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Environmental exposure to low-doses of ionizing radiation. Effects on early nephrotoxicity in mice
Environmental Research 156 (2017) 291–296
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Environmental exposure to low-doses of ionizing radiation. Effects on early nephrotoxicity in mice
MARK
Montserrat Bellésa,b, Sergio Gonzalob, Noemí Serrab, Roser Esplugasb, Meritxell Arenasc, ⁎ José Luis Domingob, Victoria Linaresa,b, a b c
Physiology Unit, School of Medicine, IISPV, Rovira i Virgili University, Reus, Spain Laboratory of Toxicology and Environmental Health, School of Medicine, IISPV, Rovira i Virgili University, Reus, Spain Radiation Oncology Department, Sant Joan University Hospital, IISPV, Rovira i Virgili University, Reus, Spain
A R T I C L E I N F O
A B S T R A C T
Keywords: Nuclear power plants Ionizing radiation 137-cesium Kidney Mice
Nuclear accidents of tremendous magnitude, such as those of Chernobyl (1986) and Fukushima (2011), mean that individuals living in the contaminated areas are potentially exposed to ionizing radiation (IR). However, the dose-response relationship for effects of low doses of radiation is not still established. The present study was aimed at investigating in mice the early effects of low-dose internal radiation exposure on the kidney. Adult male (C57BL/6 J) mice were divided into three groups. Two groups received a single subcutaneous (s.c.) doses of cesium (137Cs) with activities of 4000 and 8000 Bq/kg bw. A third group (control group) received a single s.c. injection of 0.9% saline. To evaluate acute and subacute effects, mice (one-half of each group) were euthanized at 72 h and 10 days post-exposure to 137Cs, respectively. Urine samples were collected for biochemical analysis, including the measurement of F2-isoprostane (F2-IsoP) and kidney injury molecule-1 (KIM-1) levels. Moreover, the concentrations of 8-hydroxy-2′-deoxyguanosine (8-OHdG), a sensitive marker of oxidative DNA damage, were measured in renal tissue. Urinary excretion of total protein significantly increased at 72 h in mice exposed to Cs4000. Uric acid and lactate dehydrogenase (LDH) decreased significantly at both times post-exposure in animals exposed to Cs8000. After 72 h and 10 d of exposure to Cs4000, a significant increase in the γ-glutamil transferase (GGT) and N-acetyl-β-D-glucosaminidase (NAG) activities was observed. In turn, F2-IsoP levels increased -mainly in the Cs4000 group- at 72 h post-exposure. Following irradiation (137Cs), the highest level of KIM-1 was corresponded to the Cs4000 group at 72 h. Likewise, the main DNA damage was detected in mice exposed to Cs4000, mainly at 10 d after irradiation. The alterations observed in several biomarkers suggest an immediate renal damage following exposure to low doses of IR (given as 137Cs). Further investigations are required to clarify the mechanisms involved in the internal IR-induced nephrotoxicity.
1. Introduction
area. The huge earthquake affected the nuclear power plant in the Fukushima prefecture, resulting in large amounts of radioactive materials released into the environment (Hasegawa et al., 2015). The major nuclides released were ¹³¹I, ¹³⁴Cs, and ¹³⁷Cs, which were early detected in the topsoil, plants and water (Fushiki, 2013; Orita et al., 2016). As a direct consequence of this, almost 170,000 people were evacuated or they should stay indoors. After accidents of this tremendous magnitude, the populations living in the contaminated areas are potentially exposed to external and internal radiation (Marzano et al., 2001; Ostroumova et al., 2016). The external radiation is mainly caused by the surface contamination of the environment, while the internal contamination is basically due to the presence of radionuclides in the food chain (Aono et al., 2016; Fushiki, 2013; Suslova et al., 2015). Among these radionuclides, 137Cs,
Nowadays, more than 400 nuclear power plants are in operation around the world. Despite the strict security measures required by these facilities, four major nuclear accidents occurred in the past century: Kyshtym (Russia, 1957), Windscale Piles (UK, 1957), Three Mile Island (USA, 1979) and Chernobyl (Ukraine, 1986), while one accident occurred in the current century (Fukushima, Japan, 2011). The effects of these serious accidents on individuals and societies have been diverse and enduring (Gudzenko et al., 2015; Han et al., 2011). However, due to the specific characteristics and proximity in time, the recent accident in Fukushima has been particularly shocking. On March 11, 2011, a 9.0-magnitude earthquake struck the northeast coast of the main island of Japan, triggering a tsunami with 14–15 m-high waves hitting the
⁎
Corresponding author at: Physiology Unit, School of Medicine, IISPV, Rovira i Virgili University, Reus, Spain. E-mail address: [email protected] (V. Linares).
http://dx.doi.org/10.1016/j.envres.2017.03.034 Received 28 February 2017; Received in revised form 22 March 2017; Accepted 23 March 2017 0013-9351/ © 2017 Elsevier Inc. All rights reserved.
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diseases increases human morbidity and mortality. Therefore, renal function must be part of the immediate and long-term follow-up of individuals who have had been subjected to accidental radiation exposures. In the present study, we investigated the nephrotoxic effects of low-dose IR exposure in order to assess the initial events after irradiation. The main objective was to evaluate the diagnostic capacity of biomarkers of kidney injury and OS in predicting renal damage.
