Yuri N. Korystov

Role of arachidonic acid metabolism in thymocyte apoptosis after irradiation

The present study demonstrates that DNA fragmentation, nuclear pycnosis and trypan blue staining of irradiated thymocytes is prevented by inhibition of the lipoxygenase pathway of arachidonic acid metabolism and is not affected by cyclooxygenase inhibition. Exposed to irradiation [3H]arachidonic acid‐labeled thymocytes release radioactive products to the external medium. The process is blocked by the lipoxygenase inhibitor, nordihydroguaiaretic acid. Thus, it can be concluded that irradiation activates arachidonic acid metabolism and that lipoxygenase metabolites play an important role in thymocyte apoptosis.

Dependence of thymocyte apoptosis on protein kinase C and phospholipase A2

The effect of inhibitors and activators of protein kinase C and phospholipase A2 on radiation‐induced apoptosis of rat and mouse thymocytes has been studied. It is shown that the apoptosis is prevented by the protein kinase C inhibitor 1‐(5‐isoquinolinylsulfonyl)‐2‐methylpiperasine dihydrochloride and is potentiated by protein kinase C activator phorbol 12‐myristate 13‐acetate, calcium ionophore A23187 and concanavalin A. The protein kinase C activators initiate apoptosis in mouse but not in rat thymocytes. The inhibitor of phospholipase A2 prevents apoptosis induced by all the factors. The results obtained indicate that both protein kinase C and phospholipase A2 are involved in the thymocyte apoptosis.

Effect of the sulfhydryl reagent thimerosal on cytosolic free Ca2+ and membrane potential of thymocytes

The sulfhydryl reagent thimerosal at concentrations 5-100 microM has been found to induce a variety of changes in ion transport in rat thymocytes. In particular, [Ca2+]i increases about 10-fold from the basal level. The [Ca2+]i response to thimerosal displays a two-stage time course, with the main [Ca2+]i rise during the second stage. Evidence has been obtained for the depletion of intracellular Ca2+ pools in thimerosal-treated cells, however, Ca2+ mobilization from intracellular stores does not contribute significantly into [Ca2+]i rise. Thimerosal elicits permeability not only for Ca2+, but also for Mn2+ and Ni2+, which is Ca(2+)-dependent. We failed to get any evidence on thimerosal-induced inhibition of the plasma membrane Ca(2+)-ATPase. The induction of Ca2+ influx, rather than inhibition of Ca(2+)-ATPase, accounts for the disturbance of [Ca2+]i homeostasis in thimerosal-treated cells. Thimerosal also elicits changes in monovalent ion fluxes resulting in marked depolarization. The latter seems unrelated to the changes in [Ca2+]i and is suggested to be mediated both by increased permeability for Na+ and a decreased one for K+. Thimerosal significantly stimulates AA release from thymocytes. Evidence has been presented that AA metabolite(s), probably, LO product(s), may mediate the changes in the transport of mono- and divalent cations elicited by the sulfhydryl reagent. Prolonged treatment of thymocytes with thimerosal resulted in cell death.

Determination of reactive oxygen and nitrogen species in rat aorta using the dichlorofluorescein assay

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 (DCFH2) 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 DCFH2-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.

Distribution of the activity of the angiotensin-converting enzyme in the rat aorta and changes in the activity with aging and by the action of l-NAME

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, Nω-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.

On the role of intracellular concentration of Ca2+ and H+ in thymocyte death after irradiation

