International Journal of Medical Dentistry 179 Oral biology
THE EFFECTS OF METAL NANOPARTICLES ON EMBRYOS OF
DIFFERENT ANIMAL SPECIES. A
REVIEW
A. TEUŞAN1, N. BARANOV2, R. DMOUR3, A. VIZITIU4, I. JELIHOVSCHI5, V. TEUŞAN6
1S.R. BiologistPhDResearch Dept., Apollonia University of Iasi, “Ion Haulica”Research Institute 2PhD student Periodontology Dept., Apollonia University of Iasi
3PhD student Pharmacology Dept.,“Gr. T. Popa” University of Medicine and Pharmacy of Iasi 4Assistant PhD student Periodontology Dept., Apollonia University of Iasi,
5PhD student Microbiology Dept., “Gr. T. Popa” University of Medicine and Pharmacy, Iasi
6Profesor PhD Anatomy and Embryology Dept., University of Applied Life Sciences and Environment, Iasi Corresponding author: anca.teusan@yahoo.com
Abstract
Today nanotechnology represents a domain that is rapidly developing because nanoparticles are being used in a very large range of products with biomedical applications. Every year, new products, containing nanoparticles (NP) appear on the market. Most of the products containing such nanomaterials come to be used by consumers without a previous and careful testing. Therefore, the effects they may have upon human health should be thoroughly investigated, the toxicological potential of NP upon the reproduction function (nanoreprotoxicity) in particular, as any possible noxious effect will be relected in the new generation. Most of the research papers that exist refer on the effects of silver, gold and titanium dioxide NP on embryo development. In this review paper we present the effects of less studied metal NP (platinum, aluminium, cerium oxide, tin oxide, nickel and indium) on different species of animal embryos (Gallus domesticus – different hybrids, Danio rerio and Xenopus laevis)
Keywords: nanoparticles, embryo, nanotechnology, nanoreprotoxicity.
Nanoparticles (NP) are characterized by very small sizes, ranging between 1 and 100 nanometers (nm) and various diameters and forms. As a consequence of their low dimensions, the total surface of the particles is maximized;
the irst studies developed in this ield were
initiated in the 80’ies, in USA and Japan.
Nowadays, nanotechnology is a rapidly developing domain, as nanoparticles are largely utilized in quite various areas.
Metallic nanoparticles play an increasingly important part in the life of people, being used in a very large range of products with biomedical applications. Each year, new products, containing nanoparticles of various forms and sizes, appear
on the market. Most of the products containing such nanomaterials come to be used by consumers without a previous and careful testing.
Therefore, the effects they may have upon human health should be thoroughly investigated, known and understood, if considering mainly the toxicological potential of NP upon the reproduction function (nanoreprotoxicity), as
any possible deficiency/noxious effect will be relected in the new generation.
The present study reviews the investigations
registered up to now in this ield – namely testing
of metallic nanoparticles on the embryos taken over from different animal species. The main species involved in such experiments are birds,
rats and guinea pigs, ish and batrachians.
The toxic effects of metallic NPs are caused not only by their chemical composition, but also by the size, form and surface of particles, by their charging with different substances and by the experiment itself [1].
The conception period represents a critical moment, due to the occurrence of a restructuration process at both molecular and cytological level, so that the forming organism is sensible to any
substance that may cause modiications. More
than that, due to their extremely reduced sizes, nanoparticles may easily penetrate the biological barriers – the cellular membranes, thus
inluencing the viability of the reproductive cells
and embryo development.
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A. TEUŞAN, N. BARANOV, R. DMOUR, A. VIZITIU, I. JELIHOVSCHI, V. TEUŞAN
help the companies producing metallic NPs, while also facilitating the production of particles with a high biocompatibility level [1].
Up to now, several scientiic papers referring
to the effects of nanoparticles upon embryonic development have been issued, the biological materials used including fecundated eggs of various animal species. The most frequently studied nanoparticles are those of gold, silver, titanium dioxide, zinc oxide, a.s.o.
