terrorist threats, there is a need for high-throughput biodosimetry to rapidly assess individual doses of radiation exposure. Lipidomics and metabolomics are becoming common tools for determining global signatures after disease or other physical insult and provide a “snapshot” of potential cellular damage.

Objectives

The current study assesses changes in the nonhuman primate (NHP) serum lipidome and metabolome 7 days following exposure to ionizing radiation (IR).

Methods

Serum sample lipids and metabolites were extracted using a biphasic liquid–liquid extraction and analyzed by ultra performance liquid chromatography quadrupole time-of-flight mass spectrometry. Global radiation signatures were acquired in data-independent mode.

Results

Radiation exposure caused significant perturbations in lipid metabolism, affecting all major lipid species, including free fatty acids, glycerolipids, glycerophospholipids and esterified sterols. In particular, we observed a significant increase in the levels of polyunsaturated fatty acids (PUFA)-containing lipids in the serum of NHPs exposed to 10 Gy radiation, suggesting a primary role played by PUFAs in the physiological response to IR. Metabolomics profiling indicated an increase in the levels of amino acids, carnitine, and purine metabolites in the serum of NHPs exposed to 10 Gy radiation, suggesting perturbations to protein digestion/absorption, biological oxidations, and fatty acid β-oxidation.

Conclusions

This is the first report to determine changes in the global NHP serum lipidome and metabolome following radiation exposure and provides information for developing metabolomic biomarker panels in human-based biodosimetry.

     

Temporal dynamics of ROS biogenesis under simulated solar radiation in the cyanobacterium Anabaena variabilis PCC 7937

We studied the temporal generation of reactive oxygen species (ROS) in the cyanobacterium Anabaena variabilis PCC 7937 under simulated solar radiation using WG 280, WG 295, WG 305, WG 320, WG 335, WG 345, and GG 400 nm cut-off filters to find out the minimum exposure time and most effective region of the solar spectrum inducing highest level of ROS. There was no significant generation of ROS in all treatments in comparison to the samples kept in the dark during the first 8 h of exposure; however, after 12 h of exposure, ROS were significantly generated in samples covered with 305, 295, or 280 nm cut-off filters. In contrast with ROS, the fragmentation of filaments was predominantly seen in 280 nm cut-off filter covered samples after 12 h of exposure. After 24 h of exposure, ROS levels were significantly higher in all samples than in the dark; however, the ROS signals were more pronounced in 320, 305, 295, or 280 nm cut-off filter covered samples. In contrast, the length of filaments was reduced in 305, 295, or 280 nm cut-off filter covered samples after 24 h of exposure. Thus, fragmentation of the filament was induced by all wavelengths of the UV-B region contrary to the UV-A region where only shorter wavelengths were able to induce the fragmentation. In contrast, ROS were generated by all wavelengths of the solar spectrum after 24 h of exposure; however, shorter wavelengths of both the UV-A and the UV-B regions were more effective in generating ROS in comparison to their higher wavelengths and photosynthetic active radiation (PAR). Moreover, lower wavelengths of UV-B were more efficient than the lower wavelengths of the UV-A radiation. Findings from this study suggest that certain threshold levels of ROS are required to induce the fragmentation of filaments.     

Metabolomics reveals dose effects of low-dose chronic exposure to uranium in rats: identification of candidate biomarkers in urine samples

Introduction

Data are sparse about the potential health risks of chronic low-dose contamination of humans by uranium (natural or anthropogenic) in drinking water. Previous studies report some molecular imbalances but no clinical signs due to uranium intake.

Objectives

In a proof-of-principle study, we reported that metabolomics is an appropriate method for addressing this chronic low-dose exposure in a rat model (uranium dose: 40 mg L^?1; duration: 9 months, n = 10). In the present study, our aim was to investigate the dose–effect pattern and identify additional potential biomarkers in urine samples.

Methods

Compared to our previous protocol, we doubled the number of rats per group (n = 20), added additional sampling time points (3 and 6 months) and included several lower doses of natural uranium (doses used: 40, 1.5, 0.15 and 0.015 mg L^?1). LC–MS metabolomics was performed on urine samples and statistical analyses were made with SIMCA-P+ and R packages.

