Ion Udroiu

Clastogenicity and aneuploidy in newborn and adult mice exposed to 50 Hz magnetic fields

Purpose: To detect possible clastogenic and aneugenic properties of a 50 Hz, 650 μT magnetic field. Materials and methods: The micronucleus test with CREST (Calcinosis, Raynauds phenomenon, Esophageal dismotility, Sclerodactility, Telangectasia) antibody staining was performed on liver and peripheral blood sampled from newborn mice exposed to an ELF (Extremely Low Frequency) magnetic field during the whole intra-uterine life (21 days), and on bone marrow and peripheral blood sampled from adult mice exposed to the same magnetic field for the same period. Results: Data obtained in newborn mice show a significant increase in micronuclei frequencies. In absolute terms, most of the induced micronuclei were CREST-negative (i.e., formed by a chromosome fragment). However, in relative terms, ELF exposure caused a two-fold increase in CREST-negative micronuclei and a four-fold increase in CREST-positive micronuclei (i.e., formed by a whole chromosome). No significant effect was recorded on exposed adults. Conclusions: These findings suggest the need for investigation of aneugenic properties of ELF magnetic fields in order to establish a possible relationship to carcinogenesis.

Genotoxic sensitivity of the developing hematopoietic system.

Genotoxic sensitivity seems to vary during ontogenetic development. Animal studies have shown that the spontaneous mutation rate is higher during pregnancy and infancy than in adulthood. Human and animal studies have found higher levels of DNA damage and mutations induced by mutagens in fetuses/newborns than in adults. This greater susceptibility could be due to reduced DNA repair capacity. In fact, several studies indicated that some DNA repair pathways seem to be deficient during ontogenesis. This has been demonstrated also in murine hematopoietic stem cells. Genotoxicity in the hematopoietic system has been widely studied for several reasons: it is easy to assess, deals with populations cycling also in the adults and may be relevant for leukemogenesis. Reviewing the literature concerning the application of the micronucleus test (a validated assay to assess genotoxicity) in fetus/newborns and adults, we found that the former show almost always higher values than the latter, both in animals treated with genotoxic substances and in those untreated. Therefore, we draw the conclusion that the genotoxic sensitivity of the hematopoietic system is more pronounced during fetal life and decreases during ontogenic development.

Genotoxicity Induced by Foetal and Infant Exposure to Magnetic Fields and Modulation of Ionising Radiation Effects.

Background Few studies have investigated the toxicity and genotoxicity of extremely low frequency magnetic fields (ELF-MF) during prenatal and neonatal development. These phases of life are characterized by cell proliferation and differentiation, which might make them sensitive to environmental stressors. Although in vitro evidences suggest that ELF-MF may modify the effects of ionizing radiation, no research has been conducted so far in vivo on the genotoxic effects of ELF-MF combined with X-rays. Aim and methods Aim of this study was to investigate in somatic and germ cells the effects of chronic ELF-MF exposure from mid gestation until weaning, and any possible modulation produced by ELF-MF exposure on ionizing radiation-induced damage. Mice were exposed to 50 Hz, 65 μT magnetic field, 24 hours/day, for a total of 30 days, starting from 12 days post-conception. Another group was irradiated with 1 Gy X-rays immediately before ELF-MF exposure, other groups were only X-irradiated or sham-exposed. Micronucleus test on blood erythrocytes was performed at multiple times from 1 to 140 days after birth. Additionally, 42 days after birth, genotoxic and cytotoxic effects on male germ cells were assessed by comet assay and flow cytometric analysis. Results ELF-MF exposure had no teratogenic effect and did not affect survival, growth and development. The micronucleus test indicated that ELF-MF induced a slight genotoxic damage only after the maximum exposure time and that this effect faded away in the months following the end of exposure. ELF-MF had no effects on ionizing radiation (IR)-induced genotoxicity in erythrocytes. Differently, ELF–MF appeared to modulate the response of male germ cells to X-rays with an impact on proliferation/differentiation processes. These results point to the importance of tissue specificity and development on the impact of ELF-MF on the early stages of life and indicate the need of further research on the molecular mechanisms underlying ELF-MF biological effects.

