Exposure of Zebrafish (Danio rerio) to Titanium Dioxide Nanoparticle Causes Paraptosis: Evaluation of Ovarian Follicle Ultrastructure

Cansu Akbulut1,*, Tuğba Kotil2, Burcu Öztürk1 and Nazan Deniz Yön1

1Department of Biology, Science and Letters Faculty, Sakarya University, Serdivan, Sakarya, Turkey

2Department of Histology and Embryology, İstanbulFaculty of Medicine, İstanbul University, İstanbul, Turkey

ABSTRACT

Titanium dioxide (TiO2) is widely used nanoparticle all over the world. In this study, we have investigated the histopathological effect on zebrafish ovaries after exposure to TiO2 nanoparticles. Adult zebrafish individuals were exposed to 1, 2 and 4 mg/L TiO2 for 5 days, and then their ovaries were evaluated using light and transmission electron microscopy. Numerous degenerated follicles with cytoplasmic vacuolization, mitotic catastrophe in mitochondria , chromatin condensation, mitochondrial vesiculation and dispersion at ooplasm were observed. In mitochondria, mitotic catastrophe , vesiculation, swelling and loss of organization of cristae were detected. Here we showed that TiO2 exposure trigger paraptotic type cell death in zebrafish ovary.

Article Information

Received 27 October 2015

Revised 09 May 2016

Accepted 20 January 2017

Available online 24 May 2017

Authors’ Contribution

NDY designed the experiments and supervised the work. CA and BÖ performed light microscopic studies. TK performed electron microscopic studied. CA wrote the article.

Key words

Nanoparticle, Cell death, Titanium dioxide, Danio rerio, Ovarian follicle paraptosis.

DOI: http://dx.doi.org/10.17582/journal.pjz/2017.49.3.1077.1083

* Corresponding author: cansua@sakarya.edu.tr

0030-9923/2017/0003-1077 $ 9.00/0

Copyright 2017 Zoological Society of Pakistan

INTRODUCTION

Titanium dioxide (TiO2) is one of the most commonly used nanoparticles. They are used in a range of products including sun screen, cosmetics, coatings, paint, plastics, papers, inks, medicine, medicines, pharmaceuticals, food products, toothpaste and building materials (Wolf et al., 2003; Kaida et al., 2004; Aitken et al., 2006; Wang et al., 2007). United States Environmental Protection Agency (USEPA) estimated the annual production of TiO2 nanoparticles (nano-TiO2) to be 2000 metric tons in around 2005, with 65% of this production used in products such as cosmetics and sunscreen lotions (USEPA, 2009). TiO2 is generally not considered to be obvious toxic (Park et al., 2006). It is photocatalytic and has the potential to produce cumulative cellular damage (Bar-Ilan et al., 2013). It has been explored for use in water treatment to destroy chemicals such as polychlorinated biphenyls (PCBs), pesticides, and other complex organic contaminants. The conventional sized TiO2 is considered to be physiologically inert and poses little risk to humans, and can be used as food additives (colorants). However, when TiO2 is made at the nanoscale (particle size <100 nm), its biological and environmental effects deserve our emerging attention (Chen et al., 2011). It is inevitable for TiO2 NPs to aggregate in water due to its strong interparticle self absorption properties. Dispersed NPs which resulted in toxic effects on the growth of zebrafish (Ispas et al., 2009). Aquatic organisms can also exposed nanoparticles via food chain. Zhu et al. (2010) provide that nanoscale TiO2 particles (nTiO2) can transfer from Daphnia magna to Danio rerio by dietary exposure.

A new type programmed cell death, paraptosis has been recently identified. Paraptosis, type III programmed cell death is morphlogically different from apoptosis and necrosis. Paraptosis, has been characterized by chromatin condensation, cytoplasmic vacuolization, numerous small vacuoles created in the cytoplasm, widened perinuclear space, and mitochondrial vesiculation and mitochondrial swelling (Sperandio et al., 2000; Danaila et al., 2013). Differently from apoptosis, features such as membrane blebbing and chromatin condensation aren’t seen in paraptosis. Also, this non-apoptotic programmed cell death doesn’t involve apoptotic markers such as caspase activation, apoptotic body formation (Sperandio et al., 2000). In paraptosis like programmed cell death mitotic catastrophe is seen (Bröker et al., 2005; Caruso et al., 2011).

