Effect of nanoparticles on human cells from healthy individuals and patients with respiratory diseases. Item Type Thesis Authors Osman, Ilham F. Rights <a rel="license" href="http://creativecommons.org/licenses/ by-nc-nd/3.0/"><img alt="Creative Commons License" style="border-width:0" src="http://i.creativecommons.org/l/by- nc-nd/3.0/88x31.png" /></a><br />The University of Bradford theses are licenced under a <a rel="license" href="http:// creativecommons.org/licenses/by-nc-nd/3.0/">Creative Commons Licence</a>. Download date 23/09/2021 14:42:50 Link to Item http://hdl.handle.net/10454/4864 University of Bradford eThesis This thesis is hosted in Bradford Scholars – The University of Bradford Open Access repository. Visit the repository for full metadata or to contact the repository team © University of Bradford. This work is licenced for reuse under a Creative Commons Licence. EFFECT OF NANOPARTICLES ON HUMAN CELLS FROM HEALTHY INDIVIDUALS AND PATIENTS WITH RESPIRATORY DISEASES ILHAM F. OSMAN MBBS, Khartoum University (Sudan) MSC, Manchester University (UK) PhD UNIVERSITY OF BRADFORD 2010 i Abstract Ever increasing applications of nanomaterials (materials with one or more dimension less than 100 nm) has raised awareness of their potential genotoxicity. They have unique physico–chemical properties and so could have unpredictable effects. Zinc oxide (ZnO) and titanium dioxide (TiO 2) are widely used in a number of commercial products. There are published studies indicating that some forms of these compounds may be photo-clastogenic in mammalian cells. What has not been investigated before is the effect of nanoparticles from these compounds in human germ cells. Thus the present study has examined their effects in the presence and absence of UV light in human sperm and compared responses to those obtained with human lymphocytes using the Comet assay to measure DNA damage. The effect of nanoparticles (40-70nm range) was studied in human sperm and lymphocytes in the dark, after pre-irradiation with UV and simultaneous irradiation with UV. The studies do provide some evidence that there are photo-genotoxic events in sperm and lymphocytes in the absence of overt toxicity. The cytotoxic and genotoxic potentials of ZnO and TiO 2 as well as their effect on phosphotyrosine expression, were examined in the human epithelial cervical carcinoma cells (Hela cells). This was done to try and determine the underlying molecular events resulting from their exposure to ZnO and TiO 2 nanoparticles occurring at the same time as DNA is damaged. Concentration- and time-dependent cytotoxicity, and an increase in DNA and cytogenetic damage with increasing nanoparticle concentrations were reported in this study. Mainly for zinc oxide, genotoxicity was clearly associated with an increase in tyrosine phosphorylation. Nanotechnology has raced ahead of nanotoxicology and little is known of the effects of nanoparticles in human systems, let alone in diseased individuals. Therefore, the effects of TiO 2 nanoparticles in peripheral blood lymphocytes from patients with respiratory diseases (lung cancer, chronic obstructive pulmonary disease (COPD) and asthma) were compared with those in healthy individuals using genotoxic endpoints to determine whether there are any differences in sensitivity to nano-chemical insult between the patient and control groups. The results have shown concentration dependent genotoxic effects of TiO 2 in both respiratory patient and control groups in the Comet assay and an increasing pattern of cytogenetic damage measured in the micronucleus assay without being statistically significant except when compared with the untreated controls of healthy individuals. Furthermore, modulation of ras p21 expression was investigated. Regardless of TiO 2 treatment, only lung cancer and COPD patients expressed measurable ras p21 levels that showed modulation as the result of nanoparticle treatment. Results have suggested that both ZnO and TiO 2 nanoparticles can be genotoxic over a range of concentrations without either photoa-ctivation or being cytotoxic. Key words , Nanoparticles, TiO 2, ZnO, Sperm, Lymphocyte, Comet assay, Tyrosine phosphorylation, Micronucleus assay, Respiratory diseases, lung cancer, COPD, asthma, healthy controls and ras oncoprotein. i Table of contents Page Abstract…………………………………………………………………………………...i Contents …………………………………………………………………………...…….ii Acknowledgements……………………………………………………….…..………..vii List of figures……………………………………………………….....………………viii List of tables……………………………………………………………..……………...xi Abbreviations……………………………………………………..……….…………..xiii Chapter 1 Introduction 1. Introduction…………………………………………………………...………………2 1.1 History of nanotechnology……………………………………………………..…....3 1.2. Properties of nanoparticles…………………………………………………….