Research Article

Impact of Manganese Cobalt Ferro-Magnetite Composite Nanoparticles on Hypothalamic- Pituitary-Gonadal Axis Disturbance Induced by Vanadium Exposure in Adult Male Rat

Mohamed M Rezk* and Tarek F Mohammaden

Department of Isotopes Geology, Nuclear Materials Authority, Egypt

Submission:May 01, 2019;Published:July 11, 2019

*Corresponding author:Mohamed M Rezk, Department of Isotopes Geology, Nuclear Materials Authority Cairo, Egypt

How to cite this article: Md M Rezk, Tarek F M. Impact of Manganese Cobalt Ferro-Magnetite Composite Nanoparticles on Hypothalamic-Pituitary-Gonadal Axis Disturbance Induced by Vanadium Exposure in Adult Male Rat. Open Acc J of Toxicol. 2019; 4(1):555629. DOI: 10.19080/OAJT.2019.04.555629.

Abstract

Vanadium which used in various industrial applications, might lead to pathological and irreversible damage effect in tissues and organs where it can accidently enter. Rats were distributed into four groups discriminated as; control, vanadium (V), manganese cobalt ferro-magnetite composite nanoparticles (MNPs) and V-MNPs. The rats underwent four decapitations after the 3^rd, 7^th, 10^th and 15^th day. The oral administration by 20mg/Kg b.wt. of vanadium lead to its accumulation in hypothalamus brain area and tests causing a disturbance in hypothalamic-pituitary-gonadal axis appears in the significant decrease of testosterone, FSH and LH hormone levels and GSH level, also in the significant increase of MDA level which may backed to vanadium ability to inhibit the adenylate cyclase, disturb the mitochondrial function and down-regulated the steroidogenic enzymes. On the other hand, the IP administration of 5% MNPs showed its hormone enhancement ability for testosterone, FSH and LH which appears at the end of the experiment, also it induces a significant decrease in MDA and increase in GSH levels. MNPs effects could be due to the essentiality of the three involved metal oxide to the body. So, The Mn-Co-Fe nanoparticle seems as good candidate performs preventive and/or reactionary roles against vanadium exposure hazards.

Keywords: Vanadium; Ferro-Magnetite; Nanoparticles; Rats, Hypothalamus; Tests

Introduction

Vanadium occupies the 21^st rank of the elemental abundance in the Earth’s crust and used in various applications such as; steel fabrication, oil industry, energy storage etc.… [1]. However, receiv­ing high and undesirable doses of vanadium might lead to patho­logical and irreversible changes in tissues and organs where it can enter the body via the lungs and, more commonly, the stomach. When it reaches the blood, vanadium is distributed to the body tissues and bones leading to some gastrointestinal symptoms e.g. diarrhea, vomiting, general dehydration with weight reduction, intestinal inflammation and the characteristic green tongue [2] Other bio-hazards include; hematological and biochemical alter­ations [3], loss of body weight and nephrotoxicity [4] and immu­notoxicity and behavioral toxicity [5]. Moreover, vanadium has been classified within the group 2B carcinogen [6].

Recently, utilization of metal and metal-oxide magnetic nanoparticles for biohazard and biomedicine treatment attracted more attention particularly they are easily cleaned by the body macrophages which engulf these magnetic nano particles (MNPs) via the phagocytosis [7,8].

The ferromagnetic transition metals oxides (ferrite) are typ­ical magnetic nano-particles (MNPs), its properties can be en­hanced by its conjunction with other divalent metallic ions [9]. Iron, cobalt and manganese are essential trace nutrients play an important role in the general health and fertility. Iron (Fe) is a physiological component in the cells and tissues of the male re­productive system and about 70% of the total- body iron is associ­ated with hemoglobin [10]. The major biological activity of cobalt (Co) is synthesizing of vitamin B12 as well as a small number of other cobalt-containing enzymes [11] Manganese (Mn) metal is required for normal mammalian physiological functions, where it is a cofactor for varieties of brain enzymes such as; glutamine synthetase and mitochondrial superoxide dismutase as well as transferases and hydrolases. Also, it is necessary for the normal growth and development of bone, cartilage, connective tissue, and the reproductive system [12].