Less tested have been, for example, the platinum nanoparticles. However, a research team from a Dannish university tested this type of nanoparticles on bird embryos (Gallus domesticus – the ROSS hybrid). The study focused on the evolution and development of embryos’ brain, upon which an in ovo intervention had been performed. The quantities used by this team
ranged between 1 and 20 µg/ml, and the diameter
of platinum nanoparticles was between 2 and 19 µm. Inoculation was made in the median region of the egg, the nanoparticles being introduced directly in the albumen (white of the egg) [2].
The results of such experiments showed that platinum nanoparticles did not affect the growth and development of hen embryos, however a phenomenon of cell apoptosis was observed at the level of the brain nervous tissue. Scientists consider that such experiments should be repeated, because these platinum nanoparticles can be loaded with cytostatic drugs for the treatment of brain cancer, so special care should be given to their possible toxic effects.
Another less studied type of nanoparticles refers to the copper ones. The studies made by a research team from Philippines [3] organized an experiment to investigate the effects of copper nanoparticles upon the metabolic rate of bird embryos. In this respect, fecundated eggs from the Lohmann hybrid have been used. At the end of the experiment it was observed that the inoculation of copper nanoparticles in suspension during several days of incubation caused a
signiicant decrease of oxygen consumption in
the experimental groups, comparatively with the control ones, while lipid oxidation was much more reduced than in normal situations. Also, the weight of some internal organs (heart, liver, intestines) was lower in the experimental groups, comparatively with the reference. As a conclusion,
the results of such an experiment show that, whichever the selected inoculation day, copper nanoparticles (CuNanoP) modify the metabolic rate of embryos and affect the normal weight of the internal organs, but neither immunoglobulin concentration nor its gene expression is affected.
Another species selected by researchers for studying the effects of metallic nanoparticles is
the zebra-ish (Danio rerio) from the Cypriniformes
order, Cyprinidae family. It is a small-size ish, grown in aquvarium, preferred for scientiic
investigations because its females may lay hundreds of eggs, within intervals of 1 up to 3 days, and also because this species has similar genes with the human ones.
Therefore, a team from the College of Environmental Science and Engineering of Tianjin, China, developed an experiment to determine the ecotoxicological potential of three types of NPs of metallic oxides released in the aquatic medium. The researchers exposed the embryos to NPs of zinc oxide (ZnO), titanium dioxide (TiO2) and aluminium (Al2O3), for 96 hours, and observed the effects produced, on following: the survival ratio of embryos, the hatching ratio and the malformations manifested
in ish spawn [4]. The experiment showed that
NPs affects aquatic organisms in a different manner, depending on their chemical composition. All types of NPs tested manifested toxic effects upon embryos, but the most toxic ones – causing severe ulcerations of the embryo tissue and drastically reducing their survival rate - being the zinc oxide (nZnO) nanoparticles.
Recently, a series of studies on the degree of toxicity of zinc oxide, titanium dioxide, cerium dioxide and tin dioxide particles prepared in pure water and synthetic sea water [5] evidenced
the same toxic effects of zinc oxide NPs upon ish
embryos (Danio rerio), but only when pure water was administered, the lethal concentration
observed appearing at doses of 3.5-9.1 mg/l. The
toxicity levels at an exposure time of 24 hr were 22.7 times higher when administered in larva stage, comparatively with those registered for embryos – at the same exposure time (24 hr).
International Journal of Medical Dentistry 181 THE EFFECTS OF METAL NANOPARTICLES ON EMBRYOS OF DIFFERENT ANIMAL SPECIES. A REVIEW
Beijing, evidenced the toxicity of copper nanoparticles upon the embryo development of
ish (Danio rerio). The observation made was that,
after a 24 hr exposure, ish embryos lost their
hatching capacity and also that severe malformations appeared in larva stages. At high
concentrations (>0.1 mg/l) of copper nanoparticles,
the embryos stopped their evolution in the gastrula stage, so that this value came to be considered as the lethal dose for the analyzed species. Lower
concentrations (0.006 – 0.03 mg/l) permitted
embryos survival and their hatching [6].
