A fluorescent probe for sulfur dioxide reveals intracellular reductive stress triggered by natural antioxidants

Abstract

Sulfur dioxide (SO₂), a pivotal gasotransmitter, is intricately linked to cellular redox homeostasis. Here, we developed the red-emissive DCM-MQE probe for rapid and sensitive detection of intracellular SO₂. Using this probe, we have demonstrated that natural antioxidants significantly elevate SO₂ levels in both cells and tumor-bearing mice, and confirmed that antioxidants modulate SO₂-mediated redox homeostasis through induction of reductive stress.

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Cellular redox homeostasis, maintained through the precise balance of oxidants and reductants, is fundamental to biological function. This equilibrium is primarily governed by the glutathione (GSH/GSSG) redox couple, which orchestrates electron transfer reactions critical for signaling, metabolism, and defence against oxidative damage.^1,2 Disruption of this balance—particularly an excessive reductive environment termed reductive stress—is increasingly linked to pathologies including cancer, inflammation, metabolic disorders, and neurodegenerative diseases. Unlike oxidative stress, reductive stress arises from an overabundance of reducing equivalents (e.g., elevated NAD(P)H, thiols and sulfides), leading to aberrant redox signaling and cellular dysfunction.^3–6 Understanding the molecular mechanisms driving reductive stress is thus essential for unravelling redox biology and developing targeted therapies for redox-related diseases.

Among reductive sulfur species, sulfur dioxide (SO₂) and its derivatives (sulfite/bisulfite, HSO₃⁻/SO₃²⁻) occupy a unique niche. Despite sulfur's higher oxidation state and weaker reducing capacity compared to H₂S or thiols, SO₂ plays pivotal roles in mammalian physiology.^7,8 Endogenous SO₂ production in mammals occurs via a defined enzymatic cascade: L-cysteine is oxidized by cysteine dioxygenase (CDO) to L-cysteinesulfinate, transaminated to β-sulfinylpyruvate by aminotransferase, and finally decomposed to pyruvate and SO₂.^9–11 Critically, reductive stress induced by natural antioxidants elevates cellular thiol levels, which may perturb SO₂ metabolism—yet the causal relationship remains poorly defined.^12–14 This gap stems from a lack of tools capable of specifically, sensitively, and dynamically monitoring SO₂ fluctuations in living systems. Existing probes often suffer from interference by reactive sulfur species (RSS) like H₂S, inadequate selectivity, or limited applicability in vivo, hindering mechanistic studies of antioxidant-driven reductive stress^15–21

Herein, we address this challenge by designing DCM-MQE (Scheme 1), a red-emitting fluorescent probe for SO₂ with exceptional specificity and biocompatibility. DCM-MQE exhibits a >100-fold fluorescence enhancement at 660 nm upon SO₂ addition, enabling high-contrast imaging with minimal background. Its response resolves SO₂ from biological interferents (including H₂S and cellular RSS). Leveraging this probe, we demonstrate for the first time that natural antioxidants (ellagic acid, quercetin and myricetin) from traditional Chinese medicine (TCM) induce reductive stress in tumor cells by elevating endogenous SO₂ levels. By monitoring SO₂ dynamics in live HeLa cells and in vivo models, we establish a direct link between antioxidant exposure, SO₂ upregulation, and reductive stress in cancer (Scheme 1). This work not only provides a transformative tool for redox biology but also unveils SO₂ as a key mediator in antioxidant-driven tumor modulation, opening avenues for novel redox-targeted anticancer strategies.

Scheme 1 Molecular mechanism of SO₂ detection and the antioxidant-induced reductive stress pathway. (A) Molecular structure and reaction mechanism of DCM-MQE with SO₂. (B) Pathway of natural antioxidant-induced reductive stress and SO₂ elevation. Natural antioxidants (ellagic acid, quercetin, and myricetin) enter cells and disrupt redox homeostasis, inducing reductive stress. This stress upregulates cellular thiol levels (e.g., glutathione, cysteine). Elevated thiols promote the conversion of sulfur-containing amino acids (e.g., cysteine) to SO₂ derivatives through enzymatic processes. The resulting SO₂ is detected by DCM-MQE, which generates a fluorescence signal proportional to SO₂ concentration.