with a half-life of about 30 years, is the main contributor to exposure of the individuals living in the areas around the accidents (Orita et al., 2016). Although the effects of external exposure to high and low doses of ionizing radiation (IR) have been extensively studied, nowadays information on the health effects of internal irradiation is scarce. A rapid absorption and a widespread systemic distribution of 137Cs have been reported following chronic oral ingestion (Manens et al., 2016). In contaminated areas, the incidence of thyroid cancer and bone, as well as digestive and nervous disorders increases in the children population (Ericson and Kallen, 1994; Sholl et al., 2017). On the other hand, a few animal studies have assessed the biological effects of internal exposure to 137Cs. Biological consequences were observed on small intestinal or secretor functions (Dublineau et al., 2007) and vitamin D3 and cholesterol metabolism in rats (Souidi et al., 2006; Tissandie et al., 2006). Moreover, in a recent study conducted in our laboratory, mice exposed internally to 137Cs showed impaired learning, and spatial memory, as well as increased anxiety (Heredia et al., 2016). The harmful effects of radiation can be present in various organs. However, from the standpoint of serious damage, the kidney is probably the most radiosensitive of the abdominal organs (Fuma et al., 2016; Robbins and Zhao, 2004). The tubular epithelial cells appear to be more sensitive to the radiation in comparison to epithelial cells from other tissues. Kidney irradiation induces radiation nephropathy characterized by a reduction in renal function, which is associated with structural alterations in glomerular and tubular cells (Ilhan et al., 2016; Romanenko et al., 2012). It is well known that cancer radiotherapy and accidental exposure to high-doses of external IR induce renal injury (El-Gazzar et al., 2016). Notwithstanding, information on the nephrotoxic effects of internal exposure to IR is certainly scarce. During the period subsequent to the Chernobyl accident, the incidence of malignant renal tumors in Ukraine increased from 4.7 to 10.7 per 100,000 inhabitants (Jargin, 2015), while an association of low-dose IR on renal cell carcinomas (RCCs) was observed in subjects living in 137Cs-contaminated areas of Ukraine (Morell-Quadreny et al., 2011). 137Cs constitutes 80–90% of the internal exposure in people living in radio-contaminated areas, being mainly (approx. 90%) excreted through the kidneys (Ebner et al., 2016; Romanenko et al., 2001). Most of the radiation-induced damage to biomolecules in aqueous media is caused by the formation of free radicals resulting from the radiolysis of water (Ekici et al., 2016). The generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) predominantly mediates the harmful effects of IR (El-Gazzar et al., 2016). In turn, IR-induced oxidative stress (OS) may produce ROS, which are reported to be the main cause of tissue injury. Therefore, if ROS/RNS are not scavenged, they can lead to widespread lipid, protein and DNA damage (Havas, 2017; Ozyurt et al., 2014) Overproduction of free radicals reacts with cell membrane fatty acids and proteins impair their function. As a result of this, various investigations have suggested that the measurement of F2-isoprostane (F2-isoP) levels is a reliable and useful approach to assess lipid peroxidation and OS in vivo (Alonso et al., 2010; Clayton et al., 2014). Upon kidney function disorders, alterations in urinary levels of parameters such as albumin, creatinine or urea can be used as biomarkers of kidney failure (Belles et al., 2007). Recently, a novel biomarker for renal injury is the Kidney Injury Molecule-1 (KIM-1). KIM-1 is a type I transmembrane glycoprotein discovered in renal tubular epithelial cells. KIM-1 is undetectable in healthy kidneys, but it is highly expressed and can be found at very high levels after acute kidney injury (Nan-Ya et al., 2015; Pianta et al., 2017; Yin and Wang, 2016). Partial or total body exposures to radiation doses as low as 5 Gy in a single fraction, can lead to radiation nephropathy, while exposure to even lower doses of radiation have been associated with renal disease. As a consequence of the nuclear accident of Chernobyl and more recently in that of Fukushima, the effects of low-dose IR, especially internal exposure, are at the forefront of everyone's attention. Renal
2. Materials and methods 2.1. Animals Male C57BL/6J mice (2 months of age) were purchased from Charles River (Criffa, Barcelona, Spain). Housing conditions included a temperature of 22 ± 2 °C, relative humidity of 50 ± 10% and 12 h light-dark cycle. Food (Panlab rodent chow, Panlab, Barcelona) and tap water were offered ad libitum throughout the experiment. The experimental protocol was approved by the Animal Care and Use Committee of the Universitat Rovira i Virgili (Tarragona, Catalonia, Spain) following the “Principles of Laboratory Animal Care”, being carried out in accordance with the European Union Directive 2010/63/EU for animal experiments. 2.2. Groups and treatment After a quarantine period of two weeks, mice were randomly classified into three experimental groups (n=16 per group). Two groups received a single subcutaneous (s.c.) dose of cesium (137Cs, provided by CIEMAT, Madrid, Spain) with activities of 4000 (Cs4000) and 8000 (Cs8000) Bq/kg bw, respectively. The 137Cs doses were based on the estimate of the 137Cs dietary intake of the surrounding populations during the years following the Chernobyl accident (Belles et al., 2016; Grison et al., 2012; Lestaevel et al., 2008). A third group (control group) received a single s.c. injection of 0.9% saline. To evaluate the acute and subacute renal effects, mice (half of the animals in each group) were euthanized at 72 h and 10 d post-exposure to 137Cs, respectively. Twenty-four hours before being sacrificed, animals were individually housed in plastic metabolism cages. Urines (24 h) were collected to determine various biochemical parameters, KIM-1 and F2isoP levels. After urine collection, animals were sacrificed with an overdose of ketamine–xylazine given intraperitoneally. Kidneys were removed and cleared of the adhering tissues. Kidney tissue was used to quantify DNA damage. 2.3. Urine biochemical analysis In fresh urine, the 24 h volume, and the concentrations of urea, uric acid, levels of creatinine, total protein, as well as lactate dehydrogenase (LDH), N-acetyl-β-D-glucosaminidase (NAG) and γ-glutamil-transferase (GGT) activities were analyzed using a Cobas Mira automatic analyzer (Roche Pharmaceuticals, Basel, Switzerland) (Belles et al., 2007). The levels of 8-iso-prostaglandin F2α (8-iso-PGF2α), one isomer of the F2-isoP family, were analyzed in urine for assessment of oxidant stress in vivo. In the present study, urine samples were stored in aliquots of 1 mL containing 10 μL of butylated hydroxytoluene at −80 °C until analysis. The measurement of urinary F2-isoprostane (8iso-PGF2α) concentrations was performed based on the protocol of the competitive enzyme-linked immunosorbent assay (ELISA) kit from Oxford Biomedical Research (Deltaclon, Spain). The level of 8-isoPGF2α in each urine sample was normalized to that creatinine level [8iso-PGF2α (ng)/creatinine in urine (mg)] (Alonso et al., 2010). For the measurement of KIM-1 in urine (stored at −20 °C), ELISA test kit for the detection of KIM-1 in mouse (Aviscera Bioscience, Deltaclon, Spain) was used according to the manufacturer's instructions. The amount of KIM-1 in each urine sample was normalized to that creatinine level [Kim-1 (μg)/creatinine in urine (g)]. 292
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2.4. DNA damage quantification
Table 2 Urinary parameters in mice at 10 d after exposure to internal radiation.
8-hydroxy-2′-deoxyguanosine (8-OHdG) is an oxidized derivative of deoxyguanosine, which is generated by hydroxyl radicals, singlet oxygen, and one-electron oxidants in cellular DNA. Currently, 8OHdG is widely accepted as a sensitive marker of oxidative DNA damage. In the current investigation, kidney samples were washed in 0.9% saline and homogeneized in 0.2 M sodium phosphate pH 6.25 buffer (1:20, w/v) in a Potter-Elvehjem homogenizer, with a Teflon pestle (Braun, Melsungen, Germany). Total DNA was extracted using the commercially available DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany). The concentration of DNA in each sample was determined using an EPOCH™ 2 Microplate Espectrophotometer (BioTek, Winooski, Vermont, USA). The level of 8OH-dG was measured using EpiQuik™ 8-OHdG DNA Damage Quantification Direct Kit (Epigentek, Farmingdale, NY, USA) according to the manufacturer's instructions.
137
0
4000
8000
Urinary volume (mL/24 h) Creatinine (mg/24 h) Urea (mg/24 h) Total protein (mg/24 h) Uric acid (mg/24 h) Lactate dehydrogenase (LDH) (U/24 h/kg)
1.17 ± 0.22 0.82 ± 0.06 1.96 ± 0.13 0.35 ± 0.06 0.159 ± 0.016a 1.14 ± 0.08a
1.03 ± 0.11 0.70 ± 0.05 1.96 ± 0.38 0.31 ± 0.04 0.066 ± 0.004b 1.06 ± 0.21a
0.85 ± 0.12 0.76 ± 0.12 1.75 ± 0.22 0.23 ± 0.04 0.047 ± 0.007b 0.56 ± 0.07b
Cs activity (Bq/kg bw)
Values are expressed as mean ± S.E.M. Statistics: Values in the same row not showing a common superscript (a,b) are significantly different at p < 0.05.
