טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentAvezov Bivas Katherine
SubjectOxidative Stress and Induced by Cigarette Smoke and
Hypoxia: Different Types of Tissue Damage and
Cellular Mechanisms of Defense
DepartmentDepartment of Medicine
Supervisors Clinical Professor Dror Aizenbud
Dr. Lena Lavie
Professor Abraham Reznick
Full Thesis textFull thesis text - English Version


Abstract

Oxidative stress represents an imbalance between the load of chemically reactive oxidants as reactive oxygen and nitrogen species (ROS and RNS) and the ability of biological systems to detoxify them. Disturbances in the normal redox states can cause cellular damage, inducing the development of many seemingly unrelated diseases. Endogenous ROS are natural byproducts of mitochondrial oxygen metabolism and activated leukocytes. Exogenous free radicals are derived from ionizing radiation and environmental pollutants. Biological systems utilize antioxidant detoxifying mechanisms. However, insufficient or inhibition of antioxidants levels, tilt the balance towards oxidative stress.

The current study investigated the effects of exogenously and endogenously derived oxidative stress on cells and tissues and their anti-oxidant mechanisms.  Cigarette smoke (CS) and its oxidant components (RNS, saturated and unsaturated aldehydes) represented an environmental source of ROS. ROS effects on the saliva and the salivary enzyme Lactate-dehydrogenase (LDH) and oral epithelial cells were investigated. Intermittent hypoxia (IH), the hallmark of obstructive sleep apnea (OSA), represented an endogenous source of ROS, evolving from dysfunctional mitochondria and activated leukocytes. The effects of IH on oral epithelial tissue, the formation of endothelial cell colony forming units (EC-CFUs) and their paracrine ability were investigated.

The HaCaT keratinocyte cell line representing oral keratinocytes, and human saliva from 7 consenting volunteers exposed to CS, unsaturated-aldehyde (acrolein) and saturated aldehyde (acetaldehyde) were investigated. Salivary and intracellular protein carbonyl modification (by Western blot) and salivary enzymatic dysfunction were increased after CS and acrolein exposure. Also, HaCaT oxidative stress (investigated by 2,7dichlorodihydrofluorescein (DCF) assay) was increased and antioxidant-glutathione (GSH) levels were diminished.

HaCaT cells migratory abilities were determined by a cross-scratch technique. Cells were exposed to 28 IH-cycles (5-20% oxygen) during 12-hours using the BioSpherix-OxyCycler-C42 system. Control cells were maintained in normoxic conditions (NOX) or in sustained hypoxia (SH) (5% oxygen) for same durations. In parallel experiments, 25 μM acrolein were added to each treatment. The scratch closure, measured after 24h was the slowest under IH, and was further attenuated by acrolein addition.

EC-CFUs were developed in culture using peripheral mononuclear cells from 15 healthy consenting volunteers. The cells were exposed to 14 IH-cycles during 6 hours for 3-consecutive days. Control cells were maintained under NOX and SH for same durations. Compared to NOX, IH-dependent increase in EC-CFUs numbers, proliferation, ROS production and OS markers was noted. EC-CFUs conditioned media from NOX, IH and SH treatments were collected and used to determine endothelial tube formation in EA.hy926 endothelial cell cultures. Generation of vessel-like structures was significantly higher using EC-CFUs conditioned media from IH and SH treatments compared to NOX. Adding the antioxidant N-acetyl-cysteine (NAC) and NADPH-oxidase inhibitors prevented tube formation.

Unsaturated-aldehydes were identified as the main CS ingredient inducing protein carbonylation, leading to structure alteration and dysfunction, as well as decreasing intracellular antioxidant-GSH levels and increasing oxidative stress in keratinocytes. Conversely, IH-associated oxidative stress was found to promote vascular function and paracrine capacities in EC-CFU, while NAC and NADPH-oxidase inhibitors prevented tube formation. Collectively, these findings emphasize the complex tissue-specific role of ROS in damage vs. repair.