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Shorter exposures to harder X-rays trigger early apoptotic events in Xenopus laevis embryos.

Dong J, Mury SP, Drahos KE, Moscovitch M, Zia RK, Finkielstein CV - PLoS ONE (2010)

Bottom Line: Our research challenges the current dogma of dose-dependent induction of apoptosis and establishes a new parallel paradigm to the photoelectric effect in biological systems.Overall, our data establish that the energy of the incident photon is a major contributor to the outcome of the biological system.These results suggest that biological organisms display properties similar to the photoelectric effect in physical systems and provide new insights into how radiation-mediated apoptosis should be understood and utilized for therapeutic purposes.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics, Virginia Polytechnic Institute and State University, Blacksburg, Virginia, United States of America.

ABSTRACT

Background: A long-standing conventional view of radiation-induced apoptosis is that increased exposure results in augmented apoptosis in a biological system, with a threshold below which radiation doses do not cause any significant increase in cell death. The consequences of this belief impact the extent to which malignant diseases and non-malignant conditions are therapeutically treated and how radiation is used in combination with other therapies. Our research challenges the current dogma of dose-dependent induction of apoptosis and establishes a new parallel paradigm to the photoelectric effect in biological systems.

Methodology/principal findings: We explored how the energy of individual X-ray photons and exposure time, both factors that determine the total dose, influence the occurrence of cell death in early Xenopus embryo. Three different experimental scenarios were analyzed and morphological and biochemical hallmarks of apoptosis were evaluated. Initially, we examined cell death events in embryos exposed to increasing incident energies when the exposure time was preset. Then, we evaluated the embryo's response when the exposure time was augmented while the energy value remained constant. Lastly, we studied the incidence of apoptosis in embryos exposed to an equal total dose of radiation that resulted from increasing the incoming energy while lowering the exposure time.

Conclusions/significance: Overall, our data establish that the energy of the incident photon is a major contributor to the outcome of the biological system. In particular, for embryos exposed under identical conditions and delivered the same absorbed dose of radiation, the response is significantly increased when shorter bursts of more energetic photons are used. These results suggest that biological organisms display properties similar to the photoelectric effect in physical systems and provide new insights into how radiation-mediated apoptosis should be understood and utilized for therapeutic purposes.

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Whole-mount TUNEL assay exposes apoptotic cells in irradiated embryos.A. Pre-MBT embryos were exposed to different energies 20 kV, 30 kV, 40 kV and 48 kV to equal a total dose of ∼65 Gy. Non-irradiated embryos are referred to as “control”. Six hours after the MBT, embryos were fixed in MEMFA as described in the “Materials and Methods” section and photographed. B. TUNEL staining was performed on fixed embryos to detect DNA fragmentation. Embryos were treated as described in (A). Intense TUNEL staining was detected in the animal pole portion of the embryos. The embryos shown in B are representative of the TUNEL staining observed following analysis of ∼80 embryos of which 20% were stained. Arrowhead points to labeled nuclei. An, animal pole. Scale bar, 250 µm.
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pone-0008970-g006: Whole-mount TUNEL assay exposes apoptotic cells in irradiated embryos.A. Pre-MBT embryos were exposed to different energies 20 kV, 30 kV, 40 kV and 48 kV to equal a total dose of ∼65 Gy. Non-irradiated embryos are referred to as “control”. Six hours after the MBT, embryos were fixed in MEMFA as described in the “Materials and Methods” section and photographed. B. TUNEL staining was performed on fixed embryos to detect DNA fragmentation. Embryos were treated as described in (A). Intense TUNEL staining was detected in the animal pole portion of the embryos. The embryos shown in B are representative of the TUNEL staining observed following analysis of ∼80 embryos of which 20% were stained. Arrowhead points to labeled nuclei. An, animal pole. Scale bar, 250 µm.

