Concentration-dependent effects of reductive pulmonary inhalants on ultrafine particle-induced oxidative stress: Insights for health risk assessment

Abstract

The impact of reductive pulmonary inhalants on ultrafine particles (UFPs)-induced pulmonary oxidative stress remains a crucial consideration, yet the concentration-dependent effects of these inhalants have remained unexplored. Here we synthesized composite UFPs simulating atmospheric UFPs, primarily composed of metals and quinones. We subjected these UFPs to varying concentrations (0–7000 μM) of two reductive pulmonary inhalants, N-acetylcysteine and salbutamol, to assess their influence on oxidative potential, measured through the dithiothreitol assay (OP^DTT). Simultaneously, we analysed the soluble metal content of UFPs to uncover potential relationships between oxidative potential and metal solubility. Our results unveil a dual role played by these inhalants in shaping the OP^DTT of composite UFPs. Specifically, OP^DTT generally increased as inhalant concentrations rose from 0 to 300 μM. However, an intriguing reversal occurred when concentrations exceeded 500 μM, resulting in a decline in OP^DTT. Relative to untreated UFPs, these inhalants induced promotion and inhibition effects within concentration ranges of 100–500 and >1000 μM, respectively. While no significant correlation emerged between OP^DTT and soluble metal content as inhalant concentrations ranged from 0 to 7000 μM, noteworthy positive correlations emerged at lower inhalant concentrations (e.g., N-acetylcysteine at 0–300 μM). These findings provide insights into the potential influence of reductive pulmonary inhalants on health risks associated with UFP exposure, further underscoring the need for continued research in this critical area.

Graphical abstract

Highlights

• •Reductive inhalants dual-affect ultrafine particle (UFP)-induced oxidative stress.
• •Reductive inhalants at lower levels promote UFP-induced oxidative stress.
• •Reductive inhalants at higher levels inhibit UFP-induced oxidative stress.
• •UFP oxidative will mutually affects metal dissolution at certain inhalant levels.

1. Introduction

Exposure to particulate matter (PM) correlates with millions of annual premature deaths globally [1]. Ultrafine particles (UFPs) are an important PM fraction, defined by their smaller diameter (≤100 nm) but larger specific surface area than PM_2.5 or PM₁₀ [2]. Anthropogenic UFPs in the environment are typically generated as by-products of fossil fuel combustion, the condensation of semi-volatile substances, and industrial emissions [3]. The negligible mass but substantial contribution of UFPs to the total number of atmospheric particles has been the focus of interest in recent years [4], given the threat of UFPs to human health. Moreover, UFPs can readily absorb toxic pollutants and deeply penetrate the lungs, resulting in prolonged retention and chronic lung damage [3,5,6].

Oxidative stress is an important mechanism of UFP-induced lung disease [3]. The chemical composition of PM has been shown to influence reactive oxygen species (ROS) generation in vitro and in vivo [[7], [8], [9]]. Once the PM is deposited in the lungs, redox-active PM components may interact with each other or the physiological components in lung fluid to generate ROS [10]. Redox-active PM components [11,12], such as polycyclic aromatic hydrocarbons (PAHs), PAH derivatives (e.g., quinones), and metals, have also been observed in UFPs and may contribute to the toxic effects of oxidative stress in lungs.

Antioxidant therapy to counteract oxidative stress has been investigated in the context of nutrition and epidemiology [13]. In treating respiratory disease, reductive pulmonary inhalants in lung fluid may act as antioxidants and reduce the pulmonary oxidative stress induced by PM [[14], [15], [16]]. In both in-vitro cellular and in-vivo murine models, Wang et al. [15] found that N-acetylcysteine (NAC), a commonly used inhalant, reduced ROS levels and the PM-induced inflammatory response. Lin et al. [16] conducted a murine model experiment in which NAC was similarly shown to decrease PM-mediated oxidative stress. However, there was no consistent conclusion regarding the effects of the reductive substances in lung fluid on PM-induced oxidative stress. Studies based on in-vitro acellular assays showed increased ROS generation mediated by redox-active PM components with the reductant (e.g., vitamin C) depleting in lung fluid [17,18]. The enhancement of oxidative stress by some antioxidants was reported by Childs et al. [19], who found that supplements of vitamin C (12.5 mg kg⁻¹) and NAC (10 mg kg⁻¹) resulted in a significant increase in the oxidative stress of healthy individuals after acute exercise. Another study suggested that the antioxidant concentration is an important determinant of antioxidant or pro-oxidant effects [20], as it influences the redox potential of the systems involved in ROS generation [[21], [22], [23]]. Accordingly, the concentration of reductive pulmonary inhalants in lung fluid may determine the level of lung oxidative stress induced by UFPs. However, this hypothesis has not been tested, although it has important implications regarding knowing pulmonary inhalants affect UFP-induced oxidative stress in the lung.

