Abstract

Nanomaterials are part of an industrial revolution to develop lightweight but strong materials for a variety of purposes. Singlewall carbon nanotubes are an important member of this class of materials. They structurally resemble rolled-up graphite sheets, usually with one end capped; individually they are about 1 nm in diameter and several microns long, but they often pack tightly together to form rods or ropes of microscopic sizes. Carbon nanotubes possess unique electrical, mechanical, and thermal properties and have many potential applications in the electronics, computer, and aerospace industries. Unprocessed nanotubes are very light and could become airborne and potentially reach the lungs. Because the toxicity of nanotubes in the lung is not known, their pulmonary toxicity was investigated. The three products studied were made by different methods and contained different types and amounts of residual catalytic metals. Mice were intratracheally instilled with 0, 0.1, or 0.5 mg of carbon nanotubes, a carbon black negative control, or a quartz positive control and euthanized 7 d or 90 d after the single treatment for histopathological study of the lungs. All nanotube products induced dose-dependent epithelioid granulomas and, in some cases, interstitial inflammation in the animals of the 7-d groups. These lesions persisted and were more pronounced in the 90-d groups; the lungs of some animals also revealed peribronchial inflammation and necrosis that had extended into the alveolar septa. The lungs of mice treated with carbon black were normal, whereas those treated with high-dose quartz revealed mild to moderate inflammation. These results show that, for the test conditions described here and on an equal-weight basis, if carbon nanotubes reach the lungs, they are much more toxic than carbon black and can be more toxic than quartz, which is considered a serious occupational health hazard in chronic inhalation exposures. Key Words: carbon nanotubes; pulmonary toxicity; epithelioid granulomas; nanotube toxicity. President Clinton established the National Nanotechnology Initiative in 2000 to lead this country into the next industrial revolution Carbon nanotubes structurally resemble rolled-up graphite sheets with one end capped. These tiny tubes can have single or multiple walls. Single-wall carbon nanotubes (NTs), unlike graphite or carbon black, possess highly desirable electrical, mechanical, and thermal properties NTs can be produced by deposition of carbon atoms vaporized from graphite by electric arc or by laser onto metal particles. More recently, they have been produced by chemical vapor deposition (CVD). High-pressure CO conversion (HiPco™, Rice University, TX) is a CVD process and is a more advanced method that uses carbon monoxide as carbon source; up to 97% of the carbon in the HiPco product ends up in NTs The study was conducted on three NT products made by different methods and containing different types or amounts of residual metals; they were raw (RNT) and purified (PNT) iron-containing HiPco products of Rice, and CarboLex's nickel-containing electric-arc product (CNT). For the present study, we used intratracheal instillation, an accepted route of exposure commonly used to screen dusts for potential pulmonary toxicity MATERIALS AND METHODS Animals and animal care. Male mice (B6C3F 1 , 2 months old), free of known rodent pathogens, were obtained from Charles River (Indianapolis, IN). The types of pathogens screened can be found in Charles River's Rodent Health Monitoring Summary (Charles River, 1998). The animals were housed in groups of 4 or 5 in polycarbonate cages (with HEPA air filters) in the AAALAC-accredited vivarium at the Johnson Space Center (JSC). Animals were allowed to acclimate at this facility (with a 12-h light-dark cycle) for at least one week before being used in the study. The mice had free access to tap water and Purina Formulab Chow No. 50008 (Ralston Purina Co., St. Louis, MO). They were cared for and used humanely according to NASA Animal Care and Use Program guidelines. The animals weighed about 30 g when the dust treatments were administered. Materials. The raw (RNT, Determination of metal content of nanotube samples. The metal content of the NT and carbon black samples used in the present study was determined in our laboratory. Two to four samples of NTs or Printex 90 were placed in crucibles and ashed in a muffle furnace at 550°C for 3 h. The ash from each sample was dissolved in 1 ml of hot concentrated nitric acid and was then diluted to 30 ml with deionized water. The diluted solutions were first screened FIG. 1. (A) Pouring HiPco NT (raw) between containers (courtesy of PULMONARY TOXICITY OF CARBON NANOTUBES IN MICE for the presence of metals using ICP/MS (PE Sciex Elan 6000, Perkin Elmer, Norwalk, CT). Of the 70 elements scanned, only Fe, Ni, Y, Al, Cu, Mo, Zn, and Co were found in measurable concentrations. Quantitative analyses for these 8 metals were then conducted with the same instrument. Metals detected at weight percentage Ն 0.01% are shown in Preparation of fine-dust suspensions. NTs are neither water soluble nor wettable, and fine particle suspensions suitable for instillation must be prepared with a nontoxic dispersion vehicle Intratracheal instillation. After being anesthetized with 3 to 5% isoflurane in a small chamber, individual mice were secured on an inclined plastic platform and anesthetization continued via a small nose cone. The trachea was exposed by a 1-cm incision on the ventral neck skin for instillation of the dust suspension Lung collection and histopathological examination. Seven or 90 days after instillation of the test