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Abstracts / Toxicology L

n goat (Mohammad et al., 1997). Inhibition of butyrilcholinesterasenduced less significant changes of pH in plasma and blood.

Conclusions: Metrifonate inhibited cholinesterases activity atll tested concentrations, but this was more prominent in bloodnd plasma samples than in erythrocytes. A lack of inhibition byhlormephos may be attributed to its chemical structure and aecessary metabolic activation to oxon.

eference

ohammad, F.K., Faris, G.A., al-Kassim, N.A., 1997. A modified electrometric methodfor measurement of erythrocyte acetylcholinesterase activity in sheep. Vet.Hum. Toxicol. 39 (6), 337–339.

oi:10.1016/j.toxlet.2009.06.551

17inetic of inhibition of esterases by non-stable compounds:MSF as a model

orge Estévez 1, Adolfo García-Pérez 2, José Barril 3, Eugenioilanova 3,∗

Instituto de Bioingeniería, Unidad de Toxicología y Seguridaduímica, Elche, Spain, 2 GlaxoSmithKline, Tres Cantos, Spain,Universidad Miguel Hernández, Instituto de Bioingeniería, Elche,pain

he kinetic analysis of esterase inhibition by acylating compoundsorganophosphorus carbamates and sulfonylfluorides) is some-imes unable to yield consistent results by fitting simple inhibitioninetic models to experimental data of complex systems. In thisork kinetic data were obtained for phenylmethylsulfonylfluoride

PMSF) tested at different concentrations incubated for up to 3 hith soluble fraction of chicken peripheral nerve. PMSF is a protease

nd esterase inhibitor causing protection or potentiation of delayedeuropathy and it is instable in water solution. A kinetic modelquation was deduced assuming a multienzymatic system withhree different molecular phenomena occurring simultaneously:1) inhibition; (2) spontaneous chemical hydrolysis of the inhibitor;3) ongoing inhibition (inhibition during the substrate reaction). Ahree-dimensional fit of the model was applied for analysing thexperimental data. The best fitting model is compatible with a resis-ant component (17%) and two sensitive enzymatic entities (42%nd 41%). The corresponding second order rate constants of inhi-ition (ki = 95.8 × 103 and 7.0 × 103 M−1 min−1, respectively) andhe chemical hydrolysis constant of PMSF (kh = 0.0631 min−1) wereimultaneously estimated. These parameters were similar to thoseeduced in fixed time inhibition experiments. The consistency ofesults in both experiments was considered an internal validationf the methodology. The results were also consistent with a signif-cant ongoing inhibition. The proportion of enzymatic componentshowed in this work is similar to those previously observed in inhi-

ition experiments with mipafox and paraoxon, demonstrating thathis kinetic approach gives consistent results in complex enzymaticystems.

oi:10.1016/j.toxlet.2009.06.552

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189S (2009) S57–S273 S213

18-glycoprotein induction as a cellular protection tool against

enobiotics toxicity�

enata Silva 1,∗, Anabela Cordeiro-da-Silva 2,3, Félix Carvalho 1,aria de Lourdes Bastos 1, Helena Carmo 1, Fernando Remião 1

REQUIMTE-Faculty of Pharmacy, University of Porto, Toxicologyepartment, Porto, Portugal, 2 Faculty of Pharmacy, University oforto, Biochemistry Department, Porto, Portugal, 3 IBMC – Institute ofolecular and Celular Biology, University of Porto, Porto, Portugal

he Caco-2 cell line is one of the most widely used human cell cul-ure models and has been accepted as a reliable in vitro model forhe rapid screening of the intestinal drug absorption and excretion

ediated by P-glycoprotein (P-gp) Recent in vivo studies performedy our group suggested the induction of de novo synthesis of P-gps a cellular protection tool against paraquat poisoning. Thus, theain objective of the present work was to evaluate Caco-2 cells

-gp expression and activity when exposed to doxorubicin (a P-p inducer), and to correlate these changes with paraquat toxicffects. Caco-2 cells were exposed to a range of doxorubicin concen-rations (0.1–100 �M), for a maximum period of 96 h, to establishhe inducer cytotoxic profile. Cytotoxicity was evaluated at differ-nt time points by the MTT assay. P-gp expression and transportctivity were evaluated by flow cytometry, using a CD243-FITC con-ugated antibody and rhodamine 123 as a P-gp fluorescent subtract,espectively. Exposure of these cells to doxorubicin resulted in aignificant increase of P-gp expression and in an increase of itsctivity as soon as 6 h. Based on these results, paraquat cytotoxic-ty profile was evaluated in the presence or absence of the selectedoxorubicin concentrations. The observed results were importanto characterize these cells in order to study the induction mecha-ism to protect cells from toxic compounds like paraquat, as it wasossible to reduce paraquat toxicity by a previous exposure of theells to doxorubicin or by a co-exposure to both of drugs.

cknowledgments: Renata Silva acknowledges FCT for her PhD grantSFRH/BD/29559/2006).

Selected for Oral Presentation.

oi:10.1016/j.toxlet.2009.06.553

19ffect of 10 oxime cholinesterase reactivators on cyclosarin-

ntoxicated rats

ana Zdarova Karasova ∗, Jiri Kassa, Kamil Musilek, Miroslavohanka, Kamil Kuca

Faculty of Military Health Sciences, Department of Toxicology, Hradecralove, Czech Republic

he therapeutical efficacies of seven acetylcholinesterase reac-ivators based on oxime were compared in in vivo (rat model)tudy. In this group, there were tested currently available oximesobidoxime, HI-6) and newly synthesized reactivators. These reac-ivators were considered for treatment against intoxication causedy cyclosarin.

The activities of AChE and BChE were assessed by stan-ard spectrophotometric Ellman’s method with acetylthiocholiner butyrylthiocholine iodides as substrates and 5,5′-dithiobis(2-itrobenzoic) acid as a chromogen, modified in wavelength 436 nmbecause of influence caused by hemoglobin).