Fayyaz 1
Humna Fayyaz
October 29, 2011
Biology 303- 501
Dr. Bert Ely
Genotoxicity of Radioactive Contamination on Plant Genomes
With an estimate of ten new “nuclear nations” by the year 2013, nuclear energy is gaining
popularity due to the negative economic and environmental impacts of other forms of energy
production. Governments worldwide are realizing that they are contributing to global climate
change through their growing carbon footprints, directly related to the amount of unoxidized
hydrocarbons in the atmosphere and the emission of greenhouse gasses in order to maintain
human activities (Wiedmann, 2008). Thus, the increasing concern to reduce carbon dioxide
levels in the environment has led nations to turn to nuclear energy as an alternative to burning
fossil fuels. However, the use of nuclear energy is not free from concerns, as a potential accident
can be a serious perturbation of its surrounding environment for a long period of time. Most
recently, in a port town in Okuma, Japan, in March 2011, a nuclear accident transpired at the
Fukushima-Daiichi Nuclear Power Plant (NPP) (Antola et al., 2011). The accident was a direct
result of the Tohoku earthquake, ranking 9.0 on the Moment Magnitude Scale (Mw), and the
following tsunami, with waves reaching 14.0 meters (Antola et al., 2011). The limits of the
power plant were far exceeded—thus, leading to the accumulation of radioactive heat,
meltdowns of the fuel rods inside of the reactors, and explosions of two of the six reactors during
the following days (Antola et al., 2011). Radioactive byproducts of nuclear fission, like Iodine131 and Cesium-137, were released into the atmosphere, which lead to the contamination of
nearby water systems and wildlife. Disastrous nuclear fallouts, like Fukushima-Daiichi,
demonstrate the need for more studies on radioactive contamination and its effect on the
environment. Because no studies have yet been published on the consequences of Fukushima,
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most of the hypotheses have been based on research collected from the Chernobyl accident in
1986, the most severe release of radioactive contamination in the history of the nuclear power
industry (Mousseau and Moller, 2010). Along with a reduction in the abundance and biodiversity
of animals, research conducted at the Chernobyl Nuclear Power Plant (ChNPP) has revealed the
deleterious effects of irradiation on the genome, consisting of both the genes and non-coding
sequences of DNA and RNA, of exposed organisms (Kuchma et al., 2011). In plant populations,
specifically, studies have confirmed that exposure to radiation has led to an increase in mutation
rates and overall genomic instability.
Releasing at least one hundred times the amount of radionuclides into the environment than
the atomic bombings at Hiroshima and Nagasaki combined, the Chernobyl disaster resulted in
the release of 50 million curies (MCi) of noble gases (such as xenon and krypton) and roughly 50
MCi of other radionuclides into the atmosphere directly following the explosion of the fourth
reactor (Ginzburg et al., 2011). Having longer half-lives, some radionuclides, like Cesium-137
and Strontium-90, entered the food chain and water systems and remained inconsistent in their
dispersion patterns. Because of variability of the pollution and the fear of the local population
being exposed, the Ukranian Ministry of Emergencies administered a 30-km (19 mi) Exclusion
Zone (Mouseeau et al., 2011). Most of the contamination studies are conducted in reference to
biotic and abiotic wildlife in this set perimeter, allowing for the use of organisms outside the
region as controls. To quantify the amount of radiation in the area, gamma-spectrometry methods
were utilized to determine different characteristics of the radionuclides, including their
composition, surface activity, and space distribution (Kovalchuk et al., 2011). These were
measured by portable gamma-dosimeters and gamma-spectrometers taken into the field. Not
only relying on instruments to quantify the irradiation, the studies also consist of soil sample
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analyses of 137Cs activity per unit area as a quantifier of radiation in a certain area (Kuchma et
al., 2011).
