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 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 11  |  Issue : 4  |  Page : 142-149

Complexity of chromosomal aberrations and gene expression changes in human blood lymphocytes after exposure to alpha particle radiation


1 Department of Human Genetics, Sri Ramachandra Institute of Higher Education and Research, Chennai, Tamil Nadu, India
2 Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Mumbai, Maharashtra, India

Date of Submission14-Oct-2020
Date of Acceptance03-Dec-2020
Date of Web Publication30-Dec-2020

Correspondence Address:
Prof. Perumal Venkatachalam
Department of Human Genetics, Sri Ramachandra Institute of Higher Education and Research, Chennai, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jrcr.jrcr_55_20

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  Abstract 


Background: Targeted alpha therapy (TAT) is emerging as an effective treatment modality of cancer especially for micrometastasis, lymphoproliferative malignancies, and palliative approaches of bone cancer. Human blood lymphocytes may encounter alpha (a) exposure while traversal of targeted a particle emitting radio isotopes to the tumor site, due to their nonspecificity and release of radio isotopes from the legends used for targeting. Such radiation effects to lymphocytes may be implicated in short and long term health effects during TAT. Aims and Objectives: To see the effects of a-particle in blood lymphocytes and to compare their complexity of aberration with both and X-rays. Materials and Methods: Chromosomal aberrations such as dicentric chromosome, micronuclei, nucleoplasmic bridge (NPB) in the peripheral blood lymphocytes were scored for both a alpha particle and X rays. Then, the chromosome aberrations (CA) frequency was correlated with the gene expression (FDXR, CDKN1A and GADD45A) to both the type of radiations. Results: CA induced by a radiation was complex and highly dispersed when compared to low LET radiation. Moreover, magnitude of NPB was significantly higher in case of a radiation than radiation. A dose dependent increase in gene expression (FDXR, CDKN1A and GADD45) was observed after a radiation, which however, was higher in case of a radiation than X rays. Conclusion: These results provide better understanding about effects of a radiation on human lymphocytes, which may be significant implications in developing better TAT strategies for cancer.

Keywords: Alpha particles, chromosomal aberration, gene expression


How to cite this article:
Kanagaraj K, Rajan V, Pandey BN, Venkatachalam P. Complexity of chromosomal aberrations and gene expression changes in human blood lymphocytes after exposure to alpha particle radiation. J Radiat Cancer Res 2020;11:142-9

How to cite this URL:
Kanagaraj K, Rajan V, Pandey BN, Venkatachalam P. Complexity of chromosomal aberrations and gene expression changes in human blood lymphocytes after exposure to alpha particle radiation. J Radiat Cancer Res [serial online] 2020 [cited 2021 Jan 17];11:142-9. Available from: https://www.journalrcr.org/text.asp?2020/11/4/142/305727




  Introduction Top


Applications of ionizing radiation in the management as well as treatment of cancer patients play an important role. Initially, low linear energy transfer (LET) radiation such as gamma (η) and X-rays were predominantly used in local tumor control; which however was limited with exerting radio-resistance, poor response of hypoxic tumor cells and limitation to treat tumor at micro-metastatic sites.[1] In this regard, localized effective killing of cancer cells, lesser side effects, lower possibility of radio resistance, better killing of radio-resistant hypoxic cancer cells are some of the major advantages of particulate therapy than conventional radiotherapy. Among the targeted radio-nuclide therapy strategies, α-particle based targeted therapy has gained increased attention of clinicians over β-particles and Auger particles, because of its combined merits of being high LET radiation and very limited range in tumor tissue. The number of particle track transversals through a tumor cell required to kill the cell is considerably lower for α-particles compared with β-particles, and a single α-particle transversal can kill a cell.[2],[3] Thus, targeted alpha therapy (TAT) particle could result in enhanced killing of cancer cells with lower side effects if they could be targeted effectively to the tumor site.

In TAT, despite being tumor as target, surrounding normal tissues may manifest DNA damage, which possibly will result in to compromise the delivery of required dose and eventually the therapeutic efficacy. Such radiation induced DNA damages during TAT might pose increase in risk to the development of second malignancies.[4],[5] Lymphocytes being major cellular components of blood, are likely to encounter α radiation exposure while TAT. During TAT, blood lymphocytes would get exposed to α particle for three major reasons: First, in route to targeted tumor site, lymphocytes would be encountered with α radiation from radio-isotopes. Second, nonspecificity of α emitting radiopharmaceuticals would irradiate normal tissues including lymphocytes. Third, radioisotopes are likely to be gets detached from legends due to recoil energy of the isotopes which would result in radiation exposure to lymphocytes while circulation in blood till radio-isotopes are cleared from the body. While TAT is well accepted with better therapeutic benefits, the knowledge about effects of α radiation on human blood lymphocytes is limited in literature.