Both quinones and metals contribute to the toxicity of anthropogenic UFPs [24]. UFPs generated by coal combustion can become enriched in metals such as Na, Fe, and Al [25], which, together with carbonaceous materials, increase their toxicity and reactivity. Hence, in the present study, composite UFPs, mainly consisting of metals and quinones as prepared in our recent work [26], were used to investigate the concentration-dependent effects of reductive pulmonary inhalants on UFP-induced oxidative stress in the lung. In-vitro acellular dithiothreitol (DTT) assays were conducted to measure the change in the oxidative potential (OP) of UFPs in response to changes in the concentrations of the reductive pulmonary inhalants. The correlation between the OP and the corresponding soluble metal concentration at the studied inhalant concentration range was then determined. Our findings provide further insights into UFP-induced pulmonary toxicity and the therapeutic safety of reductive pulmonary inhalants.

2. Materials and methods

2.1. Composite UFP preparation

Composite UFP samples were selected from the artificial UFPs prepared in our recent work [26] and shown to have a relatively high susceptibility to the effects of reducing agents in lung fluid. Briefly, the UFP samples were prepared by absorbing 1,4-naphthoquinone (NQ) or anthraquinone (AQ) on commercial nanoparticles of CuO, Fe₂O₃, and MnO₂ (nCuO, nFe₂O₃, and nMnO₂, respectively). The characteristics of the nCuO, nFe₂O₃, and nMnO₂ are listed in Table 1. The infrared absorption spectra of the composite UFP samples resembled those of NQ and AQ, indicating the successful absorption of these compounds onto nCuO, nFe₂O₃, and nMnO₂ (Fig. 1). The composite UFP samples consisting of nCuO, nFe₂O₃, and nMnO₂ with NQ and AQ are referred to in the following sections as UFP_Cu/NQ, UFP_Fe/NQ, UFP_Mn/NQ, UFP_Cu/AQ, UFP_Fe/AQ, and UFP_Mn/AQ, respectively.

Table 1.

Physiochemical properties of the nanoparticles of CuO, Fe₂O₃, and MnO₂.

No.	Particles	Componentsa	Average particle size (nm)b	Specific surface area (m2 g−1)c
1	nCuO	100% CuO	20.0	1.55
2	nFe2O3	100% Fe2O3	60.6	5.75
3	nMnO2	Mainly MnO2, subordinately quartz and birnessite, rarely goethite	30.5	15.7

^aComponents are analysed by X-ray diffraction.

^bAverage particle size is calculated by the Scherrer equation based on X-ray diffraction analysis.

^cSpecific surface area is analysed according to the BET method.

Fig. 1.

Fourier Transform Infrared (FTIR) spectra of composite ultrafine particles (UFPs) of nCuO, nFe₂O₃, and nMnO₂ with NQ (a) or AQ (b). In panel a, composite UFPs of nCuO, nFe₂O₃, and nMnO₂ with NQ were represented as UFP_Cu/NQ, UFP_Fe/NQ, and UFP_Mn/NQ, respectively. In panel b, composite UFPs of nCuO, nFe₂O₃, and nMnO₂ with AQ were represented as UFP_Cu/AQ, UFP_Fe/AQ, and UFP_Mn/AQ, respectively.

