Mobilization of Iron into Cell from Ambient Particulate Matter and Its Possible Participations to DNA Single Strand Break
Heesang Song1,†, Suk Hee Sung1,2,†, Young-Woo Jin2, Chong Soon Kim2, Yoon Shin Kim3, Won-Gi Bang1 and Namhyun Chung1,*
1College of Life and Environmental Sciences, Korea University, Seoul 136-701, Korea
2Radiation Health Research Institute, Seoul 132-703, Korea
3Institute of Environmental and Industrial Medicine, Hanyang University, Seoul, 133-791, Korea Received February 20, 2004; Accepted May 24, 2004
Ambient particulate matter (PM) contains various transition metals. Ferritin level of A549 cells treated with PM increased up to about six-fold of untreated control cells. To compare extents of DNA damaging capacities of Fe and other metals, the induction of DNA single strand break (SSB) was measured in the presence of hydrogen peroxide and each metals. Extents of DNA SSB with iron(II), copper(II), and copper(I) were higher than those of nickel(II), chromium(III), and manganese(II). All these results suggested that Fe and other metals in ambient PM are easily mobilized into lung cells upon inhalation to cause oxidative damage and that extent of the damage cannot be assessed by only amount of metals but by both amount of and damage extent by metals.
Key words: ferritin, transition metal, standard reference material, fenton reaction, oxidative damage
Numerous epidemiological studies have shown that elevated level of ambient air pollution is associated with increases of asthma, chronic obstructive pulmonary disease, and cardiovascular disease as well as lung cancer.1,2) Ambient air particles are a complex mixture of organic and inorganic compounds, including soluble and free transition metals.1) Recently, research on the toxic effect of the transition metals as well as organic substances of particulate matter (PM) has increased.3,4) Such studies have shown that adverse health effects from particle exposure can also result from metal- mediated generation of reactive oxygen species (ROS),which can cause severe oxidative stress within cells or tissue through the oxidation of nucleic acids, proteins, and lipids.5,6)
Ambient PM contains measurable concentrations of transition metals such as Fe, Cu, Ni, V, Co, and Cr in different amounts, forms, and oxidative states. These metals can be involved in the reduction of hydrogen peroxide to form hydroxyl radical by Fenton reaction.5,7) The cationic iron and copper are the two most commonly studied transition metals.8,9) In addition, other metals (cobalt, chromium, nickel, and vanadium) produces ROS and generates DNA strand breaks in the presence of hydrogen peroxide.10-12)
Bioavailibility of transition metals is an essential factor for carcinogenesis and tumorigenesis within the cell.13) We have already reported that in vitro mobilization, thus possibly bioavailability, of metals from PM is greatly affected by the
presence of chelator and reductant,14) an indication that the mobilizable metals from PM can cause biological damages in vivo. However, accumulation of metals within the cell via mobilization has not been shown. Therefore, the present study was undertaken to demonstrate the uptake of mobilized metals from PM into the cells via induction of ferritin synthesis based on the finding that treatment of animals or cells with iron compounds invariably induces ferritin synthesis.15) Although the metals can be mobilized within cell, the toxicity of metals in PM still needs to be examined. Several articles have reported the kind and amount of metals as indices of toxicity measurement; however, the extent of biological damage that can be done by the metals has yet to be addressed. Thus, we also attempted to estimate the relative toxicity of various metals, employing DNA single strand break (SSB) as a representative tool. We found that metals could be mobilized from PM and that each of the metals generated different degrees of biological damage.
Materials and Methods
Particulate matter. Standard reference materials (SRM) 1648 and 1649a, which contained 3.9 and 3% iron by weight, respectively, were purchased from the National Institute of Standard & Technology (Gaithersburg, MD, USA). SRM 1648 and 1649a are urban air PM and urban air dust/organics samples, respectively. Approximately 50 and 30% of the particulates in SRM 1648 and 1649a, respectively, are less than 10µm in mean diameter.16) Particle with a mean diameter less than 10µm is considered to be respirable. Urban PMs were collected from Seoul metropolitan area using Hi-Volume
*Corresponding author
Phone: +82-2-3290-3026; Fax: +82-2-3290-3503 E-mail: [email protected]
†Equally contributed to this article
of each metal contained in SRMs and PMs from Seoul and their mobilized amounts under various conditions were reported in our previous paper.14)
Reagents. Ferric chloride, cuprous chloride, and ferrous chloride were purchased from Sigma Chemical Co. (St. Louis, MO, USA), cobalt chloride, vanadium oxide, chromium chloride, nickel chloride, and zinc chloride from Junsei Chemical Co. (Tokyo, Japan), cupric chloride from Merck &
Co., Inc. (Whitehouse Station, NJ, USA), and manganese chloride and lead chloride from Kanto Corp. (Portland, OR, USA). Hydrogen peroxide used as a catalytic oxidant was obtained from Wako Pure Chemical Ind., Ltd. (Tokyo, Japan).