In the current study, the toxic effects of TiO2 nanoparticles (<150 nm particle size) were evaluated with light and transmission electron microscopy at zebrafish ovary tissue. Zebrafish is a well known vertebrate model for reproduction and development studies. The zebrafish’s hardiness makes them excellent stress test subjects, as they can survive fairly severe environmental changes without succumbing, surviving long enough to show developmental defects.

MATERIALS AND METHODS

Experimental design

Adult, one year old, zebrafish individuals were obtained from Sakarya University Aquaculture Lab., Esentepe, Turkey were raised in a computer-controlled incubation chamber at 14 h light/10 h dark photoperiod, 28.5 ± 1°C temperature, 7.0 ± 0.5 pH and 61% humidity. They were fed with Artemia sp. TetraMin© Hauptfutter (Tetra Werke, Germany) twice a day. After one week of adaptation period zebrafish were divided into four groups (n=15), one control and 3 experimental groups for 1, 2 and 4 mg/L TiO2 treatment. For investigating the effects of TiO2, fishes were anesthesized with ice water and ovary tissues were dissected after 5 day of the exposure.

Titanium dioxide nanoparticle (<150 nm particle size) was obtained from Sigma Aldrich CAS number 13463-67-7.

Histological sgtudies

The ovaries were fixed in neutral formaldehyde for 24 h. After fixation, tissues were dehydrated in ascending concentrations of ethanol, equilibrated in xylene. The tissues were then embedded in paraffin wax and cut into 5-7 μm sections on a Leica microtome. The sections were mounted on glass slides and stained with hematoxylin and eosin before examination under a Olympus light microscope.

Electron microscopy

For transmission electron microscopy, ovarian tissues were fixed by immersion in a solution containing 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M phosphate buffer at pH 7.4 for 4 h. The ovaries were fixed further overnight at 4°C using 2% glutaraldeahyde in 0.1 M phosphate buffer at pH 7.4. After an additional fixation with 1% OsO4 and pre-embedding staining with 1% uranyl acetate, ovaries were dehydrated and embedded in Embed 812 resin. The sectioning was performed using a Leica Ultracut ultramicrotome. Thick sections were stained with tolouidine blue and visualized in a Olympus light microscope to select the area of interest. There after, thin sections were collected and counterstained with 1% uranyl acetate and lead citrate and examined with Jeol transmission electron microscope.

RESULTS

Zebrafish ovary is an extremely dynamic organ in which the follicles undergo asynchronous development. The development of zebrafish oocytes is divided into four stages, based on morphological features (Koç et al., 2008).

Control group

In control group, normal ovarian histology was observed. All development stages were monitored. In primary growth phase multiple nucleoli were observed at the germinal vesicle of oocytes (Fig. 1d). In cortical alveoli stage, oocytes were identified due to growing cortical alveoli surrounding the nucleus (Figs. 1b, 1d, 1e). In this stage, increase in size of the oocytes were seen. Zona radiata structure began to emerge. Zona radiata and follicular epithelium structures were clearly monitored (Figs. 1a, 1c, 1d). During vitellogenic stage, the oocytes began to increase in size, due to accumulation of yolk. Zona radiata structure was thicker (Fig. 1f). In mature oocytes, the nucleus was dissolved and the ooplasm which consists of yolk bodies were also monitored (Fig. 1e).

1mg/L TiO2 exposed group

Degeneration and structural changes at mitochondria in both follicular epithelium and ooplasm were monitored (Fig. 2a, 2c, 2d). Deterioration of the integrity of mitochondria was observed (Fig. 2a, 2c, 2d). In mitochondria, swelling and loss of organization of cristae were detected (Fig. 2d). Deterioration at microvilli structure of zona radiata were also established (Fig. 2a). Numerous cytoplasmic vacuoles in ooplasm of the follicles have been identified (Fig. 2b, 2e). In follicles, cytoplasmic vacuolization which is one of the sign of paraptotic cell death were observed (Fig. 2b, 2g). In cortical alveolus stage, unification at cortical alveoli were monitored (Fig. 2b, 2g) and eosinophilic granues were detected (Fig. 2h). Nucleolus organization were lost at primary oocytes (Fig. 2f).

