Method for Synthesis of Oxidized Derivatives and Metabolites for Comparative Toxicity Testing

[Speaker] Sergei V. Redkozubov:1
[Co-author] Dmitrii I. Nezhinskii:1, Anastasiia A. Boitsova:1, Ekaterina A. Karpova:1, Iuliia A. Zhukovskaia:1
1:Biology, Volgograd State Medical University, Russia

Testing of medicinal substances to determine the toxicity should be performed in standard conditions in accordance with the Guidelines Protocols to confirm safety. However, statistics in Pharmacovigilance show that the using of medicinals is often carried out in conditions that are far from standard prescription. Moreover, it is necessary to predict the possibility of the formation of toxic derivatives in the manufacturing of the drug, its storage and possible modifications while using (for example when it is swallowed with chlorinated water). A separate problem is the adequate in vitro modeling of the metabolic activation of the substance in conditions far from the physiological norm.
Methods. For metabolic activation, a complex of different oxidizing enzymes linked with the well of the plate was used. H2O2 was added as the oxidative substrate in 0.1% concentration. ABTS (2,2'-azino-bis (3-ethylbenzthiosolin-6-sulphonic acid) served as a positive control and competitively oxidized substrate.
Chemical oxidation was carried out by gaseous chlorine in a chamber or by adding 1.0% H2O2 in an acidic medium. The residue of oxidant was neutralized in the chamber by gaseous hydrogen at an excess pressure of 0.5 Bar.
The amount of absorbed chlorine was determined from the discoloration of a number of control wells with a solution of methyl orange in a concentration of 0.05% to 0.001 %. ABTS also was used as an indicator of oxidation by H2O2 and reduction in the hydrogen chamber.
Comparative toxicity of derivatives was tested in the culture of infusoria Paramecium primaurelia after autogamy.
Results. A set of two indicators and available oxidizers was proposed. It allows confirming and measuring the effectiveness of chemical oxidation. The technique allows to model in vitro metabolic activation by a set of various oxidative enzymes, linked with the well. As a result, a preparation of oxidized derivatives of the starting compound was obtained. It is suitable for further toxicological tests without additional sample preparation.
Conclusions. The method can be used to predict the change in the toxicity of water-soluble substances during their oxidation and metabolic activation.

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