The present study was designed to investigate the toxic haz­ards effects of vanadium ions administration (as ammonium metavanadate, Am.V) on the hypothalamic-pituitary-gonadal axis and reproductive system and to demonstrate the attenuation and mitigation effects of Mn, Co, Fe composite nano-particles (MNPs) regarding the hazardous which raised by vanadium administra­tion.

Materials and methods

Materials

a) The employed dose of Am.V was 20mg/kg. rat that meets 1/5 of LD50 [13] and was prepared by dissolving the adequate weight of Am.V (Sigma Co.) in suitable volume of the double distilled water.

b) For ferrite (Co_0.5Mn_0.5Fe₂O₄) preparation, the ferric ni­trate (Loba Co.), Cobalt nitrate (Merck Co.), Manganese nitrate (Sigma Co.) were mixed in stesiometric amounts, then citric acid solution was addded to the solution (molar ratio of 1:1.5) to synthesiz the desired MNPs using the non-conventional ci­trate precursor method, where every 5g of the prepared mix­ture were suspended in 100ml saline to get 5% MNPs solution [14].

Animals grouping and treatment

Rattus rattus rats (110±20g) were purchased from the Re­search National Center, Giza, Egypt. They were maintained under standard laboratory ambient conditions and housed in cages (six rats/cage) at a constant temperature (approximately 25°C) with 12h light/dark cycle. Food and water were given ad libidum. The ethics-rule of animals using and care was followed in this study.

Rats were randomly grouped into four groups; G1 (the con­trol group), G2 (animals were daily oral ingested by stomach tube with 1ml Am.V by dose 20mg/kg. B.wt. for two weeks), G3 (animals were daily IP injected for two weeks with 1ml of 5% of MNPs), G4 (animals were daily oral ingested by the used dose of Am.V accompanied with IP injection of 5% MNPs for two weeks). The rats of the different groups were decapitated after the 3^rd, 7^th, 10^th and 15^th day (6 rats/period) during the treatment process.

The hypothalamus was divided into two hemispheres, also the testes were separated after the rats’ autopsy [15] and each part was separately weighted and stored at -30 °C till using. Blood samples were collected after decapitation and centrifuged after standing for 15 minutes in a water bath at 35°C and finally stored at -30°C till using.

Vanadium Estimation

To estimate vanadium in the desired tissues, one hypothala­mus hemisphere and one teste were separately placed in Teflon beakers with equal volumes of nitric acid (90%), perchloric acid (70%) and hydrogen peroxide (32%) then, the mixtures were heated on hot plate at 130°C till complete dryness. After cool­ing, the contents were transferred into 25ml volumetric flasks and diluted up to volume using double distilled water [16,17]. In the resulted solutions, vanadium was measured by the graphite atomic absorption spectrometer (AAS; Thermal-Jarrel Ash Model 12 Spectrophotometer). Vanadium concentration (in μg/g tissue) was determined using the Fisher Certified Standard diluted to ap­propriate concentrations in 2% HNO₃.

Hormones Estimation

Using the radioimmunoassay (RIA) and according to the method described by [18] the Testosterone, FSH and LH were measured

Estimation of Brain Reduced Glutathione (GSH) And Malondialdehyde (MDA)

The second half of hypothalamus and the other teste were individually homogenized in ice-cold isotonic potassium chloride (1.2%) then centrifuged for 10minutes at 3000rpm where the GSH and MDA were determined in the clear supernatant accord­ing to methods described by [19,20] respectively.

Statistical Analysis

The obtained data were statistically analyzed using one-way analysis of variance (ANOVA) by the statistical package for the so­cial science (SPSS) program version 20.

Results

Comparing to the control (G1), vanadium content gradually increased in both G2 and G4 reaching its maximum value at 15 days. On the other hand, vanadium content showed a significant decrease in G4 in relative to G2 at each decapitation that point to the effect of the contemporaneous administration of MNPs with Am.V (Figure 1).

