Protein oxidation in the fish Danio rerio (Cyprinidae) fed with single- and multi-walled carbon nanotubes
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The increase in the production of carbon nanotubes (CNT) arises potential scenarios of exposure to these nanomaterials for several organisms including aquatic species. Experiments were conducted to determine the toxicity of single-walled (SWCNT) and multi-walled (MWCNT) carbon nanotubes to the fish Danio rerio (Cyprinidae) exposed to these CNT via diet (500 mg/kg) during 28 days. Induction of oxidative stress by CNT was evaluated through protein carbonyl groups (immunohistochemistry). Higher levels of carbonyl groups were registered in several organs (liver, brain, pancreas and muscle) of fish exposed to SWCNT and MWCNT. Overall, data indicate that CNT administered through diet can in fact induce toxicological responses in aquatic organisms as fish. The measurement of irreversible protein oxidative damage through immunohistochemistry seems to be a valuable tool for nanotoxicology.
KeywordsNanotoxicology Protein carbonyl groups Nanotechnology Oxidative damage Protein oxidation
In 1991, Sumio Iijima was the first to demonstrate the existence of tubular carbon structures, which were later called carbon nanotubes (CNT) (Ijima 1991). This discovery together with the fullerenes C60 prompted the development of nanosciences and their technological applications (nanotechnologies) (Kroto et al. 1985). CNT are classified according to their tubular structure in single-walled nanotubes (SWCNT) and multi-walled nanotubes (MWCNT). Conceptually, it can be considered that SWCNT are composed of a single folded graphene layer, whereas MWCNT are constituted by several concentric graphene folded layers (da Rocha et al. 2013).
The extent of applications of nanomaterials makes the nanoscience clearly a multi-disciplinary subject. Nanomaterials are natural or synthetic substances with 50% or more of their size distribution in the range up to 100 nm. In this size range, significant changes in chemical and physical properties of molecules are exhibited, many of which may have a technological utilization (Colvin 2003; Oberdörster et al. 2005).
CNT show magnetic and optical properties, and high mechanical strength and electrical conductivity (Huczko 2002). Currently these nanomaterials have an annual production in tons, meaning that soon they probably will be present in the environment (Pérez et al. 2009). Because they are lipophilic in its pure form, CNT are one of the least biodegradable nanomaterials with a tendency to bioaccumulate (Bystrzejewska-Piotrowska et al. 2009). Poland et al. (2008) noted similarities between CNT with asbestos toxicity mechanisms. And other authors have proposed that the geometric shape of CNT should be one of the key factors of its cytotoxicity promoting necrosis and apoptosis (Jia et al. 2005).
In vivo assays with CNT in various aquatic animals showed: (1) induction of oxidative damage (Smith et al. 2007; Fraser et al. 2011; da Rocha et al. 2013); (2) an increase in mortality rates (Templeton et al. 2006; Roberts et al. 2007); (3) CNT accumulation in the digestive tract (Petersen et al. 2008, 2009); and (4) reproduction impairment (Cheng et al. 2007; Scott-Fordsmand et al. 2008). In fact, in vivo studies are of utmost importance to provide data about nanomaterials effects on the environment and organisms, a key regulatory issue. Velzeboer et al. (2011) stated that aquatic sediments are a major sink of manufactured nanomaterials such as fullerenes and CNT and these may cause adverse effects in benthic species or organisms that fed sediments. More recently, in vitro assays combined with docking simulation indicated that in this context, single-walled carbon nanotubes can induce mitotoxicity through the modulation of the ADP/ATP transport (ANT-1). ANT-1 is an important player in the onset of mitochondrial permeability transition pore (MPTP), event that triggers mitochondrial dysfunction and apoptosis (González-Durruthy et al. 2016).
The objective of this study was to investigate potential damages to the fish Danio rerio by nanotubes offered via diet, aiming to evaluate potential exposure to these CNT under an environmental realistic condition. It is also important to stress that oxidative protein damage was analyzed through the detection of carbonyl groups by immunohistochemistry. As longitudinal slices were employed, several organs were simultaneously evaluated by this technique, an approach that, to the best of our knowledge, was not previously applied.
2 Materials and methods
2.1 Carbon nanotubes employed in the assays
Single-walled (SWCNT) and multi-walled carbon nanotubes (MWCNT) were purchased from commercial supplier (SES Research: SWCNT lot ps-09607 and MWCNT lot gs-1802), with 10–30 nm diameter and 99.9% purity. To ensure the absence of metal catalysts, it was employed the technique described by Chen et al. (2004), doing an acid bath to remove traces of metals, using a solution containing sulfuric and nitric acid (3:1, v/v) mixed and sonicated for 6 h and further centrifuged for 3000×g for 20 min. The centrifugation procedure was repeated five times, and then, CNT samples were further oven dried for 48 h at 50 °C.
