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Environ. Sci. Technol. 2009, 43, 3335–3342

Communication of Radiation-Induced Signals in Vivo between DNA Repair Deficient and Proficient Medaka (Oryzias latipes) C . M O T H E R S I L L , * ,† R . W . S M I T H , † T. G. HINTON,‡ K. AIZAWA,§ AND C. B. SEYMOUR† McMaster University, 1280 Main Street West, Hamilton, Ontario L8S 4K1, Canada, Savannah River Ecology Laboratory, University of Georgia, Drawer E, Aiken, South Carolina 29802, Athens, Georgia, and Pharmaceuticals and Medical Devices Agency, Shin-Kasumigaseki Building, 3-3-2 Kasumigaseki, Chiyoda-ku, Tokyo 100-0013, Japan

Received December 11, 2008. Revised manuscript received February 17, 2009. Accepted March 11, 2009.

Radiation-induced bystander effects are established consequences of exposure to ionizing radiation. The operation of this mechanism has been seen in vitro and also between fish, mammals, and plants in vivo where stress signals from treated organisms induce responses in neighbors. In vitro research shows that DNA repair deficient cells produce more toxic bystander responses. To test this in vivo two strains of Japanese medaka were tested. One is a mutant, repair deficient strain (ric2) and the other, the wildtype repair proficient strain (CAB). Irradiated fish swam with unirradiated partners in a strain mix and match protocol. The data suggest that medaka produce signals, when exposed to radiation, that induce unirradiated fish of the same strain swimming with them to produce an altered response to that seen in bystanders to sham irradiated fish. More apoptosis was seen in bystanders to repair deficient fish. When the strains are mixed, the bystanders of either strain respond like the donor strain. Measurements of Bcl-2 and cmyc proteins in the explants confirmed these observations. A possible role for p53 was also identified in that the use of reporters with mutant p53 demonstrated that CAB signals killed all the reporter cells by apoptosis. Use of a similar but p53 wildtype cell line had no such effect. The data add to the body of knowledge showing that bystander signals operate at hierarchical levels of organization greater than the individual and may therefore have relevance in radioecology and (eco)systems biology.

Introduction Many animals and plants communicate alarm, stress, and other information using chemical signals (1-5). The molecules involved are usually small volatile and present in very low but highly effective concentrations (6, 7). Radiation exposure seems to result in similar stress signal production (8-10). It now appears that bystander effects induced by * Corresponding author phone: (905)525-9140 ext 26227; fax: (905)522-5982; e-mail: [email protected]. † McMaster University. ‡ University of Georgia. § Pharmaceuticals and Medical Devices Agency. 10.1021/es8035219 CCC: $40.75

