Quinones and Aromatic Chemical Compounds in Particulate Matter Induce Mitochondrial Dysfunction: Implications for Ultrafine Particle Toxicity

Tetramethylrhodamine methyl ester (TMRM), propidium iodide (PI), sucrose, HEPES buffer salts, EGTA, ascorbic acid, succinate, malate, glutamate, carbonyl cyanide m-chlorophenylhydrazone (CCCP), alamethacin (Ala), and tetraphenylphosphonium chloride were from Sigma (St. Louis, MO). The annexin V–fluorescein isothio-cyanate (FITC) kit was obtained from Trevigen (Gaithersburg, MD). 3,3′-Dihexyl-oxabarbocyanine iodide (DiOC₆), 10 N-nonylacridine orange (NAO), Calcium Green-5N, and hydroethidine (HE) were obtained from Molecular Probes (Eugene, OR). The PAH working standard (no. 8310) was purchased from Cerilliant Corporation (Round Rock, TX). All organic solvents used were of Fisher optima grade (Fisher Scientific, Hampton, NH), and the solid chemicals were of analytical reagent grade.

DEPs were obtained from M. Sagai (National Institute of Environment Studies, Tsukuba, Ibaraki, Japan). These particles were collected from a 4JB1-type light-duty, 2.74-L, four-cylinder Isuzu diesel engine (Isuzu Automobile Co., Tokyo, Japan) under a load of 6 kilogram meter onto a cyclone impactor (Kumagai et al. 1997). The particles were scraped from the glass fiber filters and stored as a powder under nitrogen gas. The particles consist of aggregates in which individual particles are < 1 μm in diameter. The chemical composition of these particles, including PAH and quinone analysis, has been previously described (Li et al. 2000). DEP methanol extracts were prepared by suspending 100 mg particles in 25 mL methanol, followed by sonication and centrifuging the suspension at 2,000 rpm for 10 min at 4°C (Hiura et al. 1999). The supernatant was transferred to a preweighed polypropylene tube and dried under nitrogen gas. The tube was reweighed to determine the methanol-extractable phase. The dried extract was dissolved in DMSO, and aliquots stored at −80°C in the dark.

DEPs (1.2 g) were sonicated in 200 mL methylene chloride, and the extract was filtered with a 0.45-μm nylon filter in a Millipore filtration system (Li et al. 2000). The methylene chloride extract was concentrated by rotoevaporation, and asphaltenes (insoluble, aromatic chemicals with nitrogen, oxygen, and sulfur heteroatoms) were precipitated by adding 25 mL hexane and shaking. The contents were left overnight in the freezer and then centrifuged, and the supernatant was collected. The precipitate was washed twice with hexane, and the washings were combined with the first hexane extract, concentrated, and dried over anhydrous sodium sulfate. The extract thus prepared was subjected to gravity-fed silica gel column chromatography. Three columns (1.5 × 50 cm) were packed with 26 g activated silica gel between 1 cm anhydrous sodium sulfate and conditioned with hexane. The extract was split into three equal aliquots and applied to each column. Aliphatic, aromatic, and polar fractions were successively eluted at 1.5 mL/min with 70 mL hexane, 150 mL hexane:methylene chloride (3:2, vol/vol), and 90 mL methylene chloride:methanol (1:1, vol/vol), respectively. The elution of the aromatic fraction was monitored by ultraviolet light at 365 nm. The respective eluates were combined and concentrated by rotoevaporation and made up to 1 mL in a 4-mL graduated vial, the aliphatic fraction in hexane and the others in methylene chloride. The vials were tightly sealed with a silicone-lined cap and stored at −80°C until use. The weight of the fractions was determined in a microbalance after evaporating off the hexane or methylene chloride from a known sample volume. Alkanes in the aliphatic fraction were characterized by gas chromatography (Varian 3400 with an SPI injector; Varian Inc., Palo Alto, CA) equipped with a flame ionization detector and a DB-5 column (30 m, 0.25 mm inner diameter, 0.25 μm film). The fractions were dried with N₂ gas and redissolved in DMSO for in vitro biologic studies.

