The goal of this study was to determine the amount of reactive oxygen species (ROS) that arises inside cells irradiated in medium containing blood serum using the 2'7'-dichlorofluorescein (DCF) assay. DCF fluorescence in cells and medium was recorded on an MF44 Perkin Elmer fluorimeter, and fluorescence in cells only was recorded on a Partec flow-through cytometer. Human larynx tumor HEp-2 cells and lympholeukosis P388 cells were irradiated with X rays at a dose rate of 1.12 Gy/min. The factors (temperature, pH, serum concentration) affecting the oxidation of 2'7'-dichlorofluorescin (DCFH) to DCF were studied, and errors in the dichlorofluorescein assay of ROS were minimized. The amount of ROS registered by the DCF assay in cells was found to depend on the concentration of serum in the medium during irradiation. In the presence of 10% serum, radiation had no effect on the amount of detectable ROS. The effect of radiation on the formation of intracellular ROS was almost completely abolished if the irradiated medium was removed immediately after radiation exposure. The increase in the formation of ROS in cells irradiated in medium with a low serum content is due mainly to the radiolytic products of water that arise in medium and oxidize DCFH located in cells.
A method for the determination of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in macroscopic sections of vessels has been developed on the basis of the dichlorofluorescein (DCF) assay. DCF was measured by fluorescence in extracts of vessels. The main artifact of the method is the oxidation of dichlorodihydrofluorescein (DCFH(2)) which is released from vessels together with DCF during the extraction procedure. This problem was resolved by decreasing pH during the extraction. The optimal conditions and the time for aorta incubation with DCFH(2)-DA and for the extraction of DCF from aorta have been determined. The ROS/RNS production in different aorta segments and the dependence of ROS/RNS production on rat age have been studied. It was shown that thoracic aorta sections produced the same amounts of ROS/RNS and the intermediate between the thoracic and the abdominal aorta part produced ROS and RNS by 14% more than the thoracic aorta. It was found that ROS/RNS production in aorta increases with rat age: the doubling time of ROS/RNS production rate is 113 days from birth.
The activity of the angiotensin-converting enzyme (ACE) of the inner surface (the endothelium surface) of rat aorta sections has been studied depending on their distance from the aortic arch, age of rats, and the duration of treatment of rats with the NO synthase inhibitor, N (ω)-nitro-L-arginine (L-NAME). The activity of ACE of aorta sections was determined by measuring the hydrolysis of hippuryl-L-histidyl-L-leucine and was expressed as picomoles of Hip-His-Leu hydrolyzed per minute per square millimeter of the endothelium surface. It was found that the ACE activity considerably varies along the aorta of young rats. This variability decreases with increasing age of rats and by the action of L-NAME. The average ACE activity in the aorta increases with the age of rats and with increasing time of L-NAME treatment. Enalapril normalizes the distribution of the ACE activity along the aorta and decreases the average ACE activity. The changes in the distribution of the ACE activity along the aorta and in the average ACE activity in the aorta with increasing age of the rat and by the action of L-NAME may play a role in the development of atherosclerosis of vessels on aging and the inhibition of formation of nitric oxide.
In the present study, the activity of ACE (angiotensin-converting enzyme) in the aorta of senescent rats and rats treated with the NOS (NO synthase) inhibitor L-NAME (NG-nitro-L-arginine methyl ester) or dexamethasone and the effect of low doses of ethanol (0.2-1.2 g/kg of body weight, daily for 8-12 days) on this activity were studied. We found that ACE activity increased with age and in response to L-NAME and dexamethasone treatment. Ethanol at a dose of 0.4 g/kg of body weight per day decreased ACE activity in the aorta of aged rats and of rats treated with L-NAME or dexamethasone to the level of activity in young control rats. The optimal ethanol dose (the dose inducing a maximum decrease in ACE activity) increased with increasing doses of dexamethasone: 0.4 g/kg of body weight per day at 30 μg of dexamethasone/kg of body weight and 0.8 g/kg of body weight per day at 100 μg of dexamethasone/kg of body weight. It was also found that optimal doses of ethanol increased the number of cells in the thymus of rats treated with dexamethasone. The optimal dose of ethanol of 0.4 g/kg of body weight per day, which induced a maximum decrease in ACE activity in rat aorta, corresponded to a dose of 30 g of ethanol/day, which, according to epidemiological data, produces a maximum decrease in the incidence of cardiovascular disease in humans. In conclusion, the decrease in ACE activity in vessels may be one of the main mechanisms of the beneficial effects of low doses of ethanol on human health.
The irradiation-induced changes in the MDR of tumor cells should be taken into account when combining radiotherapy with chemotherapy. It was assumed that the dependence of multidrug resistance of HEp-2 cells on radiation dose and cell density is determined by changes in the amount of reactive oxygen species in the cells.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.