(0.066 ± 0.004 versus 0.145 ± 0.013, p=0.005). At 72 h and 10 d after IR, the Cs8000 group showed a significant decrease in the activity of LDH with respect to mice in the control (0.79 ± 0.08 versus 1.63 ± 0.22, p=0.006 and 0.56 ± 0.07 versus 1.14 ± 0.08, p=0.0002, respectively) and the Cs4000 groups (0.79 ± 0.08 versus 1.10 ± 0.23, p=0.027 and 0.56 ± 0.07 versus 1.06 ± 0.21, p=0.0002, respectively). For the rest of urinary parameters no significant differences were noted between groups at 72 h and 10 d post-exposure. The effects of exposure to 137Cs in the GGT and NAG activities are depicted in Fig. 1(A-B). A significant increase in the GGT activity was observed in the animals at 72 h and 10 d after being exposed to Cs4000 with respect to the controls (9.72 ± 0.78 versus 6.18 ± 0.49, p=0.0117 and 11.45 ± 1.15 versus 5.18 ± 0.43, p=0.0002, respectively). However, for both times post-irradiation (72 h and 10 d postexposure), the activity of GGT decreased significantly in mice exposed to Cs8000 observing similar levels to those in control group (5.84 ± 0.33 versus 9.72 ± 0.78, p=0.0013 and 4.87 ± 0.49 versus
2.5. Data analysis All data are expressed as the mean ± standard error mean (S.E.M). Statistical analyses were performed using Graph Pad Prism® software version 5.01 (San Diego, California, USA). If variances were homogeneous, ANOVA was used followed by the Bonferroni's post-test correction for multiple comparisons. If the variances were not homogeneous, the Kruskal–Wallis test was then used. Differences between groups were analyzed using the Mann–Whitney U-test. Moreover, the paired t-test was used to compare the two different point of time tested. Statistical significance was set at p < 0.05. 3. Results Neither deaths, nor remarkable signs of external toxicity were observed in the groups given internal low doses of IR as 137Cs. Urinary parameters at 72 h and 10 d post-exposure to 137Cs are summarized in Tables 1 and 2, respectively. In comparison with the control and the Cs8000 group, the excretion of total protein was significantly enhanced in the Cs4000 group at 72 h post-exposure (0.46 ± 0.05 versus 0.26 ± 0.06, p=0.033 and 0.46 ± 0.05 versus 0.25 ± 0.030, p=0.004, respectively). However, this increase was restored 10 d after irradiation (0.31 ± 0.04 versus 0.46 ± 0.05, p=0.047). At 10 d post-exposure no differences between groups were noted. After 72 h of irradiation, uric acid levels in the Cs8000 group were significantly lower than those in the control and the Cs4000 group (0.071 ± 0.007 versus 0.131 ± 0.012, p=0.001 and 0.071 ± 0.007 versus 0.145 ± 0.013, p=0.0003, respectively). Moreover, at 10 d post-exposure, all irradiated mice showed significantly lower levels of uric acid than control animals (0.066 ± 0.004 versus 0.159 ± 0.016, p=0.0022 and 0.047 ± 0.007 versus 0.159 ± 0.016, p=0.002, respectively). A significant decline in the uric acid concentrations was also observed in the Cs4000 group between 72 h and 10 d after exposure Table 1 Urinary parameters in mice at 72 h after exposure to internal radiation. 137
0
4000
8000
Urinary volume (mL/24 h) Creatinine (mg/24 h) Urea (mg/24 h) Total protein (mg/24 h) Uric acid (mg/24 h) Lactate dehydrogenase (LDH) (U/24 h/kg)
0.87 ± 0.18 0.90 ± 0.06 1.52 ± 0.18 0.26 ± 0.06a 0.131 ± 0.012a 1.63 ± 0.22a
0.89 ± 0.14 0.72 ± 0.08 1.54 ± 0.14 0.46 ± 0.05b 0.145 ± 0.013a 1.10 ± 0.23a
0.90 ± 0.14 0.74 ± 0.10 1.39 ± 0.16 0.25 ± 0.03a 0.071 ± 0.007b 0.79 ± 0.08b
Cs activity (Bq/kg bw)
Fig. 1. γ-glutamil-transferase (GGT) (A) and N-acetyl-β-D-glucosaminidase (NAG) (B) activities in urine of mice at 72 h and 10 d post-exposure to internal radiation. Values are expressed as mean ± S.E.M. *p < 0.01, **p < 0.01 and ***p < 0.001 indicate significant differences between groups at 72 h and 10 d post-exposure. $p < 0.05 indicates significant differences between 72 h and 10 d for Cs8000 group.
Values are expressed as mean ± S.E.M. Statistics: Values in the same row not showing a common superscript (a,b) are significantly different at p < 0.05.
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Fig. 4. Percentage of 8-hydroxy-2′-deoxyguanosine (8-OHdG) in renal tissue in mice at 72 h and 10 d post-exposure to internal radiation. Values are expressed as mean ± S.E.M. ** p < 0.01 indicates significant differences between groups at 10 d post-exposure.
Fig. 2. Urinary F2-isoprostane (8-iso-PGF2α) levels in mice at 72 h and 10 d postexposure to internal radiation. Values are expressed as mean ± S.E.M. **p < 0.01 and *** p < 0.001 indicate significant differences between groups at 10 d post-exposure. $ $ p < 0.01 and $$$p < 0.001 indicate significant differences between 72 h and 10 d for each activity of 137Cs.
0.0029 ± 0.0002, p=0.0043). On the other hand, no significant differences between animals exposed to Cs8000 and the control group were noted.
