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Morphological assessment of influence of carbon nanofibers on digestive organs of Wistar rats upon oral administration.

Introduction

Recently, carbon nanofibers (CNF) have been drawing more and more attention [1-3]. They have wide field of application in technology ranging from textile industry with nanofabric production [4] to sorption of poison and targeted drug delivery [5]. But, according to the data of researchers, there is a danger to the environment and human health in the application of nanomaterials, including nanofibers [6-8]. Carbon nanotubes and nanofibers can be more toxic than the quartz fibers. As one ofthe scientific works illustrates [9] single-layer nanotube perceptible cytotoxicity can be observed under 6-h exposition and it increases up to 35% while growth of tube concentration to 11.3 pg per cm2.

The purpose ofthis work was to study the morphological changes in the digestive tract of animals while three types of CNF are administered orally.

Materials and Methods

All animal experiments were performed in compliance with the principles of humanity as set forth in the directives of the European Community (86/609/EEC) and the Declaration of Helsinki. Before starting the work all the animals were kept under the same conditions. During at least 10 days before the experiment starts, the rats received a full food ration in accordance with the Rules of the work using experimental animals.

To determine the effect of CNF on the morphology of the visceral organs resulted by oral administration, we used Wistar male rats with the weight of 300 g. They consume the CNF with food in dose of 500 mg per kilogram of a body weight within 14 days. CNF were mixed with food that is ordinary animal feeding stuff; the process of eating was monitoring.

Animals were divided into four experimental groups, each is consisting of 10 animals; the groups are as follows:

"Control"--the animals that received no minerals;

"KM2" group contains the rats, treated by CNF of KM2-56-BR type;

"BR" is a group where the rats were treated by CNF of 56-BR type;

"OBR" are the rats, treated by CNF of 56-OBR type.

CNF were synthesized at the Boreskov Institute of Catalysis SB Ras, Novosibirsk, by catalytic decomposition of propane-butane mixture ([C.sub.3]-[C.sub.4]) over a 90%NiO+10%[Al.sub.2][O.sub.3] heterogeneous catalyst, prepared by nitrates of nickel and aluminum coprecipitation method. Synthesis was performed on a rotor reactor facility under the 500[degrees]C temperature. Carbon product yield made up 24 g per a gram of catalyst (cat). The morphology of carbon fibers corresponds with coaxial conic type. The obtained sample, 56BR, was divided into three parts. The second part has been treating with concentrated nitric acid for 30 min, which allowed removing the dispersed particles of nickel from the carbon material (56-OBR). The third part of 56BR original material was exposed to wet milling in a suspension with water in ceramic grinding mill for 24 hr to obtain KM2-56BR CNF type. According to TEM, the average diameter of the carbon fibers in all three samples was 85 nm and it does not change during processing, while the length of the fibers has a little decreasing being comminuted. The length of fibers varies in the range of 5-50 pm. Characteristics of different CNF are given in Table 1.

CNF are the aggregates of various sizes (Figure 1).

CNF aggregates size particle analysis was performed with the Analysette 22 NanoTech (Fritsch Company) laser particle analyzer. Biopsy sampling was held after rats' intramuscular narcotization by 0.5 ml of a 5% ketamine solution. The blocks were fixed in "Histoline" (Element, Russia) 10% buffered neutral formalin solution, dehydrated in ethanol with increasing concentration and embedded in "Histomix Extra" (BioVitrum, Russia) paraffin. Semifine sections were prepared from these blocks, then the sections were colored by hematoxylin eosin, examined under "Zeiss Axio Observer A1" (Zeiss, Germany) light microscope; to make photos "Axio Cam 3" (Zeiss, Germany) and "AxioVision 4.2" program of computer morphometry were used.

Results and Discussion

Administered with animal feedstuff CNF were not detected as the individual structures and were presented by the aggregates of different sizes (see Figure 2).

For your convenience, the results of laser granulometry are summarized in Table 2.