The role of intracellular Ca2+ and H+ concentrations in radiation‐induced interphase death of rat thymocytes has been studied. In response to concanavalin A treatment in the Ca2+‐containing medium, or to the CaCl2 treatment in the Ca2+‐free medium, the [Ca2+]i rise in irradiated cells was as in the non‐treated cells. No changes in the level of [Ca2+]i and pHi were found within l h after irradiation of thymocytes with a dose of 6 Gy. 15 μM 5‐(N‐ethyl‐N‐isopropyl)‐amiloride. an inhibitor of Na+/H+ exchange, did not affect the DNA fragmentation. The fragmentation was prevented by 2–4 μM (1 ‐[bis(4‐chlorophenyl)methyl]‐3‐[2‐(2,4‐dichlorophenyl)]‐2‐[(2,4‐dichlorophenyl)‐methoxy]‐ethyl)‐1 ‐H‐imidazoliumchloride, an inhibitor of calmodulin. The above data indicate that triggering of interphase death in irradiated thymocytes is not mediated by changes in either [Ca2+]i or pHi. Such changes seem to be involved in intermediate steps of the interphase death process.

Flavonoids decrease the radiation-induced increase in the activity of the angiotensin-converting enzyme in rat aorta

ABSTRACT The basic factor of cardiovascular diseases is atherosclerosis, which is due largely to an increase in the activity of the angiotensin‐converting enzyme (ACE) in vessels. Flavonoids diminish the risk of cardiovascular diseases and the flavonoid taxifolin normalizes the activity of ACE. We examined the efficiency of seven flavonoids in preventing an increase in ACE activity in aorta of rats exposed to ionizing radiation. It was shown that the activity of flavones and flavonols decreases with an increase in the number of OH groups in the A and B rings, respectively. The reduction in the activity of flavonoids within the classes correlates with a decrease in their lipophilicity. Flavanonols (taxifolin) are more active than flavonols, and flavonols are more active than flavones.

About the Role of Extracellular Radiation-Induced Oxidants in Cell Oxidative Stress at Irradiation Determined with the Dichlorofluorescein Assay

