Quercetin and histamine effects on free radical reactions in rat erythrocytes

The effects of quercetin and histamine separately or in combination on the free radical state of rat erythrocytes were estimated in vitro. Quercetin (0.1; 0.5; 3.0; 5.0 mM) or histamine (0.01; 10.0 μM) were added to whole blood separately or in combination. The content of hydroperoxides, TBA-active products and carbonyl groups of proteins in erythrocytes after hemolysis was determined. The greatest influence of quercetin and histamine on erythrocytes state indicators was revealed under their combined action, when the level of TBA-active products and the content of carbonyl groups of proteins were found to be increased substantially.

A ctivation of lipid peroxidation is one of the triggering mechanisms of stress damage with the disorder of cellular metabolism, which is primarily associated with the damaged cellular and subcellular membranes. There is an antioxi dant defense system in the body to neutrali ze excess lipid peroxidation and maintain a steady in tracellular concentration of free radicals and lipoper oxidum [1]. The unregulated oxidative modification of lipids, proteins, and nucleic acids induced by mul tiple oxidants has been implicated in the pathogene sis of many diseases [2,3].
Quercetin activates Na + K + 2Cl − cotransporter 1, leading to elevation of the cytosolic Cl − concentra tion, which downregulates gene expression of epi thelial Na + channel. As a result of the aforementioned actions, polyphenols show various beneficial effects on body functions including regulation of blood pres sure via diminution of vascular contraction and renal Na + reabsorption by influencing epithelial Na + chan nels gene expression and Na + ,K + ATPase activity [4].
Quercetin is an antioxidant that effectively regulates energetic metabolism in the myocardium, reduces its requirement in oxygen, stabilizes the cytoplasmic membrane, and causes antiarrhythmic and anabolic effects. The drug is able to reduce the concentration of free radicals and toxic peroxidation products [1]. Quercetin has been reported to reduce the amount of histamine in the blood. However, the mechanism of reducing the amount of histamine in the blood is unknown. Quercetin absorbs free radicals and has protective effects against oxidative stress-mediated neuronal damage [5]. Published adequate scientific data for safety assessment in regard to the longterm use (>12 weeks) of high supplemental quercetin doses (≥1000 mg) are currently not available. Based on animal studies involving oral quercetin applica tion some possible critical safety aspects could be identified such as the potential of quercetin to en hance nephrotoxic effects in the pre-damaged kid ney or to promote tumor development, especially in estrogendependent cancer. Furthermore, animal and human studies with single time or shortterm supplemental quercetin application revealed interac tions between quercetin and certain drugs leading to altered drug bioavailability [6]. Quercetin reduced significantly erythrocytes lipid peroxidation levels and the susceptibility to hemolysis induced by the free radical generator AAPH. Chronic quercetin supplementation has antioxidant potential before and after a strenuous eccentric exercise thus making the erythrocytes capable of better cope with an oxidative insult [7]. The effect of quercetin on free radical pro cesses against the background of allergic reactions, where histamine has the leading effect, remains un known.
The seminal work on histamine was published in 1910, but histamine was not identified as a me diator of anaphylactic reactions until 1932. Research later showed that histamine is a major mediator responsible for the symptoms of allergic rhinitis, with its activities mediated through 4 G protein coupled receptors. Most of histamine's effects are exerted through the H₁ receptor, but some effects are through the H₂ and H₃ receptors, and possibly also through the H₄ receptor [8,9]. There are reports that erythrocytes contain histamine receptors, but they do not know exactly what function they play [10]. It is known that histamine regulates the release of reactive oxygen species by blood neutrophils. Thus, histamine is associated with free radical processes in the body. There are reports that histamine chal lenge reduces magnesium levels in erythrocytes while plasma levels remain unchanged. This hista mineinduced decrease in magnesium levels occurs regardless of the diagnosis of asthma, and it is not correlated with the degree of bronchial hyperreac tivity [11].