2 mg/L TiO2 exposed group

Besides cytoplasmic vacuolization, opening between zona radiata and follicular epithelium were monitored (Fig. 3a). Chromatin condensation at follicular epithelium (Fig. 3a) and degenerated microvilli were also detected (Fig. 3b). Mitochondrial swelling (Fig. 3c) and loss of organization of cristae were observed (Fig. 3c, 3d). Oocytes were begining to shrink (Fig. 3e, 3h). In cortical alveolus stage, vacuolization were severe (Fig. 3f) and many vacuoles were monitored under zona radiata layer (Fig. 3e). In vitellogenic oocyte, unification at cortical alveoli were detected (Fig. 3g).

4 mg/L TiO2 exposed group

Degeneration and vacuolization at follicular epithelium were observed (Fig. 4a, 4b). Opening between zona radiata and follicular epithelium was also seen at this group too (Fig. 4a, 4b). Microvilli structure were degenerated (Fig. 4b). Multilamellar vesicle formation were detected at ooplasm and follicular epithelium cytoplasm (Fig. 4b, 4e). Cytoplasmic vacuolization were monitored at both follicular epithelium and ooplasm (Fig. 4a, 4b, 4e, 4g, 4h). Disrupted mitochondria were established. Mitotic catastrophe, the signature characteristic of paraptosis were monitored at mitochondria (Fig. 4c). Altered morphology and loss of organization of cristae were detected at mitochondrium (Fig. 4d). In cortical alveolus stage, dispersion at ooplasm were monitored (Fig. 4f). Unification at the cortical alveoli structure were detected at follicles (Fig. 4g, 4h).

DISCUSSION

Nanoparticles are potential aquatic pollutants and entering waterways. They also have risks human health via water and aquatic systems (Moore, 2006). Evaluation the toxicological effects of these materials has a great importance in aquatic organisms.

Nanoparticle exposure cause histopathological changes in tissues. Chen et al. (2011) evaluated the effects of TiO2 nanoparticles on growth and some histological parameters of zebrafish after a long term exposure (2-6 months). They detected distinct nanoparticle accumulation and morphological alternation at gill. They monitored a hyperplasia-like thickening of the primary lamellae in gill filaments.

Nanoparticle exposure aslo inhibit reproduction. In particular TiO 2 nanoparticles have been used in crop production, dietary supplements, food additives, food packaging components, medicine, toothpastes, sunscreens, cosmetics, and waste water treatment. This widespread use of TiO 2 nanoparticles has inevitably led to harmful biological responses in humans and animals (Zhao et al., 2014). Yön and Akbulut (2014) conducted a study about histopathological effects of bisphenol A on zebrafish ovary and they showed bisphenol A slowed down oogenesis in zebrafish. Similarly, Wang et al. (2011) investigated chronic exposure effects of TiO 2 nanoparticles on zebrafish reproduction. They used female individuals and provided that chronic exposure of zebrafish to 0.1 mg/L TiO 2, can significantly impair zebrafish female reproduction. They found 29.5% reduction in the cumulative number of zebrafish eggs after 13 weeks of nTiO 2 exposure. The distribution of follicular developmental stages was skewed by the TiO 2 treatment toward the immature stage, as evident by an increase in stage I follicles and some reduction in other stages, especially, stage IV follicles. Distinctively we investigated histological and ultrastructural effects of TiO 2 nanoparticle. We proved that TiO 2 nanoparticle exposure inhibit oogenesis via causing cell death, which is also corroborated by Wang et al. (2011).

Zhu et al. (2008) examined developmental and toxic effects of TiO 2 exposure for 96 h at 1-500 mg/L concentration on zebrafish embryo and larvae. They proved that neither nanoTiO 2 nor TiO 2 bulk showed any toxicity to zebrafish embryos and larvae. Toxic response may be different in embryo. This response may be due to three-layered acellular envelope, called chorion which protects embryo before hatching.

Many studies have shown that TiO 2 treatment cause cell death in many cells. Cell death in human bronchial epithelial cells induced by titanium dioxide exposure was proved by Chen et al. (2008). Nanoparticle aggregates also cause oxidative stress in zebrafish embryos. Faria et al. (2014) exhibited TiO 2 aggregates impaired embryo growth and generated oxidative stress in the absence of solar simulated radiation. Cell death can be trigger by oxidative stress formation. Disruption of antioxidant defense mechanisms and generation of reactive oxygen species by cytoplasmic degeneration and nuclear destruction were reported on hepatocytes treated by TiO 2 nanoparticles (Alarifi et al., 2013).

