Nanoparticulate carbon black in cigarette smoke induces DNA cleavage and Th17-mediated emphysema
Rebuttal to the commentary by Chaudhuri, et al.
Chaudhuri, et al. suggest in their commentary that our conclusion in the manuscript by You, et al.,(1) namely, "Because no medical means of removing accumulated lung nCB exists, our findings underscore the need for all individuals and societies to minimize the production of and exposure to smoke-related particulate air pollution and industrial nCB," "is a somewhat far-reaching statement," and that it is "not justified based on the results of the study". However, we, the authors of You, et al., wholeheartedly stand behind our formerly published conclusions and we will underscore that in this rebuttal. Chaudhuri, et al., stated in their commentary that:
(1) "The identification of black particles in emphysematous lungs as CB is not conclusive." We will show that the assignment of structure is conclusive using Chaudhuri, et al. own criteria for micrographic assignment.
(2) "Cigarette smoke is unlikely to result in the formation of CB." We will show evidence that CB can form in a flame at 700°C.
(3) "Mouse instillation study results cannot be extrapolated to represent exposure to CB industrial production workers and downstream users." We will highlight the relevance of our results and further suggest that the exposure to CB in industries can be persistent and pernicious.
As Chaudhuri, et al., state, by quoting the ASTM standards, "it can be difficult to distinguish CB from soot." We concur; the distinguishing can be difficult as the x-ray diffraction patterns and Raman spectra between soot and CB are very similar. Chaudhuri, et al., further quoting the ASTM standards, state that, "in carbon black aggregates, the dimensions of the particle necks are smaller than the diameter of the primary particles. Whereas, in some soot, the necks have similar dimensions to the primary particles, thereby making it difficult to distinguish one primary particle from the next [emphasis added]." In fact, we noted this same property of CB in our manuscript by You, et al.: "The nCB, although listed to be 15 nm, is more precisely described by the manufacturer to have 15-nm CB particles that are arranged in clusters of 3–5 particles, much as grape clusters, so the actual size is ∼50–75 nm diameter clusters"(1). Therefore, using the ASTM definition cited by Chaudhuri et al.(2), we compare three carbon materials in Figure 1. First, Figure 1a shows diesel soot as described by Chaudhuri, et al., and cited as Figure 1 in their commentary as an example of soot bearing thicker necks between primary particles, where we have highlighted the neck sizes. Second, Cabot CB from an image published by three of the commentary's authors (Chaudhuri, Levy and McCunney) in 2012(3), where we again highlight the comparative neck sizes. And third, the nCB isolated from a human smoker’s lung as disclosed by You, et al.,(1) again showing the comparative primary particle and neck sizes.
In all cases, the primary particle sizes are similar. To differentiate between CB and soot, Chaudhuri, et al., cite neck size relative to primary particle size as a main method of determining soot from CB. However, in the image they cite (Figure 1a), there is necking between primary particles of one-fourth to two-thirds the diameter of the primary particles. By contrast, in the authors’ own published work, they have micrographs of CB (Figure 1b)(3) that show that the ratio of neck to primary particle diameter is larger than in the soot of Figure 1a. Our micrograph (Figure 1c)(1) shows relative necks between primary particles that are commensurate or smaller in size as compared to the Cabot CB sample in Figure 1b. Therefore, using Chaudhuri, et al., own criteria, distinguishing between soot and CB by neck size is highly ambiguous, such that neck size analysis alone cannot be used to refute that the material we isolated from human lung is nCB. For the same reason, the suggestion that the human lung-isolated carbon is soot is untenable.
Chaudhuri, et al., further suggest that cigarette temperature is too low to produce nCB. However, there are reports that CB, albeit slightly larger CB than what is produced from a burning cigarette, can form in a flame where the core temperature is 700-900 °C.(4) Chaudhuri, et al. also note that CB is quenched differently in a cigarette (in air), than in the commercial process (in reducing water). However, some of the CB potentially formed in a cigarette might be quenched in the liquid film lining the respiratory tract, which is also reducing. Finally, while the more oxidized forms of carbon black and soot are more easily cleared from the lung as we demonstrated, it is known that insoluble CB persists in lung tissue, making it plausible that unoxidized nCB produced during smoking can accumulate and persist in the lung.(5)
CB as used in industry becomes easily airborne and is pernicious in its filling of air and work environments.6 Larger aggregate sizes can help to lessen these effects; nonetheless, the health and safety cautions noted by You et al. are worth maintaining.
