Antioxidant Properties of Egg White Hydrolysate Prevent Mercury-Induced Vascular Damage in Resistance Arteries

Aim: We investigated the antioxidant protective power of egg white hydrolysate (EWH) against the vascular damage induced by mercury chloride (HgCl2) exposure in resistance arteries. Methods: Male Wistar rats received for 60 days: (I) intramuscular injections (i.m.) of saline and tap water by gavage – Untreated group; (II) 4.6 μg/kg of HgCl2 i.m. for the first dose and subsequent doses of 0.07 μg/kg/day and tap water by gavage – HgCl2 group; (III) saline i.m. and 1 g/kg/day of EWH by gavage – EWH group, or (IV) the combination of the HgCl2 i.m. and EWH by gavage – EWH + HgCl2 group. Blood pressure (BP) was indirectly measured and dose-response curves to acetylcholine (ACh), sodium nitroprusside (SNP), and noradrenaline (NE) were assessed in mesenteric resistance arteries (MRA), as in situ production of superoxide anion, nitric oxide (NO) release, vascular reactive oxygen species (ROS), lipid peroxidation, and antioxidant status. Results: Egg white hydrolysate prevented the elevation in BP and the vascular dysfunction after HgCl2 exposure; restored the NO-mediated endothelial modulation and inhibited the oxidative stress and inflammatory pathways induced by HgCl2. Conclusion: Egg white hydrolysate seems to be a useful functional food to prevent HgCl2-induced vascular toxic effects in MRA.


INTRODUCTION
The imbalance between the excessive formation of reactive oxygen species (ROS) and limited antioxidant defenses is one of the most harmful mechanisms that induce deleterious cardiovascular effects (Liguori et al., 2018). Oxidative damage has been implicated in vascular injury and endothelial dysfunction (Halliwell, 2007), a proliferation of vascular smooth muscle cells (VSMC), increase in vascular tone, migration of inflammatory mediators, vascular remodeling (Szasz et al., 2007), and hypertension (Touyz and Briones, 2011;Montezano and Touyz, 2014;Montezano et al., 2015;Incalza et al., 2018).
Several studies point out the beneficial effects of consuming different natural antioxidant compounds and bioactive food ingredients, such as protein-derived peptides against oxidative imbalance (Serafini and Peluso, 2016;Peluso and Serafini, 2017;Chikara et al., 2018). Bioactive peptides from egg white hydrolysate (EWH) have shown important antioxidant properties, preventing dysfunction in hypertensive and obese experimental models (Manso et al., 2008;Garces-Rimon et al., 2019). We propose to use EWH obtained after hydrolysis of hen egg white with pepsin for 8 h, which has previously demonstrated antioxidant, anti-inflammatory, and angiotensin-converting enzyme (ACE) inhibitory properties Moreno-Fernandez et al., 2018a,b).
Metals, such as mercury (Hg), are dangerous pollutants in the ecosystem, found in different physical and chemical forms. Their toxic effects are dose and time-dependent and can be characterized as a risk factor for the development of cardiovascular diseases by the promotion of oxidative stress (Genchi et al., 2017). Hg participates in the Fenton reaction, increasing ROS production and also leading to the depletion of important antioxidant enzymes due to their affinity for the sulfhydryl radicals (Valko et al., 2006;Su et al., 2008).
Studies have been shown that 30-day exposure to a low concentration of mercury chloride (HgCl 2 ) induces oxidative stress and activation of cyclooxygenase (COX) and angiotensin II pathways, promoting vascular changes, such as endothelial dysfunction and increased reactivity (Wiggers et al., 2008;Peçanha et al., 2010). It has been found that EWH improves the Hg-induced damage in parameters related to memory deficits (Rizzetti et al., 2016a), peripheral nervous disorders (Rizzetti et al., 2016b), male reproductive dysfunction (Rizzetti et al., 2017b), and conductance arteries injury (Rizzetti et al., 2017a). In aorta of long-term Hg-exposed rats, EWH prevented the high vascular reactivity and endothelial dysfunction promoted by the metal. However, small arteries, such as resistance mesenteric arteries (MRA), represent the primary vessels that are involved in the regulation of arterial blood pressure (BP) as well as blood flow within the organ.