Published on Web 03/30/2009

 2009 American Chemical Society

radiation and of intense interest in the fields of radiobiology and radiation protection may be another manifestation of this process which appears so widespread in nature. Bystander effects are detected in cells not themselves exposed to ionizing radiation but which receive signals from irradiated cells and respond in various ways leading to harmful effects or induction of protective responses. They have been documented in vitro and in vivo, and there are many excellent reviews in the field (11-14). These effects have been recorded since 1915 (15) but studied much more intensely since the mid 1990s (16-19). One reason for this is the need to evaluate cancer risks from low doses of radiation (20-24). The recent development of novel molecular biological tools which can detect very subtle changes in the genome and the epigenome has made this a realistic goal (25-27). Microbeams, which allow precise targeting of single cells or cellular organelles with radiation have also facilitated the growth of this field (28, 29). The major bystander effects recorded are gene and protein induction, DNA double strand breaks, sister chromatid exchanges, micronuclei, mutations, chromosome aberrations, growth delay, apoptosis, and neoplastic transformation. All these effects can be seen at very low doses in the environmentally relevant range (below 10mGy). The “dose response” typically saturates, for a given set of conditions; typically, an “on-off” type of response occurs (30, 31). The effect is increased if higher numbers of cells are exposed to the radiation dose (17) and can be titrated, suggesting a transmissible “factor” which is produced by cells receiving the actual dose. Previous work by our group, using live rainbow trout (Oncorhynchus mykiss) and zebrafish (Danio rerio), in vitro cell culture, and established fish cell lines, has confirmed bystander effects in fish. Using a reporter cell system, it has been shown that tissues of rainbow trout and zebrafish vary in the type and severity of the response induced by transfer of the signals from the irradiated tissue to a reporter cell line (9, 10). Even though communication of bystander effects between animals in vivo can be demonstrated in fish and mammals (8), it has not been possible to determine the mechanism. The recent BEIR 7 report on the biological effects of ionizing radiation (32) concluded that it was too early to assess whether bystander effects had any relevance for risk assessment. The existing in vivo work with mice suggests that signal production at least is dependent on genotype, with apoptosis prone genotypes producing factors which initiate apoptosis and cancer prone genotypes releasing factors which, if anything, stimulate growth of reporter cells (33). Additionally, a proteomics study of the rainbow trout gills revealed specific changes to the proteome in bystander fish (34). The expression of three proteins (hemopexin, Rho GDI dissociation inhibitor, and pyruvate dehydrogenase), all of which possess protective and/or restorative functions, were increased in bystander fish. In directly irradiated fish the tumorogenic associated protein annexin II was the only protein to be increased. It is important to stress that this protective response was seen in the live fish receiving the signal despite the fact that the same signals produce a toxic effect in the in vitro reporter assay used by our laboratory. This means that various receiving cells can respond differently to the same signal. Care in interpretation of data is called for when using in vitro readouts to avoid hasty conclusions about the protective or toxic nature of the signals. It has been demonstrated in vitro that the severity of the bystander response is greater when DNA repair is compromised (35, 36). When DNA repair proficient signals were VOL. 43, NO. 9, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 1. Apoptosis Related Protein Expression in X-rayed (and Sham X-rayed) Wild Type Medaka (CAB) and Repair Deficient Mutants (ric2) and Bystander Medakaa (a) Bystander Medaka Are the Same Strain As X-rayed (or Sham X-rayed) % cmyc positive cells treatment group untreated control X-ray bystander to X-rayed fish sham X-ray bystander to sham X-ray

CAB 32.5 ( 4.4d 91.5 ( 4.1b 23.3 ( 3.1e 27.8 ( 2.8de 30.1 ( 5.0e

% Bcl 2 positive cells

Ric2 52.8 ( 4.8c 100.0 ( 0.0a 85.8 ( 7.1b 53.3 (4.6c 49.8 ( 5.4c

CAB 50.0 ( 5.7C 57.0 ( 7.4B 83.5 ( 3.5A 50.8 ( 5.2C 51.3 ( 5.1C

Ric2 0D 0D 0D 0D 0D

(b) Bystander Medaka Are the Opposite Strain As X-rayed (or Sham X-rayed) bystander pairing (bystander strain/treatment strain) CAB/untreated control ric 2 CAB/X-rayed ric 2 CAB/sham X-rayed ric 2 Ric 2/untreated control CAB Ric 2/X-rayed CAB Ric 2/sham X-rayed CAB

% cmycpositivecells 42.3 (5.9c 88.8 (4.3a 43.0 (4.2c 44.8 (8.5c 67.5 (12.0b 41.0 (5.0c

% bcl 2positivecells 21.5 (3.5B 34.5 (4.4A 22.5 (4.4B 8.5 (3.9C 22.8 (5.7B 8.5 (4.7C

a Data are presented as mean % positive cells ( SD (n ) 4 individuals per group), scored 7 days after explant set up. Small and large case lettering indicates similarities and statistical differences in cmyc and bcl 2 expression, respectively, between the medaka strains and the treatments.

