Ascorbic acid prevents zinc oxide nanoparticle–induced intracellular oxidative stress and inflammatory responses

Abstract

Exposure to zinc oxide nanoparticles (ZnO NPs) promotes acute pulmonary toxicity through oxidative stress and inflammation. Furthermore, dissolved zinc from ZnO NPs induces the formation of intracellular reactive oxygen species (ROS). We previously reported that supplemental ascorbic acid (AA) inhibits ZnO NP-induced acute pulmonary toxicity in a rat model; however, the mechanism of this action remains unclear. Therefore, we investigated the effects of AA on ZnO NP-induced cytotoxicity in human lung carcinoma A549 cells. AA was found to suppress intracellular production of ROS, and thus reduce the subsequent inflammation of ZnO NPs. However, intracellular Zn²⁺ concentrations were higher in AA-treated cells than in AA-untreated cells. AA was found to react with Zn²⁺ but not with the ZnO NPs themselves. These results suggest the possibility that AA-chelated extracellular Zn²⁺ and the Zn–AA complex was readily taken up into cell. Even if the intracellular Zn²⁺ level was high, cytotoxicity might be reduced because the Zn–AA complex was stable. Co-treatment of AA to A549 inhibited ROS production and subsequent intracellular inflammatory responses. These results are consistent with those previously reported from an in vivo model. Thus, two possibilities can be considered about the cytotoxicity-reducing the effect of AA: antioxidant efficacy and chelating effect.

Keywords

Nanoparticles metal toxicity oxidative stress inflammation antioxidant

Introduction

Zinc oxide nanoparticles (ZnO NPs) are widely manufactured for applications ranging from catalysis to cosmetics. With increasing ZnO NP production, more workers are being exposed to ZnO NPs. Therefore, it is necessary to understand ZnO NP-induced toxicity and consider how to reduce the risk of toxicity.

Widespread and expanding production and use of ZnO NPs increases the potential for their release to the environment. Ma et al. reported that toxic effects of ZnO NPs in both aquatic and terrestrial species were within a broad range of taxa, and for certain species (such as Daphnia magna), the toxicity can occur at concentrations as low as less than 1 mg/l. This suggests that ZnO NPs, if reaching a sufficient level in natural environments, can cause significant risk to the environmental biota (Ma et al., 2013).

The in vivo toxic effects of ZnO NPs were studied using different treatment routes. Oral, inhalation, and intratracheal administration routes have also been used to evaluate the acute toxicity of ZnO NPs. In our previous study, intratracheal administration of ZnO NPs induced pulmonary oxidative stress and inflammation in rats, and Zn²⁺ released from ZnO NPs was associated with pulmonary toxicity (Fukui et al., 2012). Thus, it is known that the release of Zn²⁺ and subsequent induction of oxidative stress are known to be contributing factors to the mechanism of ZnO NP-induced toxicity. However, there are no reports on preventive measures for this toxicity.

Since oxidative stress is a known contributor to the pulmonary toxicity induced by ZnO NPs, we investigated the antioxidant l-ascorbic acid (AA) as a potential preventative agent. AA is a water-soluble radical scavenger, known to protect against membrane oxidation and injury. Moreover, AA has been shown to chelate heavy metals (e.g. cadmium, mercury, and zinc) to inhibit metal-derived oxidative stress (Tajmir-Riahi, 1991). AA has also been reported to inhibit intracellular oxidative stress induced by nanoparticles. For example, reports have shown that AA treatment reduces intracellular lipid peroxidation induced by talc nanoparticles in A549 cells and inhibits reactive oxygen species (ROS) generation (and subsequent activation of apoptosis) induced by silica nanoparticles (Ahmad et al., 2012; Akhtar et al., 2010). AA potentially prevents not only oxidative stress but also inflammation. In addition, AA was found to decrease the secretion of pro-inflammatory cytokines (IL-1α and IL-6) in human keratinocytes stimulated by UVA irradiation (Tebbe et al., 1997).

