Comparative study for carcinogenicity of 7 different multi-wall carbon nanotubes with different physicochemical characteristics by a single intraperitoneal injection in male Fischer 344 rats

Abstract

The present study comparatively examined carcinogenicity of 7 different multi-wall carbon nanotubes (MWCNTs) with different physicochemical characteristics. Physicochemical characteristics of MWCNTs (referred to as M-, N-, WL-, SD1-, WS-, SD2- and T-CNTs in the present study) were determined using scanning electron and light microscopes and a collision type inductively coupled plasma mass spectrometer. Male Fischer 344 rats (10 weeks old, 15 animals per group) were administered MWCNTs at a single intraperitoneal dose of 1 mg/kg body weight, and sacrificed up to 52 weeks after the commencement. Fibers of M-, N-, WL- and SD1-CNTs were straight and acicular in shape, and contained few agglomerates. They were relatively long (38-59% of fibers were longer than 5 μm) and thick (33% to more than 70% of fibers were thicker than 60 nm). All of these 4 MWCNTs induced mesotheliomas at absolute incidences of 100%. Fibers of WS-, SD2- and T-CNTs were curled and tightly tangled to form frequent agglomerates. They were relatively short and thin (more than 90% of measured fibers were thinner than 50 nm). WS- CNT did not induce mesothelioma, and only one of 15 rat given SD2- or T-CNT developed tumor. Any correlations existed between the metal content and neither the size or form of fibers, nor the carcinogenicity. It is thus indicated that the physicochemical characteristics of MWCNTs are critical for their carcinogenicity. The straight and acicular shape without frequent agglomerates, and the relatively long and thick size, but not the iron content, may be critical factors. The present data can contribute to the risk management, practical use and social acceptance of MWCNTs.

INTRODUCTION

Multi-wall carbon nanotubes (MWCNTs), a fruit of recently developing nanotechnology, have attracted attention by their promising characteristics allowing potential applicability in a wide range of industrial fields, and their practical use has partly started. A structural similarity to asbestos raised concerns, however, on the safety of MWCNTs such that these engineered nanomaterials may cause hazards, especially carcinogenicity, as asbestos that induces pleural mesothelioma and lung cancer in humans (Niklinski et al., 2004) and pleural and/or peritoneal mesotheliomas in animals depending on the route of administration (Davis, 1976; Bolton et al., 1982). Large efforts have been being made to assess risks of MWCNTs, and as parts of such efforts we have previously shown that MWCNTs in fact induce mesotheliomas in both p53-heterozygous C57BL/6 mice and Fischer 344 rats by administration into peritoneal or scrotal cavities (Takagi et al., 2008a; Sakamoto et al., 2009). Later, carcinogenicity of MWCNTs has been confirmed even when it is administered via more human-relevant routes in Fischer 344 rats (Suzui et al., 2016; Kasai et al., 2016).

It is crucial to elucidate the mechanisms underlying and factors involving the onset and magnitude of the carcinogenicity and the other toxicity of MWCNTs in order to precisely assess their risks and thereby ensure their safe use in human environments. Certain types of MWCNTs have been shown to exert no or little inflammatory or carcinogenic effects (Muller et al., 2009; Murphy et al., 2011; Nagai et al., 2011, 2013; Poland et al., 2008; Varga and Szendi, 2010), and a variety of factors have been suggested as determinants of carcinogenicity and toxicity of MWCNTs, and such factors are dimensional properties, including length and diameter, rigidity, surface properties and functionalization, biodurability and concomitant metal (Donaldson et al., 2013; Fenoglio et al., 2008; Lanone et al., 2013; Nagai et al., 2011, 2013; Toyokuni, 2013; Hamilton et al., 2017). Direct evidence is still limited, however, to indicate different carcinogenicity of different MWCNTs with different profiles regarding aforementioned factors.

