Forensic application of nuclear versus mitochondrial DNA and the formation of cholesterol oxidation biomarkers in human cells after gamma-irradiation

  • Corey Goodwin

    Student thesis: Doctoral Thesis


    Radiological crimes or terrorism could result in the exposure of biological evidence or individuals, including the perpetrators of the crime, to ionising radiation, such as gammaradiation. Cellular damage via gamma-radiation is by direct ionisation, or more predominantly, by indirect oxidation of critical biomolecules due to reactive oxygen species (ROS) induction and oxidative stress. Subsequent degradation of deoxyribonucleic acid (DNA) evidence can impede genotyping efforts when the gamma-radiation dose is sufficiently high (> 1 kGy); this may impact mitochondrial DNA (mtDNA) more than nuclear DNA (nuDNA) due to its greater susceptibility to radiation-induced oxidation. Oxidation biomarkers are therefore candidate indicators of gamma-radiation exposure. This includes metabolic and ROS-derived cholesterol oxidation products (COPs), which are implicated in the oxidative cell stress response and as biomarkers for related pathologies. At gamma-radiation doses that are nonlethal or relevant to acute radiation syndromes (< 10 Gy), such biomarkers may have application for radiation injury triage, elimination of suspects in radiological crime, and elucidating mechanisms of cellular damage and adaptation. This thesis consisted of two distinct objectives: (1) to compare the impact of high-dose (1 to 50 kGy) gamma-irradiation on the degradation of both nuDNA and mtDNA using forensic techniques; and (2) to evaluate the formation and biological significance of COPs after lowdose (1 mGy to 10 Gy) gamma-irradiation of human cells. For this purpose, a real-time polymerase chain reaction (PCR) protocol was developed for the evaluation of mtDNA degradation (Chapter 2), which was compared to that of nuDNA, after gamma-irradiation, based on autosomal short tandem repeat (STR) genotypes and indices of DNA integrity (Chapter 3). A method to quantify various sterols/COPs by gas chromatography-mass spectrometry (GC-MS) was also developed (Chapter 4) and applied to approximate their formation in gamma-irradiated cells (Chapter 5). The real-time PCR method developed for mtDNA quantification comprised three human mtDNA targets of increasing length (86, 190 and 452 base pairs). This method was demonstrated to be capable of quantifying each target from 1x108 to 10 copies with a coefficient of determination (R2) ≥ 0.999 and includes an internal PCR control assay. The developed assays are human specific, sensitive (quantification limit of 10 copies), accurate (within 10 % relative error, for most target dilutions), and precise (within ± 10 % at 95 % confidence, for most target dilutions). Each target was used to calculate three mtDNA integrity indices comprising the quantity ratio of intermediate/short, long/intermediate and long/short amplicons. This enabled comparison to peak height ratios for like-sized autosomal STR amplicons as indicators of DNA integrity after gamma-irradiation. Gamma-irradiation improved GlobalFiler STR genotypes from liquid (hydrated) and dried (dehydrated) blood at lower doses, resulting in significant peak height increases at 1 to 5 kGy (by ~ 20-45 %) for hydrated samples and 1 to 10 kGy (by ~ 15-35 %) for dehydrated samples. This suggested potential for gamma-radiation to remove certain inhibitory effects. Degradation of STR genotypes was significant from 25 to 50 kGy (~ 30-70 % decrease in peak heights) for hydrated samples and at 50 kGy (~ 37 % decrease in peak heights) for dehydrated samples. However, full DNA profiles were attainable up to the highest dose examined of 50 kGy, with allelic dropout occurring for approximately 10 to 15 % of hydrated sample alleles at 50 kGy and most cases of subthreshold alleles being due to heterozygote peak imbalance. From the calculation of integrity indices, gamma-irradiation of liquid and dried blood revealed that cell hydration exacerbated overall DNA damage as compared to desiccated cells, especially for mtDNA. Significant levels of degradation did not occur until 5 kGy for hydrated mtDNA and 10 kGy for hydrated nuDNA, while the same for dehydrated cells did not occur until 5 to 25 kGy for mtDNA (depending on the integrity index) and 25 kGy for nuDNA. Corresponding integrity indices were near 45 to 85 % lower for hydrated nuDNA and 55 to 90 % lower for hydrated mtDNA at 50 kGy. Significant differences between nuDNA and mtDNA integrity arose as early as after 1 kGy; dehydrated mtDNA had near 40 to 60 % lower integrity than dehydrated nuDNA, while hydrated mtDNA had near 30 to 75 % lower integrity than hydrated