2.2 Characterization of carbon nanotubes
The technique chosen to characterize these materials was mirroring Raman spectroscopy. This technique characterizes carbonaceous materials by identifying the types of links and provides information regarding the disorder of the crystal lattice of the material and identifying the various crystalline and amorphous forms present in the sample, and therefore exhibiting characteristic peaks in the spectra in the region between 1000 and 1800 cm−1. Raman analysis were performed at room temperature in Via Renishaw Raman spectrometer, in the range of 0–2500 cm−1 using a laser of 532- and 785-nm wavelength.
2.3 Experimental design
Adult zebrafish D. rerio (Cyprinidae) from both sexes were bought from a local store and acclimated to laboratory conditions for 3 weeks. The photoperiod was fixed at 12 h light/12 h dark, with water temperature maintained at 26 °C and the pH between 6.8 and 7.0. All procedures in this study were approved by the Committee of Ethics for Animal Use in Experimentation (CEUA FURG-no. Pq004/2013). Fish were fed twice a day, with a commercial food Brand Tetra®. The daily average amount of food eaten by fish was previously determined. This information was employed to establish the final CNT quantity to be added to the food. It was used 60 fish, equally divided in three treatments: 20 fish received the control diet (no CNT added), 20 fish were given feed containing single-walled carbon nanotubes (SWCNT) and 20 fish were given feed containing multi-walled carbon nanotubes (MWCNT), both CNT at a dose of 500 mg/kg of food, according a previous study that tested histopathological responses in rainbow trout exposed to CNT or fullerene, another carbon nanomaterial (Fraser et al. 2011). As positive control for oxidative damage, another group of fish was exposed to peroxide hydrogen (1 mM) during 36 h, under the same conditions used for the fish of the treatments cited above. Fish exposed to CNT from the negative and positive control groups were maintained individually in a single aeration flask containing 300 mL of freshwater at a temperature of 26 °C, with a photoperiod of 12 h light/12 h dark. Each fish received half of the total amount of food at 10:00 h in the morning and the other half in the afternoon at 16:00 h. The food was offered during 1 day. The next day, fish were not fed, and after each single aeration, flask was cleaned and water was changed. This procedure was followed for 28 days, after the animals were euthanized with an overdose of methane sulfonate—Tricaine (TMS, MS222 ≥ 250 mg/L).
After euthanasia, whole fish were preserved in methacarn fixing solution (methanol/chloroform/acetic acid, 6:3:1) for 12 h. After, they were stored in 70% ethanol. An automatic processor PT05 LUPE was used, where the samples underwent serial dehydration (alcohol 70, 80, 90, 96%, absolute), diaphanization or clarification (xylene baths) and finally impregnating in Paraplast (Sigma-P3558). Fish in Paraplast blocks were cut (5 μm) with microtome LUPE MRP03. Samples were mounted in slides and stained with hematoxylin and eosin (H&E) for immunohistochemistry. H&E staining followed the protocol described Carson and Hladik (2009). The slides were washed by xylene (two baths of 15 min) to remove the paraffin and after hydration were bathed in a series of absolute alcohol, 96, 90, 80, 70% and water, and subsequently stained with H&E and dehydrated again.
For the detection of protein carbonyls groups, a common indicator of irreversible protein damage via oxidative stress, it was used histological slides without H&E staining employing the immunohistochemistry kit protocol (Oxidative Stress Detection Kit; Millipore). The test involves chemical derivatization of carbonyl groups with 2,4-dinitrophenylhydrazine (DNPH) to generate 2,4-dinitrophenylhydrazone (DNP). The DNP-derivatized proteins were detected by a specific antibody that binds to the DNP molecule, with a subsequent incubation with a secondary antibody conjugated to biotin and streptavidin–HRP and stained using a 3,3′-diaminobenzidine (DAB) as substrate.
2.5 Statistical analysis
Scoring of oxidative stress in terms of protein carbonyl groups was done using the Bernet index (Bernet et al. 1999) and then analyzed through Kruskal–Wallis test (Zar 1999). As whole fish were employed for immunohistochemistry detection, several organs (gill, olfactory bulb, brains, liver, intestine, muscle, eyes and pancreas) were analyzed as described.