In this context, the present study was conducted to comparatively evaluate the carcinogenicity of different MWCNTs with different physicochemical characteristics in rats. For this purpose, we conducted the carcinogenicity study by using a single intraperitoneal dose of 1 mg/kg body weight, and an observation period of 52 weeks. This dose and periods were decided based on our previous work indicating that they can develop mesotheliomas at the high incidence in male Fischer 344 rats (Sakamoto et al., 2009).

MATERIALS AND METHODS

Test materials

In the present study, 7 different MWCNTs were used. They are called herein as M-, N-, WL-, SD1-, WS-, SD2-, T-CNTs. M-, N-, SD1-, SD2- and T-CNTs were obtained as gifts from Mitsui, Nikkiso, Showa Denko and Toda Kogyo, respectively, while WL and WS-CNTs were purchased from Wako as dry bulk powders (Table 1).

Table 1. MWCNTs used in the present study.

MWCNT	Source	Purity	Length	Diameter
		%	μm	nm
M-CNT	Mitsui (MWNT-7, lot number 05072001k28)	(> 95) a)	< 1-4 : 46%	20-50 : 36%
			5-20 < : 54%	60-100 < : 64%
			mean : 6.65	mean : 66.8
			(13)	(40-50)
N-CNT	Nikkiso	(> 98)	< 1-4 : 62%	30-50 : 67%
			5-20 < : 38%	60-100 < : 33%
			mean : 5.48	mean : 59.2
			(NS)	(30)
WL-CNT	WAKO	(ND) b)	1-4 : 41%	10-50 : 59%
			5-20 < : 59%	60-100 < : 41%
			mean : 7.31	mean : 70.9
			(0.5-10)	(85-200)
SD1-CNT	Showa-Denko	(NS)	< 1-4 : 62%	30-100 : 11%
			5-20 : 38%	100 < : 89%
			mean : 4.51	mean : 177.4
			(8)	(150)
WS-CNT	WAKO	(NS)	ND c)	20-50 : 92%
				60-100 : 8%
				mean : 44.5
			(0.5-2)	(40-70)
SD2-CNT d)	Showa-Denko	(NS)	ND	<10-30 : 100%
			(3)	mean : 13.5
				(10-15)
T-CNT	Toda Kogyo	(85-94)	ND	10-50 : 93%
				60-100 : 7%
				mean : 35.8
			(0.732)	(21)

Values resulted from the determination in the present study without the exceptions below. ^a) Values in the parentheses were the data supplied by the manufacturer. ^b) NS represents that the data were not supplied by the manufacturer. ^c) ND represents that the value was not determined. ^d) According to the manufacturer, SD2-CNT forms big and tight agglomerates.

Characterization of test materials and their preparation

For the measurement of the distribution of lengths and diameters, MWCNTs were suspended in ethanol at a concentration of 0.1 mg/mL, dispersed by the ultrasonication and rendered to the measurement by SEM (Quanta 250, Scanning Electron Microscope, EFI, Tokyo, Japan).

Metal contents in MWCNTs were determined by a collision type inductively coupled plasma mass spectrometer (ICP-MS, 7500ce, Agilent Technologies Inc., Santa Clara, CA, USA). Metals in each MWCNT were eluted by nitric acid with heating by microwave (Speedwave 4, BERGHOF Products + Instruments GmbH, Eningen, Germany) at 200°C. The elution was diluted by purified water and filtered by syringe filter (Millex®-AA, Millipore Co., Bedford, MA, USA). The resultant filtrates were used for the ICP-MS analysis.

For the carcinogenicity studies, MWCNTs were suspended in 2% carboxymethyl cellulose (CMC) (Kanto Chemical Co. Inc., Tokyo, Japan) solution at concentrations of 1 mg/mL, sterilized by an autoclave at 120°C for 20 min and vigorously mixed by hand-shaking immediately prior to the administration. In order to characterize the conditions of MWCNT fibers in suspensions used for the intraperitoneal injection (see below), the suspensions were observed light microscopically at the magnification of x 400.