2.2. Physiological buffer and reductive pulmonary inhalants

Phosphate-buffered saline (PBS) ( ×1; pH 7.4) was used as the physiological buffer. The studied reductive pulmonary inhalants were NAC and salbutamol (ALB), both employed in clinical settings. NAC and ALB were diluted with PBS to obtain final concentrations of 0, 100, 200, 300, 500, 1000, 2000, 3000, 5000, and 7000 μM to test their effects on the OP of UFPs in lung fluid.

2.3. UFP extraction

For each treatment, 20 mg of a UFP sample was resuspended in 20 mL of PBS containing different concentrations (0–7000 μM) of reductive pulmonary inhalant (ALB or NAC) and incubated for 4 h in a thermostatic box at 37 °C on a reciprocating shaker (SHA-B; Saidelisi, Shanghai, China). The suspensions were then filtered through a 0.22-μm cellulose microporous membrane; the filtrates were used for DTT assay and dissolved metal analysis. Each treatment was tested in triplicate.

2.4. Chemical analysis

2.4.1. DTT assay

In this study, the OP of UFPs was measured using the DTT assay, a widely used acellular method sensitive to metals and quinones [27]. The analytical procedures were modified from previous studies [27,28]. Briefly, 50–100 μL of the filtrate was incubated at 37 °C with 1.0 mL of 0.1 M potassium phosphate and 100 μL of 0.5 mM DTT for 0, 5, 10, 15, or 20 min, after which 100 μL of 1.0 mM 5,5′-dithiobis-(2-nitrobenzoic acid) was added. The yellow reaction products were detected using an ultramicro ultraviolet–visible (UV–vis) spectrophotometer (NanoDrop One; Thermo Fisher Scientific, Waltham, MA, USA) at 412 nm. The OP of each UFP sample was expressed as the DTT consumption rate (OP^DTT), determined from the linear regression slope of the data points, and normalized to the mass of the UFPs. The R² values derived from linear regression analysis were all >0.9. Blank controls were included with each batch.

2.4.2. Metal analysis

Metals dissolved from UFPs into extracts were determined using inductively coupled plasma-mass spectrometry (Agilent 780; Agilent Technologies, Santa Clara, CA, USA). The detection limit was 0.02–0.22 μg L⁻¹, and the recovery rate was 85–105%.

2.5. Statistical analysis

The data are presented as the mean ± standard deviation. A two-way analysis of variance and Tukey's multiple comparison tests were performed using GraphPad Prism 8.0 software (GraphPad Software Inc., San Diego, CA, USA). Statistical differences were defined at p < 0.05, p < 0.01, and p < 0.001. Associations among quantitative variables were assessed based on Pearson's product moment correlation coefficient (r), calculated using Microsoft Office Excel 2016 (Microsoft Corp., Redmond, WA, USA). Illustrations were drawn using GraphPad Prism 8.0 and Adobe Illustrator CC 2015.3 (Adobe Systems Inc., San Jose, CA, USA).

3. Results

3.1. OP^DTT of composite UFPs of nCuO with quinones (UFP_Cu)

In general, the OP^DTT of UFP_Cu increased, either significantly or non-significantly, at lower concentrations and decreased at higher concentrations (>500 μM) of reductive pulmonary inhalants (Fig. 2). For example, the OP^DTT of UFP_Cu/NQ increased significantly at ALB concentrations of 200–300 μM (p < 0.01), whereas a non-significant increase was measured at NAC concentrations of 0–500 μM (p > 0.05) (Fig. 2a); the increase in OP^DTT of UFP_Cu/AQ was not significant at NAC and ALB concentrations of 0–200 μM (p > 0.05) (Fig. 2b).

Fig. 2.

OP^DTT of the UFP_Cu/NQ (a) and UFP_Cu/AQ (b) in physiological buffer with different concentrations of reductive pulmonary inhalants. OP^DTT: oxidative potential analysed by the dithiothreitol assay; UFP_Cu/NQ and UFP_Cu/AQ: the composite UFPs of nCuO with 1,4-NQ and AQ, respectively; NAC: N-acetylcysteine; ALB: salbutamol.

