Histopathological and ultra structural effects of nanoparticles on rat testis following 90 days (Chronic study) of repeated oral administration
© Thakur et al.; licensee BioMed Central Ltd. 2014
Received: 13 August 2014
Accepted: 8 October 2014
Published: 14 October 2014
Nanoparticles (Ag NPs) have recently received much attention for their possible applications in biotechnology and biomedical. However, little is known about the toxicity in reproductive organs of animal model following exposure to nanoparticles.
This study therefore, tried to examine the effects of nanoparticles with a diameter range of 5-20 nm on the histology of the testis of wistar rats and correlate it with Transmission Electron Microscopy results.
Materials and methods
Sixteen wistar rats were randomly divided into two groups of 8 rats each. Each group received the following via gavage technique for 90 days: Control Group (Group-1)-tap water; Experimental group (Group 2) - nanoparticles (20ug/kg/day). After ninety days (chronic study), rats were sacrificed and testis tissues was processed for histology and transmission electron microscopic study.
There was significant difference between the observations of group-1 and group 2. The changes observed in the testis were disarray of the spermatogenic cells and disorientation of the testis. These changes were observed to have been disappearing from normal histological features. Detailed structural damages were observed with TEM analysis, such as depletion of germ cells, germinal cells necrosis, especially in spermatogonia and Leydig cells had an abnormal fibroblast-like appearance, abnormal space between neighboring sertoli cells, mitochondria, lost cristae and vacuolated (none energized) with those animals exposed to nanoparticles.
It seems that nanoparticles have acute and significant effects on spermatogenesis and number of spermatogenic cells. More experimental investigations are necessary to elucidate better conclusion regarding the safety of nanoparticles on male reproduction system.
Nanotechnology is a relatively new science; however it already has numerous applications in everyday life, ranging from consumer goods to medicine. Despite the wide application of nanomaterials, there has been a serious lack of information concerning the impact of Nanoparticles on human health and the environment. It is believed that the chemical nature, particle size, morphology, and surface chemistry of nanoparticles are key parameters that influence their toxicity, thus the field of nanotoxicology still lacks the necessary information and clarifications for achieving true risk assessment .
Nanoparticles (Ag NPs), is one of the most popular nanomaterial to have been used in material science, such as one of the constituent elements of dental alloys, catheters, implant surfaces and for treating of wounds and burns related infections, as well as in drug delivery in cancer and retinal therapies ,. But, there are some concerns about the safety of using nanoparticles in biomedical and in other industries ,. One concern is about the probable impacts of nanoparticles on remote organs.
Even though it is known that nanoparticles are able to penetrate into reproductive tissue through biological barriers, they may damage various cells: for example, they could reduce sperm numbers, viability and alter cell functions, as well as interfere with embryo development . Although the potential toxic effect of nanoparticles on the reproductive sphere is conceivable, further insights are needed in order to clarify this issue. Theoretically, nanoparticles may have some negative effect on human health and environment and their probable impact(s) on the male reproductive functions is remained to be clarified.
A study by Kim et al. reported, that male rats when treated orally with a varying dose of 56 nm nanoparticles for 13 weeks, resulted in decrease in body weight, accumulation of nanoparticles and enlargement of testis(particularly left), and some hepatocyte toxicity was also observed .
Further recent confirmation of the potential toxic effects of nanoparticles on spermatogenesis was reported in a study of intravenous administration of nanoparticles in male rats, which observed a size, dose and time-dependent decrease of the epididymal sperm count, increased levels of DNA damage in germ cells and a change in testis seminiferous tubule morphometry ,.
Studies on the oral effect of a fixed dose of 1 mg/kg with varying size of nanoparticles on rats for 2 weeks resulted in no significant change in body weight but accumulation in testes was found in rats treated with smaller sized nanoparticles. Study with a varying dose of 42 nm nanoparticles, both alkaline phosphatase and aspartate transaminase serum levels were found to be increased .
Finally, the question: 'How do nanoparticles cross the blood testis barrier (BTB)?, plays a vital role in explaining nanoparticles toxicity on spermatogenesis. In comparison with studies involving nanoparticles penetration of the blood brain barrier (BBB), studies involving the BTB are few. Despite the known toxic effects of nanoparticle (SNP), there are a few studies about its influence on reproduction. The motivation of our study was to examine the chronic effects of orally administered single dose of nanoparticles (SNPs) on rat spermatogenesis and seminiferous tubules morphology.
Materials and methods
Preparation of nanoparticles
Preparation of 5 ×103 mol/l of nanospheres (puress, Fluka) was done by citrate-reduction route  according to the following: 50 ml of 1 mM nitrate was heated to near boiling temperature followed by the addition of 10 ml of 41 mM trisodium citrate solution, stirred vigorously with continuous heating for about 6 minutes. Then, 25 ml of the stock solution was added to 100 ml of double distilled water. The solution was heated until it begins to boil where 5 ml of 1% of sodium citrate was added with vigorous magnetic stirring. Heating was continued till the color of the solution gradually changed to yellow. Then, it was continued for another 15 minutes, after which the solution was removed from the heater and stirred for a further 15 minutes. The electronic UV-Visible absorption spectra were recorded on a Thermo Scientific, Evolution 201 series spectrophotometer, using a quartz cell with a path length of 1 cm.