PAH content in each fraction was determined by an HPLC-fluorescence method that detects a signature group of 16 PAHs (Li et al. 2003). Quinone content was analyzed as described by Cho et al. (2004). Briefly, quinones in the samples were derivatized and evaporated to approximately 50 μL under nitrogen; then, 100 mg zinc, anhydrous tetrahydrofuran, and 200 μL acetic anhydride were added to samples. After heating at 80°C for 15 min, samples were cooled to room temperature and an additional 100 mg zinc was added, followed by an additional 15 min of heating. The reaction was quenched with 0.5 mL water and 2 mL pentane. After centrifugation at 750 × g for 10 min, the pentane layer was evaporated to dry and the residue was reconstituted in 50–100 μL dry acetonitrile. 1,2-Naphthoquinone (NQ), 1,4-NQ, phenanthraquinone (PQ), and anthraquinone (AQ) were analyzed by the electron-impact gas chromatography/mass spectrometry technique using an HP MSD mass spectrometer (Hewlitt Packard, Palo Alto, CA) equipped with an automatic sampler (Cho et al. 2004).

RAW 264.7 cells were cultured in a 5% carbon dioxide in Dulbecco modified Eagle medium (DMEM) containing 10% fetal calf serum, 5,000 U/mL penicillin, 500 μg/mL streptomycin, and 2 mM l-glutamine. For exposure to DEP extracts and its fractions, aliquots of 3 × 10⁶ cells were cultured in six-well plates in 3 mL medium at 37°C for the indicated time periods.

Cells were stained with fluorescent dyes diluted in DMEM, except for annexin V and PI, which were prepared in a commercial binding buffer (Trevigen). The following dye combinations were added for 15–30 min at 37°C in the dark: a) 0.25 μg/mL annexin V plus 47.5 μg/mL PI in 500 μL binding buffer (assessment of apoptosis); b) 20 nM DiOC₆ plus 2 μM HE (assessment of ΔΨm and mostly O₂^•− production, respectively); c) 100 nM NAO plus 2 μM HE (to assess cardiolipin mass and O₂^•− production, respectively). Flow cytometry was performed using a FACScan (Becton Dickinson, Mountain View, CA) equipped with a single 488-nm argon laser. DiOC₆, NAO, and annexin V-FITC were analyzed using excitation and emission settings of 488 nm and 535 nm (Fl-1 channel); PI, 488 nm and 575 nm (Fl-2 channel); and HE, 518 nm and 605 nm (Fl-3 channel). Forward and side scatter were used to gate out cellular fragments.

We removed livers from Balb/c mice and isolated mito-chondria by a standard differential centrifugation procedure as previously described (Xia et al. 2002). Briefly, liver tissue was homogenized with four strokes of a Teflon pestle in buffer A (250 mM sucrose, 1 mM EGTA, and 5 mM HEPES, pH 7.4) on ice. After centrifugation at 1,000 × g for 10 min at 4°C, the supernatant was removed and recentrifuged at 10,000 × g for 10 min. The pellet was sequentially washed with buffer A and buffer B (buffer A without EGTA). The pellet was resuspended in buffer B and used within 5 hr after isolation. Mitochondrial protein content was determined by the Bradford method (Xia et al. 2002).

Most of the isolated mitochondrial experiments were conducted in a fiberoptic spectrofluorimeter (Ocean Optics, Dunedin, FL), which uses a closed, continuously stirred cuvette at room temperature (Korge et al. 2002). Mitochondria were added to the cuvette at 0.1 mg/mL in a standard buffer containing 250 mM sucrose and 5 mM HEPES, pH 7.4. Substrates, Ca²⁺, PI, inhibitors, and fluorescent indicators were added at the indicated concentrations as shown for each experiment.

Mitochondria (0.1 mg/mL) were incubated in swelling buffer containing 250 mM sucrose, 5 mM HEPES (pH 7.4), 2 μM rotenone, 1 mM PI, and 4.2 mM succinate at room temperature. Mitochondria were then exposed to different chemicals.

Changes in mitochondrial volume were estimated by measuring 90° light scatter with excitation and emission wavelengths set at 520 nm (Walter et al. 2000). Changes in matrix volume were reported as a percentage of maximum (100%) swelling induced by 10μg Ala at the end of each run.

TMRM was included at 400 nM, and ΔΨm was estimated at a wavelength of 570 nm (Korge et al. 2002). Decrease in ΔΨm was expressed as percentage decrease in TMRM fluorescence compared with the effect of 1 μM CCCP (100%) in fully energized mitochondria. Light scattering was recorded simultaneously with TMRM fluorescence. In some experiments, ΔΨm was estimated using an ion-selective electrode to measure tetraphenylphosphonium ion (TPP⁺) distribution with a Flex-Ref electrode and Duo 18 recording system (World Precision Instruments, Sarasota, FL). TPP⁺ was added to a final concentration of 3 μM, and the mitochondria were energized by adding succinate at 4.2 mM.