3.1 Characterization of the SWCNT and MWCNT
3.2 Dietary exposure to carbon nanotubes
Mortality rate was 15% in the group fed with rations containing SWCNT. The group fed with MWCNT showed no mortality and the same was registered for the negative control group, whereas no mortality rate was recorded for the positive control exposed to hydrogen peroxide. It was observed that food with or without CNT was equally accepted by fish, and no apparent differences in the feeding behavior were detected.
The physicochemical properties of the CNT are important to their possible applications; however, these properties can also be responsible to generate toxicity, where generation of oxidative stress is one of the reported mechanisms (Pacurari et al. 2008; Patlolla et al. 2011; Tsukahara and Haniu 2011). However, some previous reports indicated that the generation of reactive oxygen species (ROS) induced by CNT was in fact related to the transition metals released by these nanomaterials (Liu et al. 2013). For this reason, it was employed a protocol to eliminate trace metals from the tested CNT. Other authors that employed purified CNT have reported ROS generation and activation of molecular signaling associated with oxidative stress, among them the activator protein-1 (AP-1) and nuclear factor kB (NF-kB) and MAPK (Pacurari et al. 2008), responses that in fact can alter the redox balance in biological environments (Liu et al. 2013). Moreover, carboxylated carbon nanotubes showed higher interaction with mitochondria proteins than pristine carbon nanotubes, meaning that the presence of functional groups are a key factor determining potential toxicity of these nanomaterials (González-Durruthy et al. 2016).
The pro-oxidant condition with high levels of intracellular ROS allows its reaction with cellular macromolecules, including DNA, lipids and proteins, and disrupts homeostasis of the intracellular medium after CNT exposure. da Rocha et al. (2013) reported higher mRNA levels of Nrf2, a transcription factor that is activated in pro-oxidant conditions, after exposure of zebrafish D. rerio to SWCNT through i.p. injection.
Redox homeostasis is maintained through a balance between the reactive oxygen species generated by cellular functions and the antioxidants (enzymatic and non-enzymatic) present in the organisms (Halliwell 1994). However, when this balance is disrupted, several molecules can suffer oxidation, including proteins at specific amino acid residues (cysteine, methionine, histidine and tyrosine) (Halliwell and Gutteridge 2007). Also, the reaction of by-products of lipid peroxidation such as malondialdehyde with cysteine and lysine residues can lead to oxidative modifications of protein by introducing carbonyl groups (Stadtman 1993; Halliwell 1994; Halliwell and Gutteridge 2007). In present study, protein carbonyl groups detected by immunohistochemistry showed to be a good option to evaluate responses in organs that usually are not considered in nanotoxicology studies as kidney, stomach and eyes.
In fact, considering the irreversible oxidative damage as it is carbonyl groups in proteins, no conspicuous differences were observed between fish exposed to SWCNT and MWCNT. Except for higher carbonyl levels (in respect to control group) in intestine of fish exposed to SWCNT and eyes in fish exposed to MWCNT (Fig. 5), both CNT types seemed to induce similar pro-oxidant effects. As mentioned previously, authors such as González-Durruthy et al. (2016) postulated that in terms of mitochondrial toxicity, the key toxicophore of CNT is functional groups as carboxyl groups and a similar statement has been done by Madani et al. (2013).
As conclusion, obtained data indicate that CNT administered through diet can in fact induce toxicological responses in aquatic organisms as fish. This response was obtained with pristine carbon nanotubes (without functional groups as carboxyl or hydroxyl), nanomaterials with low water solubility. Under an ecotoxicological context, it seems reasonable that exposure via of aquatic organisms to these CNT should be by ingestion. Further studies using carboxylated CNT are necessary to evaluate properly the potential environmental risk of these kinds of nanomaterials.
André L. da R. Seixas received a graduate fellowship from Instituto Nacional de Ciência e Tecnologia de Nanomateriais de Carbono (MCTI/CNPq). Marlize Ferreira-Cravo received a post doc fellowship from FAPERGS/CAPES. Ana C. Kalb received a post doc fellowship from CAPES. José M. Monserrat is a research fellow from CNPq (Process No. 307880/2013-3). The authors would like to thank the laboratory technicians working at the Instituto de Ciências Biológicas (ICB) FURG and at the Laboratório de Materiais Cerâmicos (LACER), Universidade Federal do Rio Grande do Sul (UFRGS) Porto Alegre, RS and the undergraduate students Lennon Flores Brongar and Astaruth Nayara Vicente. The logistic and material support from the Nanotoxicology Network (MCTI/CNPq, Proc. 552131/2011-3) was essential for the execution of present study. The support from CNPq (Universal Project No. 479053/2012-0) given to José M. Monserrat is also acknowledged.
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