טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentAuslander Meirav
SubjectIdentification of Environment-Affected Genes in Fish
DepartmentDepartment of Civil and Environmental Engineering
Supervisors Professor Emeritus Peter Neumann
Dr. Moshe Tom
Full Thesis text - in Hebrew Full thesis text - Hebrew Version


Abstract

Anthropogenic impact on natural biota is a major environmental threat and its monitoring is an essential tool for designing and adjusting environmental policy. A sentinel organism inhabiting an examined habitat can serve as probe of environmental impact. The subject sentinel of this research is the fish Lithognathus mormyrus (the striped sea bream). Alterations in the levels of gene transcript expression are widely used as experimental and diagnostic biomarkers in a variety of biological fields, including the evaluation of the biological effects of environmental pollutants.A major tool for simultaneous evaluation of multi-gene expression levels is the cDNA microarray, the main research tool in this study.

Individuals of L. mormyrus were exposed to a series of pollutants. A microarray was constructed using 4608 L. mormyrus hepatic cDNA cloned from the pollutant exposed fish. The clones were sequenced and assembled into a microarray with 1494 unique and annotated clones.

The toxicity of many xenobiotics including environmental pollutants is associated with the formation of free radicals including reactive oxygen species (ROS). Hence, the constructed microarray was used to identify changes in hepatic gene expression profile upon exposure to tert-butyl hydroperoxide (t-BHP). t-BHP is a pro-oxidant hepatotoxic agent, frequently used as a model to study the mechanism of cellular alterations resulting from free radical action.

169 unique clones were differentially expressed, after fish exposure to different injected t-BHP. Consistent changes in the expression of 7-dehydrocholesterol reductase ) 7-DHCR ( and two non-annotated sequences (Gene bank accession numbers DQ849829 and DQ849868) suggested their  suitability as potential biomarkers for exposure to ROS in fish.

Some of the other differentially expressed transcripts elucidated in response to t-BHP treatments were of ROS related genes including aldehyde dehydrogenase 3A2 and NADPH oxidase flavocytochrome b small subunit p22phox, ras-related C3 botulinum toxin substrate 1 (Rac1) and cytochrome P4501A1. Others  were associated   with various pathways and functions including the immune system, oxireductases, lipid and cholesterol metabolism, proteases, protease inhibitors, metal-related proteins and signal transduction.

No significant changes in 4-hydroxy-2-trans-nonenal (HNE) - protein adducts, markers of ROS related lipid peroxidation, were revealed after t-BHP exposure. However, leukocyte infiltrations to the liver a sensitive early hepatic response to t-BHP, was caused by all experimental treatments and confirmed the physiological effectiveness of the t-BHP treatment .

A guideline for using a cDNA as a bio-monitoring tool is outlined in the discussion section of the dissertation.