added to repair deficient cells, these cells “acquired” repair proficiency, suggesting that repair proficiency was a bystander signal inducible response. This was applicable for a wide range of repair defects. Both of these findings suggest that bystander biology is highly involved in both the expression of repair capacity and in the underlying cellular DNA damage sensing and repair mechanisms. To address whether the severity of the bystander response is greater when DNA repair is compromised in vivo, we chose two strains of medaka; CAB are a wild-type strain, while ric2 were selected for radiosensitivity from a batch of nitrosourea mutated hatchlings (37). Selection involved breeding parents whose eggs showed reduced hatchability after exposure to 10.2 Gy radiation. The mutants were found to be DNA double strand break repair deficient, and immediate apoptosis was the dominant response to radiation exposure. We used both strains in mix-match protocols to test the effect of signals on the same or other strain of fish. Also signals from irradiated fish of both strains were added to two different reporter cell lines with either wild-type or mutant p53. P53 is known to be a “gatekeeper”, which “directs” DNA damaged cells either to apoptosis or to DNA repair (38). Expressions of bcl-2, an antiapoptotic protein, and cmyc (a proto-oncogene with both proliferation and death inducing functions depending on circumstances) which in our hands always functions as a pro-apoptotic protein (9, 10, 12, 33) were measured in both tissue explants and reporter cells. This research specifically addresses the hypothesis that bystander signals are stronger when emitted by a repair deficient system.

Materials and Methods Fish and Husbandry. Both strains of fish were raised under identical, standard, laboratory conditions according to University of Georgia AUP A2007-10148-0. Upon reaching maturity, the fish were shipped by air to McMaster University. Each genotype was then held separately in glass aquaria, containing 40 L dechlorinated Hamilton tap water, maintained at 26 °C, and supplied with constant aeration. The aquaria were equipped with a recirculating water filtration system, incorporating mechanical, chemical, and biological filter components. Feeding was twice daily with a commercially available tropical fish food. 3336

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Fish were irradiated or sham irradiated using a FAXITRON X-ray unit. After 2 h for signal build up they were placed in partitioned tanks with a mesh separating them from unirradiated partner fish in same and mixed genotype protocols. After a further 2 h all groups plus untreated handling controls were sacrificed, and the caudel fin was taken for processing to determine signal production using the tissue explants and HaCaT and HPV-G cells to expression of pro and anti apoptotic proteins (39-46). Irradiation Protocol. The experimental design is shown in Figure S1. Briefly, there were 5 groups of animals with 4 individuals per group. The choice of sample number was determined by the technical limitations on the speed of tissue culture needed to ensure that loss of viability did not occur due to time taken to plate cells. [The first group (referred to as irradiated fish or X-R) was placed inside a portable X-ray machine (Faxitron X-ray Corporation cabinet X-ray system; Wheeling, IL, USA) and irradiated at 112 kVp (kilovolts peak) for 5 min. This delivered an average whole body dose of approximately 0.5 Gy, as previously calibrated using thermoluminescent dosimeters (TLDs) (2). The second group, sham-irradiated fish or Sh X-R (i.e., to control for the handling and confinement stress associated with administering the X-ray dose), was placed into the X-ray machine, but it was not turned on. Immediately after irradiation (actual or sham), these fish were placed into 1 side of a partitioned experimental container, (partitioned by a mesh of approx 2 mm size) and held for 2 h, to allow any signals to accumulate in the water. Following this, naive fish (i.e., non X-rayed fish) were then placed on the other side of the partition. These were groups 3 and 4, which are referred to as “bystander fish (BX-R)” (placed with group 1 fish) and “sham bystander fish (Sh BXR)” (placed with group 2 fish). Thus medaka in groups 3 and 4 received no direct radiation but were statically housed with irradiated or sham fish for a further 2 h period. These bystander fish were therefore exposed to any bystander signal which was already present in the water as well any subsequently emitted by the irradiated (or sham irradiated) fish. The final fifth group of fish were placed into the experimental containers for 2 h but were otherwise untreated. These are labeled control (C). All experiments were carried out in 500 mL of water, which was continuously aerated and maintained at 26 °C.

