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Effect of Iron Oxide Nanoparticles on Vascular Function and Nitric Oxide Production in Acute Stress-Exposed Rats

1 Institute of Normal and Pathological Physiology, Centre of Experimental Medicine, Slovak Academy of Sciences, Bratislava, Slovak Republic; 2 Institute of Pharmacology and Clinical Pharmacology, Faculty of Medicine, Comenius University, Bratislava, Slovak Republic 3 Department of Animal Physiology and Ethology, Faculty of Natural Sciences, Comenius University, Bratislava, Slovak Republic 4 Institute of Measurement Science, Slovak Academy of Sciences, Bratislava, Slovak Republic 5 Institute of Robotics and Cybernetics, Faculty of Electrical Engineering and Information Technology, Slovak University of Technology, Bratislava, Slovak Republic; 6 Department of Nuclear Physics and Biophysics, Faculty of Mathematics, Physics and Informatics, Comenius University, Bratislava, Slovak Republic;


Introduction
The last decade of investigations of metal nanoparticles (NPs), including superparamagnetic iron oxide nanoparticles (IONs), have revealed their potential for various biomedical applications, from magnetic resonance imaging contrast agents including detection of bacteria or viruses to drug delivery systems and the usage of magnetic hyperthermia in cancer treatment (Andrade et al. 2020, Poller et al. 2018. However, the properties and biological effects of IONs vary depending on their shape, size, surface modification, etc. In general, smaller nanoparticles (1-20 nm) seem to enter and exit the cell more efficiently and infiltrate organs better than larger ones, spherical NPs are less toxic than rod-shaped ones and positively charged NPs exhibit much higher rates of endocytosis and remain in the cell longer than negative or neutrally charged ones (Oh and Park 2014).
Bare IONs are toxic mainly due to the induction of haemolysis, oxidative stress and DNA breakdown (Andrade et al. 2020, Dalzon et al. 2020. Thus, various coatings have been developed to reduce ION cytotoxicity. Hydrophilic polymer polyethylene glycol (PEG) is a neutral biocompatible polymer, which improves the dispersion of IONs in water, improves the biodistribution of NPs in an organism and increases blood circulation time (Yoffe et al. 2013).
The PEGylation of nanoparticles has also been shown to reduce hemotoxic effects, haemolysis, liver uptake and immunogenicity (Lee et al. 2005, Suk et al. 2016. Intravenous application is the common route for IONs in therapeutic applications, such as drug delivery, nanocarriers and diagnostic tools (Dukhinova et al. 2019). The main ways to eliminate IONs are through renal and hepatic excretions (Almeida et al. 2011, Longmire et al. 2008. However, the iron released from decomposed IONs can also enter cellular iron metabolism (Poller et al. 2018) and thus can affect iron homeostasis. Elevated iron levels results in the elevation of hepatic hepcidin release, which is the main regulator of iron homeostasis as well as in the elevation of ferritin synthesis, which is a main iron-storage protein, in order to recover optimal iron concentrations in the tissues (De Domenico et al. 2010). In addition, ferritin levels can be altered in oxidative stress conditions (Torti and Torti 2002) which is often induced by NPs.
Endothelium, the inner monolayer of blood vessels, produces various substances collectively termed endothelium-derived factors, which modulate vascular functions and thus blood pressure (Bernatova 2014). Endothelial cells are directly exposed to IONs when Stress is considered a risk factor associated with the development of various chronic diseases, including cardiovascular diseases. Despite the variability of stressors and their effects on organisms, sudden stressors can activate the autonomic nervous system, which is associated with increased blood pressure (BP) and heart rate (HR). On the other hand, NO is a major depressor factor that attenuates BP increases via attenuation of sympathetic nervous system (SNS) activation along with the renin-angiotensin-aldosterone system (Kumagai et al. 2012).
Vascular and/or endothelial function can also be altered by stress (Puzserova and Bernatova 2016). Despite considerable differences between in vitro and in vivo studies that have investigated the influence of stressors on the production of endothelium-derived factors (Bernatova 2014), endothelium-derived NO has been suggested as a stress-relieving molecule (Stefano et al. 2006). Thus, stress may be a factor that considerably modifies the effect of treatment via modulation of vascular functions and feedback effect on the autonomic nervous system, as demonstrated by acute pharmacological adrenalectomy (Bencze et al. 2020). In addition, excessive NO production due to the activation of inducible NO synthase (NOS) contributes to vascular hypo-reactivity, reduces systemic resistance and decreases blood pressure (El Hadi et al. 2020).
In this study, we used acute air-jets to induce stress reactions associated with prominent BP increase during air jet to investigate the modulatory effect of repeated acute stress on ION administration. Air-jet stress results in psycho-emotional stress, which causes a characteristic defence reaction with increases in BP, HR and renal sympathetic nerve activity , Kanbar et al. 2007, Yamazato et al. 2006. It is well known that acute stress alters neurohumoral mechanisms, which leads to changes in vascular function and thus stress alters blood flow and distribution (Wang et al. 2005). All these factors should be taken into account during IONs application in humans.
The femoral artery is an important branch of the iliac artery that irrigates lower-limb skeletal muscles and peripheral tissues. It is a unique blood vessel with long conduction, highflow resistance and a striking relevance for medical interventions (Mewissen 2005). Femoral artery tone regulation depends on endothelium-derived relaxing and contracting factors (Bencze et al. 2015, Dobias et al. 2016, Líšková et al. 2007, thus representing an ideal artery for studying vascular responses in various experimental approaches. One such approach involve the inhibition of NOS leading to the impairment of NO release, which in turn may unmask the effect of endothelium-derived contracting factors (EDCFs) (Liskova et al. 2011). This is of particular importance because the application of N ω -nitro-L-arginine methyl ester (L-NAME allows the assessment of the L-NAME-sensitive component of concentration-response curves induced by acetylcholine (ACh). The L-NAME-sensitive component has NO-dependent and EDCFs-dependent part and thus after L-NAME application the isolated femoral arteries react with enhanced concentration-dependent contractions (Liskova et al. 2011) or impaired concentration-response curves of endothelium-dependent ACh-induced relaxations (Puzserova et al. 2013). Due to intravenous administration of IONs in clinical applications, it is of great importance to study the effect of IONs on endothelial cells as nanoparticles per se or iron released from them may penetrate the vascular wall and thus change vascular functions.
Furthermore, it is reasonable to investigate the joint influence of acute stress on vascular function and NOS activity as diseases, hospital environments and/or intravenous applications of IONs are always associated with certain levels of stress in patients.
Thus, in this study we investigated the effect of i.v. administration of IONs on vascular functions of the femoral arteries of normotensive rats, NOS activity in the tissues (kidneys, liver, brainstem and hypothalamus) and on BP at rest (100 min-post infusion, at the end of the experiment). We also investigated acute stress-induced BP and HR responses in ION-treated rats because of the possibility of stress conditions during the use of IONs in medical practice.
In addition, we determined the effect of IONs on gene expression of hepcidin (HAMP) and ferritin (FTH1) in the liver and kidneys (involved in excretion of IONs) as well as the effect of repeated acute stress on ION-originated iron content in these organs and blood.