Dicyanomethylene-4H-pyran (DCM) derivatives represent a class of classic donor–π–acceptor (D–π–A) fluorophores exhibiting red emission and large Stokes shifts. They are a universal scaffold to construct activity-based sensing probes by regulating the intramolecular charge transfer (ICT) process between the donor and acceptor of the DCM core, triggering obvious fluorogenic signals.^22,23 To enable selective detection of SO₂ and its derivatives, we designed the probe DCM-MQE. It is readily synthesized via a two-step reaction (Scheme S1) and characterized by ¹H NMR, ¹³C NMR, and MS (Fig. S1 and S2). Through a sulfite-promoted addition reaction, the quinoline moiety can be transformed into an electron-rich enamine moiety, hence resulting in a favourable D–π–A structure with a typical ICT effect.^24–26

UV-vis absorption spectroscopy revealed that upon SO₂ addition, the probe's maximum absorption band red-shifted from 365 nm to 561 nm, accompanied by a distinct color change from yellow to purple (Fig. 1A and Fig. S3, S4). In contrast to the non-fluorescent DCM-MQE, a bright red fluorescence was observed in the presence of Na₂SO₃ (Fig. 1B and Fig. S3). Notably, treatment with 10 µM Na₂SO₃ resulted in a 100-fold increase in emission intensity at 661 nm (λ_ex = 561 nm), demonstrating that DCM-MQE functions as a NIR “turn-on” probe for Na₂SO₃ with high contrast and a significant Stokes shift. Sulfite reduces the methylquinoline moiety of DCM-MQE, converting it into a typical D–π–A structure, which likely accounts for the observed hyperchromic effect and fluorescence enhancement.

Fig. 1 (A) UV-Vis absorption and (B) fluorescence emission (λ_ex = 561 nm) spectra of DCM-MQE before and after reaction with sodium sulfite. (C) and (D) Concentration-dependent fluorescence response curve of DCM-MQE to Na₂SO₃ (0–1 µM). Error bars represent standard deviations from triplicate experiments.

DCM-MQE exhibits an ultrafast reaction rate with SO₂, reaching maximum fluorescence at 661 nm within <3 s (Fig. S5). As shown in Fig. 1C and D, the emission intensity ratio (F/F₀) shows excellent linearity with Na₂SO₃ concentration (0–1 µM), where F and F₀ represent the fluorescence intensities at 661 nm with and without Na₂SO₃, respectively. The limit of detection (LOD) was calculated to be 6 nM using the 3σ/k method,²⁷ significantly below cellular SO₂ thresholds.

Selectivity assays (Fig. S6) against biologically relevant species including K⁺, Na⁺, Ca²⁺, NO₂⁻, CO₃²⁻, acetate anion (AcO⁻), ClO⁻, H₂O₂, dithiothreitol (DTT), vitamin C (VC), NADH, NADPH, cysteine (Cys), and glutathione (GSH), SO₄²⁻, S₂₋, SO₃²⁻ revealed minimal interference. Only Na₂SO₃ induced a significant fluorescence enhancement and color change, while the others caused negligible fluorescence increase. Such specific recognition is actually dependent on the ultrafast fluorogenicity of DCM-MQE for SO₂, thereby enabling it to be differentiated from other nucleophilic reactive species and reductants via drastic distinction in response kinetics.^28,29 Competition experiments confirmed minimal interference from other species, enabling specific Na₂SO₃ detection in complex environments.