nuDNA, at 50 kGy. This was suggestive of increased oxidation originating from both water radiolysis and mitochondrial function. Given the persistence of more extensive damage to mtDNA than nuDNA when cells were dehydrated, albeit to an overall lesser extent than hydrated samples, residual moisture within air-dried cells may permit the same oxidative effects as hydrated cells, but in a restricted capacity. Other mechanisms may also exist to confer differential radiosensitivity between organelles, such as varied structural arrangement or frequency of ionisation events. To monitor the formation of sterols/COPs after gamma-irradiation, methods were devised for the isolation and analysis of 7α-hydroxycholesterol (HC), 7β-HC, 7-ketocholesterol (KC), 24- HC, 25-HC and 27-HC, as well as the cholesterol precursor, lathosterol, by GC-MS. While the recovery of spiked sterols was about 10 % greater in the presence of cell matrix (~ 80-115 % without matrix and ~ 90-125 % with matrix), cell medium reduced the linearity of calibration curves from a R2 ranging 0.993-0.998 without matrix to 0.985-0.996 with matrix effects. Further, a loss in sensitivity was attained in the presence of cell medium, with quantification limits of 0.025 to 0.05 nanograms (ng) without matrix and 0.033 to 0.167 ng with matrix effects. However, the accuracy and precision of measurements was within 10-20 % for most analytes regardless of matrix type. Production of potential biodosimetric markers (sterols/COPs) in peripheral blood mononuclear cells (PBMCs), as well as Jurkat cell lines, revealed a high degree of variation between replicates, indicative of variation due to the analysis method employed despite the suitable precisions previously ascertained. While PBMC sterols frequently resulted in change to measured concentrations greater than ± two-fold after irradiation, a near equal distribution of samples exhibiting increased and decreased sterol levels prevented any significance pertinent to biodosimetry. Jurkat cells did not demonstrate the same level of inconsistency in results, emphasising a potential susceptibility of PBMCs to matrix effects during analysis and/or interindividual variation in the sterol radiation response. A significant and consistent hyperproduction of 24-HC and lathosterol in wild type Jurkat cells, near or beyond a 100 % increase of that for unirradiated control cells, was demonstrated at doses of 10 and/or 100 mGy. This effect was reversed when the mitochondrial translocator protein (TSPO) was overexpressed in Jurkat cells, resulting in significantly lower concentrations of 24-HC and lathosterol, near or beyond a 50 % loss relative to unirradiated control cells, at both 10 and 100 mGy doses. Thus, these sterols may function in the cellular adaptive response to radiation, representing potential biodosimetric markers for gammaradiation exposure that is dependent on TSPO expression level. An increase in dose to 5000 mGy saw these effects diminish or reverse once more, suggesting higher doses or dose rates to impact COP biosynthesis / cell response, or a sustained response that is independent of the sterols examined. However, given large errors associated with measurements, this work requires repetition. In summary, this thesis has demonstrated that elevated damage to mtDNA targets, such as the hypervariable regions (HVRs), can be expected after high-dose gamma-irradiation, as compared to nuDNA targets. However, at the doses examined, gamma-radiation alone is not sufficient to reduce the forensic efficacy of STR genotypes to the extent that HVR sequencing would be warranted. Thus, it is possible for STR genotyping to maintain practical value in a radiological crime scene, at least where gamma-radiation is the only source of DNA damage. Furthermore, this thesis revealed that low doses of gamma-radiation do not elicit a correlative sterol response in human PBMCs that could be detected by the methods employed. Hence, the sterols extracted from these cells did not provide value for biodosimetry or radiation injury triage. However, the identification of consistent dose-dependent changes to sterol formation, isolated from cultured Jurkat cells, suggests potential for indicative changes in alternate human cell types that may differentially express TSPO. The sterols identified to respond to gamma-radiation are valuable targets for future studies of radiation biomarkers, as well as studies of cell death and adaptation to ionising radiation and/or oxidative stress
    Date of Award2019
    Original languageEnglish
    SupervisorDennis Mcnevin (Supervisor), Michelle Gahan (Supervisor), Michelle Gahan (Supervisor) & Dennis Mcnevin (Supervisor)

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