Nanomaterials are frontier technological products used in different manufactured goods. Because of their unique physicochemical, electrical, mechanical, and thermal properties, single-walled carbon nanotubes (SWCNT) are finding numerous applications in electronics, aerospace devices, computers, and chemical, polymer, and pharmaceutical industries. SWCNT are relatively recently discovered members of the carbon allotropes that are similar in structure to fullerenes and graphite. Previously, we (47) have reported that pharyngeal aspiration of purified SWCNT by C57BL/6 mice caused dose-dependent granulomatous pneumonia, oxidative stress, acute inflammatory/cytokine responses, fibrosis, and decrease in pulmonary function. To avoid potential artifactual effects due to instillation/agglomeration associated with SWCNT, we conducted inhalation exposures using stable and uniform SWCNT dispersions obtained by a newly developed aerosolization technique (2). The inhalation of nonpurified SWCNT (iron content of 17.7% by weight) at 5 mg/m(3), 5 h/day for 4 days was compared with pharyngeal aspiration of varying doses (5-20 microg per mouse) of the same SWCNT. The chain of pathological events in both exposure routes was realized through synergized interactions of early inflammatory response and oxidative stress culminating in the development of multifocal granulomatous pneumonia and interstitial fibrosis. SWCNT inhalation was more effective than aspiration in causing inflammatory response, oxidative stress, collagen deposition, and fibrosis as well as mutations of K-ras gene locus in the lung of C57BL/6 mice.

We have found that under appropriate conditions, an allele-specific polymerase chain reaction (PCR) can achieve a sensitivity suitable for measuring specific, infrequent mutations in single cell systems or in animal tissues. Using the 12th codon GC-to-AT mutation in the rat c-Ha-ras gene as a model system, we have defined conditions that allow for measurement of mutations present at frequencies as low as one in 10(5) gene copies. Our approach involved the use of PCR primers that created a single mismatch with the mutated allele (GAA) but created a double mismatch with the wild-type allele (GGA). Five out of the six such double-mismatch primers we tested permitted amplification of the mutant allele (GAA) with a high degree of specificity. The specificity of the assay was further enhanced by using a two-step PCR cycle consisting of a denaturation step (1 min incubation at 94 degrees C) and an annealing/extension step (1 min incubation at 50 degrees C) in the presence of 10% (vol/vol) glycerol. Reconstruction experiments using genomic DNA demonstrate that this procedure cna measure the presence of 30 copies of the transforming ras allele present amongst 3 x 10(6) copies of the wild-type allele.

Denaturing gradient gel electrophoresis (DGGE) was used to separate and isolate the products of DNA amplification by polymerase chain reaction (PCR). The strategy permitted direct enumeration and identification of point mutations created by T4, modified T7, Klenow fragment of polymerase I, and Thermus aquaticus (Taq) DNA polymerases. Incorrectly synthesized sequences were separated from the wild type by DGGE as mutant/wild-type heteroduplexes and the heteroduplex fraction was used to calculate the average error rate (mutations per base duplication). The error rate induced in the 104-base-pair low-temperature melting domain of exon

The hallmark geometric feature of single-walled carbon nanotubes (SWCNT) and carbon nanofibers (CNF), high length to width ratio, makes them similar to a hazardous agent, asbestos. Very limited data are available concerning long-term effects of pulmonary exposure to SWCNT or CNF. Here, we compared inflammatory, fibrogenic, and genotoxic effects of CNF, SWCNT, or asbestos in mice 1 yr after pharyngeal aspiration. In addition, we compared pulmonary responses to SWCNT by bolus dosing through pharyngeal aspiration and inhalation 5 h/day for 4 days, to evaluate the effect of dose rate. The aspiration studies showed that these particles can be visualized in the lung at 1 yr postexposure, whereas some translocate to lymphatics. All these particles induced chronic bronchopneumonia and lymphadenitis, accompanied by pulmonary fibrosis. CNF and asbestos were found to promote the greatest degree of inflammation, followed by SWCNT, whereas SWCNT were the most fibrogenic of these three particles. Furthermore, SWCNT induced cytogenetic alterations seen as micronuclei formation and nuclear protrusions in vivo. Importantly, inhalation exposure to SWCNT showed significantly greater inflammatory, fibrotic, and genotoxic effects than bolus pharyngeal aspiration. Finally, SWCNT and CNF, but not asbestos exposures, increased the incidence of K-ras oncogene mutations in the lung. No increased lung tumor incidence occurred after 1 yr postexposure to SWCNT, CNF, and asbestos. Overall, our data suggest that long-term pulmonary toxicity of SWCNT, CNF, and asbestos is defined, not only by their chemical composition, but also by the specific surface area and type of exposure.

Lung cancer rates among men and particularly among women, almost all of whom are non-smokers, in Xuan Wei County, China are among the highest in China and have been causally associated with exposure to indoor smoky coal emissions that contain very high levels of polycyclic aromatic hydrocarbons (PAHs). As such, this population provides a unique opportunity to study the pathogenesis of PAH-induced lung cancer that is not substantially influenced by the large number of other carcinogenic constituents of tobacco smoke. Aldo-keto reductases (AKRs) activate PAH dihydrodiols to yield their corresponding reactive and redox-active o-quinones, which can then generate reactive oxygen species that cause oxidative DNA damage. We therefore examined the association between single nucleotide polymorphisms (SNPs) in four genes (AKR1C3-Gln5His, NQO1-Pro187Ser, MnSOD-Val16Ala and OGG1-Ser326Cys) that play a role in the generation, prevention or repair of oxidative damage and lung cancer risk in a population-based, case-control study of 119 cases and 113 controls in Xuan Wei, China. The AKR1C3-Gln/Gln genotype was associated with a 1.84-fold [95% confidence interval (CI) = 0.98-3.45] increased risk and the combined OGG1-Cys/Cys and Ser/Cys genotypes were associated with a 1.93-fold (95% CI = 1.12-3.34) increased risk of lung cancer. Subgroup analysis revealed that the effects were particularly elevated among women who had relatively high cumulative exposure to smoky coal. SNPs in MnSOD and NQO1 were not associated with lung cancer risk. These results suggest that SNPs in the oxidative stress related-genes AKR1C3 and OGG1 may play a role in the pathogenesis of lung cancer in this population, particularly among heavily exposed women. However, due to the small sample size, additional studies are needed to evaluate these associations within Xuan Wei and other populations with substantial exposure to PAHs.