Alterations in Apoptotic Cell Populations, Protein Markers, and Gene Expression Patterns in Rats with Sulfur Mustard-induced Pulmonary Injuries
-
Tao Liu
,
Na Zhang
,
Xin Shu
,
Xiaoxuan Hu
,
Jingtong Li
,
Yuxu Zhong
,
Jinyuan Tang
,
Xiaoji Zhu
Abstract
Sulfur mustard (SM) is a potent chemical warfare agent that causes severe cutaneous, ocular, and pulmonary injuries, with respiratory tract damage being the most life-threatening. Despite its well-documented toxicity, the cellular mechanisms driving SM-induced apoptosis remain poorly understood. This study seeks to elucidate the apoptotic pathways involved in SM-induced pulmonary injury using a rat model.
We induced acute lung injury through two delivery methods: intraperitoneal injection (8 mg/kg) and intratracheal instillation (2 mg/kg) of SM, with both doses representing 1 LD50. We assessed apoptosis-related proteins and gene expression through TUNEL staining, immunohistochemistry, and quantitative real-time PCR analyses.
Intraperitoneal administration of SM resulted in significantly elevated expression of apoptotic markers including annexin A1, annexin A2, cytochrome C, caspase-12, and JNK3, in alveolar epithelial cells compared to intratracheal delivery. Both TUNEL assays and immunohistochemical staining confirmed these findings. These results indicate that intraperitoneal SM exposure triggers more severe apoptotic responses in alveolar epithelial cells than intratracheal exposure at equivalent doses.
These findings demonstrate that intraperitoneal models can effectively identify apoptosis-related molecular targets suitable for therapeutic development.
1. Kumar R, Sinha DM, Lankau BR, Sinha NR, Tripathi R, Gupta S, et al. Differential gene expression and protein-protein interaction network profiling of sulfur mustard-exposed rabbit corneas employing RNA-seq data and bioinformatics tools. Exp Eye Res. 2023;235:109644.
2. Xiao Z, Liu F, Cheng J, Wang Y, Zhou W, Zhang Y. B-Raf inhibitor vemurafenib counteracts sulfur mustard-induced epidermal impairment through MAPK/ERK signaling. Drug Chem Toxicol. 2023;46(2):226-35.
3. Jost P, Muckova L, Pejchal J. In vitro stress response induced by sulfur mustard in lung fibroblasts NHLF and human pulmonary epithelial cells A-549. Arch Toxicol. 2020;94(10):3503-14.
4. Sabnam S, Rizwan H, Pal S, Pal A. CEES-induced ROS accumulation regulates mitochondrial complications and inflammatory response in keratinocytes. Chem Biol Interact. 2020;321:109031.
5. Zhao Z, Yan X, Li L, Shu Y, He J, Wang L, et al. Proliferating stem cells are acutely affected by DNA damage induced by sulfur mustard. DNA Cell Biol. 2022;41(8):716-726.
6. Jamshidi V, Halabian R, Saeedi P, Bagheri H, Nobakht Motlagh Ghoochani BF. Accelerating synergistic effects of preconditioned mesenchymal stem cells with Crocin and dexamethasone in pulmonary epithelial cells injury. Toxicol Res (Camb). 2023;12(3):369-80.
7. Sabnam S, Pal A. Relevance of Erk1/2-PI3K/Akt signaling pathway in CEES-induced oxidative stress regulates inflammation and apoptosis in keratinocytes. Cell Biol Toxicol. 2019;35(6):541-64.
8. Dan Yu, Bei YY, Li Y, Han W, Zhong YX, Liu F, et al. In vitro the differences of inflammatory and oxidative reactions due to sulfur mustard induced acute pulmonary injury underlying intraperitoneal injection and intratracheal instillation in rats. Int Immunopharmacol. 2017;47:78-87.
9. Jiang YY, Li ZS, Yu D, Xie JW, Zhu XJ, Zhong YX. Changes in inflammatory factors and protein expression in sulfur mustard(1LD50)-induced acute pulmonary injury in rats. Int Immunopharmacol. 2018;61:338-45.