Considering the significance of high LET radiobiological effects under various exposure conditions including TAT, we have previously examined the relative biological effectiveness (RBE) and radiation induced genomic instability in the human blood lymphocytes and skin fibroblasts after α irradiation.[6],[7] Results were consistent with the published literature[8] that the α-particle from241Am was more potent in inducing chromosome aberrations (CA) in directly exposed as well as their unexposed bystander cells. In the present study, we have extended the analysis to examine distribution and complexity of α-particles induced CA (dicentric chromosome [DC], micronuclei [MN], and nucleoplasmic bridge [NPB]) in blood lymphocytes; which were correlated with expression changes in redox sensitive, cell cycle and DNA damage genes, (FDXR, GADD45-A, CDKN1A). These results were also compared with low LET radiations.


  Materials and Methods Top


Collection of blood samples

The study was approved by the Institutional Ethics Committee of Sri Ramachandra Institute of Higher Education and Research, Chennai, India. After obtaining written informed consent, about 10 ml of venous blood was collected from healthy volunteers (n = 2) into a heparinized vacutainer (BD Vacutainer, New Jersey, USA) the lymphocytes were isolated using Ficoll-histopaque (Himedia, Mumbai, India) method.

In vitro irradiation of lymphocytes to α-particles

The α-irradiation (241Am) of isolated lymphocytes was carried out in specially designed irradiation dish using α-irradiator developed at Bhabha Atomic Research Center (BARC), Mumbai[9] as explained earlier.[6] In order to get the lymphocytes attached to the Mylar membrane (Goodfellow, Huntingdon, UK), the dishes were pretreated with × 2 concentration (40 μg/ml) of phytohemagglutinin (PHA) (Gibco, Grand Island, USA). The lymphocytes were seeded into Mylar dish and incubated at 37°C for 30 min to allow cells to attach. Then, the media were removed carefully, fresh media without PHA was added and lymphocytes were exposed to α-particle to the doses of 0.05, 0.10, 0.25, 0.5, 0.75, and 1 Gy at a dose rate 1.5 μGy/s.

In vitro irradiation of lymphocytes to η-rays

The isolated lymphocytes were aliquoted into cryovials and exposed to η-rays using a60Co teletherapy unit (Bhabhatron II Panacea Med. Tech. Pvt. Ltd, Bangalore, India), at BARC, Mumbai. The distance between the source and the sample was 100 cm and the lymphocytes were exposed to different doses (0.05, 0.1, 0.25, 0.5 and 1 Gy) at a dose rate of 1 Gy/min. The lymphocytes were irradiated at room temperature (28°C ± 3°C). An aliquot of sample was sham irradiated and used as control.

In vitro irradiation of lymphocytes to X-rays

The isolated lymphocytes were aliquoted into cryovial and exposed to 6 MV LINAC PRIMAS Siemens, Germany) X-rays with a dose rate of 3 Gy/min at Dr. Kamakshi Memorial Hospital, Chennai. One aliquot of the unexposed sample was used as a control.

The irradiated lymphocytes from different types of radiation were incubated at 37°C for 2 h followed by their culture for CA and MN assay as mentioned below.

Culture methodology to prepare metaphase chromosomes

α and η irradiated lymphocyte used to prepare metaphase chromosomes and then to analyze CA. The lymphocyte were supplemented with RPMI-1640 medium (Gibco, Grand Island, USA) with 20% Fetal bovine serum (FBS) (Himedia, Mumbai, India) and Colcimed (0.01 μg/ml) (Gibco, Grand Island, USA) was added to the culture at 24th h. Then, the culture was terminated after 50 h from initiation, the cells were treated with hypotonic solution (0.075 M of KCL) for 20 minutes and the cells were fixed using carnoys fixative. After three consecutive washes with fixative a clear pellet was obtained, to the pellet 1 ml of fresh fixative is added and casted on to a pre-chilled glass slide and stained using 2% giemsa stain (Himedia, Mumbai, India). The stained slides were mounted with DPX (Himedia, Mumbai, India) and metaphase with 46 chromosomes and above considered for scorning as described in International Atomic Energy Agency 2011[10] technical note and scored under × 100 magnification.

