Reflex-Replikation: Artefakte statt DNA-Strangbrüche (Forschung)

H. Lamarr , München, Mittwoch, 06.07.2016, 15:23 (vor 2843 Tagen) @ H. Lamarr

Die in diesem Zusammenhang beschränkte Einsatzmöglichkeit des COMET-Assays machte Schär gleich zu Anfang seiner Präsentation in einer vorweggenommenen "provokativen Conclusion" (bezogen auf HF und ELF) klar, in dem er sinngemäß sagte: "EMF makes no DNA damage, but EMF makes some biological effect that looks like DNA damage in the COMET assay. So, COMET is not the right thing to look on EMF effects." ("EMF verursacht keine DNA-Schäden, aber EMF bewirkt irgendeinen biologischen Effekt, der im COMET-Assay wie eine DNA-Schädigung aussieht. Daher ist der COMET-Assay nicht das richtige Mittel, um nach EMF-Effekten zu suchen.") Bei den Effekten meinte er dabei wohl hauptsächlich die für ihn evidenten ELF-Effekte und weniger die vernachlässigbaren HF-Effekte.

In der Zusammenfassung "Final Report Summary - SEAWIND" (Sound Exposure and Risk Assessment of Wireless Network Devices) gab 2012 Primo Schär detaillierter Auskunft über die Probleme, die er bei der Replikation von DNA-Brüchen infolge EMF-Einwirkung beobachtete, und deren Ursache er im untauglichen Kometentest verortete (falsch-positive Resultate). Dies ist deshalb von Bedeutung, weil der Kometentest einer von zwei DNA-Strangbruchdetektoren war, auf die sich die Ergebnisse der Wiener "Reflex"-Studien stützen. Was immer die Forscher in Wien durch ihre Mikroskope beim Betrachten der "Kometenschweife" gesehen haben, DNA-Bruchstücke und damit alarmierende Belege für DNA-Strangbrüche waren es gemäß Schär nicht.

Genotoxicity Testing in cultured human cells (in vitro)

Evidence for a potential genotoxic effect of wEMF [weak EMF; Anm. Spatenpauli] at 0.1 to 2 W/kg was obtained mostly from the so-called "Alkaline Comet assay", a classical experimental test to evaluate and quantify the level of DNA damage, in particular DNA strand-breaks and alkaline labile sites, in cell populations. In the SEAWIND project, we made use of this assay to re-visit the potential of wEMFs to induce DNA damage in in vitro cultured human cells. In addition to the standard test, we applied a modified version of the assay that allows a specific and more sensitive detection of oxidative DNA base lesions. Moreover, to address potential wEMF effects more comprehensively, the SEAWIND project not only tested previously reported observations but extended the investigation to a wider range of wEMF signals and modulations that have become important for wireless data transfer in recent years and to the exploration of potential co-genotoxic effect.

The first series of experiments focused on the replication of previously reported observations with the alkaline Comet assay that indicated an induction of DNA damage by mobile phone-specific UMTS and GSM signal modulations in primary human fibroblast and immortalized human trophoblast cells (HTR-8/SVneo cells). Despite considerable efforts to optimize the sensitivity of our assays, however, these effects could not be confirmed in the two SEAWIND partner laboratories. Likewise, the subsequent systematic testing of potential effects of the WiFi, RFID and CW exposure signals in the Comet assays failed to produce evidence for DNA damage induction.

To validate these results and to address the hypothesis that wEMFs might alter intracellular levels of reactive oxygen species (ROS), we systematically assessed the wEMF signals for induction of oxidative DNA damage. For this purpose, we applied the enzyme-modified version of the Comet assay, designed to specifically detect a common oxidative base modification with high sensitivity. As the standard alkaline Comet analyses, however, this assay produced no evidence for increased oxidative DNA base lesions in the two cell types tested. Consistent with this lack of effect in the enzyme-modified Comet assay, we did not observe any EMF-induced change in intracellular ROS using a highly-sensitive, fluorescence microscopy-based method that traps the short-living ROS and converts it into a more persistent fluorescence signal. Notably, this assay was negative, even if it was performed under 50Hz EMF exposure that previously produced a measurable effect in the alkaline Comet assay. Thus, as both the intracellular ROS detection and the oxidative Comet assay are very sensitive methods, we conclude that ROS-mediated oxidative DNA damage is not an important contributor to potential genotoxic effects related to wEMF exposure under the experimental conditions.

The development and application of standardized experimental procedures, including the blinded exposure and evaluation as well as the independent confirmation of key findings in the two partner laboratories, was an important conceptual strength of this part of the SEAWIND project, meant to avoid eventual inconsistencies in reproducibility that often occurred in this field of research. Minor variations of experimental conditions, due to differences in the technical infrastructure between laboratories, however, are difficult to eliminate and may have contributed to the contradictory outcome of similar experiments in the past. We addressed the issue of experimental variation, focusing on the consistency of results generated by different Comet analysis pipelines. This showed, not unexpectedly, that the methods used in the two partner laboratories produced slightly different results from otherwise identical experiments or even data sets. Thus, minor technical issues like this may add up in the multistep protocol of the Comet assay and culminate in an overall experimental variation that, if the effect under investigation is small, may produce small positive results in some analyses and negative outcomes in others. This is a negligible issue if the agent assayed is clearly DNA toxic and affects homogeneously most cells in a population with a consistent dose-dependency. EMF exposure, however, has never produced strong dose-dependent responses and the data underlying positive effects (e.g. ELF-EMF exposed human cells) suggest that only a subpopulation cells in a culture may be affected. All considered, the inconsistent nature of EMF effects in the Comet assay strongly suggests that this type of exposure does not damage the chemical structure of the DNA. It may, however, affect certain aspects of cell physiology in a way that alters the steady-state level of naturally occurring DNA strand breaks in some cells. Cells undergoing DNA synthesis, for instance, have intrinsically increased levels of DNA strand breaks that are picked up by the Comet assay, and there is evidence from ELF-EMF studies that the dynamics of such processes might be influenced under exposure.

In conclusion, considering both the results reported in the literature and the observations of the biological in vitro part of the SEAWIND project, there is no evidence for a direct DNA damaging potential of wEMFs. However, wEMF exposure might impact cellular processes that, in combination with other environmental stressors, could result in molecular readouts resembling those of genotoxins. Such effects could explain the occasionally reported positive result with the Comet assay.

--
Jedes komplexe Problem hat eine Lösung, die einfach, naheliegend, plausibel – und falsch ist.
– Frei nach Henry Louis Mencken (1880–1956) –

Tags:
Schär, Comet-Assay, DNA, Kometenschweif, DNA-Strangbrüche, Enzyme