Thus, we investigate whether the antioxidant properties of EWH have a protective power against vascular damage caused by exposure to HgCl 2 in MRA and the underlying pathways involved.

Animals and Experimental Groups
The experiments were conducted in compliance with the Principles of Laboratory Animal Care (National Institutes of Health publication 80-23, revised 1996) and in agreement with the guidelines by the Brazilian Societies of Experimental Biology and approved by the Ethics Committee on Animal Use Experimentation of the Federal University of Pampa, Uruguaiana, Rio Grande do Sul, Brazil (protocol 05/2014). The rats (Male Wistar rats; age 3 mo; 250-300 g) were kept in controlled light, temperature, and humidity conditions (12/12 h light/ dark cycle; 23 ± 2°C; 50 ± 10%, respectively) with free access to food and water and randomly submitted to one of the following protocols for 60 days: (I) intramuscular injections (i.m.) of saline solution 0.9% and tap water by gavage -Untreated group; (II) 4.6 μg/kg of HgCl 2 i.m. for the first dose and subsequent doses of 0.07 μg/kg/day, to cover daily loss, using the model described previously (Rizzetti et al., 2017c) and tap water by gavage -HgCl 2 group; (III) saline solution 0.9% i.m. and EWH from pepsin for 8 h diluted in tap water (1 g/kg/ day), by gavage, according to the model prior reported (Rizzetti et al., 2016a) -EWH group, or (IV) the combination of the HgCl 2 i.m. and EWH by gavage -EWH + HgCl 2 group. Bodyweight, food, and water intakes were measured once a week.
This experimental model of chronic controlled exposure to a low concentration of Hg has a total metal concentration in the blood at the end of the treatment of 3.04 ng/ml (Rizzetti et al., 2017c), which is within the safety limit established by US Environmental Protection Agency's (5.8 ng/ml) and similar to the blood Hg concentration of people exposed to the metal by the workplace or through diet (Rice, 2004). Moreover, to make the model more similar to the human exposure condition, we carry out a simultaneous treatment of Hg and EWH, considering that humans are rarely entirely free of any level of exposure to this metal. So our concern was to investigate the benefits of EWH during continuous and concurrent exposure to Hg.

Systolic Blood Pressure
Systolic Blood Pressure (SBP) was recorded once a week by tail plethysmography (ADInstruments Pty Ltd., Bella Vista, NSW, Australia) according to Rizzetti et al. (2017c). The animals were preheating the animals at 37°C for 10 min to make the pulsations of the caudal artery detectable, followed by 10 sequential cycles of tail inflation-deflation. The SBP was considered the average of all measures.

Vascular in situ Nitric Oxide and Reactive Oxygen Species Detection
Measurements of NO and ROS levels in MRA segments were performed as previously published by Martin et al. (2012) and Avendaño et al. (2014), respectively. Briefly, the MRA rings were incubated for 45 min with 4,5-diaminofluorescein diacetate (DAF-2, 10 μM) to assess the release of NO. The first collection of the medium measured the basal release of NO, and after the segments were incubated with NE 0.1 nM and relaxed with ACh 10 μM, the medium was collected again, and the induced release of NO was measured by fluorescence method (excitation at 492 nm and emission at 515 nm -SpectraMax M5 Molecular Devices, Sunnyvale, CA, United States). The results are expressed as arbitrary units/g tissue as a percentage of fluorescence obtained for Untreated rats. The release of NO is the result of fluorescence evoked by ACh subtracted from the basal release of NO.