Culture methodology to prepare binucleated cells

Cytokinesis arrested binucleate assay was employed to analyze the induced MN and NPB, re initiation was similar to that CA assay until the addition of cytochalsin-B at 44th h of culture initiation, 6 μg/ml of Cytochalasin-B (Cayman chemicals, Michigan, United states state/country) was added to each culture and incubated for another 28 h. At the end of 72 h of culture initiation, the cells were harvested; cast onto clean slides, stained with 5% Giemsa and around 1000 binucleated cells were scored for each dose point.[10]

Gene expression

The lymphocytes were isolated from whole blood using density gradient method as described previously and were made to attach to irradiation dishes and irradiated to α particle and X-rays. Then, the cells were detached by mild aspiration with fresh media and irradiated cells were incubated for 24 h RNA was isolated using Trizol method. In berief, 1 ml of Trizol (Gibco Grand Island, USA) was added to the irradiated lymphocytes and stored in -80°C. Isolated RNA was quantified using Nanodrop™ spectrophotometer (Thermo Scientific, MA USA), was converted in c-DNA using High-capacity cDNA reverse transcription kit (Gibco, Grand Island, USA) as per manufacture's instruction. TaqMan primer and probes (Applied bisytem, MA USA) for FDXR, GADD45 and CDKN1A genes were used to quantify relative expression using real-time polymerase chain reaction (PCR). 18SrRNA was used as housekeeping gene to normalize the expression of those genes. Real-time PCR reaction was performed with Qiagen Rotor Gene-Q PCR using Universal Taqman master mix by the following manufacturer's recommendations. Relative fold induction was calculated by the ΔΔ computed tomography method and 18sRNA was used for normalization.[11],[12]

Statistical analysis

The aberration frequency and standard error were calculated as follows:

Aberration frequency = number of aberrations/total number of cells analyzed

Standard error = √ number of aberrations/total number of cells analyzed.

Distribution of chromosome aberrations

The distribution of DC/MN in cells was studied by the method described by Papworth and adopted by Savage (1975).[13] This is done by the standard u-test using the formula



Where N is the total number of cells scored, d is the coefficient of dispersion (N - 1) σ2/Y, where Y is the mean number of observed aberrations, σ2/Y is the relative variance and Var d is the variance of d given by 2 (N - 1) (1 - 1/NY). This method makes use of the fact that the variance (σ) equals the mean (Y), i.e., the variance divided by mean is equal to 1, so that u = 0. A macro has been developed using Excel to obtain the dispersion index “u” for different dose points in the curve.

Details on calculation of dispersion index

Mean aberration frequency (CA/MN) obtained between the doses, between different types of irradiation were compared using Student's t-test. The dispersion index (”u” value) was calculated to analyze the complexity of induced aberrations and the dose-response for the aberrations with the type of radiation was obtained using linear, linear quadratic function using “dose estimate” software (Developed by Health Protection Agency, UK).


  Results Top


Frequency and distribution of dicentric chromosome in lymphocytes exposed a-particle and -radiation

About 500–1000 metaphases were analyzed to record the number of DC at each dose point. As there was no significant difference among the aberration frequency between the donors, the pooled data (number of cells analyzed and the aberration) at each dose was used for further analysis. The DC frequency and its distribution among cells with increasing dose of α and η-irradiation are given in [Table 1] and [Table 2].
Table 1: Frequency and distribution of dicentric chromosome in lymphocytes exposed α-particle radiation

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Table 2: Frequency and distribution of dicentrics in lymphocytes exposed γ-radiation

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A dose dependent and significant (P = 0.003) increase in the frequency of DC was observed at all the doses for both type of irradiation when compared to that of its unexposed control samples. It is important to note that α- irradiation induced higher magnitude and complex aberration [Figure 1] in the lymphocytes than that of induced by equal doses of η-irradiation. The complexity is also evidenced by more number of cells with multiple DC in single cells, cells in which chromosomes with multiple centromere (complex aberration); nonetheless, majority of the cells after η-irradiation showed single DC [Figure 2]. The complexity of induced aberration is also evidenced from the distribution index of aberrations measured using “u.” Thus, the distribution “u” value for the aberrations varied between +3.62 and +35.2 at different doses for α-irradiation while it was -0.143 and 2.0 after η-irradiation.
Figure 1: Comparison on the frequencies of dicentric chromosome obtained in lymphocytes exposed to α and γ radiation