Among blood cells, erythrocytes remain the least studied in terms of allergic reactions, where the leading role is played by histamine. The effect of quercetin on free radical processes in erythrocytes also remains unknown. It is known that erythro cytes are important components of the blood that perform important functions to maintain homeosta sis (transport gases, perform a protective function, etc.). One mechanism through which oxygen tension and therefore RBCs affect vascular function is the contractile force of smooth muscle cells. Chang and Detar reported that a reduction in oxygen pressure led to reduced contractile tension of helical strips cut from the aorta, femoral arteries, and small arteries from skeletal muscle. Additionally, Taggart et al saw a decrease in smooth muscle force in hypoxia indi cating smooth muscle response to oxygen may be independent of factors from the endothelium. Poten tial mechanics of smooth muscle response to oxygen tension are increased ATPsensitive K + efflux or de creased voltagesensitive Ca 2+ influx. Alternatively, low oxygen tension affects the endothelial produc tion of nitric oxide (NO) by eNOS, which requires oxygen as a substrate along with Larginine. Many researchers have shown diminished relaxation by en dothelial cells under low oxygen tension. Specifical ly, the apparent k m of eNOS for O 2 is 4 μM and NOS activity begins to slow down once oxygen tensions start falling below 1% (7.6 torr or 10 μM). In sum mary, through the management of oxygen delivery RBCs effect eNOS function and smooth muscle con tractility [12]. As quercetin can reduce the amount of histamine in the blood, it is important to study the combined effect of these substances on erythro cytes, in particular on their free radical state, which is a universal process of membrane destruction. The intensity of free radical processes reflects the degra dation of erythrocytes. Therefore, such studies will expand knowledge about the safe usage of quercetin in patients with allergic reactions.
Objective. To study the effect of quercetin and histamine on the intensity of lipid peroxidation and oxidative protein modification in rat erythrocytes.

materials and methods
The nonlinear white ratsmales body weighing 180-220 g were used for the experimental studies .
(Rattus norvegicus f. Domesticus). Chloroform was used for euthanasia. Animals were treated in ac cordance with the requirements of the European Convention for the Conservation of Vertebrate Ani mals Used for Experimental and Scientific Purposes (Strasbourg, France 1986) and in accordance with the General Principles of Animal Work approved by the First National Congress on Bioethics (Kyiv, Ukraine, 2001). After decapitation of the animals, blood was collected in a glossy cup with heparin. An adequate amount of quercetin solutions were added to whole blood to a final concentration of 0.1; 0.3; 0.5; 1; 3; 5 mM. Concentrations of 1 and 3 mM are therapeutic and used in pharmacy (single dose for oral administration). The quercetin (brand PFA) was dissolved in warm saline at 37°C (oral use of querce tin dissolved in warm water used in medicine). In other experiments, a histamine solution (0.01% his tamine dihydrochloride solution used as a stock so lution; manufacturer -Limited Liability Company "Immunolog", Ukraine, Vinnytsya) added to the blood to final concentration 0.01; 0.1; 1; 10 μM. 0.9% NaCl used to prepare the solutions. In the series of experiments, histamine (minimum and maximum test concentrations: 0.01 and 10 μM) and quercetin (at a concentration of 0.1; 0.5; 3; 5 mM) added to the blood in various possible combinations. Quercetin concentrations for this chosen as follows: minimum and maximum concentrations; where 0.5; 3 mm are those concentrations that did not change the content of lipid hydroperoxides in plasma of blood in our previous in vitro experiments. Blood erythrocytes of 5 rats (n = 5) were used in each experimental group. The blood of intact animals and the blood to which saline was added were used as controls. They were incubated 5 min, then centrifuged at 3000 rotation for 10 min. Selected for analysis erythrocytes were laundered. The water was used for their hemolysis (1 part of erythrocytes and 5 parts of water). The content of hydroperoxides, TBA-active products, and carbonyl groups of protein were determined (al dehyde and ketone derivatives of neutral and basic character measured on the spectrophotometer UL AB102UV (China) at 370 and 430 nm, respectively) in the selected samples [1315]. The intensity of the oxidative modification of proteins was determined by the method of O. Yu. Dubinina in the modifica tion of E. F. Meschyshen [15]. Protein content was determined by Lowry [16].
In vitro experiments can not reflect the whole chain of effects of quercetin and histamine on all biochemical interactions of the body, in particular, on erythrocytes, but they give a comprehensive re sponse to the effects of these substances directly on the state of the studied indicators (namely the state of free radical processes).
Analyses of all data were conducted in Excel10 for Windows. The values of parameters are given as mean (m)± standard error (m) and the degree of probability of difference (P) between indicators where appropriate. The significance of the changes of variables with abnormal distribution by Friedman test was performed. A Student's ttest was performed to determine the statistical significance. Significance was considered to be a difference in the confidence index P ≥ 0.95; P ≥ 0.99; P ≥ 0.999.