11.45 ± 1.15, p=0.0006, respectively) (Fig. 1A). Similarly, a significant increase in the NAG activity was noted at 72 h after irradiation in the Cs4000 group when compared with the control (3.48 ± 0.13 versus 2.37 ± 0.25, p=0.0014) and the Cs8000 groups+ (3.48 ± 0.13 versus 2.57 ± 0.11, p=0.0001). Ten days after irradiation, the activity of NAG decreased significantly in mice exposed to Cs8000 compared to the Cs4000 group (2.08 ± 0.06 versus 3.23 ± 0.23, p=0.020) (Fig. 1B). Ionizing radiation induced changes in the urinary levels of 8-isoPGF2α at 10 d post-exposure (Fig. 2). In both groups of irradiated mice (Cs4000 and Cs8000), the levels of 8-iso-PGF2α were significantly increased compared with control animals (79.14 ± 3.62 versus 41.43 ± 4.34, p < 0.0001 and 65.14 ± 2.43 versus 41.43 ± 4.34, p=0.0005, respectively). Mice irradiated with Cs8000 showed significant lower levels than those exposed to Cs4000 (65.14 ± 2.43 versus 79.14 ± 3.62, p=0.0075). Furthermore, at 10 d post-exposure, 8-isoPGF2α levels were significantly enhanced in the Cs4000 and Cs8000 groups compared to the observed levels at 72 h (79.14 ± 2.43 versus 48.38 ± 2.67, p < 0.0001 and 65.14 ± 2.43 versus 50.00 ± 2.40, p=0.0016, respectively). The urinary levels of KIM-1 (Fig. 3) were significantly higher at 72 h post-exposure in mice irradiated at the lower 137Cs activity (Cs4000) when compared to control animals (0.49 ± 0.06 versus 0.22 ± 0.02, p=0.015). However, this significant increase was restored at 10 d after irradiation (0.29 ± 0.04 versus 0.49 ± 0.06, p=0.038). No differences between groups were noted at 10 d post-exposure. The percentage of 8-OHdG in renal tissue was higher in mice exposed to Cs4000 in comparison to the controls (Fig. 4)being statistically significant 10 d after exposure (0.0045 ± 0.0003 versus
4. Discussion Very serious nuclear accidents such as those occurred in Chernobyl and Fukushima, imply that many radionuclides are released into the environment (Hachiya et al., 2014; Lucchini et al., 2017). Even for those individuals who were not initially exposed to radioactivity, the radionuclides can reach their bodies some years later through the food chain (Beresford et al., 2016; Burger et al., 2000). Radiation-induced damage can result in adverse health effects within hours or days. Delayed effects are even observable a number of months after exposure (Kamiya et al., 2015). The renal tissue is considered critical in humans due its functions of filtration, reabsorption and retention of the tracer by the tubules (Ilhan et al., 2016). Moreover, experimental and clinical investigations have demonstrated that kidneys are highly sensitive to radiation injuries (Canyilmaz et al., 2016). Alterations in biochemical indices for renal function can be an early warning of alterations in these organs, ultimately leading to loss of functional integrity (El-Gazzar et al., 2016). Renal toxicity is one of the well known (acute) side effects of IR exposure, and can cause glomerular filtration rate impairment and proteinuria. Likewise, after the Fukushima nuclear disaster, human biochemical analysis detected an increase in the urinary excretion of proteins (Satoh et al., 2016). An increase of proteinuria is associated with renal pathology (Alonso et al., 2010; Jarad and Miner, 2009). In the current study, urine analysis showed an increase in the protein content of mice exposed to Cs4000 at 72 h. It has been reported that external irradiation in a rat model leads to the development of proteinuria (Imig et al., 2016). Furthermore, the current results showed low levels of uric acid, mainly in the Cs8000 group at 72 h and 10 d. Uric acid is a powerful non-enzymatic antioxidant, which has been identified as a radiation marker in urine of mice, being considered a natural free radical scavenger (Laiakis et al., 2014). The harmful effects of IR in biological systems are mainly mediated through the production of ROS in cells as result of water radiolysis (Reiter et al., 2011). Data on LDH activity in urine facilitate the detection of renal damage (Moon et al., 2013). In the present investigation, we observed that Cs8000 exposure caused a decreased in LDH activity, which indicates that after irradiation, the kidney shows a generally reduced metabolic enzyme activity, which is concomitant with a reduced kidney function. On the other hand, GGT is associated with incident vascular events (Fraser et al., 2007). It has been reported that the renal functional impairment caused by irradiation is partly mediated by vascular damage (Kruse et al., 2009). GGT is a key enzyme in the metabolism of GSH, being potentially useful for studying OS-related issues in both
Fig. 3. Urinary Kidney Injury Molecule-1 (KIM-1) levels in mice at 72 h and 10 d postexposure to internal radiation. Values are expressed as mean ± S.E.M. *p < 0.05 indicates significant differences between groups at 72 h post-exposure. $p < 0.05 indicates significant differences between 72 h and 10 d for Cs4000 group.
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Acknowledgements
epidemiological and clinical settings (Djavaheri-Mergny et al., 2002; Kowalczyk-Pachel et al., 2016). In the present study, GGT activity was mostly affected in mice exposed to Cs4000 at 72 h and 10 d. It has also been reported that the extracellular cleavage of GGT induces the production of ROS, suggesting that GGT plays a pro-oxidant role (Alonso et al., 2010). Increased urinary NAG excretion is one of the most sensitive biomarkers of renal toxicity, having been recommended for the detection of alterations in tubular function (Fiseha and Tamir, 2016). A relationship between urinary NAG excretion and renal oxidative damage has been reported (Oktem et al., 2006). In the present study, the urinary NAG activity increased significantly 72 h after Cs4000 irradiation. It agrees with the results of a previous investigation conducted in our laboratory, which showed an increase of NAG activity in urine after internal exposure to uranium (Belles et al., 2007). ROS and OS may contribute to radiation-induced cytotoxicity, as well as metabolic and morphologic alterations in humans and animals (El-Missiry et al., 2007; Zhao and Robbins, 2009). Measurement of F2IsoP, products of free-radical-catalyzed peroxidation of arachidonic acid, has been accepted as an accurate biomarker of OS and as a consequence of tissue damage (Leung et al., 2015; Silbert et al., 2016; Tucker et al., 2013). We have observed that urinary levels of 8-isoPGF2α were enhanced in all irradiated mice, mainly at Cs4000 and at 10 d post-exposure. To the best of our knowledge, data on the effects of internal IR on urinary levels of F2-IsoP are currently available. However, our results are consistent with those of previous studies on the effects of radiotherapy in renal function analyzing the urinary F2IsoP excretion (Machon et al., 2012). KIM-1 is the most upregulated protein in the proximal tubules following renal ischemic or toxic injury in humans and experimental animals (Baek et al., 2015; Pianta et al., 2017). Upregulation of KIM-1 has been observed in renal biopsies after radiotherapy in pediatric patients (Nepal et al., 2008). Recently, no significant increases in the urinary levels of KIM-1 were reported in children who underwent radiotherapy of the abdominal cavity (Yang et al., 2015). However, there is a lack of information about the effects of IR on the expression or urinary levels of KIM-1. Our results show an increase in the urinary levels of KIM-1 in irradiated mice, mainly in the group of Cs4000 at 72 h after exposure. Overproduction of free radicals that react with cell membrane fatty acids and proteins impairs their function. DNA is highly susceptible to oxidative damage, while 8-OHdG is a useful tool used as an indicator for OS and oxidative DNA (Kawanishi et al., 2016; Ozyurt et al., 2014). Moreover, a number of investigations showed that cellular DNA is the primary target for the toxic effects of IR, inducing strand breaks or base deletion (Glebova et al., 2015; Kavanagh et al., 2013; Yoshikawa et al., 2015). The current findings reveal that the internal radiation-induced damage in the kidney of irradiated mice with Cs4000 of activity, had significant increases in the percentage of 8-OHdG, being significant at 10 d post-exposure. The results of previous studies in which irradiationinduced oxidative DNA damage was demonstrated, support the results of this investigation. It has been reported that a single low dose wholebody irradiation in rats produced an increase in the kidney levels of 8OHdG (Ozyurt et al., 2014). Similarly, localized kidney irradiation in rats led to a marked, dose-independent increase, in glomerular and tubular cell nuclear DNA oxidation (Robbins et al., 2002). In summary, the results of the present study show that very low doses of internal IR induced not only early renal histologic injury and acute OS, but also caused DNA damage. While DNA alterations have been reported in laboratory animals exposed to very high doses of irradiation, to date, no data at such low internal doses were available. Anyhow, further investigations are required in order to better understand the long-term consequences of these effects, as well as the mechanisms involved in IR-induced nephrotoxicity.