To study the genetic effect of irradiation, mutation rates in Pinus sylvestris L., or Scots pine
trees, from the Chernobyl Exclusion Zone were evaluated based on Polymerase Chain Reaction
(PCR) based molecular markers (Kuchma et al., 2011). Amplified fragment-length
polymorphisms (AFLPs) and microsatellite markers were used in order to analyze changes in
mutation rates between control pines and exposed pines, as well as to detect somatic mutation
events, or acquired mutations, in different regions of the tree genome. The Scots pine tree was
specifically used in this study due to its high level of genetic diversity, strong responses to stress,
and its high sensitivity to radiation (Kuchma et al., 2011). Fresh needles were collected from five
different types of Scots pine in different areas surrounding the ChNPP consisting of different
ages and irradiation levels—representative of both acute and chronic exposure. 843 samples
were genotyped for nine microsatellite loci, or short segments of DNA consisting of numerous
tandem repeats of short motifs (Kuchma et al., 2011). The presence of the specific motifs makes
this technique sensitive to extensive length variation in the different samples. The DNA was
amplified using SPAC primers, copies of the particular target sequence were generated using the
template and primer, and thermal cycling activated the PCR. Microsatellite mutation events were
detected in the experimental groups consisting of the chronic and acute radiation but none were
found in the control plots. LOP5, PtTX3107 and PtTX2146 are the three microsatellite loci
where the repeat motifs were found, all representing deletions. Figure 1 displays a deletion found
at the LOP5 locus. A greater number of mutations were found at the plots of trees exposed to
chronic exposure, compared to those that were exposed to initial acute radiation; a 1.7-fold
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increase in radiation exposure was found in the chronically exposed pines when compared to
those of the acutely exposed (Kuchma et al., 2011).
Fig. 1. Sequences of three alleles at the LOP5 locus. Sample 12 shows a deletion, a loss of 20 nucleotides.
The second technique used in this study was the AFLP, also a PCR-based marker of
mutational events in an organism’s genome. However, unlike the microsatellite method, it is
lacking the specificity of the short tandem repeats; therefore it is non-specific to what is being
amplified (Gaudeul et al., 2004). Having the capacity to amplify hundreds of fragments at one
time, AFLP-PCR can simultaneously screen for polymorphisms throughout many different
regions of the genome, thus, making it more sensitive to the genome in its entirety, when
compared to the microsatellite method (Mueller et al., 1999). After testing with the AFLP
technique, twelve mutations were found in the control, while an average of thirty-three mutations
were found in the chronically exposed group (Kuchma et al., 2011). Unlike in the test with
microsatellites, insertions, as well as deletions, were found. The mutation rate for the
microsatellite loci in irradiated groups exposed to initial acute radiation was 2.8 x 10-4, while the
groups exposed to chronic exposure had mutation rates of 7.1 x 10-4 and 3.3 x 10-4, showing no
statistically significant difference between irradiated and control groups. For the AFLP loci, the
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mutation rates were estimated to be 1.06 x 10-3 for the control group, and 3.99 x 10-3 and 3.74 x
10-3 for the groups chronically exposed to irradiation, showing an average of 3.5 times higher
mutation rate in the exposed group than in the control group—therefore, indicating a high level
of DNA damage caused by chronic exposure to radiation (Kuchma et al., 2011).
In another study, Arabidopsis thaliana and Nicotiana tabacum, transgenic plants were
grown in the areas established in the Chernobyl Exclusion zone, and a dose-effect relationship
was established between the extent of radioactive pollution and their homologous recombination
frequency (HRF) (Kovalchuk et al., 1999). Because the transgenic plants were engineered so that
recombination events restore the expression of beta-glucuronidase, the recombination events
were localized as blue sections on the white plants after histochemical staining (Kovalchuk et al.,
1999). Thus, because the exposure to radiation causes breaks in both double and single stranded
DNA, which are then repaired by homologous recombination, this technique allows for the study
of the relationship between exposure and the frequency of HRE (Kovalchuk et al., 1999).
Using gamma-spectrometry and beta-gamma radiometer devices, the soil samples from
the three plots of land (including a control plot with little to no contamination) were analyzed
and the absorbed level of radiation was estimated. Soil samples were assayed for their 137Cs and
90
Sr activity through the use of beta and gamma spectrometry instruments, and
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Fig.2. Representation of homologous recombination events in A. thaliana and N. tabacum plants. A and B represent
the HRF v. the extent of soil population in the plants, while C and D are the images after histochemical staining for
beta-glucuronidase on the plants grown in the experimental plots in Narodichi (Kovalchuk et al., 1999).
a strong positive correlation between the level of 137Cs in the soil and the HRFs in the A. thaliana
transgenic plants was observed. The control plants had 0.60 (± 0.05) recombination events per
plant which increased to 1.10 (± 0.05) recombination events at an exposure of 3.1 Ci /km2. The
number of recombination events increased, as expected, at 40 Ci/km2 to about 3.20 (± 0.15)
recombination events per plant. Similarly, laboratory results showed an increase in HRF from a
control level of 0.38 (± 0.06) recombination events per plant to 0.52 (± 0.12) at a radionuclide
level of 1.5 Ci/km2 and a more significant increase from 0.90 (± 0.15) recombination events per
plant at 3.3 Ci/km2 to 2.40 (± 0.20) at 40 Ci/km2.
