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NANOTECHNOLOGIES Effects of Multiwalled Carbon Nanotubes Received Orally during 6 Days on the Gastrointestinal Tract
K. S. Golokhvast1,2, V. V. Chaika1, L. V. Kuznetsov3, K. V. Elumeeva3, M. I. Kusaikin4, A. M. Zakharenko4, N. N. Kiselev1, A. M. Panichev1, G. V. Reva1, V. V. Usov1, I. V. Reva1, T. Yamamoto1, and A. N. Gul’kov1 Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 155, No. 6, pp. 753-757, June, 2013 Original article submitted March 8, 2012 The effects of multiwalled carbon nanotubes on epitheliocytes of different compartments of the gastrointestinal tract and urothelium of different compartments of the renal nephron were studied in CBA mice. The nanotubes affected mouse gastrointestinal mucosa and renal urothelium. The cell reaction in the macula densa of the renal distal tubules and the immune system reaction to oral nanotubes were detected. A possible effect of nanotubes administered orally on the renal filtration function was hypothesized. Key Words: multiwalled carbon nanotubes; nanotoxicity; CBA mice
Contacts of humans with nanoparticles become inevitable because of rapid development of nanotechnologies [2-4]. Research in this direction is mainly focused on the mutually related problems: evaluation of the biological and toxic effects of nanoparticles and the prospects of their use as the means for drug delivery and with diagnostic purposes [1,11]. The priority problems in nanotoxicology are studies of the basic regularities of manifestations of the nanoparticles biological and toxic effects depending on particle shape, size, form factor, initial material, surface area, surface charge, admixtures, and other physicochemical characteristics of their structure and 1
Far-Eastern Federal University, Vladivostok; 2Vladivostok Affiliated Department of Far-Eastern Center of Respiratory Pathology and Physiology, Siberian Division of the Russian Academy of Medical Sciences, Institute of Medical Climatology and Rehabilitative Treatment; 3G. K. Boreskov Institute of Catalysis, Siberian Division of the Russian Academy of Sciences, Novosibirsk; 4G. B. Elyakov Pacific Institute of Organic Biochemistry, Far-Eastern Division of the Russian Academy of Sciences, Vladivostok, Russia. Address for correspondence:
[email protected]. G. V. Reva
the mechanisms of their effects on cells and tissues [6,7,9]. Not less important are studies evaluating the doses, routes of administration, concentrations of nanoparticles in the target organ, and duration of exposure [5,8,10]. We studied the response of the gastrointestinal mucosal structures of CBA mice to oral administration of the multiwalled carbon nanotubes and their penetration through the epithelial barrier, absorption in the intestine, and reaction of the kidneys.
MATERIALS AND METHODS The study was carried out on CBA mice (n=60; vivarium of G. B. Elyakov Pacific Institute of Organic Biochemistry). The material from different gastrointestinal compartments and the kidneys was studied after oral administration of the nanotubes for 1, 2, 3, 4, 5, and 6 days (Table 1). Animals of all groups received standard ration under standard light, thermal, and humidity conditions.
0007-4888/13/15560788 © 2013 Springer Science+Business Media New York
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TABLE 1. Experimental and Control Groups Day of study Group
Total 1
2
3
4
5
6
Control (intact mice)
5
5
5
5
5
5
30
Experimental
5
5
5
5
5
5
30
Total:
10
10
10
10
10
10
60
In order to rule out estrogen effects on proliferative activity of the gastrointestinal mucosa epitheliocytes, experiments were carried out only on males. Multiwalled carbon nanotubes (SC3-2-FCC 3-E3p), 18-20 nm in diameter, with specific surface (SBET)=130 m2/g, were obtained at G. K. Boreskov Institute of Catalysis [8]. In order to remove the catalyst, the nanotubes were boiled in 15% HCl and then washed in distilled water until neutral reaction. The nanotubes used in the experiment had technological admixtures: 0.21% Fe, 0.12% Co, 0.004% Ca, 0.005% Al, and 0.08% Cl. The initial nanotubes were coiled wire 30-50 μ in size. After preliminary disintegration (1 min) in a Bandelin Sonopulse 3400 ultrasonic, they were administered to animals with fodder. The animals received fodder with nanotubes at the same time of the day in accordance with the circadian rhythms (once daily as a challenge, 500 mg/kg). Attempts at administration through a tube failed, because waterinsoluble nanotubes blocked even the largest of the available tubes. The material for the study was collected daily during the same hours (12.00) in accordance with Regulations for Studies with the Use of Experimental Animals, 12.08.1977. Biopsy specimens of the stomach were collected from the cardial, fundal, and antral compartments in accordance with the WHO golden standard. Semithin sections of all gastrointestinal compartments and the kidneys were stained with hematoxylin and eosin. The materials were analyzed and processed under an Axio Observer A1 microscope (Carl Zeiss) and Olympus Bx51 microscope with a CD 25 digital camera.