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Environmental exposure to low-doses of ionizing radiation. Effects on early nephrotoxicity in mice
MARK
Montserrat Bellésa,b, Sergio Gonzalob, Noemí Serrab, Roser Esplugasb, Meritxell Arenasc, ⁎ José Luis Domingob, Victoria Linaresa,b, a b c
Physiology Unit, School of Medicine, IISPV, Rovira i Virgili University, Reus, Spain Laboratory of Toxicology and Environmental Health, School of Medicine, IISPV, Rovira i Virgili University, Reus, Spain Radiation Oncology Department, Sant Joan University Hospital, IISPV, Rovira i Virgili University, Reus, Spain
A R T I C L E I N F O
A B S T R A C T
Keywords: Nuclear power plants Ionizing radiation 137-cesium Kidney Mice
Nuclear accidents of tremendous magnitude, such as those of Chernobyl (1986) and Fukushima (2011), mean that individuals living in the contaminated areas are potentially exposed to ionizing radiation (IR). However, the dose-response relationship for effects of low doses of radiation is not still established. The present study was aimed at investigating in mice the early effects of low-dose internal radiation exposure on the kidney. Adult male (C57BL/6 J) mice were divided into three groups. Two groups received a single subcutaneous (s.c.) doses of cesium (137Cs) with activities of 4000 and 8000 Bq/kg bw. A third group (control group) received a single s.c. injection of 0.9% saline. To evaluate acute and subacute effects, mice (one-half of each group) were euthanized at 72 h and 10 days post-exposure to 137Cs, respectively. Urine samples were collected for biochemical analysis, including the measurement of F2-isoprostane (F2-IsoP) and kidney injury molecule-1 (KIM-1) levels. Moreover, the concentrations of 8-hydroxy-2′-deoxyguanosine (8-OHdG), a sensitive marker of oxidative DNA damage, were measured in renal tissue. Urinary excretion of total protein significantly increased at 72 h in mice exposed to Cs4000. Uric acid and lactate dehydrogenase (LDH) decreased significantly at both times post-exposure in animals exposed to Cs8000. After 72 h and 10 d of exposure to Cs4000, a significant increase in the γ-glutamil transferase (GGT) and N-acetyl-β-D-glucosaminidase (NAG) activities was observed. In turn, F2-IsoP levels increased -mainly in the Cs4000 group- at 72 h post-exposure. Following irradiation (137Cs), the highest level of KIM-1 was corresponded to the Cs4000 group at 72 h. Likewise, the main DNA damage was detected in mice exposed to Cs4000, mainly at 10 d after irradiation. The alterations observed in several biomarkers suggest an immediate renal damage following exposure to low doses of IR (given as 137Cs). Further investigations are required to clarify the mechanisms involved in the internal IR-induced nephrotoxicity.
1. Introduction
area. The huge earthquake affected the nuclear power plant in the Fukushima prefecture, resulting in large amounts of radioactive materials released into the environment (Hasegawa et al., 2015). The major nuclides released were ¹³¹I, ¹³⁴Cs, and ¹³⁷Cs, which were early detected in the topsoil, plants and water (Fushiki, 2013; Orita et al., 2016). As a direct consequence of this, almost 170,000 people were evacuated or they should stay indoors. After accidents of this tremendous magnitude, the populations living in the contaminated areas are potentially exposed to external and internal radiation (Marzano et al., 2001; Ostroumova et al., 2016). The external radiation is mainly caused by the surface contamination of the environment, while the internal contamination is basically due to the presence of radionuclides in the food chain (Aono et al., 2016; Fushiki, 2013; Suslova et al., 2015). Among these radionuclides, 137Cs,
Nowadays, more than 400 nuclear power plants are in operation around the world. Despite the strict security measures required by these facilities, four major nuclear accidents occurred in the past century: Kyshtym (Russia, 1957), Windscale Piles (UK, 1957), Three Mile Island (USA, 1979) and Chernobyl (Ukraine, 1986), while one accident occurred in the current century (Fukushima, Japan, 2011). The effects of these serious accidents on individuals and societies have been diverse and enduring (Gudzenko et al., 2015; Han et al., 2011). However, due to the specific characteristics and proximity in time, the recent accident in Fukushima has been particularly shocking. On March 11, 2011, a 9.0-magnitude earthquake struck the northeast coast of the main island of Japan, triggering a tsunami with 14–15 m-high waves hitting the
⁎
Corresponding author at: Physiology Unit, School of Medicine, IISPV, Rovira i Virgili University, Reus, Spain. E-mail address: [email protected] (V. Linares).
http://dx.doi.org/10.1016/j.envres.2017.03.034 Received 28 February 2017; Received in revised form 22 March 2017; Accepted 23 March 2017 0013-9351/ © 2017 Elsevier Inc. All rights reserved.
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diseases increases human morbidity and mortality. Therefore, renal function must be part of the immediate and long-term follow-up of individuals who have had been subjected to accidental radiation exposures. In the present study, we investigated the nephrotoxic effects of low-dose IR exposure in order to assess the initial events after irradiation. The main objective was to evaluate the diagnostic capacity of biomarkers of kidney injury and OS in predicting renal damage.