10. Liu L, Hu XX, Zhang N, Zhong YX, Zhu XJ, Liu T. Oxidative stress reactions in rats with pulmonary injuries induced by sulfur mustard (1LD50). Hum Exp Toxicol. 2024;43:9603271241308772.
11. Hu XX, Zhang N, Zhong YX, Liu T, Zhu XJ. Mechanisms of apoptosis and pulmonary fibrosis resulting from sulfur mustard-induced acute pulmonary injury in rats. Int J Toxicol. 2025;44(4):314-27.
12. Neff TA, Guo RF, Neff SB, Sarma JV, Speyer CL, Gao HW, et al. Relationship of acute lung inflammatory injury to Fas/FasL system. Am J Pathol. 2005;166(3):685-94.
13. Rosenthal DS, Velena A, Chou FP, Schlegel R, Ray R, Benton B, et al. Expression of dominant-negative Fas-associated death domain blocks human keratinocyte apoptosis and vesication induced by sulfur mustard. J Biol Chem. 2003;278(10):8531-40.
14. Simbulan-Rosenthal CM, Ray R, Benton B, Soeda E, Daher A, Anderson D, et al. Calmodulin mediates sulfur mustard toxicity in human keratinocytes. Toxicology. 2006;227(1-2):21-35.
15. Steinritz D, Emmler J, Hintz M, Worek F, Kreppel H, Szinicz L, et al. Apoptosis in sulfur mustard treated A549 cell cultures. Life Sci. 2007;80(24-25):2199-201.
16. Sourdeval M, Lemaire C, Deniaud A, Taysse L, Daulon S, Breton P, et al. Inhibition of caspase-dependent mitochondrial permeability transition protects airway epithelial cells against mustard-induced apoptosis. Apoptosis. 2006;11(9):1545-59.
17. Mosayebzadeh M, Ghazanfari T, Delshad A, Akbari H. Evaluation of Apoptosis in the Lung Tissue of Sulfur Mustard-exposed Individuals. Iran J Allergy Asthma Immunol. 2016;15(4):283-8.
18. Blaha M, Kohl J, DuBose D, Bowers W Jr, Walker J. Ultrastructural and histological effects of exposure to CEES or heat in a human epidermal model. In Vitr Mol Toxicol. 2001;14(1):15-23.
19. Hasan M, Kumolosasi E, Jantan I, Jasamai M, Nazarudin N. Knockdown of Annexin A1 induces apoptosis, causing G2/M arrest and facilitating phagocytosis activity in human leukemia cell lines. Acta Pharm. 2021;72(1):109-22.
20. Vago JP, Nogueira CRC, Tavares LP, Soriani FM, Lopes F, Russo RC, et al. Annexin A1 modulates natural and glucocorticoid-induced resolution of inflammation by enhancing neutrophil apoptosis. J Leukoc Biol. 2012;92(2):249-58.
21. Li GW, He SH, Chang LJ, Lu H, Zhang HW, Zhang H, et al. GADD45α and annexin A1 are involved in the apoptosis of HL-60 induced by resveratrol. Phytomedicine. 2011;18(8-9):704-9.
22. Wu MH, Sun YQ, Xu FP, Liang YQ, Liu H, Yi YM. Annexin A2 silencing inhibits proliferation and epithelial-to-mesenchymal transition through p53-dependent pathway in NSCLCs. J Cancer. 2019;10(5):1077-85.
23. Wang CY, Chen CL, Tseng YL, Fang YT, Lin YS, Su WC, et al. Annexin A2 silencing induces G2 arrest of non-small cell lung cancer cells through p53-dependent and -independent mechanisms. J Biol Chem. 2012;287(39):32512-24.
24. Li DW, Qi XD, Zhang CH, Sun WP. Annexin A2 degradation contributes to dopaminergic cell apoptosis via regulating p53 in neurodegenerative conditions. Neuroreport. 2021;32(15):1263-8.