Changes in extramitochondrial Ca²⁺ concentration were followed by measuring Calcium Green-5N (1 μM, salt form) fluorescence at excitation and emission wavelengths of 475 and 530 nm, respectively. Individual Ca²⁺ additions were calibrated by adding known quantities of Ca²⁺ to the buffer in the presence of mitochondria and CCCP to block Ca²⁺ uptake. Addition of chemical materials did not exhibit autofluorescence in our spectrofluorimetry assays.

Mitochondrial respiration was carried out in the fiberoptic spectrofluorimeter in the presence of different substrates: succinate, 4.2 mM (complex II); malate/pyruvate/glutamate, 5 mM each (complex I); tetramethyl-p-phenylenediamine (TMPD) and ascorbate, 0.2 mM and 2.5 mM, respectively (complex IV) (Korge et al. 2002). The addition of 2 μM CCCP was used as an inducer of maximal respiration. The partial pressure of O₂ in the buffer was continuously recorded by a fiber-optic oxygen sensor (Foxy Al-300; Ocean Optics, Dunedin, FL).

UFPs were collected using the Versatile Aerosol Concentration Enrichment System (VACES) in Downey, California, as previously described by Li et al. (2003). Highly concentrated liquid particle suspensions were obtained by connecting the concentrated output flow from the VACES to a liquid impinger (BioSampler; SKC West Inc., Fullerton, CA). Particles were injected into the BioSampler in a swirling flow pattern so that they could be collected in a small volume of water by a combination of inertial and centrifugal forces.

For chemical analysis, we collected two reference filter samples in parallel with the VACES. The first sample was collected on a Teflon filter (47 mm, polytetrafluoroethylene, 2μm pore; Gelman Science, Ann Arbor, MI). Mass concentrations were determined by weighing the Teflon filter before and after each field test in a Mettler 5 Microbalance (Mettler-Toledo Inc., Highstown, NJ). Laboratory and field blanks were used for quality assurance. The Teflon filters were then analyzed by X-ray fluorescence for measurement of trace-element and metal concentrations. The second collection was done on two 47-mm quartz filters (Pallflex Corp., Putnam, CT). These filters were used for measurement of inorganic ions as well as for determining PAH, elemental carbon (EC), and organic carbon (OC) concentrations. A slice of approximately 0.2 cm² from each filter was placed in a platinum boat containing manganese dioxide. The sample was acidified with an aliquot of HCl and heated to 115°C to form CO₂ as an index of particle-associated carbon. The boat was then inserted into a dual-zone furnace, where MnO₂ oxidized OC at 550°C and EC at 850°C. A flame ionization detector converted the CO₂ combustion product to CH₄ for detection. The remaining filter was divided in two equal parts: one half was analyzed by means of ion chromatography to determine the concentrations of particulate sulfate and nitrate; the other half was analyzed by a HPLC-fluorescence method for detection of a group of signature PAHs as previously described (Li et al. 2003).

The experiments were reproduced four times, except where otherwise stated. Results were analyzed by Student’s t-test, and changes were considered significant at p < 0.05.

Previous data from our laboratory showed that crude organic DEP extracts mimic the effects of intact particles in ROS production and cytotoxicity (Hiura et al. 1999). Mitochondria play a key role in DEP-induced toxicity, as shown by an early decrease in ΔΨm, loss of inner membrane mass, caspase 9 activation, and onset of apoptosis (Hiura et al. 2000). To clarify which organic chemicals play a role in this cytotoxicity, the crude extract was fractionated by silica gel chromatography, as previously described (Li et al. 2000). Elution with increasingly polar solvents resulted in the recovery of aliphatic, aromatic, and polar fractions in the amounts shown in Table 1. Although the aromatic fraction was enriched for PAHs (Table 2), the polar fraction was devoid of this chemical group but contained an abundance of quinones (Table 3). No quinones were present in the aromatic fraction (Table 3).

