Impacts of Antioxidants on Hydroxyl Radical Production from Individual and Mixed Transition Metals in a Surrogate Lung Fluid

Abstract

Inhalation of ambient particulate matter causes morbidity and mortality in humans. One hypothesized mechanism of toxicity is the particle-induced formation of reactive oxygen species (ROS) - including the highly damaging hydroxyl radical ((·)OH) - followed by inflammation and a variety of diseases. While past studies have found correlations between ROS formation and a variety of metals, there are no quantitative measurements of (·)OH formation from transition metals at concentrations relevant to 24-hour ambient particulate exposure. This research reports specific and quantitative measurements of (·)OH formation from 10 individual transition metals (and several mixtures) in a cell-free surrogate lung fluid (SLF) with four antioxidants: ascorbate, citrate, glutathione, and uric acid. We find that Fe and Cu can produce (·)OH under all antioxidant conditions as long as ascorbate is present and that mixtures of the two metals synergistically increase (·)OH production. Manganese and vanadium can also produce (·)OH under some conditions, but given that their ambient levels are typically very low, these metals are not likely to chemically produce significant levels of (·)OH in the lung fluid. Cobalt, chromium, nickel, zinc, lead, and cadmium do not produce (·)OH under any of our experimental conditions. The antioxidant composition of our SLF significantly affects (·)OH production from Fe and Cu: ascorbate is required for (·)OH formation, citrate increases (·)OH production from Fe, and both citrate and glutathione suppress (·)OH production from Cu. MINTEQ ligand speciation modeling indicates that citrate and glutathione affect (·)OH production by changing metal speciation, altering the reactivity of the metals. In the most realistic SLF (i.e., with all four antioxidants), Fe generates approximately six times more (·)OH than does the equivalent amount of Cu. Since levels of soluble Fe in PM are typically higher than those of Cu, our results suggest that Fe dominates the chemical generation of (·)OH from deposited particles in the lungs.

Figure 1

^·OH production from 1.0 µM (10 nmol) of individual transition metals in four antioxidant solutions: a) no antioxidants added, b) Asc only, c) Asc + Cit, d) Asc + Cit + GSH + UA (i.e., All AOs). Each bar is the mean ± σ (n ≥ 3).

Figure 2

^·OH production under various antioxidant compositions from 1.0 µM (10 nmol) of a) Fe(II) or b) Cu(II). Each bar is the mean ± σ (n ≥ 3).

Figure 3

MINTEQ speciation modeling results for 10 nmol Fe(II) or Cu(II) for three antioxidant compositions: a,d) Asc, b,e) Asc + Cit and c,f) Asc + Cit + GSH. Numbers under the graphs represent the amount of ^·OH produced for each experimental condition.

Figure 4

^·OH from binary mixtures of Fe(II) + Cu(II) compared to the sum of ^·OH production from the corresponding individual solutions of Fe and Cu. The error in the sum of the individual Fe(II) and Cu(II) results (stacked bars) is the propagated standard deviation. For each pair, the sum of the individual solutions is significantly different (P<0.01) from the value in the binary mixture.

Figure 5

^·OH production from SLF extracts of ambient PM_2.5 samples (×; only available for the Asc + Cit conditions; data from Vidrio et al. 2009) and from laboratory solutions containing either a binary mixture of Fe and Cu (dark bars) or a mixture of six TMs (light bars), all at the concentrations measured in the corresponding ambient sample. SLF-soluble Fe, Cu, Mn, V, Co, and Zn levels in each sample were as follows: 2/18/2007 – 2.8, 0.05, 0.43, 0.06, 0.04 and 0.06 nmol, respectively; 4/23/2007 – 6.1, 0.67, 0.56, 0.02, 0.02, and 1.1 nmol, respectively; 5/13/2007 – 16.4, 2.0, 0.75, 0.02, 0.02, and 1.2 nmol, respectively. Asterisks (*, **) indicate laboratory solution means (binary and six-metal mixtures) that are significantly different at P<0.05 and P<0.01, respectively.