with a half-life of about 30 years, is the main contributor to exposure of the individuals living in the areas around the accidents (Orita et al., 2016). Although the effects of external exposure to high and low doses of ionizing radiation (IR) have been extensively studied, nowadays information on the health effects of internal irradiation is scarce. A rapid absorption and a widespread systemic distribution of 137Cs have been reported following chronic oral ingestion (Manens et al., 2016). In contaminated areas, the incidence of thyroid cancer and bone, as well as digestive and nervous disorders increases in the children population (Ericson and Kallen, 1994; Sholl et al., 2017). On the other hand, a few animal studies have assessed the biological effects of internal exposure to 137Cs. Biological consequences were observed on small intestinal or secretor functions (Dublineau et al., 2007) and vitamin D3 and cholesterol metabolism in rats (Souidi et al., 2006; Tissandie et al., 2006). Moreover, in a recent study conducted in our laboratory, mice exposed internally to 137Cs showed impaired learning, and spatial memory, as well as increased anxiety (Heredia et al., 2016). The harmful effects of radiation can be present in various organs. However, from the standpoint of serious damage, the kidney is probably the most radiosensitive of the abdominal organs (Fuma et al., 2016; Robbins and Zhao, 2004). The tubular epithelial cells appear to be more sensitive to the radiation in comparison to epithelial cells from other tissues. Kidney irradiation induces radiation nephropathy characterized by a reduction in renal function, which is associated with structural alterations in glomerular and tubular cells (Ilhan et al., 2016; Romanenko et al., 2012). It is well known that cancer radiotherapy and accidental exposure to high-doses of external IR induce renal injury (El-Gazzar et al., 2016). Notwithstanding, information on the nephrotoxic effects of internal exposure to IR is certainly scarce. During the period subsequent to the Chernobyl accident, the incidence of malignant renal tumors in Ukraine increased from 4.7 to 10.7 per 100,000 inhabitants (Jargin, 2015), while an association of low-dose IR on renal cell carcinomas (RCCs) was observed in subjects living in 137Cs-contaminated areas of Ukraine (Morell-Quadreny et al., 2011). 137Cs constitutes 80–90% of the internal exposure in people living in radio-contaminated areas, being mainly (approx. 90%) excreted through the kidneys (Ebner et al., 2016; Romanenko et al., 2001). Most of the radiation-induced damage to biomolecules in aqueous media is caused by the formation of free radicals resulting from the radiolysis of water (Ekici et al., 2016). The generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) predominantly mediates the harmful effects of IR (El-Gazzar et al., 2016). In turn, IR-induced oxidative stress (OS) may produce ROS, which are reported to be the main cause of tissue injury. Therefore, if ROS/RNS are not scavenged, they can lead to widespread lipid, protein and DNA damage (Havas, 2017; Ozyurt et al., 2014) Overproduction of free radicals reacts with cell membrane fatty acids and proteins impair their function. As a result of this, various investigations have suggested that the measurement of F2-isoprostane (F2-isoP) levels is a reliable and useful approach to assess lipid peroxidation and OS in vivo (Alonso et al., 2010; Clayton et al., 2014). Upon kidney function disorders, alterations in urinary levels of parameters such as albumin, creatinine or urea can be used as biomarkers of kidney failure (Belles et al., 2007). Recently, a novel biomarker for renal injury is the Kidney Injury Molecule-1 (KIM-1). KIM-1 is a type I transmembrane glycoprotein discovered in renal tubular epithelial cells. KIM-1 is undetectable in healthy kidneys, but it is highly expressed and can be found at very high levels after acute kidney injury (Nan-Ya et al., 2015; Pianta et al., 2017; Yin and Wang, 2016). Partial or total body exposures to radiation doses as low as 5 Gy in a single fraction, can lead to radiation nephropathy, while exposure to even lower doses of radiation have been associated with renal disease. As a consequence of the nuclear accident of Chernobyl and more recently in that of Fukushima, the effects of low-dose IR, especially internal exposure, are at the forefront of everyone's attention. Renal
2. Materials and methods 2.1. Animals Male C57BL/6J mice (2 months of age) were purchased from Charles River (Criffa, Barcelona, Spain). Housing conditions included a temperature of 22 ± 2 °C, relative humidity of 50 ± 10% and 12 h light-dark cycle. Food (Panlab rodent chow, Panlab, Barcelona) and tap water were offered ad libitum throughout the experiment. The experimental protocol was approved by the Animal Care and Use Committee of the Universitat Rovira i Virgili (Tarragona, Catalonia, Spain) following the “Principles of Laboratory Animal Care”, being carried out in accordance with the European Union Directive 2010/63/EU for animal experiments. 2.2. Groups and treatment After a quarantine period of two weeks, mice were randomly classified into three experimental groups (n=16 per group). Two groups received a single subcutaneous (s.c.) dose of cesium (137Cs, provided by CIEMAT, Madrid, Spain) with activities of 4000 (Cs4000) and 8000 (Cs8000) Bq/kg bw, respectively. The 137Cs doses were based on the estimate of the 137Cs dietary intake of the surrounding populations during the years following the Chernobyl accident (Belles et al., 2016; Grison et al., 2012; Lestaevel et al., 2008). A third group (control group) received a single s.c. injection of 0.9% saline. To evaluate the acute and subacute renal effects, mice (half of the animals in each group) were euthanized at 72 h and 10 d post-exposure to 137Cs, respectively. Twenty-four hours before being sacrificed, animals were individually housed in plastic metabolism cages. Urines (24 h) were collected to determine various biochemical parameters, KIM-1 and F2isoP levels. After urine collection, animals were sacrificed with an overdose of ketamine–xylazine given intraperitoneally. Kidneys were removed and cleared of the adhering tissues. Kidney tissue was used to quantify DNA damage. 2.3. Urine biochemical analysis In fresh urine, the 24 h volume, and the concentrations of urea, uric acid, levels of creatinine, total protein, as well as lactate dehydrogenase (LDH), N-acetyl-β-D-glucosaminidase (NAG) and γ-glutamil-transferase (GGT) activities were analyzed using a Cobas Mira automatic analyzer (Roche Pharmaceuticals, Basel, Switzerland) (Belles et al., 2007). The levels of 8-iso-prostaglandin F2α (8-iso-PGF2α), one isomer of the F2-isoP family, were analyzed in urine for assessment of oxidant stress in vivo. In the present study, urine samples were stored in aliquots of 1 mL containing 10 μL of butylated hydroxytoluene at −80 °C until analysis. The measurement of urinary F2-isoprostane (8iso-PGF2α) concentrations was performed based on the protocol of the competitive enzyme-linked immunosorbent assay (ELISA) kit from Oxford Biomedical Research (Deltaclon, Spain). The level of 8-isoPGF2α in each urine sample was normalized to that creatinine level [8iso-PGF2α (ng)/creatinine in urine (mg)] (Alonso et al., 2010). For the measurement of KIM-1 in urine (stored at −20 °C), ELISA test kit for the detection of KIM-1 in mouse (Aviscera Bioscience, Deltaclon, Spain) was used according to the manufacturer's instructions. The amount of KIM-1 in each urine sample was normalized to that creatinine level [Kim-1 (μg)/creatinine in urine (g)]. 292
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2.4. DNA damage quantification
Table 2 Urinary parameters in mice at 10 d after exposure to internal radiation.
8-hydroxy-2′-deoxyguanosine (8-OHdG) is an oxidized derivative of deoxyguanosine, which is generated by hydroxyl radicals, singlet oxygen, and one-electron oxidants in cellular DNA. Currently, 8OHdG is widely accepted as a sensitive marker of oxidative DNA damage. In the current investigation, kidney samples were washed in 0.9% saline and homogeneized in 0.2 M sodium phosphate pH 6.25 buffer (1:20, w/v) in a Potter-Elvehjem homogenizer, with a Teflon pestle (Braun, Melsungen, Germany). Total DNA was extracted using the commercially available DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany). The concentration of DNA in each sample was determined using an EPOCH™ 2 Microplate Espectrophotometer (BioTek, Winooski, Vermont, USA). The level of 8OH-dG was measured using EpiQuik™ 8-OHdG DNA Damage Quantification Direct Kit (Epigentek, Farmingdale, NY, USA) according to the manufacturer's instructions.
137
0
4000
8000
Urinary volume (mL/24 h) Creatinine (mg/24 h) Urea (mg/24 h) Total protein (mg/24 h) Uric acid (mg/24 h) Lactate dehydrogenase (LDH) (U/24 h/kg)
1.17 ± 0.22 0.82 ± 0.06 1.96 ± 0.13 0.35 ± 0.06 0.159 ± 0.016a 1.14 ± 0.08a
1.03 ± 0.11 0.70 ± 0.05 1.96 ± 0.38 0.31 ± 0.04 0.066 ± 0.004b 1.06 ± 0.21a
0.85 ± 0.12 0.76 ± 0.12 1.75 ± 0.22 0.23 ± 0.04 0.047 ± 0.007b 0.56 ± 0.07b
Cs activity (Bq/kg bw)
Values are expressed as mean ± S.E.M. Statistics: Values in the same row not showing a common superscript (a,b) are significantly different at p < 0.05.