Compared with treatments consisting of UFP_Cu/NQ without added reductive pulmonary inhalants, the OP^DTT was significantly higher in treatments containing 300 or 500 μM ALB (p < 0.001) (Fig. 2a). For UFP_Cu/AQ, the OP^DTT increased following the addition of NAC or ALB at a concentration of 100 μM (p < 0.05) and 200 μM (p < 0.001) (Fig. 2b). By contrast, reductive pulmonary inhalant concentrations >1000 μM caused a decrease in the OP^DTT of UFP_Cu.

3.2. OP^DTT of composite UFPs of nFe₂O₃ with quinones (UFP_Fe)

When the concentration of NAC and ALB was raised from 200 to 300 μM, the OP^DTT of UFP_Fe/NQ increased significantly, from 3.39 ± 0.47 to 6.17 ± 0.66 pmol min⁻¹ μg⁻¹ (p < 0.001) and from 4.71 ± 0.37 to 6.44 ± 0.63 pmol min⁻¹ μg⁻¹ (p < 0.01), respectively (Fig. 3a). By contrast, the OP^DTT decreased significantly at NAC and ALB concentrations >300 μM (p < 0.05) and 500 μM (p < 0.001), respectively. For UFP_Fe/AQ, the increase in the OP^DTT was not significant (p > 0.05) at NAC and ALB concentrations of 100–500 μM and 300–500 μM, respectively (Fig. 3b). However, the OP^DTT decreased significantly when the reductive pulmonary inhalant concentration was >500 μM (p < 0.01).

Fig. 3.

OP^DTT of the UFP_Fe/NQ (a) and UFP_Fe/AQ (b) in physiological buffer with different concentrations of reductive pulmonary inhalants. OP^DTT: oxidative potential analysed by the dithiothreitol assay; UFP_Fe/NQ and UFP_Fe/AQ: the composite UFPs of nFe₂O₃ with 1,4-NQ and AQ, respectively; NAC: N-acetylcysteine; ALB: salbutamol.

Compared with untreated UFP_Fe/NQ, the OP^DTT was enhanced when the particles were incubated with NAC or ALB at concentrations of 300–500 μM but reduced at concentrations >1000 μM (Fig. 3a). For UFP_Fe/AQ, the addition of 0–7000 μM of either NAC or ALB inhibited their OP^DTT (Fig. 3b).

3.3. OP^DTT of composite UFPs of nMnO₂ with quinones (UFP_Mn)

The OP^DTT of UFP_Mn/NQ and UFP_Mn/AQ increased significantly when the NAC and ALB concentration increased from 0 to 100 μM (p > 0.05) (Fig. 4), whereas the OP^DTT of UFP_Mn/NQ decreased significantly at NAC and ALB concentrations >500 μM and >200 μM, respectively (p < 0.001) (Fig. 4a). The OP^DTT of UFP_Mn/AQ also decreased significantly (p < 0.05) at NAC and ALB concentrations of <300 μM and <200 μM, respectively (Fig. 4b).

Fig. 4.

OP^DTT of the UFP_Mn/NQ (a) and UFP_Mn/AQ (b) in physiological buffer with different concentrations of reductive pulmonary inhalants. OP^DTT: oxidative potential analysed by the dithiothreitol assay; UFP_Mn/NQ and UFP_Mn/AQ: the composite UFPs of nMnO₂ with 1,4-NQ and AQ, respectively; NAC: N-acetylcysteine; ALB: salbutamol.

For both UFP_Mn/NQ and UFP_Mn/AQ, compared with the untreated controls, the OP^DTT increased in response to NAC concentrations <2000 μM and decreased at NAC concentrations >2000 μM (Fig. 4). Similarly, the OP^DTT of UFP_Mn/NQ and UFP_Mn/AQ increased in response to ALB at concentrations <500 μM (Fig. 4a) and <300 μM (Fig. 4b), respectively, but for both UFPs the OP^DTT was inhibited by higher ALB concentrations.