Study of the effects of 0.15 terahertz radiation on genome integrity of adult fibroblasts: Effects of Terahertz Radiation on Genome Integrity

The applications of Terahertz (THz) technologies have significantly developed in recent years, and the complete understanding of the biological effects of exposure to THz radiation is becoming increasingly important. In a previous study, we found that THz radiation induced genomic damage in fetal fibroblasts. Although these cells demonstrated to be a useful model, exposure of human foetuses to THz radiation is highly improbable. Conversely, THz irradiation of adult dermal tissues is cause of possible concern for some professional and nonprofessional categories. Therefore, we extended our study to the investigation of the effects of THz radiation on adult fibroblasts (HDF). In this work, the effects of THz exposure on HDF cells genome integrity, cell cycle, cytological ultrastructure and proteins expression were assessed. Results of centromere‐negative micronuclei frequencies, phosphorylation of H2AX histone, and telomere length modulation indicated no induction of DNA damage. Concordantly, no changes in the expression of proteins associated with DNA damage sensing and repair were detected. Conversely, our results showed an increase of centromere‐positive micronuclei frequencies and chromosomal nondisjunction events, indicating induction of aneuploidy. Therefore, our results indicate that THz radiation exposure may affect genome integrity through aneugenic effects, and not by DNA breakage. Our findings are compared to published studies, and possible biophysical mechanisms are discussed. Environ. Mol. Mutagen. 59:476–487, 2018.

Genomic damage induced by 1‐MHz ultrasound in vitro

Genotoxic effects of therapeutic ultrasound are poorly documented, when compared with the wide use of this physical agent. The aim of this work was to investigate the clastogenic and aneugenic potential of 1 MHz ultrasound, employing intensities (200 and 300 mW/cm2) above the cavitational threshold, but in the range of those normally used in therapeutics. Both normal fibroblasts (AG01522) and tumoral cells (MCF‐7) were sonicated. While no effects on viability were noted, significant increases of CREST‐negative micronuclei (indicative of clastogenesis) and CREST‐positive micronuclei (indicative of aneuploidy) were detected. Clastogenesis was confirmed by increases of γ‐H2AX foci, while increases of spindle anomalies confirmed the induction of aneuploidy. Our results confirm previous works that showed ultrasound‐induced DNA breakage. Moreover, our experiments show that the known effect of ultrasound‐induced damage to microtubules is also able to damage the mitotic spindle and induce aneuploidy. On the overall, this work highlights the importance to further investigate the potential risks related to therapeutics US. Environ. Mol. Mutagen. 59:60–68, 2018.

The Phylogeny of the Spleen

The spleen can exert different functions: hematopoiesis, immune response, blood filtration, and blood storage. Presence and importance of each function vary among different species and, within a single species, vary during ontogeny. During the course of evolution, loss of hematopoietic functions was accompanied by increased specialization of other functions. Increase in the complexity of immunological features can be seen in the passage from scattered lymphocytes to segregation of the white pulp, organization of T- and B-lymphocyte regions and, finally, appearance of germinal centers. The red pulp remained essentially the same in all nonmammalian vertebrates, with a microarchitecture that concentrates blood and allows its filtration. In our view, this high splenic hematocrit was eventually co-opted in teleosts and mammals to develop a new function: the storage of red blood cells. Finally, mammalian species evolved different features of the red pulp, increasing the specialization of the filtration function (pulp sinuses) and/or storing function (muscularization).

Long-term genotoxic effects in the hematopoietic system of prenatally X-irradiated mice

Abstract Purpose: To investigate the genotoxic effects of prenatal X-irradiation in mice and the possible presence of late genomic instability. Materials and methods: Pregnant mice were exposed to 0, 1 or 2 Gy at embryonic day 11.5. Blood smears were obtained from pups at birth and on post-natal day 11, 21, 42 and 140. Hematological data (diameter of erythrocytes, percentage of reticulocytes and Granulocyte-to-Lymphocyte ratio [GLR]) and genotoxicity (micronucleated erythrocytes, micronucleated reticulocytes, CREST-positive and negative micronuclei) were assessed. Results: Prenatal irradiation caused perinatal reticulocytosis (which ended on postnatal day 11) and a dose-dependent increase of GLR (indicative of myeloid skewing) on postnatal days 42 and 140. Two temporally distinct genotoxic effects were observed: an early, acute damage (still detectable at birth and soon after) and a late, long-term damage. Conclusions: Increases in micronuclei frequencies and GLR observed from day 42 on are both ascribable to DNA damage. Time of appearance of this late effect may be linked to the shift of hematopoiesis from spleen to bone marrow and to cell-extrinsic factor such as the microenvironment. This study confirms that ionizing radiation can induce long-term genotoxic effects in the hematopoietic system and shows that prenatal irradiation determines genomic instability in blood-forming tissues of adult mice.

