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Monitoring exposure to tobacco-specific nitrosamines

Institution: University of California, Riverside
Investigator(s): Thomas Morton, Ph.D.
Award Cycle: 1999 (Cycle 8) Grant #: 8IT-0058 Award: $74,999
Subject Area: Cancer
Award Type: Inno Dev & Exp Awards (IDEAS)

Initial Award Abstract
Two kinds of disease-causing chemicals are found in tobacco smoke. One kind is called Polynuclear Aromatic Hydrocarbons (PAH). PAH is usually associated with products of burning (so-called "tar") and is not unique to tobacco. The other kind is called Tobacco-Specific NitrosAmines (TSNA). TSNA occurs in unburned tobacco and enters the body when hot smoke passes through a cigarette.

PAH is associated with one form of lung cancer, and TSNA is associated with another. The form that is caused by PAH used to be the most common lung cancer that was seen. Nowadays, however, both forms are equally common. Could this change be a result of changes in smoking habits? Could exposure to TSNA increase a result of the measures taken (filters or low-tar cigarettes, for example, or "tobacco-heating" cigarettes) to reduce PAH?

To answer this kind of question, we need to find some way to measure TSNA exposure. One way would be to develop a blood test to tell how much TSNA a person has taken in. Because we don't have many ideas how to do this, a fundamental set of experiments is proposed, to see if the most important known TSNA (known as the N-Nitroso Ketone, or NNK) sticks to a type of blood cell known as a platelet. The outside of cells like platelets is called the plasma membrane. Each type of cell has distinctive, natural markers on the outside surface of its plasma membrane. It seems plausible that one of the markers characteristic of platelets may become tagged by NNK. If this is the case, then it should be possible to develop sensitive techniques to measure how much NNK has stuck to the outside of platelets.

We don't know yet whether NNK sticks to the outside of any cell. To find out, the first experiments need to explore ways to detect small amounts of chemicals that react with the natural cell-surface markers. This project examines how easily natural cell-surface markers on platelets can be tagged by radioactive chemicals. This will tell how effectively the measurement method works, because it will tell if we can separate the sort of marker that gets tagged from the many other markers that should not be tagged. This work is proceeding in 4 stages:

(1) Take a methodology that we have recently developed for water-soluble enzymes and adapt it to look for proteins that occur on the surface of a cell. The target cells will be blood platelets, which are responsible for clotting (and are also involved in heart disease); (2) Use this methodology to examine if platelets attach radiolabeled NNK in such a way that it cannot be washed off; (3) Separate the proteins and use a newly developed, ultrasensitive technique (called AMS) to identify those that are tagged; (4) Clip the tagged proteins into smaller pieces, so that they can be identified in future. With the data from this study one can then decide whether it will be possible to develop a test (called an immunoassay) to measure the extent to which platelet surfaces react with natural NNK.

Final Report
The objective of this project is to measure the extent to which the tobacco carcinogen NNK binds irreversibly to the surfaces of platelets, the cells responsible for blood clotting. NNK is the most abundant member of a class of chemicals that occur naturally in tobacco leaf known as Tobacco Specific NitrosAmines (TSNAs), all of which are carcinogenic. The experimental procedure involves separating the outer membranes of these cells and then exposing them to a solution of radiolabeled NNK. The excess radioactive NNK then has to be separated from these membranes, so that it is possible to measure how much has become chemically attached.

Because a very small amount of the NNK sticks to the membranes, it is necessary to use an extremely sensitive technique to measure the radioactivity that remains after the excess has been removed. To do this, we make use of radiocarbon as the isotope with which the NNK is radiolabeled and analyze how much remains at the end by Accelerator Mass Spectrometry (AMS). AMS has a much greater sensitivity than does counting of radioactive decay. This sensitivity permits the tagged membranes to be purified by dissolving them in detergent (SDS) and separating the different proteins by PolyAcrylamide Gel Electrophoresis (PAGE). The separated proteins from SDS-PAGE can then be analyzed by AMS in order to find out how much radiocarbon has become attached to them. Our results, as of this writing, show that roughly one molecule of NNK becomes attached per platelet cell under the conditions we have been using.