The Role of 20-HETE, COX, Thromboxane Receptors, and Blood Plasma Antioxidant Status in Vascular Relaxation of Copper-Nanoparticle-Fed WKY Rats

Recently, the addition of copper nanoparticles (NPs) in a daily diet (6.5 mg/kg) was studied in different animal models as a possible alternative to ionic forms. Male Wistar–Kyoto rats (24-week-old, n = 11) were fed with copper, either in the form of carbonate salt (Cu6.5) or metal-based copper NPs (NP6.5), for 8 weeks. The third group was fed with a half dose of each (NP3.25 + Cu3.25). The thoracic aorta and blood plasma was studied. Supplementation with NP6.5 decreased the Cu (×0.7), Cu/Zn-ratio (×0.6) and catalase (CAT, ×0.7), and increased Zn (×1.2) and superoxide dismutase (SOD, ×1.4). Meanwhile, NP3.25 + Cu3.25 decreased the Cu/Zn-ratio (×0.7), and CAT (×0.7), and increased the daily feed intake (×1.06). Preincubation with either the selective cyclooxygenase (COX)-2 inhibitor, or the non-selective COX-1/2 inhibitor attenuated vasodilation of rat thoracic aorta in the NP6.5 group exclusively. However, an increased vasodilator response was observed in the NP6.5 and NP3.25 + Cu3.25 group of rats after preincubation with an inhibitor of 20-hydroxyeicosatetraenoic acid (20-HETE) formation, and the thromboxane receptor (TP) antagonist. Significant differences were observed between the NP6.5 and NP3.25 + Cu3.25 groups of rats in: dietary intake, acetylcholine-induced vasodilation, and response to COX-inhibitors. Copper NPs in a standard daily dose had more significant effects on the mechanism(s) responsible for the utilization of reactive oxygen species in the blood plasma with the participation of prostanoids derived from COX-2 in the vascular relaxation. Dietary copper NPs in both doses modified vasodilation through the vasoconstrictor 20-HETE and the TP receptors.


Introduction
Copper fluctuations in a diet may have either pro-or antioxidant effects on animal or human health, dependent on the daily dose [1][2][3][4]. A high intake of copper (including the recommended daily dose) may induce oxidation of lipids and proteins in cells that are potentiated in situations of a high-risk susceptibility to toxic compounds, such as in diabetes mellitus or hypertension [5,6]. Many of the major enzymes of biological processes are influenced by copper intake, including Cu-Zn superoxide dismutase (SOD), Acetylcholine (chloride), indomethacin, noradrenaline (hydrochloride), and NS-398 were obtained from Sigma-Aldrich (St. Louise, MO, USA); copper as carbonate (purity ≥ 99%) from Poch (Gliwice, Poland); SQ-29,548, furegrelate, and HET0016 from Cayman Chemical (Ann Arbor, MI, USA). Stock solutions (10 mM) of these drugs were prepared in distilled water, except for noradrenaline, which was dissolved in NaCl (0.9%) + ascorbic acid (0.01% w/v) solution; HET0016, SQ-29,548, and indomethacin were dissolved in ethanol; 1400 W in methanol; and NS-398 in DMSO. The solvent concentration was less than 0.01% (v/v).

Metal-Based Copper Nanoparticles
Copper NPs (99.9% purity powder, 40-60 nm size, 12 m 2 /g SSA, spherical morphology, 0.19 g/cm 3 bulk density, 8.9 g/cm 3 true density) were purchased from Sky Spring Nanomaterials (Inc., Houston, TX, USA). Stock solution (5 g/L) was prepared in a rapeseed oil, and about 9% of NPs dissolved as Cu (II) ions; thus, the final suspension contained both NPs and released copper species. The zeta potential of the copper NP suspension was determined to be −30.3 mV (in PBS) and −38.3 mV (pH 5), and the size was 104 nm (in rapeseed oil) determined by dynamic light scattering with a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK) [12].

Experimental Protocol
24-week-old normotensive Wistar-Kyoto (WKYs/NCrl) rats from Charles River (Sulzfeld, Germany) were allocated randomly to 3 groups (n = 11) and were fed individually for 8 weeks with experimental diets under standard laboratory conditions [1]. Exclusively male rats were studied, to enable comparison with the previous experimentations. The rats were fed with copper in a standard daily dose of 6.5 mg/kg, either as carbonate salt (Cu 6.5 ) or metal NPs (NP 6.5 ). Moreover, the third group (NP 3.25 + Cu 3.25 ) was fed with 3.25 mg of copper NPs plus 3.25 mg of copper carbonate. Animal pellets were prepared according to the American Institute of Nutrition, and the copper NPs were dissolved in pure rapeseed oil (5 g/L) and mixed with the diet weekly [1].