25. Inturi S, Tewari-Singh N, Jain AK, Roy S, White CW, Agarwal R. Absence of a p53 allele delays nitrogen mustard-induced early apoptosis and inflammation of murine skin. Toxicology. 2013;311(3):184-90.
26. Tournier C, Hess P, Yang DD, Xu J, Turner TK, Nimnual A, et al. Requirement of JNK for stress-induced activation of the cytochrome c-mediated death pathway. Science. 2000;288(5467):870-4.
27. Gurzov EN, Eizirik DL. Bcl-2 proteins in diabetes: mitochondrial pathways of β-cell death and dysfunction. Trends Cell Biol. 2011;21(7):424-31.
28. Hitomi J, Katayama T, Taniguchi M, Honda A, Imaizumi K, Tohyama M. Apoptosis induced by endoplasmic reticulum stress depends on activation of caspase-3 via caspase-12. Neurosci Lett. 2004;357(2):127-30.
29. Fakhrinnisa TA, Susilo I, Mustika A, Sofyan MS. Effect of intravenous glutamine on caspase-12 expression in the apoptosis of the glomerular epithelial cells of male rats exposed to cisplatin. Asian Pac J Cancer Prev. 2021;22(2):457-62.
30. Peyrou M, Hanna PE, Cribb AE. Cisplatin, gentamicin, and p-aminophenol induce markers of endoplasmic reticulum stress in the rat kidneys. Toxicol Sci. 2007;99(1):346-53.
31. Zhang Q, Liu J, Chen S, Liu J, Liu L, Liu G, et al. Caspase-12 is involved in stretch-induced apoptosis mediated endoplasmic reticulum stress. Apoptosis. 2016;21(4):432-42.
32. Brown GC, Borutaite V. Regulation of apoptosis by the redox state of cytochrome c. Biochim Biophys Acta. 2008;1777(7-8):877-81.
33. Pérez-Mejías G, Guerra-Castellano A, Díaz-Quintana A, De la Rosa MA, Díaz-Moreno I. Cytochrome c: surfing off of the mitochondrial membrane on the tops of complexes III and IV. Comput Struct Biotechnol J. 2019;17:654-60.
34. Pohl C, Papritz M, Moisch M, Wübbeke C, Hermanns MI, Uboldi C, et al. Acute morphological and toxicological effects in a human bronchial coculture model after sulfur mustard exposure. Toxicol Sci. 2009;112(2):482-9.
35. Sharma DR, Sunkaria A, Bal A, Bhutia YD, Vijayaraghavan R, Flora SJ, et al. Neurobehavioral impairments, generation of oxidative stress and release of pro-apoptotic factors after chronic exposure to sulphur mustard in mouse brain. Toxicol Appl Pharmacol. 2009;240(2):208-18.
2. Xiao Z, Liu F, Cheng J, Wang Y, Zhou W, Zhang Y. B-Raf inhibitor vemurafenib counteracts sulfur mustard-induced epidermal impairment through MAPK/ERK signaling. Drug Chem Toxicol. 2023;46(2):226-35.
3. Jost P, Muckova L, Pejchal J. In vitro stress response induced by sulfur mustard in lung fibroblasts NHLF and human pulmonary epithelial cells A-549. Arch Toxicol. 2020;94(10):3503-14.
4. Sabnam S, Rizwan H, Pal S, Pal A. CEES-induced ROS accumulation regulates mitochondrial complications and inflammatory response in keratinocytes. Chem Biol Interact. 2020;321:109031.
5. Zhao Z, Yan X, Li L, Shu Y, He J, Wang L, et al. Proliferating stem cells are acutely affected by DNA damage induced by sulfur mustard. DNA Cell Biol. 2022;41(8):716-726.
6. Jamshidi V, Halabian R, Saeedi P, Bagheri H, Nobakht Motlagh Ghoochani BF. Accelerating synergistic effects of preconditioned mesenchymal stem cells with Crocin and dexamethasone in pulmonary epithelial cells injury. Toxicol Res (Camb). 2023;12(3):369-80.