Poole Dorset, UK), 5 mL penicillin:streptomycin solution, 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. The skin samples were cut into three separate equal sized pieces 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 26 °C. Medium Transfer and Clonogenic Reporter Assay. Two days later the medium from the cultures was harvested, filtered to ensure no cell debris was present, and added to clonogenic reporter cells seeded at densities where approximately 100 cells are expected to survive. The cell lines used were HaCaT or HPV-G immortalized keratinocytes and have been used in the laboratory for over 10 years. The end point of the reporter bioassay is colony formation where colonies with 50 or more cells are deemed to have survived. Immunostaining for Bcl 2 and cMyc Activity in Explant Cultures. The explant cultures were replenished with Clonetics serum free medium (Clonetics Corp., San Diego, CA) and allowed to grow for a further week. After this they were fixed in 10% formalin and then immunostained using antibodies against cmyc or bcl-2 Staiming was performed using the Vectastain ABC kit. More detailed information can be found in the Supporting Information. Statistics. Data are expressed as means ( standard deviation of pooled data for two completely separate experiments (n ) 4 fish in each experiment). Comparison of cmyc and bcl 2 expression and % apoptotic bodies was by Analysis of Variance, followed by Least Square Difference, using the Statistix analytical software; P < 0.05 was considered statistically significant.

Results

FIGURE 1. a. Percentage of apoptotic bodies expressed in skin cells from X-rayed (and sham X-rayed) wild-type medaka (CAB) and repair deficient mutants (ric2) and bystander medaka paired with X-rayed or sham X-rayed fish of the same strain. Data are presented as mean % positive cells ( SD (n ) 4 individuals per group) scored in explants 7 days after set up. Lettering indicates similarities and statistical differences in % apoptotic bodies. b. Percentage of apoptotic bodies expressed in skin cells from X-rayed (and sham X-rayed) wild-type medaka (CAB) and repair deficient mutants (ric2) and bystander medaka paired with X-rayed or sham X-rayed fish of the opposite strain. Data from CAB bystander medaka paired with treated ric 2 medaka are shown in gray, and data from ric 2 bystander medaka paired with treated CAB medaka are shown in black. Data are presented as mean % positive cells ( SD (n ) 4 individuals per group) scored in explants 7 days after set up. Lettering indicates similarities and statistical differences in % apoptotic bodies. Sampling Protocol. Following the 2 h holding period, fish were sacrificed by cephalic concussion, and the skin was removed asceptically, in a biological safety cabinet over ice, placed in 1 mL complete growth medium, and then transported (on ice) 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% fetal bovine serum, 1 microgram/mL hydrocortisone (Sigma,

Bcl-2 and cmyc Expression and Apoptosis in X-rayed and Bystander Medaka. Table 1a summarizes the immunocytochemistry data for the explants from X-rayed CAB and ric2 medaka and from homologous bystander treatments, i.e. X-rayed CAB/bystander CAB and X-rayed ric2/bystander ric2 (and the sham X-rayed equivalents). Neither sham X-ray nor sham bystander treatment elevated cmyc or bcl2 expression above control levels in either strain of medaka. Similarly the sham treatments of both medaka strain had no effect on the percentage of apoptotic bodies (Figure 1a). This confirms the handling and confinement stress associated with delivering the X-ray dose or manipulating the X-rayed and bystander fish did not exert a significant effect on the immunohistochemistry or on apoptosis in the medaka. The expression of cmyc, a pro-apoptotic protein, was consistently greater in ric2 cell cultures, when compared with the equivalent CAB cultures, and was elevated above control levels in both X-rayed (where expression reached 100%) and X-rayed ric2/ric2 bystander medaka. Cmyc expression increased in X-rayed CAB medaka but reduced in X-rayed CAB/ CAB bystander medaka. Bcl 2, an antiapoptotic protein, was elevated above control levels in X-rayed and bystander CAB medaka (expression being the greatest in bystander fish) but was not detected in ric2 medaka, irrespective of treatment. As a result there was a greater percentage of apoptotic bodies found in ric2 cultures, in comparison to the corresponding CAB cell cultures (Figure 1a). Direct X-ray exposure and exposure to the X-ray induced bystander signal from the same strain of medaka elevated the percentage of apoptotic bodies in both CAB and ric2 medaka; this elevation was equal in ric2 fish but X-ray treatment had the greatest effect in CAB fish (Figure 1a). Cmyc and bcl 2 expression in bystander CAB and ric2 medaka paired with sham irradiated fish of the opposite strain VOL. 43, NO. 9, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 2. Apoptosis Related Protein Expression Data for the HaCaT Reporter Cells Treated with Explant Media from X-rayed (and Sham X-rayed) and Bystander (and Sham Bystander) Wild Type Medaka (CAB) and Repair Deficient Mutants (ric2)a (a) Bystander Fish Are the Same Strain as X-rayed (or Sham X-rayed)b % cmyc positive cells treatment group untreated control X-ray bystander to X-rayed fish sham X-ray bystander to sham X-ray