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Figure 2: Comparison of dicentric chromosome distribution in lymphocytes exposed to α and γ radiation at different doses: (a) 0.05, (b) 0.1, (c) 0.2, (d) 0.5 and (e) 1 Gy

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Frequency and distribution of micronuclei in lymphocytes exposed a-particle and -radiation

The complexity and spectrum of α-particle induced DC was confirmed by using another end point the MN formation, as the assay is simple and rapid and permit more number of cells to be analyzed. That at each dose around 2000 binucleated cells were analyzed and the number of MN were recorded at each dose. Moreover, since there was no significant difference among the MN frequency between the donors at equal dose, the data were pooled and used for further analysis. MN frequency, the distribution pattern and the “u” at each dose for α-particle irradiation and η-irradiation is given in [Table 3] and [Table 4].
Table 3: Frequency and distribution of micronucleus in lymphocytes exposed α-particle radiation

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Table 4: Frequency and distribution of micronucleus in lymphocytes exposed γ-radiation

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Both type of aberrations shows a dose depend increase in the MN frequency; at the same time, the fold increase and cells with multiple MN, is more after α-particle irradiation than that of obtained with η-irradiation. The u value varies between +8.11 and +34.73 after α-irradiation; whereas it is between +1.28 and +8.09 in lymphocytes exposed to η-irradiation [Figure 3]. The NPB, another type of aberration was observed only in the α- irradiated lymphocytes; the frequency of NPB obtained at different doses of α-irradiation is shown in [Table 5]. The complexity is evidenced by more number of cells with multiple MN in single cells (complex aberration) and presence of NPB while majority of the cells after X-irradiated lymphocytes showed only one MN and without NPB.
Figure 3: Comparison of micronuclei distribution in lymphocytes exposed to α and γ radiation at different doses: (a) 0.05, (b) 0.1, (c) 0.2, (d) 0.5 and (e) 1 Gy

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Table 5: Frequency of nucleoplasmic bridge obtained in lymphocytes exposed to α and γ radiation

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Gene expression

The total RNA was isolated from the lymphocytes collected from the healthy volunteers (n = 2), exposed to α-particle and X-rays. The expression level of FDXR, GADD45A, CDKN1A genes was examined in lymphocytes after exposure to α-particle at dose (0.01, 0.1 and 0.5 Gy). [Figure 4] shows the fold change in the expression level of genes in the lymphocytes after exposure to α-radiation. A dose-dependent increase in the fold change was observed for all the genes; of the many genes examined, FDXR have showed a significant increase in fold change at 1 Gy dose point when compared to other genes (P < 0.003). [Figure 5] shows the expression level of FDXR, GADD45A, CDKN1A obtained from lymphocytes exposed to 0.5 Gy of X-rays. For the same dose (0.5 Gy) α-particle irradiated lymphocytes showed a 7 folds increase in expression for FDXR which was statistically significant (P = 0.001) when compared to that obtained for X-irradiated lymphocytes; GADD45A and CDKN1A it showed a 1.7–3.4 folds increase in expression when compared to X-rays. Thus, consistent with induced CA, fold expression of genes also remains higher α-particle irradiation.
Figure 4: Fold change in the expression of genes in lymphocytes exposed to α particle

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Figure 5: Fold change in the expression of genes in lymphocytes exposed to equal dose (0.5 Gy) α particle and X-rays

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  Discussion Top


Many reviews listed out the potential of α-particle targeted therapy strategies and have presented novel possibilities for their use in the treatment of cancer in specific to treat leukemia's and metastatic disease.[2],[3],[14],[ 15] Interestingly, α-particles offer key advantages over β-particles and Auger particles because of the combination of their high LET and limited range of penetration in tissue.[16] The relatively short α-particle tracks have a limited range in tissue and are on the order of a few cell diameters, thereby confining cytotoxic effects to a relatively small area.[17] Nonetheless, the advanced technology is not devoid of challenges; limited or delayed diffusion of the α-radio-immunocomplex and nonhomogeneous activity distributions in the targeted tumors, resulting in inhomogeneous absorbed dose distributions, are challenges.[14] Moreover, when the radioisotope, travels through the blood, and collects in certain tissues in the body, there is a potential for side effects including the development of second malignancy. The therapeutic effect of radio-labeled antibodies is thus dependent on the tumor's vascular supply, as a means to infiltrate the solid tumor.[18] Keeping that in view and to examine the normal tissue toxicity, we have investigated the frequency and complexity of chromosomal aberration (DC and MN) and gene expression changes in lymphocytes in vitro irradiated to241Am α- particles.