Animals and treatment

Male Fischer 344 rats were purchased at the age of 4 weeks old from Charles River Inc. (Kanagawa, Japan) and maintained in our laboratory until use in the present study at the age of 10 weeks old. MWCNTs in suspension (or vehicle) were intraperitoneally injected to rats (10-15 animals per group) at a single dose of 1 mg/kg body weight. The experiment was terminated 52 weeks after injection, when all the surviving animals were sacrificed under isoflurane anesthesia and examined as described below. Animals that died or sacrificed due to moribundity before the scheduled sacrifice were similarly examined.

Pathological assessment

At autopsy, rats were macroscopically examined throughout the body, including celomic cavities. All major organs, including the diaphragm with the attached mesenteriolum, were resected, fixed in 10% neutrally buffered formalin and processed routinely to prepare paraffin-embedded sections. Mesothelioma was diagnosed in combination of histological and the following immunohistochemical assessments.

For the immunohistochemical studies, primary antibodies used were C-ERC Mesothelin (No.28001, Immuno-Biological Laboratories Co. Ltd., Fujioka-shi, Gunma, Japan, 1:100) as a marker for mesothelial cells, E-cadherin (sc-7870, Santa Cruz Biotechnology, Dallas, TX, USA, 1:300) as a marker for epithelial cells, Vimentin (N1521, DAKO, Glostrup, Denmark, 1:100) as a marker for mesenchymal cells and CDKN2A/P16INK4A (CDKN2A, ab-54210, Abcam plc, Cambridge, UK, 1:100) as a marker for genomic alteration of the tumors. To enhance the immunostaining for antibodies, deparaffinized sections were placed in a thermoresistant beaker filled with a citrate buffer, pH 6.0, or Tris-EDTA buffer, pH 9.0, and a heat epitope retrieval procedure was performed using a microwave oven (500 W). The immunoperoxidase staining was done using the Envision (K1491, DAKO) as a secondary antibody and visualized with 3,3’-diaminobenzidine (K3486, DAKO), while nuclei were lightly counterstained with hematoxylin.

Ethical considerations

The experimental protocol was approved by the Experiments Regulation Committee and the Animal Experiment Committee of the Tokyo Metropolitan Institute of Public Health prior to its execution and monitored at every step during the experimentation for its scientific and ethical appropriateness, including concern for animal welfare, with strict obedience to the National Institutes of Health Guideline for the Care and Use of Laboratory Animals, Japanese Government Animal Protection and Management Law, Japanese Government Notification on Feeding and Safekeeping of Animals and other similar laws, guidelines, rules, etc. provided domestically and internationally.

RESULTS

Physicochemical characteristics of MWCNTs used in the present study

Fibers of M-, N-, WL- and SD-1 CNTs were relatively straight and acicular in shape, and contained few agglomerates (Fig. 1), by which we could determine their lengths and diameters in our laboratories on approximately 300 fibers using the SEM images. The distribution of their lengths formed Gaussian curves, and 38-59% of fibers were longer than 5 μm (Fig. 2, Table 1). The distribution of their diameters also formed Gaussian shapes, and 33% to more than 70% of fibers were thicker than 60 nm, and especially, the ratio of fibers less than 100 nm of SD1-CNT was only 11% (Fig. 2, Table 1). These MWCNTs were relatively long and thick, and the order of the length was WL- ≈EM- > SD1- ≈EN-CNT, while that of the thickness was SD1- >> WL - > M- > N-CNT.

Fig. 1

SEM images of MWCNTs.

Fig. 2

Distribution of lengths and diameters of M-, N-, WL- and SD1- CNTs.