material, each mouse was injected intraperitoneally with a lethal dose (0.1 ml) of pentobarbital sodium solution (Nembutal, Abbott, North Chicago, IL). Body weights were determined in the 90-d groups. An incision on the neck skin was made to expose the trachea for inserting a catheter; formalin (10% in neutral phosphate buffer) was allowed to drip by gravity (from a 25-ml syringe barrel hanging 1.5 feet above the neck) through the catheter into the lung for about 10 min. The trachea was then tied, and the isolated lung was placed in a glass vial containing about 10 ml of the same fixative RESULTS Effects of Carbon Nanotubes All animals treated with 0.1 mg per mouse (low dose, LD) of CNT (containing Ni and Y) showed no overt clinical signs. However, 5 of the 9 mice treated with 0.5 mg (high dose, HD) of this product died (2/4 in the 7-d group and 3/5 in the 90-d group). All deaths occurred 4 to 7 days after instillation of the CNT. The deaths were generally preceded by lethargy, inactivity, and body-weight losses. These symptoms were also seen in the HD mice that survived. Mice in the HD 90-d CNT group (including those that died within the first week) lost about 27% of their body weight (pretreatment: 30.9 Ϯ 1.1 g; posttreatment: 22.5 Ϯ 0.9 g) by the first week. Symptoms in the two surviving mice disappeared after one week, and the animals started to gain weight. The iron-containing NTs (RNT and PNT) did not cause deaths in the mice. Mild signs of inactivity, hypothermia (felt cold to the touch), piloerection, and occasionally shivering (when provoked by picking up and laying down) were most noticeable 8 to 12 h after treatment with the HD RNT; these symptoms disappeared soon after this time. These clinical signs were not observed in the mice treated with PNT. Body weight losses, seen in the first week with the HD CNT, were not observed with RNT or PNT. The distribution of black particles in lungs 90 d after they were instilled with 0.5 mg of CB or NTs is illustrated in Most of these microscopic nodules were located beneath the bronchial epithelium and were present throughout most of the lung fields. Some appeared to extend into the bronchi as polyps. The granulomas consisted of macrophages laden with black particles, and had very few lymphocytes, neutrophils, eosinophils, or other inflammatory cells. The macrophages had abundant granular cytoplasm with indistinct borders, a characteristic of activated macrophages or epitheloid cells. The black particles were almost entirely contained within these granulomas. Some of the lungs from HD 90-d NT-treated groups appeared grossly abnormal Effects of Serum, Carbon Black, and Quartz As expected, heat-inactivated mouse serum did not produce any clinical signs, or gross and microscopic lesions (Figs 2A and 3A). The mice of the negative (CB) or positive (quartz) control groups also did not show any clinical signs that could be attributed to treatment. Aside from the presence of black particles that appeared predominantly in alveoli, the lungs of the mice in CB groups were microscopically normal (Figs 3B and 4A). The lungs of the LD quartz groups were also normal. Quartz at HD induced an increase in the number of alveolar macrophages in the lungs, and some of these cells contained particles. Quartz also produced mild to moderate alveolar and interstitial inflammation. The results for the 7-d and 90-d groups were generally similar. One of the mice in the 7-d group had a low-grade granulomatous reaction DISCUSSION The present study shows that all three NT products, regardless of the type and amount of metal impurities they contained, induced dose-dependent lung lesions characterized chiefly by interstitial granulomas. Our finding that the purified NTs (PNTs), which were prepared by rigorous treatment (45-h reflux) with concentrated acids (2 M to 3 M nitric acid) to remove metal impurities We observed mortality (5/9) in animals treated with the high dose of CNT, suggesting that any subsequent intratracheal studies with CNT should not involve doses this high. The CNT product contains substantial amounts of nickel and yttrium. Nickel and its compounds are highly toxic. Although both types of carbon particles (CB and NTs) were taken up by alveolar macrophages, their fate and reactions in the lung tissue were very different. CB-laden macrophages scattered in the alveolar space, but NT-laden macrophages moved rapidly to centrilobular locations, where they entered alveolar septa and clustered to form epithelioid granulomas. It is well known that, if the lung is not dust-overloaded, dustladen macrophages on the alveolar surface will migrate upward and be carried by the escalator/mucociliary system up the trachea, and cleared into the esophagus. However, when dusts enter the interstitial or subepithelial space, they are very difficult to clear from the lung. Thus, if a biopersistent dust is irritating or toxic, the lesions resulting from the persistent interaction between the cells and the dust trapped in the interstitium will generally worsen with time, as is the case with NTs. As seen in the present study, the lung lesions of HD 90-d groups were generally more pronounced than those of the HD 7-d groups in animals treated with NTs. These findings indicate that NTs and CB have different intrinsic toxicities in the lungs. The findings that all four NT products (three in our study and one in Warheit's study) were capable of inducing granulomas in mice or rats, together with the findings (in our studies and others) that granulomas were not observed in rodents exposed to carbon black, point to the fundamental difference between the unique physicochemical properties of NTs and those of CB.