Mobilization of Iron from Urban Particulates Leads to Generation of Reactive Oxygen Species in Vitro and Induction of Ferritin Synthesis in Human Lung Epithelial Cells

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Abstract
Many of the biochemical effects of asbestos in cultured cells have been shown to be due to iron, which can be as high as 27% by weight. Urban air particulates also contain iron, and some of the pathological effects after inhalation may be due to reactive oxygen species produced by iron-catalyzed reactions. Two standard reference material (SRM) urban air particulate samples were used for the studies described here. SRM 1648 (3.9% iron by weight) was collected in the St. Louis, MO, area, and SRM 1649 (3% iron by weight) was collected in the Washington, DC, area. To determine if iron associated with urban particulates could be mobilized, as it is from asbestos, SRMs 1648 and 1649 were incubated with 1 mM citrate or EDTA, in the presence or absence of ascorbate. Iron was mobilized from both particulates by either chelator, especially in the presence of ascorbate. Citrate, in the presence of ascorbate, mobilized 30.9 nmol of Fe/mg of SRM 1648 and 65.1 nmol of Fe/mg of SRM 1649 in 24 h. EDTA, in the presence of ascorbate, mobilized 53.8 nmol of Fe/mg of SRM 1648 and 98.8 nmol of Fe/mg of SRM 1649 in 24 h. To determine whether reactive oxygen species were being produced by the particulate iron, each particulate was incubated with φX174 RFI DNA in the presence or absence of ascorbate. Single-strand breaks (SSBs) were produced by either particulate, but only in the presence of ascorbate. Incubation of SRM 1648 or 1649 (0.5 mg/mL) with DNA in the presence of ascorbate and citrate resulted in 20% or 34% DNA with SSBs, respectively. Incubation of SRM 1648 or 1649 (0.1 mg/mL) with DNA in the presence of ascorbate and EDTA resulted in 26% or 45% DNA with SSBs, respectively. To determine if iron associated with urban particulates could be mobilized by human lung epithelial cells (A549), cells were treated with particulates and the amount of the iron storage protein ferritin was determined at the end of treatment. The 6.4- or 8.4-fold increase in ferritin observed in cells treated with SRM 1648 or 1649, respectively, over that of control (untreated) cells strongly suggested that iron was mobilized in the cultured cells. If similar mobilization and reactivity of the iron occurs in the lung, this may explain some of the pathological effects of urban particulates.