Embryos from the same ish species have been
also used for testing the toxic effects of nickel nanoparticles, an experiment developed by a research team from the University of Clarkson, U.S.A. [7]. Subjected to testing were nanoparticles with three different dimensions (30, 60 and, respectively, 100 nm), as well as particle aggregates with sizes of 60 nm and dendritic structure. At the same time, an experimental group was subjected to the contact with water soluble nickel salts. The mortality ratio of embryos and the malformations observed in the end of the incubation process were recorded, the conclusion being that the nickel particle aggregates were the most toxic, whereas the nanoparticles in suspension caused separation of
the skeletal musculous ibers, thinning of the
intestinal epithelium and also some malformations of the nervous system were observed. The low-concentrated nickel salts showed no toxic effects and did not affect the digestive apparatus of embryos. Based on these results, the authors outlined that the process of nanoparticle
coniguration (form and size) affects much more
the organism which comes into contact with them, rather than their chemical composition [7].
The effects of indium and tin oxide nanoparticles have been tested by a Swiss research team (University of Applied Sciences and Arts, Northwestern Switzerland), the species they used as a biological material being again
Danio rerio. The scope of the study was to observe
the effects these nanoparticles may have both upon the environment and the living organisms, indium oxide being used especially for the fabrication of electronic devices. This oxide may enter the environment quite accidentally, during the fabrication of some electronic products or in
cases in which these are not recycled in a controlled manner [8].
Interpretations of the obtained results evidenced that, at inoculations made with
concentrations of 247 µg/l, apoptosis and oxidative
stress phenomena appear at the level of the endoplasmatic reticule of the hepatic cells. The tests have been performed on hepatocytes belonging to this species, cultivated in vitro or either separately, on embryos. The oxidative effects have been clearly evidenced for the hepatic cells and less obviously in the case of embryos.
Iron oxide nanoparticles are used in several domains, especially the biomedical ones, yet scarce information is available on their impact upon the aquatic medium and aquatic organisms. A study realized at the University of Science and Technology of Tianjin, China, [9] showed that a
maximum concentration of 100 mg/l iron oxide
nanoparticles in suspension may have the following effects: high mortality, delayed hatching and malformations (tissular ulcerations – in 32.5 % of the embryos, pericardial edema), for an exposure time of 168 hr. Such effects may be associated with the manner of nanoparticle aggregation and also with their mode of sedimentation.
Another team of investigators analyzed the effects of silica nanoparticles upon aquatic
organisms. The study was realized on ish embryos
and spawn, the administered doses being of 25,
50, 100, 200 µg/ml), while exposure – lasting
between 4 and 96 hr - occurred when the fertilization process was over [10]. Apart from the teratogenic effects, investigations on the etiology of alevines and evolution of the locomotory system were also performed. The observation made in the end of the experiment was that, with the increase of doses, the ratio of dead embryos also increases. As to the possible malformations, several cases of embryos with pericardial edema, edema of the viteline sac and deformations of the tail or of the cephalic capsule were also mentioned. The behaviour of alevines registered hyperactivity
when exposed to doses of 25 and 50 µg/ml, while those exposed to higher doses (of 100 and 200 µg/
ml) showed remarkable hypoactivity, but only in the periods of reduced light intensity.
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A. TEUŞAN, N. BARANOV, R. DMOUR, A. VIZITIU, I. JELIHOVSCHI, V. TEUŞAN
on using nanoparticles of various forms (nanospheres, nanosticks, nanoprisms and submicronic particles) [11]. The most severe effects were obtained when exposing the embryos to elongated NPs (nanosticks), a case in which a high ratio of dead embryos was recorded. The ratio of dead embryos registered after a 120 hr exposure time was of 100%. Also the behaviour of the alevines was evaluated: they hatched from eggs exposed for only 96 hr in the respective suspensions. The activity level of alevines decreases inversly proportionally with the exposure doses.
