Exposure to acute levels of waterborne aluminium modifies the legacy of early life stage irradiation, including the communication of radiation-induced bystander signals, in adult rainbow trout.

PURPOSE: To determine if the legacy of early life stage irradiation seen in rainbow trout is modified in adult fish by acute exposure to waterborne aluminium (Al). METHODS: Two-year-old trout which had been irradiated as either eggs 48 h after fertilization, eyed eggs, yolk sac larvae or as first feeders, were exposed to 100 or 200 µg l(-1) Al. These fish were then paired with untreated fish. Gill and skin samples from both the irradiated and Al-exposed, and the untreated bystander fish, were then assayed for the production of toxic or pro-death signals, using the HPV-G reporter cell line. RESULTS: Depending on the life stage, irradiated and the tissue assayed acute Al exposure modified both the radiation legacy, and the radiation-induced bystander effect in untreated fish, either by reducing reporter cell survival or increasing reporter cell growth. CONCLUSIONS: The effects of early irradiation and adult Al exposure were complex but clearly demonstrated that both the direct radiation effects and the communication of a radiation-induced bystander effect can be modified by a second environmental stressor.

Radiation induced bystander effects in mice given low doses of radiation in vivo.

The 'bystander effect' phenomenon has challenged the traditional framework for assessing radiation damage by showing radiation induced changes in cells which have not been directly targeted, but are neighbors to or receive medium from directly hit cells. Our group performed a range of single and serial low dose irradiations on two genetically distinct strains of mice. Bladder explants established from these mice were incubated in culture medium, which was used to measure death responses in a keratinocyte reporter system. The study revealed that the medium harvested from bladder tissues' (ITCM) from acutely irradiated C57BL6 but not Balb/c mice, was able to induce clonogenic death. Administration of a priming dose(s) before a challenge dose to both C57BL6 and Balb/c mice stimulated reporter cell survival irrespective of the time interval between dose(s) delivery. When ITCM corresponding to both strains of mice was measured for its calcium mobilization inducing ability, results showed an elevation in intracellular calcium levels that was strain dependent. This indicates that genotype determined the type of bystander signal/response that was produced after exposure to low and acute doses of radiation. However, serial exposure conditions modified bystander signal production to induce similar effects that were characterized by excessive growth.

Irradiation of rainbow trout at early life stages results in legacy effects in adults.

PURPOSE: Communication of signals from irradiated to non-irradiated fish has been demonstrated by our group for adults. Major questions are however, whether the effects persist for significant lengths of time (meaning there are memories or legacies of the exposure) and whether they are induced in young animals or very early stages in the life cycle. METHODS: To address these questions we used a reporter cell clonogenic bioassay to detect the effects of radiation exposure and of 'bystander' signals, emitted from irradiated fish, on non-irradiated fish. The legacy of radiation exposure or receipt of bystander signals was investigated in rainbow trout irradiated as eggs at 48 h, eyed eggs at one month, yolk sac larvae (YSL) at two months and juveniles at three months after fertilisation. The irradiated and bystander fish together with shams and unhandled husbandry controls were grown on in a hatchery and examined as they reached each of the remaining life stages. They were also re-examined as one-year-olds with and without further irradiation and finally examined as sexually mature two-year-olds. RESULTS: The data indicate a clear legacy effect of irradiation at any early life stage in the adult fish. CONCLUSION: The data suggest that bystander signals can be transmitted in vivo and once induced are persistent during the animals' lifespan.

Serum serotonin levels determine the magnitude and type of bystander effects in medium transfer experiments.

Serotonin has been shown to be involved in the production of bystander signals by irradiated cells. In this study we examined the levels of serotonin in 10 different batches of commercially available fetal calf serum and correlated the serotonin levels with the toxicity of medium harvested from irradiated cells (ICCM) using a standard medium transfer colony-forming assay. The serotonin levels in the serum varied widely between batches, and the levels correlated directly with the toxicity of the harvested ICCM. Three serum samples had levels of serotonin below 25 ng/ml, and these did not show medium transfer bystander effects. Exposure of serum samples to normal daylight reduced serotonin levels significantly. We suggest that serum batch variability may underlie much of the observed interlaboratory variation in the ability to produce bystander effects and further suggest that serum batches should be protected from light and prescreened for their ability to produce a bystander effect using a positive control cell line.

Injection of resperpine into zebrafish, prevents fish to fish communication of radiation-induced bystander signals: confirmation in vivo of a role for serotonin in the mechanism.

Serotonin (5-HT) has been implicated as a potential modulator of the bystander effect in cell cultures. To assess the relevance of serotonin in vivo experiments were done with the zebrafish (Danio rerio). This species, when irradiated, transmits bystander signals to non-irradiated fish. The animals were injected with reserpine, an inhibitor of serotonin at a dose of 80mg/kg of body mass. The results show that reserpine treated fish had only 27% of the serotonin in non-treated fish. Skin tissue samples were collected from the fish and assayed for bystander signal production using a reporter bioassay. Reserpine prevented the production and communication of signals between fish. Intracellular calcium flux, identified as a bystander response in the reporter cells confirmed this. Medium harvested from tissues of X-rayed fish and their bystanders, showed an oscillating pattern of calcium flux. Samples from X-rayed fish pretreated with reserpine produced a chaotic pattern of random fluctuations in the reporter cells, while their bystander fish led to increased calcium, but no oscillations. These results suggest that 5-HT is involved in bystander signalling in zebrafish, and by decreasing the amount of available 5-HT the bystander effect can be blocked.