Scientific Papers

Difference in carcinogenicities of two different vapor grown carbon fibers with different physicochemical characteristics induced by intratracheal instillation in rats | Particle and Fibre Toxicology


Characteristics of carbon fibers

The primary particle morphology, dispersion state, and iron content of carbon fibers

To determine the differences in the physicochemical properties of VGCF™-H and MWNT-7, an investigation of the primary particle morphology and the average hydrodynamic diameter of the carbon fibers was performed by scanning electron microscopy (SEM) and dynamic light scattering (DLS). The VGCF™-H fiber was a diameter of 148 ± 52 nm, a length of 5.2 ± 2.7 μm and a surface area of 15 m2/g. The MWNT-7 fiber was a diameter of 75 ± 20 nm, a length of 9.0 ± 6.1 μm and a surface area of 25 m2/g. Additional file 1: Fig. S1 shows the distribution of the carbon fiber length and diameter of MWNT-7 and VGCF™-H used in this study. Both VGCF™-H and MWNT-7 fibers were straight in shape. The average hydrodynamic diameters of MWNT-7 and VGCF™-H were 656 ± 34 nm and 783 ± 43 nm, 514 ± 40 nm and 589 ± 23 nm, and 561 ± 63 nm and 613 ± 29 nm in the 0.008 mg/mL (for 0.128 mg/kg), 0.04 mg/mL (for 0.64 mg/kg), and 0.2 mg/mL (for 3.2 mg/kg) doses, respectively. The average hydrodynamic diameters of the fibers did not change when passed through the microsprayer aerosolizer (data not shown). Iron contents of MWNT-7 and VGCF™-H fiber was 4200 ppm and 9.7 ppm, but no other elements were detected for VGCF™-H fibers (Additional file 1: Table S1).

Dispersion state of carbon fibers in saline solution containing 0.3% Kolliphor P188

Figure 1 shows representative SEM images of carbon fibers in the prepared test material solution. SEM observation was performed to evaluate the dispersion state of the carbon fibers in the solution. VGCF™-H fibers were found to be shorter and thicker than MWNT-7 fibers, and both the MWNT-7 and VGCF™-H fibers dispersed as single straight fibers with no aggregation. These profiles were observed at all doses.

Fig. 1
figure 1

Representative scanning electron microscopic images of MWNT-7 (A) and VGCF™-H (B) fibers in the prepared solution. VGCF™-H fibers were shorter and thicker than MWNT-7 fibers. The scale bar indicates 1 μm

Survival and body weight

Figure 2 and 3 shows a Kaplan–Meier survival curve for male and female rats. Male rats in the 0.64 and 3.2 mg/kg MWNT-7 groups began to die 56 and 53 weeks into the experimental period, which was notably earlier than those in the VGCF™-H groups (Fig. 2). In a previous report describing a 2 year carcinogenicity study, the final survival rate of male and female F344 rats were 66.0–88.0% and 68.0–86.0%, respectively [14]. No significant change in the survival rates of male and female rats in the nontreatment and control groups (82.9% and 74.3%, and 62.9%, and 80.0%, respectively) was reported compared to the results obtained in previous studies, suggesting that the intratracheal instillation process had no effect on the survival rate. The final survival rates of male rats in the MWNT-7 groups were 70.0, 40.0, and 2.5%, and survival rates of 65.0, 87.5, and 75.0% observed in the VGCF™-H groups subjected to 0.128, 0.64, and 3.2 mg/kg, respectively. Importantly, the survival rates of male rats in the 0.64 and 3.2 mg/kg MWNT-7 groups were lower than those of all other groups. On the other hand, no clear decrease in the survival rate was observed in the male VGCF™-H groups, although the survival rate of the VGCF™-H group that received 0.128 mg/kg was slightly lower than that of the control group. Females in the 3.2 mg/kg MWNT-7 group began to die at week 65 in the experimental period (Fig. 3), which was earlier than the females in the other groups. The final survival rate at week-104 of the experimental period were 70.0, 65.0, and 35.0% for the female MWNT-7 groups, and 72.5, 77.5, and 77.5% for the female VGCF™-H groups at 0.128, 0.64, and 3.2 mg/kg, respectively. The survival rates of the 0.64 and 3.2 mg/kg MWNT-7 groups were therefore markedly lower than those of the VGCF™-H group. The survival rate of the all-female MWNT-7 groups was lower than that of the control group. The survival rate of the male MWNT-7 groups was lower than that of females, and no decreasing trends were observed in the survival rates of the male VGCF™-H groups at any dose, as were observed in the male MWNT-7 groups.