As we can see, the aggregates have a big specific surface area and can adsorb biomolecules and disrupt normal biochemical and physiological processes. Light microscopy among the experimental groups of animals, treated CNF with feeding, found out a number of differences in the stomach tissues histological structure in comparison with Control group (Figures 3a and 3b).

Overall, in comparison with Control group a mucosal thickening of stomach can be observed in 56-BR, KM2, and 56-OBR groups, which is a consequence of mechanical stimulation by CNF. Submucous membrane, serosal membrane, and smooth muscles have no pathology in all groups. The vessels are full-blooded, but stasis is not registered.

Dystrophic changes and necrosis in the surface layers of mucous can be observed in rats of KM2 group (Figure 2a). The nucleuses are not traced, cells' borders are obliterated. Multiple lymphoid follicles are found in the submucous membranes in the rats of 56-OBR group (Figure 2b). Morphometric parameters of gastric epithelial cells in the experimental groups are shown in Table 3.

Light microscopy of the histological structure of intestine revealed a number of differences in the experimental groups from control (see Figures 4a and 4b).

An intense production of mucus is observed in mucosal membranes of rats from KM2, 56-BR, and 56-OBR groups. There are many goblet cells with vacuoles full of mucous in the epithelium (see Figures 4a and 4b). The vessels are full-blooded. Hypertrophy of the lymphoid follicles is recognized in the submucosal layer in group number 56-BR and 56-OBR. The other pathology was not discovered. Morphometric parameters of the intestine epithelial cells in the Control group and in experimental groups are given in Table 4.

Liver tissues in all experimental groups do not have any distinguishing features in comparison with Control group. There were no pathological sins discovered, neither dystrophy and necrosis nor edema and inflammation. We can only notice that CNF administration causes more pronounced vessels plethora and erythrocytes infiltration (see Figure 5).

Morphometric parameters of the hepatocytes in "Kulikovskoe" control and experimental group are shown in Table 5.

Conclusions

As can be seen from the results of our investigation, different types of CNF have a pronounced effect on the histological structure of the digestive organs, if it being administered orally.

Thus, submucous membrane and serosal membrane of stomach, smooth muscles are without pathological changes in all groups. However, we observed thickening of mucous in all experimental groups, which is KM2, 56-BR, and 56-OBR, against the Control group.

A great number of goblet cells with vacuoles full of mucus are found in intestine of rats from all experimental groups. It is resulting in intense mucus production (see Figures 3a and 3b). The vessels are full-blooded, because of the mechanical stimulation by nanofibers.

Liver tissue gave less apparent response. We only can notice more pronounced vessels plethora and erythrocytes infiltration on CNF administrating.

There is also a reaction of the immune system: the hypertrophy of lymphoid follicles in submucosal layer is caused by the introduction of CNF 56-BR and 56-OBR.

Apparent toxic property, which is related, probably, with the mechanical damage of tissues, as well as the other observed effects, proved itself through the dystrophic changes and necrosis of mucous in KM2 group. Probably, it can be related with the less size of CNF, occurred after grinding, and big surface area of the aggregates (up to 8983.11 [cm.sup.2]/[cm.sup.3]).

Generally our results are consistent with the data of other researchers [10,11]. The study [6] is devoted to study the dependence of the toxicity of nanotubes and nanofibers on the ratio of length and diameter and the presence ofvarious functional groups on their surface. The toxicity of multiwall nanotubes appeared to be lower than toxicity of nanofibers [7,11]. Notably that the nanotubes' toxicity increases with the increase in the ratio of length and diameter.

Undoubtedly, that this work requires further study because of the high importance of the technological process of promising nanomaterials.

The work was supported by the Scientific Fund of Far Eastern Federal University (#13-06-0318-m_a, #14-08-02-24_i) and Grant of the Ministry of Education and Science of the Russian Federation (#14.594.21.0006).

References

[1.] Huang X (2009) Fabrication and properties of carbon fibers. Materials 2: 2369-2403.