Results

The data confirmed our previous results and showed that discrimination was both dose and time related. Uranium exposure was revealed in rats contaminated for 9 months at a dose as low as 0.15 mg L^?1. Eleven features, including the confidently identified N1-methylnicotinamide, N1-methyl-2-pyridone-5-carboxamide and 4-hydroxyphenylacetylglycine, discriminated control from contaminated rats with a specificity and a sensitivity ranging from 83 to 96 %, when combined into a composite score.

Conclusion

These findings show promise for the elucidation of underlying radiotoxicologic mechanisms and the design of a diagnostic test to assess exposure in urine, in a dose range experimentally estimated to be above a threshold between 0.015 and 0.15 mg L^?1.

     

Effect of UV-B radiation on the synthesis of UV-absorbing compounds in a terrestrial cyanobacterium, Nostoc flagelliforme

Effects of two intensities (1 and 5 W?m^?2) of UV-B radiation on the synthesis of UV-absorbing compounds in a terrestrial cyanobacterium Nostoc flagelliforme were investigated. UV-B radiation resulted in lower biomass. Short period (less than 12 h) of UV-B radiation caused an increase of chlorophyll a content, but subsequent duration of treatment (more than 24 h) resulted in a rapid decrease. N. flagelliforme synthesized UV-absorbing compounds such as scytonemin and mycosporine-like amino acids (MAAs) in response to UV-B radiation. Upon 48 h of exposure to UV-B radiation, scytonemin content in cells increased by 103.8 and 164.0 % at 1 and 5 W?m^?2, respectively. Oligosaccharide-linked mycosporine-like amino acids increased by 145.5 % after 12 h at 5 W?m^?2 and 114.5 % after 48 h at 1 W?m^?2 UV-B radiation. HPLC analysis showed that nine MAAs existed in N. flagelliforme cells both from liquid suspension culture and field colony. But the concentration and kinds of them were different. At the two distinct levels of UV-B radiation, the content of particular MAAs increased, declined, or remained unchanged. Moreover, the appearance of two new MAAs was observed.     

The effect of low dose rate on metabolomic response to radiation in mice

Metabolomics has been shown to have utility in assessing responses to exposure by ionizing radiation (IR) in easily accessible biofluids such as urine. Most studies to date from our laboratory and others have employed γ-irradiation at relatively high dose rates (HDR), but many environmental exposure scenarios will probably be at relatively low dose rates (LDR). There are well-documented differences in the biologic responses to LDR compared to HDR, so an important question is to assess LDR effects at the metabolomics level. Our study took advantage of a modern mass spectrometry approach in exploring the effects of dose rate on the urinary excretion levels of metabolites 2 days after IR in mice. A wide variety of statistical tools were employed to further focus on metabolites, which showed responses to LDR IR exposure (0.00309 Gy/min) distinguishable from those of HDR. From a total of 709 detected spectral features, more than 100 were determined to be statistically significant when comparing urine from mice irradiated with 1.1 or 4.45 Gy to that of sham-irradiated mice 2 days post-exposure. The results of this study show that LDR and HDR exposures perturb many of the same pathways such as TCA cycle and fatty acid metabolism, which also have been implicated in our previous IR studies. However, it is important to note that dose rate did affect the levels of particular metabolites. Differences in urinary excretion levels of such metabolites could potentially be used to assess an individual’s exposure in a radiobiological event and thus would have utility for both triage and injury assessment.     

UV-B susceptibility and photoreactivation in embryonic development of the two-spotted spider mite, <Emphasis Type="Italic">Tetranychus urticae</Emphasis>

Developmental errors are often induced in the embryos of many organisms by environmental stress. Ultraviolet-B radiation (UV-B) is one of the most serious environmental stressors in embryonic development. Here, we investigated susceptibility to UV-B (0.5 kJ m^?2) in embryos of the two-spotted spider mite, Tetranychus urticae, to examine the potential use of UV-B in control of this important agricultural pest worldwide. Peak susceptibility to UV-B (0% hatchability) was found in T. urticae eggs 36–48 h after oviposition at 25 °C, which coincides with the stages of morphogenesis forming the germ band and initial limb primordia. However, hatchability recovered to?~?80% when eggs irradiated with UV-B were subsequently exposed to visible radiation (VIS) at 10.2 kJ m^?2, driving photoreactivation (the photoenzymatic repair of DNA damage). The recovery effect decreased to 40–70% hatchability, depending on the embryonic developmental stage, when VIS irradiation was delayed for 4 h after the end of exposure to UV-B. Thus UV-B damage to T. urticae embryos is critical, particularly in the early stages of morphogenesis, and photoreactivation functions to mitigate UV-B damage, even in the susceptible stages, but immediate VIS irradiation is needed after exposure to UV-B. These findings suggest that nighttime irradiation with UV-B can effectively kill T. urticae eggs without subsequent photoreactivation and may be useful in the physical control of this species.     