Referring to the testosterone, the continuous Am.V admin­istration (G2) accompanied with gradual decreasing in its level reaching the minimum level on the 15^th day. In G3, the testoster­one level revealed faint increasing particularly with the 15th day. On the other hand, the testosterone level in G4 went downward till the 7^th day then, turned to the gradual increasing until ending of the experiment period (Figure 2A). To great extent, the FSH lev­el similarly behaved as the testosterone except the non-significant change at the 7^th day in G3 (Figure 2B). Comparing to control (G1), the LH hormone level showed a similar trend like the FSH in G2 and G3, but in G4 its level continued in decreasing with increasing of the administration time and reached the minimum level on the 15^th day (Figure 2C).

In general, the mitigation effect of MNPs on the investigated parameters is clearly observed when we take in account the com­parison between G2 and G4.

Comparing to control (G1), the GSH level showed a marked decreasing in the 2^nd, 3^rd, and 4^th decapitations of G2 and this de­creasing was more significant with the continuous Am.V admin­istration. The same behavior was observed in G4 but with less­er decreasing particularly on the 3^rd and 4^th decapitations which reasonably attributed to the enhancement effect of MNPs (Figure 3A)., while no significant change was observed on G3 (Figure 3).

On the other hand, the testes MDA level showed significant increasing in both G2 and G4 while approximately no change was observed in G3(Figure 3B). It is worth to mention that increasing of MDA under the effect of parallel Am.V and MNPs administration was lesser than its increasing under the effect of the Am.V admin­istration only.

Relative to the control (G1), the hypothalamus GSH level suf­fered continuous decreasing with the Am. V. administration (Fig­ure 4A) while in G4 (Am. V.+MNPs administration), the GSH re­stored its level in the 1^st and 2^nd decapitations approximately as was in the control group but unexpectedly it backed to decreasing in the 3^rd and 4^th decapitations. However, its level in G4 was always greater than in G2. In G3 (MNPs administration), the hypothala­mus GSH level has no significant change comparing to the control over all the experiment period.

Referring to the hypothalamus MDA level, all the administra­tion (G2, G3 and G4) showed no significant changes relative to the control (Figure 4B). While the MDA level was steadily increased from the 2^nd to 4^th decapitations but its level showed no chang­es in G3 over the experiment period. Although it did not impose an effect on the MDA level as the unique administration, but the MNPs caused decreasing of MDA level in G4 versus its level in G2 (Figure 4B).

Discussion

With the physiological conditions, a considerable proportion of vanadate (V⁺⁵) is reduced to vanadyl (V⁺⁴) under glutathione ef­fect until reaching to the equilibrium balance between the two va­nadium species [21]. Vanadium can exist as cationic, neutral, and anionic species depending on the initial metal concentration and the medium pH.

In this work the oral administration of vanadium showed its ability to enter and accumulate in the hypothalamus brain area as well as in the testes of the adult male albino rats which agrees with other studies demonstrated the capability of vanadium to access the blood-brain barrier and accumulate in rat’s brain as well as in its testes [22-24]. Also, the ability of the individual magnetite, cobalt and manganese nano particles to access the blood-brain barrier were noticed by many studies [25-29].The significant de­creasing of vanadium ions content after the IP injection of MNPs with vanadium gavages could be ascribed to complexation and/or the electrostatic interaction between the adsorbent (MNPs) and the adsorbate vanadium species [30].

It is Known that the hypothalamic-pituitary-gonadal axis is starting by the secretion of gonadotrophin hormone from hypo­thalamus, which stimulates the anterior pituitary gland to secrete FSH and LH, then LH together with testicular auto- and paracrine factors is responsible for the regulation and the balance of the sex hormone production and gametogenesis [31].