7. Sabnam S, Pal A. Relevance of Erk1/2-PI3K/Akt signaling pathway in CEES-induced oxidative stress regulates inflammation and apoptosis in keratinocytes. Cell Biol Toxicol. 2019;35(6):541-64.
8. Dan Yu, Bei YY, Li Y, Han W, Zhong YX, Liu F, et al. In vitro the differences of inflammatory and oxidative reactions due to sulfur mustard induced acute pulmonary injury underlying intraperitoneal injection and intratracheal instillation in rats. Int Immunopharmacol. 2017;47:78-87.
9. Jiang YY, Li ZS, Yu D, Xie JW, Zhu XJ, Zhong YX. Changes in inflammatory factors and protein expression in sulfur mustard(1LD50)-induced acute pulmonary injury in rats. Int Immunopharmacol. 2018;61:338-45.
10. Liu L, Hu XX, Zhang N, Zhong YX, Zhu XJ, Liu T. Oxidative stress reactions in rats with pulmonary injuries induced by sulfur mustard (1LD50). Hum Exp Toxicol. 2024;43:9603271241308772.
11. Hu XX, Zhang N, Zhong YX, Liu T, Zhu XJ. Mechanisms of apoptosis and pulmonary fibrosis resulting from sulfur mustard-induced acute pulmonary injury in rats. Int J Toxicol. 2025;44(4):314-27.
12. Neff TA, Guo RF, Neff SB, Sarma JV, Speyer CL, Gao HW, et al. Relationship of acute lung inflammatory injury to Fas/FasL system. Am J Pathol. 2005;166(3):685-94.
13. Rosenthal DS, Velena A, Chou FP, Schlegel R, Ray R, Benton B, et al. Expression of dominant-negative Fas-associated death domain blocks human keratinocyte apoptosis and vesication induced by sulfur mustard. J Biol Chem. 2003;278(10):8531-40.
14. Simbulan-Rosenthal CM, Ray R, Benton B, Soeda E, Daher A, Anderson D, et al. Calmodulin mediates sulfur mustard toxicity in human keratinocytes. Toxicology. 2006;227(1-2):21-35.
15. Steinritz D, Emmler J, Hintz M, Worek F, Kreppel H, Szinicz L, et al. Apoptosis in sulfur mustard treated A549 cell cultures. Life Sci. 2007;80(24-25):2199-201.
16. Sourdeval M, Lemaire C, Deniaud A, Taysse L, Daulon S, Breton P, et al. Inhibition of caspase-dependent mitochondrial permeability transition protects airway epithelial cells against mustard-induced apoptosis. Apoptosis. 2006;11(9):1545-59.
17. Mosayebzadeh M, Ghazanfari T, Delshad A, Akbari H. Evaluation of Apoptosis in the Lung Tissue of Sulfur Mustard-exposed Individuals. Iran J Allergy Asthma Immunol. 2016;15(4):283-8.
18. Blaha M, Kohl J, DuBose D, Bowers W Jr, Walker J. Ultrastructural and histological effects of exposure to CEES or heat in a human epidermal model. In Vitr Mol Toxicol. 2001;14(1):15-23.
19. Hasan M, Kumolosasi E, Jantan I, Jasamai M, Nazarudin N. Knockdown of Annexin A1 induces apoptosis, causing G2/M arrest and facilitating phagocytosis activity in human leukemia cell lines. Acta Pharm. 2021;72(1):109-22.
20. Vago JP, Nogueira CRC, Tavares LP, Soriani FM, Lopes F, Russo RC, et al. Annexin A1 modulates natural and glucocorticoid-induced resolution of inflammation by enhancing neutrophil apoptosis. J Leukoc Biol. 2012;92(2):249-58.
21. Li GW, He SH, Chang LJ, Lu H, Zhang HW, Zhang H, et al. GADD45α and annexin A1 are involved in the apoptosis of HL-60 induced by resveratrol. Phytomedicine. 2011;18(8-9):704-9.
