No / Lymphocytes have many ion channels. These ion channels contribute to T cell-mediated autoimmune and/or inflammatory responses and therefore are targets for pharmacological immune modulation [1]. Lymphocytes are also suitable surrogate cells for cancer [2] and other diseases states [3] where inflammation is associated with increasing disease incidence. Non-steroidal anti-inflammatory drugs (NSAIDs), such as aspirin, have been associated with anti-tumour effects in cancers [4]. We recently compared DNA damage caused by the nanoparticle forms (NPs) of the NSAIDs, aspirin and ibuprofen and their bulk forms in peripheral blood lymphocytes of patients with respiratory diseases and healthy individuals in the Comet and micronucleus assays [5]. In this present study, we investigate electrophysiological changes from lymphocytes after NP exposure and compare these results with their DNA damage. 10 ml peripheral blood was collected from patients and healthy control individuals. Ethical permission was obtained from the Bradford Ethics Committee REC ref no: 09/H1313/37, ReDA no: 1202, and the University of Bradford ref no: 0405/8. Ibuprofen USP was purchased from Albermarle Europe sprl (Belgium). Pharmcoat 606 (HPMC) was kindly donated by Shinetsu (Japan). Aspirin and sodium lauryl sulphate were purchased from Sigma. Kollidon 30 (PVP K-30) was purchased from BASF (UK). Bulk and nano compound suspensions of aspirin and ibuprofen (IBU) were kindly prepared by Lena Nanoceutics (Bradford, UK). Whole blood collected from healthy individuals and cancer patients were treated for 30 mins with 500µg/ml of IBU bulk and nano forms separately. Whole-cell currents were recorded with normal patch clamping technique. The extracellular solution contained (in mM) the following: NaCl 125; KCl 5; MgCl2 1; CaCl2 2.5; HEPES 10; pH 7.4. The electrode internal solution contained (in mM) the following: KF 120; MgCl2 2; HEPES 10; EGTA 10; and CaCl2 1, pH 7.4. All experiments were carried out at room temperature. Compared with untreated cells, lymphocytes treated with IBU in NP form had lower whole-cell currents and the activities of ion channels were inhibited by 20% compared to those in bulk form. This result is mirrored by the DNA damage which occurred in lymphocytes after exposure to nanoparticles [5]. Although the intracellular biochemical mechanisms and ion channels involved in our nanoparticle toxicity remain to be determined, this study provides direct evidence that 500 μg/ml IBU in nano form can cause membrane damage to lymphocytes after a relatively short exposure. Such cytotoxicity of nanoparticles in lymphocytes may be associated with early membrane damage. Further detailed investigation is needed to explain the changes of lymphocytes in response to different concentrations of NPs in real time. / Poster communications

A method is presented for 160hydroxylase (SAH) induction in cultured human lymphocytes. SAH, a microsomal-associated enzyme, effects the oxidative conversion of 17pestradiol to estriol, which competes for cytoplasmic binding sites. 17,-estradiol and estrone are known mammary carcinogens, while estriol and its epimers have been suggested to have anticarcinogenic properties. To substantiate genetic variations of hydroxylase activity, an analysis of estrogen-induced cultured human lymphocytes was conducted to evaluate the frequency distribution of low, intermediate, and high SAH activity. Frequency analysis indicated that the control population distribution of SAH activity does not corroborate a proposed trimodal expansion of human SAH activity. A log normal distribution of SAH activity does exist, which suggests a polygenic mode of genetic control. SAH activity in a population of breast cancer patients and relatives of breast cancer patients showed no statistical difference from the SAH activity in the control population.

Cultured human lymphocytes may be assayed for aryl hydrocarbon hydroxylase (AHH) in whole cell preparations. The optimum assay conditions are pH 8.5, and 1.5 mM Mg++. The reaction is linear with time and cell number, and is inhibited by CO. Estradiol may inhibit induction of AHH by 3-methylcholanthrene, but is a poor competitor for the enzyme. A Caucasian population was assayed for AHH activity. The distribution was lognormal; no difference was found in cultured cells from males and females or smokers and nonsmokers. Cells from relatives of lung cancer patients showed higher activity. An American Indian population showed no difference from the Caucasian population in enzyme level. No linkage was found between AHH and 16a-hydroxylase.

