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Multi-walled carbon nanotube instillation impairs pulmonary function in C57BL/6 mice

Xiaojia Wang*, Pranita Katwa, Ramakrishna Podila, Pengyu Chen, Pu Chun Ke, Apparao M Rao, Dianne M Walters, Christopher J Wingard and Jared M Brown*

Particle and Fibre Toxicology 2011, 8:24  doi:10.1186/1743-8977-8-24

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Response to Comment on Article by Wang et al.: Multi-walled carbon nanotube instillation impairs pulmonary function in C57BL/6 mice

Jared Brown   (2011-12-07 10:28)  Brody School of Medicine, East Carolina University email

Dear Dr. Oberd��rster,

Thank-you for reading our manuscript entitled, ��Multi-Walled Carbon Nanotube Instillation Impairs Pulmonary Function in C57BL/6 Mice��, that was published in Particle and Fibre Toxicology (PFT 8:24). We acknowledge that our study, as with all experimental research, has limitations, including the aspiration of high doses of multi-walled carbon nanotubes (MWCNT). However, our study was performed with the intention of identifying potential mechanisms of toxicity associated with MWCNT exposure and was not intended as a regulatory study. Dose rates are always inherently different between instillation/aspiration techniques and inhalation, and there are advantages and disadvantages of both delivery techniques (Driscoll, et al, 2000). Additionally, we did not examine acute effects, but rather looked at a 30-day time point when acute effects have resolved. Because health effects in humans have not been documented, our study, and many others, has been conducted to preemptively delineate potential toxicological hazards associated with MWCNTs before human health effects are realized. Given the limited human exposure data, current lack of observed human health effects, and incomplete understanding of nanotoxicological mechanisms within the lung, extrapolation of ours and others findings in a human context would be premature. Nonetheless, impairment of pulmonary function was observed at high doses in the absence of any challenge to the lung such as methacholine or allergen. While the lower doses of MWCNT had little effect on pulmonary function, one could surmise that individuals with pre-existing respiratory conditions may indeed display significant impairment of lung function following exposure to lower doses of MWCNTs.

In regard to lung function testing, all procedures were approved by the IACUC and EKG was measured on all animals to monitor anesthetic depth and potential complications that could have arisen. All mice maintained a normal EKG pattern and heart rate during functional measurements and we followed a standard protocol, widely used for forced oscillation techniques, in order to compare between groups and across experiments in our lab.

Clearly more work in the emerging field of nanotoxicology remains to be done and we look forward to additional mechanistic studies published in Particle and Fibre Toxicology.

Literature Cited:

Driscoll, K., D. Costa, et al. (2000). Intratracheal instillation as an exposure technique for the evaluation of respiratory tract toxicty: Uses and limitations. Toxicological Sciences 55: 24-35.

Competing interests

No competing interests

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Comment on article by Wang et al.: Multi-Walled Carbon Nanotube Instillation Impairs Pulmonary Function in C57BL/6 Mice

Gunter Oberdorster   (2011-11-04 14:49)  University of Rochester

I write this letter to express some concerns with the recent article by Wang et al. (PFT 8:24) which appears online as a provisional pdf in Particle and Fibre Toxicology. In this study, mice aspirated very high doses of 27, 54, and 108 µg per mouse. The authors justify the doses pointing out that these have been used by others in previous studies (Shvedova et al., 2005; Porter et al., 2010). However, a critical reading of the earlier studies would have revealed that an aspirated dose of 20 µg in a mouse is equivalent to 20 days of inhalation exposure for 8 hours/day by humans at an airborne concentration of 5 mg/m3. Or, by the other estimate, 10 µg MWCNTs per mouse aspirated is equivalent to a deposited dose in the alveolar region of a worker performing light work for 30 days at 400 µg/m3, a concentration that was found at an occupational setting (Han et al., 2008). There is a huge difference in terms of dose rate between delivery of the same dose by an instantaneous bolus vs. inhalation over several weeks. The large difference in dose rate and implications thereof are neither considered nor discussed. Even if Wang et al. would have simulated a one 8 hr. human exposure, which would be an aspirated dose of about 1 µg per mouse, there would still be a substantial difference in the delivered dose rate (a fraction of a second aspiration of the whole dose vs. 28,800 seconds inhalation of the same dose). Dose and dose rate are important determinants for acute effects which differentiates bolus type delivery to the lung from inhalation, as discussed by Driscoll et al. (2000).
I am not suggesting, as some may think, that high dose studies have no value and should not be published. What I am asking for is that the selected dose levels be critically discussed and put into perspective with respect to realistic real-world exposure scenarios, including consideration of dose rate. A critical interpretation and discussion of results for relevancy is essential in order to avoid confusion when extrapolating results from in vivo and in vitro studies to humans. Would an aspirated dose of 1 µg MWCNTs per mouse have shown any of the observed effects?
Because only the right lung was lavaged and analyzed for assessing lung lavage inflammatory parameters and the left lung was used to perform histological examination, it may explain the great variation observed in the results because aspiration, similar to intratracheal instillation, does not necessarily result in equal distribution of the instillate/aspirate to both lungs. This could explain why there were no dose-response results in many of the endpoints; indeed, sometimes there was a reverse dose-response relationship, sometimes the mid-dose was highest and responses to low and high dose not significant. Possible reasons for this are not discussed in the paper, but may include significant differences in lung burdens per unit tissue weight between right and left lung.
A different concern pertains to the lung function testing which was performed under anesthesia with additional chemical paralysis of the animals: no information is given as to whether, and how, anesthesia depth was assessed (e.g., via recording of electrocardiogram, blood pressure, or electro-encephalogram) or blood pO2 and pCO2 were monitored to avoid undue suffering of the animals and imbalance of acid-base conditions that can affect responses. Did the animal use and care committees of the authors' institutions really approve this type of lung function testing on paralyzed animals without requiring some additional control measures? If respective monitoring was done, it should have been described in the paper. Ventilating all dose groups of mice with the same frequency and end-expiratory pressure does not mean that they were all ventilated equally well, especially if there were significant changes in resistance or compliance. Regarding lung function results, only the highest aspiration dose (108 µg/mouse!) showed a significant response; what does this mean, given that such dose/dose-rate will never occur in a realistic inhalation scenario?
A final note, PFT with its very respectable impact factor of 4.9 has become a widely read and cited journal, and articles appearing in PFT are likely to have significant impact. Thorough reviews by editorial board members are essential to identify high quality submissions and to assure continued success of the journal.

Literature Cited:
Driscoll, K., D. Costa, et al. (2000). Intratracheal instillation as an exposure technique for the evaluation of respiratory tract toxicty: Uses and limitations. Toxicological Sciences 55: 24-35.
Han, J. H., E. J. Lee, et al. (2008). Monitoring multiwalled carbon nanotube exposure in carbon nanotube research facility. Inhal Toxicol 20(8): 741-749.
Porter, D. W., A. F. Hubbs, et al. (2010). Mouse pulmonary dose- and time course-responses induced by exposure to multi-walled carbon nanotubes. Toxicology 269(2-3): 136-147.
Shvedova, A. A., E. R. Kisin, et al. (2005). Unusual inflammatory and fibrogenic pulmonary responses to single-walled carbon nanotubes in mice. Am J Physiol Lung Cell Mol Physiol 289(5): L698-708.
Wang, X., Katwa, P., Podila, R., et al. (2011). Multi-walled carbon nanotube instillation impairs pulmonary function in C57BL/6 mice. Particle and Fibre Toxicology 8:24, 2011.

Competing interests

None declared

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