Ionising radiation is commonly utilized in our daily lives in small amounts e.g. X-rays. However, radiation exposure results in harmful effects on DNA structure, leading to base modifications (oxidation, alkylation), cross-link formation or bulky lesions. Repair mechanisms exist to correct these modifications, however, misrepairs may lead to increases in mutations such as base substitutions and single and double-stranded breaks, the latter being the most important.
At present, mouse models are used to measure the effects of exposure to radiation. From this, the risk of gaining harmful mutations is calculated (Bouffler et al, 2006) .This is performed by identifying markers in the mouse genome (Neel, 1990, Sankaranarayanan, 2001). Phenotypical changes can be seen with the different doses of radiation exposure, e.g. fur colour Is dose-dependent. Unfortunately, the frequency of these mutations is very low (about 1 in 100 000) and hence large amounts of radiation needs to be used. Despite this, these experiments were useful as they showed that the mutations occurred at or near the sites of exposure to ionising radiation.
Subsequently, it was discovered that the mouse and human genomes contain tandem repeat DNA loci (TRDLs) which have a higher mutation frequency than the mouse genome markers (Bois 1999), which can be used to induce mutations. The higher mutation frequency of these genes in mice are evidence that these mutations are “untargeted”, that is, the mutations are not limited to the sites of exposure to ionising radiation. Further experiments have shown that exposure to ionising radiation can induce mutations in cells many years after exposure (Little, 1994). Although the exact mechanism of untargeted and delayed mutations is not fully understood, it is thought that radiation exposure results in genome instability. The regions of higher mutation in the human genome are known as “minisatellites”, and in mice, “expanded single tandem repeats (ESTRs)”. The difference in nomenclature is due to the different structures of these regions in mice and humans (Bouffler et al, 2006).
Mammalian TDRLs consist of microsatellites which are 500 base pairs long and repeat sizes of 1-4 base pairs. ESTRs are 0.5-16 kilobases long with repeat sizes of 4-9 base pairs. Human minisatellites are 0.5-10 kilobases long and the repeat sizes are 9 – 60 base pairs (Bois 1999, Ellegren, 2004, Vergnaud, 2000). TDRLs can make up about 10% of human genes. The research significance of these regions of the genome is that any additions or subtractions occur in the repeat units as a whole, and hence are useful to assess the risks of exposure to radiation.
Minisatellites are highly variable sequences (Vergnaud,2000). In humans, they are located in the sub-telomeric locations of the gene (Bouffler, 2006). Not only is this less so in pigs, rats and mice (Jeffreys, 1999), but these genomes also have higher mutation rates. Two approaches can be used to assess mutation rates in human minisatellites. These are pedigrees, where mutations are identified in complete pedigree trees. and the small pool PCR (SP-PCR) method (Tamaki, 1999). The DNA is obtained from lymphocytes and locus-specific probes are used to identify the minisatellite regions. A mutation is a new segment of DNA which does not arise from the parents’ genes and this is easily ascertained because of the high mutation frequencies of these minisatellite loci. For the pedigree method, it is important to avoid errors from non-paternity and other human errors. In the PCR technique, DNA from sperm are diluted and amplified, so that mutations can be detected. Therefore, the pedigree method identifies mutations in the maternal germline and the PCR technique, in the paternal germline. The mutation rate per generation is calculated as follows:
In the case of human minisatellites, a greater number (approximately four fold more) of paternal mutations than maternal mutations have been detected, (Bouffler, 2006). This difference has also been fairly consistent with human microsatellites (Yauk, 2004). Why this occurs is not understood. Studies involving minisatellites have also revealed that mutation rates vary between somatic and germline DNA, with the high frequencies only being seen in the germline DNA (Buard, 2000). A possible explanation could be the “gene conversion-like” processes which occur in the germline DNA, which cause many of the mutations. (Boufler 2006 ). Many additional mutations also occur in germline DNA e.g. DNA double strand breaks, recombination from meiosis at genetic hotspots. (Buard, Shone, Jeffreys, 2000). Nearly half of the minisatellites in the human genome (Denoeud F, 2003) occur in the coding sequences, therefore resulting in alterations to protein structure. The HRAS gene codes for the HRAS protein, a GTPase, which controls cell division in the presence of growth factors. Hence minisatellites within this gene will alter gene expression and thus increase the risk of familial cancer. In addition, minisatellites in the introns can alter gene splicing, due to sequence overlap between the minisatellite repeat and the splice regions. The actual purpose of minisatellites is not known, however the multiple minisatellite loci, heritability and their existence in the genes of higher mammals propose that they do serve an important purpose.
WWII Hiroshima and Nagasaki atomic bomb
Mutations in minisatellite loci were studied in the genomes of families that were exposed to ionising radiation during the WWII Hiroshima and Nagasaki atomic bomb explosions (Kodaira, 2004). The sample size studied consisted of the children of 30 fathers and 32 mothers who were exposed to the ionising radiation, and 60 children of unexposed parents. The majority of the 62 children from exposed parents were born a decade after the explosions. The Dosimetry System 86 (DS86) was utilized to process the radiation exposed by the parents. The average parental exposure was 1.9Gy and this was seen mostly in the cases where only the mother was exposed (Bouffler, 2006). The initial experiments identified low mutation rate sequences in the genomes of the exposed group (Kodaira, 1995). Subsequently, single-locus probes identified very high mutation rate minisatellite sequences (Kodaira, 2004). However, dramatically-changed mutation rates were not found. Possible reasons could be the insufficient number of exposed individuals, the large number of exposed groups consisting of exposed maternal lines and unexposed paternal lines, possible correction of any mutations in the genome before conception, or finally, a genuinely low mutation rate caused by the exposure to ionising radiation.