Several studies indicated that TiO 2 has direct effects on ovarian functions. In a study that investigation the effects of TiO 2 on rat ovarium showed mitochondrial swelling and loss of cristae, condensation of nuclear material and apoptosis (Wang et al., 2011). These results are consistent with our study.

CONCLUSION

In this study we found TiO2 nanoparticle exposure inhibit oogenesis and cause cellular cell death in zebrafish. Paraptosis is a programmed cell death type which has been recently defined. There are few information about it and many the details haven’t been illuminated. As far as known about paraptosis we can say that our data are exactly match feature of paraptosis and TiO2 exposure cause paraptosis type cell death in zebrafish ovaries.

Statement of conflict of interest

Authors have declared no conflict of interest.

REFERENCES

Aıtken, R.J., Chaudhry, M.Q., Boxall, A.B.A. and Hull, M., 2006. Manufacture and use of nanomaterials: current status in the UK and global trends. Occup. Med., 56: 300-306. https://doi.org/10.1093/occmed/kql051

Alarıfı, S., Alı, D., Al-Doaıss, A.A., Alı, B.A., Ahmed, M. and Al-Khedhaıry, A.A., 2013. Histologic and apoptotic changes induced by titanium dioxide nanoparticles in the livers of rats. Int. J. Nanomed., 8: 3937–3943.

Amatruda, J.F., Shepard, J.L., Stern, H.M. and Zon, L.I., 2002. Zebrafish as a cancer model system. Cancer Cell, 1: 229–231. https://doi.org/10.1016/S1535-6108(02)00052-1

Bar-Ilan, O., Chuang, C.C., Schwahn, D.J., Yang, S., Joshı, S., Pedersen, J.A., Hamers, R.J., Peterson, R.E. and Heıdeman, W., 2013. TiO2 nanoparticle exposure and illumination during zebrafish development: mortality at parts per billion concentrations. Environ. Sci. Technol., 47: 4726-4733. https://doi.org/10.1021/es304514r

Broker, L.E., Kryut, F.A.E. and Gıaccone, G., 2005. Cell death independent of caspases: A review. Clin. Cancer Res., 11: 3155-3162. https://doi.org/10.1158/1078-0432.CCR-04-2223

Caruso, R., Fedele, F., Lucıuanò, R., Branca, G., Parısı, C., Paparo, D. and Parısı, A., 2011. Mitotic catastrophe in malignant epithelial tumors: The Pathologist’s viewpoint. Ultrastruct. Pathol., 35: 66–71. https://doi.org/10.3109/01913123.2010.543753

Chen, E., Ruvalcaba, M., Araujo, L., Chapman, R. and Chun Chın, W., 2008. Ultrafine titanium dioxide nanoparticles induce cell death in human bronchial epithelial cells. J. exp. Nanosci., 3: 171-183. https://doi.org/10.1080/17458080802412430

Chen, J., Dong, X., Xın, Y. and Zhao, M., 2011. Effects of titanium dioxide nano-particles on growth and some histological parameters of zebrafish (Danio rerio) after a long term exposure. Aquat. Toxicol., 101: 493–499. https://doi.org/10.1016/j.aquatox.2010.12.004

Danaıla, L., Popescu, I., Paıs, V., Rıga, D., Rıga, S. and Paıs, E., 2013. Apoptosis, paraptosis, necrosis, and cell regeneration in posttraumatic cerebral arteries. Chirurgia, 108: 319-324.

Daughton, C.G., 2004. Non-regulated water contaminants: emerging research. Environ. Impact Assess. Rev., 24: 711–732. https://doi.org/10.1016/j.eiar.2004.06.003

Farıa, M., Navas, J.M., Soares, A.M.V.M. and Barata, C., 2014. Oxidative stress effects of titanium dioxide nanoparticle aggregates in zebrafish embryos. Sci. Tot. Environ., 470: 379-389. https://doi.org/10.1016/j.scitotenv.2013.09.055

Ispas, C., Andreescu, D., Patel, A., Goıa, D.V., Andreescu, S. and Wallace, K.N., 2009. Toxicity and developmental defects of different sizes and shape nickel nanoparticles in zebrafish. Environ. Sci. Tech., 43: 6349–6356. https://doi.org/10.1021/es9010543

Kaıda, T., Kobayashı, K., Adachı, M. and Suzukı, F., 2004. Optical characteristics of titanium oxide interference film and the film laminated with oxides and their applications for cosmetics. J. Cosmet. Sci., 55: 219–220.