Hams F-12 cell culture medium, 0.05% trypsin with 0.53 mM EDTA, 0.25% trypsin, and fetal bovine serum were obtained from Jeil Biotechservices Inc. (Seoul, Korea). Closed-circular, superhelical φΧ 174 RF I DNA was obtained from New England Biolabs (Beverly, MA, USA). DNA was removed from the shipping buffer by ethanol precipitation as described by Maniatis et al.17) and redissolved in 50 mM NaCl, pH 7.5.
Ultra pure water (Milli-Q grade, organic free) was used to prepare 50 mM NaCl and various metal-containing solutions.
All other solutions were then prepared using Chelex-treated NaCl. All metal-containing and hydrogen peroxide solutions were prepared immediately before use.
Cell culture. Complete growth medium was composed of Ham’s F-12 cell culture medium, 50µg/ml gentamicin, 10%
fetal bovine serum, and 1.176 g NaHCO3/l of medium (pH 7.4). A human lung epithelial cell line of A549, with characteristics of alveolar epithelial type II cells, was used because the epithelial type II cells are one of the two target cell populations for the development of cancer after PM exposure.18) The A549 cells (ATCC CCL185) were obtained from American Type Culture Collection (Rockville, MD, USA). Upon arrival, cells were thawed, resuspended in the complete growth medium, and grown in a model VS-9108MS water-jacketed incubator (Vision Scientific Co., Seoul, Korea) at 37oC in an atmosphere of 5% CO2 with 95% humidity.
Before the cells reached confluence, they were dislodged with 0.05% trypsin containing 0.53 mM EDTA, resuspended in the complete growth medium, and plated. For storage, the cells were dislodged during logarithmic growth period, resuspended in freezing medium as 1-ml aliquots at 2 × 106 cell/ml, and frozen in liquid nitrogen. Aliquots of A549 cells were then removed from liquid nitrogen, thawed, and subcultured in the complete growth medium.
Treatment of cells with particles. Particles were suspended in sterile 14 mM NaHCO3 (pH 7.4) immediately before use, vortexed for 1 min, and diluted to the appropriate concentration with complete growth medium. pH of the complete growth medium containing the particles was 7.5.
cm2). After 24 h, the complete growth medium containing particles not associated with cells and/or endocytized was removed. The cells were rinsed once with 0.15 M phosphate- buffered saline (pH 7.4) and dislodged with 0.25% trypsin without EDTA, since EDTA is known to mobilize iron from particulate air pollution.16) The cells were stored at −80oC in 1 ml of ultra pure water containing 0.1 mM phenylmethanesulfonyl fluoride.
Determination of ferritin concentration. The harvested and frozen cells were lysed using the method of Chao et al.18) The stored cells were lysed by repeated freezing in liquid nitrogen and thawing in a 37oC water bath. After three cycles, the cell lysate was centrifuged at 12,000 g for 30 min at 4oC in a Beckman L8-80M ultracentrifuge to remove PM and cell debris. A portion of 12,000 g supernatant was used to determine the concentration of ferritin through sandwich enzyme-linked immunosorbent assay (ELISA).19) Briefly, the antibody to a human liver ferritin (Boehringer Mannheim Biochemicals, Indianapolis, IN) was used as the capture antibody to coat the flat-bottomed microtiter plate. Standard human liver ferritin (Calbiochem, San Diego, CA, USA) or cell lysate was then added to the microplate for the binding of ferritin to the antibody. The conjugate of peroxidase and antibody to the human liver ferritin was then added to serve as detectors to determine the amount of ferritin bound to the capture antibody. Tetramethylbenzidine (Sigma Chemical Co.) was then added to serve as a substrate for the peroxidase, and absorbance of the oxidation product of tetramethylbenzidine was determined at 450 nm. The standard curve, using human liver ferritin, was linear at 450 nm against the amount of standard human liver ferritin between 0 and 7.5 ng. Total protein in the cell lysate was determined using bicinchoninic acid (Pierce Biotechnology Inc.). The results were expressed as ng of ferritin/ìg of total protein.
Analysis of oxidative DNA damage by transition metal.
Metal-dependent induction of DNA SSB was conducted using the modified method of Lloyd et al.10) φX 174 RF I DNA (0.5µg) was incubated with hydrogen peroxide (75 µM) and each of the transition metal for 30 min at ambient temperature.