Experimental Procedures
Intraperitoneal injections of ketamine (100 mg/kg BW) and xylazine (10 mg/kg BW) were used for anesthesia, followed by exsanguination [13]. Blood was centrifuged at 3000× g for 10 min to separate the plasma, which was further stored at −80 • C until analysis. The thoracic aorta was dissected and kept in an ice-cold Krebs-Henseleit buffer.

Blood Analysis
Copper and zinc were measured by the ICP-OES method. Bovine liver was used as a certified reference material (NIST1577C) for quality control. The units are expressed as µM. Superoxide dismutase activity was measured with Ransod and Ransel diagnostic kits (Randox); meanwhile, catalase (CAT) was determined by the enzymatic decomposition of hydrogen peroxide into water and oxygen. Data are expressed in U/mL. The sums of reduced glutathione (GSH) and oxidized glutathione (GSSG) were determined using an enzymatic method (Cell Biolabs) [14]. The units are expressed as µM. The total antioxidant potency FRAP (Ferric Reducing Antioxidant Power) was measured colorimetrically at 594 nm through the reduction of Fe(III) to Fe(II) by antioxidants present in the sample. Data are expressed as µM. The malondialdehyde (MDA) generates the MDA-TBA adduct, which was quantified with a fluorometric assay kit (ab118970) at Ex/Em = 532/553 nm. Data are expressed as mM.

Data Analysis and Statistics
Vascular relaxation was expressed as a percentage of the contractile response to noradrenaline NA (0.1 µM). The CCRCs were analyzed by a nonlinear regression model (log agonist vs. response), which determined the area under the curve (dAUC), the maximal response (E max , %), and the potency (pEC 50 = −logEC 50 ). The Gaussian distribution of residuals and homoscedasticity of variance were tested for all data with n = 11; "n" refers to independent values, not replicates. The group comparison was performed by either a parametric (t-test or ANOVA) or non-parametric test (Mann-Whitney U-test or Kruskal-Wallis test). Results are expressed as the means ± SEM (for CCRCs) and means ± SD. Due to the small group sizes (n < 12), outliers detected by Grubbs' test were included in a data set [13]. The level of significance was when * p < 0.05.

The General Characterization of WKY Rats
These results are presented in Figure 1A-D. Experimental supplementation with NP 6.5 neither changed the body weight (BW) gain (×1.0, Figure 1C), nor the dietary intake (×1.02, Figure 1D). In the NP 3.25 + Cu 3.25 group of rats, BW gain was not changed in a significant way (×1.4, Figure 1C), contrary to the significant increase in daily feed intake (×1.06, p = 0.0008, Figure 1D). No significant difference was observed in the BW gain between Nutrients 2021, 13, 3793 4 of 11 NP 3.25 + Cu 3.25 and NP 6.5 supplemented rats (×1.4, Figure 1C), as opposed to the increased daily feed intake (×1.04, p = 0.0462, Figure 1D).
The vasodilator response to acetylcholine is presented in Figures 3-7. Results are expressed as E max (%), pEC 50 and AUC, see Table 1. The vasodilator response to acetylcholine is presented in Figures 3-7. Results are expressed as Emax (%), pEC50 and AUC, see Table 1.  Values are based on the concentration-response curves shown in Figures 3-7. Data are expressed as means ± SEM where n represents the number of animals. * p < 0.05 compared with the control conditions, # p < 0.05 compared with the Cu6.5 group, $ p < 0.05 compared with the NP3.25 + Cu3.25 group as determined by one-way ANOVA followed by Tukey's post hoc test.