Fibers of WS-, SD2- and T-CNTs were curled and tightly tangled to form frequent agglomerates (Fig. 1), by which we could not determine their lengths. Their manufacturers provided the data saying that the lengths of WS-, SD2- and T-CNTs were approximately 0.5-2, 3 or 0.732 µm, respectively (Table 1). On the other hand, we could barely determine their diameters using the SEM images, and the diameters of more than 90% of measured fibers were thinner than 50 nm (Fig. 3 and Table 1). These MWCNTs were relatively short and thin, and the order of the thickness was WS- ≥ T- > SD2-CNT.

Fig. 3

Distribution of diameters of SD2-, WS- and T-CNTs. Lengths of these MWCNTs could not be measured because of their agglomerate nature.

Figure 4 shows representative light microscopic appearances of the MWCNTs used in a 2% CMC suspension that was to be administered to rats. M-, N-, WL-, and SD1-CNT fibers were well dispersed and showed straight and acicular shape or rod-like shape with varied sizes in association with few agglomerates. In contrast, WS-, SD2- and T-CNT were curled and tangled to form frequent agglomerates with varied sizes.

Fig. 4

Light microscopical images of MWCNTs in suspensions administered to rats (x 400).

The quantitative analysis of metal contents of the MWCNTs by ICP-MS showed that Fe, Al, Cr, Mn, Ni, Cu, Zn, Mo, Ba and Pb were detected, and the contents of these metals varied among the 7 MWCNTs. WL-CNT contained the most metal, and in SD1-CNT the contents of all metals were lower than the limit of quantitation. The contents of Fe and Al were relatively higher than those of the other metals. Fe contents ranged from 0.18 to 59.14 μg/mg with the order of WL- > T- > SD2- > M- > WS-CNT, and Al contents were from 0.23 to 28.78 μg/mg with the order of T- > SD2- > N- > WL- > WS-CNT (Table 2). In addition to the 10 metals above, the contents of B, Co, As, Cd, Sb, Bi were also measured, but they were all under the limit of quantitation. Any correlations existed between the metal contents and neither the sizes or forms, nor the carcinogenicities of MWCNTs (see below).

Table 2. Metal content in MWCNTs.

MWCNTs	Metal content (μg/mg CNT)
	Fe	Al	Cr	Mn	Ni	Cu	Zn	Mo	Ba	Pb
	(0.1) a)	(0.1)	(0.01)	(0.01)	(0.01)	(0.01)	(0.02)	(0.01)	(0.01)	(0.01)
M-CNT	3.2	ND	0.02	ND	ND	ND	0.03	ND	ND	ND
N-CNT	ND b)	0.5	ND	ND	ND	ND	ND	ND	0.01	ND
	[0.002-0.005 ppm] c)
WL-CNT	59.1	0.3	0.02	0.05	0.07	0.09	0.17	ND	0.02	0.13
	[< 4.7%]
SD1-CNT	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND
WS-CNT	0.2	0.2	0.03	ND	4.89	ND	0.02	ND	0.02	ND
	[< 0.1%]
SD2-CNT	11	22.5	ND	0.02	ND	ND	ND	1.98	ND	ND
T-CNT	48.6	28.8	0.01	0.13	0.01	ND	ND	ND	ND	ND

^a) Values in the parentheses were limit of quantitation. ^b) Less than limit of quantitation. ^c) Values in the brackets were the data supplied by the manufacturer.

Carcinogenicity of MWCNTs used in the present study

Control rats healthily survived until the end of the experimental period of 52 weeks, and no particular pathological symptoms or signs, including macroscopical or histological changes, were observed in these animals (Fig. 5, Table 3).

Fig. 5

Survival curves.

Table 3. Survival data and macroscopic and histological changes regarding the development of mesotheliomas.