Letter to the Editor: On the Growth of Hematopoietic Stem Cells and Childhood Leukemias

Understanding of the etiology of pediatric leukemias is constantly improving, as well as knowledge of the ontogeny of hematopoiesis. However, the peculiar incidence rates by age of acute leukemias remain enigmatic. Recently, some authors indicated the importance of elucidating the interplay between development of hematopoiesis and onset of pediatric leukemias [1, 2]. The article of Werner et al. [3] provides a mathematical model showing that ontogenic growth influences the behavior of mutations within hematopoietic stem cells (HSCs) that lead to leukemia. The authors clearly show how mutant fitness is affected by changes in population size and consequently the effects of mutations on the HSC population vary with age. Although the article gives an important contribution, we think that two issues have not been considered, and that their inclusion by the authors would give a further advancement of the model. The first issue is on the expansion of the HSCs pool. The authors correctly indicate that this is witnessed (in mice and humans) by accelerated telomere shortening during development. However, this HSC expansion is seen as an allometric growth, and its end is supposed at 20 years (when body growth ceases). We think that this phenomenon is more complex. During postnatal development, the bone marrow grows proportionally to the body growth (Fig. 1A), but, in the meantime, there is also a gradual conversion of red (hematopoietic) marrow into yellow (nonhematopoietic) marrow. While at birth almost all bone marrow is hematopoietic, in adult only half of it is hematopoietically active [4]. This determines a peculiar growth curve of the red marrow (Fig. 1B); in fact, it is noteworthy that a 3years-old child owns 1.2 kg of red marrow, compared to the 1.5 kg of an adult (Fig. 1C), despite a fivefold difference in body weight [5]. It seems reasonable that HSC expansion follows the same pattern of the red marrow. A confirmation to this seems to come from the HSC kinetics analyzed by telomere shortening [6] (Fig. 1D). In fact, the curve of HSC replications is significantly correlated with red marrow growth (R 5 0.9251, p 5 .0382) and not with bone marrow growth (R 5 0.5828, p 5 .2366), as analyzed by Pearson’s correlation coefficient. Therefore, it is probable that growth of the HSC pool is not proportional to body growth. Moreover, it is not sure that HSCs expansion stops concurrently with body growth: it is possible that it happens earlier. Studies on mice have shown that the HSC population switches from a fast-dividing state to a quiescent one well before the animal stops to grow [7, 8]. The second issue is less related to mathematical formulas, but could be included in Werner’s model for a further development. It seems evident that there are specific differences not only between fetal and adult HSCs, but also between these and the infant ones [7, 8]. HSCs comprise subpopulations that differ in their relative contribution to myeloand lymphopoiesis [9]. Beside the abovementioned differences in proliferation, variations in the proportion of HSC subpopulations during development has been demonstrated [8]. Studies on the kinetics of the HSC subtypes throughout human development could give interesting indications on the differences between myeloid and lymphoid pediatric leukemias. Dipartimento di Scienze, Universit a degli Studi Roma Tre, Roma, Italy

