Sub-cytotoxic concentrations of ionic silver promote the proliferation of human keratinocytes by inducing the production of reactive oxygen species

Abstract

Silver-containing preparations are widely used in the management of skin wounds, but the effects of silver ions on skin wound healing remain poorly understood. This study investigated the effects of silver ions (Ag⁺) on the proliferation of human skin keratinocytes (HaCaT) and the production of intracellular reactive oxygen species (ROS). After treating HaCaT cells with Ag⁺ and/or the active oxygen scavenger N-acetyl cysteine (NAC), cell proliferation and intracellular ROS generation were assessed using CCK-8 reagent and DCFH-DA fluorescent probe, respectively. In addition, 5-bromo-2-deoxyUridine (BrdU) incorporation assays, cell cycle flow cytometry, and proliferating cell nuclear antigen (PCNA) immunocytochemistry were conducted to further evaluate the effects of sub-cytotoxic Ag⁺ concentrations on HaCaT cells. The proliferation of HaCaT cells was promoted in the presence of 10⁻⁶ and 10⁻⁵ mol/L Ag⁺ at 24, 48, and 72 h. Intracellular ROS generation also significantly increased for 5–60 min after exposure to Ag⁺. The number of BrdU-positive cells and the presence of PCNA in HaCaT cells increased 48 h after the addition of 10⁻⁶ and 10⁻⁵ mol/L Ag⁺, with 10⁻⁵ mol/L Ag⁺ markedly increasing the cell proliferation index. These effects of sub-cytotoxic Ag⁺ concentrations were repressed by 5 mmol/L NAC. Our results suggest that sub-cytotoxic Ag⁺ concentrations promote the proliferation of human keratinocytes and might be associated with a moderate increase in intracellular ROS levels. This study provides important experimental evidence for developing novel silver-based wound agents or dressings with few or no cytotoxicity.

Keywords

ionic silver / human keratinocyte / cell proliferation / reactive oxygen species / active oxygen scavenger / NAC

References

[1] Moyer CA, Brentano L, Gravens DL, Margraf HW Jr, Monafo WW Jr. Treatment of large human burns with 0.5 per cent silver nitrate solution. Arch Surg 1965; 90(6): 812–867

[2] Fox CL Jr. Silver sulfadiazine—a new topical therapy for Pseudomonas in burns. Therapy of Pseudomonas infection in burns. Arch Surg 1968; 96(2): 184–188

[3] Chopra I. The increasing use of silver-based products as antimicrobial agents: a useful development or a cause for concern? J Antimicrob Chemother 2007; 59(4): 587–590

[4] Swathy JR, Sankar MU, Chaudhary A, Aigal S, Anshup T, Pradeep. Antimicrobial silver: an unprecedented anion effect. Sci Rep 2014; 4: 7161

[5] Mijnendonckx K, Leys N, Mahillon J, Silver S, Van Houdt R. Antimicrobial silver: uses, toxicity and potential for resistance. Biometals 2013; 26(4): 609–621

[6] Lansdown A, Williams A. Bacterial resistance to silver-based antibiotics. Nurs Times 2007; 103(9): 48–49

[7] Burd A, Kwok CH, Hung SC, Chan HS, Gu H, Lam WK, Huang L. A comparative study of the cytotoxicity of silver-based dressings in monolayer cell, tissue explant, and animal models. Wound Repair Regen 2007; 15(1): 94–104

[8] Berger TJ, Spadaro JA, Chapin SE, Becker RO. Electrically generated silver ions: quantitative effects on bacterial and mammalian cells. Antimicrob Agents Chemother 1976; 9(2): 357–358

[9] Arora S, Jain J, Rajwade JM, Paknikar KM. Interactions of silver nanoparticles with primary mouse fibroblasts and liver cells. Toxicol Appl Pharmacol 2009; 236(3): 310–318

[10] Zanette C, Pelin M, Crosera M, Adami G, Bovenzi M, Larese FF, Florio C. Silver nanoparticles exert a long-lasting antiproliferative effect on human keratinocyte HaCaT cell line. Toxicol In Vitro 2011; 25(5): 1053–1060

[11] Poon VKM, Burd A. In vitro cytotoxity of silver: implication for clinical wound care. Burns 2004; 30(2): 140–147

[12] Morones-Ramirez JR, Winkler JA, Spina CS, Collins JJ. Silver enhances antibiotic activity against gram-negative bacteria. Sci Transl Med 2013; 5(190): 190ra81

[13] Thomas GW, Rael LT, Bar-Or R, Shimonkevitz R, Mains CW, Slone DS, Craun ML, Bar-Or D. Mechanisms of delayed wound healing by commonly used antiseptics. J Trauma 2009, 66:82–90,  discussion 90–91

[14] Michaels JA, Campbell B, King B, Palfreyman SJ, Shackley P, Stevenson M. Randomized controlled trial and cost-effectiveness analysis of silver-donating antimicrobial dressings for venous leg ulcers (VULCAN trial). Br J Surg 2009; 96(10): 1147–1156

[15] White R, Cutting K, Ousey K, Butcher M, Gray D, Flanagan M, Donnelly J, McIntosh C, Kingsley A, Fletcher J, Chadwick P, Gethin G, Beldon P. Randomized controlled trial and cost-effectiveness analysis of silver-donating antimicrobial dressings for venous leg ulcers (VULCAN trial) (Br J Surg 2009; 96: 1147-1156). Br J Surg 2010; 97(3): 459–460