Animals and treatment
All the procedures used in this study were approved by the State Veterinary and Food Administration of the Slovak Republic in accordance with the European Union Directive 2010/63/EU. Wistar-Kyoto (WKY) male rats 12-16 weeks old, n = 6-7 per group, were used in this study. Rats were housed under standard conditions at 22-24°C in a 12-h light/dark cycle and fed with pelleted diet Altromin formula 1324, variant P (Altromin Spezialfutter, Lage, Germany) and tap water ad libitum.
A day before the experiment, all rats had two catheters implanted under 2.5-3.5% isoflurane anaesthesia, as described previously (Behuliak et al. 2013). All rats were also pretreated with meloxicam (Meloxidolor, Le Vet Beheer B.V., Oudewated, Nederland) at 2 mg/kg i.m. before surgery to prevent post-surgical pain. Fine-bore polyethylene catheters (Smiths Medical International Ltd, Kent, UK) were inserted into the left carotid artery (internal diameter 0.58 mm) to determine BP and HR and the jugular vein (internal diameter 0.28 mm) for i.v.
administration of IONs (suspended in saline) or saline (in control and stressed rats), respectively. Catheters were exteriorised in the interscapular region, and rats were allowed to recover from anaesthesia for approximately 20-24 h.
Rats were divided into four groups: control (Cont), ultra-small superparamagnetic iron oxide (Fe3O4) nanoparticle (ION)-treated rats (IONs), repeated acute air-jet stress-exposed rats (Str) and rats co-exposed to IONs and repeated air-jet stress (IONs + Str). Control and stressed rats were given 10-min infusions of saline starting approximately 30 min from the beginning of BP recording (Figure 1). IONs and IONs + Str rats were given 10-min infusions of PEG-coated IONs. Commercially available PEG-coated IONs were purchased from Sigma-Aldrich (Bratislava, Slovakia, cat. No. 747408). IONs concentration was 1 mg Fe/ml and they were dispersed in water. The size of IONs confirmed by the transmission electron microscope was 28-32 nm, the zeta potential was -12 mV, polydispersity index was 0.1 and the hydrodynamic size was about 45 nm (declared by the manufacturer). IONs were autoclaved at 121C for 30 min and before i.v. application they mixed with sterile saline to reach a final dose of 1 mg of Fe/kg of body weight. Properties of IONs before their administration to rats and their presence in the tissues (proven by bio-magnetometric method) are shown in other study (Skratek et al.