Kıng, K.L. and Cıdlowskı, J.A., 1995. Cell cycle and apoptosis: common pathways to life and death. J. cell. Biochem., 58: 175-180. https://doi.org/10.1002/jcb.240580206

Koç, N.D., Aytekın, A. and Yuce, R., 2008. Ovary maturation stages and histological investigation of ovary of the zebrafish (Danio rerio). Braz. Arch. Biol. Tech., 51: 513-522. https://doi.org/10.1590/S1516-89132008000300010

Moore, M.N., 2002. Biocomplexity: the post-genome challenge in ecotoxicology. Aquat. Toxicol., 59: 1–15. https://doi.org/10.1016/S0166-445X(01)00225-9

Moore, M.N., Depledge, M.H., Readman, J.W. and Leonard, P., 2004. An integrated biomarker-basedstrategy for ecotoxicological evaluation of risk in environmental management. Mutat. Res., 552: 247–268. https://doi.org/10.1016/j.mrfmmm.2004.06.028

Moore, M.N., 2006. Do nanoparticles present ecotoxicological risks for the health of the aquatic environment? Environ. Int., 32: 967–976. https://doi.org/10.1016/j.envint.2006.06.014

Park, J.H., Kım, S. and Bard, A.J., 2006. Novel carbon-doped TiO2 nanotube arrays with high aspect ratios for efficient solar water splitting. Nano Lett., 6: 24–28. https://doi.org/10.1021/nl051807y

Sperandıo, S., deBelle, I. and Bredesen, D.E., 2000. An alternative, nonapoptotic form of programmed cell death. Proc.natl. Acad. Sci. U.S.A., 97: 14376- 14381. https://doi.org/10.1073/pnas.97.26.14376

USEPA United States Environmental Protection Agency, 2009. Nanomaterial case studies: Nanoscale titaniumdioxide in water treatment and in topical sunscreen. EPA/600/R-09/057. Washington, DC.

Wang, J.J., Sanderson, B.J. and Wang, H., 2007. Cyto- and genotoxicity of ultrafine TiO2 particles in cultured human lymphoblastoid cells. Mutat. Res., 628: 99–106. https://doi.org/10.1016/j.mrgentox.2006.12.003

Wang, J., Zhu, X., Zhang, X., Zhao, Z., Lıu, H., George, R., Wılson-Rawls, J., Chang, Y. and Chen, Y., 2011. Disruption of zebrafish (Danio rerio) reproduction upon chronic exposure to TiO2 nanoparticles. Chemosphere, 83: 461–467. https://doi.org/10.1016/j.chemosphere.2010.12.069

Wolf, R., Matz, H., Orıon, E. and Lıpozencıc, J., 2003. Sunscreens: The ultimate cosmetic. Acta Dermatovenerol. Croat., 11: 158–162.

Yön, N.D. and Akbulut, C., 2014. Histological Changes in Zebrafish (Danio rerio) Ovaries Following Administration of Bisphenol A. Pakistan J. Zool., 46: 1153-1159.

Zhu, X., Zhu, L., Duan, Z., Qı, R., Lı, Y. and Lang, Y., 2008. Comparative toxicity of several metal oxide nanoparticle aqueous suspensions to zebrafish (Danio rerio) early developmental stage. J. Environ. Sci. Hlth. A Tox. Hazard Subst. Environ. Eng., 43: 278-284. https://doi.org/10.1080/10934520701792779

Zhu, X., Wang, J., Zhang, X., Chang, Y. and Chen, Y., 2010. Trophic transfer of TiO2 nanoparticles from daphnia to zebrafish in a simplified freshwater food chain. Chemosphere, 79: 928–933. https://doi.org/10.1016/j.chemosphere.2010.03.022

Zhao, X., Sheng, L., Wang, L., Hong, J., Yu, X., Sang, X., Sun, Q., Ze, Y. and Hong, F., 2014. Mechanisms of nanosized titanium dioxide-induced testicular oxidative stress and apoptosis in male mice. Part. Fibre Toxicol., 11: 1-14. https://doi.org/10.1186/s12989-014-0047-3