The malondialdehyde (MDA) levels that represent the lipid peroxidation were measured following Ohkawa et al. (1979) protocol with modifications by the colorimetric method. Briefly, thiobarbituric acid (TBA) 0.8%, acetic acid buffer (pH 3.2) plus sodiumduodecilsulphate (SDS) 8% were added to the samples and the color reaction was measured against blanks (532 nm -SpectraMax M5 Molecular Devices, Sunnyvale, CA, United States). The data were expressed as nmol of MDA per gram of tissue.

Statistical Analyses
Values are expressed as means ± SEM. The results were analyzed using a two-way ANOVA. When two-way ANOVA showed a statistical significance, the Fisher post hoc test was applied (Graph Pad Prism 6.0 Software, San Diego, CA). To compare the effects between groups (endothelium removal and drugs) on the response to NE, some results are expressed as the differences of areas under the concentration-response curves (dAUC) in the control and experimental situations. The differences were expressed as the % of the AUC of the corresponding control situation. The results were considered statistically significant for values of p < 0.05.
Egg white hydrolysate intake prevented the increased contractile responses to NE and the reduced endotheliumdependent vasodilator response to ACh induced by HgCl 2 exposure. The endothelium-independent vasodilator response to NPS in MRA was not affected in all treatments (Figures 1A-C).
Denuded endothelium or NOS inhibitor incubation (L-NAME) increased the contractile response to NE in all groups except in HgCl 2 group as evidenced by the dAUC values (Figures 2A-E,a-e). These findings show the absence of endothelial participation in the vasoconstrictor response to NE in this group. MRA segments from HgCl 2 exposure animals that received EWH showed similar effects of endothelium removal or L-NAME compared to the Untreated group (Figures 2A-E,a-e), suggesting that EWH prevented this reduced endothelial modulation by NO. In agreement, ACh-induced NO release was lower in MRA from Hg-treated rats. On the other hand, rats receiving EWH alone and those receiving the combination of HgCl 2 and EWH had a greater percentage of NO release when compared to the Untreated group (Figure 2a'). This finding suggests that EWH was able to induce NO production by NOS.
The cofactor for NO synthesis BH4 incubation had a smaller contractile response to NE in HgCl 2 -treated rats, showing lower BH4 bioavailability and, possibly, an uncoupled state of endothelial nitric oxide synthase (eNOS) in this vessel. EWH intake maintained BH4 bioavailability in MRA from HgCl 2treated rats, indicating the improvement of eNOS and NO synthesis (Figures 3A-E).
The NAD(P)H oxidase inhibitor VAS2870 reduced the contractile response to NE in MRA only in HgCl 2 group (Figures 3a-e). EWH prevented the increased ROS participation from NAD(P)H oxidase on contractile response to NE. The superoxide dismutase mimetic TEMPOL reduced the contractile response to NE in MRA only from HgCl 2 -exposed rats; thus, the NE responses remained unchanged in the other groups (Figures 3a'-e'). We also observed significantly higher superoxide anion production in arteries of rats exposed to HgCl 2 ; EWH prevented this effect (Figure 4). Chronic Hg treatment for 60 days increased ROS levels and lipid peroxidation in MRA from exposed rats (Figures 5A,B) and reduced the total antioxidant capacity and the NPHS levels in the Hg-exposed rats (Figures 5C,D). EWH was able to prevent the oxidative stress in MRA of HgCl 2 -treated rats, balancing the pro-oxidant and antioxidant status (Figures 5A-D).
Indomethacin reduced the response to NE in MRA segments from only in HgCl 2 -treated rats (Figures 6A-E) indicating that the enhanced COX pathway participation in these responses was prevented by EWH.

DISCUSSION
Intake of EWH as a functional food was able to reverse the increase in SBP induced by chronic exposure to HgCl 2 at low concentrations, which is related to the reduction of contractile responses and the vascular dysfunction induced by the metal in MRA. These effects were associated, at least in part, with the capacity of EWH to produce NO from eNOS and with its antioxidant and anti-inflammatory properties. The bioactive peptides of EWH protect against high concentrations of ROS from NAD(P)H oxidase and, possibly, activation of inflammatory COX in MRA in HgCl 2 -treated rats, thus normalizing the NO modulation in the vasculature.
