Myeloperoxidase Clearance of Carbon Nanotubes Figure 3. Assessment of the volume of SWCNT aggregates in the lung tissue sections from w/t and MPO k/o mice. a. Representative images of the lung tissue sections. Insert - higher magnifigation (2.56 zoom) of a field illustrating the presence of SWCNT (green punctuate spots pointed by white arrows). b. Quantitation of SWCNT aggregates (SWCNT volume/total lung volume) using their specific absorbance (750–850 nm), * p,0.05, vs w/t 1 day post exposure, # p,0.05, vs w/t 28 days post exposure. c. Assessment of SWCNT aggregates - number per microscopic field - using an automated IN Cell Analyser 1000 microscope, * p,0.05, vs w/t 1 day post exposure, # p,0.05, vs w/t 28 days post exposure. doi:10.1371/journal.pone.0030923.g003 Contribution of other heme-peroxidases to the clearance of SWCNT is quite possible. Degradation of SWCNT by MPO during the peroxidase cycle whereby the reaction with H2O2 yields a reactive intermediate, compound I, which oxidizes chloride by a single 2-electron transfer to produce the hypochlorous acid (HOCl). Both oxidants – reactive MPO intermediates and hypochlorous acid – are involved in the oxidative degradation of nanotubes [14] due to the breakage of C-C and C-H bonds [17]. Reactive intermediates can be also generated by other heme- peroxidases such as eosinophil peroxidase, lactoperoxidase, thyroid peroxidase as well as by hemoproteins with peroxidase activity (hemoglobin, myoglobin, cytochromes P450). In fact, the ability of horseradish peroxidase (HRP) and lactoperoxidase to oxidatively biodegrade SWCNT in vitro has been documented [11,12,18]. While it is possible that alternative peroxidase pathways may participate in pulmonary biodegradation of SWCNT the results of the current study - demonstrating a significantly lower clearance of SWCNT from the lungs of MPO k/o mice vs w/t animals - are supportive of the essential role of MPO in this process. While this study utilized SWCNT, it is highly likely that multi-walled carbon nanotubes, fullerenes, graphene and other carbonaceous particles may also undergo MPO- catalyzed modifications and degradation [19]. In contrast to in vitro incubation systems, non-covalent coating of SWCNT with proteins and lipids in vivo [20,21] occurring during their interactions with components of biofluids will inevitably affect recognition patterns and metabolic pathways of the particles. Our recent studies indicate that in the lung, SWCNT are effectively coated with the major surfactant lipids and proteins. Hence, the nature of the nanomaterial ‘‘coronation’’ by proteins and/or lipids may impact on the degree of recognition and biodegradation by immune-competent cells. The major bactericidal function of MPO via oxidative attack on pathogens has been well characterized [22,23]. The ability of MPO to oxidatively modify organic xenobiotics and drugs has been also documented [24,25]. Here, we describe a hitherto unknown role of MPO in the body to oxidatively biodegrade carbonaceous particles, specifically SWCNT. One can presume that this is one of the essential and very ancient catalytic roles of the enzyme aimed at oxidative degradation and clearance of different types of carbonaceous materials to which animals and humanoids have been exposed from primordial times – such as PLoS ONE | www.plosone.org 4 March 2012 | Volume 7 | Issue 3 | e30923