RAW 264.7 cells were treated with these chemicals and assessed for evidence of apoptosis (Figure 1). Figure 1A and 1B show representative flow cytometry panels of an experiment that was performed in triplicate. The results demonstrate the induction of annexin V⁺/PI⁻ (lower right) and annexin V⁺/PI⁺ (upper right) cells by the crude extract. These represent early and late apoptotic events, respectively, and can be combined with live (annexin V⁻/PI⁻, lower left) and dead (annexin V⁻/PI⁺, upper left) cells to provide a graphic display of cellular viability/toxicity (Figure 1C). This presentation format demonstrates that the polar fraction is considerably more toxic than the aromatic fraction, whereas the aliphatic fraction has no effect on cell viability (Figure 1C).

To explore mitochondrial perturbation, we assessed ΔΨm and ROS production by dual-color DiOC₆/HE fluorescence (Hiura et al. 1999). DiOC₆ reflects ΔΨm, whereas HE measures mostly O₂^•− production. This analysis shows that although the aliphatic fraction was inactive, the aromatic and polar fractions induced the appearance of DiOC₆^low/HE^high subpopulations (Figure 2A). These effects were dose dependent (not shown), with the polar being more active than the aromatic fraction at comparable dose levels (Figure 2). To test whether O₂^•− production is related to inner membrane damage, we also performed dual-color NAO/HE fluorescence (Figure 2B). NAO binds to the inner membrane phospholipid, cardiolipin. Although NAO fluorescence is ΔΨm sensitive, a decrease in fluorescence reflects inner membrane damage. Both polar and aromatic compounds led to a decrease in inner membrane mass, whereas the aliphatic fraction was inactive (Figure 2). Cells with damaged mitochondria also showed increased HE fluorescence, which is in accordance with increased O₂^•− production by cells with reduced ΔΨm (Figure 2A). Overall, the polar fraction was more active than the aromatic fraction in its ability to induce these mitochondrial effects (Figure 2). Taken together, these results demonstrate that the aliphatic, aromatic, and polar fractions exert differential toxic effects on mitochondria and cellular viability.

To further explore the action of functionalized DEP chemical groups on mitochondrial function, we performed a series of studies in isolated liver mitochondria. First, ΔΨm was recorded with a TPP⁺ electrode after the addition of phosphate and succinate to the mitochondrial preparation (Kushnareva and Sokolove 2000). The addition of CCCP, a protonophore uncoupler, led to a quick dissipation of the ΔΨm (Figure 3A). Although the carrier (DMSO) and the aliphatic fraction were inactive (Figure 3A,B), the crude extract as well as the polar fraction induced a dose-dependent decline in ΔΨm (Figure 3C,D). The polar material was more potent and induced a faster rate of depolarization (Figure 3D).

If mitochondria are well polarized, addition of a large Ca²⁺ load leads to matrix Ca²⁺ uptake and PTP opening (Korge et al. 2002). PTP opening leads to mitochondrial swelling, which can be followed by using 90° light scatter in a spectrophotometer (Figure 4A, a). In mitochondria that had not been subjected to a Ca²⁺ load, addition of a small and nondepolarizing polar dose (1–2.5 μg/mL; Figure 3) caused spontaneous induction of mitochondrial swelling (Figure 4B, c and d). Compared with the lack of response to the DMSO carrier, these results were statistically significant (p < 0.01). In contrast, higher doses of the polar fraction (≥5 μg/mL) caused a statistically significant (p < 0.01) inhibition of Ca²⁺-induced mitochondrial swelling (Figure 4A). The same effect (p < 0.01) was seen with the crude DEP extract (not shown). This inhibition of swelling can be attributed to the ΔΨm-reducing effects of these higher concentrations. This is similar to the ΔΨm dissipation by CCCP, which prevents the rise in matrix Ca²⁺ required for PTP opening. If, on the other hand, matrix Ca²⁺ is already elevated, ΔΨm depolarization promotes PTP opening because the PTP open probability is voltage dependent and increases with depolarization. To test this theory, isolated mitochondria were preexposed to a small Ca²⁺ load (10 μM) that is insufficient to induce PTP opening, and then exposed to a higher polar concentration range. This led to a dose-dependent induction of mitochondrial swelling at all doses tested (Figure 4C). DMSO and the aliphatic fraction had no effect on mitochondrial swelling (not shown).

To confirm that mitochondrial swelling induced by the crude extract and polar fraction was due to PTP opening, we examined the effects of the PTP inhibitor CsA (Figure 5). Similar to its effect on Ca²⁺-induced swelling, CsA added before the addition of the polar fraction (Figure 5A, a) abrogated polar-induced mitochondrial swelling in a statistically significant fashion (p < 0.01) (Figure 5B). Ca²⁺-dependent mitochondrial swelling by the polar fraction was confirmed by prior addition of EGTA, which led to a significant reduction in the rate and magnitude of mitochondrial swelling in the presence of 1 μg/mL of the polar material (Figure 5C, b vs. c).