2020, unpublished results).
The experiments were performed in a quiet room to avoid any non-specific stimuli affecting BP and HR, which were recorded with the sampling rate of 1 kHz. During the experiments, the conscious rats were placed in a plastic box with dark walls and a transparent lid 27 × 14 × 9 cm in size, which allowed the rats free movement. An arterial catheter was attached to the BP-recording device PowerLab system (ADInstruments, Bella Vista, Australia), and BP was allowed to stabilise for at least 15 min. BP and HR were recorded during the entire experiment, basal mean arterial pressure (MAP) and HR were calculated as the average values of a 200-sec segment between 16-20 min of the recording and end MAP and HR were calculated as the average values of a 200-sec segment between 138-142 min of the experiment.
The peak values of MAP and HR were also calculated during each air-jet (AJ) session as the maximal values of the given variable during air-jet compared with the average pre-AJ value determined during 200-sec segments preceding the corresponding AJ session ( Figure 1).
Repeated acute AJ stress was produced by a 5-sec pulse of air at the forehead of the rat in the Str and IONs + Str groups as described previously , Nakamoto et al. 2007). Each rat in these two groups was exposed to three sessions of 5-sec AJ: 1) 10 min before the infusion, 2) 10 min after the infusion and 3) 90 min after the infusion. At the end of the protocol, rats were exposed to brief CO2 anaesthesia and decapitated within 5 min of the final MAP recording. Trunk blood for determination of IONs was collected into Eppendorf test tubes and kept at -80 C. The NOS activity was determined in fresh tissue homogenates of the brain stem, hypothalamus, kidney and liver. The remaining parts of dissected kidneys and liver were frozen in liquid nitrogen and kept at -80 C for further analyses. Femoral arteries were stored in fridge at 4 °C and used the same day for vessel myography.

Plasma corticosterone
Plasma corticosterone was determined in 50 µl blood samples collected from the carotid artery (before the end of experiment) into ethylenediaminetetraacetic acid-coated test tubes and centrifuged for 10 min (850 g, 4°C). Plasma samples were stored at -80C until further analysis.
Plasma corticosterone levels were determined using the Corticosterone Rat/Mouse 125 I radioimmunoassay kit (DRG Instruments GmBH, Germany), according to the manufacturer's instructions. All samples were measured in a single assay with the intra-assay variation coefficient of 4%. The assay sensitivity was 6.3 ng/ml.
[3H]-L-citrulline was eluted by 1.5 ml of deionised water and determined by liquid scintillation counting (TriCarb, PerkinElmer). NO synthase activity was expressed as pkat per gram of protein. The protein concentration was determined using the Lowry method. NaHCO3, 1.18 KH2PO4, 0.03 Na2EDTA, 2.5 CaCl2.2H2O, 5.5 glucose, 37°C, pH 7.4) and bubbled with 95% O2 and 5% CO2. The inner arterial diameter of femoral arteries was set to 90% of the diameter predicted for the pressure of 100 mmHg in the wire myograph. After 30 min of stabilisation, the arteries achieved their basal tones. To test the viability of isolated arteries, the arteries were incubated in a depolarising solution (125 mmol/l K + , NaCl was exchanged for an equimolar concentration of KCl) for 2 min. After being washed out, the experimental protocol was carried out as described in another study (Puzserova et al. 2013).