3.4. Relationship between OP^DTT and the soluble metal concentration of composite UFPs

The soluble metal concentrations of the composite UFPs at different concentrations of NAC and ALB (0–7000 μM) are reported in Table 2. Correlation analyses were performed between the OP^DTT and the soluble metal concentration of each composite UFP treated with NAC or ALB in the studied concentration range (0–7000 μM) (Table 3). Overall, with the NAC or ALB varied at 0–7000 μM, there was no significant correlation between the OP^DTT and the soluble metal concentration of the composite UFPs (p > 0.05). However, for some composite UFPs, a significantly positive correlation was determined between the OP^DTT and the soluble metal concentration when the OP^DTT increased in response to an increase in the ALB concentration. Specifically, the OP^DTT of UFP_Cu/NQ was significantly positively correlated with the soluble Cu concentration as the ALB concentration increased from 0 to 500 μM (r = 0.8932, p < 0.05). Similarly, a correlation between the OP^DTT and the soluble Mn concentration of UFP_Mn/NQ was positive notably as the ALB concentration varied from 0 to 300 μM (r = 0.9970, p < 0.01). However, there was no significant relationship between the OP^DTT and the soluble metal concentration of any of the studied composite UFPs when the OP^DTT decreased in response to further increases in the concentration of either reductive pulmonary inhalant (p > 0.05).

Table 2.

Soluble metal concentrations of UFPs at different concentrations of N-acetylcysteine (NAC) and salbutamol (ALB) (μM).^a

Reductive pulmonary inhalants	Concentration (μM)	UFPCu/NQ		UFPCu/AQ		UFPFe/NQ		UFPFe/AQ		UFPMn/NQ		UFPMn/AQ
		AVE	SD	AVE	SD	AVE	SD	AVE	SD	AVE	SD	AVE	SD
NAC	0	181.19	3.06	225.25	6.42	306.07	4.91	258.57	18.29	1152.26	146.51	1313.93	308.78
	100	226.20	33.87	340.23	46.19	284.47	22.66	231.37	17.19	1409.63	81.49	1376.33	192.47
	200	246.97	17.99	355.28	5.56	297.53	10.55	244.13	21.76	1586.21	200.75	1306.69	212.67
	300	252.97	11.84	312.43	54.51	354.40	17.60	243.57	7.80	1760.93	173.25	1329.57	98.83
	500	256.63	6.05	338.83	13.54	364.27	11.02	230.57	19.93	1800.87	96.44	1586.23	55.77
	1000	251.27	10.38	342.47	12.23	372.67	8.49	229.90	2.16	1806.43	91.42	1448.27	65.97
	2000	241.50	17.56	360.67	8.99	381.87	8.92	248.27	12.17	1894.90	34.30	1256.90	46.80
	3000	238.73	17.56	-b	-b	366.83	15.40	-b	-b	1693.13	124.14	1296.37	101.19
	5000	-b	-b	-b	-b	366.00	25.83	-b	-b	1738.73	85.06	1287.13	25.71
	7000	-b	-b	-b	-b	362.30	7.88	-b	-b	1741.33	53.17	-b	-b
ALB	0	181.19	6.42	125.25	6.42	256.07	4.91	258.57	18.29	1018.92	47.41	1480.59	85.60
	100	187.03	6.15	166.47	4.82	244.62	14.58	233.24	26.93	1216.51	136.54	1801.82	26.33
	200	225.92	9.34	294.63	7.99	212.09	17.83	244.08	37.28	1261.22	63.07	1772.46	213.25
	300	242.47	12.44	301.70	8.36	228.70	17.49	224.07	8.09	1360.87	54.11	1924.77	117.81
	500	264.73	14.03	299.70	7.23	231.57	20.13	236.90	17.27	1393.57	14.01	1980.90	42.69
	1000	251.10	8.25	-b	-b	205.50	6.90	218.77	10.97	1298.80	49.30	1859.07	53.91
	2000	260.67	4.33	-b	-b	207.93	8.20	-b	-b	1345.83	67.74	1894.57	51.32
	3000	253.83	6.79	-b	-b	216.73	6.73	-b	-b	1341.57	53.10	1965.63	58.03
	5000	249.00	4.83	-b	-b	207.33	10.27	-b	-b	1176.40	110.34	2006.27	52.70
	7000	-b	-b	-b	-b	178.00	3.83	-b	-b	1295.83	6.63	-b	-b

^aAnalysing the concentration of the dissolved Cu, Fe, and Mn from the UFP_Cu, UFP_Fe, and UFP_Mn, respectively, and the N-acetylcysteine and salbutamol were the studied reductive pulmonary inhalants.