Chernobyl Disaster
Mutation frequencies in minisatellite loci were studied in families who were exposed to ionising radiation during the Chernobyl disaster. These families were located in the countryside of Belarus and the Ukraine (Dubrova, Grant, 2002). The sample size consisted of 127 children from exposed parents in Mogilev in Belarus. The control individuals were 120 Caucasian children of unexposed parents in the UK. High mutation frequencies were found within minisatellite sequences, through the use of 2 multilocus and 8 single-locus probes. This showed that the mutation frequency was higher in the exposed group. The mutation frequency also directly correlated to increased exposure to cesium-137, although the individuals were also exposed to other forms of ionising radiation. Another discrepancy is that the control individuals were of a different ethnicity to the exposed individuals, therefore, it cannot be proven that the increase in mutation rate is purely due to radiation exposure (Bouffler, 2006). Hence, to validate these conclusions, mutation frequencies at minisatellite loci were studied in exposed and unexposed individuals who were born in same countryside districts of the Ukraine (Dubrova, Grant, 2002). To ensure the highest degree of validity possible, the exposed and unexposed groups were made sure to have the same ethnicity, lifestyles and maternal age. Single-locus probes was used to identify any germline mutations. However, a significant increase in mutation frequency was only found in the paternal germline. This could be due to the greater early exposure immediately after the Chernobyl incident. In terms of the analogous maternal mutation rates, this could be a result of non-irradiation of the mothers during the meiotic phase of pregnancy in order to result in mutation at the minisatellite loci. These studies support the hypothesis that increased exposure to ionising radiation results in raised germline mutation frequency (Boufller, 2006).
Nuclear Weapons Testing
Semipalatinsk was an area where nuclear weapons testing was executed by the Soviet Union from 1949-1989 . As in the other studies, the frequency of mutations at minisatellite loci were analysed in forty families resident near this area (Dubrova 2002). The main contact with the ionising radiation occurred via the surface explosions executed from 1949-1956. At present, the level of radioactivity in this region is minimal. The control sample comprised 28 families resident in the Taldy Kurgan region in Kazakhstan, where there was no exposure to ionising radiation. It was ensured that all the individuals being analysed were of similar ethnicity, maternal age, occupation and smoking. The eight single-locus probes were used once again for this study. The experiment concluded that exposure to ionising radiation nearly doubled the minisatellite mutation frequency in the irradiated families. Also, the consequence of the reduced exposure during the lack of surface explosions post-1950s resulted in minisatellite mutation frequency not linking with the year that the parents were born. Hence, there is the possibility of confirming that the initiation of minisatellite mutation frequency is dose-dependent, and that exposure to ionising radiation is directly responsible for the raised minisatellite mutation frequency in the exposed group (Bouffler, 2006).
Chemotherapy and Radiotherapy
The very methods used to treat cancer patients, could ironically and potentially have the ability to induce harmful mutations that could also affect neighbouring non-cancerous cells. Hence, genetic experimentation is vital in assessing the level of mutation initiation to the germline caused by chemotherapy and radiotherapy. Once again, minisatellite mutation frequency was analysed in male cancer patients. This was performed using the SP-PCR method (May, 2000). DNA from sperm were diluted and amplified to identify novel mutations within one male patient (Jeffreys, 1994), and therefore requires a much smaller sample size than the pedigree method. However, the disadvantage of the SP-PCR method is that there is a high variation frequency within each locus (Tamaki, 1999). This makes it unsuitable for assessing the differences in mutation frequency between the exposed cohort and controls. As such, this method is used specifically to assess mutation frequencies before and after chemotherapy or radiotherapy in only one man. Another disadvantage of SP-PCR, is that only minisatellite alleles that are less than 5kb can be amplified. Initial experiments concluded that cyclophosphamide, etopside and vincristine did not alter the mutation frequency of the MS205 minisatellite (Armour, 1999). However, cyclophosphamide was shown to initiate alterations to germ cell mutations specifically during post-meiosis. On the other hand, etoposide induces mutations specifically to germ cells during meiosis (Vilarino-Guell, 2003), which creates a narrow window for assessment. Hence, these drugs are meosis stage-specific. A further study involved sperm from ten individuals post-chemotherapy for Hodgkin’s disease (Zheng, 2000). No raised mutation frequency was detected in the patients treated with vinblastine or a combination of adriamycin and bleomycin, which correlates with similar findings with these drugs on mouse models. However, procarbazine, which was shown to affect cells in the pre-meiotic stage in mouse models, raised the genome mutation rate majorly in humans (Zheng, 2000).
In the case of radiotherapy, again, no changes in mutation frequency was seen in the B6.7 and CEB1 minisatellite loci (May, 2000). The dosage delivered to the patients were 15 courses of X ray exposure (0.4-0.8Gy). This is near the doubling dose exposure for mouse models (UN. And Sanakaranarayanan, 2000). However, the results obtained could be due to the fact that the exposure to radiation in this case is fractionated, as opposed to one large dose.
In conclusion, the results of these different studies have not all been consistent with each other. Possible improvements could be to increase the sample sizes. Perhaps a novel method of calculating the effect of ionising radiation could be explored. In addition, transgenerational mutations have been detected in mice, but not in humans. This reflects the possibility that the mechanism of ESTR mutation initiation in mouse models could be different from that for human minisatellite loci. However, the mutations in mice were only seen after high-dose exposure. Research is currently taking-place to understand the possibility of a stress-like response that needs to be activated before transgenerational mutations are seen in mice. Results from these studies could be useful in understanding this process in humans, and hence our understanding of disease processes related to exposure to ionising radiation.
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