[16] Vermeulen H, van Hattem JM, Storm-Versloot MN, Ubbink DT. Topical silver for treating infected wounds. Cochrane Database Syst Rev 2007; (1): CD005486

[17] Storm-Versloot MN, Vos CG, Ubbink DT, Vermeulen H. Topical silver for preventing wound infection. Cochrane Database Syst Rev 2010; (3): CD006478

[18] Aziz Z, Abu SF, Chong NJ. A systematic review of silver-containing dressings and topical silver agents (used with dressings) for burn wounds. Burns 2012; 38(3): 307–318

[19] Gordon O, Vig Slenters T, Brunetto PS, Villaruz AE, Sturdevant DE, Otto M, Landmann R, Fromm KM. Silver coordination polymers for prevention of implant infection: thiol interaction, impact on respiratory chain enzymes, and hydroxyl radical induction. Antimicrob Agents Chemother 2010; 54(10): 4208– 4218

[20] Foldbjerg R, Olesen P, Hougaard M, Dang DA, Hoffmann HJ, Autrup H. PVP-coated silver nanoparticles and silver ions induce reactive oxygen species, apoptosis and necrosis in THP-1 monocytes. Toxicol Lett 2009; 190(2): 156–162

[21] Miyayama T, Arai Y, Suzuki N, Hirano S. Mitochondrial electron transport is inhibited by disappearance of metallothionein in human bronchial epithelial cells following exposure to silver nitrate. Toxicology 2013; 305: 20–29

[22] Allen RG, Tresini M. Oxidative stress and gene regulation. Free Radic Biol Med 2000; 28(3): 463–499

[23] Yamamoto A, Honma R, Tanaka A, Sumita M. Generic tendency of metal salt cytotoxicity for six cell lines. J Biomed Mater Res 1999; 47(3): 396–403

[24] Martin KR, Barrett JC. Reactive oxygen species as double-edged swords in cellular processes: low-dose cell signaling versus high-dose toxicity. Hum Exp Toxicol 2002; 21(2): 71–75

[25] Sena LA, Chandel NS. Physiological roles of mitochondrial reactive oxygen species. Mol Cell 2012; 48(2): 158–167

[26] Sauer H, Wartenberg M, Hescheler J. Reactive oxygen species as intracellular messengers during cell growth and differentiation. Cell Physiol Biochem 2001; 11(4): 173–186

[27] Samuni Y, Goldstein S, Dean OM, Berk M. The chemistry and biological activities of N-acetylcysteine. Biochim Biophys Acta. 2013;1830(8): 4117–4129

[28] Bateman DN, Dear JW, Thanacoody HK, Thomas SH, Eddleston M, Sandilands EA, Coyle J, Cooper JG, Rodriguez A, Butcher I, Lewis SC, Vliegenthart AD, Veiraiah A, Webb DJ, Gray A. Reduction of adverse effects from intravenous acetylcysteine treatment for paracetamol poisoning: a randomised controlled trial. Lancet 2014; 383(9918): 697–704

[29] Tominaga H, Ishiyama M, Ohseto F, Sasamoto K, Hamamoto T, Suzuki K, Watanabe M. A water-soluble tetrazolium salt useful for colorimetric cell viability assay. Anal Commun 1999; 36(2): 47–50

[30] Bosq J, Bourhis J. Bromodeoxyuridine (BrdU). Analysis of cellular proliferation. Ann Pathol 1997; 17(3): 171–178 (in French)

[31] Chen AC, Arany PR, Huang YY, Tomkinson EM, Sharma SK, Kharkwal GB, Saleem T, Mooney D, Yull FE, Blackwell TS, Hamblin MR. Low-level laser therapy activates NF-κB via generation of reactive oxygen species in mouse embryonic fibroblasts. PLoS One 2011; 6(7): e22453

[32] Burdon RH. Superoxide and hydrogen peroxide in relation to mammalian cell proliferation. Free Radic Biol Med 1995; 18(4): 775–794

[33] Ruiz-Ginés JA, López-Ongil S, González-Rubio M, González-Santiago L, Rodrµguez-Puyol M, Rodrµguez-Puyol D. Reactive oxygen species induce proliferation of bovine aortic endothelial cells. J Cardiovasc Pharmacol 2000; 35(1): 109–113

[34] Egelkrout EM, Mariconti L, Settlage SB, Cella R, Robertson D, Hanley-Bowdoin L. Two E2F elements regulate the proliferating cell nuclear antigen promoter differently during leaf development. Plant Cell 2002; 14(12): 3225–3236

[35] Koundrioukoff S, Jónsson ZO, Hasan S, de Jong RN, van der Vliet PC, Hottiger MO, Hübscher U. A direct interaction between proliferating cell nuclear antigen (PCNA) and Cdk2 targets PCNA-interacting proteins for phosphorylation. J Biol Chem 2000; 275(30): 22882–22887

Acknowledgements

This study was supported by the Independent Research Subject Project Type I of the State Key Laboratory of Trauma, Burns, and Combined Injury (No. SKLZZ201226) and open research fund of State Key Laboratory of Trauma, Burns, and Combined Injury (No. SKLZZ201504).

Compliance with ethics guidelines

Xiaodong Duan, Daizhi Peng, Yilan Zhang, Yalan Huang, Xiao Liu, Ruifu Li, Xin Zhou, and Jing Liu declare that they have no conflict of interest. This manuscript does not involve a research protocol requiring approval from a relevant institutional review board or ethics committee.

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