RESULTS No pathological shifts were found in the gastrointestinal mucosa of intact controls. In the experimental group, mast cells migrating to the lamina propria were identified after 1 day of oral administration of nanotubes. This could cause hyperpermeability of the connective tissue and de-
veloping edema. Increase of the secretory activity and hypertrophy of the gastric glandular epithelium were detected. On day 2, the nanotubes were identified in the gastrointestinal wall in the mucosal barrier zone on the surface of the gastroduodenal mucosa (Fig. 1, a, b). The nanotubes formed a layer with a clear-cut borderline adjacent to the epitheliocyte apical surface (Fig. 1, c-e). Nanotubes and their aggregations (round and oval) up to 10-20 μ in size were detected. On day 3, the nanotubes were found in the deep layers and were detected not only in the epitheliocyte cytoplasm, but in the nuclei as well. This was paralleled by reduction of nucleus basophilia. On days 3-6, the mucosal epitheliocytes of various gastrointestinal compartments contained nanotubes diffusely scattered in the cytoplasm; the nanotubes were also identified in the epitheliocyte nuclei (Fig. 2, a-d). The microcirculatory bed of the gastrointestinal mucosa lamina propria was unchanged in controls and experimental mice. No pathological changes in the kidneys or filtration barrier reaction to the nanotubes were detected in intact mice and in mice after 1 and 2 days of the challenge, but 3 days later, analysis of biopsy specimens from the kidneys showed hyperemia in the vascular glomeruli, Bowman-Shumlyansky capsule lumen remaining unchanged. The vascular glomerular cells and the macula densa were basophilic (Fig. 2, e, f). The tubular wall in the renal cortical parenchyma had normal structure on day 3, but starting from day 4, nuclear and cytoplasmic basophilia increased, which could indicate strained function of the filtration system. The nuclei acquired a flat shape, and by day 6 became oval; the nuclear/cytoplasmic proportion increased significantly. Infiltration by erythrocytes developed in the regions adjacent to the tubules. Lymphoid tissue was seen in the renal parenchyma and connective tissue; follicles were forming, with reactive zones identified in some of them. Solitary neutrophils were identified among lymphocytes in the lymphoid tissue. On day 6, in addition to changes in the gastrointestinal mucosal barrier structures, the lymphoid follicles in the mucosal lamina propria were enlarged. In the spleen, the red to white pulp ratio in experimental mice shifted towards the predominance of the white pulp, Malpighian bodies increasing in size after 6 days of observation. On day 1 of the experiment, the nanotubes were identified at the level of the esophageal wall mucosal barrier in the cardial, fundal, and antral portions of the stomach. On days 2 and 3, the nanoparticles penetrated through the mucosa and epithelial barriers in the duodenum and small and large intestinal walls.
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Fig. 1. The gastrointestinal mucosa in experimental mice, day 2 of experiment. a) gastric wall (arrow: mast cell); b) duodenal mucosal surface (arrow: nanotubes in epitheliocyte apical part); c) small intestinal mucosal surface); d, e) large intestinal mucosal surface. Hematoxylin and eosin staining, ×800.
Stage 2 of the nanotubes passage through the epithelial barrier was the epithelial cytoplasm. First, the nanotubes occupied the border zone in the epithelial apical part and then reached the basement membrane, where they formed a line parallel to the basal membrane. The migration rate of nanotubes administered orally through the intestinal mucosal epithelium was higher than through gastric mucosal epitheliocytes. No
receptor recognition was observed under conditions of a short-term experiment, only nanoparticle contamination in epitheliocytes was seen. This was paralleled by secretory hyperactivity of the glandular epithelium, a defense reaction of the mucosa. Identification of mast cells in the lamina propria of small intestinal microvillous mucosa could result from the reaction to oral nanotubes. The histophysiol-
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Fig. 2. The gastrointestinal mucosa in experimental mice. a) esophageal wall (day 4); b) gastric wall, pyloric portion; c) duodenal villus (day 5); d) small intestine (day 6); e, f) renal bodies (days 3 and 6, respectively). Hematoxylin and eosin staining, ×800. MD: macula densa cells.
ogy of mast cells suggested that they were involved in attraction of macrophages (by means of production of appropriate cytokines) and induced (by secreting local homeostasis regulators) changes in the microcirculatory vascular lumen permeability for nanoparticles elimination through the portal vein system into the
liver for detoxification. No nanotubes were identified in renal structures. The cell reaction in the macula densa of the distal tubular wall manifesting by more intense basophilia and hypertrophy was an indirect evidence of their effects on the water-salt metabolism in mice.
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Generalized reaction of the immune system to oral administration of nanotubes was realized at the expense of hypertrophy of lymphoid structures in the lamina propria of the gastrointestinal mucosa, follicle hypertrophy in the renal capsule and parenchyma, and Malpighian body hypertrophy in the splenic white pulp. We hypothesized that lymphoid tissue hypertrophy in the gastrointestinal wall and spleen resulted from induction of the immune response by epitheliocytes contaminated with carbon nanotubes with subsequent antigen presentation by effector immunocytes and migration of effector immunocytes. On the whole, we can speak about the absence of a pronounced toxic effect of multiwalled carbon nanotubes on CBA mice under conditions of a short-term experiment. However, certain immunogenic activity of the nanomaterial and increase of the migration activity of cells, manifesting by lymphoid infiltration, are worthy of note. The study was supported by the Foundation Promoting the Development of Minor Ventures in Research and Technology (U.M.N.I.K. Program) and Analytical Departmental Program “The Develop-
ment of Research Potential of Higher School (20092011)”.
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