SINGLE WALLED CARBON NANOTUBES INDUCE PEROXIDASE ACTIVITY IN WHOLE BLOOD János Fent and Susan Lakatos Department of Pathophysiology, Laboratory Institute of Health Defence, Military Medical Centre of Hungarian Defence Forces H-1134 Róbert Károly krt. 44, Budapest, HUNGARY, latyakos24@gmail.com
Experimental
Introduction • Single walled carbon nanotubes (SWCNT) can get directly into the systemic circulation and might be toxic • In vivo defence mechanism against the cytotoxicity of SWCNTs is not completely understood yet • Literature data indicate that cytoxicity of carbon nanotubes can be mitigated by peroxidases (e.g. Zhao et al. (2011) J Phys Chem 115, 9536-9544; Vlasova et al., (2012) Toxicol Appl Pharmacol, 264,131-142). • Formerly we presented (3rd Virtual Nanotechnology Poster Conference, 2013, (P1312)) that SWCNTs do not induce the CD 63 activation marker expression on the surface of granulocytes.
AIM
Determination of enzyme activities from the cell supernatants
Materials • citrated human blood • SWCNT and PEG-SWCNT --- Sigma Aldrich • fMLP – physiological activator of granulocytes Dispersion of nanotubes into physiological medium • Sonication of nanotubes in distilled water • Stabilization of dispersion with bovine serum albumin • Sonication again • Adjusting physiological ionic strength and pH Sonication again Ex vivo cell activation with nanotubes
• Peroxidase activity with TMB as substrate in 50 mM citric acid+100 mM Na2HPO4 buffer, pH 5. The reaction was terminated with 200 mM glycine, pH 10.4 • β-glucuronidase activity with phenolphtalein β-glucuronide as substrate in 100 mM Na-acetate buffer, pH 4.5 The reaction was terminated with 4 M H2SO4
• Citrated human blood is incubated with 0.1 mg/ml Absorbancies were corrected for the nanotube dispersion at room temperature for 20 „blackness” of the given carbon min after cytochalasin-B pretreatment nanopearticles, estimated from the • Cell supernatant: centrifugation 100g, 5º C spectral properties of the reaction mixture.
Aim of this study was to reveal the ex vivo effects of SWCNT and PEGSWCNT exerted on human granulocytes
Results Peroxidase activity in whole blood N=14
Peroxidase activity of isolated leukocytes in serum-free buffer N=29
Peroxidase activity of isolated leukocytes in serum N=54
β-glucuronidase activity in whole blood N=21
• The release of the azurophil granules of granulocytes can be characterized not only by the appearance of myeloperoxidase activity but by the appearance of the β-glucuronidase activity and the expression of CD63 activation marker. • The SWCNTs induced peroxidase activity in whole blood can not be attributed to the release of azurophil granules, since there is no CD63 activation marker expression neither detectable βglucuronidase activity.