A similar trend was observed with the other transgenic plant used in the study, N.
tabacum. A double-exponential regression analysis was performed for the correlation between
the recombination frequency in the two transgenic plant populations and the level of radioactive
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contamination in the soil—the correlation between the two was found to be strong and
significant (r= 0.9564, P< 0.05, n=6.) The deleterious effects of the increase of the HRFs in A.
thaliana and N. tabacum were verified by the depression of mitotic activity in the plant roots and
by a fourteen-fold increase in the total chromosomal aberration frequency (Kovalchuk et al.,
1999).
Another article concentrated on chromosomal aberrations in Allium cepa (onion plants) to
measure the genotoxicity of the radiation in soils surrounding the ChNPP (Kovalchuk et al.,
1998). The Allium test is best used to identify and monitor changes in the onion genome by
screening for negative effects, like chromosomal aberrations and micronuclei, of specific metal
and chemical ions; in the case of this study, Cesium and Strontium were observed (Kovalchuk et
al., 1998). The mitotic index was measured by examining 400 cells and squashing the root tips in
45% acetic acid. The data provides an indirect relationship between the mitotic index of the
Allium cepa cells and the sample radiation—as the radiation level increased, the mitotic index
decreased (Kovalchuk et al., 1998). From 582 Bq/kg of radiation to 2287 Bq/kg, the mitotic
index decreased from 41.3% (±4.6) to 30.5% (±5.3). An even more significant decrease was seen
from 2543 Bq/kg of radiation to 6549 Bq/kg. Thus, a strong negative relationship was found
between the mitotic index and the extent of the Cesium-137 radiation (r= 0.9615, n=6, P< 0.05)
(Kovalchuk et al., 1998).
The second facet of the study monitored chromosomal aberrations in irradiated Allium
cepa by examining the anaphase and telophase stages of 500 normal cells (Kovalchuk et al.,
1998). The chromosomal aberrations found include cells with bridges, c-mitoses (leading to the
possibility of aneuploidy,) vagrant chromosomes, multipolar anaphases, and fragments in both
anaphase and telophase. A strong positive significant correlation was observed between the
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intensity of Cesium-137 in the soil and the frequency of chromosomal aberrations (Kovalchuk et
al., 1998).
Fig. 3. Root tip cells of Allium cepa displaying chromosomal aberrations. A: normal anaphase, B: presence of
bridge, C: fragment in anaphase cell D: fragment in telophase, E: vagrant chromosomes. The graph displays the
percent of the abnormalities found in relation to the radioactive activity in the soil samples.
In conclusion, studies of the genetic impact of radioactive contamination have taken
place to better assess the effect of nuclear fallouts in the past and for improved management of
the pollution in future accidents. The monitoring of the genotoxicity of irradiation in sessile
organism, like forest trees and immobile plants, reveal much about their genetic response to
radioactive exposure. In the first study (Kuchma et al., 2011), the genetic reaction of Scots pine
forest trees to the contamination in the years following the Chernobyl disaster was studied and
the response of trees with acute and chronic exposure were compared. The low number of
microsatellite mutations leads to the hypotheses that plants in the surrounding areas of the
ChNPP have an improved-DNA repair capacity as an effect of chronic exposure to radiation. In a
second study conducted by Kovalchuk et al., chronic exposure to low doses of radiation in soil
was correlated with an increase in the homologous recombination frequency in plants, leading to
many chromosomal aberrations and deleterious effects of the plant’s genome. The results from
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this study may be able to be applied to other eukaryotes having similar patterns of exposure in
the future, possibly even humans. Because of the limited number of sites of radioactive
accidents, studies at Chernobyl are necessary in order to answer questions about the risks of
nuclear energy. Because approximately 29,000 people still reside in Zone-2 of the Chernobyl
Exclusion Zone, studies of the short and long-term genetic consequences of radioactive
contamination are relevant and necessary when considering the potential hazards related to the
production and use of nuclear energy.
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