Annika Reddig

Analysis of DNA Double-Strand Breaks and Cytotoxicity after 7 Tesla Magnetic Resonance Imaging of Isolated Human Lymphocytes

The global use of magnetic resonance imaging (MRI) is constantly growing and the field strengths increasing. Yet, only little data about harmful biological effects caused by MRI exposure are available and published research analyzing the impact of MRI on DNA integrity reported controversial results. This in vitro study aimed to investigate the genotoxic and cytotoxic potential of 7 T ultra-high-field MRI on isolated human peripheral blood mononuclear cells. Hence, unstimulated mononuclear blood cells were exposed to 7 T static magnetic field alone or in combination with maximum permissible imaging gradients and radiofrequency pulses as well as to ionizing radiation during computed tomography and γ-ray exposure. DNA double-strand breaks were quantified by flow cytometry and automated microscopy analysis of immunofluorescence stained γH2AX. Cytotoxicity was studied by CellTiter-Blue viability assay and [3H]-thymidine proliferation assay. Exposure of unstimulated mononuclear blood cells to 7 T static magnetic field alone or combined with varying gradient magnetic fields and pulsed radiofrequency fields did not induce DNA double-strand breaks, whereas irradiation with X- and γ-rays led to a dose-dependent induction of γH2AX foci. The viability assay revealed a time- and dose-dependent decrease in metabolic activity only among samples exposed to γ-radiation. Further, there was no evidence for altered proliferation response after cells were exposed to 7 T MRI or low doses of ionizing radiation (≤ 0.2 Gy). These findings confirm the acceptance of MRI as a safe non-invasive diagnostic imaging tool, but whether MRI can induce other types of DNA lesions or DNA double-strand breaks during altered conditions still needs to be investigated.

DNA double-strand breaks and micronuclei in human blood lymphocytes after repeated whole body exposures to 7T Magnetic Resonance Imaging.

PURPOSE To examine the extent of genetic damage, assessed from deoxyribonucleic acid (DNA) double-strand breaks (DSBs) and micronuclei (MN) in peripheral blood mononuclear cells obtained from individuals repeatedly exposed to 7T Magnetic Resonance Imaging (MRI). MATERIALS AND METHODS The study protocol was approved by the local ethics committee. Informed consent was obtained from 22 healthy, non-smoking, non-alcoholic male individuals, who had never undergone radio-/chemo-therapy, scintigraphy, and had not undergone X-ray examination one year prior blood withdrawal. Eleven participants were repeatedly exposed to 7T and 3T MRI while working with/around scanners or frequently participating as 7T and lower field MRI research subjects (mean age 34±7years). The other half was never exposed to 7T or lower field MRI and served as controls (mean age 33±9years). The damage in lymphocytes was assessed using anti-γH2AX immunofluorescence staining of DNA DSBs and by quantification of MN. Isolated cells were further exposed in vitro to 7T MRI either alone or in the presence of the DNA damaging drug etoposide, to determine if there is any additional combined effect. The kinetics of DNA damage repair were examined. RESULTS The mean base-level of γH2AX foci/cell and incidence of MN between repeatedly exposed and control group were not significantly different (P=0.618 and P=0.535, respectively). The additional in vitro exposure of cells to 7T MRI had no significant impact on MN frequencies and γH2AX foci at 1, 20 and 72h after exposure. CONCLUSION Frequently repeated 7T MRI exposure did not result in a detectable increase in genotoxicity indices and alterations of DNA repair kinetics.

Assessment of modulated cytostatic drug resistance by automated γH2AX analysis

The efficacy of many chemotherapeutic agents relies on the preferential destruction of rapidly dividing cancer cells by inducing various kinds of DNA damage. The most deleterious type of DNA lesions are DNA double‐strand breaks (DSB), which can be detected by immunofluorescence staining of phosphorylated histone protein H2AX (γH2AX). Furthermore, γH2AX has been suggested as clinical pharmacodynamic biomarker in chemotherapeutic cancer treatment. A great challenge in treating neoplastic diseases is the varying response behavior among cancer patients. Thus, intrinsic or drug‐induced overexpression of efflux pumps often leads to multiple drug resistance (MDR) and treatment failure. In particular, inter‐individual differences in expression levels of efflux pumps, such as the permeability glycoprotein (P‐gp), were shown to correlate with cancer progression. Several efficient cytostatic drugs, including the DSB‐inducing agent etoposide (ETP) are known P‐gp substrates. In this respect, modulation of MDR by P‐gp inhibitors, like the immunosuppressives cyclosporine A (CsA) and rapamycin (Rapa) have been described. Here, we investigated the application of γH2AX focus assay to monitor the impact of CsA and Rapa on ETP‐induced cytotoxicity in human peripheral blood mononuclear cells. Evaluation of γH2AX foci was performed by the automated fluorescence microscopy and interpretation system AKLIDES. Compared to ETP treatment alone, our results revealed a significant rise in γH2AX focus number and percentage of DSB‐positive cells after cells have been treated with ETP in the presence of either CsA or Rapa. In contrast, DSB levels of cells incubated with CsA or Rapa alone were comparable to focus number of untreated cells. Our results successfully demonstrated how automated γH2AX analysis can be used as fast and reliable approach to monitor drug resistance and the impact of MDR modulators during treatment with DSB‐inducing cytostatics.