The Hg is a well-known environmental risk factor for cardiovascular diseases (Virtanen et al., 2007). Acute Hg exposure promotes the reduction of myocardial contractility and inhibition of myosin ATPase activity (Vassallo et al., 1999). Moreover, subchronic exposure to Hg, at doses similar to human exposure, increases vascular reactivity of resistance and conductance vessels in rats (Wiggers et al., 2008;Peçanha et al., 2010). A prolonged exposure at low doses of HgCl 2 for 60 days increased SBP and vascular dysfunction in conductance arteries; these effects were related to stimulation of ACE activity, NAD(P)H oxidase-mediated oxidative stress and activation of COX-2 inflammatory pathway in these vessels (Rizzetti et al., 2017a,c). Our results demonstrate that resistance arteries are also affected by prolonged exposure to Hg, which could better explain the high SBP values observed, since the main vascular site responsible for vascular resistance and maintenance of BP is the resistance arteries (Oparil et al., 2018). Our purpose was to investigate if the mechanisms involved in this increment of SBP could be blocked by EWH treatment. The Hg exerts its toxic effects on the cardiovascular system through oxidative stress caused by the production of superoxide anion from NAD(P)H oxidase and, possibly, inflammatory mediators derived from COX. Although we have not verified the specific participation of the COX-2 pathway in the current study, previous work of our group, at the same model, evidenced the participation of this pathway in increasing vascular reactivity in conductance arteries using a selective COX-2 inhibitor (NS 398), and the relationship between ROS and COX-2-derived prostanoids. Moreover, the vascular functional findings showed a reduction in the COX-2 participation in the cardiovascular system of Hg-treated rats after EWH intake, proving its antiinflammatory property observed in vitro (Rizzetti et al., 2017a).
Thus, in this study, we suggest that inflammatory mediators derived from COX-2 are also involved in increasing vascular reactivity in resistance arteries. However, future investigations using selective inhibitors of this pathway may explore these findings.
Besides, we verified for the first time the possible involvement of eNOS uncoupling in HgCl 2 -induced negative actions on vascular tissue; this could be due to high vascular oxidative stress caused by inflammatory stimuli from the COX pathway. Our findings demonstrated the protective effect of EWH on resistance arteries, which blocked the mechanisms involved in the increment of blood pressure by Hg.
Egg white hydrolysate derived from hydrolysis with pepsin for 8 h has several biological activities, such as antioxidant, free radical scavenger, ACE inhibitor, vascular-relaxing, and anti-inflammatory . Fourteen of its constituent peptides have been identified (FRADHPFL, RADHPFL, YAEERYPIL, YRGGLEPINF, ESIINF, RDILNQ, IVF, YQIGL, SALAM, and FSL; Davalos et al., 2004;Miguel et al., 2004;Garcés-Rimón et al., 2016) and their biological actions have been previously reported in vitro or in vivo studies (Miguel et al., 2006(Miguel et al., , 2007Garces-Rimon et al., 2019). However, these peptides and amino acids act individually, cooperatively, and Frontiers in Physiology | www.frontiersin.org 6 November 2020 | Volume 11 | Article 595767 FIGURE 3 | Effects of EWH on NO cofactor modulation, participation of reactive oxygen species (ROS) from NAD(P)H oxidase and participation of superoxide anion in vasoconstrictor responses to NE in MRA from rats exposed to low concentrations of HgCl 2 for 60 days. Concentration-response curve to NE in the absence (Ct), presence of the endothelial nitric oxide synthase (eNOS) cofactor (BH4; A-D), the presence of the NAD(P)H synthase inhibitor (VAS2870; a-d) and the presence of the superoxide anion scavenger mimetic (TEMPOL; a'-d') in mesenteric segments from rats Untreated (A), treated with HgCl 2 (B), with EWH (C), and with EWH + HgCl 2 (D). The results (mean ± SEM) are expressed as a percentage of the response to 120 mmol/l KCl. Differences in the area under the concentration-response curves (dAUC) in mesenteric segments in the presence and the absence of BH4, VAS2870 and TEMPOL of the four experimental groups (E,e,e'); n of each group in parenthesis, one-way ANOVA, *p < 0.05 vs. Untreated and # vs. HgCl 2 .