^bNot analysed due to the not detectably corresponding OP^DTT.

Table 3.

Pearson's product moment correlation coefficients (r) between OP^DTT and soluble metal concentration of UFPs.^a

No.	UFP type	N-acetylcysteine	Salbutamol
1	UFPCu/NQ	0.0047	0.2999
2	UFPCu/AQ	−0.0848	−0.1244
3	UFPFe/NQ	−0.0071	0.5782
4	UFPFe/AQ	0.2980	0.7485
5	UFPMn/NQ	0.0203	−0.0069
6	UFPMn/AQ	0.3985	−0.5918

^aCorrelation analysis was performed between the OP^DTT and the soluble metal concentration of each composite UFP in the studied concentration range of N-acetylcysteine (NAC) and salbutamol (ALB) of 0–7000 μM.

4. Discussion

Knowledge concerning the effect of reductive agents in lung fluid on PM-induced oxidative stress is inconclusive. While the reductive pulmonary inhalant concentration plays an important role in determining PM-induced ROS levels in the lung, to our knowledge, the concentration-dependent effects of the reductive pulmonary inhalants in lung fluid on the OP of the UFPs remained unknown. This study demonstrated the dual effects of two reductive pulmonary inhalants (NAC and ALB) on the OP^DTT of composite UFPs as the concentrations of those agents increased from 0 to 7000 μM. Specifically, the OP^DTT of some composite UFPs increased at lower NAC and ALB concentrations, especially in the range of 0–300 μM. However, at NAC and ALB concentrations >500 μM, the OP^DTT of most composites decreased. Correspondingly, the reductive pulmonary inhalants exhibited stimulatory effects at concentrations of 100–500 μM but inhibitory effects at concentrations >1000 μM.

Various PAHs and quinones have been identified in UFPs and served as important contributors to PM-induced ROS generation and oxidative stress [11,12]. The potential mechanisms may involve enzymatic conversion and redox cycling of these redox-active compounds. For example, the oxidative damage of PAHs has been partly attributed to their metabolic process by cytochrome P450 in lung tissues. Specifically, it is generally believed that cytochrome P450 metabolism converts PAHs to their oxidized form, such as quinones [29]. These converted and original quinones in UFPs can generate O₂•⁻ by redox cycling of quinoids and their semiquinone radicals (reactions (1)–(4)) [30,31]. The subsequent production of H₂O₂ in aqueous environment in the presence of O₂ (reaction (5)) and HO• generation catalyzed by the redox-active metal elements bound to PM through Fenton or Fenton-like reactions (reaction (6)) [3,32]. In addition, reducing substances in lung fluid may promote ROS generation during quinone redox cycling by donating electrons (reactions (1)–(3)) and by maintaining Fenton or Fenton-like reactions by reducing oxidized metal elements (reaction (7)) [18,31,33]. However, reducing substances that lose their electrons may behave as free radicals and thus exert pro-oxidant effects [34]. For example, the transformation of ascorbic acid into ascorbyl radicals and of β-carotene into carotenoid radicals in the presence of oxygen and ferrous ions has been reported [18,35,36]. Thus, the sulfhydryl groups (-SH) in NAC and the phenolic hydroxyl groups (Ph-OH) in ALB may be transformed into sulfhydryl radicals (-S·) [37] and phenolic hydroxyl radicals (Ph-O⋅) [38], respectively. Similar reactions would link the increased OP^DTT of UFPs to the generation of ROS and other radicals in the presence of low concentrations of reductive pulmonary inhalants, whereas high concentrations would lower the OP^DTT of UFPs via the gradual neutralization of ROS (reactions (8)–(9)) [34].

