The Effect of Gold Nanoparticle on Luteinizing Hormone, Follicle Stimulating Hormone, Testosterone and Testis In Male Rat. (2024)

- Biomedical Research (2015) Volume 26, Issue 2

Mohammadreza Behnammorshedi^*, Habibollah Nazem, Masoud Saleh moghadam

Department of Biology, Payame Noor University, I.R. of Iran

Corresponding Author:

Mohammadreza Behnammorshedi
Department of Biology Payame Noor University I.R. of Iran

Accepted date: February 09, 2015

Abstract

Nanoparticles for their special and unique features are widely used in different industries such as medicine and pharmaceutical. In addition to positive aspects of gold nanoparticles, its toxicity in environment is unavoidable. This study was conducted on 40 Wistar rat, divided in 5 groups including a control, a sham and three other experimental groups. The control group was treated daily with 0.5 cc saline solution, the sham group had food and water, and three other groups received intraperitoneal injection of 0.5 cc gold nanoparticles at different doses (100, 50, 25 ppm). After 10 days of treatment, animals were anesthetized; blood samples were collected from the heart and luteinizing hormone (LH), follicle–stimulating hormone (FSH), and testosterone (T) were measured. Testes were removed and preserved in formaldehyde (10%) solution. For statistical analysis, the analysis of variance (ANOVA) and Dunnett test were performed by SPSS software, version19. Results showed that gold nanoparticles have significant effect on hormones in concentrations of 100 ppm. LH, FSH and testosterone were significantly increased compared to (P<0.05). Seminiferous tubular degeneration was also observed in the testes. These findings suggest that gold nanoparticles may cause an increase in hormones in female and in male with subsequent increase in sterility.

Keywords

Zinc oxide nanoparticle, testosterone, LH, FSH

Introduction

Nanomedicine has wide range of applications. One of themost important difficulties of nano medicine is to understandits effectiveness on environment and also to determinethe possible toxicity of nanoscale materials [1]. Nanoparticles can be beneficial or be damaging at the same time.Materials could be toxic and hazardous when they are convertedinto nano form [2]. Additionally, the small size ofnanoparticles allows them to overcome the defense barriersof the body without facing any obstacles [3]. Nanoparticlescan be hazardous to health for two reasons; firstly, thenanoparticles can easily be absorbed through skin andepithelial cells and secondly, new and unknown nanoparticlesexert toxic effects. Nano particles are so small thatthey can penetrate cell membrane. Larger particles couldcarry a foreign substance between “DNA” strands [4].Nowadays, different kinds of coatings such as albumin,dextran, polyethylene glycol, polyethylene oxide, asparticacid, etc., are being used to exploit the beneficial applicationsof nanoparticles in biological systems. Using of suchcoatings can be resulted in the stability of nanoparticles inbiological solutions, in circulation, and their distribution intissues. Then can also facilitate entering nanoparticles intocells and reduce their toxic effects [5,6]. Gold nanoparticles range from 3 to 100 nanometers. These nanoparticlescan be used as potential detectors in designing of biosensors.The methods, which might be used are optical absorptionspectroscopy, florescence, Raman scattering, magneticforce and electric current methods.

These particles can be used to detect DNA, proteins, microorganisms,etc. Detection of DNA using Au nanoparticlesis 10 times more sensitive and 100000 times morespecific than the conventional genomic detection systems[7]. Goodman showed that cationic gold nanoparticles aremoderately toxic, whereas anionic gold nanoparticles arequite non-toxic [8], while gold nanoparticles of diameter1.4 nm are highly toxic [9], AU15MS compounds arequite non-toxic. Particles of 1-2 nm sizes have high toxicityfor all gold nanoparticles compounds and particleswith the sizes larger than 15 nm are moderately non-toxic.The nanoparticles centrifuged during a 24 hours period atthe temperature of 4°C produced a dark red solution.Nanoparticles of 12, 4, 18 nm diameter are non-toxic totreat leukemia cells [11]. The aim of present study was toinvestigate the effects of gold nanoparticles toxicity onthe changes in concentrations of luteinizing hormone(LH), follicle-stimulating hormone (FSH) and testosterone(TSS) in the blood and testes.

Materials and Methods

Materials

The materials used in this study consisted of goldnanoparticles, acetone, and ethanol. Chloroform whichwas used for the preparation of nanoparticles and othermaterials used in this study included saline, ketamine,hematoxylin, eosin (Merck, Germany), entellan glue(Merck, Germany), xylol (Part-Teb and Teb-Al-Sadiq,IR.Iran), methanol (Part-Teb and Teb-Al-Sadiq, IR.Iran),formaldehyde (Part-Teb and Teb-Al-Sadiq, IR.Iran) andexperimental kit (DB52181, Germany).