Fig. 2
figure 2

Kaplan–Meier survival plot for the male rats in the nontreatment, control, MWNT-7, and VGCF™-H groups in each dose

Fig. 3
figure 3

Kaplan–Meier survival plot for the female rats in the nontreatment, control, MWNT-7, and VGCF™-H groups in each dose

Body weights fluctuated slightly after instillation of MWNT-7 or VGCF™-H (data not shown), but no significant changes were observed in either group at week 104 of the experimental period (Table 1).

Table 1 Body weights at week 104 of the experimental period

Evaluation of inflammation and tissue damage by carbon fibers

Analysis of inflammatory cells in the PLF

Table 2 shows the cellular component of the PLF at week 13 of the experimental period. No significant changes were observed in the nontreatment and control groups, with both males and females unaffected in terms of the studied parameters. The number of white blood cells (WBC) and differential leukocytes increased significantly in the male MWNT-7 groups, except for basophils. No significant changes were observed in the PLF from males and females in the VGCF™-H groups, except for a significant increase in the numbers of lymphocytes and eosinophils in the male 3.2 mg/kg VGCF™-H group. A significant increase was observed in the WBC and differential leukocytes of the female MWNT-7 groups, except for neutrophils and basophils, while no significant increase was observed in either WBC or differential leukocytes in the female VGCF™-H groups at week 13 of the experimental period.

Table 2 Cellular components in the PLF at week 13 of the experimental period

Table 3 shows the cellular components of the PLF at week 104 of the experimental period. No significant increase was observed in the WBC or the differential leukocytes in all male VGCF™-H groups at week 104. Many of the male animals in the MWNT-7 group that received 3.2 mg/kg died by 104 weeks of the experiment, and the cellular components of the PLF could only be measured in one rat, with a significant decrease observed in the lymphocytes of the 0.64 mg/kg male MWNT-7 group thought to be the result of error as the changes were not dose dependent. There was a significant increase in the monocytes of the 3.2 mg/kg MWNT-7 group; however, no other significant changes were observed in any of the female groups.

Table 3 Cellular components in the PLF at week 104 of the experimental period

A lower inflammatory response was observed in the VGCF™-H groups than the MWNT-7 groups. The data indicating the cellular component in the PLF at week 104 of the experimental period were less variable and the association between these results and carcinogenicity was unclear.

Analysis of clinical chemistry in the PLF

Table 4 shows the clinical chemistry data in the PLF at week 13 of the experimental period. The biomarkers μ-TP and μ-ALB were increased significantly in the male MWNT-7 group exposed to 3.2 mg/kg, while only μ-ALB was increased significantly in the 0.64 mg/kg male VGCF™-H group, and this was assumed to be the result of errors as the changes were not dose-dependent.

Table 4 Clinical chemistry in the PLF at week 13 of the experimental period

Table 5 shows the clinical chemistry data in the PLF at week 104 of the experimental period. No significant increase was observed for LDH, μ-TP, or μ-ALB in any of the male and female MWNT-7 and VGCF™-H groups. Many of the male animals in the group that received 3.2 mg/kg of MWNT-7 died by 104 weeks of the experiment, and the levels of these markers were therefore measured in only one rat. The results indicate that the pulmonary damage associated with VGCF™-H was lower than that for MWNT-7.

Table 5 Clinical chemistry in the PLF at week 104 of the experimental period

Distribution of carbon fibers

Amount of VGCF™-H and MWNT-7 fibers in the lung

To investigate lung toxicity relationships between the instillation of carbon fibers and lung toxicity such as inflammation and carcinogenicity, the lung burden of the carbon fibers was examined. Tables 6 and 7 show the amount of carbon fibers in the lungs of both male and female rats at weeks 13 and 104 of the experimental period. Table 6 shows the amount of carbon fibers in the lungs of each animal were observed to have increased in a dose-dependent manner by week 13 in the male MWNT-7 and VGCF™-H. The lung weight of males in both the MWNT-7 and VGCF™-H groups changed slightly with dose; however, the amount of carbon fibers per lung tended to be similar to the amount of carbon fibers administered. Interestingly, although the amount of carbon fibers in the lungs increased in a dose-dependent manner in both the male MWNT-7 and VGCF™-H groups, the amount of VGCF™-H observed was less than half that seen in the MWNT-7 group at week 104 of the experimental period in males that were subjected to 0.64 mg/kg. The amount of fibers in the lung could not be accurately measured in the male 3.2 mg/kg MWNT-7 group because many animals had died before week 104.