Given its robust response to Na₂SO₃in vitro, we evaluated DCM-MQE for imaging Na₂SO₃ in live cells. MTT assays confirmed negligible cytotoxicity below 10 µM (Fig. S7). Confocal imaging in HeLa cells (Fig. 2A and B) showed no fluorescence after treatment with 5 µM DCM-MQE alone, and a rapid, dose-dependent fluorescence enhancement upon incubation with Na₂SO₃ (10 min), intensifying with increasing concentrations (0–500 µM). It demonstrates excellent cell permeability and fast intracellular response kinetics (completed within ∼10 min), enabling real-time Na₂SO₃ imaging in live cells (Fig. 2C and D).

Fig. 2 (A) Concentration-dependent confocal fluorescence images and (B) quantitative analysis of the fluorescence intensity of HeLa cells. (C) Time-dependent confocal fluorescence images and (D) quantitative analysis of the fluorescence intensity of HeLa cells. Cells were pre-incubated with DCM-MQE for 1 h, followed by treatment with Na₂SO₃ (0–500 µM) for 30 min (A) and (B) or Na₂SO₃ (100 µM) for 0–15 min. Scale bar: 20 µm. Error bars represent standard deviations from triplicate experiments.

After confirming the high sensitivity and selectivity of DCM-MQE for Na₂SO₃, we employed this probe to monitor SO₂ levels in HeLa cells during treatment with natural antioxidants (ellagic acid, quercetin, and myricetin). Notably, these antioxidants themselves exhibited negligible interference with the fluorescence signal of DCM-MQE, ensuring that the observed changes in fluorescence were solely attributable to SO₂ generation (Fig. S8). Treatment with ellagic acid or quercetin (0–2 µg mL⁻¹, 6 h) induced a concentration-dependent increase in fluorescence, which declined at 5 µg mL⁻¹. Similarly, myricetin (0–5 µg mL⁻¹, 6 h) enhanced fluorescence, but this effect diminished at 10 µg mL⁻¹ (Fig. 3 and Fig. S9–S13). Further elevation of antioxidant concentrations led to decreased fluorescence, which may be attributed to the inhibition of sulfhydryl-containing amino acid oxidation processes, resulting in reduced SO₂ generation (Fig. S9–S13).

Fig. 3 Confocal fluorescence images and quantitative fluorescence intensity analysis of HeLa cells treated with natural antioxidants. Cells were pre-incubated with natural antioxidants for 6 h, followed by treatment with DCM-MQE for 1 h. Scale bar: 20 µm. Error bars represent standard deviations from triplicate experiments.

To validate the mechanism, we used HDX – an inhibitor of aspartate aminotransferase³⁰ that blocks SO₂ generation from L-cysteinesulfinate – which significantly reduced the fluorescence compared to antioxidant-treated groups. Collectively, these results demonstrate that ellagic acid, quercetin, and myricetin induce reductive stress in HeLa cells, elevating intracellular thiol levels that undergo subsequent oxidation to generate SO₂ (Fig. 3).

To further validate natural antioxidant-induced reductive stress in vivo, we conducted comprehensive experiments in tumor-bearing mice using DCM-MQE for SO₂ imaging (Fig. S14 and S15). First, we established the probe's ability to detect both endogenous and exogenous SO₂ derivatives at tumor sites. Mice were divided into three groups: (1) Control (PBS injection), (2) HDX inhibitor (HDX pre-injection), and (3) Sulfite group (Na₂SO₃ pre-injection). Fluorescence intensity was weaker in the HDX group than the control but stronger in the Na₂SO₃ group, diminishing over time—confirming DCM-MQE's ability to image SO₂ derivatives in vivo.