Chaudhuri, et al., additionally contend that the “dose used in the mouse study is significantly higher than doses used in similar toxicology studies”, but this argument is irrelevant for several reasons. First, the 6 mg total dose of CB used in our mouse studies was not an arbitrary quantity, but rather was derived based on the actual quantity of CB found in lung tissue of heavy smokers with emphysema. We calculated the percentage of residual carbon black per gram of wet lung tissue, which ranged between 0.7 and 0.95% of lung weight. Based on these values, we dosed our studies in mice to yield a comparable amount of CB delivered to mouse lungs. Secondly, our studies were not intended to represent “toxicology studies”, rather we sought to determine if CB could activate the same inflammatory pathways that we had found to be activated in response to cigarette smoke—and indeed this is precisely what we found. Chaudhuri, et al., also claim that the “large bolus dose” is likely a spurious factor in CB-induced lung inflammation. However, contrary to this argument, we showed that delivering the same (6 mg) dose of soluble, hydrophilic CB over the same six-week challenge period failed to cause emphysematous changes in the lungs, indicating that bolus dosing is irrelevant to the induction of lung inflammation as we observed in response to nCB. Instead, the key factors underlying the initiation of the destructive lung inflammation seen with nCB inhalation remain exactly what we originally reported: nCB insolubility and small particle size. These factors are as relevant to the nCB produced during cigarette smoking as through industrial-scale production.
As authors, we well-realized the implications of our claims upon the CB industry. Hence, before publication, we worked exceedingly hard to discover several methods to use CB while mitigating the deleterious biological effects in the lungs. As we suggested, by either making the CB surfaces more hydrophilic, or making the primary particle sizes larger, we were able to minimize the biological harms. Hence, realizing the important implications of this study in an industry that serves huge sectors of materials businesses, we strove to present a safe method for working with CB.
In summary, we the authors of You, et al., stand behind all of our data and original findings. Moreover, we reiterate that because no medical means of removing accumulated lung nCB exists, our findings underscore the need for all individuals and societies to minimize the production of and exposure to smoke-related particulate air pollution and industrial nCB.
References
(1) You, R., et al. Nanoparticulate carbon black in cigarette smoke induces DNA cleavage and Th17-mediated emphysema. eLife 4, e09623 (2015).
(2) ASTM. ASTM. Standard Practice for Sampling and Testing of Possible Carbon Black Fugitive Emissions or Other Environmental Particulate, or Both. . Vol. Designation: D6602– 03b (ASTM International, 100 Barr Harbour Dr., P.O. box C-700 West Conshohocken, Pennsylvania USA 2010).
(3) Levy, L., Chaudhuri, I.S., Krueger, N. & McCunney, R.J. Does carbon black disaggregate in lung fluid? A critical assessment. Chem Res Toxicol 25, 2001-2006 (2012).
(4) Nadimpalli, N.K.V., Buddhiraju, V.S. & Runkana, V. Modeling and simulation of carbon black synthesis in an aerosol flame reactor. Advanced Powder Technology 22, 141-149 (2011).
(5) http://www.ec.gc.ca/ese-ees/default.asp?lang=En&n=2cf34283-1.
(6) Kuhlbusch, T.A., Neumann, S. & Fissan, H. Number size distribution, mass concentration, and particle composition of PM1, PM2.5, and PM10 in bag filling areas of carbon black production. J Occup Environ Hyg 1, 660-671 (2004).
Figure 1. (a) Diesel soot sample cited in the Chaudhuri, et al. commentary as Figure 1. Scale bar 50 nm. Reproduced, with permission from ASTM D6602 - 03b(2010)e1 (Historical Version) Standard Practice for Sampling and Testing of Possible Carbon Black Fugitive Emissions or Other Environmental Particulate, or Both,; © copyright ASTM International, 100 Barr Harbor Drive, West Conshohocken, PA 19428 (b) CB made by Cabot Corporation, taken from a Chaudhuri, Levy and McCunney 2012 paper that teaches the micrographic qualities of CB. Reprinted with permission from Levy, L., Chaudhuri, I.S., Krueger, N. & McCunney, R.J. Does carbon black disaggregate in lung fluid? A critical assessment. Chem Res Toxicol 25, 2001-2006 (2012). © Copyright 2012, American Chemical Society. Scale bar 50 nm. (c) CB isolated from human lungs of smokers from You, et al.1 Scale bar 10 nm. The arrows were added to all three images here to guide the reader.
https://cdn.elifesciences.org/annotations-media/2690525560-001-original.jpg