Mentions: We then employed an independent assay to test whether the apoptotic response that results from energy-dependent exposure correlates with DNA damage. With this in mind, we used a highly sensitive indicator of DNA fragmentation in situ, whole-mount TUNEL (terminal deoxynucleotidyl transferase-mediated nicked-end labeling) staining, a method that allows the detection of apoptotic cells at high frequency in early embryos (Fig. 6) [33], [34]. Accordingly, embryos were exposed to various energies and times for which the system is above (≥30 kV, ∼65 Gy) or below (∼20 kV) a threshold response defined in our experimental sample (Figs. 2, 3 and 5). Treated embryos were analyzed for DNA fragmentation 8 h after the MBT (Fig. 6.A). In agreement with previous reports [33], the presence of TUNEL-positive embryos in control samples (non-irradiated) was limited to less than 3% of all embryos analyzed. This event, therefore, represents the normally occurring programmed cell death that is an essential part of embryonic development and that is expected to occur after gastrula stages and during the maturation of the nervous system [33]. Over 95% of embryos treated with energies equal to or greater than 30 kV exhibited an extensive pattern of TUNEL-positive cells after the MBT in late blastula that persisted through later stages of development (Fig. 6.B). In no case did we detect the appearance of extensive TUNEL-positive cells in embryos exposed to 20 kV; instead, these embryos closely resembled control samples. Two observations are of note here. First, greater incoming energy correlates with the detection of more TUNEL-positive cells on the animal pole of embryos, thus, indicating a larger extent of double strand breaks due to apoptosis (Fig. 6.B). Second, dying cells appear to be randomly distributed in some cases during normal gastrulation and in 20 kV-irradiated embryos (Fig. 6.B, arrowhead), an observation that has been previously reported in gastrulating newt, Cynops pyrrhogaster, chicken, Xenopus and mouse embryos as well [27], [33], [35], [36]. Whereas this event might be part of the normal developmental process in the embryo, the loss of a specific subset of cells as noticed by localized positive TUNEL-staining could well explain some of the phenotypic defects observed in a reduced number of tailbuds and revealed 20 h after exposure of embryos to 20 kV (Fig. 6.B, arrowhead).


Shorter exposures to harder X-rays trigger early apoptotic events in Xenopus laevis embryos.

Dong J, Mury SP, Drahos KE, Moscovitch M, Zia RK, Finkielstein CV - PLoS ONE (2010)

Whole-mount TUNEL assay exposes apoptotic cells in irradiated embryos.A. Pre-MBT embryos were exposed to different energies 20 kV, 30 kV, 40 kV and 48 kV to equal a total dose of ∼65 Gy. Non-irradiated embryos are referred to as “control”. Six hours after the MBT, embryos were fixed in MEMFA as described in the “Materials and Methods” section and photographed. B. TUNEL staining was performed on fixed embryos to detect DNA fragmentation. Embryos were treated as described in (A). Intense TUNEL staining was detected in the animal pole portion of the embryos. The embryos shown in B are representative of the TUNEL staining observed following analysis of ∼80 embryos of which 20% were stained. Arrowhead points to labeled nuclei. An, animal pole. Scale bar, 250 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2813296&req=5