Transmission electron microscopy [Tecnai 12BT FE1 120 kV TEM] at a voltage of 120 kV was used to study the particles size, and morphology of the nanoparticles where the aqueous dispersion of the nanoparticles was drop cast onto a carbon coated copper grid, and the grid was air dried at room temperature before viewing under the microscope, and the diameter was determined from the micrographs.
Animal and treatment
Male wistar rats (150-200 gms & 10 -12 weeks old) of the wistar strain were obtained from the Animal House, Haffkin Institute. They were maintained on a standard pellet diet and tap water and were kept in polycarbonate cages with woodchip bedding under a 12:12 h light: dark cycle and room temperature 22-24°C. Rats were acclimatized to the environment for 2 weeks prior to experimental use. This study was conducted following the guidelines of the Animal Ethics Committee, MGM Institute of Health Sciences. Rats were randomly divided into 2 groups (n =08). The animals were assigned to each experimental group, with 8 animals per group. Animals in each group were marked with picric acid on their head, abdomen, tail, back, left upper leg and left hind leg for easy identification and were subjected to the following treatment:
Group 1 - Control group of animals were administered double distilled water orally using oral gavage technique once a day for 90 days.
Group 2 -Experimental group of animals were administered nanoparticles (20/kg of 5-20 nm particle size) orally using the oral gavage technique once a day for 90 days.
These studies were carried out at the Pathology laboratory of MGM College, Kamothe. At the end of the feeding schedule (90 days) the animals were sacrificed by an overdose of chloroform. The tissue of interest, testis, were immediately fixed in 10% buffered neutral formalin solution. The tissue embedded in molten paraffin with the help of metallic blocks, covered with flexible plastic moulds and kept under freezing plates to allow the paraffin to solidify. Cross sections (5 µm thick) of the fixed tissue were cut using microtome (Leica PM 2125, Germany). These sections were then stained with hematoxylin and eosin method (Gurr ) and visualized under light microscope (Magnus, India) to study the microscopic architecture of the testis.
Transmission electron microscopic analysis
Testis tissues was cut into small 1 mm3 pieces, and immediately fixed in modified Karnovsky's fixative (4% glutaraldehyde +4% paraformaldehyde +0.2% picric Acid +0.02% calcium chloride +0.2 M cacodylate buffer), dehydrated in graded acetone solutions, and embedded in resin (araldite). Rinsing, post fixation, dehydration and infiltration was carried out in the KOS microwave tissue processor. Semi-thin sections were cut at 1 µm in thickness with a glass knife using Leica Ultracut R Ultramicrotome. They were stained with 1% toluidine blue dissolved in 1% borax for approximately 30 m at 40-50°C, washed, dried and examined under the light microscope for general orientation. Ultra thin sections, below 100 nm in thickness were obtained from the selected areas using Leica Ultracut R Ultramicrotome (Leica Microsystems, Milton Keynes, England). The grids were then stained with 2% alcoholic uranyl acetate for 10 min in the dark, thoroughly washed in milliq water and allowed to air dry before examination. The sections were then viewed and photographed using the FEI TECNAI™ Transmission Electron microscope (120kv) TEM was carried out at National Institute For Research In Reproductive Health (NIRRH), an ICMR institute, Parel.
Synthesis of nanoparticles
Body and organ weights
No deaths were observed and no other signs of toxicity were apparent in male rats treated orally with nanoparticles (20 µg /kg body weight) for 90 days. All rats were weighed weekly and also testis from nanoparticles exposed rats showed no significant differences in weight in comparison to the control groups (data not shown). Furthermore, no behavioral differences were seen.
Control group/group 1
Experimental group (nanoparticles Treated/Group 2)
Group 2 rats fed with 20 /kg/day for 90 days showed following observations:- H & E stained sections of the testis in group 2 treated with nanoparticles (Figure2c) showed histological changes that were at variance with those obtained in the control. There was disorganization of the normal appearance of the testis with overall different degrees of atrophy in the seminiferous tubules. At the same time a disorganization of the germinal epithelium, with loss of the spermatogenic cells specially spermatocytes and spermatids and exfoliation of the germ cells was also observed. In the seminiferous tubular lumen and almost all tubules showed severe atrophy as they were devoid of epithelium, with only sertoli cells and spermatogonia present within the depleted tubules. Sperms were hardly seen. The spermatogenic cells showed degeneration and/or necrosis. Testis treated with nanoparticles confirmed the atrophic changes found in the capsule and wall of the seminiferous tubules which was observed in H & E stained sections.
Electron microscopic observation
Basal lamina and spermatogonia
Sertoli cell cytoplasm and nucleus
Spermatocytes and round spermatids
Elongating and elongated spermatid
Several particle features, such as type, size, zeta potential, dispersion/agglomeration status, as well as potential interaction with biomolecules, influence nanoparticles toxicity and hence their effects in humans. Generally, size (and hence the surface/mass ratio) has been considered as the most important factor for the toxicity of nanoparticles . In the present study, we attempted to use a nanosize particle to study the toxicity. Chithrani and Chan  reported that small particle size does not necessarily lead to better uptake and hence increased toxicity . Additionally, it has been reported that the surface chemistry of nanoparticles influence interparticle interactions, hence particle distribution and in effect transport across membranes  and genotoxicity .
