טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentWeiner Dana
SubjectThe Effect of Aldehydes, Reactive Nitrogen Species and
Cigarette Smoke on a-Amylase Activity and Protein
Modification in Human Saliva
DepartmentDepartment of Medicine
Supervisors Professor Abraham Reznick
Clinical Professor Yishai Levy
Full Thesis textFull thesis text - English Version


Abstract

Cigarette smoke (CS) is associated with a variety of human pathologies including cardiovascular disease and cancer. CS contains over 4800 different chemicals and contains oxidants such as reactive oxygen , nitrogen species (ROS/RNS) and volatile aldehydes. Exposure to these molecules is probably the major cause of damage to biomolecules in saliva. Saliva is the first body fluid to encounter CS. Saliva is known for its protective functions against deleterious agents. Our working hypothesis was that when CS encounters saliva, there is an increase in ROS, RNS and aldehydes, which may cause protein alterations. Thus, external addition of antioxidants may neutralize these effects. Our aims were: To determine the interaction of aldehydes and RNS donors with human saliva by measuring carbonyl formation, α- amylase activity and protein nitration in comparison to exposure to whole CS and to elucidate the mechanisms by which antioxidants (glutathione and EGCG) may reduce the interactions of aldehydes and RNS with human saliva.

The collection of saliva was from 3 volunteers. In in vitro studies, saliva was exposed to CS, aldehydes and RNS. To determine protein carbonyl formation we employed the carbonyl assay, using DNPH reagent and the Thermochemiluminescence (TCL) analysis. Tyrosine nitration was analyzed by WB.

Our main findings were that exposure of saliva to CS and unsaturated aldehydes, caused a similar marked reduction in α-amylase activity and TCL ratio and similar increase in carbonyl formation, whereas acetaldehyde, had only a mild effect. Preincubation with glutathione partially inhibited the effects of CS and unsaturated aldehydes but had no effect on saliva exposed to acetaldehyde. Exposure of saliva to RNS donors produced a decrease in α-amylase activity and in TCL ratio and an increase in carbonyl formation, but to a lesser extent than CS. Preincubation with EGCG did not produce protection against CS and RNS damage to α-amylase but did protect against oxidation. We also found basal tyrosine nitration of salivary proteins, and exposure to CS, aldehydes and RNS produced a decrease in tyrosine nitration, whereas this phenomenon was inhibited by preincubation with glutathione and EGCG.

In conclusion, the effect of CS on saliva is multifactorial, with various agents in CS affecting salivary components via different mechanisms. The α-amylase activity loss, caused by CS, aldehydes and RNS along with increase in oxidation, may have significant implications regarding the damage caused to the oral cavity and thus may play a role in the carcinogenic effects of CS.