MWCNT	Effective number of rats	Survivors at the end of the experiment	Hemorrhagic ascite	Mesothelioma
				Incidence	Invasion	Metastasis	Osteoid change
Vehicle control	10	10 (100) a)	0	0	0	0	0
M-CNT	12	1 (8)	11 (92)	12 (100)	12 (100)	8 (67)	7 (58)
N-CNT	10	4 (40)	8 (80)	10 (100)	9 (90)	4 (40)	4 (40)
WL-CNT	15	0 (0)	10 (67)	15 (100)	14 (93)	10 (67)	10 (67)
SD1-CNT	14	3 (21)	12 (86)	14 (100)	11 (79)	7 (50)	8 (57)
WS-CNT	14	14 (100)	0	0	0	0	0
SD2-CNT	14	14 (100)	0	1 (7) b)	0	0	0
T-CNT	13	13 (100)	0	1 (8) b)	0	0	0

^a) Values of other than those for the effective number of rats, are numbers of corresponding rats followed by values in the parentheses showing those percentages. ^b) These mesotheliomas were considered spontaneous tumors seen in aged F344 rats; details are given in the text.

In the M-, N-, WL-, and SD1-CNT groups, 4, 5, 4 and 1 rats died, and 7, 1, 11 and 10 animals became moribund 32-50, 39-52, 26-43 and 36-50 weeks after the commencement, while 1, 4, 0 and 3 rats survived until the scheduled sacrifice, respectively (Fig. 5, Table 3). Among these 4 MWCNTs, trends of survival or survival rates (Table 3) were not significantly different, and causes of deaths or becoming moribund were mesotheliomas in all cases. In contrast, all rats survived without severe problems throughout the experimental period in the WS-, SD2- or T-CNT groups (Fig. 5, Table 3).

At autopsy, various sizes, shapes and extents of tumorous nodules were observed in abdominal cavities of M-, N-, WL- and SD1-CNT-treated rats (Fig. 6). The whitish and spherical nodules either existed focally or spread diffusely along both the visceral and parietal peritonea, most frequently on the serosal surfaces of the diaphragm, stomach with omentum majus, liver and ligament, including mesenterium (Fig. 6). Hemorrhagic ascites frequently accompanied (Table 3). These macroscopical features were observed similarly among rats treated with the among rats treated with M-, N-, WL-, or SD1-CNT, regardless of the autopsy timing (after premature death, early sacrifice due to moribundity, or at the scheduled sacrifice). In contrast, no particular macroscopic changes were observed, with the exception of black spots due to the deposition of MWCNT fibers, in rats treated with WS-, SD2- or T-CNT (Fig. 6).

Fig. 6

Representative macroscopic apperrances of the abdominal cavities. Inset: diaphragm. Arrow: deposited MWCNT fibers.

Mesotheliomas were histologically diagnosed in all rats treated with M-, N-, WL- or SD1-CNT, in association with frequent invasion to muscular tissues of the diaphragm and abdominal walls, and metastases toward mediastinal lymph nodes and distant thoracic tissues, or osteoid changes were seen in approximately half of such animals (Table 3). The mesotheliomas typically consisted of enlarged, pleomorphic epithelioid cells with prominent nuclear membrane and nucleoli, and basophilic cytoplasm, and occasionally of elongated or spindle-form cells (Fig. 7). These mesothelioma cells grew to form dome- or convex lens-shaped nodules on the serosal surface, projecting toward the abdominal cavity, and spherical nodules with a fibrous matrix in the central region (Fig. 7). Representative immunohistochemical characteristics of the mesotheliomas are shown in Fig. 7. Mesothelin and E-cadherin proteins were expressed only in the superficial region, and not in most parts of the tumors. Vimentin, in the cytoplasm, was diffusely positive in the tumors. Nucleic signal of CDKN2A (p16) is a marker of genetic alteration in tumorous lesions, and were clearly detected diffusely in the mesotheliomas. The histological and immunohistochemical characteristics of mesotheliomas were similar among animals treated with the among rats treated with M-, N-, WL-, or SD1-CNT, regardless of the autopsy timing (after premature death, early sacrifice due to moribundity, or at the scheduled sacrifice). In contrast, mesotheliomas were developed in no rats in the WS-CNT group, and only 1 rat each in the SD2- and T-CNT groups (Table 3). Unlike counterparts seen in M-, N-, WL- and SD1-CNT-treated rats, however, these mesotheliomas located in the limited area of the surface of the testis, epididymis and their adjacent tissues, without spread toward the peritonea, and thus considered not to be related to the administration of MWCNTs, but to be spontaneous mesotheliomas that are seen in aged male Fischer 344 rats at the incidence of 4-5% (Goodman et al., 1979; Haseman et al., 1998; Blackshear et al., 2014).