Selenium (Se) is an essential micronutrient, being a component of more than twenty seleno-proteins in humans. Previous studies suggested that increased intake of Se may reduce the risk of degenerative diseases including cancer; however, excessive intake can be toxic. Wheat is one of the major dietary sources of Se in humans, mainly in the form of L-selenomethionine (Se-met) but the impact of this source of Se on human health at the genome level was previously unexplored. This PhD project aimed to (a) determine the safe dose-range and bio-efficacy of Se-met in vitro; (b) identify the optimal concentration of Se-met for reduction of genome damage in vitro; (c) investigate the optimal concentration of Se-met for improving resistance to gamma radiation or hydrogen peroxide induced genome damage in vitro; d) determine the bioavailability and bioefficacy of Se in vivo, in the form of either Semet or wheat biofortified with Se; e) identify the nutrients and food groups that are correlated with Se intake/status and f) identify the nutrients, food groups and plasma mineral concentrations that are correlated to baseline lymphocyte DNA damage. The in vitro study was performed on the peripheral blood lymphocytes isolated from six males and cultured with media supplemented with Se-met in a series of Se concentrations from 3 to 3850 μg Se/l while keeping the total methionine (i.e. Se-met + L-methionine) concentration constant. Baseline genome stability of lymphocytes and the extent of DNA damage induced by 1.5 Gy γ-ray or 7.5 μM hydrogen peroxide (H₂O₂) were investigated using the Cytokinesis-block Micronucleus Cytome (CBMNCyt) assay and the alkaline Comet assay with and without glycosylase (Fpg or Endo III) treatment after 9 days of culture. Results showed that high Se concentrations (≥1880 μg Se/l) caused strong inhibition of cell division, extensive DNA damage and increased cell death indicating cytotoxicity and genotoxicity. Baseline frequency of nucleoplasmic bridges (NPBs) and nuclear buds (NBud) declined significantly as Se concentration increased from 3 μg Se/l to 430 μg Se/l (P trend = 0.03 and 0.008, respectively); however, a significant trend of increase in Comet DNA damage was also observed (P trend <0.05) in lymphocytes. Selenium concentration (≤ 430 μg Se/l) had no significant effect on baseline frequency of micronuclei (MN) or DNA oxidation and had no protective effect against γ-ray-induced or H₂O₂-induced genome damage in lymphocytes. A randomised double-blind placebo-controlled intervention trial was conducted on healthy South Australian males (n = 62, age (mean ± SD) 56 ± 7.0 years) with Se dosage increased every 8 weeks for a total duration of 24 weeks. This study compared the bioavailability, by using plasma Se concentration as the biomarker, and bioefficacy of Se, by using platelet glutathione peroxidase (GPx) activity and lymphocyte DNA damage as biomarkers, from wheat process-fortified with Se-met (PROFORT) and high-Se wheat biofortified with Se (BIOFORT) compared to non-fortified normal (CONTROL) wheat. It was found that increased Se intake from BIOFORT wheat increased plasma Se concentration effectively in a dose-response manner from a baseline of 122 μg/l up to 190 μg/l (P<0.001). Increased Se intake from PROFORT wheat also increased plasma Se with a plateau at 140 μg/l, being therefore less effective than BIOFORT wheat (P<0.001). There was no significant change in Se status in the CONTROL group. Improved plasma Se concentrations had no effect on platelet GPx activity or lymphocyte DNA damage in either of the intervention groups. Results from the food frequency questionnaire (FFQ) survey (n = 173) and plasma Se concentration survey (n = 179) suggested that the study population screened for participation in the in vivo trial described above had a mean plasma Se concentration (± SD) of 102 (± 12) μg/l and a mean (± SD) estimated Se intake of 165 (± 68) μg/d. This is a higher estimated Se intake than found in previous Australian studies. The major dietary sources of Se were found to be bread/cereals, fish/seafood and meat. However, increased intake of nuts/seeds, which are rich in Se, may have undesirable effects on lymphocyte DNA oxidation in this Se-replete population. In conclusion, the in vitro studies suggest that (1) Se-met at higher concentrations at greater or equal to 1880 μg Se/l is cytotoxic; (2) Se-met may improve specific genome stability biomarkers such as nucleoplasmic bridge and nuclear bud at concentrations up to 430 μg Se/l, but further studies are needed to verify this effect. The in vivo studies in older men showed that Se from BIOFORT wheat is more effective in raising plasma Se concentration than Se from wheat process-fortified by the addition of Se-met, when both wheat products were subjected to strong heat. However, the platelet GPx activity and lymphocyte DNA damage appeared not to be modified by improved Se status. This work contains two publications: 1) "The effect of selenium, as selenomethionine, on genome stability and cytotoxicity in human lymphocytes as measured by the cytokinesis-block micronucleus cytome assay". Mutagenesis 2009 May;24(3):225-32. 