CAB 100 ( 0.0a 100 ( 0.0a 100 ( 0.0a 100 ( 0.0a 100 ( 0.0a

% Bcl 2 positive cells

Ric2 46.3 ( 3.1b 100.0 ( 0.0a 100.0 ( 0.0a 45.0 ( 3.7a 45.0 ( 3.4b

CAB 0 0 0 0 0

Ric2 0 0 0 0 0

(b) Bystander Medaka Are the Opposite Strain as X-rayed (or Sham X-rayed) bystander pairing (bystander strain/treatment strain)

% cmyc positive cells

% bcl 2 positive cells

100 (0.0 100 (0.0a 100 (0.0a 25.8(3.4c 82.8(5.4b 24.0(3.9c

0.0(0.0B 0.0(0.0B 0.0(0.0B 3.3(3.3B 14.5(4.7A 2.3(2.2B

CAB/untreated control ric 2 CAB/X-rayed ric 2 CAB/sham X-rayed ric 2 Ric 2/untreated control CAB Ric 2/X-rayed CAB Ric 2/sham X-rayed CAB

a

a Data are presented as mean % positive cells ( SD (n ) 4 individuals per group), scored in HaCaT reporter colonies stained 10 days after set up. Small and large case lettering indicates similarities and statistical differences in cmyc and bcl 2 expression, respectively. b Bcl 2 expression was not detected in HaCaT cells treated with either CAB or ric 2 explant media. Small case lettering indicates similarities and statistical differences in cmyc expression. For samples from CAB fish, there were very few cells, so it was not possible to count 200 cells.

(i.e., heterologous bystander pairings) was not different to pairing with an equivalent untreated fish (Table 1b). The expression of both cmyc and bcl 2 was elevated in bystander fish of both strains when paired with X-rayed fish of the opposite strain, the increase being greatest in CAB medaka paired with X-rayed ric2 medaka (Table 1b). Despite this cells from CAB and ric2 bystander fish paired with X-rayed medaka of the opposite strain showed equally elevated percentages of apoptotic bodies (Figure 1b). Bcl-2 and cmyc Expression, and Apoptosis in HaCaT Clonogenic Reporter Cells. Irrespective of fish treatment, media from CAB and ric2 medaka explants failed to induce bcl 2 expression in HaCaT reporter cells (Table 2a). Media from CAB medaka failed to reduce the incidence of cmyc expression or apoptotic bodies in HaCaT cells below 100%, again irrespective of treatment (Table 2a). Although media from X-rayed ric2 and from an X-rayed ric2/ric2 bystander pairing also failed to reduce cmyc expression below 100%, media from untreated and sham treated control ric2 fish caused an equal decline in the percentage of cmyc positive HaCaT cells (Table 2a). Media from all ric2 fish also reduced the incidence of HaCaT apoptotic bodies below 100%, the decline from treatment with media from X-rayed ric2 and from X-rayed ric2/ ric2 bystander pairing being less than from treatment with media from untreated or sham treated ric2 medaka (Figure 2a). CAB bystander medaka paired with ric2 medaka failed to induce bcl 2 expression in HaCaT cells, irrespective of the treatment given to the ric2 fish (Table 2b). CAB bystander medaka also failed to reduce the incidence of cmyc expression or the incidence of apoptotic bodies in HaCaT cells below 100%, irrespective of the treatment given to the ric2 medaka (Table 2b). Bystander ric2 medaka paired with X-rayed CAB medaka reduced cmyc expression (Table 2b). Cmyc expression was reduced even further if the bystander ric2 medaka were paired with untreated or sham X-rayed CAB medaka. The incidence of bcl 2 expression in HaCaT cells was only increased above zero, by bystander ric2 medaka paired with X-rayed CAB medaka. The percentage of apoptotic bodies was also reduced below 100% by bystander ric2 medaka, the reduction being greatest when the bystander fish were paired with untreated or sham X-rayed CAB medaka (Figure 2b). 3338

