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Supra-physiological doses of testosterone affect membrane oxidation of human neutrophils monitored by the fluorescent probe C11-BODIPY581/591

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Abstract

The purpose of this study was to determine the effects of supra-physiological doses of testosterone (TES) on membrane oxidation of activated human neutrophils in vitro using an innovative and sensitive technique: the real-time detection with the fluorescence probe C11-BODIPY581/591. Methodological controls were performed with the lipid-soluble and powerful antioxidant astaxanthin at different neutrophil density cultures. Neutrophils from nine healthy young men (23.4 ± 2.5 years, 174.4 ± 7.0 cm height, and 78.3 ± 7.0 kg weight) were isolated and treated with 0.1 or 10 μM TES for 24 h and subsequently labeled with the free radical-sensitive probe C11-BODIPY581/591 for monitoring membrane oxidation after neutrophil activation with phorbol-12-myristate-13-acetate (PMA). First-order exponential decay kinetic indicated that both 0.1 and 10 μM TES severely increased baseline membrane oxidation in non-activated human neutrophils (compared to control). However, similar kinetics of membrane oxidation were observed in control and 0.1 μM TES-treated neutrophils after PMA activation, whereas chemical activation did not alter the baseline higher rates of membrane oxidation in 10 μM TES-treated neutrophils. The data presented here support the hypothesis that TES exerts distinct effects on the membrane oxidation of human neutrophils, depending on its dose (here, 102 to 104-fold higher than physiological levels in men) and on PMA activation of the oxidative burst. Furthermore, this paper also presents an innovative application of the free radical-sensitive probe C11-BODIPY581/591 for monitoring (auto-induced) membrane oxidation as an important parameter of viability and, thus, responsiveness of immune cells in inflammatory processes.

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Abbreviations

AAS:

Androgen-anabolic steroids

AST:

Astaxanthin

C11-BODIPY581/591 :

4,4-Difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid

DMSO:

Dimethylsulfoxide

HEPES:

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

PMA:

Phorbol-12-myristate-13-acetate

ROS/RNS:

Reactive oxygen/nitrogen species

TBARS:

Thiobarbituric acid reactive substances

TES:

Testosterone

VSMCs:

Vascular smooth muscle cells

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Acknowledgments

The authors are indebted to Dr. Rosemari Otton, and M.Sc. Douglas Marin Popp for providing neutrophil samples and resources during preliminary studies at Laboratório de Fisiologia Celular, campus Analia Franco, Universidade Cruzeiro do Sul, São Paulo, Brazil, and the assistance of Dr. Douglas Ganini for neutrophil isolation in further experiments. Dr. Marcelo Paes de Barros is also indebted to Prof. Rui Curi, at Department of Biophysics and Physiology, University of São Paulo, Brazil, for chemicals and laboratory facilities during neutrophil isolation. This research is supported by Fundação de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP 2009/12342-8), Programa de Suporte à Pós-graduação de Instituições de Ensino Particulares, da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (PROSUP/CAPES, Brazil), and Conselho Nacional de Desenvolvimento Científico e Tecnológico (Bolsas Produtividade em Pesquisa, Nivel 2, #312404/2009-3, CNPq, Brazil).

Conflict of interest

All authors of the present manuscript declare that there is no any actual or potential conflict of interest including any financial, personal or other relationships with other people or organizations that could inappropriately influence, or be perceived to influence our work.

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Correspondence to Marcelo P. Barros.

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Communicated by William J. Kraemer.

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de Souza-Junior, T.P., Yamada, A.K., Simão, R. et al. Supra-physiological doses of testosterone affect membrane oxidation of human neutrophils monitored by the fluorescent probe C11-BODIPY581/591 . Eur J Appl Physiol 113, 1241–1248 (2013). https://doi.org/10.1007/s00421-012-2538-y

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