Table 6 Amount of carbon fibers in the lungs of male rats
Table 7 Amount of carbon fibers in the lungs of female rats

Table 7 shows the amount of carbon fibers and the lung weight of females at weeks 13 and 104 of the experimental period. The amount of carbon fibers in the lung was observed to increase in a dose-dependent manner by week 13, with equivalent amounts observed in the lungs of rats receiving different doses of MWNT-7 and VGCF™-H. The amount of carbon fibers in the lungs of females in the 3.2 mg/kg VGCF™-H group was less than 1/7 that of the MWNT-7 group at week 104.

These results suggested that the lungs were cleared off the VGCF™-H fibers more rapidly than the MWNT-7 fibers in both males and females.

Additional file 1: Tables S2 and S3 shows the results of converting the amount of carbon fiber in the lung to the surface area of the carbon fiber. The total surface area of carbon fibers in the lung was greater for MWNT-7 than for VGCF™-H in both sexes up to 13 weeks after instillation. At 104 weeks of instillation, the difference was even greater, VGCF™-H at 0.64 mg/kg in males was less than 1/4 that of MWNT-7, and VGCF™-H at 0.64 mg/kg and 3.2 mg/kg in females were less than 1/2 and 1/12 that of MWNT-7, respectively. These results are similar to Tables 6 and 7, suggesting that VGCF™-H has a lower biopersistence than MWNT-7.

Number of VGCF™-H and MWNT-7 fibers in the PLF

Table 8 shows the number of carbon fibers in the PLF of males and females at weeks 13 and 104 of the experimental period, which can be utilized to investigate the relationship between carcinogenicity and the number of carbon fibers in the pleural cavity. The numbers of MWNT-7 and VGCF™-H fibers in the pleural cavity were observed to increase in a dose-dependent manner, with less than 1/10 and 1/30 VGCF™-H fibers observe as compared to MWNT-7 groups at all doses for both male and female rats at week 13. Most importantly, the increase in the number of MWNT-7 and VGCF™-H fibers in the pleural cavity observed at week 104 of the experimental period was dose-dependent increases and the number of VGCF™-H fibers was less than 1/8 that seen in the MWNT-7 groups that received 0.128 and 0.64 mg/kg. The number of fibers in the PLF could only be measured in one animal in the 3.2 mg/kg male MWNT-7 group because most of the animals were dead by week 104 of the experimental period.

Table 8 Number of carbon fibers in the PLF

These results indicated that the carbon fibers were retained in the PLF for 104 weeks, and that the VGCF™-H fibers were less easily transferred from the lungs to the pleural cavity as compared to the MWNT-7 fibers.

Changes in lung weights

No significant change was observed in the lung weight of the male MWNT-7 and VGCF™-H groups; however, the weight of the lungs from the 3.2 mg/kg female MWNT-7 group were found to have increased significantly at week 104 of the experimental period (Table 9). The lung weights of males in the 3.2 mg/kg MWNT-7 groups could not be measured because most of the animals in this group were dead by week 104 of the experimental period.

Table 9 Lung weights at week 104 of the experimental period

Gross pathological examination

Gross pathological examination was performed on dead, euthanized, and animals surviving at week 104 of the experimental period. Most of the dead or euthanized animals in the MWNT-7 groups had nodules or small granules adhered to the thoracic wall, heart, or diaphragm, with clear/red fluid in their pleural or pericardial cavities. One image of a male rat in the MWNT-7 group that died in week 45 of the experimental period shows small white granules (Fig. 4A yellow arrows), white nodules (Fig. 4A green arrows), and black deposits in the thoracic area and the interior of the lung. The granules and nodules had spread around the lungs and reached the heart epicardium, diaphragm, and thoracic wall. One image of a male rat from the VGCF™-H group that died at week 60 shows white nodules (Fig. 4B green arrows) and swelling (Fig. 4B red arrows) around the lungs and the heart. These profiles were confirmed in animals that survived until week 104, male animals that died since week 46 of the experimental period, and in female MWNT-7 groups that died since week 66. Most importantly, these profiles were confirmed only in two animals, one of which was euthanized at week 60 and one in the male 3.2 mg/kg VGCF™-H group that died 102 weeks into the experiment.