To the best of our knowledge, this work is the first to investigate the effect of bio distribution of nanoparticles in testicular cells of wistar rat having exposure for 90 days. No abnormal effects like physical, body weight and behavioural were noted in both control and treated male rats. Previously, the liver and kidneys have been described as the primary organs for distribution, whether the exposure was orally ,-, intravenously ,, subcutaneously  or through inhalation . Two other oral exposure studies reported high distribution of to the testis for 28 days exposure and sub-chronic ,,.
Data from the present study suggest that continuous exposure of male rats to nanoparticles for 90 days causes histopathological changes such as epithelial cell sloughing, atrophic changes and decrease in germ cell numbers due to cytotoxicity were the factors seen in treated animals. Degenerative changes in the seminiferous tubules and decrease of spermatozoa in the testis, epididymis and vas deferens are the evidence for genotoxicity. Degenerative changes in the seminiferous tubules indicate that nanoparticles may directly interfere in the process of spermatogenesis.
In 2011, Tiwari and colleagues used 4 doses of nanoparticles 4, 10, 20 and 40 mg/kg which were injected intravenously and they concluded that nanoparticles in doses less than 10 mg/kg are safe for biomedical application and has no side effects, but doses more than 20 mg/kg are toxic . This study is contradictory to our findings but cannot be held against as the size range of the nano particles used is not mentioned and dose used in the study was different than that used in our present study. It may be emphasized that even a low dose of SNP (20 ug/kg) ,, as used in the present study, is capable of produced ultra structural changes. However, It would have been interesting if these ultrastructrual changes could be correlated to any functional changes if any produced by these nanoparticles. However, in absence of such data, it may be speculated that it is the size rather than dose that has a major impact on the degree of tissue damage.
However, another study mentioned that after 2 weeks of intravenous administration of nanoparticles in mice, no obvious acute toxicity were observed with the dose of 30 mg/kg, while inflammatory reactions in lung and liver cells were induced in mice treated with the dose of 120 mg/kg . It should be added, that besides the doses and size of particles, the duration of exposure and route of administrationof nanoparticles may be responsible for the large variation in the toxicity profile of nanoparicles observed in various studies . The oral route should cause less toxicity as compared to the parenteral routes of administration as the bioavailability of nanoparticles would be the least via this route. The oral route exposes the nanoparticles to thigh first pass metabolism resulting in low systemic levels that may produce toxicity subsequently.
Spermatogenesis is a highly orchestrated and dynamic process resulting in the continual production of spermatozoa in mammals. Our transmission electron microscopic results indicated that severe cellular changes occurred in the cytoplasm of spermatogenia, primary and secondary spermatocytes, round and elongating spermatids and Sertoli cells in the experimental group, which indicates the alterations in the function of these cells . Sertoli cells from the blood-testis barrier which is important in maintaining sperm formation and the micro-environment around the germ cells. They also involved in production of of growth factors and nutrients , which serve as energy sources and aid the maturation of sperm that enter into the lumen. In our study, we found huge accumulation of nanoparticles in the sertoli cell cytoplasm which might have induce toxicity in these cells which would have affected the nutritional intake and normal maturation of spermatogenic cells at various stages. Nanoparticles also induced degenerative changes in basement membrane which maintains the structural and functional integrity of testicular tissues. Altered basement membrane structure might lead to severe functional impairment of the testis . The ultra structural observations suggested that all animals exposed to nanoparticles had apoptosis in different germinal cells such as spermatogonia, spermatocytes and spermatids leading to depletion of germ cells. The germ cells apoptosis that occurred in the testicular epithelium served as a major cause to reduce the germ cell population in the nanoparticles treated animals. Nanoparticles injured or disrupted the functions of basement membrane, sertoli cells and spermatogonial cells resulting in an increase in the elimination of the germ cell numbers via apoptosis . Observation of detached germ cells, deformed spermatocytes and spermatids, may be due to the marked disruption of the sertoli-germ cells interaction. The disruption in this physical interaction might have led to the sloughing of the germ cells from seminiferous epithelium .
The present Study clearly indicates that nanoparticles (20 ug/kg) have deleterious effects on testicular structure of rats. Ultra structural observations presented the evidence of severely impaired and apoptotic germ cells in the testis. Therefore, nanoparticles cannot be considered as a therapeutic hope drugs that require exposure for longer duration. Further studies are required for dose lower than the one used in the present study (20 ug/kg) for strengthening this observation. From the results of this study, we concluded that nanoparticles led to a deleterious effect on the testis that increased progressively with time. The results obtained from this study provided important clues about the impact of nanoparticles on reproductive health and would be of great value in assessing its health hazards.
This research was supported by the MGM Institute of Health Sciences (MGMIHS) and National Institute of Research in Reproductive Health (NIRRH).
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