Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-09-25
2001-04-17
Chin, Christopher L. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S002000, C435S004000, C435S007250, C435S025000, C435S375000, C435S383000, C436S063000, C436S071000, C436S503000, C436S804000
Reexamination Certificate
active
06218130
ABSTRACT:
FIELD OF THE INVENTION
This invention provides a method for evaluation of cytotoxic effects of oxidative stress on cells while avoiding the need for growth of the cells in tissue culture. The methods of the invention make it possible to test agents such as potential drugs or other bioactive agents, such as pesticides, which may be introduced into the environment to predict cytotoxic effects on animal populations. Using the methods of the invention, it is also possible to obtain predictive information about cellular response of the intended individual toward a given bioactive agent.
BACKGROUND OF THE INVENTION
The cells of all mammalian tissues require oxygen for respiration and oxidative metabolism. Unfortunately, the cellular reduction of oxygen results in the formation of reactive oxygen species (ROS) which are cytotoxic.
The membranes of all mammalian cells contain a phospholipid bilayer in which are imbedded various proteins that regulate drug transport, signal transduction and cellular metabolism. Phosphatidylcholine (PC) is the major membrane phospholipid. The PC contains an abundance of polyunsaturated fatty acids (PUFA) which are excellent free radical traps. Free radicals generated by cellular monooxygenases, which are imbedded in the membrane's phospholipid bilayer, produce membrane injury by interacting with the PUFA's of membrane-associated PC. The cell will die unless the free radical-induced membrane damage (alterations in PC structure) is rapidly repaired, since required cellular functions such as chemical transport, signal transduction and metabolism are disrupted. Therefore, the cell rapidly activates (by enzyme translocation) PC hydrolysis and biosynthesis to repair the injured membrane. In the healthy cell, reversible injury is repaired by rapid (5-10 minute) and significant increases (about 2-3 fold) in cellular PC biosynthesis. This process can be measured by determining the cellular incorporation of labeled choline into PC. However, free radical-induced membrane injury is only reversible as long as the rate of membrane injury is not greater than the cell's rate of membrane repair. If cells are continuously injured for extended periods, the cell's ability to make PC decreases, resulting in irreversible injury and cell death.
The “free radical” theory of cell injury has been proposed for many years to explain how cell death is produced by various conditions such as alcoholic liver disease (ALD), tissue dysfunction associated with aging, traumatic brain injury, drug-induced tissue injury and reperfusion cell damage. Cellular levels of free radicals increase when cellular content of oxidants and antioxidants increase and decrease, respectively. The resulting injury is known as oxidative stress.
Free radicals are very reactive and can not move far from their site of formation, since they will readily interact with various cellular components such as proteins and phospholipids. The P450-dependent monooxygenases that are imbedded in the phospholipid bilayer of cellular membranes are a major source of reactive oxygen species such as superoxide anion and hydrogen peroxide that are generated during the cellular metabolism of various agents.
It is believed that the interaction of redox active iron, superoxide anion and hydrogen peroxide produce the toxic hydroxyl radical by a Haber-Weiss reaction. Cell injury occurs, in part, when the hydroxyl radical interacts with the polyunsaturated fatty acids (PUFA's) of membrane phospholipids such as PC. Free radical-induced membrane injury is repaired if PC hydrolysis and biosynthesis are rapidly increased. However, cell death occurs if PC metabolism is not increased.
The above theory of cell injury as outlined is reasonable. However, determining the validity of this theory is difficult. The primary problem is that free radicals are very reactive, short-lived chemical entities. Therefore, it is difficult to measure the cellular level of free radicals and the effect of free radicals on cell functions. One way to overcome this problem is to incubate isolated hepatocellular fractions with labeled bioactive agents such as carbon tetrachloride and bromotrichloromethane (BTM) and thereafter determine the covalent binding of the trichloromethyl radical (.CCl
3
) to cellular components such as phospholipids and proteins. The trichloromethyl radical is rapidly bound covalently to the PUFA's of cellular PC. This free radical interaction can not be detected by measuring lipid peroxidation, since hydrogen abstraction has not occurred. Nevertheless, lipid peroxidation is routinely used to assess the reaction of free radicals with PUFA's, As a result, most investigators have concluded that the interaction of free radicals with cellular PC is a late, rather than early, event in the pathogenic sequence of cell death. The instant invention provides a much improved method for measuring the interaction of free radicals with cellular PC.
Previous studies have shown that oxidative stress can be induced and demonstrated in cultured cells by measurement of the incorporation of labeled choline into phosphatidylcholine. The use of cells grown in cell culture present several problems for the investigator. First, the propagation of the cells outside of the initial, natural host results in changes in the cells. The farther in time and/or generation that the cells of the culture are from the host that supplies the cells, the more likely it is that the cells have undergone changes that alter the oxidative response. Furthermore, the culturing of some cells is often difficult, costly and time-consuming. Finally, it is quite expensive to culture cells from an individual to get a reading of how the individual host cells (as opposed to the cells generated in culture as representative of the species and cell type) will respond to a given bioactive agent.
Previous studies in tissue culture have shown that the initial response to oxidative stress is an increase in cellular phosphatidylcholine (PC) biosynthesis, which represents the cell's attempt to repair damage. In the long term, however, there is a decrease in PC biosynthesis because the cell's repair function is damaged, eventually causing cell death.
Ferrali, et al (
Biochem Pharm.,
Vol 38, No. 11, pp 1819-1825 (1989)) showed that they could not demonstrate the adverse effects of allyl alcohol and acrylic acid in erythrocytes. They did manage to show deleterious effects on the cells arising from exposure to acreolein. It was suggested that the damage to the cells resulted from the effects of iron delocalization but did not provide definitive results in tests for oxidative stress from allyl alcohol. However, using the methods taught therein, they were unable to show effect on erythrocytes and concluded that the enzyme required for oxidative damage from allyl alcohol was not seen because the cells lacked alcohol dehydrogenase. Hence, their method did not give definitive results in tests for oxidative stress which was known to occur. This problem has been solved using the method of the invention when studying suspensions of erythrocytes.
SUMMARY OF THE INVENTION
This invention provides means of measuring oxidative response of cells without recourse to preparation of cell culture. The process consists of:
1) preparing suspensions of cells from a living host in isotonic solutions,
2) preparing samples of test materials in isotonic solution containing tagged choline,
3) adding the cells suspension prepared in step 1 to the samples prepared in step 2,
4) incubating the product of step 3 with shaking for 2-90 minutes,
5) extracting and drying the lipid phase from the product of step 4, and
6) subjecting the product of step 5 to a scintillation counter to measure choline which has been incorporated into phosphatidylcholine (PC).
During the practice of step 5, the lipid phase may be subjected to repeated washings.
An increase in incorporation of choline into PC in the short term indicates oxidative stress or free radical induced damage.
DETAILED DESCRIPTION OF THE INVENTI
Chin Christopher L.
Grun James L.
Hendricks Glenna
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