Combustion-derived nanoparticulate induces the adverse vascular effects of diesel exhaust inhalation

Abstract

Aim: Exposure to road traffic and air pollution may be a trigger of acute myocardial infarction, but the individual pollutants responsible for this effect have not been established. We assess the role of combustion-derived-nanoparticles in mediating the adverse cardiovascular effects of air pollution.

Methods and results: To determine the in vivo effects of inhalation of diesel exhaust components, 16 healthy volunteers were exposed to (i) dilute diesel exhaust, (ii) pure carbon nanoparticulate, (iii) filtered diesel exhaust, or (iv) filtered air, in a randomized double blind cross-over study. Following each exposure, forearm blood flow was measured during intra-brachial bradykinin, acetylcholine, sodium nitroprusside, and verapamil infusions. Compared with filtered air, inhalation of diesel exhaust increased systolic blood pressure (145 ± 4 vs. 133 ± 3 mmHg, P< 0.05) and attenuated vasodilatation to bradykinin (P= 0.005), acetylcholine (P= 0.008), and sodium nitroprusside (P< 0.001). Exposure to pure carbon nanoparticulate or filtered exhaust had no effect on endothelium-dependent or -independent vasodilatation. To determine the direct vascular effects of nanoparticulate, isolated rat aortic rings (n= 6-9 per group) were assessed in vitro by wire myography and exposed to diesel exhaust particulate, pure carbon nanoparticulate and vehicle. Compared with vehicle, diesel exhaust particulate (but not pure carbon nanoparticulate) attenuated both acetylcholine (P< 0.001) and sodium-nitroprusside (P= 0.019)-induced vasorelaxation. These effects were partially attributable to both soluble and insoluble components of the particulate.

Conclusion: Combustion-derived nanoparticulate appears to predominately mediate the adverse vascular effects of diesel exhaust inhalation. This provides a rationale for testing environmental health interventions targeted at reducing traffic-derived particulate emissions.

Figure 1

Exposure chamber and particle filtration system for clinical studies. Diesel exhaust was generated from an unloaded diesel engine using gas oil. More than 90% of the exhaust was shunted away, and the remaining part was diluted with air and fed at 75 L/min into the exposure chamber at steady-state concentration. Diesel exhaust particulate was removed for the control filtered exhaust exposure by passing dilute exhaust through a teflon filter. Carbon nanoparticles were generated using a Palas GFG 1000 spark discharge generator. NP, nanoparticles; T, temperature; RH, relative humidity; CO, carbon monoxide; SO₂, sulphur dioxide; NO_x, nitrogen oxides; CPC, condensation particle counter; SMPS, scanning mobility particle sizer.

Figure 2

Forearm blood flow 6–8 h after exposure to diesel exhaust or air. Infused (solid line) and non-infused (dashed line) forearm blood flow in healthy subjects, 6–8 h following diesel (filled circle) or air (open circle) exposure, during intra-brachial infusion of bradykinin, acetylcholine, sodium nitroprusside, or verapamil: for all dose responses P < 0.0001. For diesel exposure (filled circle) vs. air (open circle); bradykinin (**P= 0.005), acetylcholine (**P= 0.008), sodium nitroprusside (***P< 0.001), and verapamil (P= 0.08).

Figure 3

Forearm blood flow 6–8 h after exposure to filtered diesel or air. Infused (solid line) and non-infused (dashed line) forearm blood flow in healthy subjects 6–8 h, following filtered diesel (grey circle) or air (open circle) exposure, during intra-brachial infusion of bradykinin, acetylcholine, sodium nitroprusside, or verapamil: for all dose responses P< 0.0001. For filtered diesel exposure (grey circle) vs. air (open circle); bradykinin, acetylcholine, sodium nitroprusside, and verapamil (P> 0.05 for all comparisons).

Figure 4

Direct vascular effects of particles in rat aortic rings in vitro. Effect of diesel exhaust particles (filled circle), carbon nanoparticles (u) (both 100 µg/mL), or vehicle (open circle) (Krebs buffer) on (A) phenylephrine contraction in mN, (B) phenylephrine contraction as a percentage of maximum contraction (C) acetylcholine, and (D) sodium-nitroprusside-induced vasodilatation. Diesel exhaust particles did not affect contraction to phenylephrine (P= 0.64), but inhibited vasorelaxation to acetylcholine (***P< 0.001) and sodium nitroprusside (*P= 0.019) compared with control. Although there was an apparent rightward shift in the dose–response curve to acetylcholine and sodium nitroprusside with carbon nanoparticles, vasorelaxation was not significantly affected (P= 0.139 and P = 0.224, respectively). Effect of (E) aqueous and (F) organic separation of diesel exhaust particles on acetylcholine responses. (E) Aqueous-washed diesel exhaust particles (grey square), and aqueous extract from diesel exhaust particles (open square), inhibit vasodilation to acetylcholine (**P< 0.01 for both), with the combined effects of both constituents being equal to that of untreated particles (filled circle). (f) Organic-washed diesel exhaust particles (s) inhibited acetylcholine induced vasodilatation (*P= 0.010); the magnitude of this effect was not different from that of untreated diesel exhaust particles (filled circle) (P= 0.103). The organic extract of diesel exhaust particles (inverted triangle) had no effect on vasorelaxation to acetylcholine (P= 0.645). n= 6 for all groups.

Figure 5

Reversal of the effects of particles in vitro by superoxide dismutase. (A) Diesel exhaust particles, (B) carbon nanoparticles, (C) Krebs-washed diesel exhaust particles, (D) soluble extract from Krebs-washed diesel exhaust particles, and (E) dichloromethane-washed diesel exhaust particles. Effect of (filled triangle) particles (100 µg/mL), (grey circle) superoxide dismutase (100 U/mL), (grey triangle) particles + superoxide dismutase together, or vehicle (open circle) (Krebs buffer) on acetylcholine-induced vasodilatation. Superoxide dismutase reversed the actions of all particles (*P< 0.05, **P< 0.01, ***P< 0.001) compared with particles alone, except nCB where the reversal by superoxide dismutase was not significantly affected (P= 0.18); n= 6–9 for all groups. (f) The hydroxyl radical scavenger mannitol had no effect on the action of diesel exhaust particles on acetylcholine-induced relaxation (^nsP= 0.84: mannitol + diesel exhaust particles compared with diesel exhaust particles alone). (filled triangle) Diesel exhaust particles (10 µg/mL), (diamond) mannitol (5 mM), (inverted triangle) diesel exhaust particles + mannitol together, or vehicle (open circle) (Krebs buffer); n= 6 for all groups.