We have previously demonstrated that supplying AA in water reduces acute pulmonary toxicity (including oxidative stress, inflammation, and injury) induced by ZnO NPs in rat lungs (Fukui et al., 2015). However, the mechanism by which AA acts to prevent ZnO NP-induced toxicities (i.e. ROS trapping and/or Zn²⁺ chelating) is still unclear. Clarifying AA’s prevention mechanism is important for reducing the occupatonal health risk of ZnO NP-induced toxicities. Therefore, the present study aimed to evaluate the preventative role of AA in ZnO NP-induced toxicity and to determine its mechanism of action.

Materials and methods

ZnO NPs and chemicals

ZnO NPs were purchased from Ishihara Sangyo Kaisha, Ltd (Osaka, Japan); the product name was FZO-50 (lot No. 0010). According to the manufacturer’s material data sheets, the primary particle size was 21 nm, the purity was 97.4%, and the specific surface area was 49.6 m²/g. Zinc chloride (ZnCl₂) and AA were purchased from Wako Pure Chemical Industries, Ltd (Osaka, Japan).

Cell culture

Human lung carcinoma A549 cells were purchased from the Riken BioResource Center (Tsukuba, Ibaraki, Japan). The cells were cultured in Dulbecco’s modified Eagle medium (DMEM; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin (100 units/ml), streptomycin (100 µg/ml), and amphotericin B (250 ng/ml) (Nacalai Tesque Inc., Kyoto, Japan), abbreviated herein as DMEM-FBS.

Cells were cultured in DMEM-FBS at 37°C, in an atmosphere that contained 5% CO₂. For cellular experiments, cells were seeded at 2 × 10⁵ cells/ml in a six-well multidish (Corning Inc., Corning, NY, USA) and incubated for 24 h. The medium was then replaced by ZnO dispersion, with or without 5 mM AA, this concentration of AA was the no observed adverse effect level in preliminary examination, and incubated for another 6 h. The fresh medium was also replaced for the control cells.

Preparation of the ZnO dispersion for in vitro evaluations

The ZnO NP powder (10 mg/ml) was dispersed in a bovine serum albumin (BSA) solution (10 mg/ml; Nacalai Tesque Inc.) using sonication in an ultrasonic bath for 30 min with shaking. The ZnO–BSA dispersion was serially diluted 10 times with DMEM-FBS for use in the cellular experiments. The size distribution of ZnO NPs in the dispersion was measured by dynamic light scattering (DLS) using a Zetasizer Nano system (Malvern Instruments Limited, Malvern, UK).

Measurement of intracellular ROS level

The intracellular ROS level was determined by the 2′,7′-dichlorofluorescein diacetate (DCFH-DA; Sigma-Aldrich) method. A 5 mM DCFH-DA stock solution in dimethyl sulfoxide (DMSO) was prepared and stored at −20°C. When used in an experiment, this stock solution was diluted 500-fold with serum-free medium. After exposure of the cells to the ZnO dispersion with or without AA for 6 h, the medium was changed to serum-free DMEM that included 10 µM DCFH-DA and incubated for 30 min at 37°C. The cells were then washed with phosphate-buffered saline (PBS), trypsinized with 0.25% trypsin, washed again with PBS, and resuspended in 500 µl of PBS. The cell samples were excited with a 488-nm argon ion laser in a Cytomics FC500 flow cytometry system (Beckman Coulter Inc., Brea, CA, USA), and the emission of 2′,7′-dichlorofluorescein was recorded at 525 nm. Data were collected from at least 5000 gated events.

Real-time polymerase chain reaction

The expression of target genes was determined by real-time polymerase chain reaction (PCR). Total RNA was isolated from cells using the RNeasy mini kit (Qiagen GmbH, Hilden, Germany). cDNA synthesis was carried out with a high-capacity cDNA reverse transcription kit (Life Technologies Corp., Carlsbad, CA, USA). Real-time PCR was conducted with a StepOne real-time PCR system (Life Technologies Corp.). Gene expression levels were analyzed by the ΔΔCt method. PCR amplification of cells was analyzed using TaqMan^® gene expression assays (Life Technologies Corp.), with the human β-actin gene used as an endogenous control. The gene expression assays for heme oxygenase-1 (ho-1), interleukin-8 (il-8), and metallothionein-2 (mt-2) were designated as Hs01110250_m1, Hs00174103_m1, and Hs02379661_g1, respectively.

