pathways and induction of mutations, the irradiated and nonirradiated cells had to be in contact with each other through gap junctions. Hall and colleagues demonstrated the same bystander phenomenon for cell killing, induction of mutations (Zhou and others 2000), micronuclei formation (Hall 2000), and malignant transformation (Sawant and others 2001a); the magnitude of the bystander effect increased with the number of α-particles traversing the nuclei (Sawant and others 2001a).

For malignant transformation, the frequency when only 10% of the cells were traversed by an α-particle was as great as when every cell was exposed to an α-particle; furthermore, nonirradiated cells did not have to be in contact with irradiated cells. However, the same group subsequently reported that gap junctions appeared to be required for another bystander effect resulting in cell lethality in nonhit cells (Sawant and others 2002). The group also showed that irradiating the cytoplasm with α-particles (Wu and others 1999) induced mutations (small deletion and base-pair alteration mutations) that resembled those occurring spontaneously, not the larger deletions observed when the nucleus was irradiated to induce mutations in both irradiated and nonirradiated cells (Zhou and others 2000). Lorimore and colleagues (1998) have observed a similar bystander effect: delayed chromosomal aberrations associated with genomic instability when cells were exposed to α-particles. Prise and colleagues (1998) have observed a bystander effect for genomic instability associated with the formation of micronuclei 20–30 generations after individual cells were irradiated with a charged-particle microbeam. Their subsequent studies with primary human fibroblasts (Belyakov and others 2001) showed that even though a single cell had been targeted, an additional 80–110 cells had micronuclei; the yield of cells that had excess micronuclei was independent of the number of charged particles delivered to the targeted cell.

The molecular mechanisms proposed for the bystander effects described above are speculative (see Chapter 1DSB Signal Transduction and Inducible Repair” for a discussion of possible repair and signal transduction pathways that may be involved). Activation of the p53-mediated DNA damage response pathway in bystander cells has led to speculation (Grosovsky 1999) that reduced replication fidelity or increased recombinational activity might lead to the genetic effects that occur in these cells. However, α-particle-induced chromosomal instability was reported to be independent of the p53 status of the cells (Kadhim and others 1996). The bystander phenomenon may involve the diffusion of cytokines or long-lived reactive oxygen species (ROS; Narayanan and others 1997, 1999; Lorimore and others 1998; Wu and others 1999; Azzam and others 2002; Morgan 2003a, 2003b) including any products formed by reaction with hydrated electrons or OH radicals (Ward 2002). Also, the diffusion of paracrine proapoptotic or antiapoptotic factors induced by upregulation of p21 (Chang and others 2000) may be involved. Because CDC2 is downregulated by α-particles, there may be reduced phosphorylation of connexin 43 by CDC2 and thus increased membrane permeability (Azzam and others 1998). This hypothesis is supported by the observation that membrane signaling is involved in the bystander effect for sister-chromatid exchanges and HPRT mutations induced indirectly by α-particles (Nagasawa and others 2002).

Regardless of the molecular mechanisms involved, the bystander effects observed with high-LET particles may have important implications for low doses of high-LET radiation. According to Sawant and others (2001a), “These results, if applicable in vivo, would have significant consequences in terms of radiation risk extrapolation to low doses, implying that the relevant target for radiation oncogenesis is larger than an individual cell, and that the risk of carcinogenesis would increase more slowly, if at all, at higher doses—an effect seen in vivo, as well as epidemiologically. Thus, a simple linear extrapolation of radiation risk from high doses (where they can be measured) to lower doses (where they must be inferred) would be of questionable validity.” In other words, it is speculated that there could be a convex, downward-curving dose-response relationship at low doses, and that extrapolation of data from high doses could lead to an underestimate of the effect at low doses of high-LET radiation.

A most critical question, however, is whether these types of bystander effects exist for low-LET radiation doses <100 mGy, which are the focus of this report. For α-particles and other high-LET radiation used in bystander studies, the dose to the nucleus was calculated to be 130–500 mGy per α-particle traversal, depending on the size and shape of the cell and its nucleus (Azzam and others 1998); that is, a flattened cell nucleus would have a much lower dose from high-LET radiation than a spherical rounded cell nucleus because of the geometry of the nucleus in relation to the radiation source (Clutton and others 1996a, 1996b). For low-LET radiation (assuming an RBE of 3), the dose corresponding to that from the high-LET radiation would be 0.39–1.5 Gy. Because the bystander effect resulting from an α-particle traversal through an irradiated cell was lower by a factor of 3–5 than the direct effect on the irradiated cell and because the magnitude of the bystander effect appeared to increase as the number of traversals through the cell increased (Sawant and others 2001a), one might expect that the same type of bystander effect would not be observed in the low-dose range <100 mGy for low-LET radiation. In fact, data indicate that the bystander effect for induced expression of p53 was much greater and persisted much longer after α-irradiation than after X-irradiation (Hickman and others 1994).

In human keratinocytes, a bystander effect for cell lethality that required cell-to-cell contact with gap junctions has been reported for γ-ray doses of 500 mGy and above (Mothersill and Seymour 1997). In the same dose range, a bystander effect that did not require cell-to-cell contact was



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