2) "Increased consumption of wheat biofortified with selenium does not modify biomarkers of cancer risk, oxidative stress or immune function in Australian males" Environmental Molecular Mutagenesis. 2009 July; 50 (6):489-501 The latter one was not able to be published in a journal of higher impact factor due to part of the data had been published elsewhere. Both articles are attached in Appendix. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1523459 / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2010

No / Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit COX enzyme activity, a significant mechanism of action of NSAIDs. Inflammation is associated with increasing cancer incidence. Recent pre-clinical and clinical studies have shown that NSAID treatment could cause an anti-tumour effect in cancers. Such studies are lengthy and expensive. The present study, however, examined DNA damage in the Comet and micronucleus assays in peripheral blood lymphocytes of patients with respiratory diseases and healthy individuals using the nanoparticle (NP) and bulk versions of the NSAIDs, aspirin and ibuprofen. Lymphocytes are suitable surrogate cells for cancers and other disease states. DNA damage decreased in lymphocytes from healthy individuals, asthma, COPD and lung cancer patient groups after treatment with aspirin nano-suspension (ASP N) and ibuprofen nano-suspension (IBU N) compared to their bulk version (micro-suspension) in both assays. However, when ASP N was compared to untreated lymphocytes in all groups in the Comet assay, DNA damage significantly decreased in all groups, except the asthma group. When IBU N was compared to untreated lymphocytes, in healthy individuals and the lung cancer group, DNA damage decreased, but increased in asthma and COPD groups. Similarly, micronuclei (MNi) increased after ASP N and IBU N in the healthy individual and lung cancer groups, and decreased in asthma and COPD groups. Also shows that whilst there are basic similarities with different genetic endpoints in terms of nano and bulk forms, but highlights some differences between the disease states examined. Furthermore, lymphocyte responses after IBU N and ibuprofen bulk were investigated by patch-clamp experiments demonstrating that IBU N inhibited ion channel activity by 20%. This molecular epidemiology approach mirrors pre-clinical and clinical findings, and provides new information using nanoparticles.

No / Malaria is a mosquito-borne infectious disease caused by the genus Plasmodium. It causes one million deaths per year in African children under the age of 5 years. There is an increasing development of resistance of malarial parasites to chloroquine and other currently used anti-malarial drugs. Some plant products such as the indoloquinoline alkaloid cryptolepine have been shown to have potent activity against P. falciparum in vitro. On account of its toxicity, cryptolepine is not suitable for use as an antimalarial drug but a number of analogues of cryptolepine have been synthesised in an attempt to find compounds that have reduced cytotoxicity and these have been investigated in the present study in human sperm and lymphocytes using the Comet assay. The results suggest that cryptolepine and the analogues cause DNA damage in lymphocytes, but appear to have no effect on human sperm at the assessed doses. In the context of antimalarial drug development, the data suggest that all cryptolepine compounds and in particular 2,7-dibromocryptolepine cause DNA damage and therefore may not be suitable for pre clinical development as antimalarial agents.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. It has commonly been associated with exposure to tobacco-derived carcinogens and alcohol consumption. Pembrolizumab has shown to be effective in the treatment of many types of cancers such as melanoma, non-small cell lung cancer, due to its antiproliferative, immunoregulatory properties. The aim of this study was to investigate the effects of naked Pembrolizumab and Pembrolizumab liposome on the level of DNA damage, gene, and protein expressions in peripheral lymphocytes from HNC patients and compared to the healthy individuals by using the Comet and micronucleus assays. Western blotting and real-time polymerase chain reaction were performed to assess the potential of improving the repair mechanisms after treatment with naked Pembrolizumab and Pembrolizumab liposome. According