Determination of vascular functions
Briefly, after the washing and stabilisation of the basal tone, 5-HT-induced contraction (10 -6 mol/l) was determined. It was followed by determination of the total endothelium-dependent ACh-induced relaxations by cumulatively adding ACh at the final concentrations 10 -9 to 10 -5 mol/l. After 20 min of washing out, the same approach was applied again in the presence of the non-specific NO synthase inhibitor L-NAME (3.10 -4 mol/l, 30 min pre-incubation) to determine 5-HT contraction in the presence of NO synthase inhibitor as well as the NO-independent component of endothelium-dependent relaxation. After repeating washing, the third concentration-response curve was performed by cumulatively adding exogenous NO donor (SNP) in concentrations of 10 -9 to 10 -5 mol/l to determine the endothelium-independent relaxations produced by exogenous NO on VSMCs.
Pharmacodynamic characteristics -i.e. half-maximal effective concentration (EC50, expressed as -log mol/l) and maximal relaxation (Emax, expressed as a percentage of preconstriction) -were calculated for each ACh-and SNP-induced concentration-response curve according to the Hill equation using the fitting model described in another studies (Liskova et al. 2014, Liskova et al. 2010. The L-NAME-sensitive component of ACh-induced relaxation was calculated as the difference between the AUC of total ACh-induced relaxation and AUC of the NO-independent ACh-induced relaxation, i.e. AUCL-NAME-sensitive = AUCTotalACh -AUCNOindependent. All chemicals used in the study were obtained from Sigma-Aldrich (Bratislava, Slovakia) unless stated otherwise.

Determination of the ION-originated iron content
After dissection, the tissues were cleaned out of the connecting tissue, washed out of blood in the saline solution and dried of saline solution using filtration paper. Trunk blood was collected into Eppendorf test tubes. Tissues as well as blood were frozen in the liquid nitrogen and kept at -80C until further analyses. Before determination of relative iron content, tissue samples were mounted on 18 cm long and 0.2 mm in diameter copper wire. Blood samples were prepared as described previously and 10 µl was dropped on paper (Kluknavsky et al. 2020).

Statistical analysis
Statistical analysis was performed by two-way ANOVA (with IONs and stress as the independent factors) or two-way ANOVA for repeated measures ANOVA and Student's t-test, where appropriate. ANOVA analyses were followed by the Bonferroni post-hoc test, except the analysis of the release of EDCFs in the IONs group after ACh-administration, which was analysed by one-way ANOVA and Dunnett's test. The values were found to significantly differ when p < 0.05. The data were presented as mean ± standard error of the mean (SEM). GraphPad Prism 5.0 (GraphPad Software, Inc., San Diego, CA, USA) was used for the statistical analyses.

Mean arterial pressure, heart rate and plasma corticosterone
The mean arterial pressure (MAP) and HR of all rats at the beginning of the experiment were 132 ± 2 mmHg and 330 ± 6 bpm (n = 27), and no significant differences among the groups in these parameters were observed. MAP was reduced significantly at the end of the experiment vs. basal values in rats co-exposed to IONs and air-jet stress (IONs + Str) ( Figure 2A). HR was reduced significantly in control (Cont), ION-treated rats (IONs) and acute air-jet stress-exposed rats (Str) groups vs. the respective basal values ( Figure 2B).
MAP in the Cont, Str and IONs groups at the end of the experiment was 102 ± 4%, 99 ± 1% and 99 ± 2% of basal MAP, respectively, which were not significantly altered compared to basal MAP within each group. The MAP of rats in the IONs + Str group decreased significantly at the end of the experiment to 88 ± 4% of basal MAP (p < 0.02). HR in the Cont, Str and IONs groups at the end of the experiment was 89 ± 3%, 90 ± 2% and 88 ± 2% (p < 0.05 vs. basal for all), respectively. HR in the IONs + Str was 94 ± 2% of basal HR (n.s.). The peak values of MAP and HR during the second and the third air-jet (AJ) sessions were calculated as a percentage change of the response found during the first AJ ( Figure 2C, D). ANOVA analysis showed that IONs significantly increased the pressor responses to the stress produced by air-jet (AJ2 and AJ3) when evaluated as the main effect of the IONs + Str co-application (F(1,10) = 14.07, p < 0.004), without the significant changes in peak HR responses.
Plasma corticosterone was significantly elevated in rats exposed to stress when determined as the main effect of stress in both stress-exposed groups (F(1,20) = 9.6, p < 0.006 vs. unstressed rats), while IONs did not affect this parameter (Figure 3).