Discussion
Our previous studies revealed differences in the vascular tone regulation and the antioxidant status of rats supplemented with copper NPs (of 40-60 nm size) [1,2,12,17,18]. Of great importance is that in the previous experiments, both the age (either 4, 5, or 6 weeks) of Wistar Han IGS rats and the duration of feed intake (either 4 or 8 weeks) were what differentiated these studies from the one presented now, which can be described as 24 + 8 (24 weeks of age + 8 weeks of experimental feeding); and this was carried out on

Discussion
Our previous studies revealed differences in the vascular tone regulation and the antioxidant status of rats supplemented with copper NPs (of 40-60 nm size) [1,2,12,17,18]. Of great importance is that in the previous experiments, both the age (either 4, 5, or 6 weeks) of Wistar Han IGS rats and the duration of feed intake (either 4 or 8 weeks) were what differentiated these studies from the one presented now, which can be described as 24 + 8 (24 weeks of age + 8 weeks of experimental feeding); and this was carried out on Wistar-Kyoto (WKY) rats, as a control for the spontaneously hypertensive rat (SHR) model which was also analyzed, but will be described elsewhere. Based on the previous results, antioxidant status and the participation of arachidonic acid metabolites were further investigated in the regulation of the vasodilator response induced by acetylcholine. Three different diets (i) standard with copper carbonate (Cu 6.5 ), (ii) with metal copper NPs (NP 6.5 ), and (iii) half dose of each (NP 3.25 + Cu 3.25 ) were prepared in the form of pellets, and given daily to rats in order to study the physiological properties of dietary copper NPs.
Experimental supplementation with NP 6.5 neither modified the body weight nor the feed intake, which is in agreement with our previous results 4 + 8 (4 weeks of age + 8 weeks of experimental feeding), and 5 + 4 (5 weeks of age + 4 weeks of experimental feeding) [2,3,12,17]. However, in the NP 3.25 + Cu 3.25 group of rats, we observed a significant increase in the daily feed intake compared to Cu 6.5 and NP 6.5 , and an increase of body weight gain (not significant), which might be explained by the higher feed intake, and may become significant when more rats per group are studied. The observed increase in feed intake is difficult to explain and merits further investigation. Experimental treatment with NP 6.5 markedly reduced blood plasma Cu and increased Zn, which resulted in a decreased Cu/Zn-ratio. This is opposite to the 4 + 8 study, when Zn remained unchanged. However, in the same study, Cu and the Cu/Zn-ratio also decreased [18]. In the NP 3.25 + Cu 3.25 group of rats, the Cu/Zn-ratio decreased significantly, which was due to a decrease in Cu and an increase in Zn (both results were not significant). There was no statistically significant difference between NP 3.25 + Cu 3.25 and NP 6.5 in the Cu, Zn, and Cu/Zn-ratio. We have now observed, for the first time, increased activity of SOD and decreased CAT in NP 6.5 supplemented rats. This is contrary to our previous studies (4 + 8, and 6 + 8), when the activity of SOD was not modified and CAT increased [2,19]. However, another experiment from our research group (5 + 4) pointed to a decrease in CAT [12]. Increased SOD (result not significant) and decreased CAT were also observed for the NP 3.25 + Cu 3.25 group. Increased SOD indicates an effective means of scavenging superoxide anion, whereas decreased CAT points to possible enzyme depletion in response to the increased oxidative stress and intensified scavenging of hydrogen peroxide. Another enzyme of hydrogen peroxide degradation, glutathione peroxidase, was not modified in this study, and this is opposite to a significant decrease in the 5 + 4 study [12]. Copper in the form of NPs neither modified FRAP nor MDA. These findings are not entirely in agreement with our previous results (4 + 8 and 6 + 8), when FRAP increased [2,19], and MDA either increased (5 + 4 and 7 + 8) [1,12] or was not modified (4 + 8, and 6 + 8) [2,3,19]. We previously reported that replacing Cu 6.5 with NP 6.5 and reducing the dose (from a standard 6.5 mg/kg to a 3.25 mg/kg in the diet of rats 5 + 4) had particularly unfavorable effects on the respiratory system, causing adverse changes to the lungs. Surprisingly, these treatments also had a positive effect on the redox status of the liver and brain [20]. Moreover, the addition of copper NPs into the rat diet (5 + 4 and 7 + 8) reduced protein oxidation and nitration [1,12], as well as DNA oxidation and methylation. Meanwhile, lowering the daily dose increased the oxidation of proteins and DNA methylation [12]. In our study, neither NP 6.5 nor NP 3.25 + Cu 3.25 modified the acetylcholine-induced vasodilation compared to Cu 6.5 control. However there was a tendency to increased vasodilation in NP 6.5 supplemented rats. This stays in agreement with the 4 + 8 study [2], and is in opposition