[2.] Feng L, Xie N, Zhong J (2014) Carbon nanofibers and their composites: a review of synthesizing properties and applications. Materials 7: 3919-3945.

[3.] Chand S (2000) Review carbon fibers for composites. Journal of Materials Science 35: 1303-1313.

[4.] Krichevskiy GY (2011) Nanobiochemical Technologies and Production of the Next Generation Fibers, Textile and Clothes. Moscow: Izvestiya, p. 528.

[5.] He C, Nie W, Feng W (2014) Engineering of biomimetic nanofibrous matrices for drug delivery and tissue engineering. Journal of Materials Chemistry B 2(45): 7828-7848.

[6.] Magrez A, Kasas S, Salicio V, Pasquier N, Seo JW, et al. (2006) Cellular toxicity of carbon-based nanomaterials. Nano Letters 6: 1121-1135.

[7.] Horvath L, Magrez A, Schwaller B, Forro L (2011) Toxicity study of nanofibers. Supramolecular Structure and Function 10: 133-149.

[8.] Delorme MP, Muro Y, Arai T, Banas DA, Frame SR, et al. (2012) Ninety-day inhalation toxicity study with a vapor grown carbon nanofiber in rats. Toxicol Sciences 128(2): 449-460.

[9.] Andreev GB, Minashkin BM, Nevskiy IA, Putilov AV (2008) The materials produced by nanotechnology: potential risk in production and use. Russian Chemical Journal LII(5): 32-38.

[10.] Hammel E, Tang X, Trampert M, Schmitt T, Mauthner K, et al. (2004) Carbon nanofibers for composite applications. Carbon 42: 1153-1158.

[11.] Murray AR, Kisin ER, Tkach AV, Yanamala N, Mercer R, et al. (2012) Factoring-in agglomeration of carbon nanotubes and nanofibers for better prediction of their toxicity versus asbestos. Particle and Fibre Toxicology 9: 10.

Kirill Sergeevich Golokhvast (1) *, Nina Valentinovna Sayapina (2), Tatyana Anatolievna Batalova (2), Alexander Alexandrovich Sergievich (2), Ilya Vladimirovich Mishakov (3), Alexey Anatolievich Vedyagin (3), Mikhail Alexandrovich Novikov (4), Alexander Ivanovich Agoshkov (1), Valery Ivanovich Petukhov (1), Vladimir Alexandrovich Drozd (1), Sergey Maksimovich Ugay (1), Vladimir Viktorovich Chaika (1)

(1) Far Eastern Federal University, Sychanova Street 8, 690950 Vladivostok, Russia

(2) Amur State Medical Academy Gorkogo Street 95, 675000 Blagoveshchensk, Russia

(3) Boreskov Institute of Catalysis SB RAS, Lavrentyev Academician Avenue, 5, 630090 Novosibirsk, Russia

(4) Eastern Siberian Center of Human Ecology SB RAMS, Bte 1170, 665827 Angarsk, Russia

* Corresponding author: Golokhvast KS, Far Eastern Federal University, Sychanova Street 8, 690950 Vladivostok, Russia

Received: January 23, 2015; Accepted: February 23, 2015; Published: April 13, 2015

Table 1: Physical and chemical parameters of the CNF under experiment

Specification    CNF diameter     CNF length   CNF specific surface
                     (nm)         ([micro]m)    area ([m.sup.2]/g)

56-BR           From 20 to 200       5-50              105
KM2-56-BR       (85 in average)                        103
56-OBR                                                 125

Table 2: Physical and chemical parameters of the CNF under
experiment

Specification   Mean diameter of the CNF   CNF aggregates specific
                 aggregates ([micro]m)          surface area
                                           ([cm.sup.2]/[cm.sup.3])

56-BR                    14.52                     7549.34
KM2-56-BR                25.22                     8983.11
56-OBR                     --                        --

Table 3: Morphometric parameters of gastric epithelial
cells in the experimental groups ([micro]m)

Groups         Nucleus              Nucleus
                length               width