NO synthase activity
In the kidneys, NOS activity decreased in the IONs group by about 22% (p < 0.03) vs.
controls ( Figure 4A), while no changes were found in the liver ( Figure 4B). ION administration elevated NOS activity in the hypothalamus and brainstem by 34% (p < 0.03) and about 62% (p < 0.01), respectively, compared to the controls ( Figure 4C, D). In the IONs + Str group, NOS activity was significantly elevated in the kidneys 40% (p < 0.01) and liver 20% (p < 0.05) and reduced in the brainstem 55% (p < 0.001) and hypothalamus 38% (p < 0.001) vs. the IONs group. Stress alone had no effect on NOS activity in any tissue investigated vs. control Similarly, no differences in NOS activity were found between Str and IONs + Str groups ( Figure   4).

Contraction responses of the femoral arteries
The mean internal diameter of all arterial segments used in this study was 717 ± 7 µm and did not differ significantly among the groups. The maximal depolarisation-induced contractions produced by the high concentration of potassium (125 mmol/l K + ) in Cont, IONs, Str and IONs + Str were 8.24 ± 1.27, 7.09 ± 1.25, 9.58 ± 0.57 and 9.31 ± 1.14 mN/mm, respectively, and no significant differences were observed among the groups.
Maximal serotonin (5-HT)-induced contractions in the absence of N ω -nitro-L-arginine methyl ester (L-NAME) were similar among the groups ( Figure 5). Pre-incubation of the arteries with L-NAME significantly enhanced the 5-HT-induced contraction of femoral arteries in the Cont, Str and IONs + Str groups compared to the 5-HT-induced contraction in absence of L-NAME by approximately 36%, 38% and 18% (p < 0.05 for all groups), respectively. A correspondingly significant increase was not found in the IONs group.

Relaxation responses of the femoral arteries
The acetylcholine (ACh)-induced and sodium nitroprusside (SNP)-induced concentrationresponse curves of 5-HT-precontracted femoral arteries are shown in Table 1 and Figure 6.
Maximal ACh-induced relaxations (Emax) were not altered by stress, IONs or their joint application vs. control (Table 1, Figure 6A).
relaxations in absence of L-NAME ( Figure 6B) and allowed the calculation of Emax L-NAME, which was significantly reduced in the IONs group compared to total relaxation (i.e. Emax). In addition, Emax L-NAME for the IONs + Str group was significantly elevated compared to the IONs group (Table 1). EC50 L-NAME was shifted to higher concentrations in all groups investigated vs.
EC50 without L-NAME (Table 1). The L-NAME-sensitive component, calculated from the Emax values as a difference between Emax and Emax L-NAME, increased in the femoral arteries of the IONs group, but it returned to the level present in the Cont group in the femoral arteries of rats co-exposed to IONs + Str (Table 1). The area under the curve (AUC) calculated from the cumulative concentration response curves to ACh in the presence of L-NAME (i.e. the L-NAME-resistant component) was significantly reduced while the L-NAME-sensitive component was significantly elevated in ION-treated rats vs. the respective control values.
Moreover, the L-NAME-sensitive component of the IONs + Str group was significantly reduced compared to that of the IONs group ( Figure 6D). Pre-treatment of the arteries with the L-NAME led also to the significant reduction of relaxations at the highest ACh-concentration only in the IONs group vs. the maximal relaxation in this group at lower ACh-concentration ( Figure 6B), suggesting the release of EDCFs.

The maximal relaxations induced by cumulative concentrations of the exogenous NO
donor SNP were not altered significantly in any group vs. the control group (Table 1, Figure   6C). The sensitivity of the femoral arteries to SNP expressed as EC50 was significantly decreased in the IONs and IONs + Str groups vs. the control group (Table 1).

IONs-originated iron contents
ION-originated iron was present mainly in whole blood, lower amounts were present in the liver and kidneys (Figure 7). In blood ( Figure 7A) and liver ( Figure 7B), stress reduced IONsoriginated iron content by approximately 92% and 69 % vs. IONs group (p < 0.04 for both). On the other hand, ION-originated iron content increased significantly in the kidneys ( Figure 7C) by 296% (p < 0.03).

Gene expressions
Expression of the HAMP gene in the liver was significantly elevated when determined as the main effect of ION-administration (F(1,17) = 6.00, p < 0.03) ( Figure 8A). Despite tendency of higher FTH1 gene expression in the liver of ION-treated rats was observed, this did not reach statistically significant effect vs. the control group. In contrast, stress significantly elevated FTH1 gene expression in the liver by 122% vs. control (p< 0.006, Figure 8B). In the kidneys, no changes in the FTH1 gene expression were observed ( Figure 8C).