Fig. 4
figure 4

Representative gross pathological image of thoracic area in the male MWNT-7 group that died at week 45 of the experimental period (A) and an image of the male VGCF™-H group that died at week 60 of the experimental period (B). The small white granules (yellow arrows) and white nodule (green arrows) had spread around the lungs, and reached the heart epicardium, diaphragm, and thoracic wall (A). The white nodule (green arrows) and the swelling (red arrows) present around the lungs and heart (B). The scale bar indicates 10 mm

Histopathological examination

Neoplastic lesions

Table 10 shows details of the neoplastic lesions that were observed in the histopathology of dead, euthanized, and surviving rats 104 weeks after intratracheal instillation. No significant increase was observed in the incidence of adenoma, adenocarcinoma, and combined them in the lungs and bronchia of males or females in the MWNT-7 and VGCF™-H groups as a result of any dose. Adenosquamous carcinoma was observed in the lung/bronchia of one male and one female from the 3.2 mg/kg MWNT-7 groups, but none was observed at any dose in the VGCF™-H group. The incidences of malignant mesothelioma in the thoracic cavity 1 in 30 (3.3%), 18 in 30 (60.0%), and 37 in 39 (94.9%) in males that received 0.128, 0.64, and 3.2 mg/kg MWNT-7, and there was a significant increase in them compared to control group. However, the incidences in the thoracic cavity were 0 in 30 (0%), 0 in 30 (0%), and 2 in 30 (6.7%) in males that received 0.128, 0.64, and 3.2 mg/kg VGCF™-H, and there were not statistically differences from that of control group. Interestingly, the incidences of mesothelioma of the dead and euthanized rats were 1 in 12 (8.3%), 15 in 24 (62.5%), and 37 in 39 (94.9%) in the groups that received 0.128, 0.64, and 3.2 mg/kg of MWNT-7, respectively, while only 2 in 10 (20%) of the male rats that received 3.2 mg/kg in VGCF™-H group. This result suggested that malignant mesothelioma was developed early after carbon fiber instillation. The incidence of malignant mesothelioma in the thoracic cavity was 2 in 30 (6.7%), 1 in 30 (3.3%), and 22 in 30 (73.3%) in female rats that received 0.128, 0.64, and 3.2 mg/kg MWNT-7, and there was a significant increase in them compared to control group. No malignant mesothelioma was observed in any of the female VGCF™-H groups at any dose. The development of malignant mesothelioma in the thoracic cavity was dose-dependent increase in both male and female MWNT-7 groups. Most malignant mesothelioma had spread to the surface of the lungs (Fig. 5B for the MWNT-7 group, Fig. 5E for the VGCF™-H group), heart epicardium (Fig. 5C in MWNT-7, Fig. 5F in VGCF™-H group), thoracic wall, and diaphragm and had invaded each organ. Mesothelium hyperplasia of the heart increased significantly compared to control group in the male groups that received 0.64 mg/kg and 3.2 mg/kg and female groups that received 3.2 mg/kg MWNT-7 but was not observed in the VGCF™-H groups. Interestingly, the alveolar hyperplasia, bronchiolo alveolar hyperplasia, and mesothelium hyperplasia on the lung/bronchia increased significantly compared to control group in the MWNT-7 groups, but not in the VGCF™-H groups. The mesothelial hyperplasia of the diaphragm increased significantly compared to control group in both the 0.64 mg/kg and 3.2 mg/kg male and female MWNT-7 groups and the male 3.2 mg/kg VGCF™-H group but was not observed in any of the female VGCF™-H groups at any dose.

Table 10 Summary of histopathological findings for neoplastic lesions at 104-weeks of the experimental period
Fig. 5
figure 5

Representative histological images of the lung and heart in the MWNT-7 and VGCF™-H group. Deposition of MWNT-7 fibers (arrow in A) and the alveolar hyperplasia (A). B, C indicates the malignant mesothelioma in the MWNT-7 group. It indicates the deposition of VGCF™-H fibers (arrow in D) in the lung (D). E, F indicates the malignant mesothelioma in the VGCF™-H group. The scale bar indicates 50 μm

There were no significant differences from the incidences of the other neoplastic lesions compared to control group in both the male and female MWNT-7 and VGCF™-H groups (Tables 11 and 12).

Table 11 Histopathological findings: summary of neoplastic (benign) or neoplastic (malignant) in male
Table 12 Histopathological findings: summary of neoplastic (benign) or neoplastic (malignant) in female

Non-neoplastic and pre-neoplastic lesions

Table 13 exhibits the non-neoplastic lesions and pre-neoplastic lesions obtained from the dead, euthanized, and surviving rats via histopathology at 104 weeks following intratracheal instillation. Deposits comprising fibers and inflammatory cells that infiltrated the lungs and bronchia increased significantly compared to control group in both the male and female MWNT-7 and VGCF™-H groups. Pleura fibrosis, chronic inflammation, granuloma formation on the lung/bronchia, and pleural fibrosis in the diaphragm increased significantly compared to control group in the MWNT-7 groups, but not in the VGCF™-H groups.

Table 13 Summary of histopathological findings for non-neoplastic or pre-neoplastic lesions at 104-weeks of the experimental period



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