to the results, Comet assay and micronucleus assay showed a significantly decreased DNA damage in the lymphocytes from HNC patients after being treated with naked Pembrolizumab and pembrolizumab liposome. Furthermore, the results have shown that naked Pembrolizumab and pembrolizumab liposomes (10 μg/ml) greatly decreased the oxidative stress produced by H2O2. Both forms of pembrolizumab have also demonstrated improving the repair mechanisms in lymphocytes from HNC patients by modulating the expression of P53, P21, and Bcl-2 at mRNA and protein levels. This study suggested that Pembrolizumab naked and liposome could have an antioxidant role alongside other actions in the treatment of HNSCC. However, further studies on Cancer cell lines and in vivo observation are required to validate the anticancer potential of pembrolizumab naked with liposome in HNC. / Ministry of Higher Education and Scientific Research, Libya

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: This study’s focus is on the determination and quantization of radiation damage on a cellular level due to the decay of the Auger electron-emitting 123I and the replication of this energy deposition using Geant4 Monte Carlo simulations. The relatively short half-life of 123I (13.2 hours) makes it ideal for studies of Auger electrons which induce biological damage similar to that of high linear energy transfer radiations, when permitted to deposit their energy in close proximity to DNA. Due to small cellular dimensions, direct dose measurements are impossible but estimates may be made from Monte Carlo simulations. In this investigation the thymidine analogue 5-[123I]-iodo-2-deoxyuridine (123IUdR) was used to incorporate the 123I into the cellular DNA of T-lymphocytes from two human donors. Radiation induced micronuclei were numerated in binucleated cells using fluorescence microscopy. The energy deposition per decay of 123I was calculated within a spherical geometry, having the same size and density as a human lymphocyte, using the open source Geant4 toolkit. The absorbed energy per disintegration was used to convert the incorporated 123I activity (Bq) into absorbed dose (Gy) values, in order to compare the biological damage caused by the radioactive iodine to 60Co γ-radiation. A linear relationship between micronuclei frequency and 123I activity could be established. The linear dose-response noted for Auger electrons in the study is indicative of the high-LET nature of these particles. Using the linear-quadratic dose-response curve for micronuclei frequencies following exposure to graded doses of 60Co γ-rays, the relative biological effectiveness (RBE) of the DNA incorporated 123I estimated in this work was found to range from 19 ± 10 to 32 ± 7 for lymphocyte donor 1 and 15 ± 6 to 42 ± 11 for donor 2. The dose limiting RBE (RBEM) for lymphocyte donor 1 and 2 are respectively 34 ± 8 and 50 ± 15 and follows the expected shift in terms of the inherent radiosensitivity of the donors. We also considered the inclusion of the S-phase fraction of the lymphocytes in the dosimetry calculations. The resultant RBEs of the dose points of lymphocyte donor 1 ranges from 4 ± 2 to 7 ± 2, and those of donor 2 ranges from 3 ± 1 to 9 ± 2. The RBEM for lymphocyte donor 1 and 2 are respectively 7 ± 2 and 11 ± 3. The inclusion of the S-phase fraction reduces the calculated RBEs significantly and these observed RBE values relate well to those obtained in studies with fibroblasts and 125IUdR. / AFRIKAANSE OPSOMMING: Hierdie studie fokus op die bepaling en kwantisering van stralingskade op 'n sellulêre vlak as gevolg van die verval van 123I wat Auger elektrone afgee, asook die simulering van hierdie energie afsetting met behulp van die Geant4 Monte Carlo program. Die relatiewe kort half-leeftyd van 123I (13.2 uur) maak dit ideaal vir studies van Auger elektrone wat biologiese skade soortgelyk aan dié van 'n hoë lineêre-energie-oordrag uitstraling veroorsaak, indien die energie van die elektrone naby sellulêre DNA geabsorbeer word. As gevolg van die klein sellulêre dimensies is direkte dosis metings egter onmoontlik, maar skattings kan gemaak word met behulp van Monte Carlo simulasies. Die timidien analoog 5-[123I]-jodo-2-deoxyuridien (123IUdR) was in hierdie ondersoek gebruik om die 123I in die DNA van menslike T-limfosiete in te bou. Mikrokerne in dubbel-kernige selle wat vorm as gevolg van die Auger elektrone was getel met behulp van fluoressensie mikroskopie. Die energie afsetting per 123I verval was bereken binne ‘n sferiese geometrie, met dieselfde grootte en digtheid as 'n menslike limfosiet, met behulp van die Geant4 sagteware. Die geabsorbeerde energie per verval was gebruik om die geïnkorporeerde 123I aktiwiteit (Bq) om te skakel na ‘n waarde van geabsorbeerde dosis (Gy), ten einde die