Two recent papers in this Journal address the issue of applying the dichlorofluorescein assay to measure so-called reactive oxygen species (ROS) after irradiating cells, with diametrically opposed conclusions (1, 2). A number of related earlier papers [e.g. ref. (3)] are based on this assay, which relies on the oxidation by ROS of dichlorodihydrofluorescein (DCFH2) to fluorescent dichlorofluorescein (DCF). While not seeking to adjudicate in the argument (1, 2) of whether the assay as applied in individual protocols is measuring species originating in extracellular medium or not—more experimental studies are desirable—it does seem remarkable that in most of the over 2000 studies in biology using this assay, little attention has been paid to the chemical parameters that define what the assay reports. Most notable is the general lack of discussion as to which ROS are being measured, which must reflect the reactivities of individual ROS toward the probe, and the chemical mechanisms involved in transformation of the DCFH2 probe to the measured DCF. An overview of the use of this and related probes (4) concluded with an aide memoire of questions we should attempt to answer before using such probes, or at least before drawing definitive conclusions from the results. For brevity, only some of the headings from that aide are listed below, together with brief notes specific to the DCF assay that it is hoped will help in the resolution of controversies arising from the application of the technique, and be useful in designing future studies. Probe reactivity. ROS is a crude and increasingly inadequate descriptor of over 20 species, both radical and non-radical entities, and many not oxygen-centered; some examples and pathways in biology are included in the review in ref. (4), to which the reader is also referred for supporting references in the discussion below. Superoxide ( ) and its product H2O2 • O2 are major examples; other oxidants include products of the reaction of with nitric oxide, including and . General radical damage • • • O NO CO 2 2 3 will result in thiyl radical production, e.g. of that from glutathione, GS. Of these species, and H2O2 do not react directly with DCFH2 under • O2 biologically relevant conditions at a significant rate, but other radical oxidants (e.g. , and GS) all oxidize DCFH2 rapidly (rate con• • NO CO 2 3 stants k 107 108 M 1 s 1 at pH 7–8) to form a radical (DCFH) rather than to form DCF directly, by the rule of spin conservation. The final product, DCF, is almost as reactive as DCFH2 toward some radical oxidants. It is possible that the reactivity of radical-based oxidants toward DCFH2 is less influenced by a reduction in temperature, as used in some studies (2), than peroxidase-facilitated oxidation as described below. Catalyst. DCFH2 is oxidized efficiently by H2O2 providing a catalyst is available. Hemes are typical and cytochrome c is of major importance since it is released from mitochondria to cytosol during apoptosis, and so in some circumstances an increase in DCF signal might reflect this phenomenon rather than changes in ROS production. Oxidation by H2O2 and cytochrome c involves typical peroxidase behavior, in which oxidation proceeds via DCFH rather than to DCF directly. DCF, as a phenol, is further degraded by peroxidases. While pH indicators might be omitted in media for fluorescence measurements, typical indicators such as phenol red are good peroxidase substrates and could conceivably act as ‘‘enhancers’’ of the peroxidase cycle on oxidation to phenoxyl radicals and generation of superoxide by GS [cf. refs. (5, 6)]. Hypochlorous acid (e.g. from irradiated PBS) is an inefficient oxidant of DCFH2 (7), but it markedly enhances the peroxidase action of cytochrome c (8). Reactions of probe intermediates. The initial oxidation product DCFH reacts rapidly with oxygen (k 5 108 M 1 s 1 at pH 7.4) to generate the superoxide radical, i.e. more ROS, along with DCF, the species measured in the assay. Thus the assay involves chain amplification of the signal to an extent that could depend on several parameters (e.g. pH, O2, NO, cytochrome c and superoxide dismutase levels, and reaction of both and DCFH with antioxidants). • O2 Probe distribution. DCFH2 is a weak acid with pKas for dissociation of the two phenolic moieties of 7.9 and 9.2 and another pKa of 4; DCF has pKas of 0.9, 3.5 and 5.2. These parameters control leakage of DCFH2 and DCF from cells to media, and pH-induced concentration gradients, including variations in probe distribution across intracellular organelles of different pH, e.g. lysosomes compared to mitochondria. They also define pH-dependent absorbance of the excitation light in fluorescence measurements (unless the excitation wavelength is at an isosbestic point), pH-dependent fluorescence quantum yield, and pH-dependent reactivity (phenolates are often oxidized much more rapidly than phenols). Effects of antioxidants. The competition for reaction of ROS with DCFH2 compared to endogenous antioxidants would be easily assessed by comparing the products of rate constant concentration, but hardly any studies measure intracellular loading of DCFH2. In the author’s laboratory we found that exposing V79 fibroblasts to 10 M DCFH2 diacetate for 15 min at 37 C resulted in an average intracellular loading of 300 M DCFH2, from which it was estimated that only 4% of • NO2 (for example) would react with the probe. Hence the assay is probably usually used under ‘‘non-saturating’’ conditions, and small variations in probe loading or antioxidant levels may influence the signal independently of ROS flux. Photochemical reactions. Like many dyes, light activates DCF to a triplet state reactive toward thiols and NADH, both reactions leading to superoxide generation, so high light intensities in fluorescence instrumentation may have unwanted consequences. In this context even room lighting can similarly generate superoxide from riboflavin in some cell culture media, leading to e.g. nitric oxide depletion by peroxynitrite-mediated chain oxidation (9). For brevity, only the key headings in the earlier review (4) are addressed above. Other factors that will influence probe response include radiation-induced nitric oxide synthase activity (10), which might enhance DCF formation because NO ‘‘diverts’’ superoxide from H2O2 formation to form, via peroxynitrite, the highly reactive and . • • NO CO 2 3 In conclusion, measurements using the DCF assay for ROS can reflect a number of parameters. It is hoped this brief outline of these factors will be useful to researchers making use of the assay. Overall, the method has very much the status of caveat emptor, and studies of oxidative/nitrosative stress are best supplemented by additional methodology such as measurements of 8-oxoguanosine or nitrotyrosine. In the wider context, the widespread use of chemical probes without considering their chemical reactivities or the mechanisms involved might be viewed as a paradigm for the place of radiation chemistry in 21st century radiobiology; it is certainly a salutary illustration of the need for multidisciplinary input in radiobiological research.