Fig. 7

Representative hitstological and immunohistochemical appearances of mesotheliomas developed on the diaphragms in rats treated with M-, N-, WL- and SD1-CNTs.

In the M-, N-, WL- and SD1-CNT groups, inflammatory changes were seen throughout the peritonea in association with mesotheliomas, and MWCNT fibers were scattered as single, isolated fibers in the submesothelial area of the peritonea regardless of the presence of mesotheliomas. In some case of mesotheliomas, MWCNT fibers were observed in the tumor tissue (data not shown). Granulomas were also formed on the peritonea to include aggregations of fiber-laden macrophages, but this granuloma formation was positionaly independent from the development of mesothelioma. Other lesions, such as necrosis, in tumor nodules and/or neoplastic lesions in other organs were not observed in rats treated with MWCNTs (data not shown). In contrast, no particular changes were observed in the peritonea of WS-, SD2- or T-CNT-treated animals, with the exception of small number of granulomas that included MWCNT fibers and fiber-laden macrophages. These granulomas were basically similar to those seen in groups treated with the other 4 types of MWCNTs, but associated with more collagen fibers (Fig. 8).

Fig. 8

Representative histological appearances of granulomas on the parietal peritonea in rats treated with WS-, SD2- and T-CNTs.

DISCUSSION

The above results indicate that certain MWCNTs are carcinogenic as has been reported, but that not all MWCNTs are. One might criticize our use of an intraperitoneal route to administer the MWCNTs, because it is irrelevant to the human situation. The present study was conducted, however, to compare different carcinogenic potencies of different MWCNTs, and to seek what are factors to be involved in such difference. That is we dealt with the hazard of MWCNTs, not their risk. For such a purpose, the most sensitive and the easiest way should be used (Takagi et al., 2008b).

M-CNT is the MWCNT that we demonstrated to cause carcinogenicity for the first time in the world (Takagi et al., 2008a; Sakamoto et al., 2009). While in these of our studies and those of others (Nagai et al., 2011, 2013) studies M-CNT was administered intraperitoneally and induced mesotheliomas in rodents, a recently published inhalation study revealed that M-CNT induced lung cancers (Kasai et al., 2016). Based on these data, the International Agency for Research on Cancer has categorized M-CNT into group 2B, “possibly carcinogenic to humans,” while the other types of MWCNTs were thrown into group 3, “not classifiable as to their carcinogenicity to humans” (Grosse et al., 2014). As one of such “the other types of MWCNTs,” N-CNT was shown to induce both mesotheliomas and lung cancers (Suzui et al., 2016), while SD1-CNT invaded into the thoracic cavity and caused inflammatory changes (Xu et al., 2014), in rats by an intratracheal instillation. No chronic data for SD1-CNT, or no intraperitoneal data for N-CNT or SD1-CNT were available, and the present study revealed them for the first time. SD2-CNT did not cause any inflammatory or carcinogenic effects on the mesothelial in rats when administered either intratracheally (Xu et al., 2014) or intraperitoneally (Nagai et al., 2011, 2013), which are in line with the present results. Data for the toxicity or carcinogenicity of WL-, WS- or T-CNT are not available in the literature, and thus were revealed for the first time in the present study.