biologiese skade wat veroorsaak word deur die radioaktiewe jodium-123 met kobalt-60 gamma straling te vergelyk. ‘n Lineêre verwantskap tussen die mikrokerne frekwensies en die 123I aktiwiteit is vasgestel. Hierdie verwantskap vir Auger elektrone is 'n aanduiding van die hoë lineêre-energie-oordrag van hierdie deeltjies. Die lineêr-kwadratiese dosis-effek krommes vir mikrokerne frekwensies na blootstelling aan 60Co γ-strale was gebruik om die relatiewe biologiese doeltreffendheid (RBE) van die DNA geïnkorporeerde 123I te beraam. RBE waardes wissel van 19 ± 10 tot 32 ± 7 vir limfosiete van skenker 1 en 15 ± 6 tot 42 ± 11 vir skenker 2. Die dosis beperkte RBE (RBEM) vir limfosiet skenker 1 en 2 is onderskeidelik 34 ± 8 en 50 ± 15 en volg die verwagte skuif in terme van die inherente radiogevoeligheid van die skenkers. Die fraksie van limfosiete wat in S-fase was tydens die blootstelling aan 125IUdR was ingesluit in verdere dosimetrie berekeninge. Die gevolglike RBEs van die dosispunte van limfosiete van skenker 1 wissel van 4 ± 2 tot 7 ± 2 en dié van skenker 2 wissel van 3 ± 1 tot 9 ± 2. Die RBEM vir limfosiet skenker 1 en 2 is onderskeidelik 7 ± 2 en 11 ± 3. Die insluiting van die S-fase fraksie verminder die berekende RBEs aansienlik en die RBE waardes waargeneem hou goed verband met die wat in studies met fibroblaste en 125IUdR verkry is.

Cigarette smoke condensates from all cigarettes tested were found to be potent inducers of AHH enzyme in cultured human lymphocytes and, with the exception of Kent Lights and Carlton CSC's, all were found to be toxic under the experiment conditions. Most of the AHH inducing activity was found in basic and neutral fractions of the lAl standard cigarettes. A radiometric assay of BP metabolites in cultured human lymphocytes was developed in which we were able to separate the primary metabolites and the secondary metabolites from the parent compound (BP) by neutral alumnia HPLC. The primary metabolites were further separated by a selective enzyme hydrolysis and/or reverse phase HPLC.

Cancer is one of the leading causes of death across the globe which needs appropriate and cost-effective treatment. Several recent studies have suggested that dietary intake of various flavonoids such as myricetin have a protective effect against different types of cancers and cardiovascular diseases. The present study was conducted to investigate the genoprotective and genotoxic effects of myricetin nano and bulk forms on the lymphocytes from pre-cancerous and multiple myeloma cancer patients compared to those from healthy individuals. Also, to investigate the protective potential of myricetin bulk and nano against the oxidative stress produced in vitro by 2- amino-1-methyl-6 phenylimidazo [4, 5-b] pyridine and reactive oxygen species- induced DNA damage using the Comet assay, micronucleus assay, cellular reactive oxygen species and glutathione detection assay, Western blotting, real-time polymerase chain reaction and immunofluorescence. Lymphocytes from the patient groups showed significantly higher levels of basal DNA damage compared to the lymphocytes from healthy individuals which was observed throughout the in vitro treatment. Myricetin in both forms has not induced any significant DNA damage in all of the investigative groups at selective lower concentrations; in fact, the results demonstrate a reduction in DNA damage upon treating with myricetin nano in lymphocytes from pre-cancerous patients demonstrated by significant reduction in micronuclei formation in mononucleated cells. DNA repair capacity of myricetin bulk and nano was determined by co-treating the drugs with hydrogen peroxide. Myricetin significantly reduced the oxidative stress related damage caused by hydrogen peroxide, where myricetin nano seemed to be more effective employing the Comet assay. In the presence of myricetin bulk and nano, the damaging effects of 2- amino-1-methyl-6 phenylimidazo [4,5-b] pyridine were considerably decreased, where myricetin nano was more effective. This could be because nanoparticles have a larger surface area which could improve their reactivity and also the reduction in size of the particles could improve the anti-cancer properties of this compound. Myricetin has shown genoprotective and anti-oxidant effects by demonstrating the potential to reduce DNA damage caused by over-production of reactive oxygen species and oxidative stress. It has also shown anti-cancer potential in the lymphocytes from multiple myeloma patients by regulating the apoptosis related proteins, dependent on oxidative stress. Therefore, this study suggests that myricetin supplementation in our regular diet with enhanced bioavailability could have potential health beneficial effects and possibly protect against various diseases including cancer.