Evaluation of AA uptake in A549 cells

The concentration of AA in A549 cells was measured using a high-performance liquid chromatography system (HPLC) system equipped with a UV detector (SPD-10AV, 263 nm; Shimadzu, Japan) and NH₂ column (Wakosil 5 NH2, 5 µm, 250 × 4.6 mm; Wako Pure Chemical Industries). PBS (40 mM)/methanol (1/9, v/v) was eluted at a flow rate of 1 ml/min. The cells were treated with 1 and 5 mM AA for 6 h. After treatment, the cells were washed with PBS, trypsinized with 0.25% trypsin, washed again with PBS, and resuspended in 100 µl of PBS. The cells were then diluted with methanol (1/4, v/v) and mixed vigorously for 1 min using a vortex mixer, followed by centrifugation (20,400 × g, 10 min). An aliquot of the resulting upper layer was injected into the HPLC system for analysis.

Measurement of intracellular Zn²⁺ levels

Intracellular Zn²⁺ was detected using the fluorescent ZnAF-2 DA reagent (Sekisui Medical Co. Ltd, Tokyo, Japan) (Hirano et al., 2002). For use in cellular experiments, the ZnAF-2 DA stock solution (5 mM in DMSO) was diluted 500-fold with a serum-free medium. After exposure to the ZnO dispersion for 6 h, with or without AA treatment, the medium was changed to serum-free DMEM that included 10 µM ZnAF-2 DA and incubated for 30 min at 37°C. The cells were then washed with PBS, trypsinized with 0.25% trypsin, washed again with PBS, and resuspended in 500 µl of PBS. The cell samples were excited with a 488-nm argon ion laser in a Cytomics FC500 flow cytometry system, and the emission of ZnAF-2 was recorded at 525 nm. Data were collected from at least 5000 gated events.

The reactivity of AA with ZnO and Zn²⁺

To evaluate the reactivity of AAs with ZnO, AA was dissolved in PBS at a concentration of 40 µM. ZnO NPs were then added to the AA solution at a concentration of 0, 50, 100, and 1000 µM. The AA absorbance at 260 nm was measured using a scanning spectrophotometer (model UV-3100PC UV–VIS–NIR; Shimadzu). Changes in soluble zinc concentration in the ZnO DMEM-FBS dispersions, with or without AA, were measured at 0 and 6 h after preparation using 2-(5-bromo-2-pyridylazo)-5-[N-n-propyl-N-(3-sulfopropyl) amino] phenol disodium salt dehydrate (5-Br-PAPS; Dojindo Laboratories, Kumamoto, Japan). The ZnO NPs dispersions were centrifuged at 16,000 × g for 20 min, and the supernatant was carefully collected. After suitable dilution of the supernatant, the supernatant (400 µl), APS (100 µl, 5 mM), DTCS (100 µl, 1 mM), and 5-Br-PAPS (50 µl, 1 mM) were added to a reaction buffer (2.0 ml, prepared by dissolving 6 g of sodium hydroxide in 500 ml of 0.5 M HEPES, pH 7.8). The resulting solution was mixed well, incubated for 10 min at room temperature, and its absorbance at 555 nm was measured using a DU530-spectrophotometer (Beckman Coulter Inc., Miami, FL, USA). The reactivity of AA with Zn²⁺ was evaluated both directly and indirectly. For the direct measurement, AA was dissolved in PBS at a concentration of 40 µM. ZnCl₂ was then added to the AA solution at concentrations of 0, 10, 20, 50, 100, 200, 400, and 1000 µM. The absorbance of AA at 260 nm was then measured using the scanning spectrophotometer. For the indirect measurement, 10 µM ZnCl₂ and 50 µM 5-Br-PAPS were dissolved in PBS. AA was then added to yield concentrations ranging from 0 to 40 µM. The absorbance of 5-Br-PAPS at 555 nm was then measured using the scanning spectrophotometer.