(0.066 ± 0.004 versus 0.145 ± 0.013, p=0.005). At 72 h and 10 d after IR, the Cs8000 group showed a significant decrease in the activity of LDH with respect to mice in the control (0.79 ± 0.08 versus 1.63 ± 0.22, p=0.006 and 0.56 ± 0.07 versus 1.14 ± 0.08, p=0.0002, respectively) and the Cs4000 groups (0.79 ± 0.08 versus 1.10 ± 0.23, p=0.027 and 0.56 ± 0.07 versus 1.06 ± 0.21, p=0.0002, respectively). For the rest of urinary parameters no significant differences were noted between groups at 72 h and 10 d post-exposure. The effects of exposure to 137Cs in the GGT and NAG activities are depicted in Fig. 1(A-B). A significant increase in the GGT activity was observed in the animals at 72 h and 10 d after being exposed to Cs4000 with respect to the controls (9.72 ± 0.78 versus 6.18 ± 0.49, p=0.0117 and 11.45 ± 1.15 versus 5.18 ± 0.43, p=0.0002, respectively). However, for both times post-irradiation (72 h and 10 d postexposure), the activity of GGT decreased significantly in mice exposed to Cs8000 observing similar levels to those in control group (5.84 ± 0.33 versus 9.72 ± 0.78, p=0.0013 and 4.87 ± 0.49 versus
2.5. Data analysis All data are expressed as the mean ± standard error mean (S.E.M). Statistical analyses were performed using Graph Pad Prism® software version 5.01 (San Diego, California, USA). If variances were homogeneous, ANOVA was used followed by the Bonferroni's post-test correction for multiple comparisons. If the variances were not homogeneous, the Kruskal–Wallis test was then used. Differences between groups were analyzed using the Mann–Whitney U-test. Moreover, the paired t-test was used to compare the two different point of time tested. Statistical significance was set at p < 0.05. 3. Results Neither deaths, nor remarkable signs of external toxicity were observed in the groups given internal low doses of IR as 137Cs. Urinary parameters at 72 h and 10 d post-exposure to 137Cs are summarized in Tables 1 and 2, respectively. In comparison with the control and the Cs8000 group, the excretion of total protein was significantly enhanced in the Cs4000 group at 72 h post-exposure (0.46 ± 0.05 versus 0.26 ± 0.06, p=0.033 and 0.46 ± 0.05 versus 0.25 ± 0.030, p=0.004, respectively). However, this increase was restored 10 d after irradiation (0.31 ± 0.04 versus 0.46 ± 0.05, p=0.047). At 10 d post-exposure no differences between groups were noted. After 72 h of irradiation, uric acid levels in the Cs8000 group were significantly lower than those in the control and the Cs4000 group (0.071 ± 0.007 versus 0.131 ± 0.012, p=0.001 and 0.071 ± 0.007 versus 0.145 ± 0.013, p=0.0003, respectively). Moreover, at 10 d post-exposure, all irradiated mice showed significantly lower levels of uric acid than control animals (0.066 ± 0.004 versus 0.159 ± 0.016, p=0.0022 and 0.047 ± 0.007 versus 0.159 ± 0.016, p=0.002, respectively). A significant decline in the uric acid concentrations was also observed in the Cs4000 group between 72 h and 10 d after exposure Table 1 Urinary parameters in mice at 72 h after exposure to internal radiation. 137
0
4000
8000
Urinary volume (mL/24 h) Creatinine (mg/24 h) Urea (mg/24 h) Total protein (mg/24 h) Uric acid (mg/24 h) Lactate dehydrogenase (LDH) (U/24 h/kg)
0.87 ± 0.18 0.90 ± 0.06 1.52 ± 0.18 0.26 ± 0.06a 0.131 ± 0.012a 1.63 ± 0.22a
0.89 ± 0.14 0.72 ± 0.08 1.54 ± 0.14 0.46 ± 0.05b 0.145 ± 0.013a 1.10 ± 0.23a
0.90 ± 0.14 0.74 ± 0.10 1.39 ± 0.16 0.25 ± 0.03a 0.071 ± 0.007b 0.79 ± 0.08b
Cs activity (Bq/kg bw)
Fig. 1. γ-glutamil-transferase (GGT) (A) and N-acetyl-β-D-glucosaminidase (NAG) (B) activities in urine of mice at 72 h and 10 d post-exposure to internal radiation. Values are expressed as mean ± S.E.M. *p < 0.01, **p < 0.01 and ***p < 0.001 indicate significant differences between groups at 72 h and 10 d post-exposure. $p < 0.05 indicates significant differences between 72 h and 10 d for Cs8000 group.
Values are expressed as mean ± S.E.M. Statistics: Values in the same row not showing a common superscript (a,b) are significantly different at p < 0.05.
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Fig. 4. Percentage of 8-hydroxy-2′-deoxyguanosine (8-OHdG) in renal tissue in mice at 72 h and 10 d post-exposure to internal radiation. Values are expressed as mean ± S.E.M. ** p < 0.01 indicates significant differences between groups at 10 d post-exposure.
Fig. 2. Urinary F2-isoprostane (8-iso-PGF2α) levels in mice at 72 h and 10 d postexposure to internal radiation. Values are expressed as mean ± S.E.M. **p < 0.01 and *** p < 0.001 indicate significant differences between groups at 10 d post-exposure. $ $ p < 0.01 and $$$p < 0.001 indicate significant differences between 72 h and 10 d for each activity of 137Cs.
0.0029 ± 0.0002, p=0.0043). On the other hand, no significant differences between animals exposed to Cs8000 and the control group were noted.