Control   5.11 [+ or -] 0.46   4.46 [+ or -] 0.46
KM2       5.41 [+ or -] 0.6    4.69 [+ or -] 0.56
BR        5.15 [+ or -] 0.68   4.49 [+ or -] 0.44
OBR       5.49 [+ or -] 0.61   4.73 [+ or -] 0.53

Groups          Nucleus                Cell
                 area                 length

Control   16.29 [+ or -] 2.58   11.55 [+ or -] 1.5
KM2       18.62 [+ or -] 3.17   12.22 [+ or -] 1.37
BR        16.85 [+ or -] 3.18   11.6 [+ or -] 1.55
OBR       18.59 [+ or -] 3.51   12.18 [+ or -] 1.31

Groups           Cell                   Cell
                 width                  area

Control   9.05 [+ or -] 1.46    69.47 [+ or -] 14.74
KM2       10.24 [+ or -] 1.31   85.05 [+ or -] 15.24
BR        9.32 [+ or -] 1.06    73.3 [+ or -] 12.29
OBR       9.76 [+ or -] 1.04    78.95 [+ or -] 14.61

Table 4: Morphometric parameters of the intestine epithelial
cells in the experimental group of animals (pm)

Groups         Nucleus              Nucleus
                length               width

Control   4.96 [+ or -] 0.66   3.81 [+ or -] 0.65
KM2       5.63 [+ or -] 1.07   4.12 [+ or -] 0.85
BR         6.2 [+ or -] 0.74    4.5 [+ or -] 0.77
OBR       5.36 [+ or -] 0.74    4.1 [+ or -] 0.7

Groups          Nucleus                Cell
                 area                 length

Control   12.84 [+ or -] 2.86   14.34 [+ or -] 2.97
KM2          16 [+ or -] 3.7     16.5 [+ or -] 2.1
BR        18.54 [+ or -] 3.34   17.68 [+ or -] 1.74
OBR       15.15 [+ or -] 2.56    14.7 [+ or -] 1.6

Groups           Cell                  Cell
                width                  area

Control   8.64 [+ or -] 2.28   60.67 [+ or -] 14.7
KM2       7.25 [+ or -] 1.52   76.71 [+ or -] 11.13
BR        6.79 [+ or -] 1.08   85.32 [+ or -] 10.81
OBR       5.77 [+ or -] 0.57   68.16 [+ or -] 5.56

Table 5: Morphometric parameters of the hepatocytes
in experimental groups of animals (pm)

Groups        Nucleus              Nucleus
               length               width

Cotrol   6.23 [+ or -] 0.56   5.67 [+ or -] 0.52
KM2      6.59 [+ or -] 0.57   5.89 [+ or -] 0.4
BR       6.61 [+ or -] 0.63   5.85 [+ or -] 0.72
OBR      6.45 [+ or -] 0.39   5.83 [+ or -] 0.4

Groups         Nucleus                Cell
                area                 length

Cotrol   26.73 [+ or -] 4.07    15.9 [+ or -] 1.47
KM2      29.13 [+ or -] 3.78   15.32 [+ or -] 1.05
BR       28.89 [+ or -] 5.48   15.75 [+ or -] 1.3
OBR       28.5 [+ or -] 3.4     15.1 [+ or -] 1.36

Groups          Cell                   Cell
                width                  area

Cotrol    13.3 [+ or -] 1.73   150.56 [+ or -] 22.1
KM2      13.29 [+ or -] 1.02   145.59 [+ or -] 12.9
BR       13.48 [+ or -] 2.13   148.68 [+ or -] 17.71
OBR      12.84 [+ or -] 1.36   139.74 [+ or -] 21.86
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Title Annotation:Research Article
Author:Golokhvast, Kirill Sergeevich; Sayapina, Nina Valentinovna; Batalova, Tatyana Anatolievna; Sergievic
Publication:Biology and Medicine
Article Type:Report
Date:Apr 1, 2015
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