Statistical analysis

Data are presented as mean ± SD of at least three separate experiments. Statistical evaluations were performed via analysis of variance using the Dunnett test for multiple comparisons. Individual calculation methods are described in each figure legend.

Results

AA decreased the cytotoxicity caused by ZnO NPs

The secondary particle size of ZnO NPs in the ZnO water dispersion was measured by DLS. The average particle size was 90.2 nm (based on the number). The intracellular ROS levels in A549 cells exposed to ZnO NPs with or without AA treatment were measured by the DCFH method (Figure 1). Intracellular ROS levels increased remarkably in cells exposed to ZnO NPs but not to AA. In contrast, AA treatment was found to inhibit the ZnO NP-induced increase in intracellular ROS levels. Further, ZnO NPs were found to induce expressions of ho-1, a major oxidative stress response enzyme protein (Figure 2(a)), and il-8, one of the human chemotactic factors (Figure 2(b)) in the A549 cells. Interestingly, elevation in these gene expression levels was not observed in cells exposed to both ZnO NPs and AA.

AA uptake in A549 cells

To evaluate AA uptake in A549 cells, intracellular concentration was measured (Figure 3). AA was taken up by cells following 6 h of treatment, in a dose-dependent manner.

Effect of AA on intracellular Zn²⁺ levels

Gene expression of mt-2, which is known to be induced by metals, in A549 cells, increased following exposure to ZnO NPs. However, the co-treatment of ZnO NPs and AA inhibited the increase in mt-2 gene expression (Figure 4(a)). The Zn²⁺ level was measured in cells exposed to ZnO NPs, in the presence or absence of AA treatment (Figure 4(b)). Exposure to ZnO NPs significantly increased intracellular Zn²⁺ levels, regardless of AA treatment. In particular, Zn²⁺ levels in cells treated with AA were remarkably higher than those in cells not treated with AA.

Reactivity between AA and ZnO or Zn²⁺

We examined the reasons for the increase in the intracellular Zn²⁺ level with AA co-treatment. There are two possibilities for this result: (1) co-treatment with AA induced Zn²⁺ release from ZnO NPs and (2) co-treatment with AA enhanced cellular uptake of Zn. To confirm these possibilities, we examined whether AA accelerates Zn²⁺ release of ZnO NPs. We also examined whether AA induces cellular uptake of Zn²⁺. AA exhibits an absorption maximum in the 244–265 nm range (Nováková et al., 2008). In this study, we used a slightly broader range (200–290 nm) to measure AA absorption. The interaction between AA and ZnO NPs was evaluated by measuring AA absorbance at 260 nm (Figure 5(a)). When the concentration of ZnO NPs was 0 and 1 mM, no significant changes were observed in AA absorbance. There were no remarkable changes in Zn²⁺ concentration in ZnO DMEM-FBS dispersions with or without AA added 0 and 6 h after preparation (Table 1). However, the AA absorbance at 260 nm (and the AA absorbance curve) responded to changes in Zn²⁺ concentration in PBS buffer (pH 7.0; Figure 5(b) and (c)). The absorbance of 5-Br-PAPS complexed with zinc was measured at 555 nm (Figure 5(d)). This highly sensitive reagent is used to determine zinc concentration in solution; the 5-Br-PAPS–Zn complex is known to be red in color, with an absorption maximum at 554 nm (Makino et al., 1982). 5-Br-PAPS–Zn absorbance was detected at increasing AA concentrations.

Table 1.

Soluble zinc concentration changes in ZnO NPs DMEM-FBS dispersion.^a

		Concentration of Zn²⁺ (µg/ml)
		0 h	6 h
ZnO DMEM-FBS dispersion		17.8	14.6
	SD	0.616	0.407
ZnO DMEM-FBS dispersion with adding 5 mM AA		16.46	15.00
	SD	0.782	0.481

ZnO NPs: zinc oxide nanoparticles; DMEM-FBS: Dulbecco’s modified Eagle medium–fetal bovine serum.