11.45 ± 1.15, p=0.0006, respectively) (Fig. 1A). Similarly, a significant increase in the NAG activity was noted at 72 h after irradiation in the Cs4000 group when compared with the control (3.48 ± 0.13 versus 2.37 ± 0.25, p=0.0014) and the Cs8000 groups+ (3.48 ± 0.13 versus 2.57 ± 0.11, p=0.0001). Ten days after irradiation, the activity of NAG decreased significantly in mice exposed to Cs8000 compared to the Cs4000 group (2.08 ± 0.06 versus 3.23 ± 0.23, p=0.020) (Fig. 1B). Ionizing radiation induced changes in the urinary levels of 8-isoPGF2α at 10 d post-exposure (Fig. 2). In both groups of irradiated mice (Cs4000 and Cs8000), the levels of 8-iso-PGF2α were significantly increased compared with control animals (79.14 ± 3.62 versus 41.43 ± 4.34, p < 0.0001 and 65.14 ± 2.43 versus 41.43 ± 4.34, p=0.0005, respectively). Mice irradiated with Cs8000 showed significant lower levels than those exposed to Cs4000 (65.14 ± 2.43 versus 79.14 ± 3.62, p=0.0075). Furthermore, at 10 d post-exposure, 8-isoPGF2α levels were significantly enhanced in the Cs4000 and Cs8000 groups compared to the observed levels at 72 h (79.14 ± 2.43 versus 48.38 ± 2.67, p < 0.0001 and 65.14 ± 2.43 versus 50.00 ± 2.40, p=0.0016, respectively). The urinary levels of KIM-1 (Fig. 3) were significantly higher at 72 h post-exposure in mice irradiated at the lower 137Cs activity (Cs4000) when compared to control animals (0.49 ± 0.06 versus 0.22 ± 0.02, p=0.015). However, this significant increase was restored at 10 d after irradiation (0.29 ± 0.04 versus 0.49 ± 0.06, p=0.038). No differences between groups were noted at 10 d post-exposure. The percentage of 8-OHdG in renal tissue was higher in mice exposed to Cs4000 in comparison to the controls (Fig. 4)being statistically significant 10 d after exposure (0.0045 ± 0.0003 versus
4. Discussion Very serious nuclear accidents such as those occurred in Chernobyl and Fukushima, imply that many radionuclides are released into the environment (Hachiya et al., 2014; Lucchini et al., 2017). Even for those individuals who were not initially exposed to radioactivity, the radionuclides can reach their bodies some years later through the food chain (Beresford et al., 2016; Burger et al., 2000). Radiation-induced damage can result in adverse health effects within hours or days. Delayed effects are even observable a number of months after exposure (Kamiya et al., 2015). The renal tissue is considered critical in humans due its functions of filtration, reabsorption and retention of the tracer by the tubules (Ilhan et al., 2016). Moreover, experimental and clinical investigations have demonstrated that kidneys are highly sensitive to radiation injuries (Canyilmaz et al., 2016). Alterations in biochemical indices for renal function can be an early warning of alterations in these organs, ultimately leading to loss of functional integrity (El-Gazzar et al., 2016). Renal toxicity is one of the well known (acute) side effects of IR exposure, and can cause glomerular filtration rate impairment and proteinuria. Likewise, after the Fukushima nuclear disaster, human biochemical analysis detected an increase in the urinary excretion of proteins (Satoh et al., 2016). An increase of proteinuria is associated with renal pathology (Alonso et al., 2010; Jarad and Miner, 2009). In the current study, urine analysis showed an increase in the protein content of mice exposed to Cs4000 at 72 h. It has been reported that external irradiation in a rat model leads to the development of proteinuria (Imig et al., 2016). Furthermore, the current results showed low levels of uric acid, mainly in the Cs8000 group at 72 h and 10 d. Uric acid is a powerful non-enzymatic antioxidant, which has been identified as a radiation marker in urine of mice, being considered a natural free radical scavenger (Laiakis et al., 2014). The harmful effects of IR in biological systems are mainly mediated through the production of ROS in cells as result of water radiolysis (Reiter et al., 2011). Data on LDH activity in urine facilitate the detection of renal damage (Moon et al., 2013). In the present investigation, we observed that Cs8000 exposure caused a decreased in LDH activity, which indicates that after irradiation, the kidney shows a generally reduced metabolic enzyme activity, which is concomitant with a reduced kidney function. On the other hand, GGT is associated with incident vascular events (Fraser et al., 2007). It has been reported that the renal functional impairment caused by irradiation is partly mediated by vascular damage (Kruse et al., 2009). GGT is a key enzyme in the metabolism of GSH, being potentially useful for studying OS-related issues in both
Fig. 3. Urinary Kidney Injury Molecule-1 (KIM-1) levels in mice at 72 h and 10 d postexposure to internal radiation. Values are expressed as mean ± S.E.M. *p < 0.05 indicates significant differences between groups at 72 h post-exposure. $p < 0.05 indicates significant differences between 72 h and 10 d for Cs4000 group.
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Acknowledgements
epidemiological and clinical settings (Djavaheri-Mergny et al., 2002; Kowalczyk-Pachel et al., 2016). In the present study, GGT activity was mostly affected in mice exposed to Cs4000 at 72 h and 10 d. It has also been reported that the extracellular cleavage of GGT induces the production of ROS, suggesting that GGT plays a pro-oxidant role (Alonso et al., 2010). Increased urinary NAG excretion is one of the most sensitive biomarkers of renal toxicity, having been recommended for the detection of alterations in tubular function (Fiseha and Tamir, 2016). A relationship between urinary NAG excretion and renal oxidative damage has been reported (Oktem et al., 2006). In the present study, the urinary NAG activity increased significantly 72 h after Cs4000 irradiation. It agrees with the results of a previous investigation conducted in our laboratory, which showed an increase of NAG activity in urine after internal exposure to uranium (Belles et al., 2007). ROS and OS may contribute to radiation-induced cytotoxicity, as well as metabolic and morphologic alterations in humans and animals (El-Missiry et al., 2007; Zhao and Robbins, 2009). Measurement of F2IsoP, products of free-radical-catalyzed peroxidation of arachidonic acid, has been accepted as an accurate biomarker of OS and as a consequence of tissue damage (Leung et al., 2015; Silbert et al., 2016; Tucker et al., 2013). We have observed that urinary levels of 8-isoPGF2α were enhanced in all irradiated mice, mainly at Cs4000 and at 10 d post-exposure. To the best of our knowledge, data on the effects of internal IR on urinary levels of F2-IsoP are currently available. However, our results are consistent with those of previous studies on the effects of radiotherapy in renal function analyzing the urinary F2IsoP excretion (Machon et al., 2012). KIM-1 is the most upregulated protein in the proximal tubules following renal ischemic or toxic injury in humans and experimental animals (Baek et al., 2015; Pianta et al., 2017). Upregulation of KIM-1 has been observed in renal biopsies after radiotherapy in pediatric patients (Nepal et al., 2008). Recently, no significant increases in the urinary levels of KIM-1 were reported in children who underwent radiotherapy of the abdominal cavity (Yang et al., 2015). However, there is a lack of information about the effects of IR on the expression or urinary levels of KIM-1. Our results show an increase in the urinary levels of KIM-1 in irradiated mice, mainly in the group of Cs4000 at 72 h after exposure. Overproduction of free radicals that react with cell membrane fatty acids and proteins impairs their function. DNA is highly susceptible to oxidative damage, while 8-OHdG is a useful tool used as an indicator for OS and oxidative DNA (Kawanishi et al., 2016; Ozyurt et al., 2014). Moreover, a number of investigations showed that cellular DNA is the primary target for the toxic effects of IR, inducing strand breaks or base deletion (Glebova et al., 2015; Kavanagh et al., 2013; Yoshikawa et al., 2015). The current findings reveal that the internal radiation-induced damage in the kidney of irradiated mice with Cs4000 of activity, had significant increases in the percentage of 8-OHdG, being significant at 10 d post-exposure. The results of previous studies in which irradiationinduced oxidative DNA damage was demonstrated, support the results of this investigation. It has been reported that a single low dose wholebody irradiation in rats produced an increase in the kidney levels of 8OHdG (Ozyurt et al., 2014). Similarly, localized kidney irradiation in rats led to a marked, dose-independent increase, in glomerular and tubular cell nuclear DNA oxidation (Robbins et al., 2002). In summary, the results of the present study show that very low doses of internal IR induced not only early renal histologic injury and acute OS, but also caused DNA damage. While DNA alterations have been reported in laboratory animals exposed to very high doses of irradiation, to date, no data at such low internal doses were available. Anyhow, further investigations are required in order to better understand the long-term consequences of these effects, as well as the mechanisms involved in IR-induced nephrotoxicity.
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