Apparatus

The device used in this study included XRTEM (JEM-200CX) and optical microscope (OLYMPUS CX 21 FS1,Turkey and Japan), centrifuge (Hettich,ununiversal), microtome(LIETZ 1512, Germany), tissue bath (BI, Germany),four1 (BEHZAD NOVIN, IR.Iran), and opticalmicroscope (OLYMPUS CX 21, Turkey and Japan).

Synthesis of gold nanoparticles

Trisodium citrate, a reducing agent was used as a nucleationfactor (Turkevich method). Usually a mixture ofHAuCl₃ is heated to boil, stirred and then the aqueoussolution of sodium citrate is added to it. The solution iskept at boiling temperature. Following the addition ofsodium citrate the purple color appeared, which is becomingred. A step called delay or induction phase is occurredbefore the change of color, which resulted in the formationof precursors such as actone decarboxylic acid that isa product of the oxidation of citrate.

Animals

Suspension of purchased gold nanoparticles was administeredto rats with different doses via oral gavage. Fourtyadult Wistar rats weighing 250-200 grams at the age of 8weeks were used to perform the test. The animals werepurchased from Pasteur Institute of Iran, Tehran, IR Iranand divided into 5 groups including controls and threeexperimental groups. Eight rats were included in eachgroup and all groups were kept in polypropylene cageswith sawdust-covered floors and water bottles. Animalswere kept under controlled conditions of temperature (22± 10 °C), humidity (60 ± 10%), and light (12 hoursbrightness, 12 hours darkness), with free access to waterand food (Figure 1-2). All the rats were kept in the nestwith similar conditions for 2 weeks prior to start of theexperiments for adaptiation to their environment and diet.All procedures followed were in accordance with theethical committee. Treated animals were marked by signsand were fed by oral gavage for10 days at the doses of100, 50, 25 ppm and grouped accordingly. To test thehormones by tumor markers, blood sampling was done after 10 days of treatment. Blood samples were takenfrom the heart. The samples taken from the animals wereplaced in the special centrifuge tubes. Testes were removedand placed in a formaldehyde (10%) solution andsent to the laboratory for hematoxylin-eosin staining.

Data analyses

Data obtained from Electrochemiluminesence device wassaved and analyzed using SPSS software, version19.Analysis of variance (ANOVA) test was performed bySPSS, and then the outputs were transferred to the Excelprogram. The results were presented as mean and standarddeviation (SD). The data were normally distributed,therefore, repeated measures ANOVA was used to comparethe enzymatic results among the groups before andafter the experiment. ANOVA and Dunnett tests wereapplied to compare the groups together in each periods oftime. The Level of significance was considered less than0.05.

Results

Electron microscope study of gold nanoparticles

Transmission electron microscope (TEM) is much largerthan the light microscope. In this microscope, unlike thelight microscope, the beam shines from top-to-bottom,however the principles of its operation are similar to thelight microscope. The TEM has a long column and electronbeam source is located at the top of this piston. Electronbeams after passing through the sample, hit a film ora screen (which is made of fluorescent material) and causingimage formation. Images of electron microscope areblack and white, because some beams do not pass throughthe sample and create black dots. Transmission eelectronmicroscope (TEM) image of synthesized gold nanoparticlesis shown in Figure 1. An increased surface to volumeratio of nanoparticles by reducing the size of the smallernanoparticles could play a good role in the immobilizationprocesses. According to the results of the TEM studies,the diameter range of 10 nm had been found for goldnanoparticles[12].

Analysis of LH

Table 1-3 show P > 0.05. However, no significant differencebetween sham and controls was evident. To comparethe LH levels, animals all groups were compared with thecontrol. The mean level of LH was increased in the animalsreceiving nanoparticles compared to the control.According to Dunnett's test for comparison, the level ofLH in animals receiving 100 ppm was significant comparedto the control (P = 0.004) (Fig. 2).

Analysis of FSH

No significant difference was evident between sham andcontrols (P > 0.05). Therefore, to compare the FSH levels inthe serum, all the animals were compared with controls. Themean level of FSH was increased in animals receivingnanoparticles in comparison with the controls. Table 4-3ahow comparison among groups (Dunnett’s test). The levelof FSH in the animals receiving 100 ppm was found to besignificant compared to the controls (P = 0.036) (Fig. 3).

Testis

In the control animals, no detectable changes were seen inthe interstitial cells of Leydig, seminiferous tubules, andtesticular cell lineages. In the animals, which receivednanoparticles at a dose of 100 ppm, degeneration ofseminiferous tubules was visible (Figure 3b).