Testing of titanium dioxide nanoparticles was performed in a university of Turkey, the biological material employed being the Xenopus
laevis embryo, a batrachian (frog) species [12],
whose eggs were introduced in water with 7 different concentrations, varying between 5 and 320 ppm, evaluation being performed after 96 hr of exposure. In the end, the ratio of living embryos was calculated and a series of determinations on the amounts of acetylcolinesterase, carboxylesterase, glutation-S-transferase, glutation reductase, lactate dehydrogenase and aspartate-aminotransferase was made. Neither of the tested concentrations led to anomalies, nor did they affect hatching or the normal development of embryos. More than that, no significant modifications in the previously mentioned enzymes were observed.
At present, these (titanium dioxide) nanoparticles are studied in several countries.
However, most of the scientiic studies are based
on the effects of silver and gold nanoparticles of various sizes and forms. Most frequently, the toxic effects of these nanomaterials are induced by their mode of agglomeration in the suspension medium. Other important factors to be had in view when testing these nanoparticles are their dimension and form.
The animal species most frequently employed for this type of experiments (with reference to the teratogenic effects that may appear on exposure to media containing variable NP amounts) are: Gallus domesticus, Danio rerio, Xenopus laevis.
The review of the literature issued in the ield
permits the conclusion that the tested metallic nanoparticles have rather destructive effects
upon cellular and tissular integrity, affecting the embryonary development of the mentioned species.
References
1. Taylor U, Barchanski A, Kues W, Barcikowski S, Rath D. Impact of Metal Nanoparticles on Germ Cell Viability and Functionality. Reprod Domest Anim. 2012; 47 (S4): 359–368.
2. Prasek M, Sawosz E, Jaworski S, Grodzik M, Ostaszewska T, Kamaszewski M, Wierzbicki M,
Chwalibog A. Inluence of nanoparticles of plati -num on chicken embryo development and brain morphology. Nanoscale Res Lett. 2013; 8:251. 3. Pineda L, Sawosz E, Vadalasetty KP, Chwalibog
A. Effect of copper nanoparticles on metabolic rate and development of chicken embryos. Anim. Feed Sci. Technol. 2013; 186(1-2):125–129. 4. Zhu X, Zhu L, Duan Z, Qi R, Li Y, Lang Y.
Compa-rative Toxicity of Several Metal Oxide Nanoparticle
Aqueous Suspensions to Zebraish (Danio rerio)
Early Developmental Stage. J Environ Health Sci. 2008; Part A(43): 278–284.
5. Wehmas LC, Anders C, Chess J, Punnoose A,
Pere-ira CB, Greenwood JA, Tanguay RL. Comparative metal oxide nanoparticle toxicity using embryonic
zebraish. Toxicol Rep. 2015 (2): 702–715.
6. Bai W, Tian W, Zhang Z, He X, Ma Y, Liu N, Chai Z. Effects of copper nanoparticles on the
develop-ment of zebraish embryos. J Nanosci Nanotech-nol. 2010;10(12):8670-6.
7. Ispas C, Andreescu D, Patel A, Goia DV, Andreescu S, Wallace KN. Toxicity and developmental defects of different sizes and shape nickel nanoparticles in
zebraish. Environ Sci Technol. 2009; 43(16): 6349–
6356.
8. Brun NR, Christen V, Furrer G, Fent K. Indium and indium tin oxide induce endoplasmic reticulum
stress and oxidative stress in zebraish (Danio rerio). Environ Sci Technol. 2014;48(19):11679-87.
9. Zhu X, Tian S, Cai Z. Toxicity Assessment of Iron
Oxide Nanoparticles in Zebraish (Danio rerio)
Early Life Stages. PLoS ONE. 2012; 7(9): e46286. 10. Duan J, Yu Y, Shi H, Tian L, Guo C, Huang P, Zhou
X, Peng S, Zhiwei S. Toxic Effects of Silica
Nanopar-ticles on Zebraish Embryos and Larvae. PLoS One.
2013; 8(9): e74606.
11. Hua J, Vijver MG, Richardson MK, Ahmad F,
Peijnenburg WJ. Particle-speciic toxic effects of
differently shaped zinc oxide nanoparticles to zebra-fish embryos (Danio rerio). Environ Toxicol Chem. 2014; 33(12), 2859-68.