Discussion
This study investigated the effect of intravenously administered ultra-small PEG-coated IONs and its interaction with acute stress on vascular function, NOS activity, BP and HR and expression of HAMP and FTH1 genes in Wistar-Kyoto rats (WKY). The main findings are that IONs modified the vascular function of the femoral arteries. In these arteries IONs 1) increased the L-NAME-sensitive component of ACh-induced relaxations, 2) decreased the sensitivity of the vascular smooth muscle cells (VSMCs) to exogenous NO and 3) prevented the increase of 5-HT-induced contractions after L-NAME, and 4) induced EDCFs release in L-NAME pretreated arteries. IONs also enhanced NOS activity in the brainstem and hypothalamus and reduced NOS activity in the kidneys. Notably, the combination of IONs with repeated acute air-jet stress had no effects on the overall endothelium-dependent relaxations but altered NOS activity in the tissues and IONs-originated iron content in blood and tissues compared to IONs alone. In addition, in IONs + Str group IONs increased AJ-induced pressor responses, but decreased MAP determined 100-min post-infusion compared to basal value, which was not found in any other group of rats. In the groups of rats exposed to acute air-jet stress, we found   McBryde et al. 2018, Pecháňová et al. 2006, Drábková et al. 2020. Thus, we assume that enhanced NOS activity in the given brain structures may lead to sympathoinhibition possibly manifested in decreased NOS activity in the kidneys of ION-treated rats, leading to maintenance of unaltered levels of BP. Interestingly, in the IONs + Str group, stress blocked the increase of NOS activity in both brain structures and prevented the decrease in NOS activity in the kidneys compared to the IONs group. We assume that the simultaneous influence of two independent stressors could cause dysregulation of BP regulatory systems, which led to a temporary decrease of the resting MAP in the IONs + Str group. In addition, the decrease of MAP at rest in the IONs + Str group may, at least partially, results from elevated NOS activity in the liver. It is well known that the high production of inducible NOS-derived NO and/or other vasodilators in the liver leads to reduced systemic vascular resistance and hypotension (El Hadi et al. 2020).
Thus, our results suggest that IONs have a complex effect on BP regulation, which includes the hypothalamus, brainstem, kidneys, liver and vasculature. Our findings also point to the importance of acute stress during ION-administration that might reduce the amount of IONs in circulation. In particular, IONs i.v. administration resulted in greater BP rise after air-jet stress (AJ2 and AJ3, Figure 2C) that may alter the blood flow leading to elevated IONs decomposition and/or iron storage in the liver and to the decrease of IONs-originated iron in blood of IONs + Str rats. Our findings of elevated HAMP gene expression suggest elevated systemic iron levels in IONs-exposed rats. Despite the organism lacks regulated mechanism of excretion of iron itself, IONs bigger than 6 nm can be excreted via hepatobiliary clearance. Furthermore, if IONs are decomposed to the particles smaller than 6 nm, renal excretion of IONs can also participate in reduction of ION-derived iron in blood (Zhang et al. 2016). Because we did not measured IONs in urine and feces, we cannot exclude any of these routes.

Conclusions
Collectively, our results indicate that single i.v. administration of a low dose of PEG-coated IONs altered mechanisms of vascular wall function of increased NO and/or EDCFs release in vascular wall of femoral arteries, as manifested by enhanced L-NAME-sensitive component of ACh-induced relaxation, by the reduction of sensitivity to exogenous NO and by the absence of augmentation of 5-HT-induced contractions after NOS inhibition. These effects of IONs were absent reduced when rats were exposed also to repeated acute stress despite acute stress itself having no effect on these parameters. In addition, joint exposure of ION-treated rats to repeated acute stress reduced ION-originated iron in blood and liver, affected NOS activity and altered the mechanisms of BP regulation, resulting in enhanced pressor responses to stress, but reduced MAP at rest. Our findings thus point to considerable involvement of acute stress in clearance and/or tissue distribution of IONs associated with alterations in vascular function and BP regulation after a single i.v. IONs application. Tables   Table 1. Parameters of endothelium-independent and endothelium-dependent relaxations induced by sodium nitroprusside and acetylcholine, respectively.