The polar fraction contains a number of chemicals, among which the quinones participate in the generation of oxidative stress and covalent protein modification (Penning et al. 1999). We tested a number of DEP quinones (Table 3) for their effects on mitochondrial swelling, including PQ, 1,2-naphthaquinone, and AQ. PQ induced statistically significant (p < 0.01) mitochondrial swelling with slower kinetics than did the Ca²⁺ load stimulus (Figure 5D). This effect was totally suppressed by CsA, indicating that quinones stimulate PTP activity in a Ca²⁺-dependent fashion (Figure 5D). Similar results were obtained with 1,2-naphthaquinone, whereas a nonredox-cycling quinone, AQ, was inactive (not shown). These results suggest that redox-cycling quinones play a role in the cytotoxic effects of DEPs on the mitochondrion.

All considered, the data presented indicate that polar chemicals induce mitochondrial swelling due to PTP opening. This involves direct action on the PTP at low doses, as well as rapid-onset ΔΨm depolarization at higher doses, provided that the matrix Ca²⁺ concentration is already elevated. In the absence of Ca²⁺ loading, higher polar doses inhibit mitochondrial swelling, most likely due to interference in Ca²⁺ accumulation as a result of ΔΨm depolarization.

Mitochondrial uncoupling increases mitochondrial respiration, which can be assessed by measuring oxygen consumption with an oxygen-sensing electrode (Figure 6). Although the polar fraction increased mitochondrial respiration as a consequence of its depolarizing effect (not shown), the induction of maximal respiration by CCCP in the presence of succinate showed that subsequent addition of the polar fraction caused a slowing of respiration (Figure 6A). The crude DEP extract had the same effect, whereas the aromatic or aliphatic fractions did not affect maximal mitochondrial respiration (Figure 6A). These findings indicate that the polar fraction and crude DEPs interfere in the function of complex II in the inner membrane. Similar results were obtained when using malate/glutamate/pyruvate, which are substrates for complex I (not shown). However, there was no effect when ascorbate and TMPD were used, implying that complex IV was not affected by the polar chemicals (Figure 6B). We propose that exogenous quinones present in the polar fraction might compete with the ubiquinones, which play a critical role in electron transfer in the inner membrane complexes. Transfer of those electrons to molecular dioxygen could explain O₂^•− production.

Treatment with the aromatic fraction induced a dose-dependent ΔΨm decrease in isolated liver mitochondria at doses > 10 μg/mL (not shown). Unlike that observed with the polar fraction (Figure 3D), this depolarization was incomplete compared with CCCP (not shown). In addition, the aromatic fraction induced spontaneous mitochondrial swelling in a dose-dependent fashion (Figure 7A, b–f). In non-Ca²⁺-loaded mitochondria, this effect started at aromatic doses ≥10 μg/mL (Figure 7A), whereas lower doses (e.g., 5 μg/mL) actually inhibited Ca²⁺-induced swelling (Figure 7B). This is the opposite from the effect observed with the polar fraction, which interfered in mitochondrial swelling at high doses but induced spontaneous swelling at low doses (Figure 4B,C). Taken together, these data suggest that differences in the chemical composition of the aromatic and polar fractions lead to differential effects on mitochondrial function.

PAHs are the main components of the aromatic fraction and are capable of inducing apoptosis (Li et al. 2000). To test if PAHs exert an effect on isolated mitochondria, we used a commercial source composed of 16 DEP PAHs to conduct the swelling assay. This demonstrated that the PAH mix can induce slow-onset swelling in non-Ca²⁺-loaded mitochondria, which mimics the effects of the aromatic fraction (Figure 8). This swelling effect was incomplete and was partially but statistically significantly (p < 0.05) inhibited by CsA (Figure 8B). CsA exerted the same effect on the induction of swelling by the aromatic fraction (Figure 8A).

