Generation of Hydroxyl Radicals from Dissolved Transition Metals in Surrogate Lung Fluid Solutions

Abstract

Epidemiological research has linked exposure to atmospheric particulate matter (PM) to several adverse health effects, including cardiovascular and pulmonary morbidity and mortality. Despite these links, the mechanisms by which PM causes adverse health effects are poorly understood. The generation of hydroxyl radical (.OH) and other reactive oxygen species (ROS) through transition metal-mediated pathways is one of the main hypotheses for PM toxicity. In order to better understand the ability of particulate transition metals to produce ROS, we have quantified the amounts of .OH produced from dissolved iron and copper in a cell-free, surrogate lung fluid (SLF). We also examined how two important biological molecules, citrate and ascorbate, affect the generation of .OH by these metals. We have found that Fe(II) and Fe(III) produce little .OH in the absence of ascorbate and citrate, but that they efficiently make .OH in the presence of ascorbate and this is further enhanced when citrate is also added. In the presence of ascorbate, with or without citrate, the oxidation state of iron makes little difference on the amount of .OH formed after 24 hours. In the case of Cu(II), the production of .OH is greatly enhanced in the presence of ascorbate, but is inhibited by the addition of citrate. The mechanism for this effect is unclear, but appears to involve formation of a citrate-copper complex that is apparently less reactive than free, aquated copper in either the generation of HOOH or in the Fenton-like reaction of copper with HOOH to make .OH. By quantifying the amount of .OH that Fe and Cu can produce in surrogate lung fluid, we have provided a first step into being able to predict the amounts of .OH that can be produced in the human lung from exposure to PM containing known amounts of transition metals.

Figure 1

Formation of ·OH from a surrogate lung fluid containing 200 nmol ascorbate (Asc) and/or 300 nmol citrate (Cit) as a function of extraction time.

Figure 2

Production of ·OH from 200 nmol Fe (II), Fe (III), or Cu (II) in surrogate lung fluid solution containing 10,000 nmol HOOH along with 10,000 nmol ascorbate (Asc) and/or 10,000 nmol citrate (Cit). Bars represent the mean values (n = 3). Error bars are 1 σ values calculated from replicate experiments.

Figure 3

Production of ·OH from 200 nmol Fe (II), Fe (III), or Cu (II) in a surrogate lung fluid solution containing 200 nmol ascorbate (Asc) and/or 300 nmol citrate (Cit) (and no initial HOOH). Bars represent the mean values (n = 3) with error bars of 1 σ.

Figure 4

Production of ·OH from 200 nmol Cu (II) with various initial HOOH concentrations in a surrogate lung fluid solution containing 200 nmol ascorbate (Asc) and 300 nmol citrate (Cit). Bars represent the mean value (n = 3) with error bars of 1 σ.

Figure 5

Production of ·OH from mixtures of Fe (II) and Cu (II) with and without 200 nmol ascorbate (Asc) and 300 nmol citrate (Cit). Bars represent the mean value (n = 3) with error bars of 1 σ.