FIGURE 4 | Effects of EWH on local anion superoxide production in mesenteric of rats exposed to low concentrations of HgCl 2 for 60 days. Superoxide Anion production in mesenteric from rats Untreated, treated HgCl 2 , with EWH and with EWH + HgCl 2 levels in mesenteric of all groups; n of each group in parenthesis, one-way ANOVA, *p < 0.05 vs. Untreated, # vs. HgCl 2 .
Frontiers in Physiology | www.frontiersin.org 7 November 2020 | Volume 11 | Article 595767 synergistically; during their passage through the gastrointestinal tract, some modifications due to new hydrolysis should be considered (Miner-Williams et al., 2014). Thus, we do not know if the effects observed in this study are due to a specific peptide or the sum of all its constituent peptides' effects.
Although most studies conducted in recent years have focused on the isolation of peptide sequences released during hydrolysis, it has recently been proven that the administration of complete hydrolysates could be more relevant than the administration of a single isolated peptide since a more significant biological effect could be achieved (Liu et al., 2017). Moreover, we consider that hydrolysates could be more attractive products for developing functional foods from a technological and organoleptic point of view.
In the present study, we demonstrated improvements in resistance arteries function and, consequently, in SBP levels of Hg-treated rats after the EWH treatment. The mechanisms involved on cardiovascular beneficial effects observed after consumption of EWH are probably due to its vascular-relaxing, antioxidant and anti-inflammatory effects. The antihypertensive capacity of EWH we show here was previously reported in experimental models of spontaneously hypertensive rats (SHR) (Miguel et al., 2006) and was attributed, at least in part, to the vasodilator peptides whose N-terminal position exhibits amino acids Arg or Tyr (Miguel et al., , 2005. It has been previously reported that alterations in eNOS gene increase the susceptibility to cardiovascular diseases in individuals after Hg exposure by modulating NO levels (de Marco et al., 2012). Exposure to Hg at low concentrations has deleterious vascular effects on aorta, coronary and basilar arteries related to greater vascular reactivity caused by the reduction of NO bioavailability in these vessels (Wiggers et al., 2008;Peçanha et al., 2010;Botelho et al., 2019). Moreover, a study in mesenteric arteries from rats exposed for 30 days to HgCl 2 showed eNOS protein expression upregulation possibly due to a compensatory mechanism against metal-induced endothelial dysfunction (Wiggers et al., 2008). Our results indicate that lower NO bioavailability and endothelial dysfunction persists in MRA of long-term Hg exposure. Moreover, we showed that lower NO bioavailability occurs, at least in part, due to less NO release in resistance arteries.
Considering previous and present findings, we could hypothesize that the increased eNOS protein expression in MRA can accompany the reduction of this isoform activity or that its expression and activity may be increased. However, this enzyme could be decoupled and producing ROS, which would support the increase in oxidative stress associated with reducing the NO release observed in this study. In this condition, eNOS shifts from NO production to overproducing superoxide anion, increasing oxidative stress in the vasculature (Antoniades et al., 2006). Frontiers in Physiology | www.frontiersin.org 8 November 2020 | Volume 11 | Article 595767 Here we observed lower BH4 bioavailability in MRA of Hg-treated rats, which represents an important cofactor for the production of NO by this enzyme. This finding suggests that Hg induces eNOS uncoupling by inhibiting its cofactor. In any case, the alteration of eNOS protein expression induced by exposure to Hg increases, oxidative stress, and vascular damage are being protected by the ingestion of EWH. In this sense, NO bioavailability could also be related to oxidative stress imbalance due to overproduction of vascular ROS, generation of peroxynitrite, and reduction of antioxidant reserves (Faria et al., 2018). Interestingly, oxidative stress has also been reported as an important mechanism responsible for lower NO bioavailability induced by Hg (Wiggers et al., 2008;Rizzetti et al., 2017c); it is also involved in Hg-induced vascular damage in our study. Besides, oxidative stress can directly modify eNOS protein or its cofactor BH4, leading to enzymatic dysfunction in vascular tissue (Dumitrescu et al., 2007).