22. Wu MH, Sun YQ, Xu FP, Liang YQ, Liu H, Yi YM. Annexin A2 silencing inhibits proliferation and epithelial-to-mesenchymal transition through p53-dependent pathway in NSCLCs. J Cancer. 2019;10(5):1077-85.
23. Wang CY, Chen CL, Tseng YL, Fang YT, Lin YS, Su WC, et al. Annexin A2 silencing induces G2 arrest of non-small cell lung cancer cells through p53-dependent and -independent mechanisms. J Biol Chem. 2012;287(39):32512-24.
24. Li DW, Qi XD, Zhang CH, Sun WP. Annexin A2 degradation contributes to dopaminergic cell apoptosis via regulating p53 in neurodegenerative conditions. Neuroreport. 2021;32(15):1263-8.
25. Inturi S, Tewari-Singh N, Jain AK, Roy S, White CW, Agarwal R. Absence of a p53 allele delays nitrogen mustard-induced early apoptosis and inflammation of murine skin. Toxicology. 2013;311(3):184-90.
26. Tournier C, Hess P, Yang DD, Xu J, Turner TK, Nimnual A, et al. Requirement of JNK for stress-induced activation of the cytochrome c-mediated death pathway. Science. 2000;288(5467):870-4.
27. Gurzov EN, Eizirik DL. Bcl-2 proteins in diabetes: mitochondrial pathways of β-cell death and dysfunction. Trends Cell Biol. 2011;21(7):424-31.
28. Hitomi J, Katayama T, Taniguchi M, Honda A, Imaizumi K, Tohyama M. Apoptosis induced by endoplasmic reticulum stress depends on activation of caspase-3 via caspase-12. Neurosci Lett. 2004;357(2):127-30.
29. Fakhrinnisa TA, Susilo I, Mustika A, Sofyan MS. Effect of intravenous glutamine on caspase-12 expression in the apoptosis of the glomerular epithelial cells of male rats exposed to cisplatin. Asian Pac J Cancer Prev. 2021;22(2):457-62.
30. Peyrou M, Hanna PE, Cribb AE. Cisplatin, gentamicin, and p-aminophenol induce markers of endoplasmic reticulum stress in the rat kidneys. Toxicol Sci. 2007;99(1):346-53.
31. Zhang Q, Liu J, Chen S, Liu J, Liu L, Liu G, et al. Caspase-12 is involved in stretch-induced apoptosis mediated endoplasmic reticulum stress. Apoptosis. 2016;21(4):432-42.
32. Brown GC, Borutaite V. Regulation of apoptosis by the redox state of cytochrome c. Biochim Biophys Acta. 2008;1777(7-8):877-81.
33. Pérez-Mejías G, Guerra-Castellano A, Díaz-Quintana A, De la Rosa MA, Díaz-Moreno I. Cytochrome c: surfing off of the mitochondrial membrane on the tops of complexes III and IV. Comput Struct Biotechnol J. 2019;17:654-60.
34. Pohl C, Papritz M, Moisch M, Wübbeke C, Hermanns MI, Uboldi C, et al. Acute morphological and toxicological effects in a human bronchial coculture model after sulfur mustard exposure. Toxicol Sci. 2009;112(2):482-9.
35. Sharma DR, Sunkaria A, Bal A, Bhutia YD, Vijayaraghavan R, Flora SJ, et al. Neurobehavioral impairments, generation of oxidative stress and release of pro-apoptotic factors after chronic exposure to sulphur mustard in mouse brain. Toxicol Appl Pharmacol. 2009;240(2):208-18.
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| Issue | Articles in Press | |
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| Keywords | ||
| Acute pulmonary injury Apoptosis Equal lethal dose Sulfur mustard | ||
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How to Cite
1.
Liu T, Zhang N, Shu X, Hu X, Li J, Zhong Y, Tang J, Zhu X. Alterations in Apoptotic Cell Populations, Protein Markers, and Gene Expression Patterns in Rats with Sulfur Mustard-induced Pulmonary Injuries. Iran J Allergy Asthma Immunol. 2025;:1-9.