to the 7 + 8 study, when NP 6.5 potentiated the vasodilator response induced by acetylcholine in a significant way [1]. Surprisingly, in the present study, NP 6.5 potentiated that response compared to NP 3.25 + Cu 3.25 . Preincubation with NS-398 (COX-2 inhibitor) diminished that response, which was completely abolished with indomethacin (COX-1/2 inhibitor). These results suggest participation of COX-2 in NP-induced response, which is a dose dependent mechanism. In another experiment, conducted by Cendrowska-Pinkosz et al. [21], NP 6.5 in the diet of rats did not change the acetylcholinesterase level (an enzyme that catalyzes the breakdown of acetylcholine) in the blood compared to Cu 6.5 (7 + 8), so the observed changes might not be due to enzyme depletion nor surplus. Even though we did not currently report any changes in acetylcholine-induced vasodilation followed by NP 6.5 and NP 3.25 + Cu 3.25 intake (compared to the control Cu 6.5 ), the contribution of nitric oxide and arachidonic acid metabolites cannot be ruled out. We observed that iNOS inhibition with 1400 W did not modify that response in aortas from all three studied groups, which is contrary to the previous study (7 + 8), when the overproduction of NO from iNOS was engaged in vascular relaxation in the NP 6.5 group of rats [1]. Considering that the sensitivity of the arteries to nitric oxide was not altered (study with an exogenous NO donor sodium nitroprusside) [2], arachidonic acid derivatives may also be responsible for the vascular tone regulation of copper NPs, as we suggested previously [18]. Preincubation with either COX-2 or COX-1/2 inhibitors attenuated the vasodilator response in NP 6.5 , indicating the involvement of a vasodilatory net effect of prostanoids origin from COX-2. This was neither observed for the control (Cu 6.5 ) nor when the dose was reduced by half (in the NP 3.25 + Cu 3.25 group). The results with NP 6.5 are contrary to our previous report (4 + 8), which had revealed a decreased sensitivity of the smooth muscles to prostanoids (no significant change in acetylcholine-induced response after COX-1/2 inhibition) [2]. Given these results, the participation of another vasoconstrictor agent in vascular relaxation was studied. 20-Hydroxyeicosatetraenoic acid (20-HETE) is a potent metabolite of arachidonic acid. This reaction is mediated by cytochrome P450 and can be further metabolized by COX to 20-hydroxy compounds. Preincubation with a 20-HETE inhibitor potentiated the vascular relaxation in both NP 6.5 and NP 3.25 + Cu 3.25 groups, indicating that 20-HETE is involved in acetylcholine-induced relaxation. However, this was not observed in young rats (7 + 8) [1]. Finally, we analyzed the contribution of the potent vasoconstrictor thromboxane-A 2 in the acetylcholine-induced response. However, the present results showed that thromboxane-A 2 is not an important vasoconstrictor candidate engaged in the vascular tone regulation of supplemented rats (studies with the thromboxane-A 2 synthesis inhibitor, furegrelate). Through the activation of thromboxane-A 2 receptors (TP), prostacyclin, prostaglandins, isoprostanes, and 20-HETE participate in the endothelial dysfunction associated with cardiovascular risk factors [15]. In this study, when the TP were blocked with SQ-29,548, we observed increased vasodilation in both groups of copper NPs supplemented rats (NP 6.5 and NP 3.25 + Cu 3.25 ). These data point towards a potent vasoconstrictor that acts on the TP, suggesting 20-HETE as a possible candidate. However, other vasoconstrictors should also be taken under consideration in further studies [16]. As there is an interplay between vasoconstrictor and vasodilator factors, vasodilators should also be analyzed; this will be done in another study.
These data point to some significant changes induced by metal copper NPs, which are age-dependent and are observed between younger (12-week-old) and older (32-week-old) male WKY rats. Indeed, age-related changes in blood vessels elasticity/stiffness might be exacerbated by daily diet [22]. Many recent studies have focused on dietary intervention to improve vascular health and delay the onset of vascular aging [23]. Dietary intervention may improve not only the vascular/cardiovascular impairment, but also vascular cognitive impairment and dementia. However, so far, only certain vitamins (vitamin E, folate), multinutrient formulations, and unsaturated fatty acids have shown some initial promise [24].

Conclusions
Our findings have shown that increased oxidative stress accompanies copper NP intake, which further modulates vascular relaxation with the participation of 20-HETE, through the thromboxane-A 2 receptors. When a daily dose of copper NPs was decreased by half, the interplay between COX metabolites was also modified; however, the Cu/Znratio and CAT remained unchanged compared to the higher dose. Further studies should concentrate on animals with metabolic disorders.