^aThe soluble zinc concentration in the dispersions included 0.1 mg/ml of ZnO NPs with or without 5 mM AA at measured at 0 and 6 h after preparation. The absorbance of 5-Br-PAPS at 555 nm was measured by a spectrofluorophotometer.

Discussion

ZnO NPs are known to induce strong oxidative stress in vivo and in vitro. The oxidative stress triggered by intratracheal administration of ZnO NPs has been associated with lung inflammation and injury in a rat model (Fukui et al., 2012). ZnO NPs release Zn²⁺ into the culture media and exhibit increased levels of intracellular ROS levels associated with inflammation and corresponding tissue damaging effects including cell death (Cuzzocrea et al., 2000; Heng et al., 2011; Huang et al., 2010; Kim et al., 2010a, 2010b; Pawliczak, 2003; Sharma et al., 2012; Song et al., 2010; Tsou et al., 2010; Xia et al., 2011). Thus, Zn²⁺ release and intracellular ROS generation are important predictors of cytotoxicity associated with ZnO NPs exposure. In order to prevent ZnO NP-induced cytotoxicity, inhibition of ROS production must be addressed.

We previously reported that AA prevents pulmonary lipid peroxidation, inflammation, and injury in rat lung resulting from intratracheal administration of ZnO NPs (Fukui et al., 2012). In the present study, A549 cells exposed to ZnO NPs and treated with AA exhibited decreased intracellular ROS levels compared to cells not treated with AA. Furthermore, increases in ho-1 and il-8 gene expressions were also inhibited by AA treatment. These in vitro results are in agreement with those previously obtained in vivo.

There are two known AA membrane transporters, SVCT1 and SVCT2, in human lungs cells (Rivas et al., 2008; Savini et al., 2008; Tsukaguchi et al., 1999). In this study, AA was readily taken up by A549 cells. Since AA is widely known to be a radical-scavenging antioxidant and a zinc chelator (Tajmir-Riahi, 1991), this agent is believed to be an ideal candidate to prevent the oxidative stress triggered by ZnO NPs exposure.

The expression of metallothioneins depends on their binding to specific heavy metals (e.g. zinc, cadmium, copper, and mercury) (Kägi et al., 1984). For example, mt-2 gene expression is highly inducible by zinc (Karin and Richards, 1984). In the present study, mt-2 gene expression levels increased in cells exposed to ZnO NPs. This effect was markedly inhibited by AA co-treatment. These findings suggest that AA reduced intracellular levels of Zn²⁺ by inhibiting the release of Zn²⁺ from ZnO NPs, most likely via chelation. However, intracellular Zn²⁺ levels significantly increased upon treating the cells exposed to ZnO NPs with AA. With this result seemingly contradicting the above hypothesis, we next focused on investigating the reaction of AA with ZnO and Zn²⁺.