Our results showed that both the α-particle and η-radiation induced significantly higher amount of radiation specific DC in the irradiated lymphocytes; however, the frequency of induced DC and the complex aberrations (cell with more number of DC, tricentrics, tetra centric, etc.,) is high after α-particle irradiation than that of observed in η-irradiated lymphocytes [Figures 1 and 2]. The complexity of the α-particle-induced DNA damage was further confirmed by the presence of NPB in the CBMN cells, which was not induced by η-irradiation [Table 4]. Higher frequency and complexity of DC and NPB is attributed to the energy of the α-particle; thus, the alpha particles tendency to create dense tracks of ionization that creates clusters of DNA damage, whereas the damage caused by η-radiation is more sparsely distributed.[19] The maximum rate of double-stranded DNA breaks (DSB) occurs at LETs of 100–200 keV/μm, a range that includes the LET of most α-particle. These breaks result in CA and impairment of the reproductive integrity of a cell. The DSB created by α-particle radiation have been found to be highly complex, more resistant to normal repair, and thus more genotoxic than DSB caused by other modalities. Staaf et al.,[20] has reported a similar results in blood lymphocytes exposed to mixed beam of low and high LET radiations; that is mixed beam irradiation and α-irradiated lymphocyte showed more complex aberration that of induced by X-irradiation. It was mentioned that in addition to directly damaging DNA, α-particles have been demonstrated to cause chromosomal instability even in the descendants of nonirradiated stem cells in the vicinity of irradiated cells. This “bystander effect” reflects the potential for interaction between irradiated and nonirradiated cells in the production of genetic damage.[21] Recently, we have reported that both the magnitude bystander response and its persistence in the progeny of the irradiated lymphocytes was longer for α-particle irradiation that that of X-irradiation.[6]

In addition to those CA, higher fold change in expression of FDXR1, CDKN1A, and GADD45 was observed in blood lymphocytes exposed to α-particle irradiation that that of X-irradiation. Because chromosomal changes are associated with cancer induction this result may imply that the cancer risk of exposure to mixed beams/high LET in case of particulate therapy in radiation oncology may be higher than expected based on the additive action of the individual dose components.

The technique used to attach lymphocytes uniformly in the Mylar membrane introduced by Bauchinger and Schmid, 1999[8] has been followed in this study. As the cells are uniformly attached, the distribution of induced DC was expected to be Poisson (u value between ± 1.96). However, obtained results showed that in lymphocytes exposed to α-particles showed over dispersion for all doses from 0.05 to 1 Gy; the range is extremely high when compared to that of obtained in η-irradiated lymphocytes. Such as over-dispersion of DC after α irradiation has been published literature.[8],[22] Nikjoo et al. (2008) has been shown that the level of clustered DNA DSB, and the degree of their complexity, increases with the LET and this can explain the known increase of RBE with LET. Complex and clustered DSB are more difficult to repair than simple DSB,[17],[23] hence those breaks can convert into complex chromosomal changes. DuFrain et al.,[24] were previously reported induced CA frequency in blood lymphocytes after α-particle radiation and compared with that of low LET radiations. Higher magnitude of CA, RBE values for high LET radiation has been reported by many others.[20],,[25],[26],[27],[28]


  Conclusion Top


Majority of the studies were conducted to establish dose-response curve or dosimetry, and to calculate RBE as the value for α-particle are highly variable. Thus the commonality in all the studies was that α-particle irradiated induced complex aberration in the normal lymphocytes. However, distribution and frequency of CA and its correlation with gene expression changes were not studied previously. A comparison of effect of α radiation on counterpart tumor cells considering the biological complexities of bystander/abscopal effects would be required for better understanding of such effects during TAT. Our results provide novel insight about effects of α radiation on human lymphocytes, which may have significant implications in short- and long-term health effects during TAT.

Financial support and sponsorship

This study was supported by Council of Scientific and Industrial Research, Government of India (File No. 09/949/0005/18-EMR-I).

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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