Next, we evaluated SO₂ induction by natural antioxidants. Tumor-bearing mice received subcutaneous injections of ellagic acid, quercetin, or myricetin (5 µg mL⁻¹) for 5 h, followed by DCM-MQE administration. Fluorescence imaging revealed that all antioxidant-treated groups displayed markedly stronger signals than the HDX inhibitor group (Fig. 4). This enhancement, consistent across ellagic acid, quercetin, and myricetin treatments, directly demonstrated elevated SO₂ generation in HeLa tumors during antioxidant exposure. Collectively, these in vivo results confirm that DCM-MQE effectively images SO₂ dynamics and that natural antioxidants trigger SO₂ production through reductive stress pathways in tumors. Notably, all HDX-supplemented control groups exhibited reduced fluorescence, indicating that in vivo, antioxidants upregulate SO₂ by similarly enhancing biothiol levels, with SO₂ generation resulting from their oxidation (Fig. 4).

Fig. 4 In vivo fluorescence imaging of antioxidant-induced SO₂ dynamics in HeLa tumor-bearing mice. (A) Time-dependent fluorescence imaging of mice. HeLa tumor-bearing mice were intravenously injected with a natural antioxidant (ellagic acid, quercetin and myricetin). After 5 h, the experimental group received PBS, while the control group received inhibitor HDX. After 30 min, DCM-MQE was intravenously injected, and whole-animal fluorescence images were acquired. (B) and (D) Quantitative analysis of fluorescence intensity of tumor treated with ellagic acid (B), quercetin (C), and myricetin (D). Error bars represent standard deviations from triplicate experiments.

Sulfur dioxide is an important gaseous signaling molecule closely associated with cellular redox homeostasis. In this work, we developed a red-emitting DCM-MQE probe for rapid and sensitive detection of intracellular SO₂. Utilizing this probe, we demonstrated that natural antioxidants significantly elevated SO₂ levels in both cells and tumor-bearing mice, and confirmed that antioxidants modulate SO₂-mediated redox homeostasis by inducing reductive stress. We propose that SO₂ may emerge as a novel reductive stress target, exhibiting significant concentration changes, distinct differences, and enhanced specificity compared to biological thiols under reductive stress conditions.

All authors have given approval to the final version of the manuscript. S. L.: writing – original draft and formal analysis. L. X.: conceptualization, methodology, writing – original draft, and formal analysis. H. L.: visualization, resources, and writing – original draft. Y. L.: investigation, funding acquisition. L. S.: supervision, writing – review and editing, and funding acquisition. G. L.: conceptualization, writing – review and editing. S. Y. supervision, conceptualization, writing – review and editing, and funding acquisition.

This work was supported by the National Natural Science Foundation of China (No. 22374044 and 22271089), the Feature Innovation Projects in Ordinary Universities in Guangdong Province (2022KTSCX228), and Hunan Province College Students Research Learning and Innovative Experiment Project (S202410542130) for financial support.

Conflicts of interest

There are no conflicts to declare.

Data availability

All data generated or analyzed during this study are included in this published article and supplementary information (SI). Supplementary information is available. See DOI: https://doi.org/10.1039/d5cc05915a.