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Clonogenic Survival Determined Using the HaCaT Reporter Cell Line. Figure 3 shows the comparison between the effect of CAB and ric2 signals where the medium from each explant was harvested and transferred to a reporter cell line - the mutant p53 expressing HaCaT line. Any CAB signals, even from control fish, virtually abolished the keratinocyte cells plating efficiency. Nothing in the CAB explant cultures suggested that they were secreting a signal which would be lethal to HaCaT cells. The mechanism of cell death appears to be apoptosis as confirmed by staining of the few aborted colonies seen on the culture plates with cmyc antibodies and morphological examination (Figure S2). The signals emitted by X-rayed ric2 medaka completely kill the HaCaT reporter cells. In contrast to CAB medaka, the untreated controls and sham X-ray treated ric2 fish do not have this effect. However, attempts to study bystander signals from ric2 fish housed with ric2 fish are difficult to interpret because it appears the signals from the sham and X-ray bystander fish both induced significant increases in apoptosis in the HaCaT reporters compared with the never treated controls or sham X-rayed fish. This is presumably due to handling stress compared to the untreated controls. When ric2 fish swam with untreated, sham X-rayed or X-rayed CAB fish similar results to ric2 -ric2 combinations occurred. In the reverse experiment, treated, sham or untreated ric2 fish swam with CAB fish. All the signals from the CAB bystander fish again caused complete abortion of colony formation in the HaCaT cell line. From these results we concluded the following: 1. CAB fish tissue secretes a signal into culture medium which completely prevents growth of HaCaT cells. 2. The detected signal secreted by the tissues into culture medium is determined by the strain receiving the signal in vivo and not by the strain emitting the signal. This is not to say that both fish may not emit signals. An exchange of signals is possible, with perhaps one dominating the other, depending on the circumstances. In previous experiments with repair deficient and proficient cell lines from a variety of mammalian sources, no difference in the response of HaCaT and HPV-G cells had been found (16, 40). However this was the first time we used HaCaT cells as reporters for fish; usually HPV-G cells were

FIGURE 3. Clonogenic survival of the HaCaT reporter cell line treated with explant media from X-rayed (and sham X-rayed) wild-type medaka (CAB) and repair deficient mutants (ric2) and bystander medaka paired with X-rayed or sham X-rayed fish of the same and of the opposite strain. Data are presented as mean % clonogenic survival ( SD. Lettering indicates similarities and statistical differences. * indicates zero clonogenic survival.

FIGURE 2. a. Percentage of apoptotic bodies expressed in HaCaT reporter cells following treatment with explant media from X-rayed (and sham X-rayed) wild-type medaka (CAB) and repair deficient mutants (ric2) and bystander medaka paired with X-rayed or sham X-rayed fish of the same strain. Data are presented as mean ( SD (n ) 4 individuals per group) scored 7 days after set up. Lettering indicates similarities and statistical differences in % apoptotic bodies. b. Percentage of apoptotic bodies expressed in HaCaT reporter cells following treatment with explant media from X-rayed (and sham X-rayed) wild-type medaka (CAB) and repair deficient mutants (ric2) and bystander medaka paired with X-rayed or sham X-rayed fish of the opposite strain. Data from CAB bystander medaka paired with treated ric 2 medaka are shown in gray and data from ric 2 bystander medaka paired with treated CAB medaka are shown in black. Data are presented as mean ( SD (n ) 4 individuals per group) scored 7 days after set up. Lettering indicates similarities and statistical differences in % apoptotic bodies. used because of their wild-type p53 status (39, 41). To test the hypothesis that lack of expression of wild-type p53 might be implicated in the HaCaT response, tissues from irradiated and control CAB and ric2 medaka fish were grown as before, but the harvested medium was used to compare HPV-G and HaCaT cells. The results are shown in Figure 4. Clearly HPV-G cells do form colonies when exposed to medium from irradiated or control CAB or ric2 medium. A clear effect of repair deficiency in the ric 2 strain can be seen since the

FIGURE 4. Clonogenic survival or HPV-G and HaCaT reporter cells treated with explant media from X-rayed CAB and ric 2 medaka. Data are presented as mean % clonogenic survival ( SD (n ) 4 individuals per group). Lettering indicates similarities and statistical differences. survival is much lower (40%) in HPV-G cells treated with ric2 medium. Examination of the colonies (Table 3) reveals high cmyc staining in the ric2 medium exposed colonies compared to the CAB medium exposed colonies.

