FREE ACCESS

CELLULAR RESPONSES TO TRITIUM EXPOSURE IN RAINBOW TROUT: HTO- AND OBT-SPIKED FEED EXPOSURE EXPERIMENTS

Publication: CNL Nuclear Review27 May 2015https://doi.org/10.12943/CNR.2015.00059

Abstract

Biological effects were evaluated in rainbow trout (Oncorhynchus mykiss) exposed to tritiated water (HTO) or food spiked with organically bound tritium (OBT). An HTO exposure study was conducted using a tritium activity concentration of 7000 Bq/L, and an OBT exposure study was conducted using a tritium activity concentration of 30 000 Bq/L. Following 140 days of in vivo HTO exposure, liver, heart, spleen, kidney, and brain cells did not show statistically significant differences in viability; kidney, liver, and spleen cells did not show significant differences in DNA double-strand break repair activity compared with control cells. Membrane fatty acid composition analysis was conducted on liver cells and no effects of HTO exposure could be detected. Following 140 days of in vivo OBT exposure, viability and DNA double-strand break repair activity were not statistically different from controls in liver, heart, spleen, kidney, and brain cells. Changes, however, were noted in the fatty acid composition of liver and muscle tissues.
For both studies, all measurements were performed on each tissue and on a fraction of the same tissue that was exposed to a gamma 4 Gy dose in vitro to test for adaptive responses, and no effects were observed except for fatty acid composition. The findings demonstrated that membrane fatty acid composition is a sensitive marker and that microscopic evaluation of gamma-H2AX foci is more sensitive than the flow cytometric approach. These studies are the first to correlate uptake and depuration with biological health indicators in edible fish for tritium exposures within worldwide drinking water guidelines.

Résumé

Les effets biologiques ont été évalués en examinant la truite arc-en-ciel (Oncorhynchus mykiss) exposée à de l’eau tritiée (HTO) ou de la nourriture contenant du tritium lié aux composés organique (OBT). Une étude sur l’exposition au HTO a été menée selon une activité volumique du tritium de 7000 Bq/L, et une étude sur l’exposition à l’OBT a été réalisée selon une activité volumique du tritium de 30 000 Bq/L. Après 140 jours d’exposition in vivo au HTO, il n’y avait aucune différence statistiquement significative dans la viabilité des cellules du foie, du cœur, de la rate, du rein et du cerveau; il n’y avait non plus aucune différence statistiquement significative en ce qui concerne la réparation des cassures double brin (CDB) de l’ADN dans les cellules du rein, du foie et de la rate et les cellules témoins. Une analyse de la composition en acides gras de la membrane a été menée sur des cellules hépatiques et aucun effet d’exposition au HTO n’a été détecté. Après 140 jours d’exposition in vivo à l’OBT, il n’y avait aucune différence statistiquement significative dans la viabilité et l’activité de réparation des CDB de l’ADN des cellules du foie, du cœur, de la rate, du rein et du cerveau et les cellules témoins. Des changements ont toutefois été observés dans la composition en acides gras des tissus hépatiques et musculaires.
Dans le cadre des deux études, toutes les mesures ont été effectuées sur chacun des tissus et sur une partie du même tissu qui a été exposée à une dose de rayons gamma de 4 Gy in vitro afin d’étudier les réactions adaptatives; aucun effet n’a été constaté, sauf dans la composition en acides gras. D’après les résultats, la composition en acides gras de la membrane constitue un marqueur sensible, et l’évaluation microscopique par des foyers gamma-H2AX est plus sensible que par méthode de la cytométrie de flux. Ces études sont les premières à établir un lien entre l’absorption et l’élimination des indicateurs biologiques de la santé des poissons comestibles exposés au tritium conformément aux normes mondiales d’eau potable.

1. Introduction

Adam-Guillermin et al. [1] published an overview of the literature dealing with the effects of radionuclides released by nuclear power plants (including tritium) on DNA, development, and reproduction of aquatic organisms and noted a lack of data on the effects of tritium in fish. In fact, very few studies regarding the effects of tritium exposure in fish have been published to date. Most studies were conducted using a single fish species, e.g., medaka (Oryzias latipes) [24]. The medaka studies reported detrimental effects of tritium on developmental and reproductive endpoints at dose rates that are much higher than the ones used in the present studies.
Rainbow trout (Oncorhynchus mykiss) were exposed to tritiated water (HTO) or food spiked with organically bound tritium (OBT) (in the form of radio-labelled arginine, leucine, and lysine) for a period of up to 140 days to determine the rate of OBT formation of the 2 forms of tritium in edible fish tissue [5]. In both studies, conducted separately (in different years), some of the tissues obtained from those fish were used to evaluate if the activity concentration levels in fish tissues could be associated with measureable molecular or cellular changes. Because of the lack of data on the effects of tritium exposure in fish, especially at environmentally relevant levels, several different tissues (namely brain, heart, kidney, liver, and spleen) were harvested and biomarkers (cellular viability, quantity of DNA strand breaks (comet), expression of DNA double-strand break repair activity (gamma-H2AX) and membrane fatty acid composition) were evaluated at 130–148 days with and without subsequent in vitro exposure to a high dose (4 Gy) of 60Co gamma radiation (delivered at a dose rate of about 8 Gy/min) to assess the biological effects of these types of tritium exposures. This allowed the characterization of the effects of the tritium exposures and also the effects that these exposures have on the cellular response to a subsequent high dose. Measurements, on tissues exposed to 4 Gy, were conducted to test for adaptive responses as adaptive responses have been previously reported in frogs [6, 7].

2. Materials and Methods

2.1. Study design

The work described in this document was conducted under an animal care protocol approved by the Chalk River Animal Care Committee. The study designs (for the HTO and the OBT exposure studies), fish housing system, fish food preparation, fish handling, fish feeding regime, and fish weight measurement information are described in Kim et al. [5]. The HTO (in water) exposure study was conducted using a tritium activity concentration of 7000 Bq/L (to match the current Canadian drinking water limit) and the OBT-spiked feed exposure study was conducted using a tritium activity concentration of 30 000 Bq/L (fish in both studies were expected to be exposed to similar tritium levels, assuming 25% incorporation of the tritiated amino acids found in the feed) [811].
For both studies (conducted in different years), a control group containing a similar fish biomass was housed in an identical recirculating aquaculture system and fed the same diet as the corresponding test tank (with no tritiated amino acids in the case of the OBT exposure study) for the duration of the experiment. A summary of the quantities of feed given to the fish can be found in Kim et al. [5]. Both studies were conducted using 2-year-old juvenile trout. In the HTO study, 22 fish were placed in the control tank (total biomass of 1893 g, average fish weight of 86 g) and 23 fish were placed in the test tank (total biomass 1894 g, average weight of 82 g). After periodic sampling, at the end of the 140-day exposure period, 12 fish were left in the control tank (total biomass of 5088 g, average fish weight of 424 g) and 15 fish remained in the test tank (total biomass of 5318 g, average fish weight of 356 g). Some of these fish were used to obtain the data presented here and some were used to measure tissue HTO and OBT activity concentrations at that time point and after 30 days of depuration. For the OBT study, 31 fish were placed in the control tank (total biomass of 5766 g, average fish weight of 186 g) and 31 fish were placed in the test tank (total biomass of 5790 g, average fish weight of 283 g). At the end of the 140-day exposure period, 13 fish were left in the test tank (the average weight for the 5 fish used was 316 g) and 13 fish were left in the control tank (the average weight for the 5 fish used was 343 g). Again, some of these fish were used to obtain the data presented here and some were used to measure tissue HTO and OBT activity concentrations at that time point and after 30 days of depuration.
Fish were weighed and measured periodically to evaluate growth and condition. Fish condition was determined by measuring the standard length (from tip of the snout to base of the tail) and fish weight. The average weight per fish in the test tank was compared with the average weight per fish in the control tank.
As reported in Kim et al. [5], fish were harvested after 7–10, 70–79, and 130–148 days of exposure for measurement of HTO and OBT activity concentrations in muscle tissue and for biomarkers evaluation for both studies. The 130–148 days of exposure data are reported here. Test fish and control fish cell viability and DNA double-strand break repair activity measurements were not found to be statistically different after 7–10 or 70–79 days of exposure (data not presented). Fatty acid composition measurements were not conducted at these time points. No biomarker measurements were conducted during the depuration or at the conclusion of the 30-day depuration period.
Following 140 days of HTO exposure, the HTO and OBT activity concentrations in muscle tissue were 7920 ± 113 and 1550 ± 103 Bq/L, respectively. The control fish muscle tissue HTO and OBT activity concentrations were 490 ± 13 and 498 ± 182 Bq/L, respectively. These HTO and OBT analyses were conducted at a time when the tritium plume, from site activities, was elevated. This translated into a higher detection limit of about 250 Bq/L (normally 30–50 Bq/L) for the HTO exposure study. As both the test and the control tanks were in the same room, the test tank could have contributed to an elevation of the control tank tritium content.
After 140 days of OBT exposure, the HTO and OBT activity concentrations in muscle tissue were 47 ± 6 and 39370 ± 4210 Bq/L, respectively. The control fish muscle tissue HTO and OBT activity concentrations were 30 ± 6 and 220 ± 56 Bq/L, respectively. The detection limit was 30–50 Bq/L and no contribution from the test tank to the control tank was expected as the test fish were also in background water (and not tritiated water).
The levels listed above, and the values corresponding to the other time points, are taken from Kim et al. [5]. Five control fish and five test fish were sampled after about 140 days of HTO exposure (between days 130 and 142, simply to manage the work load) and after about 140 days of OBT exposure (between days 142 and 148, again to manage the work load) and used for biomarker evaluation as described below. It should be noted that 1 control fish was always paired with a test fish for sampling and analysis.

2.2. Tissue harvesting and preparation of cell suspensions for biomarker evaluation

The tissues specified in Tables 1 and 2 were aseptically collected. Cells in these tissues were mechanically separated in sterile phosphate buffered saline (PBS) by straining through sterile mesh (<1 mm) into a sterile tissue culture dish and reconstituted in 4 mL of PBS. The PBS was made with 137 mmol/L NaCl (Cat. # AC-8304, Anachemia Ltd.), 2.7 mmol/L KCl (Cat. # 790305, Fisher Scientific Canada), 6.3 mmol/L anhydrous Na2HPO4 (dibasic) (Cat. # S-0876, Sigma Chemical Company), 1.5 mmol/L KH2PO4 (monobasic) (Cat. # AC-7718, Anachemia Ltd.); was pH adjusted to 7.4 using dilute HCl; and was filter-sterilized using a 0.2 μm filter.
Table 1.
Table 1. Tissues used for biomarker evaluation during the HTO exposure study.
Table 2.
Table 2. Tissues used for biomarker evaluation during the OBT-spiked feed exposure study.
In both the HTO and OBT exposure studies, the cells from each tissue were evenly divided and transferred into 2 sterile tubes for each of the 5 control fish and 5 test fish. Immediately following this processing, 1 set of these duplicate samples was exposed to a high dose (4 Gy, at a dose rate of approximately 8 Gy/min) of 60Co gamma radiation in a GammaCell 220 (MDS Nordion). The second set of duplicate samples was brought to the GammaCell 220 but was not exposed. All samples were then placed on ice and aseptically divided for various tests as outlined in Tables 1 and 2 and described in Section 3.

3. Biomarker Evaluation Assays

3.1. Cellular viability assay

Cellular viability was assessed using propidium iodide (PI) according to the method described in Coder [12]. This was performed at room temperature approximately 1 hour following in vitro exposure to ionizing radiation using cells harvested from trout liver, heart, spleen, brain, and kidney. In a sterilized biological safety cabinet (BSC), 40 μL of cells suspended in PBS were diluted in 1 mL of sterile L-15 medium (Leibovitz) (Cat. # L4386, Sigma Aldrich) and filtered, using a 30 μm filter, into a Vi-cell sample cup (Cat. # 723908, Beckman Coulter Canada). Each cell suspension was mixed with 20 μL of PI (1.0 mg/mL, Cat. # P4864, Sigma Aldrich) and incubated in the dark at room temperature for 30 minutes. To ensure that this method could distinguish between live and dead cells, control samples (from in-house cell line cultures) containing various proportions of live and dead cells were also prepared.
The samples were processed on a Beckman Cell Lab Quanta SC MPL flow cytometer equipped with a 22 mW, 488 nm diode laser. The electronic volume (EV) was used as a trigger and the red PI (FL-3) fluorescence signal was acquired. The data were analysed using Beckman Cell Lab Quanta SC MPL Analysis software. Cells were gated based on size using the EV signal. Amongst the gated cell population, 2 cell populations were found. The dead cell population was discriminated from the live cell population based on FL-3 signal intensity. Dead cells showed a much higher signal compared with live cells because their compromised membranes allowed PI to enter the cells. An example is presented in Figure 1.
Figure 1.
Figure 1. Gating strategy used to evaluate cell viability. EV represents electronic volume and FL3 was used to collect red fluorescence.

3.2. Comet assay

The alkaline comet assay was conducted according to the method outlined in the product manual [13]. This assay was conducted using cells harvested from trout liver, spleen, and kidney. Immediately after the in vitro irradiation, 20 μL of each cell suspension were diluted in 1 mL of sterile PBS and 20 μL of this dilution were mixed with 200 μL of molten (37 °C) LMAgarose (Cat. # 4250-050-02, Trevigen Inc.) in a sterilized BSC. Seventy-five microlitres of the agarose-cell suspension were spread evenly across the surface of a comet slide (Cat. # 12-544-3, Fisher Scientific Canada), which was then placed in the dark at 4 °C for 30 minutes. Afterwards, each slide was transferred into cold lysis solution (Cat. # 4250-050-01, Trevigen Inc.) containing 10% dimethyl sulfoxide (Cat. # BP231-1, Fisher Scientific Canada) and incubated in the dark at 4 °C for 30 minutes. Each slide was then transferred into alkaline solution (prepared fresh by combining 0.6 g of NaOH pellets (Cat. # S318-500, Fisher Scientific Canada), 250 μL of EDTA (200 mM, pH 8.0) (Cat. # E-478, Fisher Scientific Canada), and 49.75 mL of double distilled-deionized water per 50 mL of solution) and incubated at room temperature in the dark for an additional 30 minutes.
Following removal from the alkaline solution, each slide was immersed in electrophoresis buffer and subjected to electrophoresis (at 1 Volt/cm and 0.3 A) in the dark at 4 °C for 30 minutes. The electrophoresis buffer (prepared fresh) consisted of 12 g of NaOH pellets (Cat. # S318-500, Fisher Scientific Canada) and 2 mL of EDTA (0.5 M, pH 8.0) (Cat. # E-478, Fisher Scientific Canada) dissolved in 1000 mL of double distilled-deionized water. Next, each slide was rinsed gently with cold (4 °C) deionized water, immersed in ice-cold methanol (Cat. # A412-1, Fisher Scientific Canada), and placed in the dark at 4 °C for 5 minutes. Each slide was again gently rinsed with cold (4 °C) deionised water before it was placed in absolute ethanol (Cat. # P006-EAAN, Commercial Alcohols Inc.) in the dark at room temperature for 5 minutes. Each slide was removed from the ethanol and left to dry at room temperature in the dark overnight.
After 50 μL of SYBR Green I working solution was evenly applied to each dry slide, the slides were placed in the dark at room temperature for 30 minutes. The SYBR Green 1 working solution was prepared by diluting SYBR Green I stain (10 000× in dimethyl sulfoxide) (Cat. # S9430, Sigma Aldrich) to 1000× in TE buffer (10 mM Tris HCl (Cat. # 819638 ICN Biochemicals), 1 mM EDTA (pH 7.5) (Cat. # E-5134 Sigma Aldrich). Finally, a cover slip was mounted over each slide and at least 100 cells were scored at 400× magnification using a fluorescent microscope. The scoring criteria (derived from da Silva et al. [14]) are presented in Table 3.
Table 3.
Table 3. Comet assay scoring criteria.
The percentage of cells in each class was determined and Equation (1) was used to calculate the comet index of each sample.
(1)

3.3. Gamma-H2AX measurements

Because histones are conserved between species, gamma-H2AX measurements can be carried out in trout using other species’ antibodies (e.g., Liu et al. [15]). Prior to conducting this work, the radiation-induced gamma-H2AX response (a quick increase followed by a decrease to background levels within a few hours) was confirmed in catfish cells (data not presented). This was not done in rainbow trout; however, increases in the number of foci were observed in trout as a result of high dose exposure to gamma radiation, suggesting that the staining was likely specific.
The gamma-H2AX assay was conducted using cells harvested from trout liver, heart, spleen, brain, and kidney. A fraction of suspension from each cell type was exposed to a 4 Gy dose and the cells were then immediately fixed in ice-cold 70% ethanol. After about 3 days of storage at −20 °C, the samples were centrifuged at 300× g for 8 minutes. Following removal of the supernatant and resuspension of the cell pellet, each sample was mixed with 10 mL of wash buffer (composed of PBS containing phosphatase inhibitors (10 mM sodium fluoride (Cat. # AC42432-0050, Fisher Scientific Canada), 1 mM sodium molybdate (VI) dihydrate (Cat. # AC20637-0050, Fisher Scientific Canada), 1 mM sodium metavanadate (V) (Cat. # 590088-5G, Sigma Aldrich), and 0.2% Triton X-100 (Cat. # T8787, Sigma Chemical Company), filter-sterilized using a 0.2-micron filter). Cells were then placed at room temperature for 10 minutes and centrifuged at 300× g for 8 minutes. After the supernatant was aspirated and the cell pellet was resuspended, each sample was mixed with 100 μL of antibody incubation buffer (prepared aseptically using filter-sterilized PBS and 0.2% Triton X-100; sterile 8% mouse serum (Cat. # M5905-10ML, Sigma Aldrich); and sterile 0.44 μg/mL fluorescein isothiocyanate (FITC) conjugated H2AX antibody (anti-phospho-histone H2AX (ser 139), FITC conjugate (mouse monoclonal IgG1) (Cat. # 16-202A, Millipore Canada)). The samples were incubated in the dark at 37 °C for 2 hours and mixed at 15 minute intervals. The samples were then stored overnight in the dark at 4 °C. Following removal from storage, each sample was mixed with 10 mL of wash buffer (without phosphatase inhibitors) and centrifuged at 300× g for 8 minutes. The supernatant was removed and the cell pellet was resuspended. Each sample was used to measure gamma-H2AX by means of fluorescence microscopy (foci) and flow cytometry (mean fluorescence intensity) as described below.

3.4. Expression of gamma-H2AX evaluated by fluorescence microscopy

Seven microlitres of each cell suspension were mixed on a microscope slide with 14 μL of 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI) (diluted to 0.025 μg/mL) (Cat. # D1306, Life Technologies Inc.). A cover slip was mounted on the slide and the cells were examined using a fluorescent microscope (630× or 100× magnification). The appropriate DAPI filter set was used to locate the DAPI-stained cell nuclei and then a FITC filter set was used to assess the FITC signal of the gamma-H2AX antibody. A minimum of 50 cells was scored based on FITC signal intensity according to the criteria presented in Table 4. These criteria were used (instead of counting the number of foci for each cell) to speed up scoring time. This approach enabled the use of a 4-position counter to count the number of cells in each scoring category for the entire slide and did not require the scorer to break eye contact with the slide for each cell (as would have been necessary to record the number of foci).
Table 4.
Table 4. Scoring criteria for the intensity of FITC signal associated with the gamma-H2AX antibody.
The percentage of cells with scores of 0, 1, 2, and 3 were determined and Equation (2) was used to calculate the relative gamma-H2AX signal.
(2)
The proportion of responding cells corresponds to the number of cells with a signal (scores 1, 2, and 3) divided by the total number of cells.

3.5. Expression of gamma-H2AX evaluated by flow cytometry

This protocol was developed according to Huang et al. [16]. Each cell suspension was diluted in 1000 μL of wash buffer (without phosphatase inhibitors). Three-hundred microlitres of each suspension were treated with 5 μL of RNAse (10 mg/mL, Cat. # R6513-50MG, Sigma Aldrich), filtered into a separate well in a 96-well plate, mixed with 15 μL of 7-amino-actinomycin D (7-AAD) (Cat. # IM3422, Beckman Coulter Canada), and incubated in the dark at room temperature for 30 minutes. Each sample was processed on the flow cytometer mentioned earlier.
The data were analyzed using Cell Lab Quanta SC MPL Analysis software. Cells were gated based on size using EV. Amongst the gated cell population, cells in G0 and G1 phases (nondividing cells) were discriminated based on 7-AAD signal intensity (red fluorescence signal collected using the FL-3 channel). G0 and G1 cells show a lower 7-AAD signal compared with dividing cells (S, G2, and M phases) because they contain less DNA to which 7-AAD can bind. At this point, using a gate on nondividing cells and a gate on dividing cells, it was verified that the mean gamma-H2AX signal (FITC green fluorescence observed using the FL-1 channel) was higher in the cells with a higher DNA content (dividing cells) compared with the nondividing cells. The mean gamma-H2AX signal (FL-1 signal) was obtained using a third gate that included nondividing and dividing cells and excluded DNA fragments (signal lower than the G0 and G1 signal) and cell aggregates (signal higher compared with the S, G2, and M signal). An example is presented in Figure 2.
Figure 2.
Figure 2. Gating strategy used to evaluate the mean gamma-H2AX signal. EV represents electronic volume, FL3 was used to collect red fluorescence, and FL1 was used to collect green fluorescence.
The mean signal was corrected for the proportion of dividing and nondividing cells as follows:
(3)
Controls were prepared along with the samples to be analyzed. They comprised nonirradiated, unstained cells taken from each tissue (liver, heart, spleen, brain, and kidney).

3.6. Membrane fatty acid composition analysis

Liver tissues were analyzed following HTO exposure and liver and muscle tissues were analyzed following OBT exposure. The muscle tissue was added for the OBT exposure study, because changes in muscle tissues were noted in frogs [7]. For each sample, after an hour at room temperature, 1 mL of suspended cells was aseptically collected in a sterile tube and stored at −20 °C. The samples were later shipped to the Lipidomic Laboratory (Lipinutragen, Bologna, Italy) for membrane fatty acid composition analysis.
The samples were processed by membrane pellet isolation, trituration with 2:1 chloroform:methanol (v/v), 2-phase extraction, derivation to fatty acid methyl ester (FAME), and analyses using gas chromatography as described in Ferreri et al. [17]. The following fatty acids were considered in the analysis: 14:0, 16:0, 16:1, 18:0, 18:1 trans, 18:1 (9cis), 18:1 (11cis), 18:2 omega-6, 18:2 (isomeric), 20:0, 20:1, 20:2 omega-6, 20:3 omega-6, 20:4, 20:4 trans, 20:3 omega-3, 22:0, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). The mean percentages of total fatty acids ± standard deviation (SD) were calculated for each sample group.
For both studies, the main fatty acids present in the trout liver were also compared with literature data. It was confirmed, using the obtained data, that the composition was representative of 2-year-old fish [18]. For the HTO exposure, fish were smaller compared with the OBT exposures. Using larger fish contributed to reducing the inter-individual variability.

3.7. Statistical analysis

Unless stated otherwise, the statistical analysis was conducted using the student’s t test comparing 2 groups of data. In cases where the equal variance test failed for the t test, a Mann–Whitney rank sum test was performed. The groups were said to be statistically different when p ≤ 0.05. For the membrane fatty acid composition analysis, statistical analysis was conducted by unpaired t test (95% confidence, 2-tailed, software GraphPad Prism 5).

4. Results and Discussion

Rainbow trout were chronically exposed to HTO or OBT, and biomarkers were evaluated to examine the biological effects of the tritium exposure alone or as an adapting regime when followed by a subsequent acute challenge dose of gamma radiation. When working with fish cell cultures and fish primary cultures, the authors’ laboratory normally uses a 4 Gy challenge dose instead of the 2 Gy challenge dose traditionally used in human cell studies. Recognizing the differences in sensitivities between life stages, species, and tissues, this choice was justified based on literature reporting that mature fish are generally more resistant to ionising radiation compared with humans [19, 20].
It should also be pointed out that the OBT levels in fish muscle tissue for the OBT exposure study were considerably higher compared with the muscle tissue OBT levels observed in the HTO exposure study at the 130–148 day sampling time points. The HTO levels in fish muscle tissues were, however, much lower for the OBT exposure study compared with the HTO exposure study.
All of the subsequent results are based on measurements taken from 5 control and 5 test fish at the end of the exposure period. To provide some context to the biomarker evaluation results, some information about the fish internal tritium exposure history at this sampling time point is helpful. The HTO and OBT levels observed in fish muscle tissue have been previously provided in Kim et al. [5].

4.1. Fish condition and behaviour of rainbow trout

Fish condition was determined by measuring the standard length and fresh weight of each fish. The conditions of all fish in each tank for the HTO and OBT exposures are shown in Figures 3 and 4, respectively. In both studies, the fish growth rates were comparable in both the control and test tanks. One observation that was made for both studies was that the fish in the test tank were more active compared with the fish in the control tank. Such levels of tritium exposures may therefore play a stimulatory role in muscles. Such observations were also made in amphibians at similar levels of tritium exposure (data not presented).
Figure 3.
Figure 3. HTO exposure study. Comparison of fish condition (based on standard length and weight) between the control tank and the test tank.
Figure 4.
Figure 4. OBT exposure study. Comparison of fish condition (based on standard length and weight) between the control tank and the test tank.

4.2. HTO and OBT exposure effects on cell viability

4.2.1. HTO exposure

Statistical analysis was carried out on the data obtained from 5 control fish and 5 fish exposed in vivo to HTO for 130–142 days. In this case, the cellular viability was evaluated in 5 different tissues (brain, heart, kidney, liver, and spleen).
Figure 5 indicates that, for the control tissues, exposure to 4 Gy of 60Co gamma radiation did not contribute to a decrease in viability (defined as cells that excluded propidium iodide) 1 hour post-exposure. Similar to the control tissues, exposure to 4 Gy in the HTO-exposed (test) tissues did not contribute to a decrease in viability. Loss of viability due to a 4 Gy exposure was not expected to be observed in that time frame and no time course analysis was conducted to assess longer term effects.
Figure 5.
Figure 5. HTO exposure study. The percentage of viable cells in brain, heart, kidney, liver, and spleen tissues after 130–142 days of HTO exposure (“test”) with and without challenge irradiation. Each bar represents an average for 5 fish and each error bar represents 2 σ.
No differences in the percentage of viable cells between the HTO-exposed fish and the control fish were observed. The brain tissue showed the highest viability (>90%), followed by kidney (>75%), spleen (>70%), heart (>65%), and liver (>55%). With the exception of the brain samples, it was noted that the tritium-exposed fish showed higher variability (larger error bars on measurements) in the percentage of viable cells compared with the control fish.

4.2.2. OBT-spiked feed exposure

For the OBT-spiked feed exposures, the viability assay was also conducted in brain, heart, kidney, liver, and spleen tissues. Statistical analysis was carried out on the data obtained from 5 control and 5 test fish exposed to OBT in vivo for 142–148 days (Figure 6). Again, as expected, exposure to 4 Gy of 60Co gamma radiation did not contribute to a decrease in viability 1 hour post-exposure in control and test tissues. Data resulting from the in vivo exposure to tritium were also examined. Based on the ratios of the OBT-exposed (test) fish to the control fish for a given tissue, no differences in the percentage viability between the tritium-exposed fish and the control fish were observed. The brain tissue showed the highest viability (about 90%), followed by kidney and liver (about 60%), and spleen and heart (about 40%). The differences in cellular viability noted between the controls for the HTO and OBT studies (conducted in different years) may be due to fish physiological differences, but could also be due to differences in the timing of the dissections and tissue processing.
Figure 6.
Figure 6. OBT- spiked feed study. The percentage of viable cells in brain, heart, kidney, liver, and spleen tissues after 142–148 days of OBT exposure (“test”) with and without challenge irradiation. Each bar represents an average for 5 fish and each error bar represents 2 σ.

4.3. DNA strand breaks (comet assay)

4.3.1. HTO exposure

The comet assay has been used to demonstrate the role of DNA repair, allowing the detection of the initial radiation-induced DNA breaks and providing a measure of the quantity of DNA double-strand breaks at a given time following that exposure. Following the changes in comet indices over time provides a way to evaluate how quickly the cells are repairing those breaks [21]. In this study, the comet assay was conducted in 3 different tissues (kidney, liver, and spleen) on 5 fish from the control tank and 5 fish from the HTO-exposure (test) tank. Measurements were taken 1 hour after exposure to the high dose of gamma radiation. Figure 7 indicates that, for the control fish, exposure to 4 Gy of 60Co gamma radiation contributed to an increase in the comet index in all tissues (although this was statistically significant only in the liver tissue). The same result was observed for the HTO-exposed test fish. Because of the small difference between the unexposed and 4 Gy-exposed measurements, it became clear that this assay was not going to be very sensitive. This is likely due to relatively low cell viability (about 75% for kidney, about 70% for spleen, and about 55% for liver). Adam-Guillermin et al. [1] used the comet and gamma-H2AX assays and reported no effects in fish cells exposed to a dose rate below 1 mGy/day. The dose rates produced in this HTO exposure study were well below this value.
Figure 7.
Figure 7. HTO exposure study. Comet average indices for kidney, liver, and spleen cells after 130–142 days of exposure. Each bar represents an average for 5 fish and each error bar represents 2 σ.
Despite the test limitations, it was noted that the values obtained between the 0 Gy control and HTO-exposed (test) samples were similar in the kidney and liver, but were higher in the HTO-exposed samples as compared with the control samples in the spleen (p = 0.028, student’s t test, unpaired), suggesting a higher level of endogenous DNA damage in response to chronic HTO exposure. In the 4 Gy series, the control and test sample indices were very similar for kidney and liver tissues, but were higher in the spleen tissue when the challenge dose was preceded by chronic tritium exposure (p = 0.003, student’s t test, unpaired), also suggesting more DNA damage in the spleen test samples after exposure to a high dose.

4.3.2. OBT-spiked feed exposure

Again, the comet assay was conducted in kidney, liver, and spleen on 5 fish from the control tank and 5 fish from the OBT exposure (test) tank following a 142–148 day exposure period (Figure 8). For this study, given the low dose rate compared to the previously reported comet and gamma-H2AX assay detection limit in fish [1], no marked effects were expected to be observed. The data obtained for the spleen cannot be considered for analysis as the majority of the cells were not viable (Figure 6). For the other tissues, relatively low cell viability (about 60% for the kidney and liver) and large inter-individual variability were noted. Although an increasing trend was observed between associated fish, no statistical differences were noted between the control group and the control group that received a 4 Gy dose. Furthermore, despite systematically higher indices in test fish compared with control fish, no significant differences in responses were noted between control and test fish in kidney and liver tissues.
Figure 8.
Figure 8. OBT-spiked feed study. Comet average indices for kidney and liver cells after 142–148 days of exposure. Each bar represents an average for 5 fish and each error bar represents 2 σ.

4.4. Expression of DNA double-strand break repair activity (gamma-H2AX assay)

4.4.1. HTO exposure

Gamma-H2AX allows one to follow the rapid DNA double-strand break repair kinetics [22]. It was used in this study to determine if the DNA breaks (quantitatively evaluated using the comet assay) seemed to be targeted for repair and to assess its usefulness as a biomarker. Statistical analysis was carried out on the data obtained from 5 control fish and 5 fish exposed in vivo to HTO. The gamma-H2AX data presented here can be linked to the comet data, as the samples were placed on ice while the comet slides were prepared. The samples were therefore fixed about 30 min following the 4 Gy irradiation. After in vivo exposure to tritiated water for 130–142 days, a tendency for the gamma-H2AX expression (scoring and percentage of responding cells (cells with foci), as obtained by microscopic evaluation) to be higher after a 4 Gy 60Co gamma radiation exposure (Figures 9 and 10) was observed. Despite the relatively low proportion of viable cells, this was statistically significant for most scoring data (Figure 9) and some of the data for the percentage of responding cells (Figure 10). This indicates that exposure to a 4 Gy dose increases the level of DNA double-strand damage (and potentially apoptosis) and that the repair enzymes are quickly recruited at the double-strand break. For the flow cytometric analysis, using normalized values (Figure 11), no significant differences in signal intensity were noted between the unchallenged (0 Gy) and the challenged (4 Gy-exposed) cells. This result was unexpected, as differences were seen microscopically. This lack of difference in the mean signal intensity is believed to be due to the presence of different degrees of background fluorescence in cells. This background signal, which is not associated with the presence of foci, probably reached levels corresponding to other cells with foci presenting a lower level of background fluorescence. The flow cytometric analysis of gamma-H2AX foci was, therefore, not a sensitive approach. Microscopic analysis remained the most sensitive methodology.
Figure 9.
Figure 9. HTO exposure study. Microscopic relative gamma-H2AX signal score in brain, heart, kidney, liver, and spleen cells after 130–142 days of exposure. Each bar represents an average for 5 fish and each error bar represents 2 σ. *Equal variance test failed for the t test; therefore, a Mann–Whitney rank sum test was performed on these data.
Figure 10.
Figure 10. HTO exposure study. Microscopic gamma-H2AX foci positive cells for brain, heart, kidney, liver, and spleen after 130–142 days of exposure. Each bar represents an average for 5 fish and each error bar represents 2 σ.
Figure 11.
Figure 11. HTO exposure study. flow cytometric normalized mean gamma-H2AX signal for brain, heart, kidney, liver, and spleen cells after 130–142 days of exposure. Each bar represents an average for 5 fish and each error bar represents 2 σ.
Overall (Figures 911), no statistically significant differences were noted between fish exposed to HTO in vivo (test) and controls. In addition, no significant differences in response to a 4 Gy dose were noted between control and test fish.

4.4.2. OBT-spiked food exposure

The gamma-H2AX assay was conducted in 5 different tissues (brain, heart, kidney, liver, and spleen) and statistical analysis was carried out on the data obtained from 5 control fish and 5 test fish. The scoring and percentage of responding cells (cells with foci) and the microscopic data for 142–148 days are presented in Figures 12 and 13. For microscopic scoring, statistical differences were noted between the in vitro 0 Gy and 4 Gy groups for all tissues. However, no significant differences in cell response were noted between test and control fish within each of the in vitro 0 Gy and 4 Gy groups. There was a tendency for a higher proportion of responding cells in the in vitro 4 Gy group compared with the 0 Gy group and this was statistically significant for all control tissues and all test tissues except for brain. For the flow cytometric analysis, the results presented in Figure 14 showed no significant differences. This is most likely due to the high noise level created by the varying cellular levels of background fluorescence. Again, the flow cytometric analysis of gamma-H2AX foci was not sensitive enough.
Figure 12.
Figure 12. OBT-spiked feed study. Microscopic relative gamma-H2AX signal score in brain, heart, kidney, liver, and spleen cells after 142–148 days of exposure. Each bar represents an average for 5 fish and each error bar represents 2 σ.
Figure 13.
Figure 13. OBT-spiked feed study. Microscopic gamma-H2AX foci positive brain, heart, kidney, liver, and spleen cells after 142–148 days of exposure. Each bar represents an average for 5 fish and each error bar represents 2 σ.
Figure 14.
Figure 14. OBT-spiked feed study. Flow cytometric normalized mean gamma-H2AX signal in brain, heart, kidney, liver, and spleen cells after 142–148 days of exposure. Each bar represents an average for 5 fish and each error bar represents 2 σ.

4.5. Tissue fatty acid composition analysis

4.5.1. HTO exposure

The data were obtained from 5 control and 5 test fish. Because both groups were given the exact same feed, there were no differences in fatty acid composition of the feed between the control and the test groups. The liver fatty acid composition analysis revealed large inter-individual variability and no statistically significant differences between test and control fish. The results are summarized in Table 5.
Table 5.
Table 5. HTO exposure study. Fatty acid methyl ester (FAME) composition of each rainbow trout liver after 130–142 days of exposure.

Note: Values are expressed as percentages of total fatty acids. Each value represents the mean of 5 fish. The fatty acid peaks recognized by standard references correspond to 99.5% of the total gas chromatograph peak areas. Errors are expressed as standard deviations. EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; UI, unsaturation index [(%monoenoic × 1) + (%dienoic × 2) + (%trienoic × 3) + (%tetraenoic × 4) + (%pentaenoic × 5) + (%hexaenoic × 6)]; PI, peroxidation index [(%monoenoic × 0.025) + (%dienoic × 1) + (%trienoic × 2) + (%tetraenoic × 4) + (%pentaenoic × 6) + (%hexaenoic × 8)].

4.5.2. OBT-spiked feed exposure

Data were obtained from 5 control and 5 test fish. Again, because both groups were given the exact same feed, there were no differences in fatty acid composition of the feed between the control and the test groups. The percentages of total FAME for each liver sample group (n = 5) are presented in Table 6. The percentages of total FAME for each muscle sample group (n = 5) are presented in Table 7.
Table 6.
Table 6. OBT-spiked feed study: fatty acid methyl ester (FAME) composition of each rainbow trout liver after 142–148 days of exposure.

Note: Values are expressed as percentages of total fatty acids. Each value represents the mean of 5 fish. EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; UI, unsaturation index [(%monoenoic × 1) + (%dienoic × 2) + (%trienoic × 3) + (%tetraenoic × 4) + (%pentaenoic × 5) + (%hexaenoic × 6)]; PI, peroxidation index [(%monoenoic × 0.025) + (%dienoic × 1) + (%trienoic × 2) + (%tetraenoic × 4) + (%pentaenoic × 6) + (%hexaenoic × 8)].

*
Indicates statistical differences. Errors are expressed as standard deviations.
Table 7.
Table 7. OBT-spiked feed study: fatty acid methyl ester (FAME) composition of each rainbow trout muscle after 142–148 days of exposure.

Note: Values are expressed as percentages of total fatty acids. Each value represents the mean of 5 fish. EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; UI, unsaturation index [(%monoenoic × 1) + (%dienoic × 2) + (%trienoic × 3) + (%tetraenoic × 4) + (%pentaenoic × 5) + (%hexaenoic × 6)]; PI, peroxidation index [(%monoenoic × 0.025) + (%dienoic × 1) + (%trienoic × 2) + (%tetraenoic × 4) + (%pentaenoic × 6) + (%hexaenoic × 8)].

*
Indicates statistical differences. Errors are expressed as standard deviations.

4.5.3. Effect of in vivo OBT exposure and in vitro 4 Gy 60Co gamma radiation exposure on trout liver tissue membrane fatty acid composition

The most abundant saturated fatty acid (SFA) present in liver was palmitic acid (16:0, representing 13%–25% of the total fatty acids). No significant changes in 16:0 were noted between the control and test fish. However, a reduction was observed for this type of fatty acid as well as for stearic acid (18:0, representing 3%–8% of the total fatty acids) and docosamic acid (22:0, representing <1% of the total fatty acids), leading to a reduction in total SFA. A reduction was also noted between test fish and test fish liver tissue exposed to 4 Gy 60Co gamma radiation. The reduction was not consistently observed between control fish and control liver tissue exposed to 4 Gy 60Co gamma radiation. Reductions in SFA can happen in 3 ways: reducing biosynthesis ab initio, increasing the transformation of SFA into monounsaturated fatty acids (MUFA) by activation of desaturases or increasing beta-oxidation, and scission of fatty acids for energy production (increasing the metabolic rate).
The MUFA data seem to be in favour of an increase in desaturation activity (desaturases). The most relevant MUFA is oleic acid (18:1, 9 cis), which represents 18%–25% of the total fatty acids. No corresponding increases in oleic acid were noted; however, palmitoleic acid (16:1, derived from palmitic acid and not attributable to dietary intake) and vaccenic acid (18:1 delta 11, cis, derived from the elongation and desaturation of palmitic acid) had a tendency to increase as the SFAs decreased. The behaviour of SFAs and MUFAs is summarized by the SFA/MUFA ratios, which changed significantly in the groups exposed to 4 Gy 60Co gamma radiation and to OBT-spiked food.
Because the present study does not take into account inter-individual differences in growth and metabolic rates, it is difficult to draw conclusions on the origin and consequences of the observed differences. However, parallels may be drawn with other studies. For example, the influence of the SFA/MUFA ratio on membrane properties has been studied [23] and, in this case, could point to changes (most likely increases) in fluidity and permeability. It is also worth noting that the increase of MUFA in tissue phospholipids has been correlated to the reduction of metabolic rate in some animal species [24]. However, other studies conducted in trout suggest that this may not always be the case [25, 26].
Polyunsaturated fatty acids (PUFA) represent a large family that includes linoleic acid (18:2 omega-6), arachidonic acid (20:4 omega-6), other intermediates of the omega-6 (like 20:2 omega-6 and 20:3 omega-6), eicosapentaenoic acid (20:5 omega-3), and docosahexaenoic acid (22:6 omega-3). The most abundant PUFA is docosahexaenoic acid (DHA), which represents 19%–31% of the total fatty acids for which no significant changes were observed.
Only one statistically significant change was noted. Linoleic acid (representing 7%–10% of the total fatty acids) was reduced in test fish compared with control fish exposed to 4 Gy of 60Co gamma radiation. The reduction of this fatty acid constituent cannot indicate an effect of the oxidative conditions associated with irradiation because the peroxidation randomly affects all PUFA fatty acids [27, 28]. Instead, it may be postulated that the reduction of the linoleic acid component can be correlated with the rearrangement of the lipid tissue content (in particular to the change of the SFA/MUFA ratio in the tissue phospholipids). If this were the case, this could suggest the induction of a metabolic shift due to the radiation exposure. The data do not exclude a connection between irradiation and an induced turnover of phospholipids that influence cell signaling. It is known that PUFA levels in membrane lipids correlate with p53 levels [29, 30] and also that gamma irradiation is connected with p53 induction and the apoptotic response. However, to date, no systematic research has been performed on membrane lipid remodelling due to irradiation conditions and the related membrane signalling.
A tendency for a reduction in trans-fatty acid content was also observed between control fish and test fish exposed to 4 Gy of 60Co gamma radiation. Such a change could indicate a reduction in thiol compounds (sulphur-containing antioxidants) that are known to be responsible for S-centered radical production under irradiation that causes the formation of endogenous trans-fatty acids [31].

4.5.4. Effect of in vivo OBT exposure and in vitro 4 Gy 60Co gamma radiation exposure on trout muscle tissue membrane fatty acid composition

The most abundant SFA in muscle is palmitic acid (16:0, representing 13%–26% of the total fatty acids). No significant changes in 16:0 were noted between control and test fish. However, a reduction observed for stearic acid (18:0) contributed to a reduction in total SFAs. A reduction in total SFA was also noted between test fish and test fish muscle tissue exposed to 4 Gy of 60Co gamma radiation. The reduction was not consistently observed between control fish and control fish muscle tissue exposed to 4 Gy of 60Co gamma radiation. The MUFA data seem to be in favour of an increase in desaturation activity (desaturases). The most relevant MUFA is oleic acid (18:1, 9 cis; representing 17%–23% of the total fatty acids). No corresponding increases in oleic acid were noted; however, palmitoleic acid (16:1, derived from palmitic acid) and vaccenic acid (18:1 delta 11, cis; derived from the elongation and desaturation of palmitic acid) had a tendency to increase as the SFAs decreased. The behaviour of SFAs and MUFAs are summarized by the SFA/MUFA ratios. Such trends could indicate a change in membrane performance due to radiation exposure. In addition, MUFAs (which are not so readily oxidizable compared with PUFAs) can play a protective role, as they produce favourable membrane properties.
The most abundant PUFA is docosahexaenoic acid (DHA, representing 15%–30% of the total fatty acids). Unlike the liver tissue, a tendency for a reduction in omega-3 between control and test fish was noted in muscle tissue. This could be connected to the oxidative status in the tissue and the consumption of PUFA omega-3 (which contain more double bonds than the omega-6 fatty acids). This reduction could be also due to a mobilization of PUFA omega-3 from muscle toward other tissues like liver, perhaps due to a higher metabolic requirement of liver mitochondria and an increase in beta-oxidation [32].
The lack of significant differences between the nonirradiated and the 4 Gy-irradiated tissues was noted throughout. This phenomenon has already been observed in sea bream (with significant differences observed only at 5 kGy irradiation) [33]. Such a lack of response could also be due to the lack of time allowed for changes to take place.

5. Conclusions

This study is unique. A few studies have been previously conducted to evaluate tritium uptake in fish; however, all were conducted using small fish or were part of monitoring programs [3437]. This study was primarily designed to evaluate tritium (particularly the organically bound fraction) uptake, under controlled conditions, in edible fish [5]. As none of the previously cited studies reported biological effects and because biological effect studies in fish exposed to tritium have generally been conducted at higher doses than the present study [24], some tissues were reserved for this purpose.
Throughout both studies reported here, no statistically significant differences in fish general condition, cell viability, DNA strand breaks, and DNA double-strand break repair activity were noted between the control and test fish. For cell viability, quantity of DNA breaks (comet), and gamma-H2AX expression, no indications of adaptive responses were noted, as the response to the 4 Gy dose was the same for exposed fish as for control fish. Membrane fatty acid composition analysis was conducted on the liver cells and no effects of HTO exposure could be detected. However, some significant changes to membrane fatty acid composition of muscle and liver tissues were noted following OBT exposure, suggesting a potential lipid remodelling of liver and muscle tissues in response to the environmental conditions. Because no statistical differences in other biomarkers (cellular viability, quantity of DNA strand breaks (comet), and expression of DNA double-strand break repair activity (gamma-H2AX)) were detected in fish, changes in lipid composition may be an early and sensitive indicator of tritium effects. Although further research is needed to understand in vivo lipid remodelling, the data could point to a link between radiation exposure and cell signalling. Furthermore, it suggests that the membrane fatty acid asset could be the point of initiation for cell signalling.

Acknowledgments

The work was conducted under the CNL Research and Development program. The assistance for analytical and statistical treatments of the fatty acid analysis by Dr. Valentina Sund and Mr. Simone Deplano (Lipinutragen, SRL, Bologna, Italy) is gratefully acknowledged. The authors would also like to thank Dr. Dominic, Bureau of the Department of Animal and Poultry Science, University of Guelph, Ontario, for his assistance with the preparation of the fish feed.

REFERENCES

[1]
Adam-Guillermin C., Pereira S., Della-Vedova C., Hinton T., and Garnier-Laplace J., 2012, “Genotoxic and Reprotoxic Effects of Tritium and External Gamma Irradiation on Aquatic Animals,” Reviews of Environmental Contamination and Toxicology, 220, pp. 67–103.
[2]
Hyodo-Taguchi Y. and Egami N., 1977, “Damage to Spermatogenic Cells in Fish Kept in Tritiated Water,” Radiation Research, 71(3), pp. 641–652.
[3]
Etoh H. and Hyodo-Taguchi Y., 1983, “Effects of Tritiated Water on Germ Cells in Medaka Embryos,” Radiation Research, 93(2), pp. 332–339.
[4]
Hyodo-Tagushi Y. and Etoh H., 1993, “Vertebral Malformations in Medaka (Teleost Fish) after Exposure to Tritiated Water in the Embryonic Stage,” Radiation Research, 135(3), pp. 400–404.
[5]
Kim S.B., Shultz C., Stuart M., McNamara E., Festarini A., and Bureau D.P., 2013, “Organically Bound Tritium (OBT) Formation in Rainbow Trout (Oncorhynchus mykiss): HTO and OBT-Spiked Food Exposure Experiments,” Applied Radiation and Isotopes, 72, pp. 114–122.
[6]
Audette-Stuart M., Kim S.B., McMullin D., Festarini A., Yankovich T.L., Carr J., and Mulpuru S., 2011, “Adaptive Response in Frogs Chronically Exposed to Low Doses of Ionizing Radiation in the Environment,” Journal of Environmental Radioactivity, 102(6), pp. 566–573.
[7]
Audette-Stuart M., Ferreri C., Festarini A., and Carr. J., 2012, “Fatty Acid Composition of Muscle Tissue Measured in Amphibians Living in Radiologically Contaminated and Non-contaminated Environments,” Radiation Research, 178, pp. 173–181.
[8]
Fauconneau B. and Arnal M., 1985, “In vivo Protein Synthesis in Different Tissues and the Whole Body of Rainbow Trout (Salmo gairdnerii R). Influence of Environmental Temperature,” Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 82(1), pp. 179–187.
[9]
McMillan D.N. and Houlihan D.F., 1988, “The Effect of Refeeding on Tissue Protein Synthesis in Rainbow Trout,” Physiological Zoology, 61(5), pp. 429–441.
[10]
Garatun-Tjeldstø O., Otterå H., Julshamn K., and Austreng E., 2006, “Food Ingestion in Juvenile Cod Estimated by Inert Lanthanide Markers – Effects of Food Particle Size,” ICES Journal of Marine Science, 63(2), pp. 311–319.
[11]
National Research Council, 2011, Nutrient Requirements of Fish and Shrimp, The National Academies Press, Washington, DC.
[12]
Coder D.M., 1997, “Assessment of Cell Viability,” Current Protocols in Cytometry, pp. 9.2.1–9.2.14.
[13]
Trevigen® Instructions, 2002, “CometAssay™ Reagent Kit for Single Cell Gel Electrophoresis Assay Catalog # 4250-050-K,” Trevigen, Inc.
[14]
da Silva J., de Freitas T.R.O., Marinho J.R., Speit G., and Erdtmann B., 2000, “An Alkaline Single-Cell Gel Electrophoresis (Comet) Assay for Environmental Biomonitoring with Native Rodents,” Genetics and Molecular Biology, 23(1), pp. 241–245.
[15]
Liu M., Tee C., Zeng F., Sherry J.P., Dixon B., Bols N.C., and Duncker B.P., 2011, “Characterization of p53 Expression in Rainbow Trout,” Comparative Biochemistry and Physiology, Part C, 154(4), pp. 326–332.
[16]
Huang X., Halicka H.D., and Darzynkiewicz Z., 2004, “Detection of Histone H2AX Phosphorylation on Ser-139 as an Indicator of DNA Damage (DNA Double-Strand Breaks),” Current Protocols in Cytometry, pp. 7.27.1–7.27.7.
[17]
Ferreri C., Kratzsch S., Brede O., Marciniak B., and Chatgilialoglu C., 2005, “Trans Lipid Formation Induced by Thiols in Human Monocytic Leukemia Cells,” Free Radical Biology and Medicine, 38(9), pp. 1180–1187.
[18]
Görgün S. and Akpinar M.A., 2007, “Liver and Muscle Fatty Acid Composition of Mature and Immature Rainbow Trout (Oncorhynchus mykiss) Fed Two Different Diets,” Biologia, 62(3), pp. 351–355.
[19]
Hinton T.G., Alexakhin R., Balonov M., Gentner N., Hendry J., Prister B., Strand P., and Woodhead D., 2007, “Radiation-Induced Effects on Plants and Animals: Findings of the United Nations Chernobyl Forum,” Health Physics, 93(5), pp. 427–440.
[20]
R. Swarup, S.N. Mishra and V.P. Jauhari, 1992, Encyclopaedia of Ecology, Environment and Pollution Control: Marine Environment and Analysis, Mittal Publications, New Delhi, India.
[21]
Olive P.L., 2009, “Impact of the Comet Assay in Radiobiology,” Mutation Research/Reviews in Mutation Research, 681(1), pp. 13–23.
[22]
Kuo L.J. and Yang L.X., 2008, “γ-H2AX – A Novel Biomarker for DNA Double-Strand Breaks,” In vivo, 22(3), pp. 305–309.
[23]
G. Cevc, 1993, Phospholipids Handbook, Marcel Dekker, Inc., New York.
[24]
Hulbert A.J. and Else P.L., 2000, “Mechanisms Underlying the Cost of Living in Animals,” Annual Review of Physiology, 62, pp. 207–235.
[25]
Guderley H., Kraffe E., Bureau W., and Bureau D.P., 2008, “Dietary Fatty Acid Composition Changes Mitochondrial Phospholipids and Oxidative Capacities in Rainbow Trout Red Muscle,” Journal of Comparative Physiology B, 178(3), pp. 385–399.
[26]
Martin N., Bureau D.P., Marty Y., Kraffe E., and Guderley H., 2013, “Dietary Lipid Quality and Mitochondrial Membrane Composition in Trout: Responses of Membrane Enzymes and Oxidative Capacities,” Journal of Comparative Physiology B, 183(3), pp. 393–408.
[27]
Niki E., Yoshida Y., Saito Y., and Noguchi N., 2005, “Lipid Peroxidation: Mechanisms, Inhibition, and Biological Effects,” Biochemical and Biophysical Research Communications, 338(1), pp. 668–676.
[28]
Porter N.A., Caldwell S.E., and Mills K.A., 1995, “Mechanisms of Free Radical Oxidation of Unsaturated Lipids,” Lipids, 30(4), pp. 277–290.
[29]
Zhang X.H., Zhao C., and Ma Z.A., 2007, “The Increase of Cell-Membranous Phosphatidylcholines Containing Polyunsaturated Fatty Acid Residues Induces Phosphorylation of p53 through Activation of ATR,” Journal of Cell Science, 120(23), pp. 4134–4143.
[30]
Midgley C.A., Owens B., Briscoe C.V., Thomas D.B., Lane D.P., and Hall P.A., 1995, “Coupling between Gamma Irradiation, p53 Induction and the Apoptotic Response Depends Upon Cell Type in vivo,” Journal of Cell Science, 108(5), pp. 1843–1848.
[31]
Chatgilialoglu C., Ferreri C., Lykakis I.N., and Wardman P., 2006, “Trans-Fatty Acids and Radical Stress: What Are the Real Culprits?,” Bioorganic & Medicinal Chemistry, 14(18), pp. 6144–6148.
[32]
Clarke S.D., 2001, “Nonalcoholic Steatosis and Steatohepatitis. I. Molecular Mechanism for Polyunsaturated Fatty acid Regulation of Gene Transcription,” American Journal of Physiology: Gastrointestinal and Liver Physiology, 281(4), pp. G865–G869.
[33]
Erkan N. and Özden Ö., 2007, “The Changes of Fatty Acid and Amino Acid Compositions in Sea Bream (Sparus aurata) during Irradiation Process,” Radiation Physics and Chemistry, 76(10), pp. 1636–1641.
[34]
Komatsu K., Higuchi M., and Sakka M., 1981, “Accumulation of Tritium in Aquatic Organisms through a Food Chain with Three Trophic Levels,” Journal of Radiation Research, 22(2), pp. 226–241.
[35]
Rodgers D.W., 1986, “Tritium Dynamics in Juvenile Rainbow Trout, Salmo gairdneri,” Health Physics, 50(1), pp. 89–98.
[36]
Hunt G.J., Bailey T.A., Jenkinson S.B., and Leonard K.S., 2010, “Enhancement of Tritium Concentrations on Uptake by Marine Biota: Experience from UK Coastal Waters,” Journal of Radiological Protection, 30(1), pp. 73–83.
[37]
D. Eaton and C.E. Murphy, Jr., 1992, “Tritium Uptake by Fish in a Small Stream (U),” Westinghouse Savannah River Company, WSRC-TR-92-193 (Rev. 1).

Information & Authors

Information

Published In

CNL Nuclear Review cover image
CNL Nuclear Review
Volume 5Number 1June 2016
Pages: 155 - 172

History

Received: 11 March 2015
Accepted: 21 May 2015
Published as e-First: 27 May 2015
Published online: 27 May 2016

Permissions

Request permissions for this article.

Key Words

  1. Tritium
  2. HTO and OBT exposure
  3. HTO and OBT concentration
  4. fish
  5. cellular responses

Mots clés :

  1. exposition au HTO et à l’OBT
  2. teneur en HTO et en OBT
  3. poisson
  4. réactions cellulaires

Authors

Affiliations

Amy Festarini amy.festarini@cnl.ca
Canadian Nuclear Laboratories, Chalk River, ON K0J 1J0, Canada.
Carmen Shultz
Canadian Nuclear Laboratories, Chalk River, ON K0J 1J0, Canada.
Marilyne Stuart
Canadian Nuclear Laboratories, Chalk River, ON K0J 1J0, Canada.
Sang Bog Kim
Canadian Nuclear Laboratories, Chalk River, ON K0J 1J0, Canada.
Carla Ferreri
Department of Chemical Sciences and Materials Technologies of the National Research Council of Italy, Bologna, Italy.

Notes

A correction was made to the e-First version of this paper on 9 June 2016 prior to final issue publication. The current online and print versions are identical and both contain the correction.

Metrics & Citations

Metrics

Other Metrics

Citations

Cite As

Export Citations

If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download.

There are no citations for this item

View Options

View options

Full Text

Open Full Text

PDF

Download PDF

Get Access

Media

Figures

Other

Tables

Share Options

Share

Share the article link

Share with email

Share on social media