Indoor air pollution poses a significant health risk worldwide. WHO estimates suggest that up to 6.5% of the annual disease burden in developing nations is attributable to the burning of solid fuels in the indoor environment [24
]. Smoke from cooking stoves burning biomass fuels contains carbon monoxide, fine particulates, nitrogen dioxide and hydrocarbons; all at concentrations far in excess of what is considered unsafe in outdoor air [7
]. In this study we investigated whether fine particles derived from the burning of the biofuel dung cake also displayed high levels of intrinsic oxidative activity relative to traffic and industrial derived PM. We wanted to examine the hypothesis that the health effects associated with exposure to biofuel derived PM were not solely a function of the high exposure concentrations but also because of their high content of redox active components.
It has been proposed that the capacity of inhaled particles to elicit inflammation and injury in the lung, as well as systemically, may be related to their capacity to cause oxidative stress [17
]. In this working paradigm, inhaled particles generate oxidative stress through three inter-related pathways: first, by directly introducing oxidising species into the lung, such as redox active transition metals [16
] or quinones [19
] absorbed onto their surface. Second, by introducing surface absorbed PAHs that can undergo bio-transformation in vivo
into quinones species through the action of the cytochrome P450, epoxide hydrolase and dihydrodial dehydrogenase detoxification pathway [26
] and finally by stimulating inflammatory cells to undergo the oxidative burst. In this final case, activation of inflammatory cells may be triggered by endotoxin on the surface of inhaled particles [27
], futile phagocytic processing of PM [28
], or by the up-regulation of redox sensitive transcription factors directing the synthesis of pro-inflammatory cytokines [29
]. The integrated sum of all these processes can be considered the 'total' oxidative activity of the particle.
In this study we measured PM oxidative activity using an in vitro
screening procedure that assessed the capacity of PM associated pro-oxidant components (metals and quinones) to deplete physiologically relevant antioxidants, ascorbate, urate and reduced glutathione from a synthetic model of the RTLF [21
]. This 'intrinsic' activity, measured in a cell free system, only reflects the oxidative activity attributable to redox active metals and quinone compounds and not 'latent' activities that may be associated with PAHs or endotoxin. With this caveat, we found PM samples derived from the combustion of dung cake to be significantly more active, on an equal mass basis, than either metal-rich ROFA or PAH-rich vehicle exhaust PM, despite the greater surface area of these samples. This activity was manifest by the capacity of the PM suspensions to deplete both AA and GSH from synthetic RTLF. Notably, the endotoxin and PAH content of dung cake and other biofuels have been shown to be high [24
] suggesting that their 'total' oxidative activity is likely to be far in excess of that associated with traffic derived PM. This very high oxidative activity in animal dung combustion particles supports studies demonstrating increased pulmonary toxicity in mice following instillation of particles derived from dried municipal sewage combustion, relative to coal alone [31
The depletion of both AA and GSH from the model was prevented by co-incubation with the metal chelator diethylenetriaminepentaacetate (DTPA) indicating that the losses observed were driven by redox active metals such as Fe, Cu, Ni, and Cr. DTPA has five acetate groups linked to a molecular backbone that permits it to form tight complexes with a broad range of metals, preventing them from catalysing damaging oxidation reactions. Desferoxamine (DFO) was not used in these studies as it has been reported to reduce chelated Cu that would have resulted in interpretive difficulties when examining its protective role in mixtures of soluble metals [32
]. The contribution of superoxide and hydrogen peroxide to the observed antioxidant losses was examined in co-incubation experiments using the antioxidant enzymes Cu, Zn superoxide dismutase (SOD) and catalase (CAT). Limited protection was observed with these enzymatic antioxidants suggesting that the contribution of these reactive oxygen species to the ascorbate and glutathione losses was minor compared with those attributable to their direct oxidation during the reduction of Fe3+
Thus we conclude that AA and GSH oxidation occurred predominately by their direct reduction of Fe3+
. The superoxide formed by the subsequent oxidation of ferric and cupric ions could undergo dismutation to hydrogen peroxide, reduce Fe3+
, or oxidise ascorbate, urate or glutathione within the synthetic RTLF. As the reaction rate between Fe3+
and superoxide (1.5 × 108
) greatly exceeds that its dismutation at physiological pH (5.4 × 105
, pH7.4) it seems likely that the former reaction predominated, especially as little loss of ascorbate or glutathione could be attributed to superoxide or hydrogen peroxide production. Interestingly, we saw no evidence of urate depletion, despite the importance of this antioxidant in protecting the airway against oxidant gases [33
], peroxynitrite [34
] and hydroxyl radicals [35
]. The rate of the reaction of urate with hydroxyl radicals (7.2 × 109
, pH 6–7) is broadly similar to that of both ascorbate (1.6 × 109
– 1.1 × 1010
, pH 7–7.4) and glutathione (9.0 × 109
– 1.3 × 1010
, pH 8 and 7.8, respectively). Thus the absence of UA depletion in this model supported the contention that superoxide dismutation to hydrogen peroxide was not occurring to any great extent, with little evidence of hydroxyl radical generation. Whilst the redox potentials of UA and AA at pH7 (E°' = 590 and 282 mV respectively) [36
] may suggest that the urate radical could be reduced back to UA at the expense of AA we do not believe that this occurred, as removal of urate from the RTLF had no impact on the observed rate of ascorbate depletion (data not shown).