Analysis of testosterone (T)

No significant difference was evident in animals betweenthe sham and controls (P > 0.05). T levels in the serum,animals of all the groups were compared with the controls.The mean level of T was found to be decreased withan increased dose of nanoparticles. The mean levels of Twere decreased in the animals those received nanoparticlesin comparison to the controls. According to Dunnett'stest for comparison among groups, the level of T in theanimals receiving 100 ppm was found to be significantcompared to the control. (P = 0.001) (Fig. 4).

Discussion

The science of nanotechnology is based on the fact thatmaterials reduced to the nanoscale possess special andextraordinary properties [24]. When the bulk materials areconverted into smaller structures or dimensions (such aslength, width or thickness) in the range of nanometers orless, unique particles with unpredictable properties couldbe significantly different compared to the bulk materialcharacteristics [13]. In a study conducted by Rezaei et al.,(2012), the effects of silver nanoparticles on LH, FSH andtestosterone in the male rats was investigated. The dosesused were 200, 100, 50, 25 mg per kg body weight perday orally for 28 days. In our study, significant changeswere not found in hormone concentrations up to 100 mg;however, a significant decrease in testosterone concentrationwas evident at higher doses (i.e. 200 mg) in comparisonwith controls, which could be caused by inhibitoryeffect of silver nanoparticles on the function of cells thatproduce testosterone. Results of out study also showedthat silver nanoparticles decreased the levels of LH, FSHand testosterone in male rats [14], Silver nanoparticlesalso reduced Leydig cell mitochondrial secretory activity.Furthermore, silver nanoparticles caused an increase inoxygen free radicals, such as superoxidase and increasedoxidation of molecules such as proteins [15].

Another study investigated the effect of silver nanoparticlesconsumption at concentrations of 95, 65, 35, 20, and5 ppm instead of drinking water. After 3 and 6 months, 3animals from each group were selected and the levels ofsex hormones were evaluated. It was found that the levelof testosterone increased with “testicl* hyperactivity”. Itwas also found that nanoparticles could cause alterationsin endocrine glands function [16] with decrease in thelevel of testosterone. Our study also determined the effectsof iron oxide nanoparticles on the number and motility of sperm cells. Seventy-five (75) male mice were dividedinto 5 groups with 15 mice each in control and 4expremental groups. Iron oxide nanoparticles dissolved indistilled water at doses of 40, 20, 10 and 5 mg / kg andinjected intraperitoneally. At the end of the experimentalperiod; spermatogenesis, sperm samples from the tail ofthe epididymedes were studies. Percentages of motile andimmotile spermatozoa, as well as sperm cells count weremade for each group of mice. Based on the results of thisstudy it could be suggested that the iron oxide nanoparticleshave a negative effect on sperm count and motilityand this negative effect is directly linked with higherdoses of iron oxide nanoparticles [17]. Carlson et al.,(2008) found that the nanoparticles can affect the leydigcell mitochondrial activity and thus reduce its secretionactivity. In addition, nanoparticles also caused an increasein oxygen free radicals, such as superoxidase and oxidationof molecules such as proteins [18], eventually reducethe leydig cell counts and decrease in the production oftestosterone, which is consistent with our findings. Anotherassumption is that the nanoparticles could affect thegene expression of the protein that is involved in thetransport of cholesterol into the inner membrane of mitochondriaand increased the synthesis of steroids. It is alsopossible that nanoparticles by reducing the gene expressionof the mitochondrial membrane protein Star, inhibitthe cholesterol transportation into the inner membrane ofmitochondria, and eventually inhibit the conversion ofcholesterol to pregnenolone and reduced the level of testosterone.Karpenko (2013) studied toxic effects of ceriumoxide nanoparticles on sex hormones and concludedthat nanoparticle reduced glandular and testosterone secretion,which was increased after 70 days of treatment[19]. Our study also showed that gold nanoparticles reducedthe level of testosterone.

Nonoparticles can penetrate the blood-testis barrier andadversely affect the sperm cells [20]. Researchers showedthat titanium oxide nanoparticles might produce infertilesperms and abnormal Leydig cells [21]. Nanoparticlescould also accumulate in the Leydig, sertoli, and spermatogeniccells [22].

Conclusion

According to the findings, it could be concluded that thenegative effects of nanoparticles are greater than the positiveeffects, but in general, regardless of the dual effects,the mechanism of their action is to some extent similar.Nanoparticles also showed significant negative effects onthe body. At the cellular level, the free radicals is increasedand caused significant tissue damage. Our studyshowed that different doses of gold nanoparticles exertnegative impact on testicular hormone secretion . By generatingfree radicals and reactive oxygen species,nanoparticles caused a considerable damage to the testis.

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