Q + e⁻ + H⁺ → QH• (1)

QH• + e⁻ + H⁺ → QH₂ (2)

QH₂ + O₂ → QH• + O₂•⁻ + H⁺

or QH₂ + O₂ → Q•⁻ + O₂•⁻ + 2H⁺ (3)

Q•⁻ + O₂ ⇋ Q + O₂•⁻ (4)

O₂•⁻ + 2H⁺ → H₂O₂ + O₂ (5)

H₂O₂ + Meⁿ⁺ → Meⁿ⁺¹ + OH⁻ + HO• (6)

Meⁿ⁺¹ + e⁻ → Meⁿ⁺ (7)

HO• + e⁻ → OH⁻ (8)

O₂•⁻ + 4H⁺ + 3e⁻ → 2H₂O (9)

The soluble metal fraction of PM is widely considered to be the main contributor to PM-induced ROS generation, with metal dissolution from PM, in turn, enhancing ROS generation [18,27,39]. Strong positive correlations were determined between the soluble metal fraction and the OP of PM [40,41]. In the present study, a correlation analysis was performed to estimate the relationship between the OP^DTT of the composite UFPs and their soluble metal concentration. As the NAC and ALB concentrations increased from 0 to 7000 μM, there was no significant correlation between the OP^DTT and the soluble metal concentration. Interestingly, as the OP^DTT increased in response to an increasing ALB concentration, the correlations for some composite UFPs became significantly positive, indicating that within this ALB concentration range, the soluble metal concentration was an important contributor to the OP^DTT and that metal release was enhanced by ROS generation. However, at relatively high concentrations of NAC and ALB, the soluble metal concentration remained constant and correlated weakly with the OP^DTT. This result can be partially attributed to the inhibition of ROS generation by the high concentrations of the reductive pulmonary inhalants.

To a certain degree, our findings demonstrate the importance of the reductive pulmonary inhalant concentration in affecting the oxidative stress induced by UFPs in lung fluid. At lower doses, reductive pulmonary inhalants may not exert antioxidant effects; instead, they could potentially exacerbate the oxidative stress instigated by UFPs, unless administered at higher concentrations. A previous model-based analysis by Lakey et al. [42] also indicated that PM-induced oxidative stress in the lung was potentially increased by lower concentrations of antioxidants in lung fluid (e.g., 40 μM ascorbate, 207 μM uric acid, 109 μM glutathione, and 0.7 μM α-tocopherol). Nutritional studies have likewise shown that antioxidant concentration is an essential determinant of the anti- or pro-oxidant effects of these agents [20,21]. Consequently, it is imperative to consider both the concentration and type of reductive pulmonary inhalants and their combined impact when designing respiratory disease therapies to mitigate UFP-induced oxidative stress. Failing to do so may inadvertently lead to detrimental effects stemming from the treatment.

5. Conclusion

In this study, an increase in the OP^DTT of composite UFPs was observed at lower concentrations of reductive pulmonary inhalant and a decrease at higher concentrations. Moreover, at lower concentration ranges, a significant positive correlation emerged between the OP^DTT of the composite UFPs and their soluble metal concentration. This correlation implied a potential contribution of the soluble metal fraction to the OP^DTT and a possible promotion of further metal release via ROS generation. To a certain extent, our findings can help clarify the concentration-dependent effects of reductive pulmonary inhalants on UFP-induced oxidative stress. They also highlight the importance of proper dosing of reductive pulmonary inhalants, either alone or as a combination therapy, to avoid inadvertent harmful effects and decrease the risk of UFPs deposited in the lung.

Moreover, recognizing the variability in UFP exposure levels to humans in the actual environment, our future investigations will incorporate diverse ratios of UFPs to lung fluid. This approach aims to simulate the actual UFP exposure level to humans in different situations, thereby yielding more comprehensive insights into the concentration-dependent effects of reductive pulmonary inhalants on UFP-induced oxidative stress.

CRediT authorship contribution statement

Ya-Qi Yu: Investigation, Methodology, Data Curation, Formal Analysis, Visualization, Funding Acquisition, Writing - Original Draft. Tong Zhu: Conceptualization, Resources, Project Administration, Supervision, Funding Acquisition, Writing - Review & Editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.