On the Measurement of DNA Double-Strand Breaks by Neutral Elution

Abstract

Neutral elution is a sensitive and convenient method for measuring double-strand breaks in cellular DNA, but results obtained with this method are controversial, particularly as regards form of dose-response relationship. We pointed out in a recent publication (1) that explanation for different results obtained by neutral elution compared with neutral sedimentation remains obscure. The possibility exists that types of radiation damage other than double-strand breaks might affect neutral elution behavior. In a recent Letter to Editor, Hutchinson (2) stated that single-strand breaks might alter neutral elution because of configurational changes in DNA and/or increased susceptibility to shear but the necessary experiments to determine effect of single-strand breaks on neutral elution have not been performed. These necessary experiments must test whether single-strand breaks can modulate elution behavior of DNA containing a significant number of double-strand breaks, and we have performed such experiments. We induced double-strand breaks in DNA of human P3 epithelioid cells using 125I decay exactly as described previously (3), then, after allowing for accumulations of specific numbers of decays, we induced various numbers of single-strand breaks in cells by using treatments known to produce many single-strand breaks and few or no double-strand breaks: H202 (4) and bromodeoxyuridine photolysis (5), as well as y radiation. In no case (three measurements for each single-strand breakinducing treatment at different levels of induced singleand double-strand breaks) did extra single-strand breaks have any measurable effect on elution profiles (manuscript in preparation). Thus we have experimental evidence that neutral elution is not affected by concomitant singlestrand breaks, and this explanation for discrepancy between neutral sedimentation and neutral elution appears to be invalid. With respect to effect of protein crosslinked to DNA, more rigorous DNA lysis and cleaning procedures were described by Okayasu and Iliakis (6), who obtained double-strand break dose responses without a shoulder and, in some cases, straight elution profiles following these treatments. We have repeated those experiments as closely as possible using P3 cells, but saw neither exponential profiles nor dose responses without shoulders that Okasayu and Iliakis obtained. Therefore, DNA-to-protein crosslinking does not appear to be a determinant of neutral elution behavior in all types of cells. Thus question of different form of dose response measured by elution and sedimentation is still not resolved. Hutchinson's critique of neutral elution did not mention several positive aspects of assay or generally negative aspects of neutral sedimentation covered very thoroughly by van der Schans (7). The validity of elution assay is supported by fact that singleto doublestrand break ratios caused by different agents (which may differ by more than two orders of magnitude) detected by neutral elution correspond well with results obtained by other methods. Neutral sedimentation is beset with problems of irreproducibility caused in part by susceptibility of DNA to shear (7), as well as insensitivity. (Equivalent sensitivity to that of neutral elution has been achieved (8) but only by using extraordinarily laborious sample-handling procedures and 4-day sedimentation times.) Although neutral sedimentation, unlike neutral elution, is based upon mathematical theory, extent to which sedimentation theory is both untested and problematic in range of molecular weights relevant to low-dose studies in mammalian cells (9) should not be ignored. Despite unresolved probms, neutral elution remains only practical means of determining double-strand breaks at relatively low radiation doses.