7-Hydroxycoumarin modulates the oxidative metabolism, degranulation and microbial killing of human neutrophils Luciana M. Kabeya a,⇑ , Carolina N. Fuzissaki a , Silvia H. Taleb-Contini a,1 , Ana Maria da C. Ferreira b , Zeki Naal a , Everton O.L. Santos a , Andréa S.G. Figueiredo-Rinhel a , Ana Elisa C.S. Azzolini a , Roberta B. Vermelho a , Alberto Malvezzi b,2 , Antonia T. -do Amaral b , João Luis C. Lopes a , Yara Maria Lucisano-Valim a a Departamento de Física e Química, Faculdade de Ciências Farmacêuticas de Ribeirão Preto da Universidade de São Paulo. Av. do Café s/n, 14040-903 Ribeirão Preto, SP, Brazil b Departamento de Química Fundamental, Instituto de Química da Universidade de São Paulo. Av. Prof. Lineu Prestes 748, 05508-900 São Paulo, SP, Brazil article info Article history: Received 16 April 2013 Received in revised form 14 July 2013 Accepted 17 August 2013 Available online 28 August 2013 Keywords: 7-Hydroxycoumarin Neutrophil Degranulation Myeloperoxidase Candida albicans Prooxidant abstract In the present study, we assessed whether 7-hydroxycoumarin (umbelliferone), 7-hydroxy-4-methyl- coumarin, and their acetylated analogs modulate some of the effector functions of human neutrophils and display antioxidant activity. These compounds decreased the ability of neutrophils to generate super- oxide anion, release primary granule enzymes, and kill Candida albicans. Cytotoxicity did not mediate their inhibitory effect, at least under the assessed conditions. These coumarins scavenged hypochlorous acid and protected ascorbic acid from electrochemical oxidation in cell-free systems. On the other hand, the four coumarins increased the luminol-enhanced chemiluminescence of human neutrophils stimu- lated with phorbol-12-myristate-13-acetate and serum-opsonized zymosan. Oxidation of the hydroxyl- ated coumarins by the neutrophil myeloperoxidase produced highly reactive coumarin radical intermediates, which mediated the prooxidant effect observed in the luminol-enhanced chemilumines- cence assay. These species also oxidized ascorbic acid and the spin traps a-(4-pyridyl-1-oxide)-N-tert- butylnitrone and 5-dimethyl-1-pyrroline-N-oxide. Therefore, 7-hydroxycoumarin and the derivatives investigated here were able to modulate the effector functions of human neutrophils and scavenge reac- tive oxidizing species; they also generated reactive coumarin derivatives in the presence of myeloperox- idase. Acetylation of the free hydroxyl group, but not addition of the 4-methyl group, suppressed the biological effects of 7-hydroxycoumarin. These findings help clarify how 7-hydroxycoumarin acts on neu- trophils to produce relevant anti-inflammatory effects. Ó 2013 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Neutrophils are professional killers of invading microorganisms and also contribute to the regulation of inflammatory processes. These cells secrete types of chemokines, cytokines, lipid mediators, granule proteins, and reactive oxygen species (ROS) that serve as signaling molecules for subsequent recruitment of inflammatory cells. These molecules help regulate the initiation of specific T and B cell immunity and the resolution phase of inflammation [1–3]. Thus, modulating the effector functions of neutrophils is a promising therapeutic strategy to manage inflammation. Coumarin and its derivatives have arisen as promising natural products with potent anti-inflammatory effect and low toxicity to humans [4–8]. They constitute a large class of phenolic 0009-2797/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cbi.2013.08.010 Abbreviations: AAPV, methoxy-succinyl-Ala-Ala-Pro-Val-chloromethylketone; ABAH, 4-aminobenzoic acid hydrazide; Asc, ascorbic acid; CL, chemiluminescence; CL-luc, lucigenin-enhanced chemiluminescence; CL-lum, luminol-enhanced chemiluminescence; DMPO, 5-dimethyl-1-pyrroline-N-oxide; DMPOX, 5,5-dimethyl-1-pyrrolidone-2- oxyl-1; DMSO, dimethyl sulfoxide; DPPH, 1,1-diphenyl-2-picrylhydrazyl radical; EPR, electron paramagnetic resonance; n-fMLP, n-formyl-methionyl-leucyl-phenylalanine; HBSS, Hank’s balanced saline solution; HBSS-gel, Hank’s balanced saline solution supplemented with gelatin; HRP, horseradish peroxidase; IC 50 , concentration inhibiting a biological response by 50%; LDH, lactate dehydrogenase; MPO, myeloperoxidase; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; NADPH, reduced form of nicotinamide adenine dinucleotide phosphate; PI, propidium iodide; PKC, protein kinase C; PMA, phorbol-12-myristate-13-acetate; 4-POBN, a-(4-pyridyl-1-oxide)-N- tert-butylnitrone; ROS, reactive oxygen species; SAAVNA, N-succinyl-Ala-Ala-Val-p-nitroanilide; SOD, superoxide dismutase; SOZ, serum-opsonized zymosan. ⇑ Corresponding author. Address: Departamento de Física e Química, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo. Av. do Café s/n, Bairro Monte Alegre, 14040-903 Ribeirão Preto, SP, Brazil. Tel.: +55 16 36024212; fax: +55 16 36024880. E-mail address: luciana_kabeya@yahoo.com.br (L.M. Kabeya). 1 Present address: Unidade de Biotecnologia, Universidade de Ribeirão Preto (UNAERP), Avenida Costábile Romano 2201, Bairro Ribeirânea, 14096-380 Ribeirão Preto, SP, Brazil. 2 Present address: Universidade Nove de Julho (UNINOVE), Avenida Dr. Adolpho Pinto 109, Barra Funda, São Paulo, SP 01156-050, Brazil. Chemico-Biological Interactions 206 (2013) 63–75 Contents lists available at ScienceDirect Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint