Environ. Sci. Technol. 2006, 40, 6859-6864
Communication of Radiation-Induced Stress or Bystander Signals between Fish in Vivo C . M O T H E R S I L L , * ,†,‡ C . B U C K I N G , † R. W. SMITH,† N. AGNIHOTRI,‡ A. O’NEILL,‡ M. KILEMADE,‡ AND C . B . S E Y M O U R †,‡ Department of Medical Physics and Applied Radiation Sciences, McMaster University, 1280 Main Street West, Hamilton, Ontario, Canada L8S 4K1, and Juravinski Cancer Centre, 699 Concession Street, Hamilton, Ontario, Canada L8V 5C2
We report data in this paper suggesting that fish irradiated to 0.5 Gy total body dose can release factors into the water that signal other unexposed fish and cause induction of bystander effects expressed as increased cell death in a reporter system. Radiation-induced bystander effects, resulting in the appearance of radiation damage or induction of typical radiation responses in unirradiated cells and tissues are now an established consequence of exposure to low doses of ionizing radiation, however little work has been done in vivo or in species other than humans or mice. In these experiments rainbow trout were irradiated and then paired with unirradiated fish for two hours. Additionally, unirradiated fish were placed in water which had previously been used to hold irradiated fish for 2 h. Sham-irradiated fish and absolute control fish were also examined all using blind protocols. Following a two h incubation period, at these various exposure regimes, the fish were killed by a blow to the head and dissected. Five organs were removed from each fish and tissue explants were cultured using an established technique. After 2 days, the culture medium was harvested and used in a reporter assay to determine whether a bystander effect had been induced. The explants were cultured on in Clonetics growth medium for a further 14 days then fixed for assay of radiation response proteins. The responses varied according to the cell type in the original explants, with the gill and fin showing the most pronounced response. The results suggest that communication signals leading to a typical radiation response can be passed between fish and seem to involve secretion of a chemical messenger into the water.
Introduction Bystander effects, as described here, are biological effects detected in cells not themselves exposed to ionizing radiation, but which receive signals from irradiated cells and respond as if they had received the dose. There are many excellent reviews of the field (e.g., 1-4). In the radiobiology field, bystander effects have been recorded since 1921 (5) but have * Corresponding author phone: 905 525 9140, ext. 26227; fax: 905 522 5983; e-mail:
[email protected]. † McMaster University. ‡ Juravinski Cancer Centre. 10.1021/es061099y CCC: $33.50 Published on Web 09/20/2006
2006 American Chemical Society
enjoyed renewed interest for 2 reasons: first, there is great concern about the biological effects and mechanisms of low doses of radiation (6-8); and second, there are novel molecular biological tools which can detect very subtle changes in the genome and the epigenome (9-11). Microbeams, which allow precise targeting of single cells or cellular organelles with radiation have also facilitated the growth of this research field (12, 13). The major bystander effects recorded are gene and protein induction, mutations, growth delay, apoptosis, and neoplastic transformation. All these effects can be seen at very low doses in the environmentally relevant range (below 10 mGy) and there is some evidence for a threshold around 2-3 mGy (14, 15). The “dose response” is typically saturated, with no increase in effect with dose for a given set of conditions, however, the effect is increased if higher numbers of cells are exposed to the radiation dose (16), and can be titrated, suggesting a transmissible “factor” which is produced by cells receiving the actual dose. While there are hundreds of papers in the literature concerning bystander effects in vitro in mammalian cells, and even a few describing in vivo effects in mice (see reviews 1-4), there are few data concerning lower vertebrates or invertebrates. However, recent concerns about environmental radiation effects on biota means this field is now critically important. Outside the radiation field, chemical signaling in fish and invertebrates is very well accepted (see for example 17-20), and given the ubiquity of bystander effects in mammalian systems, it would be surprising if they were not found in fish. Previous work by our group has confirmed that bystander effects do indeed occur in vitro in cultures of fish and crustacean tissues and in fish cell lines, (21-24). Using a reporter system, we have also shown that tissues of fish (rainbow trout) vary in the type and severity of the response induced by transfer of the signals from the irradiated tissue to a reporter cell line (25). Rainbow trout are excellent models for looking at the relevance of low radiation doses and bystander effects in nonhuman biota. They are well-established laboratory models and there are cell lines and molecular biological tools available to aid experimental investigations. They are also regarded as “sentinal species” in the aquatic ecosystems because of their sensitivity to water quality (26). They are also directly assimilated in the human food chain and can act as surrogates for other commercially important Salmonids, e.g., Atlantic and Pacific Salmon (Salmo salar and Oncorhynchus species, respectively) and Brown Trout (S. trutto). An additional advantage for these studies is that in vivo irradiation of rainbow trout should permit the putative bystander factors to be secreted into the water. This makes the chemistry involved in identifying the signal molecules much simpler. All attempts to date to identify the signal have failed. This is mainly because of the difficulty of detecting biological molecules in the background of culture medium containing serum. Even though induction of bystander effects in vivo can be demonstrated in fish and mammals (25, 27, 28), it has so far not been possible to demonstrate both induction of the effect and organism response to the effect in vivo due to the technical problem of transferring the unknown signal to another animal and measuring its effect in that recipient. This makes applied scientists, such as clinicians and environmental protection agencies, doubt the significance of the bystander effect. It is often suggested that it is an artifact of cell culture, and the recent BEIR 7 report on the biological VOL. 40, NO. 21, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Schematic illustrating the design of the experiment and the subsequent processing of tissue samples to detect the signal. effects of ionizing radiation (29) concluded that it was too early to assess whether bystander effects had any relevance for risk assessment. The existing in vivo work suggests that signal production at least is dependent on genotype, with apoptosis prone genotypes producing factors which initiate apoptosis in reporter cell lines and cancer prone genotypes releasing factors which, if anything, stimulate growth of the same reporter cells (27, 28). In this paper we describe an experiment where fish which had not been irradiated were allowed to swim with irradiated fish or in water previously occupied by irradiated fish. Bystander effects were looked for in 5 tissues from all these animals and in parallel shamirradiated control groups. We hypothesized that explants obtained from unirradiated fish exposed in vivo to irradiated fish or to water previously containing irradiated fish would produce bystander effects. Additionally, we predicted that explants taken from the external organs we examined would produce greater effects than those from our chosen internal organs. The latter hypothesis was based on the suggested chemical nature of the bystander factor, which is quantitatively involved in producing the cellular effects, and thus might be expected to bind to the first tissues it encountered (2).
Materials and Methods Experimental Animals. Juvenile freshwater rainbow trout (Oncorhynchus mykiss, W), of both genders and ranging in mass 85-125 g, were obtained from Humber Springs Trout Farm (Orangeville, ON). The animals were acclimated to laboratory conditions for a minimum of 21 days before experimentation. The animals were housed at a density of 1 g mass 10 L-1, and supplied with flow-through dechlorinated Hamilton (ON) city tap water [Na+ ) 0.6; Cl- ) 0.7; K+ ) 0.05; Ca2+ ) 0.5; Mg2+ ) 0.1; titration alkalinity (to pH 4.0) ) 1.9 meq L-1; total hardness ) 140 mg L-1 as CaCO3; pH 8.0]. The water was temperature-controlled to approximate seasonal conditions (10-13 °C). The fish were fed commercial fish food (Martin Mills, ON) at a 2% of body mass ration every day, however feeding was suspended for one week prior to experimentation. Irradiation Protocol. The experimental design is shown in Figure 1. Briefly, there were 7 groups of fish. The first 6860
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group (referred to as irradiated fish) were placed inside a portable X-ray machine (Faxitron X-ray Corporation cabinet X-ray system; Wheeling, IL) and irradiated at 100 kVp (kilovolts peak) for 5 min. This delivered an average dose of 0.5 Gy to the entire animal, which was previously calibrated using thermoluminescence discs (TLDs). The second group of fish (sham-irradiated fish) was placed into the X-ray machine which, in this case, was not turned on. Immediately after irradiation (sham or actual), these two groups were placed into separate static partitioned holding tanks and held for 2 h to accumulate any signals in the water. Following this, both the sham and irradiated fish were transferred to another static partitioned tank, and naive fish were placed with them. These were groups 3 and 4, which are referred to as “partner fish” (placed with group 1 fish) and “sham partner fish” (placed with group 2 fish). Fish in groups 3 and 4 received no direct radiation, and were statically housed with irradiated and sham fish for 2 h. The fifth and sixth groups of fish were statically housed for 2 h in the water that groups 1 and 2 previously occupied and were referred to as water-borne exposed fish and sham water-borne exposed fish, respectively. The seventh group of fish served as additional controls. They were placed into the static partitioned holding tanks for 2 h, but were otherwise untreated. The partitioned tanks were 25 L in volume, and continuously aerated for the duration of the experiment. Sampling Protocol. Following the 2 h holding period, fish were sacrificed by cephalic concussion and skin, fin, gill, kidney, and spleen were removed asceptically in a biological safety cabinet over ice, and placed in 1 mL of complete growth medium and transported to the tissue culture laboratory. All tissue was obtained and treated according to guidelines at McMaster University and the procedures were also covered by AUP 06-21-01. The growth medium used for all experiments and all cells was RPMI-1640. This was supplemented with 10% Foetal bovine serum, 1 µg/mL hydrocortisone (Sigma Chemical Co., Poole, Dorset, UK), 5 mL of penicillin/ streptomycin solution, 1 mL (100 I.U.) of insulin (Sigma tissue culture tested, Poole, Dorset, UK), and 5 mL of L-glutamine solution. Hepes buffer (12.5 mL of IM solution) was added to help maintain pH. Except where indicated, all reagents were obtained from Gibco Biocult, USA.
TABLE 1. Means ( Standard Errors of the Mean (n ) 8) for the Clonogenic Survival % of HPV-G Reporter Cells Exposed to Medium Harvested from Each of Five Organs from Seven Treatment Groups of Rainbow Trouta treatment
gill
fin
skin
spleen
kidney
irradiated directly sham-irradiatedb partner of irradiated fish partner of sham placed in water from irradiated fish placed in water from sham
25.1 ( 5.2b 99.1 ( 9.6a 27.6 ( 9.6b 92.8 ( 6.7a 27.4 ( 6.6b 92.8 ( 6.7a
58.0 ( 2.4b 98.7 ( 3.4a 58.0 ( 9.0b 92.9 ( 2.3a 33.5 ( 5.6b 96.7 ( 5.8a
39.7 ( 4.7b 95.7 ( 2.4a 59.4 ( 11.9b 96.7 ( 5.8a 44.0 ( 7.6b 92.9 ( 2.3a
23.8 ( 8.4c 83.3 ( 4.2a 81.4 ( 10.0a 82.5 ( 7.1a 35.1 ( 6.3b,c 82.5 ( 7.0a,b
23.1 ( 8.3c 76.8 ( 3.5a,b 22.9 ( 10.6c 84.4 ( 9.5a 37.2 ( 14.7b,c 72.4 ( 10.6a,b
a Data are normalized to 100% for the untreated control fish. Online a, b, and c are the results of the Bonferroni analysis; similar letters indicate statistically similar groups. T-test p values are presented in Table 2. b Untreated control fish were never handled during the experiment and serve as a control for the sham-irradiated group to ensure the sham protocol did not induce significant bystander effects due to stress.
Tissue Explant Technique. Explants of rainbow trout skin organs were established as described previously (33). Briefly, tissues were dissected asceptically and 3 equal-size pieces were incubated for 30 min at 19 °C in 1 mL of 0.25% w/v trypsin (Gibco Biocult, Irvine, Scotland), containing 10 mg/ mL collagenase. At the end of the incubation time the explants were washed in growth medium and plated as single explants in the center of 25 cm2 growth area, 40 mL volume flasks (NUNC, UDEN, Denmark) in 2 mL of growth medium. Flasks were left undisturbed for 48 h at 19 °C in a refrigerated incubator. All tissue was handled according to biosafety guidelines at McMaster University. Clonogenic Reporter Cell Line. The reporter HPV-G cells are adherent epithelial cells derived originally from a human foreskin primary culture and immortalized by the HPV virus (30). They were obtained as a gift from Prof. J. DiPaolo, NIH, Bethesda, MD, and have been used in our laboratory as a reporter system for bystander signal production in a wide range of experiments (e.g., 24, 27, 31-33). The cell line was grown in RPMI 1640 supplemented as above, in T75 flasks (NUNC Inc, Uden, Netherlands) and subcultured into T24 flasks (40 mL volume) for experiments. The cells are nontumorigenic, have about 30% wild-type p53 expression (30) and have a normal epithelial pattern of cobblestone density inhibited cell growth. They are used because when exposed to autologous medium harvested from irradiated cells, they give a stable bystander effect of approximately 40% reduction in plating efficiency over a very wide range of doses and exposure conditions (33). This allows comparison of bystander inducing signal strengths even when the HPV-G cells are exposed to signals from other cell lines or from explants. Clonogenic Assay for Bystander Activity using HPV-G Cells as Reporters. Flasks of HPV-G cells which were 8590% confluent and that had received a medium change the previous day were chosen. Cells were removed from the flask using 0.25 %w/v trypsin/ 1 mM EDTA solution (1:1). When the cells had detached they were resuspended in medium, and an aliquot was counted using a Coulter counter model Z2 set at a threshold calibrated for the cell line using a hemeocytometer. Flasks were seeded with 500 cells and left to attach for 6 h before receiving the medium harvested from the explants. Medium was harvested 48 h after the explants were set up. Cells were then left to form colonies, stained using carbol fuschin after 9 days, by which time survivors had grown to form macroscopic colonies. These were counted and the % surviving fraction determined. The untreated control plating efficiency of this line is approx 25%. Medium Transfer. The technique used has been described in detail in ref 34. Briefly, medium was poured off donor flasks (containing explants or control HPV-G cells). This was filtered through a 0.22 µm filter to ensure that no cells could still be present in the transferred medium. HPV-G donors were always carried as positive and negative controls with fish explants to check that control or irradiated donor medium was giving expected results. Culture medium was then
removed from the flasks designated to receive the explant medium and the filtrate was immediately added to these recipient flasks. Standard plating efficiency controls were also set up. The explants from which the medium had been harvested were incubated for a further 14 days in Clonetics serum free Keratinocyte Growth Medium (Clonetics Corp. San Diego, CA) Immunostaining for bcl-2 and cmyc Activity in Explant Cultures. Explant cultures were fixed in 10% unbuffered formalin and stored at 21 °C until processed. Processing always took place within 7 days of fixation. The culture was processed in situ on the flask bottom. Cultures were stained for expression of bcl-2 and cmyc. The bcl-2 and cmyc primary antibodies used were mouse monoclonals obtained from DAKO Cytomation and Novocastra laboratories, respectively. All were recommended for immunohistochemistry with mouse tissues but have been previously shown to work for rainbow trout (35, 36). Immunochemistry was performed using an appropriate Vectastain ABC kit (Burlingame, CA). Diaminobendizine (DAB) was used to express the positive reaction and cultures were lightly counterstained with Mayers Haematoxylin. At least three flasks were stained for each antibody and over 200 cells were scored over 5 fields using an Olympus image analysis system (ProDiscovery). The detection threshold for positivity was set using positive control sections from positive tissue blocks obtained from the Cell Pathology service. Positive and negative control sections were carried with every immunocytochemistry run to correct for run variability. This method was established in the laboratory several years ago and is discussed fully in ref 35. Statistical Analysis. Data are presented as mean ( standard error of the mean. The data were generated from 4 independent experiments involving 8 fish [4 × 2] per group. Comparison of the effects of direct X-irradiation and the bystander effect on HPV-G colony forming ability was done by Analysis of Variance followed by Benferroni’s analysis, using Statistix analytical software. F and t-tests performed using InStat software were also used where appropriate. In all cases P values