Because EDTA binds iron and other metals, tracking dye without EDTA was added. Samples were immediately loaded, without further treatment and purification, onto 0.7 % agarose gel and subjected to electrophoresis (50 mA) for ~2 h to separate closed-circular, superhelical (form II) DNA from DNA with SSB (form I). The gel was then stained with ethidium bromide, trans-illuminated with UV light and photographed. The negatives were analyzed using AlphaImager model 1220 and AlphaEase stand alone software (Alpha Innotech Co. San Leandro, CA, USA). The area obtained upon integration of the peaks for the
representative forms of DNA (i.e., I and II) was then used to calculate the percentage of DNA with SSB in each lane. To determine the fraction of form II remaining, the area of form II was divided by the total area (i.e., total DNA) of that lane. All samples were then compared by expressing each sample as a percentage of the control DNA (i.e. DNA treated under identical conditions minus PMs) and subtracted from 100% to give the percentage of DNA with SSB. Data represented means ± standard deviation for the analyses of triplicate samples. Where not apparent, the error bars were within the range obscured by the data point.
Results
Effect of PM on ferritin induction in human lung epithelial A549 cells. To determine mobilization of iron from the particles after treatment of A549 cells, ferritin levels were determined at regular intervals during 24 h treatment and compared with those of untreated controls. The amount of ferritin remained constant in untreated cells for 24 h.
However, in cells treated with 100µg/cm2 PMs, the amount of ferritin began to increase after 2 h, reached a maximum after 16 h with SRMs and 12 h with PMs collected from Seoul, and remained constant up to 24 h (Fig. 1 and 2). Ferritin levels increased about six-fold in A549 cells when treated with SRM 1648 or 1649a, compared to that of control, untreated cells (Fig. 1). Fig. 2 also showed that ferritin levels increased about six-fold in A549 cells treated with PMs collected in Seoul, compared to that of control during 24 h, although no difference in ferritin level was observed with different sizes of PM (Fig. 2).
Measurement of DNA single strand breaks in the presence of various transition metals. Agarose gel electrophoretic pattern of DNA SSBs induced by iron(II) is shown in Fig. 3. Metal-untreated control sample rarely catalyzed the formation of DNA SSBs in the presence of
hydrogen peroxide (Fig. 3, lane 1). A negligible amount of DNA SSB was observed with the metal alone in the absence of hydrogen peroxide when incubated with DNA for 30 min (Fig. 3, lane 2). DNA SSB level increased with increasing concentrations of iron(II) in the presence of hydrogen peroxide (Fig. 3, lane 3-9). These results demonstrate that the induction of DNA SSB is dependent upon iron(II) concentration. Fig. 4 shows variations in the increase of DNA SSB level with increasing concentrations of each metal in the presence of hydrogen peroxide. The results indicate that all nine transition-metal ions tested had a capacity to induce DNA SSB. A steady increase in the yield of SSB was observed with iron(II), copper(II), and copper(I) ion up to 20 µM, where maximum yield extents of SSB were 83.1, 52.9, and 48.6%, respectively (Fig. 4). The latter two metals induced even 100% of SSB with a higher metal concentration of 100µM (data not shown). Although the extent of SSBs was much lower, the other metals except vanadium (V) and cobalt Fig. 1. Effect of SRMs on induction of ferritin synthesis in
human lung epithelial A549 cells. 20,000 human lung epithe- lial cell/cm2 were treated with SRMs (100 µg/cm2).
Fig. 2. Effect of PM on induction of ferritin synthesis in human lung epithelial A549 cells. 20,000 human lung epithe- lial cell/cm2 were treated with PMs (100 µg/cm2).
Fig. 3. Agarose gel electrophoretic pattern of DNA with SSBs induced by iron (II). Experimental details are described in Materials and Methods. I. form I DNA, i.e. DNA with SSB;
II. form II DNA, i.e. closed-circular, superhelical DNA. Lane 1, control DNA treated with hydrogen peroxide only; lane 2, con- trol DNA treated with 0.1 µM of iron (II) only; lane 3~9, DNA + 75µM of hydrogen peroxide + 0.1, 0.4, 1, 3, 6, 10, and 20 µM of iron (II), respectively.
(II) also generated significant extent of DNA SSB. While the extent of SSB with iron(III) and chromium(III) remained relatively constant with increasing concentration up to 1 mM, nickel(II) and manganese(II) formed increasingly higher
extent of DNA SSBs with increasing metal ion concentration up to 5 mM (data not shown), suggesting that each metal has a different responsive range of concentration toward DNA SSB.
The extent of DNA SSB was compared at the same metal concentration of 1µM (Fig. 5), which may be a possible concentration of metals in vivo. 11.8% of SSB with copper(I) was much lower than that with copper(II). Yield of SSB with manganese(II) at the same concentration was higher than that with copper(I), which is notable since manganese(II) is not considered as a Fenton reagent.