Several reasons can explain the negative effect that imposed on the investigated hormones by vanadium exposure and accu­mulation. Decreasing of FSH, LH and Testo levels are functions for the hypothalamic-pituitary-gonadal axis disturbance that might be caused by vanadium administration due to its redox reaction leading to tissue damage [32]. Also, the hormones decreasing could be ascribed to ability of vanadium to inhibit Adenylate cy­clase which aids in formation of the second messenger cyclic ad­enosine monophosphate (cAMP) that performers an important role in controlling the cells responsiveness to the hypothalamic hormones [33]. Moreover, declining of FSH, LH and Testo is rea­sonably happened due to the down-regulation of the steroidogen­ic enzymes (17b-HSD, 3b-HSD) by the effect of vanadium admin­istration where those enzymes have a major role the hormones formation [23] Finally, vanadium could induce a mitochondrial over oxidation affecting on the cytochrome P450 substrates that have a regulatory function in Leydig cells [34-36].

As a consequence of vanadium accumulation in the hypo­thalamus and testes, a significant elevation in MDA level together with a significant decrease in GHS level were observed. Such dis­turbance in the hypothalamic-pituitary-gonadal axis is likely as­cribed to the oxidative stress and tissue damage happened due to the effect of vanadium poisonous. One or more mechanism can be involved in this disturbance. Like other heavy metals, poisoning of vanadium results in tissue damage and production of reactive ox­ygen species (ROS) causing a peroxidation of structural lipids and an alteration of the antioxidative activity of enzymes like SOD, CAT and GPX [37,38]. The high polyunsaturated fatty acids of the brain and the testes are vulnerable to the free radical attacks hence, the hormones levels suffer disturbance [39], Gutteridge [40-42,24]. Moreover, the vanadium IP injection may also be involved in the endocrinological processes and disturbs them [24] as well as af­fects the thyroid function, growth, gonadal function, adrenal hor­mones, prolactin, glucose homeostasis, calcium-phosphorus me­tabolism or thymulin activity [32].

On contrary to the effect of vanadium, the IP injection of MNPs resulted in significant increase in hormones (FSH, LH, Testo) as well as the GSH level in both hypothalamus and testes, while de­creasing of MDA level in both organs were observed.

The essential roles of Fe, Mn and Co in several body’s biologi­cal functions were the corner stone in their selection in this work. Iron has many diverse biological functions, including reversible binding of gases, enzyme catalysis, and electron transport. Fur­thermore, iron plays an important role in developing spermato­zoa and providing an extra layer of protection to the testicular tissue where it is found in a large quantity in Sertoli and Leydig testes cells [43,44] and enhances the spermatogenesis process [45,46]. Also, as the iron quantified in bovine seminal plasma as the sperm’s motility characteristics getting better [47,48].

Manganese is an essential nutrient necessary for varieties of metabolic functions including those involved in normal human development, activation of certain metalloenzymes (Prasad et al., 2014), nervous system function, reproductive hormone function and the antioxidant enzymes that protect cells from damage due to free radicals [49] IOM(2011) [50]. Also, manganese has the ability to stimulate secretion of the rat LH [25]. On the other hand, cobalt contributes in the cobalamin formation which is the func­tion unit of Vit B12. This vitamin increases the sperm production in men who have low sperm counts, increases the functionality of reproductive organs and decreases homocysteine toxicity [22] and decreases the inflammation-induced semen impairment by controlling nuclear factor-κB activation [51-53].

Conclusion

According to the executed work and the conducting results in this study, we can come to some conclusions.

a) Exposure to vanadium doses greater than the permissible levels disturbs the Hypothalamic-Pituitary-Gonadal Axis and testes leading to verified health risks.

b) The Mn-Co-Fe composite nanoparticles was presented as new effective ferrite (MNPs) able to enter the hypothalamus and testes enhancing their performance and mitigating the disturbance they were suffered due to vanadium pollution.

c) Further researches on this MNP are recommended to inves­tigate its capabilities in treating with other metals poisoning pollutants which could affect the biological functions of the different organs.

The Mn-Co-Fe composite nanoparticle seems as good candidate performs preventive and/or reactionary roles against vanadium exposure hazards for workers in facilities or activities containing vanadium handling.

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