On the Growth of Hematopoietic Stem Cells and Childhood Leukemias

Understanding of the etiology of pediatric leukemias is constantly improving, as well as knowledge of the ontogeny of hematopoiesis. However, the peculiar incidence rates by age of acute leukemias remain enigmatic. Recently, some authors indicated the importance of elucidating the interplay between development of hematopoiesis and onset of pediatric leukemias [1, 2]. The article of Werner et al. [3] provides a mathematical model showing that ontogenic growth influences the behavior of mutations within hematopoietic stem cells (HSCs) that lead to leukemia. The authors clearly show how mutant fitness is affected by changes in population size and consequently the effects of mutations on the HSC population vary with age. Although the article gives an important contribution, we think that two issues have not been considered, and that their inclusion by the authors would give a further advancement of the model. The first issue is on the expansion of the HSCs pool. The authors correctly indicate that this is witnessed (in mice and humans) by accelerated telomere shortening during development. However, this HSC expansion is seen as an allometric growth, and its end is supposed at 20 years (when body growth ceases). We think that this phenomenon is more complex. During postnatal development, the bone marrow grows proportionally to the body growth (Fig. 1A), but, in the meantime, there is also a gradual conversion of red (hematopoietic) marrow into yellow (nonhematopoietic) marrow. While at birth almost all bone marrow is hematopoietic, in adult only half of it is hematopoietically active [4]. This determines a peculiar growth curve of the red marrow (Fig. 1B); in fact, it is noteworthy that a 3years-old child owns 1.2 kg of red marrow, compared to the 1.5 kg of an adult (Fig. 1C), despite a fivefold difference in body weight [5]. It seems reasonable that HSC expansion follows the same pattern of the red marrow. A confirmation to this seems to come from the HSC kinetics analyzed by telomere shortening [6] (Fig. 1D). In fact, the curve of HSC replications is significantly correlated with red marrow growth (R 5 0.9251, p 5 .0382) and not with bone marrow growth (R 5 0.5828, p 5 .2366), as analyzed by Pearson’s correlation coefficient. Therefore, it is probable that growth of the HSC pool is not proportional to body growth. Moreover, it is not sure that HSCs expansion stops concurrently with body growth: it is possible that it happens earlier. Studies on mice have shown that the HSC population switches from a fast-dividing state to a quiescent one well before the animal stops to grow [7, 8]. The second issue is less related to mathematical formulas, but could be included in Werner’s model for a further development. It seems evident that there are specific differences not only between fetal and adult HSCs, but also between these and the infant ones [7, 8]. HSCs comprise subpopulations that differ in their relative contribution to myeloand lymphopoiesis [9]. Beside the abovementioned differences in proliferation, variations in the proportion of HSC subpopulations during development has been demonstrated [8]. Studies on the kinetics of the HSC subtypes throughout human development could give interesting indications on the differences between myeloid and lymphoid pediatric leukemias. Dipartimento di Scienze, Universit a degli Studi Roma Tre, Roma, Italy

Letter to the Editor: On the Growth of Hematopoietic Stem Cells and Childhood Leukemias: Growth of HSCs and Childhood Leukemias

Understanding of the etiology of pediatric leukemias is constantly improving, as well as knowledge of the ontogeny of hematopoiesis. However, the peculiar incidence rates by age of acute leukemias remain enigmatic. Recently, some authors indicated the importance of elucidating the interplay between development of hematopoiesis and onset of pediatric leukemias [1, 2]. The article of Werner et al. [3] provides a mathematical model showing that ontogenic growth influences the behavior of mutations within hematopoietic stem cells (HSCs) that lead to leukemia. The authors clearly show how mutant fitness is affected by changes in population size and consequently the effects of mutations on the HSC population vary with age. Although the article gives an important contribution, we think that two issues have not been considered, and that their inclusion by the authors would give a further advancement of the model. The first issue is on the expansion of the HSCs pool. The authors correctly indicate that this is witnessed (in mice and humans) by accelerated telomere shortening during development. However, this HSC expansion is seen as an allometric growth, and its end is supposed at 20 years (when body growth ceases). We think that this phenomenon is more complex. During postnatal development, the bone marrow grows proportionally to the body growth (Fig. 1A), but, in the meantime, there is also a gradual conversion of red (hematopoietic) marrow into yellow (nonhematopoietic) marrow. While at birth almost all bone marrow is hematopoietic, in adult only half of it is hematopoietically active [4]. This determines a peculiar growth curve of the red marrow (Fig. 1B); in fact, it is noteworthy that a 3years-old child owns 1.2 kg of red marrow, compared to the 1.5 kg of an adult (Fig. 1C), despite a fivefold difference in body weight [5]. It seems reasonable that HSC expansion follows the same pattern of the red marrow. A confirmation to this seems to come from the HSC kinetics analyzed by telomere shortening [6] (Fig. 1D). In fact, the curve of HSC replications is significantly correlated with red marrow growth (R 5 0.9251, p 5 .0382) and not with bone marrow growth (R 5 0.5828, p 5 .2366), as analyzed by Pearson’s correlation coefficient. Therefore, it is probable that growth of the HSC pool is not proportional to body growth. Moreover, it is not sure that HSCs expansion stops concurrently with body growth: it is possible that it happens earlier. Studies on mice have shown that the HSC population switches from a fast-dividing state to a quiescent one well before the animal stops to grow [7, 8]. The second issue is less related to mathematical formulas, but could be included in Werner’s model for a further development. It seems evident that there are specific differences not only between fetal and adult HSCs, but also between these and the infant ones [7, 8]. HSCs comprise subpopulations that differ in their relative contribution to myeloand lymphopoiesis [9]. Beside the abovementioned differences in proliferation, variations in the proportion of HSC subpopulations during development has been demonstrated [8]. Studies on the kinetics of the HSC subtypes throughout human development could give interesting indications on the differences between myeloid and lymphoid pediatric leukemias. Dipartimento di Scienze, Universit a degli Studi Roma Tre, Roma, Italy