Discussion This study had a very simple objective: to build on previous data from our group and others, showing that bystander signals, tested in terms of their toxic or mutagenic effects, were stronger when emitted or received by repair deficient cells. This was previously shown using medium transfer, microbeams, and alpha particles using a number of cell types (35, 36). Our aim was to use an in vivo model where repair competent, X-rayed fish were housed with unexposed, repair deficient fish, and vice versa. A further aim was to look at the inducibility by one strain of the repair profile characteristic VOL. 43, NO. 9, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 3. Apoptosis Related Protein Expression Data for the HPV-G Reporter Cells from Wild Type Medaka (CAB Strain) and Repair Deficient Mutants (ric2)a Ric2 treatment group C X-R Sh X-R BX-R Sh BX-R

cmyc (%) b

12.3( 2.5 91.3 ( 10.3a 12.7 ( 6.7b 100.0 ( 0.0a 16.7( 8.3b

CAB Bcl 2 (%)

cmyc (%)

Bcl 2 (%)

0 0 0 0 0

2.3 ( 2.5 43.0 ( 6.2A 2.7 ( 3.8C 11.0 ( 3.6B 1.3 ( 1.2C C

7.7 ( 2.5iii 38.3 ( 8.7ii 6.7 (2.3iii 67.0 ( 7.5i 11.0 ( 4.0iii

a HPV-G cells were treated with 5 treatment groups: untreated control (C CAB and C ric2), X-rayed (X-R CAB and X-R ric2), bystander fish to X-rayed fish (BX-R CAB and BX-R ric2 P), sham fish X-ray (Sh X-R CAB and Sh X-R ric2), and sham bystander fish (Sh BX-R CAB and Sh BX-R ric2). The X-ray was a single acute exposure to 0.5Gy. Data are presented as mean % positive cells ( SD for cmyc positive cells and Bcl 2 positive cells scored in HPV-G reporter colonies stained 10 days after set up. Numbering and lettering indicates similarities and statistical differences in cmyc expression.

of the other strain. In vitro experiments had suggested that this happened (47-50). Our in vivo results proved more complicated to interpret. CAB and ric2 strain medaka show large differences when the tissues are explanted and the skin cells from each strain tested for expression of bystander signals. Tissue from the ric2 genotype shows a clear increase in apoptotic cells and increased expression of cmyc in cells growing from explants of directly irradiated fish and also from bystander ric2 fish which were paired with the X-rayed fish for 2 h. The effect was also seen in fish introduced into water from irradiated fish, suggesting as was found before (9, 10, 34) that the signal communicated between fish is water borne. The antiapoptotic protein, Bcl-2, was not expressed in any of the ric2 fish (control, X-rayed, or paired) suggesting this protein is not normally expressed in these animals. In the repair proficient CAB strain, expression of both cmyc and bcl-2 proteins occurs at low levels in the untreated or sham exposed control animals. When compared with ric2, CAB fish show a significant but smaller increase in apoptotic cells and cmyc expression in the directly irradiated fish. Fish housed with irradiated fish show strong induction of bcl-2. These findings are consistent with a pro-apoptotic response in both strains of fish although the apoptotic response is stronger in the repair deficient strain. In the CAB strain an antiapoptotic protein (Bcl2) was induced in the tissues of partner fish despite an overall increase in apoptotic bodies. Ric2 partner fish do not induce this antiapoptotic response. This is interesting because it suggests an apoptotic mechanism is activated (via the cmyc protein in the ric2 radiosenstive strain) for eliminating DNA damage in a situation where repair of the most common radiation induced lesion (the DNA double strand break) is compromised (via, at least in part, the dysfunction of the antiapoptotic protein, Bcl 2). This would likely be protective at low doses where death of individual cells is a better option then carrying unrepaired DNA damage. What is surprising is that these effects are occurring in partner fish which do not have radiation induced DNA damage. It is possible that signaling is “proactive” or “preemptive” in that a signal to protect precedes actual damage. Such a proactive response has been documented recently for plants (48-50), where the signal increased reactive oxygen species (ROS). In the absence of DNA damage, ROS cause the induction of protective proteins. In the model used by our group, it is hard to see how ROS could be involved as the radicals would be unlikely to remain in the water and should not influence fish introduced to the water after the X-rayed fish was removed. However, some small stable molecule could conceivably be transmitting a signal. Viewed this way, both CAB and ric2 fish are responding to signals from X-rayed fish by inducing a protective (for their genotype) response. The largest result seen in our experiments was the response of the reporter cells chosen. The transfer of medium 3340