Influence of triacontanol and jasmonic acid on metabolomics during early stages of root induction in cultured tissue of tomato (<Emphasis Type="Italic">Lycopersicon esculentum)</Emphasis>

Influence of n-triacontanol (TRIA) and jasmonic acid (JA) on metabolic profiling during root morphogenesis was studied in Lycopersicon esculentum (cv. PKM-1). Proton nuclear magnetic resonance (¹H NMR) based metabolomics was employed to investigate the variations in metabolic profile. Chenomx NMR suite v.8.1 was used to identify and quantify metabolites based on their respective signature spectra. The levels of 47 metabolites were monitored for 72 h at specific time intervals (0, 3, 6, 9, 12, 24, 36, 48 and 72 h). Principal component analysis was performed to determine the variations in the metabolic profile between control and treatments during in vitro rhizogenesis. TRIA was observed to promote early root emergence (24 h) and also influence the metabolic variation during rhizogenesis between 9 and 24 h post exposure. Compounds such as IAA, ATP, NADPH, UDP-N-acetylglucosamine and gallate predominated at 9 h. Unlike TRIA, JA was unable to promote an early root induction. However, it influenced the synthesis of a relatively higher concentration of IAA at 6 h when compared to ATP, NADPH and trigonelline at 9 h. In the presence of both TRIA and JA (TRIA?+?JA), significant changes in the metabolic profiles were observed 24 h post exposure and the rooting was observed only after 72 h. The study suggests that TRIA may accelerate in vitro rhizogenesis of cultured tomato tissues by mainly increasing the synthesis of other growth promoting metabolites. But in the presence of JA, TRIA’s effect appears to be reduced.     

Plasma levels of alarmin HNPs 1–3 associate with lung dysfunction after cardiac surgery in children

Background

Early onset of lung injury is considerable common after cardiac surgery and is associated with increasing in morbidity and mortality, but current clinical predictors for the occurrence of this complication always have limited positive warning value. This study aimed to evaluate whether elevated plasma levels of human neutrophil peptides (HNPs) 1–3 herald impaired lung function in infants and young children after cardiac surgery necessitating cardiopulmonary bypass (CPB).

Methods

Consecutive children younger than 3 years old who underwent cardiac surgery were prospectively enrolled. Plasma concentrations of HNPs 1–3 and inflammatory cytokines were measured before, and immediately after CPB, as well as at 1 h, 12 h, and 24 h after CPB.

Results

Thirty patients were enrolled, 18 (60%) of whom were infants. Plasma levels of HNPs 1–3 and the pro-inflammatory cytokine interleukin-6 (IL-6) significantly increased immediately after CPB (P?<?0.001), while IL-8 increased 1 h after the CPB operation (P?=?0.002). The anti-inflammatory cytokine IL-10 levels were also significantly elevated immediately after CPB compared with the baseline (P?<?0.001). The stepwise multiple linear regression analysis showed that the plasma HNPs 1–3 levels immediately after CPB was independent correlated with the declined lung function, as reflected by the PaO₂/FiO₂ ratio on the first 2 days after operation (for the first day: OR, ?1.067, 95% CI, ?0.548 to ?1.574; P?<?0.001; for the second day: OR, ?0.667, 95% CI, ?0.183 to ?1.148; P?=?0.009) and prolonged mechanical ventilation time (OR, 0.039, 95% CI, 0.005 to 0.056; P?=?0.011). Plasma levels of HNPs 1–3 and IL-10 returned to the baseline values, while IL-6 and IL-8 levels remained significantly higher than baseline 24 h after CPB (P?≤?0.01).

Conclusions

Elevated HNPs 1–3 levels immediately after CPB correlate with impaired lung function, and HNPs 1–3 could serve as a quantifiable early alarmin biomarker for onset of lung injury in infants and young children undergoing cardiac surgery with CPB.