results and discussion
As we found that the saline addition to the blood caused a significant increase in the content of lipid hydroperoxides in the erythrocytes of rats (7.8 times, P ≥ 0,999). Therefore, all the study groups we had compared to the group were saline was added to the blood. Quercetin caused a decrease in the content of primary lipid peroxidation products in rat hemolysates. Moreover, the most pronounced decrease in the content of lipoperoxidation products by 84%, 88%, 88% were found at 0.3; 0.5; 1 mM concentrations, respectively (Fig. 1). This indicates a slow down intensity of free radical reactions in erythrocytes. Added histamine at 0.01 and 10 μM concentrations to whole blood reduced the content of the lipid hydroperoxides in erythrocytes by 36% and 38%, respectively (Fig. 1). Simultaneous incubation blood in a medium with quercetin at 0.1 mM con centration, and histamine with both 10 μM and of 0.01 μM concentration, caused a significant increase in the content of the lipid hydroperoxides in eryth rocytes by 190 and 177%, respectively (Fig. 1). A slightly increased content of these products was de tected by the action of quercetin and histamine in a combination at 0.5 mM and 0.01 μM concentrations, respectively. The content of primary lipoperoxida tion products decreased by 42% after simultaneous addition quercetin at the highest tested concentra tion and histamine at the lowest concentration to the blood. Quercetin at from 0.1 to 5 mM concentrations on the background action of histamine at 0.01 μM concentration caused a dosedependent accumula tion of lipid hydroperoxides in erythrocytes (Fig. 1).
Adding saline with diluted quercetin to the blood (in vitro) caused to decrease in the content of TBA-active products by 2.4 times (P ≥ 0.99). Therefore, the experimental samples compared with groups where red blood cells were in the medium with saline. The secondary products of lipid peroxi de oxidation by the action of quercetin increased at 0.3 mM concentration 107%, 0.5 mM 152%, 3 mM -70%. The content of TBK-active products was reduced by 39% by the influence of quercetin at the maximum tested concentration (5 mM) (Fig. 2). Histamine at 0.1 and 1 μM concentration led to a decrease in the content of TBK-active products in rat erythrocytes by 39 and 41%, respectively. Biogenic amine at the lowest and highest concentrations didn't cause significant changes in the content of secondary lipoperoxidation products. The content of TBK-ac tive products increased by the action combination of histamine at 10 μM concentration and quercetin. The maximum increase of content (by 211%) was detec ted by action the combination of histamine (10 μM) and quercetin at 5 mM concentration (Fig. 2). The combination histamine at 0.01 μM a concentration and quercetin at 0.1 and 0.5 mM concentrations didn't change the intensity of the processes of lipid oxide oxidation. The content of TBK-active products in rat erythrocytes (20 and 26%) was slightly re duced by the action simultaneous adding of hista mine (0.01 μM) and quercetin higher concentrations . Therefore, analyzing our results as a whole, we can conclude that quercetin causes a decrease in the content of primary lipoperoxida tion products by increasing the seconda ry products. However, quercetin at a concentration of 5 mM can suppress the intensity of lipid peroxidation processes as evidenced by the content of lipid hydroper oxides and TBA-active products. Intensification of lipid peroxidation processes indicates damage of eryth rocyte membranes by quercetin (at 0.3; 0.5; 3 mM concentration). As known from the literature, oral ly administrated of quercetin can be found at low concentrations in plasma metabolites, but it is not enough to show significant antioxidant activity. In plasma, quercetin and its metabolites are related mainly to albumin. Consumption of quercetinrich foods does not increase the antioxidant activity of the plasma albumin fraction. At the same time, as known, a diet rich with quercetin is a protective fac tor against athero sclerosis. These results suggest that the quercetin metabolites do not show their an tioxidant activity in plasma. But they are selectively accumulated in a specific target in the area of the vascular wall. However, there are other results from in vitro studies showing that quercetin is an effec tive antioxidant against lipid oxidation [17]. Probab ly quercetin at the tested concentrations, except the maximum (5 mM), binds to blood albumin and does not cause an antiradical effect on the erythrocytes of rats and leads to a violation of prooxidantantioxi dant homeostasis.