The physicochemical characteristics of MWCNTs have been thought to be one of the main factors to determine their bioreactivity, including toxicity (Donaldson et al., 2013; Fenoglio et al., 2008; Lanone et al., 2013; Nagai et al., 2011, 2013; Toyokuni, 2013; Hamilton et al., 2017). The lengths of 38.2-58.3% of carcinogenic M-, N-, WL- and SD1-CNT fibers were longer than 5 μm, and the diameters of 70% of fibers of such MWCNTs were thicker than 60 nm. The few other types of MWCNTs have been shown to have a carcinogenic potential, and their size distributions appeared to be similar to those of M-, N-, WL- and SD1-CNTs (Nagai et al., 2011; Rittinghausen et al., 2014). Long and thick fibers have been indicated to be a requisite for MWCNTs to cause inflammatory responses (Murphy et al., 2011; Poland et al., 2008). Similar findings have been reported for the potency to cause chronic inflammation, fibrosis and malignant tumors of asbestos and other man-made mineral fibers (Davis, 1986, 1988, 1989; Donaldson et al., 1989; Kamp et al., 1992; Kane, 1996; Miller et al., 1999; Pott et al., 1987; Stanton et al., 1977, 1981; Wagner et al., 1984), and the inflammatory reactivity of silver nanowire (Schinwald et al., 2012). The significant presence of the long (> 5 µm) and thick (> 60 nm) fibers is thus indicated to be one of the key factors for MWCNTs to cause carcinogenicity. It is noteworthy that Nagai et al. (2011) reported that NT145 in their study possessed physicochemical characteristics similar to SD1-CNT and induced mesothelioma when intraperitoneally given to rats. The carcinogenicity of NT145 was much weaker than M-CNT (Nagai et al., 2011), and Nagai et al. (2011) insisted that the carcinogenicity of MWCNT thicker than a particular optimal diameter may be reduced. While the present results for SD1-CNT was inconsistent with those for NT145, this discrepancy may be due to the fact that the lengths and diameters of fibers are so varied in each MWCNT sample, and the means and the standard deviations are not good parameters to compare these characteristics. Although the means and standard deviations of the lengths and diameters of SD1-CNT and NT145 were similar, their distributions may be significantly different, or the 2 MWCNTs may have some other different physicochemical characteristics responsible for their different carcinogenicities.

Another common physicochemical characteristic among carcinogenic M-, N-, WL- and SD1-CNTs was a straight and acicular shape in association with few agglomerates. This property, along with the above-mentioned long length and thickness, is also be a key factor of the carcinogenic potency of MWCNT, because they make it possible that MWCNT escapes from the lymphatic drainage system (Moalli et al. 1987; Abu-Hijleh et al., 1995), and becomes a good substrate of frustrated phagocytosis with the persistent expression of reactive oxygen species and cytokines, one of the major candidate mechanisms underlying the carcinogenicity of MWCNT and asbestos (Donaldson et al., 2010; Moalli et al., 1987; Tomatis et al., 2010; Schinwald et al., 2012).

On the other hand, non-carcinogenic WS-, T- and SD2-CNT fibers were less than 3 μm, and the diameters of most of these fibers were thinner than 50 nm.

Furthermore, these 3 non-carcinogenic MWCNTs were curled and tightly tangled to form frequent agglomerates. Short, thin and tangled MWCNTs are known not to induce malignant tumors even after a long period after the exposure (Muller et al., 2009; Nagai et al., 2013; Varga and Szendi, 2010). It is well known that thin fibers easily tangle and thus form agglomerates. As a result, these MWCNTs are cleared by macrophages and the lymphatic flow, or demarcated within granulomas (see Fig. 8), which ultimately prevents the nanotubes from exerting carcinogenicity.