     

Reproducibility of non-fasting plasma metabolomics measurements across processing delays

Introduction

Processing delays after blood collection is a common pre-analytical condition in large epidemiologic studies. It is critical to evaluate the suitability of blood samples with processing delays for metabolomics analysis as it is a potential source of variation that could attenuate associations between metabolites and disease outcomes.

Objectives

We aimed to evaluate the reproducibility of metabolites over extended processing delays up to 48 h. We also aimed to test the reproducibility of the metabolomics platform.

Methods

Blood samples were collected from 18 healthy volunteers. Blood was stored in the refrigerator and processed for plasma at 0, 15, 30, and 48 h after collection. Plasma samples were metabolically profiled using an untargeted, ultrahigh performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) platform. Reproducibility of 1012 metabolites over processing delays and reproducibility of the platform were determined by intraclass correlation coefficients (ICCs) with variance components estimated from mixed-effects models.

Results

The majority of metabolites (approximately 70% of 1012) were highly reproducible (ICCs?≥?0.75) over 15-, 30- or 48-h processing delays. Nucleotides, energy-related metabolites, peptides, and carbohydrates were most affected by processing delays. The platform was highly reproducible with a median technical ICC of 0.84 (interquartile range 0.68–0.93).

Conclusion

Most metabolites measured by the UPLC–MS/MS platform show acceptable reproducibility up to 48-h processing delays. Metabolites of certain pathways need to be interpreted cautiously in relation to outcomes in epidemiologic studies with prolonged processing delays.

     

Effects of heat stress on development,reproduction and activities of protective enzymes in Mononychellus mcgregori

Mononychellus mcgregori is a pest mite of cassava. Since its invasion into China in 2008 it has spread rapidly. In order to determine the potential distribution and to analyze its invasion, diffusion and ecological adaptation mechanisms, we investigated the effect of high-temperature stress (30, 33, 36, 39 and 42 °C) on its development and reproduction, and the activity of protective enzymes in the mite. The results indicated significant influences: (1) adults could not lay eggs after they had been exposed to 42 °C for 4 h or longer; (2) egg development was slower and egg hatchability decreased after exposure of adults to 33–42 °C for 1 h; (3) offspring development (all stages) was slower after exposure of adults to 33–42 °C for 2 h or more; and (4) polyphenol oxidase (PPO), peroxidase (POD), ascorbate peroxidase (APX) and catalase (CAT) activities in the adults increased to high levels after exposure to 33–42 °C for 1 h, and superoxide dismutase activity increased only after exposure to 42 °C for 1 h. In conclusion, exposure to high temperatures for only 1 h probably has an important impact on the mite’s population growth. The significant increase of PPO, POD, APX, and CAT activities in adults may partially explain how M. mcgregori survive exposure to a relatively high temperature.     

Environmental stress of mobile phone EM radiation on locomotor activity and melatonin circadian rhythms of rats

Biological clocks are innate timing mechanisms that regulate many behavioral and physiological parameters in most organisms. In our modern life, heavy use of mobile phones (MPs) exerts a massive stress on organisms because their electromagnetic radiation usually results in varying degrees of damage to their biological systems including the biological rhythms. In the present study, the possible effects of exposure to radiofrequency–electromagnetic radiation (RF–EMR) from MPs on two characteristic circadian rhythms, locomotor activity and melatonin hormone rhythms, were investigated. Rats were exposed to RF–EMR from MPs at 900 MHz frequency (2-h/day for 2 weeks) during nighttime (20:00–22:00 h) followed by another two weeks without exposure for recovery. Locomotor activity rhythms of the control and treated groups (n = 5/group) were daily recorded using running wheels along the experimental period. For evaluating melatonin hormone rhythm, blood samples of control and treated groups (n = 12/group), were collected at the end of exposure and recovery periods, at 6-h time intervals per day (at 4:00, 10:00, 16:00, and 22:00 h). Rats exposed to RF–EMR exhibited phase shifting as well as a significant increased acrophase level in locomotor activity. Meanwhile, a significant decrease in serum melatonin levels with retaining lower amplitude rhythmicity was observed. Ceasing exposure for two weeks did not restore melatonin levels and circadian locomotor activity rhythms. It could be concluded that, under the current conditions, exposure to RF–EMR revealed disturbances in locomotor activity and melatonin level, although they maintained rhythmicity.