pone-0008970-g006: Whole-mount TUNEL assay exposes apoptotic cells in irradiated embryos.A. Pre-MBT embryos were exposed to different energies 20 kV, 30 kV, 40 kV and 48 kV to equal a total dose of ∼65 Gy. Non-irradiated embryos are referred to as “control”. Six hours after the MBT, embryos were fixed in MEMFA as described in the “Materials and Methods” section and photographed. B. TUNEL staining was performed on fixed embryos to detect DNA fragmentation. Embryos were treated as described in (A). Intense TUNEL staining was detected in the animal pole portion of the embryos. The embryos shown in B are representative of the TUNEL staining observed following analysis of ∼80 embryos of which 20% were stained. Arrowhead points to labeled nuclei. An, animal pole. Scale bar, 250 µm.
Mentions: We then employed an independent assay to test whether the apoptotic response that results from energy-dependent exposure correlates with DNA damage. With this in mind, we used a highly sensitive indicator of DNA fragmentation in situ, whole-mount TUNEL (terminal deoxynucleotidyl transferase-mediated nicked-end labeling) staining, a method that allows the detection of apoptotic cells at high frequency in early embryos (Fig. 6) [33], [34]. Accordingly, embryos were exposed to various energies and times for which the system is above (≥30 kV, ∼65 Gy) or below (∼20 kV) a threshold response defined in our experimental sample (Figs. 2, 3 and 5). Treated embryos were analyzed for DNA fragmentation 8 h after the MBT (Fig. 6.A). In agreement with previous reports [33], the presence of TUNEL-positive embryos in control samples (non-irradiated) was limited to less than 3% of all embryos analyzed. This event, therefore, represents the normally occurring programmed cell death that is an essential part of embryonic development and that is expected to occur after gastrula stages and during the maturation of the nervous system [33]. Over 95% of embryos treated with energies equal to or greater than 30 kV exhibited an extensive pattern of TUNEL-positive cells after the MBT in late blastula that persisted through later stages of development (Fig. 6.B). In no case did we detect the appearance of extensive TUNEL-positive cells in embryos exposed to 20 kV; instead, these embryos closely resembled control samples. Two observations are of note here. First, greater incoming energy correlates with the detection of more TUNEL-positive cells on the animal pole of embryos, thus, indicating a larger extent of double strand breaks due to apoptosis (Fig. 6.B). Second, dying cells appear to be randomly distributed in some cases during normal gastrulation and in 20 kV-irradiated embryos (Fig. 6.B, arrowhead), an observation that has been previously reported in gastrulating newt, Cynops pyrrhogaster, chicken, Xenopus and mouse embryos as well [27], [33], [35], [36]. Whereas this event might be part of the normal developmental process in the embryo, the loss of a specific subset of cells as noticed by localized positive TUNEL-staining could well explain some of the phenotypic defects observed in a reduced number of tailbuds and revealed 20 h after exposure of embryos to 20 kV (Fig. 6.B, arrowhead).

Bottom Line: Our research challenges the current dogma of dose-dependent induction of apoptosis and establishes a new parallel paradigm to the photoelectric effect in biological systems.Overall, our data establish that the energy of the incident photon is a major contributor to the outcome of the biological system.These results suggest that biological organisms display properties similar to the photoelectric effect in physical systems and provide new insights into how radiation-mediated apoptosis should be understood and utilized for therapeutic purposes.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics, Virginia Polytechnic Institute and State University, Blacksburg, Virginia, United States of America.

ABSTRACT

Background: A long-standing conventional view of radiation-induced apoptosis is that increased exposure results in augmented apoptosis in a biological system, with a threshold below which radiation doses do not cause any significant increase in cell death. The consequences of this belief impact the extent to which malignant diseases and non-malignant conditions are therapeutically treated and how radiation is used in combination with other therapies. Our research challenges the current dogma of dose-dependent induction of apoptosis and establishes a new parallel paradigm to the photoelectric effect in biological systems.

Methodology/principal findings: We explored how the energy of individual X-ray photons and exposure time, both factors that determine the total dose, influence the occurrence of cell death in early Xenopus embryo. Three different experimental scenarios were analyzed and morphological and biochemical hallmarks of apoptosis were evaluated. Initially, we examined cell death events in embryos exposed to increasing incident energies when the exposure time was preset. Then, we evaluated the embryo's response when the exposure time was augmented while the energy value remained constant. Lastly, we studied the incidence of apoptosis in embryos exposed to an equal total dose of radiation that resulted from increasing the incoming energy while lowering the exposure time.

Conclusions/significance: Overall, our data establish that the energy of the incident photon is a major contributor to the outcome of the biological system. In particular, for embryos exposed under identical conditions and delivered the same absorbed dose of radiation, the response is significantly increased when shorter bursts of more energetic photons are used. These results suggest that biological organisms display properties similar to the photoelectric effect in physical systems and provide new insights into how radiation-mediated apoptosis should be understood and utilized for therapeutic purposes.

Show MeSH
Related in: MedlinePlus