Impact of in Vivo High-Field-Strength and Ultra-High-Field-Strength MR Imaging on DNA Double-Strand-Break Formation in Human Lymphocytes

Purpose To determine the impact of different magnetic field strengths (1, 1.5, 3, and 7 T) and the effect of contrast agent on DNA double-strand-break (DSB) formation in patients undergoing magnetic resonance (MR) imaging. Materials and Methods This in vivo study was approved by the local ethics committee, and written informed consent was obtained from each patient. To analyze the level of DNA DSBs, peripheral blood mononuclear cells were isolated from blood samples drawn directly before, as well as 5 minutes and 30 minutes after MR imaging examination. After performing γH2AX immunofluorescence staining, DSBs were quantified with automated digital microscopy. MR group consisted of 43 patients (22 women, 21 men; mean age, 46.1 years; range, 20-77 years) and was further subdivided according to the applied field strength and administration of contrast agent. Additionally, 10 patients undergoing either unenhanced or contrast material-enhanced computed tomography (CT) served as positive control subjects. Statistical analysis was performed with Friedman test. Results Whereas DSBs in lymphocytes increased after CT exposure (before MR imaging: 0.14 foci per cell ± 0.05; 5 minutes after: 0.26 foci per cell ± 0.07; 30 minutes after: 0.24 foci per cell ± 0.07; P ≤ .05), no alterations were observed in patients examined with MR imaging (before MR imaging: 0.13 foci per cell ± 0.02; 5 minutes after: 0.12 foci per cell ± 0.02; 30 minutes after: 0.11 foci per cell ± 0.02; P > .05). Differentiated analysis of MR imaging subgroups again revealed no significant changes in γH2AX level. Conclusion Analysis of γH2AX foci showed no evidence of DSB induction after MR examination, independent of the applied field strength and administration of gadolinium-based contrast agent.

MRI and Genetic Damage: An Update

Purpose of ReviewMagnetic resonance imaging (MRI) is generally considered to be a safe imaging technology when compared to diagnostic tools utilizing ionizing radiation. Unlike ionizing radiation, for which the detrimental biological effects, such as DNA damage, are known and well established, the potential of MRI to directly or indirectly induce genetic alterations is still not evident. This review article summarizes recent findings in MRI research related to DNA damage.Recent FindingsApplying different exposure conditions, several studies have examined the potential impact of MRI on DNA. While some authors reported increases in DNA damage, the largest studies and those with the highest field strength (up to 7 T) and highest exposure did not find a significant excess in DNA damage.SummaryThe debate about biological effects of MRI has been increasingly tackled over the last years, due to extended use of MRI systems in clinic and research. The balance of scientific evidence from available literature does not allow a final conclusion regarding any significant risk related to DNA damage induced by MRI. The risk and its impact on public health, if any, should be substantially small.

In-vivo-Analyse von DNA-Doppelstrangbrüchen in humanen mononukleären Zellen (MNZ) nach klinischen MRT-Untersuchungen

Material und Methodik: Bei insgesamt 43 Patienten (22 weiblich, 21 männlich, Altersspanne 20-77 Jahre, Durchschnittsalter 46,3 Jahre) wurde unmittelbar vor sowie 5 und 30 Minuten nach einer klinischen MRT-Untersuchung peripheres venöses Blut entnommen. Die klinischen MRT-Untersuchungen fanden an einem 1 Tesla-, 1,5 Tesla-, 3 Teslaoder 7 Tesla-Scanner, mit oder ohne Verabreichung von gadoliniumhaltigem Kontrastmittel statt und entsprachen klinisch üblichen MRT-Protokollen (Thorax, Abdomen, Becken, Herz, Muskuloskelettal). Als Referenz fungierten 10 Patienten mit CT-Untersuchungen (5 mit jodhaltigem Kontrastmittel, 5 nativ). Die mononukleären Zellen (MNZ) wurden mittels Dichtegradientenzentrifugation separiert und unmittelbar danach fixiert. Anschließend wurde die Anzahl von ?H2AX-Foci als Marker für DNA-Doppelstrangbrüche mittels Immunfluoreszenzfärbung und automatisierter Mikroskopie (AKLIDES-System, Medipan GmbH) zu den drei verschiedenen Zeitpunkten vor (t = -1 min) und nach der Exposition (t = 5 min und 30 min) bestimmt.