Histamine slows down the intensity of lipid peroxidation processes in rat erythrocytes. Thus, the content of lipid hydroperoxides decreased by the action of the biogenic amine at the maximum and mini mum concentrations. And amine led to a decrease in the content of the TBA-active products at the mean concentration. The intensity of lipoper oxidation processes lower than the norm can be re garded as a negative phenomenon, and in these con ditions, the membranes of erythrocytes may become stiffer, which will affect the passage of them through thin capillaries [18].
In general, the combination of histamine and quercetin caused an increase in the intensity of lipid peroxidation processes, except the next combina tions of concentrations: 0.01 μM histamine with 3 or 5 mM quercetin, that caused a decrease in the con tent of lipoperoxidation products. Such a decrease in the intensity of lipid peroxidation may be due to the interception of free radicals by high concentrations of quercetin. Increasing the content of both lipid hy droperoxides and TBA-active products occurs with the simultaneous action of histamine at 10 μM con centration and quercetin at 0.1 mM concentration. Thus, histamine, in high concentration, caused the formation of free radicals, but the amount of querce tin was insufficient to inactivate those radicals.
We assume that quercetin and histamine change the intensity of lipid peroxidation processes in rat erythrocytes.
We found that the content of carbonyl groups of proteins of a neutral character significantly de creased by the saline solution was added to the blood (P ≥ 0.999). Therefore, all studying groups we com pared to the group where we added saline to the blood. The content of carbonyl groups of proteins of neutral character increased by 354% by the action of quercetin (0.5 mM). However, quercetin of lower concentrations (0.1 and 0.3 mM) slightly reduces the intensity of oxidative modification of proteins (by 10 and 26%, respectively). In erythrocyte hemolysates, the content of carbonyl groups of proteins (neutral character) was reduced by more than 53% by the ac tion of quercetin (the maximum of tested concentra tion). Noticed that the content of carbonyl groups of proteins of a neutral nature didn't change by the ac tion of quercetin at therapeutic 1 and 3 mM concen trations (Fig. 3). That confirmed the positive effect of such doses of the drug on the protein structures of erythrocytes. Quercetin is known to affect enzymat ic systems and metabolic processes [19]. Therefore, these changes explained precisely by the influence on the oxidative modification of the protein struc ture, which we have proved. Clinicopharmacologi cal properties of quercetin are associated with versa tile anti-inflammatory effects: membrane stabilizing, antileukotriene, antioxidant [20]. Thus, the decrease in the intensity of oxidative modification of proteins is associated with the antioxidant effect of quercetin.
The content of carbonyl groups of proteins of a neutral character only increased at lower concen trations (0.1 μM by 273%; 0.01 μM by 209%) after added histamine to whole blood. Histamine at high concentrations (1 and 10 μM) didn't alter the inten sity of oxidative protein modification in rat eryth rocytes. (Fig. 3). It found that incubation of blood in the medium with quercetin and histamine in all studied combinations of concentrations caused a sig nificant increase in the content of carbonyl groups of neutral character in the erythrocytes of rats (Fig. 3). We found that histamine at a lower concentration against the background of quercetin, produces a more pronounced intensification of oxidative modi fication of proteins, compared to higher. Histamine may be able to react with quercetin, resulting in the formation of harmful metabolites that damage eryth rocyte proteins.
We also determined the content of carbonyl groups of proteins of basic character. The content of carbonyl groups of proteins of the basic character, as well as the content of carbonyl groups of proteins of neutral character, decreased in 1.5 times by the action of saline solution. (P ≥ 0.999). Under these conditions, control was considered the experimental group with the addition of saline. Quercetin at 0.1; 0.3; 5 mM concentrations caused a decrease of the content of carbonyl groups of basic proteins in eryth rocytes by 29%, 41%, 50%, respectively, was found. Whereas bioflavonoid (0.5 mM) caused increase this content (by 119%; Fig. 4). It noted that the tendency of changes in the content of carbonyl groups of pro teins of basic character is the same as that carbonyl groups of proteins of neutral character (Fig. 3).
Addition to the blood of rats of histamine at 0.1 and 0.01 μM concentrations caused the intensifica tion of oxidative modification of proteins (the for mation of carbonyl groups of both basic and neutral character) in erythrocytes (Figs. 3, 4).