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harvested from CAB tissue explants completely prevented any colony formation in the HaCaT cell line. This is a line of human keratinocytes originally developed by Dr Petra Boukamp (42). They have been used as a reporter line in our laboratory since 1996 and have always shown similar bystander effects to those seen with HPV-G keratinocytes but have never been used as recipients for fish cells before. When we compared HaCaT and HPV lines using CAB and ric2 tissues, medium harvested from CABs only contained the toxic factor for HaCaT cells and both gave expected results with HPV-G reporters. The HaCaT cells appear to die by apoptosis. It is possible that the effect is due to the presence of mutant p53 in HaCaT cells and occurs due to some surveillance mechanism which senses the presence of the mutant p53 and induces a suicide pathway. HPV-G cells contain reduced levels of wild-type p53 so they may escape this mechanism. However, this does not explain why medium from ric2 tissues does not induce total death of HaCaT cells since the medium from their tissues is clearly capable of inducing some apoptosis in both cell lines. Whatever the reason for this strange result, it points to a need to consider the reporter cells when using bioassays to quantify biological effects. Previous work by our group (46) showed that when medium from repair proficient cells was placed on repair deficient reporters, the deficient cells survived better. The reverse was also true. We carried this concept further in the results reported herein, by testing the hypothesis that in vivo signals from repair proficient or deficient fish might induce the phenotype of the X-rayed fish radiation response in the partner fish. The data for apoptosis induction and immunocytochemical expression of bcl-2 and cmyc support the hypothesis. This provides in vivo verification of the in vitro observations and suggests that in ric2 fish the necessary antiapoptotic genes may be present, but they fail to get the correct environmental signals to initiate protein production. However, the CAB fish respond to signals from ric2 fish by producing signals which increase apoptosis and expression of pro-apoptotic cmyc. This again supports the idea that as in the plant system (48-50) the environmental signal modulates protein expression in the absence of actual damage. Whatever the mechanisms underlying these effects in fish, mammalian cell lines, and plants, it is clear they are widespread in nature and conserved during evolution, suggesting that the bystander signal is indeed important. Both the cell line and whole organism data imply that a control mechanism exists which can respond to signals generated at the level of the individual (cell or organism) which has been affected by a stress - radiation in this case - and enable responses which have effects at a higher hierarchical level e.g. the cell population in the case of cell lines or the population in the case of whole organism

exposures. Such a mechanism could speed up adaptation in a changing environment by acting proactively in individuals which are not directly damaged and when it results in protective responses would also select for not already damaged individuals - a desirable evolutionary situation.

Acknowledgments We wish to acknowledge the following individuals: Hiroshi Mitani for supplying the fish, Dan Coughlin and Yi Yi for laboratory assistance and fish husbandry, and Chris Wood (McMaster University) for kindly allowing the use of his CFI funded fish facility. We also acknowledge the NSERC Industrial Chairs programme, the Canada Research Council Chairs Programme, and the NSERC Discovery Grants Programme. We also acknowledge support of colleagues in the EU NOTE integrated project

Supporting Information Available Detailed methods text and two figures showing schematic methods. This material is available free of charge via the Internet at http://pubs.acs.org.

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