Discussion
In the present study, we demonstrated that transition metals can be taken up into the lung cells from inhaled ambient PM and that SSB in DNA can be induced by Fenton -type reaction with these transition metals. Although it was not possible to prove with other meals, the amount of iron storage protein ferritin increased after 2 h in cells treated with PM. This result suggested that transition metals from PM might be introduced into the cells and oxidative damages also could be initiated at early periods by particles once inhaled. Additionally, ferritin levels reached maximum after 16 h with SRMs and 12 h with ambient particles collected from Seoul, and remained constant up to 24 h. These suggested that oxidative damage also can Fig. 4. Effect of transition metal concentration on DNA SSB induction. φX 174 RF I DNA (0.5 µg) was incubated with 75 µM hydrogen peroxide and various concentrations of transition metal ion as follows: A, iron(II) chloride; B, iron(III) chloride; C, cop- per(I) chloride; D, copper(II) chloride; E, chromium(III) chloride; F, nickel(II) chloride; G, vanadium(V) oxide; H, cobalt(II) chloride;
I, manganese(II) chloride for 30 min at ambient temperature.
Fig. 5. Effect of transition metals for the induction of DNA single strand break. φX 174 RF I DNA (0.5 µg) was incu- bated with 75 µM hydrogen peroxide and 1 µM of each transi- tion metal. Other experimental details are described in Materials and Methods.
continue for a long period.
Lloyd and Philips have shown that SSB is mediated by the generation of ROS in solution while double strand break (DSB) is formed by some kinds of site-specific mechanism.20) In the present experiment, to compare the extent of general oxidative damage each metal could exert, generation of SSB was chosen as a tool. The extent of SSB with iron(II) reached near maximum, but treatment with iron(III) did not generate significant extent of DNA SSB (Fig. 4). It was supposed that traces of Fe(III) might be able to react with H2O2; however, this is slower than the reaction of H2O2 with Fe(II) at physiological pH and very much depends on the ligand to the iron as shown below:
Fe(III) + H2O2 intermediate complex(es) Fe(II) + O2·−+ 2H+ In addition, generation of OH· by mixtures of certain Fe(III) chelates (e.g., ferric EDTA) and H2O2 appears to involve generation of O2·−, because it is inhibited by superoxide dismutase (SOD). The reaction is often written as above but no direct evidence for this chemistry has been obtained. By contrast, SOD has no effect on OH· generation by Fenton system in Fe(II)/H2O2 mixtures.5) On the other hand, both copper(I) and copper(II) ions generated significant levels of SSB in DNA (Fig. 4). Many previous studies reported that copper ions were more effective in promoting H2O2-dependent damage to isolated DNA than other metals.21) Indeed, copper ion forms stable complexes with DNA (association constant Cu(I)/DNA, 109 M; Cu(II)/DNA, 104 M).22) Cu(II) ions may undergo intercalation as well as complexation into purine bases.22,23) Therefore, Cu(I) and Cu(II) ions present within cellular DNA may be involved in the oxidative degradation of DNA, either by initiating the production of ROS or by modifying the evolution of initially produced radicals of DNA components.24) DNA single-strand scission was found in kidneys and lungs but not in livers of rats after parenteral administration of Cd(II) and Ni(II) chlorides. Likewise, cells cultured with Cd(II) or Fe(III)-NTA showed a metal concentration-dependent nuclear DNA fragments. DNA SSB was produced in vitro by Cr(VI) plus H2O2 or GSH plus ambient oxygen, and by Cu(II) and Ni(II) plus H2O2.13,25) The metals were mobilized from ambient particles in the presence
of corresponding chelators and irons were the major mobilized metals (Table 1), suggesting that once ambient PM are inhaled into the lung, various transition metals (especially iron) continuously mobilized at measurable amounts to induce oxidative damages such as DNA SSB.
Total assessment of ambient PM toxicity requires the consideration of both the toxicity of metals as well as that of organic pollutants from the ambient PM. Total amount of metals was often reported as an index for estimating the toxicity of the metal from ambient PM.3,4) However, considering the results of the present study as well as our previous study,14) the assessment of toxicity or damage by the metals from ambient PM should take into consideration not just the kind and amount of metals but also both the mobilizable amount of metals and the damage extent by the metals. We also conclude that transition metals in ambient particles are easily mobilized into the lung cells and that the metals induce SSB in DNA possibly by the generation of hydroxyl radical generation. Further studies on the comparison of the extent of oxidative damage with various kinds of particles from different emission sources and the effect of coexistence of transition metals on biomolecules (DNA and protein) are ongoing in our laboratory to gain better understanding on the hazardous effects of metals on ambient particles.
Acknowledgments. This research was supported in part by a grant from the Ministry of Environment G-7 project, Korea.
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V 1.05 0.60 3.48 0.87 0.64 0.52
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bEDTA = Ethylenediaminetetraacetic acid.
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