We also observed that the capacity of DTPA to inhibit ascorbate oxidation was significantly reduced in the ascorbate only incubation experiment: only 100 μM being required for full inhibition in the complete synthetic RTLF as opposed to 200 μM in the ascorbate only RTLF model. This finding may imply that either GSH or UA is limiting the bioavailability of Fe, either through chelation, as has been proposed for UA [37
], or by interfering with the capacity of AA to solubilise ferric iron from the particle surface [16
]. We are currently investigating these potential actions of UA and GSH. Irrespective of this, when the concentration of DTPA was increased in the AA only model, all AA oxidation was blocked indicating the absence of a quinone-dependent activity in the DC particles. This contention was supported by the observation that only a fraction of the measured oxidative activity was present in organic DC extracts. Whilst other groups have emphasised the importance of PM associated quinones/hydroquinones in the oxidative activity of ambient PM [19
] our data would tend to emphasise metal content as the major determinant of DC oxidative activity. Clearly the contribution of metal and organic components to PM oxidative activity may vary depending on its source. In addition it is also likely that the age and storage conditions of the filters used in this study resulted in losses of potentially reactive organic species. These cautionary caveats only further emphasise that we are probably underestimating the 'true' oxidative capacity of freshly generated DC particulates.
As these findings implicated redox active metals in the oxidation process we measured the bioavailable Fe and Cu content of the DC particles. This measurement included reduced and oxidised forms of these metals, both water-soluble and surface mobilisable through ligation to the chromogenic chelators bathophenantroline-disulphonate (BPS) [38
] and bathocuproine-disulphonate acid (BCS) [39
]. Using these approaches we detected considerable Fe and Cu content. The especially high content of Cu is likely to explain extensive glutathione oxidation observed with the dung cake samples, due to copper's high reactivity toward this antioxidant [18
], as well as the lack of reactivity of the ROFA sample toward GSH. It should be noted, however, that the variation in the content of these metals in the three DC samples did not match their observed variation in oxidative activity implying that Fe and Cu were not the sole determinates of the observed activity. The compositional data pertaining to the ROFA sample (PM2.5
), used in the current study have been described previously [23
] using ICP-MS. These analyses have confirmed the relatively high concentrations of total Fe, and low concentrations of Cu in the ROFA sample derived from the burning of heavy fuel oil (N° 5). These metals are however less abundant in the ROFA sample than either vanadium (58.6 μg/g) or nickel (10.6 μg/g). In contrast, ICP-MS analysis of both the gasoline and diesel samples revealed these metals to be below detectable limits [41
], which concurs with their low reactivity in this assay system and these measurements made using the chromogenic chelators. Parallel ICP-MS analysis of dung cake PM obtained under identical burn conditions has also subsequently revealed appreciable concentrations of the redox active metals Ni and Cr (45 and 40 μg/g PM, respectively) in these samples, but no detectable V. This elemental analysis will be described in detail in a subsequent manuscript.
The high metal content of the dung cake may reflect both the presence of biological metals; especially Fe and Cu in dung [42
], as well as metals associated with the local soil with which the animal dung is mixed to make the bricklets. These data therefore support the initial hypothesis that fine particles derived from the controlled burning of dung cake are highly oxidative in nature due to their content of redox active metals.