Calcium Green-5N is a fluorescent dye that can be used to assess the Ca²⁺ retention capacity of isolated mitochondria. The addition of small amounts of Ca²⁺ leads to a rapid matrix uptake into isolated energized mitochondria (Figure 9A). With repeated Ca²⁺ pulses, matrix Ca²⁺ eventually triggers PTP opening, which leads to depolarization and release of Ca²⁺ from the matrix (Figure 9A). This leads to a precipitous and sustained increase in fluorescence intensity (Figure 9A). This response is statistically significantly (p < 0.01) inhibited by CsA, which increased the number of Ca²⁺ pulses from 4 to 14 (Figure 9B). The aliphatic fraction had no effect on the number of Ca²⁺ pulses (Figure 9C), whereas 1 μg/mL of the polar material reduced the number of Ca²⁺ pulses required to trigger PTP transition (Figure 9D). This finding is compatible with the ability of the polar fraction to induce spontaneous mitochondrial swelling in a Ca²⁺-dependent fashion (Figure 4C). Higher polar concentrations induced immediate release of ambient accumulated Ca²⁺, which reflects its depolarizing effect (Figure 9C). Similar results were obtained with the crude DEP extract: a reduction in the required number of Ca²⁺ pulses at low doses and precipitous Ca²⁺ release at high doses (not shown).

Because we have shown that DEP quinones mimic the effect of the polar fraction in spontaneous mitochondrial swelling, we also tested these quinones in the Calcium Green-5N assay. PQ reduced the required number of Ca²⁺ applications to achieve PTP from 3, to 2, to 0 at PQ concentrations of 0.25, 1, and 5 μg/mL, respectively (Figure 9F–H). CsA could significantly (p < 0.01) increase the number of Ca²⁺ pulses required for precipitous Ca²⁺ release in the presence of PQ, suggesting PTP involvement. Similar results were obtained with 1,2-NQ but not with AQ (not shown).

Examination of the aromatic fraction in the Calcium Green-5N assay showed that doses < 10 μg/mL increased the Ca²⁺ retention capacity (Figure 10A,B). This is in keeping with the ability of the aromatic fraction to inhibit Ca²⁺-induced PTP opening in this dose range (Figure 7B). At higher doses, the aromatic fraction induced a short Ca²⁺ burst, probably related to ΔΨm depolarization, which is followed by a progressive decline in the ability of the matrix to accumulate Ca²⁺ (Figure 10C). This depolarization was incomplete and not CsA sensitive (not shown). In order to determine whether this effect is related to the PAHs present in the aromatic fraction, the DEP PAH mixture was separately tested. PAHs mimicked the effect of the aromatic fraction in the low and high dose range (Figure 10D,E). Taken together, these results confirm that the polar and aromatic DEP compounds exert fundamentally different actions on mitochondria.

A key question is whether the effects of the DEP chemicals can be reproduced with intact DEP and “real-life” ambient particles (Li et al. 2003). Intact DEPs induce apoptosis (Hiura et al. 1999), and ambient UFPs induce structural damage and lodge inside mitochondria in RAW 264.7 cells and epithelial cells (Li et al. 2003). When UFPs, collected by a particle concentrator in the Los Angeles Basin (Kim et al. 2001), were tested in the mitochondrial swelling assay, we observed spontaneous PTP opening at doses of 4.8 and 7.7 μg/mL in non-Ca²⁺-loaded mitochondria (Figure 11, b and c). Swelling was partially reversed by CsA (Figure 11, d). At a dose of 1.9 μg/mL, UFPs did not induce spontaneous PTP opening but interfered with Ca²⁺-induced swelling (not shown). This is similar to the effect of sonicated DEP, which interfered in Ca²⁺-induced mitochondrial swelling in a dose-dependent fashion but failed to induce spontaneous swelling (Table 4). This could relate to differences in the particle size (the DEP powder used here contains particle aggregates) as well as differences in the bioavailability of surface chemical compounds on these particles. The chemical composition of UFPs is shown in Table 5. In contrast to the particulate pollutants, artificial polystyrene microspheres (size < 100 nm) did not exert an effect on mitochondrial swelling, and the mitochondria remained fully responsive to Ala (Figure 11, a).

In the Calcium Green-5N assay, ambient UFPs induced instantaneous Ca²⁺ release but reduced Ca²⁺ retention capacity in a dose-dependent manner (Figure 12A vs. Figure 12C–F). CsA prevented this effect (Figure 12G). Sonicated DEPs had a similar effect that was also CsA sensitive (Table 4). In contrast, polystyrene microspheres (80 nm) had no effect on Ca²⁺ retention capacity (Figure 12B). This suggests that the effect of the ambient UFP is dependent on their content of redox-cycling chemicals. Taken together with the data shown in Figure 11, the UFP effects appear to be a summation of the effects of polar and aromatic chemical compounds.