Efeito do consumo de hidrolisado de clara de ovo sobre as alterações neurológicas, reprodutivas e cardiovasculares promovidas pela exposição crônica ao cloreto de mercúrio (HgCl2) em ratos

Abstract

Mercury (Hg) is a trace metal released into the environment implicated in the oxidative damage on several systems. In this context, the bioactive peptides from egg white hydrolysate protein (EWH) present antioxidant, free radical scavenging, anti-inflammatory, vasodilator and angiotensin-converting enzyme (ACE) inhibitory properties and may act on many diseases. Objective: This study aims to investigate whether the EWH intake acts on neuropathic and memory disorders, reproductive changes, hemodynamic and vascular dysfunctions induced by chronic intoxication to low concentrations of Hg. Material and Methods: Four groups of 8-week-old Wistar male rats were treated for 60 days with: a) Untreated (saline solution, i.m.); b) Mercury (mercury chloride, i.m. - 1st dose 4.6 μg/kg, subsequent doses 0.07 μg/kg/day); c) Hydrolysate (EWH, gavage - 1 g/kg/day); d) Hydrolysate-Mercury. To evaluate the peripheral nervous system, mechanical allodynia was assessed using Von Frey Hairs test; heat hyperalgesia by the plantar test; catalepsy by a modification of the “ring test” and spontaneous locomotor activity by a photocell activity chambers. Analyses were performed at 0, 30 and 60 days of treatment. Brain and plasma MDA, plasma NPSH and TNF-α determination and skin immunohistochemistry were performed at 60 days. To investigate central nervous system, object recognition memory test (OR) was performed to verify Short (STM) and Long-Term Memory (LTM) and Open Field (OF), Plus Maze (PM) and Tail Flick (TF) tests were performed as control for behavioral experiments. Reactive Oxygen Species (ROS) in hippocampus were determined by dichlorofluorescein diacetate (DCFH-DA) method, malondialdehyde (MDA) levels by TBARS, antioxidant power by FRAP assay and total Hg concentration by atomic fluorescence spectrometry. Histological studies in hippocampus were carried out in formaldehyde fixed sections. To analyze the reproductive system, sperm motility and count, daily sperm production and morphological studies were performed. ROS levels, lipid peroxidation and antioxidant capacity were assessed in testis and epididymis. Histological studies on testis and epididymis and immunohistochemical assay in testis were also carried out. Finally, to assess the cardiovascular system, indirect systolic blood pressure (SBP) was performed by tail-cuff plethysmography and direct SBP and diastolic blood pressure (DBP) by carotid cannulation. The vascular reactivity experiments in aorta rings were performed in an organ bath, where we analyzed the endothelial dependent and independent vasodilator responses to acetylcholine (ACh) and sodium nitroprusside (SNP) and the vasoconstrictor response to phenylephrine (Phe) in the presence and absence of endothelium, in the presence of NOS inhibitor (L- NAME), NADPH oxidase inhibitor (apocynin), superoxide dismutase (SOD), non-selective COX inhibitor (indomethacin), selective COX-2 inhibitor (NS 398), AT-1 receptors blocker (losartan). In situ superoxide anion production by the oxidative fluorescent dye dihydroethidium (DHE), SOD-1, NOX-4, p22phox, COX-2 and AT-1 mRNA levels by the quantitative PCR real time (qRT-PCR) and NOX-1 expression by the western blot were also performed in aorta tissue while the determination of angiotensin converting enzyme (ACE) activity by fluorimetric method was measured in plasma. Results: At peripheral and central nervous system, Hg induced a reduction in mechanical sensitivity threshold at 30 and 60 days and in thermal sensitivity threshold at 60 days. At the end of treatment catalepsy was developed, but there was not significant alteration in spontaneous locomotor activity. Hg also increased brain and plasma MDA, plasma NPSH and TNF- α levels and the number of Merkel cell–neurite complex in the skin. EWH prevented the development of mechanical allodynia, thermal hyperalgesia and catalepsy induced by Hg and the increase in MDA concentration in brain and plasma and in the number of Merkel cell–neurite complex in the skin. Indeed, we confirmed that the STM and LTM were impaired in adult rats exposed to Hg at low concentrations and proved that this damage is related to increased metal deposition and subsequent ROS production and apoptosis in hippocampus. In addition, we demonstrated for the first time that EWH treatment is able to prevent memory impartment induced by Hg exposure reducing Hg content, ROS production and cell death in hippocampus. At reproductive system, Hg decreased the testicular and epididymal sperm number, as well as increased sperm transit time in epididymis. The metal also diminished the percentage of morphologically normal spermatozoa compared with untreated group. This harmful effect was prevented by the EWH intake, and the Hydrolysate-Mercury group showed normal sperm function. HgCl2-treatment also increased ROS levels, lipid peroxidation and antioxidant capacity in testis and epididymis. In addition, inflammatory alterations in testis and histological changes in testicular and epididymal tissues were observed. EWH improved histological and immunohistochemical changes, probably due to antioxidant and anti-inflammatory activities. At vascular system, Hg treatment resulted in an increase in SBP, aortic vasoconstrictor response to Phe and a decrease in vasodilator response to ACh; the increased involvement of ROS from NADPH oxidase, constrictor prostanoids, mainly from COX-2 and angiotensin II in response to Phe, whereas the endothelial NO modulation of such responses was reduced; an increase in the NOX-4, p22phox, COX-2 and AT-1 mRNA levels and a decrease in the NOX-1 expression in aorta and increased ACE activity in plasma. Treatment with EWH prevented the increase in SBP and Phe responses and the endothelial dysfunction elicited by Hg in aorta. These vascular improvements were related to the decreased ACE activity and NOX isoforms activation by the EWH intake, resulting in alleviated ROS production and increased NO bioavailability in aorta. Conclusions: The EWH could be considered an ingredient for functional foods and could be used as alternative or complementary treatment tools for Hg-induced damage.