Metal impurities in the MWCNTs used were quantitatively analyzed by ICP-MS, and 10 metals were detected. The concentrations of the 10 metals varied among MWCNTs, and did not well correlated with the fiber sizes, forms and carcinogenicities of the MWCNTs. Concomitant metals, especially iron, are suspected to affect the carcinogenicity of MWCNT and asbestos, because they work as catalysts in the oxidative stress reactions, one of the major candidate underlying mechanisms (Jiang et al., 2012: Nagai et al., 2011, 2013; Toyokuni, 2013). In the present study, however, the iron contents of the MWCNTs used did not correlate well with their carcinogenic properties.

Mesotheliomas induced by M-, N-, WL- and SD1-CNTs were histologically similar to those seen in our previous studies using M-CNT (Takagi et al., 2008a; Sakamoto et al., 2009) and also in the literature for animals exposed to MWCNTs (Nagai et al., 2011; Rittinghausen et al., 2014) or asbestos and other man-made mineral fibers (Adachi et al., 2001; Blobel et al., 1985; Davis, 1976). While mesothelin was expressed only in superficial, epithelially-shaped cells of the tumors as previously shown (Sakamoto et al., 2009), E-cadherin demonstrated the similar staining pattern. Assuming that E-cadherin is an epithelial marker, it is suggested that the epithelial property of mesothelial cell is lost from the relatively early stage of the mesothelioma development due to carcinogenic MWCNTs. In contrast, most of the mesothelioma cells in the present study, losing the epithelial shape, were positive for vimentin, a marker of mesenchymal cells. It is known that vimentin is strongly expressed in human sarcomatous type mesotheliomas (Fassina et al., 2012), and thus the histological and vimentin immunohistochemical appearance indicate that mesothelial cells possess mesenchymal nature, and that the presently observed mesotheliomas correspond to human sarcomatous-type mesotheliomas. These indications are supported by the fact that CDKN2A was diffusely expressed in the nuclei of the presently observed mesotheliomas. The CDKN2A/2B gene is one of the most major targets genetically altered in mesotheliomas (Hu et al., 2010), and the homozygous deletion of the CDKN2A/2B tumor suppression gene is detected in asbestos-induced mesotheliomas, especially sarcomatous type (Nagai et al., 2011). Taken together, the present CDKN/2A immunohistochemistry indicates that mesothelin- or E-cadherin-negative tumor cells are mesenthelioma cells, and that such an alteration in the presently observed mesothelioma cells may result from the progression of MWCNT-inducing carcinogenic process through the epithelial-mesenchymal transition.

In this study, carcinogenicities in rats were evaluated by using MWCNTs suspended in 2% CMC solution, and their fiber sizes were determined using MWCNTs suspended in ethanol. However, because the obtained data on the size distribution are similar to those obtained from aqueous suspension of MWCNTs containing Tween 80 (Takaya et al., 2010), it is suggested that the size distributions are not practically different regardless of the vehicle, if MWCNTs are sufficiently dispersed. For the determination of the metal contents of MWCNTs, the elution of metals from MWCNTs requires the strong acidic condition at a high temperature (Fenoglio et al., 2008; Patole et al., 2016), and metals are not eluted in the aqueous solution under the neutral pH. It is thus suggested that the metal contents of MWCNTs in 2% CMC solution (administering to rats) are dependent on the concentration of MWCNTs. In this context, it is reasonable to assess the relationship between the physicochemical characteristics of MWCNTs and the carcinogenicities, using the data obtained by the methods using in the present study.

In conclusion, the above results indicate that the physicochemical characteristics of MWCNT are critical for their carcinogenicity. The straight and acicular shape without frequent agglomerates, and the relatively long and thick size, but not the iron content, may be critical factors.

ACKNOWREDGMENTS

This work was supported a Grants-in-Aid for Scientific Research from the Ministry of Health, Labour and Welfare of Japan (H27-Kagaku-Shitei-004 and H30-Kagaku-Shitei-004). The authors would like to thank to Mr.Tomokazu Maeno and Nobutaka Fukuori for analysis of MWCNT fiber physicochemical characteristics.

Conflict of interest

The authors declare that there is no conflict of interest.

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