…....4 1.2.1 Photo properties of ZnO and TiO 2…………………………………………………4 1.3 Applications of nanotechnology..................................................................................5 1.3. 1 Titanium dioxide (TiO 2) and zinc oxide (ZnO) nanoparticles…….…...……….7 1.3. 2 Pulmonary applications of nanomedicines…………………………….….…….7 1.4 The lung…………………………………………………………………….……....10 1.4. 1 Smoking and DNA Damage………………………………………….…..……10 1.4.2 Lung cancer…………………………………………………………………….11 1.4.2.1 Genetics of lung cancer…………………………………………….…..…..12 1.4.2.2 Lung cancer biomarkers …………………..……………………………....13 1.4.3 Chronic obstructive pulmonary disease (COPD) genetics….………………..14 1.4.3.1 ROS and COPD…………………………………………………………..14 1.4.3.2 Predisposing factors to COPD.……………………………………….......14 1.4.4 Bronchial asthma………………………………………………………….….15 1.5 Toxicity of nanoparticles…………………………………...………….……..…..16 1.5.1 Nanoparticles and generation of oxidants…………………………………....17 1.5.1.1 The lung and generation of oxidants ……………………………........….17 1.5.2 Mechanisms of toxicity of nanoparticles ………………..……………….…..18 1.5.3 Pulmonary toxicity of nanoparticles…………………………………...…......20 1.5.4 Toxicity to other body systems…………………………………………….....23 1.5.4.1 Dermatological toxicity…….…………………………………...………..23 1.5.4.2 Haematological and cardiovascular toxicity……...……………………....24 1.5.4.3 Central nervous system (CNS) toxicity…….…………...………………..24 1.5.4.4 Germ cell toxicity.………………………………………...……………...25 1.5.4.5 Bactericidal effect of nanoparticles………………………………............25 1.5.4.6 Toxicity to environmental aquatic species…………………………..…...26 1.5.4.7 Cellular effects of nanoparticles……………………...……………...…...26 1.6 DNA repair mechanisms…………………………………………………………...27 1.7 The cell cycle controls and cancer ……………………...………………………….29 1.7.1 Ras proteins ………………………………………..……………………….30 1.7.1.1 Ras role in the cell cycle regulation ……………...…………………...…....31 1.7.1.2 Ras p21 and cancer ………………………………………………………31 1.7.1.3 Ras and other signalling pathways ……………..………………...…….….32 1.7. 2 Phosphotyrosine and the cell cycle…………….…..………...……………..........33 1.8 Methods for the study of genotoxic effects.…..………………...……………..…...34 1.8.1 The Comet assay ……………………………………………………………….34 1.8.1.1 Comet assay on lymphocytes………………………………….…………...35 1.8.1.2 Comet assay on sperm…. ………………………………...…...…………...35 1.8.2 Western blotting method...……………………………………..………..……..36 ii 1.8.3 Cytokinesis-block micronucleus assay…………………….……...……………36 1.9 Aims and objectives………………………………………………………………...38 Chapter 2 Materials and methods 2.1 Materials…………………………………………………………………….……...41 2.2 Methods……………………………………………………………………….…....43 2.2.1 Methods for: The effect of zinc oxide and titanium dioxide nanoparticles in the Comet assay with UVA irradiation of human sperm and lymphocytes……….....43 2.2.1.1 Preparation of ZnO and TiO 2 nanoparticle stock suspensions…….…….....43 2.2.1.2 Blood sample collection and lymphocytes isolation…………….………... 44 2.2.1.3 Single cell gel electrophoresis (SCGE)/Comet assay……………….…….. 45 2.2.1.3.1 Alkaline Comet assay on lymphocytes………………………………... 46 2.2.1.3.1.1 Treatments and viabilities…………………….………………….…46 2.2.1.3.1.2 DNA damage and repair of H 2O2 on lymphocytes…………………47 2.2.1.3.1.3 UV irradiation of lymphocyte cells…………………………….......48 2.2.1.3.1.3.1 Non irradiation of lymphocytes (D) ……..……………….....…49 2.2.1.3.1.3.2 Pre irradiation (PI) of lymphocytes ………...……….…...…….49 2.2.1.3.1.3.3 Simultaneous irradiation (SI) of lymphocytes……………….....49 2.2.1.3.1.4 Slide preparation……………………………………….….……......50 2.2.1.3.1.5 Lysis of lymphocyte cells………………………………….……….50 2.2.1.3.1.6 DNA unwinding and electrophoresis of lymphocytes …...………...51 2.2.1.3.1.7 Neutralization………….....…………………………………...……51 2.2.1.3.1.8 Slide staining and coding…………………………….…………......51 2.2.1.3.2 Comet assay on sperm………………………….……...…………...…..52 2.2.1.3.2.1 Effect of H 2O2 on sperm ………………………...………….……...52 2.2.1.3.2.2 UV Irradiation of sperm cells………………………...………..…...53 2.2.1.3.2.2.1 Treatment of sperm cells with ZnO and TiO 2……...……...…...53 2.2.1.3.2.2.2 Non- irradiation of sperm (D) ……………...………………..…53 2.2.1.3.2.2.3 Pre irradiation (PI) of sperm …………………...…………...….53 2.2.1.3.2.2.4 Simultaneous irradiation (SI) of sperm………...……….…...... 54 2.2.1.3.2.3 Slide preparation for the sperm assay……………….……….......…54 2.2.1.3.2.4 Lysis of sperm cells………………………………………...………54 2.2.1.3.2.5 Sperm DNA unwinding and electrophoresis……………...………..55 2.2.1.3.2.6 Neutralization……………………………………………...…….....55 2.2.1.3.2.7 Slide staining and coding………………………………...…………55 2.2.2 Methods for genotoxicity and cytotoxicity of zinc oxide and titanium dioxide in Hep-2 cells…...…..……………………………..………………..……………………..56 2.2.2.1 Cell culture of the HEp-2 cell line……………………..……..……………..56 2.2.2.2