It is important to emphasize that EWH is composed of several peptides that have vasodilator effects (Miguel et al., 2007). These peptides were related to EWH capacity for increasing NO release by the rise in eNOS activity or the upregulation in protein expression of endothelial cells . Our study suggests that EWH was able to increase the NO release possibly by enhancing the eNOS function, which is the major isoform involved in the control of vascular function and blood pressure. The vasodilator power of EWH in SHR models is related to Arg or Tyr peptide content necessary in the N-terminal position for vasodilator activity .
An alternative mechanism implied on the vasodilator activity of the EWH and derived peptides is its action mediated by NO production via the bradykinin B1 receptor (Miguel et al., 2007). Others authors showed that egg ovotransferrin-derived peptides increased NO-mediated vasodilation in MRA of SHR animals through increasing eNOS expression (Majumder et al., 2013). Future studies are required to elucidate the mechanisms of action by which EWH acts on the expression and activity of eNOS.
In the present study, EWH was able to prevent oxidative stress in MRA of Hg-treated rats, avoiding superoxide anion generation from NAD(P)H oxidase and balancing pro-oxidant and antioxidant status verified by the functional experiments and the oxidative stress biomarkers in vascular tissue. Longterm Hg exposed-rats had lower NOX-4 and p22phox mRNA levels in aorta after co-treatment with EWH; this was related to lower vascular reactivity in conductance vessels (Rizzetti et al., 2017a). A similar effect could be found in MRA in the present study to explain our findings. Previous studies showed that antioxidant EWH properties decreased plasmatic MDA levels and increased the levels of low glutathione in the liver of Zucker obese animals (Garcés-Rimón et al., 2016). Restored plasma antioxidant capacity was found in high-fat/high-dextrose fed rats with metabolic syndrome after EWH intake (Moreno-Fernandez et al., 2018a). Also, normalized oxidative biomarkers were reported in neurological, reproductive and cardiovascular systems from Hg-exposed animals after EWH consumption (Rizzetti et al., 2016a(Rizzetti et al., ,b, 2017a. In summary, EWH intake promotes protective effects against the endothelial dysfunction in MRA, and consequently, the increase in SBP of long-term HgCl 2 -exposed rats. EWH benefits could be related to its NO-induced vasodilatation capacity and its antioxidant and anti-inflammatory properties. EWH reduces high ROS generation by NAD(P)H oxidase and, possibly, the activation of inflammatory COX in MRA from Hg-treated rats. Our findings strongly suggest that dietary supplementation with EWH may represent an important strategy for counteracting the effects of cardiotoxicity by pro-oxidant agents such as heavy metals.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

ETHICS STATEMENT
The animal study was reviewed and approved by Ethics Committee on Animal Use Experimentation of the Federal University of Pampa, Uruguaiana, Rio Grande do Sul, Brazil (protocol 05/2014).

AUTHOR CONTRIBUTIONS
DR, FP, DV, MM, and GW conceived and designed the experiments. AE, DR, and JP performed the experiments. AE, DR, FP, and GW analyzed the data. DV, MM, and GW contributed reagents, materials and analysis tools. AE, DR, DV, MM, and GW wrote the paper. All authors contributed to the article and approved the submitted version.