Notes and references

1. J. B. Spinelli and M. C. Haigis, Nat. Cell Biol., 2018, 20, 745–754.
2. S. Li, R. Liu, X. Jiang, Y. Qiu, X. Song, G. Huang, N. Fu, L. Lin, J. Song, X. Chen and H. Yang, ACS Nano, 2019, 13, 2103–2113.
3. M. Wang and R. J. Kaufman, Nature, 2016, 529, 326–335.
4. A. Tirla and P. Rivera-Fuentes, Angew. Chem., Int. Ed., 2016, 55, 14709–14712.
5. L. Luo, S. Chen, H. Jin, C. Tang and J. Du, Biochem. Biophys. Res. Commun., 2011, 415, 61–67.
6. X. Pan, Y. Zhao, T. Cheng, A. Zheng, A. Ge, L. Zang, K. Xu and B. Tang, Chem. Sci., 2019, 10, 8179–8186.
7. D. Lin, T. Lu, X. Wang, X. Ye and Z. Liu, Chem. Sci., 2024, 15, 4824–4832.
8. H. Niu, Y. Zhang, F. Zhao, S. Mo, W. Cao, Y. Ye and Y. Zhao, Chem. Commun., 2019, 55, 9629–9632.
9. S. X. Du, H. F. Jin, D. F. Bu, X. Zhao, B. Geng, C. S. Tang and J. B. Du, Acta Pharmacol. Sin., 2008, 29, 923–930.
10. B. C. Dickinson and C. J. Chang, Nat. Chem. Biol., 2011, 7, 504–511.
11. B. Yang, J. Xu and H. L. Zhu, Free Radical Biol. Med., 2019, 145, 42–60.
12. W. Zhang, F. Huo, Y. Yue, Y. Zhang, J. Chao, F. Cheng and C. Yin, J. Am. Chem. Soc., 2020, 142, 3262–3268.
13. W. Zhang, F. Huo, F. Cheng and C. Yin, J. Am. Chem. Soc., 2020, 142, 6324–6331.
14. V. S. Lin, W. Chen, M. Xian and C. J. Chang, Chem. Soc. Rev., 2015, 44, 4596–4618.
15. X. Yang, Y. Zhou, X. Zhang, S. Yang, Y. Chen, J. Guo and X. Li, Chem. Commun., 2016, 52, 10289–10292.
16. W. Xu, C. L. Teoh, J. Peng, D. Su, L. Yuan and Y. T. Chang, Biomaterials, 2015, 56, 1–9.
17. Z.-Y. Lian, P.-D. Mao, K.-S. Jin, W.-N. Wu, L.-Y. Bian, Y. Wang, Y.-C. Fan, Z.-H. Xu and T. D. James, Sens. Actuators, B, 2025, 441, 137933.
18. W.-N. Wu, Y.-F. Song, X.-L. Zhao, Y. Wang, Y.-C. Fan, Z.-H. Xu and T. D. James, Chem. Eng. J., 2023, 464, 142553.
19. K. Li, L.-L. Li, Q. Zhou, K.-K. Yu, J. S. Kim and X.-Q. Yu, Coord. Chem. Rev., 2019, 388, 310–333.
20. H. Wang, T. Zhu, W. Li, Z. Hu, X. Yang, Q. Zhang, P. Huang, J. Hu and Z. Feng, Anal. Chem., 2025, 97, 16439–16448.
21. Q. Liu, Z. Dong, C. Chang, S. Chen, P. Sun, W. Shu, C. Zeng and W. Chi, Anal. Chem., 2025, 97, 4144–4150.
22. H. Li, J. Wang, H. Kim, X. Peng and J. Yoon, Angew. Chem., Int. Ed., 2024, 63, e202311764.
23. Q. Liu, C. Sun, R. Dai, C. Yan, Y. Zhang, W.-H. Zhu and Z. Guo, Coord. Chem. Rev., 2024, 503, 215652.
24. Y. Bin, L. Huang, J. Qin, M. He, S. Zhao, Y. Huang and J. Zhao, Anal. Chem., 2025, 97, 9798–9808.
25. L. Zeng, T. Chen, B.-Q. Chen, H.-Q. Yuan, R. Sheng and G.-M. Bao, J. Mater. Chem. B, 2020, 8, 1914–1921.
26. W. Gong, C. Zhang, X. Zhang and Y. Shen, Spectrochim. Acta, Part A, 2022, 278, 121386.
27. J. You, H. Liu, Q. Pan, A. Sun, Z. Zhang and X. Shi, J. Anal. Test., 2023, 7, 69–78.
28. L. Yuan, W. Lin and Y. Yang, Chem. Commun., 2011, 47, 6275–6277.
29. J. Li, X. Yu, D. Shu, H. Liu, M. Gu, K. Zhang, G. Mao, S. Yang and R. Yang, Anal. Chem., 2024, 96, 7723–7729.
30. S. Yang, X. Wen, X. Yang, Y. Li, C. Guo, Y. Zhou, H. Li and R. Yang, Anal. Chem., 2018, 90, 14514–14520.

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Footnote

† These authors contributed equally.

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