We considered two possible explanations for the observed increase in the intracellular Zn²⁺ level resulting from AA treatment: increased intracellular Zn²⁺ dissolution from ZnO NPs and facilitate intracellular Zn²⁺ uptake. We believed that if AA directly acts to promote Zn²⁺ dissolution from ZnO NPs inside cells, it would also react with ZnO in culture medium. Although AA is known to react with Zn²⁺, it is unclear whether AA directly reacts with ZnO. Wang et al. reported that AA increased the endocytosis processes of ZnO NPs, which resulted from the increase in concentration of Zn²⁺ in human gastric epithelial cells. Moreover, they reported that the acidity of AA facilitates the dissolution rate of the ZnO NPs in cells. Unquestionably, it becomes the predominating mechanism for the increase of Zn ions, hence aggravating cytotoxicity (Wang et al., 2014). To address this question, we confirmed that the absorbance of AA at 260 nm was unaffected when ZnO (0–1000 µM in PBS buffer, pH 7.0) was added. Further, the concentration of Zn²⁺ in ZnO DMEM-FBS dispersions with AA was unchanged after 6 h. These data suggest that AA does not react with ZnO NP dissolutions in the culture medium. Thus, the extracellular or cytoplasmic Zn²⁺ dissolution from ZnO NPs is most likely not augmented by AA. However, AA absorbance at 260 nm increased in accordance with Zn²⁺ concentration in PBS. Similarly, Maniyar et al. (2012) reported that the absorbance of AA in solution containing nickel sulfate at pH 7.0 and 7.4 was higher than the absorbance of AA alone. Thus, the absorbance of an AA–metal chelate complex is most likely more pronounced than that of unchelated AA at the culture medium pH. Our results therefore suggest the latter explanation of increased intracellular Zn²⁺ concentration to be the most likely that AA chelates extracellular Zn²⁺ released from ZnO NPs and the resulting Zn–AA complexes become incorporated into cells. Intracellular Zn²⁺ can also result from ZnO NPs becoming incorporated into cells and their subsequent release of ZnO. While intracellular Zn²⁺ is the source of ZnO NP-induced cytotoxicity, its deleterious effects decrease when AA forms Zn–AA complexes inside cells. However, the effect of the AA on cell death caused by ZnO NPs was not able to evaluate because influence of the Zn²⁺ was too large.

Zinc has an important role in antioxidant mechanisms in vivo. AA has been reported to enhance the uptake of Zn²⁺ during oxidative stress and suppress its uptake when Zn²⁺ deficiencies are detected (Agte et al., 2004). In this study, the cellular uptake of Zn²⁺ might be accelerated with the induction of oxidative stress caused by Zn²⁺. The chelation of Zn²⁺ with AA most likely inhibits oxidative stress and inflammation, regardless of the increase in intracellular uptake of Zn²⁺. Concentration of free Zinc is likely low in these cells.

The Zn–AA complexes accumulated in the in vitro situation, while these complexes may be eliminated from the lung in the in vivo situation. Metal chelate complexes are known to be mobilized into urine or feces by chelating agents in vivo (Andersen, 1989; Lihm et al., 2013).

Conclusion

In conclusion, the oxidative stress and inflammatory response induced by ZnO NPs were prevented by co-treatment with AA in human lung carcinoma A549 cells. These results are consistent with those previously reported from an in vivo model. Two possibilities can be considered regarding the reduced cytotoxicity effect of AA. One possibility is that intracellular ROS generated by ZnO NPs were removed by AA. Another possibility is that Zn²⁺ was trapped by AA as a metal-chelator antioxidant effect and/or the chelating effect may be involved in the ZnO NP-induced oxidative stress and inflammation reduction by AA. In order to ensure the role of AA as a metal chelator and antioxidant, it is necessary to evaluate the protective effect of other metal chelators and antioxidants. These findings contribute to our continued efforts toward the prevention and treatment of nanoparticle-induced toxicity.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was partially supported by the Long-range Research Initiative program organized by the Japan Chemical Industry Association.

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Ascorbic acid prevents zinc oxide nanoparticle–induced intracellular oxidative stress and inflammatory responses

Abstract

Keywords

Introduction

Materials and methods

ZnO NPs and chemicals

Cell culture

Preparation of the ZnO dispersion for in vitro evaluations

Measurement of intracellular ROS level

Real-time polymerase chain reaction

Evaluation of AA uptake in A549 cells

Measurement of intracellular Zn2+ levels

The reactivity of AA with ZnO and Zn2+

Statistical analysis

Results

AA decreased the cytotoxicity caused by ZnO NPs

AA uptake in A549 cells

Effect of AA on intracellular Zn2+ levels

Reactivity between AA and ZnO or Zn2+

Discussion

Conclusion

Footnotes

Declaration of Conflicting Interests

Funding

References

Measurement of intracellular Zn²⁺ levels

The reactivity of AA with ZnO and Zn²⁺

Effect of AA on intracellular Zn²⁺ levels

Reactivity between AA and ZnO or Zn²⁺